Occurrence and distribution of phthalate esters and microplastics in wastewater treatment plants in Taiwan and their toxicological risks.

Phthalate esters (PAEs) are fat soluble synthetic chemicals, usually regarded as plasticizers for being added in numerous plastic products. Thus, environmental and health hazards of PAEs are associated with increasing plastic pollution. In this study, PAEs from sludge samples collected from water, sewage, and industrial treatment plants (N = 17) were analyzed using Gas Chromatography/Mass Spectrometry. Microplastics (MPs) were also quantified and correlated with PAEs. Results showed the highest average PAE concentrations in sewage treatment plants. The greatest ΣPAEs concentration were found in sewage treatment plant (STP4) with 32,414 µg/kg dw, while the lowest found in water treatment plant (WTP3) with 2062 µg/kg dw. Among different PAEs, di-(2-ethyl hexyl) phthalate (DEHP) contributes the highest. Similarly, DEHP, di-n-octyl phthalate (DnOP) and diisononyl phthalate (DiNP) significantly correlated with the total PAEs indicating their large contribution to sludge contamination. The abundance of microplastics in sludge ranged between 1 and 7 MP/g, highest at ITP6, but not detected in some stations. While microplastics may potentially increase PAEs, there was no significant relationship between ΣPAEs and MP abundance. The estimated human daily intake of DEHP and di-n-butyl phthalate (DnBP) when contaminated sludge be used showed low toxicological risks to exposed adults. This research presents the sludge characteristics, PAEs, and microplastic concentrations in different wastewater treatment plants in Taiwan. PAE contamination was highly contributed by domestic and industrial wastes shown by their significant amounts in STP and ITP. Results further provide evidence for potential sludge recycling (WTP sludge) and application to soil.

Annotation of differential protein expression in the hypothalami of layer-type Taiwan country chickens in response to acute heat stress.

The hypothalamus is the coordinating center for maintaining temperature homeostasis. In this study, global protein expression in the hypothalami of layer-type Taiwan country chickens in response to acute heat stress was investigated. Twelve 30-week-old female TCCs were divided into three acute heat-stressed groups, namely acute heat stress at 36 °C for 4 h with 0 h (without recovery, H4R0), 2 h (H4R2), or 6 h (H4R6) of recovery. A control group was maintained at 25 °C. Hypothalamus samples were collected at the end of each time point for proteomic analysis. The analysis results revealed that 134 protein spots representing 118 distinct proteins exhibited differential expressions after acute heat stress treatment. Results of gene ontology analysis showed that most of the differentially expressed proteins are involved in carbohydrate metabolism, cellular processes, actin cytoskeleton organization, and responses to stimuli. Functional pathway analysis results suggested that the proteins are associated with networks of carbon metabolism, glycolysis, and gluconeogenesis. Upregulation of the expression of triosephosphate isomerase, phosphoglycerate kinase, pyruvate kinase, alpha-enolase, glycogen phosphorylase (brain form), phosphoglucomutase, L-lactate dehydrogenase A chain and downregulation of 6-phosphogluconolactonase expression indicated an increase in the glycolytic activity and glucose supply for ATP production in the hypothalami in response to heat stress. By contrast, upregulated expressions of heat shock protein 90 alpha, glutathione S-transferase 2s, peroxiredoxin-1, and dihydropyrimidinase-like 2 suggested that acute heat stress adversely affects the hypothalamus; thus, it induces mechanisms that prevent oxidative damage and endoplasmic reticulum stress. In conclusion, acute heat stress induces differential protein expression in the hypothalami of the L2 strain Taiwan country chickens, which may manifest detrimental effects. Furthermore, differential expression is a critical response in the hypothalamus for the regulation of thermotolerance.

Proteomic analysis of the hypothalamus of broiler-type Taiwan country chickens in response to acute heat stress.

The hypothalamus is a critical center for regulating heat retention or dissipation. This study investigated global protein changes in the hypothalamus of broiler-type Taiwan country chickens (TCCs) after acute heat stress. Twelve TCC hens aged 30 weeks were allocated to groups subjected to acute heat stress at 38°C for 2 hr without recovery, with 2 hr of recovery, and with 6 hr of recovery; a control group was maintained at 25°C. Hypothalami were collected for protein expression analysis at the end of each time point. The results showed 114 protein spots differentially expressed after acute heat stress. Most of the differentially expressed proteins were involved in cellular processes, metabolism, transport, and cellular component organization. Functional annotation analysis suggested that these proteins were related to cellular defensive responses against heat and oxidative stress, detoxification and toxin export/delivery, cytoskeleton integrity, oxygen transport, and neural development. The results of this study suggest that acute heat stress damages the hypothalamus of broiler-type TCCs through oxidative stress and provokes a series of responses to stabilize protein structures, degrade misfolded proteins, and remodel cytoskeletons for attenuating the detrimental effects by acute heat stress.

Profiling of differential gene expression in the hypothalamus of broiler-type Taiwan country chickens in response to acute heat stress.

Acute heat stress severely impacts poultry production. The hypothalamus acts as a crucial center to regulate body temperature, detect temperature changes, and modulate the autonomic nervous system and endocrine loop for heat retention and dissipation. The purpose of this study was to investigate global gene expression in the hypothalamus of broiler-type B strain Taiwan country chickens after acute heat stress. Twelve 30-week-old hens were allocated to four groups. Three heat-stressed groups were subjected to acute heat stress at 38 °C for 2 hours without recovery (H2R0), with 2 hours of recovery (H2R2), and with 6 hours of recovery (H2R6). The control hens were maintained at 25 °C. At the end, hypothalamus samples were collected for gene expression analysis. The results showed that 24, 11, and 25 genes were upregulated and 41, 15, and 42 genes were downregulated in H2R0, H2R2, and H2R6 treatments, respectively. The expressions of gonadotropin-releasing hormone 1 (GNRH1), heat shock 27-kDa protein 1 (HSPB1), neuropeptide Y (NPY), and heat shock protein 25 (HSP25) were upregulated at all recovery times after heat exposure. Conversely, the expression of TPH2 was downregulated at all recovery times. A gene ontology analysis showed that most of the differentially expressed genes were involved in biological processes including cellular processes, metabolic processes, localization, multicellular organismal processes, developmental processes, and biological regulation. A functional annotation analysis showed that the differentially expressed genes were related to the gene networks of responses to stress and reproductive functions. These differentially expressed genes might be essential and unique key factors in the heat stress response of the hypothalamus in chickens.

Vertical profile, sources, and equivalent toxicity of polycyclic aromatic hydrocarbons in sediment cores from the river mouths of Kaohsiung Harbor, Taiwan.

Six sediment cores collected at four contaminated river mouths and two harbor entrances in Kaohsiung Harbor (Taiwan) were analyzed to evaluate the sources and potential toxicity of polycyclic aromatic hydrocarbons (PAHs). PAHs presented the wide variations ranging from 369±656 to 33,772±14,378 ng g(-1) at the six sampling sites. The composition of PAHs presented a uniform profile reflecting the importance of atmospheric input from vehicle exhausts or coal combustion in the river mouths. PAHs diagnostic ratios indicated a stronger influence of coal combustion in the Salt River mouth and the prevalence of petroleum combustion and mixed sources in the other rivers and harbor entrances. PAHs toxicity assessment using the mean effect range-median quotient (m-ERM-q: 0.011-1.804), benzo[a]pyrene-toxicity equivalent (TEQ(carc): 22-2819 ng TEQ g(-1)), and dioxin-toxicity equivalent (TEQ(fish): 37-5129 pg TEQ g(-1)) identified the Salt River mouth near the industrial area of the harbor as the most affected area.

Acute thoracic epidural hematoma following spinal manipulative therapy: case report and review of the literature.

Spinal epidural hematoma is a rare complication of chiropractic manipulation. This study reports a case of thoracic spinal epidural hematoma following spinal manipulative therapy in the absence of predisposing factors. The effectiveness and safety of chiropractic treatment in chronic spinal pain and a literature review are also presented.

Identification of early transcripts related to male development in chicken embryos.

Early transcripts related to male development in chicken embryos and their expression profiles were examined. A total of 89 and 127 candidate male development transcripts that represented 83 known and 119 unknown non-redundant sequences, respectively, were characterized in an embryonic day 3 (E3; Hamburger and Hamilton Stage 20: HH20) male-subtract-female complementary DNA library. Of 35 selected transcripts, quantitative reverse transcription-polymerase chain reaction validated that the expression levels of 25 transcripts were higher in male E3 whole embryos than in females (P < 0.05). Twelve of these transcripts mapped to the Z chromosome. At 72 wk of age, 20 and 4 transcripts were expressed at higher levels in the testes and brains of male than in the ovaries and brains of female chickens (P < 0.05), respectively. Whole mount and frozen cross-section in situ hybridization, as well as Western blotting analysis further corroborated that riboflavin kinase (RFK), WD repeat domain 36 (WDR36), and EY505808 transcripts; RFK and WDR36 protein products were predominantly expressed in E7 male gonads. Treatment with an aromatase inhibitor formestane at E4 affected the expression levels at E7 of the coatomer protein complex (subunit beta 1), solute carrier family 35 member F1, LOC427316 and EY505812 transcripts across both sexes (P < 0.05), similar to what was observed for the doublesex and mab-3 related transcription factor 1 gene. The interaction effects of sex by formestane treatment were observed in 15 candidate male development transcripts (P < 0.05). Taken together, we identified a panel of potentially candidate male development transcripts during early chicken embryogenesis; some might be regulated by sex hormones.

Laying traits and underlying transcripts, expressed in the hypothalamus and pituitary gland, that were associated with egg production variability in chickens.

The objective was to characterize the potential laying traits and underlying transcripts expressed in the hypothalamus and pituitary gland that were associated with egg production variability in five genetic stocks of chickens: two commercial lines, Red- (n=12) and Black-feather (n=14) Taiwan country chickens (TCCs); two selected lines of TCCs, B (high body weight/comb size; n=17) and L2 (high-egg production; n=14); and a commercial single comb White Leghorn (WL; n=17). Six laying traits, age at first egg, clutch length, pause length, oviposition lag within clutch, follicle rapid growth period, and rate of yolk accumulation were measured. The significance of differential values among five chicken stocks and correlation coefficients between laying traits and number of eggs to 50 weeks of age or laying rate after first egg, and the expression level of 33 transcripts were determined. Longer clutch length and shorter oviposition lag within clutch contributed to a higher number of eggs to 50 weeks of age or laying rate after first egg in L2 (P<0.05) and WL strains (P<0.05). However, their rate of yolk accumulation (P<0.05) and follicle rapid growth period (P<0.05) were different, indicating the accumulation of different alleles after long-term, independent selection. Across all five strains, numbers of eggs to 50 weeks of age were positive correlated with average clutch length (P<0.05) as well as the rate of yolk accumulation (P<0.05). Expressions of PLAG1, STMN2, PGDS, PARK7, ANP32A, PCDHA@, SCG2, BDH and SAR1A transcripts contributed to number of eggs to 50 weeks of age (P<0.05) or laying rate after first egg (P<0.05). Analysis of correlation coefficients indicated that PLAG1 additionally played roles in decreasing average pause length. Two transcripts, PRL and GARNL1, specifically contributed to number of eggs to 50 weeks of age or laying rate after first egg by reducing oviposition lag within clutch (P<0.05) and/or increasing average clutch length (P<0.05), respectively. Expression level of NCAM1, contributed to laying rate after first egg by association with a shorter oviposition lag within clutch (P<0.05). The current study attributed egg production phenotype in five strains into several laying traits; correlations between these traits and expression levels of underlying transcripts expressed in the hypothalamus and pituitary gland were also established.

Expression of 25 high egg production related transcripts that identified from hypothalamus and pituitary gland in red-feather Taiwan country chickens.

Expression levels of 33 high egg production candidate transcripts in Red-feather Taiwan country chickens (TCCs) were examined by quantitative reverse-transcription (RT) polymerase chain reactions (PCR) in this study. Candidate transcripts were previously identified from a L2-B (L2-subtract-B) hypothalamus/pituitary gland subtractive cDNA library. In this subtractive cDNA library, two divergently selected strains of TCCs, B and L2 were used. These two strains were originated from one single population and were further subjected (since 1982) to the selections of body weight/comb size (B) and eggs to 40wk of age (L2), respectively. Hypothalamuses and pituitary glands that sampled from Red-feather TCCs were previously grouped into high (Red-high; n=20) and low (Red-low; n=20) egg productions based on the rate of lay after 1st egg (hen-day laying rate; %). Rates of lay after 1st egg (mean+/-S.E.) in the Red-high and the Red-low subpopulations were 72.2+/-0.6 and 23.0+/-3.5, respectively (P<0.01). Quantitative RT-PCR validated that 25 candidate transcripts were significantly higher expressed in the Red-high than in the Red-low hens. These transcripts were ANP32A, BDH, CDC42, CNTN1, COMT, CPE, CTNNB1, DIO2, EIF4E, GARNL1, HSPCA, LAPTM4B, MBP, NAP1L4, NCAM1, PARK7, PCDHA@, PGDS, PLAG1, PRL, RAD21, SAR1A, SCG2, STMN1 and UFM1. Among these transcripts, 15 (79.0%), 13 (68.4%), and 12 (63.2%) genes were annotated to involve in cellular physiological process (GO:0050875), metabolism (GO:0008152) and cell communication (GO:0007154). Identified transcripts that related to high egg production are most active in focal adhesion, adherens junction, MAPK signaling, tight junction and cell adhesion pathways.

Identification of transcripts related to high egg production in the chicken hypothalamus and pituitary gland.

To identify transcripts related to high egg production expressed specifically in the hypothalamus and pituitary gland of the chicken, two subtracted cDNA libraries were constructed. Two divergently selected strains of Taiwan Country Chickens (TCCs), B (sire line) and L2 (dam line) were used; they had originated from a single population and were further subjected (since 1982) to selection for egg production to 40 wk of age and body weight/comb size, respectively. A total of 324 and 370 clones were identified from the L2-B (L2-subtract-B) and the B-L2 subtracted cDNA libraries, respectively. After sequencing and annotation, 175 and 136 transcripts that represented 53 known and 65 unknown non-redundant sequences were characterized in the L2-B subtracted cDNA library. Quantitative reverse-transcription (RT)-PCR was used to screen the mRNA expression levels of 32 randomly selected transcripts in another 78 laying hens from five different strains. These strains included the two original strains (B and L2) used to construct the subtracted cDNA libraries and an additional three commercial strains, i.e., Black- and Red-feather TCCs and Single-Comb White Leghorn (WL) layer. The mRNA expression levels of 16 transcripts were significantly higher in the L2 than in the B strain, whereas the mRNA expression levels of nine transcripts, BDH, NCAM1, PCDHA@, PGDS, PLAG1, PRL, SAR1A, SCG2 and STMN2, were significantly higher in two high egg production strains, L2 and Single-Comb WL; this indicated their usefulness as molecular markers of high egg production.

Proteomic analysis of hypothalamic proteins of high and low egg production strains of chickens.

Two slow-growth local chicken strains, derived from a common base population, were bi-directionally selected over twenty generations for carcass traits (B strain) and egg production (L2 strain). The objective of the present study was to identify hypothalamic proteins associated with high egg production (by taking advantage of the similar genetic background of these two strains). Prior to and during egg laying, hypothalamic proteins of B and L2 hens were analyzed with two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Approximately 430 well-resolved spots, ranging from 10 to 40 kDa, pH 5-9, were quantified by image processing. Eight protein spots differed in quantity between B and L2 strains at either stage. Using LC-MS/MS, we identified six of eight protein spots, including proteins known for regulating gene expression, signal transduction and lipid metabolism. The mRNA expression levels of these six proteins were then evaluated by quantitative RT-PCR in five strains of hens, including B, L2 and another three commercial strains; heterogeneous nuclear ribonucleoprotein H3 (HNRPH3) was higher in L2 than in the B strain (consistent with the findings in 2-DE). Increased levels of HNRPH3 mRNA were also present in the hypothalamus of high-egg-yield White Leghorn layers, but were absent in other domestic commercial strains with low egg production rates. In conclusion, the expression level of HNRPH3 may be a new molecular marker to screen for high egg production in slow-growth local chickens.