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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.

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.

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

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.

To describe fluocinolone acetonide implant dissociations in the Multicenter Uveitis Steroid Treatment (MUST) Trial.Randomized clinical trial with extended follow-up.Review of data collected on the first implant in the eye(s) of participants. Dissociation was defined as the drug pellet no longer being affixed to the strut and categorized as spontaneous or surgically related.A total of 250 eyes (146 patients) had at least 1 implant placed. Median follow-up time after implant placement was 6 years (range 0.5-9.2 years). Thirty-four dissociations were reported in 30 participants. There were 22 spontaneous events in 22 participants; 6-year cumulative risk of a spontaneous dissociation was 4.8% (95% confidence interval [CI]: 2.4%-9.1%). The earliest event occurred 4.8 years after placement. Nine of 22 eyes with data had a decline in visual acuity ≥5 letters temporally related to the dissociation. Thirty-nine implant removal surgeries were performed, 33 with replacement. Twelve dissociations were noted during implant removal surgeries in 10 participants (26%, 95% CI 15%-48%); 5 of these eyes had a decline in visual acuity ≥5 letters after surgery. The time from implant placement to removal surgery was longer for the surgeries at which dissociated implants were identified than for those without one (5.7 vs 3.7 years, P.001). Overall, visual acuity declined 15 or more letters from pre-implant values in 22% of affected eyes; declines were frequently associated with complications of uveitis or its treatment.There is an increasing risk of dissociation of Retisert implants during follow-up; the risk is greater with removal/exchange surgeries, but the risk of both spontaneous and surgically related events increases with longevity of the implants. In 22% of affected eyes visual acuity declined by 15 letters. In the context of eyes with moderate to severe uveitis for years, this rate is not unexpected.

OBJECTIVE To characterize the pathways to care that infertile couples requiring varicocele repair (VR) pursue prior to presenting to a male fertility clinic. METHODS An IRB-approved single center retrospective review of patients undergoing VR after presentation to an academic male fertility clinic was performed. Patients whose charts included partner histories were assessed for duration of attempting conception, prior workup, and assisted reproductive technology (ART) use. RESULTS A total of 405 couples were included. At presentation, mean age was 34.4 (SD ± 6.5) years for men and 31.1 (SD ± 4.3) years for women (P < .0001). A couple's first visit to a physician was a gynecologist in 59% (198/334) of couples, a reproductive endocrinologist (REI) in 25% (83/334) of cases, with 14% (47/334) presenting without a previous female workup and were self-referred, and 2% (6/334) seeing both a gynecologist and REI prior to presentation. On average, couples attempted pregnancy for 22.3 (SD ± 21.1, range 0-120) months prior to presentation. Eighteen percent of couples underwent ART prior to presentation. Couples who had undergone ART had lower pre-VR total motile sperm counts compared to couples not pursuing ART prior to presentation (P = 0.031). The majority (70.4%) of females had no abnormality in their workup, making varicocele the only correctable factor for infertility in the couple. CONCLUSIONS Our findings show a significant delay in referral of infertile men requiring VR. Eighteen percent of couples underwent costly ART prior to an inexpensive male workup. In an era of medical cost containment, early referral to a male infertility specialist is imperative.

Cellulolytic bacteria have the potential to perform lignocellulose hydrolysis and fermentation simultaneously. The metabolic pathways of these bacteria, therefore, require more comprehensive and quantitative understanding. Using isotope tracer, gas chromatography-mass spectrometry, and metabolic flux modeling, we decipher the metabolic network of Clostridium thermocellum, a model cellulolytic bacterium which represents as an attractive platform for conversion of lignocellulose to dedicated products. We uncover that the Embden–Meyerhof–Parnas (EMP) pathway is the predominant glycolytic route whereas the Entner–Doudoroff (ED) pathway and oxidative pentose phosphate pathway are inactive. We also observe that C. thermocellum's TCA cycle is initiated by both Si- and Re-citrate synthase, and it is disconnected between 2-oxoglutarate and oxaloacetate in the oxidative direction; C. thermocellum uses a citramalate shunt to synthesize isoleucine; and both the one-carbon pathway and the malate shunt are highly active in this bacterium. To gain a quantitative understanding, we further formulate a fluxome map to quantify the metabolic fluxes through central metabolic pathways. This work represents the first global in vivo investigation of the principal carbon metabolism of C. thermocellum. Our results elucidate the unique structure of metabolic network in this cellulolytic bacterium and demonstrate the capability of isotope-assisted metabolite studies in understanding microbial metabolism of industrial interests.

Recently, a new type of limb-girdle muscular dystrophy (LGMD type 2Z) has been identified due to a missense mutation in POGLUT1 (protein O-glucosyltransferase- Rumi), an enzyme capable of adding glucose to a distinct serine residue of epidermal growth factor-like repeats containing a C-X-S-X-(P/A)-C consensus sequence such as Notch receptors. Affected patients demonstrate reduced Notch signaling, decreased muscle stem cell pool and hypoglycosylation of α–dystroglycan, leading to LGMD phenotype. Here we report the generation and characterization of an iPSC line (CSCRMi001-A) from a LGMD-2Z patient with missense mutation in POGLUT1 which can be used for in vitro disease modeling.

Summary High-throughput screening of the host transcriptional response to various viral infections provides a wealth of data, but utilization of microarray and next generation sequencing (NGS) data for analysis can be difficult. The Host Transcriptional Response DataBase (HoTResDB), allows visitors to access already processed microarray and NGS data from non-human primate models of viral hemorrhagic fever to better understand the host transcriptional response. Availability HoTResDB is freely available at http://hotresdb.bu.edu

Background The risk of optic disc haemorrhages on visual outcome in idiopathic intracranial hypertension (IIH) is unknown. We report the type and frequency of optic disc haemorrhages and other funduscopic abnormalities at baseline in the study eye of the 133 subjects enrolled in the Idiopathic Intracranial Hypertension Treatment Trial completing 6 months of follow-up. Methods We reviewed optic disc photographs to tabulate the frequency and type of optic disc haemorrhages, other funduscopic abnormalities and papilloedema grades of the study eye at baseline and analyse if their presence is associated with a poor visual outcome. Results 27.2% of subjects had nerve fibre layer haemorrhages in at least one eye. Five of seven, 71% of subjects that met criteria for treatment failure, had nerve fibre layer haemorrhages in at least one eye (Fisher9s exact test: p=0.02). There was a good correlation between presence of nerve fibre layer haemorrhages and Frisen grade (Spearman9s correlation, p=0.002; r=0.271). Subjects with nerve fibre layer haemorrhages had a higher cerebrospinal fluid pressure (40.0 mm water, p=0.04). There was poor correlation between nerve fibre layer haemorrhages at baseline and the perimetric mean deviation change at 6 months. Cotton wool spots were present in 4% of subjects, exudates in 3% and pseudodrusen in 4%. Conclusions Nerve fibre layer haemorrhages are common in patients with IIH with mild visual loss and correlate with the severity of the papilloedema. They occur more frequently in treatment failure subjects and therefore may be associated with poor visual outcomes. Trial registration number NCT01003639, Post-results.

The NfnAB (NADH-dependent reduced ferredoxin: NADP+ oxidoreductase) and Rnf (ion-translocating reduced ferredoxin: NAD+ oxidoreductase) complexes are thought to catalyze electron transfer between reduced ferredoxin and NAD(P)+. Efficient electron flux is critical for engineering fuel production pathways, but little is known about the relative importance of these enzymes in vivo. In this study we investigate the importance of the NfnAB and Rnf complexes in Clostridium thermocellum for growth on cellobiose and Avicel using gene deletion, enzyme assays, and fermentation product analysis. The NfnAB complex does not seem to play a major role in metabolism, since deletion of nfnAB genes had little effect on the distribution of fermentation products. By contrast, the Rnf complex appears to play an important role in ethanol formation. Deletion of rnf genes resulted in a decrease in ethanol formation. Overexpression of rnf genes resulted in an increase in ethanol production of about 30%, but only in strains where the hydG hydrogenase maturation gene was also deleted.