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The development of sensitive and specific diagnostic tools for hepatitis B virus (HBV) and hepatitis C virus (HCV) remains crucial for effective disease management and control. In this study, we utilized CRISPR-Cas12 and CRISPR-Cas13 systems for the detection of HBV (DNA virus) and HCV (RNA virus), respectively. We designed and tested multiple guide RNAs (gRNAs) targeting both viruses, confirming successful cleavage of target sequences through gel electrophoresis and a fluorescent reporter assay. Using optimized gRNAs, we developed a lateral flow assay (LFA) for sensitive detection of HBV and HCV, demonstrating a concentration-dependent signal increase. Importantly, no cross-reactivity was observed with other viral targets. To further enhance sensitivity, we employed a dual-enzyme approach, combining Cas12 and Cas13 in a single reaction, which significantly improved detection limits for both viruses. Finally, we developed a dual antigen detection LFA strip capable of simultaneously detecting both HBV and HCV in a single sample. This approach holds promise for point-of-care (POC) diagnostics where the specific viral infection is unknown. This study addresses the current limitations in CRISPR-Cas based diagnostics, namely, the need for ultrasensitive detection methods and the ability to detect multiple antigens using a single test strip. Our findings demonstrate the feasibility of using CRISPR-Cas systems for highly sensitive and specific detection of HBV and HCV, paving the way for potential POC application., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Syeda Najidah Shahni et al.)

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need for simple, low-cost, and scalable diagnostics that can be widely deployed for rapid testing. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have emerged as a promising technology, but its implementation in clinical laboratories has been limited by the requirement of a separate amplification step prior to CRISPR-associated (Cas) enzyme-based detection. This article reports the discovery of two novel Cas12 enzymes (SLK9 and SLK5-2) that exhibit enzymatic activity at 60°C, which, when combined with loop-mediated isothermal amplification (LAMP), enable a real-time, single-step nucleic acid detection method [real-time SHERLOCK (real-time SLK)]. Real-time SLK was demonstrated to provide accurate results comparable to those from real-time quantitative RT-PCR in clinical samples, with 100% positive and 100% negative percent agreement. The method is further demonstrated to be compatible with direct testing (real-time SLK Direct) of samples from anterior nasal swabs, without the need for standard nucleic acid extraction. Lastly, SLK9 was combined with either Alicyclobacillus acidoterrestris AacCas12b or with SLK5-2 to generate a real-time, multiplexed CRISPR-based diagnostic assay for the simultaneous detection of SARS-CoV-2 and a human-based control in a single reaction, with sensitivity down to 5 copies/μL and a time to result of under 30 minutes., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Mesenchymal stem cell (MSC) transplantation alleviates metabolic defects in diseased recipient cells by intercellular mitochondrial transport (IMT). However, the effect of host metabolic conditions on IMT and thereby on the therapeutic efficacy of MSCs has largely remained unexplored. Here we found impaired mitophagy, and reduced IMT in MSCs derived from high-fat diet (HFD)-induced obese mouse (MSC-Ob). MSC-Ob failed to sequester their damaged mitochondria into LC3-dependent autophagosomes due to decrease in mitochondrial cardiolipin content, which we propose as a putative mitophagy receptor for LC3 in MSCs. Functionally, MSC-Ob exhibited diminished potential to rescue mitochondrial dysfunction and cell death in stress-induced airway epithelial cells. Pharmacological modulation of MSCs enhanced cardiolipin-dependent mitophagy and restored their IMT ability to airway epithelial cells. Therapeutically, these modulated MSCs attenuated features of allergic airway inflammation (AAI) in two independent mouse models by restoring healthy IMT. However, unmodulated MSC-Ob failed to do so. Notably, in human (h)MSCs, induced metabolic stress associated impaired cardiolipin-dependent mitophagy was restored upon pharmacological modulation. In summary, we have provided the first comprehensive molecular understanding of impaired mitophagy in obese-derived MSCs and highlight the importance of pharmacological modulation of these cells for therapeutic intervention. A MSCs obtained from (HFD)-induced obese mice (MSC-Ob) show underlying mitochondrial dysfunction with a concomitant decrease in cardiolipin content. These changes prevent LC3-cardiolipin interaction, thereby reducing dysfunctional mitochondria sequestration into LC3-autophagosomes and thus impaired mitophagy. The impaired mitophagy is associated with reduced intercellular mitochondrial transport (IMT) via tunneling nanotubes (TNTs) between MSC-Ob and epithelial cells in co-culture or in vivo. B Pyrroloquinoline quinone (PQQ) modulation in MSC-Ob restores mitochondrial health, cardiolipin content, and thereby sequestration of depolarized mitochondria into the autophagosomes to alleviate impaired mitophagy. Concomitantly, MSC-Ob shows restoration of mitochondrial health upon PQQ treatment (MSC-ObPQQ). During co-culture with epithelial cells or transplantation in vivo into the mice lungs, MSC-ObPQQ restores IMT and prevents epithelial cell death. C Upon transplantation in two independent allergic airway inflammatory mouse models, MSC-Ob failed to rescue the airway inflammation, hyperactivity, metabolic changes in epithelial cells. D PQQ modulated MSCs restored these metabolic defects and restored lung physiology and airway remodeling parameters., (© 2023. The Author(s).)

Circulating cell-free mitochondrial DNA (cf-mtDNA) has been found in the plasma of severely ill COVID-19 patients and is now known as a strong predictor of mortality. However, the underlying mechanism of mtDNA release is unexplored. Here, we show a novel mechanism of SARS-CoV-2-mediated pro-inflammatory/pro-apoptotic mtDNA release and a rational therapeutic stem cell-based approach to mitigate these effects. We systematically screened the effects of 29 SARS-CoV-2 proteins on mitochondrial damage and cell death and found that NSP4 and ORF9b caused extensive mitochondrial structural changes, outer membrane macropore formation, and the release of inner membrane vesicles loaded with mtDNA. The macropore-forming ability of NSP4 was mediated through its interaction with BCL2 antagonist/killer (BAK), whereas ORF9b was found to inhibit the anti-apoptotic member of the BCL2 family protein myeloid cell leukemia-1 (MCL1) and induce inner membrane vesicle formation containing mtDNA. Knockdown of BAK and/or overexpression of MCL1 significantly reversed SARS-CoV-2-mediated mitochondrial damage. Therapeutically, we engineered human mesenchymal stem cells (MSCs) with a simultaneous knockdown of BAK and overexpression of MCL1 (MSCshBAK+MCL1) and named these cells IMAT-MSCs (intercellular mitochondrial transfer-assisted therapeutic MSCs). Upon co-culture with SARS-CoV-2-infected or NSP4/ORF9b-transduced airway epithelial cells, IMAT-MSCs displayed functional intercellular mitochondrial transfer (IMT) via tunneling nanotubes (TNTs). The mitochondrial donation by IMAT-MSCs attenuated the pro-inflammatory and pro-apoptotic mtDNA release from co-cultured epithelial cells. Our findings thus provide a new mechanistic basis for SARS-CoV-2-induced cell death and a novel therapeutic approach to engineering MSCs for the treatment of COVID-19.

A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT ( C as13 A ssisted S aliva-based & S martphone I ntegrated T esting), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct<35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19., Competing Interests: Author RB is employed by 360 Diagnostic and Health Services, author DJG is employed by Noodle Analytics Pvt Ltd. and author GDJ is employed by Valerian Chem Pvt. Ltd. A patent for the saliva-based and RNA extraction free detection of SARS-CoV2 using CRISPR Diagnostics has been filed. TA, MJ, JI, GJ, and RK are the inventors and applicants in the patent. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Azmi, Faizan, Kumar, Raj Yadav, Chaudhary, Kumar Singh, Butola, Ganotra, Datt Joshi, Deep Jhingan, Iqbal, Joshi and Ahmad.)

Introduction: Bowling is an immensely popular, but scarcely researched sport associated with overuse injuries in its participants. The purpose of this study was to investigate and report on the incidence of common upper extremity complaints in elite bowling athletes., Materials and Methods: All Malaysian national level bowlers (n=39) were evaluated via questionnaire on their upper limb symptoms. A focused, relevant clinical examination was performed on each subject to exclude de Quervain's tenosynovitis, tennis and golfer's elbow, carpal tunnel syndrome and trigger finger. The athletes were then allowed to resume bowling for two hours before completing another symptom-related questionnaire., Results: Pain was the predominantly observed symptom, with a predilection for the wrist, ring and middle fingers, and thumb. De Quervain's tenosynovitis was found in 53.8% (n=21) of the subjects, with 52.4% and 42.9% of them experiencing pain during and after training, respectively. Other repetitive injury-related disorders were also considerably more common than in their non-playing limb and the general population., Conclusion: The incidence of de Quervain's tenosynovitis was exceptionally high in this population. Further studies on sports kinematics are needed to prevent long term morbidities in these athletes., (© 2021 Malaysian Orthopaedic Association MOA. All Rights Reserved.)

Introduction: Brain tumour (BT) is a tremendous burden on patients, families as well as the surrounding communities, especially the healthcare services. It can be classified into either a benign slow growing tumour (non-cancerous) and malignant tumour (cancerous). The purpose of this study was to determine the incidence and pattern of brain tumour admitted to the Neurosurgery Department in Hospital Sultanah Nurzahirah (HSNZ), Terengganu, Malaysia., Methods: This is a retrospective study of incidence and pattern of BT admitted to the Neurosurgery Department in HSNZ. Data was collected from the yearly census of BT registered from 2013 to 2018., Results: A total number of 386 new cases of primary BT were registered. The number of cases of BT was found to be lowest among children (0 to 10 years old) with only 4.4% but at peak among elderly aged between 51 to 60 years old (26.2%). As for gender, males constituted about 44.5% (n=172) whereas females accounted for 55.5% (n= 214) of the cases. In total, meningioma was found to have the highest incidence (27.2%) followed by metastases brain tumour (18.1%) and glioma (17.4%)., Conclusions: This study has shown that the incidence of BT was led by meningioma which had a high prevalence among the elderly population, followed by metastasis BT and gliomas.

There is increasing popularity among researchers, scholars, and policymakers concerning the efficacy of prayer as a coping strategy for job stress. This study examines the moderating effects of prayer and age on the relationship between job stress and nurses' well-being in UMMC. Three hundred (300) Muslim nursing staff working at the University of Malaya Medical Centre were sampled. Data were collected via questionnaires. The findings of this study show that the effect of job stress on well-being is significant for nurses and that prayer of nurses contributed to alleviating job stress and enhancing well-being. This study is limited to nurses in one public hospital in a developing country. Thus, it would be more interesting if the study could be extended to other public and private institutions in a Muslim country, and a comparison could be done between other religions as well.

Hepatitis B virus (HBV) infection is one of the major causes of liver diseases, which can lead to hepatocellular carcinoma. The role of HBV envelope proteins is crucial in viral morphogenesis, infection, and propagation. Thus, blocking the pleiotropic functions of these proteins especially the PreS1 and PreS2 domains of the large surface protein (LHBs) is a promising strategy for designing efficient antivirals against HBV infection. Unfortunately, the structure of the LHBs protein has not been elucidated yet, and it seems that any structure-based drug discovery is critically dependent on this. To find effective inhibitors of LHBs, we have modeled and validated its three-dimensional structure and subsequently performed a virtual high-throughput screening against the ZINC database using RASPD and ParDOCK tools. We have identified four compounds, ZINC11882026, ZINC19741044, ZINC00653293, and ZINC15000762, showing appreciable binding affinity with the LHBs protein. The drug likeness was further validated using ADME screening and toxicity analysis. Interestingly, three of the four compounds showed the formation of hydrogen bonds with amino acid residues lying in the capsid binding region of the PreS1 domain of LHBs, suggesting the possibility of inhibiting the viral assembly and maturation process. The identification of potential lead molecules will help to discover more potent inhibitors with significant antiviral activities., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Mahboubeh Mehmankhah et al.)

Lipid exchange among citrem nanoparticles and an ethanol micellar solution containing soy phosphatidylcholine was investigated in situ by coupling small angle X-ray scattering with a microfluidic device. The produced soy phosphatidylcholine/citrem nanoparticles have great potential in the development of hemocompatible nanocarriers for drug delivery.

This study has been undertaken to investigate the isolation and identification of EPEC strains from paediatric diarrhoeal patients. The study was carried out in the department of Microbiology, Sir Salimullah Medical College & Mitford Hospital, Bangladesh during January to December, 2011. Total 272 samples were studied. Samples from patients with diarrhoea were collected from two tertiary care hospital. At first Esch. coli were isolated from these specimens using standard microbiological techniques and then EPEC strains were identified on the basis of presence of bundle forming pilus (bfpA) gene. Virulence of EPEC strains were determined by detection of bfpA gene and observing localized adherence (LA) in HeLa cell adherence assay. Esch. coli was isolated and identified from all the 272 samples from patients using standard microbiological techniques. Among 272 samples 20(7.35%) isolates were identified as EPEC on the basis of presence of bfpA gene detected by polymerase chain reaction. EPEC strains were identified from those 240 samples, from which Esch. coli had been isolated only. Out of twenty EPEC strains, 17 strains (85%) showed a pattern of localized adherence in Hela cell adherence assay. EPEC strains can be identified by bfpA gene detection and by adherence assays. HeLa cell adherence assay is the most specific method for detection of EPEC strains which has bfpA gene, responsible for localized adherence (LA) in HeLa cell line. Rapid and reliable detection of EPEC is required for successful microbiological surveillance and for treatment of EPEC mediated diarrhoeal disease.

Shigella flexneri is the most common cause of bacterial dysentery in low-income countries. Despite this, S. flexneri remains largely unexplored from a genomic standpoint and is still described using a vocabulary based on serotyping reactions developed over half-a-century ago. Here we combine whole genome sequencing with geographical and temporal data to examine the natural history of the species. Our analysis subdivides S. flexneri into seven phylogenetic groups (PGs); each containing two-or-more serotypes and characterised by distinct virulence gene complement and geographic range. Within the S. flexneri PGs we identify geographically restricted sub-lineages that appear to have persistently colonised regions for many decades to over 100 years. Although we found abundant evidence of antimicrobial resistance (AMR) determinant acquisition, our dataset shows no evidence of subsequent intercontinental spread of antimicrobial resistant strains. The pattern of colonisation and AMR gene acquisition suggest that S. flexneri has a distinct life-cycle involving local persistence.

The National Stroke Registry (NSR) was established in 2009 under National Neurology Registry (NNeuR) . The main objectives of NSR were to describe the demographic and disease pattern of stroke patients in Malaysia, to examine the risk factors and evaluate the specified treatment and outcomes. This prospective observational study was carried out from August 2009 until December 2010 using a standardized case report form which involved two participating hospital, namely Hospital Sultanah Nur Zahirah, Kuala Terengganu and Hospital Seberang Jaya, Pulau Pinang. There were 1018 patients registered. Ischemic stroke accounted for the majority of cases (73.3%). The most common risk factor was hypertension (75.5 %), followed by diabetes mellitus, previous stroke or Transient Ischemic Attack (TIA), hyperlipidemia and active smoker: 45.6%, 25.1%, 22.4%, and 19.4%, respectively. Overall, our stroke management, based on nine stroke key performance indicators (KPI) still needs to be improved. There was a total of 121 mortality cases with the main contributing factor was massive cerebral bleed (21.6%). In conclusion, the findings highlight the important of primary and secondary stroke management. Further and continuous observation with more site date provider (SDP) involvement is needed to get a more comprehensive data on stroke in Malaysia.

Endosomal sorting complexes required for transport (ESCRTs) promote the invagination of vesicles into the lumen of endosomes, the budding of enveloped viruses, and the separation of cells during cytokinesis. These processes share a topologically similar membrane scission event facilitated by ESCRT-III assembly at the cytosolic surface of the membrane. The Snf7 subunit of ESCRT-III in yeast binds directly to an auxiliary protein, Bro1. Like ESCRT-III, Bro1 is required for the formation of intralumenal vesicles at endosomes, but its role in membrane scission is unknown. We show that overexpression of Bro1 or its N-terminal Bro1 domain that binds Snf7 enhances the stability of ESCRT-III by inhibiting Vps4-mediated disassembly in vivo and in vitro. This stabilization effect correlates with a reduced frequency in the detachment of intralumenal vesicles as observed by electron tomography, implicating Bro1 as a regulator of ESCRT-III disassembly and membrane scission activity.

A subset of proteins that transit the endosomal system are directed into the intralumenal vesicles of multivesicular bodies (MVBs). MVB formation is critical for a variety of cellular functions including receptor down-regulation, viral budding, antigen presentation, and the generation of lysosome-related organelles. Entry of transmembrane proteins into the intralumenal vesicles of a MVB is a highly regulated process that is positively modulated by covalent modification of cargoes with ubiquitin. To identify additional MVB sorting signals, we examined the previously described ubiquitination-independent MVB cargo Sna3. Although Sna3 ubiquitination is not essential, Sna3 MVB sorting is positively modulated by its ubiquitination. Examination of MVB sorting determinants within a form of Sna3 lacking all lysine residues identified two critical regions: an amino-terminal tyrosine-containing region and a carboxyl-terminal PPAY motif. This PPAY motif interacts with the WW domains of the ubiquitin ligase Rsp5, and mutations in either the WW or, surprisingly, the HECT domains of Rsp5 negatively impacted MVB targeting of lysine-minus Sna3. These data indicate that Rsp5 function is required for MVB targeting of Sna3 in a capacity beyond cargo ubiquitination. These results uncover a series of determinants impacting Sna3 MVB sorting, including unexpected roles for Rsp5.

In eukaryotes, the multivesicular body (MVB) sorting pathway plays an essential role in regulating cell surface protein composition, thereby impacting numerous cellular functions. Vps4, an ATPase associated with a variety of cellular activities, is required late in the MVB sorting reaction to dissociate the endosomal sorting complex required for transport (ESCRT), a requisite for proper function of this pathway. However, regulation of Vps4 function is not understood. We characterize Vta1 as a positive regulator of Vps4 both in vivo and in vitro. Vta1 promotes proper assembly of Vps4 and stimulates its ATPase activity through the conserved Vta1/SBP1/LIP5 region present in Vta1 homologues across evolution, including human SBP1 and Arabidopsis thaliana LIP5. These results suggest an evolutionarily conserved mechanism through which the disassembly of the ESCRT proteins, and thereby MVB sorting, is regulated by the Vta1/SBP1/LIP5 proteins.

Retroviral RNA encapsidation is mediated by specific interactions between viral Gag proteins and cis-acting packaging sequences in genomic RNA. Feline immunodeficiency virus (FIV) RNA encapsidation determinants have been shown to be discrete and noncontinuous, comprising one region at the 5' end of the genomic mRNA (R-U5) and another region that mapped within the proximal 311 nt of gag. To aid comparative understanding of lentiviral encapsidation and refinement of FIV vector systems, we used RNase protection assays (RPAs) of cellular and virion RNAs to investigate in detail the gag element. mRNAs of subgenomic vectors as well as of full-length molecular clones were optimally packaged into viral particles and resulted in high-titer FIV vectors when they contained only the proximal 230 nucleotides (nt) of gag. Further 3' truncations of gag sequences progressively diminished encapsidation and transduction. Deletion of the initial ninety 5' nt of the gag gene abolished mRNA packaging, demonstrating that this segment is indispensable for encapsidation. Focusing further on this proximal sequence, we found that a deletion of only 13 nt at the 5' end of gag impaired encapsidation of subgenomic vector and proviral RNAs.

Forty-one patients with 42 elbow dislocations, of which 13 had acute simple dislocations, 21 had fracture-dislocations and 8 had neglected elbow dislocations, were evaluated with regard to limitation of motion, pain, instability and residual neurovascular deficit. All patients with acute simple dislocation were treated with closed reduction, but the duration of immobilization before commencement of active motion varied. Open-reduction was indicated for failed closed reduction and neglected-dislocations. Internal fixation as a primary procedure was only performed for displaced fracture-dislocation. Excision of radial head or tricepsplasty were performed if the reduction was impossible in neglected elbow dislocation. Despite a good results in acute simple dislocation, 33.07% had flexion contracture of between 5 to 30 degrees. In fracture dislocation, satisfactory results were seen in whom the elbow was immobilized for three weeks or less. Neglected-dislocation is associated with poor functional outcome, 37.5% were good and 62.5% were poor. Prolonged immobilization after injury was strongly associated with an unsatisfactory result. The results indicate that early properly supervised active motion is a key factor in the rehabilitation of elbow dislocation.