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Paralyzed muscles can be reanimated following spinal cord injury (SCI) using a brain-computer interface (BCI) to enhance motor function alone. Importantly, the sense of touch is a key component of motor function. Here, we demonstrate that a human participant with a clinically complete SCI can use a BCI to simultaneously reanimate both motor function and the sense of touch, leveraging residual touch signaling from his own hand. In the primary motor cortex (M1), residual subperceptual hand touch signals are simultaneously demultiplexed from ongoing efferent motor intention, enabling intracortically controlled closed-loop sensory feedback. Using the closed-loop demultiplexing BCI almost fully restored the ability to detect object touch and significantly improved several sensorimotor functions. Afferent grip-intensity levels are also decoded from M1, enabling grip reanimation regulated by touch signaling. These results demonstrate that subperceptual neural signals can be decoded from the cortex and transformed into conscious perception, significantly augmenting function.
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Retroalimentación Sensorial/fisiología , Percepción del Tacto/fisiología , Tacto/fisiología , Adulto , Interfaces Cerebro-Computador/psicología , Mano/fisiopatología , Fuerza de la Mano/fisiología , Humanos , Masculino , Corteza Motora/fisiología , Movimiento/fisiología , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Angiogenesis, the formation of new blood vessels by endothelial cells (ECs), is an adaptive response to oxygen/nutrient deprivation orchestrated by vascular endothelial growth factor (VEGF) upon ischemia or exercise. Hypoxia is the best-understood trigger of VEGF expression via the transcription factor HIF1α. Nutrient deprivation is inseparable from hypoxia during ischemia, yet its role in angiogenesis is poorly characterized. Here, we identified sulfur amino acid restriction as a proangiogenic trigger, promoting increased VEGF expression, migration and sprouting in ECs in vitro, and increased capillary density in mouse skeletal muscle in vivo via the GCN2/ATF4 amino acid starvation response pathway independent of hypoxia or HIF1α. We also identified a requirement for cystathionine-γ-lyase in VEGF-dependent angiogenesis via increased hydrogen sulfide (H2S) production. H2S mediated its proangiogenic effects in part by inhibiting mitochondrial electron transport and oxidative phosphorylation, resulting in increased glucose uptake and glycolytic ATP production.
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Factor de Transcripción Activador 4/metabolismo , Aminoácidos Sulfúricos/deficiencia , Sulfuro de Hidrógeno/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor de Transcripción Activador 4/antagonistas & inhibidores , Factor de Transcripción Activador 4/genética , Aminoácidos Sulfúricos/metabolismo , Animales , Cistationina gamma-Liasa/metabolismo , Modelos Animales de Enfermedad , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isquemia/metabolismo , Isquemia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neovascularización Fisiológica , Condicionamiento Físico Animal , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Monogenic diseases are well-suited paradigms for the causal analysis of disease-driving molecular patterns. Spinal Muscular Atrophy (SMA) is one such monogenic model caused by mutation or deletion of the Survival of motor neuron 1 (SMN1) gene. Although several functions of the SMN protein have been studied, single functions and pathways alone do not allow to identify critical disease-driving molecules. Here, we analyzed the systemic characteristics of SMA employing proteomics, phosphoproteomics, translatomics and interactomics from two mouse models with different disease-severities and genetics. This systems approach revealed sub-networks and proteins characterizing commonalities and differences of both models. To link the identified molecular networks with the disease-causing SMN protein, we combined SMN-interactome data with both proteomes creating a comprehensive representation of SMA. By this approach, disease hubs and bottlenecks between SMN and downstream pathways could be identified. Linking a disease-causing molecule with widespread molecular dysregulations via multiomics is a concept for analyses of monogenic diseases.
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This commentary discusses a comprehensive history of the first-ever use of pertinent words directly related to DNA, such as desoxyribose, deoxyribose, desoxyribonucleic acid, and deoxyribonucleic acid. With almost 100 years of the identification and nomenclature of desoxyribose sugar and desoxyribonucleic acid, the term "desoxy" continues to see limited use. We hope that whenever young researchers come across the sporadic occurrence of "desoxy" in any published text, they will not consider it a mistake.
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ADN , Terminología como AsuntoRESUMEN
Many bacterial histidine kinases work in two-component systems that combine into larger multi-kinase networks. NahK is one of the kinases in the GacS Multi-Kinase Network (MKN), which is the MKN that controls biofilm regulation in the opportunistic pathogen Pseudomonas aeruginosa. This network has also been associated with regulating many virulence factors P. aeruginosa secretes to cause disease. However, the individual role of each kinase is unknown. In this study, we identify NahK as a novel regulator of the phenazine pyocyanin (PYO). Deletion of nahK leads to a fourfold increase in PYO production, almost exclusively through upregulation of phenazine operon two (phz2). We determined that this upregulation is due to mis-regulation of all P. aeruginosa quorum-sensing (QS) systems, with a large upregulation of the Pseudomonas quinolone signal system and a decrease in production of the acyl-homoserine lactone-producing system, las. In addition, we see differences in expression of quorum-sensing inhibitor proteins that align with these changes. Together, these data contribute to understanding how the GacS MKN modulates QS and virulence and suggest a mechanism for cell density-independent regulation of quorum sensing. IMPORTANCE Pseudomonas aeruginosa is a Gram-negative bacterium that establishes biofilms as part of its pathogenicity. P. aeruginosa infections are associated with nosocomial infections. As the prevalence of multi-drug-resistant P. aeruginosa increases, it is essential to understand underlying virulence molecular mechanisms. Histidine kinase NahK is one of several kinases in P. aeruginosa implicated in biofilm formation and dispersal. Previous work has shown that the nitric oxide sensor, NosP, triggers biofilm dispersal by inhibiting NahK. The data presented here demonstrate that NahK plays additional important roles in the P. aeruginosa lifestyle, including regulating bacterial communication mechanisms such as quorum sensing. These effects have larger implications in infection as they affect toxin production and virulence.
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Biopelículas , Piocianina , Histidina Quinasa/genética , Histidina Quinasa/metabolismo , Percepción de Quorum , Factores de Virulencia/metabolismo , Bacterias/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/metabolismo , Antibacterianos/farmacologíaRESUMEN
BACKGROUND: The use of naltrexone plus bupropion to treat methamphetamine use disorder has not been well studied. METHODS: We conducted this multisite, double-blind, two-stage, placebo-controlled trial with the use of a sequential parallel comparison design to evaluate the efficacy and safety of extended-release injectable naltrexone (380 mg every 3 weeks) plus oral extended-release bupropion (450 mg per day) in adults with moderate or severe methamphetamine use disorder. In the first stage of the trial, participants were randomly assigned in a 0.26:0.74 ratio to receive naltrexone-bupropion or matching injectable and oral placebo for 6 weeks. Those in the placebo group who did not have a response in stage 1 underwent rerandomization in stage 2 and were assigned in a 1:1 ratio to receive naltrexone-bupropion or placebo for an additional 6 weeks. Urine samples were obtained from participants twice weekly. The primary outcome was a response, defined as at least three methamphetamine-negative urine samples out of four samples obtained at the end of stage 1 or stage 2, and the weighted average of the responses in the two stages is reported. The treatment effect was defined as the between-group difference in the overall weighted responses. RESULTS: A total of 403 participants were enrolled in stage 1, and 225 in stage 2. In the first stage, 18 of 109 participants (16.5%) in the naltrexone-bupropion group and 10 of 294 (3.4%) in the placebo group had a response. In the second stage, 13 of 114 (11.4%) in the naltrexone-bupropion group and 2 of 111 (1.8%) in the placebo group had a response. The weighted average response across the two stages was 13.6% with naltrexone-bupropion and 2.5% with placebo, for an overall treatment effect of 11.1 percentage points (Wald z-test statistic, 4.53; P<0.001). Adverse events with naltrexone-bupropion included gastrointestinal disorders, tremor, malaise, hyperhidrosis, and anorexia. Serious adverse events occurred in 8 of 223 participants (3.6%) who received naltrexone-bupropion during the trial. CONCLUSIONS: Among adults with methamphetamine use disorder, the response over a period of 12 weeks among participants who received extended-release injectable naltrexone plus oral extended-release bupropion was low but was higher than that among participants who received placebo. (Funded by the National Institute on Drug Abuse and others; ADAPT-2 ClinicalTrials.gov number, NCT03078075.).
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Trastornos Relacionados con Anfetaminas/tratamiento farmacológico , Bupropión/administración & dosificación , Metanfetamina , Naltrexona/administración & dosificación , Administración Oral , Adolescente , Adulto , Anciano , Bupropión/efectos adversos , Preparaciones de Acción Retardada , Método Doble Ciego , Quimioterapia Combinada , Femenino , Humanos , Inyecciones , Masculino , Cumplimiento de la Medicación , Metanfetamina/orina , Persona de Mediana Edad , Naltrexona/efectos adversos , Antagonistas de Narcóticos , Adulto JovenRESUMEN
Amyloid precursor protein (APP) plays a pivotal role in the pathology of Alzheimer's disease (AD). Since the fragmentation of the membrane-bound APP that results in the production of amyloid-ß peptides is the starting point for amyloid toxicity in AD, it is important to investigate the structure and dynamics of APP in a near-native lipid-bilayer environment. However, the reconstitution of APP into a stable and suitable membrane-mimicking lipid environment is a challenging task. In this study, the 99-residue C-terminal domain of APP is successfully reconstituted into polymer nanodiscs and characterized using size-exclusion chromatography, mass spectrometry, solution NMR, and magic-angle spinning solid-state NMR. In addition, the feasibility of using lipid-solubilizing polymers for isolating and characterizing APP in the native Escherichia. coli membrane environment is demonstrated.
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Precursor de Proteína beta-Amiloide , Nanoestructuras , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Nanoestructuras/química , Escherichia coli , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Resonancia Magnética Nuclear BiomolecularRESUMEN
Diabetes mellitus (DM) is characterized by chronic hyperglycemia, and despite intensive glycemic control, the risk of heart failure in diabetic patients remains high. Diabetes-induced heart failure (DHF) presents a unique metabolic challenge, driven by significant alterations in cardiac substrate metabolism, including increased reliance on fatty acid oxidation, reduced glucose utilization, and impaired mitochondrial function. These metabolic alterations lead to oxidative stress, lipotoxicity, and energy deficits, contributing to the progression of heart failure. Emerging research has identified novel mechanisms involved in the metabolic remodeling of diabetic hearts, such as autophagy dysregulation, epigenetic modifications, polyamine regulation, and branched-chain amino acid (BCAA) metabolism. These processes exacerbate mitochondrial dysfunction and metabolic inflexibility, further impairing cardiac function. Therapeutic interventions targeting these pathways-such as enhancing glucose oxidation, modulating fatty acid metabolism, and optimizing ketone body utilization-show promise in restoring metabolic homeostasis and improving cardiac outcomes. This review explores the key molecular mechanisms driving metabolic remodeling in diabetic hearts and advanced methodology, highlighting the latest therapeutic strategies to mitigate the progression of DHF. Understanding these emerging pathways offers new opportunities to develop targeted therapies that address the root metabolic causes of heart failure in diabetes.
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Cyclin dependent kinase 4 and 6 inhibitors such as abemaciclib are routinely used to treat metastatic estrogen receptor positive (ER+) breast cancer. However, adaptive mechanisms inhibit their effectiveness and allow for disease progression. Using ER+ breast cancer cell models, we show that acquired resistance to abemaciclib is accompanied by increase in metastatic potential. Mass spectrometry-based proteomics from abemaciclib sensitive and resistant cells showed that lysosomal proteins including CTSD (cathepsin D), cathepsin A and CD68 were significantly increased in resistant cells. Combination of abemaciclib and a lysosomal destabilizer, such as hydroxychloroquine (HCQ) or bafilomycin A1, resensitized resistant cells to abemaciclib. Also, combination of abemaciclib and HCQ decreased migration and invasive potential and increased lysosomal membrane permeability in resistant cells. Prosurvival B cell lymphoma 2 (BCL2) protein levels were elevated in resistant cells, and a triple treatment with abemaciclib, HCQ, and BCL2 inhibitor, venetoclax, significantly inhibited cell growth compared to treatment with abemaciclib and HCQ. Furthermore, resistant cells showed increased levels of Transcription Factor EB (TFEB), a master regulator of lysosomal-autophagy genes, and siRNA mediated knockdown of TFEB decreased invasion in resistant cells. TFEB was found to be mutated in a subset of invasive human breast cancer samples, and overall survival analysis in ER+, lymph node-positive breast cancer showed that increased TFEB expression correlated with decreased survival. Collectively, we show that acquired resistance to abemaciclib leads to increased metastatic potential and increased levels of protumorigenic lysosomal proteins. Therefore, the lysosomal pathway could be a therapeutic target in advanced ER+ breast cancer.
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Aminopiridinas , Bencimidazoles , Neoplasias de la Mama , Proteínas , Humanos , Femenino , Neoplasias de la Mama/metabolismo , Lisosomas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
The underlying cause of Spinal Muscular Atrophy (SMA) is in the reduction of survival motor neuron (SMN) protein levels due to mutations in the SMN1 gene. The specific effects of SMN protein loss and the resulting pathological alterations are not fully understood. Given the crucial roles of the SMN protein in snRNP biogenesis and its interactions with ribosomes and translation-related proteins and mRNAs, a decrease in SMN levels below a specific threshold in SMA is expected to affect translational control of gene expression. This review covers both direct and indirect SMN interactions across various translation-related cellular compartments and processes, spanning from ribosome biogenesis to local translation and beyond. Additionally, it aims to outline deficiencies and alterations in translation observed in SMA models and patients, while also discussing the implications of the relationship between SMN protein and the translation machinery within the context of current and future therapies.
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Atrofia Muscular Espinal , Humanos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/terapia , Atrofia Muscular Espinal/metabolismo , Ribosomas/metabolismo , ARN Mensajero/metabolismo , MutaciónRESUMEN
Subclinical bioprosthetic valve thrombosis (BPVT) is a relatively common finding in asymptomatic patients during follow-up imaging. However, its clinical significance is unclear. Data from registries associate BPVT with elevated valve gradients, thromboembolic complications, recurrence, and valve degeneration. Given the dynamic nature of the disease process, management is challenging. The duration of anticoagulation is unpredictable, and the need for frequent monitoring of BPVT, even in subclinical scenarios, is unclear. Our report is shedding the light on the clinical implications BPVT.
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Estenosis de la Válvula Aórtica , Válvula Aórtica , Bioprótesis , Prótesis Valvulares Cardíacas , Recurrencia , Trombosis , Reemplazo de la Válvula Aórtica Transcatéter , Humanos , Reemplazo de la Válvula Aórtica Transcatéter/efectos adversos , Reemplazo de la Válvula Aórtica Transcatéter/instrumentación , Válvula Aórtica/cirugía , Válvula Aórtica/diagnóstico por imagen , Válvula Aórtica/fisiopatología , Estenosis de la Válvula Aórtica/cirugía , Estenosis de la Válvula Aórtica/diagnóstico por imagen , Estenosis de la Válvula Aórtica/fisiopatología , Resultado del Tratamiento , Trombosis/etiología , Trombosis/diagnóstico por imagen , Anciano de 80 o más Años , Masculino , Diseño de Prótesis , Femenino , Anticoagulantes/uso terapéutico , Anciano , Factores de TiempoRESUMEN
In the face of escalating salinity stress challenges in agricultural systems, this review article delves into the harmonious partnership between hydrogen sulfide (H2S) and nitric oxide (NO) as they collectively act as formidable defenders of plants. Once considered as harmful pollutants, H2S and NO have emerged as pivotal gaseous signal molecules that profoundly influence various facets of plant life. Their roles span from enhancing seed germination to promoting overall growth and development. Moreover, these molecules play a crucial role in bolstering stress tolerance mechanisms and maintaining essential plant homeostasis. This review navigates through the intricate signaling pathways associated with H2S and NO, elucidating their synergistic effects in combating salinity stress. We explore their potential to enhance crop productivity, thereby ensuring food security in saline-affected regions. In an era marked by pressing environmental challenges, the manipulation of H2S and NO presents promising avenues for sustainable agriculture, offering a beacon of hope for the future of global food production.
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Sulfuro de Hidrógeno , Óxido Nítrico , Óxido Nítrico/metabolismo , Sulfuro de Hidrógeno/metabolismo , Estrés Salino , Plantas/metabolismo , Estrés Fisiológico , SalinidadRESUMEN
Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7 Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate.
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Biomasa , Proteínas Fúngicas , Genoma Fúngico , Lignina , Saccharomycetales , Sordariales , Lignina/metabolismo , Sordariales/genética , Sordariales/enzimología , Sordariales/metabolismo , Hidrólisis , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deshidrogenasas de Carbohidratos/metabolismo , Deshidrogenasas de Carbohidratos/genética , Celulosa/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Celulasa/metabolismo , Celulasa/genéticaRESUMEN
Myxobacteria are non-photosynthetic bacteria distinguished among prokaryotes by a multicellular stage in their life cycle known as fruiting bodies that are formed in response to nutrient deprivation and stimulated by light. Here, we report an entrained, rhythmic pattern of Myxococcus macrosporus fruiting bodies, forming consistently spaced concentric rings when grown in the dark. Light exposure disrupts this rhythmic phenotype, resulting in a sporadic arrangement and reduced fruiting-body count. M. macrosporus genome encodes a red-light photoreceptor, a bacteriophytochrome (BphP), previously shown to affect the fruiting-body formation in the related myxobacterium Stigmatella aurantiaca. Similarly, the formation of M. macrosporus fruiting bodies is also impacted by the exposure to BphP-specific wavelengths of light. RNA-Seq analysis of M. macrosporus revealed constitutive expression of the bphP gene. Phytochromes, as light-regulated enzymes, control many aspects of plant development including photomorphogenesis. They are intrinsically correlated to circadian clock proteins, impacting the overall light-mediated entrainment of the circadian clock. However, this functional relationship remains unexplored in non-photosynthetic prokaryotes. Genomic analysis unveiled the presence of multiple homologs of cyanobacterial core oscillatory gene, kaiC, in various myxobacteria, including M. macrosporus, S. aurantiaca and M. xanthus. RNA-Seq analysis verified the expression of all kaiC homologs in M. macrosporus and the closely related M. xanthus, which lacks bphP genes. Overall, this study unravels the rhythmic growth pattern during M. macrosporus development, governed by environmental factors such as light and nutrients. In addition, myxobacteria may have a time-measuring mechanism resembling the cyanobacterial circadian clock that links the photoreceptor (BphP) function to the observed rhythmic behavior.
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Luz , Myxococcus , Myxococcus/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genéticaRESUMEN
The present study focuses on the pathological and molecular characterization of African swine fever virus (ASFV) associated with an outbreak in wild boars in two national parks in southern India in 2022-2023. Significant mortality was observed among free-ranging wild boars at Bandipur National Park, Karnataka, and Mudumalai National Park, Tamil Nadu. Extensive combing operations were undertaken in both national parks, spanning an area of around 100 km2, originating from the reported epicenter, to estimate the mortality rate. Recovered carcasses were pathologically examined, and ASFV isolates was genetically characterized. Our findings suggested spillover infection of ASFV from nearby domestic pigs, and the virus was equally pathogenic in wild boars and domestic pigs. ASFV intrusion was reported in the Northeastern region of the country, which borders China and Myanmar, whereas the current outbreak is very distantly located, in southern India. Molecular data will help in tracing the spread of the virus in the country.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Brotes de Enfermedades , Sus scrofa , Animales , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/aislamiento & purificación , India/epidemiología , Porcinos , Fiebre Porcina Africana/virología , Fiebre Porcina Africana/epidemiología , Fiebre Porcina Africana/mortalidad , Sus scrofa/virología , Brotes de Enfermedades/veterinaria , Filogenia , Animales Salvajes/virologíaRESUMEN
Inflammation and associated disorders have been a major contributing factor to mortality worldwide. The augmented mortality rate and emerging resistance against the approved therapeutics necessitate the discovery of novel chemistries destined for multiple clinical settings. Cellular factories including endophytic fungi have been tapped for chemical diversity with therapeutic potential. The emerging evidence has suggested the potential of bioactive compounds isolated from the endophytic fungi as putative agents to combat inflammation-associated disorders. The review summarizesand assists the readers in comprehending the structural and functional aspects of the medicinal chemistries identified from endophytic fungi as anticancer, antiobesity, antigout, and immunomodulatory agents.
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Hongos , Humanos , Hongos/química , Antineoplásicos/farmacología , Antineoplásicos/química , Animales , Endófitos/química , Endófitos/metabolismo , Estructura Molecular , Fármacos Antiobesidad/farmacología , Fármacos Antiobesidad/química , Fármacos Antiobesidad/aislamiento & purificación , Productos Biológicos/química , Productos Biológicos/farmacología , Productos Biológicos/aislamiento & purificación , Factores Inmunológicos/farmacología , Factores Inmunológicos/químicaRESUMEN
The current study is the first to describe the temporal and differential transcriptional expression of two lytic polysaccharide monooxygenase (LPMO) genes of Rasamsonia emersonii in response to various carbon sources. The mass spectrometry based secretome analysis of carbohydrate active enzymes (CAZymes) expression in response to different carbon sources showed varying levels of LPMOs (AA9), AA3, AA7, catalase, and superoxide dismutase enzymes pointing toward the redox-interplay between the LPMOs and auxiliary enzymes. Moreover, it was observed that cello-oligosaccharides have a negative impact on the expression of LPMOs, which has not been highlighted in previous reports. The LPMO1 (30 kDa) and LPMO2 (47 kDa), cloned and expressed in Pichia pastoris, were catalytically active with (kcat/Km) of 6.6×10-2 mg-1 ml min-1 and 1.8×10-2 mg-1 ml min-1 against Avicel, respectively. The mass spectrometry of hydrolysis products of Avicel/carboxy methyl cellulose (CMC) showed presence of C1/C4 oxidized oligosaccharides indicating them to be Type 3 LPMOs. The 3D structural analysis of LPMO1 and LPMO2 revealed distinct arrangements of conserved catalytic residues at their active site. The developed enzyme cocktails consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant LPMO1/LPMO2 resulted in significantly enhanced saccharification of steam/acid pretreated unwashed rice straw slurry from PRAJ industries (Pune, India). The current work indicates that LPMO1 and LPMO2 are catalytically efficient and have a high degree of thermostability, emphasizing their usefulness in improving benchmark enzyme cocktail performance. KEY POINTS: ⢠Mass spectrometry depicts subtle interactions between LPMOs and auxiliary enzymes. ⢠Cello-oligosaccharides strongly downregulated the LPMO1 expression. ⢠Developed LPMO cocktails showed superior hydrolysis in comparison to CellicCTec3.
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Oxigenasas de Función Mixta , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Oxigenasas de Función Mixta/química , Polisacáridos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/química , Hidrólisis , Celulosa/metabolismo , Regulación Fúngica de la Expresión Génica , Oligosacáridos/metabolismo , Clonación MolecularRESUMEN
The present study reports a highly thermostable ß-glucosidase (GH3) from Rasamsonia emersonii that was heterologously expressed in Pichia pastoris. Extracellular ß-glucosidase was purified to homogeneity using single step affinity chromatography with molecular weight of ~ 110 kDa. Intriguingly, the purified enzyme displayed high tolerance to inhibitors mainly acetic acid, formic acid, ferulic acid, vanillin and 5-hydroxymethyl furfural at concentrations exceeding those present in acid steam pretreated rice straw slurry used for hydrolysis and subsequent fermentation in 2G ethanol plants. Characteristics of purified ß-glucosidase revealed the optimal activity at 80 °C, pH 5.0 and displayed high thermostability over broad range of temperature 50-70 °C with maximum half-life of ~ 60 h at 50 °C, pH 5.0. The putative transglycosylation activity of ß-glucosidase was appreciably enhanced in the presence of methanol as an acceptor. Using the transglycosylation ability of ß-glucosidase, the generated low cost mixed glucose disaccharides resulted in the increased induction of R. emersonii cellulase under submerged fermentation. Scaling up the recombinant protein production at fermenter level using temporal feeding approach resulted in maximal ß-glucosidase titres of 134,660 units/L. Furthermore, a developed custom made enzyme cocktail consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant ß-glucosidase resulted in significantly enhanced hydrolysis of pretreated rice straw slurry from IOCL industries (India). Our results suggest multi-faceted ß-glucosidase from R. emersonii can overcome obstacles mainly high cost associated enzyme production, inhibitors that impair the sugar yields and thermal inactivation of enzyme.
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Eurotiales , beta-Glucosidasa , Hidrólisis , beta-Glucosidasa/química , BiomasaRESUMEN
Endophytic fungi, as plant symbionts, produce an elaborate array of enzymes for efficient disintegration of lignocellulosic biomass into constituent monomeric sugars, making them novel source of lignocellulolytic CAZymes with immense potential in future biorefineries. The present study reports lignocellulolytic enzymes production potential of an endophytic halotolerant Penicillium oxalicum strain isolated from Citrus limon, under submerged and solid-state fermentation (SmF & SSF, respectively), in the presence and absence of salt (1 M NaCl). The comparative QTOF-LC/MS-based exoproteome analysis of the culture extracts unveiled differential expression of CAZymes, with the higher abundance of GH6 and GH7 family cellobiohydrolase in the presence of 1 M salt. The strain improvement program, employing cyclic mutagenesis and diploidization, was utilized to develop hyper-cellulase producing mutant strains of P. oxalicum. The enzyme production of the developed strain (POx-M35) was further enhanced through statistical optimization of the culture conditions utilizing glucose mix disaccharides (GMDs) as an inducer. This optimization process resulted in the lignocellulolytic cocktail that contained high titers (U/mL) of endoglucanase (EG) (146.16), cellobiohydrolase (CBHI) (6.99), ß-glucosidase (ß-G) (26.21), xylanase (336.05) and FPase (2.02 U/mL), which were 5.47-, 5.54-, 8.55-, 4.96-, and 4.39-fold higher when compared to the enzyme titers obtained in wild HP1, respectively. Furthermore, the lignocellulolytic cocktails designed by blending secretome produced by mutant POx-M35 with xylanases (GH10 and GH11) derived from Malbranchea cinnamomea resulted in efficient hydrolysis of unwashed acid pretreated (UWAP) rice straw slurry and mild alkali deacetylated (MAD) rice straw. This study underscores the potential of bioprospecting novel fungus and developing an improved strain for optimized production and constitution of lignocellulolytic cocktails that can be an important determinant in advancing biomass conversion technologies.
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Lignina , Oryza , Penicillium , Penicillium/enzimología , Penicillium/metabolismo , Penicillium/genética , Oryza/microbiología , Lignina/metabolismo , Hidrólisis , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , FermentaciónRESUMEN
Introduction: Apart from nocturia, few reports have been published on the relationship between lower urinary tract symptoms (LUTS) and sleep disturbances in patients visiting urology outpatient clinics. This study assessed the association between our population's LUTS and sleep disturbances. Methods: This was a prospective observational study. A total of 123 male patients with a history of LUTS aged more than 40 years were recruited from urology outpatient clinic. International Prostate Symptom Score was utilized to assess LUTS. To determine the quality of sleep, the Pittsburgh Sleep Quality Index (PSQI) was used. Berlin questionnaire (BQ) was used for screening obstructive sleep apnea. Results: A total of 123 participants were enrolled in this study. The mean age of the participants was 61 ± 11.1 years. Nocturia >3 episodes were significantly more in patients with PSQI >5 (P < 0.05). There was a greater prevalence of severe LUTS in patients with PSQI >5 (P < 0.05). The association between LUTS and BQ score showed an increased prevalence of severe symptoms in patients with high BQ. Patients with PSQI >5 had more severe LUTS (53% of patients) compared to patients with PSQI ≤5 (5% of patients) (P = 0.000). Patients with PSQI >5 had overall poorer quality of life (QOL) scores, with QOL being 5 and 6 in 18% and 4.8% of the patients, respectively. Conclusions: There is a significant association between the prevalence of nocturia, moderate-to-severe LUTS, and the existence of sleep disorders. Therefore, screening for sleep disturbances may be performed on male patients who present with LUTS.