Cigarette smoke extract induces foam cell formation by impairing machinery involved in lipid droplet degradation.

The formation of foam cells, lipid-loaded macrophages, is the hallmark event of atherosclerosis. Since cigarette smoking is a risk factor for developing atherosclerosis, the current study investigated the effects of cigarette smoke extract (CSE) on different events like expressions of genes involved in lipid influx and efflux, lipophagy, etc., that play vital roles in foam cell formation. The accumulation of lipids after CSE treatment U937 macrophage cells was examined by staining lipids with specific dyes: Oil red O and BODIPY493/503. Results showed an accumulation of lipids in CSE-treated cells, confirming foam cell formation by CSE treatment. To decipher the mechanism, the levels of CD36, an ox-LDL receptor, and ABCA1, an exporter of lipids, were examined in CSE-treated and -untreated U937 cells by real-time PCR and immunofluorescence analysis. Consistent with lipid accumulation, an increased level of CD36 and a reduction in ABCA1 were observed in CSE-treated cells. Moreover, CSE treatment caused inhibition of lipophagy-mediated lipid degradation by blocking lipid droplets (LDs)-lysosome fusion and increasing the lysosomal pH. CSE also impaired mitochondrial lipid oxidation. Thus, the present study demonstrates that CSE treatment affects lipid homeostasis by altering its influx and efflux, lysosomal degradation, and mitochondrial utilization, leading to the formation of lipid-loaded foam cells. Moreover, the current study also showed that the leucine supplement caused a significant reduction of CSE-induced foam cell formation in vitro. Thus, the current study provides insight into CS-induced atherosclerosis and an agent to combat the disease.

Cloning, expression and characterization of PURase gene from Pseudomonas sp. AKS31.

Polyurethane (PUR) is a soil and aquatic contaminant throughout the world. Towards bioremediation, in a previous study, a soil bacterium, Pseudomonas sp. AKS31, capable of efficiently degrading PUR was isolated. Polyurethanase (PURase) enzyme is capable of cleaving the ester bond of PUR and is considered as a key regulator of PUR biodegradation. Hence, for a high yield, easy purification, and further characterization, the aim of this study was to clone and overexpress the PURase gene of this isolate. The current study also investigated structural aspects of this enzyme through predictive bioinformatics analyses. In this context, the PURase gene of the isolate was cloned and expressed in E. coli using pET28(a)+ vector. The obtained recombinant protein was found insoluble. Therefore, first, the protein was made soluble with urea and purified using nickel-NTA beads. The purified enzyme exhibited substantial activities when tested on the LA-PUR plate. Bioinformatics-based analysis of the protein revealed the presence of a lipase serine active site and indicated that this PURase belongs to the Family 1.3 lipase. Hence, the present study shows that active PURase can be produced in large quantities using a prokaryotic expression system and thus, provides an effective strategy for in-vitro PUR-degradation.

Isolation of a soil bacterium for remediation of polyurethane and low-density polyethylene: a promising tool towards sustainable cleanup of the environment.

A soil bacterium, designated strain AKS31, was isolated on the plastic polyurethane (PUR) and based on the molecular and biochemical analysis was tentatively assigned to the genus Pseudomonas. Preliminary studies suggested that strain AKS31 had the capability of biodegrading polyurethane (PUR) and low-density polyethylene (LDPE). This observation was confirmed by the analysis of the biodegradation products. The hydrolyzed products of PUR analyzed sequentially by High-Performance Liquid Chromatography (HPLC) and Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) showed the presence of diethylene glycol suggesting the presence of an esterase. A gene that could be involved in producing an esterase-like activity (PURase gene) was identified after the amplification and sequencing of a PCR product. Fourier Transformed Infrared (FTIR) spectrophotometric analysis of AKS31-treated LDPE film revealed the incorporation of hydroxyl groups suggesting the involvement of a hydroxylase in the degradation of LDPE. It is established that plastics form microplastics and microbeads in soils which negatively impact the health of living organisms and there have been concentrated research efforts to remediate this problem. Microcosm studies revealed that when strain AKS31 was bioaugmented with soil both the polymers were degraded during which time the heterotrophic plate counts, soil respiration and soil organic carbon content increased but this was not the case with the control nonbioaugmented microcosm. The results demonstrate that the strain AKS31 may have the potential in biodegradation of PUR and LPDE present as plastic microbeads and thereby improving soil health. Further studies in this direction are warranted. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02592-9.

Edible marine algae: a new source for anti-mycobacterial agents.

Tuberculosis is a dreaded disease, which causes innumerable death worldwide. The emergence of drug resistance strains makes the situation devastating. Therefore, for better management of public health, it is mandatory to search for new anti-mycobacterial agents. In this context, the current study investigated two edible marine algae, Ulva lactuca and Ulva intestinalis, for the probable source of new anti-mycobacterial agents. To test the anti-mycobacterial activity, alcoholic extracts of these two algae were spotted on the Mycobacterium smegmatis lawn. Upon incubation, clear zone was observed at the spots. It indicated that these two extracts have anti-mycobacterial activity. In addition, their anti-biofilm property was also tested. It was found that both the extracts inhibit the mycobacterial biofilm development as well as they can disperse the preformed mycobacterial biofilm. Since these two are capable of dispersing preformed mycobacterial biofilm, it is possible that in the presence of either of these two extracts, isoniazid and rifampicin can kill biofilm encapsulated mycobacterium in combinatorial therapy. Consistent with the hypothesis, rifampicin and isoniazid killed mycobacteria that were present in biofilm. Thus, these two extracts augment the activity of rifampicin and isoniazid upon biofilm dispersal. Moreover, treatment of different cell lines with these two extracts exhibited no or little cytotoxic effects. Thus, these two agents have the potential to be good therapeutic agents against mycobacterial diseases.

Cigarette smoke affects ESCRT-mediated vacuolar activity in Saccharomyces cerevisiae.

Cigarette smoking is a risk factor for developing chronic obstructive pulmonary disease and protein aggresome formation is considered to be a hallmark event for the disease. Since dysfunction of lysosome-mediated protein degradation leads to enhanced accumulation of misfolded proteins and subsequent aggresome formation, we examined the effect of cigarette smoke extract (CSE) on ESCRT-mediated sorting in S. cerevisiae as this process is necessary for the functioning of the vacuole, the lysosomal equivalent in yeast. An operational ESCRT pathway is essential for ion homeostasis and our observation that exposure to CSE caused increased sensitivity to LiCl indicated CSE-induced impairment of ESCRT function. To confirm the inhibition of ESCRT function, the targeting of carboxypeptidase S (CPS), which reaches the vacuole lumen via the ESCRT pathway, was examined. Treatment with CSE resulted in the mislocalization of GFP-tagged CPS to the vacuolar membrane, instead of the vacuolar lumen, confirming defective functioning of the ESCRT machinery in CSE-treated cells. Further analysis revealed that CSE-treatment inhibited the recruitment of the ESCRT-0 component, Vps27, to the endosome surface, which is a key event is for the functioning of the ESCRT pathway. This lack of endosomal recruitment of Vps27 most likely results from a depletion of the endosomally-enriched lipid, phosphatidylinositol 3-phosphate (PI3-P), which is the target of Vps27. This is supported by our observation that the presence of excess leucine, a known activator of the lipid kinase responsible for the generation of PI3-P, Vps34, in the medium can rescue the CSE-induced ESCRT misfunctioning. Thus, the current study provides an insight into CSE-induced aggresome formation as it documents that CSE treatment compromises vacuolar degradation due to an impairment of the ESCRT pathway, which likely stems from the inhibition of Vps34. It also indicates that leucine has the potential to attenuate the CSE-induced accumulation of misfolded proteins.

Nitrogen dynamics of the aquatic system is an important driving force for efficient sewage purification in single pond natural treatment wetlands at East Kolkata Wetland.

At East Kolkata Wetlands, though the domestic city sewage is purified very rapidly, the mechanisms of treatment remains inadequately explored. In this context, the present study investigated nitrogen dynamics of the single pond treatment systems during purification and explored its potential role in sewage treatment. For this purpose the concentrations of different forms of nitrogen present both in water and soil at different time points of purification were measured. The organic nitrogen content decreased sharply, in the early phase, with an increase in ammonium concentration. Notably the reduction in organic nitrogen was significantly higher than the increase in NH4+ which can be attributed to the volatilization of NH4+ under alkaline pH. This volatilization results in reduced oxygen demand. The nitrate-N concentration decreased sharply from soil with a concomitant increase in water column. However the reduction of nitrate in soil was significantly higher than the increase in water column. It indicated the occurrence of denitrification under anoxic condition wherein nitrate serves as terminal electron acceptor. Additionally a part of the nitrate supported planktonic growth. Thus it describes another mechanism of reducing oxygen demand. The initial NH4+-N concentration in the soil was very low and it increased gradually during purification due to increasing soil cation exchange capacity. Thus by trapping NH4+ ion soil contributes towards preventing contamination of water. Thus at EKW, the cumulative activities in water and soil involved in nitrogen dynamics lead to overall reduction of the oxygen demand and contribute towards efficient sewage purification.

Bioactive compound from Pseudomonas synxantha inhibits the growth of Mycobacteria.

Tuberculosis is a dreaded disease and the current situation demands new anti-tubercular agent(s) for the management of public health. Towards this direction, we obtained a contaminant organism on a Mycobacterium smegmatis lawn having growth inhibitory activity against the later. In the current study, efforts were targeted to identify this organism and characterize the bioactive compound from this isolate that inhibited the growth of Mycobacteria. The result revealed that the organism is a strain of Pseudomonas synxantha. Biophysical analyses including (1)H and (13)C NMR, ESI-mass spectroscopy, FTIR showed that the bioactive compound is a long chain aliphatic hydrocarbon with a terminal alyl bond and intermediate electronegative atom. The compound exhibited strong growth inhibitory activities against M. smegmatis and Mycobacterium tuberculosis strains H37Ra, H37Rv and BCG. Further experiments showed that both P. synxantha and its secretory metabolites are capable of inducing hemolysis of human blood. Thus the results of this study clearly indicate that the bioactive compound produced by P. Synxantha has biosurfactant activities as well as anti-myco-bacterial properties.

Antibacterial activity of long-chain fatty alcohols against mycobacteria.

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis and results in innumerable deaths across the world. The emergence of multidrug-resistant and extremely drug-resistant tuberculosis strains and its coinfection with HIV has made tuberculosis more difficult to treat. Therefore, new antimycobacterial agent(s) for both therapy and disinfection are urgently required. In this context the present study describes the antibacterial property of long-chain fatty alcohols against mycobacteria. The antimycobacterial activities of alcohols with chain length ranging from C(5) to C(13) were examined against Mycobacterium smegmatis mc(2) 155 and M. tuberculosis H(37)R(v). The best activity was found with one with a C(10) chain length. This bactericidal activity can partly be attributed to its ability to damage the robust and complex cell envelope of Mycobacteria. Moreover, our study reveals the ability of decanol to attenuate biofilm formation by M. smegmatis. This knowledge can be used to develop new therapeutics and disinfectants against mycobacteria.

Epicatechin ameliorates ionising radiation-induced oxidative stress in mouse liver.

The current study was intended to evaluate the hepatoprotective effect of Epicatechin (EC) against radiation-induced oxidative stress, in terms of inflammation and lipid peroxidation. Swiss albino mice were administered with EC (15 mg/kg body weight) for three consecutive days before exposing them to a single dose of 5-Gy (60)Co gamma (γ) irradiation. Mice were necropsied and livers were taken for immunohistochemistry, western blot analysis and biochemical tests for the detection of markers of hepatic oxidative stress. Nuclear translocation of nuclear factor kappa B (NF-κB) and lipid peroxidation were increased whereas the activities of superoxide dismutase (SOD) and catalase (CAT), reduced glutathione (GSH) content and ferric reducing antioxidant power (FRAP) were diminished upon radiation exposure compared to control. Translocation of NF-κB from cytoplasm to nucleus and lipid peroxidation were found to be inhibited whereas an increase in SOD, CAT, GSH and FRAP was observed in the mice treated with EC prior to irradiation. Thus, pre-treatment with EC offers protection against γ-radiation induced hepatic alterations.

Identification and characterization of a FYVE domain from the early diverging eukaryote Giardia lamblia.

The morphology of the endomembrane system of Giardia lamblia appears to be significantly different from higher eukaryotes. Therefore, the molecular mechanisms controlling vesicular trafficking are also likely to be altered. Since FYVE domain is a known regulator of endosomal trafficking, the authors used BLAST search to identify FYVE domain(s) in G. lamblia. A 990 amino acid long putative FYVE domain-containing ORF was identified, which contains all the conserved sequence elements in the ligand binding pocket. Phylogenetic analysis reveals that this domain is significantly diverged. The authors have shown that the corresponding gene is expressed in G. lamblia trophozoites and cysts. In spite of this phylogenetic divergence, in vitro biochemical assay indicates that this domain preferentially binds to phosphatidylinositol 3-phosphate {PtdIns(3)P}and in vivo expression of the GFP-tagged G. lamblia FYVE domain in S. cerevisiae, displays its selective localization to PtdIns(3)P-enriched endosomes. This is the first study to characterize a PtdIns(3)P effector protein in this early-diverged eukaryote.

Isolation of a Pseudomonas aeruginosa strain from soil that can degrade polyurethane diol.

Polyurethane diol (PUR-diol), a synthetic polymer, is widely used as a modifier for water-soluble resins and emulsions in wood appliances and auto coatings. Non-biodegradability of polyurethanes (PUR) and PUR-based materials poses a threat to environment that has led scientists to isolate microbes capable of degrading PUR. However, the bio-degradation of PUR-diol has not yet been reported. In this study, we report isolation of a soil bacterium that can survive using PUR-diol as sole carbon source. PUR-diol degradation by the organism was confirmed by thin layer chromatographic analysis of the conditioned medium obtained after the growth wherein a significant reduction of PUR-diol was observed compared to non-inoculated medium. To quantify the PUR-diol degradation, a sensitive assay based on High Performance Thin Layer Chromatography has been developed that showed 32% degradation of PUR-diol by the organism in 10 days. Degradation kinetics showed the maximal depletion of PUR-diol during logarithmic growth of the organism indicating a direct relation between the growth and PUR-diol degradation. Mutagenic study and GC-MS analysis revealed that esterase activity is involved in this degradation event. The ribotyping and metabolic fingerprinting analysis showed that this organism is a strain of Pseudomonous aeruginosa (P. aeruginosa). It has also been observed that this strain is able to degrade Impranil DLN™, a variety of commercially available PUR. Therefore this study identifies a new bacterium from soil that has the potential to reduce PUR-related waste burden and adds a new facet to diverse functional activities of P. aeruginosa.

Vitamin C prevents cigarette smoke induced atherosclerosis in guinea pig model.

AIM: Cigarette smoking is a major risk for developing atherosclerosis; however, the underlying mechanism is poorly understood. This paucity of knowledge is largely attributed to the lack of an animal model; therefore, our efforts were targeted towards establishing cigarette smoke (CS)-induced atherosclerosis in guinea pig. To understand the mechanism, we investigated apoptosis, an event implicated in atherosclerosis, in the aorta of CS-exposed animals. Since a major deleterious effect of CS is oxidative stress, we also examined the effect of vitamin C, an antioxidant, on CS- induced atherosclerosis. METHODS AND RESULTS: Guinea pigs on a diet with or without vitamin C supplement were exposed to CS for different time periods. Aortal sections from these animals were examined for atherosclerotic changes by staining with H&E and Oil red O. Atherogenic changes were observed in sections obtained from CS-exposed guinea pigs only. TUNEL assay showed the occurrence of apoptosis in CS-exposed guinea pig aorta. Our results revealed that CS-induced apoptosis could contribute to the progression but not to the initiation of the disease. Immunohistochemical analysis documents that CS-induced apoptosis in aortal sections is mediated at least in part by an increased Bax/Bcl2 ratio. In contrast, CS-exposed guinea pigs fed with vitamin C-supplemented diet exhibit little or no atherogenic changes. This anti-atherosclerotic activity of vitamin C can be attributed partly to its ability to inhibit CS-induced apoptosis and platelet activation. CONCLUSION: Exposure of guinea pigs to cigarette smoke causes the development of atherosclerosis, which can be prevented by vitamin C supplement.

Sewage treatment in a single pond system at East Kolkata Wetland, India.

East Kolkata Wetland (EKW), a Ramsar site, greatly contributes towards purification of city sewage employing single pond system. However, the underlying mechanism remains unknown. Therefore to gain an insight, in this study efforts have been made to understand the rate of biodegradation and the time dependent changes of different physicochemical factors and their interactions that are involved in the process. For this purpose, different parameters such as BOD, COD, faecal coliforms etc. have been measured at different time intervals during the purification process. The results reveal that biodegradation rate at EKW pond is very high and wastewater gets stabilized within 10 days of retention. The higher rate of biodegradation in pond system at EKW (k = 0.7 day(-1)) than in laboratory based in vitro experiment (k = 0.12 day(-1)) reveals the important contribution from other environmental components that are unique for this system. The results also demonstrate the significant influence (P< or =0.01) of temperature, pH and dissolved oxygen on the purification of waste water. Thus the current study provides an insight about the optimal pathway of gradual improvement of wastewater quality in the single pond system at EKW and may serve to explore the inherent mechanism to a great extent.

cAMP stringently regulates human cathelicidin antimicrobial peptide expression in the mucosal epithelial cells by activating cAMP-response element-binding protein, AP-1, and inducible cAMP early repressor.

Little is known about the regulation of the innate host defense peptide cathelicidin at the mucosal surfaces. Expression is believed to be transcriptionally regulated, and several cis-acting elements have been identified in the cathelicidin putative promoter. However, the trans-acting factors have not been clearly defined. We have recently reported that bacterial exotoxins suppress cathelicidin expression in sodium butyrate-differentiated intestinal epithelial cells (ECs), and this may be mediated through inducible cAMP early repressor. Here we have shown that cAMP-signaling pathways transcriptionally regulate cathelicidin expression in various ECs. cAMP-response element-binding protein (CREB) and AP-1 (activator protein-1) bind to the cathelicidin putative promoter in vitro. Additionally, transcriptional complexes containing CREB, AP-1, and cathelicidin upstream regulatory sequences are formed within ECs. We have also shown that these complexes may activate cathelicidin promoter and are required for its inducible expression in ECs. This is underscored by the fact that silencing of CREB and AP-1 results in failure of ECs to up-regulate cathelicidin, and hepatitis B virus X protein may use CREB to induce cathelicidin. On the other hand, inducible cAMP early repressor competes with CREB and AP-1 for binding to the cathelicidin promoter and represses transcription, thus functioning as a counter-regulatory mechanism. Finally, both CREB and AP-1 were shown to play major roles in the regulation of cathelicidin in sodium butyrate-differentiated HT-29 cells. This is the first report of a detailed mechanistic study of inducible cathelicidin expression in the mucosal ECs. At the same time, it describes a novel immunomodulatory function of cAMP.

The bacteriophage lambda DNA replication protein P inhibits the oriC DNA- and ATP-binding functions of the DNA replication initiator protein DnaA of Escherichia coli.

Under the condition of expression of lambda P protein at lethal level, the oriC DNA-binding activity is significantly affected in wild-type E. coli but not in the rpl mutant. In purified system, the lambda P protein inhibits the binding of both oriC DNA and ATP to the wild-type DnaA protein but not to the rpl DnaA protein. We conclude that the lambda P protein inhibits the binding of oriC DNA and ATP to the wild-type DnaA protein, which causes the inhibition of host DNA synthesis initiation that ultimately leads to bacterial death. A possible beneficial effect of this interaction of lambda P protein with E. coli DNA initiator protein DnaA for phage DNA replication has been proposed.

Gal80 confers specificity on HAT complex interactions with activators.

Several yeast transcription activators have been shown to interact with and recruit histone acetyltransferase complexes to promoters in chromatin. The promiscuity of activator/HAT interactions suggests that additional factors temporally regulate these interactions in response to signaling pathways. In this study, we demonstrate that the negative regulator, Gal80, blocks interactions between the SAGA and NuA4 HAT complexes and the Gal4 activator. By contrast, Gal80 did not inhibit SAGA and NuA4 interaction with another activator Gcn4. The function of Gal80 prevented Gal4 targeting of SAGA and displaced SAGA targeted by Gal4 to a promoter within a nucleosome array. In the same set of experiments, targeting of SAGA by Gcn4 was unaffected by Gal80. These studies demonstrate that the specificity of HAT/activator interactions can be dictated by cofactors that modulate activation domain function in response to cellular signals.