Neutralizing antibodies to interferon alfa arising during peginterferon therapy of chronic hepatitis B in children and adults: Results from the HBRN Trials.

BACKGROUND AIMS: Pegylated interferon-α (PegIFNα) is of limited utility during immunotolerant or immune active phases of chronic hepatitis B infection but is being explored as part of new cure regimens. Low/absent levels of IFNα found in some patients receiving treatment are associated with limited/no virological responses. The study aimed to determine if sera from participants inhibit IFNα activity and/or contain therapy-induced anti-IFNα antibodies. APPROACH RESULTS: Pre-treatment, on-treatment, and post-treatment sera from 61 immunotolerant trial participants on PegIFNα/entecavir therapy and 88 immune active trial participants on PegIFNα/tenofovir therapy were screened for anti-IFNα antibodies by indirect ELISA. The neutralization capacity of antibodies was measured by preincubation of sera±recombinant human IFNα added to Huh7 cells with the measurement of interferon-stimulated gene (ISG)-induction by qPCR. Correlations between serum-induced ISG inhibition, presence, and titer of anti-IFNα antibodies and virological responses were evaluated. Preincubation of on-treatment serum from 26 immunotolerant (43%) and 13 immune active (15%) participants with recombinant-human IFNα markedly blunted ISG-induction in Huh7 cells. The degree of ISG inhibition correlated with IFNα antibody titer ( p < 0.0001; r = 0.87). On-treatment development of anti-IFNα neutralizing antibodies (nAbs) was associated with reduced quantitative HBsAg and qHBeAg declines ( p < 0.05) and inhibited IFNα bioactivity to 240 weeks after PegIFNα cessation. Children developed anti-IFNα nAbs more frequently than adults ( p = 0.004) but nAbs in children had less impact on virological responses. CONCLUSIONS: The development of anti-IFNα nAbs during PegIFNα treatment diminishes responses to antiviral therapy. Understanding how and why anti-IFNα antibodies develop may allow for the optimization of IFN-based therapy, which is critical given its renewed use in HBV-cure strategies.

A combined experimental-computational approach uncovers a role for the Golgi matrix protein Giantin in breast cancer progression.

Our understanding of how speed and persistence of cell migration affects the growth rate and size of tumors remains incomplete. To address this, we developed a mathematical model wherein cells migrate in two-dimensional space, divide, die or intravasate into the vasculature. Exploring a wide range of speed and persistence combinations, we find that tumor growth positively correlates with increasing speed and higher persistence. As a biologically relevant example, we focused on Golgi fragmentation, a phenomenon often linked to alterations of cell migration. Golgi fragmentation was induced by depletion of Giantin, a Golgi matrix protein, the downregulation of which correlates with poor patient survival. Applying the experimentally obtained migration and invasion traits of Giantin depleted breast cancer cells to our mathematical model, we predict that loss of Giantin increases the number of intravasating cells. This prediction was validated, by showing that circulating tumor cells express significantly less Giantin than primary tumor cells. Altogether, our computational model identifies cell migration traits that regulate tumor progression and uncovers a role of Giantin in breast cancer progression.

Development and validation of a universal primer pair for the taxonomic and phylogenetic studies of vertebrates.

BACKGROUND: Recent studies in the field of molecular identification have described 16S rRNA gene as a highly informative fragment of mitochondrial DNA for species discrimination. This study presents a newly developed universal primer pair yielding an approximately 350 bp fragment of mitochondrial 16S rRNA, variable enough to encompass and identify all vertebrate classes. METHODS AND RESULTS: The primers were designed by aligning and analyzing over 1500 16S rRNA sequences downloaded from the NCBI nucleotide database. A total of 93 vertebrate species, spanning 27 orders and 55 families, were PCR-amplified to validate the primers. All the target species were successfully amplified and identified when aligned with reference sequences from the NCBI nucleotide database. Using the Kimura 2-parameter model, low intra-species genetic divergence of the target region was observed - from 0 to 4.63%, whereas relatively higher inter-species genetic divergence was observed, ranging from 4.88% to 69.81%. Moreover, the newly developed primers were successfully applied to a direct PCR protocol, making the workflow very cost-effective, time-saving and less laborious in comparison to conventional PCR. CONCLUSIONS: The short length, high variability and conserved priming sites of the target fragment across all vertebrate species make it a highly desirable marker for species identification and discrimination.

Successful application of modified crude DNA extraction from muscle tissues for various types of PCR amplifications.

BACKGROUND: One of the most challenging aspects of nucleic acid amplification tests is the extraction of genomic DNA. However, achieving satisfactory quality and quantity of genomic DNA is not always easy, while the demand for rapid, low-cost and less laborious DNA isolation methods is ever-increasing. METHODS AND RESULTS: We have developed a rapid (⁓2 min) crude DNA extraction method leading to direct-PCR that requires minimum reagents and laboratory equipment. It was developed by eliminating the time-consuming purification steps of DNA extraction, by processing the sample in optimized amounts of Taq KCl PCR buffer and DNARelease Additive/Proteinase K in only two minutes and carrying out amplification using conventional Taq DNA polymerase. The DNA preparation method was validated on muscle tissue samples from 12 different species as well as 48 cooked meat samples. Its compatibility was also successfully tested with different types of PCR amplification platforms extensively used for genetic analysis, such as simplex PCR, PCR-RFLP (Restriction Fragment Length Polymorphism), multiplex PCR, isothermal amplification, real-time PCR and DNA sequencing. CONCLUSIONS: The developed protocol provides sufficient amount of crude DNA from muscle tissues of different species for PCR amplifications to identify species-of-origin via different techniques coupled with PCR. The simplicity and robustness of this protocol make nucleic acid amplification assays more accessible and affordable to researchers and authorities for both laboratory and point-of-care tests.

Nanoparticles in plant resistance against bacterial pathogens: current status and future prospects.

Nanoparticles (NPs) serve immense roles in various fields of science. They have vastly upgraded conventional methods in the fields of agriculture and food sciences to eliminate growing threats of crop damage and disease, caused by various phytopathogens including bacteria, fungi, viruses, and some insects. Bacterial diseases resulted in mass damage of crops by adopting antibacterial resistance, which has proved to be a major threat leading to food scarcity. Therefore, numerous NPs with antibacterial potentials have been formulated to overcome the problem of antibiotic resistance alongside an increase in crop yield and boosting plant immunity. NPs synthesized through green synthesis techniques have proved to be more effective and environment-friendly than those synthesized via chemical methods. NPs exhibit great roles in plants ranging from enhanced crop yield to disease suppression, to targeted drug and pesticide deliveries inside the plants and acting as biosensors for pathogen detection. NPs serves major roles in disruption of cellular membranes, ROS production, altering of DNA and protein entities and changing energy transductions. This review focuses on the antibacterial effect of NPs on several plant bacterial pathogens, mostly, against Pseudomonas syringe, Ralstonia solanacearum, Xanthomonas axonopodis, Clavibacter michiganensisand Pantoea ananatis both in vivo and ex vivo, thereby minimizing their antibacterial resistance and enhancing the plants acquired immunity. Therefore, NPs present a safer and more reliable bactericidal activity against various disease-causing bacteria in plants.

Effect of naringenin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm.

Our objectives were to explore the effect of naringenin addition in the semen extender on the post-thaw 1) sperm quality, 2) fertility-associated gene expression, and 3) fertilization potential of buffalo bull sperm. In experiment 1, semen samples (n = 32) from four Nili-Ravi buffalo bulls were pooled (n = 8) and diluted with the tris-citric acid (TCF-EY) extender containing different concentrations of naringenin, i.e., placebo (DMSO), 0 (control), 50, 100, 150 and 200 µM naringenin. After dilution, semen samples were packed in 0.5 mL French straws, cryopreserved and analyzed for post-thawed sperm quality and gene expression. Computer-assisted Semen Analysis, Hypo-osmotic Swelling test, Normal Apical Ridge assay, Rhodamine 123, Acridine orange, Propidium iodide staining and Thiobarbituric Acid Reactive Substances assay were performed to assess sperm motility parameters, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. Results revealed that total and progressive motility, plasma membrane functionality, acrosome integrity, mitochondrial membrane potential, DNA integrity and viability were higher (P < 0.05) with 50, 100 and 150 µM naringenin compared to 200 µM naringenin, placebo and control groups. Moreover, all naringenin-treated groups improved catalase activity, and reduced lipid peroxidation compared to placebo and control groups (P < 0.05). Relative expression levels of SPACA3 and PRM1 genes were higher (P < 0.05) with 150µM naringenin compared to all groups except 100 µM (P > 0.05). No difference (P > 0.05) in the expression level of BCL2 gene was observed among all groups. Furthermore, BAX gene was expressed higher (P < 0.05) in the 200 µM naringenin group, whereas no difference (P > 0.05) in expression was noticed among the remaining groups. In addition, ROMO1 gene was expressed lower (P < 0.05) in all naringenin-treated groups compared to the control. In experiment 2, the in vivo fertility of semen doses (n = 400; 200/group) containing optimum concentration of naringenin (150 µM; depicted better in vitro sperm quality in experiment 1) was compared with control during the breeding season. Buffaloes were inseminated 24 h after the onset of natural estrus and palpated transrectal for pregnancy at least 60 days post-insemination. The fertility rate of 150 µM naringenin group was higher (P = 0.0366) compared to the control [57.00 ± 0.03% (114/200) vs. 46.50 ± 0.04% (93/200), respectively]. Taken together, it is concluded that naringenin supplementation in semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm, more apparently at 150 µM concentration.

IgA potentiates NETosis in response to viral infection.

IgA is the second most abundant antibody present in circulation and is enriched at mucosal surfaces. As such, IgA plays a key role in protection against a variety of mucosal pathogens including viruses. In addition to neutralizing viruses directly, IgA can also stimulate Fc-dependent effector functions via engagement of Fc alpha receptors (Fc-αRI) expressed on the surface of certain immune effector cells. Neutrophils are the most abundant leukocyte, express Fc-αRI, and are often the first to respond to sites of injury and infection. Here, we describe a function for IgA-virus immune complexes (ICs) during viral infections. We show that IgA-virus ICs potentiate NETosis-the programmed cell-death pathway through which neutrophils release neutrophil extracellular traps (NETs). Mechanistically, IgA-virus ICs potentiated a suicidal NETosis pathway via engagement of Fc-αRI on neutrophils through a toll-like receptor-independent, NADPH oxidase complex-dependent pathway. NETs also were capable of trapping and inactivating viruses, consistent with an antiviral function.

Ameliorative effect of crocin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm.

Buffalo bull sperm suffer more cryoinjuries due to lipid peroxidation of high structural polyunsaturated fatty acid contents than cattle sperm. Consequently, the post-thaw fertilization potential of buffalo bull sperm is compromised. Crocin is a carotenoid known for its antioxidant potential through scavenging reactive oxygen species. Objectives of the current study were to investigate the effect of crocin addition in the semen extender on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm. Semen samples (n = 32) from four Nili-Ravi buffalo bulls were extended with tris-citric acid extender containing different concentrations of crocin (0 mM; control, 0.5, 1, 1.5 and 2 mM). The extended semen was packed in 0.5 mL French straws (25 × 106 sperm/straw) and cryopreserved in liquid nitrogen. Computer-assisted semen analysis, hypo-osmotic swelling test, normal apical ridge assay, Rhodamine 123, acridine orange, propidium iodide staining, and thiobarbituric acid reactive substances assay were performed to assess sperm motility parameters, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability, and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. The fertility of semen doses containing the most potent concentration of crocin (based on optimum post-thaw semen quality) was compared with control during the breeding season. Buffaloes (n = 400; 200/group) were inseminated 24 h after the onset of oestrus and transrectally palpated for pregnancy at least 60 days post-insemination. Results revealed that 0.5 and 1 mM crocin improved sperm post-thaw total motility, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential and viability, and 1 and 1.5 mM crocin enhanced catalase activity and reduced lipid peroxidation compared to control (p < .05). Moreover, 1 mM crocin improved sperm post-thaw progressive motility, kinematics, and DNA integrity, and 1.5 mM crocin enhanced plasma membrane integrity than control (p < .05). Expression levels of SPACA3, PRM1 and BCL2 genes were higher (p < .05) with 1 mM crocin compared to other groups. In contrast, no difference (p > .05) was noticed in expressions of BAX and ROMO1 genes among all groups. The fertility rate of semen doses containing the most potent concentration (1 mM) of crocin was higher (p = .0465) compared to control (56 ± 0.03% vs. 46 ± 0.04%, respectively). In conclusion, 1 mM crocin in the semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm.

Phytochemical-mediated regulation of aflatoxigenic fungi contamination in a shifting climate and environment.

Mycotoxin contamination poses a significant problem in developing countries, particularly in northern Pakistan's fluctuating climate. This study aimed to assess aflatoxin contamination in medicinal and condiment plants in Upper Dir (dry-temperate) and Upper Swat (moist-temperate) districts. Plant samples were collected and screened for mycotoxins (Aflatoxin-B1 and Aflatoxin-B-2). Results showed high levels of AFB-1 (11,505.42 ± 188.82) as compared to AFB-2 (846 ± 241.56). The maximum contamination of AFB-1 in Coriandrum sativum (1154.5 ± 13.43 ng to 3328 ± 9.9 ng) followed by F. vulgare (883 ± 9.89 ng to 2483 ± 8.4 ng), T. ammi (815 ± 11.31 ng to 2316 ± 7.1 ng), and C. longa (935.5 ± 2.12 ng to 2009 ± 4.2 ng) while the minimum was reported in C. cyminum (671 ± 9.91 ng to 1995 ± 5.7 ng). Antifungal tests indicated potential resistance in certain plant species (C. cyminum) while A. flavus as the most toxins contributing species due to high resistance below 80% (54.2 ± 0.55 to 79.5 ± 2.02). HPLC analysis revealed hydroxyl benzoic acid (5136 amu) as the dominant average phytochemical followed by phloroglucinol (4144.31 amu) with individual contribution of 8542.08 amu and 12,181.5 amu from C. cyaminum. The comparison of average phytochemicals revealed the maximum concentration in C. cyminum (2885.95) followed by C. longa (1892.73). The findings revealed a statistically significant and robust negative correlation (y = - 2.7239 × + 5141.9; r = - 0.8136; p < 0.05) between average mycotoxins and phytochemical concentrations. Temperature positively correlated with aflatoxin levels (p < 0.01), while humidity had a weaker correlation. Elevation showed a negative correlation (p < 0.05), while geographical factors (latitude and longitude) had mixed correlations (p < 0.05). Specific regions exhibited increasing aflatoxin trends due to climatic and geographic factors.

LAMP3/CD63 Expression in Early and Late Endosomes in Human Vaginal Epithelial Cells Is Associated with Enhancement of HSV-2 Infection.

Herpes simplex virus 2 (HSV-2) is a lifelong sexually transmitted virus that disproportionately infects women through heterosexual transmission in the vaginal tract. The vaginal epithelium is known to be highly susceptible to HSV-2 infection; however, the cellular mechanism of HSV-2 uptake and replication in vaginal epithelium has not been extensively studied. Previously, we observed that lysosomal-associated membrane protein-3 (LAMP3/CD63) was among the highly upregulated genes during HSV-2 infection of human vaginal epithelial cell line VK2, leading us to posit that LAMP3/CD63 may play a role in HSV-2 infection. Consequently, we generated two gene-altered VK2-derived cell lines, a LAMP3-overexpressed (OE) line and a LAMP3 knockout (KO) line. The wild-type VK2 and the LAMP3 OE and KO cell lines were grown in air-liquid interface (ALI) cultures for 7 days and infected with HSV-2. Twenty-four hours postinfection, LAMP3 OE cells produced and released significantly higher numbers of HSV-2 virions than wild-type VK2 cells, while virus production was greatly attenuated in LAMP3 KO cells, indicating a functional association between LAMP3/CD63 expression and HSV-2 replication. Fluorescence microscopy of HSV-2-infected cells revealed that HSV-2 colocalized with LAMP3 in both early endosomes and lysosomal compartments. In addition, blocking endosomal maturation or late endosomal/lysosomal fusion using specific inhibitors resulted in reduced HSV-2 replication in VK2 cells. Similarly, LAMP3 KO cells exhibited very low viral entry and association with endosomes, while LAMP3 OE cells demonstrated large amounts of virus that colocalized with LAMP3/CD63 in endosomes and lysosomes. IMPORTANCE Collectively, these results showed that HSV-2 is taken up by human vaginal epithelial cells through an endosomal-lysosomal pathway in association with LAMP3, which plays a crucial role in the enhancement of HSV-2 replication. These findings provide the basis for the future design of antiviral agents for prophylactic measures against HSV-2 infection.

OXGR1 is a candidate disease gene for human calcium oxalate nephrolithiasis.

PURPOSE: Nephrolithiasis (NL) affects 1 in 11 individuals worldwide, leading to significant patient morbidity. NL is associated with nephrocalcinosis (NC), a risk factor for chronic kidney disease. Causative genetic variants are detected in 11% to 28% of NL and/or NC, suggesting that additional NL/NC-associated genetic loci await discovery. Therefore, we employed genomic approaches to discover novel genetic forms of NL/NC. METHODS: Exome sequencing and directed sequencing of the OXGR1 locus were performed in a worldwide NL/NC cohort. Putatively deleterious, rare OXGR1 variants were functionally characterized. RESULTS: Exome sequencing revealed a heterozygous OXGR1 missense variant (c.371T>G, p.L124R) cosegregating with calcium oxalate NL and/or NC disease in an autosomal dominant inheritance pattern within a multigenerational family with 5 affected individuals. OXGR1 encodes 2-oxoglutarate (α-ketoglutarate [AKG]) receptor 1 in the distal nephron. In response to its ligand AKG, OXGR1 stimulates the chloride-bicarbonate exchanger, pendrin, which also regulates transepithelial calcium transport in cortical connecting tubules. Strong amino acid conservation in orthologs and paralogs, severe in silico prediction scores, and extreme rarity in exome population databases suggested that the variant was deleterious. Interrogation of the OXGR1 locus in 1107 additional NL/NC families identified 5 additional deleterious dominant variants in 5 families with calcium oxalate NL/NC. Rare, potentially deleterious OXGR1 variants were enriched in patients with NL/NC compared with Exome Aggregation Consortium controls (χ2 = 7.117, P = .0076). Wild-type OXGR1-expressing Xenopus oocytes exhibited AKG-responsive Ca2+ uptake. Of 5 NL/NC-associated missense variants, 5 revealed impaired AKG-dependent Ca2+ uptake, demonstrating loss of function. CONCLUSION: Rare, dominant loss-of-function OXGR1 variants are associated with recurrent calcium oxalate NL/NC disease.

CRISPR Cas9 mediated knockout of sex determination pathway genes in Aedes aegypti.

The vector role of Aedes aegypti for viral diseases including dengue and dengue hemorrhagic fever makes it imperative for its proper control. Despite various adopted control strategies, genetic control measures have been recently focused against this vector. CRISPR Cas9 system is a recent and most efficient gene editing tool to target the sex determination pathway genes in Ae. aegypti. In the present study, CRISPR Cas9 system was used to knockout Ae. aegypti doublesex (Aaedsx) and Ae. aegypti sexlethal (AaeSxl) genes in Ae. aegypti embryos. The injection mixes with Cas9 protein (333 ng ul-1) and gRNAs (each at 100 ng ul-1) were injected into eggs. Injected eggs were allowed to hatch at 26 ± 1°C, 60 ± 10% RH. The survival and mortality rate was recorded in knockout Aaedsx and AaeSxl. The results revealed that knockout produced low survival and high mortality. A significant percentage of eggs (38.33%) did not hatch as compared to control groups (P value 0.00). Highest larval mortality (11.66%) was found in the knockout of Aaedsx female isoform, whereas, the emergence of only male adults also showed that the knockout of Aaedsx (female isoform) does not produce male lethality. The survival (3.33%) of knockout for AaeSxl eggs to the normal adults suggested further study to investigate AaeSxl as an efficient upstream of Aaedsx to target for sex transformation in Ae. aegypti mosquitoes.

Synthesis and Characterization of MIPs for Selective Removal of Textile Dye Acid Black-234 from Wastewater Sample.

Herein, a molecularly imprinted polymer (MIP) was prepared using bulk polymerization and applied to wastewater to aid the adsorption of targeted template molecules using ethylene glycol dimethacrylate (EGDMA), methacrylic acid (MAA), acid black-234 (AB-234), 2,2'-azobisisobutyronitrile (AIBN), and methanol as a cross linker, functional monomer, template, initiator, and porogenic solvent, respectively. For a non-molecularly imprinted polymer (NIP), the same procedure was followed but without adding a template. Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and a surface area analyzer were used to determine the surface functional groups, morphology and specific surface area of the MIP and NIP. At pH 5, the AB-234 adsorption capability of the MIP was higher (94%) than the NIP (31%). The adsorption isotherm data of the MIP correlated very well with the Langmuir adsorption model with Qm 82, 83 and 100 mg/g at 283 K, 298 K, and 313 K, respectively. The adsorption process followed pseudo-second-order kinetics. The imprinted factor (IF) and Kd value of the MIP were 5.13 and 0.53, respectively. Thermodynamic studies show that AB-234 dye adsorption on the MIP and NIP was spontaneous and endothermic. The MIP proved to be the best selective adsorbent for AB-234, even in the presence of dyes with similar and different structures than the NIP.

Rapid Detection of Cd2+ Ions in the Aqueous Medium Using a Highly Sensitive and Selective Turn-On Fluorescent Chemosensor.

Herein, a novel optical chemosensor, (CM1 = 2, 6-di((E)-benzylidene)-4-methylcyclohexan-1-one), was designed/synthesized and characterized by 1H-NMR and FT-IR spectroscopy. The experimental observations indicated that CM1 is an efficient and selective chemosensor towards Cd2+, even in the presence of other metal ions, such as Mn2+, Cu2+, Co2+, Ce3+, K+, Hg2+,, and Zn2+ in the aqueous medium. The newly synthesized chemosensor, CM1, showed a significant change in the fluorescence emission spectrum upon coordination with Cd2+. The formation of the Cd2+ complex with CM1 was confirmed from the fluorometric response. The 1:2 combination of Cd2+ with CM1 was found optimum for the desired optical properties, which was confirmed through fluorescent titration, Job's plot, and DFT calculation. Moreover, CM1 showed high sensitivity towards Cd2+ with a very low detection limit (19.25 nM). Additionally, the CM1 was recovered and recycled by the addition of EDTA solution that combines with Cd2+ ion and, hence, frees up the chemosensor.

Comparative evaluation of different moulting methods on productive performance, egg quality, and antibody response of leghorn hens.

The aim of the study was to compare three moulting procedures and their effect on productive performance, egg quality, and antibody response of leghorn hens. For this, a total of 324 laying hens were distributed into three treatment groups having 12 replicates of 9 birds each according to completely randomized design. Treatments consisted of three moulting procedures based on feed and light restriction. The targeted weight at the end of moulting was 1450-1470 g. As soon as the moulting procedure is complete, the comparative analysis of post-moult productive performance (feed intake, egg production, egg weight, egg mass, feed per dozen eggs, feed per kg egg mass, livability), egg quality characteristics (egg weight, egg length and width, shape index, surface area, volume, albumen height, weight, Haugh unit score, yolk width, height, index, egg shell pore number, shell weight, thickness, breaking strength), and antibody response against Newcastle disease and avian influenza (H-9) were evaluated. Birds subjected to moulting procedure 3 (8 days fasting and gradual decrease in light) showed improvement in productive performance, egg geometry and quality traits, and antibody response against Newcastle disease virus. Birds experienced moulting procedure 1 (11 days fasting) had improved feed intake, egg production, and livability. However, birds moulted with procedure 2 (6 days fasting) revealed intermediate result in all the studied parameters. In conclusion, moulting through feed and light restriction with 8 days fasting and gradual reduction in light has potential to improve performance of leghorn hens.

Differential Effects of Reactive Oxygen Species on IgG versus IgM Levels in TLR-Stimulated B Cells.

It is becoming increasingly evident that reactive oxygen species (ROS) have critical roles as "second messengers" in cell signaling. In B cells, ROS can be generated either as a byproduct of mitochondrial respiration, as a result of the endoplasmic reticulum stress response induced by high production of Igs, or by the activation of NADPH oxidase (NOX) complexes. Having previously shown that costimulation of B cells via TLR 9 and the TLR-related receptor RP105 drives maturation of human peripheral blood B cells into Ig-producing cells, we aimed to study the role of ROS generated during this vital process. To this end, the ROS levels were either reduced by the NOX inhibitor VAS2870 or by the ROS scavenger N-acetyl cysteine (NAC). We revealed that TLR9/RP105-mediated stimulation of human B cells involved a rapid activation of NOX. Moreover, VAS2870 blocked the TLR9/RP105-induced B cell activation and thereby all Ig production. Importantly, we showed that ROS targeted by NAC was selectively required for IgG but not for IgM production. The endoplasmic reticulum stress response in the TLR9/RP105-stimulated cells was higher in IgG+ than in IgG- cells and was reduced by NAC in IgG+ cells only. Of note, we revealed that substantially higher levels of IgG than IgM were produced per cell and that IgG+ cells produced significantly higher ROS levels than IgG- cells. Taken together, our results imply that NAC-targeted ROS may be particularly important for sustaining the high Ig production in IgG+ B cells.

Bacillus subtilis: As an Efficient Bacterial Strain for the Reclamation of Water Loaded with Textile Azo Dye, Orange II.

The azo dye orange II is used extensively in the textile sector for coloring fabrics. High concentrations of it are released into aqueous environments through textile effluents. Therefore, its removal from textile wastewater and effluents is necessary. Herein, initially, we tested 11 bacterial strains for their capabilities in the degradation of orange II dye. It was revealed in the preliminary data that B. subtilis can more potently degrade the selected dye, which was thus used in the subsequent experiments. To achieve maximum decolorization, the experimental conditions were optimized whereby maximum degradation was achieved at: a 25 ppm dye concentration, pH 7, a temperature of 35 °C, a 1000 mg/L concentration of glucose, a 1000 mg/L urea concentration, a 666.66 mg/L NaCl concentration, an incubation period of 3 days, and with hydroquinone as a redox mediator at a concentration of 66.66 mg/L. The effects of the interaction of the operational factors were further confirmed using response surface methodology, which revealed that at optimum conditions of pH 6.45, a dye concentration of 17.07 mg/L, and an incubation time of 9.96 h at 45.38 °C, the maximum degradation of orange II can be obtained at a desirability coefficient of 1, estimated using the central composite design (CCD). To understand the underlying principles of degradation of the metabolites in the aliquot mixture at the optimized condition, the study steps were extracted and analyzed using GC-MS(Gas Chromatography Mass Spectrometry), FTIR(Fourier Transform Infrared Spectroscopy), 1H and carbon 13 NMR(Nuclear Magnetic Resonance Spectroscopy). The GC-MS pattern revealed that the original dye was degraded into o-xylene and naphthalene. Naphthalene was even obtained in a pure state through silica gel column isolation and confirmed using 1H and 13C NMR spectroscopic analysis. Phytotoxicity tests on Vigna radiata were also conducted and the results confirmed that the dye metabolites were less toxic than the parent dye. These results emphasize that B. subtilis should be used as a potential strain for the bioremediation of textile effluents containing orange II and other toxic azo dyes.

Separation of Mandelic Acid by a Reactive Extraction Method Using Tertiary Amine in Different Organic Diluents.

Mandelic acid is a valuable chemical that is commonly used in the synthesis of various drugs, in antibacterial products, and as a skin care agent in cosmetics. As it is an important chemical, various methods are used to synthesize and extract this compound. However, the yields of the used processes is not significant. A dilute aqueous solution is obtained when using several production methods, such as a fermentation, etc. In this study, the reactive extraction of mandelic acid from aqueous solutions using tri-n-octylamine extractant at 298.15 K was investigated. Dimethyl phthalate (DMP), methyl isobutyl ketone (MIBK), 2-octanone, 1-octanol, n-pentane, octyl acetate, and toluene were used as diluents. The batch extraction results of the mandelic acid experiments were obtained for the development of a process design. Calculations of the loading factor (Z), distribution coefficient (D), and extraction efficiency (E%) were based on the experimental data. The highest separation yield was obtained as 98.13% for 0.458 mol.L-1 of tri-n-octylamine concentration in DMP. The overall extraction constants were analyzed for the complex of acid-amine by the Bizek approach, including K11, K12, and K23.

The Antibacterial and Larvicidal Potential of Bis-(2-Ethylhexyl) Phthalate from Lactiplantibacillus plantarum.

Lactic acid bacteria produce a variety of antibacterial and larvicidal metabolites, which could be used to cure diseases caused by pathogenic bacteria and to efficiently overcome issues regarding insecticide resistance. In the current study, the antibacterial and larvicidal potential of Bis-(2-ethylhexyl) phthalate isolated from Lactiplantibacillus plantarum BCH-1 has been evaluated. Bioactive compounds were extracted by ethyl acetate and were fractionated by gradient column chromatography from crude extract. Based on FT-IR analysis followed by GC-MS and ESI-MS/MS, the active compound was identified to be Bis-(2-ethylhexyl) phthalate. Antibacterial potential was evaluated by disk diffusion against E. coli (12.33 ± 0.56 mm inhibition zone) and S. aureus (5.66 ± 1.00 mm inhibition zone). Larvicidal potency was performed against Culex quinquefasciatus Say larvae, where Bis-(2-ethylhexyl) phthalate showed 100% mortality at 250 ppm after 72 h with LC50 of 67.03 ppm. Furthermore, after 72 h the acetylcholinesterase inhibition was observed as 29.00, 40.33, 53.00, 64.00, and 75.33 (%) at 50, 100, 150, 200, and 250 ppm, respectively. In comet assay, mean comet tail length (14.18 ± 0.28 µm), tail DNA percent damage (18.23 ± 0.06%), tail movement (14.68 ± 0.56 µm), comet length (20.62 ± 0.64 µm), head length (23.75 ± 0.27 µm), and head DNA percentage (39.19 ± 0.92%) were observed at 250 ppm as compared to the control. The current study for the first time describes the promising antibacterial and larvicidal potential of Bis-(2-ethylhexyl) phthalate from Lactiplantibacillus plantarum that would have potential pharmaceutical applications.

Phytochemical Composition, Antibacterial, Antioxidant and Antidiabetic Potentials of Cydonia oblonga Bark.

Cydonia oblonga is a medicinal plant that is used to treat a number of health complications in traditional medication systems. The objective of this study was to evaluate the phytochemical composition, and antibacterial, antioxidant, and ant-diabetic potentials of methanolic extracts of Cydonia oblonga bark. The Cydonia oblonga bark extraction was fractionated through HPLC and seven purified fractions labeled as F1, F2, F3, F4, F5, F6, and F7 were obtained. The HPLC-UV analysis of methanolic extract showed the presence of a number of possible compounds. The GC-MS and HPLC analysis confirmed the presence of the following bioactive compounds in the crude extract and purified fractions: malic acid, mandelic acid, quercetin, caffeic acid, catechin hydrate, as morin (HPLC analysis), BIS-(2-ethylhexyl)phthalate and diisooctyl phthalate (F1), carbamide (F2, used as fertilizer), octasiloxane and dimethylsiloxanecyclictrimer (F3), silicic acid and cyclotrisiloxane (F4), 6-AH-cAMP, 4H-cyclopropa[5',6']benz[1',2',7,8]azule, and 4-(4-chlorophenyl)-3-morpholinepyrol-2-yl)-butenedioic acid (F5), isopropyamine (F6), and 1-propylhydrazine (F7). The extract and purified fractions were then tested for biological activities. All the purified fractions and methanolic extract showed effective antibacterial activity; however, the highest activity was recorded for methanolic extract against Staphylococcus aureus and Streptococcus pneumonia. Antioxidant evaluation of methanolic extract and purified fractions against DPPH showed strong % inhibition of the synthetic free radical. The methanolic extract exhibited 87.41 ± 0.54% inhibition whereas fractions showed: F1, 85.45 ± 0.85; F2, 65.78 ± 0.68; F3, 58.61 ± 0.58; F4, 80.76 ± 0.59; F5, 571.29 ± 0.49; F6, 85.28 ± 0.94; and F7, 48.45 ± 0.62% inhibition. Ascorbic acid (standard) was used as a control with 94.88 ± 0.56% inhibition at a maximum concentration of 1000 µg/mL. The α-glucosidase inhibition assay of methanolic extract and purified fractions at a maximum concentration of 1000 µg/mL showed activities as: methanolic extract, 78.21 ± 0.67; F1, 55.01 ± 0.29; F2, 56.10 ± 0.24; F3, 62.44 ± 1.03; F4, 70.52 ± 0.15; F5, 62.18 ± 0.92; F6, 72.68 ± 0.2; and F7, 57.33 ± 0.05% inhibition. α-Amylase % inhibition of methanolic extract and purified fractions were noted as: methanolic extract, 77.98 ± 0.57; F1, 79.72 ± 0.02; F2, 79.72 ± 0.02; F3, 82.16 ± 0.48; F4, 77.37 ± 0.28; F5, 72.14 ± 0.30; F6, 74.24 ± 0.29; and F7, 56.58 ± 0.10 at the highest concentration of 1000 µg/mL. Acarbose (standard) showed 87.65 ± 0.71% inhibition of α-glucosidase and 85.99 ± 0.44% inhibition of α-amylase at the highest concentration of 1000 µg/mL. It was found that all biological activities of methanolic extract and purified fractions might be attributed to the fact that they are rich sources of phenolic and flavonoids along with other bioactive compounds. The total phenolic and flavonoid contents of methanolic extract were recorded higher as compared to purified fractions (TPC = 70% and TFC = 69%). Amongst the purified fractions, fraction 6 exhibited the highest TPC value (64%), and purified fraction 1 exhibited the highest value of TFC (58%). Recent research demonstrated that Cydonia oblonga may be considered an antibacterial medicinal plant. The result of the present study revealed that it might be utilized for the isolation of bioactive phytochemicals that can lead to new opportunities in the discovery of new antibiotics.