Role of cell-substrate association during plant biomass solubilization by the extreme thermophile Caldicellulosiruptor bescii.

Caldicellulosiruptor species are proficient at solubilizing carbohydrates in lignocellulosic biomass through surface (S)-layer bound and secretomic glycoside hydrolases. Tapirins, surface-associated, non-catalytic binding proteins in Caldicellulosiruptor species, bind tightly to microcrystalline cellulose, and likely play a key role in natural environments for scavenging scarce carbohydrates in hot springs. However, the question arises: If tapirin concentration on Caldicellulosiruptor cell walls increased above native levels, would this offer any benefit to lignocellulose carbohydrate hydrolysis and, hence, biomass solubilization? This question was addressed by engineering the genes for tight-binding, non-native tapirins into C. bescii. The engineered C. bescii strains bound more tightly to microcrystalline cellulose (Avicel) and biomass compared to the parent. However, tapirin overexpression did not significantly improve solubilization or conversion for wheat straw or sugarcane bagasse. When incubated with poplar, the tapirin-engineered strains increased solubilization by 10% compared to the parent, and corresponding acetate production, a measure of carbohydrate fermentation intensity, was 28% higher for the Calkr_0826 expression strain and 18.5% higher for the Calhy_0908 expression strain. These results show that enhanced binding to the substrate, beyond the native capability, did not improve C. bescii solubilization of plant biomass, but in some cases may improve conversion of released lignocellulose carbohydrates to fermentation products.

Engineering Caldicellulosiruptor bescii with Surface Layer Homology Domain-Linked Glycoside Hydrolases Improves Plant Biomass Solubilization.

Extremely thermophilic Caldicellulosiruptor species solubilize carbohydrates from lignocellulose through glycoside hydrolases (GHs) that can be extracellular, intracellular, or cell surface layer (S-layer) associated. Caldicellulosiruptor genomes sequenced so far encode at least one surface layer homology domain glycoside hydrolase (SLH-GH), representing six different classes of these enzymes; these can have multiple binding and catalytic domains. Biochemical characterization of a representative from each class was done to determine their biocatalytic features: four SLH-GHs from Caldicellulosiruptor kronotskyensis (Calkro_0111, Calkro_0402, Calkro_0072, and Calkro_2036) and two from Caldicellulosiruptor hydrothermalis (Calhy_1629 and Calhy_2383). Calkro_0111, Calkro_0072, and Calhy_2383 exhibited ß-1,3-glucanase activity, Calkro_0402 was active on both ß-1,3/1,4-glucan and ß-1,4-xylan, Calkro_2036 exhibited activity on both ß-1,3/1,4-glucan and ß-1,4-glucan, and Calhy_1629 was active only on arabinan. Caldicellulosiruptor bescii, the only species with molecular genetic tools as well as already a strong cellulose degrader, contains only one SLH-GH, Athe_0594, a glucanase that is a homolog of Calkro_2036; the other 5 classes of SLH-GHs are absent in C. bescii. The C. bescii secretome, supplemented with individual enzymes or cocktails of SLH-GHs, increased in vitro sugar release from sugar cane bagasse and poplar. Expression of non-native SLH-GHs in vivo, either associated with the S-layer or as freely secreted enzymes, improved total carbohydrate solubilization of sugar cane bagasse and poplar by up to 45% and 23%, respectively. Most notably, expression of Calkro_0402, a xylanase/glucanase, improved xylose solubilization from poplar and bagasse by over 70% by C. bescii. While Caldicellulosiruptor species are already prolific lignocellulose degraders, they can be further improved by the strategy described here. IMPORTANCE Caldicellulosiruptor species hold promise as microorganisms that can solubilize the carbohydrate portion of lignocellulose and subsequently convert fermentable sugars into bio-based chemicals and fuels. Members of the genus have surface layer (S-layer) homology domain-associated glycoside hydrolases (SLH-GHs) that mediate attachment to biomass as well as hydrolysis of carbohydrates. Caldicellulosiruptor bescii, the most studied member of the genus, has only one SLH-GH. Expression of SLH-GHs from other Caldicellulosiruptor species in C. bescii significantly improved degradation of sugar cane bagasse and poplar. This suggests that this extremely thermophilic bacterium can be engineered to further improve its ability to degrade specific plant biomasses by inserting genes encoding SLH-GHs recruited from other Caldicellulosiruptor species.

Biochemical and Regulatory Analyses of Xylanolytic Regulons in Caldicellulosiruptor bescii Reveal Genus-Wide Features of Hemicellulose Utilization.

Caldicellulosiruptor species scavenge carbohydrates from runoff containing plant biomass that enters hot springs and from grasses that grow in more moderate parts of thermal features. While only a few Caldicellulosiruptor species can degrade cellulose, all known species are hemicellulolytic. The most well-characterized species, Caldicellulosiruptor bescii, decentralizes its hemicellulase inventory across five different genomic loci and two isolated genes. Transcriptomic analyses, comparative genomics, and enzymatic characterization were utilized to assign functional roles and determine the relative importance of its six putative endoxylanases (five glycoside hydrolase family 10 [GH10] enzymes and one GH11 enzyme) and two putative exoxylanases (one GH39 and one GH3) in C. bescii. Two genus-wide conserved xylanases, C. bescii XynA (GH10) and C. bescii Xyl3A (GH3), had the highest levels of sugar release on oat spelt xylan, were in the top 10% of all genes transcribed by C. bescii, and were highly induced on xylan compared to cellulose. This indicates that a minimal set of enzymes are used to drive xylan degradation in the genus Caldicellulosiruptor, complemented by hemicellulolytic inventories that are tuned to specific forms of hemicellulose in available plant biomasses. To this point, synergism studies revealed that the pairing of specific GH family proteins (GH3, -11, and -39) with C. bescii GH10 proteins released more sugar in vitro than mixtures containing five different GH10 proteins. Overall, this work demonstrates the essential requirements for Caldicellulosiruptor to degrade various forms of xylan and the differences in species genomic inventories that are tuned for survival in unique biotopes with variable lignocellulosic substrates. IMPORTANCE Microbial deconstruction of lignocellulose for the production of biofuels and chemicals requires the hydrolysis of heterogeneous hemicelluloses to access the microcrystalline cellulose portion. This work extends previous in vivo and in vitro efforts to characterize hemicellulose utilization by integrating genomic reconstruction, transcriptomic data, operon structures, and biochemical characteristics of key enzymes to understand the deployment and functionality of hemicellulases by the extreme thermophile Caldicellulosiruptor bescii. Furthermore, comparative genomics of the genus revealed both conserved and divergent mechanisms for hemicellulose utilization across the 15 sequenced species, thereby paving the way to connecting functional enzyme characterization with metabolic engineering efforts to enhance lignocellulose conversion.

Identifying the Contributions of Maternal Factors and Early Childhood Externalizing Behavior on Adolescent Delinquency.

We examined whether childhood externalizing group subtypes were uniquely related to maternal depression and victimization and whether these subtypes differentially predicted adolescent delinquency. Data were drawn from the Longitudinal Study on Child Abuse and Neglect (LONGSCAN) consortium (N = 1091; 51.3% female, 52.2% African American). Latent class analysis indicated three groups at age 4 (titled "well-adjusted," "hyperactive/oppositional," and "aggressive/rule-breaking"). Caregiver victimization and depression significantly predicted group membership such that aggressive/rule-breaking group had higher levels of maternal depression and victimization although the well-adjusted group had higher levels of maternal victimization relative to the hyperactive/oppositional group. Further, membership in higher externalizing groups at age four is associated with greater risk of adolescent delinquency at age 16. These findings underscore the need to address maternal risk factors in the treatment of childhood disruptive behavior and provide evidence of the continuity of disruptive behaviors from early childhood to adolescence.

The biology and biotechnology of the genus Caldicellulosiruptor: recent developments in 'Caldi World'.

Terrestrial hot springs near neutral pH harbor extremely thermophilic bacteria from the genus Caldicellulosiruptor, which utilize the carbohydrates of lignocellulose for growth. These bacteria are technologically important because they produce novel, multi-domain glycoside hydrolases that are prolific at deconstructing microcrystalline cellulose and hemicelluloses found in plant biomass. Among other interesting features, Caldicellulosiruptor species have successfully adapted to bind specifically to lignocellulosic substrates via surface layer homology (SLH) domains associated with glycoside hydrolases and unique binding proteins (tapirins) present only in these bacteria. They also utilize a parallel pathway for conversion of glyceraldehyde-3-phosphate into 3-phosphoglycerate via a ferredoxin-dependent oxidoreductase that is conserved across the genus. Advances in the genetic tools for Caldicellulosiruptor bescii, including the development of a high-temperature kanamycin-resistance marker and xylose-inducible promoter, have opened the door for metabolic engineering applications and some progress along these lines has been reported. While several species of Caldicellulosiruptor can readily deconstruct lignocellulose, improvements in the amount of carbohydrate released and in the production of bio-based chemicals are required to successfully realize the biotechnological potential of these organisms.

Parsing in vivo and in vitro contributions to microcrystalline cellulose hydrolysis by multidomain glycoside hydrolases in the Caldicellulosiruptor bescii secretome.

Six multidomain glycoside hydrolases (GHs), CelA (Athe_1867), CelB (Athe_1859), CelC (Athe_1857), CelD (Athe_1866), CelE (Athe_1865), and CelF (Athe_1860) are encoded in the Caldicellulosiruptor bescii glucan degradation locus (GDL). Each GH was affinity-tagged, overexpressed, and purified from recombinant C. bescii for side-by-side characterization in vitro and to examine the contribution of each of these enzymes to microcrystalline cellulose hydrolysis in vivo. All six recombinant GDL GHs were glycosylated, and deletion of glycosyltransferase Athe_1864 eliminated this posttranslational modification. A simplex centroid mixture experimental design revealed that in vitro optimal mixtures of the GDL GHs were predominantly CelA, CelC, and CelE, had low to moderate proportions of CelB and CelD, and minimal CelF. The best binary mixture contained CelA + CelB in a 3:2 molar ratio, whereas the best ternary mixture was composed of CelA + CelC + CelE in equimolar amounts. Neither the native C. bescii secretome nor cocktails of GDL GHs in vitro exceeded 25% of cellulose hydrolysis observed for wild-type C. bescii in vivo. C. bescii deletion strains lacking specific GDL GHs could be restored to wild-type degradation levels with the exogenous addition of either 5 µg/ml of recombinant GDL GH cocktails based on the natural secretome or mixtures optimized in vitro. Also, the addition of CelA up to 100 µg/ml provided no significant additional benefit. These results suggest that the C. bescii secretome is naturally balanced to achieve optimal synergy for cellulose degradation. They also reinforce the importance of microbial contributions to microcrystalline cellulose hydrolysis and suggest that mass action effects from glucan fermentation shift equilibria to drive degradation.

Neuroprotective coordination of cell mitophagy by the ATPase Inhibitory Factor 1.

The mitochondrial ATPase Inhibitory Factor 1 (hereafter referred to as IF1) blocks the reversal of the F1Fo-ATPsynthase to prevent detrimental consumption of cellular ATP and associated demise. Herein, we infer further its molecular physiology by assessing its protective function in neurons during conditions of challenged homeostatic respiration. By adopting in vitro and in vivo protocols of hypoxia/ischemia and re-oxygenation, we show that a shift in the IF1:F1Fo-ATPsynthase expression ratio occurs in neurons. This increased IF1 level is essential to induce accumulation of the PTEN-induced putative kinase 1 (PINK-1) and recruitment of the mitophagic ubiquitin ligase PARK-2 to promote autophagic "control" of the mitochondrial population. In IF1 overexpressing neurons ATP depletion is reduced during hypoxia/ischemia and the mitochondrial membrane potential (ΔYm) resilient to re-oxygenation as well as resistant to electrogenic, Ca(2+) dependent depolarization. These data suggest that in mammalian neurons mitochondria adapt to respiratory stress by upregulating IF1, which exerts a protective role by coordinating pro-survival cell mitophagy and bioenergetics resilience.

Emergency department management of gastrointestinal foreign body ingestion.

There are over 150,000 reports to American Poison Centers every year due to foreign body ingestions, and many patients will be directed to emergency departments for evaluation and management. This comprehensive review evaluates the current literature related to gastrointestinal foreign body diagnosis and management. A discussion of the utility of various imaging modalities is presented, along with a description of high-risk ingestions and the evidence behind society guidelines and management strategies. Finally, controversies in the management of esophageal impactions are reviewed, including the use of glucagon.

Whither the genus Caldicellulosiruptor and the order Thermoanaerobacterales: phylogeny, taxonomy, ecology, and phenotype.

The order Thermoanaerobacterales currently consists of fermentative anaerobic bacteria, including the genus Caldicellulosiruptor. Caldicellulosiruptor are represented by thirteen species; all, but one, have closed genome sequences. Interest in these extreme thermophiles has been motivated not only by their high optimal growth temperatures (≥70°C), but also by their ability to hydrolyze polysaccharides including, for some species, both xylan and microcrystalline cellulose. Caldicellulosiruptor species have been isolated from geographically diverse thermal terrestrial environments located in New Zealand, China, Russia, Iceland and North America. Evidence of their presence in other terrestrial locations is apparent from metagenomic signatures, including volcanic ash in permafrost. Here, phylogeny and taxonomy of the genus Caldicellulosiruptor was re-examined in light of new genome sequences. Based on genome analysis of 15 strains, a new order, Caldicellulosiruptorales, is proposed containing the family Caldicellulosiruptoraceae, consisting of two genera, Caldicellulosiruptor and Anaerocellum. Furthermore, the order Thermoanaerobacterales also was re-assessed, using 91 genome-sequenced strains, and should now include the family Thermoanaerobacteraceae containing the genera Thermoanaerobacter, Thermoanaerobacterium, Caldanaerobacter, the family Caldanaerobiaceae containing the genus Caldanaerobius, and the family Calorimonaceae containing the genus Calorimonas. A main outcome of ANI/AAI analysis indicates the need to reclassify several previously designated species in the Thermoanaerobacterales and Caldicellulosiruptorales by condensing them into strains of single species. Comparative genomics of carbohydrate-active enzyme inventories suggested differentiating phenotypic features, even among strains of the same species, reflecting available nutrients and ecological roles in their native biotopes.

Complete Genome Sequences of Two Thermophilic Indigenous Bacteria Isolated from Wheat Straw, Thermoclostridium stercorarium subsp. Strain RKWS1 and Thermoanaerobacter sp. Strain RKWS2.

Reported here are complete genome sequences for two anaerobic, thermophilic bacteria isolated from wheat straw, i.e., the (hemi)cellulolytic Thermoclostridium stercorarium subspecies strain RKWS1 (3,029,933 bp) and the hemicellulolytic Thermoanaerobacter species strain RKWS2 (2,827,640 bp). Discovery of indigenous thermophiles in plant biomass suggests that high-temperature microorganisms are more ubiquitous than previously thought.

Complete Genome Sequences of Caldicellulosiruptor acetigenus DSM 7040, Caldicellulosiruptor morganii DSM 8990 (RT8.B8), and Caldicellulosiruptor naganoensis DSM 8991 (NA10).

The genome sequences of three extremely thermophilic, lignocellulolytic Caldicellulosiruptor species were closed, improving previously reported multiple-contig assemblies. All 14 classified Caldicellulosiruptor spp. now have closed genomes. Genome closure will enhance bioinformatic analysis of the species, including identification of carbohydrate-active enzymes (CAZymes) and comparison against other Caldicellulosiruptor species and lignocellulolytic microorganisms.

Fermentative conversion of unpretreated plant biomass: A thermophilic threshold for indigenous microbial growth.

Naturally occurring, microbial contaminants were found in plant biomasses from common bioenergy crops and agricultural wastes. Unexpectedly, indigenous thermophilic microbes were abundant, raising the question of whether they impact thermophilic consolidated bioprocessing fermentations that convert biomass directly into useful bioproducts. Candidate microbial platforms for biomass conversion, Acetivibrio thermocellus (basionym Clostridium thermocellum; Topt 60 °C) and Caldicellulosiruptor bescii (Topt 78 °C), each degraded a wide variety of plant biomasses, but only A. thermocellus was significantly affected by the presence of indigenous microbial populations harbored by the biomass. Indigenous microbial growth was eliminated at ≥75 °C, conditions where C. bescii thrives, but where A. thermocellus cannot survive. Therefore, 75 °C is the thermophilic threshold to avoid sterilizing pre-treatments on the biomass that prevents native microbes from competing with engineered microbes and forming undesirable by-products. Thermophiles that naturally grow at and above 75 °C offer specific advantages as platform microorganisms for biomass conversion into fuels and chemicals.

Hepatitis B Virus Reactivation Following Treatment of HNSCC With Cisplatin.

Hepatitis B Virus (HBV) reactivation is a known complication of intense immunosuppression with B-cell depleting monoclonal antibody therapy and transplantation immunosuppression. HBV reactivation has occurred following treatment with chemotherapy regimens for hematologic malignancies and solid tumors. There are 2 prior case reports of HBV reactivation following cisplatin monotherapy for head and neck squamous cell carcinoma (HNSCC). Here, we present a case of a 49-year-old Caucasian male with a past medical history of laryngeal squamous cell carcinoma (SCC). There are no consensus guidelines on how to define hepatitis B reactivation. There are guidelines on when to initiate prophylaxis with Entecavir while on immunosuppressive therapy with risk according to medication category and hepatitis B surface antigen/hepatitis B core antibody IgG serology. CDC recommends screening everyone. American Society of Clinical Oncology (ASCO) now with a recent update in 2020 recommends screening everyone. There is a definite role of immunosuppression in HBV reactivation, however, there is also direct enhancement by cisplatin of viral replication by creating endoplasmic reticulum stress which increases HBV DNA indirectly. Finally, cytotoxicity enhances HBV reactivation and immune reconstitution post withdrawing immunosuppressive treatment. Because of the effects of chemotherapy, aka cisplatin goes beyond immunosuppression-related reactivation of HBV, our recommendations are in line with CDC and ASCO to screen all patients for HBV before onset of chemotherapy and start Entecavir/Tenofovir Disoproxil Fumarate before the onset of chemotherapy for HBV-positive patients.

Mycoplasma pneumoniae COVID-19 Delta Variant Co-Infection Mimicking COVID-19 ARDS.

The Delta variant of COVID-19 has been associated with severe disease causing a surge in the second half of 2021. Atypical pathogens can be present in those in particular with severe ARDS and can contribute to excess morbidity and mortality. We must maintain a high level of suspicion for these pathogens as this can present an opportunity to dramatically improve the prognosis of a patient with COVID-19 ARDS. However, lend caution to Mycoplasma IgM serology as this can be a false-positive. If suspicion remains high for Mycoplasma pneumoniae infection, sputum polymerase chain reaction (PCR) for M pneumoniae is the gold standard for diagnosis. We present the case of a 42-year-old female with COVID-19 Delta variant presumed ARDS who had co-infection with M pneumoniae confirmed by endotracheal sputum aspirate PCR with rapidly improving oxygenation and extubation within 4 days of effective antibiotic therapy.

Treatment-Associated Acute Lymphoblastic Leukemia Following Autologous Hematopoietic Stem Cell Transplant and Lenalidomide Maintenance in Patients With Multiple Myeloma.

Secondary malignancies including leukemia are an increasing concern in patients with prior primary malignancies treated with alkylating agents or topoisomerase II inhibitors. These can also be referred to as therapy-related leukemia. Therapy-related leukemia most commonly results in myelodysplastic syndrome or acute myeloid leukemia. The alkylating agent can cause chromosomal aberrations typically manifest as deletions in chromosome 11 or loss of part of complete loss of chromosomes 5 and 7. Conversely, acute lymphoblastic leukemia (ALL) has been described following maintenance therapy with immunomodulatory (IMiD) drugs pomalidomide, thalidomide, and lenalidomide. We present a case of a 71-year-old man with a history of multiple myeloma (MM) maintained on lenalidomide after stem cell transplant who presented with treatment-associated ALL. At time of leukemic presentation, chromosomal analysis showed a near-triploid clone consistent with masked double low hyplodiploidy which is associated with a poor prognosis. The patient had a deletion of the long arm of chromosome 5 which has been described in prior case reports with ALL secondary to lenalidomide therapy. There are explicit mechanisms in the literature, which have been attributed to development of ALL after exposure to thalidomide or lenalidomide. At time of submission, there are 20 cases described in the literature linking ALL to IMiD drugs. We describe a case and review the mechanisms of lenalidomide-associated ALL.

Refractory Torsades de Pointes Due to Dofetilide Overdose.

Dofetilide, a class III antiarrhythmic, is widely used in the treatment of cardiac arrhythmias. Antiarrhythmic drugs can have a long duration of action that prolongs the QT interval. This causes bradycardia that predisposes to R-on-T phenomenon subsequently leading to torsades de pointes (TdP). This necessitates constant monitoring to prevent or treat ventricular arrhythmias or bradycardia associated with cardiac medications. Although extremely rare, dofetilide overdose has been described in the literature. However, no evidence found in the current literature required prolonged intervention after the initial acute stabilization, leading to scarcity of data for treatment of ongoing dofetilide overdose. We present the case of an intentional dofetilide overdose in a 61-year-old Caucasian woman with a history of congestive heart failure, atrial fibrillation, stage IIIb chronic kidney disease, diabetes mellitus type II, hypothyroidism, morbid obesity, and hypertension that required extensive interventions for refractory TdP that lasted 4 days. Therapeutic as well as excess dosage of dofetilide can lead to TdP, which is usually controlled by decreasing the dose or terminating drug administration. If the arrhythmia is not resolved, guidelines recommend management with activated charcoal if ingestion is within 15 minutes, followed by administration of 2 g IV (intravenous) magnesium and addressing the electrolyte imbalance. However, if the arrhythmia is persistent due to ongoing dofetilide toxicity, isoproterenol is given as a bridge to overdrive pacing and dopamine is used as an alternative to isoproterenol.

Genome-Scale Metabolic Model of Caldicellulosiruptor bescii Reveals Optimal Metabolic Engineering Strategies for Bio-based Chemical Production.

Metabolic modeling was used to examine potential bottlenecks that could be encountered for metabolic engineering of the cellulolytic extreme thermophile Caldicellulosiruptor bescii to produce bio-based chemicals from plant biomass. The model utilizes subsystems-based genome annotation, targeted reconstruction of carbohydrate utilization pathways, and biochemical and physiological experimental validations. Specifically, carbohydrate transport and utilization pathways involving 160 genes and their corresponding functions were incorporated, representing the utilization of C5/C6 monosaccharides, disaccharides, and polysaccharides such as cellulose and xylan. To illustrate its utility, the model predicted that optimal production from biomass-based sugars of the model product, ethanol, was driven by ATP production, redox balancing, and proton translocation, mediated through the interplay of an ATP synthase, a membrane-bound hydrogenase, a bifurcating hydrogenase, and a bifurcating NAD- and NADP-dependent oxidoreductase. These mechanistic insights guided the design and optimization of new engineering strategies for product optimization, which were subsequently tested in the C. bescii model, showing a nearly 2-fold increase in ethanol yields. The C. bescii model provides a useful platform for investigating the potential redox controls that mediate the carbon and energy flows in metabolism and sets the stage for future design of engineering strategies aiming at optimizing the production of ethanol and other bio-based chemicals. IMPORTANCE The extremely thermophilic cellulolytic bacterium, Caldicellulosiruptor bescii, degrades plant biomass at high temperatures without any pretreatments and can serve as a strategic platform for industrial applications. The metabolic engineering of C. bescii, however, faces potential bottlenecks in bio-based chemical productions. By simulating the optimal ethanol production, a complex interplay between redox balancing and the carbon and energy flow was revealed using a C. bescii genome-scale metabolic model. New engineering strategies were designed based on an improved mechanistic understanding of the C. bescii metabolism, and the new designs were modeled under different genetic backgrounds to identify optimal strategies. The C. bescii model provided useful insights into the metabolic controls of this organism thereby opening up prospects for optimizing production of a wide range of bio-based chemicals.

Transcriptional Regulation of Plant Biomass Degradation and Carbohydrate Utilization Genes in the Extreme Thermophile Caldicellulosiruptor bescii.

Extremely thermophilic bacteria from the genus Caldicellulosiruptor can degrade polysaccharide components of plant cell walls and subsequently utilize the constituting mono- and oligosaccharides. Through metabolic engineering, ethanol and other industrially important end products can be produced. Previous experimental studies identified a variety of carbohydrate-active enzymes in model species Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor bescii, while prior transcriptomic experiments identified their putative carbohydrate uptake transporters. We investigated the mechanisms of transcriptional regulation of carbohydrate utilization genes using a comparative genomics approach applied to 14 Caldicellulosiruptor species. The reconstruction of carbohydrate utilization regulatory network includes the predicted binding sites for 34 mostly local regulators and point to the regulatory mechanisms controlling expression of genes involved in degradation of plant biomass. The Rex and CggR regulons control the central glycolytic and primary redox reactions. The identified transcription factor binding sites and regulons were validated with transcriptomic and transcription start site experimental data for C. bescii grown on cellulose, cellobiose, glucose, xylan, and xylose. The XylR and XynR regulons control xylan-induced transcriptional response of genes involved in degradation of xylan and xylose utilization. The reconstructed regulons informed the carbohydrate utilization reconstruction analysis and improved functional annotations of 51 transporters and 11 catabolic enzymes. Using gene deletion, we confirmed that the shared ATPase component MsmK is essential for growth on oligo- and polysaccharides but not for the utilization of monosaccharides. By elucidating the carbohydrate utilization framework in C. bescii, strategies for metabolic engineering can be pursued to optimize yields of bio-based fuels and chemicals from lignocellulose. IMPORTANCE To develop functional metabolic engineering platforms for nonmodel microorganisms, a comprehensive understanding of the physiological and metabolic characteristics is critical. Caldicellulosiruptor bescii and other species in this genus have untapped potential for conversion of unpretreated plant biomass into industrial fuels and chemicals. The highly interactive and complex machinery used by C. bescii to acquire and process complex carbohydrates contained in lignocellulose was elucidated here to complement related efforts to develop a metabolic engineering platform with this bacterium. Guided by the findings here, a clearer picture of how C. bescii natively drives carbohydrate utilization is provided and strategies to engineer this bacterium for optimal conversion of lignocellulose to commercial products emerge.

Coronavirus disease 2019: International public health considerations.

On December 31, 2019, the Chinese government announced an outbreak of a novel coronavirus, recently named COVID-19. During the following weeks the international medical community has witnessed with unprecedented coverage the public health response both domestically by the Chinese government, and on an international scale as cases have spread to dozens of countries. While much regarding the virus and the Chinese public health response is still unknown, national and public health institutions globally are preparing for a pandemic. As cases and spread of the virus grow, emergency and other front-line providers may become more anxious about the possibility of encountering a potential case. This review describes the tenets of a public health response to an infectious outbreak by using recent historical examples and also by characterizing what is known about the ongoing response to the COVID-19 outbreak. The intent of the review is to empower the practitioner to monitor and evaluate the local, national and global public health response to an emerging infectious disease.

COVID-19: A review of therapeutics under investigation.

The COVID-19 outbreak has disrupted global health care networks and caused thousands of deaths and an international economic downturn. Multiple drugs are being used on patients with COVID-19 based on theoretical and in vitro therapeutic targets. Several of these therapies have been studied, but many have limited evidence behind their use, and clinical trials to evaluate their efficacy are either ongoing or have not yet begun. This review summarizes the existing evidence for medications currently under investigation for treatment of COVID-19, including remdesivir, chloroquine/hydroxychlorquine, convalescent plasma, lopinavir/ritonavir, IL-6 inhibitors, corticosteroids, and angiotensin-converting enzyme inhibitors.