Characterization and phylogenomics of the complete mitochondrial genome of the polyzoic cestode Gangesia oligonchis (Platyhelminthes: Onchoproteocephalidea).

The order Onchoproteocephalidea (Eucestoda) was recently erected to accommodate the hook-bearing tetraphyllideans and the proteocephalideans, which are characterized by internal proglottization and a tetra-acetabulate scolex. The recognized subfamilies in the Proteocephalidae appeared to be non-monophyletic based on 28S recombinant DNA (rDNA) sequence data. Other molecular markers with higher phylogenetic resolution, such as large mitochondrial DNA fragments and multiple genes, are obviously needed. Thus the mitochondrial genome of Gangesia oligonchis, belonging to the putative earliest diverging group of the Proteocephalidae, was sequenced. The circular mitogenome of G. oligonchis was 13,958 bp in size, and contained the standard 36 genes: 22 transfer RNA genes, two rRNA genes and 12 protein-coding genes, as well as two major non-coding regions. A short NCR and a large NCR (lNCR) region were 216 bp and 419 bp in size, respectively. Highly repetitive regions in the lNCR region were detected with that of 11 repeat units. The mitogenome of G. oligonchis shared 71.1% nucleotide identity with Testudotaenia sp. WL-2016. Phylogenetic analyses of the complete mitochondrial genomes with Bayesian inference and maximum likelihood methods indicated that G. oligonchis formed a sister clade with Testudotaenia sp. WL-2016 with maximum support. The ordinal topology is (Caryophyllidea, (Diphyllobothriidea, (Bothriocephalidea, (Onchoproteocephalidea, Cyclophyllidea)))). The mitogenomic gene arrangement of G. oligonchis was identical to that of Testudotaenia sp. WL-2016. Both mitogenomic and nuclear sequence data for many more taxa are required to effectively explore the inter-relationships among the Onchoproteocephalidea.

[Clinicopathological characteristics and prognosis of diffuse midline gliomas with histone H3K27M mutation: an analysis of 30 cases].

Objective: To analyze the clinicopathological characteristics and prognosis of diffuse midline glioma (DMG) with H3K27M mutation. Methods: Thirty cases of DMG were collected in Guangdong Sanjiu Brain Hospital from October 2016 to May 2018. The patients' clinicopathological data including age, tumor site and histological grade, treatment and follow-up data were collected and analyzed. Results: There were 21 males and 9 females, with a mean age of 26 years (range 5-53 years). Fourteen tumors were located in thalamus, 12 in brainstem (one involved both thalamus and brainstem), and one each in hypothalamus, fourth ventricle, and sellar region, respectively. Two cases presented as diffuse intracranial lesions. Three cases (10.0%) were of WHO grade Ⅰ, 10 cases (33.3%) were grade Ⅱ, eight cases (26.7%) were grade Ⅲ, and nine cases (30.0%) were grade Ⅳ.All patients with gradeⅠ tumors were older than 20 years. Histologically, all were pilocytic astrocytoma-like. Immunohistochemical staining demonstrated that all tumors were IDH1 negative. Twenty-eight tumors showed diffuse expression of H3K27M, and two showed focal expression. Twenty-one tumors(100.0%, 21/21) showed absent expression of H3K27me3. Sixteen tumors (57.1%, 16/28) showed strongly positive expression of p53, and ATRX was negative in eight tumors (38.1%, 8/21). The Ki-67 proliferation index ranged from 5% to 40%. Eight cases (including two cases of H3K27M expression of individual cells) showed K27M mutation in H3F3A gene. Intracranial and spinal cord dissemination occurred in six cases (20.0%, 6/30). Median progression-free survival (PFS) was 9.5 months and median overall survival (OS) was 34 months. Mean PFS was 11.2 months and mean OS was 24.3 months. Compared with adults (>20 years old), children/adolescents (no more than 20 years old) had significantly shorter median OS (8 months vs. 34 months, P=0.013). There was no significant difference in PFS and OS between DMGs located in the brain stem/thalamus and other sites within midline (P>0.05). There was no significant difference in PFS and OS between WHO grade ⅠDMGs and WHO grade Ⅱ-Ⅳ DMGs (P>0.05). Conclusions: DMGs occur more commonly in children and adolescents with male predominance. DMGs present with WHO Ⅰ-Ⅳ tumors morphologically, and pilocytic astrocytoma-like lesions with WHO Ⅰ are more common in adults. Expression of H3K27M but not H3K27me3 is helpful for diagnosis of DMG. The prognosis of children/adolescents is significantly worse than that of adults, whereas histological grade and tumor location do not affect prognosis.

Sequencing, characterization and phylogenomics of the complete mitochondrial genome of Dactylogyrus lamellatus (Monogenea: Dactylogyridae).

Despite the worldwide distribution and pathogenicity of monogenean parasites belonging to the largest helminth genus, Dactylogyrus, there are no complete Dactylogyrinae (subfamily) mitogenomes published to date. In order to fill this knowledge gap, we have sequenced and characterized the complete mitogenome of Dactylogyrus lamellatus, a common parasite on the gills of grass carp (Ctenopharyngodon idella). The circular mitogenome is 15,187 bp in size, containing the standard 22 tRNA genes, 2 rRNA genes, 12 protein-encoding genes and a long non-coding region (NCR). There are two highly repetitive regions in the NCR. We have used concatenated nucleotide sequences of all 36 genes to perform the phylogenetic analysis using Bayesian inference and maximum likelihood approaches. As expected, the two dactylogyrids, D. lamellatus (Dactylogyrinae) and Tetrancistrum nebulosi (Ancyrocephalinae), were closely related to each other. These two formed a sister group with Capsalidae, and this cluster finally formed a further sister group with Gyrodactylidae. Phylogenetic affinity between Dactylogyrinae and Ancyrocephalinae was further confirmed by the similarity in their gene arrangement. The sequencing of the first Dactylogyrinae, along with a more suitable selection of outgroups, has enabled us to infer a much better phylogenetic resolution than recent mitogenomic studies. However, as many lineages of the class Monogenea remain underrepresented or not represented at all, a much larger number of mitogenome sequences will have to be available in order to infer the evolutionary relationships among the monogeneans fully, and with certainty.

miR-508-5p acts as an anti-oncogene by targeting MESDC1 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer associated mortality. Accumulating evidence has shown that microRNAs (miRNAs) act as critical factors for tumor recurrence and metastasis. MiR-508-5p has been reported as a down-regulated miRNA in the primary gastric cancer tissues. However, the role of miR-508-5p on HCC has not been well elucidated. In this study, we observed that miR-508-5p was downregulated in HCC tissues when compared to the non-tumorous tissues. We then demonstrated that overexpression of miR-508-5p attenuated HepG2 cells proliferation and invasion and induced cell apoptosis in vitro. Furthermore, our further investigations revealed that mesoderm development candidate 1 (MESDC1) is a potential target of miR-508-5p, as well as miR-508-5p overexpression downregulated MESDC1 expression. Overexpression of MESDC1 promoted HepG2 cells migration, invasion and proliferation in vitro. In addition, miR-508-5p markedly suppressed the tumor growth in xenograft model, while MESDC1 promoted the tumor growth in xenograft model. This study provides new insight into molecular mechanisms that miR-508-5p acts as a tumor suppressor by targeting MESDC1 in HCC progression.

Benign mixed tumor of the vagina.

Mixed tumor of the vagina is a rare benign tumor containing both epithelial and mesenchymal components. The authors report the case of a 35-year-old woman who presented with a painless mass that had descended from the vagina in the last six months. Physical examination showed the presence of a solitary, non-tender nodule that was 3x3x2 cm in size and seemed to originate from the posterior wall of the lower vagina. The tumor was removed and pathologically examined. The findings were indicative of a mixed tumor of the vagina, and were in agreement with previous cases. No recurrence or progression occurred during the ten-month follow-up. As this tumor shares common features with some other tumors, its differential diagnosis is very important. Moreover, due to the rarity of this disease, gynecologists and pathologists need to familiarize themselves with the features in order to avoid a misdiagnosis.

Evaluation of mango saponin in broilers: effects on growth performance, carcass characteristics, meat quality and plasma biochemical indices.

OBJECTIVE: The objective of the present study was to determine whether mango saponin (MS) could be used as a feed additive in broiler chicks by evaluating growth performance, carcass characteristics, meat quality, and plasma biochemical indices. METHODS: A total of 216 1-d-old Arbor Acres male broiler chicks were randomly assigned into three dietary treatments supplemented with 0 (control), 0.14% (MS 0.14%), or 0.28% (MS 0.28%) MS. Each treatment had six replicates (cages) with 12 chicks each. The feeding trial lasted for six weeks. RESULTS: Compared with the control, dietary supplemented with 0.14% or 0.28% MS increased average daily weight gain of chicks in the grower (22 to 42 d) and the whole (1 to 42 d) phases, and the final body weight of chicks on d 42 was higher in MS supplemented groups (p<0.05). Lower L45 min* (lightness) and L24 h* values, lower b24 h* (yellowness) value, and higher a45 min* (redness) and a24 h* values of the breast muscle were observed in chicks fed with 0.28% MS on d 42 (p<0.05). The total antioxidant capacity in plasma increased in MS 0.14% group on d 21 (p<0.001). Lower contents of plasma total cholesterol and triglyceride were observed in chicks fed with 0.28% MS on d 21 and d 42, whereas the group supplemented with 0.14% MS only decreased plasma triglyceride content on d 21 (p<0.05). The glucose content in plasma decreased in MS 0.28% group on d 42 (p<0.001). CONCLUSION: Overall, MS could be used as a feed additive in broiler chicks, and the supplemental level of 0.28% MS in diet could improve growth performance, meat quality, and plasma lipid metabolism in broiler chicks.

Individual and Joint Acute Toxicities of Selected Insecticides Against Bombyx mori (Lepidoptera: Bombycidae).

As widely used pesticides, organophosphate, pyrethroid, and neonicotinoid insecticides have different modes of action. In the present study, we evaluated individual and joint acute toxicities of two organophosphates, two pyrethroids, and two neonicotinoids against the second-instar silkworm by feeding silkworm with the insecticide-treated mulberry leaves. The 96-h lethal concentration 50 (LC(50)) values of chlorpyrifos, acephate, imidacloprid, thiamethoxam, cypermethrin, and deltamethrin against silkworm were 3.45 (2.95-4.31), 44.45 (39.34-48.56), 1.27 (1.19-1.35), 2.38 (2.19-2.54), 0.36 (0.30-0.43), and 0.037 (0.033-0.041) mg/liter, respectively. Moreover, the 96-h LC(50) values of 50:50 binary mixtures of insecticides against silkworm ranged from 0.048 (0.043-0.054) to 3.52 (2.09-4.51) mg/liter. In addition, the combination coefficient (Q) values of all tested mixtures ranged from 0.36 to 3.37. According to the obtained Q values, the binary mixture of deltamethrin-chlorpyrifos showed antagonistic effects at 96-h interval, while the other binary mixtures had additive effects. Taken together, our results provided valuable guidelines in assessing the ecological risk of these insecticide mixtures against silkworm.

Effects of Dietary Octacosanol on Growth Performance, Carcass Characteristics and Meat Quality of Broiler Chicks.

Octacosanol, which has prominent physiological activities and functions, has been recognized as a potential growth promoter in animals. A total of 392 1-d-old male Arbor Acres broiler chicks with similar body weight were randomly distributed into four dietary groups of seven replicates with 14 birds each supplemented with 0, 12, 24, or 36 mg octacosanol (extracted from rice bran, purity >92%)/kg feed. The feeding trial lasted for six weeks and was divided into the starter (day 1 to 21) and the grower (day 22 to 42) phases. The results showed that the feed conversion ratio (FCR) was significantly improved in broilers fed a diet containing 24 mg/kg octacosanol compared with those fed the control diet in the overall phase (day 1 to 42, p = 0.042). The average daily gain and FCR both showed linear effects in response to dietary supplementation of octacosanol during the overall phase (p = 0.031 and 0.018, respectively). Broilers fed with 24 or 36 mg/kg octacosanol diet showed a higher eviscerated yield, which increased by 5.88% and 4.26% respectively, than those fed the control diet (p = 0.030). The breast muscle yield of broilers fed with 24 mg/kg octacosanol diet increased significantly by 12.15% compared with those fed the control diet (p = 0.047). Eviscerated and breast muscle yield increased linearly with the increase in dietary octacosanol supplementation (p = 0.013 and 0.021, respectively). Broilers fed with 24 or 36 mg/kg octacosanol diet had a greater (p = 0.021) pH45min value in the breast muscle, which was maintained linearly in response to dietary octacosanol supplementation (p = 0.003). There was a significant decrease (p = 0.007) in drip loss value between the octacosanol-added and the control groups. The drip loss showed linear (p = 0.004) and quadratic (p = 0.041) responses with dietary supplementation of octacosanol. These studies indicate that octacosanol is a potentially effective and safe feed additive which may improve feed efficiency and meat quality, and increase eviscerated and breast muscle yield, in broiler chicks. Dietary supplementation of octacosanol at 24 mg/kg diet is regarded as the recommended dosage in the broilers' diet.

Swine leukocyte antigen class II genes (SLA-DRA, SLA-DRB1, SLA-DQA, SLA-DQB1) polymorphism and genotyping in Guizhou minipigs.

The swine leukocyte antigen (SLA) complex harbors highly polymorphic gene clusters encoding glycoproteins that are involved in responses to vaccines, infectious disease, and production performance. Pigs with well-defined SLA class II genes are useful for the study of disease, immunology, and vaccines. In this study, we analyzed four SLA class II genes (SLA-DRA, SLA-DRB1, SLA-DQA, SLA-DQB1) in 22 founder Guizhou minipigs using a sequence-based typing method. Twelve alleles were detected, compared with the SLA class II allele sequences in the GenBank, and one of twelve alleles was found to be novel in Guizhou minipigs. There are four SLA II haplotypes, and one of them has been previously reported in Meishan pigs. Furthermore, based on sequence information of these alleles, we developed a simple SLA typing method implemented to SLA-typing for unknown offspring of Guizhou minipigs, relying on designed twelve sequence specific primers that could discriminate between each other. According to the combination of sequence-based typing and PCR-SSP, we were able to rapidly check SLA typing of Guizhou breeding stock and identified four SLA haplotypes in the herd. Therefore, SLA-defined Guizhou minipigs will be useful as animal models for xenotransplantation and immunological research.

Application of low-gossypol cottonseed meal in laying hens' diet.

An experiment was conducted to evaluate the application of dietary low-gossypol cottonseed meal (LCSM) in layers' diets. A total of 432 40-week-old Hy-line W36 laying hens were allocated to one of the six dietary treatments with 6 replicates of 12 birds each. The control group was fed a corn-soybean meal basal diet, and the 4 experimental diets consisted of a basal diet with 50, 98.3, 144.2, or 189 g/kg LCSM, respectively (correspondingly 25%, 50%, 75%, 100% dietary protein provided by soybean meal were replaced by LCSM). The sixth group was fed a basal diet supplemented with free gossypol (FG group). The feeding trial lasted for 12 weeks. The results showed that no significant difference was observed on egg production or feed conversion ratio (FCR, feed/egg, g/g) among all groups, but feed intake and egg weight were significantly decreased in the 189 g/kg LCSM group in weeks 46 to 51 (P < 0.05). A significant decrease was not observed in shell strength, shell thickness, and yolk color in all periods, but Haugh unit, albumen height, and egg white protein content were reduced in 189 g/kg LCSM group in weeks 46 to 51 (P < 0.05). No significant differences were found between the control and FG group. There was no obvious difference on plasma levels of total protein, blood urea nitrogen, uric acid, and activities of albumen, alanine aminotransferase, aspartate aminotransferase and the histopathology of liver, kidney, and oviduct in all groups. In conclusion, decreasing feed intake, egg weight, and egg quality were observed in the 189 g/kg LCSM group. However, the adverse effect was not relevant to the presence of FG in LCSM. Dietary LCSM supplementation of 98.3 g/kg was recommended with optimum FCR and without adverse effect on egg production, quality, and health of layers.

Bioefficacy comparison of organic manganese with inorganic manganese for eggshell quality in Hy-Line Brown laying hens.

This study was aimed at investigating the bioefficacy of organic compared with inorganic manganese (Mn) for eggshell quality. An amino acid-Mn complex or Mn sulfate monohydrate was used as the organic or inorganic Mn source. A total of six hundred forty-eight 50-wk-old layers (Hy-Line Brown) were divided into 9 groups; each group consisted of 6 replicates with 12 layers each. The feeding trial lasted 12 wk. During the first 4 wk of the feeding trial, the groups were fed a basal diet, which met the nutrient requirements of the layers, except for Mn. During the following 8 wk, 9 levels of Mn (inorganic Mn: 0, 25, 50, 100, and 200 mg/kg; organic Mn: 25, 50, 100, and 200 mg/kg) were used to supplement, respectively, in the basal diet on an equimolar basis. An exponential regression model was applied to calculate the bioefficacy of organic Mn compared with the inorganic Mn. Dietary supplementation with either organic or inorganic Mn did not influence egg production and feed efficiency of (P > 0.05), and eggshell quality did not exhibit a significant response to dietary supplementation with Mn sources at 56 and 58 wk (P > 0.05). Dietary supplementation with either organic Mn or inorganic Mn significantly enhanced the thickness, breaking strength, and elastic modulus of the eggshells compared with the control group at the end of 62 wk (P < 0.05). At the end of 62 wk, the bioefficacy of organic Mn was 357% (shell thickness), 406% (breaking strength), 458% (elastic modulus), and 470% (eggshell Mn), as efficacious as inorganic Mn at equimolar levels. This study suggests that organic Mn enhances eggshell quality in aged laying hens compared with inorganic Mn.

Effects of dietary pyrroloquinoline quinone disodium on growth, carcass characteristics, redox status, and mitochondria metabolism in broilers.

The potential benefits of supplementing pyrroloquinoline quinone disodium (PQQ·Na2) in the diet of broiler chicks were explored. We first examined the effect of different levels of dietary PQQ·Na2 on growth performance, carcass characteristics, and plasma biochemical parameters (trial 1). A total of 490 1-day-old male Arbor Acres (AA) broiler chicks were randomly divided into 5 dietary groups supplemented with 0, 0.05, 0.1, 0.2, or 0.4 mg PQQ·Na2/kg feed. As the 0.2 mg/kg PQQ·Na2 supplement gave the best performance, we then investigated whether this level of PQQ·Na2 influenced the redox status of plasma samples and mitochondrial-related metabolism (trial 2). A total of 120 1-day-old male AA chicks were randomly divided into 2 groups supplemented with 0 or 0.2 mg PQQ·Na2/kg diet. In trial 1, birds fed a diet containing 0.2 mg PQQ·Na2/kg showed lower feed conversion ratio compared with those fed the control diet in the overall study (d 1 to 42, P=0.039). Breast muscle yield (d 42) increased quadratically in response to dietary PQQ·Na2 supplementation (P=0.021). Analysis of plasma biochemical parameters revealed that feeding broiler chicks with ≤0.4 mg/kg PQQ·Na2 did not cause adverse health effects. In trial 2, birds fed 0.2 mg/kg PQQ·Na2 again showed improved feed efficiency than the control birds in the grower and overall phases (P=0.038 and 0.016, respectively). In addition, dietary PQQ·Na2 supplementation resulted in a higher anti-oxidative capacity (P=0.001), lower redox potential (P=0.008), and higher hepatic citrate synthase activity (P=0.002). In contrast, no difference in hepatic mitochondrial DNA copy number was observed between the 2 experimental groups (P>0.1). These results indicate that PQQ·Na2 is a potentially effective feed additive for improving feed efficiency, stimulating breast muscle development, and maintaining redox status in broiler chicks. Enhancement of mitochondria efficiency, rather than modulating mitochondria numbers, may underlie the growth-promoting effect of PQQ·Na2.

Dietary Protein Sources Affect Internal Quality of Raw and Cooked Shell Eggs under Refrigerated Conditions.

This study was conducted to evaluate the effects of various protein sources (soybean meal, SBM; cottonseed protein, CSP; double-zero rapeseed meal, DRM) on the internal quality of refrigerated eggs. A total of 360 laying hens (32 wk of age) were randomly allotted to six treatment groups (five replicates per treatment) and fed diets containing SBM, CSP, or DRM individually or in combination with equal crude protein content (SBM-CSP, SBM-DRM, and CSP-DRM) as the protein ingredient(s). A 6×3 factorial arrangement was employed with dietary types and storage time (0 d, 2 wk, and 4 wk) as the main effects. After 12 wk of diet feeding, a total of 270 eggs were collected for egg quality determination. The egg Haugh unit (HU) in the CSP, SBM-DRM, and DRM groups were significantly lower than those in the SBM and SBM-CSP groups. The hardness and springiness of the cooked yolk in the CSP group were significantly higher than those in the other treatment groups. A lower HU, lower yolk index and higher albumen pH were observed in the DRM group compared to the SBM and SBM-CSP groups when the eggs were stored to 4 wk, and the HU was improved in the CSP-DRM group compared to the DRM group (p<0.05). Higher yolk hardness was observed in the CSP group compared to the other groups during storage (p<0.05), but the hardness of the cooked yolk in the SBM-CSP and CSP-DRM groups showed no difference in comparison to the SBM group. In conclusion, CSP may ameliorate the negative effects of DRM on the HU of refrigerated eggs, and SBM or DRM may alleviate the adverse effects of CSP on yolk hardness.

Updated morphology, histopathology and molecular phylogeny of Myxobolus hearti, cardiac myxosporea in gibel carp, Carassius gibelio (Bloch).

The original description of Myxobolus hearti is supplemented with new data on spore morphology, histopathology and molecular phylogeny. Myxobolus hearti are found in the heart ventricle of the gibel carp, Carassius gibelio (Bloch), where they form whitish oval or irregularly shaped plasmodia. Mature spores are oval or shortly ellipsoidal in frontal view, lemon-shaped in sutural view and eye-shaped in apical view. The spores are 14.12 ± 0.35 (13.6-15) µm long (mean ± SD), 11.85 ± 0.34 ± 0.36 (11-12) µm wide and 7.32 ± 0.36 (7-8) µm thick. The two polar capsules are equal in size, 6.11 ± 0.29 (6-7) µm long and 3.89 ± 0.31(3-4) µm wide, and are long pyriform in shape. Polar filaments have six or seven coils situated perpendicular to the longitudinal axis of the polar capsules. Histopathology indicates that the plasmodia are encased by the host connective tissue, and no inflammatory responses are found in the heart ventricles. Phylogenetic analysis based on the 18S small-subunit ribosomal DNA sequences indicates that M. hearti is, genetically, most similar to Henneguya doneci, a gill-infecting species.

Manganese supplementation enhances the synthesis of glycosaminoglycan in eggshell membrane: a strategy to improve eggshell quality in laying hens.

This study investigated the effect of dietary Mn supplementation on eggshell quality, ultrastructure, glycosaminoglycan (GAG), and uronic acid content, and mRNA and protein expression of Galß1,3-glucuronosyltransferase (GlcAT-I). A total of 216 layers (Hy-Line Grey) at age of 50 wk were divided into 3 groups. In the first 8 wk of the 12-wk feeding trial, all groups were fed a basal diet that met all layer nutrient requirements except for Mn. In the last 4 wk, each group was fed 1 of 3 diets supplemented with Mn levels at 0, 25, or 100 mg Mn/kg. Dietary Mn deficiency did not affect the egg performance of layers. Dietary Mn supplementation significantly improved the breaking strength, thickness, and fracture toughness of eggshells (P < 0.05). In photographs of eggshell ultrastructure, the size of mammillary cones and cracks in the outer surface were decreased by dietary Mn supplementation. The contents of GAG and uronic acids in eggshell membrane were significantly increased by dietary Mn addition (P < 0.05). This result was further confirmed by increased mRNA expression and protein expression of GlcAT-I when Mn was added to the diet. This study suggests that dietary Mn supplementation can improve eggshell quality by enhancing the GAG and uronic acid synthesis in the eggshell glands, which can affect the ultrastructure of eggshells.

Intracellular localization and association of MHC class I with porcine invariant chain.

The objective was to investigate the intracellular localization and association of pig major histocompatibility complex (MHC) class I subunits with invariant chain (Ii). Pig MHC class I subunit cDNAs were cloned by RT-PCR and eukaryotic expression plasmids of α and ß2m were constructed with fusions to red or enhanced green fluorescent protein (pDsRed2-N1-α, pEGFP-N1-α, pDsRed2-N1-ß2m, and pEGFP-N1-ß2m). A pig Ii mutant with a deleted CLIP region (DCLIP-Ii) was constructed by overlap extension PCR. Wild-type Ii and mutant Ii were cloned into pEGFP-C1 (pEGFP-C1-Ii, pEGFP-C1-DCLIP-Ii). The recombinant plasmids of MHC I subunits and pEGFP-C1-Ii (pEGFP-C1-ΔCLIP-Ii) were transiently cotransfected into COS-7 cells with Lipofectamine 2000. Immunofluorescence microscopy was performed to detect expression and intracellular localization of Ii and MHC I subunits, and immunoprecipitation was used to analyze their association. Our results indicated that pig Ii associates with integrated MHC I subunits to form oligomers, but cannot associate with single MHC I subunits. Furthermore, deletion of the Ii CLIP sequence blocks association with integrated MHC I subunits. Thus, pig Ii cannot associate with a single MHC I molecule, the α or ß2m chain, but Ii and the integrated MHC I molecule can form complexes that colocalize in the endomembranes of COS-7 cells. The Ii of CLIP plays a key role in assembly of Ii and MHC I.

Trimethylamine deposition in the egg yolk from laying hens with different FMO3 genotypes.

The A/T polymorphism at position nt 1,034 of the chicken FMO3 cDNA sequence is associated with elevated levels of trimethylamine (TMA) in the egg yolk, which is responsible for the fishy egg flavor. This study was aimed to investigate yolk TMA deposition in eggs from different FMO3 genotype (AA, AT, TT) laying hens fed a high level of choline, and the relationship between egg yolk TMA contents and yolk acceptability. A total of 132 genotyped laying hens, 38 wk of age, were assigned to 1 of the 4 dietary treatments: 1) a control group of AA hens fed 370 mg of choline/kg of diet (practical choline level, provided by 500 mg of choline chloride/kg of diet); 2) AA hens were fed 2,960 mg of choline/kg of diet (higher dietary choline level, provided by 4,000 mg of choline chloride/kg of diet); 3) AT hens were fed 2,960 mg of choline/kg of diet; and 4) TT hens were fed 2,960 mg of choline/kg of diet. All layers were fed a 370 mg of choline/kg added diet for a period of 1-wk adaptation followed by a 6-wk trial period. A remarkable increase in yolk TMA concentration of TT hens (P < 0.001) caused by dietary choline addition at 2,960 mg/kg was observed. The relationship between TMA concentration in the egg yolk (µg/g, y) and deposition time (1~42 d, x) for TT hens is y = 0.0005x(3) - 0.0419x(2) + 1.0924x + 0.4323 (R(2) = 0.9259). The fish-flavor scores of egg yolks rose steadily (R(2) = 0.9324) as the TMA concentration increased, and there was also a corresponding decrease in the acceptance score (R(2) = 0.8276). The eggs were acceptable when the yolk TMA concentrations were less than 4.516 µg/g of yolk.

Effect of ß-hydroxy-ß-methylbutyrate calcium on growth, blood parameters, and carcass qualities of broiler chickens.

Beta-hydroxy-ß-methylbutyrate (HMB), the metabolite of leucine, plays an important role in muscle protein metabolism. To investigate the effect of dietary HMB calcium (HMB-Ca) on growth performance, breast muscle development, and serum parameters in broiler chickens, a total of two hundred seventy 1-d-old Arbor Acres male broiler chicks were randomly allotted into 3 dietary treatments supplemented with 0, 0.05%, or 0.1% HMB-Ca during the starter (1 to 21 d) and grower (22 to 42 d) period. The results showed that broilers fed 0.1% HMB-Ca diet had higher ADG during the starter or the whole period, and gain 148 g more BW than the chicks fed the control diet at 42 d of age (P < 0.05). At 21 d of age, birds receiving 0.1% HMB-Ca had more breast muscle yield, less abdominal fat than the control, and more dressing percentage than birds fed the control or 0.05% HMB-Ca diet (P < 0.05). At 42 d of age, 0.1% HMB-Ca increased breast muscle yield than the control and decreased abdominal fat compared with the control or 0.05% HMB-Ca group (P < 0.05). In comparison with the control, feeding 0.1% HMB-Ca increased the triiodothyronine, thyroxine, triiodothyronine/thyroxine ratio and decreased the serum uric acid level at d 21 (P < 0.05). At 42 d of age, serum thyroxine level was elevated in the 0.05% HMB-Ca treatment, and the uric acid concentration was significantly decreased by the 0.1% HMB-Ca-supplemented diet (P < 0.05). Dietary HMB-Ca did not affect the growth hormone or insulin content. This study suggested that dietary supplementation of HMB-Ca improved growth performance, stimulated the breast muscle development, and decreased the abdominal fat deposition in broiler chickens, and the favorable effects were more pronounced in the starter phase. The growth promotion effect of HMB-Ca may be partly related to the increased serum thyroid hormones in broiler chickens.

Relapse of hyperthyroidism after hemithyroidectomy in concurrent medullary thyroid cancer and Graves' disease.

We present a rare case of concurrent medullary thyroid cancer (MTC) and relapse of Graves' disease (GD). A 26-year-old Chinese female suffered from GD for 1 year. Physical examination demonstrated mildly diffuse goiter with a palpable 1.0 cm nodule in the right thyroid lobe and exophthalmos. Before hemithyroidectomy, calcitonin was elevated and thyroglobulin was normal. After surgery, histopathological findings proved coexistence of MTC and GD. 16 months later, GD relapsed and radioactive iodine 131 therapy was given. Meanwhile, postoperative monitoring showed well controlled calcitonin and carcinoembryonic antigen. Our lesson demonstrates although concomitant of MTC and GD is very rare, yet after hemithyroidectomy for the single nodular MTC lesion, remnant thyroid lobe could be the source of GD relapse very soon. More importantly, the relapse of GD and the following radioiodine therapy and radiation exposure could have been prevented if a better therapy plan of total thyroidectomy was adopted in the first place, instead of hemithyroidectomy (Tab. 1, Fig. 2, Ref. 11).

RNA is favourable for analysing EGFR mutations in malignant pleural effusion of lung cancer.

Malignant pleural effusion (MPE) is a useful specimen allowing for the evaluation of EGFR status in nonsmall cell lung cancer (NSCLC). However, direct sequencing of genomic DNA from MPE samples was found not to be sensitive for EGFR mutation detection. To test whether EGFR analysis from RNA is less prone to interference from nontumour cells that have no or lower EGFR expression, we compared three methods (sequencing from cell-derived RNA versus sequencing and mass-spectrometric analysis from genomic DNA), in parallel, for EGFR mutation detection from MPE samples in 150 lung adenocarcinoma patients receiving first-line tyrosine kinase inhibitors (TKIs). Among these MPE samples, EGFR mutations were much more frequently identified by sequencing using RNA than by sequencing and mass-spectrometric analysis from genomic DNA (for all mutations, 67.3 versus 44.7 and 46.7%; for L858R or exon 19 deletions, 61.3 versus 41.3 and 46.7%, respectively). The better mutation detection yield of sequencing from RNA was coupled with the superior prediction of clinical efficacy of first-line TKIs. In patients with acquired resistance, EGFR sequencing from RNA provided satisfactory detection of T790M (54.2%). These results demonstrated that EGFR sequencing using RNA as template greatly improves sensitivity for EGFR mutation detection from samples of MPE, highlighting RNA as the favourable source for analysing EGFR mutations from heterogeneous MPE specimens in NSCLC.