A2 Strain Research Articles

Targeted Screening of Lactiplantibacillus plantarum Strains Isolated from Tomatoes and Its Application in Tomato Fermented Juice.

This study explores the functional attributes of Lactiplantibacillus plantarum (L. plantarum) strains isolated from fermented tomato juice, focusing on their physiological, biochemical, and probiotic characteristics. The identified 66 gram-positive strains included 36 L. plantarum ones, which exhibited robust growth in acidic environments (pH 2.0-5.0) and utilization of various carbohydrates. Notably, seven strains outperformed a commercial strain in extreme acidic conditions. Antioxidant activity varied, with strain A24 showing the highest hydroxyl radical scavenging ability, while strains with high surface hydrophobicity had lower DPPH scavenging activity, indicating no direct correlation between these properties. Strains also showed strain-specific differences in carbohydrate utilization and antibiotic resistance, with some resistant to gentamicin and ciprofloxacin. Survival rates under simulated gastrointestinal conditions were strain-specific, with some strains demonstrating high survival rates, indicating their potential as probiotics. Furthermore, 13 strains used as fermentation starters in tomato juice significantly enhanced antioxidant activity and reduced pH and total soluble solids, indicating efficient sugar utilization and lactic acid production. These findings suggest that L. plantarum strains are well-suited for functional food fermentation and probiotic applications, with strain-specific traits offering versatility for use in acidic food products and probiotic formulations.

Screening, identification, engineering, and characterization of Bacillus-derived α-amylase for effective tobacco starch degradation

In this study, two high-performing α-amylase-producing strains, CK3–5 and A8–1 were successfully isolated and characterized, which were taxonomically confirmed as Bacillus velezensis through whole-genome sequencing and bioinformatics. Bioinformatic sequence analysis and molecular docking revealed the catalytic triad (Asp173-Glu208-Asp274) essential for α-amylase function. Through metabolic engineering, the recombinant strain BAX-5/PT17amy(A8–1)SP002 was developed, which exhibited the highest α-amylase activity of 1440 U/mL upon fermentation optimization, marking a 9.2-fold enhancement over the wild-type strain A8–1, and it successfully degraded 6 % of the starch in the tobacco leaves within 48 h, while the content of 13 harmful substances, including acetamide, pyridine, and acetonitrile, was reduced by 8.6 % to 25.2 %. This study reveals a novel α-amylase gene from B. velezensis and establishes an efficient expression system in B. amyloliquefaciens, offering valuable insights for industrial α-amylase production.

Actinobacteria Isolated from Soils of Arid Saharan Regions Display Simultaneous Antifungal and Plant Growth Promoting Activities.

Application of actinobacteria has grown exponentially in recent years in sustainable agricultural. Most actinobacterial inoculants are tailored to function as either biocontrol agents or biofertilizers. Hence, there is the need to obtain and include multifunctional actinobacterial strains in inocula formulations. In this research, 90 actinobacterial isolates were isolated from rhizospheric and non-rhizospheric soils of Algerian Saharan arid regions and were screened for their activity against the phytopathogenic fungi Alternaria alternata, Aspergillus flavus, Botrytis cinerea, Fusarium oxysporum, and Fusarium solani. Five isolates that inhibited at least three of these fungi were characterized according to morphological, environmental and biochemical parameters, and were preliminarily identified as Streptomyces enissocaesilis A1, Streptomyces olivoverticillatus A5, Streptomyces erumpens A6, Streptomyces cavourensis A8, and Streptomyces microflavus A20. These strains were then screened for plant growth promoting activities. All strains produced siderophores, hydrocyanic acid, ammonia and the auxin indole-3-acetic acid (IAA) and were capable of solubilizing phosphate. The highest producer of siderophores (69.19 percent siderophore units), ammonia (70.56μgmL-1) and IAA (148.76μgmL-1) was strain A8, A20, and A5, respectively. These findings showed that the five actinobacteria are multipurpose strains with simultaneous antifungal and plant growth promoting activities and have the potential to be used for sustainable agricultural practices, particularly in arid regions.

Longer interval between maternal RSV vaccination and birth increases placental transfer efficiency.

Respiratory Syncytial Virus (RSV) is associated with significant neonatal and infant morbidity and mortality. Maternal bivalent RSVpreF RSV vaccination to protect neonates and infants was approved in September 2023 for administration between 32+0 and 36+6 weeks to protect neonates and infants. This approved timeframe is narrower than the 24-36 week window evaluated in the clinical trial, due to the possible association between preterm birth and vaccine administration. Currently, data are lacking on how maternal vaccine timing within the approved window affects the transfer of antibodies from mother to fetus, critical information that could influence clinical practice. We sought to examine how gestational age at vaccination and time elapsed from maternal RSV vaccination to delivery impacted transfer of maternal antibodies measured in the umbilical cord at delivery and in peripheral blood of 2-month infants. We also examined differences in maternal and cord RSV antibody levels achieved by vaccination versus natural RSV infection. A prospective cohort study was conducted at two academic medical centers between September 20, 2023 and March 21, 2024, enrolling 124 individuals who received the RSV vaccine during pregnancy. Infant capillary blood was collected at 2 months of age from 29 of the infants. Maternal and cord IgG levels achieved by RSV vaccination were compared to those associated with maternal natural RSV infection, using banked blood from 20 maternal:cord dyads collected prior to the availability of the maternal RSV vaccine. Levels of IgG against RSV strain A2 and B fusion (F) and attachment (G) proteins and against pertussis toxin (as a comparator antigen from a vaccine routinely administered earlier in pregnancy) were measured using a Binding Antibody Multiplex Assay. Differences in titers between vaccination and natural infection were examined using Wilcoxon rank sum test. Differences in cord:maternal transfer ratios and 2-month infant antibody levels by timing of maternal vaccination were evaluated by Kruskal-Wallis testing. Maternal RSV vaccination resulted in significantly higher maternal and cord anti-F RSV antibody levels than natural infection (5.72 vs 4.82 log 10 MFI, p < 0.0001 maternal; 5.81 vs 5.03 log 10 MFI, p < 0.0001 cord). Maternal vaccination 2-3 weeks and 3-4 weeks prior to delivery was associated with significantly lower cord:maternal transfer ratios than were observed when vaccination occurred > 5 weeks prior to delivery (p = 0.03 for 2-3 weeks, p = 0.007 for 3-4 weeks), and significantly lower transfer ratios than observed for pertussis vaccination administered prior to 30 weeks' gestation (p = 0.008 for 2-3 weeks, p = 0.03 for 3-4 weeks, similar at > 4 weeks). Vaccine administration earlier in the approved 32-36 week window (at least 5 weeks prior to delivery) results in the highest transplacental transfer of maternal antibodies to the neonate. These results should inform the counseling of pregnant individuals on optimal vaccination timing.

Organic matter degradation in the deep, sulfidic waters of the Black Sea: insights into the ecophysiology of novel anaerobic bacteria

BackgroundRecent studies have reported the identity and functions of key anaerobes involved in the degradation of organic matter (OM) in deep (> 1000 m) sulfidic marine habitats. However, due to the lack of available isolates, detailed investigation of their physiology has been precluded. In this study, we cultivated and characterized the ecophysiology of a wide range of novel anaerobes potentially involved in OM degradation in deep (2000 m depth) sulfidic waters of the Black Sea.ResultsWe have successfully cultivated a diverse group of novel anaerobes belonging to various phyla, including Fusobacteriota (strain S5), Bacillota (strains A1T and A2), Spirochaetota (strains M1T, M2, and S2), Bacteroidota (strains B1T, B2, S6, L6, SYP, and M2P), Cloacimonadota (Cloa-SY6), Planctomycetota (Plnct-SY6), Mycoplasmatota (Izemo-BS), Chloroflexota (Chflx-SY6), and Desulfobacterota (strains S3T and S3-i). These microorganisms were able to grow at an elevated hydrostatic pressure of up to 50 MPa.Moreover, this study revealed that different anaerobes were specialized in degrading specific types of OM. Strains affiliated with the phyla Fusobacteriota, Bacillota, Planctomycetota, and Mycoplasmatota were found to be specialized in the degradation of cellulose, cellobiose, chitin, and DNA, respectively, while strains affiliated with Spirochaetota, Bacteroidota, Cloacimonadota, and Chloroflexota preferred to ferment less complex forms of OM. We also identified members of the phylum Desulfobacterota as terminal oxidizers, potentially involved in the consumption of hydrogen produced during fermentation. These results were supported by the identification of genes in the (meta)genomes of the cultivated microbial taxa which encode proteins of specific metabolic pathways. Additionally, we analyzed the composition of membrane lipids of selected taxa, which could be critical for their survival in the harsh environment of the deep sulfidic waters and could potentially be used as biosignatures for these strains in the sulfidic waters of the Black Sea.ConclusionsThis is the first report that demonstrates the cultivation and ecophysiology of such a diverse group of microorganisms from any sulfidic marine habitat. Collectively, this study provides a step forward in our understanding of the microbes thriving in the extreme conditions of the deep sulfidic waters of the Black Sea.Exyq52VguUrmavAnxe2qURVideo

Changes in Soil Zinc Fractions Upon Inoculation of Zinc Solubilizing Bacteria (ZnSB) Under Rice Rhizospheric Soil

ABSTRACT Zinc (Zn) is an essential micronutrient that is found in almost all classes of enzymes. Its deficiency is a well-documented problem among cereal crops that causes decreased crop yields and nutritional quality. Therefore, an experiment was implemented in a randomized block design which included the treatments of bacterial inoculation consisting of T1 (Control), T2 (100% RDF;100:40:20), T3(100% RDF + 25 kg ZnSO4.7 H2O), T4(100%RDF+ ZnSB1– Bacillus sp. strain BAU_A2 @750 ml ha−1), T5 (100%RDF+ ZnSB2– Priestia megaterium strain BAU_A3 @750 ml ha−1), T6 (100%RDF+ ZnSB3-Bacillus subtilis strain BAU_A5@750 ml ha−1). The mineral NPK was applied as per treatments. The results indicated the maximum values of water soluble exchangeable Zn and organically bounded Zn in treatments consisting ZnSB1, ZnSB2 and ZnSB3, respectively. The WS+EXCH-Zn was significantly increased by 68.57%, 28.26%, 18%, 98.14%, 41.30%, 30.00% and 128.12%, 55.55%, 37.25%, 43%, 62.22% and 43.13% in first year (2018–19) and second year (2019–2020) under the soil treated with ZnSB1-Peribacillus simplex strain BAU_A1, ZnSB2-Bacillus sp. strain BAU__A2 and ZnSB3-Bacillus subtilis strain BAU_A5 along with 100%RDF over control, application of recommended dose of fertilizer with and without application of 25 kg ZnSO4 ha−1, respectively. However, the maximum decrement in Mn-oxide-bounded zinc (1.12 mg kg−1 soil) was observed with T6 which was significantly higher by 158%, 194.4%, 185.71% over control, 100% RDF with and without application of 25 kg ZnSO4 ha−1, which showed the solubilization of zinc with the activity of ZnSB in soil. The study may be beneficial to mitigate the zinc deficiency in soil and malnutrition in children.

Mutations in the F protein of the live-attenuated respiratory syncytial virus vaccine candidate ΔNS2/Δ1313/I1314L increase the stability of infectivity and content of prefusion F protein.

Respiratory syncytial virus (RSV) is the leading viral cause of bronchiolitis and pneumonia in infants and toddlers, but there currently is no licensed pediatric vaccine. A leading vaccine candidate that has been evaluated for intranasal immunization in a recently completed phase 1/2 clinical trial is an attenuated version of RSV strain A2 called RSV/ΔNS2/Δ1313/I1314L (hereafter called ΔNS2). ΔNS2 is attenuated by deletion of the interferon antagonist NS2 gene and introduction into the L polymerase protein gene of a codon deletion (Δ1313) that confers temperature-sensitivity and is stabilized by a missense mutation (I1314L). Previously, introduction of four amino acid changes derived from a second RSV strain "line 19" (I79M, K191R, T357K, N371Y) into the F protein of strain A2 increased the stability of infectivity and the proportion of F protein in the highly immunogenic pre-fusion (pre-F) conformation. In the present study, these four "line 19" assignments were introduced into the ΔNS2 candidate, creating ΔNS2-L19F-4M. During in vitro growth in Vero cells, ΔNS2-L19F-4M had growth kinetics and peak titer similar to the ΔNS2 parent. ΔNS2-L19F-4M exhibited an enhanced proportion of pre-F protein, with a ratio of pre-F/total F that was 4.5- to 5.0-fold higher than that of the ΔNS2 parent. The stability of infectivity during incubation at 4°C, 25°C, 32°C and 37°C was greater for ΔNS2-L19F-4M; for example, after 28 days at 32°C, its titer was 100-fold greater than ΔNS2. ΔNS2-L19F-4M exhibited similar levels of replication in human airway epithelial (HAE) cells as ΔNS2. The four "line 19" F mutations were genetically stable during 10 rounds of serial passage in Vero cells. In African green monkeys, ΔNS2-L19F-4M and ΔNS2 had similar growth kinetics, peak titer, and immunogenicity. These results suggest that ΔNS2-L19F-4M is an improved live attenuated vaccine candidate whose enhanced stability may simplify its manufacture, storage and distribution, which merits further evaluation in a clinical trial in humans.

Decomposed chicken feather: A biostimulant to lettuce (Lactuca sativa) growth

It is crucial to produce plant growth biostimulants from waste and renewable resources. A study was carried during 2020 at the Soil and Water Research Institute, Karaj, Iran to evaluate the effect of decomposed chicken feather as a biostimulant on lettuce (Lactuca sativa L.) growth. A total of 29 soil samples from 15 Iranian regions were used to isolate and identify the bacteria and 31 strains that were able to produce keratinase and capable of feather degrading were isolated. Then, 8 isolates that were able to degrade the feather during 7 days were selected as the superior strains. Based on the results Bacillus siamensis c11, Bacillus methylotrophicus gh1 and Bacillus methylotrophicus a2 were identified as the new strains that can produce keratinase enzyme. Then, the effect of foliar spraying of the solutions produced from feather degradation by the new strains (c11, gh1, and a2) on the growth of lettuce was investigated. The results showed that the solution produced by methylotrophicus gh1 strain significantly increased the fresh and dry weight of the shoot and root (respectively, 25.9, 36.9, 34.1 and 51.9% increase compared to the control). It is concluded that the microbial solution of these three microbes as a consortium could be quite a new addition on one hand, could also reduce the human allergy as a via- medium of waste disposal.

Elimination of Curtobacterium sp. strain A7_M15, a contaminant in Prunus rootstock tissue culture production, using reduced graphene oxide–silver–copper and silver–selenium nanocomposites

BackgroundBacterial contamination poses a high risk to the successful establishment and maintenance of plant tissue cultures. The aim of this study was to identify the isolates representing the frequent bacterial contaminants of Prunus rootstock tissue cultures and to determine the most effective concentration of nanomaterials for Curtobacterium sp. strain A7_M15 elimination without a negative impact on explants.ResultsSix Curtobacterium sp. strains were isolated and identified, and the whole-genome sequence was obtained for strain A7_M15. Two nanocomposites, reduced graphene oxide–copper–silver and silver–selenium, with the highest bactericidal activity were selected for elimination of Curtobacterium sp. contamination in Gisela 5 rootstock tissue cultures. Both nanocomposites showed 100% inhibition of bacterial plaque formation on culture medium at concentrations of 100, 200 and 400 mg L-1 Ag (2 ×–8 × MBC). The quantity of Curtobacterium sp. on culture medium assessed using cfu enumeration was reduced by 92% and 74% in comparison to the positive control after treatment with reduced graphene oxide–silver–copper and silver–selenium at a concentration of 200 mg L-1 Ag, respectively. None of the tested concentrations resulted in a decrease in Curtobacterium sp. quantity in explants. Curtobacterium sp. was detected in donor Gisela 5 plants, indicating an endophytic character of this bacterium. The dry weight of explants was not negatively affected by the application of nanocomposites regardless of concentration, and no detrimental effect of either nanocomposite at 100 or 200 mg L-1 Ag on the surface covered by plants was observed.ConclusionsReduced graphene oxide–silver–copper and silver–selenium nanocomposites at 200 mg L-1 Ag effectively limited the Curtobacterium sp. presence in micropropagated Prunus rootstock without causing phytotoxicity; therefore, those treatments could be offered as prevention with a high activity against bacterial contamination in plant tissue cultures.Graphical

Bacillus velezensis A2 Can Protect against Damage to IPEC-J2 Cells Induced by Zearalenone via the Wnt/FRZB/β-Catenin Signaling Pathway.

Zearalenone (ZEA) has adverse effects on human and animal health, and finding effective strategies to combat its toxicity is essential. The probiotic Bacillus velezensis A2 shows various beneficial physiological functions, including the potential to combat fungal toxins. However, the detailed mechanism by which the Bacillus velezensis A2 strain achieves this protective effect is not yet fully revealed. This experiment was based on transcriptome data to study the protective mechanism of Bacillus velezensis A2 against ZEA-induced damage to IPEC-J2 cells. The experiment was divided into CON, A2, ZEA, and A2+ZEA groups. This research used an oxidation kit to measure oxidative damage indicators, the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) method to detect cell apoptosis, flow cytometry to determine the cell cycle, and transcriptome sequencing to screen and identify differentially expressed genes. In addition, gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were adopted to screen out relevant signaling pathways. Finally, to determine whether A2 can alleviate the damage caused by ZEA to cells, the genes and proteins involved in inflammation, cell apoptosis, cell cycles, and related pathways were validated using a quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot methods. Compared with the CON group, the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in the ZEA group increased significantly (p < 0.01), while the levels of antioxidant enzyme activity, total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC), and catalase (CAT) decreased significantly (p < 0.01). Compared with the ZEA group, the A2+ZEA group showed a significant decrease in ROS and MDA levels (p < 0.01), while the levels of T-SOD, GSH-PX, T-AOC, and CAT increased significantly (p < 0.01). TUNEL and cell cycle results indicated that compared with the ZEA group, the A2+ZEA group demonstrated a significant decrease in the cell apoptosis rate (p < 0.01), and the cell cycle was restored. Combining transcriptome data, qRT-PCR, and Western blot, the results showed that compared with the CON group, the mRNA and protein expression levels of Wnt10 and β-catenin increased significantly (p < 0.01), while the expression level of FRZB decreased significantly (p < 0.01); compared with the ZEA group, the expression levels of these mRNA and proteins were reversed. Bacillus velezensis A2 can increase the antioxidant level, reduce inflammatory damage, decrease cell apoptosis, and correct the cell cycle when that damage is being caused by ZEA. The protective mechanism may be related to the regulation of the Wnt/FRZB cell/β-catenin signaling pathway.

DsRNA sequencing revealed a previously missed terminal sequence of a +ssRNA virus that infects dinoflagellate Heterocapsa circularisquama.

Heterocapsa circularisquama RNA virus (HcRNAV) is the only dinoflagellate-infecting RNA virus cultured. However, only two strains of HcRNAV have been registered with complete genome sequences (strains 34 and 109 for UA and CY types, respectively). To extend the genomic information of HcRNAV, we performed full-genome sequencing of an unsequenced strain of HcRNAV (strain A8) using the fragmented and primer-ligated double-stranded RNA (dsRNA) sequencing (FLDS) method. The complete genome of HcRNAV A8 with 4457 nucleotides (nt) was successfully determined, and sequence alignment of the major capsid protein gene suggested that A8 was a UA-type strain, consistent with its intraspecific host specificity. The complete sequence was found to be 80 nt longer at the 5' terminus than the registered sequences of HcRNAV strains (34 and 109), suggesting that FLDS is more reliable for determining the terminal sequence than conventional methods (5' Rapid Amplification of cDNA End). Our study contributes to a better understanding of dinoflagellate-infecting viruses with limited sequence data.

Phenotypic Characterization and Draft Genome Sequence Analyses of Two Novel Endospore-Forming Sporosarcina spp. Isolated from Canada Goose (Branta canadensis) Feces.

In an attempt to isolate new probiotic bacteria, two Gram-variable, spore-forming, rod-shaped aerobic bacteria designated as strain A4 and A15 were isolated from the feces of Canada geese (Branta canadensis). Strain A4 was able to grow in high salt levels and exhibited lipase activity, while A15 did not propagate under these conditions. Both were positive for starch hydrolysis, and they inhibited the growth of Staphylococcus aureus. The strains of the 16S rRNA sequence shared only 94% similarity to previously identified Sporosarcina spp. The ANI (78.08%) and AAI (82.35%) between the two strains were less than the species threshold. Searches for the most similar genomes using the Mash/Minhash algorithm showed the nearest genome to strain A4 and A15 as Sporosarcina sp. P13 (distance of 21%) and S. newyorkensis (distance of 17%), respectively. Sporosarcina spp. strains A4 and A15 contain urease genes, and a fibronectin-binding protein gene indicates that these bacteria may bind to eukaryotic cells in host gastrointestinal tracts. Phenotypic and phylogenetic data, along with low dDDH, ANI, and AAI values for strains A4 and A15, indicate these bacteria are two novel isolates of the Sporosarcina genus: Sporosarcina sp. A4 sp. nov., type strain as Sporosarcina cascadiensis and Sporosarcina sp. A15 sp. nov., type strain Sporosarcina obsidiansis.

Selenium nanoparticles coated bacterial polysaccharide with potent antimicrobial and anti-lung cancer activities

Bacterial exopolysaccharides are homopolymeric or heteropolymeric polysaccharides with large molecular weights (10–1000 kDa). Exopolysaccharides' functional uses and potential have revolutionized the industrial and medicinal industries. Hence, the aim of the present study was to optimize the production of bacterial exopolysaccharide and apply it as a capping agent for selenium nanoparticles synthesis. Exopolysaccharide (EPS) producing Lactic acid bacteria (LAB) were isolated from dairy products then biochemically characterized and assessed for their potential antimicrobial effect. The most potent EPS producer was identified as Lactiplantibacillus plantarum strain A2 with accession number OP218384 using 16S rRNA sequencing. Overall, FTIR data of the extracted EPS revealed similarity with amylopectin spectrum. 1H NMR spectrum revealed an α-anomeric configuration of the glycosidic linkage pattern in the polysaccharides while the 13C NMR spectrum can also be separated into two main portions, the anomeric carbons region (δ 98–102 ppm) and the non-anomeric carbons region (δ 60–81 ppm). Antimicrobial activity of the produced EPS showed maximum activity against Staphylococcus aureus, MRSA, Enterobacter aerogenes, Klebsiella pneumoniae and Candida albicans respectively. The EPS capsule layer surrounding the bacterial cells was detected by TEM study. Optimization of EPS production was evaluated using Taguchi design, trial 23 reported the highest biomass yield and EPS output (6.5 and 27.12 g/L respectively) with 2.4 and 3.3 folds increase (from the basal media) respectively. The optimized exopolysaccharide was used as a capping and stabilizing agent for selenium nanoparticles (EPS-SeNPs) synthesis. Zeta potential, size and PDI of the synthesized nanoparticles were − 19.7 mV, 45–65 nm and 0.446 respectively with strong bactericidal and fungicidal effect against the tested pathogens. Complete microbial growth eradication was recorded after 6, 8 and 10 h against Staphylococcus aureus, Candida albicans and Klebsiella pneumoniae respectively. EPS-SeNPs showed a potent antioxidant effect reached 97.4% and anticancer effect against A549 lung cancer cell line (IC50 reached 5.324 µg/mL). EPS-SeNPs inhibited cancerous cell growth at S phase. Moreover, molecular studies revealed the anti-apoptotic activity of Bcl2's was inhibited and Bax was activated. The present investigation successfully synthesized selenium nanoparticles through bacterial EPS with significantly high antimicrobial and anticancer activity.

Study on the Effect of Bee Strain and Insemination Method on the Hygienic Behavior of Bee Colonies Tested Using the Pricking Method

The process of artificial and natural insemination was carried out on the queen bees of the local and Carniolan strains in order to study their hygienic behavior and the Pricking method for the strains, as well as the cross-insemination results between them. The study results showed that the local honey bee strain T1 outperformed the other studied strains in its hygienic behavior, as the detection rate of the dead brood by Pricking was (44.16, 93.16, and 100) % respectively, after (1, 6, and 24) hours of conducting the study. The same strain also outperformed all other strains in removing the dead brood with a percentage ranging from (0.16, 22.66, and 100) % respectively, after (1, 6, and 24) hours of conducting the study. Moreover, the artificial insemination method generally outperformed the natural insemination method in the hygienic behavior of the bee colonies, as the detection rate of the brood in the artificially inseminated bee strains A2 was (40.91, 91.00, 97.33, and 100) % respectively, after (1, 6, 24, and 36) hours of conducting the study. A2 also outperformed the A1 insemination method in terms of the percentage of removing the dead brood by Pricking, with a percentage ranging from (0.00, 17.08, 96.91, and 100) % respectively, after (1, 6, 24, and 36) hours of conducting the study.

New Anti-RSV Nucleoprotein Monoclonal Antibody Pairs Discovered Using Rabbit Phage Display Technology.

Human respiratory syncytial virus (hRSV) is one of the major contagious viruses and causes complicated respiratory issues, especially in young children. The sensitive and fast detection of hRSV is critical for taking the most effective actions. In the present study, rabbit antibodies against the hRSV nucleoprotein (NP) were developed using phage display technology. A female rabbit was immunized with an hRSV strain A2 recombinant NP. A Fab library was built and sorted during two successive panning rounds for strain B and the A2 NP (recombinant preparations), respectively. The choice of candidates was performed using ELISA on the two NP strains. The obtained library was 3 × 106 cfu/mL, with an insertion rate of >95%. The two panning rounds permitted an enrichment factor of 100. ELISA screening allowed us to obtain 28 NP-specific Fab candidates. Among them, 10 retained candidates were reformatted into rabbit full IgG; thereafter, pairing tests on the recombinant strains and native lysate samples were performed. After the pairing tests on the recombinant strains, 53 pairs were identified. Eleven pairs were identified as being able to detect RSVs from native lysates. This work presents new high-potential monoclonal antibodies mAbs (mAbs), which would benefit from lateral flow testing data with patient materials.

SYNTHESIS AND ANTIVIRAL ACTIVITY OF COPOLYMERS OF OXYCINNAMIC ACID WITH N-VINYLAMIDES

Radical copolymerization of coumarin with N-vinylamides (N‑vinylpyrrolidone, N-methyl-N-vinylacetamide, N-vinylformamide) was used to prepare copolymers of various natures and varied molecular masses. After the subsequent reactions in polymer chains, water-soluble copolymers of salts of oxycinnamic acid and its hydrazides were obtained. The synthesized copolymers of salts and hydrazides of oxycinnamic acid with N-vinylamides have low cytotoxicity and demonstrate the pronounced antiviral activity against human respiratory syncytial virus (strain A2).

Untargeted metabolomics provide new insights into the implication of Lactobacillus helveticus strains isolated from natural whey starter in methylglyoxal-mediated browning

Hard cheeses may occasionally show a brown discolouration during ripening due to multifactorial phenomena that involve bacteria and give rise to pyrazines arising from methylglyoxal. The present work aimed at developing a novel approach to investigate the role of natural starters in browning. To this object, 11 strains of L. helveticus were incubated in a medium containing 10 % rennet casein dissolved in whey, and then growth was monitored by measuring pH and number of genomes/mL. Browning was assessed through CIELab analysis, methylglyoxal production was determined by targeted mass spectrometry, and untargeted metabolomics was used to extrapolate marker compounds associated with browning discoloration. The medium allowed the growth of all the strains tested and differences in colour were observed, especially for strain A7 (ΔE* value 15.92 ± 0.27). Noteworthy, this strain was also the higher producer of methylglyoxal (2.44 µg/mL). Metabolomics highlighted pyrazines and β-carboline compounds as markers of browning at 42 °C and 16 °C, respectively. Moreover, multivariate statistics pointed out differences in free amino acids and oligopeptides linked to proteolysis, while 1,2-propanediol and S-Lactoylglutathione suggested specific detoxification route in methylglyoxal-producing strains. Our model allowed detecting differences in browning amid strains, paving the way towards the study of individual L. helveticus strains to identify the variables leading to discoloration or to study the interaction between different strains in natural whey starters.

Unusual Differences in the Pulmonary Histopathology of Mice after Intranasal Infection with Mycelial Propagules of Histoplasma capsulatum Strains Classified as LAm A2 and NAm 2 Phylogenetic Species.

The ascomycete Histoplasma capsulatum is the causative agent of systemic respiratory mycosis histoplasmosis, which sometimes develops acute disseminated or chronic clinical forms, with the latter usually associated with granuloma formation. The present report shows differential histopathological changes in the pulmonary inflammatory response of mice infected intranasally with the mycelial morphotype of H. capsulatum strains with distinct genotypes, EH-46 and G-217B, classified as LAm A2 and NAm 2 phylogenetic species, respectively. Infected male BALB/c mice were sacrificed at different postinfection times, and their serial lung sections were stained with periodic acid-Schiff and analyzed via microscopy. In mice infected with the LAm A2 strain, the results showed progressive changes in the inflammatory infiltrate of the lung parenchyma during the first hours and days postinfection as well as granulomas with macrophages containing intracellular yeast cells, which prevailed at 14 and 21 days postinfection. Bronchiolar-associated lymphoid tissue was induced in mice infected with both strains, primarily in mice infected with the NAm 2 strain. Several lung sections from mice infected with the LAm A2 strain showed PAS-positive yeast cells aggregated in a perinuclear crown-like arrangement in macrophages from 3 h to 21 days postinfection. These findings highlight differences in the host pulmonary inflammatory response associated with distinct H. capsulatum species.

Virus-Like Particles Assembled Using Respiratory Syncytial Virus Matrix Protein Elicit Protective Immunity in Mice.

Heterologous virus-like particle (VLP) assembly involving influenza or the Newcastle disease virus matrix protein (M) has been extensively used to explore the efficacies of VLP vaccines against the respiratory syncytial virus (RSV). Here, we attempted to generate homologous RSV VLPs by expressing the pre-fusion (pre-F) or the glycoprotein (G) on the RSV M protein and evaluated their protective efficacy in mice. We generated VLPs using the baculovirus expression system in Spodoptera frugiperda (Sf9) insect cells. Recombinant baculoviruses expressing the RSV pre-F, G, and M antigens were inoculated into Sf9 cells, and particles were self-assembled. Mice were immunized with either pre-F or G-expressing VLPs, and immune parameters were assessed to determine protection. Our findings show that successful VLP assembly can be achieved by utilizing recombinant baculoviruses expressing the RSV pre-F or G proteins with the native matrix protein. Mice immunized with either pre-F or the G antigen-expressing VLPs elicited robust serum-mediated virus neutralization. VLP immunization evoked Th1-biased RSV-specific antibody responses in the sera of mice. Following challenge infection with the RSV A2 strain, immunized mice experienced lesser eosinophil and IL-4 accumulation in the lungs, though a substantial increase in TNF-α secretion was observed from CD4+ T cells. Interestingly, splenic antibody-secreting cell responses were substantially enhanced against RSV F antigen, but not against the RSV G antigen following immunization and challenge infection. Immunizing mice with the VLPs significantly inhibited pulmonary histopathology development, as indicated by the diminished inflammatory immune cell influx and mucin secretion. Combined, these vaccine-induced immune responses contributed to successfully inhibiting the RSV replication in the lungs of mice and demonstrated that RSV VLP assembly using insect cell-derived homologous RSV matrix protein is a feasible approach.

Isolation and characterization of probiotic bacteria from fruit pulp, screening of probiotic properties and production of bacteriocin and probiotic curd

The present study was investigated to isolate probiotic lactic acid bacteria from Actinidia deliciosa (kiwi fruit) pulp. A total of eight isolates were found and two of them were probiotic LAB strains. The both A2 and A5 isolates strains were Gram-positive, catalase and oxidase negative, non-spore forming, non-motile, rod or short cocci shaped bacteria. The A2 and A5 strains both survived well in conditions of low pH, 0.4% phenol and high bile salt concentrations. They could grow at different temperatures and were resistant to different NaCl concentrations. A study was also conducted on the various probiotic and safety attributes of isolates. Bacteriocins, a kind of ribosomal protein produced by LAB, were estimated using the Folin-Lowery method. The formation of bacteriocins was influenced by various physical and chemical factors. The result demonstrates that the optimum conditions for the production of bacteriocins by the A2 strain were glucose as carbon source, pH 6.5, 2% NaCl and 1% bile salts. For the A5 strain, sucrose as a carbon source, pH 6.5, 1% NaCl and 1% bile salt were optimum conditions for bacteriocin production. Study was also conducted on exopolysaccharide (EPS) production, adhesion characteristics, antagonistic activity and antibiotic sensitivity. Probiotic curd (Dahi) formation was done and several parameters of curd like pH, total lactic acid concentration and water-holding capacity were all investigated.