REVIEW-The Biological importance of cells secreted Exosomes.

Exosomes are the extracellular vesicles secreted normally by most of the cells, containing important bioactive molecules including lipids, carbohydrates, protein, DNA and RNA resulting in cell to cell communication and many other biological activities. In this review we have focused on different insight onto exosomes to cover its basic mechanism, biogenesis, biomolecules it carries and how they are altering secondary sites. In cancerous cells these tiny bodies are reported to be secreted aberrantly and through paracrine signalling contributes in metastasis. Each type of cancer cells exosomes is unique with types of load inside, thus behave with an individual pattern to transfer cancer load from origin to other sites. Because of its involvement in cancer metastasis and its role as biomarkers in early stage disease identification and also as suitable particles for drug delivery system, Exosomes research has been focal field since last two decades. Currently exosomes are the hot area of research and because of their biologically important structure and composition some studies have also been conducted to use them as early stage biomarkers in different diseases and also by a modification these could also be a biocompatible source in drug delivery. The current researches data, results and advancement in exosome studies are quit promising and are positive indication in resolving many clinical complexities in future but still further investigations are needed to evaluate the clinical importance and applications of exosomes in detail.

Rapidly Evolving SARS-CoV-2: A Brief Review Regarding the Variants and their Effects on Vaccine Efficacies.

Since the commencement of Corona Virus Disease 2019 (COVID-19) pandemic, which has resulted in millions of mortalities globally, the efforts to minimize the damages have equally been up to the task. One of those efforts includes the mass vaccine development initiative targeting the deadly Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). So far, vaccines have tremendously decreased the rate of transmission and infection in most parts of the world. However, the repeated resurgence of different types of mutated versions of the virus, also known as variants, has somehow created uncertainties about the efficacies of different types of vaccines. This review discusses some of the interesting SARS-CoV-2 features, including general structure, genomics, and mechanisms of variants development and their consequent immune escape. This review also focuses very briefly on antigenic drift, shift, and vaccine-developing platforms.

Antibacterial Activity and Mechanisms of Plant Flavonoids against Gram-Negative Bacteria Based on the Antibacterial Statistical Model.

The antimicrobial quantitative structure-activity relationship of plant flavonoids against Gram-positive bacteria was established in our previous works, and the cell membrane was confirmed as a major site of action. To investigate whether plant flavonoids have similar antibacterial effects and mechanisms against both Gram-negative and Gram-positive bacteria, here, the minimum inhibitory concentrations (MICs) of 37 plant flavonoids against Escherichia coli were determined using the microdilution broth method, and then the correlation between their lipophilic parameter ACD/LogP or LogD7.40 value and their MIC was analyzed. Simultaneously, the correlation between the ACD/LogP or LogD7.40 value and the MIC of 46 plant flavonoids reported in the literature against E. coli was also analyzed. Both sets of results showed that there is a significant correlation between the LogP value and the MIC of plant flavonoids against Gram-negative bacteria. However, it is difficult to effectively predict the MIC of plant flavonoids against Gram-negative bacteria from their lipophilic parameters. By comparing two regression curves derived from plant flavonoids against Gram-negative and Gram-positive bacteria, it was further discovered that the antibacterial activities of most plant flavonoids against Gram-negative bacteria are stronger than those against Gram-positive bacteria when their LogP values are less than approximately 3.0, but the opposite is true when their LogP values are more than approximately 3.6. Moreover, this comparison also suggests that unlike mainly acting on the cell membrane of Gram-positive bacteria, plant flavonoids have multiple mechanisms against Gram-negative species, while the cell membrane is also an important action site among them. Combined with the correlation analyses between the enzyme inhibitory activity and the LogP value of the reported flavonoids, it was further suggested that DNA gyrase is another important target of plant flavonoids against Gram-negative bacteria.

Prevalence and antibiotics resistance status of Salmonella in raw meat consumed in various areas of Lahore, Pakistan.

This study reports the prevalence and antibiotics resistance status of Salmonella detected in raw meat from Lahore, Pakistan. Overall, N = 111 meat samples, were collected from local markets. Salmonella was recovered from 57 (51.35%) samples, including 45.83% of poultry, 60% of buffalo, 64.28% of cow, and 60% of goat meat samples. The predominant Salmonella strains were Salmonella enterica serovars; Typhimurium (45.4%), Typhi (27.2%), and Enteritidis (18.1%), identified by VITEK system and 16S rRNA gene sequencing. The isolates exhibited high resistance to Erythromycin (100%), Cefepime (98.24%), Colistin (94.73%), Azithromycin (92.98%), Tetracycline (87.71%), Polymyxin B (84.21%), Ciprofloxacin (84.21%), Trimethoprim-Sulfamethoxazole (80.70%), Nalidixic Acid (80.70%), Kanamycin (78.94%), Chloramphenicol (77.19%), Streptomycin (71.92%) and Ampicillin (64.91%). While the isolates exhibited more susceptibility to Meropenem (75.43%) and Amikacin (73.68%). N = 8 strains were designated as Multidrug Resistant (MDR) and N = 3 as Extensively Drug-Resistant (XDR) Salmonella. The PCR-based detection of resistance genes revealed the presence of blaTEM-1 gene (100%), catA1 gene (64%), and gyrA gene (18%). The whole genome sequencing (WGS) of two selected strains and subsequent downstream analysis confirmed the strains as MDR and XDR Salmonella enterica serovar Typhi. The study showed that raw meat consumed in Lahore carries a significantly high number of drug-resistant Salmonella.

Occurrence of Virulence Genes among Methicillin-Resistant Staphylococcus aureus Isolated from Subclinical Bovine Mastitis.

The occurrence of Staphylococcus aureus-induced subclinical mastitis holds significant implications for public health. This specific microorganism possesses a wide array of pathogenic factors that enable it to adhere to, colonize, invade, and infect the host. The objective of the current study was to assess the prevalence of S. aureus, determine antimicrobial resistance patterns, and identify virulence genes of methicillin-resistant S. aureus (MRSA) strains responsible for subclinical mastitis in bovines. A total of 249 milk samples were collected from various farms in the district of Faisalabad. The presence of subclinical mastitis was assessed by using the California mastitis test. Positive milk samples (n = 100) were then subjected to standard microbiological techniques for isolation and identification of S. aureus. Antibiogram analysis was conducted by using the disc diffusion method to assess antimicrobial resistance. For the molecular detection of S. aureus and its virulence genes, the polymerase chain reaction (PCR) was performed with species-specific primers. The overall prevalence of S. aureus was found to be 40% (40/100), which was confirmed through molecular detection of the nuc gene in 40/40 (100%) of samples using PCR. Antimicrobial susceptibility tests indicated the highest susceptibility to vancomycin, sulfamethoxazole/trimethoprim, erythromycin, gentamicin, ciprofloxacin, and chloramphenicol, while the highest resistance rate was observed against tetracycline. Additionally, 30% of samples (12/40) tested positive for methicillin resistance. PCR analysis revealed that 100% of MRSA-tested isolates harbored the mecA and clfA genes. Furthermore, the MRSA isolates showed the presence of pvl, hla, hlb, sec, icaA, icaD, icaB, and icaC genes at rates of 92, 75, 67, 42, 42, 75, 8, and 25%, respectively. These findings underscore the need for stricter aseptic control in dairy farms to prevent disease transmission between animals and ensure the production of safe and uncontaminated food for human consumption.

Right upper lobe intrapulmonary mature cystic teratoma. An unusual location with unusual associations and a review of the literature.

INTRODUCTION: Teratomas are rare neoplasms made up of mature embryonic germ cell layers. Extra-gonadal teratomas are rare and primarily in the mediastinum. Mature cystic teratomas (MCT) are slow-growing benign tumors accounting for a small number of mediastinal germ cell tumors. Patients with MCT are often asymptomatic, and even most symptomatic intrapulmonary teratomas present with vague and non-specific symptoms such as cough, hemoptysis, and chest pain. CASE PRESENTATION AND DISCUSSION: A Yemeni patient presented for two years with dry cough, shortness of breath, and stitching right-sided chest pain. Plain X-ray showed pleural thickening associated with two opacities in the right lung- one located centrally in the upper lobe and another peripherally- which were confirmed with a CT scan. During right-sided decortication with resection of the two masses, extensive adhesions were found between the right pleura and the chest wall. The adhesions were released, and right-sided decortication led to the removal of the peripheral cystic mass attached to the pleura. Histopathology reported the presence of squamous epithelium lining with ectodermal components such as hair follicles, sebaceous glands, keratin debris, fatty, and fibromuscular tissue. CONCLUSION: Intrapulmonary teratomas are rare tumors, with less than 100 cases reported in the past few years. All the reported cases are in the left lung lobes, yet our case is on the right side. Although the right-sided mature cystic teratoma is even scarcer, physicians should have a high index of suspicion for teratoma when encountering abnormal lesion in the right side of the lung confirming it either by CT or MRI.

Evaluation of vitamin D supplementation intake among children; cross-sectional observational study.

BACKGROUND: The purpose of this study was to review the vitamin D supplementation intake status among children in the general public, determine the vitamin D supplements practices, and the barriers that parents and children face with supplementation.   Methods: A cross-sectional observational questionnaire-based survey study design was used. A convenience sampling technique was used to collect the data. An online Rao soft sample size calculator was applied to determine the sample size of 319. The response rate of participants was expected to be 63%, the margin of error was 5% and the level of confidence was 95%.   Results: A total of 248 parents (89.1% mothers (n =203)) and 15.7% fathers (n=39) with a mean ± SD age of 35.4 ± 7.04 years, completed the study (77.7% response rate). Parents reported that the supplements used the most by children were vitamin D supplements (21.85%) and multivitamins (21.8%) followed by calcium supplements (5.6%). However, 27.8% of children in this study did not take any supplements. Of all the parents, 65% (162) of them reported sending their child outside to play while 34.67% (86) of parents had reported no outdoor activity. Approximately 184 (74.2%) parents reported the child's diet to contain multiple natural sources of vitamin D. However, 69 (27.8%) parents reported giving none of the natural sources of vitamin D to their children through the diet. Parents with higher education about 62.9% (n=156) had a higher frequency of providing vitamin D supplements to their children. Children in high-income families (43.63%) were more likely to take vitamin D supplements than those in middle- or low-income families.     Conclusion: The study concluded that challenges like the educational and financial background of parents, family-income level, and health insurance status could help aid in addressing the overall burden of vitamin D deficiency among young children.

Evaluation of adenovirus capsid labeling versus transgene expression.

Adenoviral vectors have been utilized for a variety of gene therapy applications. Our group has incorporated bioluminescent, fluorographic reporters, and/or suicide genes within the adenovirus genome for analytical and/or therapeutic purposes. These molecules have also been incorporated as capsid components. Recognizing that incorporations at either locale yield potential advantages and disadvantages, our report evaluates the benefits of transgene incorporation versus capsid incorporation. To this end, we have genetically incorporated firefly luciferase within the early region 3 or at minor capsid protein IX and compared vector functionality by means of reporter readout.

Using classification techniques for statistical analysis of Anemia.

Anemia in children is becoming a worldwide problem owing to the unawareness among people regarding the disease, its causes and preventive measures. This study develops a decision support system using data mining techniques that are applied to a database containing data about nutritional factors for children. The data set was taken from NFHS-4, a survey conducted by the Government of India in 2015-16. The work attempts to predict anemia among children and establish a relation between mother's health and diet during pregnancy and its effects on anemic status of her child. It aims to help parents and clinicians to understand the influence of an infant's feeding practices and diet on his/her health and provide guidelines regarding diet to prevent anemia. Earlier, systems were built on computer using medical experts' advicewhich was then translated into algorithms for use. However, this method was time consuming thus, artificial intelligence came into play utilizing knowledge discovery and data mining tools for predictive modeling. The two techniques, decision tree and association rule mining has been applied and compared to select more appropriate technique for this particular task and a model is proposed in the healthcare domain with the aim to reduce the risk of the blood-related disease anemia.

Immune profiles in primary squamous cell carcinoma of the head and neck.

OBJECTIVES: In this study we describe the tumor microenvironment, the signaling pathways and genetic alterations associated with the presence or absence of CD8+ T-cell infiltration in primary squamous cell carcinoma of the head and neck (SCCHN) tumors. MATERIALS AND METHODS: Two SCCHN multi-analyte cohorts were utilized, the Cancer Genome Atlas (TCGA) and the Chicago Head and Neck Genomics (CHGC) cohort. A well-established chemokine signature classified SCCHN tumors into high and low CD8+ T-cell inflamed phenotypes (TCIP-H, TCIP-L respectively). Gene set enrichment and iPANDA analyses were conducted to dissect differences in signaling pathways, somatic mutations and copy number aberrations for TCIP-H versus TCIP-L tumors, stratified by HPV status. RESULTS: TCIP-H SCCHN tumors were enriched in multiple immune checkpoints irrespective of HPV-status. HPV-positive tumors were enriched in markers of T-regulatory cells (Tregs) and HPV-negative tumors in protumorigenic M2 macrophages. TCIP-L SCCHN tumors were enriched for the ß-catenin/WNT and Hedgehog signaling pathways, had frequent mutations in NSD1, amplifications in EGFR and YAP1, as well as CDKN2A deletions. TCIP-H SCCHN tumors were associated with the MAPK/ERK, JAK/STAT and mTOR/AKT signaling pathways, and were enriched in CASP8, EP300, EPHA2, HRAS mutations, CD274, PDCD1LG2, JAK2 amplifications. CONCLUSIONS: Our findings support that combinatorial immune checkpoint blockade and depletion strategies targeting Tregs in HPV-positive and M2 macrophages in HPV-negative tumors may lead to improved antitumor immune responses in patients with TCIP-H SCCHN. We highlight novel pathways and genetic events that may serve as candidate biomarkers and novel targeted therapies to enhance the efficacy of immunotherapy in SCCHN patients.

Purification and characterization of a novel peroxidase from bitter gourd (Momordica charantia).

Peroxidase from bitter gourd was purified by three step purification scheme; ammonium sulphate fractionation, gel filtration and affinity chromatography. The enzyme was purified 42 fold with the retention of 67% of the initial activity. The enzyme exhibited its maximum activity at pH 5.6 and 40 degrees C. The enzyme retained half of its activity even after 1 h incubation at 60 degrees C. Molecular weight of the purified glycosylated bitter gourd peroxidase determined by Sephacryl S-100 and SDS-PAGE was 43 kDa. The stokes radius, diffusion coefficient and sedimentation coefficient of the purified peroxidase were 27.3 A, 8.17 x 10(-7) cm(2)/sec and 3.74 S, respectively. K(m) for o-dianisidine and ABTS were 1.3 and 4.9 mM, respectively. The activity of the enzyme was inhibited by sulfide, azide and L-cysteine. The carbohydrate content and sulfydryl groups of the enzyme were 25% (w/w) mass of the protein and 16 mmoles/mole of the protein, respectively.

Relationship of cell surface hydrophobicity with biofilm formation and growth rate: A study on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli.

OBJECTIVES: This study was designed to determine the relationship of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli isolates in multispecies biofilms and their individual phenotypic characters in biofilm consortia. MATERIALS AND METHODS: The subject isolates were recovered from different food samples and identified on the basis of growth on differential and selective media. Tube methods, Congo-red agar method, and scanning electron microscopy (SEM) were used to study biofilms phenotypes. The hydrophobicity of the strains was evaluated by the adhesion to apolar solvent. RESULTS: The results showed that E. coli dominated the pre-biofilm stage. It has been observed that E. coli adopted biofilm life much before S. aureus and P. aeruginosa. However, after adopting biofilm lifestyle, slowly and gradually, P. aeruginosa dominated the consortia and dispersed other stakeholders. The subject isolates of P. aeruginosa produce cis-2-decanoic acid to disperse or inhibit S. aureus and E. coli biofilms. Gas-chromatography and mass spectrometry results showed that cis-2-decanoic was higher in the co-culture condition and increased at late log-phase or at stationary phase. Although majority of S. aureus were unable to compete with P. aeruginosa, however, a minor population competed, survived, and persisted in biofilm consortia as small colony variants. The survivors showed higher expression of sigB and sarA genes. P. aeruginosa showed comparatively higher hydrophobic surface properties. CONCLUSION: Comparative analysis showed that cell surface hydrophobicity, growth rate, and small colony variants (SCVs) are correlated in biofilm consortia of the P. aeruginosa, S. aureus, and E. coli.

Polyclonal antibodies mediated immobilization of a peroxidase from ammonium sulphate fractionated bitter gourd (Momordica charantia) proteins.

Polyclonal antibody bound Sepharose 4B support has been exploited for the immobilization of bitter gourd peroxidase directly from ammonium sulphate precipitated proteins. Immunoaffinity immobilized bitter gourd peroxidase exhibited high yield of immobilization. IgG-Sepharose 4B bound bitter gourd peroxidase showed a higher stability against heat, chaotropic agents (urea and guanidinium chloride), detergents (cetyl trimethyl ammonium bromide and Surf Excel), proteolytic enzyme (trypsin) and water-miscible organic solvents (propanol, THF and dioxane). The activity of immobilized bitter gourd peroxidase was significantly enhanced in the presence of cetyl trimethyl ammonium bromide and after treatment with trypsin as compared to soluble enzyme.

A role of glycosyl moieties in the stabilization of bitter gourd (Momordica charantia) peroxidase.

The possible role of carbohydrate moieties in the stabilization of proteins has been investigated by using bitter gourd peroxidase as a model system. A comparative study of glycosylated and non-glycosylated isoenzymes of bitter gourd peroxidase was performed at various temperatures, pH, water-miscible organic solvents, detergents and chaotropic agent like urea. The pH-optima and temperature-optima of both glycosylated and non-glycosylated isoforms of bitter gourd peroxidase remained unchanged. The probes employed were changes in the enzyme activity and fluorescence. The glycosylated form of peroxidase retained greater fraction of enzyme activity against the exposure caused by various physical and chemical denaturants. The unfolding of both forms of enzyme in the presence of high urea concentrations, studied by fluorescence, indicated greater perturbations in the conformation of non-glycosylated preparation. The different properties examined thus indicated that glycosylation plays an important role in the stabilization of native conformation of proteins against the inactivation caused by various types of denaturants.

Characterization of the T-Cell Receptor Repertoire and Immune Microenvironment in Patients with Locoregionally Advanced Squamous Cell Carcinoma of the Head and Neck.

Purpose: Squamous cell carcinoma of the head and neck (SCCHN) is a lethal cancer with a suboptimal 5-year overall survival of approximately 50% with surgery and/or definitive chemoradiotherapy. Novel treatments are thus urgently awaited. Immunotherapy with checkpoint blockade has emerged as a promising option for patients with recurrent/metastatic SCCHN; however, it has not been investigated in the curative-intent setting yet. The purpose of this study was to investigate the T-cell receptor repertoire and the tumor microenvironment in tumor tissues of SCCHN patients with locoregionally advanced disease.Experimental Design: We performed T-cell receptor sequencing of tumor tissues from 44 patients with locoregionally advanced SCCHN prior to treatment with definitive chemoradiotherapy and correlated the T-cell clonality and the mRNA expression levels of immune-related genes with clinicopathologic parameters.Results: Clonal expansion of T cells was significantly higher in human papilloma virus (HPV)-negative compared with HPV-positive tumors, signifying more robust antigen presentation in HPV-negative tumors. The latter was supported by the higher percentage of HPV-negative tumors expressing HLA-A protein compared with HPV-positive tumors (P = 0.049). Higher GRZB levels correlated significantly with longer recurrence-free survival (log-rank, P = 0.003) independent of tumor size, nodal stage, and HPV status.Conclusions: Our findings support clonal expansion of T cells in SCCHN patients with locoregionally advanced disease and imply differences in the antigen presentation capacity between HPV-negative and HPV-positive tumors. Elevated GRZB mRNA levels may also serve as a favorable and independent predictor of outcome in SCCHN patients treated with chemoradiotherapy. These data provide rationale for the introduction of immunotherapeutic approaches in the curative-intent setting. Clin Cancer Res; 23(16); 4897-907. ©2017 AACR.

HIV antigen incorporation within adenovirus hexon hypervariable 2 for a novel HIV vaccine approach.

Adenoviral (Ad) vectors have been used for a variety of vaccine applications including cancer and infectious diseases. Traditionally, Ad-based vaccines are designed to express antigens through transgene expression of a given antigen. However, in some cases these conventional Ad-based vaccines have had sub-optimal clinical results. These sub-optimal results are attributed in part to pre-existing Ad serotype 5 (Ad5) immunity. In order to circumvent the need for antigen expression via transgene incorporation, the "antigen capsid-incorporation" strategy has been developed and used for Ad-based vaccine development in the context of a few diseases. This strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. The major capsid protein hexon has been utilized for these capsid incorporation strategies due to hexon's natural role in the generation of anti-Ad immune response and its numerical representation within the Ad virion. Using this strategy, we have developed the means to incorporate heterologous peptide epitopes specifically within the major surface-exposed domains of the Ad capsid protein hexon. Our study herein focuses on generation of multivalent vaccine vectors presenting HIV antigens within the Ad capsid protein hexon, as well as expressing an HIV antigen as a transgene. These novel vectors utilize HVR2 as an incorporation site for a twenty-four amino acid region of the HIV membrane proximal ectodomain region (MPER), derived from HIV glycoprotein gp41 (gp41). Our study herein illustrates that our multivalent anti-HIV vectors elicit a cellular anti-HIV response. Furthermore, vaccinations with these vectors, which present HIV antigens at HVR2, elicit a HIV epitope-specific humoral immune response.