Association Between Rheumatic Autoantibodies and Immune-Related Adverse Events.

BACKGROUND: Side effects of immune checkpoint inhibitors (ICIs), called immune-related adverse events (irAEs), closely resemble primary autoimmune or rheumatic diseases. We aimed to understand the clinical utility of rheumatic autoantibodies (rhAbs) for diagnosing irAEs. PATIENTS AND METHODS: Patients without pre-existing autoimmune disease (pAID) who had cancer treated with ICI(s) treatment from 1/1/2011 to 12/21/2020 and a rhAb checked were retrospectively identified. Logistic regression assessed associations between autoantibodies and irAEs, cancer outcome, and survival. Specificity, sensitivity, and positive/negative predictive values (PPV, NPV) were estimated for key rhAbs and ICI-arthritis. Kaplan-Meier analyzed objective response rate (ORR) and overall survival (OS). RESULTS: A total of 2662 patients were treated with≥1 ICIs. One hundred and thirty-five without pAID had ≥ 1 rhAb tested. Of which 70/135(52%) were female; median age at cancer diagnosis was 62 years with most common cancers: melanoma (23%) or non-small cell lung cancer (21%), 96/135 (75%) were anti-PD1/PDL1 treated. Eighty had a rhAb ordered before ICI, 96 after ICI, and 12 before and after. Eighty-two (61%) experienced an irAE, 33 (24%) with rheumatic-irAE. Pre-ICI RF showed significant association with rheumatic-irAEs (OR = 25, 95% CI, 1.52-410.86, P = .024). Pre- and post-ICI RF yielded high specificity for ICI-arthritis (93% and 78%), as did pre- and post-ICI CCP (100% and 91%). Pre-ICI RF carried 93% NPV and pre-ICI CCP had 89% PPV for ICI-arthritis. No variables were significantly correlated with ORR. Any-type irAE, rheumatic-irAE and ICI-arthritis were all associated with better OS (P = .000, P = .028, P = .019). CONCLUSIONS: Pre-ICI RF was associated with higher odds of rheumatic-irAEs. IrAEs had better OS; therefore, clinical contextualization for rhAbs is critical to prevent unnecessary withholding of lifesaving ICI for fear of irAEs.

A detailed genome-scale metabolic model of Clostridium thermocellum investigates sources of pyrophosphate for driving glycolysis.

Lignocellulosic biomass is an abundant and renewable source of carbon for chemical manufacturing, yet it is cumbersome in conventional processes. A promising, and increasingly studied, candidate for lignocellulose bioprocessing is the thermophilic anaerobe Clostridium thermocellum given its potential to produce ethanol, organic acids, and hydrogen gas from lignocellulosic biomass under high substrate loading. Possessing an atypical glycolytic pathway which substitutes GTP or pyrophosphate (PPi) for ATP in some steps, including in the energy-investment phase, identification, and manipulation of PPi sources are key to engineering its metabolism. Previous efforts to identify the primary pyrophosphate have been unsuccessful. Here, we explore pyrophosphate metabolism through reconstructing, updating, and analyzing a new genome-scale stoichiometric model for C. thermocellum, iCTH669. Hundreds of changes to the former GEM, iCBI655, including correcting cofactor usages, addressing charge and elemental balance, standardizing biomass composition, and incorporating the latest experimental evidence led to a MEMOTE score improvement to 94%. We found agreement of iCTH669 model predictions across all available fermentation and biomass yield datasets. The feasibility of hundreds of PPi synthesis routes, newly identified and previously proposed, were assessed through the lens of the iCTH669 model including biomass synthesis, tRNA synthesis, newly identified sources, and previously proposed PPi-generating cycles. In all cases, the metabolic cost of PPi synthesis is at best equivalent to investment of one ATP suggesting no direct energetic advantage for the cofactor substitution in C. thermocellum. Even though no unique source of PPi could be gleaned by the model, by combining with gene expression data two most likely scenarios emerge. First, previously investigated PPi sources likely account for most PPi production in wild-type strains. Second, alternate metabolic routes as encoded by iCTH669 can collectively maintain PPi levels even when previously investigated synthesis cycles are disrupted. Model iCTH669 is available at github.com/maranasgroup/iCTH669.

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.

Mechanism of furfural toxicity and metabolic strategies to engineer tolerance in microbial strains.

Lignocellulosic biomass represents a carbon neutral cheap and versatile source of carbon which can be converted to biofuels. A pretreatment step is frequently used to make the lignocellulosic carbon bioavailable for microbial metabolism. Dilute acid pretreatment at high temperature and pressure is commonly utilized to efficiently solubilize the pentose fraction by hydrolyzing the hemicellulose fibers and the process results in formation of furans-furfural and 5-hydroxymethyl furfural-and other inhibitors which are detrimental to metabolism. The presence of inhibitors in the medium reduce productivity of microbial biocatalysts and result in increased production costs. Furfural is the key furan inhibitor which acts synergistically along with other inhibitors present in the hydrolysate. In this review, the mode of furfural toxicity on microbial metabolism and metabolic strategies to increase tolerance is discussed. Shared cellular targets between furfural and acetic acid are compared followed by discussing further strategies to engineer tolerance. Finally, the possibility to use furfural as a model inhibitor of dilute acid pretreated lignocellulosic hydrolysate is discussed. The furfural tolerant strains will harbor an efficient lignocellulosic carbon to pyruvate conversion mechanism in presence of stressors in the medium. The pyruvate can be channeled to any metabolite of interest by appropriate modulation of downstream pathway of interest. The aim of this review is to emphasize the use of hydrolysate as a carbon source for bioproduction of biofuels and other compounds of industrial importance.

Clinical and Economic Impact of COVID-19 on Agricultural Workers, Guatemala1.

We evaluated clinical and socioeconomic burdens of respiratory disease in banana farm workers in Guatemala. We offered all eligible workers enrollment during June 15-December 30, 2020, and annually, then tracked them for influenza-like illnesses (ILI) through self-reporting to study nurses, sentinel surveillance at health posts, and absenteeism. Workers who had ILI submitted nasopharyngeal swab specimens for testing for influenza virus, respiratory syncytial virus, and SARS-CoV-2, then completed surveys at days 0, 7, and 28. Through October 10, 2021, a total of 1,833 workers reported 169 ILIs (12.0 cases/100 person-years), and 43 (25.4%) were laboratory-confirmed infections with SARS-CoV-2 (3.1 cases/100 person-years). Workers who had SARS-CoV-2‒positive ILIs reported more frequent anosmia, dysgeusia, difficulty concentrating, and irritability and worse clinical and well-being severity scores than workers who had test result‒negative ILIs. Workers who had positive results also had greater absenteeism and lost income. These results support prioritization of farm workers in Guatemala for COVID-19 vaccination.

Assessing the impact of substrate-level enzyme regulations limiting ethanol titer in Clostridium thermocellum using a core kinetic model.

Clostridium thermocellum is a promising candidate for consolidated bioprocessing because it can directly ferment cellulose to ethanol. Despite significant efforts, achieved yields and titers fall below industrially relevant targets. This implies that there still exist unknown enzymatic, regulatory, and/or possibly thermodynamic bottlenecks that can throttle back metabolic flow. By (i) elucidating internal metabolic fluxes in wild-type C. thermocellum grown on cellobiose via 13C-metabolic flux analysis (13C-MFA), (ii) parameterizing a core kinetic model, and (iii) subsequently deploying an ensemble-docking workflow for discovering substrate-level regulations, this paper aims to reveal some of these factors and expand our knowledgebase governing C. thermocellum metabolism. Generated 13C labeling data were used with 13C-MFA to generate a wild-type flux distribution for the metabolic network. Notably, flux elucidation through MFA alluded to serine generation via the mercaptopyruvate pathway. Using the elucidated flux distributions in conjunction with batch fermentation process yield data for various mutant strains, we constructed a kinetic model of C. thermocellum core metabolism (i.e. k-ctherm138). Subsequently, we used the parameterized kinetic model to explore the effect of removing substrate-level regulations on ethanol yield and titer. Upon exploring all possible simultaneous (up to four) regulation removals we identified combinations that lead to many-fold model predicted improvement in ethanol titer. In addition, by coupling a systematic method for identifying putative competitive inhibitory mechanisms using K-FIT kinetic parameterization with the ensemble-docking workflow, we flagged 67 putative substrate-level inhibition mechanisms across central carbon metabolism supported by both kinetic formalism and docking analysis.

A Single Nucleotide Change in the polC DNA Polymerase III in Clostridium thermocellum Is Sufficient To Create a Hypermutator Phenotype.

Clostridium thermocellum is a thermophilic, anaerobic bacterium that natively ferments cellulose to ethanol and is a candidate for cellulosic biofuel production. Recently, we identified a hypermutator strain of C. thermocellum with a C669Y mutation in the polC gene, which encodes a DNA polymerase III enzyme. Here, we reintroduced this mutation using recently developed CRISPR tools to demonstrate that this mutation is sufficient to recreate the hypermutator phenotype. The resulting strain shows an approximately 30-fold increase in the mutation rate. This mutation is hypothesized to function by interfering with metal ion coordination in the PHP (polymerase and histidinol phosphatase) domain, which is responsible for proofreading. The ability to selectively increase the mutation rate in C. thermocellum is a useful tool for future directed evolution experiments. IMPORTANCE Cellulosic biofuels are a promising approach to decarbonize the heavy-duty-transportation sector. A longstanding barrier to cost-effective cellulosic biofuel production is the recalcitrance of cellulose to solubilization. Native cellulose-consuming organisms, such as Clostridium thermocellum, are promising candidates for cellulosic biofuel production; however, they often need to be genetically modified to improve product formation. One approach is adaptive laboratory evolution. Our findings demonstrate a way to increase the mutation rate in this industrially relevant organism, which can reduce the time needed for adaptive evolution experiments.

Functional Analysis of H+-Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate Dikinase as Pyrophosphate Sources in Clostridium thermocellum.

The atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PPi) as a phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PPi was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H+-pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk-malate shunt cycle, and acetate cycling in generating PPi. Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum, and heterologous expression in Escherichia coli. Unexpectedly, individually targeted gene deletions of the four putative PPi sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PPi sources reduced the growth rate by 22% (0.30 ± 0.01 h-1) and the biomass yield by 38% (0.18 ± 0.00 gbiomass gsubstrate-1), this change was much smaller than what would be expected for stoichiometrically essential PPi-supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose, indicating that the unknown PPi-supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PPi altogether was falsified by enzyme assays, heterologous expression of candidate genes, and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PPi sources individually and combined play no significant PPi-supplying role, and the true source(s) of PPi, or alternative phosphorylating mechanisms, that drive(s) glycolysis in C. thermocellum remain(s) elusive. IMPORTANCE Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H+-pumping membrane-bound PPase, glycogen cycling, a Ppdk-malate shunt cycle, and acetate cycling are not significant sources of PPi supply, this study adds functional annotation of four genes and availability of an updated PPi stoichiometry from biosynthesis to the scientific domain. Together, this aids future metabolic engineering attempts aimed to improve C. thermocellum as a cell factory for sustainable and efficient production of ethanol from lignocellulosic material through consolidated bioprocessing with minimal pretreatment. Getting closer to elucidating the elusive source of PPi, or alternative phosphorylating mechanisms, for the atypical glycolysis is itself of fundamental importance. Additionally, the findings of this study directly contribute to investigations into trade-offs between thermodynamic driving force versus energy yield of PPi- and ATP-dependent glycolysis.

Increasing the Thermodynamic Driving Force of the Phosphofructokinase Reaction in Clostridium thermocellum.

Glycolysis is an ancient, widespread, and highly conserved metabolic pathway that converts glucose into pyruvate. In the canonical pathway, the phosphofructokinase (PFK) reaction plays an important role in controlling flux through the pathway. Clostridium thermocellum has an atypical glycolysis and uses pyrophosphate (PPi) instead of ATP as the phosphate donor for the PFK reaction. The reduced thermodynamic driving force of the PPi-PFK reaction shifts the entire pathway closer to thermodynamic equilibrium, which has been predicted to limit product titers. Here, we replace the PPi-PFK reaction with an ATP-PFK reaction. We demonstrate that the local changes are consistent with thermodynamic predictions: the ratio of fructose 1,6-bisphosphate to fructose-6-phosphate increases, and the reverse flux through the reaction (determined by 13C labeling) decreases. The final titer and distribution of fermentation products, however, do not change, demonstrating that the thermodynamic constraints of the PPi-PFK reaction are not the sole factor limiting product titer. IMPORTANCE The ability to control the distribution of thermodynamic driving force throughout a metabolic pathway is likely to be an important tool for metabolic engineering. The phosphofructokinase reaction is a key enzyme in Embden-Mayerhof-Parnas glycolysis and therefore improving the thermodynamic driving force of this reaction in C. thermocellum is believed to enable higher product titers. Here, we demonstrate switching from pyrophosphate to ATP does in fact increases the thermodynamic driving force of the phosphofructokinase reaction in vivo. This study also identifies and overcomes a physiological hurdle toward expressing an ATP-dependent phosphofructokinase in an organism that utilizes an atypical glycolytic pathway. As such, the method described here to enable expression of ATP-dependent phosphofructokinase in an organism with an atypical glycolytic pathway will be informative toward engineering the glycolytic pathways of other industrial organism candidates with atypical glycolytic pathways.

A plain language summary from pembrolizumab plus ipilimumab following PD-1 antibody failure in melanoma.

WHAT IS THIS SUMMARY ABOUT?: In this article, we discuss the results of our clinical study that looked at the use of two immunotherapy drugs for the treatment of advanced melanoma. Melanoma is considered advanced when it is no longer curable with surgery. WHAT HAPPENED IN THE STUDY?: The two-drug combination, pembrolizumab and ipilimumab, was given to people with melanoma who's cancer had progressed. This study looked at how effective these two drugs were in terms of controlling the melanoma, as well their safety. These results from the study were then compared to the results from previous studies looking at melanoma treatment with ipilimumab on its own, which previously had been the most commonly used drug. WHAT WERE THE RESULTS?: The study, originally published in the Journal of Clinical Oncology, showed that combination treatment with pembrolizumab and ipilimumab was more likely to be effective than ipilimumab on its own. Not all of the study participants benefited, but many of those who did benefit experienced long-term remission from their melanoma without needing more treatment. Around 1/3 of the participants in the study had their tumors shrink compared to previous studies, which showed that ipilimumab was expected to shrink 1 in 8 tumors. The rates of side effects were similar with the pembrolizumab and ipilimumab combination compared to ipilimumab alone.

A distinct lineage of giant viruses brings a rhodopsin photosystem to unicellular marine predators.

Giant viruses are remarkable for their large genomes, often rivaling those of small bacteria, and for having genes thought exclusive to cellular life. Most isolated to date infect nonmarine protists, leaving their strategies and prevalence in marine environments largely unknown. Using eukaryotic single-cell metagenomics in the Pacific, we discovered a Mimiviridae lineage of giant viruses, which infects choanoflagellates, widespread protistan predators related to metazoans. The ChoanoVirus genomes are the largest yet from pelagic ecosystems, with 442 of 862 predicted proteins lacking known homologs. They are enriched in enzymes for modifying organic compounds, including degradation of chitin, an abundant polysaccharide in oceans, and they encode 3 divergent type-1 rhodopsins (VirR) with distinct evolutionary histories from those that capture sunlight in cellular organisms. One (VirRDTS) is similar to the only other putative rhodopsin from a virus (PgV) with a known host (a marine alga). Unlike the algal virus, ChoanoViruses encode the entire pigment biosynthesis pathway and cleavage enzyme for producing the required chromophore, retinal. We demonstrate that the rhodopsin shared by ChoanoViruses and PgV binds retinal and pumps protons. Moreover, our 1.65-Å resolved VirRDTS crystal structure and mutational analyses exposed differences from previously characterized type-1 rhodopsins, all of which come from cellular organisms. Multiple VirR types are present in metagenomes from across surface oceans, where they are correlated with and nearly as abundant as a canonical marker gene from Mimiviridae Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes.

The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase.

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C5 precursors (such as ribose) during growth on other (non-C5) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PPi-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PPi-PFKs of Clostridium thermosuccinogenes and Clostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli, which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PPi-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of C. thermosuccinogenes that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its K½ value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.

PATRIC as a unique resource for studying antimicrobial resistance.

The Pathosystems Resource Integration Center (PATRIC, www.patricbrc.org) is designed to provide researchers with the tools and services that they need to perform genomic and other 'omic' data analyses. In response to mounting concern over antimicrobial resistance (AMR), the PATRIC team has been developing new tools that help researchers understand AMR and its genetic determinants. To support comparative analyses, we have added AMR phenotype data to over 15 000 genomes in the PATRIC database, often assembling genomes from reads in public archives and collecting their associated AMR panel data from the literature to augment the collection. We have also been using this collection of AMR metadata to build machine learning-based classifiers that can predict the AMR phenotypes and the genomic regions associated with resistance for genomes being submitted to the annotation service. Likewise, we have undertaken a large AMR protein annotation effort by manually curating data from the literature and public repositories. This collection of 7370 AMR reference proteins, which contains many protein annotations (functional roles) that are unique to PATRIC and RAST, has been manually curated so that it projects stably across genomes. The collection currently projects to 1 610 744 proteins in the PATRIC database. Finally, the PATRIC Web site has been expanded to enable AMR-based custom page views so that researchers can easily explore AMR data and design experiments based on whole genomes or individual genes.

Laboratory Evolution and Reverse Engineering of Clostridium thermocellum for Growth on Glucose and Fructose.

The native ability of Clostridium thermocellum to efficiently solubilize cellulose makes it an interesting platform for sustainable biofuel production through consolidated bioprocessing. Together with other improvements, industrial implementation of C. thermocellum, as well as fundamental studies into its metabolism, would benefit from improved and reproducible consumption of hexose sugars. To investigate growth of C. thermocellum on glucose or fructose, as well as the underlying molecular mechanisms, laboratory evolution was performed in carbon-limited chemostats with increasing concentrations of glucose or fructose and decreasing cellobiose concentrations. Growth on both glucose and fructose was achieved with biomass yields of 0.09 ± 0.00 and 0.18 ± 0.00 gbiomass gsubstrate-1, respectively, compared to 0.15 ± 0.01 gbiomass gsubstrate-1 for wild type on cellobiose. Single-colony isolates had no or short lag times on the monosaccharides, while wild type showed 42 ± 4 h on glucose and >80 h on fructose. With good growth on glucose, fructose, and cellobiose, the fructose isolates were chosen for genome sequence-based reverse metabolic engineering. Deletion of a putative transcriptional regulator (Clo1313_1831), which upregulated fructokinase activity, reduced lag time on fructose to 12 h with a growth rate of 0.11 ± 0.01 h-1 and resulted in immediate growth on glucose at 0.24 ± 0.01 h-1 Additional introduction of a G-to-V mutation at position 148 in cbpA resulted in immediate growth on fructose at 0.32 ± 0.03 h-1 These insights can guide engineering of strains for fundamental studies into transport and the upper glycolysis, as well as maximizing product yields in industrial settings.IMPORTANCEC. thermocellum is an important candidate for sustainable and cost-effective production of bioethanol through consolidated bioprocessing. In addition to unsurpassed cellulose deconstruction, industrial application and fundamental studies would benefit from improvement of glucose and fructose consumption. This study demonstrated that C. thermocellum can be evolved for reproducible constitutive growth on glucose or fructose. Subsequent genome sequencing, gene editing, and physiological characterization identified two underlying mutations with a role in (regulation of) transport or metabolism of the hexose sugars. In light of these findings, such mutations have likely (and unknowingly) also occurred in previous studies with C. thermocellum using hexose-based media with possible broad regulatory consequences. By targeted modification of these genes, industrial and research strains of C. thermocellum can be engineered to (i) reduce glucose accumulation, (ii) study cellodextrin transport systems in vivo, (iii) allow experiments at >120 g liter-1 soluble substrate concentration, or (iv) reduce costs for labeling studies.

Ophthalmic manifestations of coronavirus disease 2019 and ocular side effects of investigational pharmacologic agents.

PURPOSE OF REVIEW: To compile and report the ocular manifestations of coronavirus disease 2019 (COVID-19) infection and summarize the ocular side effects of investigational treatments of this disease. RECENT FINDINGS: Conjunctivitis is by far the most common ocular manifestation of COVID-19 with viral particles being isolated from tears/secretions of infected individuals. Multiple therapeutic options are being explored across a variety of medication classes with diverse ocular side effects. SUMMARY: Eye care professionals must exercise caution, as conjunctivitis may be the presenting or sole finding of an active COVID-19 infection. While no currently studied therapeutic agents have been found to reliably treat COVID-19, early vaccination trials are progressing and show promise. A video abstract is available for a more detailed summary. VIDEO ABSTRACT: http://links.lww.com/COOP/A36.

Reliability and Validity of an Adapted and Translated Version of the Mullen Scales of Early Learning (AT-MSEL) in Rural Guatemala.

BACKGROUND: A growing literature base supports the use of tests developed in high-income countries to assess children in low resource settings when carefully translated, adapted, and applied. Evaluation of psychometric properties of adapted and translated measures within populations is necessary. The current project sought to evaluate the reliability and validity of an adapted and translated version of the Mullen Scales of Early Learning (AT-MSEL) in rural Guatelama. METHODS: The reliability and validity of the AT-MSEL in rural Guatemala were analyzed for children ages 0-5 years. RESULTS: Interrater reliability coefficients (ICC = 0.99-1.0) and internal consistency (Cronbach's alpha = 0.91-0.93) were excellent for all subscales. General linear models utilizing paired data showed consistency between standard scores (p < 0.0001). Mean raw scores increased with chronological age, as expected. Across age groups, subscales were significantly, positively correlated with one another (p < 0.05 - < 0.001) with one exception, visual reception and expressive language at the 0-10 month age range (p = 0.43). CONCLUSIONS: The AT- MSEL showed strong psychometric properties in a sample of young children in rural Guatemala. Findings demonstrate that the AT-MSEL can be used validly and reliably within this specific population of children. This work supports the concept that tests developed in high-income countries can be used to assess children in low resource settings when carefully translated, adapted and applied.

Optimization of q-space sampling for mean apparent propagator MRI metrics using a genetic algorithm.

Mean Apparent Propagator (MAP) MRI is a recently introduced technique to estimate the diffusion probability density function (PDF) robustly. Using the estimated PDF, MAP MRI then calculates zero-displacement and non-Gaussianity metrics, which might better characterize tissue microstructure compared to diffusion tensor imaging or diffusion kurtosis imaging. However, intensive q-space sampling required for MAP MRI limits its widespread adoption. A reduced q-space sampling scheme that maintains the accuracy of the derived metrics would make it more practical. A heuristic approach for acquiring MAP MRI with fewer q-space samples has been introduced earlier with scan duration of less than 10 minutes. However, the sampling scheme was not optimized systematically to preserve the accuracy of the model metrics. In this work, a genetic algorithm is implemented to determine optimal q-space subsampling schemes for MAP MRI that will keep total scan time under 10 min. Results show that the metrics derived from the optimized schemes more closely match those computed from the full set, especially in dense fiber tracts such as the corpus callosum.

Enantioselective Synthesis of Isocarbostyril Alkaloids and Analogs Using Catalytic Dearomative Functionalization of Benzene.

Enantioselective total syntheses of the anticancer isocarbostyril alkaloids (+)-7-deoxypancratistatin, (+)-pancratistatin, (+)-lycoricidine, and (+)-narciclasine are described. Our strategy for accessing this unique class of natural products is based on the development of a Ni-catalyzed dearomative trans-1,2-carboamination of benzene. The effectiveness of this dearomatization approach is notable, as only two additional olefin functionalizations are needed to construct the fully decorated aminocyclitol cores of these alkaloids. Installation of the lactam ring has been achieved through several pathways and a direct interconversion between natural products was established via a late-stage C-7 cupration. Using this synthetic blueprint, we were able to produce natural products on a gram scale and provide tailored analogs with improved activity, solubility, and metabolic stability.

Characterization of the Clostridium thermocellum AdhE, NfnAB, ferredoxin and Pfor proteins for their ability to support high titer ethanol production in Thermoanaerobacterium saccharolyticum.

The thermophilic anaerobes Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are good candidates for lignocellulosic ethanol production. T. saccharolyticum has been successfully engineered to produce ethanol at high titer (70 g/L). The maximum ethanol titer of engineered strains of C. thermocellum is only 25 g/L. We hypothesize that one or more of the enzymes in the ethanol production pathway in C. thermocellum is not adequate for ethanol production at high titer. In this study, we focused on the enzymes responsible for the part of the ethanol production pathway from pyruvate to ethanol. In T. saccharolyticum, we replaced all of the genes encoding proteins in this pathway with their homologs from C. thermocellum and examined what combination of gene replacements restricted ethanol titer. We found that a pathway consisting of Ct_nfnAB, Ct_fd, Ct_adhE and Ts_pforA was sufficient to support ethanol titer greater than 50 g/L, however replacement of Ts_pforA by Ct_pfor1 dramatically decreased the maximum ethanol titer to 14 g/L. We then demonstrated that the reason for reduced ethanol production is that the Ct_pfor1 is inhibited by accumulation of ethanol and NADH, while Ts_pforA is not.