Serial Dilutions Research Articles

Serum Transcobalamin Concentration in Cats—Method Validation and Evaluation in Chronic Enteropathies and Other Conditions

Hypocobalaminemia is common in cats with chronic enteropathy (FCE). However, the disruptions in cobalamin metabolism are not fully understood and may vary across species. Cobalamin is distributed to target tissues via binding to transcobalamin (TC) in blood, which has not been evaluated in cats. Thus, an in-house sandwich-ELISA was established to evaluate serum total TC concentrations in cats with FCE. Surplus sera served to analytically validate the assay, and serum TC concentrations were compared among cats with FCE and other diseases (gastrointestinal neoplasia, cholangiohepatopathy, and other neoplastic or non-neoplastic conditions) and healthy controls. Observed-to-expected ratios for serial dilutions ranged from 72.4 to 145.6% and were 75.1–126.7% for spiking-and-recovery. Intra- and inter-assay variability was <17.7% and <17.2% and the preliminary reference interval for feline serum TC was <160–2795 aU/L (lower detection limit: 160 aU/L). Serum TC levels were significantly decreased (p = 0.0067) but not correlated with paired cobalamin concentrations in FCE. Hypertranscobalaminemia predominated with hypercobalaminemia, reaching the highest levels in advanced-stage chronic kidney disease (CKD) cases. TC variations in cobalamin deficiency states with FCE may be linked to inflammation or autoantibodies. This and possible links between serum TC variation in FCE, intracellular cobalamin availability, response to supplementation, and concurrent CKD require further exploration.

In vitro Evaluation of Antifungal Activity of Terbinafine Nano Form against Yeast Organism Isolated in Thumby Labs Ajman

The goal of this study is to evaluate the antifungal activity of terbinafine in its nano form against yeast organisms in vitro and compare its effectiveness to the normal form of terbinafine. Thirty isolated yeast organisms from Thumbay Labs were included in the study. Terbinafine was serially diluted to concentrations of 50 mg, 25 mg, 12 mg, and 6 mg for both the nano and normal forms. After preparing the serial dilutions, fungal suspensions were exposed to these dilutions for 7 minutes. The suspensions were then cultured on Sabouraud dextrose agar and incubated at 37°C overnight. Growth was measured using a colony count. The results for the normal form of terbinafine revealed growth at all concentrations, with varying colony counts with a highest growth was observed at the 6 mg concentration. In contrast, the nano form of terbinafine exhibited distinct development patterns. At the 50 mg concentration, seven samples showed no growth; at the 25 mg concentration, two samples showed no growth; and the remaining samples exhibited limited growth compared to the normal form. According to the results of this study, there were significant differences in the number of colonies between the two forms of terbinafine, with the nano form demonstrating greater efficacy. The most potent antifungal response was observed at the 50 mg concentration of nano terbinafine, which inhibited yeast growth in several samples. In conclusion, terbinafine nanoemulsions are more effective than the normal form against Candida.

From Killer to Solution: Evaluating Bioremediation Strategies on Microbial Diversity in Crude Oil-Contaminated Soil over Three to Six Months in Port Harcourt, Nigeria

The study aimed to evaluate the efficacy of various bioremediation approaches on microbial diversity in crude oil-contaminated soil over three to six months in Port Harcourt, Nigeria. The objective was to assess the impact of different bioremediation strategies on microbial populations, particularly focusing on hydrocarbon-utilizing bacteria and fungi. Microbial populations were quantified using serial dilution and microbial count techniques. The vapor phase transfer mechanism was employed to estimate hydrocarbon-utilizing bacteria and fungi. Bacterial and fungal colonies were incubated for five days, followed by biochemical tests for isolate identification. Fungal pure cultures were observed under a microscope. The study observed a significant increase in microbial populations in soil free of crude oil pollution when bioremediators such as mushrooms and earthworms were introduced. Mushrooms exhibited a 50% increase in hydrocarbon-utilizing bacteria (HUB), while earthworms showed a 55% increase in HUB over the three to six-month period. The longer lifespan and nutrient absorption capabilities of earthworms facilitated faster growth. Furthermore, significant growth in the microbial population of hydrocarbon-utilizing bacteria and fungi was observed in crude oil-polluted soil after employing bioremediation, with the highest growth observed in soil treated with mushrooms at six months, followed by earthworms at six months. Conversely, the lowest microbial population was recorded in soil polluted with 10% crude oil and remediated with earthworms at three months. The results suggest that mushrooms and earthworms effectively increase microbial populations in crude oil-polluted soil. However, mushrooms demonstrated a higher microbial population increase compared to earthworms, especially in terms of promoting the growth of hydrocarbon-utilizing bacteria (HUB) and hydrocarbon-utilizing fungi (HUF). Based on the findings, it is recommended to prioritize using mushrooms as bioremediation agents in similar environmental restoration efforts due to their superior efficacy in increasing microbial populations, particularly HUB and HUF. This study underscores the potential of mushrooms and earthworms as effective bioremediation agents for restoring microbial diversity in crude oil-contaminated soil, offering insights for sustainable environmental restoration practices in oil-affected regions like Port Harcourt, Nigeria.

Fungal dysbiosis following antibacterial monotherapy in canine otitis externa.

To evaluate evidence of Malassezia overgrowth following successful topical antibacterial monotherapy of refractory canine bacterial otitis using semi-quantitative cultures. Twenty-nine dogs with bacterial otitis were treated topically with either fluoro-quinolone [0.5% enrofloxacin (18 dogs, 19 treatment events, 25 ears) or 0.1% marbofloxacin (1 ear), with 0.1% dexamethasone] ("FQ") SID, or 143 mg/mL piperacillin/18 mg/mL tazobactam ("PT") BID (11 dogs, 14 treatment events, 19 ears) for 8 to 36 days (mean 20 days). At visits 1 (V1) and 2 (V2), ear swab tips were washed in PBS + Triton X-100 and serial dilutions spread-plated onto blood, MacConkey (37°C, 48 hours) and modified Dixon's agar (32°C, 14 days) to generate semiquantitative counts. Microbes were identified by phenotype and MALDI-TOF. Prior to treatment, Pseudomonas aeruginosa was isolated alone or in combination with other bacteria in 14 FQ-treated ears and 18 PT-treated ears; the next most frequent bacteria were Streptococcus canis (8 FQ, 2 PT) and Staphylococcus pseudintermedius (8 FQ, 1 PT). The proportions of dogs' ears (excluding cross-over treatments and contra-lateral affected ears) from which bacteria were isolated were significantly reduced by treatment with both FQ (V1, 13/15; V2, 5/15) and PT (V1, 14/14; V2, 2/14). The proportions of dogs' ears from which yeasts (Malassezia pachydermatis, Candida spp.) were isolated were significantly increased by treatment in dogs treated with PT (V1, 1/14; V2, 14/14) but not FQ (V1, 3/15; V2, 6/15). Otitis cases that receive potent antibacterial monotherapy must be monitored for yeast overgrowth.

E. coli-Assisted Eco-Friendly Production of Biogenic Silver Cobalt Oxide (AgCoO2) Nanoparticles: Methanolysis-Based Hydrogen Production, Wastewater Remediation, and Pathogen Control.

Herein, bacterial-assisted synthesis of AgCoO2 is carried out. In the first step, E. coli was separated from soil samples via the "serial dilution method." Ten milliliters of bacterial supernatant was mixed with cobalt chloride and silver nitrate hatched at 38°C for 24 h to get AgCoO2 nanoparticles (NPs). XRD results confirm the synthesis of AgCoO2 NPs while EDX results confirm the absence of any other elements than Ag, Co, and O. An average NP size of 12-26 nm was determined by TEM examination, and the surface of the particles was seen rough, irregularly shaped borders. The antibacterial activity of the constructed NPs was checked against S. aureus, E.coli, Bacillus subtilus, and Pseudomanas areguinosa using agar well diffusion method. The maximum zone of inhibition was 27 mm at 40 mg/mL against Bacillus subtilus. The performance of the synthesized NPs as photocatalysts was also assessed, and several operational parameters that control the photodegradation of the harmful dyes were tried to tune as well, and 85% degrading efficiency was obtained at 60oC for 240 min for 30 mg of catalyst dose These NPs were also used to produce hydrogen by methanolysis.

Serological Markers of Clinical Improvement in MuSK Myasthenia Gravis.

In this retrospective longitudinal study, we aimed at exploring the role of (a) MuSK-immunoglobulin G (IgG) levels, (b) predominant MuSK-IgG subclasses, and (c) antibody affinity as candidate biomarkers of severity and outcomes in MuSK-MG, using and comparing different antibody testing techniques. Total MuSK-IgGs were quantified with radioimmunoassay (RIA), ELISA, flow cytometry, and cell-based assay (CBA) serial dilutions using HEK293 cells transfected with MuSK-eGFP. MuSK-IgG subclasses were measured by flow cytometry. SAffCon assay was used for determining MuSK-IgG affinity. Forty-three serum samples were obtained at different time points from 20 patients with MuSK-MG (median age at onset: 48 years, interquartile range = 27.5-72.5; women, 16/20), with 9 of 20 (45%) treated with rituximab. A strong correlation between MuSK-IgG levels measured by flow cytometry and RIA titers was found (rs = 0.74, 95% CI 0.41-0.89, p = 0.0003), as well as a moderate correlation between CBA end-point titers and RIA titers (rs = 0.47, 95% CI 0.01-0.77, p = 0.0414). A significant correlation was found between MuSK-IgG flow cytometry levels and disease severity (rs = 0.39, 95% CI 0.06-0.64, p = 0.0175; mixed-effects model estimate: 2.296e-06, std. error: 1.024e-06, t = 2.243, p = 0.032). In individual patients, clinical improvement was associated with decrease in MuSK-IgG levels, as measured by either flow cytometry or CBA end-point titration. In all samples, MuSK-IgG4 was the most frequent isotype (mean ± SD: 90.95% ± 13.89). A significant reduction of MuSK-IgG4 and, to a lesser extent, of MuSK-IgG2, was seen in patients with favorable clinical outcomes. A similar trend was confirmed in the subgroup of rituximab-treated patients. In a single patient, MuSK-IgG affinity increased during symptom exacerbation (KD values: 62 nM vs 0.6 nM) while total MuSK-IgG and IgG4 levels remained stable, suggesting that affinity maturation may be a driver of clinical worsening. Our data support the quantification of MuSK antibodies by flow cytometry. Through a multimodal investigational approach, we showed that total MuSK-IgG levels, MuSK-IgG4 and MuSK-IgG2 levels, and MuSK-IgG affinity may represent promising biomarkers of disease outcomes in MuSK-MG.

Experimental and computational investigations of new organotin(IV) complexes derived from hydrazone ligands: Synthesis, structural analysis and biological assessment

In this article, we have presented the synthesis, structural analysis, and biological assessments of twelve new diorganotin(IV) complexes (derived from heterocyclic hydrazone ligands through condensation reaction between 9-formyl-8-hydroxyjulolidine and aromatic hydrazide derivatives) to discover potent biological agents. The synthesized compounds have been thoroughly characterized using numerous spectroscopic and physicochemical methods, including (1H, 13C and 119Sn) NMR, FT-IR, mass spectrometry, powder XRD and SEM. These scrutinizes indicate that complexes exhibited a pentacoordinated geometry, coordinating through azomethine nitrogen atom, enolic oxygen atom, phenolic oxygen atom and carbon atoms of alkyl/aryl groups of organotin derivatives. DFT (Density functional theory) studies were conducted at the B3LYP/LanL2DZ/6-311G level of hypothesis to investigate the quantum chemical aspects of specified compounds. These properties were facilitated through analyses of charge distribution and molecular orbitals. The antioxidant effectiveness of the prepared compounds was examined employing the DPPH assay and the results revealed that complex 7 exhibited strong antioxidant potential, having an IC50 value of 2.3 µM. Moreover, the prepared compounds were assessed for their effectiveness against four bacterial (B. cereus, B. subtilis, P. aeruginosa, E. coli) as well as two fungal strains (C. albicans and A. niger) using serial dilution approach. Complexes 7, 11, 14 and 15 having MIC values ranging from 0.0051-0.0048 µmol/mL have highest antimicrobial potency equivalent to reference drugs, Fluconazole (MIC = 0.0051 µmol/mL) and Ciprofloxacin (MIC = 0.0048 µmol/mL).

Incidence of Pathogens in Fruits and Vegetables in Tropical Bwari Town

Introduction: In numerous tropical regions of developing nations, the prevalence of foodborne illnesses is perceived to be more severe than in developed countries, due to insufficient food safety initiatives or the lack of a structured institutional framework for street food vendors. The study's objectives of examining microorganisms associated with fruits and vegetables, the prevailing temperature and the hygiene practices of various fruit and vegetable sellers were all met. Methods: A self-designed questionnaire completed by twenty vendors were used. Laboratory techniques like serial dilution, biochemical testing, and culture techniques were used. A handheld thermometer was used to determine the prevailing temperature, and descriptive statistics using Microsoft Excel was used to analyze data. Results and Discussion: Statistical analysis revealed that 80% of vendors had washing facilities, 70% worked in a clean environment, 20% washed their hands before handling fruits, 70% dressed correctly, and 70% kept their fruits and vegetables above the WHO temperature standard. A laboratory investigation revealed the bacteria counts of (watermelon being the highest 8.3×105, cucumber 5.4×105, carrot 2.3×105, orange 3.7×105). Microbial species encountered were Staphylococcus aureus, Escherichia coli, Salmonella species, amongst others, which increases health risk. Conclusion: The study concluded that temperatures, handling processes, and inadequate hygiene all had impact on fruits and vegetables sold in tropical Bwari area. It also established a link between temperature and the prevalence of bacteria in fruits and vegetables. As a result, the study suggests monitoring and surveillance of fruit and vegetable sellers, national vendor hygiene policies and public awareness.

New latex agglutination assay for the determination of lactoferrin in human milk

BackgroundLactoferrin (LF) in human milk has various biological properties and contributes to the prevention of preterm birth complications. Enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used methods to measure LF in human milk, but this method is time-consuming and laborious. In Japanese human milk banks, the concentration of LF in donor human milk (DHM) is measured routinely. Here, we reported a rapid, simple, and accurate method for determining LF in human milk using a new reagent based on a latex agglutination assay.MethodsWe obtained 208 human milk pools from 148 mothers, and samples were collected before and after Holder pasteurization. Milk samples were diluted 100- or 200-fold and LF concentrations were measured by a latex agglutination assay using an automated analyzer. The reagent was validated in terms of repeatability, linearity, detection limit, recovery, and comparison with ELISA.ResultsThe coefficient of variation (CV) for intra-assay precision ranged from 0.6 to 5.0% in human milk with high, medium, and low LF concentrations. The linearity was also tested by serial sample dilution and was confirmed up to 16 µg/mL with a detection limit of 0.2 µg/mL. The recovery rates in a spiked recovery test were ranged from 90 to 120% at high, medium, and low concentrations of LF. Furthermore, a strong correlation was observed between LF levels determined by the latex agglutination assay and ELISA (r = 0.978, p < 0.001, n = 255). The regression equation was y = 0.991x + 0.545 (r2 = 0.974, p < 0.001). Compared with ELISA, the latex agglutination assay reduces the measurement time by 160 min and the cost by 55%.ConclusionsThe latex agglutination assay used to determine LF in human milk is rapid, simple, and accurate enough to be used routinely. Its use may contribute to the quick and easy provision of appropriate DHM to preterm infants.

Effective combinations of silver nanoparticles and antimicrobial peptides against antibiotic-resistant bacteria

BACKGROUND: The problem of constantly growing resistance of microorganisms to the antibiotics used forces scientists to constantly search for new antimicrobial agents. AIM: To study the antimicrobial activity and toxicity of silver nanoparticles and antimicrobial peptides with a β-hairpin structure, and to characterize their combined antimicrobial action. MATERIALS AND METHODS: The antimicrobial activity of silver nanoparticles and antimicrobial peptides against antibiotic-resistant bacteria was assessed by serial dilutions in a liquid nutrient medium, and the combined antimicrobial activity – by serial dilutions according to the “checkerboard” method. The toxicity of the substances to human cells was determined using a hemolytic test. RESULTS: The studied silver nanoparticles have pronounced antimicrobial activity and low toxicity to human erythrocytes. Silver nanoparticles containing acetylcysteine are more active against gram-negative bacteria than against gram-positive ones. Synergism of the antibacterial action was detected with the combined use of silver nanoparticles stabilized with sodium oleate and protegrin-1. A synergistic antimicrobial effect was also noted for combinations of silver nanoparticles containing acetylcysteine with protegrin-1, ricaecilin and shuchin-4. CONCLUSIONS: Effective combinations of silver nanoparticles and antimicrobial peptides have been identified that act synergistically against antibiotic-resistant bacterial strains, i.e. they multiply the effect of each other. These combinations can be used as prototypes for the development of antibacterial drugs.

Effects of combined action of proline-rich peptides of human saliva with antimicrobial proteins

BACKGROUND: The substances that make up mixed saliva exhibit activity against bacteria, viruses and fungi. Among them are various cationic antimicrobial peptides: alpha- and beta-defensins, cathelicidins, histatins, but their concentration is relatively low. On the other hand, saliva widely contains a fraction of proline-rich proteins and products of their proteolysis — peptides, the functions of which currently remain poorly studied and unclear. The study of the antimicrobial activity of proline-rich proteins, in particular, when they act together with antimicrobial peptides, and the deciphering of the molecular and cellular basis for the implementation of the protective functions of proline-rich peptides is an urgent task in medicine and biology. The data obtained will help to understand the mechanisms of anti-infective protection of the oral cavity with the participation of the innate immune system, and in the future will allow the creation of drugs based on proline-rich proteins in humans and animals. AIM: Study of the individual antibacterial action of proline-rich salivary proteins, their combined action with the antimicrobial proteins lactoferrin and lysozyme, as well as with one of the most significant endogenous protective peptides — cathelicidin LL-37 on the formation of bacterial biofilms. MATERIALS AND METHODS: Human lactoferrin and egg lysozyme were used in the work. The minimum inhibitory concentrations of peptides against gram-negative and gram-positive bacteria were estimated using serial dilutions in a liquid nutrient medium. Using serial dilutions using the “chessboard” method, it was shown that proline-rich proteins can increase the antibacterial activity of innate immunity peptides present in the oral cavity. The ability of combinations of cationic proline-rich peptides with cathelicidin LL-37 to prevent the formation of bacterial biofilms was studied. RESULTS: In the presence of proline-rich peptides, the antimicrobial activity of some antimicrobial peptides and proteins present in saliva against planktonic bacteria increases. The combined action of proline-rich proteins with LL-37 increases the inhibition of bacterial biofilm formation. CONCLUSIONS: As a result of the study, it was shown that the analyzed cationic proline-rich peptides of human saliva exhibit anti-inflammatory effects, which suggests their significant role in the regulation of inflammatory processes in the oral cavity, as well as their participation in recovery processes.

Towards the identification of body fluids using RT-LAMP isothermal amplification coupled with CRISPR-Cas12a

While often necessary in sexual assault cases, confirmatory identification of body fluids can be a lengthy and/or costly process. In particular, the detection of vaginal fluid and menstrual fluid in forensic casework is limited to endpoint reverse-transcription PCR to detect fluid-specific messenger RNA (mRNA) markers as there are no robust chemical or enzymatic techniques available for these fluids. Similarly, testing for rectal mucosa is not possible with standard methods, the presence of which would provide probative value in cases of alleged anal penetration, although mRNA-based markers have recently been described. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative technique that enables detection of mRNA at a single temperature (usually 60–65℃) for 10–30 minutes and has comparable sensitivity to PCR. We describe the coupling of RT-LAMP amplification (60℃ for 30 minutes) with CRISPR-mediated fluorescent detection of the body fluid specific mRNA markers MMP3 (menstrual fluid), CYP2B7P (vaginal material), TNP1 (spermatozoa), KLK2 (semen), and MUC12 (rectal mucosa). Following temperature optimization and final selection of RT-LAMP-CRISPR assays, their specificity across circulatory blood, buccal, menstrual fluid, vaginal material, semen, and rectal mucosa was assessed. Most assays were specific for their intended target body fluid, although MMP3 and CYP2B7P were detected in some rectal mucosa samples, the latter of which has been observed previously in the literature. A preliminary sensitivity assessment in target fluids was determined by a dilution series over six logs of RNA input. A range of assay approaches were investigated to develop a protocol suitable for use in a forensic screening laboratory. This included the determination of fluorescent assay results by eye, use of lyophilised reagents, and RT-LAMP and CRISPR reactions undertaken in one-tube in a lower resource setting.

Recombinant MS087-based indirect ELISA for the diagnosis of Mycoplasma synoviae

Accurate detection is a prerequisite for effective prevention and control of Mycoplasma synoviae infection. ELISA is the most popular method for the clinical detection of M. synoviae because of its convenience, low cost, and high detection rate. However, the cross-reactivity of commercially available ELISA kits with other avian pathogen-positive sera needs to be addressed. The aim of this study was to establish an ELISA method with high specificity for the detection of anti-M. synoviae antibodies in chicken serum to evaluate the M. synoviae infection status on poultry farms. The recombinant MS087 (rMS087) protein was expressed in Escherichia coli BL21 (DE3) and purified by Ni2+ affinity chromatography. An antibody against rMS087 was generated by immunizing BALB/c mice. Bioinformatic analysis revealed that MS087 was conserved among M. synoviae strains. Western blotting and indirect immunofluorescence results indicated that MS087 was not only localized in the cytoplasm and on the membrane but also secreted by the organism. For the established ELISA method based on rMS087, the optimal antigen concentration, blocking buffer, blocking duration, serum dilution, serum incubation duration, secondary antibody dilution, secondary antibody incubation duration and colorimetric reaction duration were 2 μg/mL, 1% BSA, 3 h, 1:500, 1.5 h, 1:20,000, 2 h and 5 min, respectively. Validation of the rMS087-based ELISA revealed a cut-off value of 0.5. The coefficients of variation of both the intra-batch and inter-batch methods were less than 9%. The assay was able to differentiate positive serum against M. synoviae from antisera against nine other avian pathogens and was able to recognize M. synoviae-positive sera at a dilution of 1:1,000. Compared with the commercial ELISA method, the rMS087-based ELISA has the potential to recognize more positive sera against M. synoviae. Collectively, the rMS087-based ELISA is a reproducible, specific, and sensitive serological method for detecting antibodies against M. synoviae in chicken serum and has robust potential for large-scale serological epidemiology of M. synoviae infection on poultry farms.

ISOLATION AND MOLECULAR IDENTIFICATION OF INDIGENOUS BACTERIOCIN-PRODUCING WEISSELLA CIBARIA

Globally, over 6.22 million deaths are associated with antibiotic resistance. Bacteriocins, a set of antimicrobial peptides synthesized on the ribosomes, are widely viewed as a potential answer to this issue. This is due to their pore-forming ability and antimicrobial activity against antibiotic-resistant pathogens. The aim of this study is to isolate bacteriocin-producing Weissella cibaria and evaluate its antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi, Proteus mirabilis, Streptococcus sp., Candida sp. and Rhizopus stolonifer. Weissella cibaria man1 was isolated by inoculating deMan Rogosa Sharpe (MRS) broth with small pieces of ripe Mangifera indica (mango), 24-hour incubation at 370C, 10-fold serial dilution and plating on MRS agar. Molecular identification was achieved by DNA extraction, amplification of the 16S rRNA gene through polymerase chain reaction (PCR), agarose gel electrophoresis, gene sequencing, and BLASTN homology searches in the National Center for Biotechnology Information (NCBI). Antimicrobial activity of the bacteriocin was determined by agar well diffusion assay. Mangifera indica (mango) was found to harbor bacteriocin-producing Weissella cibaria man1. The bacteriocin (weissellicin man1) exhibited a broad spectrum of antimicrobial activity. Weissellicin man1 suppressed the growth of several target pathogens (Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Proteus mirabilis, Candida sp. and Rhizopus stolonifer) but had no inhibitory action against Escherichia coli, Streptococcus sp., Staphylococcus aureus. In conclusion, weissellicin man1 from Weissella cibaria man1 has a broad-spectrum of antimicrobial action. These findings will facilitate further evaluation of the antimicrobial potency of weissellicin man1.

Directed modification of characteristics of syntetyc analogue of proline-rich peptide ChBac3.4 through dendrimerisation

BACKGROUND: According to the World Health Organization, microbial resistance to antibiotics is one of the most serious threats to human health, food safety and economic development. Antimicrobial peptides of animal origin, to which resistance is difficult to develop, appear to be a promising option to overcome this problem. To solve one of the problems in the practical use of peptides — their rapid degradation in blood plasma — dendrimerization of linear analogues of already existing antimicrobial peptides can be used. This work shows that dendrimers of shortened analogue of the natural proline-rich peptide ChBac3.4 are superior in antimicrobial activity to the original peptide and are much more stable in blood serum. At the same time, as the original peptide, branched analogues do not have hemolytic activity. AIM: The aim of this study is to develop new structural analogues of a peptide that have improved stability in blood serum while maintaining high antimicrobial activity. To achieve this, we intend to create dendrimeric (branched) molecules by synthesizing peptides using solid-phase methods. MATERIALS AND METHODS: We will investigate the antimicrobial properties of the peptides using the method of serial dilutions in a liquid nutrient medium against both Gram-positive and Gram-negative bacteria. We will also assess the stability of the peptides by culturing them in the presence of human blood serum. RESULTS: After making modifications to the original peptide, we expect to see a significant increase in its antimicrobial activity against all strains tested. Additionally, we anticipate that the resistance of these peptides will increase many times over. CONCLUSIONS: Overall, our findings suggest that dendrimerization can effectively improve the properties of proline-rich peptides, making them more suitable for use in medical applications.

Biodegradation potentials of soil-borne fungal cultures grown on haloxyfop R methyl ester herbicide medium

The heterotrophic and haloxyfop-R methyl ester utilizing fungal counts associated with two top soil samples was determined. Five (5) fungal isolates; Aspergillus niger, Candida sp., Trichoderma sp., Fusarium sp., and Alternaria sp. Were cultured from the soil samples using serial dilution as well as pour plate techniques. These isolates were sub-cultured and screened for their ability to use haloxyfop-R methyl ester as sole source of metabolic energy as well as using the turbidimeteric method. Two of the screened fungal isolates which exhibited maximal optical density (OD) difference values; A. niger and Fusarium sp. Were selected and utilized for the subsequent degradation/shake flask experiment. Growth profile parameters which included; pH, OD, dissolved CO2 and weighed dry fungal biomass were determined during the growth profiling test at a 96 hour interval for 16 days using appropriate methods and equipment. Mean soil pH values were 5.08 ± 0.02 and 4.62 ± 0.02 for samples A and B. The mean total heterotrophic fungal count was 8.7×10³ cfu/g ± 1.5 for A and 1.6 × 10⁴ cfu/g ±1.0 for B. The mean pH value observed for A. niger in the course of the growth profile test ranged from 6.0 ± 8.7 to 7.3 ± 8.0 and 7.3 ± 5.8. The mean OD values observed for A. niger and Fusarium sp. In the course of the shake flask experiment varied from 0.66 ± 7.1 to 1.96 ± 4.5 and 1.62 ± 5.4 to1.99 ± 3.6 respectively. With reference to the detection of mean dissolved CO2 values, utilized as an indirect indicator of herbicide mineralization, the axenic cultured A. niger was the most effective amongst the growth profile fungal cultures.

Electroacupuncture preconditioning prevents urticaria by regulating inflammatory response in rats with urticaria

To observe the effect of electroacupuncture (EA) preconditioning at "Quchi" (LI11) and "Xuehai" (SP10) in prevention of urticaria. Twenty-four male SD rats were randomly divided into control, model and preconditioning of EA (Pre-EA) groups (8 rats/group). The urticaria model was established by intradermal injection of dilute allogeneic antioalbumin serum at the spots of the bilateral symmetry of the spine on the back, and followed by tail venous injection of mixture solution of egg albumin diluent, plus 0.5% Evans blue and normal saline. Ten days before the end of modeling, rats of the pre-EA group received EA stimulation of LI11 and SP10 for 20 min, once a day for 10 consecutive days. The times of rat's scratching the sensitized skin were recorded. HE staining method was used to observe the pathological changes of skin tissue, and toluidine blue staining method was used to observe the morphology of mast cells (MCs) in the skin, blood, mesentery, and peritoneal fluid, and calculate the degranulation rate. Immunohistochemical stainning was used to detect immunoglobulin E (IgE), histamine (HIS), and 5-hydroxytryptamine (5-HT) expressions in subcutaneous tissue. NOD like receptor thermal domain associated protein 3 (NLRP3) inflammasome, apoptosis related granule protein (ASC), and cysteine aspartate aminotransferase 1 (Caspase-1) protein expression levels in skin tissue were detected by Western blot. The contents of serum interleukin(IL)-1β and IL-18 were detected using ELISA method. Compared with the control group, the scratching times, amount of Evans blue exudation of the sensitized blue spots, degranulation rate of MCs in skin, blood, mesentery and peritoneal fluid, the expression levels of IgE, HIS, 5-HT in subcutaneous tissue, protein expression levels of NLRP3, ASC, Caspase-1 in skin tissue, and the contents of serum IL-1β and IL-18 were significantly increased (P<0.01, P<0.05) in the model group. In comparison with the model group, the scratching times, amount of Evans blue exudation of the sensitized blue spots, degranulation rate of MCs, the expression levels of IgE, HIS, 5-HT in subcutaneous tissue, protein expression levels of NLRP3, ASC, Caspase-1 in skin tissue, and the contents of serum IL-1β and IL-18 in EA group were significantly decreased (P<0.01, P<0.05). EA preconditioning at LI11 and SP10 can prevent and treat UR by inhibiting inflammatory response, which is related to the regulation of pyroptosis.

Comparative evaluation of PCR and loop-mediated isothermal amplification (LAMP) assays for detecting Pasteurella multocida in poultry

ABSTRACT Aims To develop a colourimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Pasteurella multocida in clinical poultry samples and compare the performance of this assay with PCR. A secondary aim was to evaluate a simple DNA extraction method that could enable LAMP-based testing in the field without the need for specialised laboratory equipment. Methods Primer sets for both LAMP and PCR were designed to amplify the KMT1 gene of P. multocida. DNA was extracted from 12 P. multocida isolates using a commercial extraction kit, and subjected to analysis using both LAMP and PCR. The analytical specificity of the LAMP assay was evaluated by testing it against a panel of 12 unrelated bacterial species, and the analytical sensitivity (limit of detection) was determined through testing of serial dilutions of the target DNA and compared to that of PCR. Subsequently, cloacal swabs (n = 40) from a commercial turkey flock were subjected to analysis using both LAMP and PCR assays, using a rapid DNA extraction method and a commercial extraction kit. Clinical sensitivity and specificity of the LAMP assay were calculated in comparison to PCR. Results A single DNA fragment of the expected size (∼ 200 base pairs), was amplified by PCR from 12 P. multocida isolates, which were also all positive by the LAMP assay. The identity of all PCR amplicons was confirmed by sequencing. Both PCR and LAMP showed similar analytical sensitivity, with a LOD of 20 pg of target DNA. As neither PCR nor LAMP assays produced positive results with 12 non-related bacterial species, the analytical specificity was assessed as 100%. However, LAMP demonstrated lower clinical specificity (94.74%) compared to PCR (100%) when 40 clinical samples were tested. None of the DNA samples extracted using the simplified DNA extraction method were amplified by either LAMP or PCR. Conclusion The LAMP assay developed in this study exhibits comparable performance to PCR in detecting P. multocida. Clinical relevance The use of a rapid and portable DNA extraction method, in conjunction with LAMP assays, could create opportunities for point-of-care testing for fowl cholera in field settings.

Understanding rhizospheric microbial dynamics in gladiolus corms through quorum sensing and quorum quenching for disease control and growth promotion

Gladiolus, a widely cultivated cut flower known for its aesthetically pleasing multicoloured spikes, has earned significant commercial popularity. A comprehensive understanding of the rhizosphere bacterial community associated with gladiolus is imperative for revealing its potential benefits. Molecular characterization is considered an effective method to gain insights into the structural and functional aspects of microbial populations. The soil characteristics and bacterial communities in the rhizosphere are typically influenced by quorum sensing (QS) and quorum quenching (QQ) mechanisms. This study aims to explore the niceties and diversity of rhizospheric bacterial populations linked with gladiolus corms, with a specific focus on understanding the dynamics of QS and QQ mechanisms in their complex interactions. The isolation of bacterial strains was achieved through the serial dilution method on nutrient agar (NA) media. The identification of the isolates was accomplished by amplifying 16 S rRNA gene sequences via polymerase chain reaction (PCR) via the use of universal primers. Sequence analysis was conducted via BLAST on the National Center for Biotechnology Information (NCBI) database. The characteristics of the isolated bacteria were elucidated via biosensors. This study identified three QS strains and five QQ strains. A consortium of quenchers was formulated utilizing five strains that demonstrated efficacy in mitigating the impact of disease on gladiolus and fostering growth. Among the three treatments—Scale, Descale, and Descale and Cut Half (DSC)—the DSC treatment emerged as the most effective. This treatment exhibited a broader range of variation in biological parameters over time, aligning with prevailing trends in the local market.

Advanced HPLC Method with Diode Array Detection Using a Phenyl-Bonded Column for Simultaneous Quantitation of Three Sunscreen Filters in a Moisturizing Sunscreen Cream for Acne-Prone Skin

This study introduces a novel, robust, and efficient method for the simultaneous quantitative determination of three sunscreen filters, namely, 4-methylbenzylidene camphor, octyl methoxycinnamate, and avobenzone, in a moisturizing sunscreen cream specifically designed for acne-prone skin. The method employs high-performance liquid chromatography with photodiode-array detection, providing a reliable separation of the analytes. Chromatographic separation was achieved using a Fortis Phenyl analytical column (150.0 × 2.1 mm, 5 μm), with isocratic elution at a flow rate of 0.4 mL/min. The mobile phase was composed of a 57/43 (v/v) mixture of acetonitrile/45 mM aqueous ammonium formate solution, ensuring sufficient resolution and peak symmetry for the target compounds. The method was validated comprehensively for critical performance parameters, including linearity, precision, accuracy, and robustness. Linearity was established across a suitable range for all three analytes, with high correlation coefficients. Precision was confirmed with intra-run and total precision coefficients of variation of ≤4.6%, while accuracy assessments yielded a percent recovery between 98.6 and 100.4, for all quality control levels. Additionally, the method was able to effectively separate the sunscreen filters from other cosmetic ingredients, such as [β-(1.3), (1.6)-D-glucan], low molecular weight (LMW) hyaluronic acid and plant extracts ensuring specificity in complex formulations. This straightforward and time efficient sample preparation process, involving methanol extraction followed by serial dilution, makes the method suitable for routine quality control in cosmetic laboratories. The method was successfully applied to the analysis of two different lots of a commercial sunscreen cream, achieving excellent recovery for all filters, ranging between 94.6% and 99.8%, thus demonstrating its reliability and applicability for the quality control of cosmetics.