Plastic Waste in India: overview, impact, and measures to mitigate: Review

India is one of the world’s large and fastest-growing economies. With the expanding development, the usage of plastic for anthropogenic activities has expanded many folds and India alone generated around 3.3 million metric tonnes of plastic in the financial year 2019. 79 percent of the plastic generated worldwide enters our land, water, and environment as waste; part of it also enters our bodies through the food chain. The industry in India states that 60 percent of what is generated is recycled and we had assumed that we had solved the problem of plastic waste by recycling, or burying it in landfills. But we were incorrect. Plastic garbage is omnipresent today. It is filling up our oceans and harming marine life and affecting all organisms in the food chain. With the development of economic growth of the country per capita consumption of plastic will only increase in the coming years and we will end up generating more plastic waste The review paper aimed to examine the major impact of plastic waste in India and how to reduce plastic consumption, considering measures such as phasing out or banning multilayered plastics that cannot be recycled, contemplating renewable raw materials, promoting the use of bioplastics, incentivizing the recycling business, and making the rules and guidelines for Extended Producer Responsibility (EPR) simple and enforceable

Assessing the Migration of BPA and Phthalic Acid from Take-out Food Containers: Implications for Health and Environmental Sustainability in India

The research investigates the escalating consumption of take-out food in India and the associated health risks stemming from the extensive use of plastic packaging. Through a comprehensive nationwide online survey, the study delved into dietary preferences, frequency of take-out food consumption, delivery service timing, and the types of packaging commonly encountered by Indian consumers. To address these concerns, the research team developed an analytical method to detect Bisphenol A (BPA) and Phthalic acid migration from food-contact materials (FCMs) into various food simulants. The investigation revealed that prolonged exposure to elevated temperatures led to increased migration of BPA and Phthalic acid, particularly in polyethylene pouches using 3% acetic acid as a food simulant, with the highest concentrations observed after 45 minutes of exposure. Additionally, a microbial bioassay demonstrated the mutagenic potential of migrated plasticizers, showcasing significant effects in mammalian systems, particularly under metabolic activation. The study underscores the substantial health risks associated with plastic packaging in take-out food, emphasizing potential implications for consumer health and calling for more extensive research and considerations regarding food packaging materials

Mechanism of Amphotericin B Resistance in Clinical Isolates of Leishmania donovani

The clinical value of amphotericin B, the mainstay therapy for visceral leishmaniasis in sodium antimony gluconatenonresponsive zones of Bihar, India, is now threatened by the emergence of acquired drug resistance, and a comprehensive understanding of the underlying mechanisms is the need of the hour. We have selected an amphotericin B-resistant clinical isolate which demonstrated 8-fold-higher 50% lethal doses (LD50) than an amphotericin B-sensitive strain to explore the mechanism of amphotericin B resistance. Fluorimetric analysis demonstrated lower anisotropy in the motion of the diphenylhexatriene fluorescent probe in the resistant strain, which indicated a higher fluidity of the membrane for the resistant strain than for the sensitive strain. The expression patterns of the two transcripts of S-adenosyl-L-methionine:C-24-�-sterol methyltransferase and the absence of ergosterol, replaced by cholesta-5,7,24-trien-3�-ol in the membrane of the resistant parasite, indicate a decreased amphotericin B affinity, which is evidenced by decreased amphotericin B uptake. The expression level of MDR1 is found to be higher in the resistant strain, suggesting a higher rate of efflux of amphotericin B. The resistant parasite also possesses an upregulated tryparedoxin cascade and a more-reduced intracellular thiol level, which helps in better scavenging of reactive oxygen species produced by amphotericin B. The resistance to amphotericin B was partially reverted by the thiol metabolic pathway and ABC transporter inhibitors. Thus, it can be concluded that altered membrane composition, ATP-binding cassette transporters, and an upregulated thiol metabolic pathway have a role in conferring amphotericin B resistance in clinical isolates of Leishmania donovani

Stage-Dependent Expression and Up-Regulation of Trypanothione Synthetase in Amphotericin B Resistant <i>Leishmania donovani</i>

<div><p>Kinetoplastids differ from other organisms in their ability to conjugate glutathione and spermidine to form trypanothione which is involved in maintaining redox homeostasis and removal of toxic metabolites. It is also involved in drug resistance, antioxidant mechanism, and defense against cellular oxidants. Trypanothione synthetase (TryS) of thiol metabolic pathway is the sole enzyme responsible for the biosynthesis of trypanothione in <i>Leishmania donovani</i>. In this study, TryS gene of <i>L. donovani</i> (LdTryS) was cloned, expressed, and fusion protein purified with affinity column chromatography. The purified protein showed optimum enzymatic activity at pH 8.0–8.5. The TryS amino acids sequences alignment showed that all amino acids involved in catalytic and ligands binding of <i>L. major</i> are conserved in <i>L. donovani</i>. Subcellular localization using digitonin fractionation and immunoblot analysis showed that LdTryS is localized in the cytoplasm. Furthermore, RT-PCR coupled with immunoblot analysis showed that LdTryS is overexpressed in Amp B resistant and stationary phase promastigotes (∼2.0-folds) than in sensitive strain and logarithmic phase, respectively, which suggests its involvement in Amp B resistance. Also, H₂O₂ treatment upto 150 µM for 8 hrs leads to 2-fold increased expression of LdTryS probably to cope up with oxidative stress generated by H₂O₂. Therefore, this study demonstrates stage- and Amp B sensitivity-dependent expression of LdTryS in <i>L. donovani</i> and involvement of TryS during oxidative stress to help the parasites survival.</p></div

Subcellular localization of LdTryS.

<p>(A) Differential digitonin permeabilization of stationary phase promastigotes with increasing concentrations of digitonin. Supernatant and pellet fractions were run on 10% SDS-PAGE and transferred on to nitrocellulose membrane for western blot analysis using anti-LdTryS (1∶3000), anti LdcTXN (1∶4000), and anti LdIscS (1∶2000). cTXN and IscS served as cytosolic and mitochondrial markers, respectively. (B) Immunofluorescence microscopy of <i>L. donovani</i> promastigote with anti-LdTryS sera: phase contrast image, DAPI stained nucleus (N) and kinetoplast (K), Mitotracker stained mitochondria, anti-TryS labeled promastigote along with its merged image with DAPI is showing TryS localization in the cytoplasm.</p

Up regulation of LdTryS in <i>L. donovani</i> promastigotes in response to H₂O₂ treatment.

<p>(A) TryS expression in <i>L. donovani</i> parasites in the presence of H₂O₂ (10–200 µM) was analysed by western blot. H₂O₂ treated parasites showed increased expression level of LdTryS, whereas the β-actin expression level did not change significantly. The experiments were repeated twice in duplicates and quantitation was done by densitometric analysis using Quantity One (Bio-Rad). Band intensity is presented as fold increase/decrease of LdTryS expression. (B) LdTryS expression level was analyzed by semiquantitative RT-PCR, and PCR product stained with ethidium bromide and photographed. PCR of α-tubulin was used as housekeeping control that showed uniform expression pattern irrespective of H₂O₂ concentration.</p

Determination of expression level of LdTryS in sensitive (S) vs. drug resistant (R) strains of <i>L. donovani.</i>

<p>(A) Semiquantitative RT-PCR analysis of LdTryS transcript in Amp B sensitive vs. resistant isolates. Ethidium bromide-stained PCR products were photographed and the image was analyzed densitometrically. α-tubulin was used as control to show uniform expression of a housekeeping gene in both Amp B sensitive and resistant promastigotes. (B) Bar graph represents quantitative real time PCR analysis of LdTryS expression level in Amp B sensitive vs. resistant isolates. Data are normalized by the target/reference ratio of the calibrator. (C) The total <i>Leishmania</i> lysates (30 µg) were electrophoresed on 10% SDS-PAGE gel and stained with coomassie brilliant blue. Lane 1 represents, protein marker; lane 2 represents, sensitive strain (S); lanes 3, and 4 represent, resistant isolates (R1, & R2). (D) Shows western blot of same coomassie gel using anti-LdTryS (1∶3000). The image was analyzed by densitometrically. Data was normalized and β-actin was used as control. The experiments were repeated twice and graphs represent the mean ± SD.</p

Enzymatic analysis of recombinant purified LdTryS.

<p>(A) pH profile using a coupled assay in a mixed buffer system. Activity is expressed as a percentage relative to the maximum activity observed with LdTryS. Kinetic properties of LdTryS with substrates GSH (B) and Spd (C) were analysed. <i>K</i>_m values were determined for each substrate by fitting data into Michaelis Menten equation and resulting Lineweaver-Burk plots. The experiments were performed three times in duplicate and data presents the mean ± SD.</p

Multiple sequence alignments of deduced amino acid sequences of TryS from <i>L. donovani</i> and other organisms.

<p>Protein sequences were aligned using the CLUSTAL W program (<a href="http://www.ebi.ac.uk/clustalw/" target="_blank">www.ebi.ac.uk/clustalw/</a>). Sequences are <i>L. donovani</i> (CAD23679), <i>L. infantum</i> (XP_001466426), <i>L. major</i> (XP_003721994), <i>L. amazonensis</i> (ABQ57409), <i>L. braziliensis</i> (XP_001565955), <i>C. fasciculata</i> (AAT99012), <i>T. brucei</i> (CAC87573), and <i>T. cruzi</i> (XP_816076). <i>Asterisks</i> indicate identical amino acids. <i>Dots</i> and <i>colons</i> indicate conserved amino acid substitutions. <i>Dashes</i> indicate gaps. <i>Closed boxes</i> at the amino terminus indicate conserved amino acids involved in amidase activity in all <i>Leishmania</i> species except <i>L. braziliensis</i>. <i>Closed dashed boxes</i> at the central region (on the 6^th, 7^th, and 8^th rows) interact with GSH. <i>Gray boxes</i> at the central region and C-terminal region indicate amino acids involved in synthetase activity and <i>gray closed</i> boxes show a. a. involved in binding triphosphate moiety of ATP. <i>Dashed lines</i> above the alignment indicate the linker regions between amidase and synthetase domain at N- and C-terminal region of the protein. A <i>Solid line</i> above the alignment indicates unique insertion in TryS of eukaryotes which is absent in prokaryotes TryS.</p

Effect of H₂O₂ on growth inhibition of <i>L. donovani</i> parasites.

<p>(A) <i>L. donovani</i> promastigotes (1×10⁶ cells/ml) culture was treated with increasing concentration of H₂O₂ (0–200 µM) up to 15 hrs and growth inhibitory effect of H₂O₂ determined by MTT assay at 3 hr intervals. The cell viability after exposure with increasing concentration of H₂O₂ was determined to optimize time of exposure and dose. (B) The intracellular ROS level was determined by quantification of DCF fluorescence. Results were normalized with cell numbers and presented relative to untreated control cells. (C) To confirm intracellular ROS production a quenching study was performed. The parasites treated with H₂O₂ in the presence of 20 µM <i>N</i>-acetyl-L-cysteine (NAC) ROS scavenger reversed the effect of H₂O_2. The experiments were repeated three times and graphs represent the mean ± SD.</p