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Despite the consideration of chromosomal mutations as the major cause of rifampicin (RIF) resistance in M. tuberculosis, the role of other mechanisms such as efflux pumps cannot be ruled out. We evaluated the role of four efflux pumps viz., MmpL2 (Rv0507), MmpL5 (Rv0676c), Rv0194 and Rv1250 in providing RIF resistance in M. tuberculosis. The real time expression of the efflux pumps was analyzed in 16 RIF resistant and 11 RIF susceptible clinical isolates of M. tuberculosis after exposure to RIF. Expression of efflux pumps in these isolates was also correlated with mutations in the rpoB gene and MICs of RIF in the presence and absence of efflux pump inhibitors. Under RIF stress, Rv0194 was induced in 8/16 (50%) RIF resistant and 2/11 (18%) RIF susceptible isolates; mmpL5 in 7/16 (44%) RIF resistant and 1/11 (9%) RIF susceptible isolates; Rv1250 in 4/16 (25%) RIF resistant and 2/11 (18%) RIF susceptible isolates; and mmpL2 was upregulated in 2/16 (12.5%) RIF resistant and 1/11 (9%) RIF susceptible isolates. This preliminary study did not find any association between Rv0194, MmpL2, MmpL5 and Rv1250 and RIF resistance. However, the overexpression of Rv0194 and mmpL5 in greater number of RIF resistant isolates as compared to RIF susceptible isolates and expression of Rv0194 in wild type (WT) resistant isolates suggests a need for further investigations. [ABSTRACT FROM AUTHOR]

Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus (Flaviviridae), is a causative agent of a severe neuroinfection. Recently, several flaviviruses have been shown to interact with host protein synthesis. In order to determine whether TBEV interacts with this host process in its natural target cells, we analysed de novo protein synthesis in a human cell line derived from cerebellar medulloblastoma (DAOY HTB-186). We observed a significant decrease in the rate of host protein synthesis, including the housekeeping genes HPRT1 and GAPDH and the known interferon-stimulated gene viperin. In addition, TBEV infection resulted in a specific decrease of RNA polymerase I (POLR1) transcripts, 18S and 28S rRNAs and their precursor, 45-47S pre-rRNA, but had no effect on the POLR3 transcribed 5S rRNA levels. To our knowledge, this is the first report of flavivirus-induced decrease of specifically POLR1 rRNA transcripts accompanied by host translational shut-off. [ABSTRACT FROM AUTHOR]

Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4) is threatening banana production worldwide. Despite quarantine efforts, the pathogen continues to spread; thus, early diagnosis plays an essential role for the proper execution of contingency plans. Here, we assess the accuracy of four PCR-based molecular methods described in the literature for the identification and detection of race 4 strains, including Subtropical (Foc STR4) and Tropical Race 4 causing Fusarium wilt of banana. We screened a total of 302 isolates using these four markers, and performed phylogenetic analyses, Vegetative Compatibility Group (VCG) testing, sequence comparison, and pathogenicity tests for selected isolates. Our results show that three out of the four markers tested are not reliable for identification of Foc STR4 and TR4, as DNA from isolates from Ecuador, pathogenic and nonpathogenic to banana, obtained from different banana cultivars, displayed cross-reaction with these methods; that is, false positives can occur during the diagnostic process for race 4. Phylogenetic analyses, VCG testing, sequence comparison, and pathogenicity tests suggest the presence of non-target F. oxysporum isolates that share genomic regions with pathogenic strains but lack true pathogenicity to banana. The findings of this work are of foremost importance for international regulatory agencies performing surveillance tests in pathogen-free areas using the current diagnostic methods. We suggest the use of a genetic locus possibly related to virulence, previously identified by T-DNA, and amplified with primers W2987F/ W2987R, for diagnosis of Foc TR4 as the most reliable alternative. We urge the adoption of a more holistic view in the study of F. oxysporum as a plant pathogen that considers the biology and diversity of the species for the development of better diagnostic tools. [ABSTRACT FROM AUTHOR]

Severe strains of Citrus tristeza virus (CTV) cause quick decline and stem pitting resulting in significant economic losses in citrus production. A immunocapture reverse-transcriptase loop-mediated amplification (IC-RT-LAMP) assay was developed in this study to detect the severe VT strains that are typically associated with severe CTV symptoms. The sensitivity of RT-LAMP assay was determined by ten-fold serial dilutions of CA-VT-AT39 RNA, in comparison to one-step RT-droplet digital (dd) PCR. RT-LAMP detected up to 0.002 ng RNA with an amplification time of 10:35 (min:sec.), equivalent to 11.3 copies as determined by one step RT-ddPCR. The RT-LAMP assay specifically detected CA-VT-AT39 RNA and did not cross react with other CTV genotypes tested (T36, T30, RB, S1 and T68). To facilitate rapid on-site detection, the RT-LAMP assay was improved by first capturing the CTV virions from citrus crude leaf sap using CTV-IgG (IC-RT-LAMP), thereby eliminating nucleic acid extraction steps. IC-RT-LAMP assay was optimized with two-fold dilutions of CTV-IgG ranging from 1:500 to 1:16,000. The IC-RT-LAMP assay detected the CA-VT-AT39 virions in all dilutions tested. The minimum amplification time was 6:45 (min:sec) with 1:500 and 1:1000 of CTV-IgG dilutions. The limit of detection of IC-RT-LAMP assay with crude leaf sap of CA-VT-AT39 was 1:320 with a maximum amplification time of 9:08 (min:sec). The IC-RT-LAMP assay was validated for VT genotype by comparing to IC-RT-qPCR using the CTV from 40 field tree samples. A 100% agreement was observed between tests, regardless of single or mixed infections of CTV VT with other genotypes. Therefore, the IC-RT-LAMP assay can serve as a useful tool in the management of potentially severe strains of CTV. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that acutely causes fever as well as severe joint and muscle pain. Chronic musculoskeletal pain persists in a substantial fraction of patients for months to years after the initial infection, yet we still have a poor understanding of what promotes chronic disease. While replicating virus has not been detected in joint-associated tissues of patients with persistent arthritis nor in various animal models at convalescent time points, viral RNA is detected months after acute infection. To identify the cells that might contribute to pathogenesis during this chronic phase, we developed a recombinant CHIKV that expresses Cre recombinase (CHIKV-3ʹ-Cre). CHIKV-3ʹ-Cre replicated in myoblasts and fibroblasts, and it induced arthritis during the acute phase in mice. Importantly, it also induced chronic disease, including persistent viral RNA and chronic myositis and synovitis similar to wild-type virus. CHIKV-3ʹ-Cre infection of tdTomato reporter mice resulted in a population of tdTomato+ cells that persisted for at least 112 days. Immunofluorescence and flow cytometric profiling revealed that these tdTomato+ cells predominantly were myofibers and dermal and muscle fibroblasts. Treatment with an antibody against Mxra8, a recently defined host receptor for CHIKV, reduced the number of tdTomato+ cells in the chronic phase and diminished the levels of chronic viral RNA, implicating these tdTomato+ cells as the reservoir of chronic viral RNA. Finally, isolation and flow cytometry-based sorting of the tdTomato+ fibroblasts from the skin and ankle and analysis for viral RNA revealed that the tdTomato+ cells harbor most of the persistent CHIKV RNA at chronic time points. Therefore, this CHIKV-3ʹ-Cre and tdTomato reporter mouse system identifies the cells that survive CHIKV infection in vivo and are enriched for persistent CHIKV RNA. This model represents a useful tool for studying CHIKV pathogenesis in the acute and chronic stages of disease. [ABSTRACT FROM AUTHOR]

Background: The quantitative fluorescent polymerase chain reaction (QF-PCR) has proven to be a reliable method for detection of common fetal chromosomal aneuploidies. However, there are some technical shortcomings, such as uncertainty of aneuploidy determination when the short tandem repeats (STR) height ratio is unusual due to a large size difference between alleles or failure due to the presence of maternal cell contamination (MCC). The aim of our study is to facilitate the implementation of the QF-PCR as a rapid diagnostic test for common fetal aneuploidies. Methods: Here, we describe an in-house one-tube multiplex QF-PCR method including 20 PCR markers (15 STR markers and 5 fixed size) for rapid prenatal diagnosis of chromosome 13, 18, 21, X and Y aneuploidies. In order to improve the aneuploidy classification of a given diallelic STR marker, we have employed a multilevel logistic regression analysis using "height-ratio" and "allele-size-difference" as fixed effects and "marker" as a random effect. We employed two regression models, one for the 2:1 height ratio (n = 48 genotypes) and another for the 1:2 height ratio (n = 41 genotypes) of the trisomic diallelic markers while using the same 9015 genotypes with normal 1:1 height ratio in both models. Furthermore, we have described a simple procedure for the treatment of the MCC, prior DNA isolation and QF-PCR analysis. Results: For both models, we have achieved 100% specificity for the marker aneuploidy classification as compared to 98.60% (2:1 ratio) and 98.04% (1:2 ratio) specificity when using only the height ratio for classification. Treatment of the MCC enables a successful diagnosis rate of 76% among truly contaminated amniotic fluids. Conclusions: Adjustment for the allele size difference and marker type improves the STR aneuploidy classification, which, complemented with appropriate treatment of contaminated amniotic fluids, eliminates sample re-testing and reinforces the robustness of the QF-PCR method for prenatal testing. [ABSTRACT FROM AUTHOR]

Background: Human papillomavirus 68 (HPV68) is a probable carcinogenic HPV genotype which is included in almost all HPV screening assays and exists as two genetically variable subtypes (HPV68a and HPV68b). Routine HPV sample testing has shown that the cobas 4800 HPV Test (Roche) provides higher false-negative rates for HPV68 status than PapilloCheck HPV-Screening (Greiner Bio-One). The aim of our study was to evaluate the efficacy of cobas 4800 in HPV68 detection. Methods: A total of 2,145 cervical/cervicovaginal samples from women aged 17–88 were tested for HPV68 status using the cobas 4800 and PapilloCheck HPV tests. Viral load was assessed by quantitative PCR in all of the HPV68-positive cases. HPV68a/b subtyping was performed with real-time PCR followed by high resolution melting curve analysis, and was subsequently confirmed by Sanger sequencing. Results: Cobas 4800 detected HPV positivity in only 13/33 HPV68 single-genotype infection cases. Viral load was comparable across both tested subgroups. HRM analysis and Sanger sequencing identified the HPV68a subtype in all of the 20 instances of cobas 4800 false negatives. HPV68a and HPV68b were detected in 3/13 and 10/13 cases identified as other HPV-positive by cobas 4800. Conclusion: The HPV68a subtype was missed by cobas 4800 in more than 85% of all HPV68a-positive cases. Therefore, commercially available assays may underestimate HPV68 prevalence. [ABSTRACT FROM AUTHOR]

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology. [ABSTRACT FROM AUTHOR]

Criniviruses accumulate in the phloem tissue and damage crops by reducing chlorophyll which is essential for plant growth and development. Tomato chlorosis crinivirus (ToCV) is vectored by several whitefly species that damage tomato crops throughout the world. In South Africa, ToCV is a poorly studied pathogen of global economic importance. Therefore, a national survey was initiated to investigate the occurrence and distribution of criniviruses infecting tomato crops in South Africa. Whitefly infested tomato crops exhibiting interveinal leaf chlorosis and chlorotic flecking symptoms were assayed for crinivirus infections using a multiplex reverse transcription polymerase reaction (RT-PCR) approach to assess for the presence of crinivirus species that are known to infect solanaceous hosts. Next-generation sequencing (NGS) was used to generate the complete genome of ToCV from South Africa. Results from the survey indicated that ToCV is presently the only crinivirus species infecting tomatoes in South Africa. Blast analysis showed that the RNA-1 segment of ToCV from South Africa (ToCR1-186) matched 99% to Spanish isolates. On the other hand, the RNA-2 (ToCR2-186) segment matched 98% to a South Korean isolate and three Spanish isolates. Although recombination events were not detected, phylogenetic studies showed inconsistencies in the grouping of RNA-1 and RNA-2 segments for some of the ToCV isolates analyzed in this study. Therefore, we suggest the possibility of intraspecific reassortment. This is the first comprehensive study and full genome sequence of ToCV from South Africa. The information generated from this study is intended to raise awareness of ToCV infections on tomato crops in South Africa. [ABSTRACT FROM AUTHOR]

Inbred laboratory mouse strains carry endogenous retroviruses (ERVs) classed as ecotropic, xenotropic or polytropic mouse leukemia viruses (E-, X- or P-MLVs). Some of these MLV ERVs produce infectious virus and/or contribute to the generation of intersubgroup recombinants. Analyses of selected mouse strains have linked the appearance of MLVs and virus-induced disease to the strain complement of MLV E-ERVs and to host genes that restrict MLVs, particularly Fv1. Here we screened inbred strain DNAs and genome assemblies to describe the distribution patterns of 45 MLV ERVs and Fv1 alleles in 58 classical inbred strains grouped in two ways: by common ancestry to describe ERV inheritance patterns, and by incidence of MLV-associated lymphomagenesis. Each strain carries a unique set of ERVs, and individual ERVs are present in 5–96% of the strains, often showing lineage-specific distributions. Two ERVs are alternatively present as full-length proviruses or solo long terminal repeats. High disease incidence strains carry the permissive Fv1n allele, tested strains have highly expressed E-ERVs and most have the Bxv1 X-ERV; these three features are not present together in any low-moderate disease strain. The P-ERVs previously implicated in P-MLV generation are not preferentially found in high leukemia strains, but the three Fv1 alleles that restrict inbred strain E-MLVs are found only in low-moderate leukemia strains. This dataset helps define the genetic basis of strain differences in spontaneous lymphomagenesis, describes the distribution of MLV ERVs in strains with shared ancestry, and should help annotate sequenced strain genomes for these insertionally polymorphic and functionally important proviruses. [ABSTRACT FROM AUTHOR]

Background and aims: Liver fibrosis is characterized by the excessive deposition of extracellular matrix (ECM) leading to impaired function and cirrhosis. Previous reports support a role for cadherin-11 (CDH11) in regulating the development of dermal and pulmonary fibrosis. In the current report, the extent to which CDH11 modulates the development of liver fibrosis induced by carbon tetrachloride (CCL4) was assessed. Methods: Wild type (WT) and CDH11 deficient (CDH11-/-) mice were treated with CCl4 or vehicle control for 8 weeks to induce liver fibrosis. Liver fibrosis was assessed by histology, collagen content, and RTPCR of fibrotic mediators. Results: Livers from WT mice treated with CCl4 had increased levels of CDH11 which localized to injured hepatocytes, hepatic stellate cells, and macrophages. Interestingly, CDH11-/- mice had decreased histological evidence of liver fibrosis, collagen deposition, α-smooth muscle actin (α-SMA) accumulation, and mRNA levels of fibrotic mediators such as Col1-α1, Snail, TGF-β and IL-6. Conclusions: These data demonstrate that CDH11 is increased during liver fibrosis, is an important regulator of liver fibrosis induced by CCL4 and suggest that CDH11 may be a potential therapeutic target for liver fibrosis. [ABSTRACT FROM AUTHOR]

p63 is a transcriptional regulator of ectodermal development that is required for basal cell proliferation and stem cell maintenance. p73 is a closely related p53 family member that is expressed in select p63-positive basal cells and can heterodimerize with p63. p73-/- mice lack multiciliated cells and have reduced numbers of basal epithelial cells in select tissues; however, the role of p73 in basal epithelial cells is unknown. Herein, we show that p73-deficient mice exhibit delayed wound healing despite morphologically normal-appearing skin. The delay in wound healing is accompanied by decreased proliferation and increased levels of biomarkers of the DNA damage response in basal keratinocytes at the epidermal wound edge. In wild-type mice, this same cell population exhibited increased p73 expression after wounding. Analyzing single-cell transcriptomic data, we found that p73 was expressed by epidermal and hair follicle stem cells, cell types required for wound healing. Moreover, we discovered that p73 isoforms expressed in the skin (ΔNp73) enhance p63-mediated expression of keratinocyte genes during cellular reprogramming from a mesenchymal to basal keratinocyte-like cell. We identified a set of 44 genes directly or indirectly regulated by ΔNp73 that are involved in skin development, cell junctions, cornification, proliferation, and wound healing. Our results establish a role for p73 in cutaneous wound healing through regulation of basal keratinocyte function. [ABSTRACT FROM AUTHOR]

Storage, manipulation and delivery of DNA fragments is crucial for synthetic biology applications, subsequently allowing organisms of interest to be engineered with genes or pathways to produce desirable phenotypes such as disease or drought resistance in plants, or for synthesis of a specific chemical product. However, DNA with high G+C content can be unstable in many host organisms including Saccharomyces cerevisiae. Here, we report the development of Sinorhizobium meliloti, a nitrogen-fixing plant symbioticα-Proteobacterium, as a novel host that can store DNA, and mobilize DNA to E. coli, S. cerevisiae, and the eukaryotic microalgae Phaeodactylum tricornutum. To achieve this, we deleted the hsdR restriction-system in multiple reduced genome strains of S. meliloti that enable DNA transformation with up to 1.4 x 105 and 2.1 x 103 CFU μg-1 of DNA efficiency using electroporation and a newly developed polyethylene glycol transformation method, respectively. Multi-host and multi-functional shuttle vectors (MHS) were constructed and stably propagated in S. meliloti, E. coli, S. cerevisiae, and P. tricornutum. We also developed protocols and demonstrated direct transfer of these MHS vectors via conjugation from S. meliloti to E. coli, S. cerevisiae, and P. tricornutum. The development of S. meliloti as a new host for inter-kingdom DNA transfer will be invaluable for synthetic biology research and applications, including the installation and study of genes and biosynthetic pathways into organisms of interest in industry and agriculture. [ABSTRACT FROM AUTHOR]

A recently introduced Multilocus Sequence Typing scheme for Treponema pallidum subsp. pallidum was applied to clinical samples collected from 2004 to 2017 from the two largest cities (Prague and Brno) in the Czech Republic. Altogether, a total of 675 samples were tested in this study and 281 of them were found PCR-positive for treponemal DNA and typeable. Most of the typed samples (n = 281) were swabs from primary or secondary syphilis lesions (n = 231), and only a minority were whole blood or tissue samples (n = 50). Swab samples from patients with rapid plasma regain (RPR) values of 1–1024 were more frequently PCR-positive (84.6%) compared to samples from patients with non-reactive RPR test (46.5%; p-value = 0.0001). Out of 281 typeable samples, 136 were fully-typed at all TP0136, TP0548, and TP0705 loci. Among the fully and partially typed samples, 25 different allelic profiles were identified. Altogether, eight novel allelic variants were found among fully (n = 5) and partially (n = 3) typed samples. The distribution of TPA allelic profiles identified in the Czech Republic from 2004 to 2017 revealed a dynamic character with allelic profiles disappearing and emerging over time. While the number of samples with the A2058G mutation was seen to increase (86.7% in 2016/2017), the number of samples harboring the A2059G mutation was found to have decreased over time (3.3% in 2016/2017). In addition, we found several allelic profile associations with macrolide resistance or susceptibility, the gender of patients, as well as patient residence. [ABSTRACT FROM AUTHOR]

Background: Global estimates for cholera annually approximate 4 million cases worldwide with 95,000 deaths. Recent outbreaks, including Haiti and Yemen, are reminders that cholera is still a global health concern. Cholera outbreaks can rapidly induce high death tolls by overwhelming the capacity of health facilities, especially in remote areas or areas of civil unrest. Recent studies demonstrated that stool specimens preserved on filter paper facilitate molecular analysis of Vibrio cholerae in resource limited settings. Specimens preserved in a rapid, low-cost, safe and sustainable manner for sequencing provides previously unavailable data about circulating cholera strains. This may ultimately provide new information to shape public policy response on cholera control and elimination. Methodology/Principal findings: Whole genome sequencing (WGS) recovered close to a complete sequence of the V. cholerae O1 genome with satisfactory genome coverage from stool specimens enriched in alkaline peptone water (APW) and V. cholerae culture isolates, both spotted on filter paper. The minimum concentration of V. cholerae DNA sufficient to produce quality genomic information was 0.02 ng/μL. The genomic data confirmed the presence or absence of genes of epidemiological interest, including cholera toxin and pilus loci. WGS identified a variety of diarrheal pathogens from APW-enriched specimen spotted filter paper, highlighting the potential for this technique to explore the gut microbiome, potentially identifying co-infections, which may impact the severity of disease. WGS demonstrated that these specimens fit within the current global cholera phylogenetic tree, identifying the strains as the 7th pandemic El Tor. Conclusions: WGS results allowed for mapping of short reads from APW-enriched specimen and culture isolate spotted filter papers this provided valuable molecular epidemiological sequence information on V. cholerae strains from remote, low-resource settings. These results identified the presence of co-infecting pathogens while providing rare insight into the specific V. cholerae strains causing outbreaks in cholera-endemic areas. [ABSTRACT FROM AUTHOR]

Development of a robust automated platform for enrichment of extracellular vesicles from low sample volume that matches the needs for next-generation sequencing could remove major hurdles for genomic biomarker discovery. Here, we document a protocol for urinary EVs enrichment by utilizing an automated microfluidic system, termed acoustic trap, followed by next-generation sequencing of microRNAs (miRNAs) for biomarker discovery. Specifically, we compared the sequencing output from two small RNA library preparations, NEXTFlex and CATS, using only 130 pg of input total RNA. The samples prepared using NEXTflex was found to contain larger number of unique miRNAs that was the predominant RNA species whereas rRNA was the dominant RNA species in CATS prepared samples. A strong correlation was found between the miRNA expressions of the acoustic trap technical replicate in the NEXTFlex prepared samples, as well as between the acoustic trap and ultracentrifugation enrichment methods. Together, these results demonstrate a robust and automated strategy for biomarker discovery from small volumes of urine. [ABSTRACT FROM AUTHOR]

Understanding the epidemiology of infectious diseases in a host population is a major challenge in forestry. Radiata pine plantations in New Zealand are impacted by a foliar disease, red needle cast (RNC), caused by Phytophthora pluvialis. This pathogen is dispersed by water splash with polycyclic infection affecting the lower part of the tree canopy. In this study, we extended an SI (Susceptible-Infectious) model presented for RNC to analyse the key epidemiological drivers. We conducted two experiments to empirically fit the extended model: a detached-needle assay and an in vivo inoculation. We used the detached-needle assay data to compare resistant and susceptible genotypes, and the in vivo inoculation data was used to inform sustained infection of the whole plant. We also compared isolations and real-time quantitative PCR (qPCR) to assess P. pluvialis infection. The primary infection rate and the incubation time were similar for susceptible and resistant genotypes. The pathogen death rate was 2.5 times higher for resistant than susceptible genotypes. Further, external proliferation of mycelium and sporangia were only observed on 28% of the resistant ramets compared to 90% of the susceptible ones. Detection methods were the single most important factor influencing parameter estimates of the model, giving qualitatively different epidemic outputs. In the early stages of infection, qPCR proved to be more efficient than isolations but the reverse was true at later points in time. Isolations were not influenced by the presence of lesions in the needles, while 19% of lesioned needle maximized qPCR detection. A primary infection peak identified via qPCR occurred at 4 days after inoculation (dai) with a secondary peak observed 22 dai. Our results have important implications to the management of RNC, by highlighting the main differences in the response of susceptible and resistant genotypes, and comparing the most common assessment methods to detect RNC epidemics. [ABSTRACT FROM AUTHOR]

Semen analysis is one of the standard diagnostic tools currently used to assess male infertility and reproductive toxicity. However, semen analysis has a limited ability to separate fertile from infertile populations. Additional methods to detect impaired fertility are needed. The purpose of the present study was to evaluate how spermatozoal RNA content varies with sociodemographic and behavior/lifestyle factors, and to determine if spermatozoal large and small RNAs discriminate normal from abnormal spermatozoa. Semen specimens were collected from 133 men aged between 18 to 55 years undergoing semen analysis as part of couple infertility evaluation while 10 proven fertile donors were recruited as control group. Semen samples were classified as normal or abnormal according to World Health Organization (WHO) 2010 criteria. Sperm RNAs were extracted after somatic cells were lysed, and the association of large or small RNA content with semen quality and sociodemographic and behavioral/lifestyle factors was evaluated using a generalized additive model and one-way ANOVA. Inverse relationship was observed between large RNA content and sperm parameters such as sperm count, density and motility. Large RNA content per sperm was significantly increased in semen samples showing abnormal number of round cells. Furthermore, sperm motility was inversely associated with spermatozoal small RNA contents. Grouping donors by the number of semen abnormalities, we observed significant increased spermatozoal large and small RNA content in men with more than two semen abnormalities. Alcohol consumption was strongly associated with increased large RNA per sperm concentration after adjustment for age and BMI. Our study demonstrates a strong relationship between spermatozoal large RNA content and poor semen characteristics that may lead to a role in the assessment of male fertility, and may be used as an endpoint for reproductive toxicology risk assessment. [ABSTRACT FROM AUTHOR]

Non-natural amino acids (nnAA) contain unique functional moieties that greatly expand the available tool set for protein engineering. But incorporation of nnAAs requires the function of an orthogonal aminoacyl tRNA synthetase/tRNA pair. Stable cell lines expressing these components have been shown capable of producing gram per liter levels of antibodies with nnAAs. However, little has been reported on the genetic makeup of these cells. To gain a better understanding of the minimal requirements for efficient nnAA incorporation we developed qPCR methods for the quantitation of the key components. Here we describe the development of qPCR assays for the quantification of tRNApyl and pylRS. qPCR was chosen because it provides a large dynamic range, has high specificity for its target, and is a non-radioactive method used routinely for cell line characterization. Designing assays for tRNAs present challenges due to their short length (~72 nucleotides) and high secondary structure. These tRNA assays have a ≥ 5 log dynamic range with the tRNApyl assays being able to discern the mature and unprocessed forms of the tRNApyl. Cell line analysis showed tRNApyl was expressed at higher levels than the CHO-K1 endogenous Met and Phe tRNAs and that >88% of tRNApyl was the mature form. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5’ end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester mir-122 resulted in profound loss of viral RNA in phase II clinical trials. However, some patients accumulated in their sera a viral RNA genome that contained a single cytidine to uridine mutation at the third nucleotide from the 5’ genomic end. It is shown here that this C3U variant indeed displayed higher rates of replication than that of wild-type HCV when miR-122 abundance is low in liver cells. However, when miR-122 abundance is high, binding of miR-122 to site 1, most proximal to the 5’ end in the C3U variant RNA, is impaired without disrupting the binding of miR-122 to site 2. As a result, C3U RNA displays a much lower rate of replication than wild-type mRNA when miR-122 abundance is high in the liver. This phenotype was accompanied by binding of a different set of cellular proteins to the 5’ end of the C3U RNA genome. In particular, binding of RNA helicase DDX6 was important for displaying the C3U RNA replication phenotype in liver cells. These findings suggest that sequestration of miR-122 leads to a resistance-associated mutation that has only been observed in treated patients so far, and raises the question about the function of the C3U variant in the peripheral blood. [ABSTRACT FROM AUTHOR]

The S. cerevisiae Pop2 protein is an exonuclease in the Ccr4-Not complex that is a conserved regulator of gene expression. Pop2 regulates gene expression post-transcriptionally by shortening the poly(A) tail of mRNA. A previous study has shown that Pop2 is phosphorylated at threonine 97 (T97) by Yak1 protein kinase in response to glucose limitation. However, the physiological importance of Pop2 phosphorylation remains unknown. In this study, we found that Pop2 is phosphorylated at serine 39 (S39) under unstressed conditions. The dephosphorylation of S39 was occurred rapidly after glucose depletion, and the addition of glucose to the glucose-deprived culture recovered this phosphorylation, suggesting that Pop2 phosphorylation at S39 is regulated by glucose. This glucose-regulated phosphorylation of Pop2 at S39 is dependent on Pho85 kinase. We previously reported that Pop2 takes a part in the cell wall integrity pathway by regulating LRG1 mRNA; however, S39 phosphorylation of Pop2 is not involved in LRG1 expression. On the other hand, Pop2 phosphorylation at S39 is involved in the expression of HSP12 and HSP26, which encode a small heat shock protein. In the medium supplemented with glucose, Pop2 might be phosphorylated at S39 by Pho85 kinase, and this phosphorylation contributes to repress the expression of HSP12 and HSP26. Glucose starvation inactivated Pho85, which resulted in the derepression of HSP12 and HSP26, together with other glucose sensing mechanisms. Our results suggest that Pho85-dependent phosphorylation of Pop2 is a part of the glucose sensing system in yeast. [ABSTRACT FROM AUTHOR]

Archival tissues represent a rich resource for clinical genomic studies, particularly when coupled with comprehensive medical records. Use of these in next generation sequencing (NGS) is a priority. Nine formalin-fixed paraffin-embedded (FFPE) DNA extraction methods were evaluated using twelve FFPE samples of varying tissue types. Quality assessment included total yield, percent dsDNA, fragment analysis and multiplex PCR. After assessment, three tissue types from four FFPE DNA methods were selected for NGS downstream evaluation, targeted and whole exome sequencing. In addition, two low input library protocols were evaluated for WES. Analysis revealed average coverage across the target regions for WES was ~20-30X for all four FFPE DNA extraction methods. For the targeted panels, the highest molecular tag coverage was obtained with the Kingfisher FFPE extraction method. The genotype concordance was 99% for the commonly called variant positions between all four extraction methods with the targeted PCR NGS panel and 96% with WES. Assessing quality of extracted DNA aids in selecting the optimal NGS approach, and the choice of both DNA extraction and library preparation approaches can impact the performance of archival tissue in NGS. [ABSTRACT FROM AUTHOR]

Human sample, from patients or healthy donors, are a valuable link between basic research and clinic. Especially in translational research, they play an essential role in understanding development and progression of diseases as well as in developing new diagnostic and therapeutic tools. Stored in biobanks, fast access to appropriate material becomes possible. However, biobanking in a clinical context faces several challenges. In practice, collecting samples during clinical routine does not allow to strictly adhere to protocols of sample collection in all aspects. This may influence sample quality to variable degrees. Time from sample draw to asservation is a variable factor, and influences of prolonged storage at ambient temperature of tissues are not well understood. We investigated whether delays between 5 minutes and 3 hours, and the use of RNAlater RNA-preserving reagent would lead to a relevant drop in sample quality, measured by quantitative mRNA expression analysis. Our findings suggest that even under ambient conditions, delays up to 3 hours do not have a major impact on sample quality as long as the tissue remains intact. [ABSTRACT FROM AUTHOR]

Sputum often contains large amounts of contaminating bacterial DNA that, if not eliminated during RNA isolation, may interfere with gene expression studies. During RNA isolation only repeated DNase treatment can effectively remove contaminating bacterial DNA from samples, but this compromises RNA quality. In this study we tested alternative methods to facilitate the removal of DNA and improve the quality of RNA obtained. Sputum samples obtained from patients with chronic obstructive pulmonary disease were processed with dithiothreitol and subjected to various RNA isolation methods, yet with modified protocols. Modifications included prolonged DNase treatment or vortexing of sputum cells in the presence of beads prior to RNA isolation. Bacterial DNA contamination was tested by PCR using universal bacterial primers, while RNA quality was assessed by real-time PCR using GAPDH primers for amplicons of different length. We found that the RNeasy Plus Mini kit equipped with the gDNA eliminator spin column was able to completely eliminate bacterial DNA, if sputum cells were lysed in the presence of bashing beads. Notably, compared with the standard protocol, the modified procedure yielded better quality RNA as well, as indicated by improved threshold profiles of qPCR. Bead vortexing of cells was less effective when combined with other RNA isolation methods, and the repeated DNase treatment needed to completely remove contaminating DNA from the samples reduced the quality of RNA markedly. Bead vortexing in combination with certain RNA extraction methods greatly facilitates the isolation of sputum RNA that is free of contaminating bacterial DNA, and is suitable for downstream applications. [ABSTRACT FROM AUTHOR]

Phosphorylation of the C-terminal domain (CTD) of the large subunit of human RNA polymerase II (Pol II) is regulated during the transcription cycle by the combined action of specific kinases and phosphatases. Pol II enters into the preinitiation complex (PIC) unphosphorylated, but is quickly phosphorylated by Cdk7 during initiation. How phosphatases alter the pattern and extent of CTD phosphorylation at this early stage of transcription is not clear. We previously demonstrated the functional association of an early-acting, magnesium-independent phosphatase with early elongation complexes. Here we show that Ssu72 is responsible for that activity. We found that the phosphatase enters the transcription cycle during the formation of PICs and that Ssu72 is physically associated with very early elongation complexes. The association of Ssu72 with elongation complexes was stable to extensive washing with up to 200 mM KCl. Interestingly, Ssu72 ceased to function on complexes that contained RNA longer than 28 nt. However, when PICs were washed before initiation, the strict cutoff at 28 nt was lost. This suggests that factor(s) are important for the specific regulation of Ssu72 function during the transition between initiation and pausing. Overall, our results demonstrate when Ssu72 can act on early transcription complexes and suggest that Ssu72 may also function in the PIC prior to initiation. [ABSTRACT FROM AUTHOR]

The coastal tailed frog (Ascaphus truei) is endemic to the Pacific Northwest of North America and is listed as a species of Special Concern under the Canadian Species at Risk Act. Its range is limited to British Columbia where it occurs widely west of the Coast Mountain Ranges extending north almost to the Alaskan Panhandle. The present study focused on surveying within the Cayoosh, Bridge (Shulaps), Seton, Anderson, Carpenter, and Downton Lake drainages. Four years of previous inventory efforts using conventional time-constrained search (TCS) methods detected tailed frog at 23/292 discrete sites (7.9% detection rate) in seven watersheds. Non-invasive environmental DNA (eDNA) methods hold promise for cryptic and low-abundance species detection. We rigorously validated a quantitative real-time polymerase chain reaction (qPCR)-based tool for detecting coastal tailed frog eDNA in water samples. This eASTR4 test is highly specific and sensitive. We applied a two-step targeted eDNA analysis approach on duplicate filtered water samples from a total of 72 sites collected over five days. The first IntegritE-DNA step mitigates false negative results and tests all DNA samples for the ability to support amplification from endogenous plant chloroplast DNA as a measure of sample viability. Three DNA samples failed this step even after inhibitor clean up suggesting that these samples were poor quality and not reliable for targeted species’ DNA analyses. All other DNA samples were deemed viable and were then tested for species-specific DNA. Coastal tailed frog eDNA was detected in 55/72 (76%) discrete stream reaches; nine sites with historical known occurrence were all eDNA positive. The false negative rate for TCS compared to eDNA methods was 58%. The results expand known coastal tailed frog distribution to 24 watersheds effectively more than tripling extant occurrences and confirm a previously suspected, apparently isolated coastal tailed frog metapopulation in the Shulaps drainage. [ABSTRACT FROM AUTHOR]

This study aimed to optimise techniques for whole transcriptome and small RNA analyses on clinical tissue samples from patients with cardiovascular disease. Clinical samples often represent a particular challenge to extracting RNA of sufficient quality for robust RNA sequencing analysis, and due to availability, it is rarely possible to optimise techniques on the samples themselves. Therefore, we have used equivalent samples from pigs undergoing cardiopulmonary bypass surgery to test different protocols for optimal RNA extraction, and then validated the protocols in human samples. Here we present an assessment of the quality and quantity of RNA obtained using a variety of commercially-available RNA extraction kits on both left ventricular biopsies and blood plasma. RNA extraction from these samples presents different difficulties; left ventricular biopsies are small and fibrous, while blood plasma has a low RNA content. We have validated our optimised extraction techniques on human clinical samples collected as part of the ARCADIA (Association of non-coding RNAs with Coronary Artery Disease and type 2 Diabetes) cohort study, resulting in successful whole transcriptome and small RNA sequencing of human left ventricular tissue. [ABSTRACT FROM AUTHOR]

Transcriptomic analysis of pulmonary microvascular endothelial cells from experimental models offers insight into pulmonary arterial hypertension (PAH) pathobiology. However, culturing may alter the molecular profile of endothelial cells prior to analysis, limiting the translational relevance of results. Here we present a novel and validated method for isolating RNA from pulmonary microvascular endothelial cells (PMVECs) ex vivo that does not require cell culturing. Initially, presumed rat PMVECs were isolated from rat peripheral lung tissue using tissue dissociation and enzymatic digestion, and cells were cultured until confluence to assess endothelial marker expression. Anti-CD31, anti-von Willebrand Factor, and anti-α-smooth muscle actin immunocytochemistry/immunofluorescence signal was detected in presumed rat PMVECs, but also in non-endothelial cell type controls. By contrast, flow cytometry using an anti-CD31 antibody and isolectin 1-B4 (from Griffonia simplicifolia) was highly specific for rat PMVECs. We next developed a strategy in which the addition of an immunomagnetic selection step for CD31+ cells permitted culture-free isolation of rat PMVECs ex vivo for RNA isolation and transcriptomic analysis using fluorescence-activated cell sorting. Heterogeneity in the validity and reproducibility of results using commercial antibodies against endothelial surface markers corresponded to a substantial burden on laboratory time, labor, and scientific budget. We demonstrate a novel protocol for the culture-free isolation and transcriptomic analysis of rat PMVECs with translational relevance to PAH. In doing so, we highlight wide variability in the quality of commonly used biological reagents, which emphasizes the importance of investigator-initiated validation of commercial biomaterials. [ABSTRACT FROM AUTHOR]

Biosurveillance is a proactive approach that may help to limit the spread of invasive fungal pathogens of trees, such as rust fungi which have caused some of the world’s most damaging diseases of pines and poplars. Most of these fungi have a complex life cycle, with up to five spore stages, which is completed on two different hosts. They have a biotrophic lifestyle and may be propagated by asymptomatic plant material, complicating their detection and identification. A bioinformatics approach, based on whole genome comparison, was used to identify genome regions that are unique to the white pine blister rust fungus, Cronartium ribicola, the poplar leaf rust fungi Melampsora medusae and Melampsora larici-populina or to members of either the Cronartium and Melampsora genera. Species- and genus-specific real-time PCR assays, targeting these unique regions, were designed with the aim of detecting each of these five taxonomic groups. In total, twelve assays were developed and tested over a wide range of samples, including different spore types, different infected plant parts on the pycnio-aecial or uredinio-telial host, and captured insect vectors. One hundred percent detection accuracy was achieved for the three targeted species and two genera with either a single assay or a combination of two assays. This proof of concept experiment on pine and poplar leaf rust fungi demonstrates that the genome-enhanced detection and identification approach can be translated into effective real-time PCR assays to monitor tree fungal pathogens. [ABSTRACT FROM AUTHOR]

Tripartite motif (TRIM) proteins belong to a large family with many roles in host biology, including restricting virus infection. Here, we found that TRIM2, which has been implicated in cases of Charcot–Marie–Tooth disease (CMTD) in humans, acts by blocking hemorrhagic fever New World arenavirus (NWA) entry into cells. We show that Trim2-knockout mice, as well as primary fibroblasts from a CMTD patient with mutations in TRIM2, are more highly infected by the NWAs Junín and Tacaribe virus than wild-type mice or cells are. Using mice with different Trim2 gene deletions and TRIM2 mutant constructs, we demonstrate that its antiviral activity is uniquely independent of the RING domain encoding ubiquitin ligase activity. Finally, we show that one member of the TRIM2 interactome, signal regulatory protein α (SIRPA), a known inhibitor of phagocytosis, also restricts NWA infection and conversely that TRIM2 limits phagocytosis of apoptotic cells. In addition to demonstrating a novel antiviral mechanism for TRIM proteins, these studies suggest that the NWA entry and phagocytosis pathways overlap. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) has been circulating in California since its first detection in 2003, causing repeated outbreaks affecting public, wildlife and veterinary health. Epidemics of WNV are difficult to predict due to the multitude of factors influencing transmission dynamics among avian and mosquito hosts. Typically, high levels of WNV amplification are required for outbreaks to occur, and therefore associated viral strains may exhibit enhanced virulence and mortality in competent bird species resulting in increased mosquito infection prevalence. In our previous study, most WNV isolates made from California during 2007–08 showed increased fitness when competed in House Finches (HOFI, Haemorhous mexicanus) and Culex tarsalis Coquillett mosquitoes against COAV997-5nt, a genetically marked recombinant virus derived from a 2003 California strain. Herein, we evaluated the competitive fitness of WNV strains isolated during California epidemics in 2004, 2005, 2007, 2011 and 2012 against COAV997-5nt. These outbreak isolates did not produce elevated mortality in HOFIs, but replicated more efficiently than did COAV997-5nt based on quantification of WNV RNA copies in sera, thereby demonstrating increased competitive fitness. Oral co-infections in Cx. tarsalis resulted in similar virus-specific infection and transmission rates, indicating that outbreak isolates did not have a fitness advantage over COAV997-5nt. Collectively, WNV isolates from outbreaks demonstrated relatively greater avian, but not vector, replicative fitness compared to COAV997-5nt, similar to previously characterized non-outbreak isolates of WNV. Our results indicated that ecological rather than viral factors may facilitate WNV amplification to outbreak levels, but monitoring viral phenotypes through competitive fitness studies may provide insight into altered replication and transmission potential among emerging WNV strains. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae (Spn) is an asymptomatic colonizer of the human nasopharynx but can also cause disease in the inner ear, meninges, lung and blood. Although various mechanisms contribute to the effective clearance of Spn, opsonophagocytosis by neutrophils is perhaps most critical. Upon phagocytosis, Spn is exposed to various degradative molecules, including a family of neutrophil serine proteases (NSPs) that are stored within intracellular granules. Despite the critical importance of NSPs in killing Spn, the bacterial proteins that are degraded by NSPs leading to Spn death are still unknown. In this report, we identify a 90kDa protein in a purified cell wall (CW) preparation, aminopeptidase N (PepN) that is degraded by the NSP neutrophil elastase (NE). Since PepN lacked a canonical signal sequence or LPxTG motif, we created a mutant expressing a FLAG tagged version of the protein and confirmed its localization to the CW compartment. We determined that not only is PepN a CW-localized protein, but also is a substrate of NE in the context of intact Spn cells. Furthermore, in comparison to wild-type TIGR4 Spn, a mutant strain lacking PepN demonstrated a significant hyper-resistance phenotype in vitro in the presence of purified NE as well as in opsonophagocytic assays with purified human neutrophils ex vivo. Taken together, this is the first study to demonstrate that PepN is a CW-localized protein and a substrate of NE that contributes to the effective killing of Spn by NSPs and human neutrophils. [ABSTRACT FROM AUTHOR]

The endophytic Streptomyces sp. VV/E1, and rhizosphere Streptomyces sp. VV/R4 strains, isolated from grapevine plants were shown in a previous work to reduce the infection rate of fungal pathogens involved in young grapevine decline. In this study we cloned fragments from randomly amplified polymorphic DNA (RAPD), and developed two stably diagnostic sequence-characterized amplified region (SCAR) markers of 182 and 160 bp for the VV/E1 and VV/R4 strains, respectively. The SCAR markers were not found in another 50 actinobacterial strains isolated from grapevine plants. Quantitative real-time PCR protocols based on the amplification of these SCAR markers were used for the detection and quantification of both strains in plant material. These strains were applied on young potted plants using two methods: perforation of the rootstock followed by injection of the microorganisms or soaking the root system in a bacterial suspension. Both methods were combined with a booster treatment by direct addition of a bacterial suspension to the soil near the root system. Analysis of uprooted plants showed that those inoculated by injection exhibited the highest rate of colonization. In contrast, direct addition of either strain to the soil did not lead to reliable colonization. This study has developed molecular tools for analyzing different methods for inoculating grapevine plants with selected Streptomyces sp. strains which protect them from fungal infections that enter through their root system. These tools are of great applied interest since they could easily be established in nurseries to produce grafted grapevine plants that are protected against fungal pathogens. Finally, this methodology might also be applied to other vascular plants for their colonization with beneficial biological control agents. [ABSTRACT FROM AUTHOR]

The western corn rootworm (WCR, Diabrotica virgifera virgifera) gene, dvssj1, is a putative homolog of the Drosophila melanogaster gene, snakeskin (ssk). This gene encodes a membrane protein associated with the smooth septate junction (SSJ) which is required for the proper barrier function of the epithelial lining of insect intestines. Disruption of DVSSJ integrity by RNAi technique has been shown previously to be an effective approach for corn rootworm control, by apparent suppression of production of DVSSJ1 protein leading to growth inhibition and mortality. To understand the mechanism that leads to the death of WCR larvae by dvssj1 double-stranded RNA, we examined the molecular characteristics associated with SSJ functions during larval development. Dvssj1 dsRNA diet feeding results in dose-dependent suppression of mRNA and protein; this impairs SSJ formation and barrier function of the midgut and results in larval mortality. These findings suggest that the malfunctioning of the SSJ complex in midgut triggered by dvssj1 silencing is the principal cause of WCR death. This study also illustrates that dvssj1 is a midgut-specific gene in WCR and its functions are consistent with biological functions described for ssk. [ABSTRACT FROM AUTHOR]

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage () can serve as a platform for further protocol customisation and community engagement. [ABSTRACT FROM AUTHOR]

While aberrant cells are routinely recognized and removed by immune cells, tumors eventually escape innate immune responses. Infiltrating immune cells are even corrupted by the tumor to acquire a tumor-supporting phenotype. In line, tumor-associated macrophages are well-characterized to promote tumor progression and high levels of tumor-infiltrating macrophages are a poor prognostic marker in breast cancer. Here, we aimed to further decipher the influence of macrophages on breast tumor cells and determined global gene expression changes in three-dimensional tumor spheroids upon infiltration of macrophages. While various tumor-associated mRNAs were upregulated, expression of the cytochrome P450 family member CYP1A1 was markedly attenuated. Repression of CYP1A1 in tumor cells was elicited by a macrophage-shaped tumor microenvironment rather than by direct tumor cell-macrophage contacts. In line with changes in RNA expression profiles, macrophages enhanced proliferation of the tumor cells. Enhanced proliferation and macrophage presence further correlated with reduced CYP1A1 expression in patient tumors when compared with normal tissue. These findings are of interest in the context of combinatory therapeutic approaches involving cytotoxic and immune-modulatory compounds. [ABSTRACT FROM AUTHOR]

Human Multipotent Stromal Cells (MSCs) are a valuable resource for regenerative medicine and are widely studied. They can be isolated from a variety of tissues and differentiate into multiple cell types (multi-potent). Many reports have been published using human MSCs and to be able to compare outcome, or be able to identify differences between MSCs, several cell surface markers have been proposed. Nevertheless, still many differences remain. Gene expression is known to be different between cell stage and origin. Furthermore, cells cultured on a culture dish (2D) show different gene expression profiles as compared to cells grown on scaffolds (3D). Even the RNA extraction method and the selection of genes used for normalisation have a role in gene expression profiling. To be able to compare gene expression data from samples cultured in different dimensions and RNA extracted using a variety of protocols we set out to define a set of reference genes suitable to normalise qPCR data from a very heterogeneous sample set. Hereto, Trizol was used to extract RNA from human MSCs cultured in 3D and 2D to validate newly designed and previously published primer sets. Subsequently, RNA from fresh human MSC samples and samples stored in RLT-buffer, Trizol or RNAlater was extracted using RNeasy and Trizol methods. All samples have been used to rank the candidate reference genes according to their stability after qPCR enabling identification of the most suitable reference gene(s) for normalisation of a heterogeneous sample set. The most stably expressed reference genes indicated superior normalisation of MSC marker gene expression over the least stable reference genes. [ABSTRACT FROM AUTHOR]

Positive-strand RNA viruses generally assemble RNA replication complexes on rearranged host membranes. Alphaviruses, other members of the alpha-like virus superfamily, and many other positive-strand RNA viruses invaginate host membrane into vesicular RNA replication compartments, known as spherules, whose interior is connected to the cytoplasm. Brome mosaic virus (BMV) and its close relative, cowpea chlorotic mottle virus (CCMV), form spherules along the endoplasmic reticulum. BMV spherule formation and RNA replication can be fully reconstituted in S. cerevisiae, enabling many studies identifying host factors and viral interactions essential for these processes. To better define and understand the conserved, core pathways of bromovirus RNA replication, we tested the ability of CCMV to similarly support spherule formation and RNA replication in yeast. Paralleling BMV, we found that CCMV RNA replication protein 1a was the only viral factor necessary to induce spherule membrane rearrangements and to recruit the viral 2a polymerase (2apol) to the endoplasmic reticulum. CCMV 1a and 2apol also replicated CCMV and BMV genomic RNA2, demonstrating core functionality of CCMV 1a and 2apol in yeast. However, while BMV and CCMV 1a/2apol strongly replicate each others’ genomic RNA3 in plants, neither supported detectable CCMV RNA3 replication in yeast. Moreover, in contrast to plant cells, in yeast CCMV 1a/2apol supported only limited replication of BMV RNA3 (<5% of that by BMV 1a/2apol). In keeping with this, we found that in yeast CCMV 1a was significantly impaired in recruiting BMV or CCMV RNA3 to the replication complex. Overall, we show that many 1a and 2apol functions essential for replication complex assembly, and their ability to be reconstituted in yeast, are conserved between BMV and CCMV. However, restrictions of CCMV RNA replication in yeast reveal previously unknown 1a-linked, RNA-selective host contributions to the essential early process of recruiting viral RNA templates to the replication complex. [ABSTRACT FROM AUTHOR]

Activation induced deaminase (AID) has two distinct and well defined roles, both relying on its deoxycytidine (dC) deaminating function: one as a DNA mutator and another in DNA demethylation. In chronic lymphocytic leukemia (CLL), AID was previously shown to be an independent negative prognostic factor. While there is substantial impact on DNA mutations, effects of AID on gene expression by promoter demethylation of disease related target genes in leukemia has not been addressed. To shed light on this question, we aimed at determining genome wide methylation changes as well as gene expression changes in response to AID expression in CLL. Although we found minor differences in individual methylation variable positions following AID expression, we could not find recurrent methylation changes of specific target sites or changes in global methylation. [ABSTRACT FROM AUTHOR]

The adhesion molecule and co-receptor of receptor tyrosine kinases, CD44, is expressed in all cells of the immune system, but also in numerous non-immune cells. CD44 plays roles in the cellular response to different pathogens. The molecular actions of CD44 during these processes are by and large still unknown. The CD44 molecule undergoes a sequential proteolytic cleavage which leads to the release of a soluble intracellular domain (CD44-ICD). Previous reports had shown that the CD44-ICD is taken up into the nucleus where it enhances transcription of specific target genes. By RNA profiling we identified a CD44-dependent transcriptional increase of interferon-responsive genes, among them IFI16. IFI16 is important in the innate immune response. It senses and binds pathogenic DNA and, together with cGAS, activates the cGAS-cGAMP-STING pathway and induces the expression of genes relevant for the response, e.g. IFN-β. Our results show that the enhancement of IFI16 expression depended on CD44 cleavage. A CD44-negative tumor cell line, embryonic fibroblasts and bone marrow-derived macrophages from cd44-/- mice were reduced in their response to IFN-γ, to viral DNA fragments and to Listeria monocytogenes infection. We could rescue the deficiency of CD44 negative RPM-MC cells and cd44-/- MEFs by expressing only the soluble CD44-ICD in the absence of any other CD44 domain. Expression of the CD44-ICD carrying a mutation that prevented the uptake into the nucleus, could not rescue the absence of CD44. This molecular aspect of regulation by CD44 may explain part of the immune phenotypes of mice with cd44 gene disruption. [ABSTRACT FROM AUTHOR]

Genome-wide DNA methylation studies are becoming increasingly important in unraveling the epigenetic basis of cell biology, aging and human conditions. The aim of the present study was to explore whether different methods for extracting DNA from whole blood can affect DNA methylation outcome, potentially confounding DNA methylation studies. DNA was isolated from healthy blood donors (n = 10) using three different extraction methods (i.e. two automatic extractions methods based on magnetic beads or isopropanol precipitation, and manual organic extraction). DNA methylation was analyzed using the Infinium HumanMethylation450 Bead Chip (Infinium 450K) (n = 30 samples in total), which is a frequently used method in genome-wide DNA methylation analyses. Overall, the different extraction methods did not have a significant impact on the global DNA methylation patterns. However, DNA methylation differences between organic extraction and each of the automated methods were in general larger than differences between the two automated extraction methods. No CpG sites or regions reached genome-wide significance when testing for differential methylation between extraction methods. Although this study is based on a small sample, these results suggest that extraction method is unlikely to confound Infinium 450K methylation analysis in whole blood. [ABSTRACT FROM AUTHOR]

Associated with the pathogenesis of many cancers, including brain tumors, microRNAs (miRNAs) present promising diagnostic biomarkers. These molecules have been also studied in cerebrospinal fluid (CSF), showing great potential as a diagnostic tool in patients with brain tumors. Even though there are some biological and technological factors that could affect the results and their biological and clinical interpretation, miRNA analysis in CSF is not fully standardized. This study aims to compare several RNA extraction and miRNA quantification approaches, including high-throughput technologies and individual miRNA detection methods, thereby contributing to the optimization and standardization of quantification of extracellular miRNAs in CSF. Such knowledge is essential for the potential use of miRNAs as diagnostic biomarkers in brain tumors. [ABSTRACT FROM AUTHOR]

The NA ecognition otif (RRM) is the most common RNA binding domain across eukaryotic proteins. It is therefore of great value to engineer its specificity to target RNAs of arbitrary sequence. This was recently achieved for the RRM in Rbfox protein, where four mutations R118D, E147R, N151S, and E152T were designed to target the precursor to the oncogenic miRNA 21. Here, we used a variety of molecular dynamics-based approaches to predict specific interactions at the binding interface. Overall, we have run approximately 50 microseconds of enhanced sampling and plain molecular dynamics simulations on the engineered complex as well as on the wild-type Rbfox·pre-miRNA 20b from which the mutated systems were designed. Comparison with the available NMR data on the wild type molecules (protein, RNA, and their complex) served to establish the accuracy of the calculations. Free energy calculations suggest that further improvements in affinity and selectivity are achieved by the S151T replacement. [ABSTRACT FROM AUTHOR]

In the current study, we characterized the expression and histone modification profiles of the alternative promoters found within imprinted Igf2r, Mest, Zac1, Peg3, Snrpn and non-imprinted Myc loci. In terms of expression pattern, the alternative promoters are highly tissue-specific, which is in a stark contrast to the ubiquitous expression of the corresponding main promoters. The alternative promoters are associated with the histone modification mark H3K4me1, but not with H3K4me3, which is frequently associated with the main promoters. Phylogenetic analyses also indicated that the majority of the alternative promoters are unique to the mammalian lineage, further suggesting the recent formation of these promoters during mammalian evolution. Overall, this study suggests that the alternative promoters of imprinted loci may have been derived from enhancers in recent evolutionary times and co-evolved with the genomic imprinting mechanism. [ABSTRACT FROM AUTHOR]

The family Arenaviridae comprises three genera, Mammarenavirus, Reptarenavirus and the most recently added Hartmanivirus. Arenaviruses have a bisegmented genome with ambisense coding strategy. For mammarenaviruses and reptarenaviruses the L segment encodes the Z protein (ZP) and the RNA-dependent RNA polymerase, and the S segment encodes the glycoprotein precursor and the nucleoprotein. Herein we report the full length genome and characterization of Haartman Institute Snake virus-1 (HISV-1), the putative type species of hartmaniviruses. The L segment of HISV-1 lacks an open-reading frame for ZP, and our analysis of purified HISV-1 particles by SDS-PAGE and electron microscopy further support the lack of ZP. Since we originally identified HISV-1 in co-infection with a reptarenavirus, one could hypothesize that co-infecting reptarenavirus provides the ZP to complement HISV-1. However, we observed that co-infection does not markedly affect the amount of hartmanivirus or reptarenavirus RNA released from infected cells in vitro, indicating that HISV-1 does not benefit from reptarenavirus ZP. Furthermore, we succeeded in generating a pure HISV-1 isolate showing the virus to replicate without ZP. Immunofluorescence and ultrastructural studies demonstrate that, unlike reptarenaviruses, HISV-1 does not produce the intracellular inclusion bodies typical for the reptarenavirus-induced boid inclusion body disease (BIBD). While we observed HISV-1 to be slightly cytopathic for cultured boid cells, the histological and immunohistological investigation of HISV-positive snakes showed no evidence of a pathological effect. The histological analyses also revealed that hartmaniviruses, unlike reptarenaviruses, have a limited tissue tropism. By nucleic acid sequencing, de novo genome assembly, and phylogenetic analyses we identified additional four hartmanivirus species. Finally, we screened 71 individuals from a collection of snakes with BIBD by RT-PCR and found 44 to carry hartmaniviruses. These findings suggest that harmaniviruses are common in captive snake populations, but their relevance and pathogenic potential needs yet to be revealed. [ABSTRACT FROM AUTHOR]

Background: The World Health Organization has targeted elimination of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) by 2020. Despite distinctive decline seen in the number of VL cases in ISC, there is still a quest for development of a diagnostic test which has the utility for detection of active infection and relapse cases and as a test of cure. The present study validated the sensitivity and specificity of SYBR Green I based closed tube LAMP assay reported by us for diagnosis of VL. Methodology: The validation study was carried out at two endemic sites in India, located at Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna and Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi. Standard operating protocols were provided at the two sites for applying LAMP assay on confirmed VL cases. The diagnostic accuracy of LAMP assay was evaluated by Receiver operator curve (ROC) analysis. Furthermore, a simplified LAMP assay based on direct blood lysis, DBL-LAMP, was developed and verified for its diagnostic accuracy. Principal findings: A total of 267 eligible participants were included in the study which comprised of 179 VL cases and 88 controls. Sensitivity and specificity of the LAMP assay were 98.32% (95% C.I– 95.2–99.7%) and 96.59% (95% C.I.-90.4–99.3%), respectively. ROC curve analysis depicted no significant difference between area under curve (AUCROC) for LAMP assay and rK39 RDT, indicative of LAMP as an excellent diagnostic test. DBL-LAMP assay, performed on 67 VL and 100 control samples, yielded a sensitivity of 93.05% (95% C.I- 84.75–97%) and specificity of 100% (95% C.I.- 96.30–100%). Conclusions/Significance: The validated closed tube LAMP for diagnosis of VL will provide impetus to the ongoing VL elimination programme in ISC. The assay based on direct blood lysis promotes its scope for application in field settings by further reducing time and cost. [ABSTRACT FROM AUTHOR]

During lytic replication of Kaposi’s sarcoma-associated herpesvirus (KSHV), a nuclear viral long noncoding RNA known as PAN RNA becomes the most abundant polyadenylated transcript in the cell. Knockout or knockdown of KSHV PAN RNA results in loss of late lytic viral gene expression and, consequently, reduction of progeny virion release from the cell. Here, we demonstrate that knockdown of PAN RNA from the related Rhesus macaque rhadinovirus (RRV) phenocopies that of KSHV PAN RNA. These two PAN RNA homologs, although lacking significant nucleotide sequence conservation, can functionally substitute for each other to rescue phenotypes associated with the absence of PAN RNA expression. Because PAN RNA is exclusively nuclear, previous studies suggested that it directly interacts with host and viral chromatin to modulate gene expression. We studied KSHV and RRV PAN RNA homologs using capture hybridization analysis of RNA targets (CHART) and observed their associations with host chromatin, but the loci differ between PAN RNA homologs. Accordingly, we find that KSHV PAN RNA is undetectable in chromatin following cell fractionation. Thus, modulation of gene expression at specific chromatin loci appears not to be the primary, nor the pertinent function of this viral long noncoding RNA. PAN RNA represents a cautionary tale for the investigation of RNA association with chromatin whereby cross-linking of DNA spatially adjacent to an abundant nuclear RNA gives the appearance of specific interactions. Similarly, PAN RNA expression does not affect viral transcription factor complex expression or activity, which is required for generation of the late lytic viral mRNAs. Rather, we provide evidence for an alternative model of PAN RNA function whereby knockdown of KSHV or RRV PAN RNA results in compromised nuclear mRNA export thereby reducing the cytoplasmic levels of viral mRNAs available for production of late lytic viral protein. [ABSTRACT FROM AUTHOR]

Introduction: Amyloidosis is caused by dysregulation of protein folding resulting in systemic or organ specific amyloid aggregation. When affecting the heart, amyloidosis can cause severe heart failure, which is associated with a high morbidity and mortality. Different subtypes of cardiac amyloidosis exist e.g. transthyretin cardiac amyloidosis and senile cardiac amyloidosis. Today, diagnostics is primarily based on cardiac biopsies and no clinically used circulating blood-based biomarkers existing. Therefore, our aim was to identify circulating microRNAs in patients with different forms of amyloidosis. Methods: Blood was collected from healthy subjects (n = 10), patients with reduced ejection fraction (EF < 35%; n = 10), patients affected by transthyretin cardiac amyloidosis (n = 13) as well as senile cardiac amyloidosis (n = 11). After performing TaqMan array profiling, promising candidates, in particular miR-99a-5p, miR-122-5p, miR-27a-3p, miR-221-3p, miR-1180-3p, miR-155-5p, miR-339-3p, miR-574-3p, miR-342-3p and miR-329-3p were validated via quantitative real time PCR. Results: The validation experiments revealed a significant upregulation of miR-339-3p in patients affected with senile cardiac amyloidosis compared to controls. This corresponded to the array profiling results. In contrast, there was no deregulation in the other patient groups. Conclusion: MiR-339-3p was increased in blood of patients with senile cardiac amyloidosis. Therefore, miR-339-3p is a potential candidate as biomarker for senile cardiac amyloidosis in future studies. Larger patient cohorts should be investigated. [ABSTRACT FROM AUTHOR]

Orientia tsutsugamushi (Ots) frequently causes severe scrub typhus infections in the Asia-Pacific region. Korean investigators have demonstrated that Ots encodes five different autotransporter domain (ATD) proteins (ScaA-ScaE). ScaA functions as an adhesin and confers protective immunity in a lethal mouse model of Ots infection. Specific antibodies are detected against ScaA and ScaC in Korean scrub typhus patients. However, there is limited data on the distribution of the Sca protein genes in diverse isolates of Ots. By BLAST analysis with the conserved beta barrel autotransporter domain (ATD) regions of the sca proteins, we discovered a sixth gene scaF among 3 of 10 new partial Ots genome sequences available at NCBI GenBank (Sido, Karp, AFSC7). We designed two to seven specific TaqMan assays to detect the ATD for each of the six sca genes. The TaqMan assays among those for each sca gene which gave the greatest sensitivity and linearity with DNA log dilutions were then used for screening DNAs from Ots isolates grown in L929 mouse cells for sca genes. The sca prevalence survey was performed for all six sca genes with 178 DNAs from isolates from 12 countries. The survey results were confirmed by conventional PCR with primers from conserved regions of the passenger domains (PD) and ATD of the sca proteins. The ATD was highly conserved between the DNAs of different genotypes compared to the sca PD but each TaqMan assay was sca specific. The percentage positivity for 56 kDa and scaA genes in the 178 DNAs using Ha primers was 59.6% and 62.4%, respectively. Our scaA conventional ATD PCR assay was positive in 98.3% but scaA was present in all 178 DNAs (100%) by ATD TaqMan. scaB, scaC, scaD, scaE and scaF were detected in 33.7%, 97.8%, 93.8%, 97.2% and 43.3% isolates by ATD TaqMan, respectively. The ATDs of Ots sca genes are thus sufficiently conserved between different genotypes for molecular assay design. Four sca genes are widely distributed among diverse Ots isolates from diverse geographical areas. scaB and scaF were detected in fewer Ots isolates and absent from some available genome sequences. Whether the utility of the ScaA, ScaC, ScaD, and ScaE antigenic passenger protein domains exceeds that of the mixed 56 kDa type surface antigens of Ots now used in combination diagnostic assays needs to be determined before they can be considered as suitable alternative serological antigens for diagnosis of scrub typhus. [ABSTRACT FROM AUTHOR]

Circular RNA is a newly discovered class of non-coding RNA generated through the back-splicing of linear pre-mRNA. In the current study, we characterized two circular RNAs that had been identified through NGS-based 5’RACE experiments. According to the results, the Peg3 locus contains a 214-nucleotide-long circular RNA, circPeg3, that is detected in low abundance from the neonatal brain, lung and ovary. In contrast, the Igf2r locus contains a group of highly abundant circular RNAs, circIgf2r, showing multiple forms with various exon combinations. In both cases, the expression patterns of circPeg3 and circIgf2r among individual tissues are quite different from their linear mRNA counterparts. This suggests potential unique roles played by the identified circular RNAs. Overall, this study reports the identification of novel circular RNAs specific to mammalian imprinted loci, suggesting that circular RNAs are likely involved in the function and regulation of imprinted genes. [ABSTRACT FROM AUTHOR]