The Role of AdhE on Ethanol Tolerance and Production in Clostridium thermocellum.

Many anaerobic microorganisms use the bifunctional aldehyde and alcohol dehydrogenase, AdhE, to produce ethanol. One such organism is Clostridium thermocellum, which is of interest for cellulosic biofuel production. In the course of engineering this organism for improved ethanol tolerance and production, we observed that AdhE was a frequent target of mutations. Here, we characterized those mutations to understand their effects on enzymatic activity, as well ethanol tolerance and product formation in the organism. We found that there is a strong correlation between NADH-linked alcohol dehydrogenase (ADH) activity and ethanol tolerance. Mutations that decrease NADH-linked ADH activity increase ethanol tolerance; correspondingly, mutations that increase NADH-linked ADH activity decrease ethanol tolerance. We also found that the magnitude of ADH activity did not play a significant role in determining ethanol titer. Increasing ADH activity had no effect on ethanol titer. Reducing ADH activity had indeterminate effects on ethanol titer, sometimes increasing and sometimes decreasing it. Finally, this study shows that the cofactor specificity of ADH activity was found to be the primary factor affecting ethanol yield. We expect that these results will inform efforts to use AdhE enzymes in metabolic engineering approaches.

Dandy-Walker Malformation with Neonatal Meningitis: A Case Report.

Dandy-Walker syndrome is a rare congenital central nervous system malformation. Dandy-Walker variant is characterised by cerebellar vermian hypoplasia, cystic fourth ventricular dilatation, and normal posterior fossa volume. Various prenatal tests such as ultrasound, fetal magnetic resonance imaging, and amniocentesis can help diagnose Dandy-Walker syndrome. Here, we report a case of the Dandy-Walker variant with meningitis in a neonate admitted to the neonatal intensive care unit due to multiple petechiae on the anterior abdominal wall, accompanied by peripheral cyanosis at the time of birth. Although maximum cases are diagnosed prenatally, some cases might be missed due to inadequate antenatal examination. Magnetic imaging resonance of the brain is best for the diagnosis of Dandy-Walker syndrome postnatally. Keywords: case reports; Dandy-Walker malformation, magnetic resonance imaging, meningitis.

Atypical hemolytic uremic syndrome in a child: A rare case report.

Atypical hemolytic uremic syndrome, a rare thrombotic microangiopathy, necessitates early diagnosis and comprehensive care due to its potential severity, emphasizing the importance of a multidisciplinary approach to improve outcomes.

Immune Thrombocytopenic Purpura in an Adult Male: A Case Report.

Immune thrombocytopenic purpura (ITP) is an autoimmune disease characterized by immune-mediated destruction of platelets, resulting in a decreased blood platelet count (less than 100 x 109/L) in the absence of other known etiology of thrombocytopenia. ITP is uncommon in adult males. The signs and symptoms of ITP vary widely and are quite diverse. The degree of thrombocytopenia and bleeding are not always correlated. Timely diagnosis, intervention, and regular monitoring can easily prevent complications. We report a case of a 22-year-old male presented with gum bleeding along with purpura and ecchymosis over the upper limb, lower limb, trunk, and face.

Deuterated water as a substrate-agnostic isotope tracer for investigating reversibility and thermodynamics of reactions in central carbon metabolism.

Stable isotope tracers are a powerful tool for the quantitative analysis of microbial metabolism, enabling pathway elucidation, metabolic flux quantification, and assessment of reaction and pathway thermodynamics. 13C and 2H metabolic flux analysis commonly relies on isotopically labeled carbon substrates, such as glucose. However, the use of 2H-labeled nutrient substrates faces limitations due to their high cost and limited availability in comparison to 13C-tracers. Furthermore, isotope tracer studies in industrially relevant bacteria that metabolize complex substrates such as cellulose, hemicellulose, or lignocellulosic biomass, are challenging given the difficulty in obtaining these as isotopically labeled substrates. In this study, we examine the potential of deuterated water (2H2O) as an affordable, substrate-neutral isotope tracer for studying central carbon metabolism. We apply 2H2O labeling to investigate the reversibility of glycolytic reactions across three industrially relevant bacterial species -C. thermocellum, Z. mobilis, and E. coli-harboring distinct glycolytic pathways with unique thermodynamics. We demonstrate that 2H2O labeling recapitulates previous reversibility and thermodynamic findings obtained with established 13C and 2H labeled nutrient substrates. Furthermore, we exemplify the utility of this 2H2O labeling approach by applying it to high-substrate C. thermocellum fermentations -a setting in which the use of conventional tracers is impractical-thereby identifying the glycolytic enzyme phosphofructokinase as a major bottleneck during high-substrate fermentations and unveiling critical insights that will steer future engineering efforts to enhance ethanol production in this cellulolytic organism. This study demonstrates the utility of deuterated water as a substrate-agnostic isotope tracer for examining flux and reversibility of central carbon metabolic reactions, which yields biological insights comparable to those obtained using costly 2H-labeled nutrient substrates.

Characterization and Amelioration of Filtration Difficulties Encountered in Metabolomic Studies of Clostridium thermocellum at Elevated Sugar Concentrations.

Clostridium thermocellum, a promising candidate for consolidated bioprocessing, has been subjected to numerous engineering strategies for enhanced bioethanol production. Measurements of intracellular metabolites at substrate concentrations high enough (>50 g/L) to allow the production of industrially relevant titers of ethanol would inform efforts toward this end but have been difficult due to the production of a viscous substance that interferes with the filtration and quenching steps during metabolite extraction. To determine whether this problem is unique to C. thermocellum, we performed filtration experiments with other organisms that have been engineered for high-titer ethanol production, including Escherichia coli and Thermoanaerobacterium saccharolyticum. We addressed the problem through a series of improvements, including active pH control (to reduce problems with viscosity), investigation of different filter materials and pore sizes (to increase the filtration capacity), and correction for extracellular metabolite concentrations, and we developed a technique for more accurate intracellular metabolite measurements at elevated substrate concentrations. IMPORTANCE The accurate measurement of intracellular metabolites (metabolomics) is an integral part of metabolic engineering for the enhanced production of industrially important compounds and a useful technique to understand microbial physiology. Previous work tended to focus on model organisms under laboratory conditions. As we try to perform metabolomic studies with a wider range of organisms under conditions that more closely represent those found in nature or industry, we have found limitations in existing techniques. For example, fast filtration is an important step in quenching metabolism in preparation for metabolite extraction; however, it does not work for cultures of C. thermocellum at high substrate concentrations. In this work, we characterize the extent of the problem and develop techniques to overcome it.

Identifying the environmental and anthropogenic causes, distribution, and intensity of human rhesus macaque conflict in Nepal.

Reducing conflict between humans and wildlife is considered a top conservation priority. However, increasingly human-induced disturbances across natural landscapes have escalated encounters between humans and wildlife. In Nepal, forests have been destroyed, fragmented, and developed for human settlements, agricultural production, and urban centers for decades. As a result, human-wildlife conflict, in the form of crop-raiding, livestock predation, and injuries to humans and wildlife, is common throughout the country. In particular, crop-raiding by macaques is an increasingly common form of human-wildlife conflict. Rhesus macaques (Macaca mulatta) have been identified as a top ten crop-raiding wildlife species in Nepal. In order to better understand the nationwide distribution and intensity of human-rhesus macaque conflict (HRMC), we conducted an extensive literature review of reported incidences of HRMC during the period 2000 to 2021 in Nepal. We also created an online survey to obtain nationwide data on the location and severity of HRMC, and modeled the set of ecological factors that affect habitat suitability for rhesus macaques. An ensemble of three different species distribution model (SDM) algorithms were used to analyze these data. We found that almost 44% of Nepal's land area contains suitable habitat for rhesus macaques, with less than 8% of all suitable habitat located in protected national parks. As humans continue to alter and fragment natural landscapes, HRMC in Nepal has intensified. At present, nearly 15% of the country's land area in which human settlements are permitted, is characterized by moderate or high rates of HRMC. We argue that prioritizing programs of forest restoration, strategic management plans designed to connect isolated forest fragments with high rhesus macaque population densities, creating government programs that compensate farmers for income lost due to crop-raiding, and educational outreach that informs local villagers of the importance of conservation and protecting biodiversity, offer the most effective solutions to reduce HRMC in Nepal.