Role of miRNA dysregulation in sepsis.

BACKGROUND: Sepsis is defined as a state of multisystem organ dysfunction secondary to a dysregulated host response to infection and causes millions of deaths worldwide annually. Novel ways to counteract this disease are needed and such tools may be heralded by a detailed understanding of its molecular pathogenesis. MiRNAs are small RNA molecules that target mRNAs to inhibit or degrade their translation and have important roles in several disease processes including sepsis. MAIN BODY: The current review adopted a strategic approach to analyzing the widespread literature on the topic of miRNAs and sepsis. A pubmed search of "miRNA or microRNA or small RNA and sepsis not review" up to and including January 2021 led to 1140 manuscripts which were reviewed. Two hundred and thirty-three relevant papers were scrutinized for their content and important themes on the topic were identified and subsequently discussed, including an in-depth look at deregulated miRNAs in sepsis in peripheral blood, myeloid derived suppressor cells and extracellular vesicles. CONCLUSION: Our analysis yielded important observations. Certain miRNAs, namely miR-150 and miR-146a, have consistent directional changes in peripheral blood of septic patients across numerous studies with strong data supporting a role in sepsis pathogenesis. Furthermore, a large body of literature show miRNA signatures of clinical relevance, and lastly, many miRNAs deregulated in sepsis are associated with the process of endothelial dysfunction. This review offers a widespread, up-to-date and detailed discussion of the role of miRNAs in sepsis and is meant to stimulate further work in the field due to the potential of these small miRNAs in prompt diagnostics, prognostication and therapeutic agency.

Validation of reference gene stability for miRNA quantification by reverse transcription quantitative PCR in the peripheral blood of patients with COVID-19 critical illness.

The COVID-19 pandemic has created an urgency to study the host gene response that leads to variable clinical presentations of the disease, particularly the critical illness response. miRNAs have been implicated in the mechanism of host immune dysregulation and thus hold potential as biomarkers and/or therapeutic agents with clinical application. Hence, further analyses of their altered expression in COVID-19 is warranted. An important basis for this is identifying appropriate reference genes for high quality expression analysis studies. In the current report, NanoString technology was used to study the expression of 798 miRNAs in the peripheral blood of 24 critically ill patients, 12 had COVID-19 and 12 were COVID-19 negative. A list of potentially stable candidate reference genes was generated that included ten miRNAs. The top six were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a total of 41 patients so as to apply standard computational algorithms for validating reference genes, namely geNorm, NormFinder, BestKeeper and RefFinder. There was general agreement among all four algorithms in the ranking of four stable miRNAs: miR-186-5p, miR-148b-3p, miR-194-5p and miR-448. A detailed analysis of their output rankings led to the conclusion that miR-186-5p and miR-148b-3p are appropriate reference genes for miRNA expression studies using PaxGene tubes in the peripheral blood of patients critically ill with COVID-19 disease.

Novel pharmacokinetic behavior of intravenous busulfan in children with thalassemia undergoing hematopoietic stem cell transplantation: a prospective evaluation of pharmacokinetic and pharmacodynamic profile with therapeutic drug monitoring.

We prospectively studied the pharmacokinetics (PK) and clinical outcomes of intravenous busulfan (Bu) in 71 children with preexisting liver damage who underwent hematopoietic stem cell transplantation for thalassemia. Intravenous Bu was administered every 6 hours as part of a conditioning regimen with PK-based dose adjustment to target a conservative area under the concentration-versus-time curve (AUC) range (900-1350 microMol*min). The first-dose Bu clearance (CL) was significantly higher than the subsequent daily CL that remained unchanged in the ensuing days. One-third of patients required dose escalation based on dose 1 AUC, whereas dose reduction was needed in the subsequent days. At doses 5, 9, and 13, 78%, 81%, and 87% of patients, respectively, achieved the target range of AUC. A population PK analysis confirmed that the first-dose CL was 20% higher and that body weight was the most important covariate to explain PK variability. Patients with variant GSTA1*B had a 10% lower Bu CL than wild-type. These results suggest that the disease-specific behavior of intravenous Bu PK should be considered for PK-guided dose adjustment in patients with thalassemia, and the use of a conservative AUC range resulted in low toxicity, good engraftment, and good survival rate.

Plasmin system of Alzheimer's disease patients: CSF analysis.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder characterized by the extracellular deposit of Amyloid beta (Aß), mainly of the Amyloid beta(1-42) (Aß(1-42)) peptide in the hippocampus and neocortex leading to progressive cognitive decline and dementia. The possible imbalance between the Aß production/degradation process was suggested to contribute to the pathogenesis of AD. Among others, the serine protease plasmin has shown to be involved in Aß(1-42) clearance, a hypothesis strengthened by neuropathological studies on AD brains. To explore whether there is a change in plasmin system in CSF of AD patients, we analyzed CSF samples from AD and age-matched controls, looking at plasminogen, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) protein levels and t-PA and urokinase plasminogen activator (u-PA) enzymatic activities. We also measured Aß(1-42), total-tau and phospho-tau (181) CSF levels and sought for a possible relationship between them and plasmin system values. Our findings showed that t-PA, plasminogen and PAI-1 levels, as t-PA enzymatic activity, remained unchanged in AD with respect to controls; u-PA activity was not detected. We conclude that CSF analysis of plasminogen system does not reflect changes observed post-mortem. Unfortunately, the CSF detection of plasmin system could not be a useful biomarker for either AD diagnosis or disease progression. However, these findings do not exclude the possible involvement of the plasmin system in AD.

DJ-1 binds to Rubicon to Impair LC-3 Associated Phagocytosis.

The ability to effectively clear infection is fundamental to host survival. Sepsis, defined as dysregulated host response to infection, is a heterogenous clinical syndrome that does not uniformly clear intact bacterial or sterile infection (i.e., lipopolysaccharide). These findings were further associated with increased survival in DJ-1 deficient animals exposed to intact bacteria relative to DJ-1 deficient challenged with lipopolysaccharide. We analyzed bacterial and lipopolysaccharide clearance in bone marrow macrophages (BMM) cultured ex vivo from wild-type and DJ-1 deficient mice. Importantly, we demonstrated that DJ-1 deficiency in BMM promotes Rubicon-dependent increase in L3C-associated phagocytosis, non-canonical autophagy pathway used for xenophagy, during bacterial but not lipopolysaccharide infection. In contrast to DJ-1 deficient BMM challenged with lipopolysaccharide, DJ-1 deficient BMM exposed to intact bacteria showed enhanced Rubicon complexing with Beclin-1 and UVRAG and consistently facilitated the assembly of complete autophagolysosomes that were decorated with LC3 molecules. Our data shows DJ-1 impairs or/and delays bacterial clearance and late autophagolysosome formation by binding to Rubicon resulting in Rubicon degradation, decreased L3C-associated phagocytosis, and decreased bacterial clearance in vitro and in vivo - implicating Rubicon and DJ-1 as critical regulators of bacterial clearance in experimental sepsis.

Social context influences chemical communication in D. melanogaster males.

Chemical communication mediates social interactions in insects. For the fruit fly, D. melanogaster, the chemical display is a key fitness trait because it leads to mating. An exchange of cues that resembles a dialogue between males and females is enacted by pheromones, chemical signals that pass between individual flies to alter physiology and behavior. Chemical signals also affect the timing of locomotor activity and sleep. We investigated genetic and environmental determinants of chemical communication. To evaluate the role of the social environment, we extracted a chemical blend from individual males selected from groups composed of one genotype and compared these extracts to those from groups of mixed genotypes. To evaluate the role of the physical environment, these comparisons were performed under a light-dark cycle or in constant darkness. Here, we show that chemical signaling is affected by the social environment, light-dark cycle, and genotype as well as the complex interplay of these variables. Gene-by-environment interactions produce highly significant effects on chemical signaling. We also examined individual responses within the groups. Strikingly, the response of one wild-type fly to another is modulated by the genotypic composition of his neighbors. Chemical signaling in D. melanogaster may be a "fickle" trait that depends on the individual's social background.

A simplified, non-invasive fecal-based DNA integrity assay and iFOBT for colorectal cancer detection.

PURPOSE: Neoplasia cells exfoliated from colorectal epithelium have dysfunctional apoptotic mechanisms, thus it is possible to identify high-molecular weight DNA fragments in feces. This prospective single-center study was performed to evaluate the sensitivity and specificity of fecal-based DNA integrity versus immunological fecal occult blood test (iFOBT) and calprotectin for colorectal cancer (CRC) and adenoma detection. METHODS: Feces were collected from 204 subjects and DNA integrity was quantified by quantitative-denaturing high performance liquid chromatography (QdHPLC). Calprotectin and iFOBT were assessed using commercial kits. The diagnostic performance was calculated by receiver operating characteristic (ROC) curves analysis. RESULTS: A total of 192 fecal specimens were analyzed and 12 samples were excluded due to DNA degradation. We found long DNA (L-DNA) occurrence in feces with a sensitivity of 86% (n = 24/28) and a specificity of 81% for CRC detection. To minimize false-positive cases of the developed test, area under the curve of ROC was evaluated such that the specificity was increased to 92% with decreased sensitivity to 79%, p = 0.0001 for CRC detection. iFOBT was positive in 51% (n = 14/27) while calprotectin was positive in 75% (n = 18/27). The combination of iFOBT and L-DNA identified a greater number of CRC cases with a sensitivity of 89% and a specificity of 95%, p < 0.001. The combination also improved the sensitivity of polyps, particularly high-grade dysplasia and advanced adenoma (33%, p = 0.0015) as opposed to a single evaluation assay (17-21%). CONCLUSIONS: This study illustrates the usefulness of fecal DNA integrity assay by QdHPLC as a non-invasive, easy-to-perform, and reproducible method with a high level of sensitivity in detecting individuals with colorectal neoplasia. Combination of iFOBT and L-DNA improves the sensitivity for CRC and adenoma detection.

The educational needs of Canadian homeless shelter workers related to traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) has a higher prevalence in the homeless population. Caregivers to individuals who have TBIs may require better education surrounding screening, diagnosis and management of this disease to tailor interventions to their clients' needs. OBJECTIVE: To assess the insight and educational needs of homeless care providers in recognizing and dealing with clients who had experienced a TBI. METHODS: A survey assessing the point of views of homeless care providers across Canada regarding their level of confidence in identifying and managing symptoms of TBI. RESULTS: Eight-eight completed surveys were included. Overall, frontline workers expressed a moderate level of confidence in identifying and managing TBI, stating that educational initiatives in this context would be of high value to themselves and their clients. CONCLUSIONS: Frontline workers to homeless clients rate their educational needs on the identification and management of TBI to be high such that educational initiatives for shelter workers across Canada may be beneficial to increase their knowledge in identifying and managing the TBI-related symptoms. Improved education would not only benefit frontline workers but may also have a positive effect on health outcomes for their clients.

Quantitative denaturing high performance liquid chromatography (Q-dHPLC) detection of APC long DNA in faeces from patients with colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths. However, prevention is possible by early detection. In the present work, we have demonstrated and validated a novel quantitative method based on a DNA integrity assay and mutation in faeces of CRC patients using denaturing high performance liquid chromatography (dHPLC). METHODS: Faecal DNA (fDNA) was isolated from 28 CRC, 96 healthy and 61 patients with adenomas. Adenomatosis polyposis coli (APC)-Long-DNA and its mutations were analysed using dHPLC and the Sanger sequencing method. The diagnostic performance was assessed using receiver operating characteristic curve analysis. RESULTS: We detected APC-Long-DNA in 21/28 CRC subjects with a sensitivity of 75% and specificity of 91.7%. A cut-off ratio of 0.2317 was used for APC/ß-actin. The Q-dHPLC detection limit was 0.02 ng/injection. The average initial fDNA presence based on a single gene of ß-actin was 26.12 ± 13.39 ng/mL for healthy, and 49.61 ± 46.28 ng/mL for CRC subjects, with a sensitivity of 71.4% and a specificity of 84.4% at a cut-off value >29 ng/mL. We also detected a novel mutation at codon 1576 Lys/Glu using dHPLC. CONCLUSIONS: This study highlights a novel application of Q-dHPLC in the DNA integrity assay, which demonstrates high performance, good reproducibility, and low cost for the CRC detection using faeces. Further studies in a larger population are needed to confirm these results.

Low Contrast Dose Catheter-Directed CT Angiography (CCTA).

PURPOSE: Catheter-directed computed tomography angiography (CCTA) has been shown to reduce the contrast volumes required in conventional CTA, thus minimizing the risk of contrast-induced nephropathy (CIN). MATERIALS AND METHODS: A retrospective analysis was performed on cases where CCTA was used to assess access vessels prior to transfemoral aortic valve implantation (TAVI, n = 53), abdominal aortic aneurysm assessment for endovascular aneurysm repair (EVAR, n = 11), and peripheral vascular disease (PVD, n = 24). RESULTS: We show that CCTA can image vasculature with adequate diagnostic detail to allow assessment of lower extremity disease, anatomic suitability for EVAR, as well as potential contraindications to TAVI. Average contrast volumes for pre-TAVI, pre-EVAR, and PVD cases were 7, 11, and 28 mL, respectively. CONCLUSION: This study validates the use of CCTA in obtaining diagnostic images of the abdominal and pelvic vessels and in imaging lower extremity vasculature.

Satraplatin (JM-216) mediates G2/M cell cycle arrest and potentiates apoptosis via multiple death pathways in colorectal cancer cells thus overcoming platinum chemo-resistance.

PURPOSES: Satraplatin acts as a potent inhibitor of proliferation in castration-resistant prostate cancer, yet the basic and molecular pharmacological mechanisms are still unknown in all types of cancer including colorectal cancer (CRC). In an effort to explain the mechanism of tumour sensitivity to satraplatin, the cytotoxic effects in a panel of CRC cell lines was examined with regard to their p53 genotype in comparison with oxaliplatin. METHODS: CRC cell lines were chosen to ascertain the mechanism of satraplatin-enhanced cytotoxicity. Cells were incubated with oxaliplatin and satraplatin for 24-72 h, followed by the assessment of cell chemosensitivity with MTS. Western blot analysis was used to detect the expressions of p53-related molecules. Flow cytometry was used to monitor cell cycle perturbation while qRT-PCR to detect mRNA and miRNAs activities. RESULTS: Satraplatin treatment resulted an elevated increase in cell death in vitro compared to oxaliplatin preceded by an acute arrest at G2/M phase, along with cyclin B1 and p21(waf/cip1) up-regulation. It also exhibited fourfold higher cellular platinum accumulations compared to oxaliplatin. Satraplatin treatment induces p53-related genes and its direct microRNA target of miR-34a independently. Thus, it potentiates apoptosis via multiple death pathways including the caspase 8 cleavages and Fas protein expression. The data suggest that the loss of p53 can increase oxaliplatin resistance but not satraplatin resistance. CONCLUSION: Further molecular biology studies are needed to identify the activity of satraplatin in platinum-resistant cancer models and to determine whether this orally administered platinum analogue has synergistic effects in combination with other chemotherapy agents.

Salivary miRNAome profiling uncovers epithelial and proliferative miRNAs with differential expression across dentition stages.

Saliva's ability to mirror the internal physiological environment of an organism coupled with its facile accessibility makes it an attractive diagnostic medium. The finding of microRNAs (miRNAs) in saliva has expanded the field of biomarker discovery since these tiny non-coding RNAs affect various physiological processes and diseases. Few reports have linked miRNAs to tooth development and eruption, with none having studied this in humans. As a first initiative to describe miRNAs in saliva whose modulations may reflect developing and erupting teeth, we quantified the levels of 730 miRNAs in the saliva of children of varying dentition stages: edentulous (newborns), deciduous and permanent by megaplex stemloop reverse-transcription quantitative PCR. The three groups expressed 193, 181 and 192 miRNAs, respectively, where 125 miRNAs had consistent expression. The remaining miRNAs had inter-group variations from 5 to hundreds of fold, where most had either an increasing or decreasing trend in going from edentulous to deciduous to permanent. A literature survey of epithelial miRNAs found most were present in saliva. Moreover, many miRNAs with expression differences between groups had previously documented functions in proliferation, cell cycle, apoptosis and other cellular behaviours key to the dynamics of tooth morphogenesis. Lastly, miRNAs of the same family, such as the let-7 and miR-200 families, or transcribed from the same hairpin, had similar expression patterns. The results presented here should serve as a salivary miRNA dictionary for future studies in tooth development as well as in childhood diseases associated with modulations in saliva composition.

MiR-203 controls proliferation, migration and invasive potential of prostate cancer cell lines.

Prostate cancers show a slow progression from a local lesion (primary tumor) to a metastatic and hormone-resistant phenotype. After an initial step of hyperplasia, in a high percentage of cases a neoplastic transformation event occurs that, less frequently, is followed by epithelial to mesenchymal transition and invasion of healthy tissues (usually bones). MicroRNA-203 (miR-203) is a tumor suppressor microRNA often silenced in different malignancies. Here, we show that miR-203 is downregulated in clinical primary prostatic tumors compared to normal prostate tissue, and in metastatic prostate cancer cell lines compared to normal epithelial prostatic cells. Overexpression of miR-203 in brain or bone metastatic prostate cell lines (DU145 and PC3) is sufficient to induce a mesenchymal to epithelial transition with inhibition of cell proliferation, migration and invasiveness. We have identified CKAP2, LASP1, BIRC5, WASF1, ASAP1 and RUNX2 as new miR-203 direct target mRNAs involved in these events. Therefore, miR-203 could be a potentially new prognostic marker and therapeutic target in metastatic prostate cancer.