Identifying MiR-140-3p as a stable internal reference to normalize MicroRNA qRT-PCR levels of plasma small extracellular vesicles in lung cancer patients.

Exploration of a stably expressed gene as a reference is critical for the accurate evaluation of miRNAs isolated from small extracellular vesicles (sEVs). In this study, we analyzed small RNA sequencing on plasma sEV miRNAs in the training dataset (n = 104) and found that miR-140-3p was the most stably expressed candidate reference for sEV miRNAs. We further demonstrated that miR-140-3p expressed most stably in the validation cohort (n = 46) when compared to two other reference miRNAs, miR-451a and miR-1228-3p, and the commonly-used miRNA reference U6. Finally, we compared the capability of miR-140-3p and U6 as the internal reference for sEV miRNA expression by evaluating key miRNAs expression in lung cancer patients and found that miR-140-3p was more suitable as a sEV miRNA reference gene. Taken together, our data indicated miR-140-3p as a stable internal reference miRNA of plasma sEVs to evaluate miRNA expression profiles in lung cancer patients.

Identification of mitophagy-related biomarkers in osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a common joint disease, and existing drugs cannot cure OA, so there is an urgent need to identify new targets. Mitophagy plays an important role in OA; however, the role of mitophagy in the OA immune system is not yet clear. METHODS: In this study, differential analysis and enrichment analysis were used to identify mitophagy-related genes (MRGs) with differential expression in OA and the functional pathways involved in OA. Subsequently, two machine learning methods, RF and LASSO, were used to screen MRGs with diagnostic value and construct nomograms. At the same time, the relationship between mitophagy and OA immune response was explored by immunoinfiltration analysis. RESULTS: Forty-three differentially MRGs were identified in OA, of which six MRGs (GABARAPL2, PARL, GABARAPL1, JUN, RRAS, and SNX7) were associated with the diagnosis of OA. The ROC analysis results show that these 6 MRGs have high predictive accuracy in the diagnosis of OA. In immune infiltration analysis, we found that the abundance of significantly different immune cells in OA was mostly upregulated. In addition, the expression of diagnostic-related MRGs is correlated with changes in the abundance of immune cells in OA. CONCLUSION: This study demonstrates that six MRGs can be used as diagnostic biomarkers. The expression of diagnostic-related MRGs is correlated with changes in the abundance of immune cells in OA. At the same time, mitophagy may affect the immune microenvironment of OA by regulating immune cells, ultimately leading to the progression of OA.

Methylene blue dose-dependently induces cutaneous inflammation and heat hyperalgesia in a novel rat model.

Methylene blue (MB) has been shown to reduce mortality and morbidity in vasoplegic patients after cardiac surgery. Though MB is considered to be safe, extravasation of MB leading to cutaneous toxicity has been reported. In this study, we sought to characterize MB-induced cutaneous toxicity and investigate the underlying mechanisms. To induce MB-induced cutaneous toxicity, we injected 64 adult male Sprague-Dawley rates with 200 µL saline (vehicle) or 1%, 0.1%, or 0.01% MB in the plantar hind paws. Paw swelling, skin histologic changes, and heat and mechanical hyperalgesia were measured. Injection of 1%, but not 0.1% or 0.01% MB, produced significant paw swelling compared to saline. Injection of 1% MB produced heat hyperalgesia but not mechanical hyperalgesia. Pain behaviors were unchanged following injections of 0.1% or 0.01% MB. Global transcriptomic analysis by RNAseq identified 117 differentially expressed genes (111 upregulated, 6 downregulated). Ingenuity Pathway Analysis showed an increased quantity of leukocytes, increased lipids, and decreased apoptosis of myeloid cells and phagocytes with activation of IL-1ß and Fos as the two major regulatory hubs. qPCR showed a 16-fold increase in IL-6 mRNA. Thus, using a novel rat model of MB-induced cutaneous toxicity, we show that infiltration of 1% MB into cutaneous tissue causes a dose-dependent pro-inflammatory response, highlighting potential roles of IL-6, IL-1ß, and Fos. Thus, anesthesiologists should administer dilute MB intravenously through peripheral venous catheters. Higher concentrations of MB (1%) should be administered through a central venous catheter to minimize the risk of cutaneous toxicity.

CD99 Expression and Prognostic Impact in Glioblastoma: A Single-Center Cohort Study.

Glioblastoma is the most frequent and aggressive brain tumor in adults. This study aims to evaluate the expression and prognostic impact of CD99, a membrane glycoprotein involved in cellular migration and invasion. In a cohort of patients with glioblastoma treated with surgery, radiotherapy and temozolomide, we retrospectively analyzed tumor expression of CD99 by immunohistochemistry (IHC) and by quantitative real-time polymerase chain reaction (qRT-PCR) for both the wild type (CD99wt) and the truncated (CD99sh) isoforms. The impact on overall survival (OS) was assessed with the Kaplan-Meier method and log-rank test and by multivariable Cox regression. Forty-six patients with glioblastoma entered this study. Immunohistochemical expression of CD99 was present in 83%. Only the CD99wt isoform was detected by qRT-PCR and was significantly correlated with CD99 expression evaluated by IHC (rho = 0.309, p = 0.037). CD99 expression was not associated with OS, regardless of the assessment methodology used (p = 0.61 for qRT-PCR and p = 0.73 for IHC). In an exploratory analysis of The Cancer Genome Atlas, casuistry of glioblastomas CD99 expression was not associated with OS nor with progression-free survival. This study confirms a high expression of CD99 in glioblastoma but does not show any significant impact on survival. Further preclinical studies are needed to define its role as a therapeutic target in glioblastoma.

Application of benzothiadiazole to Cabernet Gernischt grapes (Vitis vinifera L.) for quality improvement: Effects on aroma metabolism precursors and related genes expression.

Pre-harvest spraying of benzothiadiazole (BTH) can improve the winemaking properties of grapes, especially their aroma compounds and phenolics. Limited research has explored the molecular mechanisms by which BTH influences the accumulation of grape aroma precursors during early grape development. This study investigated the effects and putative molecular mechanisms of applying 0.37 mM BTH through whole-plant spraying on the accumulation of aroma metabolism precursors and gene expression in Cabernet Gernischt grapes during ripening. The results showed that BTH treatment increased the levels of fructose, alanine, aspartate, threonine, myristic acid, myristoleic acid, palmitic acid, ß-cryptoxanthin, norisoprenoids and methoxypyrazines. Contrarily, it decreased the levels of glucose, sucrose, phenylalanine, tyrosine, leucine, valine, glycine, arginine, histidine, total unsaturated fatty acids (particularly linoleic acid), zeaxanthin, lutein, and organic acids. Additionally, BTH upregulated the expression of genes associated with the production and degradation of amino acids, fatty acids, and carotenoids while decreasing the expression of genes involved in the synthesis and degradation of soluble sugars and organic acids. Ten different metabolites, including fumaric acid, were identified as potential biological markers for distinguishing BTH-treated grapes from control grapes. The study demonstrates that BTH treatment had a substantial impact on the concentration and developmental patterns of aroma metabolism precursors. Furthermore, it altered the winemaking characteristics of Cabernet Gernischt grapes by modulating genes associated with the production and breakdown of metabolites.

Comparison of telomere length in patients with bone marrow failure syndromes and healthy controls.

INTRODUCTION: During normal aging, telomeric DNA is gradually lost in dividing somatic cells, and critically short telomeres lead to replicative senescence, apoptosis, or chromosomal instability. We studied telomere length in bone marrow failure syndromes (BMFS) compared to normal healthy population. METHODS: Peripheral blood was collected from the participants, and genomic DNA was extracted. Relative telomere length was measured using a quantitative polymerase chain reaction. Statistical analysis was performed using SPSS and GraphPad Prism 8.2 software. RESULTS: The median age of normal Indian population was 31 (0-60) years. As expected, telomere length (TL) showed a decline with age and no difference in TL between males and females. The median age of 650 patients with aplastic anemia (AA) was 30 (1-60) years. TL was significantly shorter in patients with AA compared to healthy controls (p < .001). In FA and MDS patients, TL was significantly shorter than age-matched healthy controls (p = .028; p < .001), respectively. There was no difference between the median TL in age-matched AA and FA patients (p = .727). However, patients with MDS had shorter TL than age-matched AA (p = .031). CONCLUSION: TL in BMF syndrome patients was significantly shorter than age-matched healthy controls.

Molecular monitoring of treatment efficacy in human visceral leishmaniasis.

BACKGROUND: Focused efforts of the visceral leishmaniasis elimination program have led to a drastic decline in cases, and the present challenge is disease monitoring, which this study aimed to assess. METHODS: A Leishmania kinetoplastid-targeted qPCR quantified parasite load at disease presentation, and following treatment completion (n=49); an additional 80 cases were monitored after completion of treatment. RESULTS: The parasite load at disease presentation was 13 461.00 (2560.00-37764.00)/µg gDNA, which upon completion of treatment reduced in 47 of 49 cases to 1(1-1)/µg gDNA, p<0.0001. In 80 cases that presented >2 months post-treatment, their parasite burden similarly decreased to 1(1-1)/µg gDNA except in 6 of 80 cases, which were qPCR positive. CONCLUSION: In 129 cases of visceral leishmaniasis, qPCR by quantification of parasite burden proved effective for monitoring treatment.

Quantitative Real-Time PCR Method to Evaluate Gene Expression in Zebrafish Embryos.

The fast zebrafish embryonic development offers an opportunity for the study of genes crucial for developmental processes. Several genes associated with human diseases have orthologs in zebrafish. Enhanced comprehension of a gene's function can be achieved by knowing when and where it is expressed. This knowledge also makes it possible to alter a gene-by-gene knockdown in a time- and place-specific manner. Moreover, gene expression analyses contribute greatly to teratogenicity studies in zebrafish embryos. Therefore, the importance of examining the differences between the expressions of these genes has increased day by day. The incorporation of reverse transcription (RT) as the initial step prior to thermal cycling in quantitative real-time polymerase chain reaction (RT-qPCR) has made a significant contribution to RNA research. RT-qPCR is the gold standard and an effective method for gene expression analysis. Quick readout, high sensitivity, reproducibility, as well as high potential throughput, along with reliable quantification, are just a few advantages of RT-qPCR. However, there are drawbacks to its application, such as RNA's inherent variability, impurities during RNA extraction, and variations in reverse transcription and PCR efficiencies. Implementing a precise normalization technique is crucial to account for these inaccuracies. In this chapter, the protocol for gene expression analysis by RT-qPCR for zebrafish embryos is explained.

Comparative Analysis of Differentially Expressed Genes in Chondrocytes from Rats Exposed to Low Selenium and T-2 Toxin.

The aim of this study was to construct rat models of environmental risk factors for Kashin-Beck disease (KBD) with low selenium and T-2 toxin levels and to screen the differentially expressed genes (DEGs) between the rat models exposed to environmental risk factors. The Se-deficient (SD) group and T-2 toxin exposure (T-2) group were constructed. Knee joint samples were stained with hematoxylin-eosin, and cartilage tissue damage was observed. Illumina high-throughput sequencing technology was used to detect the gene expression profiles of the rat models in each group. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis were performed and five differential gene expression results were verified by quantitative real-time polymerase chain reaction (qRT‒PCR). A total of 124 DEGs were identified from the SD group, including 56 upregulated genes and 68 downregulated genes. A total of 135 DEGs were identified in the T-2 group, including 68 upregulated genes and 67 downregulated genes. The DEGs were significantly enriched in 4 KEGG pathways in the SD group and 9 KEGG pathways in the T-2 group. The expression levels of Dbp, Pc, Selenow, Rpl30, and Mt2A were consistent with the results of transcriptome sequencing by qRT‒PCR. The results of this study confirmed that there were some differences in DEGs between the SD group and the T-2 group and provided new evidence for further exploration of the etiology and pathogenesis of KBD.

Bacteriologic Conditions of the Apical Root Canal System of Teeth with and without Posttreatment Apical Periodontitis: A Correlative Multianalytical Approach.

INTRODUCTION: This study used a correlative multianalytical approach to investigate the bacteriologic conditions in the apical root canal system of treated teeth with or without apical periodontitis and their correlation with the technical quality of the previous root canal obturation and the presence and volume of apical periodontitis lesions. METHODS: Root apexes were obtained from recently extracted root canal-treated teeth with (n = 23) and without (n = 22) apical periodontitis lesions as demonstrated by cone-beam computed tomographic examination. The root apexes were sectioned and subjected to micro-computed tomographic (micro-CT) scanning. The specimens were cryopulverized, and DNA extracted from the powder was used as a template in real-time polymerase chain reaction assays to quantify total bacteria and members of the Streptococcus genus and Actinobacteria phylum. The bacteriologic findings were compared between the 2 groups and also evaluated for associations with cone-beam computed tomographic and micro-computed tomographic data. RESULTS: Bacteria were detected in all apical canal samples except 1. The mean counts of total bacteria, streptococci, and actinobacteria did not differ significantly between teeth with or without apical periodontitis (P > .05). Streptococcus levels were significantly lower by 80% in the apical canals of teeth with small lesions compared with those without lesions (P < .05). The limit of filling >2 mm short was significantly associated with more total bacterial counts compared with canals filled 0-2 mm short (P < .05). An adequate coronal restoration was significantly associated with lesser counts of Streptococcus (P < .05). CONCLUSIONS: Comparable bacterial loads were observed in the apical canal system of treated teeth with and without apical periodontitis, suggesting that factors other than only the total bacterial levels may also influence the development and progression of apical periodontitis. Bacteria were found in the apical canal in virtually all cases with a high prevalence of streptococci and actinobacteria. Streptococci counts were significantly higher in the apical canal of teeth with inadequate restorations and teeth with no lesions. Underfilled canals showed higher bacterial counts.

Dysbiosis by Eradication of Helicobacter pylori Infection Associated with Follicular Gastropathy and Pangastropathy.

Dysbiosis plays an important role in the development of bacterial infections in the gastric mucosa, particularly Helicobacter pylori. The international guidelines for the treatment of H. pylori infections suggest standard triple therapy (STT). Nevertheless, because of the increasing resistance rates to clarithromycin, metronidazole has been widely considered in several countries. Unfortunately, the non-justified administration of antibiotics induces dysbiosis in the target organ. We characterized the gastric microbiota of patients diagnosed with follicular gastropathy and pangastropathy attributed to H. pylori infection, before and after the administration of STT with metronidazole. Dominant relative abundances of Cutibacterium were observed in pre-treatment patients, whereas H. pylori was observed at <11%, suggesting the multifactor property of the disease. The correlation of Cutibacterium acnes and H. pylori with gastric infectious diseases was also evaluated using quantitative real-time polymerase chain reaction. The dominance of C. acnes over H. pylori was observed in gastritis, gastropathies, and non-significant histological alterations. None of the microorganisms were detected in the intestinal metaplasia. Post-treatment alterations revealed an increase in the relative abundances of Staphylococcus, Pseudomonas, and Klebsiella. Non-H. pylori gastrointestinal bacteria can be associated with the initiation and development of gastric diseases, such as pathobiont C. acnes.

[Development and application of a rapid scheme for detection of respiratory virus nucleic acid].

This study aimed to develop a portable, accurate and easy-to-operate scheme for rapid detection of respiratory virus nucleic acid. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the effect of extraction-free respiratory virus treatment reagent (RTU) on viral nucleic acid treatment and the effect of ultra-fast fluorescence quantitative PCR instrument (FQ-8A) on nucleic acid amplification, respectively. RTU and FQ-8A were combined to develop a rapid detection scheme for respiratory virus nucleic acid, and the positive detection rate was judged by Ct value using a fluorescence quantitative PCR instrument, and the accuracy of the scheme in clinical samples detection was investigated. The results showed that RTU had comparable sensitivity to the automatic nucleic acid extraction instrument, its extraction efficiency was comparable to the other 3 extraction methods when extracting samples of different virus types, but the extraction time of RTU was less than 5 min. FQ-8A had good consistency in detection respiratory syncytial virus (RSV) and adenovirus (ADV) compared with the control instrument ABI-7500, with kappa coefficients of 0.938 (P < 0.001) and 0.887 (P < 0.001), respectively, but the amplification time was only about 0.5 h. The RTU and FQ-8A combined rapid detection scheme had a highly consistent detection rate with the conventional detection scheme, with a sensitivity of 91.70% and specificity of 100%, and a kappa coefficient was 0.944 (P < 0.001). In conclusion, by combining RTU with FQ-8A, a rapid respiratory virus nucleic acid detection scheme was developed, the whole process could be completed in 35 min. The scheme is accurate and easy-to-operate, and can provide important support for the rapid diagnosis and treatment of respiratory virus.

Salivary levels of five microorganisms of root caries in nursing home elderly: a preliminary investigation.

BACKGROUND: Streptococcus, Bifidobacteria, Lactobacillus and Actinomyces are acidogenic aciduria that may be associated with root caries (RC). The aim of the study was to analyze Streptococcus mutans (S. mutans), Streptococcus sobrinus (S. sobrinus), Bifidobacterium spp., Lactobacillus spp. and Actinomyces naeslundii (A. naeslundii) in the saliva of nursing home elderly, to assess the correlation between bacterial composition and RC for five putative catiogenic organisms. METHODS: In this study, we collected 43 saliva samples and divided into two groups: the root caries group (RCG, n = 21) and the caries-free group (CFG, n = 22). Bacterial DNA was extracted from the saliva samples. The presence and abundance of the five microorganisms were detected by Quantitative real-time PCR (qPCR). Spearman correlation test was performed to evaluate the relationship between the numbers of root decayed filled surfaces (RDFS) and root caries index (RCI) and salivary levels of the bacteria. RESULTS: The salivary levels of S. mutans, S. sobrinus, Bifidobacterium spp. and Lactobacillus spp. were significantly higher in RCG than in CFG (p < 0.05). RDFS and RCI (RDFS/RCI) were positively associated with salivary levels of S. mutans, S. sobrinus and Bifidobacterium spp. (r = 0.658/0.635, r = 0.465/0.420 and r = 0.407/0.406, respectively). No significant differences in presence and amounts of A. naeslundii was observed between the two groups (p > 0.05). CONCLUSION: S. mutans, S. sobrinus and Bifidobacterium spp. in saliva appear to be associated with RC in the elderly. Taken together, the findings indicate that specific salivary bacteria may be involved in the progression of RC.

Identification and Validation of Ferroptosis-Related Genes in Patients with Acute Spinal Cord Injury.

Ferroptosis plays crucial roles in the pathology of spinal cord injury (SCI). The purpose of this study was to identify differentially expressed ferroptosis-related genes (DE-FRGs) in human acute SCI by bioinformatics analysis and validate the hub DE-FRGs in non-SCI and SCI patients. The GSE151371 dataset was downloaded from the Gene Expression Omnibus and difference analysis was performed. The differentially expressed genes (DEGs) in GSE151371 overlapped with the ferroptosis-related genes (FRGs) obtained from the Ferroptosis Database. A total of 41 DE-FRGs were detected in 38 SCI samples and 10 healthy samples in GSE151371. Then, enrichment analyses of these DE-FRGs were performed for functional annotation. The GO enrichment results showed that upregulated DE-FRGs were mainly associated with reactive oxygen species and redox reactions, and the KEGG enrichment analysis indicated involvement in some diseases and ferroptosis pathways. Protein-protein interaction (PPI) analysis and lncRNA-miRNA-mRNA regulatory network were performed to explore the correlations between genes and regulatory mechanisms. The relationship between DE-FRGs and differentially expressed mitochondria-related genes (DE-MRGs) was also analyzed. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the hub DE-FRGs in clinical blood samples from acute SCI patients and healthy controls. Consistent with the bioinformatics results, qRT-PCR of the clinical samples indicated similar expression levels of TLR4, STAT3, and HMOX1. This study identified DE-FRGs in blood samples from SCI patients, and the results could improve our understanding of the molecular mechanisms of ferroptosis in SCI. These candidate genes and pathways could be therapeutic targets for SCI.

mRNA expression profiling of mitochondrial subunits in subjects with Parkinson's disease.

Introduction: Parkinson's disease (PD) is a common adult-onset neurodegenerative disorder caused by a progressive loss of dopaminergic neurons due to the accumulation of α-synuclein in the substantia nigra. Mitochondria are known to play a key role in cell respiratory function and bioenergetics. Indeed, mitochondrial dysfunction causes insufficient energy production required to satisfy the needs of several organs, especially the nervous system. However, the profiling of messenger RNA (mRNA) expression of mitochondrial subunits in PD has not been systematically investigated yet. Material and methods: We explored the mRNA expression of mitochondrial DNA (mtDNA) encoded respiratory chain (RC) subunits in 43 PD patients and 43 normal controls (NC). Next generation sequencing analysis (NGS) was used and quantitative real-time polymerase chain reaction (qRT-PCR) assay was used for confirmation of the NGS results. Results: All tested mitochondrial RC subunits were significantly over-expressed in subjects with PD compared to NC. In qRT-PCR the mean expression of all mitochondrial subunits had an expression level of at least 7 times compared to NC. Conclusions: The over-expression of mitochondrial subunits in PD subjects with respect to NC might be secondary to a degeneration-related alteration of the mitochondrial structure and/or dynamics, or to the occurrence of a compensatory mechanism. The study of specific mRNA by peripheral blood mononuclear cells may provide a further diagnostic frame for early detection PD patients.

REDD1 deletion and treadmill running increase liver hepcidin and gluconeogenic enzymes in male mice.

The iron-regulatory hormone hepcidin is transcriptionally up-regulated by gluconeogenic signals. Recent evidence suggeststhat increases in circulating hepcidin may decrease dietary iron absorption following prolonged exercise, however evidence is limited on whether gluconeogenic signals contribute to post-exercise increases in hepcidin. Mice with genetic knockout of regulated in development and DNA response-1 (REDD1) display greater glycogen depletion following exercise, possibly indicating greater gluconeogenesis. The objective of the present study was to determine liver hepcidin, markers of gluconeogenesis and iron metabolism in REDD1 knockout and wild-type mice following prolonged exercise. Twelve-week-old male REDD1 knockout and wild-type mice were randomised to rest or 60 min treadmill running with 1, 3 or 6 h recovery (n = 5-8/genotype/group). Liver gene expression of hepcidin (Hamp) and gluconeogenic enzymes (Ppargc1a, Creb3l3, Pck1, Pygl) were determined by qRT-PCR. Effects of genotype, exercise and their interaction were assessed by two-way ANOVAs with Tukey's post-hoc tests, and Pearson correlations were used to assess the relationships between Hamp and study outcomes. Liver Hamp increased 1- and 4-fold at 3 and 6 h post-exercise, compared to rest (P-adjusted < 0⋅009 for all), and was 50% greater in REDD1 knockout compared to wild-type mice (P = 0⋅0015). Liver Ppargc1a, Creb3l3 and Pck1 increased with treadmill running (P < 0⋅0001 for all), and liver Ppargc1a, Pck1 and Pygl were greater with REDD1 deletion (P < 0⋅02 for all). Liver Hamp was positively correlated with liver Creb3l3 (R = 0⋅62, P < 0⋅0001) and Pck1 (R = 0⋅44, P = 0⋅0014). In conclusion, REDD1 deletion and prolonged treadmill running increased liver Hamp and gluconeogenic regulators of Hamp, suggesting gluconeogenic signalling of hepcidin with prolonged exercise.

A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy.

PURPOSE: Pregnancy-mediated physiological and biochemical changes contribute to alterations in the pharmacokinetics of certain drugs. There is a paucity of data on the systematic evaluation of the underlying mechanisms. The objective of the current study was to examine the impact of changes in circulating and tissue hormonal concentration during the late stage of pregnancy on the activity and expression of hepatic cytochrome P450 (CYP) enzymes using a cocktail probe approach. METHODS: Freshly isolated primary human hepatocytes were incubated with third trimester physiologic (plasma) and projected liver (ten-fold higher) concentrations of female hormones: progesterone (2 µM), estradiol (0.3 µM), estriol (0.8 µM), estrone (0.2 µM), 17α-hydroxyprogesterone (0.1 µM), and human growth hormone (0.005 µM). The metabolic activity of the hepatocytes was assessed using a cocktail of isozyme-specific P450 probe substrates (CYP1A2 (phenacetin), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4 (testosterone)). A validated LC-MS/MS assay was used to measure the corresponding metabolite concentrations. CYP450 protein and mRNA levels were measured using western blot and qRT-PCR, respectively. RESULTS: Female hormones at projected third-semester hepatic concentrations significantly enhanced mRNA and protein expression and increased the metabolic activity of CYP3A4. The expression and activity of other CYP450 enzymes studied were not affected by mixtures of female hormones at concentrations used. CONCLUSION: The increased activity of CYP3A4 is consistent with the clinically observed increase in clearance of CYP3A4 substrates during pregnancy. Overall expression and activity of CYP450 isozymes are differentially regulated during pregnancy.

Strain-specific responsiveness of hepatitis D virus to interferon-alpha treatment.

Background & Aims: Pegylated interferon alpha (pegIFNα) is commonly used for the treatment of people infected with HDV. However, its mode of action in HDV-infected cells remains elusive and only a minority of people respond to pegIFNα therapy. Herein, we aimed to assess the responsiveness of three different cloned HDV strains to pegIFNα. We used a previously cloned HDV genotype 1 strain (dubbed HDV-1a) that appeared insensitive to interferon-α in vitro, a new HDV strain (HDV-1p) we isolated from an individual achieving later sustained response to IFNα therapy, and one phylogenetically distant genotype 3 strain (HDV-3). Methods: PegIFNα was administered to human liver chimeric mice infected with HBV and the different HDV strains or to HBV/HDV infected human hepatocytes isolated from chimeric mice. Virological parameters and host responses were analysed by qPCR, sequencing, immunoblotting, RNA in situ hybridisation and immunofluorescence staining. Results: PegIFNα treatment efficiently reduced HDV RNA viraemia (∼2-log) and intrahepatic HDV markers both in mice infected with HBV/HDV-1p and HBV/HDV-3. In contrast, HDV parameters remained unaffected by pegIFNα treatment both in mice (up to 9 weeks) and in isolated cells infected with HBV/HDV-1a. Notably, HBV viraemia was efficiently lowered (∼2-log) and human interferon-stimulated genes similarly induced in all three HBV/HDV-infected mouse groups receiving pegIFNα. Genome sequencing revealed highly conserved ribozyme and L-hepatitis D antigen post-translational modification sites among all three isolates. Conclusions: Our comparative study indicates the ability of pegIFNα to lower HDV loads in stably infected human hepatocytes in vivo and the existence of complex virus-specific determinants of IFNα responsiveness. Impact and implications: Understanding factors counteracting HDV infections is paramount to develop curative therapies. We compared the responsiveness of three different cloned HDV strains to pegylated interferon alpha in chronically infected mice. The different responsiveness of these HDV isolates to treatment highlights a previously underestimated heterogeneity among HDV strains.

Study of the gut microbiome as a novel target for prevention of hospital-associated infections in intensive care unit patients.

BACKGROUND: Hospital-acquired infections (HAIs) are increasing due to the spread of multi-drugresistant organisms. Gut dysbiosis in an intensive care unit (ICU) patients at admission showed an altered abundance of some bacterial genera associated with the occurrence of HAIs and mortality. In the present study, we investigated the pattern of the gut microbiome in ICU patients at admission to correlate it with the development of HAIs during ICU stay. METHODS: Twenty patients admitted to an ICU with a cross-matched control group of 30 healthy subjects of matched age and sex. Quantitative SYBR green real-time polymerase chain reaction was done for the identification and quantitation of selected bacteria. RESULTS: Out of those twenty patients, 35% developed ventilator-associated pneumonia during their ICU stay. Gut microbiome analysis showed a significant decrease in Firmicutes and Firmicutes to Bacteroidetes ratio in ICU patients in comparison to the control and in patients who developed HAIs in comparison to the control group and patients who did not develop HAIs. There was a statistically significant increase in Bacteroides in comparison to the control group. There was a statistically significant decrease in Bifidobacterium and Faecalibacterium prausnitzii and an increase in Lactobacilli in comparison to the control group with a negative correlation between Acute Physiology and Chronic Health Evaluation (APACHE) II score and Firmicutes to Bacteroidetes and Prevotella to Bacteroides ratios. CONCLUSIONS: Gut dysbiosis of patients at the time of admission highlights the importance of identification of the microbiome of patients admitted to the ICU as a target for preventing of HAIs.

Bioinformatics-Based Analysis of Key Genes in Steroid-Induced Osteonecrosis of the Femoral Head That Are Associated with Copper Metabolism.

Osteonecrosis of the femoral head (ONFH) is a common disabling disease. Copper has positive effects on cells that regulate bone metabolism. However, the relationship between copper metabolism (CM) and steroid-induced ONFH (SONFH) remains unclear. The GSE123568 dataset was downloaded from the Gene Expression Omnibus. The differentially expressed CM-related SONFH genes (DE-CMR-SONFHGs) were identified via differential analysis and weighted gene coexpression network analysis (WGCNA). Receiver operating characteristic (ROC) analysis was performed for the predictive accuracy of key genes. Targeting drugs and the copper death-related genes (CDRGs) relevant to key genes were investigated. The bioinformatics results were confirmed via quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. Two out of 106 DE-CMR-SONFHGs were identified as key genes (PNP and SLC2A1), which had diagnostic value in distinguishing SONFH from control samples and were related to various immune cell infiltrations. Eleven PMP-targeting drugs and five SLC2A1-targeting drugs were identified. The qRT-PCR, as well as WB, results confirmed the downregulation PNP and SLC2A1 and high expression of the CDRGs DLD, PDHB, and MTF1, which are closely related to these two key genes. In conclusion, PNP and SLC2A1 were identified as key genes related to SONFH and may provide insights for SONFH treatment.