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Erratic rainfall often results in intermittent drought and/or waterlogging and limits maize (Zea mays L.) productivity in many parts of the Asian tropics. Developing climate-resilient maize germplasm possessing tolerance to these key abiotic stresses without a yield penalty under optimal growing conditions is a challenge for breeders working in stress-vulnerable agro-ecologies in the region. Breeding stress-resilient maize for rainfed stress-prone ecologies is identified as one of the priority areas for CIMMYT-Asia maize program. We applied rapid cycle genomic selection (RCGS) on two multiparent yellow synthetic populations (MYS-1 and MYS-2) to improve grain yield simultaneously under drought and waterlogging conditions using genomic-estimated breeding values (GEBVs). Also, the populations were simultaneously advanced using recurrent phenotypic selection (PS) by exposing them to managed drought and waterlogging and intermating tolerant plants from the two selection environments. Selection cycles per se (C1, C2, and C3) of the two populations developed using RCGS and PS approach and their test-cross progenies were evaluated separately in multilocation trials under managed drought, waterlogging, and optimal moisture conditions. Significant genetic gains were observed with both GS and PS, except with PS in MYS-2 under drought and with GS in MYS-1 under waterlogging. Realized genetic gains from GS were relatively higher under drought conditions (110 and 135 kg ha−1 year−1) compared to waterlogging (38 and 113 kg ha−1 year−1) in both MYS-1 and MYS-2, respectively. However, under waterlogging stress PS showed at par or better than GS as gain per year with PS was 80 and 90 kg ha−1, whereas with GS it was 90 and 43 kg ha−1 for MYS-1 and MYS-2, respectively. Our findings suggested that careful constitution of a multiparent population by involving trait donors for targeted stresses, along with elite high-yielding parents from diverse genetic background, and its improvement using RCGS is an effective breeding approach to build multiple stress tolerance without compromising yield when tested under optimal conditions.

The development of blood vessels, referred to as angiogenesis, is an intricate process regulated spatially and temporally through a delicate balance between the qualitative and quantitative expression of pro and anti-angiogenic molecules. As angiogenesis is a prerequisite for solid tumors to grow and metastasize, a variety of tumor angiogenesis models have been formulated to better understand the underlying mechanisms and associated clinical applications. Studies have demonstrated independent mechanisms inducing angiogenesis in tumors such as (a) HIF-1/VEGF mediated paracrine interactions between a cancer cell and endothelial cells, (b) recruitment of progenitor endothelial cells, and (c) vasculogenic mimicry. Moreover, single-cell sequencing technologies have indicated endothelial cell heterogeneity among organ systems including tumor tissues. However, existing angiogenesis models often rely upon normal endothelial cells which significantly differ from tumor endothelial cells exhibiting distinct (epi)genetic and metabolic signatures. Besides, the existence of intra-individual variations necessitates the development of improved tumor vascular model systems for personalized medicine. In the present review, we summarize recent advancements of 3D tumor vascular model systems which include (a) tissue engineering-based tumor models; (b) vascular organoid models, and (c) organ-on-chips and their importance in replicating the tumor angiogenesis along with the associated challenges to design improved models.

In recent years, National Maize Research Program (NMRP) aimed a paradigm shift from open-pollinated varieties (OPVs) towards hybrid maize to achieve self-sufficiency in maize for food, feed, and hybrid seed within the country. In this mission, it is necessary to identify and deploy high-yielding stress-resilient maize hybrids that can cope with climate change effects, including heat stress. Under the project “Heat Tolerant Maize for Asia (HTMA)”, NMRP introduced the hybrids that performed better in previous years in different environments from International Maize and Wheat Improvement Center (CIMMYT) Hyderabad for multilocation on-farm testing. Fifteen genotypes were evaluated at two locations, two sites in Madi, Chitwan, and one in Ghorahi, Dang, along with Rampur Hybrid-8 as a heat-tolerant check, and RML-86/RML-96 and RML-95/RML-96 as normal checks. Randomized complete block design (RCBD) was used with three replicates during the spring of 2016/17. Likewise, another 20 and 18 promising hybrids were demonstrated during the winter of 2016/17 and 2017/18, respectively, in different hybrid growing pockets considering a site – a replication. Grain yield and yield attributing traits at all locations were recorded. From the across-site data analysis, selected heat-tolerant hybrids from the experiment were CAH1432, ZH15405, ZH141592, and CAH1715 which were statistically at par with promising normal hybrid RML-86/RML-96 and superior to already released heat-tolerant Rampur Hybrid-8. In 2016/17, ZH138098, ZH1620, and VH121062 were farmers’ preferred heat-tolerant hybrids. In 2017/18, Rampur Hybrid-10, ZH141592, CAH1715, and ZH15440 were preferred by farmers. The selected bestbet are taken forward for official release/registration followed by commercialization through a public-private partnership with Nepali seed companies/cooperatives. SAARC J. Agric., 19(1): 27-43 (2021)

With progressive climate change and the associated increase in mean temperature, heat stress tolerance has emerged as one of the key traits in the product profile of the maize breeding pipeline for lowland tropics. The present study aims to identify the genomic regions associated with heat stress tolerance in tropical maize. An association mapping panel, called the heat tolerant association mapping (HTAM) panel, was constituted by involving a total of 543 tropical maize inbred lines from diverse genetic backgrounds, test-crossed and phenotyped across nine locations in South Asia under natural heat stress. The panel was genotyped using a genotyping-by-sequencing (GBS) platform. Considering the large variations in vapor pressure deficit (VPD) at high temperature (Tmax) across different phenotyping locations, genome-wide association study (GWAS) was conducted separately for each location. The individual location GWAS identified a total of 269 novel significant single nucleotide polymorphisms (SNPs) for grain yield under heat stress at a p value of –5. A total of 175 SNPs were found in 140 unique gene models implicated in various biological pathway responses to different abiotic stresses. Haplotype trend regression (HTR) analysis of the significant SNPs identified 26 haplotype blocks and 96 single SNP variants significant across one to five locations. The genomic regions identified based on GWAS and HTR analysis considering genomic region x environment interactions are useful for breeding efforts aimed at developing heat stress resilient maize cultivars for current and future climatic conditions through marker-assisted introgression into elite genetic backgrounds and/or genome-wide selection.

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR–Cas immune system and a popular tool for genome editing. The commonly used Streptococcus pyogenes Cas9 (SpCas9) is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific. However, previous studies have focused on specificity of double-strand break (DSB) or indel formation, potentially overlooking alternative cleavage activities of these Cas9 variants. In this study, we employed in vitro cleavage assays of target libraries coupled with high-throughput sequencing to systematically compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9 and HiFi Cas9). We observed that all Cas9s tested could cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on target sequence and Cas9 variant. In addition, SaCas9 and engineered SpCas9 variants nick targets with multiple mismatches but have a defect in generating a DSB, while SpCas9 creates DSBs at these targets. Overall, these differences in cleavage rates and DSB formation may contribute to varied specificities observed in genome editing studies.

Aim: We report here the development of tenofovir- and curcumin-loaded lactoferrin nanoparticles (TCNPs) as an HIV-microbicide. Materials & methods: TCNPs were subjected to various physicochemical characterization experiments, followed by in vitro and in vivo experiments to assess their efficacy. Results: TCNPs had a diameter of 74.31 ± 2.56 nm with a gross encapsulation of more than 61% for each drug. Nanoparticles were effective against HIV-1 replication, with an IC50 of 1.75 μM for curcumin and 2.8 μM for tenofovir. TCNPs provided drug release at the application site for up to 8–12 h, with minimal leakage into the systemic circulation. TCNPs showed spermicidal activity at ≥200 μM and induced minimal cytotoxicity and inflammation in the vaginal epithelium as revealed by histopathological and ELISA studies. Conclusion: We demonstrated that TCNPs could serve as a novel anti-HIV microbicidal agent in rats. [Formula: see text]

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are gaining increasing importance in the field of regenerative medicine. Although therapeutic value of MSCs is now being established through many clinical trials, issues have been raised regarding their expansion as per regulatory guidelines. Fetal bovine serum usage in cell therapy poses difficulties due to its less-defined, highly variable composition and safety issues. Hence, there is a need for transition from serum-based to serum-free media (SFM). Since SFM are cell type-specific, a precise analysis of the properties of MSCs cultured in SFM is required to determine the most suitable one. Six different commercially available low serum/SFM with two different seeding densities were evaluated to explore their ability to support the growth and expansion of BM-MSCs and assess the characteristics of BM-MSCs cultured in these media. Except for one of the SFM, all other media tested supported the growth of BM-MSCs at a low seeding density. No significant differences were observed in the expression of MSC specific markers among the various media tested. In contrary, the population doubling time, cell yield, potency, colony-forming ability, differentiation potential, and immunosuppressive properties of MSCs varied with one another. We show that SFM tested supports the growth and expansion of BM-MSCs even at low seeding density and may serve as possible replacement for animal-derived serum.

In recent years, the various health benefits of Cyanobacteria microalgae – such as Arthrospira platensis, commonly called Spirulina, an edible blue-green algae – have attracted scientific attention including micro-level examinations of its bioactive components. As a whole food and nutritional supplement, it serves as a plant protein source, which has shown positive effects across a wide range of human health concerns, from malnutrition to metabolic syndrome. Spirulina bioactives, such as essential amino acids, phycocyanin, polysaccharides, carotenoids, and chlorophyll, and essential vitamins and trace minerals, are responsible for its holistic actions against oxidative stress and inflammation, and its antiviral, antibacterial, and immune-modulating effects. Various in vitro, in vivo, and ex vivo experiments have established Spirulina's mechanism of action and its effect on immunity as a proof of concept. The phenolic compounds and extracellular metabolites released from Spirulina whole food after digestion are postulated to strengthen the epithelial lining with antibacterial effects against pathogenic bacteria, adding to its prebiotic effect on the gut microbiota (like Bifidobacterium and Lactobacillus) due to its fiber content. In this study, the digestibility of Spirulina was assessed by the determination of free amino acids and peptide release during the each phase of digestion in a simulated static digestive model system. The hypothesis bridging poor gut health to low-level inflammation and metabolic syndrome, and the potential to address those issues with nutritional supplementation, such as with Spirulina, could also be beneficial in the long run to reduce comorbid illnesses, such as those associated with the currently prevailing coronavirus disease 2019 pandemic.

The present experiment was conducted to understand the effect of soil moisture status on the economic or standard heterosis in fifty maize hybrids for grain yield and flowering traits. The trials were planted at Agricultural Research Farm, Banaras Hindu University, Varanasi in alpha-lattice design with two replications. The analysis of variance revealed the significant differences for grain yield and flowering related traits such as days to 50% anthesis, days to 50% silking and anthesis-silking interval under all the moisture conditions including optimal, managed drought and managed waterlogging conditions. Significant amount of heterosis was observed over the selected check P3502 for all the traits under study; however, the magnitude and direction varied with traits and with soil moisture level. For days to 50% anthesis, hybrids ZH17192, VH112926, VH123021, ZH114250, ZH16929 and ZH16930 were showed significant negative heterosis under all the moisture conditions that explained earliness under both moisture-stress and normal conditions. Among the tested hybrids, VH123021 and ZH16929 were recorded significant negative standard heterosis for flowering traits; and significant positive heterosis for grain yield under all the three moisture conditions. Six hybrids under drought, seven hybrids under optimal and two hybrids under waterlogged condition showed positive standard heterosis for grain yield. Further evaluation of these hybrids at multi-locations and multi-year is advisable to confirm the promising findings observed in our study. This study could be valuable for development of climate-resilient maize hybrids.

Improvements in long-read data and scaffolding technologies have enabled rapid generation of reference-quality assemblies for complex genomes. Still, an assessment of critical sequence depth and read length is important for allocating limited resources. To this end, we have generated eight assemblies for the complex genome of the maize inbred line NC358 using PacBio datasets ranging from 20 to 75 × genomic depth and with N50 subread lengths of 11–21 kb. Assemblies with ≤30 × depth and N50 subread length of 11 kb are highly fragmented, with even low-copy genic regions showing degradation at 20 × depth. Distinct sequence-quality thresholds are observed for complete assembly of genes, transposable elements, and highly repetitive genomic features such as telomeres, heterochromatic knobs, and centromeres. In addition, we show high-quality optical maps can dramatically improve contiguity in even our most fragmented base assembly. This study provides a useful resource allocation reference to the community as long-read technologies continue to mature., Sequence depth and read length determine the quality of genome assembly. Here, the authors leverage a set of PacBio reads to develop guidelines for sequencing and assembly of complex plant genomes in order to allocate finite resources using maize as an example.

The aim of this study was to assess whether transversus abdominis plane (TAP) blocks can be utilized to decrease patient pain scores and narcotic use during the first 24 h following robot-assisted laparoscopic prostatectomy (RALP). 100 patients received a TAP block with a mixture of 1.3% liposomal bupivacaine, 0.5% Marcaine and 0.9% NaCl prior to RALP. This was in addition to an already established pain management regiment, which included preoperative PO acetaminophen (650 mg), celecoxib (200 mg), and tolterodine ER (4 mg). These patients were prospectively followed and then retrospectively compared to a 1:1 propensity matched group of 100 patients that did not receive a TAP but did receive the preoperative PO medications. Pain scores were assessed on a scale from 1-10 in the PACU, as well as the surgical floor at 8, 16, and 24-h post-surgery. Intra-/post-operative narcotic use and time to ambulation following arrival to the surgical floor were also analyzed. Patient receiving TAP blocks had immediate post-op pain scores of 2.23 vs 4.26 for those not receiving TAP blocks (p = 0.000). The pain scores at 8, 16, and 24 h for TAP patients were 2.68, 2.62, and 2.62 as compared to 2.89, 2.87, and 3.36 for non-TAP patients. The difference was statistically significant for immediate and 24-h pain scores (p = 0.000, 0.001, respectively). On average, TAP block patients ambulated faster than non-TAP patients, 2.68 vs 4.91 h (p = 0.000). Intra-operative narcotic use was decreased in the TAP group for each of the opioids that were used: fentanyl 177.5 vs 205mcg (p = 0.001), morphine 5.5 vs 10 mg (p = 0.000), and hydromorphone 0.75 vs 1.75 mg (p = 0.001). Narcotic usage in the PACU was limited to hydromorphone and TAP patients used 0.7 mg compared to 1.36 mg (p = 0.003) for non-TAP patients. Oral oxycodone/acetaminophen (5 mg/325 mg) was used for pain control on the surgical floor and on average TAP patients received less, 2.4 vs 5 tabs (p = 0.000). Average time to perform the TAP block was 3.5 min and total OR time for TAP vs non-TAP patients was 107.41 vs 106.58 min (p = 0.386). TAP blocks as part of a perioperative pain management protocol can be utilized during RALPs to decrease patient pain scores at two different time intervals, immediately post-operative and 24 h after surgery. Patients also ambulate sooner following surgery and require a decreased amount of narcotics during the intra-operative and post-operative periods. TAP blocks are quick, effective, and do not add a significant amount of OR time to RALPs.

Inefficient physiological transitions are known to cause metabolic disorders. Therefore, investigating mechanisms that constitute molecular switches in a central metabolic organ like the liver becomes crucial. Specifically, upstream mechanisms that control temporal engagement of transcription factors, which are essential to mediate physiological fed–fast–refed transitions are less understood. SIRT1, a NAD(+)-dependent deacetylase, is pivotal in regulating hepatic gene expression and has emerged as a key therapeutic target. Despite this, if/how nutrient inputs regulate SIRT1 interactions, stability, and therefore downstream functions are still unknown. Here, we establish nutrient-dependent O-GlcNAcylation of SIRT1, within its N-terminal domain, as a crucial determinant of hepatic functions. Our findings demonstrate that during a fasted-to-refed transition, glycosylation of SIRT1 modulates its interactions with various transcription factors and a nodal cytosolic kinase involved in insulin signaling. Moreover, sustained glycosylation in the fed state causes nuclear exclusion and cytosolic ubiquitin-mediated degradation of SIRT1. This mechanism exerts spatiotemporal control over SIRT1 functions by constituting a previously unknown molecular relay. Of note, loss of SIRT1 glycosylation discomposed these interactions resulting in aberrant gene expression, mitochondrial dysfunctions, and enhanced hepatic gluconeogenesis. Expression of nonglycosylatable SIRT1 in the liver abrogated metabolic flexibility, resulting in systemic insulin resistance, hyperglycemia, and hepatic inflammation, highlighting the physiological costs associated with its overactivation. Conversely, our study also reveals that hyperglycosylation of SIRT1 is associated with aging and high-fat–induced obesity. Thus, we establish that nutrient-dependent glycosylation of SIRT1 is essential to gate its functions and maintain physiological fitness.

Beet cyst nematodes depend on a set of secretory proteins (effectors) for the induction and maintenance of their syncytial feeding sites in plant roots. In order to understand the relationship between the beet cyst nematode H. schachtii and its host, identification of H. schachtii effectors is crucial and to this end, we sequenced a whole animal pre-infective J2-stage transcriptome in addition to pre- and post-infective J2 gland cell transcriptome using Next Generation Sequencing (NGS) and identified a subset of sequences representing putative effectors. Comparison between the transcriptome of H. schachtii and previously reported related cyst nematodes and root-knot nematodes revealed a subset of esophageal gland related sequences and putative effectors in common across the tested species. Structural and functional annotation of H. schachtii transcriptome led to the identification of nearly 200 putative effectors. Six putative effector expressions were quantified using qPCR and three of them were functionally analyzed using RNAi. Phenotyping of the RNAi nematodes indicated that all tested genes decrease the level of nematodes pathogenicity and/or the average female size, thereby regulating cyst nematode parasitism. These discoveries contribute to further understanding of the cyst nematode parasitism.

Significance Evolutionarily conserved insulin signaling is central to nutrient sensing, storage, and utilization across tissues. Dysfunctional insulin signaling is associated with metabolic disorders, cancer, and aging. Hence, the pathway components have emerged as key targets for pharmacological interventions in addition to insulin administration itself. Despite this, activation–inactivation dynamics of individual components, which exert regulatory control in a physiological context, is poorly understood. Now, with our systems-based approach, we reveal kinetic parameters, which define the flow of information through both metabolic and growth-factor arms and thus determine signaling architecture. We also provide a kinetic basis for 1) the advantage of pulsatile-fasted insulin signaling that enables fed-insulin response and 2) the detrimental impact of repeat fed-insulin inputs that causes resistance., Understanding kinetic control of biological processes is as important as identifying components that constitute pathways. Insulin signaling is central for almost all metazoans, and its perturbations are associated with various developmental disorders, metabolic diseases, and aging. While temporal phosphorylation changes and kinetic constants have provided some insights, constant or variable parameters that establish and maintain signal topology are poorly understood. Here, we report kinetic parameters that encode insulin concentration and nutrient-dependent flow of information using iterative experimental and mathematical simulation-based approaches. Our results illustrate how dynamics of distinct phosphorylation events collectively contribute to selective kinetic gating of signals and maximum connectivity of the signaling cascade under normo-insulinemic but not hyper-insulinemic states. In addition to identifying parameters that provide predictive value for maintaining the balance between metabolic and growth-factor arms, we posit a kinetic basis for the emergence of insulin resistance. Given that pulsatile insulin secretion during a fasted state precedes a fed response, our findings reveal rewiring of insulin signaling akin to memory and anticipation, which was hitherto unknown. Striking disparate temporal behavior of key phosphorylation events that destroy the topology under hyper-insulinemic states underscores the importance of unraveling regulatory components that act as bandwidth filters. In conclusion, besides providing fundamental insights, our study will help in identifying therapeutic strategies that conserve coupling between metabolic and growth-factor arms, which is lost in diseases and conditions of hyper-insulinemia.

In this work, we sequenced and annotated the genome ofStreptochaeta angustifolia, one of two genera in the grass subfamily Anomochlooideae, a lineage sister to all other grasses. The final assembly size is over 99% of the estimated genome size. We find good collinearity with the rice genome and have captured most of the gene space.Streptochaetais similar to other grasses in the structure of its fruit (a caryopsis or grain) but has peculiar flowers and inflorescences that are distinct from those in the outgroups and in other grasses. To provide tools for investigations of floral structure, we analyzed two large families of transcription factors, AP2-like and R2R3 MYBs, that are known to control floral and spikelet development in rice and maize among other grasses. Many of these are also regulated by small RNAs. Structure of the gene trees showed that the well documented whole genome duplication at the origin of the grasses (ρ) occurred before the divergence of the Anomochlooideae lineage from the lineage leading to the rest of the grasses (the spikelet clade) and thus that the common ancestor of all grasses probably had two copies of the developmental genes. However,Streptochaeta(and by inference other members of Anomochlooideae) has lost one copy of many genes. The peculiar floral morphology ofStreptochaetamay thus have derived from an ancestral plant that was morphologically similar to the spikelet-bearing grasses. We further identify 114 loci producing microRNAs and 89 loci generating phased, secondary siRNAs, classes of small RNAs known to be influential in transcriptional and post-transcriptional regulation of several plant functions.

What is the genetic architecture of local adaptation and what is the geographic scale that it operates over? We investigated patterns of local and convergent adaptation in five sympatric population pairs of traditionally cultivated maize and its wild relative teosinte (Zea mayssubsp.parviglumis). We found that signatures of local adaptation based on the inference of adaptive fixations and selective sweeps are frequently exclusive to individual populations, more so in teosinte compared to maize. However, for both maize and teosinte, selective sweeps are frequently shared by several populations, and often between the subspecies. We were further able to infer that selective sweeps were shared among populations most often via migration, though sharing via standing variation was also common. Our analyses suggest that teosinte has been a continued source of beneficial alleles for maize, post domestication, and that maize populations have facilitated adaptation in teosinte by moving beneficial alleles across the landscape. Taken together, out results suggest local adaptation in maize and teosinte has an intermediate geographic scale, one that is larger than individual populations, but smaller than the species range.

One model to study the emergence of the human trophoblast (TB) has been the exposure of pluripotent stem cells to bone morphogenetic protein 4 (BMP4) in presence of inhibitors of ACTIVIN/TGFB; A83–01 and FGF2; PD173074 (BAP), which generates a mixture of cytotrophoblast, syncytiotrophoblast, and cells with similarities to extravillous trophoblast. Here, H1 human embryonic stem cells were BAP-exposed under two O2 conditions (20% and 5%, respectively). At day 8, single nuclei RNA sequencing was used for transcriptomics analysis, thereby allowing profiling of fragile syncytial structures as well as the more resilient mononucleated cells. Following cluster analysis, two major groupings, one comprised of five (2,4,6,7,8) and the second of three (1,3,5) clusters were evident, all of which displayed recognized TB markers. Of these, two (2 and 3) weakly resembled extravillous trophoblast, two (5 and 6) strongly carried the hallmark transcripts of syncytiotrophoblast, while the remaining five were likely different kinds of mononucleated cytotrophoblast. We suggest that the two populations of nuclei within syncytiotrophoblast may have arisen from fusion events involving two distinct species of precursor cells. The number of differentially expressed genes between O2 conditions varied among the clusters, and the number of genes upregulated in cells cultured under 5% O2 was highest in syncytiotrophoblast cluster 6. In summary, the BAP model reveals an unexpectedly complex picture of trophoblast lineage emergence that will need to be resolved further in time-course studies.

The world crossed the halfway mark in urbanization in 2007. Various projections suggest that by 2030, this will increase to almost 60 percent, and to about 70 percent of the world’s population by 2050. With urban areas becoming the source of livelihoods for a majority of the population around the globe, a huge toll on natural resources is imminent. In this context, the planning of urban areas, megacities and their impact on the environment become extremely important issues of consideration. At the same time, the emerging implications for food security and nutrition are equally significant. This chapter summarizes the central questions addressed by the volume, and focuses on the contributions made. Researchers, policymakers and practitioners working on urbanization have the difficult task of understanding and responding to a complex and dynamic set of challenges in this crucial area for millions of people in the global south. Against this background, it is hoped that this volume will help to provide some useful reflections – if not complete answers – relevant to this challenging task in the global south and suggest ways for policy interventions and future research in this important area.

The stiff‐stalk heterotic group in Maize (Zea mays L.) is an important source of inbreds used in U.S. commercial hybrid production. Founder inbreds B14, B37, B73, and, to a lesser extent, B84, are found in the pedigrees of a majority of commercial seed parent inbred lines. We created high‐quality genome assemblies of B84 and four expired Plant Variety Protection (ex‐PVP) lines LH145 representing B14, NKH8431 of mixed descent, PHB47 representing B37, and PHJ40, which is a Pioneer Hi‐Bred International (PHI) early stiff‐stalk type. Sequence was generated using long‐read sequencing achieving highly contiguous assemblies of 2.13–2.18 Gbp with N50 scaffold lengths >200 Mbp. Inbred‐specific gene annotations were generated using a core five‐tissue gene expression atlas, whereas transposable element (TE) annotation was conducted using de novo and homology‐directed methodologies. Compared with the reference inbred B73, synteny analyses revealed extensive collinearity across the five stiff‐stalk genomes, although unique components of the maize pangenome were detected. Comparison of this set of stiff‐stalk inbreds with the original Iowa Stiff Stalk Synthetic breeding population revealed that these inbreds represent only a proportion of variation in the original stiff‐stalk pool and there are highly conserved haplotypes in released public and ex‐Plant Variety Protection inbreds. Despite the reduction in variation from the original stiff‐stalk population, substantial genetic and genomic variation was identified supporting the potential for continued breeding success in this pool. The assemblies described here represent stiff‐stalk inbreds that have historical and commercial relevance and provide further insight into the emerging maize pangenome.

In this work, we sequenced and annotated the genome ofStreptochaeta angustifolia, one of two genera in the grass subfamily Anomochlooideae, a lineage sister to all other grasses. The final assembly size is over 99% of the estimated genome size, capturing most of the gene space.Streptochaetais similar to other grasses in the structure of its fruit (a caryopsis or grain) but has peculiar flowers and inflorescences that are distinct from those in the outgroups and in other grasses. To provide tools for investigations of floral structure, we analyzed two large families of transcription factors, AP2-like and R2R3 MYBs, that are known to control floral and spikelet development in rice and maize among other grasses. Many of these are also regulated by small RNAs. Structure of the gene trees showed that the well documented whole genome duplication at the origin of the grasses (ρ) occurred before the divergence of the Anomochlooideae lineage from the lineage leading to the rest of the grasses (the spikelet clade) and thus that the common ancestor of all grasses probably had two copies of the developmental genes. However,Streptochaeta(and by inference other members of Anomochlooideae) has lost one copy of many genes. The peculiar floral morphology ofStreptochaetamay thus have derived from an ancestral plant that was morphologically similar to the spikelet-bearing grasses. We further identify 114 loci producing microRNAs and 89 loci generating phased, secondary siRNAs, classes of small RNAs known to be influential in transcriptional and post-transcriptional regulation of several plant functions.

Platelet-activating factor (PAF) is a potent inflammatory agonist. In Swiss albino mice, intraperitoneal injection of PAF causes sudden death with oxidative stress and disseminated intravascular coagulation (DIC), characterized by prolonged prothrombin time, thrombocytopenia, reduced fibrinogen content, and increased levels of fibrinogen degradation products. However, the underlying mechanism(s) is unknown. The PAF-R antagonist WEB-2086 protected mice against PAF-induced death by reducing DIC and oxidative stress. Accordingly, general antioxidants such as ascorbic acid, α-tocopherol, gallic acid, and N-acetylcysteine partially protected mice from PAF-induced death. N-acetylcysteine, a clinically used antioxidant, prevented death in 67% of mice, ameliorated DIC characteristics and histological alterations in the liver, and reduced oxidative stress. WEB-2086 suppressed H2O2-mediated oxidative stress in isolated mouse peritoneal macrophages, suggesting that PAF signaling may be a downstream effector of reactive oxygen species generation. PAF stimulated all three (ERK, JNK, and p38) of the MAP-kinases, which were also inhibited by N-acetylcysteine. Furthermore, a JNK inhibitor (SP600125) and ERK inhibitor (SCH772984) partially protected mice against PAF-induced death, whereas a p38 MAP-kinase inhibitor (SB203580) provided complete protection against DIC and death. In human platelets, which have the canonical PAF-R and functional MAP-kinases, JNK and p38 inhibitors abolished PAF-induced platelet aggregation, but the ERK inhibitor was ineffective. Our studies identify p38 MAP-kinase as a critical, but unrecognized component in PAF-induced mortality in mice. These findings suggest an alternative therapeutic strategy to address PAF-mediated pathogenicity, which plays a role in a broad range of inflammatory diseases.

Alpha-1-acid glycoprotein (AGP-1) is a major positive acute phase glycoprotein with unknown functions that likely plays a role in inflammation. We tested its involvement in a variety of inflammatory responses using human AGP-1 purified to apparent homogeneity and confirmed its identity by immunoblotting and mass spectrometry. AGP-1 alone upregulated MAPK signaling in murine peritoneal macrophages. However, when given in combination with TLR ligands, AGP-1 selectively augmented MAPK activation induced by ligands of TLR-2 (Braun lipoprotein) but not TLR-4 (lipopolysaccharide).In vivotreatment of AGP-1 in a murine model of sepsis with or without TLR-2 or TLR-4 ligands, selectively potentiated TLR-2-mediated mortality, but was without significant effect on TLR-4-mediated mortality. Furthermore,in vitro, AGP-1 selectively potentiated TLR-2 mediated adhesion of human primary immune cell, neutrophils. Hence, our studies highlight a new role for the acute phase protein AGP-1 in sepsis via its interaction with TLR-2 signaling mechanisms to selectively promote responsiveness to one of the two major gram-negative endotoxins, contributing to the complicated pathobiology of sepsis.

Global protein synthesis is emerging as an important player in the context of aging and age-related diseases. However, the intricate molecular networks that regulate protein synthesis are poorly understood. Here, we report that SIRT6, a nuclear-localized histone deacetylase represses global protein synthesis by transcriptionally regulating mTOR signalling via the transcription factor Sp1, independent of its deacetylase activity. Our results suggest that SIRT6 deficiency increases protein synthesis in mice. Further, multiple lines of in vitro evidence suggest that SIRT6 negatively regulates protein synthesis in a cell-autonomous fashion and independent of its catalytic activity. Mechanistically, SIRT6 binds to the zinc finger DNA binding domain of Sp1 and represses its activity. SIRT6 deficiency increased the occupancy of Sp1 at key mTOR signalling gene promoters resulting in enhanced expression of these genes and activation of the mTOR signalling pathway. Interestingly, inhibition of either mTOR or Sp1 abrogated the increased protein synthesis observed under SIRT6 deficient conditions. Moreover, pharmacological inhibition of mTOR restored cardiac function in muscle-specific SIRT6 knockout mice, which spontaneously develop cardiac hypertrophy. Overall, these findings have unravelled a new layer of regulation of global protein synthesis by SIRT6, which can be potentially targeted to combat aging-associated diseases like cardiac hypertrophy.

Heat stress resilience in maize hybrids is emerging as an important trait in germplasm targeted for cultivation in the post-rainy season spring in South Asia. One of the major challenges in targeted breeding for these agro-ecologies is the differential response of maize genotypes to heat stress across locations during the spring season. This study is targeted at identifying the major environmental variables that contributed to the genotype × environmental (GEI) yield variations observed among genotypes grown in response to heat stress. The trial dataset used for this study constitutes 46 trials × location combinations spread over a period of three years (2013-2015). Partial least square (PLS) regression analysis was implemented to decipher the important environmental variables contributing to the observed yield variation among maize trials planted during spring across locations of South Asia. The first two factors from the PLS study explained the 30 per cent yield variation across trials. The largest contributor of this variation was relative humidity (RH) and vapor pressure deficit (VPD) during flowering stage of the crop across the years.

International audience; Cyclin-dependent kinase 1 (CDK1) is essential for cell-cycle progression. While dependence of CDK activity on cyclin levels is well established, molecular mechanisms that regulate their binding are less understood. Here, we report for the first time that CDK1:cyclin-B binding is not default but rather determined by the evolutionarily conserved catalytic residue, lysine-33 in CDK1. We demonstrate that the charge state of this lysine allosterically remodels the CDK1:cyclin-B interface. Cell cycle-dependent acetylation of lysine-33 or its mutation to glutamine, which mimics acetylation, abrogates cyclin-B binding. Using biochemical approaches and atomistic molecular dynamics simulations, we have uncovered both short-range and long-range effects of perturbing the charged state of the catalytic lysine, which lead to inhibition of kinase activity. Specifically, although loss of the charge state of catalytic lysine did not impact ATP binding significantly, it altered its orientation in the active site. In addition, the catalytic lysine also acts as an intra-molecular electrostatic tether at the active site to orient structural elements interfacing with cyclin-B. Physiologically, opposing activities of SIRT1 and P300 regulate acetylation and thus control the charge state of lysine-33. Importantly, cells expressing acetylation mimic mutant of Cdc2/CDK1 in yeast are arrested in G2 and fail to divide, indicating the requirement of the deacetylated state of the catalytic lysine for cell division. Thus, by illustrating the molecular role of the catalytic lysine and cell cycle-dependent deacetylation as a determinant of CDK1:cyclin-B interaction, our results redefine the current model of CDK1 activation and cell-cycle progression.

Heat stress resilience has emerged as an important trait in maize hybrids targeted for post–monsoon spring cultivation in large parts of South Asia and many other parts of the tropics. Selection based on grain yield alone under heat stress is often misleading, and therefore an approach involving stress-adaptive secondary traits along with grain yield could help in the development of improved, stable heat stress tolerant cultivars. We attempted to identify reliable and effective secondary traits associated with heat stress tolerance in tropical maize and sources of heat stress tolerant germplasm. A panel of 99 elite maize inbred lines representing the wider genetic diversity of tropical maize and a set of 58 elite hybrids were phenotyped under natural heat stress and optimal temperature for grain yield and 15 secondary traits including 10 morpho-physiological traits and 5 yield attributes. Evaluation under natural heat stress was done during the spring season by adjusting the planting date so that the complete reproductive stage (from tassel emergence to late grain filling) was exposed to heat stress. The optimal temperature trial was planted during the monsoon season with no exposure to heat stress at any crop stage. Heat stress significantly affected most of the observed traits. Among the traits studied two yield attributing traits, i.e.- ears per plant (EPP) and kernel per row (KPR), and three morpho-physiological traits, i.e.- chlorophyll content (CC), leaf firing (LF) and tassel blast (TB) were found to be the key secondary traits associated with grain yield under heat stress. In addition, low anthesis-silking internal (ASI) is an important trait that needs to be added in the index selection for heat stress tolerance. The study identified nine promising heat stress tolerant maize inbred lines with desirable secondary traits and grain yield under severe heat stress, which could be used as source germplasm in heat stress tolerance maize breeding program.

The present study analyzed the effects of zinc oxide nanoparticles (ZnO-NPs) and zinc sulfate (ZnSO4) in the testis of six-month-old common carp Cyprinus carpio exposed to three different doses, viz., 10, 50, and 100 μg/L for 21 days. Characterization of ZnO-NPs was done after sonication, the size and shape of ZnO-NPs were determined as ∼20-30 nm spherical structure measured zeta potential of +26.0 mV. After treatment, determination of zinc (Zn) concentration in the testes revealed desired impact of the exposure. Expression of several transcription factors and few steroidogenic enzyme genes in the treated testis showed significant downregulation than the control. Measurement of oxidative stress-related enzymes such as catalase, superoxide dismutase, and glutathione-S-transferase revealed substantial elevation in the testis of treated groups when compared to control. Histological analysis of testis exhibited dose-related response, defective lumen, and slow progression of spermatogenesis. Exposure of both the forms of Zn on TM3 Leydig cell culture displayed loss of adhesion, clumping with decreased viability, and a significant increase in the apoptotic cells. In addition, comet and intracellular reactive oxygen species (ROS) assays authenticated DNA damage upon treatment with a significant increase in ROS. Histological analysis after treatment withdrawal showed revival of testis in carp to rescue the effect. Thus, the present report highlights the adverse effect of Zn on the testis function in common carp as well as evident drastically toxic in in vitro cultures.

Abalone are one of the few marine taxa where aquaculture production dominates the global market as a result of increasing demand and declining natural stocks from overexploitation and disease. To better understand abalone biology, aid in conservation efforts for endangered abalone species, and gain insight into sustainable aquaculture, we created a draft genome of the red abalone (Haliotis rufescens). The approach to this genome draft included initial assembly using raw Illumina and PacBio sequencing data with MaSuRCA, before scaffolding using sequencing data generated from Chicago library preparations with HiRise2. This assembly approach resulted in 8,371 scaffolds and total length of 1.498 Gb; the N50 was 1.895 Mb, and the longest scaffold was 13.2 Mb. Gene models were predicted, using MAKER2, from RNA-Seq data and all related expressed sequence tags and proteins from NCBI; this resulted in 57,785 genes with an average length of 8,255 bp. In addition, single nucleotide polymorphisms were called on Illumina short-sequencing reads from five other eastern Pacific abalone species: the green (H. fulgens), pink (H. corrugata), pinto (H. kamtschatkana), black (H. cracherodii), and white (H. sorenseni) abalone. Phylogenetic relationships largely follow patterns detected by previous studies based on 1,784,991 high-quality single nucleotide polymorphisms. Among the six abalone species examined, the endangered white abalone appears to harbor the lowest levels of heterozygosity. This draft genome assembly and the sequencing data provide a foundation for genome-enabled aquaculture improvement for red abalone, and for genome-guided conservation efforts for the other five species and, in particular, for the endangered white and black abalone.

Background & objectives: Infection from fluoroquinolone-resistant extra-intestinal Escherichia coli is a global concern. In this study, isolation and characterization of fluoroquinolone-resistant extra-intestinal E. coli isolates obtained from hospital samples were undertaken to detect plasmid-mediated quinolone resistance (PMQR) genes. Methods: Forty three isolates of E. coli obtained from patients with extra-intestinal infections were subjected to antibiogram to detect fluoroquinolone resistance. The mechanism of fluoroquinolone resistance was determined by the detection of PMQR genes and mutations in quinolone resistance determining region (QRDR). Results: Of the 43 isolates, 36 were resistant to nalidixic acid (83.72%) and 28 to ciprofloxacin (65.11%). Eight E. coli isolates showed total resistance to both the antimicrobials without any minimum inhibitory concentration. The detection of PMQR genes with qnr primers showed the presence of qnrA in two, qnrB in six and qnrS in 21 isolates. The gene coding for quinolone efflux pump (qepA) was not detected in any of the isolates tested. The presence of some unexpressed PMQR genes in fluoroquinolone sensitive isolates was also observed. Interpretation & conclusions: The detection of silent PMQR genes as observed in the present study presents a risk of the transfer of the silent resistance genes to other microorganisms if present in conjugative plasmids, thus posing a therapeutic challenge to the physicians. Hence, frequent monitoring is to be done for all resistance determinants.

Objectives This study sought to explore the spectrum of cardiac abnormalities in student athletes who returned to university campus in July 2020 with uncomplicated coronavirus disease 2019 (COVID-19). Background There is limited information on cardiovascular involvement in young individuals with mild or asymptomatic COVID-19. Methods Screening echocardiograms were performed in 54 consecutive student athletes (mean age 19 years; 85% male) who had positive results of reverse transcription polymerase chain reaction nasal swab testing of the upper respiratory tract or immunoglobulin G antibodies against severe acute respiratory syndrome coronavirus type 2. Sequential cardiac magnetic resonance imaging was performed in 48 (89%) subjects. Results A total of 16 (30%) athletes were asymptomatic, whereas 36 (66%) and 2 (4%) athletes reported mild and moderate COVID-19 related symptoms, respectively. For the 48 athletes completing both imaging studies, abnormal findings were identified in 27 (56.3%) individuals. This included 19 (39.5%) athletes with pericardial late enhancements with associated pericardial effusion. Of the individuals with pericardial enhancements, 6 (12.5%) had reduced global longitudinal strain and/or an increased native T1. One patient showed myocardial enhancement, and reduced left ventricular ejection fraction or reduced global longitudinal strain with or without increased native T1 values was also identified in an additional 7 (14.6%) individuals. Native T2 findings were normal in all subjects, and no specific imaging features of myocardial inflammation were identified. Hierarchical clustering of left ventricular regional strain identified 3 unique myopericardial phenotypes that showed significant association with the cardiac magnetic resonance findings (p = 0.03). Conclusions More than 1 in 3 previously healthy college athletes recovering from COVID-19 infection showed imaging features of a resolving pericardial inflammation. Although subtle changes in myocardial structure and function were identified, no athlete showed specific imaging features to suggest an ongoing myocarditis. Further studies are needed to understand the clinical implications and long-term evolution of these abnormalities in uncomplicated COVID-19., Central Illustration

Cholesterol derived from the host milieu forms a critical factor for mycobacterial pathogenesis. However, the molecular circuitry co-opted by Mycobacterium tuberculosis (Mtb) to accumulate cholesterol in host cells remains obscure. Here, we report that a functional amalgamation of WNT-responsive histone modifiers G9a (H3K9 methyltransferase) and Sirt6 (H3K9 deacetylase) orchestrate cholesterol build-up in in-vitro and in-vivo models of Mtb infection. Mechanistically, G9a, along with SREBP2, drives the expression of cholesterol biosynthesis and uptake genes; while Sirt6 represses the genes involved in cholesterol efflux. The accumulated cholesterol promotes the expression of antioxidant genes leading to reduced oxidative stress, thereby supporting Mtb survival. In corroboration, loss-of-function of G9a in vitro and in vivo by pharmacological inhibition; or utilization of BMDMs derived from Sirt6 KO mice or in vivo infection in Sirt6 heterozygous mice; hampers host cholesterol accumulation and restricts Mtb burden. These findings shed light on the novel roles of G9a and Sirt6 during Mtb infection and highlight the previously unknown contribution of host cholesterol in potentiating anti-oxidative responses for aiding Mtb survival.

While it is well known that the genome can affect social behavior, recent models posit that social lifestyles can, in turn, influence genome evolution. Here, we perform the most phylogenetically comprehensive comparative analysis of 16 bee genomes to date: incorporating two published and four new carpenter bee genomes (Apidae: Xylocopinae) for a first-ever genomic comparison with a monophyletic clade containing solitary through advanced eusocial taxa. We find that eusocial lineages have undergone more gene family expansions, feature more signatures of positive selection, and have higher counts of taxonomically restricted genes than solitary and weakly social lineages. Transcriptomic data reveal that caste-affiliated genes are deeply-conserved; gene regulatory and functional elements are more closely tied to social phenotype than phylogenetic lineage; and regulatory complexity increases steadily with social complexity. Overall, our study provides robust empirical evidence that social evolution can act as a major and surprisingly consistent driver of macroevolutionary genomic change., Shell et al. compare the molecular evolution of bee genomes across species with varying social behaviours. Their findings indicate that eusocial lineages tend to have more gene family expansion than weakly social lineages. Transcriptomic data suggests that social phenotype is a consistent driver of macroevolutionary genomic change.

We report de novo genome assemblies, transcriptomes, annotations, and methylomes for the 26 inbreds that serve as the founders for the maize nested association mapping population. The data indicate that the number of pan-genes exceeds 103,000 and that the ancient tetraploid character of maize continues to degrade by fractionation to the present day. Excellent contiguity over repeat arrays and complete annotation of centromeres further reveal the locations and internal structures of major cytological landmarks. We show that combining structural variation with SNPs can improve the power of quantitative mapping studies. Finally, we document variation at the level of DNA methylation, and demonstrate that unmethylated regions are enriched for cis-regulatory elements that overlap QTL and contribute to changes in gene expression.One sentence summaryA multi-genome analysis of maize reveals previously unknown variation in gene content, genome structure, and methylation.

An a-maize-ing set of genomes Maize is an important crop cultivated worldwide. As maize spread across the world, selection for local environments resulted in variation, but the impact on differences between the genome has not been quantified. By producing high-quality genomic sequences of the 26 lines used in the maize nested association mapping panel, Hufford et al . map important traits and demonstrate the diversity of maize. Examining RNA and methylation of genes across accessions, the authors identified a core set of maize genes. Beyond this core set, comparative analysis across lines identified high levels of variation in the total set of genes, the maize pan-genome. The value of this resource was further exemplified by mapping quantitative traits of interest, including those related to pathogen resistance. —LMZ

The utility of surface layer proteins (Hpi and SlpA) of the radiation resistant bacterium, Deinococcus radiodurans, was investigated for surface display and bioremediation of cadmium and uranium. The smtA gene, from Synechococcus elongatus (encoding the metal binding metallothionein protein), was cloned and over-expressed in D. radiodurans, either as such or as a chimeric gene fused with hpi ORF (Hpi-SmtA), or fused to the nucleotide sequence encoding the SLH domain of the SlpA protein (SLH-SmtA). The expressed fusion proteins localized to the deinococcal cell surface, while the SmtA protein localized to the cytoplasm. Recombinant cells surface-displaying the SLH-SmtA or Hpi-SmtA fusion proteins respectively removed 1.5–3 times more cadmium than those expressing only cytosolic SmtA. The deinococcal Hpi protein layer per se also contributed to U binding, by conferring substantial negative charge to deinococcal cell surface. The ORF of an acid phosphatase, PhoN was fused with the hpi or SLH domain DNA sequence and purified. Isolated Hpi-PhoN and SLH-PhoN, immobilized on deinococcal peptidoglycan showed efficient uranium precipitation (446 and 160 mg U/g biomass used respectively). The study demonstrates effective exploitation of the deinococcal S layer protein components for (a) cell surface-based sequestration of cadmium, and (b) cell-free preparations for uranium remediation.

Spring maize area has emerged as a niche market in South Asia. Production of maize during this post-rainy season is often challenged due to heat stress. Therefore, incorporating heat stress resilience is an important trait for incorporation in maize hybrids selected for deployment in this season. However, due to the significant genotype &times, environment interaction (GEI) effects under heat stress, the major challenge lies in identifying maize genotypes with improved stable performance across locations and years. In the present study, we attempted to identify the key weather variables responsible for significant GEI effects, and identify maize hybrids with stable performance under heat stress across locations/years. The study details the evaluation of a set of prereleased advanced maize hybrids across heat stress vulnerable locations in South Asia during the spring seasons of 2015, 2016 and 2017. Using factorial regression, we identified that relative humidity (RH) and vapor pressure deficit (VPD) as the two most important environmental covariates contributing to the large GEI observed on grain yield under heat stress. The study also identified reproductive stage, starting from tassel emergence to early grain-filling stage, as the most critical crop stage highly susceptible to heat stress. Across-site/year evaluation resulted in identification of six high yielding heat stress resilient hybrids.

Background Heterodera glycines, commonly referred to as the soybean cyst nematode (SCN), is an obligatory and sedentary plant parasite that causes over a billion-dollar yield loss to soybean production annually. Although there are genetic determinants that render soybean plants resistant to certain nematode genotypes, resistant soybean cultivars are increasingly ineffective because their multi-year usage has selected for virulent H. glycines populations. The parasitic success of H. glycines relies on the comprehensive re-engineering of an infection site into a syncytium, as well as the long-term suppression of host defense to ensure syncytial viability. At the forefront of these complex molecular interactions are effectors, the proteins secreted by H. glycines into host root tissues. The mechanisms of effector acquisition, diversification, and selection need to be understood before effective control strategies can be developed, but the lack of an annotated genome has been a major roadblock. Results Here, we use PacBio long-read technology to assemble a H. glycines genome of 738 contigs into 123 Mb with annotations for 29,769 genes. The genome contains significant numbers of repeats (34%), tandem duplicates (18.7 Mb), and horizontal gene transfer events (151 genes). A large number of putative effectors (431 genes) were identified in the genome, many of which were found in transposons. Conclusions This advance provides a glimpse into the host and parasite interplay by revealing a diversity of mechanisms that give rise to virulence genes in the soybean cyst nematode, including: tandem duplications containing over a fifth of the total gene count, virulence genes hitchhiking in transposons, and 107 horizontal gene transfers not reported in other plant parasitic nematodes thus far. Through extensive characterization of the H. glycines genome, we provide new insights into H. glycines biology and shed light onto the mystery underlying complex host-parasite interactions. This genome sequence is an important prerequisite to enable work towards generating new resistance or control measures against H. glycines. Electronic supplementary material The online version of this article (10.1186/s12864-019-5485-8) contains supplementary material, which is available to authorized users.

Alpha-1-acid glycoprotein (AGP-1) is a positive acute phase glycoprotein with uncertain functions. Serum AGP-1 (sAGP-1) is primarily derived from hepatocytes and circulates as 12–20 different glycoforms. We isolated a glycoform secreted from platelet-activating factor (PAF)-stimulated human neutrophils (nAGP-1). Its peptide sequence was identical to hepatocyte-derived sAGP-1, but nAGP-1 differed from sAGP-1 in its chromatographic behavior, electrophoretic mobility, and pattern of glycosylation. The function of these 2 glycoforms also differed. sAGP-1 activated neutrophil adhesion, migration, and neutrophil extracellular traps (NETosis) involving myeloperoxidase, peptidylarginine deiminase 4, and phosphorylation of ERK in a dose-dependent fashion, whereas nAGP-1 was ineffective as an agonist for these events. Furthermore, sAGP-1, but not nAGP-1, inhibited LPS-stimulated NETosis. Interestingly, nAGP-1 inhibited sAGP-1-stimulated neutrophil NETosis. The discordant effect of the differentially glycosylated AGP-1 glycoforms was also observed in platelets where neither of the AGP-1 glycoforms alone stimulated aggregation of washed human platelets, but sAGP-1, and not nAGP-1, inhibited aggregation induced by PAF or ADP, but not by thrombin. These functional effects of sAGP-1 correlated with intracellular cAMP accumulation and phosphorylation of the protein kinase A substrate vasodilator-stimulated phosphoprotein and reduction of Akt, ERK, and p38 phosphorylation. Thus, the sAGP-1 glycoform limits platelet reactivity, whereas nAGP-1 glycoform also limits proinflammatory actions of sAGP-1. These studies identify new functions for this acute phase glycoprotein and demonstrate that the glycosylation of AGP-1 controls its effects on 2 critical cells of acute inflammation.

Understanding kinetic control of biological processes is as important as identifying components that constitute pathways. Insulin signaling (IS) is central for almost all metazoans and its perturbations are associated with various diseases and aging. While temporal phosphorylation changes and kinetic constants have provided some insights, constant or variable parameters that establish and maintain signal topology are poorly understood. Our iterative experimental and mathematical simulation-based approaches reveal novel kinetic parameters of IS that encode concentration and nutrient dependent information. Further, we find that pulsatile fasting insulin rewires IS akin to memory and in anticipation of a fed response. Importantly, selective kinetic gating of signals and maximum connectivity, between metabolic and growth-factor arms under normo-insulinemic states, maintains network topology. In addition to unraveling kinetic constraints that determine cascade architecture, our findings will help in identifying novel therapeutic strategies that conserve coupling between metabolic and growth-factor arms, which is lost in diseases and conditions of hyperinsulinemia.

Wnt signaling is conserved among all species and plays a significant role in various cellular processes including reproduction. The present study identified significant involvement of wnt4a, wnt5b, and wnt8a signaling in the testicular growth of common carp,Cyprinus carpio. Predominant expression of wnt4a, wnt5b, and wnt8a was found in the gonads and Wnt4a was localized in spermatocytes and interstitial cells. Ontogeny and testicular phase-wise analysis signified the importance of wnt isofoms analyzed in this study. Specific pathway activation of Wnt signaling revealed that Wnt4a and Wnt5b act through non-canonical while Wnt8a prefers the canonical pathway. The Wnt signaling regulates several steroidogenic enzyme and testis-related genes which was confirmed by the Wnt blockade experiments. Incidentally, zinc (Zn) is an essential trace element involved in the progression of spermatogenesis in teleosts. In adult male carp, a single administration of Zn at different doses elevated the expression of Wnt and Zn transporter genes and a single-dose (30 μg/g body weight [BW]) of Zn treatment elevated steroidogenic enzyme and testis-related genes which coincided with elevated androgens. Conversely, single-dose administration of Zn chelator to the Zn administered (30 μg/g BW) fish reversed the effects emphasizing a prominent role of Zn in the testicular function perhaps through Wnt signaling. Similar effects were observed in the in vitro experiments using the Zn chelator. Bioaccumulation of Zn and histological analysis revealed the importance of Zn in progression of spermatogenesis and sperm motility. Various assays related to cell viability and proliferation exhibited the role of Zn in promoting spermatogenic cell progression. Flow cytometric analysis confirmed Zn-induced elevation of Wnt and Zn transporter genes in germ and supporting cells. Furthermore, the effects of Zn are dose-related in carp. Taken together, it seems wnt4a, wnt5b, and wnt8a play an important role in testis and exposure of Wnt inhibitor, canonical as well as non-canonical activators, and Zn confirmed that Zn regulates Wnt signaling vis-a-vis promoting spermatogenesis in the common carp.

Creating gapless telomere-to-telomere assemblies of complex genomes is one of the ultimate challenges in genomics. We use two independent assemblies and an optical map-based merging pipeline to produce a maize genome (B73-Ab10) composed of 63 contigs and a contig N50 of 162 Mb. This genome includes gapless assemblies of chromosome 3 (236 Mb) and chromosome 9 (162 Mb), and 53 Mb of the Ab10 meiotic drive haplotype. The data also reveal the internal structure of seven centromeres and five heterochromatic knobs, showing that the major tandem repeat arrays (CentC, knob180, and TR-1) are discontinuous and frequently interspersed with retroelements.

Cas9 is an RNA-guided endonuclease in the bacterial CRISPR-Cas immune system and a popular tool for genome editing. The most commonly used Cas9 variant, Streptococcus pyogenes Cas9 (SpCas9), is relatively non-specific and prone to off-target genome editing. Other Cas9 orthologs and engineered variants of SpCas9 have been reported to be more specific than wild-type (WT) SpCas9. However, systematic comparisons of the cleavage activities of these Cas9 variants have not been reported. In this study, we employed our high-throughput in vitro cleavage assay to compare cleavage activities and specificities of two natural Cas9 variants (SpCas9 and Staphylococcus aureus Cas9) and three engineered SpCas9 variants (SpCas9 HF1, HypaCas9, and HiFi Cas9). We observed that all Cas9s tested were able to cleave target sequences with up to five mismatches. However, the rate of cleavage of both on-target and off-target sequences varied based on the target sequence and Cas9 variant. For targets with multiple mismatches, SaCas9 and engineered SpCas9 variants are more prone to nicking, while WT SpCas9 creates double-strand breaks (DSB). These differences in cleavage rates and DSB formation may account for the varied specificities observed in genome editing studies. Our analysis reveals mismatch position-dependent, off-target nicking activity of Cas9 variants which have been underreported in previous in vivo studies.

Cas12a (Cpf1) is an RNA-guided endonuclease in the bacterial type V-A CRISPR-Cas anti-phage immune system that can be repurposed for genome editing. Cas12a can bind and cut dsDNA targets with high specificity in vivo, making it an ideal candidate for expanding the arsenal of enzymes used in precise genome editing. However, this reported high specificity contradicts Cas12a's natural role as an immune effector against rapidly evolving phages. Here, we employed high-throughput in vitro cleavage assays to determine and compare the native cleavage specificities and activities of three different natural Cas12a orthologs (FnCas12a, LbCas12a, and AsCas12a). Surprisingly, we observed pervasive sequence-specific nicking of randomized target libraries, with strong nicking of DNA sequences containing up to four mismatches in the Cas12a-targeted DNA-RNA hybrid sequences. We also found that these nicking and cleavage activities depend on mismatch type and position and vary with Cas12a ortholog and CRISPR RNA sequence. Our analysis further revealed robust nonspecific nicking of dsDNA when Cas12a is activated by binding to a target DNA. Together, our findings reveal that Cas12a has multiple nicking activities against dsDNA substrates and that these activities vary among different Cas12a orthologs.

Diabetic foot ulcer (DFU) is a major complication of diabetes with high morbidity and mortality rates. The pathogenesis of DFUs is governed by a complex milieu of environmental and host factors. The empirical treatment is initially based on wound severity since culturing and profiling the antibiotic sensitivity of wound-associated microbes is time-consuming. Hence, a thorough and rapid analysis of the microbial landscape is a major requirement toward devising evidence-based interventions. Toward this, 122 wound (100 diabetic and 22 nondiabetic) samples were sampled for their bacterial community structure using both culture-based and next-generation 16S rRNA-based metagenomics approach. Both the approaches showed that the Gram-negative microbes were more abundant in the wound microbiome. The core microbiome consisted of bacterial genera, including Alcaligenes, Pseudomonas, Burkholderia, and Corynebacterium in decreasing order of average relative abundance. Despite the heterogenous nature and extensive sharing of microbes, an inherent community structure was apparent, as revealed by a cluster analysis based on Euclidean distances. Facultative anaerobes (26.5%) were predominant in Wagner grade 5, while strict anaerobes were abundant in Wagner grade 1 (26%). A nonmetric dimensional scaling analysis could not clearly discriminate samples based on HbA1c levels. Sequencing approach revealed the presence of major culturable species even in samples with no bacterial growth in culture-based approach. Our study indicates that (i) the composition of core microbial community varies with wound severity, (ii) polymicrobial species distribution is individual specific, and (iii) antibiotic susceptibility varies with individuals. Our study suggests the need to evolve better-personalized care for better wound management therapies. IMPORTANCE Chronic nonhealing diabetic foot ulcers (DFUs) are a serious complication of diabetes and are further exacerbated by bacterial colonization. The microbial burden in the wound of each individual displays diverse morphological and physiological characteristics with unique patterns of host-pathogen interactions, antibiotic resistance, and virulence. Treatment involves empirical decisions until definitive results on the causative wound pathogens and their antibiotic susceptibility profiles are available. Hence, there is a need for rapid and accurate detection of these polymicrobial communities for effective wound management. Deciphering microbial communities will aid clinicians to tailor their treatment specifically to the microbes prevalent in the DFU at the time of assessment. This may reduce DFUs associated morbidity and mortality while impeding the rise of multidrug-resistant microbes.

BackgroundGenome assemblies are foundational for understanding the biology of a species. They provide a physical framework for mapping additional sequences, thereby enabling characterization of, for example, genomic diversity and differences in gene expression across individuals and tissue types. Quality metrics for genome assemblies gauge both the completeness and contiguity of an assembly and help provide confidence in downstream biological insights. To compare quality across multiple assemblies, a set of common metrics are typically calculated and then compared to one or more gold standard reference genomes. While several tools exist for calculating individual metrics, applications providing comprehensive evaluations of multiple assembly features are, perhaps surprisingly, lacking. Here, we describe a new toolkit that integrates multiple metrics to characterize both assembly and gene annotation quality in a way that enables comparison across multiple assemblies and assembly types.FindingsOur application, named GenomeQC, is an easy-to-use and interactive web framework that integrates various quantitative measures to characterize genome assemblies and annotations. GenomeQC provides researchers with a comprehensive summary of these statistics and allows for benchmarking against gold standard reference assemblies.ConclusionsThe GenomeQC web application is implemented in R/Shiny version 1.5.9 and Python 3.6 and is freely available at https://genomeqc.maizegdb.org/ under the GPL license.All source code and a containerized version of the GenomeQC pipeline is available in the GitHub repository https://github.com/HuffordLab/GenomeQC.

Alpha-1-acid glycoprotein (AGP-1) is a positive acute phase glycoprotein with uncertain functions. Serum AGP-1 (sAGP-1) is primarily derived from hepatocytes and circulates as 12 to 20 different glycoforms. We isolated a glycoform secreted from stimulated human neutrophils (nAGP-1). Its peptide sequence was identical to hepatocyte-derived sAGP-1, but nAGP-1 differed from sAGP-1 in its chromatographic behaviour, electrophoretic mobility, and glycosylation. The function of these two glycoforms also differed. sAGP-1 activated neutrophil adhesion, migration and NETosis in a dose-dependent fashion, while nAGP-1 was ineffective as an agonist for these events. Furthermore, sAGP-1, but not nAGP-1, inhibited LPS-stimulated NETosis. However, nAGP-1 inhibited sAGP-1-stimulated neutrophil NETosis. The discordant effect of the differentially glycosylated AGP-1 glycoforms was also observed in platelets where neither of the AGP-1 glycoforms alone stimulated aggregation of washed human platelets, but sAGP-1, and not nAGP-1, inhibited aggregation induced by Platelet-activating Factor (PAF) or ADP, but not by thrombin. These functional effects of sAGP-1 correlated with intracellular cAMP accumulation and were accompanied by phosphorylation of the PKA substrate Vasodialator stimulated phosphoprotein (VASP) and reduction of Akt, ERK, and p38 phosphorylation. Thus, the sAGP-1 glycoform limits platelet reactivity while nAGP-1 glycoform also limits pro-inflammatory actions of sAGP-1. These studies identify new functions for this acute phase glycoprotein and demonstrate that the glycosylation of AGP-1 controls its effects on two critical cells of acute inflammation.