Medires Publishers - Article

Abstract

The phenylketonuria due to decreasing in lysine “AAG” and in Lys/ phosphorylation (which necessary for lysosomal digestive function), and associated with sever decreasing in Trp “TGG” , followed by mitochondrial dysfunction and lysosomal dysfunction, that followed by sever decreasing in phe /hydroxylase and in Tyr/ hydroxylase , and followed by decreasing in lysine acylation and in SFACs production which followed by decreasing in RA productive pathway and associated with increasing in both of TNFa and cholesterol accumulation, which reflect decreasing in estrogen synthesis and in dopamine production, that followed by increasing in the risk of ischemic damage and diabetes, and increasing in the risk of pulmonary disease . And we can conclude that both of Lys “AAG” and Trp “TGG” are so necessary for activating mitochondrial oxidative func- tions and necessary for promoting transport system, where Mitochondria is so necessary to activate antioxidant function and is necessary to promote all of Phe /hydroxylase, Tyr /hydroxylase, and Lys /acylation production which necessary for dopamine and RA synthesis respectively, where the PKU characterized by sever decreasing in antioxidant function, and sever decreasing in both Phe/ hydroxylase & Trp/ hydroxylase production, followed by decreasing in dopamine production and sever decreasing in retinoic acid production (which due to the reduction in Lys acylation and in SFACs production ). Retionic acid “RA” synthesis is activated by lysine acylation and is regulated by lysine acylation mediated by short fatty acid synthesis, which regulate aminoacyl-tRNAs production regulated by mitochondrial enzymes and regulate the mRNA production by E coli (regulated by phenylalanine) that necessary to activate antioxidant functions and cellular biosyn- thesis. The anti-hypertensive pathway mechanism is : the Lysine ¬¬>activate ATPase & GTPase which activate both lysosomes and Mitochondrial oxidative functions, followed by activating lysine/acylation ¬¬> which activate short fatty acid synthesis ¬¬> stimulate retinoic acid “RA” synthesis - ¬¬¬> where both Lys “AAG” and Trp “TGG” activate mitochondria repair and oxidative functions, which activate, Phe/ hydroxylase, and activate Tyr/ hydroxylase which activate dopamine , and Trp oxygenases followed by protection against PKU, pulmonary disease, ischemic damage, and CVD. The activation of antihypertensive pathway associated by activating lysosomes and Mitochondrial oxidative functions, that associated by activating NR4As pathway which responsible for activating GC-beta and both of Oxytocin and Nrf2, followed by Ang2-AT2 and VEGF-A productive functions and heme oxygenase production (notice dopamine regulate heme oxygenase mediated by dopamine beta-hydroxylase which regulated by Mitochondrial oxidative functions) followed by activating anti-inflamma- tory growth and processes . Lysine Methylation has so important roles to enhance and adopt hypertension through activating RA in pervious Antihy- pertensive pathway mediated by Phe /hydroxylase synthesis and Tyr /hydroxylase production followed by activating all of dopamine, retinoic acid synthesis, and NR4As pathway Where dopamine synthesis reflect its functional pathway to activate Antioxidant functions that prevent cholesterol accumulation, and increase the estrogen production (which it’s synthesis reflect potential protection from pulmonary disease), followed by activating the NR4As pathway which regulate all of Oxytocin, Nrf2 production, followed by Ang2-AT2 and VEGF-A production, which followed by activating heme ox- ygenase and anti-inflammatory growth and processes that prevent LV-hypertrophy, and prevent coronary calcification . Also, the increasing in Lys AAA with decreasing in Lys AAG and decreasing Trp TGG will cause decreasing in lysosome proper function, that will cause Decreasing in GTPase and decreasing in the Mitochondrial oxidative functions, and will cause decreasing in Phe /hydroxylase and in Tyr /hydroxylase followed by decreasing in lysine acylation and in Trp oxy- genases , that will be associated with increasing in cholesterol accumulation, and decreasing in both of IL17 production and estrogen functional pathway , which will cause increasing in the risk of T2D, pulmonary disease, ischemic damage, and increasing in the risk of PKU and CVD. . Both Lys and Trp are so necessary to activate transport system across cells membranes and across tissues that activate antihypertensive pathway, antibacterial function, and anti-atherosclerosis. Both Lys and Trp are having the Potency to activate lysosomes and Mitochondrial oxidative functions which activate lipid and protein digestion and enhance Phe hydroxylase and Tyr hydroxylase followed by activating lysine acylation which activate SFACs production and promote the RA synthesis and enhance antioxidant functions followed by dopamine syn- thesis and protection against ischemic damage and from both of diabetes and PKU. Key words: Lysine, Phenylalanine, Tyrosine, phe/hydroxylase, Tyr /hydroxylase, Lysine acetylation, Trp 2,3-oxygenases, AT- Pase and GTPase, Dopamine, Mitochondrial membranes, Lysosomes, E coli, Nrf2, Oxytocin, VEGF-A, Angiotensin-2 (Ang2- AT2), Phenylketonuria causes, CVD, and pulmonary disease _diabetic tissues, LV hypertrophy lysosomes, E coli, Aminoacyl tRNAs, Cardiomyopathy, TNFa, NLRP3 inflammasome, Retinoic acid, and short fatty acid, Dopamine hydroxylase, Trp 2,3 oxygenases, and, Heme oxygenases.

High Lights

Lysine “AAG” and Tryptophan “TGG” are so important for activating all of Phe/hydroxylase, Tyr hydroxylase, and Lys acylation for improving own necessary mRNA production and improving both of protein synthesis and dopamine, where sever decreases in Lys AAG and Trp TGG cause sever decreases in Phe /hydroxylase and in Tyr/ hydroxylase.

• The lysosomal damage and mitochondrial dysfunction are associated with phenylketonuria, followed by decreasing in both of dopamine and estrogen synthesis , followed by an increase in TNFa which increase the risk of pulmonary disease, CVD, and increasing in Tumor cancer.

• The lysosomal dysfunction and Mitochondrial dysfunctions (due to severe Decreasing in Lys “AAG” and in Trp “TGG”) cause increasing in cholesterol accumulation and decreasing in Trp oxygenase production, followed by increasing in TNFa that cause decreasing in lysine acylation followed by decreasing in short fatty acid production and decreasing in RA synthesis, that cause di- abetes, coronary artery disease, ischemic damage, and atherogenesis diseases.

• Lys activate lysine acylation mediated lysosomal function and short fatty acid production which necessary for the necessary retinoic acid productive functional pathway.

Methods and Results

The Lysosomal dysfunction is associated with increasing in undigested polymers, and decreasing in Mitochondrial func- tions, and followed by decreasing in both of E coli and in aminoacyl tRNAs (which regulated by lysine acetylation) productive functions, and followed by decreasing in mRNAs (produced by E coli) which followed by decreasing in both anti-inflammatory processes and decreasing in heart func- tions, and associated with increasing in TNFa which activate the increasing in NLRP3 inflammasome functions that in- crease the pathogenic survival . Lysine “AAA, AAG” and Trp “TTG” necessary amino acids for activating lysosomes digestive functions, and necessary to promote both of ATPase and GTPase productive func- tions, that GTPase necessary for activating mitochondrial repairs and functions which necessary for activating Phe / hydroxylase and Tyr /hydroxylase (where Tyr hydroxylase activate dopamine production), that prevent the cholesterol accumulation and prevent the increasing in TNFa, followed by enhancing the activation of antioxidant function which me- diated by activating Phe/ hydroxylase and Tyr/ hydroxylase which regulate dopamine production, then followed by acti- vating lysosomes and both of retinoic acid and NR4As pro- ductive pathway which regulate all of GC-beta, B-adrenergic, oxytocin and Nrf2 production . Decreasing in both of lysine, and Trp, cause mitochon- drial dysfunction, and Lysosomal dysfunction followed by increasing in TNFa and appearance of incurable med- ical cases: Lysosomal dysfunction associated with deficiency in Lysine phosphorylation, followed by decreasing in mitochon- drial functions and decreasing in all of Phe/ hydroxylase, Tyr/ hydroxylase, lysine acylation, and estrogen production (which regulated by OPA1 synthase oxidative functions ) which followed by decreasing in E coli functions and de- creasing in aminoacyl-tRNAs production, then followed by decreasing in retinoic acid and protein biosynthesis mediat- ed by decreasing in mRNAs efficiency (which produced by E coli) , and increasing in both of cholesterol accumulation , and undigested polymers precipitation in blood vessels, then their level in plasma will be elevated , that will cause pulmo- nary disease and cardiovascular disease . The lysosomes dysfunction is due to lysine dysfunction which associated with decreasing in Phe/hydroxylase pro- ductive functions which reflect decreasing in mitochondrial oxidative function and in Tyr/ hydroxylase, followed by decreasing in aminoacyl tRNAs production,and decreasing in dopamine biosynthesis , that will lead to abnormal autophagy production and reduction in NR4A pathway that followed by reduction in the antioxidant functions . That, some studies reported: Defective lysosomes lead to abnormal autophagy, activation of inflammation, and reduc- tion in the infection control.[1] The Lysosomal damage reflect increasing in the undigested polymers and reflect sever decreasing in GTPase which cause Mitochondrial dysfunction, that both of cholesterol and undigested polymers will be responsible for activating TNFa production which will activate the NLRP3 inflammasome which activate pathogenic pathways. That it has been approved that: the Lysosomal damage has been implicated in activating NLRP3 inflammasome in response to crystals or particulates. [2] Also, The Lysosomal dysfunction is associated with activat- ing the NLRP3 inflammasome in chronic unpredictable mild stress-induced depressive mice. [3] So, Lysosomal damage reflect increasing in undigested polymers and in cholesterol followed by increasing in TNFa which promote the NLRP3 inflammasomes activities, which will increase the risk of CVD. The increasing in NLRP3 reflect increasing in TNFa, and re- flect severe reduction in both of lysosomal digestive functions and in mitochondrial oxidative function which cause decreasing in all of Phe /hydroxylase, in Tyr /hydroxylase, and in Lys/ acylation that can cause serious Pathogenic cas- es eg: diabetes, phenylketonuria, CVD, pulmonary disease, and ischemic damage. That some studies reported that: Ac- tivation of Inflammation in Patients with Cardiovascular Dis- ease are associated with Higher Phenylalanine to Tyrosine Ratios. [4] So increasing in inflammation reflected decreasing in lyso- somal function and in mitochondria, and reflect reduction in Lys and in Trp followed by reduction in Phe hydroxylase and Tyr hydroxylase that can reflect increasing in the risk of of CVD and PKU. Also, the lysine residues in cathepsin D were so important for phosphorylation as those in pro-cathepsin L. [5] While it has been reported that: Lysine fatty acylation pro- motes lysosomal targeting of TNF-α. [6] Where, lysine acylation production promotes lysosomal tar- geting TNFa through activating retinoic acids synthesis and reflect proper lysosomes and Mitochondrial functions, and reflect proper production of both of Phe /hydroxylase and Tyr /hydroxylase which activate antihypertensive pathway which inhibit the TNF-a productive functions. So, we can conclude that as the function of lysine fatty acy- lation biosynthesis increased, as the TNFa will be decreased or will be inhibited, that mediated by proper activation to lyso- somes and mitochondrial functions, followed by proper acti- vation to Phe /hydroxylase and Tyr /hydroxylase and proper activation to antihypertensive pathway. So lysine is the major amino acid in cathepsin D that is so important for phosphorylation through activating both ATPase and GTPase which necessary for activating mitochondrial repairs and its oxidative functions, which activate lysosome digestive functions, therefore prevent the accumulated cho- lesterol and then prevent the increasing in cholesterol and inhibit both of TNFa and NLRP3, that prevent the aorta and Blood vessels calcification. Where, studies reported that: Lysine functions Prevents Ar- terial Calcification. [7] And, the altered CNS excitability due to decreasing in lysine which followed by lysosomal dysfunction and Mitochondrial dysfunction, which followed by increasing in cholesterol and increasing in the undigested polymers which promote the in- creasing in TNFa production. That studies reported that: the Microglial activation and TNFα production mediate altered CNS excitability following peripheral inflammation. [8] And so, the lysosomal dysfunction (due to decreasing in ly- sine phosphorylation) is associated with decreasing in Lys acetylation and decreasing in Phe hydroxylase (which due to decreasing in mitochondria oxidative functions), and then associated with increasing in TNFa, and followed by increas- ing in the risk of calcification, cardiovascular disease, and pulmonary diseases. Also, studies reported that: Lysine suppresses myofibrillar protein degradation by regulating the autophagiclysosomal system through phosphorylation of Akt in C2C12 cells. [9] note that phosphorylation of Akt is the main for activating lysine Phosphorylation in lysosome that prevent or suppress myofibrillar protein degradation by regulating the lysosom- al digestive functions and activating mitochondrial oxidative functions. Also, lysosome not only is a place for cargo degradation but also plays crucial roles in regulating macrophage polarization. [10] that macrophage polarization mainly regulated by both E coli and mitochondrial functions where their functions mainly controlled by lysosomal function which regulated by the Lysine functional activities. The lysine phosphorylation which activate Phe /hydroxylase and E coli functions is mediated by activating mitochondrial repairs and functions which mainly controlled by GTPase production (that Lys & Trp are having important roles in ac- tivating GTPase production ), where mitochondrial function necessary to activate IL17 production followed by glucocor- ticoid beta and β-arrestin2 production that followed by acti- vating both of oxytocin and Nrf2 production (in availability of Leu, Cys, Tyr) to improve myocardial function through adopt- ing glycemic optimal percentages , adopt protein glycation, and form own related macrophages productive functions which adopt anti-inflammatory growth and processes. Where, studies reported that, L-Lysine is so important to Prevents Arterial Calcification (through activating lysosomal function and activating GTPase which promote mitochondria to prevent cholesterol and undigested polymers accumulation). [11] Also it has been approved that, Nrf2-mediated anti-inflam- matory polarization of macrophages. [12] Now I would like to clarify an important fact that: not only lysine important for activating the lysosome and mitochondria, but also the tryptophan is very important activator for lysosomal functions and for activating mitochondrial through its important ability to produce the GTPase which reactivate the mitochondrial repairs and functions. That it has been approved that: TRP channel 3 (TRPML3), a transient receptor potential cation channel localized to ly- sosomes. TRPML3 activation initiates lysosome exocytosis, resulting in expulsion of exosome-encased bacteria. [13] Trp has important role to activate lysosome phosphorylation by activating GTPase production which also has important role in activating mitochondrial repairs and functions, that TRP Channel has important role to Trigger Pathogens Expul- sion by activating GTPase production which has the function to activate both lysosome and mitochondria. Also, that previ- ous role of Trp to activate GTPase production can explain its role along the kynurenine (Kyn) pathway to prevents hyper- inflammation and induces long-term immune tolerance. [14] So it’s clear that both of lysine and Tryptophan amino acids are having so important roles in activating both of lysosomes digestive functions and mitochondrial oxidative functions, that both are now responsible for preventing the accumulation of undigested and undigested polymers and cholesterol , that both of Lys and Trp responsible to activate mitochondria which responsible to activate both of IL17 and estrogen, and are responsible for activating Phe /hydroxylase and Tyr /hy- droxylase production (regulated by mitochondrial oxidative functions) . The decreasing in both lysine and Tryptophan are associ- ated with lysosomal damage that cause decreasing in mito- chondrial oxidative function lead to decreasing in both of Phe /hydroxylase and Tyr/ hydroxylase that will cause decreasing in dopamine productive Functions and increasing in the inflammation that will cause phenylketonuria and cause sever decreasing in antioxidant functional pathways. Also the activation of IL17 by mitochondrial function will acti- vate GC-beta production and then will activate both of Oxy- tocin and Nrf2, where Oxytocin alleviates liver fibrosis via hepatic macrophages. [15] Where, oxytocin (synthesis) stimulates the AMP-activated protein kinase pathway (AMPK) involve the autophagic pro- cesses, and may support the renewal of mitochondria. [16] So, the Treatment with oxytocin can reduces the accumula- tion of proinflammatory cytokines and reduces immune cell infiltration (by reactivating lysosomal and Mitochondrial func- tions). That Oxytocin stimulates differentiation stem cells to cardiomyocyte lineages as well as generation of endothelial and smooth muscle cells, promoting angiogenesis. [17] Also it has been approved that: Tryptophan metabolism (Trp activate Mitochondrial oxidative functions and lysosomal function that adopt blood pressure by activating dopamine) important for treatment patients suffering from CVD. [18] So, CVD is due to sever decreasing in lysosomes in mitigat- ing region and functions that can be treated by tryptophan “TGG” and lysine “AAG” to which can activate both previous intracellular system to recover CVD. Notice that High tryptophan diet reduces extracellular dopa- mine, and can affect on the dopamine levels and functions which will be discussed later. Lysosomal dysfunction is associated with increasing in TNFa and decreasing in VEGF-A Firstly, the Lysosomal dysfunction induce apoptosis in chon- drocytes through BAX-mediated mitochondrial damage. [19] And the mitochondrial dysfunction promise the increasing and accumulation in cholesterol and pro-inflammation, that it has been approved that Chronic inflammation during IBD may be mediated by NLRP3 inflammasome activation. [20] And, free Cholesterol-loaded Macrophages Are an Abundant Source of Tumor Necrosis Factor-α and Interleukin-6. [21] And now, the Increasing in TNFa is related to increasing in cholesterol and decreasing in both of mitochondria and lyso- somes digestive function, that will cause decreasing in myo- cardial function. Where, TNF-alpha downregulates expression VEGF receptors in human endothelial cells [22] The Lysosomal dysfunction is associated with decreasing in mitochondrial function followed by increasing in both of cho- lesterol and TNFa production , where TNFa activate NLRP3 inflammasome pathway functions followed by enhancement the cardiovascular pathologies development . That studies reported that: the Lysosomal dysfunction is as- sociated with NLRP3 inflammasome activation. [23] And the TNf-a regulates transcription of NLRP3 inflam- masome components and inflammatory molecules. [24] Also, the increasing in lysosomal dysfunction reflect de- creasing in lysine followed by decreasing in GTPase and de- creasing in mitochondrial function then followed by increas- ing in undigested polymers and increasing in TNF-α which enhanced the development of a number of cardiovascular diseases. [25] Also, Patients with acute myocardial infarction had statisti- cally significant increased serum levels of PTEN & TNF-α gene activity. [26] So, it’s clear that Patients with acute myocardial infarction are having severe Decreasing in both lysosomal and mito- chondrial functions followed by increasing in the accumulat- ed cholesterol, undigested polymers, and TNFa functional activities, which originally caused by sever decreasing in both of lysine phosphorylation and Tryptophan functions, which followed by decreasing in dopamine synthesis. That both DPM and DBM are useful in acute cardiogenic cir- culatory collapse. [27] That previous study indicated that CVD characterized by severe reduction in dopamine, that’s why dopamine is useful for treating patients with CVD. And, the Myocardial Infarction is a Consequence of Mito- chondrial Dysfunction (that Mitochondrial dysfunctions due to reduction in Lys & Trp will cause increasing in TNFa and reduction in Lys /acylation that increase the risk of myocardi- al infraction). [28] And, In classical PKU, the serotonin and dopamine biosynthesis is inhibited. [29] So, it’s clear that both of CVD and PKU are characterized by severe reduction in dopamine, that means those two diseas- es are connected together in lysosomal and mitochondrial dysfunction, followed by increasing in the accumulated cho- lesterol, undigested polymers, and TNFa functional pathway, which originally caused by sever decreasing in both of lysine phosphorylation and Tryptophan functions. Also it has been reported that, the Tumor Necrosis Factor-α (TNFa) promotes and exacerbates calcification in heart valve myofibroblast populations. [30] Also, studies approved that the TNF-α antagonism is unlikely to be a beneficial therapeutic strategy in patients with acute myocardial infarction. [31] Also, the lysosomal dysfunction can Increase the concentra- tions of TNF-α which are found in acute and chronic inflammatory conditions. [32] Now, positively the Genetically-predicted TNF levels are as- sociated with coronary artery disease and inversely associ- ated with overall cancer. [33] Also, Lysine is responsible to Prevents Arterial Calcification in adenine-induced uremic rats. [34] And Nuclear ATR ly- sine-tyrosylation protects against heart failure. [35] Where, cerebral ischemia due to extensive calcified vasculopathy, disruption of the basal gangliathalamo-cortical cir- cuit, and nigrostriatal dopaminergic dysfunction are plausible pathogenic mechanisms. [36] So, lysine and Tryptophan are playing important role in Protecting from cerebral ischemia, Heart Failure, Prevents Ar- terial Calcification by enhancing lysosomal Functions and mitochondrial oxidative functions followed by activating all of Phe hydroxylase, Tyr hydroxylase and Lys acylation which activate retinoic acid synthesis (which prevents ischemic damage) followed by activating estrogen production which prevent the accumulated cholesterol and un-digestive poly- mers. [37] Where, VEGF production regulated by angiotensin-2 which controlled by mitochondrial function, which regulated by intracellular ATPase and GTPase (that GTPase production regulated by both of Lys “AAG” and Trp “TGG”) That studies approved that, mitochondrial function control delivery of nutrients and oxygen to tissues through the ex- tension of blood vessels by angiogenesis. [38] So, decreasing in lysosomal function followed by reduction in mitochondrial function will increase all of cholesterol TNFa production and reduce angiotensin-2 and VEGF-A productive functions that can be the result of of increasing the risk of cerebral ischemia, CVD, and Arterial Calcification, followed by reduction in neuroprotective effects on hypoxic motor neuro, and decreasing in the protection against memory impairment “figure 1 “ “which Explains, describes and summa- rizes the basics of the importance of lysine and tryptophan in this work”. Where, VEGF-A also has a neuroprotective effect on hypoxic motor neurons, and is a modifier of ALS (amyotrophic lateral sclerosis). [39] Also, it is important to note that: VEGF A in serum protects against memory impairment in APP/PS1 transgenic mice by blocking neutrophil infiltration. [40] That reduction in VEGFa reflect reduction in dopamine fol- lowed by memory impairment. Also IL17 production which activated by mitochondrial function is important activator to Glucocorticoids-beta synthesis via NR4As pathway (and prevent organs failure cholesterol accumulation), followed by activating B-arrestins and both of B-adrenergic and Nrf2 syn- thesis followed by activating angiotensin-2 (Ang2-AT2) and VEGF-A productive functions. [41] Also, both of Lys and Glu (Lys AAG is reversed copy of Glu GAA) are having important role in stabilizing leucine “CTT” functional pathway (which important for brain function and for increasing memory functions too) , and are important for activating mitochondrial function which activate IL 17 pro- duction which activate NR4As pathway started by activating GC-beta, followed by activating angiotensin-2 and VEGF-A production, which reflect reduction in TNFa and in choles- terol. So, Lysosomal dysfunction is associated with decreasing in mitochondria function and decreasing in VEGF-A followed by increasing in TNFa, where TNFa activate NLRP3 inflam- masome pathway functions followed by activating the cardiovascular diseases, myocardial infarction as described in “Figure 1”, (figure 1is explanation of main aspect of the importance of lysine & Trp in lysosome & Mitochondrial oxida- tive functional pathways). Both lysine and phenylalanine important for treating pulmonary disease: Firstly, pulmonary diseases characterized by mitochondrial dysfunction followed by decreasing in Estrogen and increas- ing in cholesterol accumulations, (where cholesterol is the substrate for estrogen synthesis regulated by synthase en- zyme), followed by decreasing in IL17 and GC-beta produc- tion and decreasing in NR4As pathway, That it has been approved that: Estrogen Inhibition Reverses Pulmonary Arterial Hyperten- sion and Associated with Metabolic Defects. [42] And decreasing in Estrogen reflect increasing in cholester- ol accumulation , where mitochondrial repairs and functions activated by GTPase which produced by Trp “TGG “and Lys “AAG ” , that we conclude that :the estrogen Inhibition re- flect Inhibition in mitochondrial functions and reflect sever decreasing in both Lys and in Trp, that followed by Inhibition in NR4As pathway and Inhibition in both of angiotensin-2 and VEGF-A production, followed by increasing in TNFa, and in- creasing in the risk of Pulmonary Arterial Hypertension, and arterial calcification. Also, we can conclude that lysosomal dysfunction are the main for pulmonary disease Also, mitochondrial dysfunction is a systemic phenomenon during chronic obstructive pulmonary disease COPD [43] Also, studies reported that Mitochondrial Dysfunction as a Pathogenic Mediator of Chronic Obstructive Pulmonary Disease and Idiopathic Pulmonary Fibrosis. [44] And, the absence of Prolyl-4 Hydroxylase 2 (PHD2) indicate absence of Α-Ketoglutaric acid which is a short-chain fatty acid, that reflect sever decreasing in mitochondrial function and sever decreasing in both Lys and Trp that will be the result of increasing the risk of pulmonary, PKU, ischemic dam- age diseases, and can cause more Incurable diseases. That it has been approved: the Prolyl-4 Hydroxylase 2 (PHD2) Deficiency in Endothelial Cells Induces Obliterative Vascular Remodeling and Severe Pulmonary Arterial Hyper- tension. [45] Also, studies indicated that: GTP-bound Rab7 promotes mitochondrialysosome contact site formation and tethering, while mitochondrial TBC1D15 (Rab7-GAP) recruited to mito- chondria via Fis1 drives lysosomal Rab7 GTP hydrolysis at mitochondrialysosome contact sites. [46] So, pulmonary disease characterized by Inhibition GTP bound RAB-7 which (GTP bound RAB 7 MAINLY activated by Trp and Lys) control mitochondrial function, followed by Inhibition in mitochondrial functions and accumulation in the cholesterol and increasing in TNFa functions followed by in- hibition in RA and dopamine synthesis. Also other studies approved that, Ras proteins control mito- chondrial biogenesis and function. [47] Also, Lysine acylation synthesis reflect Activation to Mitochondrial Aconitase in the Heart [48], mediated by activating ATPase and GTPase production, and followed by preventing the accumulation of cholesterol and protect from pulmonary disease. Also, alterations in the expression of Lysine Methyltransfer- ase have been linked to the genesis and progress of sev- eral diseases, including cancer, neurological disorders, and growing defects, [49] So, the availability of lysine is necessary for lysine Acetyla- tion synthesis mediated by Activating Mitochondrial Aconi- tase in the Heart through activating GTP-bound Rab7 which promotes mitochondrial and lysosome functions followed by preventing the cholesterol accumulation, and followed by activating Phe /hydroxylase production which controls the phe- nylalanine metabolic functions, and followed by activating Tyr hydroxylase, Lys acylation, and RA synthesis preventing aorta calcification. Also, the absence of lysine will inhibit lysine fatty acylation followed by Inhibition to adipocyte B-adrenergic function which will be followed by decreasing in fatty metabolic process lead to cholesterol accumulation. That the presence of lysine is necessary to activate the lysine fatty acylation of an anchoring protein mediates adipocyte adrenergic functions (which has the antioxidant function). [50] Also in the other hand, TBX5 is required for patterning of the cardiac system and maintenance of cardiomyocyte functions, where lysine is required for acetylation which required for activating TBX5 for Regulating heart development and function, “figures 1 & 3”. [51] So, both of Lysine and Trp have so important roles for acti- vating TBX5 for Regulating heart development and functions through activating lysosomes and mitochondrial function which followed by activating Lysine fatty acylation which required for activating TBX5 function and antioxidant mediated by Phe /hydroxylase and Tyr/ hydroxylase productions which considered as stable mRNAs produced by E coli for modulat- ing heart and brain functions, and necessary for controlling phenylalanine metabolic pathway. I would like to give a little note that , arginine has import- ant role in activating tRNAs and accelerate transport system pathway , that Arg is required to be included in mRNAs which produced by E coli to accelerate immune functional pathway that accelerate antioxidant and transport systematic pathway (which mainly regulated and promoted by GTPase production ) function and prevent calcification and the accu- mulation of inflammation which can be the main for lysosomal and Mitochondrial disorders. That it has been approved that: l-Arginine prevents the over-expression of alkaline phosphatase (ALP, p < 0.001) and reduces matrix calcification (p < 0.05) in VICs treated with LPS. [52] And, Homo-arginine Supplementation Prevents Left Ventric- ular Dilatation and Preserves Systolic Function in a Model of Coronary Artery Disease. [53] And, decreasing in both of lysine and Trp will decrease mitochondrial oxidative function and Will be followed by decreasing in Lys acylation which followed by decreasing in TBX5 functional activities and decreasing in heart function and development , and also the decreasing in lysosome and Mitochondrial functions will be followed by increasing in in- flammation and in cholesterol accumulation, and will cause decreasing in both Phe /hydroxylase production and Tyr /hydroxylase which cause increasing in the risk of both of PKU and the cardiovascular disease that it has been approved that : the cardiovascular phenotype of adult PKU patients is characterized by high levels of inflammatory and oxidative stress markers, endothelial dysfunction and vascular stiffness “figure 2 “. [54] Also, it has been approved that the abnormalities in patients with phenylketonuria consisted of widening and cupping of the metaphysis, an intact zone of provisional calcification. [55] And, the patient with malignant phenylketonuria (PKU) who underwent both CT and MR Imaging is reported, that CT demonstrated the characteristic calcifications of the basal ganglia. [56] Also, decreasing in pyrimidine kinases (pyrimidine synthesis regulated by mitochondrial synthetase) reflect mitochondrial dysfunction, followed by increasing in cholesterol accumula- tion and decreasing in Phe /hydroxylase, which reflect reduc- tion in antioxidant function, and reflect reduction in estrogen synthesis and decreasing in dopamine synthesis, which fol- lowed by aorta classification and pulmonary disease. Where, studies reported that: Deficiency in pyrimidine kinas- es cause cholesterol accumulation and increase the risk of coronary artery disease. [57] So, decreasing in Phe /hydroxylase reflect decreasing in both of lysine and Tryptophan will cause decreasing in ly- sosomes function and cause decreasing in mitochondrial oxidative function that will be result in decreasing in both of Phe /hydroxylase and Tyr /hydroxylase and synthesis and in Lys acylation (which required for TBX5 function), and then the decreasing in lysosome and in mitochondria function will cause increasing in cholesterol and in TNFa which will be followed by increasing phenylalanine accumulation which will be followed by increasing in calcification and pulmonary disease “Figure 1 and 2 “. Also, phenylalanine and Phe/hydroxylase controlled by lysine functions through translation and by activating mitochondrial functions, that will be the reason of protecting Against mono- crotaline-induced pulmonary vascular remodeling and lung inflammation. Where studies indicated that 4-Chloro-DL-phenylalanine protects against monocrotaline-induced pulmo- nary vascular remodeling and lung inflammation. [58] So again, the lysine is basic controller to Phenylalanine and phe/hydroxylase Functions by translation process Lys “AAA, AAG “¬¬>Phe “TTT, TTC” where, both of lysine and Tryptophan TGG are the basic activator to mitochondrial repairs and oxidative functions which activate all of Phe /hydroxylase, Tyr hydrox- ylase followed by dopamine productive functions. Lysosomal dysfunction cause decreasing in mitochon- dria and in both of lysine Methylation and Phe /hydroxylase, where Lys and Trp required for alveoli and protect from pulmonary, and phenylketonuria: As we mentioned before lysine phosphorylation is the main activator for lysosomal function, where other studies ap- proved that: the Loss of laat-1 transporter caused accumula- tion of lysine and arginine in enlarged, degradation-defective lysosomes. [59] And, lysine residues are required for phosphorylation of pro-cathepsin L and are a common feature of the site on many lysosomal proteins. [60] That, as lysine decreased as so many of lysosomal protein synthesis will be decreased and cause lysosome damage. Also, both of Lys “AAG” and Trp “TGG “are so necessary for activating mitochondrial oxidative functions which is so necessary to activate antioxidant function where PKU char- acterized by sever decreasing in antioxidant function. That, oxidative stress is involved in the pathophysiology of the tis- sue damage found in PKU. [61] Also, decreasing in Phe /hydroxylase reflect decreasing in mitochondrial oxidative functions which demonstrate the en- ergy deficit and oxidative stress which characterized PKU pathophysiology. [ 62] _And the Mitochondrial dysfunction cause cellular injury and declined antioxidant capacity. [63] _where, the mitochondrial redox homeostasis is a potential target for disease treatment, That disruption of mitochondrial redox homeostasis in mus- cle resulted in energy defect and exercise intolerance. [64] _And, Mitochondrial necessary to activate antioxidant which the step toward disease treatment. [65] Also, Tryptophan “TGG“ has the role of reducing the mRNA levels of proinflammatory cytokine genes and enhance mi- tochondrial function by increasing the mRNA levels of mito- chondrial transcription.[66] _While Dietary lysine levels modulate the lipid metabolism, mitochondrial biogenesis and immune response.[67] And, also, it’s very important to note that Lysine Acetylation has the roles of Activating Mitochondrial Aconitase in the Heart [ 68], which mediated by short fatty acid production which activate retinoic acid function that adopt heart function That Short-chain fatty acids activate acetyltransferase p300. [69] Where, and lysine acyltransferases regulate protein synthe- sis (and retinoic acid synthesis) , and are direct sensors and mediators of the cellular metabolic state.[70] Also, the short fatty acids (SCFAs) promote retinoic acid synthesis and functions, that SCFAs crosstalk with RARα in dendritic cells as a critical modulator that plays a core role in promoting Th2 cells differentiation at a state of modified/ disturbed microbiome, “figure 1, 9 ”. [71] So, we can conclude that both of Lys and Trp are so neces- sary for activating mitochondrial oxidative functions which is so necessary to activate antioxidant function by promoting Phe/ hydroxylase & Tyr/ hydroxylase production, where the PKU characterized by sever decreasing in antioxidant func- tion and sever decreasing in Phe hydroxylase production. Also Lys acylation has the importance role in activating short fatty acid chains “SFACs“ and acetyltransferase production which mediated the importance of proper metabolic path- ways, where SFACs have important roles for activating ret- inoic acid “RA” functional pathway which has important role to activate antioxidant , that’s why PKU characterized by re- duction in Phe/ hydroxylase, (and reduction in Tyr/ hydrox- ylase due to reduction in mitochondrial function ) reduction in acetyl transfers, and then reduction in SFACs production followed by reduction in both of retinoic acid function and reduction in dopamine productive functions. Therefore, the lysine (and tryptophan) has an intense role in activating immune functions and anti-inflammatory process- es, that has the necessity of the Regulation of lysine methyl- ation emerged as a critical regulator of neurological function and disease. [72] Again the meaning of the necessity of Lys methylation to be produced in vivo is reflecting the proper availability of lysosomal and mitochondrial functions that reflect proper activation to Phe/ hydroxylase productive pathway and Tyr/ hydroxylase pathway, and also reflecting the production of Lys acylation which activate short fatty acid production which necessary for activating retinoic acid synthesis which neces- sary for activating RORs functional pathway and activating antioxidant function , which followed by activating NR4As pathway that enhance full controlling the antioxidant func- tions through producing oxytocin and Nrf2, and full angio- tensin-2 and VEGF-A functional pathway which activate anti inflammatory growth and processes to protect from many viral toxins and protect from many critical diseases including mitochondrial disorders and inflammatory disorders. The Mitochondrial dysfunction and lysosomal dysfunction are reflecting reduction in lysine acylation and in Trp, which reflect decreasing in short fatty acids “SFACs” production which followed by reduction in RA (which regulated by Lys acylation & SFACs ), and followed by increasing in cholesterol which activate increasing in TNFa that play important roles in inflammatory diseases, which characterized by swelling due to absence of lysosomal and Mitochondrial functions, where Some oncologists can be confused about the swell- ing in tissue that can consider it as tumor cancer without un- derstanding the basic of swelling. Also, dopamine (regulated by Trp) may have beneficial ef- fects on the respiratory system by decreasing oedema for- mation and improving respiratory muscle function [73] That Dopamine Reverses Lung Function Deterioration After Cardiopulmonary Bypass Without Affecting Gas Exchange. [74] So, The decreasing in both of lysine “AAG” and Tryptophan ”TGG” cause decreasing in lysosomal function and in mito- chondrial oxidative function, that will cause accumulation in the undigested polymers which cause lysosomal enlarge- ment, then followed by decreasing in lysine acylation and in aminoacyl tRNAs, that will cause decreasing in Phe/ hy- droxylase and decreasing in Tyr/ hydroxylase, followed by decreasing in E coli and decreasing in Estrogen and in dopamine productive functions, followed by increasing in oedema function and reduction in respiratory functions,”figure 1& 2”. Lysine and Tryptophan as discussed before are so important for activating mitochondrial oxidative function which necessary for both of pyrimidine kinases, and estrogen synthesis (which required for pulmonary alveolar functions). That it has been approved that, estrogen receptors, are required for the formation of a full complement of alveoli in fe- male mice. [75] Now, we can understand why both of lysine and Tryptophan are so important for treating pulmonary disease, where reduction in lysine and in tryptophan will cause increasing in Left vertical size (due to increasing in cholesterol and in TNFa, and decreasing in GTPase and in estrogen which due to decreasing in mitochondrial function ), where Lys phosphorylation and Tryptophan are important for activating mitochondrial functions, mediated by activating both lyso- somes and E coli functions, and followed by activating Lys acylation and Phe /hydroxylase productions . Also, the Aztreonam lysine “AZLI” (an inhaled lysine salt for- mulation) is effective, safe and well tolerated in the treatment of acute pulmonary exacerbations of CF. Superior improve- ments in lung function and quality of life suggest AZLI may represent a new treatment approach for acute pulmonary exacerbation. [76] And for sure the decreasing in mitochondria function will be followed by decreasing in phe /hydroxylase, in Tyr /hydroxy- lase, and in Lys acylation then will be followed by accumulat- ed Phenylalanine, which followed by reduction in dopamine biosynthesis, which followed by increasing in oedema forma- tion and decreasing in respiratory muscle functions “figure 2”. Also, the Inhibition in dopamine (due to Inhibition in both of Lys /hydroxylase and Phe /hydroxylase) is Associated with and reflect the phenylketonuria disease and connected to ly- sosomal dysfunction. Where, Phenylketonuria (PKU) is an autosomal recessive disorder caused by reduction in Phe /hydroxylase, which re- quired for Tyr /hydroxylase synthesis, which is essential for dopamine production. [77] Also, adult Patients Suffering from Phenylketonuria are suf- fering from reduction in Cerebral Fluoro-l-Dopamine Uptake. [78] Where , the accumulation of phenylalanine in the blood of patients suffering from phenylketonuria, are suffering from deficiency in both of lysine hydroxylase and Phenylalanine hydroxylase will be the result of causing deficiency in mito- chondrial oxidative function and deficiency in Estrogen and RA synthesis which followed by accumulation in phenylala- nine and reduction in Tyr/ hydroxylase, and reflect reduction in all of GTPase and mitochondrial function, that will cause reduction in dopamine synthesis (and reduction in antioxi- dant and increasing in the formation of oedema formation which associated with reduction in respiratory muscle function ), ” figure : 1& 2”. [79] So, sever decreasing in Lys and Trp will cause the lysosomal damage and mitochondrial dysfunction followed by the caus- ing of phenylketonuria (or the appearance of phenylketon- uria)) which Associated with reduction in Phe/ hydroxylase and reduction in all of Tyr /hydroxylase, Lysine acylation, and estrogen synthesis, and associated with decreasing in dopamine, followed by increasing in the risk of pulmonary disease, CVD. Again, not only lysine are important to regulate mitochon- drial oxidative functions, but also tryptophan which promote GTPase production which promote mitochondrial oxidative functions, and is also is important to promote dopamine pro- duction where it has been reported that: Trp is necessary to regulates DAergic neurogenesis and Mitochondrial oxidative functions. [80] And, both of lysine and Tryptophan are having important roles in activating mitochondrial oxidative function that en- hance the Phe/hydroxylase and Tyr hydroxylase synthesis and prevent the accumulated phenylalanine and then control the reduction in oedema formation and improving the respi- ratory functions through promoting, Lys acylation, RA syn- thesis, estrogen production which improve all immune met- abolic pathway which necessary for protecting both of heart and brain function, and also are important for promoting IFNs production which will be discussed later. Also, lysine acylation necessary for activating E coli, and allows the actual effects of lysine acetylation for protein productive functions. [81] Also, as lysine necessary for activating lysosomal function, as dopamine also is very important for promoting lysosomal Functions and is strongly connected to lysine functions for promoting both of lysosomes and mitochondria function. That it has been approved that Inhibition of the dopamine transporter promotes lysosome biogenesis and ameliorates Alzheimer's disease-. [82] And so, we can conclude that: both lysine phosphorylation and Tryptophan are connected together in their functions for activating lysosomal function and mitochondrial function, followed by activating Phe/ hy- droxylase, Tyr/ hydroxylase production, and Lys acylation production, then followed by their roles in activating E coli functions, where the necessity of Lys acylation in activating retinoic acid synthesis mediated by activating SFACs pro- duction and followed by increasing in antioxidant functionsor “Figure 9 “. Lysine, phenylalanine, and tryptophan, promote dopamine synthesis and Mitochondrial functions that protect from alz- heimers disease Firstly, as we discussed previously that both lysine and Tryp- tophan are having their strong roles in regulating all of ly- sosomes, mitochondria, and E coli functions, as sever de- creasing in those amino acids can lead to sever decreasing in lysosomes and in E coli that will be the result of decreasing in heart function that lead to ji cardiac impairment. That, patients suffer from a lack of aminoacyl tRNA produc- tions and they suffer from lysosome and E coli dysfunctions. [83] Where previous study indicated decreasing in Lys will cause reduction in Lys acylation which will be followed by reduction in aminoacyl tRNAs synthesis and mainly cause reduction in lysosomal function which will be followed by in- creasing in accumulated undigested polymers and choles- terol and reduction in retinoic acids synthesis which regulat- ed by SFACs which it’s production regulated by Lys acylation function. And, the modulation of activating mitochondria and both of lysosomes and E coli (by Lys and Trp) can be a potential therapeutic for activating Lys acylation and both of Phe hy- droxylase and Tyr hydroxylase followed by activating both of RA and dopamine that are potential therapeutic strategy for age-associated cardiac impairment. Where studies approved that: the modulation of activating mitochondria and both of lysosomes and E coli can be a potential therapeutic strategy for age-associated cardiac im- pairment. [84] Also, Nuclear ATR lysine-tyrosylation protects against heart failure by activating DNA damage response. [85] And other studies approved that l-Lysine is an essential ami- no acid important for maintaining human health. [86] Now in the availability of proper mitochondrial function: the controlled Phenylalanine synthesis will stimulate the in- creasing in immune antioxidant by the Phe/ hydroxylase production (regulated by mitochondrial oxidative function), which increase the E coli functions and activate Tyr hydrox- ylase which will activate dopamine production (where dopa- mine considered as strong activator to antioxidant functional pathway). [87] And, both of Phenylalanine (Phe) and tyrosine constitute the two initial steps in dopamine biosynthesis “figure 1”. [88] Also we have to notice that, both of tryptophan and Tyr/ Hy- droxylase Regulate Dopamine Synthesis, where studies has been approved that: the Tyrosine hydroxylase (TH) catalyz- es the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. [89- 90] Also, the parkinson Diseases characterized by depletion of dopamine (which regulated by tryptophan, and by both of Phe/ hydroxylase and Tyr/ hydroxylase synthesis). [91] Also, The Dopamine (productive pathway) has the role of in- creasing the force of contraction, and increase the elevation of the beating rate, and the constriction of the coronary arteries. [92] Whereas mentioned previously both of lysine and Tryptophan activate mitochondrial function that promote both of Phe/ hydroxylase and Tyr/ hydroxylase production which activate dopamine production “figure 1&2”, while mitochon- drial synthase promote estrogen and IL17 production which activate GC-beta, followed by activating B-adrenergic and both of Oxytocin and Nrf2 production which considered as strong antioxidant and strong protection against pulmonary disease. And now we can understand that the mechanism of dopa- mine is necessary for promoting anti-hypertensive pathway by lowering and adopt blood pressure. Where, the decreasing in Phe / hydroxylase and in Tyr /hydroxylase will be fol- lowed by decreasing in dopamine and decreasing in antihy- pertensive pathway, “Figure 4&5”. Where studies approved that: Dopamine considered as promoting antihypertensive pathway, by lowering blood pressure. [93] Also, Dopamine: is considered as Important Antihyperten- sive Counterbalance Against Hypertensive Factors. [94] Where, the antihypertensive pathway mechanism is : the tryptophan and Lysine ¬¬> activate Mitochondrial oxidative functions ¬¬> will activate methylation ¬> which will activate Phe/ hydroxylase ¬¬> followed by activating Tyr/ hydroxy- lase and Lys acylation ¬> followed by activating both RA and dopamine ¬> where dopamine synthesis in vivo which nec- essary to lowering “adopt” blood pressure “figure 1& 4” , also activated antihypertensive pathway reflect the activation to NR4As pathway mediated by activating the estrogen and IL17 by mitochondrial synthase function ¬> which activate GC-beta production ¬> followed by activating both of Oxy- tocin and Nrf2 ¬> Ang2-AT2 and VEGF-A productive func- tions ¬>heme oxygenase ¬>anti-inflammatory growth and processes. Also, Tyr / hydroxylase are found in heart tissue that indi- cated the necessity of dopamine in adopting heart functions directly and indirectly by activating previous antihyperten- sive, that studies reported that: the activity of the enzyme tyr /hydroxylase showed that was present in both hearts and in tissue with total sympathetic denervation. [95] So, dopamine (regulated by tryptophan, Phe/ hydroxylase, and Tyr/ hydroxylase) are having the role of protecting heart function and brain function through activating antioxidant function and enhance mitochondrial function, followed by lowering “adopt” blood pressure. Also, it is reported that: L-lysine influences the selective brain activity in dependence on the biological significance of pain-induced behavior. [96] That L lysine has the role of activating previous antihyperten- sive pathway including the activation of Lys acylation which promote RA synthesis and reduce pain-induced behavior. So, it is clear that lysine and Tryptophan are so important to improve lysosomal function followed by enhancing the activation of mitochondrial proper functions followed by ac- tivating both of dopamine and antioxidant (by activating antihypertensive pathway) mediated by Improving both of Phe/ hydroxylase and Tyr hydroxylase y “Figure 1&4” , followed by activating NR4As pathway, which mediated by activating estrogen and IL17, that promote GC-beta and both of oxytocin and Nrf2 production which necessary for adopting antioxidant functions and protect against pulmonary disease, PKU and CVD . That, DA has direct autocrine effects on beta cells, and indi- rect paracrine effects through delta cells. [97] Where, deficiency in Phe/ hydroxylase followed by decreas- ing in both of antihypertensive pathway, and decreasing in Tyr/ hydroxylase will cause decreasing in all of lysosomal function and diseasing in mitochondrial function, and then decreasing in dopamine, which followed by the increasing in Blood pressure and decreasing in antioxidant function , that will be the result of causing PKU symptoms which is charac- terized by an accumulation of traditional cardiovascular risk , high levels of inflammatory and oxidative stress markers, endothelial dysfunction and vascular stiffness . [98] Decreasing in Retinoic acid related orphan “RORs” is fully connected to lysosomal dysfunction and mitochondrial dys- function that cause increasing in both of TNFa and choles- terol: There are strong cooperation between Lys, Trp, and RORs productive functions in activating immune functionality , that both Lys and Trp are very necessary for activating both of lysosomes and functions and mitochondrial functions which responsible for promoting RORs productive pathway that their activities reflect Inhibition to cholesterol accumulation and activate both of IL17 and estrogen productions, and also their activities consequently inhibits TNFa production (through activating lysosome and Mitochondrial functions). And, I’ve mentioned previously that absence of lysine will inhibit lysosomal function and will inhibit lysine fatty acylation synthesis which necessary for SFACs production which by itself is necessary for Regulating retinoic acid “RA” produc- tive functions , where absence of lysine will be followed by Inhibition to adipocyte B-adrenergic function, which followed by decreasing in fatty metabolic process and increasing in both of undigested polymers and cholesterol accumulation, then cause decreasing in Retinoic acid functional pathway . That retinoic acid functional pathway is fully connected with and regulated by lysosomal function and by mitochondrial function, that Mitochondrial functions is regulated by both ly- sine and Tryptophan functional productive pathways, where retinoic acid functions promote the decreasing in TNFa func- tions. That studies reported that: Retinoic acid receptor-related orphan receptor A (RORA) inhibits the transcription and ex- pression of TNF,” Figure 1”. [99] So, it’s very clear that Lys and Lys acylation are playing so important role in decreasing TNFa through the role of Lys acylation in activating SFACs production which necessary for activating retinoic acid productive functions and activating antihypertensive pathway “figure 9”. While, the short fatty acids “SFACs” production which pro- moted by lysosomal digestive functions and by mitochondri- al oxidative function (and activate Lys acylation which im- portant for SFACs production) are very important for retinoic acid synthesis, where in case of lysosomal dysfunction and Mitochondrial dysfunction will cause reduction in lipid digestion and sever reduction in short chain fatty acids “SFAC” followed by reduction in RA synthesis and increasing in oxidative processes. That it has been approved that “SCFAs” affect DCs by facilitating retinoic acid synthesis “Figure 1”. [100] That’s why the retinoic acid-related orphan receptors RORs (regulated by mitochondrial function) is play a regulatory role in lipid/glucose homeostasis and various immune functions that reflect SFACs production and RA synthesis which reflect the activation of antihypertensive pathway. That decreasing in Trp “TGG” and in Lys “AAG” will cause reduction in both of lysosomes and mitochondrial functions, and will be associated with increasing in cholesterol and in undigested polymers which promote the increasing in TNFa productive functions and reduction in both RA and dopamine synthesis. There are strong cooperation between Lys, Trp, and RORs productive functions in activating immune functionality, and in protecting against both of diabetes and calcification, that both of lysine “AAG” and Tryptophan” TGG “are having im- portant role in the decreasing all of cholesterol, TNFa, and Calcifications, followed by protection from tumor cancer. The Retinoic acid is necessary for Retinoic Receptor-Related Orphan Receptors for running several cellular metabolic functions, that reduces apoptosis and oxidative stress, evaluate cardiac development, enhances the repair of infarcted myocardium, and the all-trans retinoic acid “ATRA” is having important role in dissolving Aorta calcification by promoting fission events throughout accelerating lysine and Trp func- tions for activating mitochondrial repairs and functions Where studies approved that: Retinoic acid (RA) established several functions during cardiac development, including ac- tions in the fetal epicardium required for myocardial growth, (also, retinoic acid reduce oxidative stress), ”figure 1”. [101] And the activated acyclic retinoic acid pathway can reflect activated ribosomes and mitochondrial function for activat- ing lysine acylation which necessary for activating SFACs production followed by activating Phe/ hydroxylase, Tyr/ hy- droxylase production, and activating both of RA and estrogen synthesis which followed by activating IL 17 production from acting on IL2 & and IL6 (which means previous processes control the Dendritic cells function), followed by activating GCs-beta and both of oxytocin and Nrf2 which followed by activating angiotensin2 and VEGFa functional pathway. Where studies approved that, acyclic retinoic acid pathway can specifically block calcification in a vascular cell, ”Figure 9”. [102] I would like to mention that: it’s clear that the high level of endothelial dysfunction is the sign of mitochondrial dysfunc- tions, and reflect sever decreasing in lysine and Tryptophan and reflect increasing in cholesterol and increasing in TNFa followed by un controlling to Dendritic Cells functions and then followed by increasing in vascular stiffness. Also notice that, as both of lysosomes and mitochondrial dysfunction as the function of RORs will be decreased and associated with decreasing in antioxidant function and de- creasing in lipid metabolic pathway, as will be associated with decreasing in SFACs production , followed by increasing in TNFa and increasing in the risk of high blood pressure and increasing in the risk of heart failure, that studies approved that : the proteomic analyses of human and guinea pig heart failure (HF) were consistent with a decline in resident cardiac all-trans retinoic acid ”ATRA” “Figure 1”. [103] Also, the reduction In ROR pathway reflect Diabetic cardio- myopathy. [104] So , Reduction in RORs pathway reflect decreasing in ret- inoic acid synthesis and reflect decreasing in Lys-acylation production which followed by decreasing in SFACs production and reduction in both of lysosomes and mitochondri- al functions that reflect defect in both of Lys “AAG” and Trp “TGG” availability, followed by reduction in GTPase produc- tion and then reflect reduction in RORs subunits produc- tion (which regulated by OPA1 function ), then followed by reduction in the estrogen production and increasing in the accumulated androgen , and Associated with accumulation in both of cholesterol and undigested polymers which pro- mote the increasing in TNFa, which followed by decreasing in dopamine production and increasing in Blood pressure followed by increasing in calcifications, and cause Diabetic cardiomyopathy, and Pulmonary disease . Where, studies approved that The Retinoic acid promote the increasing in the mRNA levels and protein synthesis of matrix Gla protein, and calcification inhibitory molecule, in human coronary artery and aortic valve cells “Figure 9”. [105] The sever decreasing in Lys “AAG” and in Trp “TGG” cause decreasing in lysosomal Functions and both of in anticoagulant and in antihypertensive pathway that Cause atherogenesis, and coronary Calcifications: Lysosomes are Membrane-bound organelles with roles in processes of de- grading and recycling cellular waste, cellular signaling and energy metabolism. That sever decreasing in lysine and in tryptophan (with decreasing in dopamine) cause lysosomal storage disorders, survival of tumor cancer, autoimmune dis- orders, blood disorders, Atherosclerosis, and neurodegener- ative diseases. The lysosomal dysfunction promise the accumulated phenylalanine, where the absence of lys AAG reflect decreasing in the translations process and decreasing in the stability of the normal Phe functions that will cause Phe accumulation. Also, sever decreasing in lysosomal functions cause decreasing in mitochondrial function that cause decreasing in lysine acylation and in SFACs production, followed by de- creasing in retinoic acid synthesis and reduction in antioxidant pathway which followed by increasing in inflammation, and in cholesterol deposit in arteries that cause calcification and reduction in antihypertensive pathway. As I discussed previously, both of lysine and Tryptophan are necessary for activating mitochondrial oxidative functions and both necessary for reducing inflammation, reducing cho- lesterol, and protecting against Mitochondrial disorder and against the increasing fat deposition , through activating the SFACs production which activate RA synthesis and activate both of RORs pathway and antioxidant function , followed by decreasing or inhibiting the increasing in TNF-a , where now not only lysine phosphorylation necessary for lysosom- al Functions but also tryptophan is so necessary for pro- tecting and purifying lysosomal Functions and contribute in the SFACs production and in RA productive functions which started by activating mitochondrial functions and increasing the cellular transport system functions. Also the necessity of Trp can be started by activating mitochondrial oxidative functions which activate both Phe hydroxylase and Tyr hy- droxylase, and activate tryptophan-2,3-dioxygenase which necessary for protecting heart from ischemic damage. Where, studies approved that: Tryptophan “TRP” Channel Senses Lysosome Neutralization by Patho- gens to Trigger Their Expulsion. [106] And, as we mentioned previously that Lys which activate lysosomal function is so necessary for translating and stabiliz- ing Phe functions, that studies reported that: Involvement of lysosomes in substrate stabilization of tryptophan-2,3-dioxy- genase. [107] And other studies reported that: The tryptophan oxygenase are so necessary for protecting heart from ischemic damage, “figure 1, 9”. [108]. So, lys is very necessary to promote tryptophan functions which promote Trp dioxygenase which protect heart from ischemic damage. Also, it has been approved that: RAG-GTPases (which activated by Trp & Lys) and LAMTOR proteins are required for lysosomal leucine storage. [109] Notice Lys “AAG” is a reversed copy of Glu “GAA” where both activate Leu “TTC” function by translation processes, that means that both of Lys and Glu are stabilizing each oth- er and are very necessary for stabilizing Leu functional path- way. In other side, estrogen (regulated by mitochondrial functions) deficiency reflect diabetes and increasing in inflammations and cholesterol, followed by increasing in TNFa productive pathway, that studies reported that: Exacerbates Type 1 Di- abetes-Induced Bone TNF-α Expression and Osteoporosis. [110] So, severe Decreasing in Estrogen reflect sever decreasing in both Lys and Trp will cause sever decreasing in lysosome and Mitochondrial functions followed by decreasing in SFACs production and increasing in cholesterol accu- mulation and sever decreasing in Trp 2,3- oxygenases pro- duction, which associated by increasing in TNFa that cause diabetes, coronary artery disease, ischemic damage, pulmo- nary disease, and atherogenesis. The accumulated cholesterol is associated with undigested polymers due to lysosomal dysfunction lead to the deposit of cholesterol in blood vessels, calcification, and followed by increasing in TNFa synthesis “figure 4”. That, increasing in the deposition of cholesterol in the blood vessels in patients reflect decreasing in fat metabolism and decreasing in lysine acylation, followed by decreasing in SFACs production that followed by decreasing in RA syn- thesis and increasing in TNFa production which increase the risk of coronary artery disease and atherogenesis. That it has been reported that: the deposition of free choles- terol in the blood vessels in patients associated with coro- nary artery disease and atherogenesis. [111] So, the Deposition of cholesterol in the blood vessels not due to deficiency in estrogen biostatistics but due to sever decreasing in lysine and in Tryptophan, and reflect decreas- ing in Estrogen, that reflect lysosomal and Mitochondrial dysfunction, and reflect decreasing in lysine acylation which followed by decreasing in both of SFACs and retinoic acid synthesis which followed by decreasing in antihypertensive pathway, ” Figure 4 & 9”. The decreasing in fat metabolism will be main reason for ath- erogenesis, and sever decreasing in lysosomal function and in mitochondrial functions can be the main for decreasing in fat metabolic pathways. That lysosomal dysfunction has important role in atherogen- esis. [112] Also, the disruption of Lysosome Function will cause de- creasing or mutation to its functional pathway that will pro- mote accumulation of undigested polymers and cholesterol that will be result of promoting NF-κB production and patho- genesis including phenylketonuria, neuro degenerative diseases, aorta calcification, and tumor survival. [113] And, the Lysosome dysfunction cause neurodegenerative diseases. [114] Also, Lysosomes dysfunction contributes to the cardiovascu- lar disorders which is known as lysosomal storage disorders. [115] So now , it’s clear to me that lysosomal dysfunction is due to sever decreasing in both of Lys and Trp which contributes to mitochondrial dysfunction, that associated with increas- ing in cholesterol accumulation and followed by decreas- ing SFACs and in RA synthesis, and followed by decreasing in Phe/ hydroxylase and in Tyr/ hydroxylase productive pathway, followed by decreasing in Trp 2, 3 dioxygenases and associated with increasing in TNFa production, that will cause increasing in in the risk of cardiovascular disorders, neurodegenerative diseases, coronary calcification, isch- emic damage, and atherogenesis. Not only Lysine promote the anticoagulant, antioxidant and activate NAADP, but also dopamine has same previ- ous roles followed by anti-calcifications functions: Firstly, dopamine synthesis is a mediator and activator to VEGF productive functions for promoting anticoagulant roles. Where studies approved that: the Tailoring of the dopamine coated surface with VEGF loaded heparin/poly-L-lysine par- ticles for anticoagulation. [116] While, lysine promise the activation of Phe /hydroxylase, and Lys acylation which necessary for RA synthesis and activate antioxidant and increase anticoagulant, that studies reported lysine promise strong cooperation with RA to enhance anti- coagulant function, and to Prevent calcification “figure 5, 9”. [117] Also, Lysine-containing peptides can be used as promising antiplatelet drugs in prothrombotic conditions of the organ- ism. [118] But in the other hand, the tryptophan derivatives evaluated for antiplatelet aggregation activity “Figure 5 & 9”. [119] So, it’s clear that both of Lys and Trp are cooperating togeth- er for running anti-calcification and anti-coagulant functions that both are so important for protecting heart functions and brain functions mediated by dopamine productive functions. Both lysine and Tryptophan are necessary for transport sys- tem in neuron and in heart: Firstly, Uniqueness of Tryptophan in the Transport System in the Brain and Peripheral Tissues. [120] And, both of Lysine and tryptophan are facilitative amino acid transporters through distinct sodium-independent. [121] And, lysine, and tryptophan are possible modulators of growth, immune response, and disease resistance “Figure 5”. [122] In the other hand, lysine and Trp considered as having the roles of activating antimicrobial functions through their role of activating both lysosomal digestive functions and Mito- chondrial functions followed by activating both of RA and dopamine productive functions, and activating the proper transport functions , that it has been reported that : pep- tides composed of lysine (K)-tryptophan (W) repeats (KW) destroyed and agglutinated bacterial cells, demonstrating its potential as an antimicrobial agent “Figure 5, 9 ”. [123] Also L Arg and Trp may have distinct effects on the develop- ment of atherosclerosis. [124] The active transport is an energy-driven process, is activated by both of Lys and Trp functions, but is increased more in case of activating Trp function which activate GTPase pro- duction, that each GTPase recruits a unique set of effector on running transport systems, and those transitions help to define changes in the functionality of the membrane com- partments with which are associated, “Figure 5”. [125] So, both Lys and Trp facilitate transport system that activate amino acids transport across cells membranes through their role in activating GTPase production which activate Mito- chondrial functions (and activate lysosomal digestive func- tion) that activate antibacterial function and anti-atherosclerosis, mediated by the role of GTPase in running the cellular transport functional pathways. Pulmonary disease characterized by decreasing in tryptophan metabolic pathway: We've discussed previously that the lysosomal damage and mitochondrial dysfunction (due to sever decreasing in both of Lys and Trp) are connected to phenylketonuria and Asso- ciated with reduction in dopamine which followed by reduc- tion in Phe/ hydroxylase and reduction in Tyr /hydroxylase, which associated with decreasing in mitochondrial function, and decreasing in dopamine productions , and associated with decreasing in estrogen and followed by increasing in inflammation, that increase the risk of pulmonary disease, (depending on the % of the decreasing in Lys and Trp). Some studies approved that: DA have beneficial effects on the respiratory system by decreasing oedema formation and improving respiratory muscle function (only DA which regu- lated by lysosomal Functions and by Trp and also by proper mitochondrial function “not by TNFa” have beneficial effect on respiratory functions). [126] I would like to note that decreasing in GTPase which pro- duced by Trp TGG will cause reduction in Mitochondrial ox- idative functions and reduction in all of Phe/ hydrolase, in Tyr/ hydroxylase, in oxygenases, and then reduction in both of lysine acylation and dopamine production, and also will be followed by reduction in transport system that will cause delays to cellular metabolic function (due to reduction in GT- Pase), that will cause increasing in pathogenic survival and then progression in diseases pathways. Also, Patients with inflammatory lung disease characterized by decreasing in tryptophan metabolic pathway, that Pa- tients with acute exacerbations of COPD (AECOPD) have a unique metabolomic signature that includes a decrease in tryptophan levels. [127] Also, it has been reported that Hole hopping through tyrosine/tryptophan chains protects proteins from oxidative damage, “Figure 6, 9”. [128] Also, Histidine-tryptophan-ketoglutarate solution decreases mortality and morbidity in high-risk patients with severe pul- monary arterial hypertension. [129] Where studies reported that: the disruption of noncanonical functions of AARSs connects to various types of diseases. [130] So, it's clear again that the sever decreasing in lysosomal Functions due to sever decreasing in lysine and in Trp ( which protect from respiratory damages ) will be associated with sever decreasing in GTPase and decreasing in mitochondri- al functions, followed by decreasing in Phe /hydroxylase and in Tyr /hydroxylase production, and followed by decreasing in transport system, and increasing in cholesterol and in inflam- mation , followed by decreasing in dopamine synthesis and decreasing in antioxidant functions and in estrogen (where decreased estrogen characterized the pulmonary diseases) ,”Figure 6” , followed by increasing in the TNFa production and increasing risk of pulmonary disease , (depending on the % of the decreasing in Lys AAG and Trp “TGG” ) Lys activate lysosomal function and activate lysine acylation and short fatty acid production, followed by RA synthesis and improvement phospholipid functions: Lysine has strong role in activating GTPase and ATPase production which activate lysine phosphorylation which nec- essary for lysosomal digestive functions, while GTPase acti- vate mitochondrial function which activate all of lysine acetylation, Phe /hydroxylase which activate E coli functions, and activate Tyr/ hydroxylase followed by dopamine production. Notice that, lysine AAG is the reversed copy of glutamate Glu “GAA”, that are having same functions for activating ly- sosomal activation and activating antihypertensive pathway. While the deficiency in Lys AAG in vivo will revers deficiency in Glu GAA and consequently revers deficiency in the sta- bility function of leu CTT and in Phe TTC, so positively the severe Decreasing in Phe/ hydroxylase productive functions reflect accumulation in leu “CCT” and in Phe “TTC” and reflect sever decreasing in Lys “AAG” and in Glu “GAA” that will be followed by coagulation, calcification, & decreasing in antimicrobial function. Where, Lysine-selective molecular tweezers are cell pene- trant and concentrate in lysosomes. [131] notice that both Lys & Trp are strong activator to transport system, that’s why lysine is cell penetrant and concentrated in lysosome for ac- tivating Lys phosphorylation which promote lysosomal digestive functions, and antimicrobial functions. And, L-Lysine protects against sepsis-induced chronic lung injury “Figure 9”. [132] So, as L lysine protect against sepsis induced lung injury, as all of Glu, Leu and Phe are having the same role in protect- ing against lung injury under availability of proper translation process, and are having the same roles in activating mitochondrial oxidative function and then promoting Phe /hydrox- ylase and Tyr /hydroxylase production which can indicated their necessity in promoting Lys acylation functions, and their necessity in protecting from PKU, from pulmonary disease, and from lysosomal disorders. Lysine is so important to activate lysosomes digestive func- tion and Mitochondrial functions followed by activating Lys acetylation which activate and increase the phospholipids transport, and activate Phe/ hydroxylase production (regu- lated by mitochondrial oxidative function) which carry strong roles in promoting Tyr/ hydroxylase productions which acti- vate dopamine production, The synthesis of lysine fatty acylation (as I described before) will promotes lysosomal “digestion” targeting the TNF-α, and will activate RA synthesis which is important for activating antioxidant (and antihypertensive pathway), “figure 9”. [133] Also TEADs are long-chain fatty acylated at Conserved ly- sine residues too. [134] So, Lys activate lysine acylation mediated by lysosomal ox- idative function and short fatty acid production which nec- essary for retinoic acid productive functional pathway, and necessary for reducing TNFa, followed by protection from lung injury, and from PKU. Also, the necessity of lysine function has been indicated in this studies which reported LAAT-1 was required to reduce lysosomal cystine levels and suppress lysosome enlarge- ment. [135] Also, Lysine is a basic amino acid to activate transport sys- tem that is important on the vacuole membrane. Where, the absence of lysine destabilizes purified Ypq1 and causes it to aggregate. [136] Lysine also bind with phosphatidylinositol 3,5-bisphosphate for regulating trafficking and membrane dynamics by Direct Activation of Mucolipin Ca2+ Release Channels in the En- do-lysosome. [137] While, Trp residues of the JMD influence the electrostatic surface potential by controlling the position of neighboring ly- sine and arginine residues at the membrane–water interface. [138] that indicate there are strong cooperation between Lys and Trp for stabilizing each other in nature. That tryptophan is necessary to contribute lysine function and both are having important role in regulating membrane traffic and dynamic activities. It Is important to note that lysine-rich cluster necessary to activate the enzyme PtdIns(4,5)P2 , to establish further in- teractions with diacylglycerol and/or acidic phospholipids (for increasing phospholipids transport) , leading to the full acti- vation of PKCα .[139] While, Trp chiral forms promote formation of hydrogen bonds and/or hydration in the PO2- moiety of phosphate group, That neutral and anionic lipid bilayers are sensitive to Trp. [140] TNFa activate increasing in histone, while lysine, Trp, and both of phe/ hydroxylase and angiotensin-2 activate DNA synthesis, and alpha-amylase: As we discussed before that increasing in TNFa will reflect decreasing in both of lysosomes and Mitochondrial functions and reflect decreasing in lysine acylation and in RA synthesis that reflect decreasing in Estrogen synthesis and decreas- ing in IL 17 synthesis that will be followed by decreasing in Gcbeta synthesis and reduction in NLR4 pathway which responsible for activating Nrf2 and angiotensin2 productive pathway. Some studies reported that Histone acetylation and chroma- tin conformation are regulated separately at the TNF-alpha promoter in monocytes.[141] And Oxidative stress and TNF-a induce histone Acetylation and NF-кB/AP-1 activation in Alveolar epithelial cells: Poten- tial mechanism In gene transcription in lung inflammation. [142] And TNF-α regulates diabetic macrophage function through the histone acetyltransferase MOF. [143] While, Angiotensin II (which regulated by mitochondrial and regulate GC-beta and both of oxytocin and Nrf2) stimulates DNA and protein synthesis in vascular smooth muscle cells from human arteries. [144] Where, the activated angiotensin-2 productive pathway reflect strong role in activated lysosomal functions and Mito- chondrial oxidative functions and E coli activation which ac- tivate DNA synthesis. That studies reported that: the angiotensin-2 has strong role in activating DNA synthesis in organ culture. [145]. So, we can conclude that Lysine and Tryptophan are nec- essary for activating lysosomes and mitochondrial functions followed by activating E coli which regulate DNA synthesis in tissues, which necessary for activating anti-inflammatory growth and processes mediated by Controlling dendritic cells and activating IL 17 which activate Gc-beta production then followed by activating Nrf2 and An- giotensin2 productive functions. Also, it’s reported that Phenylalanine (while Lys stabilize the Phe functional pathway) regulates the synthesis of α-amy- lase, trypsin and lipase through mRNA translation initiation factors – S6K1 and 4EBP1. [146]. And in other hand, the Trp58 plays a critical role in substrate binding and hydrolytic activity of human salivary alpha-amy- lase. [147] So TNFa activated by cholesterol and can irregularly activate growth and the increasing in histone, while lysine and Trp are so important for activating alpha amylase, trypsin, and lipase and in other side are very important for activating phe/ hydroxylase and Tyr hydroxylase, followed by angioten- sin-2, mediated by activating E coli which activate the normal DNA synthesis followed by activating anti-inflammatory growth. Also, It looks to me that Lys and Trp are responsible for activating the normal function of human salivary alpha-amylase through activating the previous Lys &Trp functional pathway (where Lys is responsible for activating Phe through transactions). TNFa can Only activate dopamine in case of lysosomal dysfunction followed by increasing in the risk of CAD: Firstly, we’ve discussed before that the activation Phe hy- droxylase is so necessary for activating Tyr hydroxylase pro- duction which is so required for dopamine synthesis, while Phe hydroxylase synthesis is stabilized by Lys functions and lysosomal function and Mitochondrial oxidative functions, where dopamine has antioxidant function. While it has been reported that: Tumor necrosis factor-α modulates GABAergic and dopaminergic neurons. [148] While, the Systemic tumor necrosis factor-alpha decreases brain stimulation reward and increases metabolites of sero- tonin and dopamine. [149] Where TNFa which activated by undigested polymers and cholesterol (in lysosomal dysfunction) that can activate the mutated dopamine which cannot promote brain function and at same time reduce the antioxidant function. And the higher free dopamine levels (mutated or up normal dopamine molecular structure) are an independent risk fac- tor for future coronary events in CAD patients. [150] While, Lysine fatty acylation promotes lysosomal targeting of TNF-α. [151] While, Lys decreases mRNA and TNF-α protein in IL-1β-stim- ulated chondrocytes. [152] So, lysosomal dysfunction (due to the sever decreasing in Lys phosphorylation and sever decreasing in Glu amino a. functions) will cause accumulation of undigested polymers and cholesterol, which by itself promote the increasing in TNFa which activate pathogenic pathway, while the activa- tion of mutated or improper dopamine molecular structure by TNFa is due to the sever decreasing in Phe hydroxylase and Tyr hydroxylase which are so important for activating proper dopamine functions, and so it's indicated to me is reflecting the decreasing in lysosome and Mitochondrial functions, and so will increase risk of coronary artery disease. The dopa- mine when promoted by TNFa can promise its mutated mo- lecular structure which will not activate antioxidant function and may can promote mutated serotonin, that can increase the risk of coronary artery disease. Notice, the lysosomal dysfunction cause accumulation of TNFa which promote CAD which connected by Increasing in hypotension due to the decreasing GTPase which is essential for activating transport system and Mito- chondrial oxidative functions. That, Hypotension associated with patients with coronary disease. [153-154] While, Lys can treat CVD and hypotension, that Prevents Arterial Calcification in Adenine-Induced Uremic Rats. [155] Notice that: the availability of Lys AAA with decreasing in Lys AAG and Trp TGG will be the result of decreasing in GTPase, followed by decreasing in activating Mitochondrial oxidative functions, that will cause increasing in cholesterol accumu- lation and decreasing in both of IL17 and estrogen produc- tive functions, that will increase the CVD risk and T2D, that studies reported That: association of 2-AAA with future risk of T2D. And association of lysine with subsequent CVD risk. [156] So both of Trp TGG and the Lys triplet AAG as we discussed previously are so important for activating GTPase (and transport system) and activating both of mitochondrial and lysosomal digestive functions followed by preventing the cholesterol accumulation (through activating Lys acylation which activate SFACs and followed by activating retinoic acid productive function) , and then activate both of IL17 and GC-beta production followed by oxytocin and Nrf2 pro- duction through controlling dendritic cells functions . Also it’s very important to note that the proper functions of dopamine is in the availability of lysosomal proper Func- tions, and when regulated by Phe/ hydroxylase which nec- essary for Tyr/ hydroxylase production which promote dopa- mine production (but in availability of lysosomal dysfunction the dopamine will not adopt heart and brain function and will be replayed by TNFa production function which activated by cholesterol ) , where Dopamine increased pulse pressure, heart rate and circulating epinephrine € and norepinephrine (NE) levels. [157] Also, it has been approved that dopamine improve de- pressed myocardial function following acute coronary arterial embolization, at least temporarily. [158] Dopamine which promoted by both of Phe/ hydroxylase and Tyr/ hydroxylase increase antioxidants function due to the No of hydroxylase groups in phenolic rings which originally formed from Phe/ hydroxylase and Tyr/ hydroxylase. Where it has been reported that Dopamine has stronger antioxidant activity than other related compounds that is correlated to the numbers of hydroxy groups on the phenolic ring. [159] Notice Brain Disorders Due to Lysosomal Dysfunction (which reflect reduction in lysine acetylation and in both Phe/ hydroxylase and Tyr/ hydroxylase). [160] But, in case of lysosomal dysfunction (sever decreasing in Lys AAG), the TNf-a will improve dopamine synthesis which can be associated with CVD and heart failure. Where studies reported that Cardiac dopamine D1 receptor triggers ventric- ular arrhythmia in chronic heart failure. [161] Lysosomal dysfunction Cause LV hypertrophy and car- diomyopathy mediated by mitochondrial dysfunction, and increasing in TNFa: Firstly, it has been summarized that: the effects of anti-TNF-α treatment in patients with and without heart disease and de- scribes the involvement of TNF-α signaling in a number of animal models of cardiovascular diseases. [162] Also, TNF-α blockade may improve insulin resistance and lipid profiles in patients with chronic inflammatory diseases. [163] Where, the TNf-a is increased upon the lysosomal dysfunc- tion and then upon the increasing in cholesterol, that it has been approved that: the free Cholesterol-loaded Macro- phages are an Abundant Source of Tumor Necrosis Factor. [164] And the accumulation of cholesterol and pro-inflammation in myocardium will be subject to heart failure. [165] So, the lysosomal dysfunction is the Abundant Source of the accumulation of undigested polymers and cholesterol which are the Abundant Source of TNFa production, and conse- quently are the Abundant Source of improper macrophages functions, and then are the basic source of heart failure. Decreasing in GTPase followed by mitochondrial dysfunction will increase the risk of of heart failure mediated by decreasing in lysosomal functions and increasing in TNFa functions, where the decreasing in lysine and tryptophan reflect reduc- tion in GTPase and increasing in hypertension, that studies approved that: L tryptophan promise as a naturally occurring antihypertensive compound, “Figure: 9”. [166] So , the cardiomyopathy caused due to lysosomal dysfunc- tion (which subject to sever decreasing in Lys AAG and in Trp TGG ) followed by mitochondrial dysfunction, and fol- lowed by decreasing in both ATPase and GTPase which cause decreasing in mitochondrial oxidative function and in both of Phe /hydroxylase and Tyr /hydroxylase production , then will be associated with accumulation in cholesterol and in undigested polymers which subject to activating TNFa fol- lowed by decreasing in both of angiotensin-2 and VEGF-A, that will increase the improper proliferation in vertical tissue cells which cause increasing in LV hypertrophy and cause increasing in improper macrophages functions . Positively, Lysine with this triplet “AAG“ is the most effec- tive amino acid for activating lysosomes digestive functions and Mitochondrial repairs and functions (where it has been reported previously that AAA is associated with heart failure) , where ATPase, and GTPase produced by Lys and Trp are necessary for activating lysine Phosphorylation for promoting lysosome digestive functions, followed by activat- ing mitochondria and E coli functions which activate Phe/ hydroxylase, Tyr /hydroxylase, and Lys acylation production , while Trp “TGG” is so effective for promoting mitochondrial oxidative functions (through activating GTPase production) which necessary for activating dopamine and Trp 2,3,ox- ygenases production which reflect protection against isch- emic damage . Both of Lysine “AAG” and tryptophan “TGG” adopt hyper- tension, and activate antioxidant, followed by activating the protection from Type-2 Diabetes: Firstly Treatment with L-lysine seems to slow down the pro- gression of diabetic nephropathy. [167] And, phenylketonuria (which characterized by lysosomal dysfunction and Mitochondrial dysfunction, as discussed be- fore) associated with diabetes disease too. [168] So, treatment with lysine and Trp in proper arranged mo- lecular structure will reduce diabetes and phenylketonuria syndrome mediated by activating both of lysosomes and mi- tochondrial functions which activate E coli function and acti- vate nrf2, Angiotensin2 and dopamine productive pathways. Where, Tryptophan Predicts the Risk of diabetes through re- acting Mitochondrial oxidative function and re-activate proper transport system, and so will promote the Phe /hydroxylase and Tyr /hydroxylase production regulated by Mitochondrial oxidative functions, and then reactivate dopamine which con- sidered as antioxidant. Where, studies reported that: Trypto- phan Predicts the Risk for Future Type 2 Diabetes. [169] The decreased Trp reflect decreased GTPase and reduction in lysosome and Mitochondrial oxidative function, that reflect reduction in transport system pathway which will delay trans- port of amino acids and delay cellular metabolic pathway that will promote the accumulated cholesterol and undigested polymers. In general, we can conclude : lysosomal dysfunction reflect decreasing in Trp (and decreasing in dopamine syn- thesis in vivo), that associated with decreasing in GTPase which cause decreasing in both of mitochondrial OPA1 oxidative functions, and transport system, which consequently will cause reduction in Phe/ hydroxylase (phenylketonuria) , and reduction in both of Tyr hydroxylase and Lys acylation followed by accumulation in Phenylalanine which Associated with calcification in blood vessels (where calcifications due to accumulation of both of undigested polymer and cholesterol) That it has been reported that: Lysine is an lipid (a)-binding inhibitor acts as antioxidant, that Lysine harvesting is an an- tioxidant strategy and triggers underground polyamine me- tabolism. [170] And, the Accelerated lysine metabolism conveys kidney pro- tection. [171] Also, Lysosomal dysfunction and the altered activities of lysosomal cathepsins have been observed in the diabetic heart. [172] Also, Lysine (and Trp) functions slow down diabetic nephropathy Through activating lysosome and E coli followed by activat- ing mitochondrial function which followed by activating dopa- mine followed by activating estrogen and GC-beta through activating NR4As pathway which activate oxytocin and Nrf2 production. Where, Treatment with L-lysine seems to slow down the pro- gression of diabetic nephropathy. [173]. And, L-Tryptophan Suppresses Rise in Blood Glucose and Preserves Insulin Secretion in Type-2 Diabetes. “Figure 7”. [174] Lysine enhance cardiac function and protect from pul- monary disease: Lysine necessary for blood synthesis, And hemoglobin is known to contain lysine, hence a lysine deficiency would prevent hemoglobin synthesis. Also, the heme group itself must be synthesized, presumably from amino acids, and lysine may be the precursor. Lysine necessary for activating blood platelet function. [175] Lysine play imp role in activation lysosomal function and blood platelets functions, that as lysosomal dysfunction be- gin (due to sever decreasing in Lys phosphorylation) as will be followed by anemia. Nuclear ATR lysine-tyrosylation protects against heart failure by activating DNA damage response. [176] Lysine protect from pulmonary disease, that L-Lysine pro- tects against sepsis-induced chronic lung injury in male al- bino rats. [177] L-Lysine Decreases Nitric Oxide Production and Increases Vascular Resistance in Lungs Isolated from Lipopolysaccha- ride-Treated Neonatal Pigs. [ 178] Also, the Poly-l-Lysine Kills Bacteria mediated by activating the lysosomal digestive function, that it has been reported: Poly-l-Lysine Compacts DNA, Kills Bacteria, and Improves Protease Inhibition in Cystic Fibrosis Sputum. [179] Also, it’s reported that L-lysine treatment under certain con- ditions could sustain cardiac performance. .[180] While, other studies reported that: Tryptophan decreased in Chronic obstructive pulmonary disease (COPD). [181] Both Lysine and tryptophan necessary to control both of dendritic cells and MHC class I and class II, for promoting myocardial functions and anti-tumor: Dendritic cells are found in tissue such as lung mucosa, and epithelial cells, where those cells are controlled by both ly- sine and Tryptophan functions. That studies reported that Dopaminergic Signalling Enhances IL-2 Production and Strengthens Anti-Tumour Response. [182] Also, Lysine 9 (H3K9) Methyltransferase Recruitment to the Interleukin-2 (IL-2) Promoter. [183] Also, Lysine Acetylation Is a Highly Abundant and Evolution- arily Conserved Modification in Escherichia Coli. [184] So both lysine and Tryptophan enhance IL2 production through direct control to dendritic cells functions. Also it has been approved that: Lysosomes control of den- dritic cell function. [185] Also, it has been approved that: MHC Class II Presentation Is Controlled by the Lysosomal Small GTPase which pro- duced by Lys “AAG” and Trp “TGG”. [ 186] And, Dendritic cells and B cells are also APCs expressing MHC class II. CD4+ T cells recognize antigens presented on macrophages by using TCR. [187] But, follicular dendritic cells, implicated in presentation of na- tive antigen to B cells.[188] And, B cell subsets contribute to myocardial protection by inducing neutrophil apoptosis after ischemia and reperfusion. [ 189] And it has been reported that: B-cells might have potential applications in treating cardiovascular diseases.[190] So both Lys and Trp are so essential to promote dendritic cells which implicated in presentation of native antigen to B cells for contributing myocardial protection and for activating Anti-Tumors. So, it’s clear that lysine “AAG” and Trp “TGG” activate GT- Pase which control lysosome function and control both of E coli functions in heart and control Dendritic cell function are implicated in presentation of native antigen to B cells, mediated by controlling mitochondrial repairs and functions which regulate MHC Class II Presentation, that contribute to myocardial protection via activating antihypertensive and NR4As pathway. Also both Lys and Trp are contributing to activate dopamine which reduce adopt blood pressure and also activate Trp ox- ygenases which protect from ischemic damage).

Statistics

Statistical analysis could not be performed for this study, and statistical methods are not applicable. The reliability of the study is determined by evaluating the presence of three positive markers (CD105+, CD29+, and SCA-1+) and one negative marker (CD45-), each serving as a distinct indicator of BM-MSCSs.

Results

Flow cytometry was used to assess the purity of BM-MSCs. The analysis utilized a flow cytometry kit to measure the expression of specific markers: CD45-, CD29+, SCA-1+, and CD105+. The results indicated a high purity of BM-MSCs, with CD45- showing a negativity rate of 99.8%. The expression levels for CD105+, CD29+, and SCA-1+ were 97.6%, 88.3%, and 93.5%, respectively (Figure 1)

Figure 1: (Continuation). Flow cytometry result.

Discussion

In previous studies, the negativity rate for CD45- in mesen- chymal stem cells (MSCs) ranged from 97% to 99%. The positivity rates for CD105+ varied between 86% and 99%, CD29+ ranged from 75% to 99%, and the Sca-1+ positivity rate decreased to as low as 86%. In our study, the CD45- negativity rate was found to be 99.8%. The positivity rates for CD105+, CD29+, and Sca-1+ were 97.6%, 88.3%, and 93%, respectively [4-8] (Figure 1). The results of our study are consistent with findings from previous research. As highlight- ed, adhering to international standards for producing MSCs allows for their application in a variety of areas, including the treatment of vertebral marrow damage and wound healing in diabetic patients. Therefore, this study is both important and significant in advancing the use of MSCs.

Conclusion

Mesenchymal stem cells are a current issue in the world and have a wide range of experimental and clinical uses. This study provides information to physicians and researchers about how to produce mesenchymal stem cells. Therefore, the current study is important and noteworthy.

Ethics Committee Approval

The current study complies with ethical standards, which is a prerequisite. The Laboratory Animals Ethics Board of Selçuk University Experimental Medicine Research and Application Center gave its approval to the research protocol (Ethics Approval Number: 2016-43).

Conflicts of Interest

I declare no conflicts of interest.

Financial Disclosure

This study was supported by the Selcuk University Academic Staff Training Program (ÖYP) Coordination Unit (2013-ÖYP- 088)

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