Integration of chemokine signaling with non-coding RNAs in tumor microenvironment and heterogeneity in different cancers.

Chemokines are small secreted proteins that regulate the immune system by signaling through chemokine receptors to induce immune cell migration, motility, and infiltration into the tissue. Altered chemokine/receptor expression is associated with numerous inflammatory diseases, and more recently in non-immune cell diseases like cancer. Emerging new studies demonstrate that chemokines can directly modulate the tumor microenvironment (TME) to assist tumorigenesis by regulating proinflammatory signaling, immune cell infiltration,and metastasis. However, the diversity and complexity in the regulation of chemokine expression and how chemokine receptor signaling influences TME needs comprehensive understanding. One mechanistic pathway that has shown promising early results in targeting tumor progression is the non-coding RNAs (ncRNAs). These are widely expressed and designated as prime gene regulatory factors in tumors and the immune system. Notably, ncRNAs have been implicated in regulating chromatin stability, translation of cytoplasmic mRNAs, and the functional regulation of membrane-less nuclear bodies, which are significant pathways implicated in tumorigenesis. Tissue-specific patterns of expression of ncRNAs have suggested their role as potential cancer biomarkers, providing a suitable rationale for targeting them clinically. In this review, we discuss the recent findings which demonstrate the role of differential expression of chemokines and ncRNA in modulating TME during tumor progression. We also discuss the communication between tumor and immune effector cells via chemokine/ncRNAs and identify their potential as novel therapeutic targets.

Genetically Encoded FRET-Based Nanosensor for Real-Time Monitoring of A549 Exosomes: Early Diagnosis of Cancer.

Exosomes contain a plethora of unique disease biomarkers involving cellular homeostasis, infection dissemination, cancer development, and cardiac diseases. Exosomes originating from cancer cells have promising biomarkers for the early detection and assessment of the therapeutic response to cancer. The exosomal epidermal growth factor receptor (EGFR) is a potential biomarker which is overexpressed in cancer; thus, the level of EGFR expression is investigated by so many methods in a liquid and solid biopsy. The optimal method for isolating pure exosomal EGFRs has not been well understood so far. Current approaches are complicated and time-consuming, therefore hampering their clinical applications. Here, we demonstrate the creation of an innovative fluorescence resonance energy transfer (FRET) sensor, named ExoSen (exosome sensor), which can be implemented to determine the concentration of exosomal EGFRs at in vitro as well as in vivo levels. In this study, a sensing element for A549 exosomes, mitogen-inducible gene 6 (MIG6), has been employed between the FRET pair ECFP and Venus. MIG6 binding to ExoSen induced a conformational change that can be monitored by a variation in the FRET ratio. Moreover, the developed sensor, expressed in bacterial, yeast, and HEK-293T cells, demonstrates an increased FRET ratio with the addition of A549 exosomes, which can quantify the A549 exosomes noninvasively. The ExoSen enables rapid detection of A549 exosomes with great sensitivity at a concentration of 3.5 × 109 particles/mL. ExoSen is stable to pH fluctuations and provides a highly accurate, real-time optical readout in cell-based experiments by using confocal microscopy.

FRET-based probe for ratiometric detection and imaging of folic acid in real-time.

Inadequate folic acid intake is linked to diseases such as megaloblastic anemia, neural tube defects, and hyperhomocysteinemia, increasing the risk of vascular disease and thrombosis. Folic acid, a cofactor in various enzymes, can be produced by plants and bacteria, but not by humans and other animals. L-5-methyl-tetrahydrofolate (L-5-methyl-THF) is the primary dietary folate form, transported in circulation for cellular metabolism. Traditional methods of determining folic acid levels are unreliable and time-consuming. SenFol (Sensor for folic acid) is a fluorescence resonance energy transfer (FRET)-based nanosensor that we have developed by inserting folic acid-binding protein (FolT) as the folate detecting domain between the pair of enhanced cyan fluorescent protein (ECFP) and Venus. The developed sensor is highly specific, produces a quick signal, which is pH stable, and delivers precise, ratiometric readings in cell-based experiments. The projected affinity score of folic acid with FolT was -7.4 kcal/mol. The apparent affinity (Kd) of SenFol for folic acid is 28.49 × 10-9 M, with a detection range of 5 × 10-9 M to 5 × 10-7 M, and a maximum FRET ratio change of 0.45. WT SenFol, a highly efficient folic acid nanosensor, can dynamically detect intracellular folic acid content in E. coli, yeast, and HEK-293 T cells, confirming its potential.

A district-level vulnerability assessment of next COVID-19 variant (Omicron BA.2) in Uttarakhand using quantitative SWOT analysis.

COVID-19 has had an impact on the entire humankind and has been proved to spread in deadly waves. As a result, preparedness and planning are required to better deal with the epidemic's upcoming waves. Effective planning, on the other hand, necessitates detailed vulnerability assessments at all levels, from the national to the state or regional. There are several issues at the regional level, and each region has its own features. As a result, each region needs its own COVID-19 vulnerability assessment. In terms of climate, terrain and demographics, the state of Uttarakhand differs significantly from the rest of India. As a result, a vulnerability assessment of the next COVID-19 variation (Omicron BA.2) is required for district-level planning to meet regional concerns. A total of 17 variables were chosen for this study, including demographic, socio-economic, infrastructure, epidemiological and tourism-related factors. AHP was used to compute their weights. After applying min-max normalisation to the data, a district-level quantitative SWOT is created to compare the performance of 13 Uttarakhand districts. A COVID-19 vulnerability index (normalised R i ) ranging between 0 and 1 was produced, and district-level vulnerabilities were mapped. Quantitative SWOT results depict that Dehradun is a best performing district followed by Haridwar, while Bageshwar, Rudra Prayag, Champawat and Pithoragarh are on the weaker side and the normalised Ri proves Dehradun, Nainital, Champawat, Bageshwar and Chamoli to be least vulnerable to COVID-19 (normalised R i ≤ 0.25) and Pithoragarh to be the most vulnerable district (normalised R i > 0.90). Pauri Garwal and Uttarkashi are moderately vulnerable (normalised R i 0.50 to 0.75).

Design, synthesis & biological evaluation of ferulic acid-based small molecule inhibitors against tumor-associated carbonic anhydrase IX.

Carbonic anhydrase IX (CAIX) is an emerging drug target for hypoxia associated cancers. To identify potent and selective inhibitors of CAIX, a small library of ferulic acid (FA) derivatives bearing triazole moiety has been designed, synthesized and evaluated against different human CA isoforms (CAII, CAVA & CAIX). Though most of the compounds showed CAIX inhibition in the micromolar range, compound 7i selectively inhibits CAIX in the nanomolar range (IC50 = 24 nM). In silico analysis revealed binding of 7i with the catalytically important amino acid residues of CAIX. Further, cell-based studies indicate that 7i inhibits the activity of CAIX, decreases the epithelial to mesenchymal transitions, induces apoptosis, inhibits cell migration and colonization potential of cancer cells. Taken together, these results emphasized the use of 7i as a prospective pharmacological lead molecule in CAIX targeted anticancer therapeutics.

A Non-Invasive Tool for Real-Time Measurement of Sulfate in Living Cells.

Sulfur (S) is an essential element for all forms of life. It is involved in numerous essential processes because S is considered as the primary source of one of the essential amino acids, methionine, which plays an important role in biological events. For the control and regulation of sulfate in a metabolic network through fluxomics, a non-invasive tool is highly desirable that opens the door to monitor the level of the sulfate in real time and space in living cells without fractionation of the cells or tissue. Here, we engineered a FRET (fluorescence resonance energy transfer) based sensor for sulfate, which is genetically-encoded and named as FLIP-SP (Fluorescent indicator protein for sulfate). The FLIP-SP can measure the level of the sulfate in live cells. This sensor was constructed by the fusion of fluorescent proteins at the N- and C-terminus of sulfate binding protein (sbp). The FLIP-SP is highly specific to sulfate, and showed pH stability. Real-time monitoring of the level of sulfate in prokaryotic and eukaryotic cells showed sensor bio-compatibility with living cells. We expect that this sulfate sensor offers a valuable strategy in the understanding of the regulation of the flux of sulfate in the metabolic network.

FRET-based nanosensors for monitoring and quantification of alcohols in living cells.

Odorants constitute a small and chemically diverse group of molecules with ethanol functioning as a key odorant that induces reproductive toxicity and adverse chronic effects on the liver. Analytical tools designed so far for the detection of odorant molecules are relatively invasive. Therefore, a tool that can measure the corresponding rate changes of ethanol concentration in real-time is highly desirable. Here in this work, we report a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor for in vivo quantification of ethanol at the cellular level with high spatial and temporal resolution. A human odorant-binding protein (hOBPIIa) was flanked by fluorescent proteins ECFP (Enhanced Cyan Fluorescent Protein) and Venus at the N- and C-terminus respectively. The constructed FRET nanosensor was named the fluorescent indicator protein for odorants (FLIPO). FLIPO allows in vitro and in vivo determination of FRET changes in a concentration-dependent manner. The developed nanosensor is highly specific to ethanol, stable to pH changes and provides rapid detection rate response. FLIPO-42 is the most efficient nanosensor created that measures ethanol with an apparent affinity (Kd) of 4.16 µM and covers the physiological range of 500 nM to 12 µM ethanol measurement. FLIPO-42 can measure ethanol dynamics in bacterial, yeast and mammalian cells non-invasively in real time which proves its efficacy as a sensing device in both prokaryotic and eukaryotic systems. Taken together, a prototype for a set of nanosensors was established, potentially enabling the monitoring of dynamic changes of ethanol and investigate its uptake and metabolism with subcellular resolution in vivo and ex vivo. Furthermore, the advent of a set of novel nanosensors will provide us with the tools for numerous medical, scientific, industrial and environmental applications which would help to illuminate their role in biological systems.

Nigella sativa seed based nanocomposite-MnO2/BC: An antibacterial material for photocatalytic degradation, and adsorptive removal of Methylene blue from water.

Antimicrobial Nigella sativa seed-based nanocomposite, MnO2/BC, was synthesized and utilized for the water purification through adsorption, and the photocatalytic degradation. MnO2/BC was prepared by co-precipitation method, and characterized using FT-IR, XRD, SEM, TEM, TGA, and DSC techniques. The composite was investigated for inhibition of bacterial cells growth. FT-IR spectrum indicated the presence of oxygenous groups on the surface; TGA and DSC showed thermal degradation; and XRD, SEM, and TEM investigations indicated amorphous, and porous nature of MnO2/BC having particle size of 190-220 nm. The nanocomposite inhibited the growth of both Gram-positive and Gram-negative bacteria cells in water. The adsorption of Methylene blue from water was investigated in batch method in terms of amount of MnO2/BC, dye concentration, pH, time, and temperature. 1.0 g L-1 of MnO2/BC removed more than 98% of Methylene blue from aqueous solution having concentration of 10 mg L-1 and pH 7.0 at 27 °C. The maximum Langmuir adsorption capacity of MnO2/BC was 185.185 mg g-1 at 45 °C. The adsorption was an endothermic process which obeyed Freundlich isotherm, and pseudo-second order kinetics. Therefore, the Methylene blue binding onto MnO2/BC surface was site-specific partially through the weak hydrogen bonding, and electrostatic interactions. The photocatalytic activity of MnO2/BC has been investigated by degrading the Methylene blue molecules/ions in water under the sunlight and 85% of degradation was achieved during 120 min irradiation. The dye was desorbed at lower pH and regenerated MnO2/BC was used for second cycle of Methylene blue adsorption. The results obtained for this study are much better than the previous Methylene blue adsorption studies with acid washed Black cumin seeds and MnFe2O4/BC for which the capacities were 73.529 mg g-1 and 10.070 mg g-1 at 27 °C, respectively (J. Mol. liq. 2018a, 264, 275-284; J. Clean. Prod. 2018a, 200, 996-1008).

Genetically encoded FRET-based optical sensor for Hg2+ detection and intracellular imaging in living cells.

Due to the potential toxicity of mercury, there is an immediate need to understand its uptake, transport and flux within living cells. Conventional techniques used to analyze Hg2+ are invasive, involve high cost and are less sensitive. In the present study, a highly efficient genetically encoded mercury FRET sensor (MerFS) was developed to measure the cellular dynamics of Hg2+ at trace level in real time. To construct MerFS, the periplasmic mercury-binding protein MerP was sandwiched between enhanced cyan fluorescent protein (ECFP) and venus. MerFS is pH stable, offers a measurable fluorescent signal and binds to Hg2+ with high sensitivity and selectivity. Mutant MerFS-51 binds with an apparent affinity (Kd) of 5.09 × 10-7 M, thus providing a detection range for Hg2+ quantification between 0.210 µM and 1.196 µM. Furthermore, MerFS-51 was targeted to Escherichia coli (E. coli), yeast and human embryonic kidney (HEK)-293T cells that allowed dynamic measurement of intracellular Hg2+ concentration with a highly responsive saturation curve, proving its potential application in cellular systems.

Nanoscale gizmos - the novel fluorescent probes for monitoring protein activity.

Nanobiotechnology has emerged inherently as an interdisciplinary field, with collaborations from researchers belonging to diverse backgrounds like molecular biology, materials science and organic chemistry. Till the current times, researchers have been able to design numerous types of nanoscale fluorescent tool kits for monitoring protein-protein interactions through real time cellular imagery in a fluorescence microscope. It is apparent that supplementing any protein of interest with a fluorescence habit traces its function and regulation within a cell. Our review therefore highlights the application of several fluorescent probes such as molecular organic dyes, quantum dots (QD) and fluorescent proteins (FPs) to determine activity state, expression and localization of proteins in live and fixed cells. The focus is on Fluorescence Resonance Energy Transfer (FRET) based nanosensors that have been developed by researchers to visualize and monitor protein dynamics and quantify metabolites of diverse nature. FRET based toolkits permit the resolution of ambiguities that arise due to the rotation of sensor molecules and flexibility of the probe. Achievements of live cell imaging and efficient spatiotemporal resolution however have been possible only with the advent of fluorescence microscopic technology, equipped with precisely sensitive automated softwares.

Designing, construction and characterization of genetically encoded FRET-based nanosensor for real time monitoring of lysine flux in living cells.

BACKGROUND: Engineering microorganisms in order to improve the metabolite flux needs a detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer (FRET) based genetically encoded nanosensors represent a promising tool for measuring the metabolite levels and corresponding rate changes in live cells. Here, we report the development of a series of FRET based genetically encoded nanosensor for real time measurement of lysine at cellular level, as the improvement of microbial strains for the production of L-lysine is of major interest in industrial biotechnology. RESULTS: The lysine binding periplasmic protein (LAO) from Salmonella enterica serovar typhimurium LT2 strain was used as the reporter element for the sensor. The LAO was sandwiched between GFP variants i.e. cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Affinity, pH stability, specificity and metal ions effects was scrutinized for the in vitro characterization of this nanosensor, named as FLIPK. The FLIPK is specific to lysine and found to be stable with the pH within the physiological range. The calculated affinity (K d ) of FLIPK was 97 µM. For physiological applications, mutants with different binding affinities were also generated and investigated in vitro. The developed nanosensor efficiently monitored the intracellular level of lysine in bacterial as well as yeast cell. CONCLUSION: The developed novel lysine fluorescence resonance energy transfer sensors can be used for in vivo monitoring of lysine levels in prokaryotes as well as eukaryotes. The potential of these sensors is that they can be used as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular lysine during fermentation.

FRET-based genetically-encoded sensors for quantitative monitoring of metabolites.

Neighboring cells in the same tissue can exist in different states of dynamic activities. After genomics, proteomics and metabolomics, fluxomics is now equally important for generating accurate quantitative information on the cellular and sub-cellular dynamics of ions and metabolite, which is critical for functional understanding of organisms. Various spectrometry techniques are used for monitoring ions and metabolites, although their temporal and spatial resolutions are limited. Discovery of the fluorescent proteins and their variants has revolutionized cell biology. Therefore, novel tools and methods targeting sub-cellular compartments need to be deployed in specific cells and targeted to sub-cellular compartments in order to quantify the target-molecule dynamics directly. We require tools that can measure cellular activities and protein dynamics with sub-cellular resolution. Biosensors based on fluorescence resonance energy transfer (FRET) are genetically encoded and hence can specifically target sub-cellular organelles by fusion to proteins or targetted sequences. Since last decade, FRET-based genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of cellular physiology. This review, describing the design and principles of sensors, presents a database of sensors for different analytes/processes, and illustrate examples of application in quantitative live cell imaging.

Leishmania donovani modulates host miRNAs regulating cholesterol biosynthesis for its survival.

Cholesterol reduction by intracellular protozoan parasite Leishmania donovani (L. donovani), causative agent of leishmaniasis, impairs antigen presentation, pro-inflammatory cytokine secretion and host-protective membrane-receptor signaling in macrophages. Here, we studied the miRNA mediated regulation of cholesterol biosynthetic genes to understand the possible mechanism of L. donovani-induced cholesterol reduction and therapeutic importance of miRNAs in leishmaniasis. System-scale genome-wide microtranscriptome screening was performed to identify the miRNAs involved in the regulation of expression of key cholesterol biosynthesis regulatory genes through miRanda3.0. 11 miRNAs out of 2823, showing complementarity with cholesterol biosynthetic genes were finally selected for expression analysis. These selected miRNAs were differentially regulated in THP-1 derived macrophages and in primary human macrophages by L. donovani. Correlation of expression and target validation through luciferase assay suggested two key miRNAs, hsa-miR-1303 and hsa-miR-874-3p regulating the key genes hmgcr and hmgcs1 respectively. Inhibition of hsa-mir-1303 and hsa-miR-874-3p augmented the expression of targets and reduced the parasitemia in macrophages. This study will also provide the platform for the development of miRNA-based therapy against leishmaniasis.

Real-Time Monitoring of Selenium in Living Cells by Fluorescence Resonance Energy Transfer-Based Genetically Encoded Ratiometric Nanosensors.

Selenium is a component of selenoproteins, which plays a crucial role in cellular redox homeostasis, thyroid metabolism, and DNA synthesis. Selenium has pleiotropic effects like antioxidant and anti-inflammatory activities; however, excess intake of selenium can imbalance such processes. The effects of selenium on human health are numerous and complex, demanding additional research to monitor the flux rate of selenium. Here, we have created a noninvasive and highly efficient genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor, SelFS (Selenium FRET-Sensor), for real-time monitoring of selenium at the cellular and subcellular levels. The construct of the nanosensor contains a selenium-binding protein (SeBP) as the selenium-detecting element inserted between the green fluorescent protein variants enhanced cyan fluorescent protein and Venus. In the presence of selenium, SelFS brings a conformational change, which is seen in the form of FRET. In vitro studies showed that SelFS is highly specific and selective for selenium and stable at an altered pH range from 5.0 to 8.0. SelFS is a flexible and dynamic tool for the detection of selenium in both prokaryotes and eukaryotes in a noninvasive way, with a binding constant (K d) of 0.198 × 10-6 M as compared to its mutants. The developed nanosensor can provide us a reporter tool for a wide range of industrial and environmental applications, which will help us to understand its functions in biological systems.

Combined Multiomics and In Silico Approach Uncovers PRKAR1A as a Putative Therapeutic Target in Multi-Organ Dysfunction Syndrome.

Despite all epidemiological, clinical, and experimental research efforts, therapeutic concepts in sepsis and sepsis-induced multi-organ dysfunction syndrome (MODS) remain limited and unsatisfactory. Currently, gene expression data sets are widely utilized to discover new biomarkers and therapeutic targets in diseases. In the present study, we analyzed MODS expression profiles (comprising 13 sepsis and 8 control samples) retrieved from NCBI-GEO and found 359 differentially expressed genes (DEGs), among which 170 were downregulated and 189 were upregulated. Next, we employed the weighted gene co-expression network analysis (WGCNA) to establish a MODS-associated gene co-expression network (weighted) and identified representative module genes having an elevated correlation with age. Based on the results, a turquoise module was picked as our hub module. Further, we constructed the PPI network comprising 35 hub module DEGs. The DEGs involved in the highest-confidence PPI network were utilized for collecting pathway and gene ontology (GO) terms using various libraries. Nucleotide di- and triphosphate biosynthesis and interconversion was the most significant pathway. Also, 3 DEGs within our PPI network were involved in the top 5 significantly enriched ontology terms, with hypercortisolism being the most significant term. PRKAR1A was the overlapping gene between top 5 significant pathways and GO terms, respectively. PRKAR1A was considered as a therapeutic target in MODS, and 2992 ligands were screened for binding with PRKAR1A. Among these ligands, 3 molecules based on CDOCKER score (molecular dynamics simulated-based score, which allows us to rank the binding poses according to their quality and to identify the best pose for each system) and crucial interaction with human PRKAR1A coding protein and protein kinase-cyclic nucleotide binding domains (PKA RI alpha CNB-B domain) via active site binding residues, viz. Val283, Val302, Gln304, Val315, Ile327, Ala336, Ala337, Val339, Tyr373, and Asn374, were considered as lead molecules.

Biochemical and biophysical analysis of the interaction of a recombinant form of Staphylococcus aureus enolase with plasminogen.

Aim: Pathogenic invasion of Staphylococcus aureus is critically dependent on host plasminogen activation. Materials & methods: The pathophysiological implications of the interactions between S. aureus recombinant enolase and host plasminogen were investigated. The effects of mutation and small synthetic peptide inhibitors on interactions were assessed. Results: In vitro, the S. aureus recombinant enolase exists as a catalytically active fragile octamer and a robust dimer. The dimer interacts with the host plasminogen on the S. aureus surface. Conclusion: The interaction of host plasminogen and S. aureus enolase might mediate bacterial adherence to the host, activate the plasminogen with the help of plasminogen activators and prevent α2-antiplasmin-mediated inhibition of plasmin. Incorporating mutant and synthetic peptides inhibited the interactions and their associated pathophysiological consequences.

Real time optical detection of gold in living cells through genetically-encoded probe.

To study the efflux of gold (Au) in living cells, a genetically encoded fluorescence resonance energy transfer (FRET)-based sensor has been developed. The gold-sensing domain GolB from Salmonella typhimurium has been fused to the N- and C-termini of the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus respectively. In living cells, this probe is highly selective and sensitive to gold and it can withstand changes in variable pH ranges. GolSeN-25, the most efficient sensor variant, binds gold with an affinity (K d) of 0.3 × 10-6 M, covering gold concentrations of nM to µM, and can be used for non-invasive real-time in vivo gold measurement in living cells. A simple and sensitive FRET probe was designed for the detection of gold with high selectivity and can be applied to the analysis of real samples.

A GIS-based novel approach for suitable sanitary landfill site selection using integrated fuzzy analytic hierarchy process and machine learning algorithms.

Disposal of waste without treatment is the least preferable way of sustainable solid waste management (SWM). But most cities in developing nations still use open dumps, causing negative impacts on the environment and human health. This study offered a novel approach for selecting landfill sites and sustainable SWM in Aligarh city, India. This was done through data collection, selecting models for criterion weighting, and validation. In order to prepare a landfill site suitability map, a geographic information system (GIS)-based ensemble fuzzy analytic hierarchy process-support vector machine (FAHP-SVM) and fuzzy analytic hierarchy process-random forest (FAHP-RF) models were implemented. Considering the previous studies and the study area characteristics, eighteen thematic layers were selected. The result revealed that land value; distance from residential roads, hospitals and clinics, and waste bins; and normalized difference built-up index (NDBI) have a fuzzy weight greater than 0.10, indicating significant factors. In contrast, land elevation, land slope, surface temperature, soil moisture index, normalized difference vegetation index (NDVI), and urban classification have a zero fuzzy weight, indicating these criteria have no importance. The result further revealed that FAHP-RF with an area under curve (AUC) value of 0.91 is the more accurate model than FAHP-SVM. According to the final weight-based overlay result, seven potential landfill sites were identified, out of which three were determined as most suitable by considering current land cover, public opinions, and environmental and economic concerns. This research proposed a zonal division model based on landfill sites location for sustainable SWM in Aligarh city. However, the findings may provide a guideline to the decision-makers and planners for optimal landfill site selection in other cities of developing countries.

Integrated transcriptomic and regulatory network analyses uncovers the role of let-7b-5p, SPIB, and HLA-DPB1 in sepsis.

Sepsis has affected millions of populations of all age groups, locations, and sexes worldwide. Immune systems, either innate or adaptive are dysregulated due to the infection. Various biomarkers are present to date, still sepsis is a primary cause of mortality. Globally, post-operative body infections can cause sepsis and septic shock in ICU. Abnormal antigen presentation to T-cells leads to a dysregulated immune system. miRNAs are sparkly evolved as biomarkers due to their high sensitivity and efficiency. In this work, we analyzed high-throughput mRNA data collected from Gene Expression Omnibus (GEO) and linked it to significant miRNAs and TFs using a network-based approach. Protein-protein interaction (PPI) network was constructed using sepsis-specific differentially expressed genes (DEGs) followed by enrichment analyses and hub module detection. Sepsis-linked decrease transcription of the classical HLA gene such as HLA-DPB1 and its interplay with miR-let-7b-5p and transcription factor SPIB was observed. This study helped to provide innovative targets for sepsis.

A perspective on the isolation and characterization of extracellular vesicles from different biofluids.

Extracellular vesicles (EVs) are small membrane-bound particles, which include exosomes, micro vesicles (MVs) and various-sized vesicles, released by healthy and diseased cells. EVs also include other vesicular structures, such as large apoptotic bodies (1-5 µm), as well as membrane particles (50-80 nm) originating from the plasma membrane. However, exosomes are nanosize (≈30-100 nm) extracellular vesicles of endocytic origin that are bud-off by most types of cells and circulate in bodily fluids. Extracellular nanovesicles contain a large variety of biomolecules, including miRNA, RNA, DNA, proteins, signaling peptides and lipids, that can have diagnostic and therapeutic value. The spectrum of the existing scientific interest in extracellular nanovesicles is comprehensive, which ranges from understanding their functions and pathways to their potential clinical usage. EVs can be obtained from different body fluids with minimally invasive techniques (e.g., urine, plasma, serum), so they are most useful in disease diagnosis. High yield and purity contribute to the accurate diagnosis of various diseases, but damaged EVs and impurities can cause misinterpreted results. Over the last decade, a plethora of approaches have been developed for examining EVs using optical and non-optical tools. However, EV isolation methods have different yields and purities. Moreover, the isolation method that is most appropriate to maximize EVs recovery depends on the different experimental situations. This review explores the emerging use of micro and nano-technologies to isolate and characterize exosomes and microvesicles (MVs) from different biological samples, and the application of these technologies for the monitoring and diagnosis of different pathological conditions.