The bound polyphenols of foxtail millet (Setaria italica) inner shell inhibit breast cancer by promoting lipid accumulation-induced autophagic death.

Foxtail millet is a traditional excellent crop with high nutritional value in the world, belong to cereals. The bran of foxtail millet is rich in polyphenol that has antioxidant, anti-inflammatory, and anti-tumorigenic effects. Previously, we extracted bound polyphenols from the inner shell of foxtail millet bran (BPIS). Here, we report that BPIS specifically induced breast cancer cell death and elevated the autophagy level simultaneously. The addition of an autophagy inhibitor blocked BPIS-induced breast cancer cell death, indicating that excessive autophagy induced cell death. Furthermore, oil red O and BODIPY staining also confirmed that lipids, which are important inducers of autophagy, accumulated in breast cancer cells treated with BPIS. Lipidomics research revealed that glycerophospholipids were the main accumulated lipids induced by BPIS. Further study showed that elevated PCYT1A expression was responsible for glycerophospholipid accumulation, and BPIS contained ferulic acid and p-coumaric acid, which induced PCYT1A expression and breast cancer cell death. Collectively, our results revealed that BPIS resulted in autophagic death by enhancing lipid accumulation in breast cancer cells, and BPIS contains ferulic acid and p-coumaric acid, which provided new insights into developing nutraceuticals and drugs for breast cancer patients.

Tumor Cell-Intrinsic BTLA Receptor Inhibits the Proliferation of Tumor Cells via ERK1/2.

B and T lymphocyte attenuator (BTLA) is an immune checkpoint molecule that mediates the escape of tumor cells from immunosurveillance. Consequently, BTLA and its ligand herpesvirus entry mediator (HVEM) are potentially immunotherapeutic targets. However, the potential effects of BTLA on tumor cells remain incompletely unknown. Here, we show that BTLA is expressed across a broad range of tumor cells. The depletion of BTLA or HVEM promotes cell proliferation and colony formation, which is reversed by the overexpression of BTLA in BTLA knockout cells. In contrast, overexpression of BTLA or HVEM inhibits tumor cell proliferation and colony formation. Furthermore, the proliferation of a subpopulation with high BTLA was also significantly slower than that of the low BTLA subpopulation. Mechanistically, the coordination of BTLA and HVEM inhibits its major downstream extracellular regulated protein kinase (ERK1/2) signaling pathway, thus preventing tumor cell growth. This study demonstrates that tumor cell-intrinsic BTLA/HVEM is a potential tumor suppressor and is likely to have a potential antagonist for immunotherapy, thus representing a potential biomarker for the optimal cancer immunotherapeutic treatment.

Genetic Characteristics of Porcine Hemagglutinating Encephalomyelitis Coronavirus: Identification of Naturally Occurring Mutations Between 1970 and 2015.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a Betacoronavirus characterized by neurological symptoms and a worldwide prevalence. Although PHEV is one of the earliest discovered porcine coronaviruses, it remains poorly studied. The full-length genome of the earliest PHEV strain collected in 1970 in the United States (PHEV/67 N/US/1970) was determined in October 2020. Using this virus as a prototype, we comparatively analyzed all available PHEV full-length sequences during 1970-2015. In phylogenetic trees based on PHEV full-length or spike glycoprotein open reading frame genomic sequences, PHEV/67 N/US/1970 was sorted into a clade different from that of viruses isolated in the United States in 2015. Intriguingly, United States and Belgium viruses isolated in 2015 and 2005, respectively, revealed multiple deletion mutation patterns compared to the strain PHEV/67 N/US/1970, leading to a truncated or a non-functional NS2A coding region. In addition, the genomic similarity analysis showed a hypervariability of the spike glycoprotein coding region, which can affect at least eight potential linear B cell epitopes located in the spike glycoprotein. This report indicates that PHEVs in the United States underwent a significant genetic drift, which might influence PHEV surveillance in other countries.

Progressive liver injury and increased mortality risk in COVID-19 patients: A retrospective cohort study in China.

BACKGROUND: Liver injury is common and also can be fatal, particularly in severe or critical patients with coronavirus disease 2019 (COVID-19). AIM: To conduct an in-depth investigation into the risk factors for liver injury and into the effective measures to prevent subsequent mortality risk. METHODS: A retrospective cohort study was performed on 440 consecutive patients with relatively severe COVID-19 between January 28 and March 9, 2020 at Tongji Hospital, Wuhan, China. Data on clinical features, laboratory parameters, medications, and prognosis were collected. RESULTS: COVID-19-associated liver injury more frequently occurred in patients aged ≥ 65 years, female patients, or those with other comorbidities, decreased lymphocyte count, or elevated D-dimer or serum ferritin (P < 0.05). The disease severity of COVID-19 was an independent risk factor for liver injury (severe patients: Odds ratio [OR] = 2.86, 95% confidence interval [CI]: 1.78-4.59; critical patients: OR = 13.44, 95%CI: 7.21-25.97). The elevated levels of on-admission aspartate aminotransferase and total bilirubin indicated an increased mortality risk (P < 0.001). Using intravenous nutrition or antibiotics increased the risk of COVID-19-associated liver injury. Hepatoprotective drugs tended to be of assistance to treat the liver injury and improve the prognosis of patients with COVID-19-associated liver injury. CONCLUSION: More intensive monitoring of aspartate aminotransferase or total bilirubin is recommended for COVID-19 patients, especially patients aged ≥ 65 years, female patients, or those with other comorbidities. Drug hepatotoxicity of antibiotics and intravenous nutrition should be alert for COVID-19 patients.

Ubiquitin-conjugating enzyme E2T knockdown suppresses hepatocellular tumorigenesis via inducing cell cycle arrest and apoptosis.

BACKGROUND: Hepatocellular carcinoma (HCC) is now the most common primary liver malignancy worldwide, and multiple risk factors attribute to the occurrence and development of HCC. Recently, increasing studies suggest that ubiquitin-conjugating enzyme E2T (UBE2T) serves as a promising prognostic factor in human cancers, although the molecular mechanism of UBE2T in HCC remains unclear. AIM: To investigate the clinical relevance and role of UBE2T in HCC development. METHODS: UBE2T expression in HCC tissues from the TCGA database and its association with patient survival were analyzed. A lentivirus-mediated strategy was used to knock down UBE2T in HCC cells. qRT-PCR and Western blot assays were performed to check the effect of UBE2T silencing in HCC cells. Cell growth in vitro and in vivo was analyzed by multiparametric high-content screening and the xenograft tumorigenicity assay, respectively. Cell cycle distribution and apoptosis were determined by flow cytometry. The genes regulated by UBE2T were profiled by microarray assay. RESULTS: UBE2T was overexpressed in HCC tissues compared with paired and non-paired normal tissues. High expression of UBE2T predicted a poor overall survival in HCC patients. In vitro, lentivirus-mediated UBE2T knockdown significantly reduced the viability of both SMMC-7721 and BEL-7404 cells. In vivo, the xenograft tumorigenesis of SMMC-7721 cells was largely attenuated by UBE2T silencing. The cell cycle was arrested at G1/S phase in SMMC-7721 and BEL-7404 cells with UBE2T knockdown. Furthermore, apoptosis was increased by UBE2T knockdown. At the molecular level, numerous genes were dysregulated after UBE2T silencing, including IL-1B, FOSL1, PTGS2, and BMP6. CONCLUSION: UBE2T plays an important role in cell cycle progression, apoptosis, and HCC development.

Whole-genome resequencing reveals candidate mutations for pig prolificacy.

Changes in pig fertility have occurred as a result of domestication, but are not understood at the level of genetic variation. To identify variations potentially responsible for prolificacy, we sequenced the genomes of the highly prolific Taihu pig breed and four control breeds. Genes involved in embryogenesis and morphogenesis were targeted in the Taihu pig, consistent with the morphological differences observed between the Taihu pig and others during pregnancy. Additionally, excessive functional non-coding mutations have been specifically fixed or nearly fixed in the Taihu pig. We focused attention on an oestrogen response element (ERE) within the first intron of the bone morphogenetic protein receptor type-1B gene (BMPR1B) that overlaps with a known quantitative trait locus (QTL) for pig fecundity. Using 242 pigs from 30 different breeds, we confirmed that the genotype of the ERE was nearly fixed in the Taihu pig. ERE function was assessed by luciferase assays, examination of histological sections, chromatin immunoprecipitation, quantitative polymerase chain reactions, and western blots. The results suggest that the ERE may control pig prolificacy via the cis-regulation of BMPR1B expression. This study provides new insight into changes in reproductive performance and highlights the role of non-coding mutations in generating phenotypic diversity between breeds.

[A new balancer of chapped wing on chromosome 3 and a gathering line with double balancers of chapped wing (L) and Cy in Drosophila melanogaster.].

The balancers of Drosophila melanogaster are widely used in genetic research. By analyzing the phenotype of offspring from hybridization of chapped wing (L) mating with OR, 982p and e, respectively, we mapped the chapped wing mutation on chromosome 3 for the first time and demonstrated the chapped wing mutation as a new balancer of D. melanogaster with dominant wing nicking phenotype. Finally, we bred a novel gathering line with double balancers of L and Cy in D. melanogaster. The mutant L provided a legible dominant marker for the balancer of chromosome 3, and the cultivation of double balancers chapped-curly wing enriches the balancer stock, which is often used in mapping and screening.

[Analysis of radioactive distribution in the sacrum in whole-body bone scanning].

OBJECTIVE: To analyze the radiogenic distribution in the sacrum in whole-body bone scanning. METHODS: A total of 212 patients receiving whole-body bone scanning without any explicit bone metastases were divided into different age and gender groups. The radioactive distribution in the sacrum in whole-body bone scanning was analyzed statistically. RESULTS: Of these cases, 31.1% presented with thin radioactive distribution in the sacrum and 11.3% exhibited increased radioactive distribution. Normal radioactive distribution in the sacrum was found in 57.6% of the cases. In both male and female elderly patients (>70 years), the rate of normal radioactive distribution in the sacrum was obviously reduced with increased rate of thin radioactive distribution. The female elderly patients showed higher rate of increased radioactive distribution in the sacrum than male elderly patients. CONCLUSION: The radioactive distribution in the sacrum is similar between female and male patients. Elderly male patients over 70 years have generally thin radioactive distribution in the sacrum due to the presence of osteoporosis, which is also associated with latent fracture of the sacrum to result in increased radioactive distribution in the sacrum in whole-body bone scanning.

[Function of inducible nitric oxide synthase on adaptability to hypoxia in Tibetan chicken].

The inducible nitric-oxide synthase (iNOS) can be induced by hypoxia to produce NO, which regulates blood flow and improves oxygen delivery to tissues. In present study, SNPs in coding and 5'-flanking regions of iNOS gene were examined in Tibetan chicken and lowland chicken using sequence and PCR-RFLP methods, and the quantitive express of mRNA of iNOS and the enzyme activity were measured in chorioallantoic membrane of chick embryo that was incubated under hypoxic and normal conditions. The results showed that a special SNP (-870C-->T)was found in 5'-flanking region of iNOS gene. Tibetan chicken has higher frequencies in mutation allele T than lowland chicken. In hypoxic incubation, the mRNA expression of iNOS and enzyme activity were higher in Tibetan chicken than that in Dwarf chicken. It was concluded that the mutation of iNOS and hypoxic increase of its expression were important base for adaptability to hypoxia in Tibetan chicken.

[Cardiac enzymes related to high-altitude hypoxic adaptation in Tibetan chicken].

AIM: The purpose of this experiment was to investigate the cardiac physiological characteristics for adaptation to high-altitude hypoxia in chickens. METHODS: Tibetan, Dwarf Recessive White and Shouguang chickens were fed at low-and high-altitude, and measurements were made in heart weights, lactic acid (LA), lactate dehydrogenase (LDH), and succinate dehydrogenase (SDH) at the age of 10 weeks. RESULTS: The results showed that Tibetan chickens at high-altitude had lower heart weight and LA content, and similar LDH activity, and higher SDH activity when compared to Dwarf Recessive White and Shouguang chickens. CONCLUSION: It was concluded that the cardiac mechanisms of high-altitude hypoxic adaptation in Tibetan chickens were increasing neither heart weight, nor level of anaerobic metabolism, but the higher SDH activity was significant to the adaptation. The SDH was a symbol enzyme for hypoxic adaptation in Tibetan chicken.

[Polymorphisms of three major genes for reproductive traits in Tibet pigs].

Tibet pig is a special Chinese indigenous pig breed, which could survive in high-altitude areas of Tibet. In the present study, the genotypes of ESR, FSHbeta, and PRLR gene, the three known major genes of reproductive traits, were identified in 202 Tibet pig individuals. The results showed that the predominant genotypes of ESR, FSHbeta, and PRLR gene were BB, AB and AA, respectively. The genotype frequencies of the three genes were in Hardy-Weinberg equilibrium. The frequencies of desired alleles of the three genes were higher than those of other alleles, which indicated that Tibet pig had a good potential for improving reproductive traits.

[Differential gene expression of hypoxia inducible factor-1alpha and hypoxic adaptation in chicken].

Hypoxia inducible factor-1 (HIF-1) is a transcriptional factor first discovered in cancer cells under hypoxic conditions, and was demonstrated to play a key role in the organism's adaptation to changing oxygen tensions. The Tibetan chicken, a breed indigenous to the Tibetan plateau is particularly adaptable to the low oxygen tension and low temperature conditions in the high altitude environment. By comparison, the White Leghorn and ShouGuang chicken are low-altitude chicken breeds. The eggs of these three chicken breeds were incubated in a fitted hatcher that simulated hypoxic condition. The results demonstrated that the hatching rate for Tibetan chicken was significantly higher than the two low altitude breeds, and Tibetan chicken displayed higher endurance in the hypoxia environment. To a certain degree, the hypoxic condition proved fatal to hatching for the low-altitude chickens. Gene expression of HIF-1alpha was detected in brain and skeletal muscle tissues for three chicken varieties using the TaqMan probe FQRT-PCR method. The results showed that HIF-1alpha mRNA displayed tissue specific differential expression, with the highest in the brain. In addition, the expression of HIF-1alpha mRNA in the brain of Tibetan chicken embryos was similar to that of the low-altitude chickens when they were hatched under normal oxygen tensions.

Analysis of expressed sequence tags from skeletal muscle-specific cDNA library of Chinese native Xiang pig.

A Longissimus Dorsi muscle cDNA library of Xiang Pig was constructed, and 131 randomly isolated clones were sequenced in this study. The results of bioinformatics analysis showed that 131 ESTs represented 109 unique clones sequences, of which 99 showed homology to previously identified genes in humans or other mammals, 3 matched other uncharacterized expressed sequence tags (ESTs), and 7 showed no significant matches to sequences already present in DNA databases. No protein matches were found for 10 ESTs. Functional analysis of the ESTs showed that a considerable proportion of them encoded proteins involved in gene/protein expression (45.46%). Other classes included genes involved in metabolism (10.10%), cell structure/motility (10.10%), cell/organism defense (5.05%), cell signaling/communication (2.02%), and cell division (0.0%). Unclassified genes constituted the remaining 27.27%. This study reported the results of the first gene expression profile analysis of Chinese native Xiang Pig skeletal muscle cells, thereby greatly facilitating the functional study of candidate genes involved in muscle growth as well as in the improvement of meat quality in domestic pigs.

[Identification of a common deletion in different curly mutants in Drosophila melanogaster].

Curly is a easily distinguishable dominant mutant wing character. The Cy mutation is the most commonly used dominant marker for the second chromosome balancers in Drosophila melanogaster, but little is known about the Cy gene. Based on known genomic and cytogenetic information, a 102 bp deletion which is located between the Genes synaptotagmin (syt) and Activin Like Protein at 23B(Alp23B) on the Drosophila melanogaster genome (release 4) had been found to be commonly contained on Cy chromosome in three different curly strains. Meanwhile, when using the deletion as a DNA marker, the result suggested that Cy homozygote be lethal in embryo period. These results will provide some helpful information to investigate molecular mechanism of curly wings in the further study.

[Genetic analysis of feather color and shank color traits based on F-2 resource population in Tibetan chicken].

Tibetan chickens were crossed reciprocally with White Leghorn and Shou-Guang chicken respectively, and inter se matings in F1 were carried out to generate the F2 population. Feather color and shank color appearance and segregation ratio in F1 and F2 were observed. Results indicate that white feather of White Leghorn chicken and black feather of Shou-Guang chicken exhibit complete dominant heredity to hemp feather of Tibetan chicken. Hemp feather is determined by two or more alleles. Only when these two or more alleles are concurrently present, will hemp feather then be displayed. The Id/id allele that determines shank color demonstrates sex-linked inheritance, and the recessive id is expressed gradually in the homozygotes. We confirm that genotype of shank color in White Leghorn rooster used in this study is the dominant homozygote.

Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection.

The objective was to determine the effects of various methods of oocyte activation and sperm pretreatment on development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). The second polar body was extruded in the majority (>78.4%) of in vitro-matured (IVM) oocytes 4h after electrical pulse activation. In embryos generated by ICSI and sham-ICSI, a combination of an electrical pulse, with various chemical activators 4 h later, improved (P < 0.05) blastocyst formation rate compared to activation only with a pulse. Treatment with 6-dimethylaminopurine (DMAP) after electrical activation significantly increased the oocyte activation rate. The effects of exposure of sperm to repeated freeze-thaw cycles (without cryoprotectant) on oocyte activation and the effects of sperm pre-incubated with dithiothreitol (DTT) or Triton X-100 on early embryo development were also examined. Blastocyst formation rates after ICSI did not differ between motile sperm and those rendered immotile by one-time freezing and thawing without cryoprotectant. However, sperm rendered immotile by three cycles of freezing/thawing without cryoprotectant had a significantly lower blastocyst formation rate. Although oocytes injected with sperm pre-incubated with Triton X-100 had a higher normal fertilization rate than those pre-incubated with DTT or one-time frozen/thawed sperm, rates of blastocyst formation and cell numbers were similar among the three groups. In conclusion, various methods of oocyte activation and sperm preparation significantly affected the developmental capacity of early porcine embryos derived from IVM and ICSI.

[Sequencing analysis of the control region and intron1 region and characterization of two novel microsatellites in obese gene of pig].

For the first time, a 16 kb fragment of the porcine Ob gene, namely intron1, exon1, 5' region of Ob gene, was restrictively analyzed and sequenced by the primers designed in the portion of the swine Ob sequence. The first small 38 bp untranslated exon1 is located 11.1 kb upstream of the initiator ATG codon, and two novel microsatellites SW200 and SW160 are found in intron1. Promoter analysis of several putative binding sites revealed that this 300 bp promoter located at -1 to -300, including C/EBP and two Sp1, may be as effective as the longer promoter in directing leptin transcription. To examine microsatellites association with important economic traits, we statistically analyzed genotypes and alleles of the two microsatellites. Statistical analysis carried out by SAS 8.2 revealed significant positive correlation between the two microsatellites genotypes and the litter size in first parity of Erhualian.

[A new method for phylogenic analysis].

With ESTs from porcine fatty tissue and cDNA sequences from human, bovine and mouse in non-reduncdant database and dbEST in GenBank,we sampled cDNA sequences of 70 function-known genes in four species on the base of randomly sampling method, analyzed the mutation pattern of 70 x 150 bp linking sequences between them, and established an integrated phylogenic analysis method. The results showed that 391 single bases mutations were found in 70 x 150 bp linking sequences alignment among four species. The number of mutation bases between them were greatly exceeded the 1/1000 predicted in the human genome analysis. C/T(T/C) and A/G (G/A) transitions were the major types of single base mutation. The genetic relationship between pig and bovine who are both Artiodactylous is the nearest, the next is human, and the farthest is mouse. The differentiation sequence taken place in four species from the same ancestor is that mouse is the earliest one, and the latter human, and pig and bovine are the latest.

[Identification and analysis of a novel microsatellite marker flanking porcine myostatin gene (MSTN)].

In animal breeding, microsatellite marker plays an important role in constructing genetic maps, QTL mapping and function analysis of structural genes. Myostatin, also known as GDF8, is a negative regulator of skeletal muscle mass and, in swine, it is evidenced to be related to birth weight and average daily gain from 60 kg to 100 kg of body weight. In present study, by subcloning and sequencing,we identified a novel microsatellite marker which is useful for fine QTL mapping for meat traits. A BAC clone containing porcine MSTN was extracted and digested with EcoR I to recover the fragment of > 4 kb for subcloning in pGEM-3zf (+). Sequencing and alignment results showed that this subcloned fragment was not from porcine MSTN, but included a tandem repeat of (TG) 13, which is a novel microsatellite marker (GenBank accession number: AF454400) flanking MSTN. To exclude its vector origin we designed specific primers flanking this marker and successfully amplified this fragment from porcine genome. Through a pedigree analysis of a double-muscled Yorshire strain, we found that it is inherited in a co-dominant manner. We also checked the gene frequencies of this locus in 381 unrelated individuals of 7 pig breeds, namely Laiwu,Landrace, Yorkshire,Duroc, Peterian, Min and Erhualian. Only two alleles were detected, the repeating number of which are 13 (allele A) and 19 (allele B) respectively, which indicated that it is a low poly morphic microsatellite marker. In addition, the frequencies of the two alleles are different between the two types of pig breeds, while allele A is dominant in Chinese local breeds, allele B is dominant in imported breeds. Alignment with AY208121 indicate that this locus is located 42 kb downstream of porcine MSTN. We speculate that this microsatellite DNA is an important marker both in fine QTL mapping for meat traits and in the expression study of porcine MSTN.