Culture Medium Research Articles

Formate from THF-C1 metabolism induces the AOX1 promoter in formate dehydrogenase-deficient Komagataella phaffii.

In Komagataella phaffii (Pichia pastoris), formate is a recognized alternative inducer to methanol for expression systems based on the AOX1 promoter (pAOX1). By disrupting the formate dehydrogenase encoding FDH1 gene, we converted such a system into a self-induced one, as adding any inducer in the culture medium is no longer requested for pAOX1 induction. In cells, formate is generated from serine through the THF-C1 metabolism, and it cannot be converted into carbon dioxide in a FdhKO strain. Under non-repressive culture conditions, such as on sorbitol, the intracellular formate generated from the THF-C1 metabolism is sufficient to induce pAOX1 and initiate protein synthesis. This was evidenced for two model proteins, namely intracellular eGFP and secreted CalB lipase from C. antarctica. Similar protein productivities were obtained for a FdhKO strain on sorbitol and a non-disrupted strain on sorbitol-methanol. Considering a K. Phaffii FdhKO strain as a workhorse for recombinant protein synthesis paves the way for the further development of methanol-free processes in K. phaffii.

Glucose and glycogen affects Ca2+ transient during fatigue to a greater extent in the least than in the most fatigue resistant mouse FDB fibers.

The overall objective was to determine how no extracellular glucose and/or low glycogen content affect fatigue kinetics in mouse flexor digitorum brevis (FDB) single muscle fibers. High glycogen content (Hi GLY), near normal insitu level, was obtained by incubating fibers in culture medium containing glucose and insulin while low glycogen content (Lo GLY), at about 19% of normal insitu level, was achieved by incubating fibers without glucose. Neither Lo GLY nor the absence of extracellular glucose (0GLU) affected tetanic [Ca2+]i prior to fatigue. The number of contracting unfatigued fibers versus stimulus strength relationship of Lo GLY-0GLU fibers was shifted to higher voltages compared to Hi GLY fibers exposed to 5.5 mM glucose (5GLU). The relationship for Lo GLY-0GLU fibers was shifted back toward that of Hi GLY-5GLU fibers when glucose was reintroduced, whereas the removal of glucose from Hi GLY-5GLU fibers had no effect. Fatigue was elicited with one 200 ms long tetanic contraction every s for 3 min. Both Lo GLY and 0GLU increased the rate at which intracellular tetanic concentration ([Ca2+]i) declined and unstimulated [Ca2+]i increased during fatigue in the order of the least fatigue resistant > mid fatigue resistant > the most fatigue resistant fibers.

Investigation of Antimicrobial Compounds Produced by Endolichenic Fungi in Different Culture Media.

Continuous use of synthetic fungicides has led to explosive emergence of fungicide-resistant microbes. Therefore, there are urgent needs for environmentally friendly antimicrobial agents with novel modes of action. This study investigated endolichenic fungi (ELF) as a source of antimicrobial compounds against various plant pathogens. We utilized an One Strain MAny Compounds (OSMAC) approach to enhance the chemical diversity of fourteen ELF. This involved cultivation of ELF in four growth media and subsequently assessing antimicrobial activities of culture extracts. Nearly half of the culture extracts exhibited antimicrobial activity against a Gram-positive bacterium, but showed minimal activity against Gram-negative bacteria tested. Notably, culture extracts from two ELF, Chaetomium globosum and Nodulisporium sp., demonstrated significant inhibitory effects against plant pathogenic fungi. LC-MS/MS-based metabolome profiling confirmed the presence of known bioactive compounds like cyclic dipeptides and chaetoglobosins. These findings highlight the effectiveness of combining OSMAC and metabolomics for identifying antimicrobial agents for agricultural use.

The Application of Culturomics to Explore African Skin Microbiota.

Over the past 12 years, culturomics, a high-throughput culture method, has been developed, considerably widening the repertoire of known cultured bacteria. An exhaustive database, including a list of microbes isolated by culture from human skin, was recently established by performing a review of the literature. The aim of the present study was to use the culturomics approach to explore the African skin microbiota. Skin swabs from the palms of human hands were collected between January and December 2016 from healthy subjects from the villages of Dielmo and Ndiop in rural Senegal. Three culture media were selected for the isolation of bacteria in aerobic conditions. Bacterial colonies were subjected to matrix-assisted laser desorption ionization-time of flight mass spectroscopy and the 16 S rRNA gene was sequenced for unidentified colonies. A total of 176 bacterial species were isolated. This increased the repertoire of bacterial species on the skin by 14.0%, by adding 71 bacteria, including seven new species. The culturomics approach characterizing microbial diversity has significantly changed our view of the skin microbiota, raising many important questions about the host-microorganism relationship and its relevance to skin diseases. In particular, the difference between the palm microbiota of these African populations (composed mainly of the genera Staphylococcus, Arthrobacter, Bacillus, and Microbacterium) and that of Western populations, whose main genera are Staphylococcus, Propionibacterium, Micrococcus, Corynebacterium, Enhydrobacter, and Streptococcus. This study demonstrates the need to continue to explore the skin microbiome using the culturomics approach.

Berberine alters the gut microbiota metabolism and impairs spermatogenesis.

Berberine (BBR) is used to treat diarrhea clinically. However, its reproductive toxicity is unclear. This study aims to investigate the impact of BBR on the male reproductive system. Intragastric BBR administration for 14 consecutive days results in a significant decrease in the serum testosterone concentration, epididymal sperm concentration, mating rate and fecundity of male mice. Testicular treatment with testosterone propionate (TP) partially reverses the damage caused by BBR to the male reproductive system. Mechanistically, the decrease in Muribaculaceae abundance in the gut microbiota of mice is the principal cause of the BBR-induced decrease in the sperm concentration. Both fecal microbiota transplantation (FMT) and polyethylene glycol (PEG) treatment demonstrate that Muribaculaceae is necessary for spermatogenesis. The intragastric administration of Muribaculaceae intestinale to BBR-treated mice restores the sperm concentration and testosterone levels. Metabolomic analysis reveals that BBR affects arginine and proline metabolism, of which ornithine level is downregulated. Combined analysis via 16S rRNA metagenomics sequencing and metabolomics shows that Muribaculaceae regulates ornithine level. The transcriptomic results of the testes indicate that the expressions of genes related to the low-density lipoprotein receptor (LDLR)-mediated testosterone synthesis pathway decrease after BBR administration. The transcriptional activity of the Ldlr gene in TM3 cells is increased with increased ornithine supplementation in the culture media, leading to increased testosterone synthesis. Overall, this study reveals an association between a BBR-induced decrease in Muribaculaceae abundance and defective spermatogenesis, providing a prospective therapeutic approach for addressing infertility-related decreases in serum testosterone triggered by changes in the gut microbiota composition.

3D bioprinting meniscus tissue onboard the International Space Station

We bioprinted meniscus tissue on the International Space Station (ISS) using the onboard BioFabrication Facility (BFF). The three dimensional (3D) printing bioink, cells, culture media and fixative were delivered to the ISS on NG-18 and SpX-27 vehicles and stored prior to the printing operation. The meniscus tissue was fabricated from ink composed of collagens type I and II, chondroitin sulfate and mesenchymal stem cells. Following printing, the meniscus tissue was cultured for 2 weeks in growth media, then stored at 4 °C and returned to earth for analysis. The print showed good overall shape fidelity, and dimensions were comparable to control meniscus tissue printed on Earth. Young's modulus of the ISS printed meniscus was approximately 4-fold lower than the control. Histologic evaluation showed good cell distribution within the print. Though logistical challenges were encountered during payload delivery to the ISS and operational challenges limited the cell culture portion of this study, this investigation demonstrated the feasibility for 3D printed musculoskeletal tissue in microgravity. The completed meniscus tissue print is the largest tissue engineered model 3D printed on the ISS to date, the first to be 3D bioprinted using an ink similar in composition to native tissue, and the first to be fabricated on the ISS in an anatomically relevant shape. These experiments help advance the field of tissue engineering in low or microgravity where 3D bioprinting may have a role in future long term space flight or extraterrestrial habitation.

Investigation of Acetoin Biosynthesis by Bacillus subtilis ACA-DC 1176 Growing on Crude Glycerol in Flask and Bioreactor Trials

Acetoin biosynthesis by two Bacillus subtilis strains valorising crude glycerol was thoroughly explored within a pre-defined range of culture conditions and systems. B. subtilis ACA-DC 1176 stood out for its higher efficiency in acetoin production, prompting an investigation into the potential for enhanced productivity through the evaluation of diverse culture conditions and media compositions. The primary by-products of the biodiesel and corn industries, namely crude glycerol and corn steep liquor, respectively, were successfully employed as the principal carbon and nitrogen sources of the newly developed low-cost culture medium. Furthermore, the results of the various feeding strategies that were tested indicated that the conversion of 2,3-butanediol (BDO) to acetoin occurred exclusively when the concentration of glycerol was below approximately 5 g/L. This seemed to be necessary for the production of NADH, which is essential for maintaining cellular processes. Following the complete depletion of glycerol, acetic acid increased and became the predominant metabolite, while both acetoin and BDO decreased, presumably resulting in ATP generation. This is likely a mechanism employed by the cell to generate energy in the absence of a carbon source. In the fed-batch bioreactor culture, the kinetics of metabolites differed, as there was no conversion of BDO to acetoin at the final depletion of glycerol. At volumetric mass transfer coefficient (kLa) levels exceeding approximately 70 1/h, the production of acetoin was favoured over that of BDO, with the highest observed acetoin/BDO ratio reaching 4.29 g/g. Conversely, at kLa values below approximately 60 1/h, the titres of acetoin and BDO were found to be nearly equal. The final concentrations of acetoin and BDO reached 36.0 g/L and 25.5 g/L, respectively, resulting in a total yield of both (acetoin + BDO) per glycerol consumption of 0.40 g/g. To the best of our knowledge, this is the first study to focus on acetoin production from crude glycerol fermentative valorisation. The study presents new findings regarding the parameters influencing the level of BDO conversion to acetoin. However, further research is required in order to gain a comprehensive understanding of the underlying phenomena and metabolic pathways involved.

Treatment of liquid anaerobic digestate with natural zeolite and Arthrospira platensis cyanobacteria from laboratory to pilot-scale

Using liquid digestate as nutrients source for microalgae cultivation has gained interest this last decade but high ammonium content and strong turbidity can limit the growth performances. For this purpose, natural zeolite was investigated as a novel and innovative solution to mitigate liquid digestate toxicity before microalgae cultivation at both laboratory and pilot-scales. Natural zeolite (clinoptilolite) was used in adsorption columns at a ratio of 0.5 kgzeolite.L-1 to treat liquid digestate (0.45 L). After 24 h of treatment, the ammonium concentration was significantly reduced from 2273 to 115 mgN.L-1, resulting in a removal efficiency and adsorption capacity of 95% and 4.31 mg.gzeolite-1, respectively. Arthrospira platensis cells were able to proliferate in the treated digestate with relative low dilution factors (≤ 5x) whereas high dilution factors (≥ 20x) were necessary for the untreated digestate. The results obtained at laboratory-scale were successfully scaled-up to pilot-scale with similar treatment performances when detoxifying 15.5 L of anaerobic digestate. The resulting pretreated digestate was used as culture medium for Arthrospira platensis in flat panel photobioreactors without being diluted, reaching a final Arthrospira platensis concentration of 0.82 gDW.L-1 and biomass productivity of 33 mg.L-1.d-1. The findings of this study highlight the potential of natural zeolite in the development of microalgae-based processes for digestate and CO2 treatment.

Unveiling exosomes: Cutting-edge isolation techniques and their therapeutic potential.

Exosomes are one type of nanosized membrane vesicles with an endocytic origin. They are secreted by almost all cell types and play diverse functional roles. It is essential for research purposes to differentiate exosomes from microvesicles and isolate them from other components in a fluid sample or cell culture medium. Exosomes are important mediators in cell-cell communication. They deliver their cargos, such as mRNA transcripts, microRNA, lipids, cytosolic and membrane proteins and enzymes, to target cells with or without physical connections between cells. They are highly heterogeneous in size, and their biological functions can vary depending on the cell type, their ability to interact with recipient cells and transport their contents, and the environment in which they are produced. This review summarized the recent progress in exosome isolation and characterization techniques. Moreover, we review the therapeutic approaches, biological functions of exosomes in disease progression, tumour metastasis regulation, immune regulation and some ongoing clinical trials.

Maximizing chitin and chitosan recovery yields from Fusarium verticillioides using a many-factors-at-a-time approach

The extraction of chitin and chitosan presents challenges due to the complexity of the process and the influence of many variables. This study aimed to optimize chitin and chitosan extraction from Fusarium verticillioides by analyzing many additives and processing variables and modeling their yields using multiple linear regression (MLR) and evolutionary algorithms. FT-IR analysis confirmed the presence of characteristic bands in the extracted samples, and SEM analysis further revealed the microfibrillar appearance of the chitin and the dense, non-porous structure of the chitosan. The Ant Lion Optimizer (ALO) was employed to select significant factors and optimize model parameters. A transformation was applied to capture nonlinear relationships, and the fine-tuned models showed improved predictive power, with p-values of 0.00203 for chitin and 0.00884 for chitosan. Multi-objective optimization (MOO) using the Adaptive Geometry Estimation-based Multi-Objective Evolutionary Algorithm (AGE-MOEA) further identified significant factors for optimal yields, achieving 3 g of Arginine, 100 ml of culture medium volume, 7 to 11 days of incubation time, 0.2 to 1.76 ml of Oligochitin, 1.4 g of FeSO4, 1.5 g of K2HPO4, and 1 g of NaCl. Therefore, the integration of ALO and AGE-MOEA algorithms effectively modeled and optimized chitin and chitosan yields by maximizing biopolymer recovery, enabling significant industrial exploitation.

Acanthamoeba castellanii trophozoites need oxygen for normal functioning and lipids are their preferred substrate, offering new possibilities for treatment

Acanthamoebae, pathogenic free-living amoebae, can cause Granulomatous Amoebic Encephalitis (GAE) and keratitis, and for both types of infection, no adequate treatment options are available. As the metabolism of pathogens is an attractive treatment target, we set out to examine the energy metabolism of Acanthamoeba castellanii and studied the aerobic and anaerobic capacities of the trophozoites. Under anaerobic conditions, or in the presence of inhibitors of the electron-transport chain, A. castellanii trophozoites became rounded, moved sluggishly and stopped multiplying. This demonstrates that oxygen and the respiratory chain are essential for movement and replication. Furthermore, the simultaneous activities of both terminal oxidases, cytochrome c oxidase and the plant-like alternative oxidase, are essential for normal functioning and replication. The inhibition of normal function caused by the inactivity of the respiratory chain was reversible. Once respiration was made possible again, the rounded, rather inactive amoebae formed acanthopodia within 4 h and resumed moving, feeding and multiplying. Experiments with radiolabelled nutrients revealed a preference for lipids over glucose and amino acids as food. Subsequent experiments showed that adding lipids to a standard culture medium of trophozoites strongly increased the growth rate. Acanthamoeba castellanii trophozoites have a strictly aerobic energy metabolism and β-oxidation of fatty acids, the Krebs cycle, and an aerobic electron-transport chain coupled to the ATP synthase, producing most of the used ATP. The preference for lipids can be exploited, as we show that three known inhibitors of lipid oxidation strongly inhibited the growth of A. castellanii. In particular, thioridazine and perhexiline showed potent effects in low micromolar concentrations. Therefore, this study revealed a new drug target with possibly new options to treat Acanthamoeba infections.

In Vitro Rooting of <em>Gyrinops walla</em> in Activated Charcoal-Containing Semi-Solid Culture and Filter Paper-Bridged Liquid Culture Systems

Plant tissue culture is the best option in producing planting material for cultivation programmes of Gyrinops walla. Perfection of in vitro rooting protocol for this species is the most decisive step in micropropagation. In the present study the effect of physical state of substrate, different concentrations of sucrose, IBA and NAA in media containing activated charcoal on in vitro rooting was investigated. In experiment-1, 0.2% (w/v) activated charcoal was added in to the ½ MS medium fortified with 30 or 40 g/L sucrose and varying concentrations of IBA or NAA (0, 0.1, 0.5, 1.0 and 2.0 mg/L). In experiment-2, filter paper-bridged liquid medium was used with 40 g/L sugar and varying concentrations of IBA or NAA (0, 0.1, 0.5, 1.0 and 2.0 mg/L). The treatments were arranged in completely randomized design with three replicates. Percentage of rooted microshoots and number of roots per shoot were recorded. The ½ MS semi-solid medium with 0.2% activated charcoal, 40 g/L sucrose and 0.1 mg/L NAA resulted in the significantly highest rooting percentage (50%)while the significantly highest number of roots (6.0) were noted in ½ MS charcoal added (0.2%) basal medium with 40 g/L sucrose and 0.5 mg/L NAA. Shoots supported with filter paper bridge on ½ MS liquid medium, supplemented with 0.5 mg/L IBA, had the highest rooting percentage (66.7%) and the highest mean number of roots per shoot (3.0) (P<0.05). Results confirmed that in vitro rooting of G. walla could be perfected by optimizing the culture medium and its constituents.

Investigation into optimizing fermentation processes to enhance uric acid degradation by probiotics

To enhance the activity of Lactiplantibacillus plantarum FS4722 (L. plantarum FS4722) in reducing uric acid, this study optimized the culture medium components and fermentation conditions using response surface methodology. The results indicated that the optimal culture medium comprised glucose at 25.61g/L, yeast extract powder (YEP) at 26.86g/L, and nucleoside addition at 0.48g/L. The optimal fermentation conditions were: a fermentation time of 10h, an initial pH of 6.5, a fermentation temperature of 33 °C, and an inoculum size of 1.0%. Under these conditions, the nucleoside degradation ability of L. plantarum FS4722 increased by 41.9 times, enhancing its potential applications in reducing uric acid and developing anti-gout foods, health supplements, and pharmaceuticals.

Salinity as an Abiotic Stressor for Eliciting Bioactive Compounds in Marine Microalgae.

This study investigated the impact of culture medium salinity (5-50 PSU) on the growth and maximum photochemical yield of photosystem II (Fv/Fm) and the composition of carotenoids, fatty acids, and bioactive substances in three marine microalgae (Chrysochromulina rotalis, Amphidinium carterae, and Heterosigma akashiwo). The microalgae were photoautotrophically cultured in discontinuous mode in a single stage (S1) and a two-stage culture with salt shock (S2). A growth model was developed to link biomass productivity with salinity for each species. C. rotalis achieved a maximum biomass productivity (Pmax) of 15.85 ± 0.32 mg·L-1·day-1 in S1 and 16.12 ± 0.13 mg·L-1·day-1 in S2. The salt shock in S2 notably enhanced carotenoid production, particularly in C. rotalis and H. akashiwo, where fucoxanthin was the main carotenoid, while peridinin dominated in A. carterae. H. akashiwo also exhibited increased fatty acid productivity in S2. Salinity changes affected the proportions of saturated, monounsaturated, and polyunsaturated fatty acids in all three species. Additionally, hyposaline conditions boosted the production of haemolytic substances in A. carterae and C. rotalis.

Pancreatic Differentiation of Oral Minor Salivary Gland Stem Cells.

Stem cells from various sources including major salivary glands have been used to establish pancreatic differentiation in an attempt to provide new treatment options for patients with diabetes mellitus. In contrast, the potential of using the more easily accessible intraoral minor salivary glands has not been evaluated so far. Salivary stem cells were isolated from normal labial minor salivary glands that were removed during the excision of a mucocele and were attempted to differentiate into pancreatic cell lines using a culture medium enriched with activin A, retinoic acid and GLP-1.Real time RT-PCR was used to evaluate the expression of the genes of pancreatic transcription factors MafA, Ptf1a, Hb9 and Arx. Complementary, 22 labial minor salivary gland paraffin-embedded specimens were examined using immunohistochemistry for the presence of the relevant gene products of the pancreatic transcription factors Arx, MafA, Ptf1a and Pdx1. The differentiated salivary stem cells(cells of passage 3) expressed the genes of the pancreatic transcription factors MafA, Ptf1a, Hb9 and Arx even on the first day of the experiment while immunohistochemistry also confirmed the presence of the protein products of Arx, MafA, Ptf1a as well as Pdx1[> 50% of the specimens for Arx(5/8) and MafA(7/8), < 50% for Ptf1a(5/11) and Pdx1(5/11)] in ducts, mesenchymal connective tissue and acinar cells. Labial minor salivary glands may share gene and protein characteristics with pancreas suggesting a possible usefulness for pancreatic regeneration or substitution in cases of deficiency.

Polyvinyl alcohol assisted citrate based reduction of gold(III) ions: Theoretical design and experimental study on green synthesis of spherical and biocompatible gold nanoparticles

In this study, we demonstrate the synthesis of small gold nanoparticles (typically 8-10 nm) through a green synthesis approach. This method involves utilizing chloroauric acid as the gold precursor, trisodium citrate as a mild reducing and co-capping agent, and polyvinyl alcohol (PVA), as a co-capping and shape-directing agent. This novel synthesis method stands alone as a protocol that involves just mixing the reagents at room temperature and allowing the reaction to proceed at ambient temperature without any disturbance. In the absence of PVA, spherical particles are not formed and the reaction mixture slowly turns black followed by precipitation.The synthesis process was meticulously tracked using time-resolved monitoring of the growth of the localized surface plasmon resonance (LSPR) by UV-vis spectroscopy and transmission electron microscopy. The as-prepared colloidal gold solution was bright red, attributed to the small size (≤ 10 nm) and spherical geometry of nanoparticles. This colloidal solution could be stored indefinitely at room temperature without precipitation or a change in its absorption profile. The nanoparticles remained stable in adverse conditions such as 100 mM NaCl solution, 1×PBS and cell culture medium. Additionally, they could be easily loaded with drugs like doxorubicin by adsorption. The particles were thoroughly characterized using a range of techniques, including UV-vis spectroscopy, attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR), high-resolution transmission electron microscopy (HRTEM), hyperspectral-enhanced dark field microscopy (HEDFM), dynamic light scattering (DLS), and X-ray photoelectron spectroscopy (XPS). Cell viability tests conducted on 2D cell lines and 3D spheroids revealed negligible toxicity even at high concentrations. Furthermore, we demonstrate that an advanced version of conventional diffusion-limited aggregation (DLA) schemes, which allows individual clusters to move freely within a domain to form larger aggregates, can effectively replicate the intricate interactions between chloroauric acid, trisodium citrate, and PVA, the agents involved in the synthesis of gold nanoparticles.

Characterization of the Clostridioides difficile 630Δerm putative Pro-Pro endopeptidase CD1597.

Clostridioides difficile is the leading cause of antibiotic-associated infections worldwide. Within the host, C. difficile can transition from a sessile to a motile state by secreting PPEP-1, which releases the cells from the intestinal epithelium by cleaving adhesion proteins. PPEP-1 belongs to the group of Pro-Pro endopeptidases (PPEPs), which are characterized by their unique ability to cleave proline-proline bonds. Interestingly, another putative member of this group, CD1597, is present in C. difficile. Although it possesses a domain similar to other PPEPs, CD1597 displays several distinct features that suggest a markedly different role for this protein. We investigated the proteolytic activity of CD1597 by testing various potential substrates. In addition, we investigated the effect of the absence of CD1597 by generating an insertional mutant of the cd1597 gene. Using the cd1597 mutant, we sought to identify phenotypic changes through a series of in vitro experiments and quantitative proteomic analyses. Furthermore, we aimed to study the localization of this protein using a fluorogenic fusion protein. Despite its similarities to PPEP-1, CD1597 did not show proteolytic activity. In addition, the absence of CD1597 caused an increase in various sporulation proteins during the stationary phase, yet we did not observe any alterations in the sporulation frequency of the cd1597 mutant. Furthermore, a promoter activity assay indicated a very low expression level of cd1597 in vegetative cells, which was independent of the culture medium and growth stage. The low expression was corroborated by our comprehensive proteomic analysis of the whole cell cultures, which failed to identify CD1597. However, an analysis of purified C. difficile spores identified CD1597 as part of the spore proteome. Hence, we predict that the protein is involved in sporulation, although we were unable to define a precise role for CD1597 in C. difficile.

Effects of fermentation conditions (salt concentration, temperature, and pH) on Lactobacillus strains for induction of interleukin-12 in the exposed murine splenocytes

The present work aimed to assess the stresses of temperature, salt concentration, and pH on Lactobacillus gasseri 54C, Lactiplantibacillus plantarum ATCC 14917, and 11SH in fermented culture medium and their effects on interleukin-12 (IL-12) induction in murine splenocytes. First, the strains were fermented under different microbial stress conditions at salt concentrations (2, 4, and 6% w/v), temperatures (30 and 42°C), and pH levels (4, 5, and 6), as well as their combined conditions. Then, they were heat-killed, lyophilized, and exposed to murine splenocytes. The IL-12 induction was measured using the immunoassay method. It was indicated that the induction of IL-12 depended on the culture conditions, as it was increased by the fermentation strains under microbial stresses with the higher temperature (42°C), lower salt concentration (%2), and lower pH level (4). It seems that the bacterial cells' integrity is essential to stimulate the induction of IL-12.

A low concentration of choline chloride alters the developmental program of the bovine preimplantation embryo.

Choline is a known developmental programming agent of the bovine preimplantation embryo. Culture of the embryo with 1.8 mmol/L choline, a concentration much higher than in blood, alters development to cause increased weaning weight and other changes during the postnatal period. It was hypothesized here that choline exerts similar effects on the developmental program of the embryo when added at concentrations similar to those in peripheral blood (i.e., 4 mol/L). Oocytes were collected via ovum pick up and embryos were produced in vitro. Embryos were cultured until day 7 after fertilization in medium with 4 mol/L choline chloride, or, as a vehicle control, with an additional 4 mol/L sodium chloride. Blastocysts were transferred into recipients and pregnancy was diagnosed at approximately 28 d of gestation. Subsequent calves (n=37 for vehicle and n=35 for choline) were weighed at birth and at weaning. Addition of choline to culture medium did not affect the proportion of embryos that became blastocysts or the proportion of transferred blastocysts that produced a pregnancy. Birth weight was unaffected by treatment but calves derived from choline-treated embryos were heavier at time of weaning and gained more per day from birth until weaning than calves derived from embryos treated with vehicle. Results demonstrate that choline can act on the preimplantation embryo at a physiologically-relevant concentration to alter postnatal phenotype. Observations are further evidence for the importance of the first days of embryonic development for the phenotype of the resulting calf.

First report of Colletotrichum perseae Causing Anthracnose Disease of Avocado (Persea americana) in Türkiye

Colletotrichum-induced anthracnose is a major disease of avocado (Persea americana), causing significant losses in production worldwide. In 2023, a survey of avocado fruits in markets within the Kızıltepe district of Mardin, Türkiye, revealed symptoms of anthracnose, characterized by brown-black lesions and internal necrosis. To identify the causal agent, 20 symptomatic avocado fruits were collected and fungal isolates resembling Colletotrichum species were obtained. Morphological characterization, multilocus sequence analysis of the internal transcribed spacer (ITS), actin (act), the apn2-Mat1-2 intergenic spacer (ApMat), and calmodulin (cal) loci, and pathogenicity testing were performed for only two representative isolates (Pa01 and Pa02). These analyses identified the pathogen as Colletotrichum perseae. The isolates readily formed ascomata on all culture media tested, suggesting a high capacity for sexual reproduction. Pathogenicity tests confirmed that C. perseae caused characteristic dark, sunken anthracnose lesions on avocado fruits. This is the first report of C. perseae causing anthracnose in avocados in Türkiye, expanding the known geographical distribution of this pathogen. The findings highlight the need for effective disease management strategies and further research to understand the pathogen’s lifecycle, environmental conditions favoring infection, and potential for seedborne transmission.