The essential but enigmatic regulatory role of HERVH in pluripotency.

Human specific endogenous retrovirus H (HERVH) is highly expressed in both naive and primed stem cells and is essential for pluripotency. Despite the proven relationship between HERVH expression and pluripotency, there is no single definitive model for the function of HERVH. Instead, several hypotheses of a regulatory function have been put forward including HERVH acting as enhancers, long noncoding RNAs (lncRNAs), and most recently as markers of topologically associating domain (TAD) boundaries. Recently several enhancer-associated lncRNAs have been characterized, which bind to Mediator and are necessary for promoter-enhancer folding interactions. We propose a synergistic model of HERVH function combining relevant findings and discuss the current limitations for its role in regulation, including the lack of evidence for a pluripotency-associated target gene.

Investigating the Streptococcus sinensis competence regulon through a combination of transcriptome analysis and phenotypic evaluation.

Streptococcus sinensis is a recently identified member of the Mitis group of streptococci. This species has been associated with infective endocarditis; however its mechanisms of pathogenesis and virulence are not fully understood. This study aimed to investigate the influence of the competence-stimulating peptide (CSP) and the competence regulon quorum-sensing circuitry (ComABCDE) on subsequent gene transcription and expression, as well as resultant phenotypes. In this study we confirmed the native CSP identity, ascertained when endogenous CSP was produced and completed a transcriptome-wide analysis of all genes following CSP exposure. RNA sequencing analysis revealed the upregulation of genes known to be associated with competence, biofilm formation and virulence. As such, a variety of phenotypic assays were utilized to assess the correlation between increased mRNA expression and potential phenotype response, ultimately gaining insight into the effects of CSP on both gene expression and developed phenotypes. The results indicated that the addition of exogenous CSP aided in competence development and successful transformation, yielding an average transformation efficiency comparable to that of other Mitis group streptococci. Additional studies are needed to further delineate the effects of CSP exposure on biofilm formation and virulence. Overall, this study provides novel information regarding S. sinensis and provides a substantial foundation on which this species and its role in disease pathogenesis can be further investigated.

Drought tolerance of the grapevine, Vitis champinii cv. Ramsey, is associated with higher photosynthesis and greater transcriptomic responsiveness of abscisic acid biosynthesis and signaling.

BACKGROUND: Grapevine is an economically important crop for which yield and berry quality is strongly affected by climate change. Large variations in drought tolerance exist across Vitis species. Some of these species are used as rootstock to enhance abiotic and biotic stress tolerance. In this study, we investigated the physiological and transcriptomic responses to water deficit of four different genotypes that differ in drought tolerance: Ramsey (Vitis champinii), Riparia Gloire (Vitis riparia), Cabernet Sauvignon (Vitis vinifera), and SC2 (Vitis vinifera x Vitis girdiana). RESULTS: Ramsey was particularly more drought tolerant than the other three genotypes. Ramsey maintained a higher stomatal conductance and photosynthesis at equivalent levels of moderate water deficit. We identified specific and common transcriptomic responses shared among the four different Vitis species using RNA sequencing analysis. A weighted gene co-expression analysis identified a water deficit core gene set with the ABA biosynthesis and signaling genes, NCED3, RD29B and ABI1 as potential hub genes. The transcript abundance of many abscisic acid metabolism and signaling genes was strongly increased by water deficit along with genes associated with lipid metabolism, galactinol synthases and MIP family proteins. This response occurred at smaller water deficits in Ramsey and with higher transcript abundance than the other genotypes. A number of aquaporin genes displayed differential and unique responses to water deficit in Ramsey leaves. Genes involved in cysteine biosynthesis and metabolism were constitutively higher in the roots of Ramsey; thus, linking the gene expression of a known factor that influences ABA biosynthesis to this genotype's increased NCED3 transcript abundance. CONCLUSION: The drought tolerant Ramsey maintained higher photosynthesis at equivalent water deficit than the three other grapevine genotypes. Ramsey was more responsive to water deficit; its transcriptome responded at smaller water deficits, whereas the other genotypes did not respond until more severe water deficits were reached. There was a common core gene network responding to water deficit for all genotypes that included ABA metabolism and signaling. The gene clusters and sub-networks identified in this work represent interesting gene lists to explore and to better understand drought tolerance molecular mechanisms.

A comparison of heat-stress transcriptome changes between wild-type Arabidopsis pollen and a heat-sensitive mutant harboring a knockout of cyclic nucleotide-gated cation channel 16 (cngc16).

BACKGROUND: In flowering plants, the male gametophyte (pollen) is one of the most vulnerable cells to temperature stress. In Arabidopsis thaliana, a pollen-specific Cyclic Nucleotide-Gated cation Channel 16 (cngc16), is required for plant reproduction under temperature-stress conditions. Plants harboring a cncg16 knockout are nearly sterile under conditions of hot days and cold nights. To understand the underlying cause, RNA-Seq was used to compare the pollen transcriptomes of wild type (WT) and cngc16 under normal and heat stress (HS) conditions. RESULTS: Here we show that a heat-stress response (HSR) in WT pollen resulted in 2102 statistically significant transcriptome changes (≥ 2-fold changes with adjusted p-value ≤0.01), representing approximately 15% of 14,226 quantified transcripts. Of these changes, 89 corresponded to transcription factors, with 27 showing a preferential expression in pollen over seedling tissues. In contrast to WT, cngc16 pollen showed 1.9-fold more HS-dependent changes (3936 total, with 2776 differences between WT and cngc16). In a quantitative direct comparison between WT and cngc16 transcriptomes, the number of statistically significant differences increased from 21 pre-existing differences under normal conditions to 192 differences under HS. Of the 20 HS-dependent changes in WT that were most different in cngc16, half corresponded to genes encoding proteins predicted to impact cell wall features or membrane dynamics. CONCLUSIONS: Results here define an extensive HS-dependent reprogramming of approximately 15% of the WT pollen transcriptome, and identify at least 27 transcription factor changes that could provide unique contributions to a pollen HSR. The number of statistically significant transcriptome differences between WT and cngc16 increased by more than 9-fold under HS, with most of the largest magnitude changes having the potential to specifically impact cell walls or membrane dynamics, and thereby potentiate cngc16 pollen to be hypersensitive to HS. However, HS-hypersensitivity could also be caused by the extensive number of differences throughout the transcriptome having a cumulative effect on multiple cellular pathways required for tip growth and fertilization. Regardless, results here support a model in which a functional HS-dependent reprogramming of the pollen transcriptome requires a specific calcium-permeable Cyclic Nucleotide-Gated cation Channel, CNGC16.

The common transcriptional subnetworks of the grape berry skin in the late stages of ripening.

BACKGROUND: Wine grapes are important economically in many countries around the world. Defining the optimum time for grape harvest is a major challenge to the grower and winemaker. Berry skins are an important source of flavor, color and other quality traits in the ripening stage. Senescent-like processes such as chloroplast disorganization and cell death characterize the late ripening stage. RESULTS: To better understand the molecular and physiological processes involved in the late stages of berry ripening, RNA-seq analysis of the skins of seven wine grape cultivars (Cabernet Franc, Cabernet Sauvignon, Merlot, Pinot Noir, Chardonnay, Sauvignon Blanc and Semillon) was performed. RNA-seq analysis identified approximately 2000 common differentially expressed genes for all seven cultivars across four different berry sugar levels (20 to 26 °Brix). Network analyses, both a posteriori (standard) and a priori (gene co-expression network analysis), were used to elucidate transcriptional subnetworks and hub genes associated with traits in the berry skins of the late stages of berry ripening. These independent approaches revealed genes involved in photosynthesis, catabolism, and nucleotide metabolism. The transcript abundance of most photosynthetic genes declined with increasing sugar levels in the berries. The transcript abundance of other processes increased such as nucleic acid metabolism, chromosome organization and lipid catabolism. Weighted gene co-expression network analysis (WGCNA) identified 64 gene modules that were organized into 12 subnetworks of three modules or more and six higher order gene subnetworks. Some gene subnetworks were highly correlated with sugar levels and some subnetworks were highly enriched in the chloroplast and nucleus. The petal R package was utilized independently to construct a true small-world and scale-free complex gene co-expression network model. A subnetwork of 216 genes with the highest connectivity was elucidated, consistent with the module results from WGCNA. Hub genes in these subnetworks were identified including numerous members of the core circadian clock, RNA splicing, proteolysis and chromosome organization. An integrated model was constructed linking light sensing with alternative splicing, chromosome remodeling and the circadian clock. CONCLUSIONS: A common set of differentially expressed genes and gene subnetworks from seven different cultivars were examined in the skin of the late stages of grapevine berry ripening. A densely connected gene subnetwork was elucidated involving a complex interaction of berry senescent processes (autophagy), catabolism, the circadian clock, RNA splicing, proteolysis and epigenetic regulation. Hypotheses were induced from these data sets involving sugar accumulation, light, autophagy, epigenetic regulation, and fruit development. This work provides a better understanding of berry development and the transcriptional processes involved in the late stages of ripening.

Sporobolus stapfianus: Insights into desiccation tolerance in the resurrection grasses from linking transcriptomics to metabolomics.

BACKGROUND: Understanding the response of resurrection angiosperms to dehydration and rehydration is critical for deciphering the mechanisms of how plants cope with the rigors of water loss from their vegetative tissues. We have focused our studies on the C4 resurrection grass, Sporobolus stapfianus Gandoger, as a member of a group of important forage grasses. METHODS: We have combined non-targeted metabolomics with transcriptomics, via a NimbleGen array platform, to develop an understanding of how gene expression and metabolite profiles can be linked to generate a more detailed mechanistic appreciation of the cellular response to both desiccation and rehydration. RESULTS: The rehydration transcriptome and metabolome are primarily geared towards the rapid return of photosynthesis, energy metabolism, protein turnover, and protein synthesis during the rehydration phase. However, there are some metabolites associated with ROS protection that remain elevated during rehydration, most notably the tocopherols. The analysis of the dehydration transcriptome reveals a strong concordance between transcript abundance and the associated metabolite abundance reported earlier, but only in responses that are directly related to cellular protection during dehydration: carbohydrate metabolism and redox homeostasis. The transcriptome response also provides strong support for the involvement of cellular protection processes as exemplified by the increases in the abundance of transcripts encoding late embryogenesis abundant (LEA) proteins, anti-oxidant enzymes, early light-induced proteins (ELIP) proteins, and cell-wall modification enzymes. There is little concordance between transcript and metabolite abundance for processes such as amino acid metabolism that do not appear to contribute directly to cellular protection, but are nonetheless important for the desiccation tolerant phenotype of S. stapfianus. CONCLUSIONS: The transcriptomes of both dehydration and rehydration offer insight into the complexity of the regulation of responses to these processes that involve complex signaling pathways and associated transcription factors. ABA appears to be important in the control of gene expression in both the latter stages of the dehydration and the early stages of rehydration. These findings add to the growing body of information detailing how plants tolerate and survive the severe cellular perturbations of dehydration, desiccation, and rehydration.

Transcriptomic analysis of the late stages of grapevine (Vitis vinifera cv. Cabernet Sauvignon) berry ripening reveals significant induction of ethylene signaling and flavor pathways in the skin.

BACKGROUND: Grapevine berry, a nonclimacteric fruit, has three developmental stages; the last one is when berry color and sugar increase. Flavors derived from terpenoid and fatty acid metabolism develop at the very end of this ripening stage. The transcriptomic response of pulp and skin of Cabernet Sauvignon berries in the late stages of ripening between 22 and 37 °Brix was assessed using whole-genome micorarrays. RESULTS: The transcript abundance of approximately 18,000 genes changed with °Brix and tissue type. There were a large number of changes in many gene ontology (GO) categories involving metabolism, signaling and abiotic stress. GO categories reflecting tissue differences were overrepresented in photosynthesis, isoprenoid metabolism and pigment biosynthesis. Detailed analysis of the interaction of the skin and pulp with °Brix revealed that there were statistically significantly higher abundances of transcripts changing with °Brix in the skin that were involved in ethylene signaling, isoprenoid and fatty acid metabolism. Many transcripts were peaking around known optimal fruit stages for flavor production. The transcript abundance of approximately two-thirds of the AP2/ERF superfamily of transcription factors changed during these developmental stages. The transcript abundance of a unique clade of ERF6-type transcription factors had the largest changes in the skin and clustered with genes involved in ethylene, senescence, and fruit flavor production including ACC oxidase, terpene synthases, and lipoxygenases. The transcript abundance of important transcription factors involved in fruit ripening was also higher in the skin. CONCLUSIONS: A detailed analysis of the transcriptome dynamics during late stages of ripening of grapevine berries revealed that these berries went through massive transcriptional changes in gene ontology categories involving chemical signaling and metabolism in both the pulp and skin, particularly in the skin. Changes in the transcript abundance of genes involved in the ethylene signaling pathway of this nonclimacteric fruit were statistically significant in the late stages of ripening when the production of transcripts for important flavor and aroma compounds were at their highest. Ethylene transcription factors known to play a role in leaf senescence also appear to play a role in fruit senescence. Ethylene may play a bigger role than previously thought in this non-climacteric fruit.

A multi-omics approach for understanding blood digestion dynamics in Ixodes scapularis and identification of anti-tick vaccine targets.

Ixodes scapularis, the black-legged tick, is a major arthropod vector that transmits the causative agents of Lyme disease and several other pathogens of human significance. The tick midgut is the main tissue involved in blood acquisition and digestion and the first organ to have contact with pathogens ingested through the blood meal. Gene expression in the midgut before, during, and after a blood meal may vary in response to the physiological changes due to blood feeding. A systems biology approach based on RNA and protein sequencing was used to gain insight into the changes in tick midgut transcripts and proteins during blood ingestion (unfed and partially fed) and digestion (1-, 2-, 7-, and 14 days post detachment from the host) by the Ixodes scapularis female ticks. A total of 2,726 differentially expressed transcripts, and 449 proteins were identified across the time points. Genes involved in detoxification of xenobiotics, proteases, protease inhibitors, metabolism, and immunity were differentially expressed in response to blood feeding. Similarly, proteins corresponding to the same groups were also differentially expressed. Nine genes from major gene categories were chosen as potential vaccine candidates, and, using RNA interference, the effect of these gene knockdowns on tick biology was investigated. Knockdown of these genes had variable negative impacts on tick physiology, such as the inability to engorge fully and to produce eggs and increased mortality. These and additional gene targets provide opportunities to explore novel tick control strategies.

Detection of the Omicron BA.1 Variant of SARS-CoV-2 in Wastewater From a Las Vegas Tourist Area.

Importance: Interpretation of wastewater surveillance data is potentially confounded in communities with mobile populations, so it is important to account for this issue when conducting wastewater-based epidemiology (WBE). Objectives: To leverage spatial and temporal differences in wastewater whole-genome sequencing (WGS) data to quantify relative SARS-CoV-2 contributions from visitors to southern Nevada. Design, Setting, and Participants: This cross-sectional wastewater surveillance study was performed during the COVID-19 pandemic (March 2020 to February 2022) and included weekly influent wastewater samples that were analyzed by reverse transcription-quantitative polymerase chain reaction to quantify SARS-CoV-2 RNA and WGS for identification of variants of concern. This study was conducted in the Las Vegas, Nevada, metropolitan area, which is a semi-urban area with approximately 2.3 million residents and nearly 1 million weekly visitors. Samples were collected from 7 wastewater treatment plant (WWTP) locations that collectively serve the vast majority of southern Nevada (excluding the small number of septic systems) and 1 manhole serving the southern portion of the Las Vegas Strip. With Las Vegas tourism returning to prepandemic levels in 2021, it was hypothesized that visitors were contributing a disproportionate fraction of SARS-CoV-2 RNA to the largest WWTP in southern Nevada, potentially confounding efforts to estimate COVID-19 incidence in the local community through WBE. Main Outcomes and Measures: Relative SARS-CoV-2 load and variants from visitors vs the local population. Results: The Omicron BA.1 VOC was detected in the Las Vegas Strip manhole approximately 1 week before its detection at the WWTP locations (December 13, 2021) and by clinical testing (December 14, 2021). On December 13, Omicron-specific mutations represented a mean (SD) of 48.0% (4.2%) of all genomes from the Las Vegas Strip manhole and 4.1% (1.4%) of all genomes at facilities 2 and 3; by December 20, Omicron-specific mutations represented means (SD) of 82.0% (3.0%) of all genomes at the Las Vegas Strip manhole and 48.0% (2.8%) of all genomes at facilities 2 and 3, respectively. During this time, it was estimated that visitors contributed more than 60% of the SARS-CoV-2 load to the sewershed serving the Las Vegas Strip and that Omicron prevalence among visitors was 40% to 60% on December 13 and 80% to 100% on December 20th. Conclusions and Relevance: Wastewater surveillance is a valuable complement to clinical tools and can provide time-sensitive data for decision-makers and policy makers. This study represents a novel approach for quantifying the confounding effects of mobile populations on wastewater surveillance data, thereby allowing for modification of an existing WBE framework for estimating COVID-19 incidence in southern Nevada.

Identification and genome sequencing of an influenza H3N2 variant in wastewater from elementary schools during a surge of influenza A cases in Las Vegas, Nevada.

Real-time surveillance of infectious diseases at schools or in communities is often hampered by delays in reporting due to resource limitations and infrastructure issues. By incorporating quantitative PCR and genome sequencing, wastewater surveillance has been an effective complement to public health surveillance at the community and building-scale for pathogens such as poliovirus, SARS-CoV-2, and even the monkeypox virus. In this study, we asked whether wastewater surveillance programs at elementary schools could be leveraged to detect RNA from influenza viruses shed in wastewater. We monitored for influenza A and B viral RNA in wastewater from six elementary schools from January to May 2022. Quantitative PCR led to the identification of influenza A viral RNA at three schools, which coincided with the lifting of COVID-19 restrictions and a surge in influenza A infections in Las Vegas, Nevada, USA. We performed genome sequencing of wastewater RNA, leading to the identification of a 2021-2022 vaccine-resistant influenza A (H3N2) 3C.2a1b.2a.2 subclade. We next tested wastewater samples from a treatment plant that serviced the elementary schools, but we were unable to detect the presence of influenza A/B RNA. Together, our results demonstrate the utility of near-source wastewater surveillance for the detection of local influenza transmission in schools, which has the potential to be investigated further with paired school-level influenza incidence data.

The Vitis vinifera C-repeat binding protein 4 (VvCBF4) transcriptional factor enhances freezing tolerance in wine grape.

Chilling and freezing can reduce significantly vine survival and fruit set in Vitis vinifera wine grape. To overcome such production losses, a recently identified grapevine C-repeat binding factor (CBF) gene, VvCBF4, was overexpressed in grape vine cv. 'Freedom' and found to improve freezing survival and reduced freezing-induced electrolyte leakage by up to 2 °C in non-cold-acclimated vines. In addition, overexpression of this transgene caused a reduced growth phenotype similar to that observed for CBF overexpression in Arabidopsis and other species. Both freezing tolerance and reduced growth phenotypes were manifested in a transgene dose-dependent manner. To understand the mechanistic basis of VvCBF4 transgene action, one transgenic line (9-12) was genotyped using microarray-based mRNA expression profiling. Forty-seven and 12 genes were identified in unstressed transgenic shoots with either a >1.5-fold increase or decrease in mRNA abundance, respectively. Comparison of mRNA changes with characterized CBF regulons in woody and herbaceous species revealed partial overlaps, suggesting that CBF-mediated cold acclimation responses are widely conserved. Putative VvCBF4-regulon targets included genes with functions in cell wall structure, lipid metabolism, epicuticular wax formation and stress-responses suggesting that the observed cold tolerance and dwarf phenotypes are the result of a complex network of diverse functional determinants.

SARS-CoV-2 variant detection at a university dormitory using wastewater genomic tools.

In the Fall of 2020, university campuses in the United States resumed on-campus instruction and implemented wastewater monitoring for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While quantitative polymerase chain reaction (qPCR) tests were deployed successfully to detect viral RNA in wastewater across campuses, the feasibility of detecting viral variants from a residential building like a dormitory was unclear. Here, we demonstrate that wastewater surveillance from a dormitory with at least three infected students could lead to the identification of viral genomes with more than 95% coverage. Our results indicate that viral variant detection from wastewater is achievable at a dormitory and that coronavirus disease 2019 (COVID-19) wastewater surveillance programs will benefit from the implementation of viral whole genome sequencing at universities.

Urban monitoring of antimicrobial resistance during a COVID-19 surge through wastewater surveillance.

During the early phase of the COVID-19 pandemic, infected patients presented with symptoms similar to bacterial pneumonias and were treated with antibiotics before confirmation of a bacterial or fungal co-infection. We reasoned that wastewater surveillance could reveal potential relationships between reduced antimicrobial stewardship, specifically misprescribing antibiotics to treat viral infections, and the occurrence of antimicrobial resistance (AMR) in an urban community. Here, we analyzed microbial communities and AMR profiles in sewage samples from a wastewater treatment plant (WWTP) and a community shelter in Las Vegas, Nevada during a COVID-19 surge in December 2020. Using a respiratory pathogen and AMR enrichment next-generation sequencing panel, we identified four major phyla in the wastewater, including Actinobacteria, Firmicutes, Bacteroidetes and Proteobacteria. Consistent with antibiotics that were reportedly used to treat COVID-19 infections (e.g., fluoroquinolones and beta-lactams), we also measured a significant spike in corresponding AMR genes in the wastewater samples. AMR genes associated with colistin resistance (mcr genes) were also identified exclusively at the WWTP, suggesting that multidrug resistant bacterial infections were being treated during this time. We next compared the Las Vegas sewage data to local 2018-2019 antibiograms, which are antimicrobial susceptibility profile reports about common clinical pathogens. Similar to the discovery of higher levels of beta-lactamase resistance genes in sewage during 2020, beta-lactam antibiotics accounted for 51 ± 3 % of reported antibiotics used in antimicrobial susceptibility tests of 2018-2019 clinical isolates. Our data highlight how wastewater-based epidemiology (WBE) can be leveraged to complement more traditional surveillance efforts by providing community-level data to help identify current and emerging AMR threats.

Patched and Costal-2 mutations lead to differences in tissue overgrowth autonomy.

Genetic screens are used in Drosophila melanogaster to identify genes key in the regulation of organismal development and growth. These screens have defined signalling pathways necessary for tissue and organismal development, which are evolutionarily conserved across species, including Drosophila. Here, we have used an FLP/FRT mosaic system to screen for conditional regulators of cell growth and cell division in the Drosophila eye. The conditional nature of this screen utilizes a block in the apoptotic pathway to prohibit the mosaic mutant cells from dying via apoptosis. From this screen, we identified two different mutants that mapped to the Hedgehog signalling pathway. Previously, we described a novel Ptc mutation and here we add to the understanding of disrupting the Hh pathway with a novel allele of Cos2. Both of these Hh components are negative regulators of the pathway, yet they depict mutant differences in the type of overgrowth created. Ptc mutations lead to overgrowth consisting of almost entirely wild-type tissue (non-autonomous overgrowth), while the Cos2 mutation results in tissue that is overgrown in both the mutant and wild-type clones (both autonomous and non-autonomous). These differences in tissue overgrowth are consistent in the Drosophila eye and wing. The observed difference is correlated with different deregulation patterns of pMad, the downstream effector of DPP signalling. This finding provides insight into pathway-specific differences that help to better understand intricacies of developmental processes and human diseases that result from deregulated Hedgehog signalling, such as basal cell carcinoma.

Use of wastewater surveillance for early detection of Alpha and Epsilon SARS-CoV-2 variants of concern and estimation of overall COVID-19 infection burden.

A decline in diagnostic testing for SARS-CoV-2 is expected to delay the tracking of COVID-19 variants of concern and interest in the United States. We hypothesize that wastewater surveillance programs provide an effective alternative for detecting emerging variants and assessing COVID-19 incidence, particularly when clinical surveillance is limited. Here, we analyzed SARS-CoV-2 RNA in wastewater from eight locations across Southern Nevada between March 2020 and April 2021. Trends in SARS-CoV-2 RNA concentrations (ranging from 4.3 log10 gc/L to 8.7 log10 gc/L) matched trends in confirmed COVID-19 incidence, but wastewater surveillance also highlighted several limitations with the clinical data. Amplicon-based whole genome sequencing (WGS) of 86 wastewater samples identified the B.1.1.7 (Alpha) and B.1.429 (Epsilon) lineages in December 2020, but clinical sequencing failed to identify the variants until January 2021, thereby demonstrating that 'pooled' wastewater samples can sometimes expedite variant detection. Also, by calibrating fecal shedding (11.4 log10 gc/infection) and wastewater surveillance data to reported seroprevalence, we estimate that ~38% of individuals in Southern Nevada had been infected by SARS-CoV-2 as of April 2021, which is significantly higher than the 10% of individuals confirmed through clinical testing. Sewershed-specific ascertainment ratios (i.e., X-fold infection undercounts) ranged from 1.0 to 7.7, potentially due to demographic differences. Our data underscore the growing application of wastewater surveillance in not only the identification and quantification of infectious agents, but also the detection of variants of concern that may be missed when diagnostic testing is limited or unavailable.

Identification of tissue-specific, abiotic stress-responsive gene expression patterns in wine grape (Vitis vinifera L.) based on curation and mining of large-scale EST data sets.

BACKGROUND: Abiotic stresses, such as water deficit and soil salinity, result in changes in physiology, nutrient use, and vegetative growth in vines, and ultimately, yield and flavor in berries of wine grape, Vitis vinifera L. Large-scale expressed sequence tags (ESTs) were generated, curated, and analyzed to identify major genetic determinants responsible for stress-adaptive responses. Although roots serve as the first site of perception and/or injury for many types of abiotic stress, EST sequencing in root tissues of wine grape exposed to abiotic stresses has been extremely limited to date. To overcome this limitation, large-scale EST sequencing was conducted from root tissues exposed to multiple abiotic stresses. RESULTS: A total of 62,236 expressed sequence tags (ESTs) were generated from leaf, berry, and root tissues from vines subjected to abiotic stresses and compared with 32,286 ESTs sequenced from 20 public cDNA libraries. Curation to correct annotation errors, clustering and assembly of the berry and leaf ESTs with currently available V. vinifera full-length transcripts and ESTs yielded a total of 13,278 unique sequences, with 2302 singletons and 10,976 mapped to V. vinifera gene models. Of these, 739 transcripts were found to have significant differential expression in stressed leaves and berries including 250 genes not described previously as being abiotic stress responsive. In a second analysis of 16,452 ESTs from a normalized root cDNA library derived from roots exposed to multiple, short-term, abiotic stresses, 135 genes with root-enriched expression patterns were identified on the basis of their relative EST abundance in roots relative to other tissues. CONCLUSIONS: The large-scale analysis of relative EST frequency counts among a diverse collection of 23 different cDNA libraries from leaf, berry, and root tissues of wine grape exposed to a variety of abiotic stress conditions revealed distinct, tissue-specific expression patterns, previously unrecognized stress-induced genes, and many novel genes with root-enriched mRNA expression for improving our understanding of root biology and manipulation of rootstock traits in wine grape. mRNA abundance estimates based on EST library-enriched expression patterns showed only modest correlations between microarray and quantitative, real-time reverse transcription-polymerase chain reaction (qRT-PCR) methods highlighting the need for deep-sequencing expression profiling methods.

De novo identification and targeted sequencing of SSRs efficiently fingerprints Sorghum bicolor sub-population identity.

Recent plant breeding studies of several species have demonstrated the utility of combining molecular assessments of genetic distance into trait-linked SNP genotyping during the development of parent lines to maximize yield gains due to heterosis. SSRs (Short Sequence Repeats) are the molecular marker of choice to determine genetic diversity, but the methods historically used to sequence them have been burdensome. The ability to analyze SSRs in a higher-throughput manner independent of laboratory conditions would increase their utility in molecular ecology, germplasm curation, and plant breeding programs worldwide. This project reports simple bioinformatics methods that can be used to generate genome-wide de novo SSRs in silico followed by targeted Next Generation Sequencing (NGS) validation of those that provide the most information about sub-population identity of a breeding line, which influences heterotic group selection. While these methods were optimized in sorghum [Sorghum bicolor (L.) Moench], they were developed to be applied to any species with a reference genome and high-coverage whole-genome sequencing data of individuals from the sub-populations to be characterized. An analysis of published sorghum genomes selected to represent its five main races (bicolor, caudatum, durra, kafir, and guinea; 75 accessions total) identified 130,120 SSR motifs. Average lengths were 23.8 bp and 95% were between 10 and 92 bp, making them suitable for NGS. Validation through targeted sequencing amplified 188 of 192 assayed SSR loci. Results highlighted the distinctness of accessions from the guinea sub-group margaritiferum from all other sorghum accessions, consistent with previous studies of nuclear and mitochondrial DNA. SSRs that efficiently fingerprinted margaritiferum individuals (Xgma1 -Xgma6) are presented. Developing similar fingerprints of other sub-populations (Xunr1 -Xunr182) was not possible due to the extensive admixture between them in the data set analyzed. In summary, these methods were able to fingerprint specific sub-populations when rates of admixture between them are low.

RNA-Seq used to identify ipsdienone reductase (IDONER): A novel monoterpene carbon-carbon double bond reductase central to Ips confusus pheromone production.

The pinyon ips beetle, Ips confusus (LeConte) is a highly destructive pest in pine forests in western North America. When colonizing a new host tree, I. confusus beetles coordinate a mass attack to overcome the tree's defenses using aggregation pheromones. Ips confusus, as with other Ips spp. beetles, biosynthesize ipsdienol and ipsenol in a specific enantiomeric blend and ratio as aggregation pheromones. While several of the initial steps in the pheromone biosynthetic pathway have been well defined, the final steps were unknown. We used comparative RNA-Seq analysis between fed and unfed male I. confusus midgut tissue to identify candidate genes involved in pheromone biosynthesis. The 12,995 potentially unique transcripts showed a clear separation based on feeding state. Differential expression analysis identified gene groups that were tightly connected. This analysis identified all known pheromone biosynthetic genes and suggested a novel monoterpene double bond reductase, ipsdienone reductase (IDONER), with pheromone biosynthetic gene expression patterns. IDONER cDNA was cloned, expressed, and functionally characterized. The coding DNA sequence has an ORF of 1101 nt with a predicted translation product of 336 amino acids. The enzyme has a molecular weight of 36.7 kDa with conserved motifs of the medium chain dehydrogenases/reductase (MDR) superfamily in the leukotriene B4 dehydrogenases/reductases (LTB4R) family. Tagged recombinant protein was expressed and purified. Enzyme assays and GC/MS analysis showed IDONER catalyzed the reduction of ipsdienone to form ipsenone. This study shows that IDONER is a monoterpene double bond reductase involved in I. confusus pheromone biosynthesis.

Mycoplasma agassizii, an opportunistic pathogen of tortoises, shows very little genetic variation across the Mojave and Sonoran Deserts.

Mycoplasma agassizii is a common cause of upper respiratory tract disease in Mojave desert tortoises (Gopherus agassizii). So far, only two strains of this bacterium have been sequenced, and very little is known about its patterns of genetic diversity. Understanding genetic variability of this pathogen is essential to implement conservation programs for their threatened, long-lived hosts. We used next generation sequencing to explore the genomic diversity of 86 cultured samples of M. agassizii collected from mostly healthy Mojave and Sonoran desert tortoises in 2011 and 2012. All samples with enough sequencing coverage exhibited a higher similarity to M. agassizii strain PS6T (collected in Las Vegas Valley, Nevada) than to strain 723 (collected in Sanibel Island, Florida). All eight genomes with a sequencing coverage over 2x were subjected to multiple analyses to detect single-nucleotide polymorphisms (SNPs). Strikingly, even though we detected 1373 SNPs between strains PS6T and 723, we did not detect any SNP between PS6T and our eight samples. Our whole genome analyses reveal that M. agassizii strain PS6T may be present across a wide geographic extent in healthy Mojave and Sonoran desert tortoises.

Genomic evidence for reinfection with SARS-CoV-2: a case study.

BACKGROUND: The degree of protective immunity conferred by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently unknown. As such, the possibility of reinfection with SARS-CoV-2 is not well understood. We describe an investigation of two instances of SARS-CoV-2 infection in the same individual. METHODS: A 25-year-old man who was a resident of Washoe County in the US state of Nevada presented to health authorities on two occasions with symptoms of viral infection, once at a community testing event in April, 2020, and a second time to primary care then hospital at the end of May and beginning of June, 2020. Nasopharyngeal swabs were obtained from the patient at each presentation and twice during follow-up. Nucleic acid amplification testing was done to confirm SARS-CoV-2 infection. We did next-generation sequencing of SARS-CoV-2 extracted from nasopharyngeal swabs. Sequence data were assessed by two different bioinformatic methodologies. A short tandem repeat marker was used for fragment analysis to confirm that samples from both infections came from the same individual. FINDINGS: The patient had two positive tests for SARS-CoV-2, the first on April 18, 2020, and the second on June 5, 2020, separated by two negative tests done during follow-up in May, 2020. Genomic analysis of SARS-CoV-2 showed genetically significant differences between each variant associated with each instance of infection. The second infection was symptomatically more severe than the first. INTERPRETATION: Genetic discordance of the two SARS-CoV-2 specimens was greater than could be accounted for by short-term in vivo evolution. These findings suggest that the patient was infected by SARS-CoV-2 on two separate occasions by a genetically distinct virus. Thus, previous exposure to SARS-CoV-2 might not guarantee total immunity in all cases. All individuals, whether previously diagnosed with COVID-19 or not, should take identical precautions to avoid infection with SARS-CoV-2. The implications of reinfections could be relevant for vaccine development and application. FUNDING: Nevada IDEA Network of Biomedical Research, and the National Institute of General Medical Sciences (National Institutes of Health).