Alzheimer’s disease (AD), the most common neurodegenerative disease and the first cause of dementia worldwide, has no effective treatment, and its pathological mechanisms are not yet fully understood. We conducted this study to explore the proteomic differences associated with Familial Alzheimer’s Disease (FAD) in olfactory ecto-mesenchymal stem cells (MSCs) derived from PSEN1 (A431E) mutation carriers compared with healthy donors paired by age and gender through two label-free liquid chromatography-mass spectrometry approaches. The first analysis compared carrier 1  and its control , and the second compared carrier 2  with its respective control cells (C2) to evaluate whether the protein alterations presented in the symptomatic carrier were also present in the pre-symptom stages. Finally, we analyzed the differentially expressed proteins (DEPs) for biological and functional enrichment. These proteins showed impaired expression in a stage-dependent manner and are involved in energy metabolism, vesicle transport, actin cytoskeleton, cell proliferation, and proteostasis pathways, in line with previous AD reports. Our study is the first to conduct a proteomic analysis of MSCs from the Jalisco FAD patients in two stages of the disease (symptomatic and presymptomatic), showing these cells as a new and excellent in vitro model for future AD studies. Alzheimer’s (AD) is the most common neurodegenerative disease, the first cause of dementia, and one of the top seven causes of death globally. Due to the accelerated epidemiological growth of aging people, cases will triple over the next 30 years ,2. CurreAD can be classified into Late-Onset Alzheimer’s disease (LOAD) or Sporadic Alzheimer’s disease (SAD) when it occurs in people over 65 years old and is of unknown etiology, comprising 95% of all cases, and Early Onset Alzheimer’s disease (EOAD) if it occurs in individuals under 65 years of age , dysarthE quantitative proteomics (Ultra performance liquid chromatography coupled with an ion mobility time-of-flight high-definition/high-resolution mass spectrometer), which uses proteome analysis with the highest coverage to look at changes in the proteomics profile, can be used to find novel pathways, molecular mechanisms, and therapeutic targets that may significantly advance our understanding of the disease 2+ or more charges into account. There were 257,971 peptides detected in the symptomatic patient and 185,976 peptides in the presymptomatic patient; accuracy was 74.72% and 83.15%, respectively, with a maximum of 5 ppm A,E. BaseAt the protein level, 4750 proteins were identified in P1 and 3971 in P2, averaging 5.7 and 5.67 peptides per protein. The 2870 and 2361 proteins were not quantified, and 330 and 246 were reversed sequences. The pattern of the significant values in P1 vs. P2 was maintained. Finally, 1553 proteins in P1 and 1277 in P2 were tested, with an average of 10.34 and 9.69 peptides per protein and a dynamic range of 6.5 and 6 orders of magnitude, respectively. These results revealed that the normalization of injection had been carried out correctly, with both conditions being highly sensitively comparable D,H, showAdditionally, we found three unique proteins for C1, twelve for C2, and eleven and twelve for P1 and P2, respectively . All of p-value ≤ 0.05, and a coefficient of variation (CV) ≤ 0.3. We removed the reversed proteins that were false positives and the proteins in each condition in one of the three duplicates. Fold change (FC) levels of downregulated and upregulated proteins, respectively, are 0.6 and >1.5 in the differential expression. With these restrictive filters, 703 and 517 highly reliable quantified proteins remained in P1/C1 and P2/C2, respectively. Of these, 359 and 195 were differentially expressed proteins (DEPs), respectively. For P1/C1, 118 proteins were upregulated, and 241 were downregulated. For P2/C2, 75 were upregulated, and 120 were downregulated, scattered in volcano plots  with −52 FC. The most over-regulated proteins with these stringent filters were Guanylyl cyclase C and PRP4B, with 34.9 and 25.9 FC, respectively. For P2, the two genes that were most negatively regulated were SCIN and EEF1E1-BLOC1S5, with −14.9 and −9.7, respectively, and the two genes that were most positively regulated were DNM1L and COPS8, with 94.5 and 35 of FC, respectively. HBB (hemoglobin subunit beta) was the most downregulated protein with −189.5 FC, followed by retroelement silencing factor 1 protein , Parkinson’s disease (PD), Huntington’s disease (HD), prion disease (PrD), and Amyotrophic Lateral Sclerosis (ALS), were interestingly enriched according to KEGG and STRING data .STIP1, an adaptor protein that controls the ATPase cycles of other chaperones like HSP70 and HSP90. SOD1, a protein implicated in ROS detoxification, and three prolyl hydroxylases  downregulated. The only elevated proteins .As opposed to the P2 interactome B of vesiTo compare the pathways between the DEPs involved in mutation carriers, Because our results include proteins involved in some signaling pathways that are essential in the biology of NDDs, we evaluated the conditions that might be involved using GeneAnalytics and WEBGESTALT with the OMIM and DISGENET databases . This alPI4KA, S100A4, and DES; 4 in blood, DNAH6, CEP152, CRMP1, ITGA11; and 25 GeneCards inferred as ADAM10, ALB, HSPA1A, HSPA5, MAPK1, NOS2, SOD1, and STAT1), HD, and surprisingly hereditary spastic paraplegia with 24 matched genes, ALDH18A1 as a causative mutation; 13 differentially expressed in skeletal muscle as BAG2, CANX, SCARB2, and UBR5; and 12 inferred genes . In the presymptomatic case, the analysis also revealed muscle degeneration and atrophy, particularly in the lower limbs, and abnormalities in upper motor neuron disease, in which weakness, spasticity, and hyperreflexia are classic symptoms. It is essential to mention that the presymptomatic patient presented hyperreflexia. GeneAnalytics shows 30 proteins matched with FAD  and 24 with Hereditary Spastic Paraplegia . These studies imply that progenitor cells of PSEN1 (A431E) mutation carriers may be connected with the clinic in these individuals, but further investigation is necessary to confirm these findings.Using GeneAnalytics, we obtained similar results for muscular dystrophy, ALS, FAD, with 32 proteins matched  mutation and healthy donors, two proteome profiles from various phases of FAD were examined. The significance of these DEPs was investigated further using bioinformatics and bibliographic techniques, including gene ontology analysis, KEGG pathway analysis, PPI network creation, and compared with other AD databases.The symptomatic carrier (P1) expresses eleven distinct proteins, principally concerned with viability, proliferation, migration, and inflammation, which may account for the carrier’s olfactory MSCs’ observed difficulties in growth and proliferation.WDR62, MCPH1, and CDK5RAP2. Numerous neurological disorders, including Alzheimer’s disease and microcephaly, have been linked to mutations in these proteins [WDR62 and CDK5RAP2 may indirectly affect the growth of neurofibrillary tangles (NFTs) since JNK and CDK5 kinases regulate tau phosphorylation [The proteins involved in neurogenesis, brain development, self-renewal, survival, and plasticity include proteins ,46. WDR6rylation .UTY and HIST1H1B genes, may impact genes associated with the pathophysiology of Alzheimer’s disease. H1.5 influences chromatin’s accessibility and structure, and UTY, a histone demethylase, is implicated in the epigenetic regulation of gene expression and cell development. After being identified in amyloid plaques, HIST1H1B has been found to have the ability to produce amyloid-like fibers [The expression of histone H1.5, encoded by the e fibers ,49.KIF5C is a kinesin that regulates organelles’ mobility across microtubules and mitochondrial transport. In PS1(E280A) mutant carriers, KIF5C decreased in mitochondria but increased in synaptosomal-enriched fractions [SCUBE2 participates in SHH signaling and, when overexpressed, decreases glioma cell proliferation, migration, and invasion [PIGN mutations cause epileptic encephalopathy and MCAHS1 . The protein PIGN transports phosphatidylethanolamine (PE) from the ER to the first glycosylphosphatidylinositol (GPI) mannose. In fibroblasts from individuals with PSEN1 and PSEN2 mutations, elevated PE levels have been discovered in mitochondria, plasma membranes, and ER-associated mitochondrial membranes (MAMs). These modifications may increase γ-secretase activity and cause the production of Amyloid-β [ractions . SCUBE2 invasion . PIGN mumyloid-β .ATP5PF, a component of the mitochondrial ATP synthase in charge of producing ATP, has been linked to Alzheimer’s disease, neurodegeneration, and oxidative stress as early manifestations of this pathology [KIAA0825 and ANKRD36C are unknown; however, they may be connected to several activities, including inflammation [Furthermore, MCAHS1 mutations created by CRISPR/Cas9 in C. elegans result in protein aggregation and the activation of the unfolded protein response (UPR) pathway in the ER . ATP5PF,athology ,55. The ammation .RTRAF, DIP2C, and PROX1), mitochondrial function (ATP5H and TIMM13), proliferation control (CNBP and PROX1), neuronal development (PROX1 and DIP2C), and cellular transport , suggesting a potential compensatory or resistance process in the cell to maintain proper cell function. Interestingly, neurological illnesses such as autism, neurodevelopmental disorders, deafness dystonia syndrome, microcephaly, Alzheimer’s disease, and several cancer types have been associated with RTRAF, DIP2C, YARS, CNBP, BIN3, DYNLRB1, and TIMM13. In addition, DIP2 paralogues and spastic hemiplegia are connected [The unique proteins found in the presymptomatic PSEN1(A431E) mutation carrier are primarily involved in transcriptional regulation (onnected .YARS) mutations have been linked to Charcot–Marie–Tooth disease. This neuropathy causes progressive loss of muscle tissue and touch sensitivity in different body parts [CNBP [Tyrosyl-tRNA synthetase  mutant carriers may point to a complex interaction of biological mechanisms that attenuates the effects of the mutation in MSCs at the early stages of the disease.Most downregulated proteins in symptomatic carriers are distinct proteins that appear to be connected to changes in cell proliferation, cycle arrest, differentiation, susceptibility to damage, and ROS generation.Unexpectedly, the α and β chains of hemoglobin, the protein that transports oxygen and carbon dioxide in cells of the erythroid lineage , are two of the most downregulated proteins in symptomatic mutation carriers compared to the control. Hb chains, however, have been found in non-erythroid cells , in primOur results support Ferrer I. et al. , who fouKIAA1551 (RESF1-retroelement silencing factor 1) is necessary to maintain the repressive gene state in undifferentiated embryonic stem cells [RASA2, CEP192, ZGRF1, and ITGA2 reduces cell migration, adhesion, and proliferation while also causing cell cycle arrest, which may have an impact on the phenotypic and the course of the disease as it is now understood to exist. The maturation of the mitotic centrosome and the formation of the bipolar spindle both depend on the protein CEP192 . Cytosolic microtubule organizing centers (MTOCs) were in excess in CEP192 knockdown cells [ZGRF1 null cells [em cells . Downregwn cells . Severalll cells .ITGA2), an extracellular matrix receptor for laminin, collagen, fibronectin, and E-cadherin, plays a role in promoting cell proliferation and invasion in different types of cancer [ITGA2 is also one of the most downregulated proteins in the presymptomatic carrier.Integrin alpha 2 , OAS3 (2′5′-oligoadenylate synthetase 3), and IFIT3 (Interferon-induced protein containing tetratricopeptide repeats 3), whose downregulation may prevent cell migration and proliferation. It is interesting to note that, in contrast to our findings, MxA expression has been found in senile plaques and reactive microglia in AD brains [IFIT3 in lung epithelial cells [Interferon induces several proteins, including D brains , and MxAD brains . Apoptosal cells .Desmin and Dysferlin are among the top 20 most downregulated proteins that are noteworthy to mention because of their involvement in myofibrillar myopathies and muscular dystrophy, which are diseases characterized by progressive skeletal muscle weakness, atrophy, autolysis/proteolysis, and autophagy of muscle cells . SCIN and LRP1B were downregulated, but UTY, a particular symptomatic carrier protein, was upregulated.Several oncogenes are among the proteins that are downregulated in the presymptomatic carrier, which may also indicate alterations in protein trafficking, apoptosis, and proliferation. Interestingly, the symptomatic carrier was altered by four of the proteins that were most heavily downregulated in the presymptomatic carrier. SCIN, LRP1B, CPNE3, TICRR, and PPP1R7 are connected to cancer cell invasion, migration, proliferation, and apoptosis. By organizing the cytoskeleton, the calcium-dependent actin filament protein, SCIN regulates exocytosis and vesicle transit [SCIN was knocked down [ transit . Dental ked down .LRP1B and APP interact, preventing APP from being internally processed and not favoring amyloidogenic processing [LRP1B expression and protects immature and adult neurons against fibrillar and oligomeric Aβ toxicity [Since LDL receptors are ApoE receptors and control both the clearance of Aβ and the amyloidogenic processing of APP, they are connected to the pathogenesis of AD ,81. PSENocessing . Additiotoxicity . HoweverEEF1E1-BLOC1S5, AIMP3. When it was lacking in mouse embryonic stem cells (mESCs), an accumulation of DNA damage occurred, which caused p53 to become transcriptionally active and lead to a loss of stemness and differentiation potential [AIMP3 is inhibited under hypoxia, slowing cell senescence, lowering mitochondrial respiration, and activating autophagy. Additionally, it is favorably controlled by Notch3 in human placenta amnion-derived mesenchymal stem cells (hpMSCs) [The macromolecular tRNA synthase complex includes the auxiliary protein-coding gene otential . However(hpMSCs) .MYH10) inhibits the Wnt/β-Catenin pathway and decreases glioma cell migration and invasion [Non-muscle myosin II heavy chain  and mtp53 are downregulated by the knockdown of ribophorin II (RPN2), an essential subunit of the N-oligosaccharyl transferase (OST) complex, in breast cancer stem cells [RNF6 is an E3 ubiquitin ligase protein participating in IFN-antiviral response and proteasome degradation [RNF6 is silenced, which increases GSK3 activity, decreases p-GSK3 inhibition of the Wnt/β-Catenin pathway in cancer cells, and suppresses MAPK/ERK signaling, which in turn reduces cell growth and proliferation [Heat shock proteins , which caused cell growth retardation by inducing cell cycle arrest at the G1/S checkpoint [S100A16, which inhibits proliferation, migration, and invasion via the JNK/p38 MAPK pathway [Many of the overexpressed proteins in symptomatic carriers are linked to cell proliferation, migration, and cell cycle progression via Notch1, β-Catenin, NF-kB, AKT, and ERK1 signaling, and pathways closely related to AD, including eckpoint , and S10 pathway .GUCY2C or GC-C), the most upregulated protein in the symptomatic carrier, inhibits phosphodiesterase 3 (PDE3), inducing cell cycle arrest, cytostasis, and senescence via activation of p21, PKGII, and p38 MAPK [Also, Guanylate Cyclase 2C or Heat Stable Enterotoxin Receptor  has been linked to psychiatric disorders, depression, and neurodegeneration [S100A16 promotes the Wnt/β-Catenin signaling pathway and the HRD1-induced ubiquitination and degradation of GSK3 and CK1 in cases of acute renal injury [The S100 calcium-binding protein A16 (neration . S100A16l injury . ER strel injury .PRPS2) acts as an oncogene by controlling the activities of matrix metalloproteinase 9 and the expression of E-Cadherin [TSC2 (Tuberous Sclerosis Complex subunit 2 or Tuberin) negatively controls mTORC1 signaling and is crucial for autophagy and dendritic formation [A key player in the production of purines and pyrimidines, phosphoribosyl pyrophosphate synthetase 2 , are also involved in the maintenance of mitochondrial morphology and dynamics, interact with Bax and Bak to promote cell apoptosis via reactive oxygen species [n et al.  reported species , form he species , and can species .DSCAML1 plays a role in several neurodevelopmental processes, including branching, migration, synaptogenesis, and dendritic self-avoidance. Dscam2 also inhibits the deposition of synaptic vesicles through an endosomal route dependent on phosphatidylinositol-3 kinase (PI3K) [The transmembrane immunoglobulin superfamily member e (PI3K) .NAGA was proposed as a genetic risk for schizophrenia [ophrenia  because ophrenia .STAM2 , a part of the ESCRT-0 complex that regulates receptor signaling and trafficking. It plays a regulatory role in the endosomal sorting of ubiquitinated membrane proteins [SRRM2 is overexpressed in the brain and blood of people with Parkinson’s disease [SRRM2 has been connected to neurodevelopmental disorders. Additionally, it accumulates and mislocalizes from cytosolic tau NFTs to nuclear splicing speckles in cells, mice, and the brains of AD patients [SDHA takes part in both the tricarboxylic acid (TCA) cycle and the mitochondrial electron transport chain (ETC). Inflammation-promoting fumarate buildup and enhanced mitochondrial respiration have been linked to increased SDHA function [Another interesting product among the most downregulated proteins is the endosomal protein proteins . It is sproteins . In HeLaproteins . The sca disease  and is n disease . Dysfuncpatients . SDHA tafunction .PHGDH) upregulated in the subventricular zone (SVZ), a neural stem/progenitor niche, may play a part in neurogenesis. Severe neurological abnormalities in PHGDH knockout mice include congenital microcephaly and psychomotor impairment [PHGDH and the astrocytic L-serine biosynthesis pathway are diminished in the AD brain and 3xTg-AD mice, according to a recent study by Le Douce et al. [PHGDH mRNA and protein levels rise as AD pathology and symptoms worsen in 3xTg-AD and P301S tau transgenic mice (PS19) and human AD brains [Phosphoglycerate dehydrogenase , a GTPase essential for mitochondrial fission. Abnormal mitochondrial dynamics, including increased fragmentation and decreased fusion, have been seen in Alzheimer’s disease (AD). In AD neurons, elevated DNM1L activity led to mitochondrial fragmentation and dysfunction [DNM1L prevents cell death, enhances synaptic function, and increases neuronal activity [One of the most increased proteins in the presymptomatic carrier is Dynamin-related protein 1  dysfunction and apoptosis [CSF1R) is a transmembrane protein that aids in the differentiation and survival of microglia. It is elevated in post-mortem samples from AD patients [CSF1R in forebrain neurons in mice accelerated excitotoxin-induced death and neurodegeneration [poptosis . In a mopoptosis . Colony-patients ,128, andneration .However, other studies showed that pharmacological inhibition in APP/PS1 mice improved performance in memory and behavioral tasks and prevented synaptic degeneration .FKBP1A, a member of the immunophilin protein family involved in protein folding and trafficking, was protective against the cytotoxicity caused by Aβ in cultured cells when overexpressed [xpressed . Cognitixpressed .OLA1) lessens the antioxidant response, strengthening the integrated stress response and controlling protein production. SAMHD1 is likewise elevated in the symptomatic carrier (both with FC of 1.7). Its overexpression makes cells more vulnerable to cytotoxicity from thiol-depleting and peroxide antioxidants [OLA1 binds to and stabilizes HSP70 to shield cells from heat shock [CPT1C lowered cell viability, oxidative stress, and apoptosis in Aβ25-35-induced neurons and decreased APP, p-tau, and Bace-1 levels. CPT1A is an enzyme that transports fatty acids into the mitochondria to produce energy [Obg-like ATPase 1 (oxidants . But accat shock . Overexpe energy .The term “proteostasis network” (PN) describes the interaction of a complex network of processes that allow the maintenance of the conformation, concentration, and localization of proteins for their correct function. This network comprises about 2500 genes  and multiple signaling pathways that control protein biogenesis, folding, trafficking, and degradation. Any disruption in Protein Quality Control (PQC), changes in glucose uptake and metabolism, oxidative stress, transport, and abnormal autophagy or degradation systems as a result of stress can result in misfolding or accumulation of proteins that interfere with various cellular functions and disrupt the entire proteostasis network, as happens in aging and the main NDDs, including AD , causingOur findings demonstrate that the primary categories that are altered in MSCs derived from carriers of the PSEN1(A431E) mutation include proteostasis network functions like energy metabolism, processing, synthesis, and folding of proteins, cytoskeleton organization, and intracellular and vesicular transport .According to some studies, bioenergetic changes, including mitochondrial malfunction, changes in glycolysis and PPP, reduced ATP generation, and oxidative damage are early symptoms of AD ,139,140.One of the characteristics of AD is a decline in cerebral glucose absorption. The changes in glucose hypometabolism in pre-symptomatic AD are now being studied using MRI and 18F-FDG PET (Brain Fluorodeoxyglucose Positron Emission Tomography) imaging investigations ,142. A gPHGDH and ALDH18A1 are downregulated in P2. It is interesting to note that the gene for ALDH18A1 (Delta-1-Pyrroline-5-Carboxylate Synthase), a condition with symptoms comparable to those of people with PSEN1(A431E) mutation, is associated with spastic paraplegias (SPG9A and SP9B) [ALDH18A1 mutations [PGD, MAT2A, and PKM were the three proteins whose expression levels were downregulated in both mutation carriers. The pentose phosphate pathway (PPP) has three enzymes. The third enzyme is PGD (phosphogluconate dehydrogenase). In contrast to our results, an old work reported that G6PD and PGD activity increased in AD patients’ inferior temporal cortex [MAT2A (Methionine Adenosyl transferase 2A) is essential for the methylation of neurotransmitters, DNA, proteins, and lipids. As our results, Schrötter et al. [MAT2A in frontal cortex samples from AD patients than in controls. The knockdown of the APP protein family resulted in its downregulation at the RNA and protein levels but with a subsequent S-adenosylmethionine (SAM) elevation. To produce ATP and pyruvate, the pyruvate kinase (PKM) catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate to ADP. PKM2 controls the activity of γ-secretase and is necessary for cell proliferation. Contrary to our findings, Han et al. claimed that its expression is increased in brain samples from AD patients and mouse models and that its silencing led to lower levels of Aβ1-40 and Aβ1-42 and γ-secretase activity [Most DEPs participating in energy metabolism pathways were downregulated, pointing to disordered energy metabolism and a potential rise in ROS generation. Although elevated in P1, nd SP9B) . AD and utations . PGD, MAl cortex . Howeverl cortex . MAT2A , lysosome dyshomeostasis, the accumulation of ubiquitinated proteins, and changes in proteasome subunits that reduce the proteasomal activity .The function and destiny of critical molecules involved in the genesis of Alzheimer’s disease (AD) depend on endocytosis. For instance, early endosomes are essential for producing Aβ peptides, which are noticeably increased within neurons in the brains of patients with early-stage AD ,152. TheCRYAB, ERO1A, CALR, CANX, ERP29, PDIA3, PDIA4, PDIA6, BAG2, HSPA1A, HSPA5, DNAJB11, HSP90AB1, HSP90B1, HYOU1, and P4HB) were identified in the clinical data of twenty-two DEPs in protein processing in the endoplasmic reticulum pathway.Most of these events occur in the endoplasmic reticulum (ER), where most non-cytosolic proteins (secretory or membrane) are generated. A rigorous quality control system prevents around 30% of all proteins from misfolding physiologically, and chaperones, also known as heat shock proteins (HSPs), provide the first line of defense. To keep the balance between protein synthesis and degradation, they bind to unfolded proteins and keep them in a properly folded state while preventing aggregation. These proteins are grouped into various families based on their molecular weight and functions, such as cotranslational folding, refolding or degradation of misfolded proteins, and the inactivation of the Unfolded Protein Response (UPR) . SixteenAlmost all chaperone protein families are altered in AD brain tissue and animal models. Many have been overexpressed due to chronic inflammation, but some reports show that HSPs are downregulated in AD ,158,159.However, the findings that many molecular chaperones are downregulated in undifferentiated cells are noteworthy since they might play a role in the earlier clinical presentation of FAD compared to SAD.Most excitatory synapses within the brain occur on small dendritic protrusions called dendritic spines. The quantity and size of dendritic spines, whose stability is provided by the actin cytoskeleton, significantly impact synaptic strength and neuronal function . Actin dThis decrease may be brought on directly by the toxicity of Aβ oligomers or indirectly by the change of several synaptic proteins in these patients . In lineDBN1), which accumulates in dendritic spines and creates a stable pool of slowly replenishing F-actin and bundles filaments by cross-linking them together.In addition, p21-activated kinases (PAKs), essential regulators of actin assembly and subsequent spine modulation in neurons, decrease in the hippocampus of AD patients and animal models of the disease . KalirinCFL1, an actin-binding protein that depolymerizes and cleaves actin filaments to release free G-actin monomers recruited for filament elongation and branching. Aging wild-type mice have higher levels of activated CFL1. This reduction is more significant in APPPS1 mice than in aging wild-type mice. Additionally, it has been noted that AD patients’ brains had lower levels of active CFL1 [Aged APPPS1 mice exhibit lower levels of activated ive CFL1 .According to Bao et al., who discovered dramatically changed expression of cell adhesion pathway (CAM) genes in AD in cerebellar and temporal cortex samples from AD-affected people , severalOn the other hand, contactin-2 levels are decreased in AD patients’ temporal lobes  and N-caMeanwhile, single nucleotide polymorphisms (SNPs) in NCAM2 are associated as a risk factor related to AD progression in the Japanese population, according to Kimura et al. . At the CLTA, AP2B1, DNAH6, and FUS) were downregulated. In both, RYR1 was elevated, and TUBB3 and MATR3 expression varied.NDDs are primarily distinguished by a failure of Protein Quality Control (PQC) to remove or degrade misfolded proteins accumulating in various brain regions. NDD shares multiple pathophysiological processes that result in neuronal injury. The current study enriched KEGG and STRING data for both carriers for the major NDD pathways, including those for ALS, Parkinson’s, prion, Huntington’s, and Alzheimer’s . These pThe cytoskeleton’s structure, intracellular and vesicular transport, and energy metabolism are the primary functions that are impacted in MSCs derived from PSEN1 (A431E) mutant carriers. These processes are related to the proteostasis network and involve protein processing, synthesis, and folding.Some AD investigations have reported proteostasis network changes, with findings similar to ours. Vesicular transport, post-translational protein modifications, trafficking, and proteostasis were among the five classes of functionally related molecular pathways linked to AD that Rosenthal et al.  proposedJohnson and colleagues conducted a detailed proteomic analysis of more than 2000 human brain tissues from 453 brains and 400 cerebrospinal fluid samples . They in: CDH6, CAMK2D, CUTA, MARCKS, SRCAP, TXN TPI1, WARS, BASP1, ALCAM, CYCS, HNRNPU, UCHL1, CHGB, HLA-B, some related to PI3k-Akt signaling pathway , with the lysosome , with exocytosis, secretion and vesicle transport , and protein processing in ER .Additionally, Pedrero-Prieto et al.  created Furthermore, Higginbotham et al.  applied Recent research found that compared to fibroblasts from healthy donors, skin fibroblasts from FAD patients with PS1 (M146L or A246E) mutations expressed higher levels of HSPs and autophagic-lysosomal pathway proteins . Our finTwo healthy adult donors and two PSEN1A431E) mutation carriers from a Mexican-Mestizo family provided the olfactory neuroepithelium. The family carriers are siblings affected with lower limb paraparesis for three generations . Santos-1E mutatiOur patient (P1) is a 54-year-old guy with progressive motor dysfunction in the lower limbs, slight cognitive impairment, typical symptoms of upper motor neuron disease, and anosmia that has been present for seven years. The presymptomatic carrier (P2) is a 44-year-old asymptomatic female with normal cognitive function, modest hyperreflexia throughout her body, and slight hyposmia. In addition, control subjects consist of two healthy donors (without the mutation) matched by age and gender: a male 54-year-old serving as the control for the symptomatic subject and a female 42-year-old serving as the control for the presymptomatic subject.These participants provided their informed consent following the guidelines of the Bioethics Committee on Human Research  by signing a form authorizing the collection of their medical history and biological samples.2. Before obtaining the sample, we treated the dishes for two hours with poly-D-lysine (diluted in Milli-Q water at 50 mg/mL). The cells were transferred to a new plate and cultured under the same conditions when they reached 80% confluence.Nasal epithelium cells were exfoliated from the anterior region of the medial and lateral turbinate using a special toothbrush according to the methodology reported by Benítez-King et al.  and harv6 cells per tube. Mesenchymal cells were identified using the markers CD105+ , CD73+ , CD90+ , CD45- , CD14- , CD19- , and CD34- . The information was recorded using a Fortessa flow cytometer (BD) and examined using the Flowjo 7.6 application.Culture plates were trypsinized, inactivated, and supplied with DMEM/F-12 before being centrifuged at 1300 rpm for 5 min, and the supernatant was discarded. Conjugated primary antibodies were added to the pellet after it had been resuspended in PBS with 1 × 10w/v), 0.1 M DTT, and 100 mM Tris/HCl pH 7.6). The samples were sonicated ten times  at 20 °C. The supernatant was then transferred to another tube after the undisturbed cells and cell debris were separated via centrifugation at 16,000× g for 5 min at −20 °C. Following the manufacturer procedure, the protein concentration was assessed using a 2D Quant kit .At 80% confluence, MSCs cells were grown, and proteins were removed from the culture media with three cold PBS washes. Reconstituted cells were incubated at 95 °C for 5 min in an SDT lysis solution ; finally, the sample was desalted in a C18 resin column and stored at −80 °C until further use. Digestion of the samples was according to the FASP protocol described by Wiśniewski et al. [FASP digestion of the protein extract was carried out according to a modified protocol described by Wiśniewski et al. . We usedi et al.  and modii et al. .E mode to calculate the area under the curve (AUC) of the Total Ion Chromatogram and thus normalize the injection in the HPLC. A Symmetry C18 Trap V/M precolumn with dimensions of 180 mm × 20 mm, a pore size of 100°, and a particle size of 5 μm was loaded precisely with tryptic peptides in each condition. The precolumn was desalted using mobile phase A, which contained 0.1% formic acid (FA) in water and mobile phase B, which had 0.1% FA in acetonitrile, under the following isocratic gradient: 99.9% mobile phase A and 0.1% of mobile phase B at a flow of 5 μL min−1 during 3 min. Afterward, peptides were loaded and separated on an HSS T3 C18 Column with 75 μm × 150 mm, 100 A° pore size, and 1.8 μm particle size, using a UPLC ACQUITY M-Class with the same mobile phases under the following gradient: 0 min 7% B, 121.49 min 40% B, 123.15 to 126.46 min 85% B, 129 to 130 min 7% B, at a flow of 400 nL min−1 and 45 °C. Data-independent acquisition technique in the HDMSE mode was used to obtain the spectra in a mass spectrometer equipped with electrospray ionization and ion mobility separation Synapt G2-Si . The tune page for the ionization source was set with the following parameters: 2.75 kV in the sampler capillary tube, 30 V in the sampling cone, 30 V in the source offset, 70 °C for the source temperature, 0.5 Bar for the nanoflow gas, and 150 L h−1 for the purge gas flow. Two chromatograms (low and high energy) were acquired in a positive mode in a range of m/z 50–2000 with a velocity of 0.5 scans s−1. For the high-energy chromatograms, the precursor ions were fragmented in the transfer using a collision energy ramp of 19–55 V. We analyzed the generated raw files in the DriftScope v2.8 software  to eliminate ions with z = 1+ and to obtain the DrifTime from each peptide detected in the mass spectrometer to generate a .rul file to calculate specific collision energy for every peptide caught in the UDMSE mode .The resulting peptides were injected into the mass spectrometer Synapt G2-Si  in MSThe .raw files containing the MS and MS/MS intensities were normalized, aligned, compared, and relatively quantified using Progenesis QI for Proteomics software v3.0.3  against a reversed Homo Sapiens database. The results generated from the software were exported to .xmls files to verify two levels of data quality control for label-free experiments (peptide and protein level) following the figures of merit described by Souza et al., 2017   using the Kyoto Encyclopedia of Genes and Genomes (KEGG), REACTOME https://reactome.org/ and Protein ANalysis THrough Evolutionary Relationships (PANTHER) http://pantherdb.org/ databases. The protein lists were submitted with their respective gene symbol, and a comparison was made with the existing list of the human genome. For pathways enrichment, we used the top 15 significance level. KEGG Mapper-Color https://www.genome.jp/kegg/mapper/color.html/ accessed on 21 April 2023) was used for scheme pathways.For Gene Ontology (GO) terms, as well as a pathway enrichment analysis, we used WEB-based Gene SeT AnaLysis Toolkit (WEBGESTALT) “https://string-db.org/ accessed on 5 may 2023) with the following settings: Homo Sapiens database, text mining, experiments, database, co-expression, neighborhood, gene fusion, and co-occurrence as active interaction source with the highest confidence score (0.90).Interactomes of differentially expressed proteins were predicted using Search Tool for the Retrieval of Interacting Genes/Proteins STRING ver. 11.5  and WEBGESTALT with OMIM and DISGENET databases to find related diseases. UniProt and GeneCards (https://www.uniprot.org/ and https://www.genecards.org/ accessed on 3 april 2023) were used to identify protein characteristics and function.Additionally, we used GenAnalytics  mutation. The MSCs have a direct connection to the illness, a high neurogenic potential, and mesenchymal properties that enable them to differentiate into several cell lineages. They are also simple to obtain and barely invasive. These fresh MSCs from patients are ideal in vitro models for upcoming research on Alzheimer’s disease.They might also suggest that FAD patients are more vulnerable to stress due to altered energy metabolism, which might cause ROS production to rise and symptoms to manifest earlier. Our findings present a thorough proteomic analysis of MSCs from FAD patients in the early and late stages of the disease that resemble reported pathways in Sporadic Alzheimer’s disease. Although more research and validation are required to understand some altered mechanisms reported in this work, our results provide guidelines for the study and identification of new therapeutic targets and the basis for the presymptomatic diagnosis of AD.Two patents were summited to the Mexican Institute of Intellectual Property (IMPI).


HMOX1 , NQO1 , CCL2 , IL1B , IL8 , TNF  and ICAM1 was below the 2-fold threshold for all products. With respect to protein expression, IL1B  and IL8  were elevated over the 2-fold threshold, whereas CCL2, TNF, and ICAM1 were below 2-fold expression for all products. At higher doses, greater inductions were observed with all extracts; however, NGP responses were typically lower than 3R4F. In conclusion, anti-oxidative and pro-inflammatory processes were activated by all products. NGPs overall showed lower responses relative to controls than THP-1 cells exposed to 3R4F AqE.Monocytes exhibiting a pro-inflammatory phenotype play a key role in adhesion and development of atherosclerotic plaques. As an alternative to smoking, next-generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their pro-inflammatory effects on monocytes. We investigated cell viability, anti-oxidant and pro-inflammatory gene and protein expression in THP-1 monocytes after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine (nic). Treatment with 3R4F reduced cell viability in a dose-dependent manner, whereas exposure to alternative smoking products showed no difference to control. At the highest non-lethal dose of 3R4F (20%), the following notable mRNA expression changes were observed for 3R4F, HTP, and e-cig respectively, relative to control;  Tobacco smoking is a key stimulus in the development of atherosclerosis , 42, 50.2−) [2−) by NOX2 has been shown to be involved in the development of endothelial dysfunction and atherosclerosis [HMOX1) and NAD(P)H dehydrogenase (quinone 1) (NQO1) and modulates their transcription [Smoking leads to oxidative stress and mediates the development of reactive oxygen species (ROS), which are key mediators for smoke-induced vascular inflammation during atherogenesis. The family of NADPH oxidases is one of the major sources of cellular ROS. In monocytes, the NADPH oxidase isoform 2 (NOX2), also known as cytochrome b-245 beta chain (CYBB), is the main source and marker of oxidative stress which produces superoxide anions (O2−) , 32, 45.clerosis . As protclerosis , 65, 72.clerosis . After acription , 44. Thecription , 33, 39.cription . C-C motcription . In addicription , 49, 74.Over the last years, we have intensively studied the effects of smoking on the progress of atherosclerosis on a cellular level. First, we started investigating the impact of conventional smoking not only on endothelial cells but also on human monocytes. These cell types are constantly exposed and directly affected by circulating smoke-mediated toxicants and reactive oxygen species in the bloodstream and mediated important steps in the initiation and first steps of atherogenesis , 26.One strategy to reduce the harmful side effects of tobacco smoking is the development of next-generation tobacco and nicotine products (NGP) . Over thThe present study is a follow-up study. Therefore, only brief information is provided in the following sections. For detailed descriptions of the material and methods used in this study, please refer to our previous publications , 26, 27.Human monocytic THP-1 cells (ATCC# TIB-202) were used in this study. Cells were cultured for a maximum period of 8 weeks in RPMI-1640 . Every 2–3 days, THP-1 cells were split (ratio 1:8) in fresh culture media for further cultivation , 12, 70.3R4F reference cigarettes, a heated tobacco product  and a nicotine product , were used in this study (for detailed information please see Table THP-1 cells were exposed to increasing AqE dosages (0–88.3 %) of 3R4F, HTP, e-cig, and pure nicotine (control) for the indicated time period. Each sample was accompanied by a control without additional stimulation (time-matched controls) .After cultivation, monocytes were seeded in a 96-well plate , stimulated with the different test substances (0-88.3 % for 24h). Cell viability assay  was performed and luminescence emission was detected , 27.Monocytes were seeded , stimulated with AqE of 3R4F, HTP, e-cig, or pure nicotine (0-88.3 % for 24h). After treatment, total RNA was isolated (High Pure RNA Isolation Kit/Roche Diagnostics) and reverse transcription of mRNA into cDNA was performed (SuperScript II Reverse Transcriptase/Thermo Fisher Scientific). For qPCR, GoTaq qPCR Master Mix (Promega) was used with gene-specific primers , stimulated with AqE of 3R4F, HTP, e-cig, or pure nicotine (0-88.3 % for 24h). Whole-cell extracts were prepared  and protein concentration was determined (BCA Protein Assay Reagent/Perbio Science) , 27.Inflammatory biomarkers were quantified using the electrochemiluminescence-based Mesoscale Discovery (MSD) V-Plex assay platform . Protein samples were analysed using the Vascular Injury Panel 2 (human) Kit (ICAM1), Chemokine Panel 1 (human) Kit (CCL2), and Proinflammatory Panel 1 (human) Kit .Assay was performed briefly as follows: preparation of analyte detection plates following the manufacturer’s instructions; adding serial dilution of calibrators and the test samples in duplicates to the wells; incubation under shaking for 1 h; preparing SULFO-TAG labelled detection antibody solutions; washing plates; adding respective detection antibody mixture to each well; incubation under shaking for 1 h; washing plates; adding Read buffer to each well. Finally, plates were detected using the MSD Plate reader (MESO QuickPlex SQ 120).First, the putative harmful effects of different types of aqueous extracts (AqE) produced were studied. Human THP-1 monocytes were exposed to increasing dosages of 3R4F, HTP, e-cig, and nic ranging from 10 to 88.3 % for 24 h. Treatment with 30% or higher dosages of 3R4F reduced monocytic cell viability in a dose-dependent manner compared to unstimulated controls. All other test reagents did not show differences in cell viability compared to controls  as a main marker of oxidative stress in monocytes, showed a slight upregulation on mRNA level after treatment with 3R4F, HTP, and e-cig. Exposure to pure nicotine solution did not affect NOX2 in a constant manner . Pure nicotine did not result in a regulation of CCL2 , chemokine (C-X-C Motif) ligand 8 ), and the tumour necrosis factor (TNF).IL1B mRNA expression after a 24-h stimulation. IL8 expression showed comparable results as IL1B after stimulation with aqueous smoke extracts of all tobacco/nicotine products. 3R4F, HTP, and e-cig induced IL8 mRNA and protein expression. Nicotine treatment had only a minor effect on IL8 , such as the use of e-cigarettes (e-cig) and heated tobacco products (HTP), is an emerging trend in recent years. Often, considered a “safer alternative” to conventional cigarettes, little is known about their actual potentially harmful effects on the vascular endothelium. Therefore, these effects should be carefully investigated. In this study, we investigated the effect of traditional cigarettes versus next-generation tobacco and nicotine products (NGP) on human monocytes in vitro. To get detailed information about the actual deleterious impact of NGPs, this study analysed parameters of cell viability, oxidative stress, and the inflammatory state of monocytes after exposure to different smoking products.Human THP-1 monocytes were used as a model for mimicking the function and regulation of monocytes in the vasculature. The monocyte cell line THP-1 is a well-accepted model for mimicking the function and regulation of monocytes in the vasculature , 58. OneDetermination of cell viability is a common key parameter for product screening and gives first results about cytotoxicity of the tested compounds. Based on our own previous testing with primary endothelial cells and monocytes, different cigarette smoke extract dosages, and various time points, we decided to use ATP-based cell viability assays for investigation of the effects of next-generation tobacco and nicotine products in this study , 26, 27.NRF2 expression itself remains unchanged, the expression of its target genes HMOX1 and NQO1 were induced in a dose-dependent manner. Under stimulation with NGP, human monocytes typically showed a lower and rightwards shifted activation of the cellular oxidative stress response. Recent studies investigated the effects of NGPs on cellular oxidative stress and the NRF2 system in various cellular systems [Key elements in the pathogenesis of endothelial dysfunction and atherogenesis are oxidative stress and inflammatory status. Cigarette smoke itself contains different populations of free radicals causing various negative vascular effects. In addition, cigarette smoking-induced intracellular ROS production leads to oxidative stress , 59, 75, systems , 68. TheReactive oxygen species play a crucial role in the progression of atherosclerosis. Particularly the production of superoxide by NOX2, a marker of oxidative stress in monocytes contributes to the development of endothelial dysfunction and atherosclerosis . TherefoCigarette smoking results in a chronic systemic inflammatory response , 52. SevAn important aspect which needs to be carefully considered while interpreting and comparing data from smoke extract treatments is the normalization of used AqE concentrations. In the corresponding literature, various normalization factors, e.g. number of puffs, percentages, or nicotine amounts are used for the application of smoke extracts in vitro and in vivo , 66, 76.Next-generation tobacco and nicotine products (NGP) are often considered a “safer alternative” to conventional smoking which makes their actual biological impact currently of substantial interest. Various data from in vitro, animal, and human clinical studies have been published. Most of these studies show similar results about the biological impact of NGPs in relation to conventional cigarettes. This corroborates the relevance of in vitro approaches in research to characterize the molecular and functional effects of test compounds under simplified and defined in vitro conditions mimicking in vivo conditions.This study suggests that in vitro stimulation of human monocytes with conventional cigarette leads to onset of antioxidative and pro-inflammatory mechanisms at lower doses, whereas treatment with alternative smoking products leads to reduced activation of the analysed study parameters: cell viability, cellular oxidative stress response, and inflammatory state. Exposition to NGP typically shifted the response rightward to higher doses compared to 3R4F. Therefore, this study protocol can be considered a valid model system for the assessment of next-generation tobacco and nicotine products.


USP48 and SF3B1, represent very recent and infrequent genetic associations; thus, their clinical relevance remains unclear. For two other genes, GNAS and USP8, discrepancies exist among studies regarding their associated phenotypes. Finally, the phenotypes associated with BRAF and DICER1 are well defined in other settings, but not yet in sporadic PitNETs. Additional studies are required to assess the potential of these molecular alterations as druggable targets in PitNETs.Mutational hotspots have gained importance as oncological biomarkers in recent years because of their potential as predictors of clinical outcomes and/or therapeutic targets. In addition, they are easily detectable in clinical samples via Sanger or next-generation sequencing (NGS). The role of these genetic defects is less clear in pituitary neuroendocrine tumors (PitNETs), even though the most common genetic drivers of these neoplasms are located within mutational hotspots. Indeed, hotspots in six different genes are of particular importance in this context. Two of them, GNAS and USP8 are the most common genetic causes of acromegaly and Cushing’s disease, respectively. Although it has been proposed that these genetic defects could define specific clinical phenotypes, results are highly variable among studies. In contrast, DICER1 hotspot variants are associated with a familial syndrome of cancer predisposition, and only exceptionally occur as somatic changes. A small number of non-USP8-driven corticotropinomas are due to somatic hotspot variants in USP48 or BRAF; the latter is a well-known mutational hotspot in cancer. Finally, somatic variants affecting a hotspot in SF3B1 have been associated with multiple cancers and, more recently, with prolactinomas. Since the associations of BRAF, USP48, and SF3B1 hotspot variants with PitNETs are very recent, their effects on clinical phenotypes are still unknown. Further research is required to fully define the role of these genetic defects as disease biomarkers and therapeutic targets. The most common genetic drivers of pituitary neuroendocrine tumors (PitNETs) lie within mutational hotspots, which are genomic regions where variants tend to cluster. Some of these hotspot defects are unique to PitNETs, while others are associated with additional neoplasms. Hotspot variants in  Hotspots are genomic regions where variation occurs with a higher frequency than what would be expected by chance . Specifipassenger variants and only a small fraction are drivers for neoplasms [Genetic changes accumulate in the genome throughout life. The majority of them represent eoplasms . Cancer eoplasms ,6. In caeoplasms .GNAS and USP8, which are the most common genetic defects leading to PitNETs, are located in hotspots [MEN1 and TP53) are not included in this review. Pituitary neuroendocrine tumors (PitNETs) are usually benign lesions with indolent behavior that display a lower-middle tumor mutation burden, and only occasionally develop features of aggressiveness ,9. Multihotspots ,11,12. PProtein kinase is the most frequently shared domain among cancer-associated proteins, therefore representing a particularly attractive therapeutic target [BRAF , a proto-oncogene with preferential expression in neural tissues, and is the most potent activator of the RAS-GTPase (RAS)-RAF-MAPK and ERK kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling pathway (RAS-RAF-MEK-ERK pathway) [c target . The isoc target ,15,16. Tpathway) ,18,19  has been used as a marker for measurable residual disease in hairy cell leukemia [The phosphorylation of residues T599 and S602 (UniProt P15056), which flank the variant, is required for BRAF to be recruited to the cell membrane and folded into its active conformation. The p.V600E change destabilizes the inactive conformation of BRAF and promotes its active state, thereby acting as a omimetic . This wa in vivo ,26,27. I defects . In addipoptosis . In colonal load . In addileukemia .BRAF variants may also develop pituitary hormone deficiencies. Although BRAF p.V600E has not been clinically identified in this context, its expression in the embryonic anterior pituitary leads to severe hypoplasia in vivo, due to initially accelerated cell proliferation, followed by cell cycle arrest and apoptosis of progenitor cells at later stages [BRAF p.V600E is associated with the development of papillary craniopharyngiomas (PCPs), which are benign tumors most likely derived from pituitary precursors [CTNNB1-driven [BRAF p.V600E is associated with a stable genome and its detection helps to confirm the histopathological diagnosis [Recent research has demonstrated that BRAF has an important role in the development and terminal differentiation of the anterior pituitary ,31. Inder stages . In contecursors . Althoug1-driven ,35,36,37iagnosis . These tiagnosis ,39.BRAF p.V600E was identified in 9–10% of cases of non-USP8-driven Cushing’s disease (CD)  see  by two dype BRAF . Given ty tumors . BRAF p.V600E has been exploited as a therapeutic target in many neoplasms. Vemurafenib, dabrafenib, and encorafenib are adenosine triphosphate (ATP)-competitive RAF inhibitors that selectively inhibit BRAF p.V600E, but paradoxically activate RAS-RAF-MEK-ERK signaling in BRAF wild-type tumors, particularly in those with RAS activating variants [BRAF p.V600E-driven colorectal cancer, Erdheim-Chester disease, hematological malignancies, melanoma, non-small-cell lung cancer, and PTC [Thanks to the availability of BRAF inhibitors, variants ,47. Thei and PTC ,50,51,52BRAF p.V600E positive posterior pituitary tumors (two with confirmed NKX2-1-expression) treated with dabrafenib, either alone [Individual case reports of PCP treatment with drugs targeting BRAF and/or other RAS-RAF-MEK-ERK components have shown encouraging results . Very reer alone  or combier alone  or trameer alone . All tumer alone  and two er alone , althougGNAS , ultimately translates into multiple proteins, namely XLαs, ALEX, NESP55, and Gsα [At least ~100 human genes are subjected to genomic imprinting, an epigenetic mechanism that controls gene expression in a parent-of-origin and tissue-specific manner . Using d and Gsα ,62. The  and Gsα . Gsα is  and Gsα .At the molecular level, guanine nucleotide-binding proteins (G proteins) function as information transducers between the cell-membrane-bound G-protein-coupled receptors (GPCRs) and their effectors, thereby regulating the production of second messengers . G proteGNAS variants affecting residues R201 , and G227  of GNAS have been described in endocrine tumors and other human neoplasms. They have been found as somatic changes in somatotropinomas (4.4–59.5%), non-functioning PitNETs (7–10%), corticotropinomas (6%), autonomous thyroid adenomas (5%) and thyroid cancer (13% of PTC and up to 4% of follicular tumors), and occasionally, in ovarian and testicular Leydig cell tumors, prolactinomas, adrenocortical adenomas, pheochromocytomas, paragangliomas, parathyroid adenomas, and in patients with multiple endocrine tumors [Missense itNETs 7–%, cortice tumors ,96,97,98e tumors ,101,102.GNAS variants also underlie the McCune–Albright syndrome , a rare condition with sporadic presentation characterized by genetic mosaicism due to early postzygotic GNAS hotspot defects [café-au-lait skin spots, and endocrine hyperfunction  [GNAS variant during embryogenesis [ defects ,104. The others) . Ninety- others) ,107,108.ogenesis . GNAS hotspot variants cause the loss of protein function that results in increased activity of the cAMP signaling pathway, by (1) stabilizing Gsα in its active conformation, thereby mimicking the effect of extracellular growth factors by stimulating ACs, and (2) inhibiting GTPase activity and causing a constitutive activation of ACs [GNAS defects are often referred to as activating variants or gsp oncogene [GNAS is an attractive therapeutic target, although drugs with this specific effect have not been reported yet. In contrast, non-hotspot loss-of-function (LOF) GNAS variants cause Albright’s hereditary osteodystrophy [n of ACs ,110. Forystrophy .GNAS variants have been thoroughly studied in somatotropinomas. Some studies have defined a particular GNAS-associated phenotype, with patients usually being older and presenting significantly smaller tumors associated with low serum GH or IGF1 levels [GNAS-driven tumors as having a slow growth rate and a better response to pharmacological or surgical treatment compared with wild-type tumors [GNAS hotspot variants define a distinctive subgroup of somatotropinomas that display hypomethylation, limited chromosomal alterations, and activation of the GPCR pathway, although results vary among studies [GNAS variants almost always affect the maternal allele [GNAS variants [GNAS imprinting correlates with lower GNAS, SSTR2, and AIP expression, suggesting a possibly reduced response to somatostatin receptor ligands [The clinical consequences of 1 levels ,89,96,97e tumors ,112. Thee tumors . Differee tumors ,115. At  studies ,116,117.l allele . While wvariants ,120. TheThe DICER1 syndrome (MIM #601200) is an autosomal dominant condition of tumor predisposition that encompasses otherwise infrequent dysembryonic tumors, such as pleuropulmonary blastoma (PPB), cystic nephroma (CN), ovarian sex cord stromal tumor, nasal chondromesenchymal hamartoma, ciliary body and cerebral medulloepitheliomas, anaplastic kidney sarcoma, pineoblastoma, embryonal rhabdomyosarcoma (ERMS), and pituitary blastoma (PitB) . Other aDICER1 (14q32.13) variants that appear de novo in 10–20% of cases [DICER1 variants, which has been associated with earlier disease onset, more DICER1-associated tumors, and a distinctive presentation known as GLOW syndrome  [This syndrome presents usually at an early age and occasionally in young adults and is caused in most cases by germline heterozygous LOF of cases ,125,126.s tumor) ,128.DICER1 canonical transcript (NM_030621.4) encodes a widely expressed 1922 amino acid cytoplasmic enzyme (Q9UPY3-1) composed, from N- to C-terminal, of a helicase domain, a domain of unknown function (DUF283), a platform domain, a P-element-induced whimpy tested (PIWI)-Argonaute (AGO)-Zwille (PAZ) domain, a connector domain, the class 3 ribonuclease (RNase III) a and b domains, and a double-stranded RNA (dsRNA)-binding domain [The 29-exon g domain . DICER1 g domain ,131. Theg domain . The AGOg domain   [DICER1 variants have been reported in the presence or absence of germline defects in patients with PPB, CN, WT, non-epithelial ovarian tumors, cervical ERMS, PitB, prostate carcinoma, pineoblastoma, differentiated thyroid carcinoma, and testicular germ cell tumors [Most individuals carrying germline ty (LOH) . Moreovel tumors ,143,144.l tumors ,141,145.DICER1 variants, although it is not clear if any cases were caused by somatic defects [oncofetal molecular signature usually express ACTH and may present clinically silently or as CD [DICER1 screening and genetic counseling [Nineteen out of the twenty PitBs genotyped so far were due to LOF  defects ,147,148. defects ,147,148.or as CD ,149,150.unseling .RNAse IIIb variants affect metal ion binding and adjacent amino acids, specifically 1705, 1709, 1809, 1810, or 1813, which are therefore considered missense hotspots ,131. SecDICER1-associated tumors, this abnormal miRNA repertoire leads to the overexpression of the preferentially expressed antigen in the melanoma gene (PRAME) [PRAME overexpression remain unclear [PRAME overexpression has recently been explored as a potential therapeutic target for immunotherapy in various neoplasms, although not yet in patients with DICER1 LOF [In PitB and other  (PRAME) ,151. PRA (PRAME) . Aside f unclear ,151. PRACER1 LOF .DICER1 expression due to haploinsufficiency or other mechanisms correlates with bad outcomes in multiple types of cancer [DICER1 hotspot variants.Aside from its role as a tumor driver, reduced f cancer . In thesf cancer ,154. ThiSF3B1, 2q33.1, NM_012433.4) was identified in 20% of prolactinomas of a single cohort of 227 cases [SF3B1 p.R625H displayed significantly higher prolactin levels and a shorter progression-free survival, compared with SF3B1 wild-type cases. A recent Sanger sequencing-based study identified the same variant and an additional missense variant in the same residue  in 7 out of 282 prolactinomas analyzed (2.5%) [SF3B1 hotspot defects. In line with the earlier findings, SF3B1 variants were associated with a larger tumor size and increased mortality, but also with a higher Ki67 index and a need for more therapeutic interventions.Using genome sequencing in 21 patients and targeted genotyping by ddPCR in the rest, a recurrent missense somatic variant  in the splicing factor 3B subunit 1 gene (27 cases . When 15d (2.5%) . InteresSF3B1 encodes a component of the U2 small nuclear ribonucleoprotein (snRNP) complex and is therefore a component of the pre-mRNA splicing machinery. SF3B1 is involved in 3′ acceptor splice site (SS) recognition, as well as in recruiting other U2 snRNP subunits to the branch point (BP) of pre-mRNAs via interaction with the BP and U2AF2 [nd U2AF2   mRNA, resulting in stronger interaction with the pituitary-specific positive transcription factor 1 (POU1F1) and excessive prolactin secretion [DLG1 in human prolactinomas and rat somatotropinoma GH3 cells. In the latter, the variant causes an epithelial–mesenchymal transition phenotype [Recurrent somatic variants in hotspots within the fifth and ninth HEAT repeats have been found in myelodysplastic syndrome, chronic myelomonocytic leukemia, acute myeloid leukemia, myeloproliferative neoplasms, primary myelofibrosis, chronic lymphocytic leukemia, breast cancer, pancreatic ductal adenocarcinoma, uveal, mucosal, and cutaneous melanoma, and prostate cancer ,174,175. in some ,169, alt in some . In prolecretion . This vahenotype .The SF3B1 residues involved in neoplasia are crucial for maintaining the tertiary structure of the protein. Their substitution induces conformational changes in the HEAT domain that hamper the interaction of SF3B1 with the BP and with other U2 snRNP components . This reSF3B1 variants for clinical prognosis in neoplasms remains unclear, but they could represent a druggable target [SF3B1 silencing in breast cancer cell lines inhibited aberrant splicing, reducing cell proliferation, migration, and invasion, suggesting a potential antineoplastic role for SF3B1 inhibitors [SF3B1 hotspot variants.The significance of e target . SF3B1 shibitors . Examplehibitors . Intravehibitors . Oral H3hibitors . Intereshibitors . FurtherUSP8 (exon 14 in NM_005154.5) are the most frequent genetic cause of CD, being found in 21–62% of corticotropinomas [USP8 (15q21.2) encodes the 1118-amino-acid ubiquitin carboxyl-terminal hydrolase 8 (P40818-1), which belongs to the family of deubiquitinases (DUBs) [Research published over the last eight years has established that somatic defects in codons 718–720 of opinomas ,194,195.opinomas ). USP8 (s (DUBs) . Ubiquits (DUBs) .USP8 activating variants result in increased EGFR recycling, which in turn leads to a rise in POMC transcription [The mutational hotspot implicated in CD lies within the USP8 14-3-3 interacting motif (residues 715–720), which is crucial for maintaining USP8 protein integrity. The CD-associated variants lead to the loss of the 14-3-3 interaction and cleavage of USP8 just upstream of the interacting site, resulting in a C-terminal 40 kDa protein fragment with an enhanced DUB activity . The epicription ,12,183 , is another DUB that has recently been implicated in the pathogenesis of CD. Recurrent somatic missense variants affecting a hotspot in residue 415  of USP48 (p.M415I or p.M415V) have been identified in 4–23% of corticotropinomas negative for USP8 variants in the general population and in only 1% of USP8 wild-type pediatric corticotropinomas [The ubiquitin-specific protease 48, encoded by the opinomas ,195,213.USP48 variants result in increased POMC transcription by at least two possible mechanisms. The sonic hedgehog (SHH) pathway effector GLI1, which is overexpressed in this setting, potentiates the stimulatory effect of CRH on the POMC promoter [USP48 variants also lead to the inhibition of NFKB, probably via enhanced stabilization of its RELA subunit, thereby blunting its effect as a POMC negative transcriptional regulator [These variants lie within the peptidase domain of the protein, specifically in its catalytic “palm”, but do not affect its expression . The abnpromoter . Interespromoter . In addiegulator  , although there are no data in the literature on USP48-specific compounds [Corticotropinomas carrying e tumors ,43. A the tumors . Comparen levels . Unfortuompounds .GNAS and USP8 might define specific clinical phenotypes, results are highly variable among studies. In contrast, DICER1 variants are clearly associated with an aggressive phenotype, but they only exceptionally occur as somatic changes. Since BRAF, USP48, and SF3B1 hotspot variants have only recently been identified in PitNETs, their associated clinical pictures are still unknown. All these variants are easily detectable by Sanger sequencing or NGS techniques, which might become part of routine diagnostic tumor assessments in the near future. Further preclinical and clinical research protocols are required to test the efficacy and safety of compounds directed toward these molecular alterations in PitNETs. Hotspot variants associated with PitNETs have been proposed as potential disease biomarkers by multiple groups. Although


Mitochondrial dynamics, including fission and fusion processes, are essential for heart health. Mitochondria, the powerhouses of cells, maintain their integrity through continuous cycles of biogenesis, fission, fusion, and degradation. Mitochondria are relatively immobile in the adult heart, but their morphological changes due to mitochondrial morphology factors are critical for cellular functions such as energy production, organelle integrity, and stress response. Mitochondrial fusion proteins, particularly Mfn1/2 and Opa1, play multiple roles beyond their pro-fusion effects, such as endoplasmic reticulum tethering, mitophagy, cristae remodeling, and apoptosis regulation. On the other hand, the fission process, regulated by proteins such as Drp1, Fis1, Mff and MiD49/51, is essential to eliminate damaged mitochondria via mitophagy and to ensure proper cell division. In the cardiac system, dysregulation of mitochondrial dynamics has been shown to cause cardiac hypertrophy, heart failure, ischemia/reperfusion injury, and various cardiac diseases, including metabolic and inherited cardiomyopathies. In addition, mitochondrial dysfunction associated with oxidative stress has been implicated in atherosclerosis, hypertension and pulmonary hypertension. Therefore, understanding and regulating mitochondrial dynamics is a promising therapeutic tool in cardiac diseases. This review summarizes the role of mitochondrial morphology in heart diseases for each mitochondrial morphology regulatory gene, and their potential as therapeutic targets to heart diseases. Mitochondria are essential organelles for adenosine triphosphate (ATP) production and are closely linked to cellular health and energy demand. On the other hand, mitochondria release reactive oxygen species (ROS) that cause oxidative damage. These mitochondrial functions are regulated by two distinct systems: fission and fusion. Fission, facilitated by proteins such as dynamin-related protein 1 (Drp1), fission 1 (Fis1), mitochondrial fission factor (Mff), and mitochondrial division (MiD) 49 and 51, leads to mitochondrial fragmentation, which is essential for cell division and removal of damaged mitochondria. Fusion, orchestrated by mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and optic atrophy 1 (Opa1), results in interconnected networks that help repair mitochondrial DNA and distribute energy. While the roles of fission proteins are primarily associated with morphological changes, fusion proteins show diverse roles influencing mitophagy, apoptosis, and energy production. Consequently, mitochondrial morphology is strictly regulated by several key genes. Unlike other cells, the adult heart contains relatively static mitochondria that are segregated into three functional subpopulations. Despite this fixed arrangement, emerging evidence suggests that mitochondrial morphological factors play an important role in cardiac health and pathology.Mitochondrial dysfunction has been implicated in a spectrum of cardiac diseases. Defective fission has been associated with severe cardiac abnormalities, highlighting the importance of these dynamics in cardiac pathologies, including cardiomyopathies and ischemic conditions. A profound understanding of mitochondrial dynamics opens a new avenue for therapeutic approaches to heart disease. The balance between fission and fusion processes offers a promising strategy to preserve cardiac health, especially under stress.In this review, we summarize the relationship between mitochondrial morphology regulatory genes and heart diseases. We discuss the molecular biology underlying mitochondrial dynamics, clarify how defects in mitochondrial dynamics are associated with heart damage during stress, and detail the importance of considering mitochondrial dynamics as a new therapeutic target for mitochondrial morphology regulatory genes.Mitochondrial dynamics involves a continuous process of mitochondrial fission and fusion. Mitochondrial dynamics regulate mitochondrial turnover and the intracellular environment . Here, wMitochondrial fission depends on the activation of Drp1 in the cytoplasm, which causes Drp1 to translocate into the mitochondria and bind to receptors on the mitochondrial outer membrane (OMM). Mitochondrial fission produces daughter mitochondria with different membrane potentials (Δψm). This function supports the selective degradation of damaged mitochondria by autophagy and maintains cellular homeostasis ,3. DurinDrp1 gene expression. During apoptosis, RNA known as miR-30 is reduced, and Drp1 expression is increased [Drp1, a key fission protein, is regulated by multiple post-translational modifications, including phosphorylation ,23,24, uncreased . In myocncreased . Drp1 dencreased . Mitochoncreased ,35. MITOncreased .In mammals, Drp1 recruitment to the OMM depends on key adaptors: Fis1, Mff, and MiD 49 and 51 ,38,39. TMff, which is anchored to the OMM, has a putative Drp1 binding domain at its N-terminus ,38. InhiMiD49 and MiD51 are mitochondrial adaptors of Drp1 that promote fission . HoweverThe fusion process of the OMM is orchestrated by a GTPase-driven mechanism that is predominantly mediated by the homo- or hetero-oligomeric interactions of the mitofusins ,59,60. TMfn2 expression [Mfn2 expression [Mfns [Mfns are regulated at multiple levels to control fusion. The peroxisome proliferator-activated receptor γ coactivator 1-α (Pgc-1α), estrogen-related receptor-α (Err-α), and Pgc-1β increase pression , whereaspression . Severalon [Mfns ,71. Mitoon [Mfns . MITOL/Mon [Mfns , and hison [Mfns . Variouson [Mfns ,76. ThesOPA1 gene contains more than 30 exons and eight mRNAs that differ in biological function, such as maintaining mitochondrial fusion [Opa1 is the only dynamin-like protein in the IMM and is essential for mitochondrial elongation . Opa1 hal fusion ,80. Mamml fusion ,82. The l fusion .In cultured cells and neonatal cardiomyocytes, mitochondria are dynamic organelles that spread throughout the cytoplasm in a network-like pattern. In adult cardiomyocytes, however, mitochondria have a different arrangement, being more compact and tightly interconnected . Thus, tMitochondrial dynamics during heart development and in the resting state have been studied using whole-body or heart-specific knockout models targeting mitochondrial fission and fusion . Here, wDrp1 gene results in embryonic death by day E12.5 [Deletion of the ay E12.5 . In the ay E12.5 . This unay E12.5 ,110. Furay E12.5 . In adulay E12.5 . In adulay E12.5 ,111. Oveay E12.5 . Taken tIn mice, combined cardiac deletion of Mfn1 and Mfn2 results in spherical and functionally normal mitochondrial function, eventually leading to cardiac dysfunction by postnatal day 7 and death within 16 days of birth . CombineMarf (human Mfn) and Opa1 results in cardiomyopathy and reduces contractility. This effect is rescued by overexpression of human mitofusins [In Drosophila, knockdown of the cardiac genes tofusins . In conttofusins . Taken tMff-deficient mice exhibit impaired mitochondrial function and increased mitophagy, leading to dilated cardiomyopathy and heart failure by 13 weeks of age. This cascade leads to a decrease in cardiac ATP concentration, which predisposes cardiomyocytes to apoptosis, culminating in fibrotic changes and heart failure. Surprisingly, concomitant deletion of Mfn1 attenuates these pathological changes, preserved cardiac function and extended lifespan .Cardiomyocyte-specific heterozygous Opa1-deficient mice do not show cardiac abnormalities under non-stress conditions, but they show enlarged mitochondria. Furthermore, in vitro studies on these cardiomyocytes reveal a reduced sensitivity of the opening of the mitochondrial permeability transition pore (mPTP) to calcium accumulation .Several mitochondrial morphology factors have been implicated in the complex relationship between mitochondrial dynamics and cardiomyopathy, heart failure, and reperfusion injury. Here, we focus on five key molecules—Drp1, Mfn1/2, Mff and Opa1.2O2-induced mitochondrial dysfunction [Mitochondrial dynamics between fission and fusion are finely regulated in response to nutrient availability and metabolic demands. C57BL/6 mice develop hyperlipidemia and hyperglycemia when exposed to a high-fat diet. This condition is caused by activation of Drp1 at serine 616, which leads to myocardial insulin resistance, reduces contractile efficiency, and ultimately cardiomyocyte death . The accfunction . In addifunction . In advafunction . Sepsis function .Mitofusins, Mfn1 and Mfn2, are GTPase proteins essential for mediating fusion of the OMM. The prognosis for severe heart failure remains poor. Some heart failure patients do not respond to established multidisciplinary therapy and are classified as “non-responders”. Mfn1 is significantly reduced in non-responders . In patiMff acts as a receptor for Drp1 on the OMM, facilitating the fission process. Its role is highlighted in lipotoxic cardiomyopathy, where fatty acid overload coupled with ceramide accumulation increases the expression of both Drp1 and Mff, perpetuating mitochondrial dysfunction .Opa1 is critical for fusion of the inner mitochondrial membrane. Cardiomyocytes from Zucker diabetic rats show reduced Opa1 expression, which affects hypertrophy and calcium handling via the Orai1 channel . HyperglCardiac-specific overexpression of miR-122, elevated in heart failure patients, induces mitochondria-dependent cardiomyocyte apoptosis and accelerates heart failure through the activation of Drp1 by inhibiting Hand2 . Drp1 plXbp1 expression, a sarcoplasmic reticulum stress-responsive transcription factor, enhances the stress-responsive capacity of the sarcoplasmic reticulum and rescues cardiomyopathy caused by mitofusin/MARF deficiency without ameliorating cardiomyopathy caused by Opa1 deletion . MitochoCardiomyocyte-specific Yme1L deficient mice exhibit reduced Opa1 levels via activation of Oma1, leading to a shift in cardiac metabolism and, ultimately, heart failure . Ischemi2+ strongly regulates Drp1 activity. Notably, miR-499 attenuates the effects of Ca2+ overload on Drp1 function [Dynamin-related protein 1 (Drp1) plays a crucial role as an inducer of mitochondrial fragmentation in the detrimental effects of cardiomyocytes after ischemia-reperfusion (I/R) injury. In I/R injury, Drp1 translocates into the OMM and causes mitochondrial fragmentation . Mitochofunction . Drp1 hafunction  [24,30,1function ,161,162.function .Mfn1 expression in cardiomyocytes, blocks mitochondrial networks, and exacerbates apoptosis in cardiomyocytes [Mitofusin 1 and 2 (Mfn1/2) are elongation factors of mitochondrial dynamics, and their expression levels are strongly associated with the pathophysiology of I/R injury. Mitochondrial fragmentation induced by I/R also results from decreased expression of Mfn1, Mfn2, or Opa1, leading to impaired respiratory function. Consequently, it promotes I/R-induced cardiomyocyte apoptosis ,165,166.myocytes . In vitrmyocytes . In othemyocytes . Additiomyocytes . Overexpmyocytes ,172.Opa1 has been identified as playing a protective role in cardiomyocytes following I/R injury. A notable downregulation of Opa1 expression is observed in cardiomyocytes subjected to I/R injury in vivo, as well as in hypoxia-treated cardiomyocytes in vitro . Opa1 isThis section summarizes the therapeutic potential for mitochondrial morphological abnormalities. Numerous investigations have explored various agents targeting each mitochondrial regulatory factor . AlthougMelatonin inactivates I/R injury-induced phosphorylation of Drp1 at serine 616. Long-term melatonin treatment attenuates the dilated cardiomyopathy progression and reduces myocardial vulnerability to I/R injury by maintaining mitochondrial quality control. Melatonin membrane receptors are associated with regulating the SIRT6-AMPK-PGC-1α-AKT axis in this effect . HydralaSAMβA attenuates cell death through Mfn1 enhancement in ischemic heart failure . In the Irisin administration induces Opa1-mediated mitophagy and protects myocardial cells from post-myocardial infarction damage . Remote Mitochondrial dynamics, including fission and fusion processes, play an essential role in heart health and disease. Recent research has highlighted the potential of targeting these dynamics to treat various cardiac diseases, including acute myocardial infarction, hypertrophy, PAH, ischemic diseases and heart failure. However, it is important to recognize the multifaceted role of mitochondrial morphology factors, particularly fusion proteins such as Mfn2 and Opa1. Acute and chronic modulation of these proteins may have therapeutic effects or unintended adverse consequences. Although most of the current knowledge is derived from transgenic animal models, early pharmacological interventions have yielded promising results. It is essential to extend this research to human samples and to improve the diagnostic and therapeutic toolkit by integrating biopsy analysis and indirect markers of mitochondrial dynamics. This would help to refine patient risk assessment and tailor treatment strategies for cardiovascular disease.Furthermore, there is a perception that mitochondrial fusion is beneficial and fission is detrimental, but this binary view may be too simplistic. Fission plays a fundamental role in separating damaged mitochondria for removal by mitophagy, and excessive inhibition of this process may exacerbate myocardial injury. Thus, striking the right balance in the regulation of mitochondrial dynamics is critical for potential clinical applications.


Mycobacterium tuberculosis-infected BALB/c and caseum-forming C3HeB/FeJ mice. These observations have been important for testing new drug combinations in noninferiority clinical trials, in order to shorten the duration of current regimens against TB. In 2022, the World Health Organization, following the results of one of these trials, supported the use of a 4-month regimen for the treatment of drug-susceptible TB as a possible alternative to the current 6-month regimen.The lungs of tuberculosis (TB) patients contain a spectrum of granulomatous lesions, ranging from solid and well-vascularized cellular granulomas to avascular caseous granulomas. In solid granulomas, current therapy kills actively replicating (AR) intracellular bacilli, while in low-vascularized caseous granulomas the low-oxygen tension stimulates aerobic and microaerophilic AR bacilli to transit into non-replicating (NR), drug-tolerant and extracellular stages. These stages, which do not have genetic mutations and are often referred to as persisters, are difficult to eradicate due to low drug penetration inside the caseum and mycobacterial cell walls. The sputum of TB patients also contains viable bacilli called differentially detectable (DD) cells that, unlike persisters, grow in liquid, but not in solid media. This review provides a comprehensive update on drug combinations killing in vitro AR and drug-tolerant bacilli (persisters and DD cells), and sterilizing  Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB). The World Health Organization (WHO) estimated that in 2021 approximately 10.6 million people developed tuberculosis (TB), and about 1.6 million died from the active disease, making TB one of the leading causes of death worldwide [The lungs of patients with active TB and LTBI contain a spectrum of granulomatous lesions ranging from solid and well-vascularized cellular granulomas to avascular caseous granulomas ,4,5. In However, the sputum of drug-untreated patients was found to also contain viable Mtb bacilli that were not detectable as CFU ,16,17,18Overall, these observations on dormant, drug-tolerant bacilli (persister cells and DD cells) may aid in finding new drugs and in designing shorter drug combination regimens. The goal of this review is to give an update on the reported in vitro and in vivo combinations eradicating all dormant Mtb stages (persister cells and DD cells), and on their impact on the therapeutic strategies endorsed by the WHO to shorten the microbiological and therapeutical management of DS- and drug-resistant (DR)-TB.Candida spp.) [Plasmodium, Leishmania, Trypanosoma and Toxoplasma spp.) [Persister cells (persisters) were reported in several Gram-positive and Gram-negative bacteria  and in mda spp.) , and proma spp.) , where tma spp.) ,25. All Staphylococcus pyogenes cultures due to the generation of a small number of cocci “individually insensitive to penicillin, but developing normally when penicillin is destroyed” [The term “persisters” was used for the first time in 1944 by Joseph Bigger, who realized that penicillin failed to sterilize stroyed” ,26,27. WEscherichia coli with a vastly increased frequency of persistence (Hip mutants) [hip locus affects the frequency of persistence to the lethal consequences of selective inhibition of either DNA or peptidoglycan synthesis [hipA7 strain produced about 1% persisters and, after exposure to ampicillin, it generated about 1000 times more persisters than the wild-type strain [In the search for persisters genes, starting in the 1980s, Harris Moyed and coworkers isolated mutants of mutants) . The hipynthesis . The hipe strain .In 2019, Nathalie Balaban et al., spurred by increased concerns about antibiotic resistance and several papers published on the persistence phenomenon, released an international consensus statement on definitions and guidelines for research on antibiotic persistence . AntibioM. tuberculosis persisters are NR multidrug-tolerant bacilli genetically identical to drug-susceptible cells, which survive in the hostile environments of granulomas and in the sputum and adipose tissues of TB patients [patients ,37,38. Tpatients ,36,39. Rpatients ,43,44,45The primary reason for the 6-month duration of the first-line TB therapy (R-H-E-Z) is due to the ability of Mtb to generate heterogeneous subpopulations adapting to different microenvironments within granulomas. The microfluidic devices for single-cell analysis revealed that the wide diversity of tubercle bacilli can be due to the fact that the mycobacterial cell division is asymmetrical, with the old pole elongating more than the new pole ,47. AsymStaphylococcus aureus and E. coli persisters also have low ATP levels is in keeping with these observations [Other studies on single cells reported that Mtb persisters are rare low-energy cells formed stochastically during normal growth . Stochasrvations ,50. In trvations . Mycobacrvations .Mycobacterium smegmatis persisters, which carried elevated levels of the reactive oxygen species (ROS) hydroxyl radical, superoxide radical and hydrogen peroxide [rpoB and gyrA genes, respectively [Of importance, persister cells may generate genetically resistant mutants. Indeed, prolonged exposure to lethal concentrations of R or the fluoroquinolone moxifloxacin (M) induced the formation of Mtb and peroxide ,53,54. Tectively ,53. It iectively .recA, lexA, dnaE2) and DNA repair genes [Many bacteria react to ROS-generated DNA lesions caused by the bactericidal drugs quinolones, aminoglycosides and beta-lactams by inducing the SOS gene regulon, which is regulated by the transcriptional repressor LexA and the protease RecA. In Mtb, we found that treatment with M induced overexpression of SOS genes  Mtb mutants, selected with a lethal dose of R and streptomycin, showed upregulation of some toxin-antitoxins, i.e., the modules in which the toxins lead to growth arrest when labile antitoxins are degraded by proteases [Hip mutants showed up to 1000 times more persisters than the wild-type strain, a decrease in the phospholipid biosynthesis, and mutations in the cell wall lipid phthiocerol dimycocerosate, an important cell wall lipid and virulence factor. These and other genetic mechanisms suggested that Mtb persisters formation involves multiple pathways, and that hip mutants, obtained using both the reference strain H37Rv and human clinical isolates, may contribute to the tolerance of Mtb to drug exposure. Research on Mtb persisters genes will facilitate the development of more effective therapies to eradicate them from TB patients.Low numbers of Mtb persisters are present in the lag and early exponential phases but reach about 1% in the stationary phase ,59. Tranroteases ,60. Hip Several studies have reported the presence of DD Mtb cells in the sputum produced by patients with active TB. These cells are not able to form colonies on standard glycerol-based solid media (7H10 agar plates and Löwenstein-Jensen slants) but can grow in liquid media supplemented with mycobacterial culture filtrates (CF) ,18,61,62Dormant DD Mtb cells, which retain stable, low-abundant mRNAs , may plaDD-Mtb cells represented the majority of the bacilli in the sputum of 21% of the patients with DS-TB, and this level increased to 65% after 2 weeks of treatment with first-line drugs . In a stE. coli cells from growing as CFU, without killing them, and that ROS accumulation mediated by self-amplifying mechanisms continued after antibiotic removal [Mycobacteriun bovis and its vaccine derivative, the Bacillus Calmette-Guérin (BCG), are not able to do it, and do not generate DD cells because they succumb to ROS [E. coli cells died, but below that threshold, the provisions of anti-oxidants revealed a CFU population [In order to explore the biological mechanisms generating DD cells, Saito et al. found that Mtb can enter into a DD state after a sub-lethal oxidative stress damaging DNA, proteins and lipid components . This co removal . Transcr removal . Thus, Mb to ROS . The obspulation ,69.M. tuberculosis cells are likely to exist in the host at various levels of oxidative stress and replicability [1 inhibitors showed strong bactericidal activity against hypoxic NR Mtb [In summary, after the treatment of Mtb with bactericidal antibiotics, increasing and self-amplifying ROS levels may be generated. Surviving bacilli may then be detected as CFU (persister cells) if ROS levels are low; LD-MPN (DD cells) if ROS levels are intermediate; or not detected (dead cells) if ROS levels are high. cability . Overallcability . Combinac NR Mtb .+ LD-MPN and TTP values for smear-positive and smear-negative sputa [Finally, besides LD-MPN and CFU counting, in the sputum of TB patients, the presence of a greater mycobacterial load in liquid than in solid media may be detected by the mycobacterial growth indicator tube (MGIT) time to positivity (TTP) assay, a semiquantitative measure of viable Mtb that showed an inverse relationship with the CFU ,73,74. Tve sputa . These dTools to identify promising in vitro drug regimens for rapid clinical trial evaluation are needed. One such tool is the hollow fibre system model of TB (HFS-TB), a bioreactor containing tubular hollow fibres made of semi-permeable membranes, which showed a 94% predictive accuracy for clinical response rates and optimal exposures ,76. In 2Mycobacterium abscessus in 42 and 56 days, respectively [Giardia spp [A more stringent TTP threshold value was used by our group, who defined the Mtb killing of both AR and NR cells (the latter obtained in the Wayne model) as the lack of regrowth of drug-treated bacilli after 100 days of incubation in MGIT tubes (TTP > 100 days) ,78,79. Uectively . Overallrdia spp ,82 and irdia spp . It is prdia spp ,53,56,57rdia spp ,53,54, aThe ability of a drug combination to sterilize TB lesions and prevent relapses due to drug-tolerant persisters and DD cells is determined by the synergistic activities of drug components. In the current regimen used for the treatment of DS TB (R-H-E-Z), R and Z have higher sterilizing activity than H and E . In the However, the low-cost, non caseum-forming BALB/c mice model was the most extensively used for testing drug combinations. In several studies, treatment efficacy was assessed based on lung CFU counts at selected time points during treatment , and on the proportion of mice with culture-positive relapse at least 3 months after the completion of treatment (a measure of sterilizing activity) ,102,103.A more synthetic view of Overall, these data have been useful for identifying pivotal drugs/drug combinations eradicating Mtb from BALB/c mice. Some of these drugs were also tested in combination experiments for the ability to sterilize caseum-forming C3HeB/FeJ mice, and for the efficacy of shortening human TB therapy in well-controlled clinical trials.As shown above, 10H25Z150 (regimens 1–3) or R10H10E100Z150 (regimen 6) for 2 months, followed by 4 months of R10H25 (regimens 1–3) or R10H10 (regimen 6) sterilized the mice in 4.5 to 6 months. These regimens mimicked those currently being used for the treatment of human TB (R-H-E-Z). However, only regimen 6 contained E, a bacteriostatic drug. A 5-month sterilization occurred when H was replaced with M in the intensive and continuation therapy (regimen 4), or when the combination contained the new anti-TB drug Pa .The first 6 combinations (regimens 1 to 6) sterilized BALB/c mice after 4.5 to 6 months of treatment ,94,97,9910H25Z150 and R10H10E100Z150 during the entire period of treatment. In some cases, R, H and Z were administered at high doses. For instance, in regimen 7, after the initial 0.5 months of administration of 25 mg/kg of H (H25), the drug was later administered at 75 mg/kg (H75). Furthermore, R was administered at 50 mg/kg (R50) in regimen 22, and Z at 300 mg/kg (Z300) in the intensive phase of regimen 9. A common characteristic of regimens 7–22 was the use of drugs other than R, H and Z, including (i) 15 mg/kg of the long-acting rifamycin P (P15) (regimens 7–9) that was substituted for 10 mg/kg of rifampin (R10); (ii) 25 mg/kg of B ; (iii) 50 or 100 mg/kg of the nitroimidazole Pa ; iv) 100 mg/kg of M ; (v) 12.5 or 25 mg/kg of C ; and (vi) 50 mg/kg of S . Overall, Z was contained in 81% of these combinations, followed by Pa (50%), B and R (44%), M (31%), P (25%), H, C and E (13%) and S (6%).Sterilization occurred also after 3.5 to 4 months of treatment with the 16 regimens (regimens 7 to 22) listed in Sterilization was observed also following 2.5 to 3 months of treatment with the 32 regimens (regimens 23 to 54) listed in 25 and Z150, followed by Pa (6/11), P and C (3/11), M and L (2/11)  that sterilized mice in 1.5–2 months. Ten of them contained both BL (2/11) B. In thiAll these data, reported from 2006 to 2022 mostly by the Center for TB research of the Johns Hopkins University, Baltimore, Maryland, USA, coordinated by Dr. Eric Nuermberger ,102,103,The results shown in the BALB/c mice were important in designing drug combinations eradicating Mtb in the caseous lesions of C3HeB/FeJ mice, a strain that develops lung necrotic granulomas similar to those found in human TB ,85. In 210H10E100Z150 for 2 months, followed by R10H10 for 4 months , sterilized the mice in 6 months. The addition of Z at 150 mg/kg (Z150) in the continuation phase sterilized the mice in 4.5 months (regimen 2). Thus, continuing Z beyond the first two months was beneficial to prevent relapses in caseum-forming mice. However, this is not applicable to humans, because the use of R-Z beyond 2 months is hepatotoxic, as shown during the prolonged use of this combination for the treatment of LTBI [10 with P10, and E100 with M100, sterilized the C3HeB/FeJ mice in 4 months [10 was replaced with a high dose of P (P20), and 12.5 mg/kg of the anti-leprosy drug C (C12.5) was added (regimen 4). Clofazimine is a drug that seems in large part to function by inducing ROS in Mtb [20H10E100Z150 in 2.5 months (regimen 7).The administration of R of LTBI . In regi4 months . SteriliS in Mtb . The adm25M100Z150Rb10 (regimen 5) or 2 months of B25M100Z150Rb10, followed by 1 month of B25M100Rb10, (regimen 6) [Finally, due to the knowledge that a strong induction of the cytochrome P450 3A (CYP3A) caused by R and P but not rifabutin (Rb) reduced the concentration of B , it was gimen 6) . OverallThe frequent presence of Z in combinations sterilizing BALB/c and C3HeB/FeJ mice is likely related to the knowledge that this drug penetrates all TB lung lesions and kills persisters residing in caseum . This inOverall, the murine TB data were useful for the choice of drugs to be combined in human TB clinical trials, in order to shorten the current 6-month regimen (R-H-E-Z). This regimen, finalized during the 1980s, and based on seminal studies conducted by the British Medical Research Council in the second half of the 20th century, was widely adopted worldwide for more than four decades. It cured more than 95% of persons, but in some cases long-term adherence to the therapy was difficult to sustain for patients and national resource constraints, contributing to the development of genetic drug-resistance ,111,112.All the trials included Z-containing combinations with 6-month , 4-month600H300Z1500E1200 and 4 months of continuation therapy with R600H300) and a 6-month regimen as effective as the control regimen in which H was replaced by a daily dose of 400 mg of M (M400) for 2 months, followed by one weekly dose of both M400 and a high dose (1200 mg) of P (P1200) for 4 months.The first paper  was publ600H300Z1500E1200), and a 4-month regimen with 2 months (indicated as 8 weeks in the paper) of once-daily P1200H300Z1500M400 , followed by 2 months (indicated as 9 weeks in the paper) of once-daily P1200H300M400. This 4-month regimen was supported by the WHO as a possible alternative to the current 6-month regimen for DS-TB, because it showed a similar performance in terms of its efficacy and safety [The second paper  was publd safety ,116. In d safety ,116.400/200L600H300Z1500E1100) (The third paper  was publ00E1100)  was noniIn the framework of the WHO’s vision to eliminate TB as a public health problem by 2035 , the thrIt is difficult to correlate the physicochemical properties of the drugs considered , with thfu) (free drug) in caseum or the bactericidal activity in caseum, a lipidic niche for Mtb drug-tolerant persisters and DD cells [max decrease in 7 days, max decrease) [However, some observations may be drawn concerning, for instance, the interplay among the half-life in serum or tissues , the unbDD cells ,120,121.The drugs with half-lives of ≥10 h were, in decreasing order, B (5.5 months) > C (about 25 days) > Rb (45 h) > Pa (18 h) > M (11.5–15.6 h) > P (10–15 h) > Z (9–10 h) . In spitfu, fu > 10% [fu values of the hydrophilic H and Z (clogP -0.71)  were <0.01%, i.e., they could not penetrate caseum [fu values . In this view, a rational design of combinations should involve drugs that complement each other in their ability to penetrate caseous granulomas and kill drug-tolerant persisters and DD cells living inside them.As to the unbound fraction in caseum fu, , this pafu > 10% . Indeed,P -0.71)  were >99e caseum ,119. Usi1200H300Z1500M400)  in rabbit caseum of >99.9%, 13.5% and 0.5%, respectively  , which c5 months , are quiectively . Howeverectively  [110,120ectively . However400/200L600H300Z1500E1100)  that sterilized the caseum-forming C3HeB/FeJ mice in 3 months   that curt caseum . HoweverAll the data on drug activity against drug-tolerant Mtb, ranging from the most studied in vitro Wayne dormancy model  to the pIn the last two decades, this new way of seeing the TB boosted the search for novel drugs/drug combinations killing in vitro and in vivo AR cells and NR drug-tolerant cells containing persisters and DD cells. More recently, some of these combinations were tested in several DS- and DR-TB clinical trials, so far achieving in various cases the goal of shortening the duration of the current drug regimens. In 2022, the WHO, following the results of one of these trials, supported the use of a 4-month regimen for the treatment of DS-TB as a possible alternative to the current 6-month regimen ,116.