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PD1 blockade therapy, harnessing the cytotoxic potential of CD8+ T cells, has yielded clinical success in treating malignancies. However, its efficacy is often limited due to the progressive differentiation of intratumoral CD8+ T cells into a hypofunctional state known as terminal exhaustion. Despite identifying CD8+ T cell subsets associated with immunotherapy resistance, the molecular pathway triggering the resistance remains elusive. Given the clear association of CD38 with CD8+ T cell subsets resistant to anti-PD1 therapy, we investigated its role in inducing resistance. Phenotypic and functional characterization, along with single-cell RNA sequencing analysis of both in vitro chronically stimulated and intratumoral CD8+ T cells, revealed that CD38-expressing CD8+ T cells are terminally exhausted. Exploring the molecular mechanism, we found that CD38 expression was crucial in promoting terminal differentiation of CD8+ T cells by suppressing TCF1 expression, thereby rendering them unresponsive to anti-PD1 therapy. Genetic ablation of CD38 in tumor-reactive CD8+ T cells restored TCF1 levels and improved the responsiveness to anti-PD1 therapy in mice. Mechanistically, CD38 expression on exhausted CD8+ T cells elevated intracellular Ca2+ levels through RyR2 calcium channel activation. This, in turn, promoted chronic AKT activation, leading to TCF1 loss. Knockdown of RyR2 or inhibition of AKT in CD8+ T cells maintained TCF1 levels, induced a sustained anti-tumor response, and enhanced responsiveness to anti-PD1 therapy. Thus, targeting CD38 represents a potential strategy to improve the efficacy of anti-PD1 treatment in cancer.
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Linfocitos T CD8-positivos , Neoplasias , Ratones , Animales , Linfocitos T CD8-positivos/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Subgrupos de Linfocitos T/metabolismoRESUMEN
The formation of a reversible disulfide bond between the catalytic cysteine and a spatially neighboring cysteine (backdoor) in protein tyrosine phosphatases (PTPs) serves as a critical regulatory mechanism for maintaining the activity of protein tyrosine phosphatases. The failure of such protection results in the formation of irreversibly oxidized cysteines into sulfonic acid in a highly oxidative cellular environment in the presence of free radicals. Hence, it is important to develop methods to interconvert PTPs into reduced and oxidized forms to understand their catalytic function in vitro. Protein tyrosine phosphatase 4A type 1 (PTP4A1), a dual-specificity phosphatase, is catalytically active in the reduced form. Unexpectedly, also its oxidized form performs a key biological function in systemic sclerosis (SSc) by forming a kinase-phosphatase complex with Src kinases. Thus, we developed simple and efficient protocols for producing oxidized and reduced PTP4A1 to elucidate their biological function, which can be extended to study other protein tyrosine phosphatases and other recombinantly produced proteins.
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Cisteína , Fosfatasas de Especificidad Dual , Catálisis , Citoplasma , Dominios ProteicosRESUMEN
Penicillin-binding proteins (PBPs) are essential for the formation of the bacterial cell wall. They are also the targets of ß-lactam antibiotics. In Enterococcus faecium, high levels of resistance to ß-lactams are associated with the expression of PBP5, with higher levels of resistance associated with distinct PBP5 variants. To define the molecular mechanism of PBP5-mediated resistance we leveraged biomolecular NMR spectroscopy of PBP5 - due to its size (>70 kDa) a challenging NMR target. Our data show that resistant PBP5 variants show significantly increased dynamics either alone or upon formation of the acyl-enzyme inhibitor complex. Furthermore, these variants also exhibit increased acyl-enzyme hydrolysis. Thus, reducing sidechain bulkiness and expanding surface loops results in increased dynamics that facilitates acyl-enzyme hydrolysis and, via increased ß-lactam antibiotic turnover, facilitates ß-lactam resistance. Together, these data provide the molecular basis of resistance of clinical E. faecium PBP5 variants, results that are likely applicable to the PBP family.
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Antibacterianos , Hexosiltransferasas , Proteínas de Unión a las Penicilinas/genética , Proteínas de Unión a las Penicilinas/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Resistencia betalactámica/genética , Monobactamas , beta-Lactamas/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
Berberine (BBR), a traditional Chinese phytomedicine extracted from various parts of Berberis plants, is an isoquinoline alkaloid used for centuries to treat diabetes, hypercholesterolemia, hypertension, and so forth. It has recently received immense attention worldwide to treat cancer due to its potent pro-apoptotic, antiproliferative, and anti-inflammatory properties. BBR efficiently induces tumor apoptosis, replicative quiescence and abrogates cell proliferation, epithelial-mesenchymal transition, tumor neovascularization, and metastasis by modulating diverse molecular and cell signaling pathways. Furthermore, BBR could also reverse drug resistance, make tumor cells sensitive to current cancer treatment and significantly minimize the harmful side effects of cytotoxic therapies. This review comprehensively analyzed the pharmacological effects of BBR against the development, growth, progression, metastasis, and therapy resistance in wide varieties of cancer. Also, it critically discusses the significant limitations behind the development of BBR into pharmaceuticals to treat cancer and the future research directions to overcome these limitations.
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Antineoplásicos , Berberina , Resistencia a Antineoplásicos , Medicamentos Herbarios Chinos , Neoplasias , Berberina/farmacología , Berberina/uso terapéutico , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/prevención & control , Humanos , Resistencia a Antineoplásicos/efectos de los fármacos , Apoptosis/efectos de los fármacos , Metástasis de la Neoplasia , Proliferación Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/prevención & controlRESUMEN
The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of substrates by associating with >200 regulatory proteins to form specific holoenzymes. The major PP1 targeting protein in the nucleolus is RRP1B (ribosomal RNA processing 1B). In addition to selectively recruiting PP1ß/PP1γ to the nucleolus, RRP1B also has a key role in ribosome biogenesis, among other functions. How RRP1B binds PP1 and regulates nucleolar phosphorylation signaling is not yet known. Here, we show that RRP1B recruits PP1 via established (RVxF/SILK/ΦΦ) and non-canonical motifs. These atypical interaction sites, the PP1ß/γ specificity, and N-terminal AF-binding pockets rely on hydrophobic interactions that contribute to binding and, via phosphorylation, regulate complex formation. This work advances our understanding of PP1 isoform selectivity, reveals key roles of N-terminal PP1 residues in regulator binding, and suggests that additional PP1 interaction sites have yet to be identified, all of which are necessary for a systems biology understanding of PP1 function.
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Nucléolo Celular , Procesamiento Postranscripcional del ARN , Proteína Fosfatasa 1 , Holoenzimas , FosforilaciónRESUMEN
Systemic sclerosis (SSc) is a fibrotic autoimmune disease characterized by pathogenic activation of fibroblasts enhanced by local oxidative stress. The tyrosine phosphatase PTP4A1 was identified as a critical promoter of TGF-ß signaling in SSc. Oxidative stress is known to functionally inactivate tyrosine phosphatases. Here, we assessed whether oxidation of PTP4A1 modulates its profibrotic action and found that PTP4A1 forms a complex with the kinase SRC in scleroderma fibroblasts, but surprisingly, oxidative stress enhanced rather than reduced PTP4A1's association with SRC and its profibrotic action. Through structural assessment of the oxo-PTP4A1-SRC complex, we unraveled an unexpected mechanism whereby oxidation of a tyrosine phosphatase promotes its function through modification of its protein complex. Considering the importance of oxidative stress in the pathogenesis of SSc and fibrosis, our findings suggest routes for leveraging PTP4A1 oxidation as a potential strategy for developing antifibrotic agents.
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Esclerodermia Sistémica , Fibroblastos/metabolismo , Fibrosis , Humanos , Estrés Oxidativo , Esclerodermia Sistémica/patología , Tirosina/metabolismoRESUMEN
Retinoblastoma (Rb) is the most common childhood malignancy initiated by biallelic mutation in RB1 gene and driven by various epigenetic events including DNA methylation and microRNA dysregulation. Hence, understanding the key genes that are critically modulated by epigenetic modifications in RB1 -/- cells is very important to identify prominent biomarkers and therapeutic targets of Rb. In this study, we for the first time have integrated various Rb microarray NCBI-GEO datasets including DNA Methylation (GSE57362), miRNA (GSE7072) and mRNA (GSE110811) to comprehensively investigate the epigenetic consequences of RB loss in retinoblastoma tumors and identify genes with the potential to serve as early diagnostic markers and therapeutic targets for Rb. Interestingly, the GEO2R and co-expression network analysis have identified three genes namely E2F3, ESR1, and UNC5D that are significantly deregulated by modified DNA methylation, mRNA and microRNA expression in Rb tumors. Due to their recognition in all epigenetic, transcriptomic, and miRNA datasets, we have termed these genes as "common genes". The results of our integrative bioinformatics analysis were validated in vitro by studying the gene and protein expression of these common genes in Y79, WERI-Rb-1, Rb cell lines and non-tumorigenic retinal pigment epithelial cell line (hTERT-RPE). The expression of E2F3 and UNC5D were up-regulated and that of ESR1 was down-regulated in Rb tumor cells when compared to that in non-tumorigenic hTERT-RPE cells. More importantly, UNC5D, a potent tumor suppressor gene in most cancers is significantly up-regulated in Y79 and Weri Rb1 cells, which, in turn, questions its anti-cancer properties. Together, our study shows that E2F3, ESR1, and UNC5D may be crucially involved in Rb tumorigenesis and possess the potential to act as early diagnostic biomarkers and therapeutic targets of Rb.
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BACKGROUND: Single-session endoscopic stone extraction (ESE) and laparoscopic cholecystectomy (LC) has the best outcome in managing concomitant cholelithiasis (gallstone disease [GSD]) and choledocholithiasis (common bile duct stone [CBDS]). Traditional rendezvous technique with an intraoperative cholangiogram is associated with various technical (bowel distention, frozen Calot's triangle, limitation of intraoperative cholangiogram and so on) and logistical difficulties (lack of trained personnel and equipment for ESE in the operating room). We modified our approach of ESE-LC (tandem ESE-LC) to study the safety of the approach and overcome these disadvantages of the traditional rendezvous approach. METHODS: A prospective study of patients with GSD and suspected CBDS from January 2017 to December 2019 was conducted. Tandem ESE-LC involves ESE and LC under the same general anaesthesia in a single day, while ESE is performed in the endoscopic suite using carbon dioxide insufflation, a balloon/basket was used for achieving bile duct clearance and the same was confirmed with an occlusion cholangiogram. Patients were then shifted to the operating room for LC. The primary outcome included bile duct clearance and safety of the procedure. RESULTS: Of 56 patients assessed for eligibility, 42 were included in the study (median age: 53 years, 25 [60%] women). Biliary colic was the most common presenting symptom (n = 24, 57%), followed by acute cholecystitis (n = 11, 26%). The median number of stones and stone size was 1 (1-6) and 4 mm (3-10), respectively. All patients had successful bile duct clearance. Stenting was performed in 5 (12%) patients. Intraoperatively, Calot's dissection was difficult and frozen in 10 and 11 patients respectively. The cystic duct was short and wide in 13 (31%) patients. Subtotal cholecystectomy was performed in 6 (14%) patients. The median duration of postprocedural hospital stay was 1 (0-13) day. Three patients had tandem ESE-LC on a day-care basis. One patient had post-endoscopic retrograde cholangiopancretography pancreatitis, and another required percutaneous drainage for gall bladder fossa collection. No patient had retained CBDS at a median follow-up of 18 (3-28) months. CONCLUSION: Tandem ESE-LC is safe and effective method in managing concomitant GSD and CBDS.
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Mucinous cystic neoplasm (MCN) of the pancreas is a rare neoplasm affecting the elderly women. They vary in their clinical presentation and biological behavior. Spontaneous rupture of MCN is very rare and only eight cases have been reported so far in the English literature. We report a case of a young woman presenting with abdominal pain following spontaneous contained rupture of MCN managed with surgical resection. How to cite this article: Krishnamurthy G, Ganesan S, Ramas J, et al. Ruptured Mucinous Cystadenoma Pancreas: A Case Report and Review of Literature. Euroasian J Hepato-Gastroenterol 2021;11(1):45-48.
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The sequence-specific backbone assignment of the mitogen-activated protein kinase (MAPK) binding domain of the dual-specificity phosphatase 1 (DUSP1) has been accomplished using a uniformly [13C, 15N]-labeled protein. These assignments will facilitate further studies of DUSP1 in the presence of inhibitors/ligands to target MAPK associated diseases and provide further insights into the function of dual-specificity phosphatase 1 in MAPK regulation.
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Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Diabetic retinopathy (DR), a major microvascular complication of diabetes, affects most diabetic individuals and has become the leading cause of vision loss. Metabolic memory associated with diabetes retains the risk of disease occurrence even after the termination of glycemic insult. Further, various limitations associated with its current diagnostic and treatment strategies like unavailability of early diagnostic and treatment methods, variation in treatment response from patient to patient, and cost-effectiveness have driven the need to find alternative solutions. Post-transcriptional epigenetic modification of RNA mainly, N6-methyladenosine (m6A), is an emerging concept in the scientific community. It has an indispensable effect in various physiological and pathological conditions. m6A mediates its effect through the various reader, writer, and eraser proteins. Recent studies have shown the impact of m6A RNA modification on various disease conditions, including diabetes, but its role in diabetic retinopathy is still unclear. However, change in m6A levels has been observed in various prime aggravators of DR pathogenesis, such as inflammation, oxidative stress, and angiogenesis. Further, various non-coding RNAs like microRNA, lncRNA, and circRNA are also associated with DR, and m6A has been shown to affect all these non-coding RNAs. This review is concerned with the possible mechanisms through which alteration in m6A modification of RNA can participate in the DR progression and pathogenesis and its expected role in metabolic memory phenomena.
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Retinopatía Diabética/genética , Epigénesis Genética/genética , Metiltransferasas/genética , MicroARNs/genética , Retinopatía Diabética/metabolismo , Humanos , Metilación , Metiltransferasas/metabolismo , MicroARNs/metabolismoRESUMEN
Cisplatin is the most commonly used first-line drug for cancer treatment. However, many patients develop resistance to cisplatin therapy which ultimately results in therapy failure and increased mortality. A growing body of evidence shows that the hypoxic microenvironment is the prime factor underlying tumor insensitivity to cisplatin treatment. Since tumors in the majority of cancer patients are under hypoxic stress (low oxygen supply), it becomes necessary to understand the pathobiology behind hypoxia-induced cisplatin resistance in cancer cells. Here, we discuss the molecular events that render hypoxic tumors insensitive to cisplatin therapy. Furthermore, various drugs and tumor oxygenation techniques have been developed to circumvent cisplatin resistance in hypoxic tumors. However, their pharmaceutical applications are limited due to failures in clinical investigations and a lack of preclinical studies in the hypoxic tumor microenvironment. This review addresses these challenges and provides new directions for the strategic deployment of cisplatin sensitizers in the hypoxic tumor microenvironment.
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Cisplatino , Neoplasias , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Hipoxia , Neoplasias/tratamiento farmacológico , Hipoxia Tumoral , Microambiente TumoralRESUMEN
ß-sitosterol (SIT), the most abundant bioactive component of vegetable oil and other plants, is a highly potent antidiabetic drug. Our previous studies show that SIT controls hyperglycemia and insulin resistance by activating insulin receptor and glucose transporter 4 (GLUT-4) in the adipocytes of obesity induced type 2 diabetic rats. The current research was undertaken to investigate if SIT could also exert its antidiabetic effects by circumventing adipocyte induced inflammation, a key driving factor for insulin resistance in obese individuals. Effective dose of SIT (20 mg/kg b.wt) was administered orally for 30 days to high fat diet and sucrose induced type-2 diabetic rats. Metformin, the conventionally used antidiabetic drug was used as a positive control. Interestingly, SIT treatment restores the elevated serum levels of proinflammatory cytokines including leptin, resistin, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) to normalcy and increases anti-inflammatory adipocytokines including adiponectin in type 2 diabetic rats. Furthermore, SIT decreases sterol regulatory element binding protein-1c (SREBP-1c) and enhances Peroxisome Proliferator-activated receptor-γ (PPAR-γ) gene expression in adipocytes of diabetic rats. The gene and protein expression of c-Jun-N-terminal kinase-1 (JNK1), inhibitor of nuclear factor kappa-B kinase subunit beta (IKKß) and nuclear factor kappa B (NF-κB) were also significantly attenuated in SIT treated groups. More importantly, SIT acts very effectively as metformin to circumvent inflammation and insulin resistance in diabetic rats. Our results clearly show that SIT inhibits obesity induced insulin resistance by ameliorating the inflammatory events in the adipose tissue through the downregulation of IKKß/NF-κB and c-Jun-N-terminal kinase (JNK) signaling pathway.
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Adipocitos/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Regulación hacia Abajo , Quinasa I-kappa B/metabolismo , Inflamación/tratamiento farmacológico , Resistencia a la Insulina , Obesidad/complicaciones , Sitoesteroles/uso terapéutico , Adipocitos/efectos de los fármacos , Adipoquinas/sangre , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Peso Corporal/efectos de los fármacos , Citocinas/sangre , Citocinas/genética , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Dieta Alta en Grasa , Regulación hacia Abajo/efectos de los fármacos , Conducta Alimentaria , Inflamación/sangre , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Simulación del Acoplamiento Molecular , FN-kappa B/metabolismo , Obesidad/sangre , PPAR gamma/genética , PPAR gamma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Sitoesteroles/farmacología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Sacarosa , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: Acute gallbladder perforation (GBP) is associated with significant mortality and morbidity. Percutaneous drainage followed by interval cholecystectomy has been the preferred management. The outcomes of early surgery, especially by laparoscopy, have not been well studied in GBP. We present our experience in early laparoscopic cholecystectomy in GBP. METHODOLOGY: A retrospective analysis of patients admitted with GBP between April 2014 and December 2018 was done. Clinical presentation, preoperative imaging, surgical procedure, operative findings and the outcomes in these patients were analysed. Video of the surgeries was reviewed in case of the absence of data from the case records. RESULTS: Fifteen patients were treated for GBP during the study period. Eleven patients were male, and the mean age was 61 years. Fourteen patients (93.3%) had associated co-morbidities. Systemic inflammatory response syndrome, sepsis, severe sepsis and septic shock were present in 3, 3, 6 and 3 patients, respectively. The location of the collection was gallbladder fossa, pericholecystic, subhepatic and diffuse in 3, 5, 4 and 3 patients, respectively. Intraoperatively, 13 patients were detected to have perforation at the fundus of the gallbladder. Cystic duct stump was managed with clip, endoloop, suturing and external drainage in 7, 2, 5 and 1 patient, respectively. Laparoscopic cholecystectomy was completed in 12 (80%) patients. Retroinfundibular technique was used in 12 (80%) patients. There was one conversion. Two patients required endoscopic retrograde cholangiogram + bile duct stenting, and one was reexplored for cystic artery bleed. There were no mortalities. The median duration of post-operative hospital stay and drain removal was 3 (1-19) and 3 (1-6), respectively. CONCLUSION: Early laparoscopic cholecystectomy in acute GBP is feasible and can be safely performed in centres having sufficient expertise. Retroinfundibular technique of laparoscopic cholecystectomy is useful in tackling frozen Calot's triangle in GBP.
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Mitogen-activated protein kinase (MAPK; p38, ERK, and JNK) cascades are evolutionarily conserved signaling pathways that regulate the cellular response to a variety of extracellular stimuli, such as growth factors and interleukins. The MAPK p38 is activated by its specific upstream MAPK kinases, MKK6 and MKK3. However, a comprehensive molecular understanding of how these cognate upstream kinases bind and activate p38 is still missing. Here, we combine NMR spectroscopy and isothermal titration calorimetry to define the binding interface between full-length MKK6 and p38. It was shown that p38 engages MKK6 not only via its hydrophobic docking groove, but also influences helix αF, a secondary structural element that plays a key role in organizing the kinase core. It was also shown that, unlike MAPK phosphatases, the p38 conserved docking (CD) site is much less affected by MKK6 binding. Finally, it was demonstrated that these interactions with p38 are conserved independent of the MKK6 activation state. Together, the results revealed differences between specificity markers of p38 regulation by upstream kinases, which do not effectively engage the CD site, and downstream phosphatases, which require the CD site for productive binding.
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MAP Quinasa Quinasa 6/química , Proteínas Quinasas p38 Activadas por Mitógenos/química , Sitios de Unión , Activación Enzimática , Humanos , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica en Hélice alfa , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Chemotherapy and radiotherapy are mainstay treatments for cancer patients. However, their clinical outcomes are highly limited by the resistance of malignant tumors to these therapies and the incurrence of serious damages in vital organs. This in turn necessitates the development of adjunct drugs that overcomes chemo/radioresistance in refractory cancers and protects vital organs from the cytotoxic effects of cancer therapies. In recent years, Berberine (BBR), a natural isoquinoline alkaloid has garnered more attention due to its potent chemosensitizing and chemoprotective properties. BBR effectively sensitizes refractory cancers to chemotherapy and radiotherapy by ameliorating the diverse events underlying therapy resistance. Furthermore, it protects the heart, liver, lungs, and kidneys from severe damages caused by these therapies. In this review, we discuss the molecular mechanisms underlying the chemo/radiosensitizing and chemo/radioprotective potential of BBR during cancer treatment. Also, we highlight the limitations that hamper the clinical application of BBR as an adjunct drug and how novel innovations have been made in recent years to circumvent these challenges.
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Antineoplásicos/farmacología , Berberina/farmacología , Neoplasias/tratamiento farmacológico , Animales , HumanosRESUMEN
The increased incidence of diabetic retinopathy (DR) and the legacy effect associated with it has raised a great concern toward the need to find early diagnostic and treatment strategies. Identifying alterations in genes and microRNAs (miRNAs) is one of the most critical steps toward understanding the mechanisms by which a disease progresses, and this can be further used in finding potential diagnostic and prognostic biomarkers and treatment methods. We selected different datasets to identify altered genes and miRNAs. The integrative analysis was employed to find potential candidate genes (differentially expressed and aberrantly methylated genes that are also the target of altered miRNAs) and early genes (genes showing altered expression and methylation pattern during early stage of DR) for DR. We constructed a protein-protein interaction (PPI) network to find hub genes (potential candidate genes showing a greater number of interactions) and modules. Gene ontologies and pathways associated with the identified genes were analyzed to determine their role in DR progression. A total of 271 upregulated-hypomethylated genes, 84 downregulated-hypermethylated genes, 11 upregulated miRNA, and 30 downregulated miRNA specific to DR were identified. 40 potential candidate genes and 9 early genes were also identified. PPI network analysis revealed 7 hub genes (number of interactions >5) and 1 module (score = 5.67). Gene ontology and pathway analysis predicted enrichment of genes in oxidoreductase activity, binding to extracellular matrix, immune responses, leukocyte migration, cell adhesion, PI3K-Akt signaling pathway, ECM receptor interaction, etc., and thus their association with DR pathogenesis. In conclusion, we identified 7 hub genes and 9 early genes that could act as a potential prognostic, diagnostic, or therapeutic target for DR, and a few early genes could also play a role in metabolic memory phenomena.
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Transcription factor phosphorylation at specific sites often activates gene expression, but how environmental cues quantitatively control transcription is not well-understood. Activating protein 1 transcription factors are phosphorylated by mitogen-activated protein kinases (MAPK) in their transactivation domains (TAD) at so-called phosphoswitches, which are a hallmark in response to growth factors, cytokines or stress. We show that the ATF2 TAD is controlled by functionally distinct signaling pathways (JNK and p38) through structurally different MAPK binding sites. Moreover, JNK mediated phosphorylation at an evolutionarily more recent site diminishes p38 binding and made the phosphoswitch differently sensitive to JNK and p38 in vertebrates. Structures of MAPK-TAD complexes and mechanistic modeling of ATF2 TAD phosphorylation in cells suggest that kinase binding motifs and phosphorylation sites line up to maximize MAPK based co-regulation. This study shows how the activity of an ancient transcription controlling phosphoswitch became dependent on the relative flux of upstream signals.
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Factor de Transcripción Activador 2/metabolismo , Regulación de la Expresión Génica , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Transcripción Genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Factor de Transcripción Activador 2/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Células HEK293 , Humanos , Luciferasas/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Fosforilación , Unión Proteica , Dedos de ZincRESUMEN
Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for the treatment of diabetes and obesity. Ertiprotafib is a PTP1B inhibitor that reached the clinical trial stage for the treatment of diabetes. Interestingly, Ertiprotafib reduces the melting temperature of PTP1B in differential scanning fluorimetry (DSF) assays, different from most drugs that increase the stability of their target upon binding. No molecular data on how Ertiprotafib functions has been published. Thus, to gain molecular insights into the mode of action of Ertiprotafib, we used biomolecular NMR spectroscopy to characterize the molecular details of the PTP1B:Ertiprotafib interaction. Our results show that Ertiprotafib induces aggregation of PTP1B in a concentration dependent manner. This shows that the insufficient clinical efficacy and adverse effects caused by Ertiprotafib is due to its tendency to cause aggregation of PTP1B.