Synthesis, characterization, and applications of iron oxide nanoparticles.

Objective: The green synthesis method for nanoparticles is getting more attention globally, due to its lesser cost, non-hazardous, and eco-friendly nature. The novelty of the present work is to investigate the anti-bacterial and degradation activity of the green synthesized Iron Oxide NPs. Methods: In this study, the Iron Oxide NPs were synthesized through a green synthesis route from leaves of Ficus Palmata. UV-Vis confirmed Iron Oxide NP's peaks between (230-290 nm), while Fourier transforms infrared spectroscopy analysis showed that several groups were involved in reduction and stabilization. Results: Results indicated that the highest photo thermal activity was shown in light and it was almost 4 folds greater than the control. Similarly, Iron Oxide NPs showed excellent antimicrobial potential against bacterial species "Salmonella typhi" "Xanthomonas Oryzae" and "Lactobacillus" at low concentrations (150 µg/mL). Hemolytic assay results showed that the toxicity was lesser than 5% at both dark and light conditions. Moreover, we also evaluated the photo-catalytic potential of Iron Oxide NPs against methylene orange. Results indicated that almost complete degradation was noted after 90 min in the presence of continuous light. All tests were performed in triplicates. All the data was subjected to P-test (P < 0.5) using Excel and graph pad (V.5.0). Conclusion: Iron Oxide NPs holds a promising future and could be used in treating diseases, and microbial pathogenesis and also could be used as a vector in drug delivery. Moreover, they can also eradicate persistent dyes and could be used as an alternative to remediate pollutants from the environment.

Development and Validation of In-house HEp-2 Cell Slides for Detection of Antinuclear Antibodies (ANA) by Indirect Immunofluorescence.

OBJECTIVE: To develop and validate in-house HEp-2 cell slides for the detection of ANA by indirect immunofluorescence. STUDY DESIGN: Cross sectional validation study. Place and Duration of the Study: Department of Immunology, Armed Forces Institute of Pathology Rawalpindi, Punjab, Pakistan, from April to September 2022. METHODOLOGY: This study involved development of in-house HEp-2 cell slides after procuring cell lines, sub-culturing and fixing them on different slides using variety of fixatives under different protocols. After standardisation of procedure, validation of procedure was done by testing sera of 305 patients for ANA detection at 1:40 dilution on in-house HEp-2 cell slides and subsequently on commercial HEp-2 cell slides (gold standard). Indirect immunofluorescence was observed by the two observers working independently and kept blinded from the results interpreted by each other. Data were collected on a pre-designed proforma and then analysed. Sensitivity, specificity, and positive predictive value (PPV) and negative predictive values (NPV) of in-house HEp-2 cell slides were calculated. RESULT: Sera of 305 patients were tested on in-house and commercial HEp-2 cell slides. Sensitivity and specificity of in-house HEp-2 cell slides for ANA detection were 96.92% and 99.58%, respectively. PPV and NPV of in-house HEp-2 cell slides came out to be 98.43% and 99.17% respectively. CONCLUSION: In-house HEp-2 cell slides are as effective as commercial HEp-2 cell slides for the detection of ANA and can be used as cost-effective alternative. KEY WORDS: Antinuclear antibodies (ANA), Human epithelial type-2 (HEp-2), Immunofluorescence.

Investigating the Resistance Mechanism of Wheat Varieties to Fusarium Head Blight Using Comparative Metabolomics.

Fusarium head blight (FHB) is primarily caused by Fusarium graminearum and severely reduces wheat yield, causing mycotoxin contamination in grains and derived products. F. graminearum-secreted chemical toxins stably accumulate in plant cells, disturbing host metabolic homeostasis. We determined the potential mechanisms underlying FHB resistance and susceptibility in wheat. Three representative wheat varieties (Sumai 3, Yangmai 158, and Annong 8455) were inoculated with F. graminearum and their metabolite changes were assessed and compared. In total, 365 differentiated metabolites were successfully identified. Amino acids and derivatives, carbohydrates, flavonoids, hydroxycinnamate derivatives, lipids, and nucleotides constituted the major changes in response to fungal infection. Changes in defense-associated metabolites, such as flavonoids and hydroxycinnamate derivatives, were dynamic and differed among the varieties. Nucleotide and amino acid metabolism and the tricarboxylic acid cycle were more active in the highly and moderately resistant varieties than in the highly susceptible variety. We demonstrated that two plant-derived metabolites, phenylalanine and malate, significantly suppressed F. graminearum growth. The genes encoding the biosynthetic enzymes for these two metabolites were upregulated in wheat spike during F. graminearum infection. Thus, our findings uncovered the metabolic basis of resistance and susceptibility of wheat to F. graminearum and provided insights into engineering metabolic pathways to enhance FHB resistance in wheat.

Aberrant expression of myeloid antigens in patients of acute lymphoblastic leukaemia.

OBJECTIVE: To determine the immunophenotypic pattern and aberrant expression of myeloid antigens in newly diagnosed patients of acute lymphoblastic leukaemia(ALL). METHODS: This descriptive cross-sectional study was carried out in Haematology / Pathology department, Army Medical College, National University of Medical Sciences (NUMS) in collaboration with Immunology and Haematology departments of Armed Forces Institute of Pathology (AFIP), Rawalpindi from 1st January, 2019 to 31st December, 2019. Seventy-three (73) recently diagnosed patients of Acute Lymphoblastic leukaemia of all age groups and both genders were included in the study. A proforma was used to note demographic data. CBC, cytochemical stains and bone marrow examinations were carried out and assessed for morphology and percentage of blasts using a microscope. Flow cytometry was used to perform immunophenotyping on samples of peripheral blood and bone marrow, using a standard panel. RESULTS: The most commonly expressed markers were weak CD45, TdT, CD19, CD10 and HLA-DR. Weak CD45 was present in almost all blast cells and there was no remarkable difference in its positivity among various subtypes of ALL. Myeloid expression was observed in 13 (17.8%) cases. CD13 and CD33 were aberrantly expressed in 11 and 12.3 of all cases of ALL respectively. CONCLUSIONS: Expression of aberrant myeloid CD markers in acute lymphocytic leukaemia has prognostic significance and should be documented during lineage assignment of acute leukaemias while performing immunophenotyping.

HLA-DQ2 and HLA-DQ8 Alleles in Celiac Disease Patients and Healthy Controls.

OBJECTIVE: To compare HLA-DQ2 and HLA-DQ8 alleles between celiac disease patients and healthy control group. STUDY DESIGN: Observational cross-sectional study. PLACE AND DURATION OF STUDY: Department of Immunology, Armed Forces Institute of Pathology (AFIP), from April to December 2018. METHODOLOGY: Subjects were included: 100 celiac disease patients selected by non-probability consecutive sampling, and 100 healthy subjects. After collecting peripheral blood in EDTA tubes, chromosomal DNA was extracted and amplified, using sequence specific primers. Post-amplification electrophoresis was performed on two per cent agarose gel, followed by ethidium bromide staining; and specific band patterns were recorded under ultraviolet illumination to determine the HLA-DQ alleles. The subtypes of HLA-DQ2, i.e. HLA-DQ2.5 and HLA-DQ2.2 were also assessed. Frequency, percentage, mean and SD were calculated. Post-stratification Chi-square test was applied. RESULTS: The mean age of celiac disease group and healthy subjects was 14.79 ± 5.32 years and 14.71 ± 5.21 years, respectively. The frequency of HLA-DQ2 and HLA-DQ8 among celiac disease patients was 93% and 4%, respectively. Among HLA-DQ2 positive, HLA-DQ2.5 and HLA-DQ2.2 were found in 92% and 8%, respectively. Statistically significant difference (p <0.05) was observed between the celiac disease patients and healthy group. There was no significant difference observed among different age groups and gender (p >0.05). CONCLUSION: HLA-DQ2 detection reliably diagnoses celiac disease among all age groups and either gender. It can be used as an effective marker for early diagnosis of celiac disease instead of invasive procedures such as intestinal biopsy. The diagnosis can be pin-pointed by presence of HLA-DQ2.5. Key Words: Celiac disease, HLA-DQ2, HLA-DQ8, HLA-DQ2.5, HLA-DQ2.2.

Single-cell transcriptome analysis of Bisphenol A exposure reveals the key roles of the testicular microenvironment in male reproduction.

Testicular development during juvenile is crucial for subsequent male reproductive function. However, it remains poorly understood about the contribution of the testis microenvironment to human germ cell maturation. Therefore, we systematically analyzed scRNA-seq transcriptome and found the dramatic changes in cell-type composition in human testis during puberty. Then we constructed cell-cell communication networks between germ cells and somatic cells in the juvenile testis, which may be achieved via immune-related pathways. Our results showed that maturation-promoting factors are the switches of the Sertoli cells that drive sperm maturation. Furthermore, we found that Bisphenol A(BPA) enhanced the maturation and growth of germ cells through the Sertoli cell's secretory protein. Finally, our results indicate Bisphenol A would lead to the dysregulation of secreted protein expression in Sertoli cells during spermatogenesis, which in turn has direct cytotoxicity to Sertoli cells. Bisphenol A is one of the underlying causes of non-obstructive azoospermia (NOA). In summary, our results reveal the reproductive toxicity and molecular mechanism of Bisphenol A in Sertoli cells and male reproduction. Provide a reference for the toxicity of Bisphenol A to human reproduction.

Phytochemistry, antioxidant and antibacterial activities of fruit extracts of Myrsine africana L.

Myrsine africana L. a commonly consumed medicinal plant grows in forest of mountains region located at North East of Pakistan. In current study, the fruit extracts were chemically characterized and their bioactivities were determined. Higher quantity of total phenols, total flavonoids and tannins were obtained from methanolic fruit extracts. The HPLC analysis provided higher level of quercetin followed by rutin and p-coumaric acid. Whereas the GC-MS quantification had given significant level of ten saturated and unsaturated fatty acids and some of them were not reported earlier. In vitro study, lower cytotoxic behavior of fruit extracts but higher antioxidant values as well as higher zone of inhibition versus S. aureus, E. coli, K. pneumonia and B. subtilis and Mycobacterium tuberculosis were observed. The organic compounds found in fruit extracts of M. africana correlated well with its used in ethno medicines.

Impact of Homozygous C4A Deficiency on Clinical Presentation of Systemic Lupus Erythematosus.

OBJECTIVE: To investigate the association of C4A null allele (C4AQ0) with systemic lupus erythematosus (SLE) and determine the clinical presentation of SLE in relation to C4A null allele. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Armed Forces Institute of Pathology (AFIP), Rawalpindi, Immunology Department, from December 2018 to December 2019. METHODOLOGY: Patients referred to AFIP, who fulfilled American College of Rheumatology (ACR) criteria of 1997 for diagnosis of SLE were included in the study. Approval from the Institutional Ethical Review Board was taken. C4A and C4B null alleles were determined in 66 SLE patients and 40 age- and gender-matched healthy controls by polymerase chain reaction (PCR) using sequence-specific primers (PCR-SSP). Various clinical features and laboratory findings in the SLE patients were analysed in relation with C4A null allele. RESULTS: The mean age of the study population was 30.56 ±10.08 years. C4A null allele was detected in 7 (10.6%) patients; whereas, C4B null allele was detected in only two (3%) patients. SLE patients with C4A null allele had increased incidence of arthritis (100%) and renal damage (85.7%); compared to those with normal C4A allele, 57.6% and 32%, respectively. Fisher's Exact test revealed strong association of C4A null allele with arthritis and renal damage, (p = 0.039 and 0.01, respectively). CONCLUSION: Homozygous absence of C4A alleles was encountered in 10.6% of Pakistani patients of SLE and is closely related with clinical features of arthritis and renal damage. Knowledge of C4A null allele in SLE patients at diagnosis can predict disease course. Key Words: SLE, C4A null alleles, C4AQ0, Homozygous C4A deficiency.

Specific expression and alternative splicing of mouse genes during spermatogenesis.

Considering the high abundance of spliced RNAs in testis compared to other tissues, it is needed to construct the landscape of alternative splicing during spermatogenesis. However, there is still a lack of the systematic analysis of alternative RNA splicing in spermatogenesis. Here, we constructed a landscape of alternative RNA splicing during mouse spermatogenesis based on integrated RNA-seq data sets. Our results presented several novel alternatively spliced genes (Eif2s3y, Erdr1 Uty and Zfy1) in the Y chromosome with a specific expression pattern. Remarkably, the alternative splicing genes were grouped into co-expression networks involved in the microtubule cytoskeleton organization and post-transcriptional regulation of the gene expression, indicating the potential pathway to germ cell generation. Furthermore, based on the co-expression networks, we identified Atxn2l as a potential key gene in spermatogenesis, which presented dynamic expression patterns in different alternative splicing types. Ultimately, we proposed splicing regulatory networks for understanding novel and innovative alternative splicing regulation mechanisms during spermatogenesis. In summary, our research provides a systematic analysis of alternative RNA splicing and some novel spliced genes related to spermatogenesis.

Leukocyte Adhesion Deficiency Type 1 with Low Expression of CD 11b.

Leukocyte adhesion deficiency type 1 (LAD-1) is a rare autosomal recessive disorder caused by mutations in the gene that codes for CD18, the beta chain of beta-2 integrins, located on the long arm of chromosome 21. This defect results in failure of leukocyte migration to the site of infection due to the absence of surface integrins. Leukocyte adhesion deficiency should be suspected in any patient with recurrent infections, impaired wound healing, history of delayed umbilical cord separation, periodontitis, leukocytosis, recurrent soft tissue and oral infections. Diagnosis is based on the analysis of neutrophils for the surface expression of CD18, CD11a, CD11b and CD11c by flow cytometry. Here, we present a 55-day male infant with umbilical cord separation on the 10th day of life and no history of infection, who was identified with LAD-1 with low expression of CD11b. The purpose of performing LAD flow cytometric analysis in this patient was to screen him for LAD-1 as his elder brother had LAD-1 and one elder sister died undiagnosed with recurrent skin and chest infections at 8 months of age.

Genome-wide analysis of family-1 UDP glycosyltransferases (UGT) and identification of UGT genes for FHB resistance in wheat (Triticum aestivum L.).

BACKGROUND: Fusarium head blight (FHB), a devastating disease in wheat worldwide, results in yield loses and mycotoxin, such as deoxynivalenol (DON), accumulation in infected grains. DON also facilitates the pathogen colonization and spread of FHB symptoms during disease development. UDP-glycosyltransferase enzymes (UGTs) are known to contribute to detoxification and enhance FHB resistance by glycosylating DON into DON-3-glucoside (D3G) in wheat. However, a comprehensive investigation of wheat (Triticum aestivum) UGT genes is still lacking. RESULTS: In this study, we carried out a genome-wide analysis of family-1 UDP glycosyltransferases in wheat based on the PSPG conserved box that resulted in the identification of 179 putative UGT genes. The identified genes were clustered into 16 major phylogenetic groups with a lack of phylogenetic group K. The UGT genes were invariably distributed among all the chromosomes of the 3 genomes. At least 10 intron insertion events were found in the UGT sequences, where intron 4 was observed as the most conserved intron. The expression analysis of the wheat UGT genes using both online microarray data and quantitative real-time PCR verification suggested the distinct role of UGT genes in different tissues and developmental stages. The expression of many UGT genes was up-regulated after Fusarium graminearum inoculation, and six of the genes were further verified by RT-qPCR. CONCLUSION: We identified 179 UGT genes from wheat using the available sequenced wheat genome. This study provides useful insight into the phylogenetic structure, distribution, and expression patterns of family-1 UDP glycosyltransferases in wheat. The results also offer a foundation for future work aimed at elucidating the molecular mechanisms underlying the resistance to FHB and DON accumulation.

Molecular Characterization and Expression of PFT, an FHB Resistance Gene at the Fhb1 QTL in Wheat.

Fusarium head blight (FHB) is a destructive fungal disease in wheat worldwide. Efforts have been carried out to combat this disease, and the pore-forming toxin-like (PFT) gene at the quantitative trait locus (QTL) Fhb1 was isolated and found to confer resistance to FHB in Sumai 3. In this study, we characterized PFT in 348 wheat accessions. Four haplotypes of PFT were identified. The wild haplotype of PFT had higher resistance than other haplotypes and explained 13.8% of phenotypic variation in FHB resistance by association analysis. PFT was highly expressed during early flowering and increased after Fusarium graminearum treatment in Sumai 3. Analysis of the 5' flanking sequence of PFT predicted that the cis elements of the PFT promoter were related to hormones and biological defense responses. However, PFT existed not only in the FHB-resistant accessions but also in some susceptible accessions. These results suggested that FHB resistance in a diverse range of wheat genotypes is partially conditioned by PFT. The profiling of FHB resistance and the PFT locus in this large collection of wheat germplasm may prove helpful for incorporating FHB resistance into wheat breeding programs, although more work is needed to reveal the exact role of the QTL Fhb1 in conferring resistance to fungal spread.

Assessing Utilization and Environmental Risks of Important Genes in Plant Abiotic Stress Tolerance.

Transgenic plants with improved salt and drought stress tolerance have been developed with a large number of abiotic stress-related genes. Among these, the most extensively used genes are the glycine betaine biosynthetic codA, the DREB transcription factors, and vacuolar membrane Na(+)/H(+) antiporters. The use of codA, DREBs, and Na(+)/H(+) antiporters in transgenic plants has conferred stress tolerance and improved plant phenotype. However, the future deployment and commercialization of these plants depend on their safety to the environment. Addressing environmental risk assessment is challenging since mechanisms governing abiotic stress tolerance are much more complex than that of insect resistance and herbicide tolerance traits, which have been considered to date. Therefore, questions arise, whether abiotic stress tolerance genes need additional considerations and new measurements in risk assessment and, whether these genes would have effects on weediness and invasiveness potential of transgenic plants? While considering these concerns, the environmental risk assessment of abiotic stress tolerance genes would need to focus on the magnitude of stress tolerance, plant phenotype and characteristics of the potential receiving environment. In the present review, we discuss environmental concerns and likelihood of concerns associated with the use of abiotic stress tolerance genes. Based on our analysis, we conclude that the uses of these genes in domesticated crop plants are safe for the environment. Risk assessment, however, should be carefully conducted on biofeedstocks and perennial plants taking into account plant phenotype and the potential receiving environment.

Utilization of genes encoding osmoprotectants in transgenic plants for enhanced abiotic stress tolerance

Global agriculture in the context of growing and expanding populations is under huge pressure to provide increased food, feed, and fiber. The recent phenomenon of climate change has further added fuel to the fire. It has been practically established now that the global temperature has been on the increase with associated fluctuations in annual rainfall regimes, and the resultant drought and flood events and increasing soil and water salinization. These challenges would be met with the introduction and utilization of new technologies coupled with conventional approaches. In recent years, transgenic technology has been proved very effective in terms of production of improved varieties of crop plants, resistant to biotic stresses. The abiotic stresses such as salt and drought are more complex traits, controlled by many genes. Transgenic plant development for these stresses has utilized many single genes. However, much emphasis has been placed on genes catalyzing the biosynthetic pathways of osmoprotectants. This review focuses on the current status of research on osmoprotectant genes and their role in abiotic stress tolerance in transgenic plants.

Antimicrobial activity of different solvent extracted samples from the flowers of medicinally important Plumeria obstusa.

The present research work was carried out to investigate the antimicrobial (eight bacteria and one fungus) activities of different solvent (ethanol, petroleum ether, chloroform, ethyl acetate and isobutanol) extracted samples from flowers of P. obstusa by disc diffusion method. Analysis of the data revealed that all the five extracts from flowers of P. obstusa showed different ranges of antimicrobial activities. Petroleum ether fractions showed inhibitory activities against all the nine microbial species except Klebsiella pneumonia. Ethyl acetate and isobutanol fractions showed inhibitory effects against all the tested microbial species except Pseudomonas aeruginosa. Chloroform and ethanol extracts had varying levels of inhibitions against all of the tested microorganisms. The most susceptible gram positive bacterium was Bacillus subtilis which was inhibited by all the five extracts while the most resistant gram positive bacterium was Staphylococcus aureus. Erwinia carotovora was the most susceptible gram negative bacterium while Pseudomonas aeruginosa was highly resistant among the gram negative bacteria.

Characterization of the genetic structure of mango ginger (Curcuma amada Roxb) from Myanmar in farm and genebank collection by the neutral and functional genomic markers

A preliminary characterization was undertaken to describe genetic structure of mango ginger (Curcuma amada) acquired from farmers and ex situ genebank in Myanmar using neutral (rice SSR based RAPDs) and functional genomic (P450 based analog) markers. The high polymorphism (> 91 percent) depicted has displayed existence of genetic variability in the germplasm investigated. Large number of source-specific alleles (neutral-markers = 78, functional-markers = 63) was amplified which revealed that neutral regions of the mango ginger were more variable compared with the functional regions. The major fraction of the molecular variance (neutral-markers = 85 percent, functional-markers = 93 percent) was explained within germplasm acquisition sources and this tendency was also supported by the estimate of gene diversity. The genebank accessions have shown comparatively more genetic variability than farmers' accessions. The variability observed in mango ginger may possibly be associated with the long history of its cultivation under diverse ecological conditions. The two marker systems elucidated their high resolving power which detected variability even in fewer genotypes assayed. As the target sites of these markers are different, therefore, the variability detected is believed to cover diverse part of the genome together with neutral and functional regions. We found the concurrent use of the different types of molecular markers valuable to comprehend a dependable variability pattern in the germplasm assayed.