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The maintenance of intestinal homeostasis is a fundamental process critical for organismal integrity. Sitting at the interface of the gut microbiome and mucosal immunity, adaptive and innate lymphoid populations regulate the balance between commensal micro-organisms and pathogens. Checkpoint inhibitors (CPI), particularly those targeting the CTLA-4 pathway, disrupt this fine balance and can lead to inflammatory bowel disease (IBD) and immune checkpoint colitis (CPI-C). Here, we show that CTLA-4 is expressed by innate lymphoid cells (ILC) and that its expression is regulated by ILC subset-specific cytokine cues in a microbiota-dependent manner. Genetic deletion or antibody blockade of CTLA-4 demonstrates that this pathway plays a key role in intestinal homeostasis and is conserved in human IBD and CPI-induced colitis (CPI-C). We propose that this population of CTLA-4-positive ILC may serve as an important target for the treatment of idiopathic and iatrogenic intestinal inflammation.

Purpose Treatment options for corticosteroid-refractory and/or high-grade checkpoint inhibitor (CPI)-induced cutaneous adverse events (CAEs) are limited; however, anecdotal reports of biologic therapies have been successful. We aim to characterize the appropriate treatment scenarios and safety and efficacy profiles of biologics used to treat patients with CPI-induced CAEs at a single institution. Methods This is a retrospective case series of patients from January 1st, 2015 to October 20th, 2020, with CPI-induced CAEs who were treated with biologics at a single cancer center. Patients were identified using institutional electronic medical record who underwent CPI therapy with subsequent CAEs that necessitated biologic therapy. Diagnostic criteria utilized for CAEs were based on documentation by four board-certified dermatologists, in combination with detailed chart reviews and pathology findings. Primary study outcome measurements include CAE response, tumor response, and adverse events during biologics treatment. Results We identified 17 patients who fit study criteria. Sixteen patients experienced some degree of CAE improvement on biologics, with 10 of 10 patients reaching CAE resolution at 6 months post biologics. Eight patients needed new systemic treatment post biologics treatment, while 9 patients received no further treatment or stayed on the CPI. Thirteen patients tolerated biologics well with no significant adverse events or blood abnormalities, with only 2 patients experiencing biologic dose delays. Conclusion In our cohort, biologics appear to be extremely efficacious in the treatment of severe-grade and/or steroid refractory CAEs. They also appeared to be well-tolerated without overtly negative effects on tumor response. In patients with limited cancer treatment options and good tumor response to CPIs, biologics should be considered for severe-grade and/or refractory CAEs.

Our objective is to provide awareness about psychotic vulnerability in patients infected with SARS-CoV-2 and to better understand the role of steroid withdrawal in manic episodes, especially with its common usage in respiratory disease caused by SARS-CoV-2.We present the case of a patient who was hospitalized twice after discontinuing steroid therapy for SARS-CoV-2 infection and presented with a manic episode despite not having a psychiatric history.The patient tested positive on a polymerase chain reaction test for SARS-CoV-2 and developed pneumonia. Other organic differential diagnoses such as encephalitis were also investigated and excluded. Manic episodes were diagnosed according to DSM-V criteria. Subsequently, the patient was diagnosed with type I bipolar disorder.According to the protocols, supplemental oxygen therapy, prophylactic enoxaparin and intravenous (IV) steroids were administered. Steroid dosage was gradually reduced under supervision. During the acute mania, antipsychotics and benzodiazepines were administered.After discharge, the patient was admitted to the psychiatric consultation service. He first received mood stabilizer therapy and then received supportive psychotherapy.Psychotic symptoms commonly occur after the discontinuation of high-dose steroid therapy; however, controlled tapering may prevent these side effects. Only a few cases have reported concomitant SARS-CoV-2 infection and manic episodes, often with an apparent relationship with steroid withdrawal syndrome. In this case, we considered psychotic vulnerability a condition that is often underestimated. In consideration of the SARS-CoV-2 pandemic, the case may represent an underlying trigger for psychotic decompensation, which, in concert with neuroinflammation, may induce a manic episode.

Purpose: Epidermal growth factor receptor inhibitors (EGFRI) can be used with pathway inhibitors, including mitogen-activated protein kinase kinase inhibitors (MEKIs), BRAF inhibitors (BRAFIs), and checkpoint inhibitors such as programmed death-ligand 1 (PD-L1) and programmed cell death protein 1 (PD-1) to treat colorectal cancer. These can precipitate treatment-resistant acneiform eruptions, prompting dose modification or discontinuation. Predicting the likelihood of severe rash development and crafting effective treatments may promote adherence to life-saving chemotherapy. Methods: An Institutional Review Board approved retrospective chart review of patients with colorectal cancer treated with EGFRI or MEKI in combination with HER2, BRAF, PI3K, or checkpoint inhibitors between January 1, 2016 and January 1, 2020 was performed. Surrogates for rash severity were investigated, including lower extremity involvement, utilization of oral steroids or retinoids, dose modification, and incidence of superinfection. Results: Of 122 patients treated with combination therapy, 105 developed a rash, and 87 developed an acneiform eruption. Common combinations included MEKI/PD-LI, EGFRI/MEKI, and MEKI/PD-1I. Patients treated with EGFRI/MEKI developed the most severe rashes (p=0.02). Lower extremity involvement was more frequent with EGFRI/MEKI compared to alternative combinations (p=0.05). Drug holiday correlated with all rash severity surrogates, including rash grade, lower extremity involvement, oral steroid or retinoid use, and incidence of superinfection. Use of oral steroids or retinoids was associated with development of superinfection (p=0.002). Prophylactic tetracycline use did not impact rash severity or rash incidence. Conclusion: This is the first descriptive analysis to characterize acneiform eruptions for patients with colorectal cancer on combination cancer therapy. Approximately 85% of patients developed a cutaneous toxicity with, what appears, to be synergistic effects of EGFRI and MEKI combination therapy causing the most severe eruptions. Superinfection rate correlated to systemic therapy use beyond oral tetracyclines. Further investigation into the utility of prophylactic oral tetracyclines in this population is needed.

Acetogenic bacteria can fix single-carbon (C 1 ) molecules. However, improvements are needed to overcome poor product titers. Butyribacterium methylotrophicum can naturally ferment C 1 compounds into longer-chained molecules such as butyrate alongside traditional acetate.

Using the Wood-Ljungdahl pathway, acetogens can nonphotosynthetically fix gaseous C(1) molecules, preventing them from entering the atmosphere. Many acetogens can also grow on liquid C(1) compounds such as formate and methanol, which avoid the storage and mass transfer issues associated with gaseous C(1) compounds. Substrate redox state also plays an important role in acetogen metabolism and can modulate products formed by these organisms. Butyribacterium methylotrophicum is an acetogen known for its ability to synthesize longer-chained molecules such as butyrate and butanol, which have significantly higher values than acetate or ethanol, from one-carbon (C(1)) compounds. We explored B. methylotrophicum’s C(1) metabolism by varying substrates, substrate concentrations, and substrate feeding strategies to improve four-carbon product titers. Our results showed that formate utilization by B. methylotrophicum favored acetate production and methanol utilization favored butyrate production. Cofeeding of both substrates produced a high butyrate titer of 4 g/liter when methanol was supplied in excess to formate. Testing of formate feeding strategies, in the presence of methanol, led to further increases in the butyrate to acetate ratio. Mixotrophic growth of liquid and gaseous C(1) substrates expanded the B. methylotrophicum product profile, as ethanol, butanol, and lactate were produced under these conditions. We also showed that B. methylotrophicum is capable of producing caproate, a six-carbon product, presumably through chain elongation cycles of the reverse β-oxidation pathway. Furthermore, we demonstrated butanol production via heterologous gene expression. Our results indicate that both selection of appropriate substrates and genetic engineering play important roles in determining titers of desired products. IMPORTANCE Acetogenic bacteria can fix single-carbon (C(1)) molecules. However, improvements are needed to overcome poor product titers. Butyribacterium methylotrophicum can naturally ferment C(1) compounds into longer-chained molecules such as butyrate alongside traditional acetate. Here, we show that B. methylotrophicum can effectively grow on formate and methanol to produce high titers of butyrate. We improved ratios of butyrate to acetate through adjusted formate feeding strategies and produced higher-value six-carbon molecules. We also expanded the B. methylotrophicum product profile with the addition of C(1) gases, as the organism produced ethanol, butanol, and lactate. Furthermore, we developed a transformation protocol for B. methylotrophicum to facilitate genetic engineering of this organism for the circular bioeconomy.

Immune checkpoint inhibitors (ICIs) are being used to treat a growing variety of metastatic cancers. With this increased cancer heterogeneity, there is increased patient diversity. Vitiligo is one of the earliest cutaneous adverse events (CAEs) reported with this class of medications and can have a significant psychosocial and quality of life impact on patients, in particular skin of color patients. Although ICI-associated vitiligo is a known CAE, little has been reported about the course, prognosis, and management of this disease. This single-institution, retrospective study of 151 consecutive patients with ICI-induced vitiligo was conducted using the natural language processing and the electronic medical record. Results highlight the various cancer types associated with immune checkpoint inhibitor-induced vitiligo. Our cohort had mostly melanoma patients (132, 87.4%), with the most prevalent race overall being Caucasians (108, 71.5%). Our cohort of 19 (12.6%) non-melanoma patients, however, definitively challenges the previously held notion that ICI-induced vitiligo is limited to melanoma patients. Specifically, ICI-induced vitiligo in patients with angiosarcoma, Hodgkin’s lymphoma, and mycosis fungoides were not previously reported in the literature. We noted that non-melanoma patients had a statistically significant difference (p = 0.005) in median time to onset of vitiligo compared to melanoma patients with 382 days versus 209 days, respectively. Vitiligo outcomes in our cohort showed 117 patients (78%) had stable disease, while 26 (17.3%) worsened and 7 (4.7%) improved. This supports vitiligo as a predominantly non-progressive CAE. Further, potential management options including successful repigmentation with standard of care narrowband ultraviolet light B and topical corticosteroid therapy are anecdotally demonstrated in three patients. This study emphasizes the potential impact of this disease on the growing population of patients at risk of ICI-induced vitiligo as well as the potential for effective treatment.

Immune checkpoint inhibitors (CPIs) have revolutionised cancer treatment, with previously untreatable disease now amenable to potential cure. Combination regimens of anti-CTLA-4 and anti-PD-1 show enhanced efficacy but are prone to off-target immune-mediated tissue injury, particularly at the barrier surfaces. CPI-induced colitis is a common and serious complication. To probe the impact of immune checkpoints on intestinal homeostasis, mice were challenged with combination anti-CTLA-4/anti-PD-1 immunotherapy and manipulation of the intestinal microbiota. Colonic immune responses were profiled using bulk and single-cell RNA-sequencing and flow cytometry. CPI-colitis was dependent on the composition of the intestinal microbiota and was characterized by remodelling of mucosal lymphocytes with induction of polyfunctional lymphocyte responses characterized by increased expression of interferon-γ (IFNγ), other pro-inflammatory cytokines/chemokines (Il22, Il17a Ccl3, Ccl4 and Ccl9), cytotoxicity molecules (Gzmb, Gzma, Prf1, Nkg7) and the chemokine receptor Cxcr6. In comparison with mucosal lymphocytes in the steady state, polyfunctional lymphocytes from both CD4+ and CD8+ lineages upregulated costimulatory molecules and checkpoint molecules in CPI-colitis, indicating that these cells are tightly regulated. CPI-colitis was attenuated following depletion of effector lymphocytes or following blockade of the IL23/IFNγ axis. This study provides new mechanistic insights into CPI-colitis, identifying polyfunctional, cytotoxic lymphocytes as key mediators of disease. Therapeutic targeting of their effector response or regulatory networks, including the IL23/IFNγ axis likely holds the key to preventing and reversing CPI-colitis.

As treatment with immune checkpoint inhibitors (CPIs) for cancer increases, so has the incidence of immune-related cutaneous adverse events (irCAEs). These toxicities can significantly impact quality of life and may be dose-limiting. Current guidelines for irCAEs offer only corticosteroids or CPI discontinuation. Evidence supports biologic immunomodulatory therapies when corticosteroids fail or need avoidance. A review of literature from 2010 to 2020 yielded 45 articles, resulting in 185 irCAEs, including bullous pemphigoid-like eruption (n = 55), psoriasis/psoriasiform dermatitis (n = 41), and maculopapular rash (n = 31). Treatments included immunomodulators, intravenous immunoglobulin, aprepitant, acitretin, tetracyclines, and biologic agents. Overall, 92.3% of patients saw improvement or resolution of their rash. Bullous pemphigoid-like eruptions were treated with a tetracycline +/- niacinamide (94.7% success [18/19]), omalizumab (100% success [7/7]), and rituximab (100% success [10/10]). Although prospective research is required, this review provides a comprehensive list of successful, non-corticosteroid treatment options for irCAEs to improve compliance with lifesaving cancer therapy.

The lethal triad of coagulopathy, hypothermia, and acidosis is a well-known cause of severe deterioration and poor prognosis in trauma patients. The presence of this triad complicates the surgical management of a patient suffering from penetrating injury and hemorrhage. Here, we report the case and management of a 44-year-old man with multiple high-caliber gunshot wound (GSW) injuries who became severely acidotic (pH

Clostridium ljungdahlii was grown on syngas of varying composition to investigate how syngas blends used in fermentation can be tailored toward desired product formation. Experiments were conducted with liquid batch cultures exposed to the same volume of gas with varying ratios of hydrogen to carbon monoxide (H2/CO). Formation of acetate was favored by higher concentrations of hydrogen in the headspace. The maximum acetate concentration of 35.21 ± 0.84 mM was obtained using an H2/CO ratio of 2.0. Generation of more reduced metabolites was favored by greater carbon monoxide concentrations in the headspace. Maximum ethanol (7.53 ± 0.58 mM) and 2,3-Butanediol (5.20 ± 0.79 mM) concentrations were achieved under an H2/CO ratio of 0.5. Linear relationships were obtained in that describe the effect of the syngas H2/CO ratios on acetate, ethanol, and 2,3-BD formation, respectively.

Thermophilic bacteria are attractive hosts to produce bio-based chemicals. While various genetic manipulations have been employed in the metabolic engineering of thermophiles, a robust means to regulate gene expression in these bacteria (∼55 °C) is missing. Our bioinformatic search for various riboswitches in thermophilic bacteria revealed that major classes of riboswitches are present, suggesting riboswitches' regulatory roles in these bacteria. By building synthetic constructs incorporating natural and engineered purine riboswitch sequences originated from foreign species, we quantified respective riboswitches activities in repressing and up-regulating gene expression in Geobacillus thermoglucosidasius using a green fluorescence protein. The elicited regulatory response was ligand-concentration-dependent. We further demonstrated that riboswitch-mediated gene expression of adhE (responsible for ethanol production) in Clostridium thermocellum can modulate ethanol production, redirect metabolites, and control cell growth in the adhE knockout mutant. This work has made tunable gene expression feasible across different thermophiles for broad applications including biofuels production and gene-to-trait mapping.

The Wood-Ljungdahl pathway (WLP) is a natural carbon fixation pathway capable of converting one-carbon (C1) compounds (CO2, CO, formate) to two-carbon (C2) metabolite acetyl-CoA or coordinating with canonical glycolysis to convert sugar feedstocks to acetyl-CoA with high carbon yield. The catalytic inefficiency and engineering difficulty in key enzymes, however, limit the biosynthetic potential of this pathway. Here we design a synthetic acetyl-CoA bi-cycle to synergize the WLP for efficient C2 metabolite synthesis. This pathway produces an acetyl-CoA by fixation of two CO2 equivalents via three functional modules acting in series: carbon fixation, gluconeogenesis, and non-oxidative glycolysis. We examine the pathway through comprehensive in silico thermodynamic and kinetic analyses. The prototypic pathway is implemented in a syngas-fermenting Clostridium ljungdahlii DSM 13528 by expressing a heterologous phosphoketolase and coordinating with native enzymes in the host acetogen. We demonstrated the effectiveness of this synthetic pathway in carbon conversion under various growth conditions, which complements the WLP for valorization of syngas as well as sugar feedstocks with high catalytic efficiency. This study underscores the reductive acetyl-CoA bi-cycle as a practical strategy to improve carbon conversion and redox homeostasis in the acetogenic host.

Summary: Myogenic differentiation of human pluripotent stem cells (hPSCs) has been done by gene overexpression or directed differentiation. However, viral integration, long-term culture, and the presence of unwanted cells are the main obstacles. By using CRISPR/Cas9n, a double-reporter human embryonic stem cell (hESC) line was generated for PAX7/MYF5, allowing prospective readout. This strategy allowed pathway screen to define efficient myogenic induction in hPSCs. Next, surface marker screen allowed identification of CD10 and CD24 for purification of myogenic progenitors and exclusion of non-myogenic cells. CD10 expression was also identified on human satellite cells and skeletal muscle progenitors. In vitro and in vivo studies using transgene and/or reporter-free hPSCs further validated myogenic potential of the cells by formation of new fibers expressing human dystrophin as well as donor-derived satellite cells in NSG-mdx4Cv mice. This study provides biological insights for myogenic differentiation of hPSCs using a double-reporter cell resource and defines an improved myogenic differentiation and purification strategy. : Wu et al. demonstrate that, by generation of a double-reporter human embryonic stem cell line for skeletal muscle lineage, cells can be screened for governing pathways and surface markers, defining myogenic induction and purification protocol and evaluation of their regenerative potential using in vivo studies in a mouse model for muscular dystrophy.

Clostridium thermocellum is among the most efficient bacteria to convert cellulosic biomass into H2 during dark fermentation. However, despite great progress the H2 yield and rate are still not satisfactory for large scale applications. The purpose of this study was to evaluate whether in situ gas extraction using membrane bioreactors would increase H2 production from Clostridium thermocellum when compared to a conventional anaerobic fermentation setup in thermophilic conditions. C. thermocellum DSM 1313, a cellulotyic, thermophilic bacterium was grown on cellobiose and Avicel in an anaerobic-fermenter (AF) and an anaerobic-membrane-bioreactor (AnMBR). Compared to the AF, the AnMBR increased cumulative H2 production by 63%, from 25.8 to 42.1 mmols, increased the max H2 production rate by 24%, from 3.4 to 4.2 mmol h−1, and increased yield by 58%, from 0.43 to 0.68 mmol H2 per mmol hexose, on cellobiose. Likewise, on Avicel, the AnMBR increased cumulative H2 production by 59%, from 46.8 to 74.6 mmols, increased the max H2 rate by 87%, from 3.1 to 5.8 mmol h−1, and increased the yield by 59%, from 0.76 to 1.21 mmol H2 per mmol hexose. These results show that anaerobic membrane gas extraction can be an effective approach to increasing both rate and yield of fermentative H2 production.

Volumetric muscle defect, caused by trauma or combat injuries, is a major health concern leading to severe morbidity. It is characterized by partial or full thickness loss of muscle and its bio-scaffold, resulting in extensive fibrosis and scar formation. Therefore, the ideal therapeutic option is to use stem cells combined with bio-scaffolds to restore muscle. For this purpose, muscle-derived stem cells (MDSCs) are a great candidate due to their unique multi-lineage differentiation potential.In this study, we evaluated the regeneration potential of MDSCs for muscle loss repair using a novel in situ fibrin gel casting. Muscle defect was created by a partial thickness wedge resection in the tibialis anterior (TA) muscles of NSG mice which created an average of 25% mass loss. If untreated, this defect leads to severe muscle fibrosis. Next, MDSCs were delivered using a novel in situ fibrin gel casting method.Our results demonstrated MDSCs are able to engraft and form new myofibers in the defect when casted along with fibrin gel. LacZ labeled MDSCs were able to differentiate efficiently into new myofibers and significantly increase muscle mass. This was also accompanied by significant reduction of fibrotic tissue in the engrafted muscles. Furthermore, transplanted cells also contributed to new vessel formation and satellite cell seeding.These results confirmed the therapeutic potential of MDSCs and feasibility of direct in situ casting of fibrin/MDSC mixture to repair muscle mass defects. Keywords: Skeletal muscle, Volumetric muscle loss, Fibrin gel, Muscle-derived stem cells (MDSCs), Muscle repair, Bio-scaffold

Summary We develop roadmaps to transform the all-purpose energy infrastructures (electricity, transportation, heating/cooling, industry, agriculture/forestry/fishing) of 139 countries to ones powered by wind, water, and sunlight (WWS). The roadmaps envision 80% conversion by 2030 and 100% by 2050. WWS not only replaces business-as-usual (BAU) power, but also reduces it ∼42.5% because the work: energy ratio of WWS electricity exceeds that of combustion (23.0%), WWS requires no mining, transporting, or processing of fuels (12.6%), and WWS end-use efficiency is assumed to exceed that of BAU (6.9%). Converting may create ∼24.3 million more permanent, full-time jobs than jobs lost. It may avoid ∼4.6 million/year premature air-pollution deaths today and ∼3.5 million/year in 2050; ∼$22.8 trillion/year (12.7 ¢/kWh-BAU-all-energy) in 2050 air-pollution costs; and ∼$28.5 trillion/year (15.8 ¢/kWh-BAU-all-energy) in 2050 climate costs. Transitioning should also stabilize energy prices because fuel costs are zero, reduce power disruption and increase access to energy by decentralizing power, and avoid 1.5°C global warming.

Ferredoxin:NAD + oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD + . This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway in Thermoanaerobacterium saccharolyticum ; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activity in vitro with a ferredoxin-based FNOR assay. To determine its role in metabolism, the tsac_1705 gene was deleted in different strains of T. saccharolyticum . In wild-type T. saccharolyticum , deletion of tsac_1705 resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR in T. saccharolyticum . When both NADH- and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. Finally, we tested the effect of heterologous expression of Tsac_1705 in Clostridium thermocellum and found improvements in both the titer and the yield of ethanol. IMPORTANCE Redox balance plays a crucial role in many metabolic engineering strategies. Ferredoxins are widely used as electron carriers for anaerobic microorganism and plants. This study identified the gene responsible for electron transfer from ferredoxin to NAD + , a key reaction in the ethanol production pathway of this organism and many other metabolic pathways. Identification of this gene is an important step in transferring the ethanol production ability of this organism to other organisms.

Quantitative understanding of clostridial metabolism is of longstanding interest due to the importance of Clostridia as model anaerobes, biotechnology workhorses, and contributors to evolutionary history and ecosystem. Current computational methods such as flux balance analysis-based construction of clostridial metabolism in genome scale provide a fundamental framework for metabolic analysis. However, this method alone is inadequate to characterize cellular metabolic activity. Experiment-driven approaches including isotope tracer-based fluxomics in association with genetic and biochemical methods are needed to gain a more comprehensive understanding. Here we focus on typical examples where these integrated approaches have contributed to the identification of new metabolic pathways and quantification of metabolic fluxes in Clostridia. We also highlight the opportunities and challenges of cutting-edge fluxomics approaches such as machine learning modeling, deuterium tracer approach, and high throughput flux phenotyping in exploring clostridial metabolism with respect to inorganic carbon utilization, redox cofactor interconversion, and other key metabolic features.

Objective To evaluate if oocyte penetration and viability can be confirmed by an electrical resistance increase. Automated (robotic) intracytoplasmic sperm injection (ICSI) requires confirmation of oolemma penetration before sperm injection. Visual assessment using image processing algorithms have been developed but remain unreliable. We hypothesized that an increase in electrical resistance upon oolemma piercing during ICSI can serve as an objective tool to confirm oocyte penetration and viability. Design Experimental study. Setting Research laboratory in an academic center. Patients/Animals Oocytes from female mice and women undergoing oocyte retrieval procedure. Intervention Oolemma piercing attempts with the ICSI pipette were performed by advancing the pipette towards mature (metaphase II) oocytes collected from 6 to 12-week-old mice and immature (germinal vesicle stage and metaphase I) oocytes donated by women who underwent oocyte retrieval. Electrical resistance was measured using a conventional electrophysiological setup that includes an electrical resistance meter and two electrical wires located in the lumina of the holding and ICSI pipettes. Main Outcome Measure(S) The measure of interest was the change in electrical resistance (ΔR) before and after advancing the ICSI pipette in an attempt to penetrate an oocyte. The experiments of resistance measurements were done in 3 steps: Step 1 (proof of concept), penetrated vs. non-penetrated mouse oocytes. Step 2, mouse oocytes with visually intact oolemma vs. fragmented mouse oocytes. Step 3, human oocytes with visually intact oolemma vs. fragmented human oocytes. For each group, median and range (in parenthesis) of ΔR were determined in MΩ. Mann-Whitney test was performed to compare the two groups in each step. Results In Step 1, the penetrated mouse oocytes showed a statistically significant resistance increase compared to the non-penetrated ones (n = 20, median ΔR = 7.79 [2.57 – 106.00] vs. n = 15, median ΔR = 0.10 [-0.06 – 0.69], respectively. In Step 2, the mouse oocytes with visually intact oolemma showed a statistically significant resistance increase compared to the fragmented ones (n = 45, median ΔR = 6.5 [0.1 – 191.7] vs. n = 13, median ΔR = 0.1 [-0.3 – 2.2], respectively. In Step 3, the human oocytes with visually intact oolemma showed a statistically significant resistance increase compared to the fragmented ones (n = 96, median ΔR = 1.92 [-0.05 – 6.70] vs. n = 17, median ΔR = 0.11 [0.00 – 0.30], respectively. Conclusions An electrical resistance increase can serve as a reliable tool to confirm oocyte penetration and viability, independent of optical visualization. Following further validation and safety assessment, this technology can potentially be integrated into manual and robotic ICSI systems.

Volume 197, no. 8, p. 1386–1393, 2015, [https://doi.org/10.1128/JB.02450-14][1]. We reported a G470T mutation in the lactate dehydrogenase ( ldh ) gene which resulted in an R157L amino acid mutation. After review of the genome resequencing data from this strain, it appears that the mutation is in

Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase.