Biosensor for the detection of Listeria monocytogenes: emerging trends.

The early detection of Listeria monocytogenes (L. monocytogenes) and understanding the disease burden is of paramount interest. The failure to detect pathogenic bacteria in the food industry may have terrible consequences, and poses deleterious effects on human health. Therefore, integration of methods to detect and trace the route of pathogens along the entire food supply network might facilitate elucidation of the main contamination sources. Recent research interest has been oriented towards the development of rapid and affordable pathogen detection tools/techniques. An innovative and new approach like biosensors has been quite promising in revealing the foodborne pathogens. In spite of the existing knowledge, advanced research is still needed to substantiate the expeditious nature and sensitivity of biosensors for rapid and in situ analysis of foodborne pathogens. This review summarizes recent developments in optical, piezoelectric, cell-based, and electrochemical biosensors for Listeria sp. detection in clinical diagnostics, food analysis, and environmental monitoring, and also lists their drawbacks and advantages.

Virulence and genotypic characterization of Listeria monocytogenes isolated from vegetable and soil samples.

BACKGROUND: Listeria monocytogenes, a foodborne pathogen is ubiquitous to different environments including the agroecosystem. The organism poses serious public health problem. Therefore, an attempt has been made to gain further insight to their antibiotic susceptibility, serotypes and the virulence genes. RESULTS: Out of the 10 vegetables selected, 6 (brinjal, cauliflower, dolichos-bean, tomato, chappan-kaddu and chilli), 20 isolates (10%) tested positive for L. monocytogenes. The prevalence of the pathogen in the respective rhizosphere soil samples was 5%. Noticeably, L. monocytogenes was absent from only cabbage, broccoli, palak and cowpea, and also the respective rhizospheric soils. The 30 isolates + ve for pathogenicity, belonged to serogroup 4b, 4d or 4e, and all were positive for inlA, inlC, inlJ, plcA, prfA, actA, hlyA and iap gene except one (VC3) among the vegetable isolates that lacked the plcA gene. ERIC- and REP-PCR collectively revealed that isolates from vegetables and their respective rhizospheric soils had distinct PCR fingerprints. CONCLUSIONS: The study demonstrates the prevalence of pathogenic L. monocytogenes in the selected agricultural farm samples. The increase in the number of strains resistant to ciprofloxacin and/or cefoxitin seems to pose serious public health consequences.

Phylogenetic analysis of the Listeria monocytogenes based on sequencing of 16S rRNA and hlyA genes.

The discrimination between Listeria monocytogenes and Listeria species has been detected. The 16S rRNA and hlyA were PCR amplified with set of oligonucleotide primers with flank 1,500 and 456 bp fragments, respectively. Based on the differences in 16S rRNA and hlyA genes, a total 80 isolates from different environmental, food and clinical samples confirmed it to be L. monocytogenes. The 16S rRNA sequence similarity suggested that the isolates were similar to the previously reported ones from different habitats by others. The phylogenetic interrelationships of the genus Listeria were investigated by sequencing of 16S rRNA and hlyA gene. The 16S rRNA sequence indicated that genus Listeria is comprised of following closely related but distinct lines of descent, one is the L. monocytogenes species group (including L. innocua, L. ivanovii, L. seeligeri and L. welshimeri) and other, the species L. grayi, L. rocourtiae and L. fleischmannii. The phylogenetic tree based on hlyA gene sequence clearly differentiates between the L. monocytogenes, L. ivanovii and L. seeligeri. In the present study, we identified 80 isolates of L. monocytogenes originating from different clinical, food and environmental samples based on 16S rRNA and hlyA gene sequence similarity.

Time- and sex-dependent delayed effects of acute radiation exposure manifest via miRNA dysregulation.

The detrimental effects of high-dose ionizing radiation on human health are well-known, but the influence of sex differences on the delayed effects of acute radiation exposure (DEARE) remains unclear. Here, we conducted six-month animal experiments using escalating radiation doses (7-9 Gy) on male and female C57BL/6 mice. The results show that female mice exhibited greater resistance to radiation, showing increased survival at six months post-total body irradiation. LD50/30 (lethal dose expected to cause 50% lethality in 30 days) for female mice is 8.08 Gy, while for male mice it is 7.76 Gy. DEARE causes time- and sex-dependent dysregulation of microRNA expression, processing enzymes, and the HOTAIR regulatory pathway. Differential regulation of molecular patterns associated with growth, development, apoptosis, and cancer is also observed in male and female mice. These findings shed light on the molecular basis of age and sex differences in DEARE response and emphasize the importance of personalized medicine for mitigating radiation-induced injuries and diseases.

CDX-301 prevents radiation-induced dysregulation of miRNA expression and biogenesis.

Risks of radiation exposure necessitate the development of radioprophylactic drugs. We have reported the efficacy of CDX-301, a recombinantly developed human protein form of Fms-related tyrosine kinase 3 ligand (Flt3L), as a radioprophylactic and radiomitigatory agent. Here, we performed global microRNA profiling to further understand the mechanism of action of CDX-301. We find that CDX-301 administration 24 h prior to total body irradiation prevents radiation-induced dysregulation of microRNA biogenesis and expression in murine serum and spleen samples in a time- and tissue-dependent manner. Further analysis shows that activation of the HOTAIR regulatory pathway has a prominent function in radiation-induced injury responses, which is inhibited by pre-treatment with CDX-301. Moreover, CDX-301 attenuates radiation-induced dysregulation of several cellular functions such as inflammatory and immune responses. In corroboration, we also find that pre-treatment with CDX-301 restores the expression of bone marrow aplasia markers and inflammatory cytokines and growth factors, as well as the expression of genes associated with MAP kinase and TGF-ß pathways that are altered by radiation. Our findings provide new insights into CDX-301-mediated molecular and cellular mechanisms and point to a possible novel radioprotective drug for the prevention of irradiation-induced injury and hematopoietic acute radiation syndrome.

Role of Non-Coding RNAs in Post-Transcriptional Regulation of Lung Diseases.

Non-coding RNAs (ncRNAs), notably microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), have recently gained increasing consideration because of their versatile role as key regulators of gene expression. They adopt diverse mechanisms to regulate transcription and translation, and thereby, the function of the protein, which is associated with several major biological processes. For example, proliferation, differentiation, apoptosis, and metabolic pathways demand fine-tuning for the precise development of a specific tissue or organ. The deregulation of ncRNA expression is concomitant with multiple diseases, including lung diseases. This review highlights recent advances in the post-transcriptional regulation of miRNAs and lncRNAs in lung diseases such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, and idiopathic pulmonary fibrosis. Further, we also discuss the emerging role of ncRNAs as biomarkers as well as therapeutic targets for lung diseases. However, more investigations are required to explore miRNAs and lncRNAs interaction, and their function in the regulation of mRNA expression. Understanding these mechanisms might lead to early diagnosis and the development of novel therapeutics for lung diseases.

SFPQ rescues F508del-CFTR expression and function in cystic fibrosis bronchial epithelial cells.

Cystic fibrosis (CF) occurs as a result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to misfolding, trafficking defects, and impaired function of the CFTR protein. Splicing factor proline/glutamine-rich (SFPQ) is a multifunctional nuclear RNA-binding protein (RBP) implicated in the regulation of gene expression pathways and intracellular trafficking. Here, we investigated the role of SFPQ in the regulation of the expression and function of F508del-CFTR in CF lung epithelial cells. We find that the expression of SFPQ is reduced in F508del-CFTR CF epithelial cells compared to WT-CFTR control cells. Interestingly, the overexpression of SFPQ in CF cells increases the expression as well as rescues the function of F508del-CFTR. Further, comprehensive transcriptome analyses indicate that SFPQ plays a key role in activating the mutant F508del-CFTR by modulating several cellular signaling pathways. This is the first report on the role of SFPQ in the regulation of expression and function of F508del-CFTR in CF lung disease. Our findings provide new insights into SFPQ-mediated molecular mechanisms and point to possible novel epigenetic therapeutic targets for CF and related pulmonary diseases.

ATP-binding cassette (ABC) import systems of Mycobacterium tuberculosis: target for drug and vaccine development.

Nutrient procurement specifically from nutrient-limiting environment is essential for pathogenic bacteria to survive and/or persist within the host. Long-term survival or persistent infection is one of the main reasons for the overuse of antibiotics, and contributes to the development and spread of antibiotic resistance. Mycobacterium tuberculosis is known for long-term survival within the host, and develops multidrug resistance. Before and during infection, the pathogen encounters various harsh environmental conditions. To cope up with such nutrient-limiting conditions, it is crucial to uptake essential nutrients such as ions, sugars, amino acids, peptides, and metals, necessary for numerous vital biological activities. Among the various types of transporters, ATP-binding cassette (ABC) importers are essentially unique to bacteria, accessible as drug targets without penetrating the cytoplasmic membrane, and offer an ATP-dependent gateway into the cell by mimicking substrates of the importer and designing inhibitors against substrate-binding proteins, ABC importers endeavour for the development of successful drug candidates and antibiotics. Alternatively, the production of antibodies against substrate-binding proteins could lead to vaccine development. In this review, we will emphasize the role of M. tuberculosis ABC importers for survival and virulence within the host. Furthermore, we will elucidate their unique characteristics to discover emerging therapies to combat tuberculosis.

Pregnancy - associated human listeriosis: Virulence and genotypic analysis of Listeria monocytogenes from clinical samples.

Listeria monocytogenes, a life-threatening pathogen, poses severe risk during pregnancy, may cause abortion, fetal death or neonatal morbidity in terms of septicemia and meningitis. The present study aimed at characterizing L. monocytogenes isolated from pregnant women based on serotyping, antibiotic susceptibility, virulence genes, in vivo pathogenicity test and ERIC- and REP-PCR fingerprint analyses. The results revealed that out of 3700 human clinical samples, a total of 30 (0.81%) isolates [12 (0.80%) from placental bit (1500), 18 (0.81%) from vaginal swab (2200)] were positive for L. monocytogenes. All the isolates belonged to serogroup 4b, and were + ve for virulence genes tested i.e. inlA, inlC, inlJ, plcA, prfA, actA, hlyA, and iap. Based on the mice inoculation tests, 20 isolates showed 100% and 4 isolates 60% relative virulence while 6 isolates were non-pathogenic. Moreover, 2 and 10 isolates were resistant to ciprofloxacin and cefoxitin, respectively, while the rest susceptible to other antibiotics used in this study. ERIC- and REP-PCR collectively depicted that the isolates from placental bit and vaginal swab had distinct PCR fingerprints except a few isolates with identical patterns. This study demonstrates prevalence of pathogenic strains mostly resistant to cefoxitin and/or ciprofloxacin. The results indicate the importance of isolating and characterizing the pathogen from human clinical samples as the pre-requisite for accurate epidemiological investigations.

Label-free impedimetric detection of Listeria monocytogenes based on poly-5-carboxy indole modified ssDNA probe.

Listeria monocytogenes is a life threatening pathogenic bacteria concerned with human health. The accurate and rapid detection of L. monocytogenes is required for preventing of listeriosis. In this study, DNA sensing probe based on conducting polymer poly-5-carboxy indole (5C Pin) was developed for the detection of L. monocytogenes hlyA gene responsible for pathogenicity. The probe sequences (24 mer ssDNA) were covalently immobilized on 5C Pin via N-(3-dimethylaminopropyl)-N'-ethylcarbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS). The probe having ssDNA was further hybridized with the target DNA sequence. Electrochemical impedance spectroscopic study was carried out to determine the extent of DNA hybridization over the probe. Significant change was observed in the impedance spectra before and after hybridization of ssDNA immobilized over the probe with the target DNA. RCT (charge transfer resistance) was estimated from the Nyquist plot (impedance plot) for target DNA (hlyA gene) in the solution. RCT vs. logarithmic concentrations of the target (genomic) DNA plot showed a linear range (1 × 10(-4) to 1 × 10(-12)M) in case hybridization was performed under optimized conditions. The method proposed, is simple, free from any label, and highly sensitive for the detection of L. monocytogenes in environmental and clinical samples.