Prefoldins are novel regulators of the unfolded protein response in artemisinin resistant P. falciparum malaria.

Emerging Artemisinin (ART) resistance in Plasmodium falciparum (Pf) poses challenges for discovery of novel drugs to tackle ART resistant parasites. Concentrated efforts towards ART resistance mechanism indicated a strong molecular link of ART resistance with up-regulated expression of unfolded protein response pathways involving Prefoldins (PFDs). However, a complete characterization of PFDs as molecular players taking part in ART resistance mechanism, and discovery of small molecule inhibitors to block this process have not been identified to date. Here, we functionally characterized all Pf Prefoldin subunits (PFD1-6), and established a causative role played by PFDs in ART resistance by demonstrating their expression in intra-erythrocytic parasites along with their interactions with Kelch13 protein through immunoprecipitation coupled MS/MS analysis. Systematic biophysical interaction analysis between all subunits of PFDs revealed their potential to form a complex. The role of PFDs in ART resistance was confirmed in orthologous yeast PFD6 mutants, where PfPFD6 expression in yeast mutants reverted phenotype to ART resistance. We identified an FDA approved drug 'Biperiden' that restricts the formation of Prefoldin complex and inhibits its interaction with its key parasite protein substrates, MSP-1 and α-tubulin-I. Moreover, Biperiden treatment inhibits the parasite growth in ART sensitive Pf3D7 and resistant Pf3D7k13R539T strains. Ring survival assays that are clinically relevant to analyse ART resistance in Pf3D7k13R539T parasites demonstrate the potency of BPD to inhibit growth of survivor parasites. Overall, our study provides first evidence towards the role of PfPFDs in ART resistance mechanism, and opens new avenues for the management of resistant parasite.

Antiplatelet Effect of Daphnetin Is Regulated by cPLA2-Mediated Thromboxane A2 Generation in Mice.

Coumarin derivatives have been recognized for their antithrombotic, anti-inflammatory, and antioxidant properties, and daphnetin is one of the natural coumarin derivatives isolated from Daphne Koreana Nakai. Although the pharmacological value of daphnetin is well documented in diverse biological activities, its antithrombotic effect has not been studied to date. Here, we characterized the role and underlying mechanism of daphnetin in the regulation of platelet activation using murine platelets. In order to check the effect of daphnetin on platelet function, we first measured the effect of daphnetin on platelet aggregation and secretion. Collagen-induced platelet aggregation and dense granule secretion were partially inhibited by daphnetin. Interestingly, 2-MeSADP-induced secondary waves of aggregation and secretion were completely inhibited by daphnetin. It is known that 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by the positive feedback effect of thromboxane A2 (TxA2) generation, suggesting the important role of daphnetin on TxA2 generation in platelets. Consistently, daphnetin did not affect the 2-MeSADP-induced platelet aggregation in aspirinated platelets where the contribution of TxA2 generation was blocked. Additionally, platelet aggregation and secretion induced by a low concentration of thrombin, which is affected by the positive feedback effect of TxA2 generation, were partially inhibited in the presence of daphnetin. Importantly, 2-MeSADP- and thrombin-induced TxA2 generation was significantly inhibited in the presence of daphnetin, confirming the role of daphnetin on TxA2 generation. Finally, daphnetin significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets. Only cPLA2 phosphorylation, not ERK phosphorylation, was significantly inhibited by daphnetin in aspirinated platelets. In conclusion, daphnetin plays a critical role in platelet function by inhibiting TxA2 generation through the regulation of cPLA2 phosphorylation.

An Insight into Recent Advances on Platelet Function in Health and Disease.

Platelets play a variety of roles in vascular biology and are best recognized as primary hemostasis and thrombosis mediators. Platelets have a large number of receptors and secretory molecules that are required for platelet functionality. Upon activation, platelets release multiple substances that have the ability to influence both physiological and pathophysiological processes including inflammation, tissue regeneration and repair, cancer progression, and spreading. The involvement of platelets in the progression and seriousness of a variety of disorders other than thrombosis is still being discovered, especially in the areas of inflammation and the immunological response. This review represents an integrated summary of recent advances on the function of platelets in pathophysiology that connects hemostasis, inflammation, and immunological response in health and disease and suggests that antiplatelet treatment might be used for more than only thrombosis.

The GRKs Reactome: Role in Cell Biology and Pathology.

G protein-coupled receptor kinases (GRKs) are protein kinases that function in concert with arrestins in the regulation of a diverse class of G protein-coupled receptors (GPCRs) signaling. Although GRKs and arrestins are key participants in the regulation of GPCR cascades, the complex regulatory mechanisms of GRK expression, its alternation, and their function are not thoroughly understood. Several studies together with the work from our lab in recent years have revealed the critical role of these kinases in various physiological and pathophysiological processes, including cardiovascular biology, inflammation and immunity, neurodegeneration, thrombosis, and hemostasis. A comprehensive understanding of the mechanisms underlying functional interactions with multiple receptor proteins and how these interactions take part in the development of various pathobiological processes may give rise to novel diagnostic and therapeutic strategies. In this review, we summarize the current research linking the role of GRKs to various aspects of cell biology, pathology, and therapeutics, with a particular focus on thrombosis and hemostasis.

Pyk2 downstream of G12/13 pathways regulates platelet shape change through RhoA/p160ROCK.

Rho/Rho-kinase downstream of G12/13 plays an important role in the regulation of calcium-independent platelet shape change. We have previously shown that proline-rich tyrosine kinase 2 (Pyk2) is activated downstream of G12/13 pathways. In this study, we evaluated the role of Pyk2 in G12/13-induced platelet shape change. We used low concentrations of YFLLRNP, a heptapeptide binding to protease-activated receptor 1 (PAR1), or PAR4-activating peptide AYPGKF in the presence of Gαq inhibitor YM254890 to selectively stimulate G12/13 pathways. We found that G12/13-induced platelet shape change was completely inhibited in the presence of Pyk2 inhibitors AG17 and TAT-Pyk2-CT, suggesting an important role of Pyk2 in platelet shape change. In addition, AYPGKF-induced shape change in Gq -/- platelets was completely inhibited in the presence of AG17 or RhoA/p160ROCK inhibitor Y27632, confirming the role of Pyk2 in RhoA-dependent shape change. Furthermore, AYPGKF-induced platelet aggregation and dense granule secretion were inhibited by blocking Pyk2 or RhoA. Finally, G12/13-induced myosin phosphatase target subunit 1 (MYPT1) phosphorylation was inhibited by AG17, confirming that Pyk2 regulates RhoA/p160ROCK activation in platelets. These results demonstrate that Pyk2 downstream of G12/13 pathways regulates platelet shape change as well as platelet aggregation and dense granule secretion through the regulation of RhoA/p160ROCK.

Characterization of Integrin αIIbß3-Mediated Outside-in Signaling by Protein Kinase Cδ in Platelets.

Engagement of integrin αIIbß3 promotes platelet-platelet interaction and stimulates outside-in signaling that amplifies activation. Protein kinase Cδ (PKCδ) is known to play an important role in platelet activation, but its role in outside-in signaling has not been established. In the present study, we determined the role of PKCδ and its signaling pathways in integrin αIIbß3-mediated outside-in signaling in platelets using PKCδ-deficient platelets. Platelet spreading to immobilized fibrinogen resulted in PKCδ phosphorylation, suggesting that αIIbß3 activation caused PKCδ activation. αIIbß3-mediated phosphorylation of Akt was significantly inhibited in PKCδ -/- platelets, indicating a role of PKCδ in outside-in signaling. αIIbß3-mediated PKCδ phosphorylation was inhibited by proline-rich tyrosine kinase 2 (Pyk2) selective inhibitor, suggesting that Pyk2 contributes to the regulation of PKCδ phosphorylation in outside-in signaling. Additionally, Src-family kinase inhibitor PP2 inhibited integrin-mediated Pyk2 and PKCδ phosphorylation. Lastly, platelet spreading was inhibited in PKCδ -/- platelets compared to the wild-type (WT) platelets, and clot retraction from PKCδ -/- platelets was markedly delayed, indicating that PKCδ is involved in the regulation of αIIbß3-dependent interactivities with cytoskeleton elements. Together, these results provide evidence that PKCδ plays an important role in outside-in signaling, which is regulated by Pyk2 in platelets.

Role of GRK6 in the Regulation of Platelet Activation through Selective G Protein-Coupled Receptor (GPCR) Desensitization.

Platelet G protein-coupled receptors (GPCRs) regulate platelet function by mediating the response to various agonists, including adenosine diphosphate (ADP), thromboxane A2, and thrombin. Although GPCR kinases (GRKs) are considered to have the crucial roles in most GPCR functions, little is known regarding the regulation of GPCR signaling and mechanisms of GPCR desensitization by GRKs in platelets. In this study, we investigated the functional role of GRK6 and the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used GRK6 knockout mice to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense- and -granule secretion, and fibrinogen receptor activation induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6-/- platelets compared to the wild-type (WT) platelets. However, collagen-related peptide (CRP)-induced platelet aggregation and secretion were not affected in GRK6-/- platelets. Interestingly, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate Gq-coupled 5HT2A and Gz-coupled 2A adrenergic receptors, respectively, was not affected in GRK6-/- platelets, suggesting that GRK6 was involved in specific GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP and AYPGKF was restored in GRK6-/- platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, indicating that GRK6 contributed to P2Y1, P2Y12, and PAR4 receptor desensitization. Furthermore, 2-MeSADP-induced Akt phosphorylation and AYPGKF-induced Akt, extracellular signal-related kinase (ERK), and protein kinase Cδ (PKC) phosphorylation were significantly potentiated in GRK6-/- platelets. Finally, GRK6-/- mice exhibited an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery and shorter tail bleeding times, indicating that GRK6-/- mice were more susceptible to thrombosis and hemostasis. We conclude that GRK6 plays an important role in regulating platelet functional responses and thrombus formation through selective GPCR desensitization.

Nordic walking training and nutritional supplementation in pre-frail older Indians: an open-labelled experimental pre-test and post-test pilot study to develop intervention model.

BACKGROUND: Identifying and treating people in a pre-frail state may be an effective way to prevent or delay frailty and preserve their functional capacity. This study aimed to assess the efficacy of, and compliance with, a 12 week individualized nutritional supplementation (INS) and Nordic walking (NW) program in pre-frail older Indians. The primary measure is physical performance, as indicated by Fried's Frailty scale. Other measures include: cognition, as indicated by the Hindi Mental Status Examination; mood, by the Geriatric Depression Scale; and nutritional status, by the Mini Nutritional Assessment. METHODS: This is an open-labeled experimental pre-test and post-test study, which took place from October 2012 to December 2014. The study was approved by Institute Ethics committee (IEC/NP-350/2012/RP-26/2012) at the All India Institute of Medical Sciences (AIIMS), New Delhi. Participants were sixty-six pre-frail elderly, who were randomly allocated into three subgroups, namely: A (NW only), B (INS only), and C (NW and INS). One-way ANOVA was used to statistically assess differences in baseline characteristics for quantitative variables, with the Chi-Square/Fischer exact test utilized for qualitative variables. Paired t-tests were used to assess pre and post intervention difference within the group for quantitative variables, with McNemar's Chi-Square test used for qualitative variables. Kruskal Wallis test was used to assess significant intervention effects among the groups. A p-value < 0.05 was considered as statistically significant. RESULTS: There was significant effect of intervention in gait speed in group A (p = 0.001) and C (p = 0.002), but not in group B (p = 0.926). While there was no significant change in grip strength in Group A (p = 0.488) and B (p = 0.852), a statistically significant increase was observed in group C (p = 0.013). Mood significantly improved in group B (p = 0.025) and C (p = 0.021). No significant difference was noted in cognitive status across groups. Following the interventions, a total of 18.18% of pre-frail participants were classified as non-frail. CONCLUSIONS: Combining NW and INS provides a simple, pragmatic intervention with efficacy in the management of functionally vulnerable older adults, and allows their maintained independence. Future studies should replicate this readily applicable intervention in a larger cohort with a longer follow-up period. TRIAL REGISTRATION: Clinical Trial Registry-India CTRI/2016/05/006937 [Registered on: 16/05/2016]; Trial was Registered Retrospectively.

Toxicity profile of repeated doses of PEG-asparaginase incorporated into a pediatric-type regimen for adult acute lymphoblastic leukemia.

Despite having been long regarded as too toxic for adult patients, pediatric-like regimens containing L-asparaginase have resulted in improved outcomes for adults with acute lymphoblastic leukemia (ALL). To characterize the spectrum of toxicity of repeated doses of polyethylene glycolated-asparaginase (PEG-asp) in adults, we reviewed all doses (2000 IU/m(2) ) administered as part of a pediatric-inspired regimen in adult ALL at our center. Subjects aged 18-60 yr with ALL (n = 152, 69.1% male) contributed 522 dose cycles to the study. Hepatotoxicity was the most common adverse event: grades 3-4 transaminitis and hyperbilirubinemia occurred in 53.9% and 23.7% of subjects, respectively. Hepatotoxicity was reversible; no cases of fulminate hepatic failure were observed. Other toxicities affecting at least 5% of subjects were grades 3-4 triglyceridemia in 50.9%, hypofibrinogenemia (<100 mg/dL) in 47.9%, clinical pancreatitis in 12.6%, venous thromboembolism in 11.2%, allergic reaction in 7.2%, and any grade bleeding in 5.3%. PEG-asp was always discontinued after grades 3-4 pancreatitis or allergic reaction. Otherwise, toxicities did not preclude administration of additional cycles of the drug. Our results suggest that repeated PEG-asp dosing is safe in adults aged 18-60 yr, even after occurrence of a drug-related toxicity.

Mechanism of action of novel synthetic dodecapeptides against Candida albicans.

BACKGROUND: Three de novo designed low molecular weight cationic peptides (IJ2, IJ3 and IJ4) containing an unnatural amino acid α,ß-didehydrophenylalanine (∆Phe) exhibited potent antifungal activity against fluconazole (FLC) sensitive and resistant clinical isolates of Candida albicans as well as non-albicans and other yeast and filamentous pathogenic fungi. In the present study, their synthesis, susceptibility of different fungi and the mechanism of anti-candidal action have been elucidated. METHODS: The antimicrobial peptides (AMPs) were synthesized by solid-phase method and checked for antifungal activity against different yeasts and fungi by broth microdilution method. Anti-candidal mode of action of the peptides was investigated through detecting membrane permeabilization by confocal microscopy, Reactive Oxygen Species (ROS) generation by fluorometry, apoptosis and necrosis by flow cytometry and cell wall damage using Scanning and Transmission Electron Microscopy. RESULTS AND CONCLUSIONS: The MIC of the peptides against C. albicans and other yeast and filamentous fungal pathogens ranged between 3.91 and 250µM. All three peptides exhibited effect on multiple targets in C. albicans including disruption of cell wall structures, compromised cell membrane permeability leading to their enhanced entry into the cells, accumulation of ROS and induction of apoptosis. The peptides also showed synergistic effect when used in combination with fluconazole (FLC) and caspofungin (CAS) against C. albicans. GENERAL SIGNIFICANCE: The study suggests that the AMPs alone or in combination with conventional antifungals hold promise for the control of fungal pathogens, and need to be further explored for treatment of fungal infections.

The Perspectives of Platelet Proteomics in Health and Disease.

Cardiovascular thromboembolic diseases and cancer continue to be a leading cause of death and disability worldwide. Therefore, it is crucial to advance their diagnoses and treatment in the context of individualized medicine. However, the disease specificity of the currently available markers is limited. Based on analyses of a subset of peptides and matching proteins in disease vs. healthy platelets, scientists have recently shown that focused platelet proteomics enables the quantification of disease-specific biomarkers in humans. In this review, we explored the potential of accurate platelet proteomic research, which is required to identify novel diagnostic and pharmaceutical targets by comprehending the proteome variety of healthy individuals and patients for personalized and precision medicine.

Histopathological and Immunohistochemical Characterization of Sebaceous Adenoma and Epithelioma in Dogs.

Sebaceous gland tumors are neoplasms originating from the sebaceous gland and are the third most common type of skin tumor, accounting for 21-35% of all cutaneous neoplasms in dogs. According to their histopathological characteristics, sebaceous gland tumors can be classified into adenoma as a benign tumor and epithelioma as a malignant tumor. Sebaceous epithelioma is distinguished from sebaceous adenoma by containing 90% or more reserve cells. However, this simple numerical criterion is insufficient to histologically distinguish between epitheliomas and adenomas. In addition, sebaceoma in humans, a similar tumor to sebaceous epithelioma, is a term used for tumors with more than 50% of reserve cells, unlike epithelioma. Therefore, we aimed to compare and characterize the histological and immunohistochemical profiles of comprehensive sebaceous adenoma, epithelioma, and borderline tumors that have more than 50% but less than 90% of reserve cells. A total of 14 canine sebaceous tumors were diagnosed as seven adenomas, four borderline tumors, and three epitheliomas. Histologically, the sebaceous adenomas showed nodules consisting of mature sebocytes surrounded by monolayer basaloid cells. In contrast, the portion of the reserve cells was increased, the portion of lipidized cells was decreased, and the majority of lipidized cells were found to be immature in sebaceous epithelioma. In the sebaceous adenomas, necrosis was not observed and mitotic figures were rarely seen. However, necrosis and mitotic figures were highly frequent in both borderline tumor and sebaceous epithelioma. Immunohistochemistry revealed that borderline tumor and sebaceous epithelioma showed significantly higher expression against Ki-67 than sebaceous adenoma. We conclude that it is more accurate to employ the cut-off value of 50% reserve cells in humans rather than the current 90% reserve cells for classifying sebaceous gland tumors in dogs, thereby providing new insight into the characterization of the sebaceous gland tumors.

Reappraisal of the morphological and morphometric study of the psoas minor muscle with clinical and developmental insights: cadaveric study.

BACKGROUND: The Psoas Minor (PMi) is the most unstable muscle of the psoas group of the posterior abdominal muscle. This muscle has a fusiform shape and consists of a short fusiform belly continuing distally as a long tendon inserted on the pecten pubis and the iliopectineal arch. The present study was conducted to obtain more detailed information about the muscle and to expand knowledge about its morphology and morphometry. MATERIALS AND METHODS: The posterior abdominal wall of 30 adult cadavers was dissected. Anatomical variabilities in origin, insertion, length, width, and muscle-to-cone ratio were measured when PMi was found. The data collected was interpreted descriptively. RESULTS: PMi was found in 12 cases, ten bilateral and two unilateral. The origin was constant in all cases and, except for three cases, extended into the iliac fascia and the iliopubic eminence. Morphometric analysis revealed that the average length of the proximal muscle belly and distal tendons was 4.52 ± 1.35 cm and 13.05 ± 0.90 cm, respectively. The mean width of the muscle belly was 1.71 ± 0.17 cm, and that of the tendon was 0.47 ± 0.10 cm. On average, the muscle belly occupied the proximal 33.71 ± 6.15% of the total musculotendinous unit. CONCLUSIONS: Findings confirm the inconsistency of PMi in the study population. Morphological variations became more evident as the tendon approached the insertion level. The muscle's distal attachment to the iliac fascia may partially control the position, mechanical stability of the underlying iliopsoas and this circumstantial function may be clinically related to iliopsoas inflammation and pathology. However, further studies recommended to determine biomechanical validity and clinical applicability of this vestigial muscle in human.

Hand hygiene with interventions: an observational study from a tertiary care institute over 2 years.

Background: Appropriate adherence to hand hygiene (HH) practices by health care workers (HCWs) reduces the transmission of pathogens and subsequently the incidence of hospital acquired infections (HAIs), in health care settings. Strict monitoring and auditing of this simple and cost-effective intervention is very important, as it significantly contributes in reducing the HAIs. Material and methods: A retrospective observational study, evaluating the HH audits from June 2021 till May 2023 in a tertiary health care facility in North India. HH audits were conducted in the ICUs and wards daily, by the trained infection control nurses (ICNs), using direct observation method based on World health organization (WHO) hand hygiene observational forms. HH total adherence (HHTAR), partial adherence (HHPAR) and complete adherence rate (HHCAR) were analyzed in Microsoft Excel sheet. HHTAR rates were compared among different profession, moments and the month wise trend was also observed over the period. Results: A total of 24,740 HH opportunities were observed. The compliance rate for HHCAR, HHPAR and HHTAR were 20.3%, 41.5% and 61.4% respectively. Overall better compliance was reported from the ICUs, profession-specific compliance was highest among nurses (62.8%) and doctors (61.5%). Significant increase in adherence rate was appreciated post intervention 46.1% to 67.3%, (p value < 0.01). Conclusions: Continuous monitoring and reinforcement with timely feedback for intervention and regular auditing is a necessity to improve and maintain the appropriate HH practices among the HCWs. Low- and middle-income countries need to focus more on this simple and promising measure to combat the increasing HAI rates.

Shedding Light on the Cell Biology of Platelet-Derived Extracellular Vesicles and Their Biomedical Applications.

EVs are membranous subcellular structures originating from various cells, including platelets which consist of biomolecules that can modify the target cell's pathophysiological functions including inflammation, cell communication, coagulation, and metastasis. EVs, which are known to allow the transmission of a wide range of molecules between cells, are gaining popularity in the fields of subcellular treatment, regenerative medicine, and drug delivery. PEVs are the most abundant EVs in circulation, being produced by platelet activation, and are considered to have a significant role in coagulation. PEV cargo is extremely diverse, containing lipids, proteins, nucleic acids, and organelles depending on the condition that induced their release and can regulate a wide range of biological activities. PEVs, unlike platelets, can overcome tissue barriers, allowing platelet-derived contents to be transferred to target cells and organs that platelets cannot reach. Their isolation, characterization, and therapeutic efficacy, on the other hand, are poorly understood. This review summarizes the technical elements of PEV isolation and characterization methods as well as the pathophysiological role of PEVs, including therapeutic potential and translational possibility in diverse disciplines.

Role of Prednisolone in Platelet Activation by Inhibiting TxA2 Generation through the Regulation of cPLA2 Phosphorylation.

Glucocorticoids have been commonly used in the treatment of inflammation and immune-mediated diseases in human beings and small animals such as cats and dogs. However, excessive use can lead to Cushing's syndrome along with several thrombotic and cardiovascular diseases. Although it is well-known that glucocorticoids exert a significant effect on coagulation, the effect of cortisol on platelet function is much less clear. Thus, we aimed to study the effects of prednisolone, one of the commonly used glucocorticoids, on the regulation of platelet function using murine platelets. We first evaluated the concentration-dependent effect of prednisolone on 2-MeSADP-induced platelet function and found that the 2-MeSADP-induced secondary wave of aggregation and dense granule secretion were completely inhibited from 500 nM prednisolone. Since 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by TxA2 generation, this result suggested a role of prednisolone in platelet TxA2 generation. Consistently, prednisolone did not affect the 2-MeSADP-induced aggregation in aspirinated platelets, where the secondary wave of aggregation and secretion were blocked by eliminating the contribution of TxA2 generation by aspirin. In addition, thrombin-induced platelet aggregation and secretion were inhibited in the presence of prednisolone by inhibiting the positive-feedback effect of TxA2 generation on platelet function. Furthermore, prednisolone completely inhibited 2-MeSADP-induced TxA2 generation, confirming the role of prednisolone in TxA2 generation. Finally, Western blot analysis revealed that prednisolone significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets, while only cPLA2 phosphorylation, but not ERK phosphorylation, was significantly inhibited by prednisolone in aspirinated platelets. In conclusion, prednisolone affects platelet function by the inhibition of TxA2 generation through the regulation of cPLA2 phosphorylation, thereby shedding light on its clinical characterization and treatment efficacy in dogs with hypercortisolism in the future.

Hearing Loss among Children Visiting Department of Otolaryngology and HNS of a Tertiary Care Centre.

Introduction: Hearing loss is defined as the partial or total reduction in auditory acuity. Hearing loss can cause detrimental effects on speech, language, developmental, educational, and cognitive outcomes in children. This study aimed to find out the prevalence of hearing loss among children visiting the Department of Otolaryngology and HNS of a tertiary care centre. Methods: A descriptive cross-sectional study was conducted among children visiting the Department of Otolaryngology and HNS between 1 January 2022 and 31 December 2022 after obtaining ethical approval. All the patients who underwent pure tone evaluation were included in the study. A convenience sampling technique was used. The point estimate was calculated at a 95% Confidence Interval. Results: Among 3051 children, 328 (10.75%) (9.65-11.85, 95% Confidence Interval) had hearing loss. Among children with hearing loss, 170 (51.83%) of children were female. The mean age of children with hearing loss was 13.31±3.39 years. The mean pure tone average among 452 ears with hearing loss was 44.60±17.71 dB. The commonest degree of hearing loss was mild hearing loss 266 (58.85%), and the commonest type was conductive hearing loss 310 (68.58%). Among children with hearing loss, 124 (37.80%) had bilateral hearing loss. Conclusions: The prevalence of hearing loss among children visiting the Department of Otolaryngology and HNS was found to be lower than similar studies done in similar settings. Keywords: audiology; audiometry; hearing loss; outpatients; prevalence.

Chronic Otitis Media among Patients Visiting Community-Based Static Outreach Clinics.

Introduction: Chronic otitis media is a chronic inflammation of the middle ear and mastoid cavity, with recurrent ear discharges or otorrhoea through a tympanic perforation for the past 3 months. It is a common cause of hearing impairment, disability, and poor scholastic performance and can lead to fatal intracranial infections and acute mastoiditis. This study aimed to find out the prevalence of chronic otitis media among patients visiting community-based static outreach clinics. Methods: A descriptive cross-sectional study was conducted among patients visiting the community-based static outreach clinics from 1 January 2017 to 31 December 2019. The ethical approval was taken from the Ethical Review Board. The diagnosis of chronic otitis media was done using otoscopy. The records of patients coming to outreach clinics visiting for ear, nose and throat care were reviewed using a pre-designed study proforma. A systematic random sampling method was used. The point estimate was calculated at a 95% Confidence Interval. Results: Among 385 patients, 37 (9.61%) (6.67-12.55, 95% Confidence Interval) had chronic otitis media. The mean age of patients with chronic otitis media was 27.59±13.24 years, with 28 (75.67%) patients aged between 18-60 years. Among them, 30 (81.08%) had unilateral and 34 (91.89%) had a mucosal type. Conclusions: The prevalence of chronic otitis media was lower than in other studies done in similar settings. Keywords: community health services; otitis media; patients; prevalence.

Fatal Clostridium sporogenes Soft Tissue Polymicrobial Infections in Two Immunocompetent Cases: Case Report.

BACKGROUND: Clostridium sporogenes is reported rarely in literature. Reports from the skin and soft tissue infections are even less, more so in immunocompetent patients. CASE PRESENTATION: Two skin and soft tissue infections with C. sporogenes in immunocompetent patients have been presented in this study. One of the cases was following an electrical burn wound, and the other was following a bedsore. Both patients expired despite antibacterial treatment and debridement. DISCUSSION AND CONCLUSION: C. sporogenes had usually been reported after trauma particularly after penetrating and deep wound infection. More attention should be given to these patients so that the infection can be treated and diagnosed early in suspected anaerobic infections like Clostridium species.

Artemisinin resistance in P. falciparum: probing the interacting partners of Kelch13 protein in parasite.

OBJECTIVES: Artemisinin (ART) resistance in Plasmodium is threatening the artemisinin combination therapies-the first line of defence against malaria. ART resistance has been established to be mediated by the Plasmodium Kelch13 (PfK13) protein. For the crucial role of PfK13 in multiple pathways of the Plasmodium life cycle and ART resistance, it is imperative that we investigate its interacting partners. METHODS: We recombinantly expressed PfK13-p (Bric a brac/Poxvirus and zinc finger and propeller domains), generating anti-PfK13-p antibodies to perform co-immunoprecipitation assays and probed PfK13 interacting partners. Surface plasmon resonance and pull-down assays were performed to establish physical interactions of representative proteins with PfK13-p. RESULTS: The co-immunoprecipitation assays identified 17 proteins with distinct functions in the parasite life cycle- protein folding, cellular metabolism, and protein binding and invasion. In addition to the overlap with previously identified proteins, our study identified 10 unique proteins. Fructose-biphosphate aldolase and heat shock protein 70 demonstrated strong biophysical interaction with PfK13-p, with KD values of 6.6 µM and 7.6 µM, respectively. Additionally, Plasmodium merozoite surface protein 1 formed a complex with PfK13-p, which is evident from the pull-down assay. CONCLUSION: This study adds to our knowledge of the PfK13 protein in mediating ART resistance by identifying new PfK13 interacting partners. Three representative proteins-fructose-biphosphate aldolase, heat shock protein 70, and merozoite surface protein 1-demonstrated clear evidence of biophysical interactions with PfK13-p. However, elucidation of the functional relevance of these physical interactions are crucial in context of PfK13 role in ART resistance.