Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of ...single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. We demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, we also show that targeting individual genes with multiple sgRNAs per cell improves efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Last, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.
The global significance of infective endocarditis (IE) caused by Staphylococcus aureus is unknown.
To document the international emergence of health care-associated S aureus IE and ...methicillin-resistant S aureus (MRSA) IE and to evaluate regional variation in patients with S aureus IE.
Prospective observational cohort study set in 39 medical centers in 16 countries. Participants were a population of 1779 patients with definite IE as defined by Duke criteria who were enrolled in the International Collaboration on Endocarditis-Prospective Cohort Study from June 2000 to December 2003.
In-hospital mortality.
S aureus was the most common pathogen among the 1779 cases of definite IE in the International Collaboration on Endocarditis Prospective-Cohort Study (558 patients, 31.4%). Health care-associated infection was the most common form of S aureus IE (218 patients, 39.1%), accounting for 25.9% (Australia/New Zealand) to 54.2% (Brazil) of cases. Most patients with health care-associated S aureus IE (131 patients, 60.1%) acquired the infection outside of the hospital. MRSA IE was more common in the United States (37.2%) and Brazil (37.5%) than in Europe/Middle East (23.7%) and Australia/New Zealand (15.5%, P<.001). Persistent bacteremia was independently associated with MRSA IE (odds ratio, 6.2; 95% confidence interval, 2.9-13.2). Patients in the United States were most likely to be hemodialysis dependent, to have diabetes, to have a presumed intravascular device source, to receive vancomycin, to be infected with MRSA, and to have persistent bacteremia (P<.001 for all comparisons).
S aureus is the leading cause of IE in many regions of the world. Characteristics of patients with S aureus IE vary significantly by region. Further studies are required to determine the causes of regional variation.
In the carnivorous plant family Lentibulariaceae, all three genome compartments (nuclear, chloroplast, and mitochondria) have some of the highest rates of nucleotide substitutions across angiosperms. ...While the genera Genlisea and Utricularia have the smallest known flowering plant nuclear genomes, the chloroplast genomes (cpDNA) are mostly structurally conserved except for deletion and/or pseudogenization of the NAD(P)H-dehydrogenase complex (ndh) genes known to be involved in stress conditions of low light or CO2 concentrations. In order to determine how the cpDNA are changing, and to better understand the evolutionary history within the Genlisea genus, we sequenced, assembled and analyzed complete cpDNA from six species (G. aurea, G. filiformis, G. pygmaea, G. repens, G. tuberosa and G. violacea) together with the publicly available G. margaretae cpDNA. In general, the cpDNA structure among the analyzed Genlisea species is highly similar. However, we found that the plastidial ndh genes underwent a progressive process of degradation similar to the other terrestrial Lentibulariaceae cpDNA analyzed to date, but in contrast to the aquatic species. Contrary to current thinking that the terrestrial environment is a more stressful environment and thus requiring the ndh genes, we provide evidence that in the Lentibulariaceae the terrestrial forms have progressive loss while the aquatic forms have the eleven plastidial ndh genes intact. Therefore, the Lentibulariaceae system provides an important opportunity to understand the evolutionary forces that govern the transition to an aquatic environment and may provide insight into how plants manage water stress at a genome scale.
In addition to the juxtaglomerular apparatus, renin is also synthesized in renal tubular epithelium, including the collecting duct (CD). Angiotensin (Ang) II differentially regulates the synthesis of ...juxtaglomerular (inhibition) and CD (stimulation) renin. Because diabetes mellitus, a disease with high intrarenal renin-Ang system and Ang II activity, is characterized by high prorenin levels, we hypothesized that the CD is the major source of prorenin in diabetes. Renin granular content was visualized using in vivo multiphoton microscopy of the kidney in diabetic Munich-Wistar rats. Diabetes caused a 3.5-fold increase in CD renin, in contrast to less pronounced juxtaglomerular changes. Ang II type 1 receptor blockade with Olmesartan reduced CD renin to control levels but significantly increased juxtaglomerular renin. Using a fluorogenic renin assay, the prorenin component of CD renin content was measured by assessing the difference in enzymatic activity of medullary homogenates before and after trypsin activation of prorenin. Trypsinization caused no change in control renin activity but a 5-fold increase in diabetes. Studies on a CD cell line (M1) showed a 22-fold increase in renin activity after trypsinization and a further 35-fold increase with Ang II treatment. Therefore, prorenin significantly contributes to baseline CD renin. Diabetes, possibly via Ang II, greatly stimulates CD prorenin and causes hyperplasia of renin-producing connecting segments. These novel findings suggest that, in a rat model of diabetes, prorenin content and release from the CD may be more important than the juxtaglomerular apparatus in contrast to the existing paradigm.
The prorenin receptor (PRR) was originally proposed to be a member of the renin-angiotensin system (RAS); however, recent work questioned their association. The present paper describes a functional ...link between the PRR and RAS in the renal juxtaglomerular apparatus (JGA), a classic anatomical site of the RAS. PRR expression was found in the sensory cells of the JGA, the macula densa (MD), and immunohistochemistry-localized PRR to the MD basolateral cell membrane in mouse, rat, and human kidneys. MD cell PRR activation led to MAP kinase ERK1/2 signaling and stimulation of PGE
release, the classic pathway of MD-mediated renin release. Exogenous renin or prorenin added to the in vitro microperfused JGA-induced acute renin release, which was inhibited by removing the MD or by the administration of a PRR decoy peptide. To test the function of MD PRR in vivo, we established a new mouse model with inducible conditional knockout (cKO) of the PRR in MD cells based on neural nitric oxide synthase-driven Cre-lox recombination. Deletion of the MD PRR significantly reduced blood pressure and plasma renin. Challenging the RAS by low-salt diet + captopril treatment caused further significant reductions in blood pressure, renal renin, cyclooxygenase-2, and microsomal PGE synthase expression in cKO vs. wild-type mice. These results suggest that the MD PRR is essential in a novel JGA short-loop feedback mechanism, which is integrated within the classic MD mechanism to control renin synthesis and release and to maintain blood pressure.
Messenger RNA (mRNA) localization and regulated translation can spatially restrict gene expression to each of the thousands of synaptic compartments formed by a single neuron. Although cis-acting RNA ...elements have been shown to direct localization of mRNAs from the soma into neuronal processes, less is known about signals that target transcripts specifically to synapses. In Aplysia sensory-motor neuronal cultures, synapse formation rapidly redistributes the mRNA encoding the peptide neurotransmitter sensorin from neuritic shafts into synapses. We find that the export of sensorin mRNA from soma to neurite and the localization to synapse are controlled by distinct signals. The 3' UTR is sufficient for export into distal neurites, whereas the 5' UTR is required for concentration of reporter mRNA at synapses. We have identified a 66-nt element in the 5' UTR of sensorin that is necessary and sufficient for synaptic mRNA localization. Mutational and chemical probing analyses are consistent with a role for secondary structure in this process.
Salzm. ex A.St.-Hil. & Girard (Lentibulariacea
) is a highly polymorphic carnivorous plant taxonomically rearranged many times throughout history. Herein, the complete chloroplast genomes (cpDNA) of ...three
morphotypes: purple-, white-, and yellow-flowered, were sequenced, compared, and putative markers for systematic, populations, and evolutionary studies were uncovered. In addition, RNA-Seq and RNA-editing analysis were employed for functional cpDNA evaluation. The cpDNA of three
morphotypes exhibits typical quadripartite structure. Fine-grained sequence comparison revealed a high degree of intraspecific genetic variability in all morphotypes, including an exclusive inversion in the
M and
N genes in
yellow. Phylogenetic analyses indicate that
morphotypes are monophyletic. Furthermore, in contrast to the terrestrial
cpDNA, the
morphotypes retain all the plastid NAD(P)H-dehydrogenase (
) complex genes. This observation supports the hypothesis that the
s in terrestrial
were independently lost and regained, also suggesting that different habitats (aquatic and terrestrial) are not related to the absence of
s gene repertoire as previously assumed. Moreover, RNA-Seq analyses recovered similar patterns, including nonsynonymous RNA-editing sites (e.g.,
and
). Collectively, our results bring new insights into the chloroplast genome architecture and evolution of the photosynthesis machinery in the Lentibulariaceae.
Endothelial intracellular calcium (Ca(2+)(i)) plays an important role in the function of the juxtaglomerular vasculature. The present studies aimed to identify the existence and molecular elements of ...an endothelial calcium wave in cultured glomerular endothelial cells (GENC). GENCs on glass coverslips were loaded with Fluo-4/Fura red, and ratiometric Ca(2+)(i) imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in Ca(2+)(i), which propagated from cell to cell throughout the monolayer (7.9 +/- 0.3 microm/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 microm. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced DeltaCa(2+)(i) by 50%. Importantly, the gap junction uncoupler alpha-glycyrrhetinic acid or knockdown of connexin 40 (Cx40) by transfecting GENCs with Cx40 short interfering RNA (siRNA) almost completely eliminated DeltaCa(2+)(i) and the calcium wave. Breakdown of extracellular ATP using a scavenger cocktail (apyrase and hexokinase) or nonselective inhibition of purinergic P2 receptors with suramin, had similar blocking effects. Scraping cells off along a line eliminated physical contact between cells but did not effect calcium wave propagation. Using an ATP biosensor technique, we detected a significant elevation in extracellular ATP (Delta = 76 +/- 2 microM) during calcium wave propagation, which was abolished by Cx40 siRNA treatment (Delta = 6 +/- 1 microM). These studies suggest that connexin 40 hemichannels and extracellular ATP are key molecular elements of the glomerular endothelial calcium wave, which may serve important juxtaglomerular functions.
Utricularia foliosa
L., commonly known as leafy bladderwort, is a widespread aquatic carnivorous plant from the Lentibulariaceae family. The species of the
Utricularia
and
Genlisea
genera are known ...to possess the smallest nuclear genomes across angiosperms. This study reveals that
U. foliosa
have a typical chloroplast genome of 150,851 bp in length, comprised of a large single-copy region (LSC) of 82,720 bp, a small single-copy region (SSC) of 17,481 bp, and inverted repeat regions (IRs) of 25,325 bp. A total of 139 genes, 88 of which are protein coding genes, 37 tRNA genes, eight rRNA genes and six pseudogenes were identified. All plastid NAD(P)H-dehydrogenase genes are present as intact copies. Phylogenetic analyses indicate that
U. foliosa
is closely related to other suspended or affixed aquatic species belonging to the
Utricularia
sect.
Utricularia
.
The people of Karakalpakstan, along with those of the entire Aral Sea region, are facing a multitude of health problems corresponding to the drying of the Aral Sea and accompanying ecological ...consequences. In case studies of other environmental disasters, research has shown that environmental exposures may impact not only the physiological but also the psychosocial health of individuals. This research aims to determine the contribution of the environmental disaster to the psychosocial health of people living in Karakalpakstan, a semi-autonomous Republic in Uzbekistan. An interview survey was carried out by Médecins Sans Frontières, with the assistance of the McMaster Institute of Environment and Health, local Universities and local health care workers, on a random sample of 1118 individuals aged 18 years and older in three communities in Karakalpakstan in May/June 1999. The communities were chosen according to distance from the former seashore, urban/rural characteristics and ethnic composition. The survey included questions about perceived general health, the General Health Questionnaire, the somatic symptom checklist of the Symptom Check List-90, questions about perceptions of the environmental disaster, social support as well as socio-demographic and socio-economic characteristics. Findings show that 41% of all respondents reported environmental concern while 48% reported levels of somatic symptoms (SCL-90) associated with emotional distress, above the normalized cut-point. Significant differences in levels of emotional distress were reported between men and women as well as between ethnic groups. Environmental problems are commonly perceived to be the cause of somatic symptoms and are significantly related to self-rated health status.
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