The possible transport and exclusion mode of lipofuscin in rat myocardium under electron microscopy.

Lipofuscin accumulation has been observed in human coronary arteries but whether or not myocardial tissue can release lipofuscin generated within cardiomyocytes must be clarified, as this may provide indicators for future anti-ageing research. The hearts of Sprague Dawley rats, aged 6-24 months, were embedded in resin and ultrathin sections cut for electron microscopy. Lipofuscin granules were abundant in cardiomyocytes. Cardiomyocytes were seen to release lipofuscin granules into the myocardial interstitium as cytoplasmic fragments with irregular protrusions on the sarcolemma surface. The cytoplasmic fragments entering the stroma fused directly with the endothelial cells of adjacent capillaries, delivering lipofuscin to the vessel wall. These fragments were also seen to be engulfed by stromal macrophages or fused with fibroblasts, which then combined with capillary endothelial cells to deliver lipofuscin to the vessel wall. Some cytoplasmic fragments disaggregated and formed membrane-like waste, which travelled to the vessel wall from the myocardial stroma as soluble fine particles via diffusion or pinocytosis of capillary endothelial cells. Lipofuscin entered the vascular wall and endothelial cells, forming large and small protrusions or folds that transported the lipofuscin to the vascular lumen and bloodstream.

Non-additive effects between genotypes: Implications for competitive fitness assays.

Competitive fitness assays are widely used in evolutionary biology and typically rely on a reference strain to compare different focal genotypes. This approach implicitly relies on the absence of interaction between the competing genotypes. In other words, the performance of the reference strain must not depend on the competitor. This report scrutinized this assumption by competing diverged Drosophila simulans populations against a common reference strain. We detected strong evidence for interaction between the competing genotypes: (1) Frequency-dependent selection was common with opposite effects in genetically diverged populations. (2) Temporal heterogeneity of fitness estimates, which can be partially attributed to a competitor-specific delay in the eclosion of the reference strain. We propose that this inconsistent behavior of the reference strain can be considered a specific case of a genotype × environment interaction. Focal populations could modify the environment of the reference strain, either indirectly by altering the microbiome composition and food availability or directly by genotype-specific cannibalism. Our results provide new insights into the interaction of diverged genotypes and have important implications for the interpretation of competitive fitness assays.

A chromosome-level genome assembly of Cairina moschata and comparative genomic analyses.

BACKGROUND: The Muscovy duck (Cairina moschata) is an economically important duck species, with favourable growth and carcass composition parameters in comparison to other ducks. However, limited genomic resources for Muscovy duck hinder our understanding of its evolution and genetic diversity. RESULTS: We combined linked-reads sequencing technology and reference-guided methods for de novo genome assembly. The final draft assembly was 1.12 Gbp with 29 autosomes, one sex chromosome and 4,583 unlocalized scaffolds with an N50 size of 77.35 Mb. Based on universal single-copy orthologues (BUSCO), the draft genome assembly completeness was estimated to be 93.30 %. Genome annotation identified 15,580 genes, with 15,537 (99.72 %) genes annotated in public databases. We conducted comparative genomic analyses and found that species-specific and rapidly expanding gene families (compared to other birds) in Muscovy duck are mainly involved in Calcium signaling, Adrenergic signaling in cardiomyocytes, and GnRH signaling pathways. In comparison to the common domestic duck (Anas platyrhynchos), we identified 104 genes exhibiting strong signals of adaptive evolution (Ka/Ks > 1). Most of these genes were associated with immune defence pathways (e.g. IFNAR1 and TLR5). This is indicative of the existence of differences in the immune responses between the two species. Additionally, we combined divergence and polymorphism data to demonstrate the "faster-Z effect" of chromosome evolution. CONCLUSIONS: The chromosome-level genome assembly of Muscovy duck and comparative genomic analyses provide valuable resources for future molecular ecology studies, as well as the evolutionary arms race between the host and influenza viruses.

The assembly of caprine Y chromosome sequence reveals a unique paternal phylogenetic pattern and improves our understanding of the origin of domestic goat.

The mammalian Y chromosome offers a unique perspective on the male reproduction and paternal evolutionary histories. However, further understanding of the Y chromosome biology for most mammals is hindered by the lack of a Y chromosome assembly. This study presents an integrated in silico strategy for identifying and assembling the goat Y-linked scaffolds using existing data. A total of 11.5 Mb Y-linked sequences were clustered into 33 scaffolds, and 187 protein-coding genes were annotated. We also identified high abundance of repetitive elements. A 5.84 Mb subset was further ordered into an assembly with the evidence from the goat radiation hybrid map (RH map). The existing whole-genome resequencing data of 96 goats (worldwide distribution) were utilized to exploit the paternal relationships among bezoars and domestic goats. Goat paternal lineages were clearly divided into two clades (Y1 and Y2), predating the goat domestication. Demographic history analyses indicated that maternal lineages experienced a bottleneck effect around 2,000 YBP (years before present), after which goats belonging to the A haplogroup spread worldwide from the Near East. As opposed to this, paternal lineages experienced a population decline around the 10,000 YBP. The evidence from the Y chromosome suggests that male goats were not affected by the A haplogroup worldwide transmission, which implies sexually unbalanced contribution to the goat trade and population expansion in post-Neolithic period.

Analysis of whole-genome re-sequencing data of ducks reveals a diverse demographic history and extensive gene flow between Southeast/South Asian and Chinese populations.

BACKGROUND: The most prolific duck genetic resource in the world is located in Southeast/South Asia but little is known about the domestication and complex histories of these duck populations. RESULTS: Based on whole-genome resequencing data of 78 ducks (Anas platyrhynchos) and 31 published whole-genome duck sequences, we detected three geographic distinct genetic groups, including local Chinese, wild, and local Southeast/South Asian populations. We inferred the demographic history of these duck populations with different geographical distributions and found that the Chinese and Southeast/South Asian ducks shared similar demographic features. The Chinese domestic ducks experienced the strongest population bottleneck caused by domestication and the last glacial maximum (LGM) period, whereas the Chinese wild ducks experienced a relatively weak bottleneck caused by domestication only. Furthermore, the bottleneck was more severe in the local Southeast/South Asian populations than in the local Chinese populations, which resulted in a smaller effective population size for the former (7100-11,900). We show that extensive gene flow has occurred between the Southeast/South Asian and Chinese populations, and between the Southeast Asian and South Asian populations. Prolonged gene flow was detected between the Guangxi population from China and its neighboring Southeast/South Asian populations. In addition, based on multiple statistical approaches, we identified a genomic region that included three genes (PNPLA8, THAP5, and DNAJB9) on duck chromosome 1 with a high probability of gene flow between the Guangxi and Southeast/South Asian populations. Finally, we detected strong signatures of selection in genes that are involved in signaling pathways of the nervous system development (e.g., ADCYAP1R1 and PDC) and in genes that are associated with morphological traits such as cell growth (e.g., IGF1R). CONCLUSIONS: Our findings provide valuable information for a better understanding of the domestication and demographic history of the duck, and of the gene flow between local duck populations from Southeast/South Asia and China.

Protective effects of carnosic acid on retinal ganglion cells in acute ocular hypertension rats.

OBJECTIVE: To observe the protective effects of carnosic acid on rat retinal ganglion cells (RGCs) among acute ocular hypertension rats. METHODS: Sixty male SPF (specific-pathogen-free) SD rats (10 weeks) were randomly assigned to untreated group, carnosic-acid-treated group and hypertensive group with 20 rats for each. The acute ocular hypertension animal model was induced by the perfusion of normal saline solution into anterior chamber of eyes to elevate the intraocular pressure (IOP) to 110 mmHg for 60 min in the rats of the carnosic-acid-treated group and hypertensive group. Then, the carnosic acid dissolving in dimethyl sulfoxide (DMSO) was intraperitoneally injected for consecutive 7 days in the carnosic-acid-treated group, and only DMSO was used in the same way in the hypertensive group. The rats were killed 2 weeks after experiment, and retinal sections were prepared for histopathological and apoptotic retinal ganglion cells (RGCs) examination by hemotoxylin and eosin staining and TUNEL staining. Use immunofluorescence employed to examine the survival of RGCs. This study protocol was approved by the Ethic Committee for Experimental Animal of Three Gorges University. RESULTS: The retinal morphology and structure were clear in the untreated group. The edema of retinal tissue, loosely arranged RGCs and swollen nucleus were seen in the hypertensive group. In the carnosic-acid-treated group, the retinal morphology and structure were regular. The retinal nerve fiber layer (RNFL) thickness was (32.96 ± 1.63), (58.96 ± 1.57) and (50.11 ± 2.37) µm, and the apoptotic cell number was (6.92 ± 2.96), (29.85 ± 6.40) and (14.69 ± 2.98)/field, and the survived cell number was (2363.17 ± 148.45), (1308.67 ± 106.02) and (1614.17 ± 96.39)/0.235 mm2 in the untreated group, hypertensive group and carnosic-acid-treated group, respectively, showing significant differences among groups (F = 339.284, 81.583, 122.68, all at P < 0.01). Compared with the untreated group, the RNFL thickness was thickened, the number of apoptotic RGCs was much more, and the number of survived RGCs was decreased in the hypertensive group. In the carnosic-acid-treated group, the RNFL thickness was thinner, the number of apoptotic RGCs was reduced, and the number of survived RGCs was increased in comparison with the untreated group (all at P < 0.01). CONCLUSIONS: Carnosic acid plays a protective effect on RGCs by inhibiting the cell apoptosis in acute ocular hypertension rats.

Detecting the Population Structure and Scanning for Signatures of Selection in Horses (Equus caballus) From Whole-Genome Sequencing Data.

Animal domestication gives rise to gradual changes at the genomic level through selection in populations. Selective sweeps have been traced in the genomes of many animal species, including humans, cattle, and dogs. However, little is known regarding positional candidate genes and genomic regions that exhibit signatures of selection in domestic horses. In addition, an understanding of the genetic processes underlying horse domestication, especially the origin of Chinese native populations, is still lacking. In our study, we generated whole genome sequences from 4 Chinese native horses and combined them with 48 publicly available full genome sequences, from which 15 341 213 high-quality unique single-nucleotide polymorphism variants were identified. Kazakh and Lichuan horses are 2 typical Asian native breeds that were formed in Kazakh or Northwest China and South China, respectively. We detected 1390 loss-of-function (LoF) variants in protein-coding genes, and gene ontology (GO) enrichment analysis revealed that some LoF-affected genes were overrepresented in GO terms related to the immune response. Bayesian clustering, distance analysis, and principal component analysis demonstrated that the population structure of these breeds largely reflected weak geographic patterns. Kazakh and Lichuan horses were assigned to the same lineage with other Asian native breeds, in agreement with previous studies on the genetic origin of Chinese domestic horses. We applied the composite likelihood ratio method to scan for genomic regions showing signals of recent selection in the horse genome. A total of 1052 genomic windows of 10 kB, corresponding to 933 distinct core regions, significantly exceeded neutral simulations. The GO enrichment analysis revealed that the genes under selective sweeps were overrepresented with GO terms, including "negative regulation of canonical Wnt signaling pathway," "muscle contraction," and "axon guidance." Frequent exercise training in domestic horses may have resulted in changes in the expression of genes related to metabolism, muscle structure, and the nervous system.

Qing brick tea (QBT) aqueous extract protects monosodium glutamate-induced obese mice against metabolic syndrome and involves up-regulation Transcription Factor Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2) antioxidant pathway.

BACKGROUND: Qing brick tea (QBT), traditional and popular beverage for Chinese people, is an important post-fermentation dark tea. Our present study was performed to investigate the ameliorative effects of QBT aqueous extract on metabolic syndrome (Mets) in monosodium glutamate-induced obese mice and the potential mechanisms. METHOD: Monosodium glutamate-induced obese mice were used to evaluate the anti-Mets effects of QBT. Content levels of malonaldehyde (MDA), reactive oxygen species (ROS) and protein carbonylation, antioxidant enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione reductase (GR) in the skeletal muscle were assessed by commercial kits, respectively. Western blot and Q-PCR were used to detect the expressions of Transcription Factor Nuclear Factor-Erythroid 2-Related Factor 2 (Nrf2) signaling pathway and downstream antioxidant factors. In addition, activity of AKT signaling and expression of glucose transporter type 4 (GLUT4) in the skeletal muscle were investigated by western blot. RESULT: QBT treatment limited gain of body weight, waistline and LEE index, improved insulin resistance and glucose intolerance, reduced lipid level in MSG mice. Content levels of MDA, ROS and protein carbonylation in skeletal muscle of QBT group were significantly improved compared to those of MSG mice. The antioxidant enzyme activities of SOD, GPx, CAT, and GR were increased in skeletal muscle of MSG mice intervened with QBT. After 20-week QBT treatment, Nrf2 signaling pathway and downstream antioxidant factors were both increased in the skeletal muscle. In addition, QBT treatment improved insulin signaling by preferentially augmenting AKT signaling, as well as increased the protein expression of GLUT4 in the skeletal muscle. CONCLUSION: Our results showed that QBT intake was effective in protecting monosodium glutamate-induced obese mice against metabolic syndrome and involved in the Nrf2 signaling pathway in the skeletal muscle.

Comparative testing of HPV L1 protein monoclonal antibody panel for the detection of HPV in cervical exfoliated cells.

AIMS: To determine the value of a monoclonal antibody panel against a C-terminal conserved sequence polypeptide of human papillomavirus (HPV) L1 (a major capsid protein) for the detection of HPV in cervical exfoliated cells, as well as the potential of this antibody panel to be developed into an assay kit for the clinical screening of cervical cancer. METHODS: Cervical exfoliated cells were collected at a gynecology clinic. One part of each sample was sent to the Department of Pathology for HPV genotyping, and the other part was sent to the Department of Pathology for cytologic testing and then to the laboratory for immunological histological chemistry (IHC) assay in which an HPV L1 C-terminal conserved sequence polypeptide-induced mouse monoclonal antibody panel was used to detect HPV L1. RESULTS: Cervical cell samples were collected from 514 patients at the gynecology clinic; of these, 339 samples were sent for HPV genotyping, and 220 were HPV positive (64.90%, 220/339). Moreover, the duplicate samples from these 339 patients were sent for IHC assay, and 229 samples were positive (67.55%, 229/339). The IHC result was concordant with that obtained by HPV genotyping (Kappa = 0.743, p < 0.001). CONCLUSION: This study showed that use of the HPV L1 C-terminal conserved sequence polypeptide-induced mouse monoclonal antibody panel was of great value for the detection of HPV in cervical cells; the resulting detection rate was comparable to that obtained using the commercial HPV genotyping kit that is currently in use in clinical practice.

Observations on the expression of human papillomavirus major capsid protein in HeLa cells.

BACKGROUND: The goal of this study was to identify the nature of the inclusion bodies that have been found in HeLa cells (cervical cancer immortal cell line) by electron microscope and to determine whether the major capsid protein (L1) of human papillomavirus (HPV) can be expressed in HPV-positive uterine cervix cancer cells. METHODS: HPV L1 protein expression in HeLa cells was detected with anti-HPV L1 multivalent mice monoclonal antibody and rabbit polyclonal anti-HPV L1 antibody by ELISA, light microscope immunohistochemistry, electron microscope immunocytochemistry and Western blotting assays. Reverse transcriptional PCR (RT-PCR) was performed to detect the transcription of L1 mRNA in HeLa cells. The immortalized human keratinocyte HeCat was used as the negative control. RESULTS: HPV L1 proteins reacted positively in the lysate of HeLa cells by ELISA assays. HRP labeled light microscope immunohistochemistry assay showed that there was a strong HPV L1 positive reaction in HeLa cells. Under the electron microscope, irregular shaped inclusion bodies, assembled by many small and uniform granules, had been observed in the cytoplasm of some HeLa cells. These granules could be labeled by the colloidal gold carried by HPV L1 antibody. The Western blotting assay showed that there was a L1 reaction strap at 80-85 kDa in the HeLa cell lysates, hence demonstrating the existence of HPV18 L1 in HeLa cells. RT-PCR assay showed that the L1 mRNA was transcribed in HeLa cells. CONCLUSIONS: The inclusion bodies found in the cytoplasm of HeLa cells are composed of HPV18 L1 protein. Since HeLa cell line is a type of cervical cancer cells, this implies that HeLa cells have the ability to express HPV L1 proteins.

[Effects of serum derived from rats undergone auricular acupuncture intervention on expression of TNF-alpha mRNA, cell adhesion factor-1 and vascular intercellular adhesion molecule-1 proteins of incubated cerebral microvascular endotheliocytes with diabetic injury].

OBJECTIVE: To observe the protective effect of serum derived from rats undergone auricular electroacupuncture (EA) stimulation on the incubated cerebral microvascular endotheliocytes with diabetic injury so as to investigate the underlying mechanism of cholinergic anti-inflammatory action. METHODS: SD rats were randomized into normal group (n = 10), diabetic model group (n = 6), auricular EA group (n = 8), vagotomy + EA group (n = 7, received ipsilateral vagotomy before auricular EA stimulation), atropine + EA group (n = 8), hexamethonium + EA group (n = 7) and alpha-bungarotoxin + EA group (n = 7). Diabetic mellitus model was established by feeding the rats with high sugar, high fat forage and intraperitoneal injection of 1% streptozotocin injection (STZ, 35 mg/kg). EA was applied to ipsilateral "Yi-Dan"-point and "Er-Shenmen" for 30 min, once daily for 10 days. Atropine (0.1 mg/kg, an anticholinergic drug), hexamethonium (10 mg/kg, an antagonist of the nicotinic acetylcholine receptors located in sympathetic and parasympathetic ganglia) and alpha-bungarotoxin (1.0 microg/kg, a type of alpha-neurotoxin that is known to bind irreversibly and competitively to the nicotinic acetylcholine receptors) were given to the rats by tail venous injection, respectively, before ipsilateral auricular EA intervention, once daily for 10 days. Blood samples from rats of each group were then collected. Normally cultured rat brain microvascular endotheliocytes were randomly divided into the same 7 groups. The diabetic-like damage model of cerebral microvascular endotheliocytes was established in the 6 groups except the normal group by adding the fluid containing glucose (20 mmol/L), insulin (100 mU/L) and oxidized low density lipoprotein (200 mg/L) to the culture medium. After 48 hours' incubation, the conditional culture solutions were collected for filtration and degerming. Morphological changes and cellular ultra-microstructure were examined using light microscope and transmission electron microscope, respectively. Tumor necrosis factor-alpha (TNF-alpha) mRNA expression of the cultured microvascular endotheliocytes was assayed using RT-PCR, and the soluble cell adhesion factor-1 (sICAM-1) and soluble vascular intercellular adhesion molecule-1 (sVCAM-1) concentrations in 1 mL culture fluid were measured using ELISA. RESULTS: Compared to the normal control group, the cultured cerebral microvascular endotheliocytes in the model group displayed a cluster-like or floating state, enlargement of the space, and increase of refractivity under light microscope, and showed swelling of the mitochondria with broken cristae and expansion of the space and even with membrane fusion or disappearance under electron microscope. This situation was relatively lighter in both auricular EA and atropine groups, and severe in the vagotomy, hexamethonium and alpha-bungarotoxin groups. TNF-alpha mRNA expression and sICAM-1 and sVCAM-1 concentrations were significantly higher in the model group than in the normal group, but significantly lower in both auricular EA group and atropine group than in the model group (P < 0.01, P < 0.05). No remarkable diffe-rences were found among the model, vagotomy, hexamethonium and alpha-bungarotoxin groups in the levels of TNF-alpha mRNA expression and sICAM-1 and sVCAM-1 concentrations (P > 0 05). CONCLUSION: Auricular EA intervention rat serum can lighten diabetic cellular injury, suppress TNFalpha mRNA expression and reduce ICAM-1 and sVCAM-1 concentrations of rat cerebral microvascular endotheliocytes, which is closely associated with the intact vagus nerve and normal nicotinic acetylcholine receptors in rats.