Ethanol preservation and pretreatments facilitate quality DNA extractions in recalcitrant plant species.

Premise: The preservation of plant tissues in ethanol is conventionally viewed as problematic. Here, we show that leaf preservation in ethanol combined with proteinase digestion can provide high-quality DNA extracts. Additionally, as a pretreatment, ethanol can facilitate DNA extraction for recalcitrant samples. Methods: DNA was isolated from leaves preserved with 96% ethanol or from silica-desiccated leaf samples and herbarium fragments that were pretreated with ethanol. DNA was extracted from herbarium tissues using a special ethanol pretreatment protocol, and these extracts were compared with those obtained using the standard cetyltrimethylammonium bromide (CTAB) method. Results: DNA extracted from tissue preserved in, or pretreated with, ethanol was less fragmented than DNA from tissues without pretreatment. Adding proteinase digestion to the lysis step increased the amount of DNA obtained from the ethanol-pretreated tissues. The combination of the ethanol pretreatment with liquid nitrogen freezing and a sorbitol wash prior to cell lysis greatly improved the quality and yield of DNA from the herbarium tissue samples. Discussion: This study critically reevaluates the consequences of ethanol for plant tissue preservation and expands the utility of pretreatment methods for molecular and phylogenomic studies.

Phylogenomic analyses using a new 1013-gene Vitaceae bait-set support major groups of North American Vitis.

A set of newly designed Vitaceae baits targeting 1013 genes was employed to explore phylogenetic relationships among North American Vitis. Eurasian Vitis taxa including Vitis vinifera were found to be nested within North American Vitis subgenus Vitis. North American Vitis subgenus Vitis can be placed into nine main groups: the Monticola group, the Occidentales group, the Californica group, the Vinifera group (introduced from Eurasia), the Mustangensis group, the Palmata group, the Aestivalis group, the Labrusca group, and the Cinerea group. Strong cytonuclear discordances were detected in North American Vitis, with many species non-monophyletic in the plastid phylogeny, while monophyletic in the nuclear phylogeny. The phylogenomic analyses support recognizing four distinct species in the Vitis cinerea complex in North America: V. cinerea, V. baileyana, V. berlandieri, and V. simpsonii. Such treatment will better serve the conservation of wild Vitis diversity in North America. Yet the evolutionary history of Vitis is highly complex, with the concordance analyses indicating conflicting signals across the phylogeny. Cytonuclear discordances and Analyses using the Species Networks applying Quartets (SNaQ) method support extensive hybridizations in North American Vitis. The results further indicate that plastid genomes alone are insufficient for resolving the evolutionary history of plant groups that have undergone rampant hybridization, like the case in North American Vitis. Nuclear gene data are essential for species delimitation, identification and reconstructing evolutionary relationships; therefore, they are imperative for plant phylogenomic studies.

Identifying Measures of Suboptimal Healthcare Interaction (SOHI) to Develop a Claims-Based Model for Predicting Patients with Inflammatory Bowel Disease at Risk for SOHI.

BACKGROUND: Understanding the demographic and clinical characteristics of patients with Inflammatory Bowel Disease (IBD) who are likely to experience poor disease outcomes may allow early interventions that can improve health outcomes. OBJECTIVES: To describe demographic and clinical characteristics of patients with ulcerative colitis (UC) and Crohn's disease (CD) with the presence of at least one Suboptimal Healthcare Interaction (SOHI) event, which can inform the development of a model to predict SOHI in members with IBD based on insurance claims, with the goal of offering these patients some additional intervention. METHODS: We identified commercially insured individuals with IBD between 01 January 2019 and 31 December 2019 using Optum Labs' administrative claims database. The primary cohort was stratified on the presence or absence of ≥ 1 SOHI event (a SOHI-defining data point or characteristic at a specific time point) during the baseline observation period. SOHI was deployed as the basis for the development of a model to predict which individuals with IBD were most likely to continue to have SOHI within a 1-year timeframe (follow-up SOHI) using insurance claims data. All baseline characteristics were analyzed descriptively. Multivariable logistic regression was used to examine the association of follow-up SOHI with baseline characteristics. RESULTS: Of 19,824 individuals, 6872 (34.7%) were found to have follow-up SOHI. Individuals with follow-up SOHI were more likely to have had similar SOHI events in the baseline period than those with non-SOHI. A significantly greater proportion of individuals with SOHI had ≥ 1 claims-based C-reactive protein (CRP) test order and ≥ 1 CRP lab results compared with non-SOHI. Individuals with follow-up SOHI were more likely to incur higher healthcare expenditures and resource utilization as compared with non-SOHI individuals. A few of the most important variables used to predict follow-up SOHI included baseline mesalamine use, count of baseline opioid fills, count of baseline oral corticosteroid fills, baseline extraintestinal manifestations of disease, proxy for baseline SOHI, and index IBD provider specialty. CONCLUSION: Individuals with SOHI are likely to have higher expenditures, higher healthcare resource utilization, uncontrolled disease, and higher CRP lab results as compared with non-SOHI members. Distinguishing SOHI and non-SOHI patients in a dataset could efficiently identify potential cases of poor future IBD outcomes.

We have developed a model for identifying suboptimal healthcare interactions (SOHI) at follow-up and used it to predict the individuals with inflammatory bowel disease (IBD) who are likely to suffer poor healthcare outcomes. Our study showed that the SOHI and non-SOHI cohorts had notable differences in clinical baseline characteristics. Compared with non-SOHI members, individuals with SOHI experienced poor IBD outcomes and incurred higher healthcare resource utilization and costs. Understanding baseline characteristics of patients with SOHI to predict follow-up SOHI can improve health outcomes by early identification of patients with IBD who are likely to experience it. This can help in targeting efforts toward additional care, resulting in greater chances of a well-managed disease.

Climate-influenced boreotropical survival and rampant introgressions explain the thriving of New World grapes in the north temperate zone.

The north temperate region was characterized by a warm climate and a rich thermophilic flora before the Eocene, but early diversifications of the temperate biome under global climate change and biome shift remain uncertain. Moreover, it is becoming clear that hybridization/introgression is an important driving force of speciation in plant diversity. Here, we applied analyses from biogeography and phylogenetic networks to account for both introgression and incomplete lineage sorting based on genomic data from the New World Vitis, a charismatic component of the temperate North American flora with known and suspected gene flow among species. Biogeographic inference and fossil evidence suggest that the grapes were widely distributed from North America to Europe during the Paleocene to the Eocene, followed by widespread extinction and survival of relicts in the tropical New World. During the climate warming in the early Miocene, a Vitis ancestor migrated northward from the refugia with subsequent diversification in the North American region. We found strong evidence for widespread incongruence and reticulate evolution among nuclear genes within both recent and ancient lineages of the New World Vitis. Furthermore, the organellar genomes showed strong conflicts with the inferred species tree from the nuclear genomes. Our phylogenomic analyses provided an important assessment of the wide occurrence of reticulate introgression in the New World Vitis, which potentially represents one of the most important mechanisms for the diversification of Vitis species in temperate North America and even the entire temperate Northern Hemisphere. The scenario we report here may be a common model of temperate diversification of flowering plants adapted to the global climate cooling and fluctuation in the Neogene.

"You'll Be Chased Away": Sources, Experiences, and Effects of Violence and Stigma among Gay and Bisexual Men in Kenya.

Gay and bisexual men in Kenya face extreme socio-political stigma which manifests in widespread violence and discrimination across socio-ecological levels. We conducted individual in-depth interviews with 60 gay and bisexual men in western and central Kenya. Interview transcripts were thematically analyzed using an inductive, phenomenological approach to qualitatively examine experiences of stigma and violence at the interpersonal and institutional levels. A total of seven primary themes and four sub-themes emerged from the data. At the interpersonal level, participants described stigma and violence from family, friends, and romantic/sexual partners with sub-themes for gay-baiting violence, blackmail, intimate partner violence, and commitment phobia. At the institutional level, participants described stigma and violence from religious, employment, educational, and healthcare institutions. This stigma and violence severely impacted the lives of participants including their mental health, physical health, sexual health, socioeconomic status, and ability to access health-promoting services. These data identify sources of stigma and describe how this stigma manifests in the everyday lives of gay and bisexual men in Kenya. Study findings and quotes from participants highlight the severity of violence, stigma, and discrimination faced by this community and emphasize the need for decriminalization of same-sex sexualities as well as interventions to support health and wellbeing.

Isolation of DNA from plant tissues using a miniaturized matrix solid-phase dispersion approach featuring ionic liquid and magnetic ionic liquid solvents.

The isolation of high-quality plant genomic DNA is a major prerequisite in many plant biomolecular analyses involving nucleic acid amplification. Conventional plant cell lysis and DNA extraction methods involve lengthy sample preparation procedures that often require large amounts of sample and chemicals, high temperatures and multiple liquid transfer steps which can introduce challenges for high throughput applications. In this study, a simple, rapid, miniaturized ionic liquid (IL)-based extraction method was developed for the isolation of genomic DNA from milligram fragments of Arabidopsis thaliana plant tissue. This method is based on a modification of vortex-assisted matrix solid-phase dispersion (VA-MSPD) in which the trihexyl(tetradecyl)phosphonium bis(trifluoromethylsulfonyl)imide ([P6,6,6,14+][NTf2-]) IL or trihexyl(tetradecyl)phosphonium tris(hexafluoroacetylaceto)nickelate(II) ([P6,6,6,14+][Ni(hfacac)3-]) magnetic IL (MIL) was directly applied to treated plant tissue (∼1.5 mg) and dispersed in an agate mortar to facilitate plant cell lysis and DNA extraction, followed by recovery of the mixture with a qPCR compatible co-solvent. This study represents the first approach to use ILs and MILs in a MSPD procedure to facilitate plant cell lysis and DNA extraction. The DNA-enriched IL- and MIL-cosolvent mixtures were directly integrated into the qPCR buffer without inhibiting the reaction while also circumventing the need for additional purification steps prior to DNA amplification. Under optimum conditions, the IL and MIL yielded 2.87 ± 0.28 and 1.97 ± 0.59 ng of DNA/mg of plant tissue, respectively. Furthermore, the mild extraction conditions used in the method enabled plant DNA in IL- and MIL-cosolvent mixtures to be preserved from degradation at room temperature.

Using hierarchical unsupervised learning to integrate and reduce multi-level and multi-paraspinal muscle MRI data in relation to low back pain.

PURPOSE: The paraspinal muscles (PSM) are a key feature potentially related to low back pain (LBP), and their structure and composition can be quantified using MRI. Most commonly, quantifying PSM measures across individual muscles and individual spinal levels renders numerous separate metrics that are analyzed in isolation. However, comprehensive multivariate approaches would be more appropriate for analyzing the PSM within an individual. To establish and test these methods, we hypothesized that multivariate summaries of PSM MRI measures would associate with the presence of LBP symptoms (i.e., pain intensity). METHODS: We applied hierarchical multiple factor analysis (hMFA), an unsupervised integrative method, to clinical PSM MRI data from unique cohort datasets including a longitudinal cohort of astronauts with pre- and post-spaceflight data and a cohort of chronic LBP subjects and asymptomatic controls. Three specific use cases were investigated: (1) predicting longitudinal changes in pain using combinations of baseline PSM measures; (2) integrating baseline and post-spaceflight MRI to assess longitudinal change in PSM and how it relates to pain; and (3) integrating PSM quality and adjacent spinal pathology between LBP patients and controls. RESULTS: Overall, we found distinct complex relationships with pain intensity between particular muscles and spinal levels. Subjects with high asymmetry between left and right lean muscle composition and differences between spinal segments PSM quality and structure are more likely to increase in pain reported outcome after prolonged time in microgravity. Moreover, changes in PSM quality and structure between pre and post-spaceflight relate to increase in pain after prolonged microgravity. Finally, we show how unsupervised hMFA recapitulates previous research on the association of CEP damage and LBP diagnostic. CONCLUSION: Our analysis considers the spine as a multi-segmental unit as opposed to a series of discrete and isolated spine segments. Integrative and multivariate approaches can be used to distill large and complex imaging datasets thereby improving the clinical utility of MRI-based biomarkers, and providing metrics for further analytical goals, including phenotyping.

Simple and efficient isolation of plant genomic DNA using magnetic ionic liquids.

BACKGROUND: Plant DNA isolation and purification is a time-consuming and laborious process relative to epithelial and viral DNA sample preparation due to the cell wall. The lysis of plant cells to free intracellular DNA normally requires high temperatures, chemical surfactants, and mechanical separation of plant tissue prior to a DNA purification step. Traditional DNA purification methods also do not aid themselves towards fieldwork due to the numerous chemical and bulky equipment requirements. RESULTS: In this study, intact plant tissue was coated by hydrophobic magnetic ionic liquids (MILs) and ionic liquids (ILs) and allowed to incubate under static conditions or dispersed in a suspension buffer to facilitate cell disruption and DNA extraction. The DNA-enriched MIL or IL was successfully integrated into the qPCR buffer without inhibiting the reaction. The two aforementioned advantages of ILs and MILs allow plant DNA sample preparation to occur in one minute or less without the aid of elevated temperatures or chemical surfactants that typically inhibit enzymatic amplification methods. MIL or IL-coated plant tissue could be successfully integrated into a qPCR assay without the need for custom enzymes or manual DNA isolation/purification steps that are required for conventional methods. CONCLUSIONS: The limited amount of equipment, chemicals, and time required to disrupt plant cells while simultaneously extracting DNA using MILs makes the described procedure ideal for fieldwork and lab work in low resource environments.

It's a Kiki!: Developmental benefits of the Kiki scene for Black gay/bisexual/transgender adolescents/emerging adults.

Younger members of the House and Ball Community (HBC) have created an emerging social scene called "Kiki" that has shared elements with the HBC. Given the growing popularity of the Kiki scene in urban communities with large numbers of Black gay, bisexual, and transgender (GBT) adolescents and emerging adults, it is important to understand the developmental benefits of the Kiki scene. We conducted individual in-depth interviews with 30 GBT adolescents and emerging adults (ages 15-24) who attended Kiki-related events and 15 older opinion leaders affiliated with the HBC. Participants described how the Kiki scene provides them with a range of supportive and affirming functions and offers a place where they can achieve important developmental milestones. The results clustered into three primary thematic areas of development: Executive Functioning Development, Social-Emotional Development, and Physical and Emotional Safety during Development. Within each of these thematic areas are sub-themes that further explicate the ways in which these functions are supported. We believe this to be one of the first papers exclusively focused on the positive aspects of the Kiki scene for adolescent and emerging adult development, and we encourage other researchers to further explore this emerging socializing force and its potential benefits to development.

Biomechanical changes in the lumbar spine following spaceflight and factors associated with postspaceflight disc herniation.

BACKGROUND CONTEXT: For chronic low back pain, the causal mechanisms between pathological features from imaging and patient symptoms are unclear. For instance, disc herniations can often be present without symptoms. There remains a need for improved knowledge of the pathophysiological mechanisms that explore spinal tissue damage and clinical manifestations of pain and disability. Spaceflight and astronaut health provides a rare opportunity to study potential low back pain mechanisms longitudinally. Spaceflight disrupts diurnal loading on the spine and several lines of evidence indicate that astronauts are at a heightened risk for low back pain and disc herniation following spaceflight. PURPOSE: To examine the relationship between prolonged exposure to microgravity and the elevated incidence of postflight disc herniation, we conducted a longitudinal study to track the spinal health of twelve NASA astronauts before and after approximately 6 months in space. We hypothesize that the incidence of postflight disc herniation and low back complaints associates with spaceflight-included muscle atrophy and pre-existing spinal pathology. STUDY DESIGN: This is a prospective longitudinal study. PATIENT SAMPLE: Our sample included a cohort of twelve astronaut crewmembers. OUTCOME MEASURES: From 3T MRI, we quantified disc water content (ms), disc degeneration (Pfirrmann grade), vertebral endplate irregularities, facet arthropathy and/ fluid, high intensity zones, disc herniation, multifidus total cross-sectional area (cm2), multifidus lean muscle cross-sectional area (cm2), and muscle quality/composition (%). From quantitative fluoroscopy we quantified, maximum flexion-extension ROM (°), maximum lateral bending ROM (°), and maximum translation (%). Lastly, patient outcomes and clinical notes were used for identifying postflight symptoms associated with disc herniations from 3T MRI. METHODS: Advanced imaging data from 3T MRI were collected at three separate time points in relation to spending six months in space: (1) within a year before launch ("pre-flight"), (2) within a week after return to Earth ("post-flight"), and (3) between 1 and 2 months after return to Earth ("recovery"). Fluoroscopy of segmental kinematics was collected at preflight and postflight timepoints. We assessed the effect of spaceflight and postflight recovery on longitudinal changes in spinal structure and function, as well as differences between crew members who did and did not present a symptomatic disc herniation following spaceflight. RESULTS: Half of our astronauts (n=6) experienced new symptoms associated with a new or previously asymptomatic lumbar disc protrusion or extrusion following spaceflight. We observed decreased multifidus muscle quality following spaceflight in the lower lumbar spine, with a reduced percentage of lean muscle at L4L5 (-6.2%, p=.009) and L5S1 (-7.0%, p=.006) associated with the incidence of new disc herniation. Additionally, we observed reduced lumbar segment flexion-extension ROM for L2L3 (-17.2%, p=.006) and L3L4 (-20.5%, p=.02) following spaceflight, and furthermore that reduced ROM among the upper three lumbar segments (-24.1%, p=.01) associated with the incidence of disc herniation. Existing endplate pathology was most prevalent in the upper lumbar spine and associated with reduced segmental ROM (-20.5%, p=.02). CONCLUSIONS: In conclusion from a 10-year study investigating the effects of spaceflight on the lumbar spine and risk for disc herniation, we found the incidence of lumbar disc herniation following spaceflight associates with compromised multifidus muscle quality and spinal segment kinematics, as well as pre-existing spinal endplate irregularities. These findings suggest differential effects of spinal stiffness and muscle loss in the upper versus lower lumbar spine regions that may specifically provoke risk for symptomatic disc herniation in the lower lumbar spine following spaceflight. Results from this study provide a unique longitudinal assessment of mechanisms and possible risk factors for developing disc herniations and related low back pain. Furthermore, these findings will help inform physiologic countermeasures to maintain spinal health in astronauts during long-duration missions in space.

Development and phylogenetic utilities of a new set of single-/low-copy nuclear genes in Senecioneae (Asteraceae), with new insights into the tribal position and the relationships within subtribe Tussilagininae.

The tribe Senecioneae is one of the largest tribes in Asteraceae, with a nearly cosmopolitan distribution. Despite great efforts devoted to elucidate the evolution of Senecioneae, many questions still remain concerning the systematics of this group, from the tribal circumscription and position to species relationships in many genera. The hybridization-based target enrichment method of next-generation sequencing has been accepted as a promising approach to resolve phylogenetic problems. We herein develop a set of single-/low-copy genes for Senecioneae, and test their phylogenetic utilities. Our results demonstrate that these genes work highly efficiently for Senecioneae, with a high average gene recovery of 98.8% across the tribe and recovering robust phylogenetic hypotheses at different levels. In particular, the delimitation of the Senecioneae has been confirmed to include Abrotanella and exclude Doronicum, with the former sister to core Senecioneae and the latter shown to be more closely related to Calenduleae. Moreover, Doronicum and Calenduleae are inferred to be the closest relatives of Senecioneae, which is a new hypothesis well supported by statistical topology tests, morphological evidence, and the profile of pyrrolizidine alkaloids, a special kind of chemical characters generally used to define Senecioneae. Furthermore, this study suggests a complex reticulation history in the diversification of Senecioneae, accounting for the prevalence of polyploid groups in the tribe. With subtribe Tussilagininae s.str. as a case study showing a more evident pattern of gene duplication, we further explored reconstructing the phylogeny in the groups with high ploidy levels. Our results also demonstrate that tree topologies based on sorted paralogous copies are stable across different methods of phylogenetic inference, and more congruent with the morphological evidence and the results of previous phylogenetic studies.

Mental Health Challenges and Needs among Sexual and Gender Minority People in Western Kenya.

Background: Sexual and gender minority (SGM) people in Kenya face pervasive socio-cultural and structural discrimination. Persistent stress stemming from anti-SGM stigma and prejudice may place SGM individuals at increased risk for negative mental health outcomes. This study explored experiences with violence (intimate partner violence and SGM-based violence), mental health outcomes (psychological distress, PTSD symptoms, and depressive symptoms), alcohol and other substance use, and prioritization of community needs among SGM adults in Western Kenya. Methods: This study was conducted by members of a collaborative research partnership between a U.S. academic institution and a Kenyan LGBTQ civil society organization (CSO). A convenience sample of 527 SGM adults (92.7% ages 18-34) was recruited from community venues to complete a cross-sectional survey either on paper or through an online secure platform. Results: For comparative analytic purposes, three sexual orientation and gender identity (SOGI) groups were created: (1) cisgender sexual minority women (SMW; 24.9%), (2) cisgender sexual minority men (SMM; 63.8%), and (3) gender minority individuals (GMI; 11.4%). Overall, 11.7% of participants reported clinically significant levels of psychological distress, 53.2% reported clinically significant levels of post-traumatic stress disorder (PTSD) symptoms, and 26.1% reported clinically significant levels of depressive symptoms. No statistically significant differences in clinical levels of these mental health concerns were detected across SOGI groups. Overall, 76.2% of participants reported ever using alcohol, 45.6% home brew, 43.5% tobacco, 39.1% marijuana, and 27.7% miraa or khat. Statistically significant SOGI group differences on potentially problematic substance use revealed that GMI participants were less likely to use alcohol and tobacco daily; and SMM participants were more likely to use marijuana daily. Lifetime intimate partner violence (IPV) was reported by 42.5% of participants, and lifetime SGM-based violence (SGMV) was reported by 43.4%. GMI participants were more likely than other SOGI groups to have experienced both IPV and SGMV. Participants who experienced SGMV had significantly higher rates of clinically significant depressive and PTSD symptoms. Conclusions: Despite current resilience demonstrated by SGM adults in Kenya, there is an urgent need to develop and deliver culturally appropriate mental health services for this population. Given the pervasiveness of anti-SGM violence, services should be provided using trauma-informed principles, and be sensitive to the lived experiences of SGM adults in Kenya. Community and policy levels interventions are needed to decrease SGM-based stigma and violence, increase SGM visibility and acceptance, and create safe and affirming venues for mental health care. Political prioritization of SGM mental health is needed for sustainable change.

Pogostemon guamensis Lorence & W.L.Wagner (Lamiaceae), a new species from Guam, Mariana Islands.

While undertaking a botanical survey of the Andersen Air Force Base on Guam (Mariana Islands) in 1994, botanists from the National Tropical Botanical Garden collected an unusual suffrutescent, non-aromatic member of the Lamiaceae family growing on limestone cliffs in the northeastern part of the island. Based on morphology and molecular data (trnLF, matK), it was determined to belong to the genus Pogostemon Desf., a genus previously unknown from the Micronesian, Melanesian, and Polynesian region. Moreover, the analysis also showed that it was not conspecific with P. cablin (patchouli), and of the species available to include in the phylogenetic analyses it is sister to P. hirsutus¸ a species from India and Sri Lanka. Differing from its congeners by its large, loose inflorescence 2.5-5 cm wide and up to 7 cm wide in fruit, it is here illustrated and described as a new species, Pogostemon guamensis Lorence & W.L. Wagner and its habitat and conservation status are discussed.

Nuclear and Chloroplast Sequences Resolve the Enigmatic Origin of the Concord Grape.

Despite the commercial importance of the Concord grape, its origin has remained unresolved for over 150 years without a comprehensive phylogenetic analysis. In this study we aimed to reconstruct the evolutionary history of the Concord grape using sequence data from four nuclear markers (AT103, GAI1, PHYA, and SQD1), six plastid markers (matK, psbA-trnH, petN-trnC, ycf1, trnL-F, and trnS-G), and the plastid genome. We sampled extensively the Vitis species native to northeastern North America as well as representative species from Europe and Asia, including the commercially important Vitis vinifera (wine grape), a native European species with hermaphroditic flowers, and its wild progenitor, V. vinifera subsp. sylvestris. We also sequenced the plastid genome of one accession of the Concord grape and compared the plastid genome data to the recently published data set of Vitis plastomes. Phylogenetic analyses of the plastid and nuclear data using maximum likelihood and Bayesian inference support the hybrid origin of the Concord grape. The results clearly pinpoint the wine grape, V. vinifera, as the maternal donor and the fox grape, Vitis labrusca, which is common in northeastern North America, as the paternal donor. Moreover, we infer that the breeding history of the Concord grape must have involved the backcrossing of the F1 hybrid with the paternal parent V. labrusca. This backcrossing also explains the higher morphological similarity of the Concord grape to V. labrusca than to V. vinifera. This study provides concrete genetic evidence for the hybrid origin of a widespread Vitis cultivar and is, therefore, promising for similar future studies focused on resolving ambiguous origins of major crops or to create successful hybrid fruit crops.

A locked immunometabolic switch underlies TREM2 R47H loss of function in human iPSC-derived microglia.

Loss-of-function genetic variants of triggering receptor expressed on myeloid cells 2 (TREM2) are linked with an enhanced risk of developing dementias. Microglia, the resident immune cell of the brain, express TREM2, and microglial responses are implicated in dementia pathways. In a normal surveillance state, microglia use oxidative phosphorylation for their energy supply, but rely on the ability to undergo a metabolic switch to glycolysis to allow them to perform rapid plastic responses. We investigated the role of TREM2 on the microglial metabolic function in human patient iPSC-derived microglia expressing loss of function variants in TREM2. We show that these TREM2 variant iPSC-microglia, including the Alzheimer's disease R47H risk variant, exhibit significant metabolic deficits including a reduced mitochondrial respiratory capacity and an inability to perform a glycolytic immunometabolic switch. We determined that dysregulated PPARγ/p38MAPK signaling underlies the observed phenotypic deficits in TREM2 variants and that activation of these pathways can ameliorate the metabolic deficit in these cells and consequently rescue critical microglial cellular function such as ß-Amyloid phagocytosis. These findings have ramifications for microglial focussed-treatments in AD.

High throughput DNA extraction of legume root nodules for rhizobial metagenomics.

A novel combination of tissue homogenization, cell lysis, and DNA purification techniques was developed for isolating total DNA from whole legume root nodules on an automated robotic system. Silica dehydrated root nodules from soy bean, Glycine max cv. Tara, and mung bean, Vigna radiata cv. Crystal, were homogenized in 96-well plates and were enzymatically and chemically lysed before being loaded into the AutoGenprep 965 for automated phenol-chloroform based DNA isolation. The resulting DNAs were of relatively high concentration, low fragmentation, and free of phenolic and polysaccharide contaminants. In contrast to manual methods, this adaptation of the Autogen Prep automated DNA extraction instrument allows for 100's to 1000's of samples to be prepared in a fraction of the time and cost.

Transcriptome analysis of the curry tree (Bergera koenigii L., Rutaceae) during leaf development.

The curry tree (Bergera koenigii L.) is a widely cultivated plant used in South Asian cooking. Next-generation sequencing was used to generate the transcriptome of the curry leaf to detect changes in gene expression during leaf development, such as those genes involved in the production of oils which lend the leaf its characteristic taste, aroma, and medicinal properties. Using abundance estimation (RSEM) and differential expression analysis, genes that were significantly differentially expressed were identified. The transcriptome was annotated with BLASTx using the non-redundant (nr) protein database, and Gene Ontology (GO) terms were assigned based on the top BLAST hit using Blast2GO. Lastly, functional enrichment of the assigned GO terms was analyzed for genes that were significantly differentially expressed. Of the most enriched GO categories, pathways involved in cell wall, membrane, and lignin synthesis were found to be most upregulated in immature leaf tissue, possibly due to the growth and expansion of the leaf tissue. Terpene synthases, which synthesize monoterpenes and sesquiterpenes, which comprise much of the curry essential oil, were found to be significantly upregulated in mature leaf tissue, suggesting that oil production increases later in leaf development. Enzymes involved in pigment production were also significantly upregulated in mature leaves. The findings were based on computational estimates of gene expression from RNA-seq data, and further study is warranted to validate these results using targeted techniques, such as quantitative PCR.

Low-copy nuclear markers in Isoëtes (Isoëtaceae) identified with transcriptomes.

PREMISE OF THE STUDY: Few genetic markers provide phylogenetic information in closely related species of Isoëtes (Isoëtaceae). We describe the development of primers for several putative low-copy nuclear markers to resolve the phylogeny of Isoëtes, particularly in the southeastern United States. METHODS AND RESULTS: We identified regions of interest in Isoëtes transcriptomes based on low-copy genes in other plants. Primers were designed for these regions and tested with 16 taxa of Isoëtes and one species of Lycopodium. Parts of the pgiC, gapC, and IBR3 gene regions show phylogenetic signal within the North American and Mediterranean clades of Isoëtes. CONCLUSIONS: Transcriptome data prove useful for identification and primer design of low-copy genes. Three new markers show potential for inferring phylogenies in regional clades of Isoëtes, and possibly across the entire genus.

miRNAome expression profiles in the gonads of adult Melopsittacus undulatus.

The budgerigar (Melopsittacus undulatus) is one of the most widely studied parrot species, serving as an excellent animal model for behavior and neuroscience research. Until recently, it was unknown how sexual differences in the behavior, physiology, and development of organisms are regulated by differential gene expression. MicroRNAs (miRNAs) are endogenous short non-coding RNA molecules that can post-transcriptionally regulate gene expression and play a critical role in gonadal differentiation as well as early development of animals. However, very little is known about the role gonadal miRNAs play in the early development of birds. Research on the sex-biased expression of miRNAs in avian gonads are limited, and little is known about M. undulatus. In the current study, we sequenced two small non-coding RNA libraries made from the gonads of adult male and female budgerigars using Illumina paired-end sequencing technology. We obtained 254 known and 141 novel miRNAs, and randomly validated five miRNAs. Of these, three miRNAs were differentially expressed miRNAs and 18 miRNAs involved in sexual differentiation as determined by functional analysis with GO annotation and KEGG pathway analysis. In conclusion, this work is the first report of sex-biased miRNAs expression in the budgerigar, and provides additional sequences to the avian miRNAome database which will foster further functional genomic research.

Reproductive biology and variation of nuclear ribosomal ITS and ETS sequences in the Calligonum mongolicum complex (Polygonaceae).

To explore the biosystematics of the Calligonum mongolicum complex (Polygonaceae), the flowering phenological period, breeding and pollination characters and seed set of the complex (Calligonum Mongolicum Turze, Calligonum chinense A. Los., Calligonum gobicum A. Los., Calligonum pumilum A. Los. and Calligonum zaidamense A. Los.) were documented in the Turpan Eremophyte Botanical Garden, China. The sequences of the nuclear ribosomal ITS and ETS region were employed to differentiate the Calligonum mongolicum complex and other species in sect. Medusae. The results showed species of the Calligonum mongolicum complex occupied overlapping flowering periods and had consistent pollination agents. Their breeding systems are all self-compatible, tend to be out-crossing and they interbreed amongst each other (out-crossing index, OCI = 4).The crosses within and amongst species had high seed sets (44 - 65%). Phylogenetic analyses of Calligonum sect. Medusae and the network analysis of nrDNA (ITS and ETS) in the complex suggest interbreeding amongst "species" within the complex and provide evidence for taxonomically merging the five species in the complex. The detected hybridisation, occurring within the complex, suggests the need to improve traditional methods of ex situ plant conservation in botanical gardens for maintaining genetic diversity of Calligonum within and amongst species from different geographic areas.