Effect of Vitamin D Supplementation on CD4 Count in HIV-Infected Children and Adolescents in North India: A Non-Randomized Comparative Study.

BACKGROUND: HIV infection is still a serious public health issue globally. Suboptimal vitamin D status is highly prevalent in HIV-infected children and adolescents throughout the world. OBJECTIVES: To evaluate the outcome of vitamin D supplementation on CD4 count in HIV-infected children and adolescents with suboptimal vitamin D status. METHODS: Vitamin D level of HIV-infected children and adolescents were measured at enrolment. Suboptimal vitamin D level was defined as 25(OH)D < 30 ng/ml. Vitamin D insufficiency and deficiency were defined as 21-29 and <20 ng/ml, respectively. Children with suboptimal vitamin D levels were supplemented with vitamin D. RESULTS: This was a single-centre, non-randomized comparative study enrolling 50 eligible participants. There were 20 patients who were vitamin D sufficient, 7 were vitamin D insufficient and 23 were found to be vitamin D deficient at enrolment. However, after supplementation, the status of sufficient remained same and 7 insufficient become sufficient, whereas in 23 deficient, 18 (78.3%) become sufficient and 5 (21.7%) become insufficient and this change was found statistically significant among the groups (χ2 = 6.52, p = 0.038). There was a significant improvement of CD4 count from baseline to 4 months in deficient group on vitamin D supplementation (p value < 0.001; 1.2-fold rise). No significant change was seen in vitamin D insufficient (p value = 0.791) and sufficient groups (p value = 0.168). CONCLUSION: Vitamin D should be supplemented in HIV-infected children on ART with low CD4 counts.

Ginger extract activates caspase independent paraptosis in cancer cells via ER stress, mitochondrial dysfunction, AIF translocation and DNA damage.

The rhizome of ginger (Zingiber officinale) a common culinary agent is also known for its medicinal activity. We have earlier reported that pure 6-shogaol, an important component of ginger induces paraptosis in triple negative breast cancer (MDA-MB-231) and non small cell lung (A549) cancer cells. However, the chemopreventive potential of the whole ginger extract in food remains to be elucidated. Here, we demonstrate for the first time that ginger extract (GE) triggers similar anticancer activity/paraptosis against the same cell lines but through different molecular mechanisms. Q-TOF LC-MS analysis of the extract showed the presence of several other metabolites along with 6-shogaol and 6-gingerol. GE induces cytoplasmic vacuolation through ER stress and dilation of the ER. Drastic decrease in the mitochondrial membrane potential and ATP production along with the excess generation of ROS contributed to mitochondrial dysfunction. Consequently, GE caused the translocation of apoptosis inducing factor to the nucleus leading to the fragmentation of DNA. Taken together, these show a novel mechanism for ginger extract induced cancer cell death that can be of potential interest for cancer preventive strategies.

Proteasomal dysfunction and ER stress triggers 2'-hydroxy-retrochalcone-induced paraptosis in cancer cells.

Chalcones are biologically active class of compounds, known for their anticancer activities. Here we show for the first time that out of the six synthetic derivatives of chalcone tested, 2'-hydroxy-retrochalcone (HRC) was the most effective in inducing extensive cytoplasmic vacuolation mediated death called paraptosis in malignant breast and cervical cancer cells. The cell death by HRC is found to be nonapoptotic in nature due to the absence of DNA fragmentation, PARP cleavage, and phosphatidylserine externalization. It was also found to be nonautophagic as there was an increase in the levels of autophagic markers LC3I, LC3II and p62. Immunofluorescence with the endoplasmic reticulum (ER) marker protein calreticulin showed that the cytoplasmic vacuoles formed were derived from the ER. This ER dilation was due to ER stress as evidenced from the increase in polyubiquitinated proteins, Bip and CHOP. Docking studies revealed that HRC could bind to the Thr1 residue on the active site of the chymotrypsin-like subunit of the proteasome. The inhibition of proteasomal activity was further confirmed by the fluorescence based assay of the chymotrypsin-like subunit of the 26S proteasome. The cell death by HRC was also triggered by the collapse of mitochondrial membrane potential and depletion of ATP. Pretreatment with thiol antioxidants and cycloheximide were able to inhibit this programmed cell death. Thus our data suggest that HRC can effectively kill cancer cells via paraptosis, an alternative death pathway and can be a potential lead molecule for anticancer therapy.

Identification of carbonylated proteins from monocytic cells under diabetes-induced stress conditions.

Diabetes is a metabolic disorder characterized by the presence of elevated glucose in the blood and enhanced oxidative stress. It affects the cellular homeostasis that leads to the development of micro-and macro-vascular complications. Monocytes are the primary immune cells present in the circulatory system. Under high-glucose conditions, the cells undergo oxidative stress and secrete reactive oxygen species. The enhanced release of reactive species is known to modify biomolecules like proteins and nucleic acids. Protein carbonylation, one of the most harmful and irreversible protein modifications, is considered as a key player in the progression of diabetes and associated complications. Hence, the present study explores the identification of carbonylated proteins from the monocytes under diabetic stress and determination of their site of modification. Combined avidin affinity chromatography and bottom-up proteomics experiments identified 13 consistently expressed carbonylated proteins. Most of the identified proteins were reported to have altered functions under diabetic conditions that contribute to the development of diabetes-associated inflammation and complications. We were able to determine oxidative stress-induced modifications on Lys, Val, Ile, Cys, Thr and Asp residues.

6-Shogaol induces caspase-independent paraptosis in cancer cells via proteasomal inhibition.

An α, ß-unsaturated carbonyl compound of ginger, 6-Shogaol (6S), induced extensive cytoplasmic vacuolation and cell death in breast cancer cell (MDA-MB-231) and non-small lung cancer (A549) cells. In the presence of autophagic inhibitors the cells continued to exhibit cytoplasmic vacuolation and cell death clearly distinguishing it from the classic autophagic process. 6S induced death did not exhibit the characteristic apoptotic features like caspase cleavage, phosphatidyl serine exposure and DNA fragmentation. The immunofluorescence with the Endoplasmic Reticulum (ER) resident protein, calreticulin indicated that the vacuoles were of ER origin, typical of paraptosis. This was supported by the increase in level of microtubule associated protein light chain 3B (LC3 I and LC3 II) and polyubiquitin binding protein, p62. The level of ER stress markers like polyubiquitinated proteins, Bip and CHOP also consistently increased. We have found that 6S inhibits the 26S proteasome. The proteasomal inhibitory activity was elucidated by a) molecular docking of 6S onto the active site of ß5 subunit and b) reduced fluorescence by the fluorogenic substrate of the chymotrypsin-like subunit. In conclusion these studies demonstrate for the first time that proteasomal inhibition by 6S induces cell death via paraptosis. So 6-shogaol may act as a template for anti-cancer lead discovery against the apoptosis resistant cancer cells.

Renal Adverse Effects of Tenofovir Containing Regimens in HIV-Infected Children and Adolescents in North India.

OBJECTIVE: To study the prevalence of abnormal renal functions among children living with HIV (CLHIV) receiving tenofovir disoproxil fumarate (TDF) containing antiretroviral therapy (ART). METHODS: A prospective, observational study was conducted among CLHIV aged 10 years to 21 years attending the pediatric HIV clinic. We included CLHIV weighing ≥ 30 kg who had been receiving TDF-containing regimens for at least 6 months, with estimated glomerular filtration rate (eGFR) > 60 ml/min/m2 at enrolment and for whom baseline laboratory parameters were available before starting ART. Clinical and laboratory parameters like serum creatinine, serum phosphate, urinary protein and glucose estimation, CD4 count and viral load were noted from records. The mean change in serum creatinine, estimated glomerular filtration rate (eGFR), creatinine clearance, serum phosphate, and presence of urinary glucose and protein by dipstick were assessed at 3- and 12-months follow-up. RESULTS: We enrolled 70 patients with mean (SD) age 14.99 (2.45) years who had been receiving TDF-based ART for a mean (SD) duration of 14.60 (12.80) months. At 3-months and 12-months follow-up, 32.85% and 41.42% patients, respectively, had eGFR below 90 mL/min/1.73m2, while 4.2% and 2.8% patients, respectively, had eGFR between 50-60 mL/min/1.73m2. One patient had creatinine clearance below 50 mL/min/1.73m2. Four patients had hypophosphatemia at the first and last follow-up respectively, and five patients had proteinuria. There was no statistically significant change in CD4 counts, serum potassium, or serum uric acid during study duration. CONCLUSION: TDF-containing ART regimen is associated with decreased eGFR, creatinine clearance and proteinuria.

Effect of the timing of antiretroviral treatment initiation on CD4 count in children and youths living with HIV in North India.

BACKGROUND: Immediate start of antiretroviral treatment (ART) among non-hospitalized outpatient children living with HIV may improve or worsen clinical outcomes due to immune reconstitution. OBJECTIVE: Role of immediate versus post-stabilization start of antiretroviral treatment in children and youths living with HIV on CD4 count and viral load suppression. METHODS: This was a single blinded, randomized controlled trial conducted on outpatients attending a tertiary care hospital associated HIV clinic in North India. We enrolled ART-naive children and youths living with HIV aged 18 months to 21 years in a 1:1 ratio. Block randomization was done using computerized software. Children and youths living with HIV were either started with ART on diagnosis immediately within 24 h (Group A) or post stabilization at 2 weeks (Group B) as per National AIDS Control Organization (NACO) India guidelines. Both groups were comparable for baseline characteristics. RESULTS: There was no significant difference seen in CD4 counts between two groups at 6 months follow up. CD4 count increased significantly in immediate group but not in post-stabilization group at 6 months. No significant changes/differences was seen in WHO clinical staging or anthropometry; one patient developed tuberculosis in both groups. Viral load at 6 months in both the groups did not differ significantly. CONCLUSION: Immediate ART in children and youths living with HIV results in significant increase in CD4 count at 6 months follow up exemplifying immunological response to ART.

Paraptosis: a unique cell death mode for targeting cancer.

Programmed cell death (PCD) is the universal process that maintains cellular homeostasis and regulates all living systems' development, health and disease. Out of all, apoptosis is one of the major PCDs that was found to play a crucial role in many disease conditions, including cancer. The cancer cells acquire the ability to escape apoptotic cell death, thereby increasing their resistance towards current therapies. This issue has led to the need to search for alternate forms of programmed cell death mechanisms. Paraptosis is an alternative cell death pathway characterized by vacuolation and damage to the endoplasmic reticulum and mitochondria. Many natural compounds and metallic complexes have been reported to induce paraptosis in cancer cell lines. Since the morphological and biochemical features of paraptosis are much different from apoptosis and other alternate PCDs, it is crucial to understand the different modulators governing it. In this review, we have highlighted the factors that trigger paraptosis and the role of specific modulators in mediating this alternative cell death pathway. Recent findings include the role of paraptosis in inducing anti-tumour T-cell immunity and other immunogenic responses against cancer. A significant role played by paraptosis in cancer has also scaled its importance in knowing its mechanism. The study of paraptosis in xenograft mice, zebrafish model, 3D cultures, and novel paraptosis-based prognostic model for low-grade glioma patients have led to the broad aspect and its potential involvement in the field of cancer therapy. The co-occurrence of different modes of cell death with photodynamic therapy and other combinatorial treatments in the tumour microenvironment are also summarized here. Finally, the growth, challenges, and future perspectives of paraptosis research in cancer are discussed in this review. Understanding this unique PCD pathway would help to develop potential therapy and combat chemo-resistance in various cancer.

Buckets associated home drowning in Indian infants and toddlers: An analysis of 18 fatalities from 2016-2022.

Cases of drowning at home of unsupervised infants and toddlers in buckets have been reported elsewhere but little research on this largely preventable death in India exists. We performed a descriptive analysis on the basis of Google search of published news report in leading Indian newspapers or news channels. Data were collected employing a pre-determined tool. Between April 2016 and March 2022, we found 18 such cases. The large majority were between 12 and 18 months of age (12/18). This little recognized source of unintentional injury is eminently avoidable, necessitating both public and parental attention and awareness.

Intravenous immunoglobulin in the management of neonatal sepsis: A randomised controlled trial.

Sepsis is a leading cause of neonatal mortality and morbidity in low and middle-income countries. We designed a double-blinded randomised controlled trial in a neonatal intensive care unit (NICU) of a tertiary care teaching hospital to determine the role of intravenous immunoglobulin (IVIG) in decreasing hospital stay. Eighty neonates with clinical features of sepsis were enrolled in the study and placebo groups to receive 500 mg/kg of IVIG for three consecutive days or a placebo. The primary outcome measure was duration of hospital stay in days. The babies in both groups were comparable in terms of birth weight, gestation and sex distribution. There was no significant difference in duration of hospital stay (days) in the study and placebo groups. We found that treatment with IVIG did not shorten the duration of hospital stay in our setting.

Corrigendum: Paraptosis: a unique cell death mode for targeting cancer.

[This corrects the article DOI: 10.3389/fphar.2023.1159409.].

Mitochondrial remodeling following fission inhibition by 15d-PGJ2 involves molecular changes in mitochondrial fusion protein OPA1.

We showed earlier that 15 deoxy Delta(12,14) prostaglandin J2 (15d-PGJ2) inactivates Drp1 and induces mitochondrial fusion [1]. However, prolonged incubation of cells with 15d-PGJ2 resulted in remodeling of fused mitochondria into large swollen mitochondria with irregular cristae structure. While initial fusion of mitochondria by 15d-PGJ2 required the presence of both outer (Mfn1 and Mfn2) and inner (OPA1) mitochondrial membrane fusion proteins, later mitochondrial changes involved increased degradation of the fusion protein OPA1 and ubiquitination of newly synthesized OPA1 along with decreased expression of Mfn1 and Mfn2, which likely contributed to the loss of tubular rigidity, disorganization of cristae, and formation of large swollen degenerated dysfunctional mitochondria. Similar to inhibition of Drp1 by 15d-PGJ2, decreased expression of fission protein Drp1 by siRNA also resulted in the loss of fusion proteins. Prevention of 15d-PGJ2 induced mitochondrial elongation by thiol antioxidants prevented not only loss of OPA1 isoforms but also its ubiquitination. These findings provide novel insights into unforeseen complexity of molecular events that modulate mitochondrial plasticity.

Inhibition of mitochondrial division through covalent modification of Drp1 protein by 15 deoxy-Delta(12,14)-prostaglandin J2.

Arachidonic acid derived endogenous electrophile 15d-PGJ2 has gained much attention in recent years due to its potent anti-proliferative and anti-inflammatory actions mediated through thiol modification of cysteine residues in its target proteins. Here, we show that 15d-PGJ2 at 1 microM concentration converts normal mitochondria into large elongated and interconnected mitochondria through direct binding to mitochondrial fission protein Drp1 and partial inhibition of its GTPase activity. Mitochondrial elongation induced by 15d-PGJ2 is accompanied by increased assembly of Drp1 into large oligomeric complexes through plausible intermolecular interactions. The role of decreased GTPase activity of Drp1 in the formation of large oligomeric complexes is evident when Drp1 is incubated with a non-cleavable GTP analog, GTPgammaS or by a mutation that inactivated GTPase activity of Drp1 (K38A). The mutation of cysteine residue (Cys644) in the GTPase effector domain, a reported target for modification by reactive electrophiles, to alanine mimicked K38A mutation induced Drp1 oligomerization and mitochondrial elongation, suggesting the importance of cysteine in GED to regulate the GTPase activity and mitochondrial morphology. Interestingly, treatment of K38A and C644A mutants with 15d-PGJ2 resulted in super oligomerization of both mutant Drp1s indicating that 15d-PGJ2 may further stabilize Drp1 oligomers formed by loss of GTPase activity through covalent modification of middle domain cysteine residues. The present study documents for the first time the regulation of a mitochondrial fission activity by a prostaglandin, which will provide clues for understanding the pathological and physiological consequences of accumulation of reactive electrophiles during oxidative stress, inflammation and degeneration.

Highly chemoselective turn-on fluorescent probe for ferrous (Fe2+) ion detection in cosmetics and live cells.

In modern society, the use of cosmetics has increased extensively; unfortunately, so-called several toxic metal salts are present as the colorant or filler in cosmetics. The ferrous ion (Fe2+) is one of the metal ions used in cosmetics as a colorant. Ferrous ion (Fe2+) is a vital component in live cells. Considering the adverse effect of high doses of ferrous ions in cosmetics and live cells, we developed a turn-on fluorescent probe PFe(II) for quantitative estimation of ferrous ion (Fe2+) in cosmetics and monitoring of labile ferrous (Fe2+) ion in live cells. The fluorescent probe PFe(II) showed a visual color change from colorless to orange in the presence of ferrous ion (Fe2+) in the cosmetics. We observed that UV-absorption increased at 390 nm upon incubation with ferrous ion (Fe2+). The probe PFe(II) has provided quantitative information on ferrous ion (Fe2+) in various cosmetics, kajol, lip balm, face foundation, mascara, eyeliner, lipliner, face makeup, sindoor, lipstick, nail polish in ppm level through the fluorescence signaling at 460 nm.The probe PFe(II) provided information on labile Fe2+ ion pool via a fluorescence imaging. It is a new addition to the diagnostic inventory for detecting ferrous ion in live cells and cosmetics.

Oxyresveratrol drives caspase-independent apoptosis-like cell death in MDA-MB-231 breast cancer cells through the induction of ROS.

Earlier studies from our laboratory have demonstrated that Oxyresveratrol (OXY), a hydroxyl-substituted stilbene, exhibits potent inhibition of human melanoma cell proliferation. The present study defines a cytotoxic effect of OXY on the highly chemo-resistant, triple-negative human breast cancer cell line MDA-MB-231. OXY-mediated cell death resulted in accumulation of cells at the sub-G1 phase of the cell cycle, induced chromatin condensation, DNA fragmentation, phosphatidylserine externalization and PARP cleavage, indicative of apoptosis. Interestingly, morphology and cell viability studies with the pan-caspase inhibitor, QVD-OPH revealed that OXY-induced cell death was caspase-independent. Docking studies also showed that OXY can bind to the S1 site of caspase-3, and could also exert an inhibitory effect on this executioner caspase. The immunoblot analysis demonstrating the absence of caspase cleavage during cell death further confirmed these findings. OXY was also observed to induce the production of reactive oxygen species, which caused the depolarization of the mitochondrial membrane resulting in translocation of Apoptosis Inducing Factor (AIF) into the nucleus. Pretreatment of the cells with N-Acetyl Cysteine antioxidant prevented cell death resulting from OXY treatment. Thus, OXY initiates ROS-mediated, apoptosis-like cell death, involving mitochondrial membrane depolarization, translocation of AIF into the nucleus, and DNA fragmentation, resulting in caspase-independent cell death in MDA-MB-231 cells. The cytotoxicity manifested by OXY was also observed in 3D cell culture models and primary cells, thereby providing a basis for the utilization of OXY as a novel template for the future design of anticancer therapeutics.

In-vitro evaluation on drug release kinetics and antibacterial activity of dextran modified polyurethane fibrous membrane.

pH stimuli drug release nanofibrous membranes of polyurethane/dextran were developed for tailoring of antibacterial wound dressings. Incorporation of dextran in polyurethane (PU) showed increment in hydrophilicity, vapour transmission rate, percentage sorption values, and biodegradability. Dextran also acts as reinforcement filler in PU matrix. Dextran induces a high degree of platelet adhesion and hemostasis potential which is essential for promoting the wound healing process. Moreover, 20 wt% dextran loaded membranes (PU/20D) exhibited enhanced cell proliferation, attachment and viability against 3T3 fibroblasts. Curcumin loaded PU/20 dextran membrane exhibited pH-controlled drug release potency and synergistic antibacterial activity against gram-positive bacteria. It is confirmed that, PU/20D membranes could promote, pH-controlled drug release and synergistic antibacterial activity for a promising wound dressing material.

Plumbagin induces paraptosis in cancer cells by disrupting the sulfhydryl homeostasis and proteasomal function.

Plumbagin (PLB) is an active secondary metabolite extracted from the roots of Plumbago rosea. In this study, we report that plumbagin effectively induces paraptosis by triggering extensive cytoplasmic vacuolation followed by cell death in triple negative breast cancer cells (MDA-MB-231), cervical cancer cells (HeLa) and non-small lung cancer cells (A549) but not in normal lung fibroblast cells (WI-38). The vacuoles originated from the dilation of the endoplasmic reticulum (ER) and were found to be empty. The cell death induced by plumbagin was neither apoptotic nor autophagic. Plumbagin induced ER stress mainly by inhibiting the chymotrypsin-like activity of 26S proteasome as also evident from the accumulation of polyubiquitinated proteins. The vacuolation and cell death were found to be independent of reactive oxygen species generation but was effectively inhibited by thiol antioxidant suggesting that plumbagin could modify the sulfur homeostasis in the cellular milieu. Plumbagin also resulted in a decrease in mitochondrial membrane potential eventually decreasing the ATP production. This is the first study to show that Plumbagin induces paraptosis through proteasome inhibition and disruption of sulfhydryl homeostasis and thus further opens up the lead molecule to potential therapeutic strategies for apoptosis-resistant cancers.

Direct readout protonophore induced selective uncoupling and dysfunction of individual mitochondria within cancer cells.

Concurrently, manipulation of mitochondrial activity and its monitoring have enormous significance in cancer therapy and diagnosis. In this context, a fluorescent probe MitoDP has been developed for validating H2S mediated protonophore (2,4-dinitrophenol, DNP) induced mitochondrial membrane potential change, ROS formation and ATP depletion in cancer cells. The extent of protonophore activation for mitochondrial dysfunction is monitored through fluorescence signalling at 450 nm. The current study provides a proof for the concept of endogenous H2S-mediated controlled and spatial release of bioactive agents, or toxins specifically in mitochondria of cancer cells.

Monitoring of topoisomerase (I) inhibitor camptothecin release from endogenous redox-stimulated GO-polymer hybrid carrier.

We have developed endogenous redox-responsive polymer conjugated GO-based hybrid nanomaterials (GO-PEGssFol-CPT) for delivery of anticancer drug camptothecin (CPT) to the cancer cells. The synthesized intermediate (PEGSSFol) and CPT loaded GO- PEGSSFol were characterized using Fourier transform infrared spectroscopy (FTIR) and 1H NMR. The morphological feature changes of TEM and AFM images have confirmed the loading of CPT on the nanocarrier and its release from the nanocarrier. The amount of CPT was loaded was found to be 14.2%. The extent of camptothecin (CPT) release from GO-BiotinPVA-CPT in the presence of different concentrations of glutathione (GSH) was monitored with the increase in the fluorescence intensity at λmax 438 nm and UV-Vis absorbance at 366 nm. The time-dependent camptothecin (CPT) release was monitored in the presence of GSH. It was noticed that CPT was completely released from GO-PEGssFol-CPT within 45 min. This release process is free from interference by other ubiquitous analytes in the living system. The constant fluorescence intensity of GO-PEGssFol-CPT against acidic pH indicated that CPT would not be released in the extracellular region of cancer cells. Therefore, such delivery system could be used to prevent unwanted cytotoxicity to the healthy cells. The GO-PEGssFol-CPT showed higher antiproliferative activity against cervical cancer cells compared to the CPT. Thus, GO-PEGssFol-CPT can be a new material to deliver the anticancer drug to the target tumor region.

Autolysin mediated adherence of Staphylococcus aureus with Fibronectin, Gelatin and Heparin.

Major autolysin (Atl) of Staphylococcus aureusis a cell surface associated peptidoglycan hydrolase with amidase and glucosaminidase domains. Atl enzymes (amidase and glucosaminidase) are known to participate in biofilm formation and also can bind with host matrices. Earlier studies demonstrated the binding of Atlwithfibronectin, thrombospondin 1, vitronectin and heat shock cognate protein Hsc70. Here, we have shown, Atl mediates attachment of S.aureus to heparin and gelatine as well. The atl mutant strain demonstrated around 2.5 fold decreased adherence with fibronectin, gelatin and heparin coated microtiter plates. The microscopic studies confirmed the reduced binding of atl mutant with them compared to its parental wild type and complemented mutant strains. Amidase and glucosaminidase were expressed as N-terminal histidine tagged proteins from Escherichia coli, purified and refolded. We found refolded amidase bind with fibronectin, gelatin and heparin; whereas refolded glucosaminidase binds with only fibronectin and heparin but not gelatin. These results reemphasize Atl as one of the crucial proteins from Staphylococcus that facilitate their binding with multiple host cellular components during colonization and infection.