Search

Carotid intima media thickness (cIMT) is a biomarker of subclinical atherosclerosis and a predictor of future cardiovascular events. Identifying associations between gene expression levels and cIMT may provide insight to atherosclerosis etiology. Here, we use two approaches to identify associations between mRNA levels and cIMT: differential gene expression analysis in whole blood and S-PrediXcan. We used microarrays to measure genome-wide whole blood mRNA levels of 5647 European individuals from four studies. We examined the association of mRNA levels with cIMT adjusted for various potential confounders. Significant associations were tested for replication in three studies totaling 3943 participants. Next, we applied S-PrediXcan to summary statistics from a cIMT genome-wide association study (GWAS) of 71 128 individuals to estimate the association between genetically determined mRNA levels and cIMT and replicated these analyses using S-PrediXcan on an independent GWAS on cIMT that included 22 179 individuals from the UK Biobank. mRNA levels of TNFAIP3, CEBPD and METRNL were inversely associated with cIMT, but these associations were not significant in the replication analysis. S-PrediXcan identified associations between cIMT and genetically determined mRNA levels for 36 genes, of which six were significant in the replication analysis, including TLN2, which had not been previously reported for cIMT. There was weak correlation between our results using differential gene expression analysis and S-PrediXcan. Differential expression analysis and S-PrediXcan represent complementary approaches for the discovery of associations between phenotypes and gene expression. Using these approaches, we prioritize TNFAIP3, CEBPD, METRNL and TLN2 as new candidate genes whose differential expression might modulate cIMT.

Inorganic pyrophosphatase catalyzes the conversion of pyrophosphate to phosphate and is often critical for driving reactions forward in cellular processes such as nucleic acid and protein synthesis. Commonly used methods for quantifying pyrophosphatase enzyme activity employ reacting liberated phosphate with a second molecule to produce absorbance changes or employing a second enzyme in coupled reactions to produce a product with a detectable absorbance. In this investigation, a novel [31P]-NMR spectroscopy-based assay was used to quantitatively measure the formation of phosphate and evaluate the activity of inorganic pyrophosphatase from the thermoacidophilic Crenarchaeota Sulfolobus islandicus. The enzymatic activity was directly measured via integration of the [31P] resonance associated with the phosphate product (δ = 2.1 ppm). Sulfolobus islandicus inorganic pyrophosphatase preferentially utilized Mg2+ as divalent cation and had pH and temperature optimums of 6.0 of 50 °C, respectively. The Vmax value was 850 μmol/min/mg and the Km for pyrophosphate was 1.02 mM. Sequence analysis indicates the enzyme is a Family I pyrophosphatase. Sulfolobus islandicus inorganic pyrophosphatase was shown to be inhibited by sodium fluoride with a IC50 of 2.26 mM, compared to a IC50 of 0.066 mM for yeast inorganic pyrophosphatase. These studies reveal that a [31P]-NMR spectroscopy-based assay is an effective method for analyzing catalysis by phosphate-producing enzymes.

Background/aim A new class of imidazo[2,1-b][1,3,4]thiadiazole compounds have recently been evaluated as inhibitors of phosphorylation of focal adhesion kinase (FAK) in pancreatic cancer. FAK is overexpressed in mesothelioma and has recently emerged as an interesting target for the treatment of this disease. Materials and methods Ten imidazo[2,1-b][1,3,4]thiadiazole compounds characterized by indole bicycle and a thiophene ring, were evaluated for their cytotoxic activity in two primary cell cultures of peritoneal mesothelioma, MesoII and STO cells. Results Compounds 1a and 1b showed promising antitumor activity with IC50 values in the range of 0.59 to 2.81 μM in both cell lines growing as monolayers or as spheroids. Their antiproliferative and antimigratory activity was associated with inhibition of phospho-FAK, as detected by a specific ELISA assay in STO cells. Interestingly, these compounds potentiated the antiproliferative activity of gemcitabine, and these results might be explained by the increase in the mRNA expression of the key gemcitabine transporter human equilibrative nucleoside transporter-1 (hENT-1). Conclusion These promising results support further studies on new imidazo[2,1-b][1,3,4]thiadiazole compounds as well as on the role of both FAK and hENT-1 modulation in order to develop new drug combinations for peritoneal mesothelioma.

Rice (Oryza sativa) is a staple food crop and serves as a model cereal plant. It contains two biosynthetic gene clusters (BGCs) for the production of labdane-related diterpenoids (LRDs), which serve important roles in combating biotic and abiotic stress. While plant BGCs have been subject to genetic analyses, these analyses have been largely confined to the investigation of single genes. CRISPR/Cas9-mediated genome editing was used to precisely remove each of these BGCs, as well as simultaneously knock out both BGCs. Deletion of the BGC from chromosome 2 (c2BGC), which is associated with phytocassane biosynthesis, but not that from chromosome 4 (c4BGC), which is associated with momilactone biosynthesis, led to a lesion mimic phenotype. This phenotype is dependent on two closely related genes encoding cytochrome P450 (CYP) mono-oxygenases, CYP76M7 and CYP76M8, from the c2BGC. However, rather than being redundant, CYP76M7 has been associated with the production of phytocassanes, whereas CYP76M8 is associated with momilactone biosynthesis. Intriguingly, the lesion mimic phenotype is not present in a line with both BGCs deleted. These results reveal directional cross-cluster phytotoxicity, presumably arising from the accumulation of LRD intermediates dependent on the c4BGC in the absence of CYP76M7 and CYP76M8, further highlighting their interdependent evolution and the selective pressures driving BGC assembly.

A simple expression and purification protocol is presented which produces a high yield and good micrographs of the apoferritin bacterioferritin B (BfrB) from Mycobacterium tuberculosis. Its 2.12 Å resolution cryo-EM structure is presented, revealing a unique C-terminal extension (164–181), and it is discussed how BfrB could serve the growing cryo-EM community in characterizing and pushing the limits of their electron microscopes and workflows., The use of cryo-EM continues to expand worldwide and calls for good-quality standard proteins with simple protocols for their production. Here, a straightforward expression and purification protocol is presented that provides an apoferritin, bacterioferritin B (BfrB), from Mycobacterium tuberculosis with high yield and purity. A 2.12 Å resolution cryo-EM structure of BfrB is reported, showing the typical cage-like oligomer constituting of 24 monomers related by 432 symmetry. However, it also contains a unique C-terminal extension (164–181), which loops into the cage region of the shell and provides extra stability to the protein. Part of this region was ambiguous in previous crystal structures but could be built within the cryo-EM map. These findings and this protocol could serve the growing cryo-EM community in characterizing and pushing the limits of their electron microscopes and workflows.

Mycobacterium tuberculosis (Mtb) is one of the deadliest pathogens encountered by humanity. Over the decades, its characteristic membrane organization and composition have been understood. However, there is still limited structural information and mechanistic understanding of the constituent membrane proteins critical for drug discovery pipelines. Recent advances in single-particle cryo-electron microscopy and cryo-electron tomography have provided the much-needed impetus towards structure determination of several vital Mtb membrane proteins whose structures were inaccessible via X-ray crystallography and NMR. Important insights into membrane composition and organization have been gained via a combination of electron tomography and biochemical and biophysical assays. In addition, till the time of writing this review, 75 new structures of various Mtb proteins have been reported via single-particle cryo-EM. The information obtained from these structures has improved our understanding of the mechanisms of action of these proteins and the physiological pathways they are associated with. These structures have opened avenues for structure-based drug design and vaccine discovery programs that might help achieve global-TB control. This review describes the structural features of selected membrane proteins (type VII secretion systems, Rv1819c, Arabinosyltransferase, Fatty Acid Synthase, F-type ATP synthase, respiratory supercomplex, ClpP1P2 protease, ClpB disaggregase and SAM riboswitch), their involvement in physiological pathways, and possible use as a drug target. Graphic Abstract Tuberculosis is a deadly disease caused by Mycobacterium tuberculosis. The Cryo-EM and tomography have simplified the understanding of the mycobacterial membrane organization. Some proteins are located in the plasma membrane; some span the entire envelope, while some, like MspA, are located in the mycomembrane. Cryo-EM has made the study of such membrane proteins feasible. Supplementary Information The online version contains supplementary material available at 10.1007/s00232-021-00179-w.

The invasion of waterhemp into northern sugarbeet growing regions has prompted producers to re-integrate inter-row cultivation into weed management programs, as no currently registered herbicides can control glyphosate-resistant waterhemp POST in crop. Inter-row cultivation was a common weed control practice in sugarbeet until the release of glyphosate-resistant sugarbeet cultivars in 2008 made the use of inter-row cultivation unnecessary. In the late 2010s, producers began again to use inter-row cultivation to remove weeds that glyphosate did not control, but producers need information on the effectiveness and safety of inter-row cultivation when used with soil-residual herbicide programs. Efficacy and tolerance field experiments were conducted in Minnesota and North Dakota from 2017 to 2019. Results from the efficacy experiment demonstrated that cultivation improved waterhemp control 11% and 12%, 14 and 28 d after treatment, respectively. Waterhemp response to cultivation was dependent on crop canopy and precipitation after cultivation. Cultivation had minimal effect on waterhemp density in three environments, but at one environment, near Galchutt, ND in 2019, waterhemp density increased 600% and 196%, 14 and 28 d after treatment, respectively. Climate data indicated that in 2019 Galchutt, ND received 105 mm of precipitation in the 14 d following cultivation and had an open crop canopy that probably contributed to further weed emergence. Results from the tolerance experiment demonstrated that root yield and recoverable sucrose were not affected by cultivation timing or number of cultivations. In one environment, cultivating reduced sucrose content by 0.8% regardless of date or cultivation number, but no differences were found in four environments. Damage/destruction of leaf tissue from in-season cultivation is probably responsible for the reduction in sucrose content. Results indicate that cultivation can be a valuable tool to control weeds that herbicide cannot, but excessive rainfall and open crop canopy following cultivation can create an environment conducive to further weed emergence. Nomenclature: Glyphosate; waterhemp; Amaranthus tuberculatus (Moq.) J.D. Sauer; sugarbeet; Beta vulgaris L.

Since the commercialization of herbicide-resistant (HR) crops, primarily glyphosate-resistant crops, their adoption has increased rapidly. Multiple herbicide resistance traits in crops such as canola (Brassica napus L.), corn (Zea mays L.), cotton (Gossypium hirsutum L.), and soybean [Glycine max (L.) Merr.] have become available in recent years, and management of their volunteers needs attention to prevent interference and yield loss in rotational crops. The objectives of this review were to summarize HR crop traits in barley (Hordeum vulgare L.), canola, corn, cotton, rice (Oryza sativa L.), soybean, sugarbeet (Beta vulgaris L.), and wheat (Triticum aestivum L.); assess their potential for volunteerism; and review existing literature on the interference of HR crop volunteers, yield loss, and their management in rotational crops. HR crop volunteers are problem weeds in agronomic cropping systems, and the impact of volunteerism depends on several factors, such as crop grown in rotation, the density of volunteers, management practices, and microclimate. Interference of imidazolinone-resistant (IR) barley or wheat volunteers can be a problem in rotational crops, particularly when IR crops such as canola or wheat are grown. HR canola volunteers are abundant in the Northern Great Plains due to high fecundity, seed loss before or during harvest, and secondary seed dormancy, and they can interfere in crops grown in rotation such as flax (Linum usitatissimum L.), field peas (Pisum sativum L.), and soybean. HR corn volunteers are competitive in crops grown in rotation such as corn, cotton, soybean, and sugarbeet, with yield loss depending on the density of HR corn volunteers. Volunteers of HR cotton, rice, soybean, and sugarbeet are not major concerns and can be controlled with existing herbicides. Herbicide options would be limited if the crop volunteers are multiple HR; therefore, recording the cultivar planted the previous year and selecting the appropriate herbicide are important. The increasing use of 2,4-D, dicamba, glufosinate, and glyphosate in North American cropping systems requires research on herbicide interactions and alternative herbicides or methods for controlling multiple HR crop volunteers.

The distribution of marine mammal species in many areas remains poorly understood, especially as observations for some taxa are rare and large-scale surveys are time consuming and extremely costly....

Tanshinones are the bioactive nor-diterpenoid constituents of the Chinese medicinal herb Danshen (Salvia miltiorrhiza). These groups of chemicals have the characteristic furan D-ring, which differentiates them from the phenolic abietane-type diterpenoids frequently found in the Lamiaceae family. However, how the 14,16-epoxy is formed has not been elucidated. Here, we report an improved genome assembly of Danshen using a highly homozygous genotype. We identify a cytochrome P450 (CYP71D) tandem gene array through gene expansion analysis. We show that CYP71D373 and CYP71D375 catalyze hydroxylation at carbon-16 (C16) and 14,16-ether (hetero)cyclization to form the D-ring, whereas CYP71D411 catalyzes upstream hydroxylation at C20. In addition, we discover a large biosynthetic gene cluster associated with tanshinone production. Collinearity analysis indicates a more specific origin of tanshinones in Salvia genus. It illustrates the evolutionary origin of abietane-type diterpenoids and those with a furan D-ring in Lamiaceae., Salvia miltiorrhiza is a medicinal plant that can produce the bioactive tanshinones. Here, the authors report the improved genome assembly and reveal the possible roles of three CYP71Ds in catalyzing the reactions leading to the formation of the characteristic furan D-ring of transhinones.

Pyrazinamide, a first-line antibiotic used against Mycobacterium tuberculosis, has been shown to act in a pH-dependent manner in vitro. Why pyrazinamide, an antitubercle prodrug discovered more than 65 years ago, exhibits this pH-dependent activity was unclear. Upon entering mycobacterial cells, pyrazinamide is deamidated to pyrazinoate by an enzymatic process and exists in an acid-base equilibrium with pyrazinoic acid. Thus, the effects of total pyrazinoic acid (pyrazinoic acid + pyrazinoate) on M. tuberculosis growth, pH homeostasis, and proton motive force over a range of pH values found in host tissues were investigated. Although M. tuberculosis was able to maintain pH homeostasis over an external pH range of 7.0 to 5.5, total pyrazinoic acid induced growth inhibition increased as culture medium pH was decreased from 7.3 to 6.4. Consistent with growth inhibition, total pyrazinoic acid increased both acidification of the bacterial cytoplasm and dissipation of membrane potential as the environmental pH decreased when added to the bacterial suspensions. The results suggest pyrazinoic acid is the active form of the drug, which acts as an uncoupler of proton motive force, likely a protonophore, providing a mechanistic explanation for the pH dependence of the drug activity.

Dolphins are among the largest and most diverse predators in marine ecosystems, but our understanding of their foraging ecology, which is crucial for ecosystem management, is poor. Delphinus delphis (common dolphins) are found in tropical and temperate waters globally. Stomach content studies indicate they are opportunistic predators that feed locally on abundant small pelagic schooling fish, but information is lacking on how their diet may vary seasonally and with ontogeny. We investigated effects of body length, sex, season, and year on D. delphis diet in the Hauraki Gulf, New Zealand, using carbon (δ13C) and nitrogen (δ15N) stable isotope analysis of 56 skin samples collected during 13 years (2004-2016). Dolphin δ15N values changed with body length, suggesting ontogenetic dietary variation. Nitrogen isotope values were higher in austral autumn/winter compared to spring/summer, whilst δ13C values decreased throughout the study period, suggesting temporal changes in primary productivity likely affecting the dolphins’ diet. Sex had a minor effect on dolphin δ13C values, with male and female isotopic niches overlapping by 62.6% and 45.2% (respectively). Our results provide a baseline for detecting future changes in the trophic ecology of D. delphis in a key habitat in New Zealand and highlight their role as a sentinel species in this coastal ecosystem.

Direct targeting of energy metabolism to defeat cancer is not a recent strategy. Although quite a few drugs use cellular metabolism for their antitumor effect, no direct inhibitors of energy metabolism have been approved by the FDA. Currently, several inhibitors of lactate dehydrogenase A (LDH-A), a key player in glycolysis, are in development. Earlier, we demonstrated the efficacy of N-hydroxyindole-based LDH-A inhibitors in different cancer types. In this study we describe the efficacy of NHI-Glc-2, which is designed to dual target cancer cells, by exploiting a simultaneous enhanced glucose uptake by overexpressed glucose transporter 1 (GLUT1) and by inhibition of LDH-A. NHI-Glc-2 inhibits LDH-A enzyme activity, PANC-1 cell growth and disrupts spheroid integrity, with an overall effect that is more pronounced when combined with gemcitabine.

Introduction: Non-small cell lung cancer (NSCLC) is a highly lethal disease. During the past 20 years, the epidermal growth factor receptor (EGFR) has been a relevant target for anticancer drug-design, and a large family of EGFR tyrosine kinase inhibitors (TKI) were designed, which improved therapeutic outcomes compared to conventional chemotherapy in NSCLC patients with specific EGFR mutations. However, resistance to these inhibitors occurs; therefore, the debate on which inhibitor should be used first is still open. Dacomitinib was approved in 2018 for the first-line treatment of NSCLC with EGFR activating mutations. Areas covered: This manuscript reviews the properties of dacomitinib, including the current information from clinical trials and its potential application as stand-alone therapy, or in combination. Expert opinion: Dacomitinib is a second-generation EGFR-TKI that has demonstrated significant improvement in overall survival in a phase III randomized study compared with gefitinib, a first-generation TKI. However, the rapid development and approval of a new generation of TKIs (osimertinib), with better clinical profiles, raises the question of which role can dacomitinib play in NSCLC. Further studies are required to evaluate the efficacy of this drug on brain metastases, as a second-line treatment after third-generation TKIs, or in combination with other types of treatments.

Background Clinical characteristics of the international population with severe asthma are unknown. Intercountry comparisons are hindered by variable data collection within regional and national severe asthma registries. We aimed to describe demographic and clinical characteristics of patients treated in severe asthma services in the United States, Europe, and the Asia-Pacific region. Methods The International Severe Asthma Registry retrospectively and prospectively collected data in patients with severe asthma (≥ 18 years old), receiving Global Initiative for Asthma (GINA) Step 5 treatment or with severe asthma remaining uncontrolled at GINA Step 4. Baseline demographic and clinical data were collected from the United States, United Kingdom, South Korea, Italy, and the Severe Asthma Web-based Database registry (including Australia, Singapore, and New Zealand) from December 2014 to December 2017. Results We included 4,990 patients. Mean (SD) age was 55.0 (15.9) years, and mean (SD) age at asthma onset was 30.7 (17.7) years. Patients were predominantly female (59.3%) and white (72.6%), had never smoked (60.5%), and were overweight or obese (70.4%); 34.9% were at GINA Step 5; and 57.2% had poorly controlled disease. A total of 51.1% of patients were receiving regular intermittent oral corticosteroids, and 25.4% were receiving biologics (72.6% for those at GINA Step 5). Mean (SD) exacerbation rate was 1.7 (2.7) per year. Intercountry variation was observed in clinical characteristics, prescribed treatments, and biomarker profiles. Conclusions Using a common data set and definitions, this study describes severe asthma characteristics of a large patient cohort included in multiple severe asthma registries and identifies country differences. Whether these are related to underlying epidemiological factors, environmental factors, phenotypes, asthma management systems, treatment access, and/or cultural factors requires further study.

Extinctions stemming from environmental change often trigger trophic cascades and coextinctions. Bottom-up cascades occur when changes in the primary producers in a network elicit flow-on effects to higher trophic levels. However, it remains unclear what determines a species' vulnerability to bottom-up cascades and whether such cascades were a large contributor to the megafauna extinctions that swept across several continents in the Late Pleistocene. The pathways to megafauna extinctions are particularly unclear for Sahul (landmass comprising Australia and New Guinea), where extinctions happened earlier than on other continents. We investigated the potential role of bottom-up trophic cascades in the megafauna extinctions in Late Pleistocene Sahul by first developing synthetic networks that varied in topology to identify how network position (trophic level, diet breadth, basal connections) influences vulnerability to bottom-up cascades. We then constructed pre-extinction (-80 ka) network models of the ecological community of Naracoorte, south-eastern Sahul, to assess whether the observed megafauna extinctions could be explained by bottom-up cascades. Synthetic networks showed that node vulnerability to bottom-up cascades decreased with increasing trophic level, diet breadth and basal connections. Extinct species in the Naracoorte community were more vulnerable overall to these cascades than were species that survived. The position of extinct species in the network - tending to be of low trophic level and therefore having relatively narrow diet breadths and fewer connections to plants - made them vulnerable. However, these species also tended to have few or no predators, a network-position attribute that suggests they might have been particularly vulnerable to new predators. Together, these results suggest that trophic cascades and naivety to predators could have contributed to the megafauna extinction event in Sahul.

Characterization of cryptic biosynthetic gene clusters (BGCs) from microbial genomes has been proven to be a powerful approach to the discovery of new natural products. However, such a genome mining approach to the discovery of bioactive plant metabolites has been muted. The plant BGCs characterized to date encode pathways for antibiotics important in plant defense against microbial pathogens, providing a means to discover such phytoalexins by mining plant genomes. Here is reported the discovery and characterization of a minimal BGC from the medicinal plant Catharanthus roseus, consisting of an adjacent pair of genes encoding a terpene synthase (CrTPS18) and cytochrome P450 (CYP71D349). These two enzymes act sequentially, with CrTPS18 acting as a sesquiterpene synthase, producing 5-epi-jinkoheremol (1), which CYP71D349 further hydroxylates to debneyol (2). Infection studies with maize revealed that 1 and 2 exhibit more potent fungicidal activity than validamycin. Accordingly, this study demonstrates that characterization of such cryptic plant BGCs is a promising strategy for the discovery of potential agrochemical leads. Moreover, despite the observed absence of 1 and 2 in C. roseus, the observed transcriptional regulation is consistent with their differential fungicidal activity, suggesting that such conditional coexpression may be sufficient to drive BGC assembly in plants.

International audience; Respiratory infections remain a major global health concern. Tuberculosis is one of the top 10 causes of death worldwide, while infections with Non-Tuberculous Mycobacteria are rising globally. Recent advances in human tissue modelling offer a unique opportunity to grow different human "organs" in vitro, including the human airway, that faithfully recapitulate lung architecture and function. Here, we have explored the potential of human airway organoids (AOs) as a novel system in which to assess the very early steps of mycobacterial infection. We reveal that Mycobacterium tuberculosis (Mtb) and Mycobacterium abscessus (Mabs) mainly reside as extracellular bacteria and infect epithelial cells with very low efficiency. While the AO microenvironment was able to control, but not eliminate Mtb, Mabs thrives. We demonstrate that AOs responded to infection by modulating cytokine, antimicrobial peptide and mucin gene expression. Given the importance of myeloid cells in mycobacteria infection, we co-cultured infected AOs with human monocyte-derived macrophages and found that these cells to interact with the organoid epithelium. We conclude that adult stem cell (ASC)-derived AOs can be used to decipher very early events of mycobacteria infection in human settings thus offering new avenues for fundamental and therapeutic research.

T-cell exhaustion is a hallmark of hepatitis C virus (HCV) infection and limits protective immunity in chronic viral infections and cancer. Limited knowledge exists of the initial viral and immune dynamics that characterise exhaustion in humans. We studied longitudinal blood samples from a unique cohort of subjects with primary infection using single cell multi-omics to identify the functions and phenotypes of HCV-specific CD8+ T cells. Early elevated IFN-γ response against the transmitted virus was associated with the rate of immune escape, larger clonal expansion, and early onset of exhaustion. Irrespective of disease outcome we discovered progenitors of early-exhaustion with intermediate expression of PD-1. Intra clonal analysis revealed distinct trajectories with multiple fates suggesting evolutionary plasticity of precursor cells. These findings challenge current paradigm on the contribution of CD8+ T cells to HCV disease outcome and provide data for future studies on T-cell differentiation in human infections.One sentence summaryProgenitors of T-cell exhaustion in acute HCV infection

Conservation management of wildlife species should be underpinned by knowledge of their distribution and abundance, as well as impacts of human activities on their populations and habitats. Common dolphins (Delphinus delphis) are subject to incidental capture in a range of Australia’s commercial fisheries including gill netting, purse seining and mid-water trawling. The impact these fishery interactions have on common dolphin populations is uncertain, as estimates of abundance are lacking, particularly for the segments of the populations at risk of bycatch and in greater need of protection. Here we used double-observer platform aerial surveys and mark-recapture distance sampling methods to estimate the abundance of common dolphins in 2011 over an area of 42,438 km2 in central South Australia, where incidental mortality of common dolphins due to fisheries bycatch is the highest. We also used the potential biological removal (PBR) method to estimate sustainable levels of human-caused mortality for this segment of the population. The estimated abundance of common dolphins was 21,733 (CV = 0.25; 95% CI = 13,809–34,203) in austral summer/autumn and 26,504 in winter/spring (CV = 0.19; 95% CI = 19,488–36,046). Annual PBR estimates, assuming a conservative maximum population growth rate of Rmax = 0.02 and a recovery factor of Fr = 0.5 for species of unknown conservation status, ranged from 95 (summer/autumn) to 120 dolphins (winter/spring), and from 189 (summer/autumn) to 239 dolphins (winter/spring) with an Rmax = 0.04. Our results indicate that common dolphins are an abundant dolphin species in waters over the central South Australian continental shelf (up to 100 m deep). Based on the 2011 abundance estimates of this species, the highest estimated bycatch of common dolphins (423 mortalities in 2004/05) in the southern Australian region exceeded the precautionary PBR estimates for this population segment. Recent bycatch levels appear to be below PBR estimates, but low observer coverage and underreporting of dolphin mortalities by fishers means that estimates of dolphin bycatch rates are not robust. The effects of cumulative human impacts on common dolphins are not well understood, and thus we recommend a precautionary management approach to manage common dolphin bycatch based on local abundance estimates.

Summary Plants produce an array of specialized metabolites with important ecological functions. The mechanisms underpinning the evolution of new biosynthetic pathways are not well‐understood. Here, we exploit available genome sequence resources to investigate triterpene biosynthesis across the Brassicaceae.Oxidosqualene cyclases (OSCs) catalyze the first committed step in triterpene biosynthesis. Systematic analysis of 13 sequenced Brassicaceae genomes was performed to identify all OSC genes. The genome neighbourhoods (GNs) around a total of 163 OSC genes were investigated to identify Pfam domains significantly enriched in these regions. All‐vs‐all comparisons of OSC neighbourhoods and phylogenomic analysis were used to investigate the sequence similarity and evolutionary relationships of the numerous candidate triterpene biosynthetic gene clusters (BGCs) observed. Functional analysis of three representative BGCs was carried out and their triterpene pathway products were elucidated.Our results indicate that plant genomes are remarkably plastic, and that dynamic GNs generate new biosynthetic pathways in different Brassicaceae lineages by shuffling the genes encoding a core palette of triterpene‐diversifying enzymes, presumably in response to strong environmental selection pressure.These results illuminate a genomic basis for diversification of plant‐specialized metabolism through natural combinatorics of enzyme families, which can be mimicked using synthetic biology to engineer diverse bioactive molecules., See also the Commentary on this article by Peters, 227: 992–994.

The architectural protein CTCF is a mediator of chromatin conformation, but how CTCF binding to DNA is orchestrated to maintain long-range gene expression is poorly understood. Here we perform RNAi knockdown to reduce CTCF levels and reveal a shared subset of CTCF-bound sites are robustly resistant to protein depletion. The ‘persistent’ CTCF sites are enriched at domain boundaries and chromatin loops constitutive to all cell types. CRISPR-Cas9 deletion of 2 persistent CTCF sites at the boundary between a long-range epigenetically active (LREA) and silenced (LRES) region, within the Kallikrein (KLK) locus, results in concordant activation of all 8 KLK genes within the LRES region. CTCF genome-wide depletion results in alteration in Topologically Associating Domain (TAD) structure, including the merging of TADs, whereas TAD boundaries are not altered where persistent sites are maintained. We propose that the subset of essential CTCF sites are involved in cell-type constitutive, higher order chromatin architecture., The architectural protein CTCF is a mediator of chromatin conformation, but how CTCF binding to DNA is regulated remains poorly understood. Here the authors find that there is a shared subset of CTCF-bound sites resistant to protein depletion in different cell lines, which are enriched at domain boundaries and chromatin loops constitutive to all cell types.

Cycling intestinal Lgr5(+) stem cells are intermingled with their terminally differentiated Paneth cell daughters at crypt bottoms. Paneth cells provide multiple secreted (e.g., Wnt, EGF) as well as surface-bound (Notch ligand) niche signals. Here we show that ablation of Paneth cells in mice, using a diphtheria toxin receptor gene inserted into the P-lysozyme locus, does not affect the maintenance of Lgr5(+) stem cells. Flow cytometry, single-cell sequencing, and histological analysis showed that the ablated Paneth cells are replaced by enteroendocrine and tuft cells. As these cells physically occupy Paneth cell positions between Lgr5(+) stem cells, they serve as an alternative source of Notch signals, which are essential for Lgr5(+) stem cell maintenance. Our combined in vivo results underscore the adaptive flexibility of the intestine in maintaining normal tissue homeostasis.

All land plants contain at least one class II diterpene cyclase (DTC), which utilize an acid-base catalytic mechanism, for the requisite production of ent-copalyl diphosphate (ent-CPP) in gibberellin A (GA) phytohormone biosynthesis. These ent-CPP synthases (CPSs) are hypothesized to be derived from ancient bacterial origins and, in turn, to have given rise to the frequently observed additional DTCs utilized in more specialized plant metabolism. However, such gene duplication and neo-functionalization has occurred repeatedly, reducing the utility of phylogenetic analyses. Support for evolutionary scenarios can be found in more specific conservation of key enzymatic features. While DTCs generally utilize a DxDD motif as the catalytic acid, the identity of the catalytic base seems to vary depending, at least in part, on product outcome. The CPS from Arabidopsis thaliana has been found to utilize a histidine-asparagine dyad to ligate a water molecule that serves as the catalytic base, with alanine substitution leading to the production of 8β-hydroxy-ent-CPP. Here this dyad and effect of Ala substitution is shown to be specifically conserved in plant CPSs involved in GA biosynthesis, providing insight into plant DTC evolution and assisting functional assignment. Even more strikingly, while GA biosynthesis arose independently in plant-associated bacteria and fungi, the catalytic base dyad also is specifically found in the relevant bacterial, but not fungal, CPSs. This suggests functional conservation of CPSs from bacteria to plants, presumably reflecting an early role for derived diterpenoids in both plant development and plant–microbe interactions, eventually leading to GA, and a speculative evolutionary scenario is presented.

Bioactive gibberellins (GAs or diterpenes) are essential hormones in land plants that control many aspects of plant growth and development. In flowering plants, 13-OH GAs (having low bioactivity-for example, GA1) and 13-H GAs (having high bioactivity-for example, GA4) frequently coexist in the same plant. However, the identity of the native Arabidopsis thaliana 13-hydroxylase GA and its physiological functions remain unknown. Here, we report that cytochrome P450 genes (CYP72A9 and its homologues) encode active GA 13-hydroxylases in Brassicaceae. Plants overexpressing CYP72A9 exhibited semi-dwarfism, which was caused by significant reduction in GA4 levels. Biochemical assays revealed that recombinant CYP72A9 protein catalysed the conversion of 13-H GAs to the corresponding 13-OH GAs. CYP72A9 was expressed predominantly in developing seeds in Arabidopsis. Freshly harvested seeds of cyp72a9 mutants germinated more quickly than the wild type, whereas stratification-treated seeds and seeds from long-term storage did not. The evolutionary origin of GA 13-oxidases from the CYP72A subfamily was also investigated and discussed here.

Terpene synthases often catalyze complex carbocation cascade reactions. It has been previously shown that single residue switches involving replacement of a key aliphatic residue with serine or threonine can "short-circuit" such reactions, presumed to act indirectly via dipole stabilization of intermediate carbocations. Here a similar switch was found in the structurally characterized ent-kaurene synthase from Bradyrhizobium japonicum. Application of a recently developed computational approach to terpene synthases, TerDockin, surprisingly indicates direct action of the introduced serine hydroxyl as a catalytic base. Notably, this model suggests alternative interpretation of previous results, and potential routes towards reengineering terpene synthase activity more generally.

Rationale: The macrolide antibiotic azithromycin reduces exacerbations in adults with persistent symptomatic asthma. However, owing to the pleotropic properties of macrolides, unintended bacteriological consequences such as augmented pathogen colonization or dissemination of antibiotic-resistant organisms can occur, calling into question the long-term safety of azithromycin maintenance therapy.Objectives: To assess the effects of azithromycin on the airway microbiota, pathogen abundance, and carriage of antibiotic resistance genes.Methods: 16S rRNA sequencing and quantitative PCR were performed to assess the effect of azithromycin on sputum microbiology from participants of the AMAZES (Asthma and Macrolides: The Azithromycin Efficacy and Safety) trial: a 48-week, double-blind, placebo-controlled trial of thrice-weekly 500 mg oral azithromycin in adults with persistent uncontrolled asthma. Pooled-template shotgun metagenomic sequencing, quantitative PCR, and isolate whole-genome sequencing were performed to assess antibiotic resistance.Measurements and Main Results: Paired sputum samples were available from 61 patients (n = 34 placebo, n = 27 azithromycin). Azithromycin did not affect bacterial load (P = 0.37) but did significantly decrease Faith's phylogenetic diversity (P = 0.026) and Haemophilus influenzae load (P < 0.0001). Azithromycin did not significantly affect levels of Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, or Moraxella catarrhalis. Of the 89 antibiotic resistance genes detected, five macrolide resistance genes and two tetracycline resistance genes were increased significantly.Conclusions: In patients with persistent uncontrolled asthma, azithromycin reduced airway H. influenzae load compared with placebo but did not change total bacterial load. Macrolide resistance increased, reflecting previous studies. These results highlight the need for studies assessing the efficacy of nonantibiotic macrolides as a long-term therapy for patients with persistent uncontrolled asthma.

Betulinic acid (BA) and its derivatives possess potent pharmacological activity against cancer and HIV. As with many phytochemicals, access to BA is limited by the requirement for laborious extraction from plant biomass where it is found in low amounts. This might be alleviated by metabolically engineering production of BA into an industrially relevant microbe such as Saccharomyces cerevisiae (yeast), which requires complete elucidation of the corresponding biosynthetic pathway. However, while cytochrome P450 enzymes (CYPs) that can oxidize lupeol into BA have been previously identified from the CYP716A subfamily, these generally do not seem to be specific to such biosynthesis and, in any case, have not been shown to enable high-yielding metabolic engineering. Here RoCYP01 (CYP716A155) was identified from the BA-producing plant Rosmarinus officinalis (rosemary) and demonstrated to effectively convert lupeol into BA, with strong correlation of its expression and BA accumulation. This was further utilized to construct a yeast strain that yields > 1 g/L of BA, providing a viable route for biotechnological production of this valuable triterpenoid.

The koala's Phascolarctos cinereus distribution is currently restricted to eastern and south‐eastern Australia. However, fossil records dating from 70 ± 4 ka (ka = 10³ yr) from south‐western Australia and the Nullarbor Plain are evidence of subpopulation extinctions in the southwest at least after the Last Interglacial (~128–116 ka). We hypothesize that koala sub‐population extinctions resulted from the eastward retraction of the koala's main browse species in response to unsuitable climatic conditions. We further posit a general reduction in the distribution of main koala‐browse trees in the near future in response climate change. We modelled 60 koala‐browse species and constructed a set of correlative species distribution models for five time periods: Last Interglacial (~128–116 ka), Last Glacial Maximum (~23–19 ka), Mid‐Holocene (~7–5 ka), present (interpolations of observed data, representative of 1960–1990), and 2070. We based our projections on five hindcasts and one forecast of climatic variables extracted from WorldClim based on two general circulation models (considering the most pessimistic scenario of high greenhouse‐gas emissions) and topsoil clay fraction. We used 17 dates of koala fossil specimens identified as reliable from 70 (± 4) to 535 (± 49) ka, with the last appearance of koalas at 70 ka in the southwest. The main simulated koala‐browse species were at their greatest modelled extent of suitability during the Last Glacial Maximum, with the greatest loss of koala habitat occurring between the Mid‐Holocene and the present. We predict a similar habitat loss between the present and 2070. The spatial patterns of habitat change support our hypothesis that koala extinctions in the southwest, Nullarbor Plain and central South Australia resulted from the eastward retraction of the dominant koala‐browse species in response to long‐term climate changes. Future climate patterns will likely increase the extinction risk of koalas in their remaining eastern ranges.

Sugarbeet growers only recently have combined ethofumesate, S-metolachlor, and dimethenamid-P in a weed control system for waterhemp control. Sugarbeet plant density, visible stature reduction, root yield, percent sucrose content, and recoverable sucrose were measured in field experiments at five environments between 2014 and 2016. Sugarbeet stand density and stature reduction occurred in some but not all environments. Stand density was reduced with PRE application of S-metolachlor at 1.60 kg ai ha–1 and S-metolachlor at 0.80 kg ha–1 + ethofumesate at 1.68 kg ai ha–1 alone or followed by POST applications of dimethenamid-P at 0.95 kg ai ha–1. Sugarbeet visible stature was reduced when dimethenamid-P followed PRE treatments. Stature reduction was greatest with ethofumesate at 1.68 or 4.37 kg ha–1 PRE and S-metolachlor at 0.80 kg ha–1 + ethofumesate at 1.68 kg ha–1 PRE followed by dimethenamid-P at 0.95 kg ha–1 POST. Stature reduction ranged from 0 to 32% 10 d after treatment (DAT), but sugarbeet recovered quickly and visible injury was negligible 23 DAT. Although root yield and recoverable sucrose were similar across herbicide treatments and environments, we caution against the use of S-metolachlor at 0.80 kg ha–1 + ethofumesate at 1.68 kg ai ha–1 PRE followed by dimethenamid-P at 0.95 kg ha–1 in sugarbeet.

Simple Summary Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers in humans, due to late diagnosis and limited treatment possibilities. Improved treatment for PDAC patients is warranted. Cyclin-dependent kinase 1 (CDK1) is a stimulator of cell cycle progression and its activity is regularly enhanced in pancreatic cancer cells. Therefore, CDK1 has been proposed as a novel drug target to treat patients with PDAC. This review describes the potential of CDK1 inhibition as a treatment for PDAC by outlining the molecular pathways influenced by CDK1 inhibition and new therapeutic strategies. Abstract The role of CDK1 in PDAC onset and development is two-fold. Firstly, since CDK1 activity regulates the G2/M cell cycle checkpoint, overexpression of CDK1 can lead to progression into mitosis even in cells with DNA damage, a potentially tumorigenic process. Secondly, CDK1 overexpression leads to the stimulation of a range of proteins that induce stem cell properties, which can contribute to the development of cancer stem cells (CSCs). CSCs promote tumor-initiation and metastasis and play a crucial role in the development of PDAC. Targeting CDK1 showed promising results for PDAC treatment in different preclinical models, where CDK1 inhibition induced cell cycle arrest in the G2/M phase and led to induction of apoptosis. Next to this, PDAC CSCs are uniquely sensitive to CDK1 inhibition. In addition, targeting of CDK1 has shown potential for combination therapy with both ionizing radiation treatment and conventional chemotherapy, through sensitizing tumor cells and reducing resistance to these treatments. To conclude, CDK1 inhibition induces G2/M cell cycle arrest, stimulates apoptosis, and specifically targets CSCs, which makes it a promising treatment for PDAC. Screening of patients for CDK1 overexpression and further research into combination treatments is essential for optimizing this novel targeted therapy.

SUMMARYCD21low age-associated or atypical memory B cells, enriched for autoantibodies and poised for plasma cell differentiation, accumulate in large numbers in chronic infections, autoimmune disease and immunodeficiency, posing the question of what checkpoints normally oppose their excessive accumulation. Here, we reveal a critical role for the calcium-NFAT-regulated transcription factors EGR2 and EGR3. In the absence of EGR2 and EGR3 within B cells, CD21low and B1 B cells accumulate and circulate in young mice in numbers 10-20 times greater than normal, over-express a large set of EGR2 ChIP-seq target genes including known drivers of plasma cell differentiation and under-express drivers of follicular germinal centers. Most follicular B cells constitutively express Egr2 proportionally to surface IgM down-regulation by self-antigens, and EGR2/3 deficiency abolishes this characteristic anergy response. These results define a key transcriptional checkpoint repressing CD21low B cell formation and inform how NFATC1 or EGR2 mutations promote B1 cell-derived chronic lymphocytic leukemias.

For decades, Mycobacterium tuberculosis has been one of the deadliest pathogens known. Despite infecting approximately one-third of the human population, no effective treatment or vaccine is available., Mycobacterium tuberculosis infections claim more than a million lives each year, and better treatments or vaccines are required. A crucial pathogenicity factor is translocation from phagolysosomes to the cytosol upon phagocytosis by macrophages. Translocation from the phagolysosome to the cytosol is an ESX-1-dependent process, as previously shown in vitro. Here, we show that in vivo, mycobacteria also translocate to the cytosol but mainly when host immunity is compromised. We observed only low numbers of cytosolic bacilli in mice, armadillos, zebrafish, and patient material infected with M. tuberculosis, M. marinum, or M. leprae. In contrast, when innate or adaptive immunity was compromised, as in severe combined immunodeficiency (SCID) or interleukin-1 receptor 1 (IL-1R1)-deficient mice, significant numbers of cytosolic M. tuberculosis bacilli were detected in the lungs of infected mice. Taken together, in vivo, translocation to the cytosol of M. tuberculosis is controlled by adaptive immune responses as well as IL-1R1-mediated signals. IMPORTANCE For decades, Mycobacterium tuberculosis has been one of the deadliest pathogens known. Despite infecting approximately one-third of the human population, no effective treatment or vaccine is available. A crucial pathogenicity factor is subcellular localization, as M. tuberculosis can translocate from phagolysosome to the cytosol in macrophages. The situation in vivo is more complicated. In this study, we establish that high-level cytosolic escape of mycobacteria can indeed occur in vivo but mainly when host resistance is compromised. The IL-1 pathway is crucial for the control of the number of cytosolic mycobacteria. The establishment that immune signals result in the clearance of cells containing cytosolic mycobacteria connects two important fields, cell biology and immunology, which is vital for the understanding of the pathology of M. tuberculosis.

Background: Critical illness frequently requires the use of broad-spectrum antimicrobials to treat life-threatening infection. The resulting impact on microbiome diversity is profound, influencing gastrointestinal fermentation endpoints, host immune response and metabolic activity including the conversion of primary bile acids to secondary bile acids. We previously observed reduced fermentation capacity in the gut microbiota of critically ill children upon hospital admission, but the functional recovery trajectory of the paediatric gut microbiome during critical illness has not been well defined. Here, we longitudinally studied the intestinal microbiome and faecal bile acid profile of critically ill children during hospitalisation in a paediatric intensive care unit (PICU). The composition of the microbiome was determined by sequencing of the 16s rRNA gene, and bile acids were measured from faecal water by liquid chromatography hyphenated to mass spectrometry. Results: In comparison to admission faecal samples, members of Clostridium cluster XIVa and Lachnospiraceae recovered during the late-acute phase (days 8-10) of hospitalisation. Patients with infections had a lower proportion of Lachnospiraceae in their gut microbiota than control microbiota and patients with admitting diagnoses. The proportion of Recovery Associated Bacteria (RAB) was observed to decline with the length of PICU admission. Additionally, the proportions of RAB were reduced in those with systemic infection, respiratory failure, and undergoing surgery. Notably, Clostridioides were positively associated with the secondary bile acid deoxycholic acid, which we hypothesised to driven by secondary bile acid induced sporulation; the ratio of primary to secondary bile acids demonstrated recovery during critical illness. Conclusion: The recovery of secondary bile acids occurs quickly after intervention for critical illness. Bile acid recovery may be induced by the Lachnospiraceae , the composition of which shifts during critical illness. Our data suggest that gut health and early gut microbiota recovery can be assessed by readily quantifiable faecal bile acid profiles.

Chronic stimulation of CD8+ T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.

A new phase of the COVID-19 pandemic has started as several SARS-CoV-2 variants are rapidly emerging globally, raising concerns for increased transmissibility. As animal models and traditional in vitro systems may fail to model key aspects of the SARS-CoV-2 replication cycle, representative in vitro systems to assess variants phenotypically are urgently needed. We found that the British variant (clade B.1.1.7), compared to an ancestral SARS-CoV-2 clade B virus, produced higher levels of infectious virus late in infection and had a higher replicative fitness in human airway, alveolar and intestinal organoid models. Our findings unveil human organoids as powerful tools to phenotype viral variants and suggest extended shedding as a correlate of fitness for SARS-CoV-2.One-Sentence SummaryBritish SARS-CoV-2 variant (clade B.1.1.7) infects organoids for extended time and has a higher fitness in vitro.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers because of diagnosis at late stage and inherent/acquired chemoresistance. Recent advances in genomic profiling and biology of this disease have not yet been translated to a relevant improvement in terms of disease management and patient’s survival. However, new possibilities for treatment may emerge from studies on key epigenetic factors. Deregulation of microRNA (miRNA) dependent gene expression and mRNA splicing are epigenetic processes that modulate the protein repertoire at the transcriptional level. These processes affect all aspects of PDAC pathogenesis and have great potential to unravel new therapeutic targets and/or biomarkers. Remarkably, several studies showed that they actually interact with each other in influencing PDAC progression. Some splicing factors directly interact with specific miRNAs and either facilitate or inhibit their expression, such as Rbfox2, which cleaves the well-known oncogenic miRNA miR-21. Conversely, miR-15a-5p and miR-25-3p significantly downregulate the splicing factor hnRNPA1 which acts also as a tumour suppressor gene and is involved in processing of miR-18a, which in turn, is a negative regulator of KRAS expression. Therefore, this review describes the interaction between splicing and miRNA, as well as bioinformatic tools to explore the effect of splicing modulation towards miRNA profiles, in order to exploit this interplay for the development of innovative treatments. Targeting aberrant splicing and deregulated miRNA, alone or in combination, may hopefully provide novel therapeutic approaches to fight the complex biology and the common treatment recalcitrance of PDAC.

Summary Antibiotic resistance is a growing problem, but bacteria can evade antibiotic treatment via tolerance and persistence. Antibiotic persisters are a small subpopulation of bacteria that tolerate antibiotics due to a physiologically dormant state. Hence, persistence is considered a major contributor to the evolution of antibiotic-resistant and relapsing infections. Here, we used the synthetically developed minimal cell Mycoplasma mycoides JCVI-Syn3B to examine essential mechanisms of antibiotic survival. The minimal cell contains only 473 genes, and most genes are essential. Its reduced complexity helps to reveal hidden phenomenon and fundamental biological principles can be explored because of less redundancy and feedback between systems compared to natural cells. We found that Syn3B evolves antibiotic resistance to different types of antibiotics expeditiously. The minimal cell also tolerates and persists against multiple antibiotics. It contains a few already identified persister-related genes, although lacking many systems previously linked to persistence (e.g. toxin-antitoxin systems, ribosome hibernation genes)., Graphical abstract, Highlights • Syn3B can form antibiotic tolerant and persister subpopulations with only 473 genes • Syn3B evolves expeditiously to gain resistance against multiple antibiotics • Syn3B contains a few systems previously linked to persistence (e.g., protease) • Syn3B lacks systems previously linked to persistence (e.g., ribosome dimerization), Microbiology ; Microbiofilms

Introduction: Our objective was to design an eccentric bicycle design to elicit delayed onset muscle soreness (DOMS). Methods: To assess the bicycle designs’ ability to elicit DOMS, fourteen, recreationally active, males performed five-minutes of eccentric bicycling at 50% of their individualized power determined from a modified six-second Wingate test. Outcome measures to assess DOMS included the Likert pain scale, creatine kinase, lactate blood concentration, and pressure algometry detection evaluated at four time points (baseline (before the eccentric bicycling), immediate post, 24 hours post, and 48 hours post). Results: The Likert pain scale was different (F = 75.88, p < 0.001) at baseline (0.14 ± 0.36) and immediate post (0.21 ± 0.43), compared to 24 hours post (3.07 ± 0.83), and 48 hours post (2.93 ± 1.07). No changes were reported for creatine kinase (F = 0.7167, p = 0.475), lactate blood concentration (F = 2.313, p = 0.107), or pressure algometry detection. Conclusions: To understand mechanisms of DOMS, there is a need for a consistent, reliable method for producing DOMS. Our eccentric bicycle design and protocol offers an alternative approach to previous eccentric ergometer designs - demonstrating the potential to elicit DOMS in one, five-minute session.

Drugs that target human thymidylate synthase (hTS) are widely used in anti-cancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces its over-expression and the development of drug resistance. We thus pursued an alternative strategy that led to the discovery of TS-dimer disrupters that bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. We performed a structural, spectroscopic and kinetic investigation of the effects of these small molecules andthe best one, E7, accelerates the proteasomal degradation of hTS in cancer cells. E7 showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) causes coronavirus disease 2019 (COVID‐19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air–liquid interface culture system which was characterized by confocal and electron microscopy and single‐cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self‐renewing fetal lung bud tip organoids. These cultures were readily infected by SARS‐CoV‐2 with mainly surfactant protein C‐positive alveolar type II‐like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS‐CoV‐2 infection and can be applied for drug screens., A human airway in vitro culture permissive to COVID‐19 demonstrates a drug‐sensitive IFN response.

Many pathogenic bacteria use the type III secretion system (T3SS), or injectisome, to secrete toxins into host cells. These protruding systems are primary targets for drug and vaccine development. Upon contact between injectisomes and host membranes, toxin secretion is triggered. How this works structurally and functionally is yet unknown. Using cryo-focused ion beam milling and cryo-electron tomography, we visualized injectisomes of Yersinia enterocolitica inside the phagosomes of infected human myeloid cells in a close-to-native state. We observed that a minimum needle length is required for injectisomes to contact the host membrane and bending of host membranes by some injectisomes that contact the host. Through subtomogram averaging, the structure of the entire injectisome was determined, which revealed structural differences in the cytosolic sorting platform compared to other bacteria. These findings contribute to understanding how injectisomes secrete toxins into host cells and provides the indispensable native context. The application of these cryo-electron microscopy techniques paves the way for the study of the 3D structure of infection-relevant protein complexes in host-pathogen interactions.

The Bermuda buckeye, Junonia coenia bergi, is the only butterfly endemic to Bermuda, but is largely unstudied. Whole-genome Illumina sequencing was used to obtain a complete circular mitochondrial genome sequence of 15,221 bp consisting of 22 tRNAs, 13 protein-coding genes, 2 rRNAs and a control region. Mitogenome structure and organization was found to be very similar to that of other Junonia butterfly mitogenomes. Excluding ambiguous nucleotides, the J. coenia bergi mitogenome is 99.1% identical to the J. coenia coenia mitogenome. Parsimony and maximum-likelihood phylogenetic reconstruction revealed the monophyly of subfamily Nymphalinae, genus Junonia, and species J. coenia.

Introduction: The microbiota is recognized for its impact on both human health and disease. The human microbiota is made up of trillions of cells, including bacteria, viruses, and fungi. The largest population of microbes reside in the gut, prompting research for better understanding of the impact of gastrointestinal microbiota in different diseases. Evidence from numerous studies has pointed out the role of commensal microbes as key determinants of cancer pathogenesis. Moreover, gut microbiota may play an important role in chemoresistance; consequently, this knowledge might be important for novel strategies to improve anticancer treatment efficacy. Areas covered: We describe the role of microbiota in different gastrointestinal cancer types (esophageal, gastric, colorectal, hepatocellular and pancreatic-biliary tract cancers). Moreover, we analyzed the impact of the microbiota on resistance to anticancer therapies, and, lastly, we focused on possibilities of microbiota modulation to enhance anticancer therapy efficacy. Expert opinion: Increasing evidence shows that gut microbiota might influence resistance to anticancer treatment, including conventional chemotherapy, immunotherapy, radiotherapy, and surgery. Therefore, a better knowledge of gut microbiota and its interactions with anticancer drugs will enable us to develop novel anticancer treatment strategies and subsequently improve the cancer patients’ outcome.

For centuries, tuberculosis has been a worldwide burden for human health, and gaps in our understanding of its pathogenesis have hampered the development of new treatments. ESX-1 is a complex machinery responsible for the secretion of virulence factors that manipulate the host response. Despite the importance of these secreted proteins for pathogenicity, only a few of them have been structurally and functionally characterised. Here, we describe a structural study of the ESX-secretion associated protein K (EspK), a 74 kDa protein known to be essential for the secretion of other substrates and the cytolytic effects of ESX-1. Small-Angle X-ray Scattering (SAXS) data show that EspK is a long molecule with a maximal dimension of 228 &Aring, It consists of two independent folded regions at each end of the protein connected by a flexible unstructured region driving the protein to coexist as an ensemble of conformations. Limited proteolysis identified a 26 kDa globular domain at the C-terminus of the protein consisting of a mixture of a-helices and b-strands, as shown by circular dichroism (CD) and SAXS. In contrast, the N-terminal portion is mainly helical with an elongated shape. Sequence conservation suggests that this architecture is preserved amongst the different mycobacteria species, proposing specific roles for the N- and C-terminal domains assisted by the middle flexible linker.

All land plants (embryophytes) must contain an ent-kaurene synthase (KS), as the ability to produce this olefin from ent-copalyl diphosphate (ent-CPP) is required for phytohormone biosynthesis. These KSs have frequently given rise to other class I diterpene synthases that catalyze distinct reactions for more specialized plant metabolism. Indeed, the prevalence of such gene duplication and neofunctionalization has obscured phylogenetic assignment of function. Here a pair of threonines is found to be conserved in all land plant KS involved in phytohormone biosynthesis, and their role in enzyme function investigated. Surprisingly, these threonines are not required, nor even particularly important for efficient production of ent-kaurene from ent-CPP. In addition, these threonines do not seem to affect protein structure or stability. Moreover, the absence of codon bias and positioning within an intron do not support a role in transcription or translation either. Despite their lack of apparent function, this pair of threonines are nevertheless completely conserved in all embryophyte KS from phytohormone biosynthesis. Thus, regardless of exact role, this serves as a diagnostic mark for such KS, enabling more confident distinction of these critical enzymes.

Tuberculosis, one of the oldest human pathogens remains a major global health threat. Recent advances in organoid technology offer a unique opportunity to grow different human “organs” in vitro, including the human airway, that faithfully recapitulate tissue architecture and function. We have explored the potential of human airway organoids (AOs) as a novel system in which to model tuberculosis infection. To this end, we adapted biosafety containment level 3–approved procedures to allow successful microinjection of Mycobacterium tuberculosis, the causative agent of tuberculosis, into AOs. We reveal that mycobacteria infected epithelial cells with low efficiency, and that the organoid microenvironment was able to control, but not eliminate the pathogen. We demonstrate that AOs responded to infection by inducing cytokine and antimicrobial peptide production, and inhibiting mucins. Given the importance of myeloid cells in tuberculosis infection, we co-cultured mycobacteria-infected organoids with human monocyte-derived macrophages, and found that these cells were recruited to the organoid epithelium. We conclude that adult stem cell–derived airway organoids can be used to model early events of tuberculosis infection and offer new avenues for fundamental and therapeutic research.

Simple Summary Despite the enormous advance in biomarker discovery, many potential biomarkers of drug activity are unable to satisfy the clinical need due to inadequate sensitivity and specificity. The nucleoside transporter hENT-1 has been studied as a potential biomarker to predict the effect of the widely used anticancer drug gemcitabine in pancreatic cancer. However, several studies showed controversial results regarding the predictive value of hENT-1, prompting new analyses with larger cohorts of patients and standardized methodologies. Improved insights on molecular mechanisms underlying hENT-1 expression and activity should also help in the identification of subsets of patients who are more likely to benefit from specific treatments and improve their clinical outcome. The establishment of such biomarker is especially valuable in pancreatic cancer, which is frequently characterized by complex disease biology and high mortality. Abstract Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive tumor characterized by early invasiveness, rapid progression and resistance to treatment. For more than twenty years, gemcitabine has been the main therapy for PDAC both in the palliative and adjuvant setting. After the introduction of FOLFIRINOX as an upfront treatment for metastatic disease, gemcitabine is still commonly used in combination with nab-paclitaxel as an alternative first-line regimen, as well as a monotherapy in elderly patients unfit for combination chemotherapy. As a hydrophilic nucleoside analogue, gemcitabine requires nucleoside transporters to permeate the plasma membrane, and a major role in the uptake of this drug is played by human equilibrative nucleoside transporter 1 (hENT-1). Several studies have proposed hENT-1 as a biomarker for gemcitabine efficacy in PDAC. A recent comprehensive multimodal analysis of hENT-1 status evaluated its predictive role by both immunohistochemistry (with five different antibodies), and quantitative-PCR, supporting the use of the 10D7G2 antibody. High hENT-1 levels observed with this antibody were associated with prolonged disease-free status and overall-survival in patients receiving gemcitabine adjuvant chemotherapy. This commentary aims to critically discuss this analysis and lists molecular factors influencing hENT-1 expression. Improved knowledge on these factors should help the identification of subgroups of patients who may benefit from specific therapies and overcome the limitations of traditional biomarker studies.

Plants can contain biosynthetic gene clusters (BGCs) that nominally resemble those found in microbes. However, while horizontal gene transmission is often observed in microbes, plants are limited to vertical gene transmission, implying that their BGCs may exhibit distinct inheritance patterns. Rice (Oryza sativa) contains two unlinked BGCs involved in diterpenoid phytoalexin metabolism, with one clearly required for momilactone biosynthesis, while the other is associated with production of phytocassanes. Here, in the process of elucidating momilactone biosynthesis, genetic evidence was found demonstrating a role for a cytochrome P450 (CYP) from the other “phytocassane” BGC. This CYP76M8 acts after the CYP99A2/3 from the “momilactone” BGC, producing a hemiacetal intermediate that is oxidized to the eponymous lactone by a short-chain alcohol dehydrogenase also from this BGC. Thus, the “momilactone” BGC is not only incomplete, but also fractured by the need for CYP76M8 to act in between steps catalyzed by enzymes from this BGC. Moreover, as supported by similar activity observed with orthologs from the momilactone-producing wild-rice species Oryza punctata, the presence of CYP76M8 in the other “phytocassane” BGC indicates interdependent evolution of these two BGCs, highlighting the distinct nature of BGC assembly in plants.