The benefits of flushing with Lemuru fish oil as a source of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on the performance of reproductive parameters in Garut ewes.

This study aimed to evaluate and analyze the effects of a flushing diet containing Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) from Lemuru (Sardinella sp) fish oil on the reproductive performance parameters of Garut ewes. Forty (n = 40) primiparous Garut ewes aged 12-14 months with an average body weight of 28.92 ± 4.94 kg were assigned into four experimental treatment groups. The experimental diets contained roughage: concentrate (30:70%) designated as control concentrate (CNT), flushing concentrate with 6% palm oil (PO), flushing concentrate with 3% palm oil mixed with 3% lemuru oil as DHA and EPA sources (PFO), and flushing concentrate with the addition of 6% lemuru oil (FO). Treatment animals were fed two weeks before and after conception and parturition (8 weeks of total flushing treatment). The addition of fish oil at either 3% (PFO) or 6% (FO) resulted in significantly higher reproductive performance of ewes by increasing the litter size, as reflected by the birth of multiple kids (P < 0.05) compared to CNT and PO. Adding fish oil (PFO and FO) also maintains gestation, resulting in increased lamb yield, especially in the FO treatment, which yields the highest lamb yield (0% single lamb birth). The lamb male ratio was also higher with fish oil supplementation (PFO and PO) (P < 0.05). This research revealed a positive effect of 6% Lemuru oil on decreasing embryo loss and increasing the proportion of twin births. These findings thus support the hypothesis that ration flushing with double the required DHA and EPA from 6% Lemuru fish oil (FO) resulted in significantly higher reproductive performance in Garut sheep.

WFS1 protein expression correlates with clinical progression of optic atrophy in patients with Wolfram syndrome.

BACKGROUND: Wolfram syndrome (WFS) is a rare disorder characterised by childhood-onset diabetes mellitus and progressive optic atrophy. Most patients have variants in the WFS1 gene. We undertook functional studies of WFS1 variants and correlated these with WFS1 protein expression and phenotype. METHODS: 9 patients with a clinical diagnosis of WFS were studied with quantitative PCR for markers of endoplasmic reticulum (ER) stress and immunoblotting of fibroblast protein extracts for WFS1 protein expression. Luciferase reporter assay was used to assess ATF-6 dependent unfolded protein response (UPR) activation. RESULTS: 6 patients with compound heterozygous nonsense mutations in WFS1 had no detectable WFS1 protein expression; 3 patients with missense variants had 4%, 45% and 48% WFS1 protein expression. One of these also had an OPA1 mutation and was reclassified as autosomal dominant optic atrophy-plus syndrome. There were no correlations between ER stress marker mRNA and WFS1 protein expression. ERSE-luciferase reporter indicated activation of the ATF6 branch of UPR in two patients tested. Patients with partial WFS1 expression showed milder visual acuity impairment (asymptomatic or colour blind only), compared with those with absent expression (registered severe vision impaired) (p=0.04). These differences remained after adjusting for duration of optic atrophy. CONCLUSIONS: Patients with WFS who have partial WFS1 protein expression present with milder visual impairment. This suggests a protective effect of partial WFS1 protein expression on the severity and perhaps progression of vision impairment and that therapies to increase residual WFS1 protein expression may be beneficial.

Evaluation of rations containing bioconverted cacao pod as fiber source for small ruminant.

This study aimed to evaluate the potential use of bioconverted cacao pod (BCP) as a substitute for forage in the total mixed ration (TMR) for a small ruminant. In the in vitro experiment, the control TMR (30% forage and 70% concentrate) was substituted with two different levels of BCP (15% and 30%) and two different types of BCP ( BCP-pc and BCP-tv). Based on the in vitro evaluation, the best ration was then chosen for the in vivo experiment, in which male goats were fed a control TMR, the TMR containing 15% BCP-pc (RC), and TMR containing 15% bioconverted palm kernel meal (RP). The results showed that TMRs with 15% BCP-pc and BCP-tv substitution had significantly lower gas production and digestibility than the control ration. However, the TMR with 15% or 30% BCP substitution showed no significant difference in rumen fermentation characteristics, methane production, and total protozoa. In the in vivo experiment, the RC showed no significant difference in all nutrient intakes, the average daily gain of animals, feed conversion ratio value, and crude fiber digestibility but reduced dry and organic matter digestibility. In comparison, the RP resulted in reduced parameters. Therefore, the study concluded that BCP-pc at a level of 15% could be used as a substitute for forage in TMR for male goats without compromising the fermentability of rumen, nutrient intakes, and their average daily gain and feed conversion ratio. Overall, this study suggests the potential of BCP-pc as an alternative feed ingredient.

Monogenic diabetes syndromes: Locus-specific databases for Alström, Wolfram, and Thiamine-responsive megaloblastic anemia.

We developed a variant database for diabetes syndrome genes, using the Leiden Open Variation Database platform, containing observed phenotypes matched to the genetic variations. We populated it with 628 published disease-associated variants (December 2016) for: WFS1 (n = 309), CISD2 (n = 3), ALMS1 (n = 268), and SLC19A2 (n = 48) for Wolfram type 1, Wolfram type 2, Alström, and Thiamine-responsive megaloblastic anemia syndromes, respectively; and included 23 previously unpublished novel germline variants in WFS1 and 17 variants in ALMS1. We then investigated genotype-phenotype relations for the WFS1 gene. The presence of biallelic loss-of-function variants predicted Wolfram syndrome defined by insulin-dependent diabetes and optic atrophy, with a sensitivity of 79% (95% CI 75%-83%) and specificity of 92% (83%-97%). The presence of minor loss-of-function variants in WFS1 predicted isolated diabetes, isolated deafness, or isolated congenital cataracts without development of the full syndrome (sensitivity 100% [93%-100%]; specificity 78% [73%-82%]). The ability to provide a prognostic prediction based on genotype will lead to improvements in patient care and counseling. The development of the database as a repository for monogenic diabetes gene variants will allow prognostic predictions for other diabetes syndromes as next-generation sequencing expands the repertoire of genotypes and phenotypes. The database is publicly available online at https://lovd.euro-wabb.org.

Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression.

Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that is involved in the regulation of the unfolded protein response (UPR), intracellular ion homeostasis, cyclic adenosine monophosphate production and regulation of insulin biosynthesis and secretion. In this study, single cell Ca(2+) imaging with fura-2 and direct measurements of free cytosolic ATP concentration ([ATP]CYT) with adenovirally expressed luciferase confirmed a reduced and delayed rise in cytosolic free Ca(2+) concentration ([Ca(2+)]CYT), and additionally, diminished [ATP]CYT rises in response to elevated glucose concentrations in WFS1-depleted MIN6 cells. We also observed that sarco(endo)plasmic reticulum ATPase (SERCA) expression was elevated in several WFS1-depleted cell models and primary islets. We demonstrated a novel interaction between WFS1 and SERCA by co-immunoprecipitation in Cos7 cells and with endogenous proteins in human neuroblastoma cells. This interaction was reduced when cells were treated with the ER stress inducer dithiothreitol. Treatment of WFS1-depleted neuroblastoma cells with the proteasome inhibitor MG132 resulted in reduced accumulation of SERCA levels compared with wild-type cells. Together these results reveal a role for WFS1 in the negative regulation of SERCA and provide further insights into the function of WFS1 in calcium homeostasis.

Vacuolar-type H+-ATPase V1A subunit is a molecular partner of Wolfram syndrome 1 (WFS1) protein, which regulates its expression and stability.

Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that also localizes to secretory granules in pancreatic beta cells. Although its precise functions are unknown, WFS1 protein deficiency affects the unfolded protein response, intracellular ion homeostasis, cell cycle progression and granular acidification. In this study, immunofluorescent and electron-microscopy analyses confirmed that WFS1 also localizes to secretory granules in human neuroblastoma cells. We demonstrated a novel interaction between WFS1 and the V1A subunit of the H(+) V-ATPase (proton pump) by co-immunoprecipitation in human embryonic kidney (HEK) 293 cells and with endogenous proteins in human neuroblastoma cells. We mapped the interaction to the WFS1-N terminal, but not the C-terminal domain. V1A subunit expression was reduced in WFS1 stably and transiently depleted human neuroblastoma cells and depleted NT2 (human neuron-committed teratocarcinoma) cells. This reduced expression was not restored by adenoviral overexpression of BiP (immunoglobulin-binding protein) to correct the ER stress. Protein stability assays demonstrated that the V1A subunit was degraded more rapidly in WFS1 depleted neuroblastoma cells compared with wild-type; however, proteosomal inhibition did not restore the expression of the V1A subunit. Cell cycle assays measuring p21(cip) showed reduced levels in WFS1 depleted cells, and an inverse association between p21(cip) expression and apoptosis. We conclude that WFS1 has a specific interaction with the V1A subunit of H(+) ATPase; this interaction may be important both for pump assembly in the ER and for granular acidification.

Utilization of Primary Health Care Under National Health Insurance in Samarinda Municipality, East Kalimantan Province, Indonesia.

Aim: In Indonesia, basic community health services are provided to all citizens through Primary Health Care (PHC) settings under the National Health Insurance (NHI) scheme. The insurance is compulsory and provides basic community health needs. Based on a gatekeeper concept, the PHC is deemed to be the first contact point for all basic healthcare needs. Despite the commencement of services through PHC settings in 2014 under this concept, utilization in PHC settings remains lower than in hospital settings. This study aimed to assess factors associated with utilization of PHC under National Health Insurance in Samarinda Municipality, East Kalimantan Province, Indonesia. Materials and Methods: The research examined the utilization of services over six months. It employed a cross-sectional method and included 382 NHI participants in 10 districts of Samarinda Municipality. Each district was divided into urban and semi-urban areas based upon local government indicators representing the whole research area. A two-stage random sampling and purposive sampling approach was implemented to select the sample. The participants were interviewed using a structured questionnaire. Chi-square and multiple logistic regressions were conducted to determine the impact of factors on the utilization of PHC. Results: Only 17.3% of participants used PHC services regularly. Three constitutive factors, type of NHI participants (Adj. OR: 2.62; p<0.005), accommodation (Adj. OR: 2.18; p<0.005) and awareness (Adj. OR: 3.27; p<0.005) most profoundly influenced the under-utilization of PHC by NHI participants. Conclusion: The study found that the type of NHI participant and the utilization factors of accommodation and awareness significantly influenced the degree of utilization of PHC facilities by NHI participants and that the differences arose from variations in knowledge and experience. Strengthening these factors will rely upon an expanded role of government and community collaboration, emphasizing the needs of NHI participants.

Use of irradiated chitosan as a matrix for slow-release urea and in vitro fermentation characteristics of slow-release urea supplementation in ruminant rations.

Background and Aim: Irradiated chitosan can be used as a matrix for slow-release urea (SRU) production. This study aimed to (1) determine the optimal formulation of irradiated chitosan matrix for controlling nitrogen release and (2) evaluate the characteristics of SRU in vitro fermentation based on irradiated chitosan as a feed supplement. Materials and Methods: In the first phase of the investigation, four chitosan-based SRU formulations with varying amounts of acrylamide (3 and 5 g) and gamma irradiation (5 and 10 kGy) were evaluated. Scanning electron microscopy, Fourier transform mid-infrared spectroscopy, and ammonia release characteristics were used to observe morphological, functional group, and ammonia release characteristics. In the second phase of research, the most effective SRU formulation was utilized as a supplement to ruminant rations based on rice straw, sorghum straw, and alfalfa. Gas production, rumen fermentation characteristics, and methane gas production were observed in vitro. Results: On the basis of surface image analysis, the four SRU formulas generate a similar appearance. Compared with untreated urea, the SRU3 formula reduced the percentage of ammonia emission by 12.85%-27.64% after 24 h of incubation (p = 0.05), as determined by the first phase study. SRU3 became the basis for the second testing phase. The addition of SRU3 did not affect the optimal gas production in vitro. SRU3 treatment produced less gas than Optigen® treatment (p = 0.05). With regard to rumen fermentation and digestibility, Optigen® yielded better results than SRU3 (p = 0.05). However, the treatment with SRU3 resulted in reduced methane production compared to that in the control (p = 0.05). Conclusion: Irradiated chitosan as an SRU matrix may control the release of ammonia in the rumen medium. The SRU3 formulation is the most effective. The addition of SRU to rice straw-based rations reduces methane production without affecting in vitro digestibility.

Perlman syndrome: overgrowth, Wilms tumor predisposition and DIS3L2.

Perlman syndrome is a rare autosomal recessively inherited congenital overgrowth syndrome characterized by polyhydramnios, macrosomia, characteristic facial dysmorphology, renal dysplasia and nephroblastomatosis and multiple congenital anomalies. Perlman syndrome is associated with high neonatal mortality and, survivors have developmental delay and a high risk of Wilms tumor. Recently a Perlman syndrome locus was mapped to chromosome 2q37 and homozygous or compound heterozygous mutations were characterized in DIS3L2. The DIS3L2 gene product has ribonuclease activity and homology to the DIS3 component of the RNA exosome. It has been postulated that the clinical features of Perlman syndrome result from disordered RNA metabolism and, though the precise targets of DIS3L2 have yet to be characterized, in cellular models DIS3L2 knockdown is associated with abnormalities of cell growth and division.

The diversity and quality of forages and their potency as herbal anthelmintic for swamp buffalo in Brebes District, Central Java.

Background and Aim: Swamp buffaloes play an important role in the rural economy of Indonesia. They consume various forages during their grazing time, including those with anti-parasitic potential. However, the information about the type and quality of forages and their potential as a natural anthelmintic for swamp buffalo is very limited. This study aimed to identify the diversity, quality, and anthelmintic potency of forages consumed by swamp buffaloes reared in Bantarkawung Subdistrict, Brebes District, Central Java Province, Indonesia. Materials and Methods: Samples of forages were obtained during three observation periods of the study, with a 12-week interval between each period. Forage diversity was evaluated by identifying its consumption by swamp buffaloes during their grazing activity in the field and feeding time in their shed. The quality of forages was analyzed using proximate analysis to measure their dry matter (DM), crude fiber (CF), crude protein (CP), crude fat (CFat), and ash contents. This is followed by the calculation of their total digestible nutrient based on the proximate analysis results. Botanical composition analysis was then conducted to measure the predominance of forages consumed by the livestock during their grazing activity. Literature reviews were carried out to explore forage's anthelmintic activity. Results: The results showed that swamp buffaloes consume nine species of forage in the shed and 47 in the grazing area, including nine legumes, 18 grass, and 20 others. Swamp buffaloes consumed forages of lower quality, which contained high CF contents and varying levels of other nutrients below their daily nutritional needs. The grazing activity allowed swamp buffaloes to consume a higher variety of forages with better nutritional quality, thereby enabling them to meet their nutritional needs. Legumes and other forages served as the major protein sources, providing CP of 20.03% DM and 11.53% DM, and CF levels of 17.01% DM and 20.35% DM, respectively. The results also showed that the consumption of these forages increased during the rainy season. The predominant species of legumes consumed were Leucaena leucocephala and Acacia spp., while Alternanthera sessilis and Merremia umbellata were the predominant species of other forages. A total of 13 of the 47 species could potentially be used as natural anthelmintic due to their secondary metabolites, namely, tannin, flavonoid, saponin, terpenoid, diterpenoid, and mimosine. These compounds exert anthelmintic effects by inhibiting egg-hatching and larval development, as well as damaging the surface structure of both larvae and adult worms, ultimately leading to the death of the parasites. Conclusion: Overall, swamp buffaloes consumed more variety of forages during grazing compared to when they were kept in sheds. While the low-variety and low-quality forages provisioned for swamp buffaloes in their shed resulted in a low nutrient intake below their daily requirement. Furthermore, daily grazing activities allowed swamp buffaloes to fulfill and supplement their need by consuming a variety of grasses, legumes, and other forages in their respective grazing areas. Some of these forages also have the potential to become natural anthelmintic because they contain secondary metabolites, such as tannins, flavonoids, saponins, terpenoids, diterpenoids, and mimosine.

Analysis of autophagy deficiency and cytotoxicity in autophagy-deficient human embryonic stem cell-derived neurons.

Autophagy, a catabolic process governing cellular and energy homeostasis, is essential for cell survival and human health. Here, we present a protocol for generating autophagy-deficient (ATG5-/-) human neurons from human embryonic stem cell (hESC)-derived neural precursors. We describe steps for analyzing loss of autophagy by immunoblotting. We then detail analysis of cell death by luminescence-based cytotoxicity assay and fluorescence-based TUNEL staining. This hESC-based experimental platform provides a genetic knockout model for undertaking autophagy studies relevant to human biology. For complete details on the use and execution of this protocol, please refer to Sun et al. (2023).1.

Depletion of WFS1 compromises mitochondrial function in hiPSC-derived neuronal models of Wolfram syndrome.

Mitochondrial dysfunction involving mitochondria-associated ER membrane (MAM) dysregulation is implicated in the pathogenesis of late-onset neurodegenerative diseases, but understanding is limited for rare early-onset conditions. Loss of the MAM-resident protein WFS1 causes Wolfram syndrome (WS), a rare early-onset neurodegenerative disease that has been linked to mitochondrial abnormalities. Here we demonstrate mitochondrial dysfunction in human induced pluripotent stem cell-derived neuronal cells of WS patients. VDAC1 is identified to interact with WFS1, whereas loss of this interaction in WS cells could compromise mitochondrial function. Restoring WFS1 levels in WS cells reinstates WFS1-VDAC1 interaction, which correlates with an increase in MAMs and mitochondrial network that could positively affect mitochondrial function. Genetic rescue by WFS1 overexpression or pharmacological agents modulating mitochondrial function improves the viability and bioenergetics of WS neurons. Our data implicate a role of WFS1 in regulating mitochondrial functionality and highlight a therapeutic intervention for WS and related rare diseases with mitochondrial defects.

NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.

Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.

The t(1;3) breakpoint-spanning genes LSAMP and NORE1 are involved in clear cell renal cell carcinomas.

By positional cloning, we identified two breakpoint-spanning genes in a familial clear cell renal cell carcinoma (CCRCC)-associated t(1;3)(q32.1;q13.3): LSAMP and NORE1 (RASSF1 homolog). Both genes are downregulated in 9 of 9 RCC cell lines. While the NORE1A promoter predominantly presents partial methylation in 6 of the cell lines and 17/53 (32%) primary tumors, the LSAMP promoter is completely methylated in 5 of 9 cell lines and in 14/53 (26%) sporadic and 4 familial CCRCCs. Expression of LSAMP and NORE1A proteins in CCRCC cell lines inhibited cell proliferation. These characteristics indicate that LSAMP and NORE1A may represent new candidate tumor suppressors for CCRCC.

Black soldier fly as feed ingredient for ruminants.

This paper is a review of some experiments using black soldier fly (BSF) and its by-product to explore their nutritional value, production potential in Indonesia and its application in the ration of ruminants. Evaluation on the effect of milk replacer, creep feed containing BSF, BSF frass and the possibility to use lactic acid bacteria from BSF as probiotics are presented. Utilization of BSF larvae in milk replacer as skim and cream milk substitute showed that there were similarity on physiological, hematological status and performance of goat kids compared to those offered goat milk or commercial milk replacer. In addition, BSF larvae can be used to substitute soybean meal in the creep feed for post weaning goat kids without any differences in weight gain and blood profiles. However, utilization of BSF frass in the fattening goat ration resulted lower digestibility of dry matter and organic matter due to the chitin content in the frass. Black soldier fly larvae grown on chicken manure harbour lactic acid bacteria (LAB) which have potential as probiotics for ruminants. In general, BSF larvae has potential as ingredient for milk replacer, creep feed, fattening ration, and source of LAB for probiotics.

Atherosclerotic Lesion of the Carotid Artery in Indonesian Cynomolgus Monkeys Receiving a Locally Sourced Atherogenic Diet.

The atherosclerotic lesion is a principal hallmark of atherosclerotic animal models. This study aimed to assess lesions of the carotid artery in Indonesian cynomolgus monkeys exposed to an IPB-1 atherogenic diet. A total of 20 adult male cynomolgus monkeys received the local IPB-1 diet for two years. Blood lipid profiles, morphology, and carotid ultrasound of monkeys were measured. Nine of them were euthanized to confirm atherosclerotic lesions. Common carotid arteries (CCA) and carotid bifurcation (BIF) samples were collected and stained using Verhoef-van Giessen and CD68 immunohistochemistry. The results reveal the presence of severe atherosclerosis plaques in six out of nine animals (66.7%) corresponding to intermediately and hyper-responsive groups. The hyper-responsive group displayed the highest response in the developing intimal area (IA) at the CCA (0.821 mm2), whereas the hyporesponsive group had the smallest IA (0.045 mm2) (p = 0.0001). At the BIF, the hyporesponsive group showed the smallest IA (p = 0.001), but there was no difference between the intermediately and hyper-responsive groups (p = 0.312). The macrophage marker CD68 was also expressed on the cartotid of the intermediately and hyper-responsive groups. These results indicate that severe atherosclerotic lesions with high infiltration of macrophages were formed in the carotid arteries of intermediately and hyper-responsive Indonesian cynomolgus monkeys fed with the local atherogenic diet IPB-1 over two years, thus confirming atherosclerosis in a nonhuman primate model.

Stimulation of follicle growth and development during estrus in Ettawa Grade does fed a flushing supplement of different polyunsaturated fatty acids.

BACKGROUND AND AIM: Flushing with the manipulation of fatty acids, particularly polyunsaturated fatty acids, like linoleic and α-linolenic acids in the ration, is a strategy to raise the nutritional status of the female mammals to improve ovarian function and follicle development. This study was designed to investigate the effectiveness of flushing supplementation with different types of polyunsaturated and saturated fatty acids in stimulating follicle growth and development during estrus in Ettawa Grade does with a low initial body condition score (BCS ≤2). MATERIALS AND METHODS: Eighteen Ettawa Grade does in the second parity, with an average body weight of 32.11±2.19 kg, were divided into three groups according to the fatty acid supplemented to their ration: (i) About 2.8% lauric acid flushing (group); (ii) 2.8% linoleic acid flushing (LA group); and (iii) 2.8% α-linolenic acid flushing (ALA group). The ration was formulated to be isocaloric (total digestible nutrient = 77%) and isonitrogenous (crude protein = 15%). The experiment was conducted for 35 days; that is, 14 days for acclimatization and synchronization of the estrous cycle and 21 days for fatty acid flushing until the appearance of the next estrus. A completely randomized design was applied. RESULTS: According to the results, none of the different fatty acids in the ration affected the nutrient intakes, BCSs, average daily gains, and plasma glucose, cholesterol, and progesterone concentrations of the three groups of does. However, the BCSs (by 0.8-0.9) and the plasma cholesterol concentrations were higher after fatty acid flushing for 21 days than before the flushing period. The ALA group had the highest number of large-sized preovulatory follicles, whereas the LAURIC group had the highest plasma estradiol concentration during estrus. All three groups had similar plasma progesterone concentrations during estrus after fatty acid flushing. CONCLUSION: Flushing supplementation with 2.8% ALA from flaxseed oil gave the best results in terms of stimulating the highest number of large-sized preovulatory follicles in Ettawa Grade does.

Prevalence and risk factors of trematode infection in swamp buffaloes reared under different agro-climatic conditions in Java Island of Indonesia.

BACKGROUND AND AIMS: This study was conducted to estimate the prevalence and intensity and to identify the associated risk factors and impact of trematode infection in swamp buffaloes reared under different agro-climatic conditions in Java, Indonesia. MATERIALS AND METHODS: A total of 580 fecal samples were collected from swamp buffaloes in five different agro-climatic areas in Banten and Central Java Provinces, Indonesia. The fecal samples were examined using the Danish Bilharziasis Laboratory Technique to determine the prevalence and intensity of trematode infection. The risk factors for infection were determined from an in-depth interview of owners/keepers, and the results were analyzed using Chi-square tests and multiple logistic regression. The infection was also correlated with swamp buffalo production parameters (body weight and body condition score [BCS]). RESULTS: From all fecal samples, the overall prevalence of trematode infection was 64.83%, which comprised Fasciola spp. (16.03%; mean eggs per gram [EPG]±SD: 1.02±0.43) and Paramphistomatidae (62.93%; mean EPG±SD: 1.01±0.66). The main risk factor for trematode infection was feeding animals with rice straw (odds ratio [OR]: 40.124); the risk of trematode infection was 40.142 times higher in buffaloes that consumed rice straw. Other risk factors included the frequency of anthelmintic treatment (OR: 4.666), age (OR: 0.449), and drinking water source (OR: 0.358). Trematode infection did not significantly affect the body weight or BCS of swamp buffaloes. CONCLUSION: Although the prevalence of trematode infection was high in swamp buffaloes, the intensity of infection was low, and the infection did not affect the animals' physical parameters.

Evaluation of a functional epigenetic approach to identify promoter region methylation in phaeochromocytoma and neuroblastoma.

The molecular genetics of inherited phaeochromocytoma have received considerable attention, but the somatic genetic and epigenetic events that characterise tumourigenesis in sporadic phaeochromocytomas are less well defined. Previously, we found considerable overlap between patterns of promoter region tumour suppressor gene (TSG) hypermethylation in two neural crest tumours, neuroblastoma and phaeochromocytoma. In order to identify candidate biomarkers and epigenetically inactivated TSGs in phaeochromocytoma and neuroblastoma, we characterised changes in gene expression in three neuroblastoma cell lines after treatment with the demethylating agent 5-azacytidine. Promoter region methylation status was then determined for 28 genes that demonstrated increased expression after demethylation. Three genes HSP47, homeobox A9 (HOXA9) and opioid binding protein (OPCML) were methylated in >10% of phaeochromocytomas (52, 17 and 12% respectively). Two of the genes, epithelial membrane protein 3 (EMP3) and HSP47, demonstrated significantly more frequent methylation in neuroblastoma than phaeochromocytoma. These findings extend epigenotype of phaeochromocytoma and identify candidate genes implicated in sporadic phaeochromocytoma tumourigenesis.

Genetic and epigenetic analysis of von Hippel-Lindau (VHL) gene alterations and relationship with clinical variables in sporadic renal cancer.

Genetic and epigenetic changes in the von Hippel-Lindau (VHL) tumor suppressor gene are common in sporadic conventional renal cell carcinoma (cRCC). Further insight into the clinical significance of these changes may lead to increased biological understanding and identification of subgroups of patients differing prognostically or who may benefit from specific targeted treatments. We have comprehensively examined the VHL status in tissue samples from 115 patients undergoing nephrectomy, including 96 with sporadic cRCC. In patients with cRCC, loss of heterozygosity was found in 78.4%, mutation in 71%, and promoter methylation in 20.4% of samples. Multiplex ligation-dependent probe amplification identified intragenic copy number changes in several samples including two which were otherwise thought to be VHL-noninvolved. Overall, evidence of biallelic inactivation was found in 74.2% of patients with cRCC. Many of the mutations were novel and approximately two-thirds were potentially truncating. Examination of these and other published findings confirmed mutation hotspots affecting codons 117 and 164, and revealed a common region of mutation in codons 60 to 78. Gender-specific differences in methylation and mutation were seen, although not quite achieving statistical significance (P = 0.068 and 0.11), and a possible association between methylation and polymorphism was identified. No significant differences were seen between VHL subgroups with regard to clinicopathologic features including stage, grade, tumor size, cancer-free and overall survival, with the exception of a significant association between loss of heterozygosity and grade, although a possible trend for survival differences based on mutation location was apparent.