16S rRNA Levels Research Articles

Screening And Identification Of Bacterial Isolate Producing Arbutin Glycoside And Improving Its Productivity By Mutagenesis

From three sources of Iraqi soil, 30 bacterial isolates were obtained. after performing the primary isolation process, 12 isolates could produce the glycoside arbutin as an indication of the reddish-brown precipitate. When secondary isolation was conducted, it was found that two isolates, A7 and C10, gave the highest production of arbutin as a function of the concentration of the resulting compound, as it reached a concentration of Arbutin 5.3 µg/ml and 8.5 µg/ml, respectively. The diagnosis process was carried out for isolate C10 based on cultural and microscopic characteristics, as the nature of growth on the liquid medium was that it floated on the surface, and the cells formed a thin membrane with each other. Pellice as for the nature of growth on a liquid media, the colonies were spread out and flat, with irregular edges, a cream color, and a wrinkled surface, while the microscopic characteristics of the bacterial isolate were that they were positive for Gram stain and the cells were rod-shaped of medium length. The biochemical tests Vitec 2 results showed that the bacterial isolate belonged to Bacillus subtilis with a probability of 98%. Upon genetic diagnosis at the 16SrRNA level, it was found that the isolate belonged to the genus Bacillus subtilis by 100% compared to what is available in the NCBI GenBank and that exposure for 30 minutes at a wavelength of 257 nanometers (20w) and at a distance of (20) cm from the dish to ultraviolet radiation resulted in to improve the production of arbutin twice as much as the wild strain.

Unveiling the consequences of early human saliva contamination on membranes for guided bone regeneration.

GBR membranes have various surface properties designed to elicit positive responses in regenerative clinical procedures; dental clinicians attempt to employ techniques to prevent the direct interaction of contaminated oral fluids with these biomaterials. However, saliva is uninterruptedly exhibited in oral surgical procedures applying GBR membranes, suggesting a persistent interaction with biomaterials and the surrounding oral tissues. This fundamental study aimed to investigate potential alterations in the physical, chemical, and key biological properties of membranes for guided bone regeneration (GBR) caused by isolated early interaction with human saliva. A reproducible step-by-step protocol for collecting and interacting human saliva with membranes was developed. Subsequently, membranes were evaluated for their physicochemical properties, protein quantification, DNA, and 16S rRNA levels viability of two different cell lines at 1 and 7 days, and ALP activity. Non-interacted membranes and pure saliva of donors were applied as controls. Qualitative morphological alterations were noticed; DNA extraction and 16S quantification revealed significantly higher values. Furthermore, the viability of HGF-1 and MC3T3-E1 cells was significantly (p < .05) reduced following saliva interaction with biodegradable membranes. Saliva contamination did not prejudice PTFE membranes significantly in any biological assay. These outcomes demonstrated a susceptible response of biodegradable membranes to isolated early human saliva interaction, suggesting impairment of structural morphology, reduced viability to HGF-1 and MC3T3-E1, and higher absorption/adherence of DNA/16S rRNA. As a result, clinical oral procedures may need corresponding refinements.

The overlooked evolutionary dynamics of 16S rRNA revises its role as the “gold standard” for bacterial species identification

The role of 16S rRNA has been and largely remains crucial for the identification of microbial organisms. Although 16S rRNA could certainly be described as one of the most studied sequences ever, the current view of it remains somewhat ambiguous. While some consider 16S rRNA to be a variable marker with resolution power down to the strain level, others consider them to be living fossils that carry information about the origin of domains of cellular life. We show that 16S rRNA is clearly an evolutionarily very rigid sequence, making it a largely unique and irreplaceable marker, but its applicability beyond the genus level is highly limited. Interestingly, it seems that the evolutionary rigidity is not driven by functional constraints of the sequence (RNA–protein interactions), but rather results from the characteristics of the host organism. Our results suggest that, at least in some lineages, Horizontal Gene Transfer (HGT) within genera plays an important role for the evolutionary non-dynamics (stasis) of 16S rRNA. Such genera exhibit an apparent lack of diversification at the 16S rRNA level in comparison to the rest of a genome. However, why it is limited specifically and solely to 16S rRNA remains enigmatic.

Effects of In Utero EtOH Exposure on 18S Ribosomal RNA Processing: Contribution to Fetal Alcohol Spectrum Disorder.

Fetal alcohol spectrum disorders (FASD) are leading causes of neurodevelopmental disability. The mechanisms by which alcohol (EtOH) disrupts fetal brain development are incompletely understood, as are the genetic factors that modify individual vulnerability. Because the phenotype abnormalities of FASD are so varied and widespread, we investigated whether fetal exposure to EtOH disrupts ribosome biogenesis and the processing of pre-ribosomal RNAs and ribosome assembly, by determining the effect of exposure to EtOH on the developmental expression of 18S rRNA and its cleaved forms, members of a novel class of short non-coding RNAs (srRNAs). In vitro neuronal cultures and fetal brains (11-22 weeks) were collected according to an IRB-approved protocol. Twenty EtOH-exposed brains from the first and second trimester were compared with ten unexposed controls matched for gestational age and fetal gender. Twenty fetal-brain-derived exosomes (FB-Es) were isolated from matching maternal blood. RNA was isolated using Qiagen RNA isolation kits. Fetal brain srRNA expression was quantified by ddPCR. srRNAs were expressed in the human brain and FB-Es during fetal development. EtOH exposure slightly decreased srRNA expression (1.1-fold; p = 0.03). Addition of srRNAs to in vitro neuronal cultures inhibited EtOH-induced caspase-3 activation (1.6-fold, p = 0.002) and increased cell survival (4.7%, p = 0.034). The addition of exogenous srRNAs reversed the EtOH-mediated downregulation of srRNAs (2-fold, p = 0.002). EtOH exposure suppressed expression of srRNAs in the developing brain, increased activity of caspase-3, and inhibited neuronal survival. Exogenous srRNAs reversed this effect, possibly by stabilizing endogenous srRNAs, or by increasing the association of cellular proteins with srRNAs, modifying gene transcription. Finally, the reduction in 18S rRNA levels correlated closely with the reduction in fetal eye diameter, an anatomical hallmark of FASD. The findings suggest a potential mechanism for EtOH-mediated neurotoxicity via alterations in 18S rRNA processing and the use of FB-Es for early diagnosis of FASD. Ribosome biogenesis may be a novel target to ameliorate FASD in utero or after birth. These findings are consistent with observations that gene-environment interactions contribute to FASD vulnerability.

Selection and Evaluation of Reference Genes for RT-qPCR Analysis in Amorphophallus Konjac Based on Transcriptome Data.

Combined with the Konjac transcriptome database of our laboratory and internal reference genes commonly used in plants, the eight candidate internal reference genes were screened and detected. They are the 25S ribosomal RNA gene (25S rRNA), 18S ribosomal RNA gene (18S rRNA), actin gene (ACT), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), ubiquitin gene (UBQ), β-tubulin gene (β-TUB), eukaryotic elongation factor 1-αgene(eEF-1α), and eukaryotic translation initiation factor 4α-1 gene (eIF-4α). The results of GeNorm, Normfinder, and BestKeeper were analyzed comprehensively. The data showed that the expression levels of 25S rRNA, 18S rRNA, and ACT at the reproductive periods, eEF-1α and eIF-4α at the nutritional periods, and eEF-1α, UBQ, and ACT at different leaf developmental periods were stable. These identified and stable internal reference genes will provide the basis for the subsequent molecular biology-related studies of Konjac.

Ampicillin treated German cockroach extract leads to reduced inflammation in human lung cells and a mouse model of Asthma.

Cockroaches can cause allergic sensitization in humans via contact with their feces or frass. Antibiotics can affect concentration of major allergen and total bacteria production in German cockroaches (Blattella germanica). This study examined the ability of antibiotic-treated German cockroaches to induce allergic airway inflammation and the effect of antibiotics on their lipopolysaccharide and Bla g1, 2, and 5 expression levels. Specifically, we measured the ability of German cockroach extract (with or without prior antibiotic exposure) to induce allergic inflammation in human bronchial epithelial cells and a mouse model of asthma. Bacterial 16S rRNA and lipopolysaccharide levels were lower in ampicillin-treated cockroaches than in the control group. The Bla g1, Bla g2, and Bla g5 expression in ampicillin-treated cockroaches decreased at both the protein and RNA levels. In human bronchial epithelial cell lines BEAS-2B exposed to the ampicillin-treated extract, expression levels of interleukin-6 and interleukin-8 were lower than that in the control group. The total cell count and eosinophil count in bronchoalveolar lavage fluid was also lower in mice exposed to the ampicillin-treated extract than in those exposed to normal cockroach extract. Mouse lung histopathology showed reduced immune cell infiltration and mucus production in the ampicillin group. Our results showed that ampicillin treatment reduced the symbiont bacterial population and major allergen levels in German cockroaches, leading to reduced airway inflammation in mice. These results can facilitate the preparation of protein extracts for immunotherapy or diagnostics applications.

Urinary Eubacterium sp. CAG:581 Promotes Non-Muscle Invasive Bladder Cancer (NMIBC) Development through the ECM1/MMP9 Pathway.

Increasing evidence points to the urinary microbiota as a possible key susceptibility factor for early-stage bladder cancer (BCa) progression. However, the interpretation of its underlying mechanism is often insufficient, given that various environmental conditions have affected the composition of urinary microbiota. Herein, we sought to rule out confounding factors and clarify how urinary Eubacterium sp. CAG:581 promoted non-muscle invasive bladder cancer (NMIBC) development. Differentially abundant urinary microbiota of 51 NMIBC patients and 47 healthy controls (as Cohort 1) were first determined by metagenomics analysis. Then, we modeled the coculture of NMIBC organoids with candidate urinary Eubacterium sp. CAG:581 in anaerobic conditions and explored differentially expressed genes of these NMIBC tissues by RNA-Seq. Furthermore, we dissected the mechanisms involved into Eubacterium sp. CAG:581 by inducing extracellular matrix protein 1 (ECM1) and matrix metalloproteinase 9 (MMP9) upregulation. Finally, we used multivariate Cox modeling to investigate the clinical relevance of urinary Eubacterium sp. CAG:581 16S ribosomal RNA (16SrRNA) levels to the prognosis of 406 NMIBC patients (as Cohort 2). Eubacterium sp. CAG:581 infection accelerated the proliferation of NMIBC organoids (p < 0.01); ECM1 and MMP9 were the most upregulated genes induced by the increased colony forming units (CFU) gradient of Eubacterium sp. CAG:581 infection via phosphorylating ERK1/2 in NMIBC organoids of Cohort 1. Excluding the favorable impact of potential contributing factors, the ROC curve of Cohort 2 manifested its 3-year AUC value as 0.79 and the cut-off point of Eubacterium sp. CAG:581 16SrRNA as 10.3 (delta CT value). Our evidence suggests that urinary Eubacterium sp. CAG:581 promoted NMIBC progression through the ECM1/MMP9 pathway, which may serve as the promising noninvasive diagnostic biomarker for NMIBC.

Quantification of polyhydroxyalkanoate accumulated in waste activated sludge

Polyhydroxyalkanoate accumulation experiments at pilot scale were performed with fullscale municipal waste activated sludge. Development of biomass PHA content was quantified by thermogravimetric analysis. Over 48 h the biomass reached up to 0.49 ± 0.03 gPHA/gVSS (n=4). Samples were processed in parallel to characterise the distribution of PHA in the biomass. Selective staining methods and image analysis were performed by Confocal Laser Scanning Microscopy. The image analysis indicated that nominally 55% of this waste activated sludge was engaged in PHA storage activity. Thus even if the biomass PHA content reached 0.49gPHA/gVSS, the accumulating fraction of the biomass was estimated to have attained about 0.64gPHA/gVSS. The combination of quantitative microscopy and polymer mass assessment enabled to distinguish the effect of level of enrichment in PHA storing bacteria and the average PHA storage capacity of the accumulating bacteria. The distribution of microbial 16S rRNA levels did not follow a measurable trend during PHA accumulation.

Suppression of methanogenesis in paddy soil increases dimethylarsenate accumulation and the incidence of straighthead disease in rice

Some soil microbes can methylate arsenic (As) and produce dimethylarsenate (DMA) as a main product. Excessive accumulation of DMA by rice plants can cause the straighthead disease, a physiological disorder leading to substantial yield losses. DMA can also be demethylated in soil, but the mechanism and the microbes involved are not well understood. We investigated the dynamics of methylated As species, including monomethylarsenate (MMA), DMA and dimethyl-monothioarsenate (DMMTA), in three paddy soils that produced the straighthead disease. The soils were incubated under flooded conditions with or without the addition of 2-bromoethanesulfonate (BES), a specific inhibitor of methanogenesis. DMA and DMMTA concentrations in porewater increased initially as soil redox potential decreased, and then decreased rapidly coinciding with the production of methane. BES addition largely suppressed methanogenesis and the disappearance of DMA and DMMTA, but not of MMA. BES addition suppressed the transcript levels of archaeal 16S rRNA and, particularly, mcrA gene encoding methyl-coenzyme reductase subunit A. Among the core genera of archaea, the absolute abundances of Methanomassiliicoccus and Methanosarcina were decreased significantly by BES in the three soils. In a pot experiment with two soils, BES addition significantly increased DMA accumulation in rice husks and the incidence of the straighthead disease in rice. The results suggest that DMA and DMMTA demethylation in paddy soil is coupled to methanogenesis with Methanomassiliicoccus and Methanosarcina likely playing an important role.

A fully integrated rapid on-chip antibiotic susceptibility test – A case study for Mycobacterium smegmatis

Antibiotic resistance is one of the most pressing scientific and societal issues of our age. There is an urgent need to develop new diagnostic technologies which can quickly determine whether an infection is susceptible or resistant to different treatments so that rational antibiotic prescribing can take place. The main objective of the study was therefore to develop a rapid, simple, cost effective and comprehensive antibiotic susceptibility/resistance test based on rapid nucleic acid profiling. To do so, we integrated a microelectrode sensor within a microfluidic chip that combined bacterial incubation, lysis, and electrochemical detection chambers in a single simple set-up. As a case study, Mycobacterium smegmatis was investigated as a surrogate organism for Mycobacterium tuberculosis. The novelty of the work lies in developed capability of performing incubation, lysis, fragmentation, and detection process in a comprehensive yet simple lab-on-a-chip device called ‘MycoCHIP’. A gold microelectrode in combination with a specifically developed nucleic acid probe sequence for the 16SrRNA region of the mycobacterial genome were employed to monitor M. smegmatis nucleic acid sequences using Differential Pulse Voltammetry (DPV) and Square-Wave Voltammetry (SWV). The results demonstrated that it was possible to detect bacterial nucleic acid sequences and distinguish antibiotic incubated (Ab-i) cells from nonincubated (Ab-n) cells on MycoCHIP with a label-free molecular detection. The antibiotic susceptibility test showed that through measuring 16SrRNA levels from M. smegmatis,sensitivity to antibiotic was apparent after 24 h incubation, with a developed protocol representing a potential approach to determining antibiotic susceptibility more quickly, reliable and economically than current methods.

Maize RNA 3'-terminal phosphate cyclase-like protein promotes 18S pre-rRNA cleavage and is important for kernel development.

Plant ribosomes contain four specialized ribonucleic acids, the 5S, 5.8S, 18S, and 25S ribosomal RNAs (rRNAs). Maturation of the latter three rRNAs requires cooperative processing of a single transcript by several endonucleases and exonucleases at specific sites. In maize (Zea mays), the exact nucleases and components required for rRNA processing remain poorly understood. Here, we characterized a conserved RNA 3′-terminal phosphate cyclase (RCL)-like protein, RCL1, that functions in 18S rRNA maturation. RCL1 is highly expressed in the embryo and endosperm during early seed development. Loss of RCL1 function resulted in lethality due to aborted embryo cell differentiation. We also observed pleiotropic defects in the rcl1 endosperm, including abnormal basal transfer cell layer growth and aleurone cell identity, and reduced storage reserve accumulation. The rcl1 seeds had lower levels of mature 18S rRNA and the related precursors were altered in abundance compared with wild type. Analysis of transcript levels and protein accumulation in rcl1 revealed that the observed lower levels of zein and starch synthesis enzymes mainly resulted from effects at the transcriptional and translational levels, respectively. These results demonstrate that RCL1-mediated 18S pre-rRNA processing is essential for ribosome function and messenger RNA translation during maize seed development.

ВКЛАД mTORC1 И GSK-3 В РЕГУЛЯЦИЮ БИОГЕНЕЗА РИБОСОМ В ПОСТУРАЛЬНОЙ МЫШЦЕ КРЫСЫ В УСЛОВИЯХ МОДЕЛИРУЕМОЙ МИКРОГРАВИТАЦИИ

The investigation was aimed to reveal a potential role of mechanistic target of rapamycin (mTORC1) and glycogen synthase kinase (GSK-3) in the regulation of ribosome biogenesis and protein synthesis in rat soleus muscle during simulated gravitational unloading. Wistar rats were subject to 7-day tail-suspension (hindlimb unloading) and treated with rapamycin (mTORC1 inhibitor) or AR-A014418 (GSK-3 inhibitor). Protein synthesis was measured with SUnSET. Phosphorylation of glycogen synthase-1 (GS-1) and ribosomal protein S6 (rpS6) was measured with the use of Western-blotting. Levels of 28S and 18S rRNA were determined using agarose gel electrophoresis. Expression of 47S pre-rRNA and c-Myc was assessed with RT-PCR. Gravitational unloading led to an increase in phosphorylation of GS-1 (a marker of GSK-3 activity) and inhibited phosphorylation of rpS6 (a marker of mTORC1 activity), expression of the ribosome biogenesis markers (total RNA РНК, 28S and 18S rRNA, 47S pre-rRNA, с-Myc) and protein synthesis in soleus muscle. GSK-3 inhibition prevented an increase in GS-1 phosphorylation and, partly, a reduction in the markers of both ribosomal biogenesis and protein synthesis. The mTORC1 inhibitor contributed to the phospho-rpS6 reduction but did not substantially influence the parameters of translational capacity and protein synthesis. To sum up, unlike mTORC1, an elevated GSK-3 activity can contribute to the inhibition of ribosome biogenesis and protein synthesis in rat soleus muscle under 7-day gravitational unloading.

Correlation between Lactobacillus and expression of E-cadherin, β-catenin, N-cadherin, and Vimentin in postmenopausal cervical lesions.

To detect the correlation between Lactobacillus vaginalis and the expression of epithelial-mesenchymal transition (EMT)-related factors, E-cadherin, β-catenin, N-cadherin, and Vimentin, in postmenopausal cervical squamous intraepithelial lesions (SILs) and cervical squamous cell carcinoma (SCC), and to explore the possible mechanism. From January 2016 to January 2020, 30 postmenopausal patients with low-grade SIL (LSIL), 18 patients with high-grade SIL (HSIL), and 30 patients with SCC who underwent colposcopy biopsy in the Outpatient Department of the First Affiliated Hospital of Inner Mongolia Medical University were selected as the experimental group, and 30 postmenopausal normal women were selected as the control group. The expression of 16SrRNA of Lactobacillus vaginalis in each group was determined by the 16S third-generation full-length amplification sequencing technique. The mRNA expression levels of E-cadherin, β-catenin, N-cadherin, and Vimentin were detected by quantitative real-time polymerase chain reaction (qPCR). The correlation between the 16SrRNA expression level of Lactobacillus vaginalis and the mRNA expression level of the EMT-related proteins was compared among all groups. (I) The progression of postmenopausal cervical SILs to cervical SCC was significantly positively correlated with age, number of pregnancies, smoking, pH value, positive rate of HPV16, and negatively correlated with total Lactobacillus 16SrRNA expression (P<0.0001). (II) The level of vaginal microbiota in postmenopausal women showed that Lactobacillus iners was dominant. With the progression of the disease, the expression levels of 16SrRNA in Lactobacillus iners and Lactobacillus total vagina decreased gradually, and the differences were statistically significant (P<0.05). (III) With the disease progresses. The expression of total Lactobacillus 16SrRNA was positively correlated with the mRNA expression of β-catenin and E-cadherin (r>0; P<0.05), and negatively correlated with the mRNA expression of Vimentin and N-cadherin (r<0; P<0.05). In postmenopausal women, Lactobacillus vaginalis interacts with HPV and is associated with the occurrence of EMT, promoting the development of cervical lesions.

Circulating Microbial Signatures and Cardiovascular Death in Patients With ESRD

IntroductionPatients with end-stage renal disease (ESRD) experience disproportionately high cardiovascular morbidity and mortality. Accumulating evidence suggests a role for the circulating microbiome in the pathogenesis of cardiovascular disease; however, little is known about its association with premature cardiovascular mortality in ESRD.MethodsIn a pilot case-control study of 17 hemodialysis patients who died of a cardiovascular event and 17 matched hemodialysis controls who remained alive during a median follow-up of 2.0 years, we compared the levels and composition of circulating microbiome, including Bacteria, Archaea, and Fungi, in serum samples by quantitative polymerase chain reaction and 16S or Internal Transcribed Spacer (ITS) ribosomal RNA (rRNA) sequencing, respectively. Associations of the circulating cell-free microbial signatures with clinical parameters and cardiovascular death were examined using the Spearman rank correlation and multivariable conditional logistic regression, respectively.ResultsBoth 16S and ITS rRNA were detectable in all (except 3 for ITS) examined patients’ serum samples. Despite no significant difference in 16S rRNA levels and α diversity between cases and controls, taxonomic analysis demonstrated differential community membership between groups, with significantly greater Actinobacteria and less Proteobacteria observed in cases than in controls at the phylum level. Proportions of Actinobacteria and Proteobacteria phyla were significantly correlated with plasma nuclear factor erythroid 2−related factor 2 (Nrf2) levels (rho = −0.41 and 0.42, P = 0.015 and 0.013, respectively) and marginally associated with risk of cardiovascular death (adjusted odds ratios [95% confidence intervals] = 1.12 [0.98−1.29] and 0.88 [0.76−1.02] for 1% increase, respectively).ConclusionAlterations of the circulating cell-free microbial signatures may be associated with higher premature cardiovascular mortality in ESRD.

Ribosomal RNA degradation induced by the bacterial RNA polymerase inhibitor rifampicin.

Rifampicin, a broad-spectrum antibiotic, inhibits bacterial RNA polymerase. Here we show that rifampicin treatment of Escherichia coli results in a 50% decrease in cell size due to a terminal cell division. This decrease is a consequence of inhibition of transcription as evidenced by an isogenic rifampicin-resistant strain. There is also a 50% decrease in total RNA due mostly to a 90% decrease in 23S and 16S rRNA levels. Control experiments showed this decrease is not an artifact of our RNA purification protocol and therefore due to degradation in vivo. Since chromosome replication continues after rifampicin treatment, ribonucleotides from rRNA degradation could be recycled for DNA synthesis. Rifampicin-induced rRNA degradation occurs under different growth conditions and in different strain backgrounds. However, rRNA degradation is never complete, thus permitting the reinitiation of growth after removal of rifampicin. The orderly shutdown of growth under conditions where the induction of stress genes is blocked by rifampicin is noteworthy. Inhibition of protein synthesis by chloramphenicol resulted in a partial decrease in 23S and 16S rRNA levels whereas kasugamycin treatment had no effect. Analysis of temperature-sensitive mutant strains implicate RNase E, PNPase, and RNase R in rifampicin-induced rRNA degradation. We cannot distinguish between a direct role for RNase E in rRNA degradation versus an indirect role involving a slowdown of mRNA degradation. Since mRNA and rRNA appear to be degraded by the same ribonucleases, competition by rRNA is likely to result in slower mRNA degradation rates in the presence of rifampicin than under normal growth conditions.

Transcriptome-based identification and validation of optimal reference genes for quantitative real-time PCR normalisation in Psathyrostachys huashanica

P. huashanica (Psathyrostachys huashanica) is a unique, endangered, national plant and an important resistance resource reservoir that would be widely exploited in wheat genetic improvements. RT-qPCR (Real-time quantitative polymerase chain reaction) is a sensitive approach for determining relative gene expression and can provide accurate and valuable information on gene function. Here, sixteen candidate reference genes (RGs), 18S rRNA (18S ribosomal RNA), EF1-α (eukaryotic elongation factor 1 alpha), UBC2 (ubiquitin-conjugating enzyme E2–2), UBC17 (ubiquitin-conjugating enzyme E2–17), α-TUB2A (alpha tubulin-2A), β-TUB3 (beta tubulin 3), ADF4 (Actin-depolymerising factor 4), ACTIN (actin), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), 60SARP (60S acidic ribosomal protein), UBQ (polyubiquitin), SamDC (S-Adenosylmethionine decarboxylase), EIF4A (eukaryotic initiation factor 4A), ARF (ADP-ribosylation factor), HIS1 (histone H1), and HIS2B (histone H2B), was evaluated in Psathyrostachys huashanica samples using four algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Based on our results, the UBC17 and 18S rRNA levels were highly stable reference genes in presence of both abiotic and biotic stressors. However, α-TUB2A, UBC and ACTIN genes were confirmed to be optimal RGs in all tested samples. To further verify the suitability of the selected RGs, we assessed the relative levels of two stress-related genes PsaCPK3 (Calcium-dependent protein kinase) and PsaHSP70–1 (heat shock protein 70–1), which are known to respond to adversity. In summary, we established a series of appropriate RGs suited for RT-qPCR-mediated identification of target genes from P. huashanica in various treatment conditions. Moreover, our work will offer a valuable resource for gene expression analysis and functional gene research in P. huashanica.

Validation of a noninvasive prognostic signature for allograft failure following BK virus associated nephropathy.

Identifying kidney transplant recipients at risk for graft failure following BK virus nephropathy (BKVN) may allow personalization of therapy. We have reported that a noninvasive composite signature of urinary cell level of plasminogen activator inhibitor-1(PAI-1) mRNA and serum creatinine level, measured at the time of BKVN diagnosis, is prognostic of graft failure. In this investigation, we determined whether the composite signature is prognostic of graft failure in an independent cohort of 25 patients with BKVN. Of the 25 patients, 8 developed graft failure and 17 did not. We measured urinary cell levels of PAI-1 mRNA, 18S rRNA, and BKV VP1 mRNA at the time of BKVN diagnosis and evaluated clinical parameters including Banff pathology scores, acute rejection, and graft function. The area under the receiver operating characteristic curve for the noninvasive composite signature was 0.95 (P<.001) for prognosticating graft failure. The previously reported threshold of -0.858 predicted graft failure with a sensitivity of 75% and a specificity of 94%. Our current study validates the use of composite signature and the threshold of -0.858 to identify those at risk for graft failure following BKVN diagnosis, and supports future studies utilizing the composite signature score to personalize treatment of BKVN.

Morphologic and molecular identification of three macroscopic Sarcocystis species infecting domestic sheep (Ovis aries) and cattle (Bos taurus) in Egypt.

In a survey study on the macroscopic species of Sarcocystis infecting domestic sheep (Ovis aries) and cattle (Bos taurus) in Egypt, the macrosarcocysts of Sarcocystis gigantea and Sarcocystis medusiformis were detected in the carcasses of 33 domestic sheep out of a total of 250 (13.20%), whereas Sarcocystis hirsuta macrosarcocysts were found in 17 out of 150 cattle (11.33%) slaughtered at the municipal abattoirs of two different provinces in Egypt. The sarcocysts of each species were thoroughly described morphologically through gross inspection, histopathologic and transmission electron microscopic (TEM) examination. By TEM, S. gigantea primary cyst wall was 6-7.5μm thick and had irregular highly branched cauliflower-like villar protrusions (VP).The VP contained microtubules (mt) and multiple electron dense granules (edg) that were dispersed inside the cores of the branched VP. Besides, the parasitophorous vacuolar membrane (PVM) had minute blister-like invaginations all over the entire surface of the sarcocyst. S. medusiformis cyst had a thin sarcocyst wall (~2μm thick) as compared to that of S. gigantea. The cyst wall had trapezoidal or nearly pyramidal VP that were surrounded by thick PVM in addition to a ground substance GS that contained electron-dense fine particles. S. hirsuta sarcocyst wall was 7-9μm thick and possessed rhomboid-shaped VP that contained microtubules (mt) and electron-dense granules (edg) of variable sizes. The edg were arranged in rows and running parallel to the longitudinal axis of the protrusions. The VP had characteristic narrow neck-like constrictions at their bases, dilated middle portions, and tapered distal ends. The detected macrosarcocysts were eventually confirmed by molecular characterization on the levels of 18S rRNA, 28S rRNA, and Cox1 sequences. Phylogenetic analyses based on the sequences of the 18S rRNA and Cox1 genetic markers gave rise to robust associations of the currently identified isolates of S. gigantea, S. medusiformis, and S. hirsuta within a major clade of Sarcocystis species with felines as presumed or known definitive hosts.

Impaired chondrocyte U3 snoRNA expression in osteoarthritis impacts the chondrocyte protein translation apparatus

Although pathways controlling ribosome activity have been described to regulate chondrocyte homeostasis in osteoarthritis, ribosome biogenesis in osteoarthritis is unexplored. We hypothesized that U3 snoRNA, a non-coding RNA involved in ribosomal RNA maturation, is critical for chondrocyte protein translation capacity in osteoarthritis. U3 snoRNA was one of a number of snoRNAs with decreased expression in osteoarthritic cartilage and osteoarthritic chondrocytes. OA synovial fluid impacted U3 snoRNA expression by affecting U3 snoRNA gene promoter activity, while BMP7 was able to increase its expression. Altering U3 snoRNA expression resulted in changes in chondrocyte phenotype. Interference with U3 snoRNA expression led to reduction of rRNA levels and translational capacity, whilst induced expression of U3 snoRNA was accompanied by increased 18S and 28S rRNA levels and elevated protein translation. Whole proteome analysis revealed a global impact of reduced U3 snoRNA expression on protein translational processes and inflammatory pathways. For the first time we demonstrate implications of a snoRNA in osteoarthritis chondrocyte biology and investigated its role in the chondrocyte differentiation status, rRNA levels and protein translational capacity.

Ginkgolide-A attenuates bacterial translocation through activating PXR and improving antimicrobial peptide Reg 3A in experimental cirrhosis

Background and aimsBacterial translocation (BT) is strongly associated with disease progression and poor outcome in cirrhotic patients. The role of Pregnane X receptor (PXR) in regulating bacterial translocation in cirrhosis is unknown. We previously showed that Ginkgolide-A (GA), a natural PXR ligand, attenuated BT in cirrhotic mice by abrogating inflammation along the gut-liver-axis, and by protecting small intestinal tight junctions (TJ). Here, we aimed to investigate the effect of GA in activating PXR and associated antimicrobial peptides (AMPs) in regulating BT in experimental cirrhosis. MethodsMale Swiss albino mice were administered CCl4 (0.5 mL/kg body-weight, i.p twice a week) for 12 consecutive weeks. After the 12th week, mice were randomized and administered with GA (100-mg/kg body-weight, oral) every-day for 2 weeks. At termination, blood, gut and liver tissues were collected for molecular studies. ResultsGA treatment to cirrhotic mice significantly increased the expression of small intestinal PXR and Regenerating family member 3 alpha (Reg3A), which were otherwise reduced in CCl4 cirrhotic mice. Moreover, compared to naive mice a significantly reduced Lactobacillus, and increased Bacteroides and Enterococcus 16s rRNA levels were observed in the small intestine and liver of cirrhotic mice. Treatment with GA to cirrhotic mice significantly reduced intestinal overgrowth and translocation of Enterococcus and Bacteroides to the liver. Furthermore, GA treatment significantly attenuated intestinal permeability and BT marker soluble-CD14 (sCD14), which were increased in CCl4 cirrhotic mice. ConclusionThe study showed for the first time that, GA treatment to cirrhotic rodents attenuates BT, by improving PXR and Reg3A expression.