Normative data for insulin-like growth factor-1 (IGF-1) in healthy children and adolescents from India.

BACKGROUND: Serum IGF-1 is an important biochemical tool to diagnose and monitor GH-related disorders. However, ethnic-specific Indian data following consensus criteria for the establishment of normative data, are not available. Our objective was to generate chronological age (CA)-, bone age (BA)- and Tanner stage-specific normative data for IGF-1 in healthy Indian children and adolescents. METHODS: A cross-sectional epidemiological study was conducted in schools and the community, which enrolled apparently healthy children and adolescents with robust exclusion criteria. The outcome measure was serum IGF-1 assessed using an electro-chemiluminescence immunoassay (ECLIA). The 2.5th, 5th, 10th, 25th, 50th (median), 75th, 90th, 95th, and 97.5th centiles for IGF-1 were estimated using generalized additive models. RESULTS: We recruited 2226 apparently healthy participants and following exclusion, 1948 (1006 boys, 942 girls) were included in the final analysis. Girls had median IGF-1 peak at CA of 13 years (321.7 ng/mL), BA of 14 years (350.2 ng/mL) and Tanner stage IV (345 ng/mL), while boys had median IGF-1 peak at CA of 15 years (318.9 ng/mL) BA of 15 years (340.6 ng/mL) and Tanner stage III (304.8 ng/mL). Girls had earlier rise, peak and higher IGF-1 values. The reference interval (2.5th-97.5th percentile) was broader during peri-pubertal ages, indicating a higher physiological variability. CONCLUSION: This study provides ethnicity-specific normative data on serum IGF-1 and will improve the diagnostic utility of IGF-1 in the evaluation and management of growth disorders in Indian children and adolescents.

C-terminal region of Rv1039c (PPE15) protein of Mycobacterium tuberculosis targets host mitochondria to induce macrophage apoptosis.

Mycobacterium tuberculosis (Mtb) genome possesses a unique family called Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) gene family, exclusive to pathogenic mycobacterium. Some of these proteins are known to play role in virulence and immune response modulation, but many are still uncharacterized. This study investigated the role of C-terminal region of Rv1039c (PPE15) in inducing mitochondrial perturbations and macrophage apoptosis. Our in-silico studies revealed the disordered, coiled, and hydrophobic C-terminal region in Rv1039c has similarity with C-terminal of mitochondria-targeting pro-apoptotic host proteins. Wild type Rv1039c and C-terminal deleted Rv1039c (Rv1039c-/-Cterm) recombinant proteins were purified and their M. smegmatis knock-in strains were constructed which were used for in-vitro experiments. Confocal microscopy showed localization of Rv1039c to mitochondria of PMA-differentiated THP1 macrophages; and reduced mitochondrial membrane depolarization and production of mitochondrial superoxides were observed in response to Rv1039c-/-Cterm in comparison to full-length Rv1039c. The C-terminal region of Rv1039c was found to activate caspases 3, 7 and 9 along with upregulated expression of pro-apoptotic genes like Bax and Bim. Rv1039c-/-Cterm also reduced the Cytochrome-C release from the mitochondria and the expression of AnnexinV/PI positive and TUNEL positive cells as compared to Rv1039c. Additionally, Rv1039c was observed to upregulate the TLR4-NF-κB-TNF-α signalling whereas the same was downregulated in response to Rv1039c-/-Cterm. These findings suggested that the C-terminal region of Rv1039c is a molecular mimic of pro-apoptotic host proteins which induce mitochondria-dependent macrophage apoptosis and evoke host immune response. These observations enhance our understanding about the role of PE/PPE proteins at host-pathogen interface.

Role of MicroRNAs as post transcription regulators of matrix metalloproteinases and their association in tuberculous meningitis.

Matrix metalloproteinases (MMPs) have a role in driving neuroinflammation in infectious as well as non-infectious diseases; however, recent reports have potentiated the role of microRNAs in regulating MMPs at post-transcriptional levels, leading to dysregulation of crucial MMP functions like tissue remodelling, blood brain barrier integrity, etc. In present study, microRNAs regulating MMPs (MMP2 and MMP3) were selected from database search followed by literature support. Expression of these microRNAs i.e., hsa-miR-495-3p, hsa-miR-132-3p and hsa-miR-21-5p was assessed by RT-PCR and the protein levels of MMPs were assessed by ELISA in the cerebrospinal fluid (CSF) of tuberculous meningitis (TBM) patients, healthy controls (HC) and non-infectious neuroinflammatory disease (NID) patients. The expression of hsa-miR-495-3p and hsa-miR-132-3p showed downregulation in TBM while hsa-miR-21-5p was overexpressed as compared to healthy controls. Moreover, MMP levels were found to be deranged with a significant increase in MMP3 levels in the TBM and NID patients compared to HC group. These observations highlight dysregulated microRNAs (hsa-miR-495-3p, hsa-miR-21-5p and hsa-miR-132-3p) levels might impair the levels of MMPs (MMP2 and MMP3) leading to neuroinflammation in TBM and NID population. These findings can further be applied to target these microRNAs for developing newer treatment modalities for better complication management.

Mycobacterium tuberculosis protein PPE15 (Rv1039c) possesses eukaryote-like SH3 domain that interferes with NADPH Oxidase assembly and Reactive Oxygen Species production.

Inhibition of Reactive Oxygen Species (ROS) is one of the strategies that Mycobacterium tuberculosis (Mtb) employs as its defence mechanism. In this study, the role of PPE15 (Rv1039c), a late-stage protein, has been investigated in modulating the cellular ROS. We discovered PPE15 to be a secretory protein that downregulates ROS generation in THP1 macrophages. Our in-silico analysis revealed the presence of a eukaryote-like SH3 (SH3e) domain in PPE15. The predicted SH3e-domain of PPE15 was found to interact with cytosolic components of NADPH Oxidase (NOX), p67phox and p47phox through molecular docking. In-vitro experiments using THP1 macrophages showed a diminished NADP/NADPH ratio, indicating reduced NOX activity. We also observed increased levels of p67phox and p47phox in the cytoplasmic fraction of PPE15 treated macrophages as compared to the plasma membrane fraction. To understand the role of the SH3e-domain in ROS modulation, this domain was deleted from the full-length PPE15 (PPE15-/-SH3). We observed an increase in cellular ROS and NADP/NADPH ratio in response to PPE15-/-SH3 protein. The interaction of PPE15-/-SH3 with p67phox or p47phox was also reduced in the cytoplasm, indicating migration of NOX subunits to the plasma membrane. Additionally, M. smegmatis expressing PPE15 was observed to be resistant to oxidative stress with significant intracellular survival in THP1 macrophages as compared to M. smegmatis expressing PPE15-/-SH3. These observations suggest that the SH3e-domain of PPE15 interferes with ROS generation by sequestering NOX components that inhibit NOX assembly at the cell membrane. Therefore, PPE15 acts like a molecular mimic of SH3-domain carrying eukaryotic proteins that can be employed by Mtb at late stages of infection for its survival. These findings give us new insights about the pathogen evading strategy of Mtb which may help in improving the therapeutics for TB treatment.

An Updated Trial Sequential Meta-analysis of Vitamin D Receptor Gene Polymorphism (Fok1, Bsm1, Taq1 and Apa1) and Risk to Tuberculosis.

Vitamin D receptor (VDR) is one of the most widely studied genes for the Tuberculosis (TB) susceptibility. Several studies have been conducted to establish some association between them but most of the time they are contradictory and underpowered. So, a trial sequential meta-analysis between VDR gene polymorphisms and TB susceptibility can provide a better understanding of the relationship. A meta-analysis was carried out using a total of 17 case-control studies which includes Fok1 (14 Studies), Bsm1 (8 Studies), Apa1 (8 Studies) and Taq1 (12 Studies) polymorphisms in the VDR gene searched from Pubmed and Google Scholar. Pooled Odds Ratio (OR) and 95% Confidence Interval (CI) were calculated using StatsDirect Version 3, using random effects model. Trial sequential analysis (TSA) was also performed to assess if the statistical significance of the meta-analysis was within monitoring boundaries. It was found that the individuals with BB genotype of Bsm1 polymorphism with OR = 0.713 (95%CI = 0.521, 0.974; p value < 0.05) and FF genotype of Fok1 polymorphism with pooled OR = 0.716 (95%CI = 0.523, 0.979; p value < 0.05) had decreased incidence of TB. Also, the aa genotype of Apa1 gene polymorphism increases susceptibility to TB with pooled OR = 1.997 (95%CI = 1.121, 3.558; p value < 0.05). All these analyses reached the required information size through TSA analysis. No statistically significant result was found for Taq1 polymorphisms and TB susceptibility. VDR polymorphisms in Fok1 and Bsm1 played protective roles against development of TB infection, while Apa1 appeared to have a significant association to TB susceptibility. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01091-3.

Expression of mammalian cell entry genes in clinical isolates of M. tuberculosis and the cell entry potential and immunological reactivity of the Rv0590A protein.

Mammalian cell entry (mce) operons play a vital role in cell invasion and survival of M. tuberculosis. Of the mce genes, the function of Rv0590A is still unknown. The present study was performed to investigate the function and immunogenic properties of the protein Rv0590A. Human leukemia monocytic cell line (THP-1) derived macrophages were infected with M. tuberculosis H37Rv at 3, 6, and 24 h of infection. The maximum colony forming units (CFU) were observed at 6 h (p < 0.005), followed by 3 h after infection. M. tuberculosis H37Rv and clinical isolates representative of Delhi/CAS, EAI, Beijing, Haarlem and Euro-American-superlineage were included in the study for expression analysis of mce1A, mce2A, mce3A, mce4A, and Rv0590A genes. Maximum upregulation of all mce genes was observed at 3 h of infection. All the five clinical isolates and H37Rv upregulated Rv0590A at various time points. Macrophage infection with M. tuberculosis H37Rv-overexpressing Rv0590A gene showed higher intracellular CFU as compared to that of wild-type H37Rv. Further, purified Rv0590A protein stimulated the production of TNFα, IFNγ, and IL-10 in macrophages. Thus, Rv0590A was found to be involved in cell invasion and showed good immunological response.

In vitro and in vivo evaluation of dual Clofazimine and Verapamil loaded PLGA nanoparticles.

Combination therapy may counter the risk caused by efflux pumps mediated resistance developed by mycobacteria with a concomitant increase of the bactericidal effect of anti-TB drugs. In the present study, combination of two drugs in a nanoformulation was prepared. Clofazimine targets type 2 NADH dehydrogenase of the electron transport chain, and Verapamil inhibits various mycobacterial efflux pumps. The nanotechnology approach was adopted to overcome limitations associated with administration of free form of drugs by using poly (D, L-lactic-co-glycolic acid) as a polymer. Nanoparticles were prepared by oil/water single emulsion solvent evaporation procedure and characterized by various techniques. The results thus highlighted that developed nanoparticles were spherical with nano range size (200-450 nm). Fourier transform infrared spectroscopy revealed successful encapsulation of drugs in developed nanoformulations. Drugs in combination showed higher encapsulation efficiency and percentage drug loading capacity as compared to individual drug nanoformulations. Also, reduced toxicity of nanoformulation was observed in hemolysis assay as compared to free drugs. Ex-vivo analysis demonstrated efficient uptake of rhodamine encapsulated nanoparticles by THP-1 cells, while in-vivo results revealed sustained drug release of nanoformulation as compared to free drugs in combination. Therefore, we were able to achieve development of a single nanoformulation encapsulating Clofazimine and Verapamil in combination. Based on these findings, future studies can be designed to explore the potential of co-encapsulated Clofazimine and Verapamil nanoparticles in management of tuberculosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01062-8.

PE_PGRS45 (Rv2615c) protein of Mycobacterium tuberculosis perturbs mitochondria of macrophages.

The PE_PGRS proteins have coevolved with the antigenic ESX-V secretory system and are abundant in pathogenic Mycobacterium. Only a few PE_PGRS proteins have been characterized, and research suggests their role in organelle targeting, cell death pathways, calcium (Ca2+ ) homeostasis and disease pathogenesis. The PE_PGRS45 (Rv2615c) protein was predicted to contain mitochondria targeting sequences by in silico evaluation. Therefore, we investigated the targeting of the Rv2615c protein to host mitochondria and its effect on mitochondrial functions. In vitro experiments showed the Rv2615c protein colocalized with the mitochondria and led to morphological mitochondrial perturbations. Recombinant Rv2615c was observed to cause increased levels of intracellular reactive oxygen species and the adenosine diphosphate-to-adenosine triphosphate ratio. The Rv2615c protein also induced mitochondrial membrane depolarization and the generation of mitochondrial superoxide. We observed the release of cytochrome C into the cytoplasm and increased expression of proapoptotic genes Bax and Bim with no significant change in anti-apoptotic Bcl2 in Rv2615c-stimulated THP1 macrophages. Ca2+ is a key signaling molecule in tuberculosis pathogenesis, modulating host cell responses. As reported for other PE_PGRS proteins, Rv2615c also has Ca2+ -binding motifs and thus can modulate calcium homeostasis in the host. We also observed a high level of Ca2+ influx in THP1 macrophages stimulated with Rv2615c. Based on these findings, we suggest that Rv2615c may be an effector protein that could contribute to disease pathogenesis by targeting host mitochondria.

Proteomic-based identification of APCS as candidate protein for diagnosis of patients exhibiting anti-tubercular drug induced liver injury.

Traditional markers evaluate anti-tubercular drug-induced liver injury (AT-DILI). However, these markers have certain limitations and studies are in progress to characterize AT-DILI at an early stage. In the present study, 40 patients were categorized and equally distributed into healthy controls, newly diagnosed tuberculosis (TB), TB without hepatotoxicity and TB with hepatotoxicity groups based on their conventional liver function tests. Relative protein quantification was performed on depleted pooled serum samples of each representative group by LC-MS/MS, and validation of shortlisted protein was done by ELISA. Levels of all analysed biochemical parameters showed a statistical increment in the hepatotoxicity group compared to the other three groups, representing AT-DILI. Comparative proteomic analysis between TB with hepatotoxicity versus TB without hepatotoxicity groups highlighted 24 significant differentially expressed proteins, including PROS1, KNG1, CFH, LCAT, APCS and ADIPOQ. Identified proteins were involved in complement activation, triglyceride-rich lipoprotein particle remodelling and pathways comprising complement, coagulation cascades and cholesterol metabolism. Based on functional relevance, the serum amyloid P component (APCS) was shortlisted for validation, and it showed a similar trend as observed in the discovery phase with 100% sensitivity and 87% specificity; however, findings need exploration in larger cohorts.

Molecular and cellular remodeling of HepG2 cells upon treatment with antitubercular drugs.

Drug-induced liver injury (DILI) is an adverse outcome of the currently used tuberculosis treatment regimen, which results in patient noncompliance, poor treatment outcomes, and the emergence of drug-resistant tuberculosis. DILI is primarily caused by the toxicity of the drugs and their metabolites, which affect liver cells, biliary epithelial cells, and liver vasculature. However, the precise mechanism behind the cellular damage attributable to first-line antitubercular drugs (ATDs), as well as the effect of toxicity on the cell survival strategies, is yet to be elucidated. In the current study, HepG2 cells upon treatment with a high concentration of ATDs showed increased perforation within the cell, cuboidal shape, and membrane blebbing as compared with control/untreated cells. It was observed that ATD-induced toxicity in HepG2 cells leads to altered mitochondrial membrane permeability, which was depicted by the decreased fluorescence intensity of the MitoRed tracker dye at higher drug concentrations. In addition, high doses of ATDs caused cell damage through an increase in reactive oxygen species production in HepG2 cells and a simultaneous reduction in glutathione levels. Further, high dose of isoniazid (50-200 mM), pyrazinamide (50-200 mM), and rifampicin (20-100 µM) causes cell apoptosis and affects cell survival during toxic conditions by decreasing the expression of potent autophagy markers Atg5, Atg7, and LC3B. Thus, ATD-mediated toxicity contributes to the reduced ability of hepatocytes to tolerate cellular damage caused by altered mitochondrial membrane permeability, increased apoptosis, and decreased autophagy. These findings further emphasize the need to develop adjuvant therapies that can mitigate ATD-induced toxicity for the effective treatment of tuberculosis.

Impact of Community Based Nutrition Intervention in the Prevention and Control of Anaemia Among Reproductive Aged Women of India.

INTRODUCTION: Maternal and child under-nutrition is particularly widespread in low and middle-income nations, increasing the overall disease burden due to poor nutritional status. The aim of this study was to develop nutrition intervention for the prevention and control of anaemia among women of reproductive age. METHODS: Community-based intervention study was conducted among 443 women of reproductive age group (15-49 years) to determine the effectiveness of a 6-month nutrition intervention package. The nutrition intervention was developed by using Precede-Proceed model and the trans-theoretical model of behavior change. Multi-channel communication approach was adopted and nutrition intervention package was provided. Assessment of haemoglobin, red blood cells, platelet, ferritin, folate, vitamin B12, haematocrit test, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red cell distribution width and total leucocyte count was compared at the baseline and endline after the intervention among the participants. The chi-square test of independence and t-test were performed. RESULTS: The only mean ferritin level shows significant improvement (p < 0.001). A significant decrease (~ 15%, p = 0.027) in anaemia was observed after the intervention. CONCLUSIONS: Improvement in anaemic status of women was observed. National schemes and programs require a more robust strategical implementation like food fortification/bio fortification and behaviour change communication at village level to enhance the availability and accessibility of fortified food.

Late stage specific Rv0109 (PE_PGRS1) protein of Mycobacterium tuberculosis induces mitochondria mediated macrophage apoptosis.

Mitochondria are the powerhouse of the cell and a critical cell signalling hub that decides the fate of the cell. Mycobacterium tuberculosis (Mtb) being a successful pathogen targets and controls the host mitochondria for pathogenesis. Various effector proteins of Mtb are also known to target host mitochondria which include few proteins of a unique Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) family exclusively present in pathogenic mycobacteria, but many of them are still uncharacterized. The present study investigates one such late expressing Rv0109 (PE_PGRS1) protein of Mtb. In-silico analysis predicted the presence of mitochondria targeting signal sequences in Rv0109 and its role in regulation of cysteine type endopeptidase (caspase) activity during apoptosis. Recombinant Rv0109 gets localized to mitochondria of THP1 macrophages as shown by confocal microscopy. Rv0109 was observed to induce mitochondrial stress which resulted in mitochondrial membrane depolarization, upregulation of mitochondrial superoxides and release of Cytochrome-C in the cytoplasm through flow cytometry. Depleted intracellular ATP was observed in THP1 macrophages in response to Rv0109. This mitochondrial stress in response to Rv0109 was observed to culminate in increased expression of pro-apoptotic Bax and Bim factors and caspase activation leading to macrophage apoptosis. Since Rv0109 is a late stage specific protein expressed within granuloma; mitochondria mediated apoptosis induced by Rv0109 may be explored for its role in granuloma maintenance and pathogen persistence.

Role of C-terminal domain of Mycobacterium tuberculosis PE6 (Rv0335c) protein in host mitochondrial stress and macrophage apoptosis.

PE/PPE proteins of Mycobacterium tuberculosis (Mtb) target the host organelles to dictate the outcome of infection. This study investigated the significance of PE6/Rv0335c protein's unique C-terminal in causing host mitochondrial perturbations and apoptosis. In-silico analysis revealed that similar to eukaryotic apoptotic Bcl2 proteins, Rv0335c had disordered, hydrophobic C-terminal and two BH3-like motifs in which one was located at C-terminal. Also, Rv0335c's N terminal had mitochondrial targeting sequence. Since, C-terminal of Bcl2 proteins are crucial for mitochondria targeting and apoptosis; it became relevant to evaluate the role of Rv0335c's C-terminal domain in modulating host mitochondrial functions and apoptosis. To confirm this, in-vitro experiments were conducted with Rv0335c whole protein and Rv0335c∆Cterm (C-terminal domain deleted Rv0335c) protein. Rv0335c∆Cterm caused significant reduction in mitochondrial perturbations and Caspase-mediated apoptosis of THP1 macrophages in comparison to Rv0335c. However, the deletion of C-terminal domain didn't affect Rv0335c's ability to localize to mitochondria. Nine Ca2+ binding residues were predicted within Rv0335c and four of them were at the C-terminal. In-vitro studies confirmed that Rv0335c caused significant increase in intracellular calcium influx whereas Rv0335c∆Cterm had insignificant effect on Ca2+ influx. Rv0335c has been reported to be a TLR4 agonist and, we observed a significant reduction in the expression of TLR4-HLA-DR-TNF-α in response to Rv0335c∆Cterm protein also suggesting the role of Rv0335c's C-terminal domain in host-pathogen interaction. These findings indicate the possibility of Rv0335c as a molecular mimic of eukaryotic Bcl2 proteins which equips it to cause host mitochondrial perturbations and apoptosis that may facilitate pathogen persistence.

Transition to adult services: the current and potential role of the UK hospital pharmacist.

PURPOSE: To explore the current and potential role for UK pharmacists in the transition to adult services for young people with chronic health problems. METHODS: UK hospital pharmacists were surveyed using an online questionnaire with closed and open questions covering their involvement in a transition programme, demography and scope of work, experiences of transition, and the barriers encountered in providing an effective transition service. RESULTS: Overall, 74 pharmacists completed the questionnaire. Most were female (70% (52/74)), had ≥6 years of experience (62% (46/74)), were paediatric pharmacists (74% (55/74)), and were based in a teaching hospital practice setting (70% (52/74)). Many participants (57% (42/74)) had a transition programme in place in their hospital; of these, 55% (23/42) were not a part of the service. Respondents identified unique skills that pharmacists could contribute to the transition service, including knowledge of medications (including formulations and unlicensed medications), awareness of medication services beyond paediatrics, commissioning of medications, and familiarity with adult services. Most commonly identified barriers to transition included 'time constraints', 'pharmacists not involved as part of the wider multidisciplinary team', and 'lack of engagement between different services'. Pharmacists noted that their ideal transition service would include specific medication-related transition, for example, adherence, counselling, and supply of medications. CONCLUSIONS: These findings support the role of hospital pharmacists as crucial members of the multidisciplinary team required for transition. The skills and knowledge of the hospital pharmacist is under-utilised within the transition service, yet pharmacists are motivated and uniquely skilled healthcare professionals who have the potential to improve medicines transition.

Correlation of Urinary Biomarkers (Interleukin-6, Transforming growth factor-ß, E-Cadherin, and MCP-1) with Conventional Parameters of Disease Progression in Patients of Posterior Urethral Valves: A Comparative Analysis.

Aims: Posterior urethral valves (PUV) are the leading cause of end-stage renal disease in boys. The study aimed to look at the ongoing renal damage and profibrotic activity by measuring the levels of Interleukin-6 (IL-6), Transforming growth factor-ß (TGF-ß), E-cadherin, and Monocyte Chemoattractant Protein-1 (MCP-1) and observing trends in subsequent follow-ups and at the same time correlating them with the established parameters of disease progression. Materials and Methods: This prospective study included 36 consecutive patients of PUV, managed over a period of 18 months. IL-6, TGF-ß, E-cadherin, and MCP-1 were measured in urine samples at the time of admission, pre-fulguration and 3 months' and 9 months' post fulguration. The observed values were correlated with the conventional parameters used in clinical practice. Results: All the biomarkers showed statistically significant trends when these values were compared on admission, postoptimization and 3 months' and 9 months' postfulguration. None of the biomarkers showed a significant correlation with renal function tests. E-Cadherin and TGF-ß showed a positive and a negative correlation with ultrasonography (USG) kidney, ureter, and bladder (KUB) respectively. E-Cadherin showed a positive correlation, whereas IL-6 and TGFß showed negative correlation respectively with micturating cystourethrogram (MCUG). IL-6 showed statistically a significant negative correlation with dimercapto succinic acid (DMSA). MCP-1 did not show any significant correlation with USG KUB, MCUG and DMSA. Conclusion: This study concludes that E-Cadherin, IL-6, TGF-ß can be promising urinary biomarkers for early detection of the ongoing renal damage in patients of PUV following valve fulguration. MCP-1 may have more complex interactions, with inflammatory markers; which warrants further research.

Elucidating the function of hypothetical PE_PGRS45 protein of Mycobacterium tuberculosis as an oxido-reductase: a potential target for drug repurposing for the treatment of tuberculosis.

Mycobacterium tuberculosis (Mtb) encodes a total of 67 PE_PGRS proteins and definite functions of many of them are still unknown. This study reports PE_PGRS45 (Rv2615c) protein from Mtb as NADPH dependent oxido-reductase having substrate specificity for fatty acyl Coenzyme A. Computational studies predicted PE_PGRS45 to be an integral membrane protein of Mtb. Expression of PE_PGRS45 in non-pathogenic Mycobacterium smegmatis, which does not possess PE_PGRS genes, confirmed its membrane localization. This protein was observed to have NADPH binding motif. Experimental validation confirmed its NADPH dependent oxido-reductase activity (Km value = 34.85 ± 9.478 µM, Vmax = 96.77 ± 7.184 nmol/min/mg of protein). Therefore, its potential to be targeted by first line anti-tubercular drug Isoniazid (INH) was investigated. INH was predicted to bind within the active site of PE_PGRS45 protein and experiments validated its inhibitory effect on the oxido-reductase activity of PE_PGRS45 with IC50/Ki values of 5.66 µM. Mtb is resistant to first line drugs including INH. Therefore, to address the problem of drug resistant TB, docking and Molecular Dynamics (MD) simulation studies between PE_PGRS45 and three drugs (Entacapone, Tolcapone and Verapamil) which are being used in Parkinson's and hypertension treatment were performed. PE_PGRS45 bound the three drugs with similar or better affinity in comparison to INH. Additionally, INH and these drugs bound within the same active site of PE_PGRS45. This study discovered Mtb's PE_PGRS45 protein to have an oxido-reductase activity and could be targeted by drugs that can be repurposed for TB treatment. Furthermore, in-vitro and in-vivo validation will aid in drug-resistant TB treatment. HIGHLIGHTSIn-silico and in-vitro studies of hypothetical protein PE_PGRS45 (Rv2615c) of Mycobacterium tuberculosis (Mtb) reveals it to be an integral membrane proteinPE_PGRS45 protein has substrate specificity for fatty acyl Coenzyme A (fatty acyl CoA) and possess NADPH dependent oxido-reductase activityDocking and simulation studies revealed that first line anti-tubercular drug Isoniazid (INH) and other drugs with anti-TB property have strong affinity for PE_PGRS45 proteinOxido-reductase activity of PE_PGRS45 protein is inhibited by INHPE_PGRS45 protein could be targeted by drugs that can be repurposed for TB treatmentCommunicated by Ramaswamy H. Sarma.

Mycobacterium tuberculosis dormancy regulon proteins Rv2627c and Rv2628 as Toll like receptor agonist and as potential adjuvant.

During latency, DosR proteins of Mycobacterium tuberculosis (M.tb) get activated and help the bacterium to remain dormant. We have shown earlier that 2 such proteins Rv2627c and Rv2628 are immunogenic and induce a TH1 kind of immune response. In this study, through in-vitro experiments we have confirmed that Rv2627c and Rv2628 proteins act as protein Toll-Like Receptor (TLR) agonist-adjuvant. Rv2627c and Rv2628 stimulated THP-1 macrophages showed an increased expression of TLR2, TLR4 and co-stimulatory molecules CD40, CD80, CD86 and antigen presenting molecule HLA-DR. Further studies also found enhanced expression of downstream signaling molecules of TLR activation like MyD88, NF-κB-p65 and pro-inflammatory cytokines. Inhibition studies using TLR blocking antibodies decreased the expression of co-stimulatory molecules, MyD88, NF-κB-p65, and pro-inflammatory cytokines. Rv2627c and Rv2628 stimulation of HEK-TLR2 reporter cell line confirmed the interaction of these proteins with TLR2. Moreover, molecular docking and simulations of Rv2627c and Rv2628 proteins with TLR2 and TLR4 showed stable interactions. The adjuvant activity of Rv2628 was further validated by a protein adjuvanted with pre-clinically validated peptides as multi-epitope vaccine construct which showed good binding with TLR2 and TLR4 and activate dendritic cells and induce sustained pro-inflammatory cytokine response by C-ImmSim analysis. We propose that our vaccine construct will produce a better immune response than BCG and can be taken up as a post-exposure therapeutic subunit vaccine along with standard TB therapy. We also anticipate that our construct can be taken up as a protein adjuvant with other vaccine candidates as these can activate macrophages through TLR signaling.

Therapeutic potential of human serum albumin nanoparticles encapsulated actinonin in murine model of lung adenocarcinoma.

Non-small cell lung cancer comprises 85% of the global lung cancer cases. Conventional chemotherapeutics possess certain limitations like systemic toxicity and drug resistance that requires the development of new therapeutic agents for successful treatment of lung cancer. Actinonin, a human peptide deformylase inhibitor, has demonstrated anti-cancerous properties in various leukemias and solid cancer types. However, it has limited therapeutic application because of its low bioavailability and systemic toxicity if administered in free form. This limitation can be overcome by using nano-delivery systems that will increase the therapeutic efficacy of actinonin. In the present study, human serum albumin actinonin nanoparticles were prepared using a desolvation technique and folic acid was conjugated to lysine residues of albumin for effective delivery to the lung. The lung adenocarcinoma model was established 24 weeks after intraperitoneal administration of urethane and chemotherapeutic efficacy of free as well as nanoencapsulated actinonin was evaluated. This study demonstrated anti-proliferative potential of folic acid conjugated human serum albumin nanoparticles encapsulating actinonin. The intraperitoneally administered nanoformulation exhibited sustain release profile of actinonin with longer half-life and mean retention time. The reduced dose frequency resulted in therapeutic efficacy comparable to free drug in vivo in terms of 100% survival and reduced tumor burden along with downregulation of epidermal growth factor receptor, folate receptor α and peptide deformylase expression in lung adenocarcinoma mice model. Therefore, actinonin encapsulated albumin nanoparticles-based therapy holds great potential as an alternative strategy to improve its anti-cancerous activity against lung adenocarcinoma.

Comparative Evaluation of Two Antibiotic Pastes for Root Canal Disinfection.

Aim and objective: To compare the efficacy of triple antibiotic paste (TAP) and double antibiotic paste (DAP) for root canal disinfection during revascularization of immature pulpless teeth. Materials and methods: A sample of 20 immature teeth in subjects aged 8-13 years was selected for a revascularization procedure. The teeth were randomly distributed in two groups corresponding to the medicament received-TAP group (n = 10) and DAP group (n = 10). Microbial samples were collected using dry paper points at the beginning of treatment and thereafter every 3 weeks till sterile reading was obtained. Microbial samples were then sent to the microbiological lab for identification. Results: A variety of opportunistic microbes were detected in samples obtained before placement of medicaments with no significant difference between the two medicament groups. The efficacy of TAP to eliminate microbes was lower in comparison to DAP after 3 weeks of placement of medicaments, whereas it was found to be more efficacious in comparison to DAP after 6 weeks and no significant difference (p> 0.05) was observed between the two groups. Conclusion: Immature permanent teeth with pulp necrosis can achieve complete root development with regenerative endodontic technique. The use of antibiotic pastes including TAP and DAP can help achieve a successful outcome with thorough decontamination of the root canal. How to cite this article: Goswami M, Baveja CP, Bhushan U, et al. Comparative Evaluation of Two Antibiotic Pastes for Root Canal Disinfection. Int J Clin Pediatr Dent 2022;15(S-1):S12-S17.