Cryo-EM structures of a human ABCG2 mutant trapped in ATP-bound and substrate-bound states.

ABCG2 is a transporter protein of the ATP-binding-cassette (ABC) family that is expressed in the plasma membrane in cells of various tissues and tissue barriers, including the blood-brain, blood-testis and maternal-fetal barriers1-4. Powered by ATP, it translocates endogenous substrates, affects the pharmacokinetics of many drugs and protects against a wide array of xenobiotics, including anti-cancer drugs5-12. Previous studies have revealed the architecture of ABCG2 and the structural basis of its inhibition by small molecules and antibodies13,14. However, the mechanisms of substrate recognition and ATP-driven transport are unknown. Here we present high-resolution cryo-electron microscopy (cryo-EM) structures of human ABCG2 in a substrate-bound pre-translocation state and an ATP-bound post-translocation state. For both structures, we used a mutant containing a glutamine replacing the catalytic glutamate (ABCG2EQ), which resulted in reduced ATPase and transport rates and facilitated conformational trapping for structural studies. In the substrate-bound state, a single molecule of estrone-3-sulfate (E1S) is bound in a central, hydrophobic and cytoplasm-facing cavity about halfway across the membrane. Only one molecule of E1S can bind in the observed binding mode. In the ATP-bound state, the substrate-binding cavity has collapsed while an external cavity has opened to the extracellular side of the membrane. The ATP-induced conformational changes include rigid-body shifts of the transmembrane domains, pivoting of the nucleotide-binding domains (NBDs), and a change in the relative orientation of the NBD subdomains. Mutagenesis and in vitro characterization of transport and ATPase activities demonstrate the roles of specific residues in substrate recognition, including a leucine residue that forms a 'plug' between the two cavities. Our results show how ABCG2 harnesses the energy of ATP binding to extrude E1S and other substrates, and suggest that the size and binding affinity of compounds are important for distinguishing substrates from inhibitors.

Standards for fecal microbiota transplant: Tools and therapeutic advances.

Fecal microbiota transplantation (FMT) has been demonstrated to be efficacious in preventing recurrent Clostridioides difficile (C. difficile) infections, and is being investigated for treatment of several other diseases including inflammatory bowel disease, cancer, obesity, liver disease, and diabetes. To speed up the translation of FMT into clinical practice as a safe and standardized therapeutic intervention, additional evidence-based technical and regulatory guidance is needed. To this end in May of 2022, the International Alliance for Biological Standardization (IABS) and the BIOASTER Microbiology Technology Institute hosted a second webinar to discuss key issues still impeding the advancement and standardization of FMT. The goal of this two-day webinar was to provide a forum for scientific experts to share and discuss data and key challenges with one another. Discussion included a focus on the evaluation of safety, efficacy, clinical trial design, reproducibility and accuracy in obtained microbiome measurements and data reporting, and the potential for standardization across these areas. It also focused on increasing the application potential and visibility of FMT beyond treating C. difficile infections.

Legacy genetics of Arachis cardenasii in the peanut crop shows the profound benefits of international seed exchange.

The narrow genetics of most crops is a fundamental vulnerability to food security. This makes wild crop relatives a strategic resource of genetic diversity that can be used for crop improvement and adaptation to new agricultural challenges. Here, we uncover the contribution of one wild species accession, Arachis cardenasii GKP 10017, to the peanut crop (Arachis hypogaea) that was initiated by complex hybridizations in the 1960s and propagated by international seed exchange. However, until this study, the global scale of the dispersal of genetic contributions from this wild accession had been obscured by the multiple germplasm transfers, breeding cycles, and unrecorded genetic mixing between lineages that had occurred over the years. By genetic analysis and pedigree research, we identified A. cardenasii-enhanced, disease-resistant cultivars in Africa, Asia, Oceania, and the Americas. These cultivars provide widespread improved food security and environmental and economic benefits. This study emphasizes the importance of wild species and collaborative networks of international expertise for crop improvement. However, it also highlights the consequences of the implementation of a patchwork of restrictive national laws and sea changes in attitudes regarding germplasm that followed in the wake of the Convention on Biological Diversity. Today, the botanical collections and multiple seed exchanges which enable benefits such as those revealed by this study are drastically reduced. The research reported here underscores the vital importance of ready access to germplasm in ensuring long-term world food security.

Living Donor Kidney Transplant in Recipients With Glomerulonephritis: Donor Recipient Biologic Relationship and Allograft Outcomes.

Using the Scientific Registry of Transplant Recipients, we examined the association between donor-recipient biologic relationship and long-term recipient and allograft survival among glomerulonephritis (GN) patients. Four GN types were studied: membranous nephropathy, IgA, lupus-associated nephritis, and focal segmental glomerulosclerosis (FSGS). We identified all adult primary living-donor recipients between 2000 and 2018 (n = 19,668): related (n = 10,437); unrelated (n = 9,231). Kaplan-Meier curves were generated for the recipient, death-censored graft survival and death with functioning graft through ten years post-transplant. Multivariable Cox proportional hazard models were used to examine the association between the donor-recipient relationship and outcomes of interest. There was an increased risk for acute rejection by 12 months post-transplant among the unrelated compared to the related group in IgA (10.1% vs. 6.5%, p<0.001), FSGS (12.1% vs. 10%, p-0.016), and lupus nephritis (11.8% vs. 9.2%; p-0.049). The biological donor-recipient relationship was not associated with a worse recipient or graft survival or death with functioning graft in the multivariable models. These findings are consistent with the known benefits of living-related-donor kidney transplants and counter the reports of the potential adverse impact of the donor-recipient biologic relationship on allograft outcomes.

TAR30, a homolog of the canonical plant TTTAGGG telomeric repeat, is enriched in the proximal chromosome regions of peanut (Arachis hypogaea L.).

Telomeres are the physical ends of eukaryotic linear chromosomes that play critical roles in cell division, chromosome maintenance, and genome stability. In many plants, telomeres are comprised of TTTAGGG tandem repeat that is widely found in plants. We refer to this repeat as canonical plant telomeric repeat (CPTR). Peanut (Arachis hypogaea L.) is a spontaneously formed allotetraploid and an important food and oil crop worldwide. In this study, we analyzed the peanut genome sequences and identified a new type of tandem repeat with 10-bp basic motif TTTT(C/T)TAGGG named TAndem Repeat (TAR) 30. TAR30 showed significant sequence identity to TTTAGGG repeat in 112 plant genomes suggesting that TAR30 is a homolog of CPTR. It also is nearly identical to the telomeric tandem repeat in Cestrum elegans. Fluorescence in situ hybridization (FISH) analysis revealed interstitial locations of TAR30 in peanut chromosomes but we did not detect visible signals in the terminal ends of chromosomes as expected for telomeric repeats. Interestingly, different TAR30 hybridization patterns were found between the newly induced allotetraploid ValSten and its diploid wild progenitors. The canonical telomeric repeat TTTAGGG is also present in the peanut genomes and some of these repeats are closely adjacent to TAR30 from both cultivated peanut and its wild relatives. Overall, our work identifies a new homolog of CPTR and reveals the unique distributions of TAR30 in cultivated peanuts and wild species. Our results provide new insights into the evolution of tandem repeats during peanut polyploidization and domestication.

Recipient and kidney graft outcomes of deceased donors with human immunodeficiency virus in the United States.

BACKGROUND: The HIV Organ Policy Equity (HOPE) act afforded transplantation of organs from donors who have HIV. Herein we compared the long-term outcomes of recipients with HIV by donor HIV testing status. METHODS: Using the Scientific Registry of Transplant Recipients, we identified all primary adult kidney transplant recipients who were HIV-positive between 1/1/16-12/31/21. Recipients were grouped into three cohorts according to the donor HIV status based on antibody (Ab) and nucleic acid testing (NAT): Donor Ab-/NAT- (n = 810), Donor Ab+ /NAT- (n = 98), and Donor Ab+/NAT+ (n = 90). We compared recipient and death-censored graft survival (DCGS) by donor HIV testing status using Kaplan-Meier curves and Cox proportional hazards regression, censored at 3 years posttransplant. Secondary outcomes were delayed graft function (DGF) and the following 1-year outcomes: acute rejection, re-hospitalization, and serum creatinine. RESULTS: In Kaplan-Meier analyses, patient survival and DCGS were similar by donor HIV status (log rank p = .667; log rank p = .388). DGF occurred more frequently in donors with HIV Ab-/NAT- testing compared with Ab+/NAT- or Ab+/NAT+ testing (38.0% vs. 28.6% vs. 26.7%, p = .028). Average dialysis time before transplant was twice as long for recipients who received organs from donors with Ab-/NAT- testing (p < .001). Acute rejection, re-hospitalization and serum creatinine at 12 months did not differ between the groups. CONCLUSIONS: Patient and allograft survival for recipients living with HIV remains comparable irrespective of donor HIV testing status. Utilizing kidneys from deceased donors with HIV Ab+/NAT- or Ab+/NAT+ testing shortens dialysis time prior to transplant.

BK DNAemia in pediatric kidney transplant recipients: Predictors and outcomes.

BACKGROUND: Pediatric data on risk factors and the clinical course of BK DNAemia are limited. We aimed to determine the effects of BK DNAemia on transplant outcomes and delineate the safety and efficacy of various treatment approaches. METHODS: This retrospective-cohort study included 161 transplants (age ≤ 21 years) performed at a single center between 1/1/2012 and 1/1/2020. We used Cox proportional models to evaluate the effects of BK DNAemia on patient survival (PS), graft survival (GS), and acute rejection (AR), using BK as a time-dependent covariate. We also assessed the effects of pharmacological intervention on BK DNAemia duration using intervention as a time-dependent covariate. RESULTS: BK-free survival was 69.1% at 1-year and 54.6% at 3-year posttransplant. After multivariate adjustment, BK DNAemia was associated with young age at transplant (aHR, age 5-<12 vs. ≥12 (years): 2.5 (1.4-4.5); p = .001) and steroid-based immunosuppression (IS) (aHR: 2.2 [1.1-4.5]; p = .03). We found no effect of DNAemia on AR (aHR: 1.25; p = .5), PS (aHR: 2.85; p = .22), and GS (aHR: 0.56; p = .41). Of 70 patients with DNAemia, 22 (31.4%) received no treatment, 20 (28.6%) received IS reduction alone, and 28 patients (40%) received treatment with at least one pharmacological agent (leflunomide, IVIG, ciprofloxacin, cidofovir). Sixty-three patients (90%) cleared DNAemia with median time to resolution of 2.4 months (IQR:1.4-5.6). We found no significant effect of BK-directed pharmacological treatment on time to resolution (aHR: 0.64;p = .13). BK-directed agents were well tolerated. CONCLUSIONS: BK DNAemia is associated with a young age at transplant and steroid-based maintenance IS. We found no effect of BK DNAemia on AR, GS, and PS.

Structure of the human multidrug transporter ABCG2.

ABCG2 is a constitutively expressed ATP-binding cassette (ABC) transporter that protects many tissues against xenobiotic molecules. Its activity affects the pharmacokinetics of commonly used drugs and limits the delivery of therapeutics into tumour cells, thus contributing to multidrug resistance. Here we present the structure of human ABCG2 determined by cryo-electron microscopy, providing the first high-resolution insight into a human multidrug transporter. We visualize ABCG2 in complex with two antigen-binding fragments of the human-specific, inhibitory antibody 5D3 that recognizes extracellular loops of the transporter. We observe two cholesterol molecules bound in the multidrug-binding pocket that is located in a central, hydrophobic, inward-facing translocation pathway between the transmembrane domains. Combined with functional in vitro analyses, our results suggest a multidrug recognition and transport mechanism of ABCG2, rationalize disease-causing single nucleotide polymorphisms and the allosteric inhibition by the 5D3 antibody, and provide the structural basis of cholesterol recognition by other G-subfamily ABC transporters.

Analysis of CACTA transposase genes unveils the mechanism of intron loss and distinct small RNA silencing pathways underlying divergent evolution of Brassica genomes.

In comparison with retrotransposons, DNA transposons make up a smaller proportion of most plant genomes. However, these elements are often proximal to genes to affect gene expression depending on the activity of the transposons, which is largely reflected by the activity of the transposase genes. Here, we show that three AT-rich introns were retained in the TNP2-like transposase genes of the Bot1 (Brassica oleracea transposon 1) CACTA transposable elements in Brassica oleracea, but were lost in the majority of the Bot1 elements in Brassica rapa. A recent burst of transposition of Bot1 was observed in B. oleracea, but not in B. rapa. This burst of transposition is likely related to the activity of the TNP2-like transposase genes as the expression values of the transposase genes were higher in B. oleracea than in B. rapa. In addition, distinct populations of small RNAs (21, 22 and 24 nt) were detected from the Bot1 elements in B. oleracea, but the vast majority of the small RNAs from the Bot1 elements in B. rapa are 24 nt in length. We hypothesize that the different activity of the TNP2-like transposase genes is likely associated with the three introns, and intron loss is likely reverse transcriptase mediated. Furthermore, we propose that the Bot1 family is currently undergoing silencing in B. oleracea, but has already been silenced in B. rapa. Taken together, our data provide new insights into the differentiation of transposons and their role in the asymmetric evolution of these two closely related Brassica species.

Long-term outcomes of crossmatch positive simultaneous liver-kidney transplantations in the United States.

The long-term outcomes of positive crossmatch (+XM) simultaneous liver-kidney (SLK) transplantations are conflicting. We examined the association between crossmatch status and SLK outcomes in recipients discharged on tacrolimus and mycophenolate with or without steroids. We analyzed the Scientific Registry of Transplant Recipients for all primary SLK recipients between 2003 and 2020 with available crossmatch and induction data. We grouped recipients according to the crossmatch status: negative crossmatch (-XM; n = 3040) and +XM (n = 407). Kaplan-Meier curves were generated to examine recipient, death-censored liver, and death-censored kidney survival by crossmatch status. Cox proportional hazard models were used to investigate the association between crossmatch status and outcomes of interest with follow-up censored at 10 years. Models were adjusted for recipient age, sex, diabetes mellitus, Model for End-Stage Liver Disease score, duration on the liver waiting list, induction immunosuppression, steroid maintenance, hepatitis C infection, donor age and sex, local vs. shared organ, cold ischemia time, and previous liver transplantation status. In the univariable analysis, crossmatch status was not associated with recipient survival (log-rank p = 0.63), death-censored liver graft survival (log-rank p = 0.05), or death-censored kidney graft survival (log-rank p = 0.11). Compared with -XM, +XM recipients had a similar 1-year liver rejection rate, but higher kidney rejection rate (4.6% vs. 8.9%, p = 0.002). In the multivariable models, +XM status was not associated with deleterious long-term recipient, liver, or kidney grafts survival. -XM and +XM SLK transplantations have comparable long-term recipient, liver graft, and kidney survival with a slightly increased risk of early kidney allograft rejection in the +XM group. Crossmatch positivity in SLK transplantations should not influence the decision to use organs from a specific donor.

Genome-Wide Reinforcement of DNA Methylation Occurs during Somatic Embryogenesis in Soybean.

Somatic embryogenesis is an important tissue culture technique that sometimes leads to phenotypic variation via genetic and/or epigenetic changes. To understand the genomic and epigenomic impacts of somatic embryogenesis, we characterized soybean (Glycine max) epigenomes sampled from embryos at 10 different stages ranging from 6 weeks to 13 years of continuous culture. We identified genome-wide increases in DNA methylation from cultured samples, especially at CHH sites. The hypermethylation almost exclusively occurred in regions previously possessing non-CG methylation and was accompanied by increases in the expression of genes encoding the RNA-directed DNA methylation (RdDM) machinery. The epigenomic changes were similar between somatic and zygotic embryogenesis. Following the initial global wave of hypermethylation, rare decay events of maintenance methylation were observed, and the extent of the decay increased with time in culture. These losses in DNA methylation were accompanied by downregulation of genes encoding the RdDM machinery and transcriptome reprogramming reminiscent of transcriptomes during late-stage seed development. These results reveal a process for reinforcing already silenced regions to maintain genome integrity during somatic embryogenesis over the short term, which eventually decays at certain loci over longer time scales.

Short-chain diamines are the physiological substrates of PACE family efflux pumps.

Acinetobacter baumannii has rapidly emerged as a major cause of gram-negative hospital infections worldwide. A. baumannii encodes for the transport protein AceI, which confers resistance to chlorhexidine, a widely used antiseptic. AceI is also the prototype for the recently discovered proteobacterial antimicrobial compound efflux (PACE) family of transport proteins that confer resistance to a range of antibiotics and antiseptics in many gram-negative bacteria, including pathogens. The gene encoding AceI is conserved in the core genome of A. baumannii, suggesting that it has an important primordial function. This is incongruous with the sole characterized substrate of AceI, chlorhexidine, an entirely synthetic biocide produced only during the last century. Here we investigated a potential primordial function of AceI and other members of the PACE family in the transport of naturally occurring polyamines. Polyamines are abundant in living cells, where they have physiologically important functions and play multifaceted roles in bacterial infection. Gene expression studies revealed that the aceI gene is induced in A. baumannii by the short-chain diamines cadaverine and putrescine. Membrane transport experiments conducted in whole cells of A. baumannii and Escherichia coli and also in proteoliposomes showed that AceI mediates the efflux of these short-chain diamines when energized by an electrochemical gradient. Assays conducted using 8 additional diverse PACE family proteins identified 3 that also catalyze cadaverine transport. Taken together, these results demonstrate that short-chain diamines are common substrates for the PACE family of transport proteins, adding to their broad significance as a novel family of efflux pumps.

Outcomes of Kidney Allograft and Recipient Survival After Liver Transplantation by Induction Type in the United States.

There are several choices for induction immunosuppression in kidney-after-liver transplantation. We used the Scientific Registry of Transplant Recipients database. We assessed all kidney-after-liver transplant recipients in the United States between 1/1/2000 and 7/31/2017 to study kidney graft and patient outcomes by induction type. We only included patients discharged on tacrolimus and mycophenolate with or without steroids and had a negative crossmatch before kidney engraftment. We grouped recipients by kidney induction type into the following 3 groups: depletional (n = 550), nondepletional (n = 434), and no antibody induction (n = 144). We studied patient and kidney allograft survival using Cox proportional hazard regression, with transplant center included as a random effect. Models were adjusted for liver induction regimen, recipient and donor age, sex, human leukocyte antigen mismatches, payor type, living donor kidney transplantation, dialysis status, time from liver engraftment, hepatitis C virus status, and the presence of diabetes mellitus at time of kidney transplantation and transplantation year. The 6-month and 1-year rejection rates did not differ between groups. Compared with no induction, neither depletional nor nondepletional induction was associated with an improved recipient or graft survival in the multivariable models. Depletional induction at the time of liver transplantation was associated with worse patient survival after kidney transplantation (hazard ratio [HR], 1.7; 95% confidence interval [CI], 1.09-2.67; P = 0.02). Living donor kidney transplantation was associated with a 48.1% improved graft survival (HR, 0.52; 95% CI, 0.33-0.82; P = 0.00). In conclusion, in the settings of a negative cross-match and maintenance with tacrolimus and mycophenolate, induction use was not associated with a patient or graft survival benefit in kidney-after-liver transplantations.

Standards efforts and landscape for rapid microbial testing methodologies in regenerative medicine.

The Standards Coordinating Body for Gene, Cell, and Regenerative Medicines and Cell-Based Drug Discovery (SCB) supports the development and commercialization of regenerative medicine products by identifying and addressing industry-wide challenges through standards. Through extensive stakeholder engagement, the implementation of rapid microbial testing methods (RMTMs) was identified as a high-priority need that must be addressed to facilitate more timely release of products. Since 2017, SCB has coordinated efforts to develop standards for this area through surveys, weekly meetings, workshops, leadership in working groups and participation in standards development organizations. This article describes the results of these efforts and discusses the current landscape of RMTMs for regenerative medicine products. Based on discussions with stakeholders across the field, an overview of traditional culture-based methods and limitations, alternative microbial testing technologies and current challenges, fit-for-purpose rapid microbial testing and case studies, risk-based strategies for selection of novel rapid microbial test methods and ongoing standards efforts for rapid microbial testing are captured here. To this end, SCB is facilitating several initiatives to address challenges associated with rapid microbial testing for regenerative medicine products. Two documentary standards are under development: an International Organization for Standardization standard to provide the framework for a risk-based approach to selecting fit-for-purpose assays primarily intended for cell and gene therapy products and an ASTM standard guide focused on sampling methods for microbial testing methods in tissue-engineered medical products. Working with the National Institute of Standards and Technology, SCB expects to facilitate the process of developing publicly available microbial materials for inter-laboratory testing. These studies will help collect the data necessary to facilitate validation of novel rapid methods. Finally, SCB has been working to increase awareness of, dialog about and participation in efforts to develop standards in the regenerative medicine field.

Primary pediatric deceased-donor kidney transplant recipients outcomes by immunosuppression induction received in the United States.

BACKGROUND: We studied the association of induction immunosuppression and pediatric deceased-donor kidney recipient and graft survival. METHODS: We utilized the SRTR to evaluate all primary pediatric deceased-donor kidney transplants from January 1st, 2000, through December 2018. We included only recipients who were maintained on tacrolimus and mycophenolate. Recipients were grouped by induction type: alemtuzumab n = 320, r-ATG n = 2091 and IL-2RA n = 2165. Recipient and allograft survival, and their predictors, were examined. Models were adjusted for age, sex, ethnicity, HLA-antigen mismatches, transplant year, steroid maintenance, pre-emptive transplantation and payor type, with the transplant center included as a random effect. RESULTS: Rejection rates at 6 months (alemtuzumab 8.6% vs r-ATG 7.8% vs IL2-RA 9.2%; P = .30) and 12 months (alemtuzumab 17.2% vs r-ATG 15.7% vs IL2-RA 16.5%; P = .70) were not significantly different between induction groups. In the multivariable models, compared to IL-2RA neither alemtuzumab nor r-ATG was associated with improved recipient [alemtuzumab (HR 1.06, P = .88); r-ATG (HR 1.03, P = .84)] or graft survival [alemtuzumab (HR 1.18, P = .32); r-ATG (HR 1.10, P = .21)]. CONCLUSION: In this large cohort of standard immunological risk primary pediatric deceased-donor kidney recipients on tacrolimus and mycophenolate maintenance, depletional induction regimens were not associated with better rejection rates, recipient, or graft survival compared to IL-2RA induction. Racial, payor type, and sex-related outcome disparities were significant in this group independent of the induction choice.

Primary pediatric live-donor-kidney transplant-recipients' outcomes by immunosuppression induction received in the United States.

OBJECTIVE: We examined the association between induction type and outcomes of live-donor pediatric kidney recipients on tacrolimus and mycophenolate maintenance. MATERIAL AND METHODS: We analyzed the SRTR standard analysis file to evaluate primary live-donor pediatric kidney recipients between 2000 and 2018. Recipients were grouped by induction type into three groups: alemtuzumab n = 289, anti-thymocyte n = 1197, and IL-2RA n = 1625. Kaplan-Meier curves were generated for recipient and death-censored graft survival. Predictors of recipient and allograft survival were examined using Cox proportional hazards models. Models were adjusted for age, sex, ethnicity, renal failure etiology, HLA-mismatches, transplant year, steroid maintenance, preemptive transplantation, payor type, and donor factors such as age, sex, and donor-recipient relationship. The transplant center was included as a random effect to account for inter-center variability. RESULTS: Rejection rates at 6 months (Alemtuzumab 9.5% vs. r-ATG 5.7% vs. IL2-RA 5.3%; P: .023) and 12 months (Alemtuzumab 14.5% vs. r-ATG 10.8% vs. IL2-RA 9%; P: .028) were significantly higher in the alemtuzumab group. PTLD rate (Alemtuzumab 0.8% vs. r-ATG 2.2% vs. IL2-RA 1%; P: .028) was significantly higher in the anti-thymocyte group. In the multivariable models, induction type did not influence patient or death-censored graft survival within ten years post-transplant. CONCLUSION: In this large cohort of standard immunological risk primary pediatric live-donor kidney recipients, as compared to IL-2RA, neither alemtuzumab nor anti-thymocyte globulin was associated with improved long-term graft or recipient survival. In the first year post-transplant, recipients of alemtuzumab induction had a higher rejection rate, while PTLD was more frequently observed in the anti-thymocyte recipients.

Construction and comparison of three reference-quality genome assemblies for soybean.

We report reference-quality genome assemblies and annotations for two accessions of soybean (Glycine max) and for one accession of Glycine soja, the closest wild relative of G. max. The G. max assemblies provided are for widely used US cultivars: the northern line Williams 82 (Wm82) and the southern line Lee. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 single-nucleotide polymorphisms (snps) per kb between Wm82 and Lee, and 4.7 snps per kb between these lines and G. soja. snp distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgression and haplotype structure. Comparisons against the US germplasm collection show placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan-gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found approximately 40-42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and approximately 32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for the domestication and improvement of soybean, serving as a basis for future research and crop improvement efforts for this important crop species.

Genome-wide analysis of epigenetic and transcriptional changes associated with heterosis in pigeonpea.

Hybrids are extensively used in agriculture to deliver an increase in yield, yet the molecular basis of heterosis is not well understood. Global DNA methylation analysis, transcriptome analysis and small RNA profiling were aimed to understand the epigenetic effect of the changes in gene expression level in the two hybrids and their parental lines. Increased DNA methylation was observed in both the hybrids as compared to their parents. This increased DNA methylation in hybrids showed that majority of the 24-nt siRNA clusters had higher expression in hybrids than the parents. Transcriptome analysis revealed that various phytohormones (auxin and salicylic acid) responsive hybrid-MPV DEGs were significantly altered in both the hybrids in comparison to MPV. DEGs associated with plant immunity and growth were overexpressed whereas DEGs associated with basal defence level were repressed. This antagonistic patterns of gene expression might contribute to the greater growth of the hybrids. It was also noticed that some common as well as unique changes in the regulatory pathways were associated with heterotic growth in both the hybrids. Approximately 70% and 67% of down-regulated hybrid-MPV DEGs were found to be differentially methylated in ICPH 2671 and ICPH 2740 hybrid, respectively. This reflected the association of epigenetic regulation in altered gene expressions. Our findings also revealed that miRNAs might play important roles in hybrid vigour in both the hybrids by regulating their target genes, especially in controlling plant growth and development, defence and stress response pathways. The above finding provides an insight into the molecular mechanism of pigeonpea heterosis.

Workshop report: Toward the development of a human whole stool reference material for metabolomic and metagenomic gut microbiome measurements.

INTRODUCTION: To date, there has been little effort to develop standards for metabolome-based gut microbiome measurements despite the significant efforts toward standard development for DNA-based microbiome measurements. OBJECTIVES: The National Institute of Standards and Technology (NIST), The BioCollective (TBC), and the North America Branch of the International Life Sciences Institute (ILSI North America) are collaborating to extend NIST's efforts to develop a Human Whole Stool Reference Material for the purpose of method harmonization and eventual quality control. METHODS: The reference material will be rationally designed for adequate quality assurance and quality control (QA/QC) for underlying measurements in the study of the impact of diet and nutrition on functional aspects of the host gut microbiome and relationships of those functions to health. To identify which metabolites deserve priority in their value assignment, NIST, TBC, and ILSI North America jointly conducted a workshop on September 12, 2019 at the NIST campus in Gaithersburg, Maryland. The objective of the workshop was to identify metabolites for which evidence indicates relevance to health and disease and to decide on the appropriate course of action to develop a fit-for-purpose reference material. RESULTS: This document represents the consensus opinions of workshop participants and co-authors of this manuscript, and provides additional supporting information. In addition to developing general criteria for metabolite selection and a preliminary list of proposed metabolites, this paper describes some of the strengths and limitations of this initiative given the current state of microbiome research. CONCLUSIONS: Given the rapidly evolving nature of gut microbiome science and the current state of knowledge, an RM (as opposed to a CRM) measured for multiple metabolites is appropriate at this stage. As the science evolves, the RM can evolve to match the needs of the research community. Ultimately, the stool RM may exist in sequential versions. Beneficial to this evolution will be a clear line of communication between NIST and the stakeholder community to ensure alignment with current scientific understanding and community needs.

Report of the 2019 NIST-FDA workshop on standards for next generation sequencing detection of viral adventitious agents in biologics and biomanufacturing.

Adventitious virus testing assures product safety by demonstrating the absence of viruses that could be unintentionally introduced during the manufacturing process. The capabilities of next-generation sequencing (NGS) for broad virus detection in biologics have been demonstrated by the detection of known and novel viruses that were previously missed using the recommended routine assays for adventitious agent testing. A meeting was co-organized by the National Institute of Standards and Technology and the U.S. Food and Drug Administration on September 18-19, 2019 in Gaithersburg, Maryland, USA, to facilitate standardization of NGS technologies for applications of adventitious virus testing in biologics. The goal was to assess the currently used standards for virus detection by NGS and their public availability, and to identify additional needs for different types of reference materials and standards (natural and synthetic). The meeting focused on the NGS processes from sample preparation through sequencing but did not thoroughly cover bioinformatics, since this was considered to be the topic of a separate meeting.