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Umer Ejaz,1 Muhammad Afzal,1 Modasrah Mazhar,1 Muhammad Riaz,1 Naveed Ahmed,2 Waleed Y Rizg,3,4 Amerh Aiad Alahmadi,4 Moutaz Y Badr,5 Rayan Y Mushtaq,6 Chan Yean Yean2 1Department of Basic and Applied Chemistry, Faculty of Science and Technology, University of Central Punjab, Lahore, 54000, Pakistan; 2Department of Medical Microbiology and Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan, 16150, Malaysia; 3Center of Innovation in Personalized Medicine (CIPM), 3D Bioprinting Unit, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 4Department of Pharmaceutics, Faculty of Pharmacy, King Abdulaziz University, Jeddah, 21589, Saudi Arabia; 5Department of Pharmaceutical Sciences, College of Pharmacy, Umm Al-Qura University, Makkah, 24381, Saudi Arabia; 6Department of Pharmaceutics, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi ArabiaCorrespondence: Chan Yean Yean, Department of Medical Microbiology and Parasitology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan, 16150, Malaysia, Email yeancyn@yahoo.com Muhammad Afzal, Department of Basic and Applied Chemistry, Faculty of Science and Technology, University of Central Punjab, Lahore, 54000, Pakistan, Email dr.afzal@ucp.edu.pkIntroduction: Silver nanoparticles (AgNPs) have been found to exhibit unique properties which show their potential to be used in various therapies. Green synthesis of AgNPs has been progressively gaining acceptance due to its cost-effectiveness and energy-efficient nature.Objective: In the current study, aqueous extract of Thymus vulgaris (T. vulgaris) was used to synthesize the AgNPs using green synthesis techniques followed by checking the effectiveness and various biological activities of these AgNPs.Methods: At first, the plant samples were proceeded for extraction of aqueous extracts followed by chromatography studies to measure the phenolics and flavonoids. The synthesis and characterization of AgNPs were done using green synthesis techniques and were confirmed using Fourier transform infra-red (FT-IR) spectroscopy, UV–visible spectroscopy, scanning electron microscope (SEM), zeta potential, zeta sizer and X-Ray diffraction (XRD) analysis. After confirmation of synthesized AgNPs, various biological activities were checked.Results: The chromatography analysis detected nine compounds accounting for 100% of the total amount of plant constituents. The FT-IR, UV–vis spectra, SEM, zeta potential, zeta sizer and XRD analysis confirmed the synthesis of AgNPs and the variety of chemical components present on the surface of synthesized AgNPs in the plant extract. The antioxidant activity of AgNPs showed 92% inhibition at the concentration of at 1000 μg/mL. A greater inhibitory effect in anti-diabetic analysis was observed with synthesized AgNPs as compared to the standard AgNPs. The hemolytic activity was low, but despite low concentrations of hemolysis activity, AgNPs proved not to be toxic or biocompatible. The anti-inflammatory activity of AgNPs was observed by in-vitro and in-vivo approaches in range at various concentrations, while maximum inhibition occurs at 1000 μg (77.31%).Conclusion: Our data showed that the potential biological activities of the bioactive constituents of T. vulgaris can be enhanced through green synthesis of AgNPs from T. vulgaris aqueous extracts. In addition, the current study depicted that AgNPs have good potential to cure different ailments as biogenic nano-medicine.Keywords: nanotechnology, medicinal plants, antioxidant, anti-inflammatory, antidiabetic

Naveed Ahmed,1 Mohmed Isaqali Karobari,2– 4 Anam Yousaf,5 Roshan Noor Mohamed,6 Sohaib Arshad,7 Syed Nahid Basheer,8 Syed Wali Peeran,9 Tahir Yusuf Noorani,4 Ali A Assiry,10 Abdulaziz S Alharbi,11 Chan Yean Yean1 1Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, 16150, Malaysia; 2Department of Restorative Dentistry & Endodontics, Faculty of Dentistry, University of Puthisastra, Phnom Penh, 12211, Cambodia; 3Center for Transdisciplinary Research (CFTR), Saveetha Dental College & Hospitals, Saveetha Institute of Medical and Technical Sciences University, Chennai, Tamil Nadu, 600077, India; 4Conservative Dentistry Unit, School of Dental Sciences, Universiti Sains Malaysia, Health Campus, Kota Bharu, Kelantan, 16150, Malaysia; 5Department of Pathology Laboratory, Pakistan Kidney and Liver Institute & Research Center, Lahore, Pakistan; 6Department of Pediatric Dentistry, Faculty of Dentistry, Taif University, Taif, 21944, Saudi Arabia; 7Periodontics Unit, School of Dental Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Kelantan, 16150, Malaysia; 8Division of Operative Dentistry, Department of Restorative Dental Sciences, College of Dentistry, Jazan University, Jazan, Saudi Arabia; 9Department of Periodontics, Armed Forces Hospital Jizan, Jizan, Kingdom of Saudi Arabia; 10Preventive Dental Science Department, Faculty of Dentistry, Najran University, Najran, Kingdom of Saudi Arabia; 11Saudi Board of Pediatric Dentistry (SB-PD), King Fahad Military Medical Complex – KFMMC, Dammam, Saudi ArabiaCorrespondence: Mohmed Isaqali Karobari, Department of Restorative Dentistry & Endodontics, Faculty of Dentistry, University of Puthisastra, Phnom Penh, 12211, Cambodia, Email dr.isaq@gmail.com Chan Yean Yean, Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, 16150, Malaysia, Email yychan@usm.myAbstract: Introduction: Ginger (Zingiber officinale) has been one of the most commonly consumed herbal medicines for a long time to treat several common diseases. Antibacterial activity, antioxidant properties and many bioactive compounds in ginger have been identified previously, which could be used as an alternative method to treat many infectious diseases.Methods: The current study evaluates ginger’s biochemical profile using qualitative and quantitative analysis and its bioactive potentials using antioxidant and antimicrobial assays against Streptococcus mutans and selective oral microbes. HPLC analysis was performed for the quantitative analysis. DPPH and disc diffusion assays were used for antioxidant and antimicrobial activities. The antimicrobial activity was checked against Streptococcus mutans, Enterococcus faecalis, Staphylococcus spp., and Lactobacillus spp. All solvents were removed by rotary evaporation before testing the dried extracts.Results: The observed IC50 value showed that distilled water extract exhibited the highest antioxidant activity (43.9), followed by ethanol extract (52.4), and the lowest activity was observed in n-butanol extract (91.2) and n-hexane (90.6). Different plant extracts have shown significant antibacterial activity (p = 0.001) against each bacterium. The highest antibacterial activity against tested bacteria was observed in n-hexane, chloroform and ethanol extracts. In comparison, the ethyl acetate, n-butanol and water extracts showed low antibacterial activity.Conclusion: This study emphasizes that Zingiber officinale (Z. officinale) against Gram-positive bacteria is an effective antimicrobial herb. Furthermore, it can be used as a potential natural source of antioxidants. Further studies on the toxicity analysis of ginger are recommended.Keywords: Streptococcus mutans, ginger, dental, oral microbes, herbal medicine, medicinal plants, healthy food

Background: The sensitivity and specificity of 18S rRNA polymerase chain reaction (PCR) in the detection of fungal aetiology of microbial keratitis was determined in thirty patients with clinical diagnosis of microbial keratitis.Methods: Corneal scrapings from patients were used for Gram stain, culture and PCR analysis. PCR was performed with primer pairs targeted to the 18S rRNA gene. The result of the PCR was compared with conventional culture and Gram staining method. The PCR positive samples were identified by DNA sequencing of the internal transcribed spacer (ITS) region of the rRNA gene. Main outcome measures were sensitivity and specificity of PCR in the detection of fungus in corneal keratitis.Results: Combination of microscopy and culture gave a positive result in 11 of 30 samples of microbial keratitis. PCR detected 10 of 11 samples that were positive by conventional method. One of the 19 samples that was negative by conventional method was positive by PCR. Statistical analysis revealed that the PCR to have a sensitivity of 90.9% and specificity of 94.7% in the detection of a fungal aetiology in microbial keratitis.Conclusion: PCR is a rapid, sensitive and useful method to detect fungal aetiology in microbial keratitis. [ABSTRACT FROM AUTHOR]

Background: Breast cancer has been the most frequently diagnosed cancer among women in Taiwan since 2003. While genetic variants play a significant role in the elevated risk of breast cancer, their implications have been less explored within Asian populations. Variant-based polygenic risk scores (PRS) have emerged as valuable tools for assessing the likelihood of developing breast cancer. In light of this, we attempted to establish a predictive breast cancer PRS tailored specifically for the Taiwanese population., Methods: The cohort analyzed in this study comprised 28,443 control subjects and 1501 breast cancer cases. These individuals were sourced from the Taiwan Precision Medicine Initiative (TPMI) array and the breast cancer registry lists at Taichung Veterans General Hospital (TCVGH). Utilizing the breast cancer-associated Polygenic Score (PGS) Catalog, we employed logistic regression to identify the most effective PRS for predicting breast cancer risk. Subsequently, we subjected the cohort of 1501 breast cancer patients to further analysis to investigate potential heterogeneity in breast cancer risk., Results: The Polygenic Score ID PGS000508 demonstrated a significant association with breast cancer risk in Taiwanese women with a 1.498-fold increase in cancer risk(OR = 1.498, 95 % CI(1.431-1.567, p=5.38×10^-68). Individuals in the highest quartile exhibited a substantially elevated risk compared to those in the lowest quartile, with an odds ratio (OR) of 3.11 (95 % CI: 2.70-3.59; p=1.15×10^-55). In a cohort of 1501 breast cancer cases stratified by PRS distribution, women in the highest quartile were diagnosed at a significantly younger age (p=0.003) compared to those in the lowest quartile. However, no significant differences were observed between PRS quartiles in relation to clinical stage (p=0.274), pathological stage (p=0.647), or tumor subtype distribution (p=0.244)., Conclusion: In our study, we pinpointed PGS000508 as a significant predictive factor for breast cancer risk in Taiwanese women. Furthermore, we found that a higher PGS000508 score was associated with younger age at the time of first diagnosis among the breast cancer cases examined., Competing Interests: Declaration of Competing Interest We thank all of the participants and investigators from the Taiwan Precision Medicine Initiative. The authors sincerely appreciate the assistance of the Center for Translational Medicine of Taichung Veterans General Hospital. This study was funded by Academia Sinica (40–05-GMM, AS-GC-110-MD02 and 236e-1100202), and National Development Fund, Executive Yuan (NSTC 111–3114-Y-001–001)., (Copyright © 2024. Published by Elsevier Ltd.)

Background and Purpose: Antimicrobial resistance (AMR) has emerged as a significant global concern. To combat this growing threat, various strategies have been employed, including the use of plant extracts and the biosynthesis of nanoparticles (NPs). The current study was designed to evaluate the phytochemical analysis of ginger ( Zingiber officinale ) extracts, characterize the silver nanoparticles (AgNPs) and to see their antibacterial potentials against multi-drug resistant (MDR) bacterial strains., Methods: The extracts were prepared and initially assessed for their phytochemical composition and antibacterial activity. Then, AgNPs were synthesized from these extracts at room temperature, and various analytical techniques, including UV-visible spectroscopy, X-ray diffraction (XRD), ATIR-FTIR, zeta sizer, scanning electron microscopy (SEM), and energy-dispersive X-ray analysis (EDXA), were used to characterize the NPs. After confirmation of prepared NPs, they were subjected to their antibacterial activity., Results: HPLC analysis demonstrated the presence of eight phytoconstituents in organic ginger extracts. The absorption spectra of the silver suspension exhibited surface plasmon resonance peaks with maxima between 420 and 448 nm. Functional groups like C-H, N-H, OH, C-O-C, C=O, and C-O were identified in both the organic and aqueous extracts of Z. officinale , playing a key role in the formation of AgNPs, as characterized by ATR-FTIR analysis. Both ginger organic and aqueous extract synthesized AgNPs crystalline structure was shown in XRD analysis and the particle size distribution showed average diameter of 200.5 nm of AgNPs from aqueous extracts. Scanning Electron Microscopy displayed spherical structure and EDA results showed the percentage of elements in synthesized AgNPs using plant extracts. Most promising antibacterial activity was obtained against Escherichia coli ie 20.83±0.53 for 100 µg/mL., Conclusion: The results of the current study showed that AgNPs synthesized from different ginger extracts have promising antibacterial properties and can be potential candidates for alternative treatment options for bacterial infections., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Ramzan et al.)

Wild birds could be a reservoir of medically relevant microorganisms, particularly multidrug-resistant Enterococcus spp. Resistant bacteria's epidemiology and transmission between animals and humans has grown, and their zoonotic potential cannot be ignored. This is the first study to evaluate the status of vancomycin resistant enterococci (VRE) in various wild bird species using meta-analysis and a systematic review. In this study, the pooled prevalence was obtained by analyzing data from published articles on the occurrence of VRE in wild bird species. It's unclear how the antibiotic resistance gene transfer cycle affects wild birds. Google Scholar and PubMed were used to conduct the research. The data and study methodology was assessed and extracted by two reviewers independently, with a third reviewing the results. Heterogeneity between study and publication bias were analyzed using the random effect model. Thirty-eight studies were included in the meta-analysis. 382 out of the 4144 isolates tested, were VRE. The pooled prevalence of VRE among wild birds was estimated at 11.0% (95% CI; 6.9 -17.2%; I 2 = 93.204%; P < 0.001). There was high variability between study (t 2 = 2.156; heterogeneity I 2 = 93.204% with chi-square (Q) = 544.413, degrees of freedom (df) = 37, and P < 0.001). Egger's test verified the funnel plot's bias, while result from the leave-one-out forest plot had no effect on the pooled prevalence., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

TRAIL (TNF-related apoptosis-inducing ligand) is a potent inducer of tumor cell apoptosis through TRAIL receptors. While it has been previously pursued as a potential anti-tumor therapy, the enthusiasm subsided due to unsuccessful clinical trials and the fact that many tumors are resistant to TRAIL. In this report, we identified heparan sulfate (HS) as an important regulator of TRAIL-induced apoptosis. TRAIL binds HS with high affinity ( K D = 73 nM) and HS induces TRAIL to form higher-order oligomers. The HS-binding site of TRAIL is located at the N-terminus of soluble TRAIL, which includes three basic residues. Binding to cell surface HS plays an essential role in promoting the apoptotic activity of TRAIL in both breast cancer and myeloma cells, and this promoting effect can be blocked by heparin, which is commonly administered to cancer patients. We also quantified HS content in several lines of myeloma cells and found that the cell line showing the most resistance to TRAIL has the least expression of HS, which suggests that HS expression in tumor cells could play a role in regulating sensitivity towards TRAIL. We also discovered that death receptor 5 (DR5), TRAIL, and HS can form a ternary complex and that cell surface HS plays an active role in promoting TRAIL-induced cellular internalization of DR5. Combined, our study suggests that TRAIL-HS interactions could play multiple roles in regulating the apoptotic potency of TRAIL and might be an important point of consideration when designing future TRAIL-based anti-tumor therapy., Competing Interests: YL, HH, CO, RD, LP, DX No competing interests declared, ZW is also an employee at Glycan Therapeutics, YX, JL is a founder of Glycan Therapeutics and has stock ownership of the company

Streptococcus pneumoniae (S. pneumoniae) is a bacterial species often associated with the occurrence of community-acquired pneumonia (CAP). CAP refers to a specific kind of pneumonia that occurs in individuals who acquire the infection outside of a healthcare setting. It represents the leading cause of both death and morbidity on a global scale. Moreover, the declaration of S. pneumoniae as one of the 12 leading pathogens was made by the World Health Organization (WHO) in 2017. Antibiotics like β -lactams, macrolides, and fluoroquinolones are the primary classes of antimicrobial medicines used for the treatment of S. pneumoniae infections. Nevertheless, the efficacy of these antibiotics is diminishing as a result of the establishment of resistance in S. pneumoniae against these antimicrobial agents. In 2019, the WHO declared that antibiotic resistance was among the top 10 hazards to worldwide health. It is believed that penicillin-binding protein genetic alteration causes β -lactam antibiotic resistance. Ribosomal target site alterations and active efflux pumps cause macrolide resistance. Numerous factors, including the accumulation of mutations, enhanced efflux mechanisms, and plasmid gene acquisition, cause fluoroquinolone resistance. Furthermore, despite the advancements in pneumococcal vaccinations and artificial intelligence (AI), it is not feasible for individuals to rely on them indefinitely. The ongoing development of AI for combating antimicrobial resistance necessitates more research and development efforts. A few strategies can be performed to curb this resistance issue, including providing educational initiatives and guidelines, conducting surveillance, and establishing new antibiotics targeting another part of the bacteria. Hence, understanding the resistance mechanism of S. pneumoniae may aid researchers in developing a more efficacious antibiotic in future endeavors.

Antibiotic resistance is a global public health concern, posing a significant threat to the effectiveness of antibiotics in treating bacterial infections. The accurate and timely detection of antibiotic-resistant bacteria is crucial for implementing appropriate treatment strategies and preventing the spread of resistant strains. This manuscript provides an overview of the current and emerging technologies used for the detection of antibiotic-resistant bacteria. We discuss traditional culture-based methods, molecular techniques, and innovative approaches, highlighting their advantages, limitations, and potential future applications. By understanding the strengths and limitations of these technologies, researchers and healthcare professionals can make informed decisions in combating antibiotic resistance and improving patient outcomes.

One of the most important breakthroughs in healthcare is the development of vaccines. The life cycle and its gene expression in the numerous virus-associated disorders must be considered when choosing the target vaccine antigen for Epstein-Barr virus (EBV). The vaccine candidate used in the current study will also be effective against all other herpesvirus strains, based on the conservancy study, which verified that the protein is present in all herpesviruses. From the screening, two B-cell epitopes, four MHC-I, and five MHC-II restricted epitopes were chosen for further study. The refined epitopes indicated 70.59% coverage of the population in Malaysia and 93.98% worldwide. After removing the one toxin (PADRE) from the original vaccine design, it was projected that the new vaccine would not be similar to the human host and would instead be antigenic, immunogenic, non-allergenic, and non-toxic. The vaccine construct was stable, thermostable, soluble, and hydrophilic. The immunological simulation projected that the vaccine candidate would be subject to a long-lasting active adaptive response and a short-lived active innate response. With IgM concentrations of up to 450 cells per mm 3 and active B-cell concentrations of up to 400 cells per mm 3 , the B-cells remain active for a considerable time. The construct also discovered other conformational epitopes, improving its ability to stimulate an immune response. This suggests that, upon injection, the epitope will target the B-cell surface receptors and elicit a potent immune response. Furthermore, the discotope analysis confirmed that our conformational B-cell epitope was not displaced during the design. Lastly, the docking complex was stable and exhibited little deformability under heat pressure. These computational results are very encouraging for future testing of our proposed vaccine, which may potentially help in the management and prevention of EBV infections worldwide.

Unlike pandemics in the past, the outbreak of coronavirus disease 2019 (COVID-19), which rapidly spread worldwide, was met with a different approach to control and measures implemented across affected countries. The lack of understanding of the fundamental nature of the outbreak continues to make COVID-19 challenging to manage for both healthcare practitioners and the scientific community. Challenges to vaccine development and evaluation, current therapeutic options, convalescent plasma therapy, herd immunity, and the emergence of reinfection and new variants remain the major obstacles to combating COVID-19. This review discusses these challenges in the management of COVID-19 at length and highlights the mechanisms needed to provide better understanding of this pandemic., (© 2022 [The Author/The Authors].)

Zinc oxide nanoparticles (ZnO NPs) are the second most prevalent metal oxide, owing to their characteristics of low cost, safe, and easily prepared. ZnO NPs have been found to exhibit unique properties which show their potential to be used in various therapies. Numerous techniques have been devised for the manufacture of zinc oxide because it is one of the nanomaterials that has received major research interest. Mushroom sources are proven to be efficient, ecologically friendly, inexpensive, and safe for humankind. In the current study, an aqueous fraction of methanolic extract of Lentinula edodes ( L. edoes ) was used to synthesize ZnO NPs. The biosynthesis of ZnO NPs was achieved by using the reducing and capping capability of an L. edodes aqueous fraction. Bioactive compounds from mushroom, such as flavonoids and polyphenolic compounds, are used in the green synthesis process to biologically reduce metal ions or metal oxides to metal NPs. Biogenically synthesized ZnO NPs were further characterized by using UV-Vis, FTIR, HPLC, XRD, SEM, EDX, zeta sizer and zeta potential analyses. The FTIR showed the functional group at the spectra in the range 3550-3200 cm -1 indicated the presence of the hydroxyl (OH) group, while bands in the range 1720-1706 cm -1 indicated C=O carboxylic stretches bonds. Furthermore, the XRD pattern of ZnO NPs created in the current study was found to be nanocrystals which are hexagonal. The SEM analysis of ZnO NPs showed spherical shapes and size distributions in the range 90-148 nm. Biologically synthesized ZnO NPs have substantial biological activities including antioxidant, antimicrobial, antipyretic, antidiabetic and anti-inflammatory potential. Biological activities showed significant antioxidant (65.7 ± 1.09), antidiabetic (85.18 ± 0.48), and anti-inflammatory potential (86.45 ± 0.60) at 300 µg inhibition in paw inflammation of (1.1 ± 0.06) and yeast-induced pyrexia (97.4 ± 0.51) at 10 mg in a dose-dependent manner. The outcomes of this research indicated that ZnO NPs significantly reduced inflammation and have the ability to scavenge free radicals and prevent protein denaturation, while also indicating their possible use in food and nutraceutical applications to treat various ailments.

Background: Enterococci are Gram-positive cocci found in the guts of humans and animals. The goal of this research is to develop a multiplex PCR assay that can detect the Enterococcus genus, four VRE genes, and three LZRE genes simultaneously., Methods: Primers used in this study were specifically designed for the detection of 16S rRNA of Enterococcus genus, van A- van B -van C -van D for vancomycin, cfr methyltransferase, and optr A , and poxt A, as well as an adenosine triphosphate-binding cassette (ABC) transporter for linezolid. A Vibrio cholerae ctx A (internal amplification control) was included. Optimization of primer concentrations and PCR components was also done. This was followed by evaluating the sensitivity and specificity of the optimized multiplex PCR., Results: Final Primer concentrations were optimized as follows: 16S rRNA is 1.0 pmol/μL, van A is 1.0 pmol/μL, optr A is 1.0 pmol/μL, cfr is 1.0 pmol/μL, poxt A is 0.1 pmol/μL, van B is 0.08 pmol/μL, ctx A is 0.07 pmol/μL, van C is 0.8 pmol/μL, and van D is 0.1 pmol/μL. Further, the optimized concentrations for MgCl 2, dNTPs and Taq DNA polymerase were 2.5 mM, 0.16 mM, and 0.75 units respectively, and an annealing temperature of 64.5 °C., Conclusions: The developed multiplex PCR is sensitive and species-specific. The development of a multiplex PCR assay that will take into account all known VRE genes and linezolid mutation is highly recommended.

Herbal products are preferable to synthetic medicines, and the use of traditional medicines is increasing day-by-day. The current study was designed to evaluate the potentials of bioactive compounds from Citrullus colocynthis by performing FTIR, HPLC, and GC-MS analyses, which explore the good concentration of the secondary metabolites, such as gallic acid (74.854 ppm), vanillic acid (122.616 ppm), and ferulic acid (101.045 ppm) with considerable bioactivities. Antimicrobial protein was estimated by performing SDS-PAGE, ranging from 15 to 70 kDa in all protein fractions. The current study also checked the cytotoxicity of the bioactive compounds in the active fraction of C. colocynthis , and to perform this activity, the groups of rats were arranged with 16 rats randomly divided into four groups (three experimental and one control) by administering various dosage of methanolic fractions in dose-dependent manner. Histopathology was conducted on the livers of the rats after 15 days of sacrifice under deep anesthesia. In liver cell slides examined at the maximum dose of 600 mg/kg, minimal morphological changes, such as slight ballooning, nuclear variation, vacuolar degeneration, and hydropic degeneration, were observed. Furthermore, the in silico analysis identified bioactive compounds as potential drug candidates.

Nasopharyngeal carcinoma (NPC) is an uncommon type of malignancy/cancer worldwide. However, NPC is an endemic disease in southeast Asia and southern China and the reasons behind the underlying for such changes are unclear. Even though the Epstein-Barr infection (EBV) has been suggested as an important reason for undistinguishable NPC, the EBV itself is not adequate to source this type of cancer. The risk factors, for example, genetic susceptibility, and environmental factors might be associated with EBV to undertake a part in the NPC carcinogenesis. Normal healthy people have a memory B cell pool where the EBV persists, and any disturbance of this connection leads to virus-associated B cell malignancies. Less is known about the relationship between EBV and epithelial cell tumors, especially the EBV-associated nasopharyngeal carcinoma (EBVaNPC) and EBV-associated gastric carcinoma (EBVaGC). Currently, it is believed that premalignant genetic changes in epithelial cells contribute to the aberrant establishment of viral latency in these tumors. The early and late phases of NPC patients' survival rates vary significantly. The presence of EBV in all tumor cells presents prospects for the development of innovative therapeutic and diagnostic techniques, despite the fact that the virus's exact involvement in the carcinogenic process is presently not very well known. EBV research continues to shed light on the carcinogenic process, which is important for a more comprehensive knowledge of tumor etiology and the development of targeted cancer therapeutics. In order to screen for NPC, EBV-related biomarkers have been widely used in a few high-incidence locations because of their close associations with the risks of NPC. The current review highlights the scientific importance of EBV and its possible association with NPC.

The emergence of genetic mutations in chromosomal genes and the transmissible plasmid-mediated colistin resistance gene may have helped in the spread of colistin resistance among various Klebsiella pneumoniae (K. pneumoniae) isolates and other different bacteria. In this study, the prevalence of mutated colistin-resistant K. pneumoniae isolates was studied globally using a systematic review and meta-analysis approach. A systematic search was conducted in databases including PubMed, ScienceDirect, Scopus and Google Scholar. The pooled prevalence of mutated colistin resistance in K. pneumoniae isolates was analyzed using Comprehensive Meta-Analysis Software (CMA). A total of 50 articles were included in this study. The pooled prevalence of mutated colistin resistance in K. pneumoniae was estimated at 75.4% (95% CI = 67.2−82.1) at high heterogeneity (I2 = 81.742%, p-value < 0.001). Meanwhile, the results of the subgroup analysis demonstrated the highest prevalence in Saudi Arabia with 97.9% (95% CI = 74.1−99.9%) and Egypt, with 4.5% (95% CI = 0.6−26.1%), had the lowest. The majority of mutations could be observed in the mgrB gene (88%), pmrB gene (54%) and phoQ gene (44%). The current study showed a high prevalence of the mutation of colistin resistance genes in K. pneumoniae. Therefore, it is recommended that regular monitoring be performed to control the spread of colistin resistance.

The continued circulation of SARS-CoV-2 virus in different parts of the world opens up the possibility for more virulent variants to evolve even as the coronavirus disease 2019 transitions from pandemic to endemic. Highly transmissible and virulent variants may seed new disruptive epidemic waves that can easily put the healthcare system under tremendous pressure. Despite various nucleic acid-based diagnostic tests that are now commercially available, the wide applications of these tests are largely hampered by specialized equipment requirements that may not be readily available, accessible and affordable in less developed countries or in low resource settings. Hence, the availability of lateral flow immunoassays (LFIs), which can serve as a diagnostic tool by detecting SARS-CoV-2 antigen or as a serological tool by measuring host immune response, is highly appealing. LFI is rapid, low cost, equipment-free, scalable for mass production and ideal for point-of-care settings. In this review, we first summarize the principle and assay format of these LFIs with emphasis on those that were granted emergency use authorization by the US Food and Drug Administration followed by discussion on the specimen type, marker selection and assay performance. We conclude with an overview of challenges and future perspective of LFI applications.

Antimicrobial resistance (AMR) is a leading cause of treatment failure for many infectious diseases worldwide. Improper overdosing and the misuse of antibiotics contributes significantly to the emergence of drug-resistant bacteria. The co-contamination of heavy metals and antibiotic compounds existing in the environment might also be involved in the spread of AMR. The current study was designed to test the efficacy of heavy metals (arsenic) induced AMR patterns in clinically isolated extended-spectrum β-lactamase (ESBL) producing bacteria. A total of 300 clinically isolated ESBL-producing bacteria were collected from a tertiary care hospital in Lahore, Pakistan, with the demographic characteristics of patients. After the collection of bacterial isolates, these were reinoculated on agar media for reidentification purposes. Direct antimicrobial sensitivity testing (AST) for bacterial isolates by disk diffusion methods was used to determine the AST patterns with and without heavy metal. The heavy metal was concentrated in dilutions of 1.25 g/mL. The collected bacterial isolates were isolated from wounds ( n = 63, 21%), urine ( n = 112, 37.3%), blood ( n = 43, 14.3%), pus ( n = 49, 16.3%), and aspirate ( n = 33, 11%) samples. From the total 300 bacterial isolates, n = 172 were Escherichia coli (57.3%), 57 were Klebsiella spp. (19%), 32 were Pseudomonas aeruginosa (10.6%), 21 were Proteus mirabilis (7%) and 18 were Enterobacter spp. (6%). Most of the antibiotic drugs were found resistant to tested bacteria. Colistin and Polymyxin-B showed the highest sensitivity against all tested bacteria, but when tested with heavy metals, these antibiotics were also found to be significantly resistant. We found that heavy metals induced the resistance capability in bacterial isolates, which leads to higher AMR patterns as compared to without heavy metal tested isolates. The results of the current study explored the heavy metal as an inducer of AMR and may contribute to the formation and spread of AMR in settings that are contaminated with heavy metals.

The epidemiological and clinical aspects of coronavirus disease-2019 (COVID-19) have been subjected to several investigations, but little is known about symptomatic patients with negative SARS-CoV-2 PCR results. The current study investigated patients who presented to the hospital with respiratory symptoms (but negative SARS-CoV-2 RT-PCR results) to determine the prevalence of bacterial pathogens among these patients. A total of 1246 different samples were collected and 453 species of bacterial pathogens were identified by culture. Antibiotic susceptibility testing was performed via the Kirby Bauer disc diffusion test. Patients showed symptoms, such as fever (100%), cough (83%), tiredness (77%), loss of taste and smell (23%), rigors (93%), sweating (62%), and nausea (81%), but all tested negative for COVID-19 by PCR tests. Further examinations revealed additional and severe symptoms, such as sore throats (27%), body aches and pain (83%), diarrhea (11%), skin rashes (5%), eye irritation (21%), vomiting (42%), difficulty breathing (32%), and chest pain (67%). The sum of n = 1246 included the following: males, 289 were between 5 and 14 years, 183 (15-24 years), 157 (25-34 years), 113 (35-49 years), and 43 were 50+ years. Females: 138 were between 5 and 14 years, 93 (15-24 years), 72 (25-34 years), 89 (35-49 years), and 68 were 50+ years. The Gram-positive organisms isolated were Staphylococcus aureus ( n = 111, 80.43%, MRSA 16.6%), E. faecalis ( n = 20, 14.49%, VRE: 9.4%), and Streptococcus agalactiae ( n = 7, 5.07%), while, Gram-negative organisms, such as E. coli ( n = 135, 42.85%, CRE: 3.49%), K. pneumoniae ( n = 93, 29.52%, CRE: 1.58%), P. aeruginosa ( n = 43, 13.65%), C. freundii ( n = 21, 6.66%), Serratia spp. ( n = 8, 2.53%), and Proteus spp. ( n = 15, 4.76%) were identified.

Background: Social anxiety disorder (SAD) is the fear of social situations where a person anticipates being evaluated negatively. Changes in autonomic response patterns are related to the expression of anxiety symptoms. Virtual reality (VR) sickness can inhibit VR experiences., Objective: This study aimed to predict the severity of specific anxiety symptoms and VR sickness in patients with SAD, using machine learning based on in situ autonomic physiological signals (heart rate and galvanic skin response) during VR treatment sessions., Methods: This study included 32 participants with SAD taking part in 6 VR sessions. During each VR session, the heart rate and galvanic skin response of all participants were measured in real time. We assessed specific anxiety symptoms using the Internalized Shame Scale (ISS) and the Post-Event Rumination Scale (PERS), and VR sickness using the Simulator Sickness Questionnaire (SSQ) during 4 VR sessions (#1, #2, #4, and #6). Logistic regression, random forest, and naïve Bayes classification classified and predicted the severity groups in the ISS, PERS, and SSQ subdomains based on in situ autonomic physiological signal data., Results: The severity of SAD was predicted with 3 machine learning models. According to the F1 score, the highest prediction performance among each domain for severity was determined. The F1 score of the ISS mistake anxiety subdomain was 0.8421 using the logistic regression model, that of the PERS positive subdomain was 0.7619 using the naïve Bayes classifier, and that of total VR sickness was 0.7059 using the random forest model., Conclusions: This study could predict specific anxiety symptoms and VR sickness during VR intervention by autonomic physiological signals alone in real time. Machine learning models can predict the severe and nonsevere psychological states of individuals based on in situ physiological signal data during VR interventions for real-time interactive services. These models can support the diagnosis of specific anxiety symptoms and VR sickness with minimal participant bias., Trial Registration: Clinical Research Information Service KCT0003854; https://cris.nih.go.kr/cris/search/detailSearch.do/13508., (©Joo Young Chun, Hyun-Jin Kim, Ji-Won Hur, Dooyoung Jung, Heon-Jeong Lee, Seung Pil Pack, Sungkil Lee, Gerard Kim, Chung-Yean Cho, Seung-Moo Lee, Hyeri Lee, Seungmoon Choi, Taesu Cheong, Chul-Hyun Cho. Originally published in JMIR Serious Games (https://games.jmir.org), 16.09.2022.)

This study develops a multi-functional hydrogel with a dual injection system based on the adhesive and self-healing properties of the byssus excretion found in mussels. Through precisely controlling the composite cross-linking hydrophobic association (HA) structure composed of A and B solutions, a high-strength, temperature-sensitive injectable hydrogel can be obtained, and it has good self-healing properties. The main composition of A solution contains the surfactant SDS, which can form amphiphilic micelles, the strength increasing component stearyl methacrylate (C18), and NIPAAm, which provides thermo-sensitivity. Solution B contains dopamine acrylate (DAA), which has self-healing properties, and ferric chloride (FeCl 3 ), which is a connecting agent. The rheological behavior shows that when the temperature is increased from 25 °C to 32 °C, the gel can be completed in seven minutes to form a composite hydrogel of NIPAAm-DAA-HA. When NMR identification was conducted on composite DAA, it was found that when comparing DAA and dopamine hydrochloride there were new peaks with specific characteristics, which confirm that this study successfully prepared DAA; swelling tests found that swelling could surpass a rate of 100%, and a higher ratio of crosslinking agent decreased the amount of moisture absorbed; the results of the compression test showed that the addition of hydrophobic micelles C18 effectively enhanced the mechanical properties of hydrogel, allowing it to withstand increased external stress; the adhesiveness results show that an increase in the catechol-Fe 3+ concentration of the NIPAAm-DAA-HA hydrogel results in an increased adhesiveness of 0.0081 kg/cm 2 on pig skin; the self-healing tests show that after taking damage, NIPAAm-DAA-HA hydrogel can be reactivated with catechol-Fe 3+ and self-heal at a rate of up to 70% after 24 h; antibacterial tests show that hydrogel has good bacterial resistance to against E. coli , staphylococcus epidermidis, and bacillus cereus; through in vitro transdermal absorption, it can be seen that the release ability of drugs within the hydrogel can reach up to 8.87 μg/cm 2 . The NIPAAm-DAA-HA hydrogel prepared by this study performed excellently in both adhesion and self-healing tests. The thermo-sensitive and antibacterial properties can be applied to the treatment of deep wounds and address some of the flaws of traditional wound dressings.

Acinetobacter baumannii (A. baumannii) are phenotypically indistinguishable from the Acinetobacter calcoaceticus−A. baumannii (ACB) complex members using routine laboratory methods. Early diagnosis plays an important role in controlling A. baumannii infections and this could be assisted by the development of a rapid, yet sensitive diagnostic test. In this study, we developed an enzyme-based electrochemical genosensor for asymmetric PCR (aPCR) amplicon detection of the blaOXA-51-like gene in A. baumannii. A. baumanniiblaOXA-51-like gene PCR primers were designed, having the reverse primer modified at the 5′ end with FAM. A blaOXA-51-like gene sequence-specific biotin labelled capture probe was designed and immobilized using a synthetic oligomer (FAM-labelled) deposited on the working electrode of a streptavidin-modified, screen-printed carbon electrode (SPCE). The zot gene was used as an internal control with biotin and FAM labelled as forward and reverse primers, respectively. The blaOXA-51-like gene was amplified using asymmetric PCR (aPCR) to generate single-stranded amplicons that were detected using the designed SPCE. The amperometric current response was detected with a peroxidase-conjugated, anti-fluorescein antibody. The assay was tested using reference and clinical A. baumannii strains and other nosocomial bacteria. The analytical sensitivity of the assay at the genomic level and bacterial cell level was 0.5 pg/mL (1.443 µA) and 103 CFU/mL, respectively. The assay was 100% specific and sensitive for A. baumannii. Based on accelerated stability performance, the developed genosensor was stable for 1.6 years when stored at 4 °C and up to 28 days at >25 °C. The developed electrochemical genosensor is specific and sensitive and could be useful for rapid, accurate diagnosis of A. baumannii infections even in temperate regions.

Background: Epstein-Barr virus (EBV) is a member of the herpesvirus family that is known to ubiquitously infect people worldwide. However, the actual prevalence of EBV infection in diseased patients in Nigeria, remains unknown. This study was thus conducted to ascertain the true prevalence., Methods: A systematic review and meta-analysis of published data was conducted according to the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). Electronic databases including PubMed, Scopus, ScienceDirect, and Google Scholar were searched for studies reporting the occurrence of EBV infection among patients with established diseases. Studies were included if they assessed EBV infection in diseased patients in Nigeria. Data were extracted and subsequently analysed using R software. Funnel plot and Egger's regression test was used to assess publication bias, while JBI prevalence tool was used to assess study quality., Results: A total of 13 studies covering 228 cases of EBV infection among 1157 diseased patients were included. Summary estimates were computed using random-effects model. The pooled prevalence of EBV infection was 20.3% (95% CI: 10.8-34.9, I 2  ​= ​92.26, p ​< ​0.001). When stratified according to the type of disease, higher estimates were obtained for patients suffering from Kaposi's sarcoma (98.7%, 95% CI: 82.2-99.9) and Nasopharyngeal malignancy (85.7%, 95% CI: 70.0-93.9). A prevalence of 13.4% (95% CI: 6.0-27.4) and 12.2% (95% CI: 4.8-27.8) was derived for the most reported patient populations, lymphoma and HIV, respectively., Conclusion: This first meta-analysis on the prevalence of EBV among Nigerian patients suffering from various diseases reveals a prevalence that emphasises the need to routinely monitor EBV infection in all EBV-associated diseases in Nigeria., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

In recent years, the idea that Vitamin C (Vit-C) could be utilized as a form of anti-cancer therapy has generated many contradictory arguments. Recent insights into the physiological characteristics of Vit-C, its pharmacokinetics, and results from preclinical reports, however, suggest that high-dose Vit-C could be effectively utilized in the management of various tumor types. Studies have shown that the pharmacological action of Vit-C can attack various processes that cancerous cells use for their growth and development. Here, we discuss the anti-cancer functions of Vit-C, but also the potential for the use of Vit-C as an epigenetic regulator and immunotherapy enhancer. We also provide a short overview of the current state of systems for scavenging reactive oxygen species (ROS), especially in the context of their influencing high-dose Vit-C toxicity for the inhibition of cancer growth. Even though the mechanisms of Vit-C action are promising, they need to be supported with robust randomized and controlled clinical trials. Moreover, upcoming studies should focus on how to define the most suitable cancer patient populations for high-dose Vit-C treatments and develop effective strategies that combine Vit-C with various concurrent cancer treatment regimens.

Human Parainfluenza Virus (HPIV) Type-1, which is an anti-sense ribonucleic acid (RNA) virus belonging to the paramyxoviridae family, induces upper and lower respiratory tract infections. The infections caused by the HPIV Type-1 virus are usually confined to northwestern regions of America. HPIV-1 causes infections through the virulence of the hemagglutinin-neuraminidase (HN) protein, which plays a key role in the attachment of the viral particle with the host's receptor cells. To the best of our knowledge, there is no effective antiviral drugs or vaccines being developed to combat the infection caused by HPIV-1. In the current study, a multiple epitope-based vaccine was designed against HPIV-1 by taking the viral HN protein as a probable vaccine candidate. The multiple epitopes were selected in accordance with their allergenicity, antigenicity and toxicity scoring. The determined epitopes of the HN protein were connected simultaneously using specific conjugates along with an adjuvant to construct the subunit vaccine, with an antigenicity score of 0.6406. The constructed vaccine model was docked with various Toll-like Receptors (TLRs) and was computationally cloned in a pET28a (+) vector to analyze the expression of vaccine sequence in the biological system. Immune stimulations carried out by the C-ImmSim Server showed an excellent result of the body's defense system against the constructed vaccine model. The AllerTop tool predicted that the construct was non-allergen with and without the adjuvant sequence, and the VaxiJen 2.0 with 0.4 threshold predicted that the construct was antigenic, while the Toxinpred predicted that the construct was non-toxic. Protparam results showed that the selected protein was stable with 36.48 instability index (II) scores. The Grand average of Hydropathicity or GRAVY score indicated that the constructed protein was hydrophilic in nature. Aliphatic index values (93.53) confirmed that the construct was thermostable. This integrated computational approach shows that the constructed vaccine model has a potential to combat laryngotracheobronchitis infections caused by HPIV-I.

The high rates of bacterial infections affect the economy worldwide by contributing to the increase in morbidity and treatment costs. The present cross-sectional study was carried out to evaluate the prevalence of bacterial infection in urinary tract infection (UTI) patients and to evaluate the antimicrobial resistance rate (AMR) in a Tertiary Care Hospital in Lahore, Pakistan. The study was conducted for the period of one year from January 2020 to December 2020. A total of 1899 different clinical samples were collected and examined for bacterial cultures using standard procedures. Samples were inoculated on different culture media to isolate bacterial isolates and for identification and susceptibility testing. A total of 1107/1899 clinical samples were positive for Staphylococcus aureus ( S. aureus ), Pseudomonas aeruginosa (P. aeruginosa) , Escherichia coli (E. coli) and other bacterial isolates. Methicillin-resistant S. aureus (MRSA) prevalence was 16.93% from these positive cases. MRSA strains were found to be highly resistant to amikacin, clindamycin, fusidic acid, gentamicin and tobramycin, while highest sensitivity was noted against vancomycin (100%) and linezolid (100%). MRSA and high rates of multidrug resistance (MDR) pose a serious therapeutic burden to critically ill patients. A systematic and concerted effort is essential to rapidly identify high-risk patients and to reduce the burden of AMR.

Accurate diagnosis to limit the spread of SARS-CoV-2 is crucial for the clinical management of this lethal infection. Recently, many low-cost and easy-to-use rapid test kits (RTK) have been developed in many countries for the massive screening of SARS-CoV-2. Thus, evaluating the accuracy and reliability of an RTK is critical. The current study was conducted on 157 individuals to evaluate the performance accuracy of rapid SARS-CoV-2 antigen detection kits using different clinical samples compared with qRT-PCR results. Nasopharyngeal swabs were collected from patients for qRT-PCR and RTK tests, and then buccal and nasal, and nasal swabs were collected for RTK tests separately. The nasal and buccal swabs showed high sensitivity (98%) and specificity (100%) compared with the qRT-PCR results. Meanwhile, for nasal, the sensitivity was 96% with 98% specificity, and nasopharyngeal swabs showed 98% sensitivity and 94% specificity. Fisher’s exact test revealed statistical significance (p < 0.05) between nasopharyngeal, nasal and buccal, and nasal swabs compared with qRT-PCR results. The study concludes that different clinical samples used for the rapid diagnosis of SARS-CoV-2 showed high sensitivities and specificities compared with qRT-PCR. The RTKs using nasal and buccal, nasopharyngeal, and nasal swabs are valuable tools for the early detection of SARS-CoV-2, especially when molecular detections are available with limited access and a high infectivity rate, when the timely detection of virus cases is urgently needed. These types of clinical samples are effective to be used by RTKs for surveillance among community and healthcare workers.

RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice ( Ager AHA/AHA ) by introducing point mutations to specifically disrupt HS-RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, Ager AHA/AHA mice phenocopied Ager -/- mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of Ager AHA/AHA mice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while Ager -/- mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in Ager AHA/AHA mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), Ager AHA/AHA mouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to Ager -/- mice., Competing Interests: ML, DX is one of the inventors for an international patent (pending, WO 2021/087462) that covers the sequence and use of anti-RAGE mAb B2, CO, CL, LT, AV, YY, XZ, DS, ES No competing interests declared, (© 2022, Li et al.)

Databases such as PubMed, Scopus and Google Scholar were searched. Data extraction and assessment of study protocol was done by two independent reviewers and the results were reviewed by a third. OpenMeta analyst and comprehensive meta-analysis (CMA) were used for the meta-analysis. The random effect model was used, publication bias and between-study heterogeneity was assessed. Seventeen studies were added to the final meta-analysis. Studies were sampled from 2000-2018 and of the 8684 isolates tested, 2824 were VRE. The pooled prevalence of VRE among poultry in Malaysia was estimated at 24.0% (95% CI; 16.7-33.1%; I 2 = 98.14%; p < 0.001). Between-study variability was high ( t 2 = 0.788; heterogeneity I 2 = 98.14% with heterogeneity chi-square ( Q ) = 858.379, degrees of freedom (df) = 16, and p < 0.001). The funnel plot showed bias which was confirmed by Egger's test and estimates from the leave-one-out forest plot did not affect the pooled prevalence. Pooled prevalence of VRE in chickens and ducks were 29.2% (CI = 18.8-42.5%) and 11.2%, CI = 9.0-14.0%) respectively. Enterococcus faecalis was reported most with more studies being reported in Peninsular Malaysia Central region and used antibiotic disc diffusion as detection method. Increased surveillance of VRE in poultry in Malaysia is required.

Antimicrobial resistance (AMR) is a global health issue that plays a significant role in morbidity and mortality, especially in immunocompromised patients. It also becomes a serious threat to the successful treatment of many bacterial infections. The widespread and irrelevant use of antibiotics in hospitals and local clinics is the leading cause of AMR. Under this scenario, the study was conducted in a tertiary care hospital in Lahore, Pakistan, from 2 August 2021 to 31 October 2021 to discover the prevalence of bacterial infections and AMR rates in COVID-19 patients admitted in surgical intensive care units (SICUs). Clinical samples were collected from the patients and we proceeded to identify bacterial isolates, followed by antibiotic susceptibility testing (AST) using the Kirby Bauer disk diffusion method and minimum inhibitory concentration (MIC). The data of other comorbidities were also collected from the patient's medical record. The current study showed that the most common pathogens were E. coli (32%) and Klebsiella pneumoniae (17%). Most E. coli were resistant to ciprofloxacin (16.8%) and ampicillin (19.8%). Klebsiella pneumoniae were more resistant to ampicillin (13.3%) and amoxycillin (12.0%). The most common comorbidity was chronic kidney disease (CKD) and urinary tract infections (UTIs). Around 17 different types of antibiotic, the carbapenem, fluoroquinolones, aminoglycoside, and quinolones, were highly prevalent in ICU patients. The current study provides valuable data on the clinical implication of antibiotics consumed by COVID-19 patients in SICUs and the AMR rates, especially with different comorbidities.

The emergence of nosocomial multidrug-resistant Klebsiella pneumoniae is an escalating public health threat worldwide. The prevalence of nosocomial infections due to K. pneumoniae was recorded up to 10%. In this systematic review and meta-analysis, which were conducted according to the guidelines of Preferred Reporting Items for Systematic Review and Meta-Analysis, 1092 articles were screened from four databases of which 47 studies fulfilled the selected criteria. By performing a random-effect model, the pooled prevalence of nosocomial multidrug-resistant K. pneumoniae was estimated at 32.8% (95% CI, 23.6-43.6), with high heterogeneity (I 2 98.29%, p -value < 0.001). The estimated prevalence of this pathogen and a few related studies were discussed, raising awareness of the spread of multidrug-resistant K. pneumoniae in the healthcare setting. The emergence of nosocomial multidrug-resistant K. pneumoniae is expected to increase globally in the future, and the best treatments for treating and preventing this pathogen should be acknowledged by healthcare staff.

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global threat with an ever-increasing death toll even after a year on. Hence, the rapid identification of infected individuals with diagnostic tests continues to be crucial in the on-going effort to combat the spread of COVID-19. Viral nucleic acid detection via real-time reverse transcription polymerase chain reaction (rRT-PCR) or sequencing is regarded as the gold standard for COVID-19 diagnosis, but these technically intricate molecular tests are limited to centralized laboratories due to the highly specialized instrument and skilled personnel requirements. Based on the current development in the field of diagnostics, the programmable clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system appears to be a promising technology that can be further explored to create rapid, cost-effective, sensitive, and specific diagnostic tools for both laboratory and point-of-care (POC) testing. Other than diagnostics, the potential application of the CRISPR-Cas system as an antiviral agent has also been gaining attention. In this review, we highlight the recent advances in CRISPR-Cas-based nucleic acid detection strategies and the application of CRISPR-Cas as a potential antiviral agent in the context of COVID-19.

Despite growing knowledge of the adverse effects of cigarette smoking on general health, smoking is one of the most widely prevalent addictions around the world. Globally, about 1.1 billion smokers and over 8 million people die each year because of cigarette smoking. Smoking acts as a source for a variety of oral and systemic diseases. Various periodontal issues such as increased pocket depth, loss of alveolar bone, tooth mobility, oral lesions, ulcerations, halitosis, and stained teeth are more common among smokers. This systematic review was conducted according to the guidelines from PRISMA, and research articles were retrieved from the Web database sources on 31 May 2021. The quality of research articles was ensured by the type of evidence from combined schema incorporating as schema-13 evidence type description, Cochrane health promotion and public health field (CHPPHF), and the health gains notation framework-14 screening question for quality assessment of qualitative and quantitative studies. Smokers have been found to have bleeding on probing, periodontal pockets, and clinical attachment loss compared to nonsmokers. Oral and respiratory cancers are among the most lethal known diseases caused by cigarette smoking and other commonly occurring sequelae such as stained teeth, periodontal diseases, etc.

Nasopharyngeal carcinoma (NPC) is an epithelial tumor with high prevalence in southern China and Southeast Asia. NPC is well associated with the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) 30 bp deletion by having its vital role in increased tumorigenicity and decreased immune recognition of EBV-related tumors. This study developed an InnoPrimers-duplex qPCR for detection of NPC blood circulating LMP1 30 bp deletion genetic biomarker for early diagnosis and treatment response prediction of NPC patients. The analytical and diagnostic evaluation and treatment response prediction were conducted using NPC patients' whole blood (WB) and tissue samples and non-NPC cancer patients and healthy individuals' WB samples. The assay was able to detect as low as 20 ag DNA per reaction (equivalent to 173 copies) with high specificity against broad reference microorganisms and archive NPC biopsy tissue and FNA samples. The diagnostic sensitivity and specificity were 83.3% and 100%, respectively. The 30 bp deletion genetic biomarker was found to be a good prognostic biomarker associated with overall clinical outcome of NPC WHO type III patients. This sensitive and specific assay can help clinicians in early diagnosis and treatment response prediction of NPC patients, which will enhance treatment outcome and lead to better life-saving.

This study is a cross-sectional, observational analysis of the COVID-19 pandemic in Africa, to understand the progression of the disease across the continent. Published data on COVID-19 from 20 January 2020 to 21 June 2021 were obtained and analyzed. Case fatality ratios, as well as case growth rates and other indices were computed. On 21 June 2021, a total of 178,210,532 confirmed cases and 3,865,978 deaths had been recorded worldwide. While the Americas recorded the highest number of cases, Southern Africa recorded the majority of African cases. Fatality rate since from 20 February 2020 to 21 June 2021 was highest in the Americas (2.63%) and low in the South Eastern Asia region (1.39%), globally increasing from 2.17% at the end of January to 6.36% in May 2020 and decreasing to a range between 2.14% to 2.30% since January 2021. In Africa, the infection rate per 100,000 persons was up to 3090.18, while deaths per 100,000 and case fatality ratio were as high as 119.64 and 5.72%, respectively, among the 20 most-affected countries. The testing rate per million population was highest in Botswana (512,547.08). Fatality appears to be increasing in some regions of Africa. The rate of infection and fatality in Africa could still likely take an upward turn. Strict control measures are required, considering the continent's weak healthcare systems.

Background: Although it has been well demonstrated that the efficacy of virtual reality therapy for social anxiety disorder is comparable to that of traditional cognitive behavioral therapy, little is known about the effect of virtual reality on pathological self-referential processes in individuals with social anxiety disorder., Objective: We aimed to determine changes in self-referential processing and their neural mechanisms following virtual reality treatment., Methods: We recruited participants with and without a primary diagnosis of social anxiety disorder to undergo clinical assessments (Social Phobia Scale and Post-Event Rumination Scale) and functional magnetic resonance imaging (fMRI) scans. Participants with social anxiety disorder received virtual reality-based exposure treatment for 6 sessions starting immediately after baseline testing. After the sixth session, participants with social anxiety disorder completed follow-up scans during which they were asked to judge whether a series of words (positive, negative, neutral) was relevant to them., Results: Of 25 individuals with social anxiety disorder who participated in the study, 21 completed the sessions and follow-up; 22 control individuals also participated. There were no significant differences in age (P=.36), sex (P=.71), or handedness (P=.51) between the groups. Whole-brain analysis revealed that participants in the social anxiety disorder group had increased neural responses during positive self-referential processing in the medial temporal and frontal cortexes compared with those in the control group. Participants in the social anxiety disorder group also showed increased left insular activation and decreased right middle frontal gyrus activation during negative self-referential processing. After undergoing virtual reality-based therapy, overall symptoms of the participants with social anxiety disorder were reduced, and these participants exhibited greater activity in a brain regions responsible for self-referential and autobiographical memory processes while viewing positive words during postintervention fMRI scans. Interestingly, the greater the blood oxygen level dependent changes related to positive self-referential processing, the lower the tendency to ruminate on the negative events and the lower the social anxiety following the virtual reality session. Compared with that at baseline, higher activation was also found within broad somatosensory areas in individuals with social anxiety disorder during negative self-referential processing following virtual reality therapy., Conclusions: These fMRI findings might reflect the enhanced physiological and cognitive processing in individuals with social anxiety disorder in response to self-referential information. They also provide neural evidence of the effect of virtual reality exposure therapy on social anxiety and self-derogation., (©Ji-Won Hur, Hyemin Shin, Dooyoung Jung, Heon-Jeong Lee, Sungkil Lee, Gerard J Kim, Chung-Yean Cho, Seungmoon Choi, Seung-Moo Lee, Chul-Hyun Cho. Originally published in JMIR Mental Health (http://mental.jmir.org), 14.04.2021.)

For more than 50 years, nasopharyngeal carcinoma (NPC) has been associated with dermatomyositis (DM), a rare idiopathic inflammatory disorder that mainly affects the skin and muscles. Although the association between these rare diseases is well-documented, the actual prevalence of NPC in DM patients remains unknown. Here, a systematic review and meta-analysis of published data was conducted in accordance with the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). Electronic databases including PubMed, Scopus, ScienceDirect, and Google Scholar were searched without year or language restrictions for studies reporting the occurrence of NPC in DM patients. The study protocol was lodged with PROSPERO (CRD42021225335). A total of 95 studies covering 303 cases of NPC among 16,010 DM patients was included. Summary estimates were calculated using the random-effects model. The pooled prevalence of NPC in DM was 3.3% (95% CI, 2.5-4.3). When stratified according to study location, higher prevalence estimates were obtained for Hong Kong (36.5%), Malaysia (27.7%), and Singapore (11.9%). There was a predominance of cases among male DM patients compared with females, and most patients were aged 40 and above. Many of the NPC cases were found to be diagnosed after the diagnosis of DM. It is therefore pertinent to screen for NPC in DM patients, especially among older DM patients in the Asian region.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which emerged in December 2019 and caused the coronavirus disease 2019 (COVID-19) pandemic, took the world by surprise with an unprecedented public health emergency. Since this pandemic began, extraordinary efforts have been made by scientists to understand the pathogenesis of COVID-19, and to fight the infection by providing various preventive, diagnostic and treatment opportunities based on either novel hypotheses or past experiences. Despite all the achievements, COVID-19 continues to be an accelerating health threat with no specifically approved vaccine or therapy. This review highlights the recent advances in COVID-19 infection, with a particular emphasis on nanomedicine applications that can help in the development of effective vaccines or therapeutics against COVID-19. A novel future perspective has been proposed in this review based on utilizing polymersome nano-objects for effectively suppressing the cytokine storm, which may reduce the severity of COVID-19 infection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Antimicrobial resistance in companion animals is a major public health concern worldwide due to the animals' zoonotic potential and ability to act as a reservoir for resistant genes. We report on the first use of meta-analysis and a systematic review to analyze the prevalence of vancomycin-resistant Enterococcus (VRE) in companion animals. Databases such as MedLib, PubMed, Web of Science, Scopus, and Google Scholar were searched. The information was extracted by two independent reviewers and the results were reviewed by a third. Two reviewers independently assessed the study protocol using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) checklist and the study quality using the Joanna Briggs Institute (JBI) critical appraisal checklist for prevalence data. OpenMeta analyst and comprehensive meta-analysis (CMA) were used for the meta-analysis. The random effect model was used, and publication bias was assessed using the Eggers test and funnel plot. Between-study heterogeneity was assessed, and the sources were analyzed using the leave-one-out meta-analysis, subgroup analysis and meta-regression. Twenty-two studies met the eligibility criteria, but because some studies reported the prevalence of VRE in more than one companion animal, they were considered as individual studies, and 35 studies were therefore added to the final meta-analysis. Sampling period of the included studies was from 1995-2018. Of the 4288 isolates tested in the included studies, 1241 were VRE. The pooled prevalence of VRE in companion animals was estimated at 14.6% (95% CI; 8.7-23.5%; I 2 = 97.10%; p < 0.001). Between-study variability was high ( t 2 = 2.859; heterogeneity I 2 = 97.10% with heterogeneity chi-square ( Q ) = 1173.346, degrees of freedom (df) = 34, and p < 0.001). The funnel plot showed bias, which was confirmed by Eggers test ( t -value = 3.97165; p = 0.00036), and estimates from the leave-one-out forest plot did not affect the pooled prevalence. Pooled prevalence of VRE in dogs and cats were 18.2% (CI = 9.4-32.5%) and 12.3%, CI = 3.8-33.1%), respectively. More studies were reported in Europe than in any other continent, with most studies using feces as the sample type and disc diffusion as the detection method. With the emergence of resistant strains, new antimicrobials are required in veterinary medicine.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began as a cluster of pneumonia cases in Wuhan, China before spreading to over 200 countries and territories on six continents in less than six months. Despite rigorous global containment and quarantine efforts to limit the transmission of the virus, COVID-19 cases and deaths have continued to increase, leaving devastating impacts on the lives of many with far-reaching effects on the global society, economy and healthcare system. With over 43 million cases and 1.1 million deaths recorded worldwide, accurate and rapid diagnosis continues to be a cornerstone of pandemic control. In this review, we aim to present an objective overview of the latest nucleic acid-based diagnostic tests for the detection of SARS-CoV-2 that have been authorized by the Food and Drug Administration (FDA) under emergency use authorization (EUA) as of 31 October 2020. We systematically summarize and compare the principles, technologies, protocols and performance characteristics of amplification- and sequencing-based tests that have become alternatives to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. We highlight the notable features of the tests including authorized settings, along with the advantages and disadvantages of the tests. We conclude with a brief discussion on the current challenges and future perspectives of COVID-19 diagnostics.

Dengue is an arbovirus disease transmitted mainly by Aedes mosquitoes. As dengue shares similar clinical symptoms with other infectious diseases, prompt and accurate diagnosis is pivotal to clinicians' decisions on appropriate management. Conventional diagnostic tests to detect the dengue-specific IgM antibody are limited in their performance and ease of use. To address these issues, we developed and evaluated a biosensor based on screen-printed carbon electrodes (SPCEs) for the detection of dengue-specific immunoglobulin M (IgM) antibodies. Various optimisations were performed in order to increase the sensitivity and specificity of the biosensor. For optimal and proper orientation of the paratope sites of goat anti-human IgM capture antibodies (GAHICA), various antibody techniques, including passive, covalent, protein A, protein G and streptavidin/biotin systems, were tested on the SPCEs. The assay reagents for the biosensor were also optimised prior to its evaluation. Analytical sensitivity evaluation was carried out using pooled sera, while analytical specificity evaluation was conducted on a panel of six non-dengue serum samples. Subsequently, diagnostic sensitivity and specificity evaluation were performed using 144 reference samples. Electrochemical current signals generated from H 2 O 2 catalysed by HRP-labelled anti-dengue detection antibodies were measured using the chronoamperometric technique. With a limit of detection (LOD) of 10 6 serum dilution, the analytical sensitivity of the developed biosensor was 10 times higher than commercial ELISA. The analytical specificity of this dengue IgM biosensor was 100%. Similarly, the biosensor's diagnostic performance was 100% for sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV). These findings suggest that the developed biosensor has a great potential to be used to diagnose dengue after seroconversion.

Background: Social anxiety disorder (SAD) is characterized by excessive fear of negative evaluation and humiliation in social interactions and situations. Virtual reality (VR) treatment is a promising intervention option for SAD., Objective: The purpose of this study was to create a participatory and interactive VR intervention for SAD. Treatment progress, including the severity of symptoms and the cognitive and emotional aspects of SAD, was analyzed to evaluate the effectiveness of the intervention., Methods: In total, 32 individuals with SAD and 34 healthy control participants were enrolled in the study through advertisements for online bulletin boards at universities. A VR intervention was designed consisting of three stages (introduction, core, and finishing) and three difficulty levels (easy, medium, and hard) that could be selected by the participants. The core stage was the exposure intervention in which participants engaged in social situations. The effectiveness of treatment was assessed through Beck Anxiety inventory (BAI), State-Trait Anxiety Inventory (STAI), Internalized Shame Scale (ISS), Post-Event Rumination Scale (PERS), Social Phobia Scale (SPS), Social Interaction Anxiety Scale (SIAS), Brief-Fear of Negative Evaluation Scale (BFNE), and Liebowitz Social Anxiety Scale (LSAS)., Results: In the SAD group, scores on the BAI (F=4.616, P=.009), STAI-Trait (F=4.670, P=.004), ISS (F=6.924, P=.001), PERS-negative (F=1.008, P<.001), SPS (F=8.456, P<.001), BFNE (F=6.117, P=.004), KSAD (F=13.259, P<.001), and LSAS (F=4.103, P=.009) significantly improved over the treatment process. Compared with the healthy control group before treatment, the SAD group showed significantly higher scores on all scales (P<.001), and these significant differences persisted even after treatment (P<.001). In the comparison between the VR treatment responder and nonresponder subgroups, there was no significant difference across the course of the VR session., Conclusions: These findings indicated that a participatory and interactive VR intervention had a significant effect on alleviation of the clinical symptoms of SAD, confirming the usefulness of VR for the treatment of SAD. VR treatment is expected to be one of various beneficial therapeutic approaches in the future., Trial Registration: Clinical Research Information Service (CRIS) KCT0003854; https://cris.nih.go.kr/cris/search/search&#95;result&#95;st01.jsp?seq=13508., (©Hyun-Jin Kim, Seulki Lee, Dooyoung Jung, Ji-Won Hur, Heon-Jeong Lee, Sungkil Lee, Gerard J Kim, Chung-Yean Cho, Seungmoon Choi, Seung-Moo Lee, Chul-Hyun Cho. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 06.10.2020.)

Vancomycin-Resistant Enterococci (VRE) are on the rise worldwide. Here, we report the first prevalence of VRE in Nigeria using systematic review and meta-analysis. International databases MedLib, PubMed, International Scientific Indexing (ISI), Web of Science, Scopus, Google Scholar, and African journals online (AJOL) were searched. Information was extracted by two independent reviewers, and results were reviewed by the third. Two reviewers independently assessed the study quality using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) checklist. OpenMeta analyst was used. The random effect was used, and publication bias was assessed using a funnel plot. Between-study heterogeneity was assessed, and the sources were analysed using the leave-one-out meta-analysis, subgroup analysis, and meta-regression. Nineteen studies met the eligibility criteria and were added to the final meta-analysis, and the study period was from 2009-2018. Of the 2552 isolates tested, 349 were VRE, and E. faecalis was reported the most. The pooled prevalence of VRE in Nigeria was estimated at 25.3% (95% CI; 19.8-30.8%; I 2 = 96.26%; p < 0.001). Between-study variability was high ( t 2 = 0.011; heterogeneity I 2 = 96.26% with heterogeneity chi-square (Q) = 480.667, degrees of freedom (df) = 18, and p = 0.001). The funnel plot showed no publication bias, and the leave-one-out forest plot did not affect the pooled prevalence. The South-East region had a moderate heterogeneity though not significant ( I 2 = 51.15%, p = 0.129). Meta-regression showed that all the variables listed contributed to the heterogeneity except for the animal isolate source ( p = 0.188) and studies that were done in 2013 ( p = 0.219). Adherence to proper and accurate antimicrobial usage, comprehensive testing, and continuous surveillance of VRE are required.

Background: Lymph node (LN) metastasis is one of the independent prognostic factors of gastric cancer (GC). The difference in survival rates and initial recurrence patterns in patients with node-positive GC with retrieved LN numbers greater than or less than 16 is worthy of further study., Methods: A total of 1314 patients with node-positive GC were enrolled. The clinicopathological characteristics, retrieved LN numbers, adjuvant chemotherapy, initial recurrence patterns, and survival differences between serosa-negative and serosa-positive GC were investigated., Results: For serosa-negative GC, patients with retrieved LN numbers ≥16 were associated with fewer tumor recurrences, locoregional recurrences, distant metastases, and better 5-year overall survival (OS) rates and disease-free survival (DFS) rates. For serosa-positive GC, patients with retrieved LN numbers ≥16 were associated with similar locoregional and distant metastasis and similar 5-year OS and DFS rates compared with those with retrieved LN numbers <16. Retrieved LN numbers fewer than 16 can cause stage migration compared with retrieved LN numbers ≥16. Multivariate analysis showed that both the retrieved LN numbers (≥ or <16) and adjuvant chemotherapy were independent prognostic factors affecting OS in serosa-negative GC, while adjuvant chemotherapy but not the retrieved LN numbers was an independent prognostic factor of OS in serosa-positive GC., Conclusion: For serosa-negative GC, retrieved LN numbers fewer than 16 can cause stage migration, a higher tumor recurrence rate and worse OS and DFS rates compared with patients with retrieved LN numbers ≥16. Due to a high tumor recurrence rate in serosa-positive GC, adjuvant chemotherapy rather than retrieved LN numbers played an important role in improving patient prognosis.

Histatin 5 (Hst 5) is an antimicrobial peptide produced in human saliva with antifungal activity for opportunistic pathogen Candida albicans . Hst 5 binds to multiple cations including dimerization-inducing zinc (Zn 2+ ), although the function of this capability is incompletely understood. Hst 5 is taken up by C. albicans and acts on intracellular targets under metal-free conditions; however, Zn 2+ is abundant in saliva and may functionally affect Hst 5. We hypothesized that Zn 2+ binding would induce membrane-disrupting pores through dimerization. Through the use of Hst 5 and two derivatives, P113 (AA 4-15 of Hst 5) and Hst 5ΔMB (AA 1-3 and 15-19 mutated to Glu), we determined that Zn 2+ significantly increases killing activity of Hst 5 and P113 for both C. albicans and Candida glabrata . Cell association assays determined that Zn 2+ did not impact initial surface binding by the peptides, but Zn 2+ did decrease cell association due to active peptide uptake. ATP efflux assays with Zn 2+ suggested rapid membrane permeabilization by Hst 5 and P113 and that Zn 2+ affinity correlates to higher membrane disruption ability. High-performance liquid chromatography (HPLC) showed that the higher relative Zn 2+ affinity of Hst 5 likely promotes dimerization. Together, these results suggest peptide assembly into fungicidal pore structures in the presence of Zn 2+ , representing a novel mechanism of action that has exciting potential to expand the list of Hst 5-susceptible pathogens.

Background: Cholera, an acute watery diarrhoeal disease caused by Vibrio cholerae serogroup O1 and O139 across the continents. Replacing the existing WHO licensed killed multiple-dose oral cholera vaccines that demand 'cold chain supply' at 2-8 °C with a live, single-dose and cold chain-free vaccine would relieve the significant bottlenecks and cost determinants in cholera vaccination campaigns. In this direction, a prototype cold chain-free live attenuated cholera vaccine formulation (LACV) was developed against the toxigenic wild-type (WT) V. cholerae O139 serogroup. LACV was found stable and retained its viability (5 × 10 6  CFU/mL), purity and potency at room temperature (25 °C ± 2 °C, and 60% ± 5% relative humidity) for 140 days in contrast to all the existing WHO licensed cold-chain supply (2-8 °C) dependent killed oral cholera vaccines., Results: The LACV was evaluated for its colonization potential, reactogenicity, immunogenicity and protective efficacy in animal models after its storage at room temperature for 140 days. In suckling mice colonization assay, the LACV recorded the highest recovery of (7.2 × 10 7  CFU/mL) compared to those of unformulated VCUSM14P (5.6 × 10 7  CFU/mL) and the WT O139 strain (3.5 × 10 7  CFU/mL). The LACV showed no reactogenicity even at an inoculation dose of 10 4 -10 6  CFU/mL in a rabbit ileal loop model. The rabbits vaccinated with the LACV or unformulated VCUSM14P survived a challenge with WT O139 and showed no signs of diarrhoea or death in the reversible intestinal tie adult rabbit diarrhoea (RITARD) model. Vaccinated rabbits recorded a 275-fold increase in anti-CT IgG and a 15-fold increase in anti-CT IgA antibodies compared to those of rabbits vaccinated with unformulated VCUSM14P. Vibriocidal antibodies were increased by 31-fold with the LACV and 14-fold with unformulated VCUSM14P., Conclusion: The vaccine formulation mimics a natural infection, is non-reactogenic and highly immunogenic in vivo and protects animals from lethal wild-type V. cholerae O139 challenge. The single dose LACV formulation was found to be stable at room temperature (25 ± 2 °C) for 140 days and it would result in significant cost savings during mass cholera vaccination campaigns.

Regulatory T cells (Tregs) are renowned for maintaining homeostasis and self-tolerance through their ability to suppress immune responses. For over two decades, Tregs have been the subject of intensive research. The immunosuppressive and migratory potentials of Tregs have been exploited, especially in the areas of cancer, autoimmunity and vaccine development, and many assay protocols have since been developed. However, variations in assay conditions in different studies, as well as covert experimental factors, pose a great challenge to the reproducibility of results. Here, we focus on human Tregs derived from clinical samples and highlighted caveats that should be heeded when conducting Tregs suppression and migration assays. We particularly delineated how factors such as sample processing, choice of reagents and equipment, optimization and other experimental conditions could introduce bias into the assay, and we subsequently proffer recommendations to enhance reliability and reproducibility of results. It is hoped that prioritizing these factors will reduce the tendencies of generating false and misleading results, and thus, help improve our understanding and interpretation of Tregs functional studies., Competing Interests: The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript or in the decision to publish the results.

Objectives : Isolation of Leptospira by culture represents a definitive growth and confirmation of the disease, yet it is hampered with its nature of slow growth. With slight modification of culture method, the study aims to isolate and characterize Leptospira spp. from patients with acute febrile illness. Methods : A total of 109 blood samples were collected from patients with acute febrile illness that presented at the Emergency Department of Hospital Universiti Sains Malaysia, Malaysia. Clinical samples were subjected to Leptospira IgM Rapid test, microscopic agglutination test (MAT), isolation by culture method, and direct real-time PCR test. For leptospiral isolation, the samples (whole blood and deposit from spun plasma) were cultured into modified Ellinghausen McCullough Johnson Harris (EMJH) media with and without 5'-fluorouracil (5-FU). In every culture positive sample, partial 16S rRNA gene sequencing was performed for molecular identification of the isolates. Phylogenetic analysis was carried out to determine the genetic relatedness among the isolates. An inhibition of 5-FU study was performed on Leptospira interrogans serovar Canicola with different concentrations to compare the growth detection of the tested Leptospira with or without 5-FU within 7 days of incubation. Results : Leptospirosis was diagnosed in 14.7% of patients with acute febrile illness. Two Leptospira spp. (n = 2/109, 1.85%) were successfully isolated from whole blood and deposit from spun plasma samples. B004 and B208 samples were positive at day 11 and day 7, respectively, in EMJH media without addition of 5-FU. Sample B004 was identified as Leptospira interrogans and B208 as Leptospira weilli . Phylogenetic analysis confirmed that both of them were within pathogenic group and they were not related. The 5-FU inhibition study revealed that additional of 5-FU at final concentration of 200 µg/mL to EMJH media demonstrated an inhibitory effect on the growth of the tested strain Conclusion : Isolation of Leptospira spp. using EMJH media without addition of 5'-fluorouracil resulted in a better outcome. Two pathogenic Leptospira isolates were successfully cultivated from patients with acute febrile illness that were genetically not related., Competing Interests: Ethical Approval: This study was approved by the Human Research Ethics Committee USM (USM/JEPeM/16090300).

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3' untranslated regions (3' UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3' UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.