Cellular RNA Content Research Articles

Genomic architecture constrains macromolecular allocation in dinoflagellates

Dinoflagellate genomes have a unique architecture that may constrain their physiological and biochemical responsiveness to environmental stressors. Here we quantified how nitrogen (N) starvation influenced macromolecular allocation and C:N:P of three photosynthetic marine dinoflagellates, representing different taxonomic classes and genome sizes. Dinoflagellates respond to nitrogen starvation by decreasing cellular nitrogen, protein and RNA content, but unlike many other eukaryotic phytoplankton examined RNA:protein is invariant. Additionally, 2 of the 3 species exhibit increases in cellular phosphorus and very little change in cellular carbon with N-starvation. As a consequence, N starvation induces moderate increases in C:N, but extreme decreases in N:P and C:P, relative to diatoms. Dinoflagellate DNA content relative to total C, N and P is much higher than similar sized diatoms, but similar to very small photosynthetic picoeukaryotes such as Ostreococcus. In aggregate these results indicate the accumulation of phosphate stores may be an important strategy employed by dinoflagellates to meet P requirements associated with the maintenance and replication of their large genomes.

T6A and ms2t6A Modified Nucleosides in Serum and Urine as Strong Candidate Biomarkers of COVID-19 Infection and Severity.

SARS-CoV-2 infection alters cellular RNA content. Cellular RNAs are chemically modified and eventually degraded, depositing modified nucleosides into extracellular fluids such as serum and urine. Here we searched for COVID-19-specific changes in modified nucleoside levels contained in serum and urine of 308 COVID-19 patients using liquid chromatography-mass spectrometry (LC-MS). We found that two modified nucleosides, N6-threonylcarbamoyladenosine (t6A) and 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A), were elevated in serum and urine of COVID-19 patients. Moreover, these levels were associated with symptom severity and decreased upon recovery from COVID-19. In addition, the elevation of similarly modified nucleosides was observed regardless of COVID-19 variants. These findings illuminate specific modified RNA nucleosides in the extracellular fluids as biomarkers for COVID-19 infection and severity.

Growth rate-dependent coordination of catabolism and anabolism in the archaeon Methanococcus maripaludis under phosphate limitation.

Catabolic and anabolic processes are finely coordinated in microorganisms to provide optimized fitness under varying environmental conditions. Understanding this coordination and the resulting physiological traits reveals fundamental strategies of microbial acclimation. Here, we characterized the system-level physiology of Methanococcus maripaludis, a niche-specialized methanogenic archaeon, at different dilution rates ranging from 0.09 to 0.003 h-1 in chemostat experiments under phosphate (i.e., anabolic) limitation. Phosphate was supplied as the limiting nutrient, while formate was supplied in excess as the catabolic substrate and carbon source. We observed a decoupling of catabolism and anabolism resulting in lower biomass yield relative to catabolically limited cells at the same dilution rates. In addition, the mass abundance of several coarse-grained proteome sectors (i.e., combined abundance of proteins grouped based on their function) exhibited a linear relationship with growth rate, mostly ribosomes and their biogenesis. Accordingly, cellular RNA content also correlated with growth rate. Although the methanogenesis proteome sector was invariant, the metabolic capacity for methanogenesis, measured as methane production rates immediately after transfer to batch culture, correlated with growth rate suggesting translationally independent regulation that allows cells to only increase catabolic activity under growth-permissible conditions. These observations are in stark contrast to the physiology of M. maripaludis under formate (i.e., catabolic) limitation, where cells keep an invariant proteome including ribosomal content and a high methanogenesis capacity across a wide range of growth rates. Our findings reveal that M. maripaludis employs fundamentally different strategies to coordinate global physiology during anabolic phosphate and catabolic formate limitation.

Reticular Dysgenesis-Associated Adenylate Kinase 2 Deficiency Impairs Purine Metabolism and Ribosomal Biogenesis during Myelopoiesis

Reticular Dysgenesis (RD) is a particularly grave form of severe combined immunodeficiency (SCID), characterized by maturation arrest of both myeloid and lymphoid lineages paired with sensorineural hearing loss. RD is caused by biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2). AK2 catalyzes the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) in the mitochondrial intermembrane space. Using a CRISPR/Cas9 AK2 biallelic knock out model in human hematopoietic stem and progenitor cells (HSPCs), we have shown that AK2 -/- HSPCs mimic the neutrophil maturation defect in RD patients. Mitochondrial respiration is compromised in AK2 -/- HSPCs, which leads to a decreased NAD +/NADH ratio resulting in reductive stress. Metabolomics analysis by LC-MS/MS showed a significant accumulation of AMP, along with increased AMP/ADP and AMP/ATP ratios in AK2 -/- HSPCs, suggesting that purine metabolism is compromised by AK2 deficiency. Purine metabolism defects, such as deficiencies in adenosine deaminase (ADA) and purine nucleotide phosphorylase (PNP), have long been recognized as a cause of SCID. Furthermore, pharmacological interference with purine metabolism is a highly effective antiproliferative strategy in cancer therapy. In this study, we sought to investigate whether impaired purine metabolism contributes to the myelopoietic defect caused by AK2 deficiency.ResultsWe explored how purine metabolism affects myelopoiesis by differentiating HSPCs in media containing no nucleosides (nucleoside-), mixed nucleosides (nucleoside+) or adenosine only (adenosine+). We observed no difference in proliferation or neutrophil maturation between nucleosides- and nucleoside+ media for both control and AK2 -/- HSPCs, suggesting that AK2 -/- HSPCs do not rely on exogenous nucleosides. Interestingly, control HSPCs cultured in adenosine+ media showed severe proliferation and neutrophil maturation defects that mimic AK2 deficiency, suggesting that purine imbalance is detrimental to myelopoiesis.Previous metabolomics analysis showed a significant accumulation of inosine monophosphate (IMP) in AK2 -/- HSPCs. Since IMP can be produced through AMP deamination by AMPD, we asked whether the IMP accumulation in AK2 -/- HSPCs is caused by converting excess AMP to IMP. An LC-MS/MS analysis showed that AMPD inhibitor (AMPDi) treatment significantly lowered IMP levels and increased AMP levels in AK2 -/- HSPCs, indicating that AMP deamination is a major source of IMP accumulation in AK2 -/- HSPCs. Furthermore, AMPDi treatment did not improve, but rather slightly aggravated neutrophil differentiation in AK2 -/- HSPCs, suggesting that the AK2 -/- neutrophil maturation defect is not caused by IMP accumulation. This raises the possibility that AK2 -/- HSPCs employ AMP deamination as a mechanism to curtail the toxicity of excess AMP.Since purine is a building block of RNA, and ribosomal RNA (rRNA) constitutes >85% of cellular RNA content, we asked whether rRNA synthesis is compromised by AK2 deficiency. Pyronin Y staining showed a significant decrease in rRNA content in AK2 -/- HSPCs. Nascent peptide synthesis rate was also decreased in AK2 -/- HSPCs, as quantified by OP-puromycin uptake. These findings are corroborated by RNA-seq analysis of AK2 -/- and control HSPCs, which showed that ribosomal subunits, ribosomal biogenesis and ribonucleoprotein complex assembly are among the top down-regulated pathways. The data suggest that defective purine metabolism in AK2 -/- HSPCs impairs ribosomal biogenesis and protein synthesis.ConclusionOur studies showed that purine imbalance in HSPCs impairs myeloid proliferation and neutrophil maturation. AK2 depletion in HSPCs leads to AMP accumulation and defective ribosomal biogenesis. AK2 -/- HSPCs convert excess AMP to IMP, possibly as a means to mitigate AMP toxicity. However, AMP deamination activities alone are not sufficient to lower AMP levels to those of control HSPCs. We are currently testing whether boosting 5'-nucleotidase activities (cNIA, cN1B and cNII) in AK2 -/- HSPCs can decrease AMP levels and rescue the neutrophil maturation defect. As purine metabolic defects are associated with diverse immune and non-immune abnormalities, further understanding of how purine metabolism governs differentiation of human HSPCs will enable us to develop novel therapeutic strategies for RD and other purine disorders. DisclosuresPorteus: CRISPR Therapeutics: Current equity holder in publicly-traded company; Allogene Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Versant Ventures: Consultancy; Ziopharm: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Graphite Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Morrison: Garuda Therapeutics: Other: founder and SAB member ; Kojin Therapeutics: Other: SAB member ; Frequency Therapeutics: Other: SAB member ; Ona Terapeutics: Other: SAB member ; Protein Fluidics: Other: SAB member .

Characterizing RNA stability genome-wide through combined analysis of PRO-seq and RNA-seq data

BackgroundThe concentrations of distinct types of RNA in cells result from a dynamic equilibrium between RNA synthesis and decay. Despite the critical importance of RNA decay rates, current approaches for measuring them are generally labor-intensive, limited in sensitivity, and/or disruptive to normal cellular processes. Here, we introduce a simple method for estimating relative RNA half-lives that is based on two standard and widely available high-throughput assays: Precision Run-On sequencing (PRO-seq) and RNA sequencing (RNA-seq).ResultsOur method treats PRO-seq as a measure of transcription rate and RNA-seq as a measure of RNA concentration, and estimates the rate of RNA decay required for a steady-state equilibrium. We show that this approach can be used to assay relative RNA half-lives genome-wide, with good accuracy and sensitivity for both coding and noncoding transcription units. Using a structural equation model (SEM), we test several features of transcription units, nearby DNA sequences, and nearby epigenomic marks for associations with RNA stability after controlling for their effects on transcription. We find that RNA splicing-related features are positively correlated with RNA stability, whereas features related to miRNA binding and DNA methylation are negatively correlated with RNA stability. Furthermore, we find that a measure based on U1 binding and polyadenylation sites distinguishes between unstable noncoding and stable coding transcripts but is not predictive of relative stability within the mRNA or lincRNA classes. We also identify several histone modifications that are associated with RNA stability.ConclusionWe introduce an approach for estimating the relative half-lives of individual RNAs. Together, our estimation method and systematic analysis shed light on the pervasive impacts of RNA stability on cellular RNA concentrations.

Proximity to injury, but neither number of nuclei nor ploidy define pathological adaptation and plasticity in cardiomyocytes

The adult mammalian heart consists of mononuclear and binuclear cardiomyocytes (CMs) with various ploidies. However, it remains unclear whether a variation in ploidy or number of nuclei is associated with distinct functions and injury responses in CMs, including regeneration. Therefore, we investigated transcriptomes and cellular as well as nuclear features of mononucleated and binucleated CMs in adult mouse hearts with and without injury. To be able to identify the role of ploidy we analyzed control and failing human ventricular CMs because human CMs show a larger and disease-sensitive degree of polyploidization.Using transgenic Myh6-H2BmCh to identify mononucleated and binucleated mouse CMs, we found that cellular volume and RNA content were similar in both. On average nuclei of mononuclear CMs showed a 2-fold higher ploidy, as compared to binuclear CMs indicating that most mononuclear CMs are tetraploid. After myocardial infarction mononucleated and binucleated CMs in the border zone of the lesion responded with hypertrophy and corresponding changes in gene expression, as well as a low level of induction of cell cycle gene expression. Human CMs allowed us to study a wide range of polyploidy spanning from 2n to 16n. Notably, basal as well as pathological gene expression signatures and programs in failing CMs proved to be independent of ploidy.In summary, gene expression profiles were induced in proximity to injury, but independent of number of nuclei or ploidy levels in CMs.

Nutrient Control of mRNA Translation.

The emergence of genome-wide analyses to interrogate cellular DNA, RNA, and protein content has revolutionized the study of control networks that mediate cellular homeostasis. mRNA translation represents the last step of genetic flow and primarily defines the proteome. Translational regulation is thus critical for gene expression, in particular under nutrient excess or deficiency. Until recently, it was unclear how the global effects of translational control are orchestrated by nutrient signaling pathways. An emerging concept of translational reprogramming addresses how to maintain the expression of specific proteins during nutrient stress by translation of selective mRNAs. In this review, we describe recent advances in our understanding of translational control principles; nutrient-sensing mechanisms; and their dysregulation in human diseases such as diabetes, cancer, and aging. The mechanistic understanding of translational regulation in response to different nutrient conditions may help identify potential dietary and therapeutic targets to improve human health.

Networking and Dynamic Switches in Biological Condensates

Cellular liquid-liquid phase separation (LLPS) plays a key role in the dynamics and function of RNA-protein condensates like stress granules. In this issue ofCell, Yang et al., Guillén-Boixet et al., and Sanders et al. use a combination of experiment and modeling to provide an exciting mechanistic insight into the relationship between stress granules and LLPS, for example, in the context of protein disorder, switchable interactions, graph theory, and multiple interacting dense phases.

Valine-Induced Isoleucine Starvation in Escherichia coli K-12 Studied by Spike-In Normalized RNA Sequencing.

Escherichia coli cells respond to a period of famine by globally reorganizing their gene expression. The changes are known as the stringent response, which is orchestrated by the alarmone ppGpp that binds directly to RNA polymerase. The resulting changes in gene expression are particularly well studied in the case of amino acid starvation. We used deep RNA sequencing in combination with spike-in cells to measure global changes in the transcriptome after valine-induced isoleucine starvation of a standard E. coli K12 strain. Owing to the whole-cell spike-in method that eliminates variations in RNA extraction efficiency between samples, we show that ribosomal RNA levels are reduced during isoleucine starvation and we quantify how the change in cellular RNA content affects estimates of gene regulation. Specifically, we show that standard data normalization relying on sample sequencing depth underestimates the number of down-regulated genes in the stringent response and overestimates the number of up-regulated genes by approximately 40%. The whole-cell spike-in method also made it possible to quantify how rapidly the pool of total messenger RNA (mRNA) decreases upon amino acid starvation. A principal component analysis showed that the first two components together described 69% of the variability of the data, underlining that large and highly coordinated regulons are at play in the stringent response. The induction of starvation by sudden addition of high valine concentrations provoked prominent regulatory responses outside of the expected ppGpp, RpoS, and Lrp regulons. This underlines the notion that with the high resolution possible in deep RNA sequencing analysis, any different starvation method (e.g., nitrogen-deprivation, removal of an amino acid from an auxotroph strain, or valine addition to E. coli K12 strains) will produce measurable variations in the stress response produced by the cells to cope with the specific treatment.

Fennel induces cytotoxic effects against testicular germ cells in mice; evidences for suppressed pre-implantation embryo development.

Foeniculum vulgare (FVE; fennel) is an aromatic plant belonging to Umbelliferae family, which is widely used in traditional societies because of its different pharmaceutical properties. To uncover the fennel-derived essential oil (FVEO)-induced effects on male reproductive potential, 24 mature male albino mice were divided into, control, 0.37, 0.75, and 1.5 mg kg-1 FVEO-received groups. Following 35 days, the animals were euthanized and the testicular tissue and sperm samples were collected. The histological alterations, tubular differentiation (TDI), spermiogenesis (SPI) indices, apoptosis ratio, and RNA damage of germinal cells were analyzed. Moreover, the sperm count, motility, viability, chromatin condensation, and DNA fragmentation were assessed. Finally, the pre-implantation embryo development including; the percentage of zygote, 2-cell embryos and blastocysts were assessed. Observations showed that the FVEO, dose dependently, increased histological damages, resulted in germ cells dissociation, depletion, nuclear shrinkage and significantly (P < .05) decreased tubular differentiation and spermiogenesis ratios. Moreover, the FVEO-received animals (more significantly in 1.5 mg kg-1 -received group) exhibited decreased sperm count, viability, and motility and represented enhanced percentage of sperms with decondensed chromatin and DNA fragmentation. Finally, the animals in FVEO-received group showed diminished zygote formation and represented decreased pre-implantation embryo development compared to control animals. In conclusion, our data showed that, FVEO albeit at higher doses, is able to adversely affect cellular DNA and RNA contents, which in turn is able to negatively affect the sperm count and morphology. All these impairments are able to negatively affect the fertilization potential as well as pre-implantation embryo development.

Sulfur restriction extends fission yeast chronological lifespan through Ecl1 family genes by downregulation of ribosome.

Nutritional restrictions such as calorie restrictions are known to increase the lifespan of various organisms. Here, we found that a restriction of sulfur extended the chronological lifespan (CLS) of the fission yeast Schizosaccharomyces pombe. The restriction decreased cellular size, RNA content, and ribosomal proteins and increased sporulation rate. These responses depended on Ecl1 family genes, the overexpression of which results in the extension of CLS. We also showed that the Zip1 transcription factor results in the sulfur restriction-dependent expression of the ecl1+ gene. We demonstrated that a decrease in ribosomal activity results in the extension of CLS. Based on these observations, we propose that sulfur restriction extends CLS through Ecl1 family genes in a ribosomal activity-dependent manner.

Total Cellular RNA Modulates Protein Activity.

RNA constitutes up to 20% of a cell's dry weight, corresponding to ∼20 mg/mL. This high concentration of RNA facilitates low-affinity protein-RNA quinary interactions, which may play an important role in facilitating and regulating biological processes. In the yeast Pichia pastoris, the level of ubiquitin-RNA colocalization increases when cells are grown in the presence of dextrose and methanol instead of methanol as the sole carbon source. Total RNA isolated from cells grown in methanol increases β-galactosidase activity relative to that seen with RNA isolated from cells grown in the presence of dextrose and methanol. Because the total cellular RNA content changes with growth medium, protein-RNA quinary interactions can alter in-cell protein biochemistry and may play an important role in cell adaptation, critical to many physiological and pathological states.

Active and Secretory IgA-Coated Bacterial Fractions Elucidate Dysbiosis in Clostridium difficile Infection.

The onset of Clostridiumdifficile infection (CDI) has been associated with treatment with wide-spectrum antibiotics. Antibiotic treatment alters the activity of gut commensals and may result in modified patterns of immune responses to pathogens. To study these mechanisms during CDI, we separated bacteria with high cellular RNA content (the active bacteria) and their inactive counterparts by fluorescence-activated cell sorting (FACS) of the fecal bacterial suspension. The gut dysbiosis due to the antibiotic treatment may result in modification of immune recognition of intestinal bacteria. The immune recognition patterns were assessed by FACS of bacterial fractions either coated or not with intestinal secretory immunoglobulin A (SIgA). We described the taxonomic distributions of these four bacterial fractions (active versus inactive and SIgA coated versus non-SIgA coated) by massive 16S rRNA gene amplicon sequencing and quantified the proportion of C.difficile toxin genes in the samples. The overall gut microbiome composition was more robustly influenced by antibiotics than by the C.difficile toxins. Bayesian networks revealed that the C.difficile cluster was preferentially SIgA coated during CDI. In contrast, in the CDI-negative group Fusobacterium was the characteristic genus of the SIgA-opsonized fraction. Lactobacillales and Clostridium cluster IV were mostly inactive in CDI-positive patients. In conclusion, although the proportion of C.difficile in the gut is very low, it is able to initiate infection during the gut dysbiosis caused by environmental stress (antibiotic treatment) as a consequence of decreased activity of the protective bacteria. IMPORTANCE C.difficile is a major enteric pathogen with worldwide distribution. Its expansion is associated with broad-spectrum antibiotics which disturb the normal gut microbiome. In this study, the DNA sequencing of highly active bacteria and bacteria opsonized by intestinal secretory immunoglobulin A (SIgA) separated from the whole bacterial community by FACS elucidated how the gut dysbiosis promotes C.difficile infection (CDI). Bacterial groups with inhibitory effects on C.difficile growth, such as Lactobacillales, were mostly inactive in the CDI patients. C.difficile was typical for the bacterial fraction opsonized by SIgA in patients with CDI, while Fusobacterium was characteristic for the SIgA-opsonized fraction of the controls. The study demonstrates that sequencing of specific bacterial fractions provides additional information about dysbiotic processes in the gut. The detected patterns have been confirmed with the whole patient cohort independently of the taxonomic differences detected in the nonfractionated microbiomes.

Regulation of ribosomal RNA expression across the lifespan is fine-tuned by maternal diet before implantation

Cells and organisms respond to nutrient deprivation by decreasing global rates of transcription, translation and DNA replication. To what extent such changes can be reversed is largely unknown. We examined the effect of maternal dietary restriction on RNA synthesis in the offspring. Low protein diet fed either throughout gestation or for the preimplantation period alone reduced cellular RNA content across fetal somatic tissues during challenge and increased it beyond controls in fetal and adult tissues after challenge release. Changes in transcription of ribosomal RNA, the major component of cellular RNA, were responsible for this phenotype as evidenced by matching alterations in RNA polymerase I density and DNA methylation at ribosomal DNA loci. Cellular levels of the ribosomal transcription factor Rrn3 mirrored the rRNA expression pattern. In cell culture experiments, Rrn3 overexpression reduced rDNA methylation and increased rRNA expression; the converse occurred after inhibition of Rrn3 activity. These observations define novel mechanism where poor nutrition before implantation irreversibly alters basal rates of rRNA transcription thereafter in a process mediated by rDNA methylation and Rrn3 factor.

土壤微生物总活性研究方法进展

PDF HTML阅读 XML下载 导出引用 引用提醒 土壤微生物总活性研究方法进展 DOI: 10.5846/stxb201410262093 作者: 作者单位: 中国科学院大学; Griffith University,中国科学院大学,中国科学院大学,南京师范大学,中国科学院大学,中国科学院大学,中国科学院大学 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金项目(41230750); 中国科学院战略先导专项B课题(XDB05010200) A reviewon the methods for measuring total microbial activity in soils Author: Affiliation: University of Chinese Academy of Sciences,,,,,,University of Chinese Academy of Sciences Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:微生物总活性是指在某一时段内微生物所有生命活动的总和,它直接决定着微生物行使生理、生态功能的能力,是微生物学研究的热点,也是难点。迄今为止,还没有建立直接测定微生物总活性的方法,只能用一些相关指标来间接反映它。目前常用的指标主要包括微生物的呼吸速率、生长速率以及胞内RNA含量等。与其它一些基质和环境相比,测定土壤中的微生物总活性更为困难。通过总结研究土壤微生物总活性常用的3种方法,在简略概括传统的土壤微生物呼吸测定法的基础上,详细介绍了放射性同位素标记法和RNA直接表征法的原理和操作流程,整理归纳了一些重要应用案例,比较分析了不同方法的优缺点,以期为选择研究土壤微生物总活性的适宜方法提供依据。 Abstract:Total microbial activity (TMA) in soils is vital for understanding the roles of microorganisms in ecosystem processes. It can be defined as the sum of physiological activities of all the microbes at a given moment. As TMA is difficult to measure directly, a series of proxies, such as respiration rates, growth rates, and cellular RNA concentration, have been proposed. Here, methods used to measure soil TMA are synthesized and compared. (1) Respiration may be the process most closely related to life activities. Thus, respiration rates are the most commonly used proxies of soil TMA. The main limitation is that current methods to determine respiration rates usually cannot accurately reflect actual respiration rates. When respiration rates are measured using CO2 production or O2 consumption rates, they indicate carbon mineralization or aerobic respiration rates, respectively. (2) Microbes with higher growth rates are usually more active. Thus, growth rates are also widely used to indicate soil TMA. As biomacromolecule synthesis is approximately proportional to microbial growth rates, incorporation of radioactive isotope labeled precursors (i.e., thymidine, leucine, and acetate) can be employed to estimate microbe growth rates. Generally, trace radioactively labeled precursors are added to slurries (traditional methods) or extracted microbial suspensions (Bååth's methods). After a brief incubation, microbes are killed and the corresponding biomacromolecules are extracted to measure their radioactivity. Thymidine and leucine incorporation are commonly used to measure heterotrophic bacterial growth rates, while acetate incorporation is used to estimate growth rates of saprotrophic fungi. Radioactive isotope labeling methods are robust tools to estimate the growth rates of soil microbes. However, one critical problem is that TMA includes both growth activity and non-growth activity, whereas these methods only reflect the former. (3) Evidently, none of the methods based on respiration rates or incorporation of radioactive isotope labeled precursors can accurately link microbial activity with their identities. However, this issue can be resolved through using methods based on RNA. RNA correlates closely with protein synthesis, which is involved in most metabolic processes. Therefore, RNA concentration is assumed an ideal indicator of microbial activity. Generally, mRNA can be used to indicate the activity of specific metabolic processes, including nitrogen fixation and denitrification, whereas rRNA is a proxy of soil TMA. As cellular concentrations of small subunit rRNA (SSU rRNA) are proportional to total cellular rRNA concentrations, SSU rRNA copies can serve as an indicator of soil TMA and the ratio of SSU rRNA copies to SSU rRNA gene copies can be used to determine average microbial activity in soil. Additionally, active microbial community structure can be illustrated using profiling methods, such as clone library, T-RFLP, and high-throughput sequencing, based on SSU rRNA. These approaches can simultaneously identify soil microbes and their activity via in situ measurements. However, there is still no adequate evidence to support the assertion that the methods based on SSU rRNA can accurately reflect microbial activity, especially for non-growth activity. In conclusion, none of these methods are perfect to determine soil TMA; and a combination of suitable methods should be selected for individual ecosystems. 参考文献 相似文献 引证文献

Cytotoxic effect of nanosilver particles on testicular tissue: Evidence for biochemical stress and Hsp70-2 protein expression.

Lastly, there are growing evidences that nanosilver (NS) particles highly induce cytotoxic impacts in vitro and in vivo. Here, we analyzed the dose dependent effect of NS on histological changes, biochemical alterations and endocrine statuses, sperm parameters as well as chaperone Hsp70-2 expression. NS particles (50-60nm) were administrated in 3 doses of 0.5, 1 and 5mg/kg, intraperitoneally, for 35 days. The 0.3mL normal saline was administrated in control-sham group. Histological alterations, sperm parameters, serum levels of LH, FSH and testosterone were evaluated. Germinal and Leydig cells RNA damage, Leydig cells steroidogenic foci, the testicular and sperm total antioxidant capacity (TAC), malondialdehyde (MDA), nitric oxide (NO) levels, immunohistochemical (IHC) expression and mRNA level of Hsp70-2 were analyzed. The NS, dose dependently, resulted in enhanced germinal cells degeneration, necrosis, seminiferous tubules atrophy and decreased serum levels of LH, FSH and testosterone. Elevated germinal and Leydig cells RNA damage associated with increased sperm abnormalities were observed in NS-treated groups. Expression of Hsp70-2 was up-regulated in 0.5mg/kg, while its expression was decreased in 1 and 5mg/kg NS-treated groups. Testicular and sperm TAC levels reduced. However, the MDA and NO levels significantly (P<0.05) increased in all NS-treated groups. No histological and biochemical changes were detected in control-sham group. In conclusion, the NS particles exert their pathological impact via affecting testicular antioxidant and endocrine statuses, which in turn lead to diminished expression of Hsp70-2. Ultimately, by this mechanism NS particles adversely impact the cellular RNA, DNA and protein contents.

Inhibition of hepatitis E virus replication by proteasome inhibitor is nonspecific.

The ubiquitin proteasome system plays important role in virus infection. A previous study showed that the proteasome inhibitor MG132 could potentially affect hepatitis E virus (HEV) replication. In this study, we found that MG132 could inhibit HEV and hepatitis C virus (HCV) replication-related luciferase activity in subgenomic models. Furthermore, treatment with MG132 in a HEV infectious model resulted in a dramatic reduction in the intracellular level of HEV RNA. Surprisingly, MG132 concurrently inhibited the expression of a luciferase gene used as a control as well as a wide range of host genes. Consistently, the total cellular RNA and protein content was concurrently reduced by MG132 treatment, suggesting a nonspecific antiviral effect.

Gene expression measurements normalized to cell number reveal large scale differences due to cell size changes, transcriptional amplification and transcriptional repression in CHO cells

Conventional approaches to differential gene expression comparisons assume equal cellular RNA content among experimental conditions. We demonstrate that this assumption should not be universally applied because total RNA yield from a set number of cells varies among experimental treatments of the same Chinese Hamster Ovary (CHO) cell line and among different CHO cell lines expressing recombinant proteins. Conventional normalization strategies mask these differences in cellular RNA content and, consequently, skew biological interpretation of differential expression results. On the contrary, normalization to synthetic spike-in RNA standards added proportional to cell numbers reveals these differences and allows detection of global transcriptional amplification/repression. We apply this normalization method to assess differential gene expression in cell lines of different sizes, as well as cells treated with a cell cycle inhibitor (CCI), an mTOR inhibitor (mTORI), or subjected to high osmolarity conditions. CCI treatment of CHO cells results in a cellular volume increase and global transcriptional amplification, while mTORI treatment causes global transcriptional repression without affecting cellular volume. Similarly to CCI treatment, high osmolarity increases cell size, total RNA content and antibody expression. Furthermore, we show the importance of spike-in normalization for studies involving multiple CHO cell lines and advocate normalization to spike-in controls prior to correlating gene expression to specific productivity (qP). Overall, our data support the need for cell number specific spike-in controls for all gene expression studies where cellular RNA content differs among experimental conditions.

Correction of gene expression data: Performance-dependency on inter-replicate and inter-treatment biases

This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies. For maximal reliability of analysis, therefore, comparisons should be performed at the cellular level. This could be accomplished using an appropriate correction method that can detect and remove the inter-treatment bias for cell-number. Based on inter-treatment variations of reference genes, we introduce an analytical approach to examine the suitability of correction methods by considering the inter-treatment bias as well as the inter-replicate variance, which allows use of the best correction method with minimum residual bias. Analyses of RNA sequencing and microarray data showed that the efficiencies of correction methods are influenced by the inter-treatment bias as well as the inter-replicate variance. Therefore, we recommend inspecting both of the bias sources in order to apply the most efficient correction method. As an alternative correction strategy, sequential application of different correction approaches is also advised.

An assessment of RNA content inPrymnesium parvum,Prymnesium polylepis,cf.Chattonellasp. andKarlodinium veneficumunder varying environmental conditions for calibrating an RNA microarray for species detection

Traditional methods of identification and enumeration can be somewhat ambiguous when identifying phytoplankton that requires electron microscopic examination to verify specific morphological features. Members of the genus Prymnesium (division Haptophyta), members of the Raphidophyceae and naked dinoflagellates are examples of such phytoplankton whose identification can be difficult. One alternative to traditional microscopy-based methods of identification is to use molecular protocols to detect target species. Methods that measure cellular DNA and RNA content can be used to estimate the number of cells present in a sample. This study investigated the variation of RNA yields in Prymnesium parvum, P.polylepis, cf. Chattonella sp. and Karlodinium veneficum cells grown under different light, temperature, salinity and inorganic nutrient conditions. This information was used to calibrate the signal intensity of a variety of oligonucleotide probes spotted onto the microarrays for the detection of toxic algae (MIDTAL), which is being developed to aid national monitoring agencies and to provide a faster means of identifying and quantifying harmful phytoplankton in water column samples.