Differentially expressed microRNAs targeting genes in key pathways in keratoconus.

Introduction: Keratoconus (KTCN) is a corneal ectasia, characterized by a progressive thinning and protrusion of the cornea, with a complex etiology involving genetic, behavioral, lifestyle, and environmental factors. Previous studies indicated that microRNAs (miRNAs) could be involved in KTCN pathogenesis. This in silico study aimed to identify precursor microRNAs (pre-miRNAs) differentially expressed in KTCN corneas and to characterize mature miRNAs and their target genes. Materials and methods: Expression levels of pre-miRNAs were retrieved from our previously obtained RNA sequencing data of 25 KTCN and 25 non-KTCN human corneas (PMID:28145428, PMID:30994860). Differential expression with FDR ≤0.01 and ≥1.5-fold changes were considered significant. Lists of target genes (target score ≥90) of mature miRNAs were obtained from miRDB. Revealed up-/downregulated miRNAs and their target genes were assessed in databases and literature. Enrichment analyses were completed applying the DAVID database. Results: From a total of 47 pre-miRNAs, six were remarkably upregulated (MIR184, MIR548I1, MIR200A, MIR6728, MIR429, MIR1299) and four downregulated (MIR6081, MIR27B, MIR23B, MIR23A) in KTCN corneas. Out of the 1,409 target genes, 220 genes with decreased and 57 genes with increased expression levels in KTCN samples vs non-KTCN samples were found. The extracellular matrix (ECM) organization, response to mechanical stimulus, regulation of cell shape, and signal transduction processes/pathways were identified as distinctive in enrichment analyses. Also, processes associated with the regulation of transcription and DNA binding were listed. Conclusion: Indicated miRNAs and their target genes might be involved in KTCN pathogenesis via disruption of crucial molecular processes, including ECM organization and signal transduction.

Macrolide and lincosamide resistance of Streptococcus agalactiae in pregnant women in Poland.

Knowing about the antibiotic resistance, serotypes, and virulence-associated genes of Group B Streptococcus for epidemiological and vaccine development is very important. We have determined antimicrobial susceptibility patterns, serotype, and virulence profiles. The antibiotic susceptibility was assessed for a total of 421 Streptococcus agalactiae strains, isolated from pregnant women and neonates. Then, 89 erythromycin and/or clindamycin-resistant strains (82 isolates obtained from pregnant women and seven isolates derived from neonates) were assessed in detail. PCR techniques were used to identify the studied strains, perform serotyping, and assess genes encoding selected virulence factors. Phenotypic and genotypic methods determined the mechanisms of resistance. All tested strains were sensitive to penicillin and levofloxacin. The constitutive MLSB mechanism (78.2%), inducible MLSB mechanism (14.9%), and M phenotype (6.9%) were identified in the macrolide-resistant strains. It was found that macrolide resistance is strongly associated with the presence of the ermB gene and serotype V. FbsA, fbsB, fbsC, scpB, and lmb formed the most recurring pattern of genes among the nine surface proteins whose genes were analysed. A minority (7.9%) of the GBS isolates exhibited resistance to lincosamides and macrolides, or either, including those that comprised the hypervirulent clone ST-17. The representative antibiotic resistance pattern consisted of erythromycin, clindamycin, and tetracycline resistance (71.9%). An increase in the fraction of strains resistant to macrolides and lincosamides indicates the need for monitoring both the susceptibility of these strains and the presence of the ST-17 clone.

Gender Influences Gut Microbiota among Patients with Irritable Bowel Syndrome.

Irritable bowel syndrome (IBS) is a chronic functional gastrointestinal disease that affects approximately 11% of the general population. The gut microbiota, among other known factors, plays a substantial role in its pathogenesis. The study aimed to characterize the gut microbiota differences between patients with IBS and unaffected individuals, taking into account the gender aspect of the patients and the types of IBS determined on the basis of the Rome IV Criteria, the IBS-C, IBS-D, IBS-M, and IBS-U. In total, 121 patients with IBS and 70 unaffected individuals participated in the study; the derived stool samples were subjected to 16S rRNA amplicon sequencing. The gut microbiota of patients with IBS was found to be more diverse in comparison to unaffected individuals, and the differences were observed primarily among Clostridiales, Mogibacteriaceae, Synergistaceae, Coriobacteriaceae, Blautia spp., and Shuttleworthia spp., depending on the study subgroup and patient gender. There was higher differentiation of females' gut microbiota compared to males, regardless of the disease status. No correlation between the composition of the gut microbiota and the type of IBS was found. Patients with IBS were characterized by more diverse gut microbiota compared to unaffected individuals. The gender criterion should be considered in the characterization of the gut microbiota. The type of IBS did not determine the identified differences in gut microbiota.

Differences in the composition of the bacterial element of the urinary tract microbiome in patients undergoing dialysis and patients after kidney transplantation.

Introduction: The development of molecular biology methods and their application in microbial research allowed the detection of many new pathogens that cause urinary tract infections (UTIs). Despite the advances of using new research techniques, the etiopathogenesis of UTIs, especially in patients undergoing dialysis and patients after kidney transplantation, is still not fully understood. Methods: This study aimed to characterize and compare the composition of the bacterial element of the urinary tract microbiome between the groups of patients undergoing dialysis (n = 50) and patients after kidney transplantation (n = 50), with positive or negative urine culture, compared to healthy individuals (n = 50). Results: Asymptomatic bacteriuria was observed in 30% of the urine cultures of patients undergoing dialysis and patients after kidney transplantation, with Escherichia coli as the most dominant microorganism (73%) detected with the use of classical microbiology techniques. However, differences in the bacterial composition of the urine samples between the evaluated patient groups were demonstrated using the amplicon sequencing. Finegoldia, Leptotrichia, and Corynebacterium were found to be discriminative bacteria genera in patients after dialysis and kidney transplantation compared to the control group. In addition, in all of urine samples, including those without bacteriuria in classical urine culture, many types of bacteria have been identified using 16S rRNA sequencing. Discussion: The revealed microbial characteristics may form the basis in searching for new diagnostic markers in treatment of patients undergoing dialysis and patients after kidney transplantation.

Changes in Nuclear Gene Expression Related to Mitochondrial Function Affect Extracellular Matrix, Collagens, and Focal Adhesion in Keratoconus.

Purpose: Mitochondrial DNA (mtDNA) abnormalities were previously found to be causative in the pathogenesis of various diseases. Here, comprehensive mitochondrial and nuclear sequence and transcript analyses, along with analyses of the methylation aspects of nuclear genes related to mitochondrial function, were performed in patients with keratoconus (KTCN) to evaluate their contribution to the KTCN pathogenesis. Methods: Blood mtDNA of 42 KTCN and 51 non-KTCN individuals was Sanger sequenced and analyzed along with the previously obtained corneal RNA-sequencing data of 20 KTCN and 21 non-KTCN individuals. In addition, the expression and methylation of mtDNA genes and 1223 mitochondria-related nuclear genes were evaluated. Results: The mtDNA sequence alterations detected in blood coincided with variants identified in transcripts of the matched corneal tissues. In KTCN corneas, 97 mitochondria-related genes were deregulated, including TGFB1, P4HB, and BCL2, which are involved in the extracellular matrix (ECM) organization, collagen formation, and focal adhesion pathways. No changes in the expression of mtDNA transcripts and no differentially methylated genes among the assessed mitochondrial-nuclear gene sets were found. Conclusions: The absence of corneal-specific mtDNA variants indicates that there is no direct relationship between mitochondrial sequence variability and KTCN phenotype in the studied individuals. However, the identified KTCN-specific transcriptomic alterations of the nuclear genes directly related to the mitochondria functioning point to their possible involvement in the ECM organization, collagen formation, and focal adhesion. Translational Relevance: The identification of abnormalities within nuclear genes regulating ECM formation, collagen synthesis, and/or focal adhesion may form the basis of future treatment strategies or predict the progression of corneal changes in KTCN.

Further evaluation of differential expression of keratoconus candidate genes in human corneas.

BACKGROUND: Keratoconus (KTCN) is a progressive eye disease, characterized by changes in the shape and thickness of the cornea that results in loss of visual acuity. While numerous KTCN candidate genes have been identified, the genetic etiology of the disease remains undetermined. To further investigate and verify the contribution of particular genetic factors to KTCN, we assessed 45 candidate genes previously indicated as involved in KTCN etiology based on transcriptomic and genomic data. METHODS: The RealTime ready Custom Panel, covering 45 KTCN candidate genes and two reference transcripts, has been designed. Then, the expression profiles have been assessed using the RT-qPCR assay in six KTCN and six non-KTCN human corneas, obtained from individuals undergoing a penetrating keratoplasty procedure. RESULTS: In total, 35 genes exhibiting differential expression between KTCN and non-KTCN corneas have been identified. Among these genes were ones linked to the extracellular matrix formation, including collagen synthesis or the TGF-ß, Hippo, and Wnt signaling pathways. The most downregulated transcripts in KTCN corneas were CTGF, TGFB3, ZNF469, COL5A2, SMAD7, and SPARC, while TGFBI and SLC4A11 were the most upregulated ones. Hierarchical clustering of expression profiles demonstrated almost clear separation between KTCN and non-KTCN corneas. The gene expression levels determined using RT-qPCR showed a strong correlation with previous RNA sequencing (RNA-Seq) results. CONCLUSIONS: A strong correlation between RT-qPCR and earlier RNA-Seq data confirms the possible involvement of genes from collagen synthesis and the TGF-ß, Hippo, and Wnt signaling pathways in KTCN etiology. Our data also revealed altered expression of several genes, such as LOX, SPARC, and ZNF469, in which single nucleotide variants have been frequently identified in KTCN. These findings further highlight the heterogeneous nature of KTCN.

Increasing Resistance and Changes in Distribution of Serotypes of Streptococcus agalactiae in Poland.

Streptococcus agalactiae is responsible for serious infections in newborn babies, pregnant women, and other patients. The aim of this study was to evaluate antimicrobial susceptibility, serotype distribution, and virulence determinants of the S. agalactiae isolates derived from clinical specimens considering the global increase of both antibiotic resistance and virulence. A total of 165 isolates were identified and serotyped by PCR techniques. Antimicrobial susceptibility was assessed by disk diffusion method, gradient diffusion method and VITEK® System. Virulence associated genes were investigated by PCR; ability to form biofilm was assessed using a microtiter plate assay. The highest observed MIC value for penicillin G was 0.12 µg/mL, seen in 8.5% of isolates. Resistance to erythromycin and clindamycin were found in 30.38% and 24.8% of the strains, respectively. The serotype III (32.73%), V (25.45%), and Ia (18.18%) were found as the most frequently represented. Previously unidentified strains in Poland, belonging to serotypes VI (three strains) and VII (one strain) were recognized. The presence of genes encoding various virulence factors as well as diverse ability to form biofilm were found. In conclusion, macrolide-resistance and decreased susceptibility to penicillin G were revealed signifying the increasing resistance among group B streptococci. Moreover, the presence of genes encoding various virulence factors and the ability to form biofilm were confirmed indicating their role in the pathomechanisms of the evaluated GBS infections.