Synthesis and Antimicrobial Evaluation of Novel Carbazole Based β-diketones and its Pyrazole Derivatives

Novel 9-ethyl-9H-carbazole-3-carboxylic acid derivatives including ester, β-diketone and pyrazole were prepared and characterized by FT-IR, 1H NMR, 13C NMR and mass spectroscopic techniques. All synthesized compounds evaluated for their in vitro antimicrobial activities against four bacteria (Escherichia coli, Pseudomonas putide, Bacillus subtilis, and Streptococcus lactis) and three fungi (Aspergillus niger, Penicillium sp and Candida albicans). Among the compounds tested, 3a, 3b, 3c, 4a, 4b, 4c, 5a and 5b exhibited pronounced antibacterial activity as compared with standard drug ampicillin. Notably, carbazole based pyrazole derivatives 5a and 5b showed potent antifungal activity against C. albicans comparable to reference drug greseofulvin. This work is licensed under a Creative Commons Attribution 4.0 International License

Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum

Extracellular vesicles (EVs), including the nano-sized exosomes, have the capacity to transfer multiple functional molecules between cells. In cell culture experiments, fetal bovine serum (FBS) is often used to supplement cell culture medium as a nutrient, but it is important to know that the FBS also contain significant quantities of EVs. The aim of the current study was to determine whether the FBS EVs can influence cultured cell phenotype, and secondly to determine the efficiency of FBS-EV elimination protocols. Firstly, FBS that had not been depleted of EVs induced a migratory phenotype in a lung cancer epithelial cell line (A549 cells), an effect that could be mimicked by isolated FBS EVs alone. FBS-derived EVs also contained RNA, which was protected from consecutive proteinase K and RNase A treatment. Comparison of common isolation protocols suggested that an 18-hour centrifugation period eliminates approximately 95% of RNA-containing FBS EVs, whereas a 1.5-hour protocol is insufficient. In conclusion, this study shows that FBS EVs substantially influence cultured cell behaviour, but also that they can be virtually removed by an 18-hour ultracentrifugation protocol

TIME-DEPENDENT DETERMINATIVE BIOCHEMICAL TRAITS FOR SALT TOLERANCE MECHANISM IN MUNGBEAN (Vigna radiata (L.) R. WILCZEK)

Mungbean is one of the commercially valuable pulse crops. Time-dependent biochemical modulations in the mungbean varieties PKV AKM 12-28 and VBN (Gg)3 exposed to 75, 100, and 125 mM NaCl were estimated, and the results were concluded through multivariate modeling. The cluster analysis gave two fairly distinct clusters that had similar biochemical responses. Results on the principal component analysis suggested that protein content (PC), total phenolic content (TPC), total flavonoid content (TFC), DPPH radical scavenging activity, ABTS radical scavenging activity, proline content (PRC), total free amino acid (TFAA) content, and malondialdehyde (MDA) contents were dominant traits in the shoot as compared to the root. These can be taken as the primary indicators to assess the effect of salt stress on mungbean varieties. The discriminant analysis had identified TFC, MDA, and total sugar content (TSC) as discriminating variables between the roots and shoots. Further, MDA and TFC were identified as discriminating variables under different salt concentrations, and TSC was identified as a discriminating variable at different exposure durations. Discriminant partial least squares analysis further identified optimum biochemical modulations in the shoots of PKV AKM 12-28 and 75 mM NaCl. The salt treatment produced a strong biochemical modulation after 30 and 45 days, which helped plants survive under salt stress. The multivariate approaches efficiently interpreted time-dependent biochemical modulations in shoots and roots of mungbean varieties under salt stress

Supply Chain Tracing and Anti-Counterfeiting with Distributed Ledger Technology

In recent times, there has been a rampant proliferation of counterfeit products that has left a trail of devastation in the manufacturing sectors. The repercussions of this extend to companies, impacting their brand reputation, revenue streams and overall profitability. Industries like agriculture, banking, electronics, and high-value deliveries uses the emergence of blockchain technology as a powerful tool to discern between authentic and counterfeit items. Its potential as a means to curtail the influx of fake products in the market is substantial. Blockchain technology, at its core, operates as a decentralized and distributed digital ledger system, meticulously recording transactions within interconnected blocks across multiple databases. The inherent security of this technology ensures the immutability of these blocks, rendering them invulnerable to alteration or hacking. By leveraging blockchain technology, consumers can independently verify the authenticity of a product, eliminating the need for reliance on third-party intermediaries. Incorporating recent technological advancements, the utilization of Quick Response (QR) codes offers a robust approach to combat the proliferation of counterfeit goods. The integration of blockchain technology with QR codes serves as a means to uphold the integrity of products. This innovative system securely stores product details and unique codes in the form of blocks,  where QR codes play a pivotal role in collecting and matching these unique codes with entries in the blockchain database. If the QR code matches with entries in the database, the user receives a confirmation of the product's authenticity; otherwise, an alert is triggered, signaling the presence of a counterfeit product

Endosomal signalling via exosome surface TGF beta-1

Extracellular vesicles such as exosomes convey biological messages between cells, either by surface-to-surface interaction or by shuttling of bioactive molecules to a recipient cell's cytoplasm. Here we show that exosomes released by mast cells harbour both active and latent transforming growth factor beta-1 (TGF beta-1) on their surfaces. The latent form of TGF beta-1 is associated with the exosomes via heparinase-II and pH-sensitive elements. These vesicles traffic to the endocytic compartment of recipient human mesenchymal stem cells (MSCs) within 60 min of exposure. Further, the exosomes-associated TGF beta-1 is retained within the endosomal compartments at the time of signalling, which results in prolonged cellular signalling compared to free-TGF beta-1. These exosomes induce a migratory phenotype in primary MSCs involving SMAD-dependent pathways. Our results show that mast cell-derived exosomes are decorated with latent TGF beta-1 and are retained in recipient MSC endosomes, influencing recipient cell migratory phenotype. We conclude that exosomes can convey signalling within endosomes by delivering bioactive surface ligands to this intracellular compartment.11Ysciescopu

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Function of Surface-Associated Protein and DNA on Extracellular Vesicles

Extracellular vesicles (EVs), including exosomes, are nano-sized, lipid bilayer enclosed vesicles that are released into the extracellular environment from by almost all cells. EVs contain biomolecules, such as proteins, lipids, and nucleic acids, and they are suggested to play vital roles in cellular communication. In addition, they are used as biomarkers and have therapeutic applications. The goal of this Ph.D. thesis was to define the localization of EV associated cargo (particularly proteins and DNA) and to determine the role of EVs in regulating biological processes. We addressed these questions by using mast cell-derived EVs (exosomes) and determining their effects on signaling pathways in primary human mesenchymal stem cells, epithelial cells, and monocytes. We have made three important discoveries. First, we showed that the protein cargo, TGFβ-1, present on the surface of EVs derived from mast cells, activated a migratory phenotype in primary human MSCs. The major form of TGFβ-1 was inactive and was associated with heparan sulfate proteoglycans. Moreover, these EVs enhanced the immunosuppressive phenotype of MSCs in a mouse model of allergic airway inflammation. EVs activated prolonged and efficient TGFβ-signaling and were retained in the endosomal compartments of MSCs during this period. Furthermore, based on the protein expression and the morphological features that were induced in lung epithelial cells, we also concluded that the epithelial-to-mesenchymal transition could be induced by these EVs. Additionally, we found that these EVs could activate the phosphorylation of proteins that are involved in EMT. Second, we showed that the surface of EVs is associated with extracellular DNA that induced the aggregation of EVs. Additionally, DNA was also present on the inside of EVs. The DNA on both the inside and outside of the EVs consisted of both mitochondrial and nuclear DNA. In this study, we were able to separate the EVs based on their density, followed by detection of the DNA that was associated with the EVs. The EV-associated DNA was able to initiate the activation of innate immune signaling by phosphorylation of interferon regulatory factor-3 in monocytes. Third, we evaluated and found that 18 hour is more efficient than 1.5 hours of ultracentrifugation in depleting EV-associated RNA (as well as DNA) from fetal bovine serum prior to its use in cell culture media. We conclude that mast cell-derived EVs harbors bioactive molecules (e.g., TGFβ-1 and DNA) on their surfaces. These EVs can affect MSCs by regulating the immune environment of the lung during inflammation. Some portion of the secreted TGFβ-1 is inactive and is attached to the surface of EVs. This might target the EVs to the acidifying compartment of early/late endosomes and lead to the activation of TGFβ-1 along with the uptake of the EVs. Additionally, EVs also carry DNA. Most of the DNA molecules were present on the surface of the EVs and were able to activate the DNA sensors in recipient cells. Thus, EVs assist in the uptake of DNA into the cytoplasm of the recipient cell, and this mechanism has implications in autoimmune disease and in the maintenance of inflammation