Epigenetic and chromatin reprogramming in mouse development and embryonic stem cells

It is well established that epigenetics and chromatin modifications are important factors that can govern gene activity and nuclear architecture. They are also proven to be essential for normal embryonic development and cell differentiation. One important event during mouse development is the establishment of epigenetic reprogramming which is believed to be essential for normal growth and development, however; the mechanism is still poorly understood. The general objective of this PhD study was to investigate the profiles and mechanisms of epigenetic and chromatin modifications during normal mouse development and in embryonic stem cells. Mouse pre- and postimplantation embryos and ES cells were used in experiments employing a range of different methodologies. The dynamics of epigenetic DNA and histone methylation were captured using laser confocal immunofluorescent microscopy and western blotting. The activity of epigenetic modifiers was monitored by real-time PCR and candidate genes were validated using siRNA technology. The present studies demonstrate that heterochromatin markers H3K9me3, H3K9me2, H4K20me2, H4K20me3, HP1α and HP1β are reprogrammed during early development. Demethylation of H3K9me2, H3K9me3 and H4K20me3 took place at two-cell stage and remethylation occurred at four-cell stage except for H4K20me3. The reestablishment of H4K20me3 was initially observed in early postimplantation embryos in extraembryonic tissue, specifically in the mural trophectoderm. In embryonic tissue, H4K20me3 was not clearly detected until in mid to late postimplantation development. The mechanism of H3K9me2 and H3K9me3 demethylation might be due to either an imbalance of epigenetic modifiers or the presence of Jmjd2a and Jmjd1a histone demethylase postfertilisation. We have also report evidence that HP1α and Suv4-20h are required in heterochromatin before the recruitment of H4K20me3 during mouse development and in ES cells. Therefore H4K20me3 removal was believed to involve the lack of prerequisite heterochromatin complexes such as HP1α and Suv4-20h enzymes. Furthermore, the presence and levels of H4K20me3 and HP1α might be strongly associated with cell differentiation and tissue maturation in mouse in vivo development but not in vitro early differentiated ES cells. Surprisingly, the results showed that chromatin modifications and their modifiers in ES cells are different from ICM and epiblast. Chromatin modifications H4K20me3 and HP1α were absent from ICM and epiblast, but were detected in ES cells. Notably, H4K20me3 and HP1α were established after early incubation of ICM into ES cell medium, but this change was not dependent on the presence of serum and leukaemia inhibiting factor. Epigenetic modifier Jmjd2a but not Jmjd1a was found in ICM. Conversely, Jmjd1a is highly expressed in ES cells while Jmjd2a was inactivated. In addition, the present studies revealed the substantial role of histone demethylases in development, as it may be important for epigenetic reprogramming. The results demonstrated that inhibition of demethylase Jmjd2a and Jmjd1a caused preimplantation embryos to arrest at the twocell stage while Jmjd2c deficient embryos failed to reach blastocyst. Thus it is possible that Jmjd2a and Jmjd1a were essential for epigenetic reprogramming while Jmjd2c is critical for cell fate establishment during blastocyst formation. In conclusion, the global chromatin signature in ES cells differs from ICM and epiblast; heterochromatin reprogramming occurs at two-cell stage; maturation of heterochromatin occurs at postimplantation; and histone demethylases Jmjd1a, Jmjd2a and Jmjd2c are important in preimplantation development. Results from the present studies could provide crucial information for developmental biology and stem cell research, and provide as a model for improvement of reproductive biotechnologies such as somatic cell reprogramming, and diagnosis of epigenetic abnormalities in early development

Fertility after deep intra-uterine AI of concentrated low-volume boar semen doses

Boar semen can be successfully frozen -highly packed- in small containers (medium-straw, MS or multiple FlatPack, MFP). The use of deep intrauterine artificial insemination (DIUAI) can make possible the deposition of small volumes of this thawed, non re-extended semen deeply intra-cornual. The fertility achieved after single or double DIU-AI per oestrus was hereby studied, with special attention to the interval between AI and spontaneous ovulation. Semen from two boars of proven fertility was frozen in MS or MFP holding 1x109 total spermatozoa. Multiparous (n=42) crossbred sows were checked for oestrous behaviour after weaning and the occurrence of spontaneous ovulation was checked with transrectal ultrasonography (TUS) to establish the interval between onset of oestrus (OO) and ovulation. Sows were subjected to DIU-AI in the following oestrus using thawed semen (MS=20 or MFP=22), inseminated without further re-extension. Sows were randomly allotted to one of 3 groups: (1) Single DIU-AI 8 h before expected ovulation (Control group, n= 19), (2) Single DIU-AI 4 h before expected ovulation (Treatment group S, n=15) and (3) Double DIU-AI 12 and 4 h before expected ovulation (Treatment group D, n=8). Pregnancy was confirmed by TUS 28 days after OO in those sows not returning to oestrus. These sows were later slaughtered (day 30 to 45 of pregnancy), noting the appearance of the reproductive tract and ovaries, numbers of live foetuses, implantation sites and of CL. Some sows (n= 9) returning to oestrus were re-inseminated (either once [n=4] or twice [n=5]) in the following oestrus with either MFP (n=5) or MS (n=4) and slaughtered 12 to 14 h post-ovulation for recovery of spermatozoa from the utero-tubal junctions (UTJ, sperm reservoir) and of tubal oocytes, to disclose the effectiveness of sperm transport. Post-thaw sperm motility was 44.3±3.21% in MFP and 42.8±0.72 % for MS (LSMean±SEM, n.s.), and did not significantly change from thawing to AI. The DIU-AI could be performed in all sows, but insertion was slow (>5 min) in 5/42 sows of which 4 returned to oestrus. Pregnancy rate averaged 35% (Group D: 25%, Group S: 40%, Control: 36%, n.s.). The interval between DIU-AI and ovulation varied largely (group C: between -13 and -3 h , for S-group: between -11 and +3h , for group D: between -17 and -4 h). Pregnancy rates clearly related to the interval DIU-AI and ovulation, being highest (60%, 12/20) when AI occurred between 8 and 4 h before ovulation. Numbers of apparent implantation sites ranged 6 to 22 and of live foetuses 2 to 11 (n.s. among groups), while fertilization rate (total number of implantations/CL) ranged 48.0 to 69.7%, being highest in group D (P<0.05). The examination of the open sows slaughtered 12 to14 h post ovulation showed low sperm numbers (approx 4,000) in the UTJs. Only 40% of oocytes had spermatozoa bound to the zona pellucida, not more than 2 spermatozoa per oocyte, and only 10 % of recovered oocytes were fertilized, irrespective of using one or two DIU-AI (n.s.). The highest (p<0.05) values for these variables were recorded when DIU-AI (either single or double [second AI]) was done between 4 to 8 h before ovulation, especially when MFP-semen was used (P<0.05). In conclusion; (1) DIU-AI can be easily performed in most sows, (2)pregnancies can be obtained by the DIU-AI of low volumes of highly concentrated frozenthawed boar semen, once or twice during oestrus, but fertility is still low, probably owing to an incomplete replenishment of the sperm reservoirs, and (3) fertility is mainly related to the interval DIU-AI and ovulation -which should be -8 to -4 h of spontaneous ovulationand to the package, MFP having shown better results in vivo

Association of ocean macroplastic debris with stranded sea turtles in the Central Gulf of Thailand

The impact of macroplastic debris (>5 mm) on marine life is a global concern but has rarely been investigated in Thailand. This study investigated the relationship between stranded sea turtles and macroplastics in the Central Gulf of Thailand. Records of stranded turtles (n = 388) from 2017-2020 were analysed retrospectively to determine their interaction with macroplastics. In addition, macroplastics collected from the gastrointestinal (GI) tracts of 30 dead stranded turtles and 13 beaches (along a 100 m transect mid-way between high and low tide) between 2019 and 2020 were investigated. Types and composition of macroplastics were identified with the use of a stereomicroscope and Fourier-transform infrared spectrometer. Green turtles Chelonia mydas comprised the majority of stranded turtles (74%, n = 251), and macroplastics (entanglement or ingestion) were the leading cause of death (n = 152). Most stranded turtles were juveniles (65%), and their stranding was significantly correlated with macroplastics (p < 0.001). Juveniles were more prone than adults to become entangled (p = 0.007), while adults had a higher ingestion rate than juveniles (p = 0.009). Plastic fibres were commonly found in the GI tracts (62%, n = 152 of 244) and beaches (64%, n = 74 of 115). Most fibres from the GI tracts (83%, n = 126 of 152) and beaches (93%, n = 68 of 74) were fishing nets made of polyethylene or polypropylene. We conclude that fishing nets are a significant cause of sea turtle stranding in the Central Gulf of Thailand, and this issue requires immediate resolution

A systematic review and meta-analysis of the global prevalence and relationships among Burkholderia pseudomallei sequence types isolated from humans, animals, and the environment

Background and Aim: Burkholderia pseudomallei, a highly pathogenic bacterium responsible for melioidosis, exhibits ecological ubiquity and thrives within soil and water reservoirs, posing significant infection risks to humans and animals through direct contact. The aim of this study was to elucidate the genetic diversity and prevalence patterns of B. pseudomallei sequence types (STs) across a global spectrum and to understand the relationships between strains isolated from different sources. Materials and Methods: We performed a systematic review and meta-analysis in this study. Extensive research was carried out across three comprehensive databases, including PubMed, Scopus, and ScienceDirect with data collected from 1924 to 2023. Results: A total of 40 carefully selected articles contributed 2737 B. pseudomallei isolates attributed to 729 distinct STs and were incorporated into the systematic review. Among these, ST46 emerged as the most prominent, featuring in 35% of the articles and demonstrating a dominant prevalence, particularly within Southeast Asia. Moreover, ST51 consistently appeared across human, animal, and environmental studies. Subsequently, we performed a meta-analysis, focusing on nine specific STs: ST46, ST51, ST54, ST70, ST84, ST109, ST289, ST325, and ST376. Surprisingly, no statistically significant differences in their pooled prevalence proportions were observed across these compartments for ST46, ST70, ST289, ST325, and ST376 (all p > 0.69). Conversely, the remaining STs, including ST51, ST54, ST84, and ST109, displayed notable variations in their prevalence among the three domains (all p < 0.04). Notably, the pooled prevalence of ST51 in animals and environmental samples surpassed that found in human isolates (p < 0.01). Conclusion: To the best of our knowledge, this study is the first systematic review and meta-analysis to investigate the intricate relationships between STs and their sources and contributes significantly to our understanding of B. pseudomallei diversity within the One Health framework

Interactions between marine megafauna and plastic pollution in Southeast Asia

Southeast (SE) Asia is a highly biodiverse region, yet it is also estimated to cumulatively contribute a third of the total global marine plastic pollution. This threat is known to have adverse impacts on marine megafauna, however, understanding of its impacts has recently been highlighted as a priority for research in the region. To address this knowledge gap, a structured literature review was conducted for species of cartilaginous fishes, marine mammals, marine reptiles, and seabirds present in SE Asia, collating cases on a global scale to allow for comparison, coupled with a regional expert elicitation to gather additional published and grey literature cases which would have been omitted during the structured literature review. Of the 380 marine megafauna species present in SE Asia, but also studied elsewhere, we found that 9.1 % and 4.5 % of all publications documenting plastic entanglement (n = 55) and ingestion (n = 291) were conducted in SE Asian countries. At the species level, published cases of entanglement from SE Asian countries were available for 10 % or less of species within each taxonomic group. Additionally, published ingestion cases were available primarily for marine mammals and were lacking entirely for seabirds in the region. The regional expert elicitation led to entanglement and ingestion cases from SE Asian countries being documented in 10 and 15 additional species respectively, highlighting the utility of a broader approach to data synthesis. While the scale of the plastic pollution in SE Asia is of particular concern for marine ecosystems, knowledge of its interactions and impacts on marine megafauna lags behind other areas of the world, even after the inclusion of a regional expert elicitation. Additional funding to help collate baseline data are critically needed to inform policy and solutions towards limiting the interactions of marine megafauna and plastic pollution in SE Asia

Unique patterns of cardiogenic and fibrotic gene expression in rat cardiac fibroblasts

Background and Aim: Cardiac fibroblasts are important for both normal and pathological states of the heart, but the knowledge in cell physiology and genomics is still poorly understood. The aims of the present study were; first, to investigate the expression of cardiac and fibrotic genes in rat cardiac fibroblasts compared to cardiomyocytes and other fibroblasts (skin and muscle fibroblasts), second, to examine the in vitro effect of serum concentration on fibroblast gene expression. The findings can potentially be applied in ischemia/reperfusion models. Materials and Methods: Rat cardiac fibroblasts were collected and cultured in different conditions, and their gene expression (21 cardiogenic genes and 16 fibrotic genes) was compared with cardiomyocytes and other fibroblasts using comparative quantitative polymerase chain reaction. We also mimicked myocardial ischemia/reperfusion by depleting and then adding a serum into the culture in conventional culture (10% serum). Results: Cardiac fibroblasts expressed most of the cardiogenic genes, but their expression levels were significantly lower than in cardiomyocytes, while almost all fibrotic genes in the cardiac fibroblasts were significantly more highly expressed than in cardiomyocytes, except matrix metallopeptidase 9 (Mmp9) which also had greater expression in other fibroblasts. After mimicking cardiac ischemia and reperfusion in vitro by starving and then adding a serum into the cardiac fibroblast culture, the results revealed that Mmp9 expression was significantly increased (>30 times) after increasing but not reducing the serum in the culture. The expression of most cardiogenic and fibrotic genes in cardiac fibroblasts tended to decrease after increasing the serum in the culture. These changes were specific to cardiac fibroblasts but no other fibroblasts. Conclusion: Cardiac fibroblasts have a distinct pattern of gene expression from other fibroblasts and cardiomyocytes. They are also sensitive to high serum concentration but not affected by serum depletion, suggesting that the process of developing cardiac fibrosis might be stimulated by reperfusion or overcirculation rather than ischemia. The cell starvation followed the adding of serum may serve as a useful model to study cardiac fibrosis cause by the change of blood flow

Modifying a mini drone for remote drug delivery for wildlife medicine

Remote drug delivery is an essential tool for administering medication to wildlife. However, the conventional method, the dart gun, has limitations in terms of injection distance, posing risks for operators. This study aimed to modify a mini drone equipped with a dart syringe and delivery system for use with large wildlife. A commercial mini drone was modified to release a syringe dart using a vertical gravity-based delivery system. The performance of the drone and delivery system was evaluated based on accuracy to the target and penetration ability through pig skin. The evaluation compared a dart with or without a plastic shell, with tests conducted both indoors and outdoors. The results indicated that the higher the drone’s flight, the more the dart tended to deviate from the target. In outdoor tests, a syringe dart without a shell showed greater accuracy than a dart with a shell. Regarding penetration ability, only a dart without a shell had a 100% success rate at a maximum height of 5 m, with an overall statistical difference (P = 0.01). In conclusion, this study represents the first scientific validation of using mini drones for remote drug injections that could be used in large wildlife medicine

The occurrence of elephant endotheliotropic herpesvirus infection in wild and captive Asian elephants in Thailand: Investigation based on viral DNA and host antibody

Background and Aim: Elephant endotheliotropic herpesvirus (EEHV) is a serious disease, threatening the life of young elephants. Many elephants have been infected with no clinical signs and may serve as carriers spreading this disease. It is important to monitor the disease through clinical signs and molecular diagnosis. In this study we investigated the occurrence of EEHV and the efficiency of different techniques used to monitor EEHV infection in various samples and populations of Asian elephants. Materials and Methods: Blood and trunk swabs were collected from live elephants, while visceral organs (lung, digestive tract, spleen, lymph nodes, and kidney) were collected from dead elephants. EEHV was detected by polymerase chain reaction (PCR) in whole blood, trunk swabs, and visceral organs as samples, while elephant anti-EEHV immunoglobulin G (IgG) in serum was detected by enzyme-linked immunosorbent assay (ELISA). A total of 162 samples were analyzed in this study: 129 from healthy, 26 from dead, and 7 from sick elephants. Results: The present study showed that the overall incidence of EEHV was 40.1% (n=65/162). Approximately 46.2% (n=12/26) and 85.7% (n=6/7) of dead and sick elephants were positive for EEHV by PCR, respectively. All sick elephants that were young and affected by EEHV clinical disease tested negative for the IgG antibody ELISA, suggesting primary EEHV infection in this group. In addition, 2.3% (n=3/129) of subclinical infections were detected using PCR, and trunk swab samples showed slightly higher sensitivity (5.3%, n=2/38) to detect EEHV than whole blood (1.2%, n=1/84). As many as, 48.4% (n=44/91) of healthy elephants were EEHV seropositive (ELISA-positive), suggesting that many elephants in Thailand had previously been infected. Overall, 30% of dead wild elephants had been infected with EEHV (n=3/10). Moreover, statistical analysis revealed no significant differences in the EEHV detection rate between different age groups or sexes (p>0.05). Conclusion: PCR is better than ELISA to detect EEHV active infection in dead/sick elephants and to monitor EEHV in young elephants. ELISA is suitable for detecting previous EEHV infection and carriers, particularly adults. Theoretically, we could use both PCR and ELISA to increase the sensitivity of testing, along with observing abnormal behavior to efficiently monitor this disease. Identification of EEHV carriers within elephant populations is important to prevent transmission to healthy individuals, especially young elephants with high mortality from EEHV. This is the first report from Thailand regarding EEHV infection in wild elephants, showing the importance of preventing disease transmission between captive and wild elephants

Molecular characterization and nucleotide substitution of antibiotic resistance genes in multidrug-resistant Escherichia coli isolated from environmental swine farms

An increase of multi-drug resistant Escherichia coli in swine husbandry have been concerned worldwide. This study aimed to determine the multidrug resistance and nucleotide substitution of beta-lactam antibiotic and tetracycline resistant genes in E. coli from swine farms in Southern Thailand. A total of 112 isolates of E. coli was isolated from 50 pig farms, which were confirmed by identified by MALDI-TOF analysis. Seventy-three isolates (65.18%) and 39 isolates (34.82%) were isolated from the feces and waste water samples, respectively. One hundred percent resistance to beta-lactam antibiotics as well as their resistant gene blaTEM was detected in isolates. Furthermore, 81% isolates were tetracycline resistance and both tetA [68.42% (13/19) in feces samples, 72.73% (8/11) in waste water samples] and tetB [10.53% (2/19) in feces samples, 18.18% (2/11) in waste water samples] genes responsible for tetracycline resistance were observed. Furthermore, 54 isolates had multi-drug resistance that presented 11 different patterns. The nucleotide substitution of genes was detected in 3 isolates of E. coli, and may consider as the point mutation. The nucleotide at 859 bp of tetA gene of the isolate WU-WW009-01 was changed from T to A. While, the isolate WU-WW004-02 showed 2 nucleotide substitution sites at the position of 266 (from A to G) and 859 (from T to G) bp. The nucleotide at 36 bp of blaTEM gene of the isolate WU-F003-02 was replaced from G to A. Findings of this study may help to control the spread of E. coli antibiotic resistance genes in the swine farms