The purpose of this study was to evaluate the effects of 635 nm diode laser with different powers on undifferentiated mesenchymal stem cells obtained from buccal fat pad. Human buccal fat stem cells were cultured in DMEM containing 10% FBS, penicillin, and streptomycin under 5% CO2 and 95% humidity. Cells were cultured in 96-well plate and 24 h later, laser irradiation with 635 nm diode laser was performed in four groups of 200, 300, 400, and 500 mW powers in addition to the control group with the same energy density of 4 J/cm2. MTT and flow cytometry assay was performed to evaluate cell proliferation and viability on 2 and 4 days after irradiation. Alizarin red assay and real-time PCR (OPN, OCN, ALP, and RUNX-2 genes) was performed to evaluate osteogenic differentiation. According to the MTT assay, none of the mentioned powers of 635 nm diode laser had significant effect on cell proliferation. Cells irradiated with power of 400 mW and 500 mW significantly showed a greater number of necrotic cells compared to the control group in Day 4. Cells irradiated with 300 mW power significantly exhibited a greater amount of nodule formation compared to all groups. Results of this study indicated that 635 nm diode laser with energy density of 4 J/cm2 has a positive effect inducing osteogenic differentiation when applying with a power of 300 mW in buccal fat pad mesenchymal stem cells.

BACKGROUND: Burkholderia pseudomallei, an environmental saprophyte bacterium, causes melioidosis in humans and animals. It was first discovered in Iran between 1967 and 1976 in small ruminants, equines, environments and humans. No subsequent studies have been conducted to determine the existence and prevalence of this pathogen in the country.
OBJECTIVE: The present study aims to monitor the presence of B. pseudomallei in the ruminant population of the Golestan province of Iran, which largely depends on pastures. The ruminants can serve as sentinels to indicate the presence of the bacteria in the environment and its potential impact on human health in the One Health triad.
METHODS: Liver and lung abscesses from domestic sheep, cattle and goats in three industrial and three conventional slaughterhouses were sampled and analysed using 23S ribosomal DNA polymerase chain reaction (rDNA PCR) with primers CVMP 23-1 and CVP-23-2 for B. pseudomallei, Burkholderia cepacia and Burkholderia vietnamiensis, as well as B. pseudomallei-specific TTS1 real-time PCR, along with microbiological and biochemical assays.
RESULTS: Out of the 97 animals sampled, only 14 (15%) tested positive for 23S rDNA PCR. However, the follow-up evaluation using TTS1 real-time PCR and microbiological and biochemical assays did not confirm the presence of B. pseudomallei in the samples.
CONCLUSIONS: Although B. pseudomallei was not detected in the current survey, conducting abattoir-based surveillance of ruminants is a cost-effective One Health approach to monitor pathogenic Burkholderia. Developing standards of clinical and laboratory good practices for Burkholderia infections is crucial for One Health surveillance.

Acute encephalitis syndrome (AES) is a major public health concern in India as the aetiology remains unknown in the majority of cases with the current testing algorithm. We aimed to study the incidence of Japanese encephalitis (JE) and determine the aetiology of non-JE AES cases to develop an evidence-based testing algorithm. Cerebrospinal fluid (CSF) samples were tested for Japanese encephalitis virus by ELISA and polymerase chain reaction (PCR). Multiplex real-time PCR was done for Dengue, Chikungunya, West Nile, Zika, Enterovirus, Epstein Barr Virus, Herpes Simplex Virus, Adenovirus, Cytomegalovirus, Herpesvirus 6, Parechovirus, Parvovirus B19, Varicella Zoster Virus, Scrub typhus, Rickettsia species, Leptospira, Salmonella species, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Plasmodium species and by ELISA for Mumps and Measles virus. Of the 3173 CSF samples, 461 (14.5%) were positive for JE. Of the 334 non-JE AES cases, 66.2% viz. Scrub typhus (25.7%), Mumps (19.5%), Measles (4.2%), Parvovirus B19 (3.9%) Plasmodium (2.7%), HSV 1 and 2 (2.4%), EBV and Streptococcus pneumoniae (2.1% each), Salmonella and HHV 6 (1.2% each) were predominant. Hence, an improved surveillance system and our suggested expanded testing algorithm can improve the diagnosis of potentially treatable infectious agents of AES in India.

OBJECTIVE: Comprehensive molecular analysis for patients with non-small-cell lung carcinoma (NSCLC) is essential for managing modern targeted therapies. This study sought to establish the feasibility of utilising real-time PCR to perform rapid and comprehensive profiling on minimal amounts of endobronchial ultrasound-guided (EBUS) aspirates as a fast, tissue-sparing route of predictive profiling.
METHODS: A volume of 500 μL of EBUS aspirate and fixative from patients with NSCLC was decanted, and 80 μL (<1% of total specimen received) was utilised for analysis. Biocartis Idylla™ cartridges for epidermal growth factor receptor (EGFR) mutations, KRAS mutations and a GeneFusion cartridge (ALK, ROS1, RET, NTRK1/2/3 rearrangements & MET 14 exon skipping) were analysed for each case to provide molecular data on the main clinically relevant targets as per UK guidelines.
RESULTS: A total of 62 cases were included; all of which had successful DNA analysis (EGFR and KRAS cartridges). RNA analysis (GeneFusion cartridge) was successful for 42 of 51 (82%) with initial approach, with 11 of 11 (100%) achieving a successful result with modified protocol. In all, 23 KRAS mutations (37%), 5 EGFR mutations (8%) and 1 ROS fusion (2%) were identified. Average time from specimen receipt to molecular read-out was 5 h.
CONCLUSIONS: Real-time PCR utilising the Idylla™ platform is rapid, utilises minimal amounts of tissue and provides accurate results. We propose this is a useful ancillary method to utilise alongside next-generation sequencing (NGS) in cases of urgent clinical requirement or EBUS aspirates with inadequate quantities of tissue for NGS.

OBJECTIVE: We sought to evaluate accurate and reproducible detection of Myxobolus cerebralis (Mc), the causative agent of whirling disease, by using nested polymerase chain reaction (nPCR) and three previously established real-time quantitative PCR (qPCR) assays: K18S (Kelley 18S), C18S (Cavender 18S), and Hsp70 (heat shock protein 70). We used a \"fit for purpose\" approach combined with intra- and interlaboratory testing to identify a molecular testing method that would be equivalent to the currently accepted nPCR procedure for Mc.
METHODS: Assay performance was compared using a combination of intra- and interlaboratory testing that used synthetic gBlocks along with naturally and experimentally infected fish tissue. North American isolates representing geographically distinct locations were also tested using all three assays.
RESULTS: The K18S and C18S assays exhibited high assay sensitivity, intra- and interlaboratory repeatability of sample replicates, and reproducible identification of all test samples across multiple laboratories. In contrast, the Hsp70 assay failed to detect several positive samples at low DNA concentrations during intra- and interlaboratory testing. The K18S assay was the only procedure that demonstrated perfect detection accuracy when testing geographically distinct Mc isolates. Results demonstrated the K18S assay is robust under variable test conditions, is more accurate than the C18S and Hsp70 assays, and provides detection capabilities equivalent to those of the currently accepted nPCR confirmation assay \"gold standard\" that is described in the American Fisheries Society-Fish Health Section (AFS-FHS) Blue Book.
CONCLUSIONS: The \"fit for purpose\" approach and preliminary completion of the World Organization for Animal Health validation pathway demonstrate that the K18S assay provides an alternate method for Mc testing. This work provides the foundation for acceptance of the K18S assay into the AFS-FHS Blue Book as a standardized test procedure for Mc.

As sturgeon breeding has proliferated, there has been a heightened demand for growth stimulators in their diets. This study aimed to determine the impact of dietary chitosan on growth performance, whole-body proximate composition, growth-related gene expression, and intestinal histology in juvenile Acipenser stellatus. A total of 180 A. stellatus juveniles with an average weight of 31.90 ± 0.73 g were fed with diets containing 0 (control), 1.5, 3.0, 4.5, and 6.0 g chitosan.kg-1 basic diet for eight weeks. The findings revealed a significant enhancement in growth performance with rising chitosan concentrations. Furthermore, chitosan supplementation upregulated the expression of the growth hormone gene in both brain and liver tissues. In liver samples, the most pronounced expression of the insulin-like growth factor-1 gene was noted at 6.0 g chitosan.kg-1, while in brain samples, peak expressions were observed in both the 4.5 and 6.0 g chitosan.kg-1 treatments. While the whole-body proximate composition remained relatively stable, there was a notable decrease in whole-body lipids with the escalation of chitosan dosage. Intestinal villi dimensions, both height and width, were amplified in the chitosan-supplemented groups compared to controls. In summation, chitosan supplementation showed promise in bolstering growth performance, refining intestinal morphology, and enhancing growth-related gene expression. Analysis of the polynomial regression of weight gain and specific growth rate revealed that the optimum dietary chitosan requirements in A. stellatus were 5.32 and 5.21 g chitosan.kg-1, respectively.

Several crustaceans including shrimps change the amount of specific free amino acids to regulate the osmotic pressure in their bodies. Kuruma shrimp Penaeus japonicus also increases the concentration of alanine (Ala) in the abdominal muscle following the increase of environmental salinity. In the present study, to elucidate the mechanisms of changes in Ala accumulation of kuruma shrimp depending on salinity, we cloned the gene encoding alanine aminotransferase (ALT), an enzyme involved in Ala biosynthesis, and examined its expression profile. It was found that the full-length kuruma shrimp ALT1 cDNA consisted of 3,301 bp, encoding 514 amino acids, and that all amino acid residues important for ALT activity were conserved. Phylogenetic analysis also indicated that the ALT gene cloned in this study was classified as ALT1. Moreover, we examined the expression levels of the ALT1 gene in the abdominal muscle and the hepatopancreas of kuruma shrimp acclimated at 17‰, 34‰, and 40‰ salinities, resulting that the mRNA levels of the ALT1 genes in both tissues of the shrimp acclimated at 40‰ were significantly higher than those at 17‰ for 12 h (p < 0.05). The mRNA levels of the ALT1 gene in the abdominal muscle of the shrimp acclimated for more than 24 h tended to increase following the increase of environmental salinity. These results indicate that ALT1 is responsible for the increase of free Ala concentration in the abdominal muscle of kuruma shrimp to regulate osmotic pressure at high salinity.

The increasing prevalence of Salmonella contamination in poultry meat emphasizes the importance of suitable predictive microbiological models for estimating Salmonella growth behavior. This study was conducted to evaluate the potential of chicken juice as a model system to predict the behavior of Salmonella spp. in cooked and raw chicken products and to assess its ability to predict cross-contamination scenarios. A cocktail of four Salmonella serovars was inoculated into chicken juice, sliced chicken, ground chicken, and chicken patties, with subsequent incubation at 10, 15, 20, and 25°C for 39 h. The number of Salmonella spp. in each sample was determined using real-time polymerase chain reaction. Growth curves were fitted into the primary Baranyi and Roberts model to obtain growth parameters. Interactions between temperature and growth parameters were described using the secondary Ratkowsky\'s square root model. The predictive results generated by the chicken juice model were compared with those obtained from other chicken meat models. Furthermore, the parameters of the chicken juice model were used to predict Salmonella spp. numbers in six worst-case cross-contamination scenarios. Performance of the chicken juice model was evaluated using the acceptable prediction zone from -1.0 (fail-safe) to 0.5 (fail-dangerous) log. Chicken juice model accurately predicted all observed data points within the acceptable range, with the distribution of residuals being wider near the fail-safe zone (75%) than near the fail-dangerous zone (25%). This study offers valuable insights into a novel approach for modeling Salmonella growth in chicken meat products, with implications for food safety through the development of strategic interventions. PRACTICAL APPLICATION: The findings of this study have important implications in the food industry, as chicken juice could be a useful tool for predicting Salmonella behavior in different chicken products and thus reducing the risk of foodborne illnesses through the development of strategic interventions. However, it is important to recognize that some modifications to the chicken juice model will be necessary to accurately mimic all real-life conditions, as multiple factors particularly those related to food processing can vary between different products.

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The present study investigates molecular-based PCR techniques to estimate the prevalence of fish pathogens in southwest Mexico where recurrent mortality in the tilapia cultures has been observed. Sample of internal organs and lesions of Nile tilapia were taken and analysed in 2018, 2019, 2020 and 2022 to detect bacterial pathogens using PCR. No samples were taken in 2021 due to the COVID-19 pandemic. The real-time PCR conditions were optimized to allow a qualitative reliable detection of the bacteria from fixed fish tissue. A total of 599 pond- and cage-cultured tilapia from the southwestern Mexican Pacific (Guerrero, Oaxaca and Chiapas states) were analysed. In this tropical region, during 2018 and 2019 water temperatures of the tilapia cultures were generally with the optimal range to grow Nile tilapia, although extreme values were recorded on some farms. Most of the tilapia sampled were apparently healthy. No Francisella sp. was detected in any sample, and Staphylococcus sp. was the most prevalent (from 0% to 64%) bacteria from the three states over time. Low prevalence of Aeromonas sp. was found, from 0% to 4.3%, although the fish pathogen Aeromonas dhakensis was not detected. Sterptococcus iniae was only detected in Chiapas in 2019 at a low prevalence (1.4%), while the major tilapia pathogen S. agalactiae was detected at a high prevalence (from 0% to 59%) in the three Mexican states. This is the first detection of these pathogenic bacteria in rural farms using real-time PCR and constitutes a great risk for tilapia aquaculture in Mexico, as well as a potential dispersion of these pathogens to other aquaculture areas.