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Engineered microorganisms have the potential to convert renewable substrates into biofuels and valuable bioproducts, which offers an environmentally sustainable alternative to fossil-fuel-derived products. Isoprenoids are a diverse class of biologically derived compounds that have commercial applications as various commodity chemicals, including biofuels and biofuel precursor molecules.

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 (PP(i)) instead of ATP as the phosphate donor for the PFK reaction. The reduced thermodynamic driving force of the PP(i)-PFK reaction shifts the entire pathway closer to thermodynamic equilibrium, which has been predicted to limit product titers. Here, we replace the PP(i)-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 (13)C labeling) decreases. The final titer and distribution of fermentation products, however, do not change, demonstrating that the thermodynamic constraints of the PP(i)-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.

While corn is considered to be a healthy food option, common agricultural practices, such as the application of soil amendments, might be introducing contaminants of concern (COC) into corn plants. The use of dredged material, which contain contaminants such as heavy metals, polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs), as a soil amendment is increasing. Contaminants from these amendments can accumulate in corn kernels harvested from plants grown on these sediments and potentially biomagnify in organisms that consume them. The extent to which secondary exposure to such contaminants in corn affect the mammalian central nervous system has been virtually unexplored. In this preliminary study, we examine the effects of exposure to corn grown in dredge amended soil or a commercially available feed corn on behavior and hippocampal volume in male and female rats. Perinatal exposure to dredge-amended corn altered behavior in the open-field and object recognition tasks in adulthood. Additionally, dredge-amended corn led to a reduction in hippocampal volume in male but not female adult rats. These results suggest the need for future studies examining how dredge-amended crops and/or commercially available feed corn may be exposing animals to COC that can alter neurodevelopment in a sex-specific manner. This future work will provide insight into the potential long-term consequences of soil amendment practices on the brain and behavior.

Bacteria are equipped with a diverse set of regulatory tools that allow them to quickly adapt to their environment. The RimK system allows for Pseudomonas spp. to adapt through post-transcriptional regulation by altering the ribosomal subunit RpsF. RimK is found in a wide range of bacteria with a conserved amino acid sequence, however, the genetic context and the role of this protein is highly diverse. By solving and comparing the structures of RimK homologs from two related but functionally divergent systems, we uncovered key structural differences that likely contribute to the different activity levels of each of these homologs. Moreover, we were able to clearly resolve the active site of this protein for the first time, resolving binding of the glutamate substrate. This work advances our understanding of how subtle differences in protein sequence and structure can have profound effects on protein activity, which can in turn result in widespread mechanistic changes.

We have developed compounds with a promising activity against Acinetobacter baumannii and Pseudomonas aeruginosa, which are both on the WHO priority list of antibiotic-resistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumannii and P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase IIα, and no cross-resistance to novobiocin. Cocrystal structures of 1 in complex with Escherichia coli GyrB24 and (S)-27 in complex with A. baumannii GyrB23 and P. aeruginosa GyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27 were improved by fine-tuning of lipophilicity. In particular, analogs of 27 with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities. Opis vira z dne 19. 1. 2023. Nasl. z nasl. zaslona. Bibliografija: str. 1423-1425. Abstract. ARRS, Farmacevtska kemija: načrtovanje, sinteza in vrednotenje učinkovin ARRS, MTAvsAMR: novi večtarčni antibiotiki proti večkratno odpornim bakterijam ARRS, Razvoj novih zaviralcev bakterijskih topoizomeraz za boj proti odpornim infekcijam iCASE studentship funded by BBSRC and Redx Pharma Plc Investigator Award from the Wellcome Trust BBSRC Institute Strategic Programme

Inflammatory bowel diseases (IBD) are chronic inflammatory diseases in which abdominal pain, bloody diarrhea, weight loss, and fatigue collectively result in diminished quality of patient life. The disappearance of intestinal helminth infections in Western societies is associated with an increased prevalence of IBD and other immune-mediated inflammatory diseases. Evidence indicates that helminths induce tolerogenic dendritic cells (tolDCs), which promote intestinal tolerance and attenuate intestinal inflammation characteristic of IBD, but the exact mechanism is unclear. Helminth-derived excretory-secretory (HES) products including macromolecules, proteins, and polysaccharides have been shown to modulate the antigen presenting function of DCs with down-stream effects on effector CD4+T cells. Previous studies indicate that DCs in helminth-infected animals induce tolerance to unrelated antigens and DCs exposed to HES display phenotypic and functional features of tolDCs. Here, we identify that nonpolar metabolites (HnpM) produced by a helminth, the murine gastrointestinal nematodeHeligmosomoides polygyrus bakeri(Hpb), induce tolDCs as evidenced by decreased LPS-induced TNF and increased IL-10 secretion and reduced expression of MHC-II, CD86, and CD40. Furthermore, these DCs inhibited OVA-specific CD4+T cell proliferation and induced CD4+Foxp3+regulatory T cells. Adoptive transfer of HnpM-induced tolDCs attenuated DSS-induced intestinal inflammation characteristic of IBD. Mechanistically, HnpM induced metabolic and transcriptional signatures in BMDCs consistent with tolDCs. Collectively, our findings provide groundwork for further investigation into novel mechanisms regulating DC tolerance and the role of helminth secreted metabolites in attenuating intestinal inflammation associated with IBD.Summary SentenceMetabolites produced byHeligmosomoides polygyrusinduce metabolic and transcriptional changes in DCs consistent with tolDCs, and adoptive transfer of these DCs attenuated DSS-induced intestinal inflammation.

Listeria monocytogenesis a remarkably well-adapted facultative intracellular pathogen that can thrive in a wide range of ecological niches.L. monocytogenesmaximizes its ability to generate energy from diverse carbon sources using a respiro-fermentative metabolism that can function under both aerobic and anaerobic conditions. Cellular respiration maintains redox homeostasis by regenerating NAD+while also generating a proton motive force (PMF). The end products of the menaquinone (MK) biosynthesis pathway are essential to drive both aerobic and anaerobic cellular respiration. We previously demonstrated that intermediates in the MK biosynthesis pathway, notably 1,4-dihydroxy-2-naphthoate (DHNA), are required for the survival and virulence ofL. monocytogenesindependent of their role in respiration. Furthermore, we found that restoration of NAD+/NADH ratio through expression of water-forming NADH oxidase (NOX) could rescue phenotypes associated with DHNA deficiency. Here we extend these findings to demonstrate that endogenous production or direct supplementation of DHNA restored both the cellular redox homeostasis and metabolic output of fermentation inL. monocytogenes. Further, exogenous supplementation of DHNA rescues thein vitrogrowth andex vivovirulence ofL. monocytogenesDHNA-deficient mutants. Finally, we demonstrate that exogenous DHNA restores redox balance inL. monocytogenesspecifically through the recently annotated NADH dehydrogenase Ndh2, independent of the extracellular electron transport (EET) pathway. These data suggest that the production of DHNA may represent an additional layer of metabolic adaptability byL. monocytogenesto drive energy metabolism in the absence of respiration-favorable conditions.

A new series of dual low nanomolar benzothiazole inhibitors of bacterial DNA gyrase and topoisomerase IV were developed. The resulting compounds show excellent broad-spectrum antibacterial activities against Gram-positive Enterococcus faecalis, Enterococcus faecium and multidrug resistant (MDR) Staphylococcus aureus strains [best compound minimal inhibitory concentrations (MICs): range

Grass pea (Lathyrus sativus L.) is a rich source of protein cultivated as an insurance crop in Ethiopia, Eritrea, India, Bangladesh, and Nepal. Its resilience to both drought and flooding makes it a promising crop for ensuring food security in a changing climate. The lack of genetic resources and the crop’s association with the disease neurolathyrism have limited the cultivation of grass pea. Here, we present an annotated, long read-based assembly of the 6.5 Gbp L. sativus genome. Using this genome sequence, we have elucidated the biosynthetic pathway leading to the formation of the neurotoxin, β-L-oxalyl-2,3-diaminopropionic acid (β-L-ODAP). The final reaction of the pathway depends on an interaction between L. sativus acyl-activating enzyme 3 (LsAAE3) and a BAHD-acyltransferase (LsBOS) that form a metabolon activated by CoA to produce β-L-ODAP. This provides valuable insight into the best approaches for developing varieties which produce substantially less toxin.

Ineffective pain control is the most commonly cited reason for misuse of prescription opioids and is influenced by genetics. In particular, the gene encoding the CYP2D6 enzyme, which metabolizes some of the most commonly prescribed opioids (e.g., tramadol, hydrocodone) to their more potent forms, is highly polymorphic and can lead to reduced concentrations of the active metabolites and decreased opioid effectiveness. Consideration of the CYP2D6 genotype may allow for predicting opioid response and identifying patients who are likely to respond well to lower potency opioids as well as those who may derive greater pain relief from non-opioid analgesics versus certain opioids. There is emerging evidence that a CYP2D6-guided approach to pain management improves pain control and reduces opioid consumption and thus may be a promising means for combating opioid misuse. Clinical practice guidelines are available for select opioids and other analgesics to support medication and dose selection based on pharmacogenetic data. This article describes the evidence supporting genotype-guided pain management as a means of improving pain control and reducing opioid misuse and clinical recommendations for genotype-guided analgesic prescribing. In addition, a “how to” guide using patient case examples is provided to demystify the process for implementing pharmacogenetics-guided pain management in order to optimize analgesia and minimize adverse effects. Optimizing pain management through genotype-guided approaches may ultimately provide safer and more effective therapy for pain control while decreasing the risk for opioid misuse.

Background Pharmacy stands increasingly on the frontline of patient care, yet current studies of clinical decision-making by pharmacists only capture deliberative processes that can be stated explicitly. Decision-making incorporates both deliberative and intuitive processes. Clinical Judgement Analysis (CJA) is a method novel to pharmacy that uncovers intuitive decision-making and may provide a more comprehensive understanding of the decision-making processes of pharmacists. Objectives This paper describes how CJA potentially uncovers the intuitive clinical decision-making processes of pharmacists. Using an illustrative decision-making example, the application of CJA will be described, including: • Scenario and associated task development around a defined judgement • Capture of pharmacists' decision-making processes and analysis using appropriate statistical methods Method An illustrative study was used, applying an established method for CJA. The decision to initiate anticoagulation, alongside appropriate risk judgements, was chosen as the context. Expert anticoagulation pharmacists were interviewed to define and then refine variables (cues) involved in this decision. Decision tasks with sixty scenarios were developed to explore the effect of these cues on pharmacists’ decision-making processes and distributed to participants for completion. Descriptive statistical and regression analyses were conducted for each participant. Results The method produced individual judgement models for each participant, for example, demonstrating that when judging stroke risk each participant's judgements could be accurately predicted using only 3 or 4 out of the possible 11 cues given. The method also demonstrated that participants appeared to consider multiple cues when making risk judgements but used an algorithmic approach based on one or two cues when making the clinical decision. Conclusion CJA generates insights into the clinical decision-making processes of pharmacists not uncovered by the current literature. This provides a springboard for more in-depth explorations; explorations that are vital to the understanding and ongoing development of the role of pharmacists.

We review extant experimental work in strategic management and argue that experiments constitute an underused methodology that has significant potential. We examine and categorize 179 experiments from 119 published articles over a 20-year period, delineating the contributions of these experiments to the strategic management literature. In doing so, we identify topic areas in which experiments have been effectively deployed as well as several literature streams that have a limited amount of prior experimental research. We discuss specific challenges of using experiments in strategy research, especially given its strong focus on the firm level of analysis. We also emphasize approaches for how experiments can be instrumental in extending management theories and accelerating behavioral microfoundations of strategy research. In light of past contributions and gaps, we discuss specific opportunities and means of designing innovative experiments, propose novel potential research questions, and provide a best practices methodological guide that scholars can use when considering experimental designs. Overall, our work documents experimental research and provides a methodological practicum, thereby offering a platform for future experiment-based research in strategic management.

Small alarmone hydrolases (SAHs) are alarmone metabolizing enzymes found in both metazoans and bacteria. In metazoans, the SAH homolog Mesh1 is reported to function in cofactor metabolism by hydrolyzing NADPH to NADH. In bacteria, SAHs are often identified in genomes with toxic alarmone synthetases for self-resistance. Here, we characterized a bacterial orphan SAH, i.e., without a toxic alarmone synthetase, in the phytopathogen Xanthomonas campestris pv.

Purpose: The impact of antiplatelet therapy with availability of CYP2C19 genotyping on bleeding in a real-world setting has not been extensively studied. Methods: Prospective, single-center, cohort study conducted between December 2015 and October 2019 with 1-year follow-up. Patients underwent percutaneous coronary intervention (PCI), CYP2C19 genotyping, and received P2Y12 inhibitor therapy. The primary outcome was time to first bleed of any severity using Bleeding Academic Research Consortium criteria. Secondary outcomes included time to first major bleed and rates of antiplatelet switching. Results: The primary outcome occurred in 697 of 2091 (33%) participants at a median of 15 days. Major bleeding occurred in 176 (8%) of patients. Compared to clopidogrel, treatment with ticagrelor or prasugrel was associated with increased risk of any bleeding (adjusted HR [aHR] 2.04, 95% CI 1.69-2.46). For patients without CYP2C19 no function alleles, treatment with prasugrel or ticagrelor was associated with increased risk of any bleeding (aHR 2.31, 95% CI 1.83-2.90). Similar associations were observed for major bleeding. No difference in ischemic events was observed. Among patients discharged on ticagrelor or prasugrel, 199 (36%) were de-escalated to clopidogrel within 1 year. De-escalation was more likely after a bleed if patients did not have a no function allele (35.9% vs 19.1%; P = .02). Conclusion: Bleeding is common in post-PCI patients on antiplatelet therapy. Patients on high potency agents had higher bleeding risk in the population at-large and in non-carriers of CYP2C19 no function alleles. Genotype-guided antiplatelet de-escalation should be further explored in prospective studies.

Parasites counteract host immunity by suppressing helper NLR proteins that function as central nodes in immune receptor networks. Understanding the mechanisms of immunosuppression can lead to strategies for bioengineering disease resistance. Here, we show that a cyst nematode virulence effector binds and inhibits oligomerization of the helper NLR protein NRC2 by physically preventing intramolecular rearrangements required for activation. A single amino acid polymorphism at the binding interface between NRC2 and the inhibitor is sufficient for this helper NLR to evade immune suppression, thereby restoring the activity of multiple disease resistance genes. This points to a novel strategy for resurrecting disease resistance in crop genomes.One sentence summaryA helper NLR is mutated to evade inhibition by a parasite effector.

Medium-chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non-canonical 1,4-reduction of an α,β-unsaturated iminium moiety. Comparison with structures of plant-derived ADHs suggest the 1,4-iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2-aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2-iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.

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.

We explore implications of asbestos for the measurement and reporting of liabilities, assets and expenses by diverse entities. We argue that entities in both public and private sectors are failing to recognise or appropriately measure liabilities related to asbestos and that the implications of asbestos for assets and expenses in financial statements are rarely reported. While we focus on recognition and measurement implications for Australian entities, we also examine relevant requirements in other jurisdictions and for sustainability reporting.

Background Nearly 300 medications contain pharmacogenomic information in their labeling approved by the U.S. Food and Drug Administration. As this number continues to grow, community pharmacists will be called on to use available pharmacogenomic data at the point of dispensing. Objective This qualitative study aimed to describe how pharmacists envision the integration of pharmacogenomic data into the current workflows of community pharmacy practice. Methods Community pharmacists from a regional supermarket chain pharmacy in the greater Pittsburgh area were interviewed using a semistructured interview guide. Participating pharmacists were presented with 3 clinical scenarios, followed by questions, to gain insight into how they envisioned the integration of pharmacogenomic data into community pharmacy workflow. The interview transcriptions were transcribed and coded. The content was analyzed to deduce the final themes. Supporting quotes were selected to illustrate each theme. Results Ten community pharmacists from 3 different pharmacy locations participated in the study. A thematic analysis produced 6 themes: (1) integrating pharmacogenomic data into the dispensing software, (2) receiving an alert for pharmacogenomic information within the dispensing software, (3) accessing pharmacogenomic clinical guidelines to guide drug–decision-making, (4) contacting the prescriber by adding a task to the call queue, (5) placing a mandatory counseling alert on medications that were adjusted using pharmacogenomic data, and (6) counseling the patient on the first refill of a medication that was adjusted using pharmacogenomic data. Conclusion This study describes how pharmacists envisioned the integration of pharmacogenomic data into community pharmacy workflow. The participants sought the integration of pharmacogenomic data into existing dispensing software, alerts for actionable prescribing changes using patient-specific pharmacogenomic data when available, and access to clinical decision support. In addition, the participants preferred to engage prescribers and receive alerts to counsel patients at prescription pick-up. These findings are key to integrating pharmacogenomic data into community pharmacy practice.

SummaryEngineering expanded effector recognition in plant immune receptors is a promising prospect for generating new disease resistant crop varieties. However, modification of plant NLR receptors has proven challenging due to the lack of understanding of their context as part of complex immune systems. Here, we demonstrate a new avenue for NLR-mediated engineering that exploits the allelic diversity in the Pik NLR pair to allow for the generation of receptors with expanded recognition specificities, which would otherwise result in constitutive cell death. This work lays the foundation for the incorporation of new effector recognition motifs into the Pik system and advances the development of designer NLRs that can be tailored to specific secreted pathogen signatures.AbstractEngineering the plant immune system offers genetic solutions to mitigate crop diseases caused by diverse agriculturally significant pathogens and pests. Modification of intracellular plant immune receptors of the nucleotide-binding leucine rich repeat (NLRs) superfamily for expanded recognition of pathogen virulence proteins (effectors) is a promising approach for engineering novel disease resistance. However, engineering can cause NLR autoactivation, resulting in constitutive defence responses that are deleterious to the plant. This may be due to plant NLRs associating in highly complex signalling networks that co-evolve together, and changes through breeding or genetic modification can generate incompatible combinations, resulting in autoimmune phenotypes. We have previously shown how alleles of the rice NLR pair Pik have differentially co-evolved, and how sensor/helper mismatching between non-co-evolved alleles triggers constitutive activation and cell death (De la Concepcion et al., 2021b). Here, we dissect incompatibility determinants in the Pik pair and found that HMA domains integrated in Pik-1 not only evolved to bind pathogen effectors but also likely co-evolved with other NLR domains to maintain immune homeostasis. This explains why changes in integrated domains can lead to autoactivation. We then used this knowledge to facilitate engineering of new effector recognition specificities overcoming initial autoimmune penalties. We show that by mismatching alleles of the rice sensor and helper NLRs Pik-1 and Pik-2, we can enable the integration of synthetic HMA domains with novel and enhanced recognition of an effector from the rice blast fungus. Taken together, our results reveal a new strategy for engineering NLRs, which has the potential to allow an expanded set of integrations and therefore new disease resistance specificities in plants.

Viruses employ a variety of strategies to escape or counteract immune responses, including depletion of cell surface major histocompatibility complex class I (MHC-I), that would ordinarily present viral peptides to CD8 + cytotoxic T cells. As part of a screen to elucidate biological activities associated with individual severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral proteins, we found that ORF7a reduced cell surface MHC-I levels by approximately fivefold. Nevertheless, in cells infected with SARS-CoV-2, surface MHC-I levels were reduced even in the absence of ORF7a, suggesting additional mechanisms of MHC-I down-regulation. ORF7a proteins from a sample of sarbecoviruses varied in their ability to induce MHC-I down-regulation and, unlike SARS-CoV-2, the ORF7a protein from SARS-CoV lacked MHC-I downregulating activity. A single amino acid at position 59 (T/F) that is variable among sarbecovirus ORF7a proteins governed the difference in MHC-I downregulating activity. SARS-CoV-2 ORF7a physically associated with the MHC-I heavy chain and inhibited the presentation of expressed antigen to CD8 + T cells. Specifically, ORF7a prevented the assembly of the MHC-I peptide loading complex and caused retention of MHC-I in the endoplasmic reticulum. The differential ability of ORF7a proteins to function in this way might affect sarbecovirus dissemination and persistence in human populations, particularly those with infection- or vaccine-elicited immunity.

Efforts to cure HIV have focused on reactivating latent proviruses to enable elimination by CD8+ cytotoxic T-cells. Clinical studies of latency reversing agents (LRA) in antiretroviral therapy (ART)-treated individuals have shown increases in HIV transcription, but without reductions in virologic measures, or evidence that HIV-specific CD8+ T-cells were productively engaged. Here, we show that the SARS-CoV-2 mRNA vaccine BNT162b2 activates the RIG-I/TLR – TNF – NFκb axis, resulting in transcription of HIV proviruses with minimal perturbations of T-cell activation and host transcription. T-cells specific for the early gene-product HIV-Nef uniquely increased in frequency and acquired effector function (granzyme-B) in ART-treated individuals following SARS-CoV-2 mRNA vaccination. These parameters of CD8+ T-cell induction correlated with significant decreases in cell-associated HIV mRNA, suggesting killing or suppression of cells transcribing HIV. Thus, we report the observation of an intervention-induced reduction in a measure of HIV persistence, accompanied by precise immune correlates, in ART-suppressed individuals. However, we did not observe significant depletions of intact proviruses, underscoring challenges to achieving (or measuring) HIV reservoir reductions. Overall, our results support prioritizing the measurement of granzyme-B-producing Nef-specific responses in latency reversal studies and add impetus to developing HIV-targeted mRNA therapeutic vaccines that leverage built-in LRA activity.

DNA damage triggers a widely conserved stress response in bacteria called the SOS response that involves two key regulators, the activator RecA and the transcriptional repressor LexA. Despite the wide conservation of the SOS response, the number of genes controlled by LexA varies considerably between different organisms. The filamentous soil-dwelling bacteria of the genus Streptomyces contain LexA and RecA homologs but their roles in Streptomyces have not been systematically studied. Here, we demonstrate that RecA and LexA are required for the survival of Streptomyces venezuelae during DNA damaging conditions and for normal development during unperturbed growth. Monitoring the activity of a fluorescent recA promoter fusion and LexA protein levels revealed that the activation of the SOS response is delayed in S. venezuelae. By combining global transcriptional profiling and ChIP-seq analysis, we determined the LexA regulon and defined the core set of DNA damage repair genes that are expressed in response to treatment with the DNA alkylating agent mitomycin C. Our results show that DNA damage-induced degradation of LexA results in the differential regulation of LexA target genes. Using Surface Plasmon Resonance, we further confirm the LexA DNA binding motif (SOS box) and demonstrate that LexA displays tight but distinct binding affinities to its target promoters, indicating a graded response to DNA damage.IMPORTANCEThe transcriptional regulator LexA functions as a repressor of the bacterial SOS response, which is induced during DNA damaging conditions. This results in the expression of genes important for survival and adaptation. Here, we report the regulatory network controlled by LexA in the filamentous antibiotic producing Streptomyces bacteria and establish the existence of the SOS response in Streptomyces. Collectively, our work reveals significant insights into the DNA damage response in Streptomyces that will promote further studies to understand how these important bacteria adapt to their environment.

Diadenosine tetraphosphate (Ap4A) is a putative second messenger molecule that is conserved from bacteria to humans. Nevertheless, its physiological role and the underlying molecular mechanisms are poorly characterized. We investigated the molecular mechanism by which Ap4A regulates inosine-5'-monophosphate dehydrogenase (IMPDH, a key branching point enzyme for the biosynthesis of adenosine or guanosine nucleotides) in Bacillus subtilis. We solved the crystal structure of BsIMPDH bound to Ap4A at a resolution of 2.45 Å to show that Ap4A binds to the interface between two IMPDH subunits, acting as the glue that switches active IMPDH tetramers into less active octamers. Guided by these insights, we engineered mutant strains of B. subtilis that bypass Ap4A-dependent IMPDH regulation without perturbing intracellular Ap4A pools themselves. We used metabolomics, which suggests that these mutants have a dysregulated purine, and in particular GTP, metabolome and phenotypic analysis, which shows increased sensitivity of B. subtilis IMPDH mutant strains to heat compared with wild-type strains. Our study identifies a central role for IMPDH in remodelling metabolism and heat resistance, and provides evidence that Ap4A can function as an alarmone.

The C19 diterpenoid alkaloids (C19 DTAs) are a large family of natural products, many of which modulate the activity of ion channels in vivo and are therefore of interest for the study of neurological and cardiovascular diseases. The complex architectures of these molecules continue to challenge the state-of-the art in chemical synthesis, particularly with respect to efficient assembly of their polcyclic ring systems. Here, we report the total syntheses of (-)-talatisamine, (-)-liljestrandisine, and (-)-liljestrandinine, three aconitine-type C19 DTAs, using a fragment coupling strategy. Key to this approach is a 1,2-addition/semipinacol rearrangement sequence which efficiently joins two complex fragments and sets an all-carbon quaternary center.

The island of Lanzarote in the Canary Islands was first settled by people from northern Africa in the first millennium BC and then colonized by Spain in the late fifteenth century. This colonial le...

This article points to gaps between academic research and the needs of accounting standard-setters. In part it attributes those gaps to the academic literature seeming to be inaccessible and oriented to ideas apparently unrelated to the policy-making issues facing standard-setters. As a means of partially reducing that perceived inaccessibility, the paper provides a way for standard-setters to identify and classify the various forms of academic accounting research so that they can evaluate their usefulness. Two prominent strands of research (agency theory/costly contracting and value relevance) are, as illustrations, analysed so that standard-setters can see how they might approach those strands. The paper suggests a users’ needs/demand driven approach to improving understanding, rather than a supply (by academics) driven approach. Finally, the paper explains how the performance metrics faced by academics can be inconsistent with the readiness expressed by standard-setters to have academics assist them. The paper provides a suggestion as to how there could be some alignment of academic performance metrics and standard-setters’ needs.

The capability to design microbiomes with predictable functions would enable new technologies for applications in health, agriculture, and bioprocessing. Towards this goal, we develop a model-guided approach to design synthetic human gut microbiomes for production of the health-relevant metabolite butyrate. Our data-driven model quantifies microbial interactions impacting growth and butyrate production separately, providing key insights into ecological mechanisms driving butyrate production. We use our model to explore a vast community design space using a design-test-learn cycle to identify high butyrate-producing communities. Our model can accurately predict community assembly and butyrate production across a wide range of species richness. Guided by the model, we identify constraints on butyrate production by high species richness and key molecular factors driving butyrate production, including hydrogen sulfide, environmental pH, and resource competition. In sum, our model-guided approach provides a flexible and generalizable framework for understanding and accurately predicting community assembly and metabolic functions., Microbiomes designed with predictable functions could enable broad applications in health, agriculture and bioprocessing. Here the authors use a model-guided approach to design diverse synthetic human gut communities for production of the health-relevant metabolite butyrate.

Why do entrepreneurs prefer to seek one equity form of funding over another? To address this question, we develop a contingency-based model of perceived funding fit that delineates several factors that influence strategic fund-seeking decisions by entrepreneurs. In prior research, entrepreneur fund-seeking has largely been explained using models that rely on rule-based approaches (e.g., the pecking order assumption) or value capture considerations. In contrast, we propose a dynamic contingency-based model that delineates several factors that influence entrepreneur perceptions of funding fit over and above transactional efficiency, including atypical value creation from the fundraising process itself and external stakeholder values. We inductively assess our model in the context of equity crowdfunding (ECF) and find that perceived funding fit can motivate some strategic fund-seekers to opt to pursue ECF, even when they have a reasonable opportunity to obtain other more established sources of funding such as angel or seed-stage venture capital. This indicates that ECF in several cases is not a funding mode of last resort as proposed in prior literature. Plain English Summary Raising capital is a complex and dynamic process. Strategic entrepreneurs seek “funding fit” for their particular ventures leading some to opt for less established forms of funding such as equity crowdfunding for a variety of reasons beyond efficiency. Prior venture funding research has largely taken the view of the investor, emphasizing what entrepreneurs must do to win the favor of angel investors and other seed funders, and deeming equity crowdfunding (ECF) a funding mode of last resort for discouraged entrepreneurs. Inductively analyzing hundreds of regulatory filings, entrepreneur interviews, public information, and media pieces about ECF-funded firms, we find evidence that in several cases, strategic entrepreneurs may prefer to opt for ECF if they perceive it to be a better fit due to novel forms of nonfinancial value. We explain our findings by proposing an emergent contingency-based model of “funding fit.”

Black patients suffer worse outcomes after percutaneous coronary intervention (PCI) than White patients. Inequities in antiplatelet prescribing may contribute to this health disparity. We compared P2Y

Introduction An estimated 64 million Medication Compliance Aids (MCAs) are dispensed by pharmacies in England each year as a method of reasonable adjustment to improve medication adherence (NICE 2009) and support medicines administration by carers (RPS 2013). Complexities exist when implementing medication changes for patients using MCAs, particularly at hospital discharge or outpatient appointments, where practices seem to vary. This National Survey is the first to determine the current policy and service provision of MCAs by acute hospitals in England. Methods An electronic survey was emailed to Chief Pharmacists via the Regional Medicines Information Services in Spring 2019. Initial non-responders were contacted by email and telephone. Results 51% (73/144) of acute hospital trusts in England responded. 77% (56/73) dispensed medication in MCAs at discharge. Of these, 62.5% would initiate MCAs and 61% supplied a different length of MCA vs non-MCA prescription (see table). 41 hospitals had designated staff completing MCAs. The median time to complete an MCA was 59.5 minutes (range 10–200). The median time from prescription receipt in pharmacy to MCA arrival on ward was 144.5 minutes (range 60–1,440). Of the 17 hospitals not providing MCAs, the majority would, upon discharge, contact the community pharmacy that provided the MCA pre-admission to update any medication changes and request the provision of a new supply of medicines. Conclusion Despite the ubiquitous nature of the MCA, there is no standard approach to the supply of these devices from acute hospitals across England. When hospitals do provide MCAs their preparation is time consuming, often requiring additional staff. A national approach to MCAs might help patients and carers, and reduce medication-related problems and costs.

Research to date on leader behaviors such as justice rule adherence, abusive supervision, and ethical leadership has found a clear linkage between such behaviors and employees’ work attitudes and p...

T-cell responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described in recovered patients, and may be important for immunity following infection and vaccination as well as for the development of an adoptive immunotherapy for the treatment of immunocompromised individuals. In this report, we demonstrate that SARS-CoV-2–specific T cells can be expanded from convalescent donors and recognize immunodominant viral epitopes in conserved regions of membrane, spike, and nucleocapsid. Following in vitro expansion using a good manufacturing practice-compliant methodology (designed to allow the rapid translation of this novel SARS-CoV-2 T-cell therapy to the clinic), membrane, spike, and nucleocapsid peptides elicited interferon-γ production, in 27 (59%), 12 (26%), and 10 (22%) convalescent donors (respectively), as well as in 2 of 15 unexposed controls. We identified multiple polyfunctional CD4-restricted T-cell epitopes within a highly conserved region of membrane protein, which induced polyfunctional T-cell responses, which may be critical for the development of effective vaccine and T-cell therapies. Hence, our study shows that SARS-CoV-2 directed T-cell immunotherapy targeting structural proteins, most importantly membrane protein, should be feasible for the prevention or early treatment of SARS-CoV-2 infection in immunocompromised patients with blood disorders or after bone marrow transplantation to achieve antiviral control while mitigating uncontrolled inflammation., Key Points • Coronavirus-specific polyfunctional T cells can be expanded from convalescent individuals for use for patients after bone marrow transplant. • SARS-CoV-2 T-cell products target structural viral proteins, including commonly recognized regions in the C terminus of membrane protein.

Many academic institutions are collecting blood samples from patients seeking treatment for coronavirus disease 2019 (COVID‐19) to build research biorepositories. It may be feasible to extract pharmacogenomic (PGx) information from biorepositories for clinical use. We sought to characterize the potential value of multigene PGx testing among individuals hospitalized with COVID‐19 in the United States. We performed a cross‐sectional analysis of electronic health records from consecutive individuals hospitalized with COVID‐19 at a large, urban academic health system. We characterized medication orders, focusing on medications with actionable PGx guidance related to 14 commonly assayed genes (CYP2C19, CYP2C9, CYP2D6, CYP3A5, DPYD, G6PD, HLA‐A, HLA‐B, IFNL3, NUDT15, SLCO1B1, TPMT, UGT1A1, and VKORC1). A simulation analysis combined medication data with population phenotype frequencies to estimate how many treatment modifications would be enabled if multigene PGx results were available. Sixty‐four unique medications with PGx guidance were ordered at least once in the cohort (n = 1,852, mean age 60.1 years). Nearly nine in 10 individuals (89.7%) had at least one order for a medication with PGx guidance and 427 patients (23.1%) had orders for 4 or more actionable medications. Using a simulation, we estimated that 17 treatment modifications per 100 patients would be enabled if PGx results were available. The genes CYP2D6 and CYP2C19 were responsible for the majority of treatment modifications, and the medications most often affected were ondansetron, oxycodone, and clopidogrel. PGx results would be relevant for nearly all individuals hospitalized with COVID‐19 and would provide the opportunity to improve clinical care.