Out-of-Pocket Expenditure for Selected Surgeries in the Cardiology Department for Ayushman Bharat Pradhan Mantri Jan Arogya Yojana (AB-PMJAY), Private Health Insurance, and Uninsured Patients in a Tertiary Care Teaching Hospital in Karnataka, India.

INTRODUCTION: Cardiovascular diseases are a major public health issue and the leading cause of mortality globally. The global economic burden of out-of-pocket expenditure (OOPE) for cardiovascular surgeries and procedures is substantial, with average costs being significantly higher than other treatments. This imposes a heavy economic burden. Government insurance schemes like Ayushman Bharat Pradhan Mantri Jan Arogya Yojana (AB-PMJAY) aim to enhance affordability and access to cardiac care. METHODOLOGY: This retrospective study analyzed OOPE incurred for top cardiac surgeries under AB-PMJAY, private insurance, and uninsured patients at a tertiary care teaching hospital in Karnataka. Data of 1021 patients undergoing common cardiac procedures from January to July 2023 were analyzed using descriptive statistics (mean, median) and the Shapiro-Wilk test for normality. The study aims to evaluate financial risk protection offered by AB-PMJAY compared to private plans and inform effective policy-making in reducing the OOPE burden for surgeries in India. RESULTS: The study analyzed OOPE across 1021 patients undergoing any of four surgeries at a tertiary care teaching hospital in Karnataka. AB-PMJAY patients incurred zero OOPE across all surgeries. Uninsured patients faced the highest median OOPE, ranging from ₹1,15,292 (1390.57 USD) to ₹1,72,490 (2080.45 USD) depending on surgery type. Despite the presence of private insurance, the median out-of-pocket expenditure ranged from ₹1,689 (20.38 USD) to ₹68,788 (829.67 USD). Significant variations in OOPE were observed within different payment groups. Private insurance in comparison with AB-PMJAY had limitations like co-payments, deductibles, and limited coverage resulting in higher OOPE for patients. DISCUSSION: The results illustrate the efficacy of AB-PMJAY in reducing the financial burden and improving the affordability of cardiac procedures compared to private insurance. This emphasizes the significance of programmmes funded by the government in reducing the OOPE burden and ensuring equitable healthcare access. The comprehensive and particular estimates of OOPE for different surgical procedures, categorized by payment methods provide valuable information to guide the development of policies that aim to reduce OOPE and progress toward universal health coverage in India.

Bendless is essential for PINK1-Park mediated Mitofusin degradation under mitochondrial stress caused by loss of LRPPRC.

Cells under mitochondrial stress often co-opt mechanisms to maintain energy homeostasis, mitochondrial quality control and cell survival. A mechanistic understanding of such responses is crucial for further insight into mitochondrial biology and diseases. Through an unbiased genetic screen in Drosophila, we identify that mutations in lrpprc2, a homolog of the human LRPPRC gene that is linked to the French-Canadian Leigh syndrome, result in PINK1-Park activation. While the PINK1-Park pathway is well known to induce mitophagy, we show that PINK1-Park regulates mitochondrial dynamics by inducing the degradation of the mitochondrial fusion protein Mitofusin/Marf in lrpprc2 mutants. In our genetic screen, we also discover that Bendless, a K63-linked E2 conjugase, is a regulator of Marf, as loss of bendless results in increased Marf levels. We show that Bendless is required for PINK1 stability, and subsequently for PINK1-Park mediated Marf degradation under physiological conditions, and in response to mitochondrial stress as seen in lrpprc2. Additionally, we show that loss of bendless in lrpprc2 mutant eyes results in photoreceptor degeneration, indicating a neuroprotective role for Bendless-PINK1-Park mediated Marf degradation. Based on our observations, we propose that certain forms of mitochondrial stress activate Bendless-PINK1-Park to limit mitochondrial fusion, which is a cell-protective response.

Comparison of the incidence of postoperative pain in single sitting root canal treatment after using two reciprocating systems and two continuous rotary systems: An in vivo study.

Aim: The aim of the present study was to compare the incidence of postoperative pain in single sitting root canal treatment after using two reciprocating and two continuous rotating file systems. Methodology: One hundred and four permanent mandibular canine and first premolar teeth were selected for the study and were divided into two groups 1 and 2 comprising 52 teeth each. Group 1 was treated using reciprocating file systems and was subdivided into two subgroups A and B using WaveOne Gold (WOG) and Reciproc Blue (RB), respectively. Group 2 was treated using continuous rotating file systems and was divided into two subgroups A and B using One Curve (OC) and Vortex Blue (VB), respectively. Treatments were performed in a single sitting. After the visit, the patients were given a prescription for ibuprofen 400 mg to be taken every 6 h if they experience pain. Participants were asked to rate the intensity of the postoperative pain on a visual analog scale according to four classes (no pain, mild pain, moderate pain, and severe pain) after 24 h, 48 h, 72 h, and 7 days. Patients were also asked to record the number of prescribed analgesic medication tablets taken at these time points. Results: There was a statistically significant difference (P = 0.00) among the reciprocating file systems and continuous file systems regarding the incidence of postoperative pain after 24, 48, and 72 h which signifies that pain incidence and duration is less with respect to reciprocation instrumentation as compared to continuous instrumentation. Conclusion: The use of Reciproc instrumentation system (WOG and RB) showed significantly less intensity and duration of posttreatment pain compared to the single-file rotary system (OC and VB) in patients with symptomatic irreversible pulpitis with apical periodontitis.

Efficacy of articaine versus mepivacaine administered as different supplementary local anesthetic techniques after a failed inferior alveolar nerve block with lidocaine in patients with irreversible pulpitis: An in vivo study.

Aim: The aim of the present study was to assess the efficacy of articaine versus mepivacaine administered as different supplementary local anesthetic techniques after a failed inferior alveolar nerve block (IANB) with lidocaine in patients with irreversible pulpitis. Materials and Methods: A total of 120 patients were included in the study. Patients were given IANB with 2 ml of 2% lidocaine hydrochloride with 1:80,000 epinephrine. Patients who showed subjective symptoms of IANB but did not secure pulpal anesthesia were randomly allocated to one of the following four groups by random sampling for supplementary local anesthesia - Group 1: buccal infiltration (BI) with 4% articaine with 1:100,000 epinephrine, Group 2: four-site intraligamentary (IL) injection with 4% articaine with 1:100,000 epinephrine, Group 3: BI with 2% mepivacaine with 1:100,000 epinephrine, and Group 4: four-site IL injection with 2% mepivacaine with 1:100,000 epinephrine. Results: Group 1 - BI with articaine provided anesthesia success in 27 cases (90%, n = 30). Group 2 - IL injection with articaine provided anesthesia success in 20 cases (66.67%, n = 30). Group 3 - BI with mepivacaine provided anesthesia success in 21 cases (70%, n = 30). Group 4 - IL injection with mepivacaine provided anesthesia success in 15 cases (50%, n = 30). A significant difference between the four groups was found. Conclusion: In patients with a mandibular first molar exhibiting symptomatic irreversible pulpitis, block injection with lidocaine along with supplemental BI with articaine allowed more pain-free treatments. Articaine showed better results when compared with mepivacaine.

Self-reported Adherence to Medication among Patients with Type II Diabetes Mellitus attending a Tertiary Care Hospital of Delhi.

INTRODUCTION: Diabetes mellitus (DM) is becoming a potential epidemic in India with more than 62 million diagnosed diabetics and an increase of nearly 2 million per year. Poor adherence to medication regimens increases the probability of adverse outcomes in type 2 diabetes patients. Therefore, improving medication adherence is a growing priority to control this epidemic. Hence, this study was conducted to determine the level of adherence to medication in Type II diabetic patients and to study the various factors affecting adherence to medication and the relationship between the severity of diabetes with the adherence categories. METHODS: A cross-sectional study was conducted at medicine outpatient department (OPD) of a tertiary care hospital, New Delhi among 200 type 2 diabetic patients for duration of 2 months using a predesigned and pretested semi-structured interview schedule and diabetes medication adherence was assessed by Morisky's medication adherence scale questionnaire. RESULTS: Out of 200 participants, 32.5% were found to have high adherence while 34.5% and 33% had moderate and low adherence. Factors found to be associated with adherence were age, educational status, longer duration of disease and presence of glucometer. Almost four-fifths of the patients (79.5%) had poor plasma glucose control. CONCLUSION: There is a need to focus on improving adherence among type 2 diabetes patients and strengthening health care systems for regular supply of medicines and provide health education to the patients and their families emphasising the need of adherence to medications.

Pathways to neurodegeneration: lessons learnt from unbiased genetic screens in Drosophila.

Neurodegenerative diseases are a complex set of disorders that are known to be caused by environmental as well as genetic factors. In the recent past, mutations in a large number of genes have been identified that are linked to several neurodegenerative diseases. The pathogenic mechanisms in most of these disorders are unknown. Recently, studies of genes that are linked to neurodegeneration in Drosophila, the fruit flies, have contributed significantly to our understanding of mechanisms of neuroprotection and degeneration. In this review, we focus on forward genetic screens in Drosophila that helped in identification of novel genes and pathogenic mechanisms linked to neurodegeneration. We also discuss identification of four novel pathways that contribute to neurodegeneration upon mitochondrial dysfunction.

A conserved human DJ1-subfamily motif (DJSM) is critical for anti-oxidative and deglycase activities of Plasmodium falciparum DJ1.

Plasmodium falciparum DJ1 (PfDJ1) belongs to the DJ-1/ThiJ/PfpI superfamily whose members are present in all the kingdoms of life and exhibit diverse cellular functions and biochemical activities. The common feature of the superfamily is the class I glutamine amidotransferase domain with a conserved redox-active cysteine residue, which mediates various activities of the superfamily members, including anti-oxidative activity in PfDJ1 and human DJ1 (hDJ1). As the superfamily members represent diverse functional classes, to investigate if there is any sequence feature unique to hDJ1-like proteins, sequences of the representative proteins of different functional classes were compared and analysed. A novel motif unique to PfDJ1 and several other hDJ1-like proteins, with the consensus sequence of TSXGPX5FXLX5L, was identified that we designated as the hDJ1-subfamily motif (DJSM). Several mutations that have been associated with Parkinson's disease are also present in DJSM, suggesting its functional importance in hDJ1-like proteins. Mutations of the conserved residues of DJSM of PfDJ1 did not significantly affect overall secondary structure, but caused both a significant loss (S151A and P154A) and gain (L168A) of anti-oxidative activity. We also report that PfDJ1 has deglycase activity, which was significantly decreased in its mutants of the catalytic cysteine (C106A) and DJSM (S151A and P154A). Episomal expression of the catalytic cysteine (C106A) or DJSM (P154A) mutant decreased growth rates of parasites as compared to that of wild type parasites or parasites expressing wild type PfDJ1. S151 appears to properly position the nucleophilic elbow containing C106 and P154 forms a hydrogen bond with C106, which could be a reason for the loss of activities of PfDJ1 upon their mutations. Taken together, DJSM delineates PfDJ1 and other hDJ1-subfamily proteins from the remaining superfamily, and is critical for anti-oxidative and deglycase activities of PfDJ1.

Deletion of Pofut1 in Mouse Skeletal Myofibers Induces Muscle Aging-Related Phenotypes in cis and in trans.

Sarcopenia, the loss of muscle mass and strength during normal aging, involves coordinate changes in skeletal myofibers and the cells that contact them, including satellite cells and motor neurons. Here we show that the protein O-fucosyltransferase 1 gene (Pofut1), which encodes a glycosyltransferase required for NotchR-mediated cell-cell signaling, has reduced expression in aging skeletal muscle. Moreover, premature postnatal deletion of Pofut1 in skeletal myofibers can induce aging-related phenotypes in cis within skeletal myofibers and in trans within satellite cells and within motor neurons via the neuromuscular junction. Changed phenotypes include reduced skeletal muscle size and strength, decreased myofiber size, increased slow fiber (type 1) density, increased muscle degeneration and regeneration in aged muscles, decreased satellite cell self-renewal and regenerative potential, and increased neuromuscular fragmentation and occasional denervation. Pofut1 deletion in skeletal myofibers reduced NotchR signaling in young adult muscles, but this effect was lost with age. Increasing muscle NotchR signaling also reduced muscle size. Gene expression studies point to regulation of cell cycle genes, muscle myosins, NotchR and Wnt pathway genes, and connective tissue growth factor by Pofut1 in skeletal muscle, with additional effects on α dystroglycan glycosylation.

Technetium uptake predicts remission and relapse in Grave's disease patients on antithyroid drugs for at least 1 year in South Indian subjects.

CONTEXT: Most of the information on remission related factors in Grave's disease are derived from Western literature. It is likely that there may be additional prognostic factors and differences in the postdrug treatment course of Grave's disease in India. AIM: To study factors which predict remission/relapse in Grave's disease patients from South India. Also to establish if technetium (Tc) uptake has a role in predicting remission. SUBJECTS AND METHODS: Records of 174 patients with clinical, biochemical, and scintigraphic criteria consistent with Grave's disease, seen in our Institution between January 2006 and 2014 were analyzed. Patient factors, drug-related factors, Tc-99m uptake and other clinical factors were compared between the remission and nonremission groups. STATISTICAL ANALYSIS USED: Mann-Whitney U-test and Chi-square tests were used when appropriate to compare the groups. RESULTS: Fifty-seven (32.7%) patients attained remission after at least 1 year of thionamide therapy. Of these, 11 (19.2%) patients relapsed within 1 year. Age, gender, goiter, and presence of extrathyroidal manifestations were not associated with remission. Higher values of Tc uptake were positively associated with remission (P- 0.02). Time to achievement of normal thyroid function and composite dose: Time scores were significantly associated with remission (P - 0.05 and P - 0.01, respectively). Patients with lower FT4 at presentation had a higher chance of remission (P - 0.01). The relapse rates were lower than previously reported in the literature. A higher Tc uptake was found to be significantly associated with relapse also (P - 0.009). CONCLUSION: The prognostic factors associated with remission in Graves's disease in this South Indian study are not the same as that reported in Western literature. Tc scintigraphy may have an additional role in identifying people who are likely to undergo remission and thus predict the outcome of Grave's disease.

Deletion of Galgt2 (B4Galnt2) reduces muscle growth in response to acute injury and increases muscle inflammation and pathology in dystrophin-deficient mice.

Transgenic overexpression of Galgt2 (official name B4Galnt2) in skeletal muscle stimulates the glycosylation of α dystroglycan (αDG) and the up-regulation of laminin α2 and dystrophin surrogates known to inhibit muscle pathology in mouse models of congenital muscular dystrophy 1A and Duchenne muscular dystrophy. Skeletal muscle Galgt2 gene expression is also normally increased in the mdx mouse model of Duchenne muscular dystrophy compared with the wild-type mice. To assess whether this increased endogenous Galgt2 expression could affect disease, we quantified muscular dystrophy measures in mdx mice deleted for Galgt2 (Galgt2(-/-)mdx). Galgt2(-/-) mdx mice had increased heart and skeletal muscle pathology and inflammation, and also worsened cardiac function, relative to age-matched mdx mice. Deletion of Galgt2 in wild-type mice also slowed skeletal muscle growth in response to acute muscle injury. In each instance where Galgt2 expression was elevated (developing muscle, regenerating muscle, and dystrophic muscle), Galgt2-dependent glycosylation of αDG was also increased. Overexpression of Galgt2 failed to inhibit skeletal muscle pathology in dystroglycan-deficient muscles, in contrast to previous studies in dystrophin-deficient mdx muscles. This study demonstrates that Galgt2 gene expression and glycosylation of αDG are dynamically regulated in muscle and that endogenous Galgt2 gene expression can ameliorate the extent of muscle pathology, inflammation, and dysfunction in mdx mice.

Independent amino acid residues in the S2 pocket of falcipain-3 determine its specificity for P2 residues in substrates.

Falcipain-3 (FP3) is an essential and drug target cysteine protease of the most lethal human malaria parasite Plasmodium falciparum. FP3 and its majority of homologs in malaria parasites prefer Leu at the P2 position in substrates and inhibitors, whereas its major host homolog cathepsin L prefers Phe. However, FP3 is much less active on peptide substrates and has negligible activity against a P2 Arg-containing substrate (Z-RR-AMC) compared to its paralog falcipain-2A (FP2A). To identify the specificity determinants, the S2/3 pocket residues of FP3 were substituted with the corresponding residues in FP2 or cathepsin L, and the wild type and mutant proteases were assessed for hydrolysis of peptide and protein substrates. Our results indicate that the S2 pocket residues I94 and P181 of FP3 are chiefly responsible for its P2 Leu preference and negligible activity for Z-RR-AMC, respectively. E243 in FP3 and the corresponding residue D234 in FP2 have a key role in Z-RR-AMC hydrolysing activity, possibly through stabilization of side chain interactions, as their substitution with Ala abolished the activity. Several FP3 mutants, which retained P2 Leu preference and showed similar or more activity than wild type FP3 on peptide substrates, degraded haemoglobin less efficiently than wild type FP3, suggesting that multiple residues contribute to haemoglobinase activity. Furthermore, P181 and E243 appear to contribute to the optimum activity of FP3 in the food vacuole milieu (≈pH 5.5). The identification of residues determining specificity of FP3 could aid in developing specific inhibitors of FP3 and its homologs in malaria parasites.

Spinal subdural hematoma with cauda equina syndrome: A complication of combined spinal epidural anesthesia.

Combined spinal-epidural anesthesia (CSE) is considered safe in lower limb surgeries. We report a case of sudden neurological deterioration in a stable postoperative patient who was given CSE for total knee replacement and low molecular weight heparin in postoperative period. On the 4(th) postoperative day, she developed sudden onset weakness in left lower limb along with bladder incontinence. Magnetic resonance imaging spine revealed a subdural hematoma at L2-L3 level. Immediate laminectomy along with cord decompression was done and patient recovered well except for a persistent foot drop on left side.

A role for Galgt1 in skeletal muscle regeneration.

BACKGROUND: Cell surface glycans are known to play vital roles in muscle membrane stability and muscle disease, but to date, roles for glycans in muscle regeneration have been less well understood. Here, we describe a role for complex gangliosides synthesized by the Galgt1 gene in muscle regeneration. METHODS: Cardiotoxin-injected wild type (WT) and Galgt1 (-/-) muscles, and mdx and Galgt1 (-/-) mdx muscles, were used to study regeneration in response to acute and chronic injury, respectively. Muscle tissue was analyzed at various time points for morphometric measurements and for gene expression changes in satellite cell and muscle differentiation markers by quantitative real-time polymerase chain reaction (qRT-PCR). Primary cell cultures were used to measure growth rate and myotube formation and to identify Galgt1 expression changes after cardiotoxin by fluorescence-activated cell sorting (FACS). Primary cell culture and tissue sections were also used to quantify satellite cell apoptosis. RESULTS: A query of a microarray data set of cardiotoxin-induced mouse muscle gene expression changes identified Galgt1 as the most upregulated glycosylation gene immediately after muscle injury. This was validated by qRT-PCR as a 23-fold upregulation in Galgt1 expression 1 day after cardiotoxin administration and a 16-fold upregulation in 6-week-old mdx muscles. These changes correlated with increased expression of Galgt1 protein and GM1 ganglioside in mononuclear muscle cells. In the absence of Galgt1, cardiotoxin-induced injury led to significantly reduced myofiber diameters after 14 and 28 days of regeneration. Myofiber diameters were also significantly reduced in Galgt1-deficient mdx mice compared to age-matched mdx controls, and this was coupled with a significant increase in the loss of muscle tissue. Cardiotoxin-injected Galgt1 (-/-) muscles showed reduced gene expression of the satellite cell marker Pax7 and increased expression of myoblast markers MyoD, Myf5, and Myogenin after injury along with a tenfold increase in apoptosis of Pax7-positive muscle cells. Cultured primary Galgt1 (-/-) muscle cells showed a normal growth rate but demonstrated premature fusion into myofibers, resulting in an overall impairment of myofiber formation coupled with a threefold increase in muscle cell apoptosis. CONCLUSIONS: These experiments demonstrate a role for Galgt1 in skeletal muscle regeneration and suggest that complex gangliosides made by Galgt1 modulate the survival and differentiation of satellite cells.

Genetic ablation of plasmoDJ1, a multi-activity enzyme, attenuates parasite virulence and reduces oocyst production.

Malaria parasites must respond to stresses and environmental signals to perpetuate efficiently during their multistage development in diverse environments. To gain insights into the parasite's stress response mechanisms, we investigated a conserved Plasmodium protein, which we have named plasmoDJ1 on the basis of the presence of a putative cysteine protease motif of the DJ-1/PfpI superfamily, for its activities, potential to respond to stresses and role in parasite development. PlasmoDJ1 is expressed in all intraerythrocytic stages and ookinetes. Its expression was increased 7-9-fold upon heat shock and oxidative stress due to H2O2 and artemisinin; its expression in a stress-sensitive Escherichia coli mutant conferred tolerance against oxidative stress, indicating that plasmoDJ1 has the potential to sense and/or protect from stresses. Recombinant plasmoDJ1 efficiently neutralized H2O2, facilitated renaturation of denatured citrate synthase and showed protease activity, indicating that plasmoDJ1 is a multi-activity protein. Mutation of the catalytic cysteine residue, but not other residues, reduced H2O2-neutralization activity by ~90% and significantly decreased chaperone and protease activities, indicating that these activities are intrinsic to plasmoDJ1. The plasmoDJ1 gene knockout in Plasmodium berghei ANKA attenuated virulence and reduced oocyst production, suggesting a major role for plasmoDJ1 in parasite development, which probably depends on its multiple activities.

Genetic ablation of plasmoDJ1, a multi-activity enzyme, attenuates parasite virulence and reduces oocyst production.

Malaria parasites must respond to stresses and environmental signals to perpetuate efficiently during their multistage development in diverse environments. To gain insights into the parasite's stress response mechanisms, we investigated a conserved Plasmodium protein, which we have named plasmoDJ1 on the basis of the presence of a putative cysteine protease motif of the DJ-1/PfpI superfamily, for its activities, potential to respond to stresses and role in parasite development. PlasmoDJ1 is expressed in all intraerythrocytic stages and ookinetes. Its expression was increased 7-9-fold upon heat shock and oxidative stress due to H2O2 and artemisinin; its expression in a stress-sensitive Escherichia coli mutant conferred tolerance against oxidative stress, indicating that plasmoDJ1 has the potential to sense and/or protect from stresses. Recombinant plasmoDJ1 efficiently neutralized H2O2, facilitated renaturation of denatured citrate synthase and showed protease activity, indicating that plasmoDJ1 is a multi-activity protein. Mutation of the catalytic cysteine residue, but not other residues, reduced H2O2-neutralization activity by ~90% and significantly decreased chaperone and protease activities, indicating that these activities are intrinsic to plasmoDJ1. The plasmoDJ1 gene knockout in Plasmodium berghei ANKA attenuated virulence and reduced oocyst production, suggesting a major role for plasmoDJ1 in parasite development, which probably depends on its multiple activities.

Association of leisure-time physical activity with cardiorespiratory fitness in Indian men.

BACKGROUND: The relationship between leisure-time physical activity (LTPA) and cardiorespiratory fitness (CRF) is not clearly understood in Indian men. It is important to elucidate whether the duration or intensity of LTPA is responsible for increasing CRF. This will help in designing better physical activity intervention strategies for improving CRF in Indian men. METHODS: Healthy nondiabetic urban Indian men with no history of coronary heart disease (CHD) were selected (n = 603; aged 22-64 years) and their energy intake and physical activity was determined using a questionnaire. Body fat (percent) was determined by leg-to-leg bioelectrical impedance analysis while CRF was measured on multistage, continuous treadmill test using Bruce protocol. RESULTS: Intensity of physical activity (METs) emerged as the best independent predictor of CRF (ß = 0.217; P < .001). Using univariate General Linear Model, it was found that CRF is more a function of LTPA intensity than LTPA duration, since LTPA duration was not related to CRF when controlled for LTPA intensity. However, LTPA intensity remained significantly associated with CRF even after adjustment for LTPA duration. CONCLUSION: LTPA of preferably higher intensity should be incorporated in the lifestyle to improve CRF and prevent CHD in Indian men.

Blocking Plasmodium falciparum development via dual inhibition of hemoglobin degradation and the ubiquitin proteasome system by MG132.

Among key potential drug target proteolytic systems in the malaria parasite Plasmodium falciparum are falcipains, a family of hemoglobin-degrading cysteine proteases, and the ubiquitin proteasomal system (UPS), which has fundamental importance in cellular protein turnover. Inhibition of falcipains blocks parasite development, primarily due to inhibition of hemoglobin degradation that serves as a source of amino acids for parasite growth. Falcipains prefer P2 leucine in substrates and peptides, and their peptidyl inhibitors with leucine at the P2 position show potent antimalarial activity. The peptidyl inhibitor MG132 (Z-Leu-Leu-Leu-CHO) is a widely used proteasome inhibitor, which also has P2 leucine, and has also been shown to inhibit parasite development. However, the antimalarial targets of MG132 are unclear. We investigated whether MG132 blocks malaria parasite development by inhibiting hemoglobin degradation and/or by targeting the UPS. P. falciparum was cultured with inhibitors of the UPS (MG132, epoxomicin, and lactacystin) or falcipains (E64), and parasites were assessed for morphologies, extent of hemoglobin degradation, and accumulation of ubiquitinated proteins. MG132, like E64 and unlike epoxomicin or lactacystin, blocked parasite development, with enlargement of the food vacuole and accumulation of undegraded hemoglobin, indicating inhibition of hemoglobin degradation by MG132, most likely due to inhibition of hemoglobin-degrading falcipain cysteine proteases. Parasites cultured with epoxomicin or MG132 accumulated ubiquitinated proteins to a significantly greater extent than untreated or E64-treated parasites, indicating that MG132 inhibits the parasite UPS as well. Consistent with these findings, MG132 inhibited both cysteine protease and UPS activities present in soluble parasite extracts, and it strongly inhibited recombinant falcipains. MG132 was highly selective for inhibition of P. falciparum (IC50 0.0476 µM) compared to human peripheral blood mononuclear cells (IC50 10.8 µM). Thus, MG132 inhibits two distinct proteolytic systems in P. falciparum, and it may serve as a lead molecule for development of dual-target inhibitors of malaria parasites.

Distinct contributions of Galgt1 and Galgt2 to carbohydrate expression and function at the mouse neuromuscular junction.

At the mammalian neuromuscular junction (NMJ), the CT (cytotoxic T cell) carbohydrate antigen [GalNAcß1,4[Neu5Ac/Gcα2,3]Galß1,4GlcNAc-] is a unique synaptic cell surface carbohydrate present in both the presynaptic and postsynaptic membranes. Here we show that Galgt1, which synthesizes the ß1,4GalNAc linkage of the CT carbohydrate on gangliosides, is required for presynaptic expression of the CT carbohydrate at the NMJ, while Galgt2, which can synthesize the ß1,4GalNAc of the CT carbohydrate on glycoproteins, is required for postsynaptic expression. Proper postsynaptic localization of the CT carbohydrate also required muscle expression of dystroglycan, a known muscle substrate for Galgt2. Transgenic overexpression of Galgt2 in skeletal myofibers altered the expression of synaptic muscle proteins and altered neuromuscular topography, which was partially NCAM-dependent, while an increase in postsynaptic AChR-rich domains was observed in both neuron- and skeletal muscle-specific Galgt2 transgenic mice. By contrast, overexpression of Galgt1 in muscle did not allow for increased expression of CT carbohydrate on the sarcolemmal membrane and instead caused muscle pathology. Loss of Galgt2 increased intracellular accumulation of acetylcholine receptors and acetylcholinesterase within skeletal myofibers, suggesting an additional role for Galgt2 in neuromuscular stability. These experiments demonstrate that Galgt1 and Galgt2 contribute in distinct ways to the expression and function of synaptic ßGalNAc-containing carbohydrates at the NMJ.

Role of extracellular matrix proteins and their receptors in the development of the vertebrate neuromuscular junction.

The vertebrate neuromuscular junction (NMJ) remains the best-studied model for understanding the mechanisms involved in synaptogenesis, due to its relatively large size, its simplicity of patterning, and its unparalleled experimental accessibility. During neuromuscular development, each skeletal myofiber secretes and deposits around its extracellular surface an assemblage of extracellular matrix (ECM) proteins that ultimately form a basal lamina. This is also the case at the NMJ, where the motor nerve contributes additional factors. Before most of the current molecular components were known, it was clear that the synaptic ECM of adult skeletal muscles was unique in composition and contained factors sufficient to induce the differentiation of both pre- and postsynaptic membranes. Biochemical, genetic, and microscopy studies have confirmed that agrin, laminin (221, 421, and 521), collagen IV (α3-α6), collagen XIII, perlecan, and the ColQ-bound form of acetylcholinesterase are all synaptic ECM proteins with important roles in neuromuscular development. The roles of their many potential receptors and/or binding proteins have been more difficult to assess at the genetic level due to the complexity of membrane interactions with these large proteins, but roles for MuSK-LRP4 in agrin signaling and for integrins, dystroglycan, and voltage-gated calcium channels in laminin-dependent phenotypes have been identified. Synaptic ECM proteins and their receptors are involved in almost all aspects of synaptic development, including synaptic initiation, topography, ultrastructure, maturation, stability, and transmission.