Multi-omics portrait of ductal carcinoma in situ in young women.

BACKGROUND: Young patients with breast ductal carcinoma in situ (DCIS) often face a poorer prognosis. The genomic intricacies in young-onset DCIS, however, remain underexplored. METHODS: To address this gap, we undertook a comprehensive study encompassing exome, transcriptome, and vmethylome analyses. Our investigation included 20 DCIS samples (including 15 young-onset DCIS) and paired samples of normal breast tissue and blood. RESULTS: Through RNA sequencing, we identified two distinct DCIS subgroups: "immune hot" and "immune cold". The "immune hot" subgroup was characterized by increased infiltration of lymphocytes and macrophages, elevated expression of PDCD1 and CTLA4, and reduced GATA3 expression. This group also exhibited active immunerelated transcriptional regulators. Mutational analysis revealed alterations in TP53 (38%), GATA3 (25%), and TTN (19%), with two cases showing mutations in APC, ERBB2, and SMARCC1. Common genomic alterations, irrespective of immune status, included gains in copy numbers at 1q, 8q, 17q, and 20q, and losses at 11q, 17p, and 22q. Signature analysis highlighted the predominance of signatures 2 and 1, with "immune cold" samples showing a significant presence of signature 8. Our methylome study on 13 DCIS samples identified 328 hyperdifferentially methylated regions (DMRs) and 521 hypo-DMRs, with "immune cold" cases generally showing lower levels of methylation. CONCLUSION: In summary, the molecular characteristics of young-onset DCIS share similarities with invasive breast cancer (IBC), potentially indicating a poor prognosis. Understanding these characteristics, especially the immune microenvironment of DCIS, could be pivotal in identifying new therapeutic targets and preventive strategies for breast cancer.

Development of a multiplex qRT-PCR assay for detection of classical swine fever virus, African swine fever virus, and Erysipelothrix rhusiopathiae.

Classical swine fever virus (CSFV), African swine fever virus (ASFV), and Erysipelothrix rhusiopathiae (E. rhusiopathiae) remain endemic in many parts of China. Co-infections make distinguishing their clinical symptoms and pathological changes difficult. This study developed a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) that can simultaneously detect CSFV, ASFV, and E. rhusiopathiae. Three sets of primers and probes were designed to target the CSFV 5΄ untranslated region, ASFV p72 gene, and E. rhusiopathiae 16sRNA gene. Multiplex qRT-PCR for simultaneous differential detection of these three pathogens was developed after optimizing reaction parameters such as annealing temperature, primer and probe concentrations, amplification cycles, etc. The multiplex qRT-PCR could detect CSFV, ASFV, and E. rhusiopathiae simultaneously but could not amplify other porcine pathogens. The assay's limit of detection (LOD) was 2.89 × 102 copies/µL for CSFV, ASFV, and E. rhusiopathiae. All correlation coefficients (R2) at higher than 0.99, and the amplification efficiency was 98, 90, and 84%, respectively. All correlation coefficients (R2) were higher than 0.99, and the efficacy of amplification was 84%. In a repeatability test utilizing standard recombinant plasmids, the intra- and inter-assay coefficients of variation (CVs) were less than 2.27 and 3.79 percent, respectively. Lastly, 150 clinical samples were used to evaluate the assay's applicability in the field. The positive rates of CSFV, ASFV, and E. rhusiopathiae were 1.33%, 0, and 3.33%, respectively. And no co-infection among the three pathogens was found. The concordance rate between the multiplex qRT-PCR and single-plex commercial PCR kits reached 100%. This study's multiplex qRT-PCR could provide a rapid, sensitive, and specific method for the simultaneous and differential detection of CSFV, ASFV, and E. rhusiopathiae.

Germinated Brown rice enhanced n-3 PUFA metabolism in type 2 diabetes patients: A randomized controlled trial.

BACKGROUND: Brown rice (BR) has been considered as a potential strategy in improving T2DM. However, there are a lack of population-based trials on the association of Germinated brown rice (GBR) and diabetes. AIMS: We aimed to explore the influence of GBR diet in T2DM patients for 3 months and whether this effect relates to serum fatty acids. METHODS: Two hundred and twenty T2DM patients have been enrolled and eligible subjects (n = 112, 61 female, 51 male) were randomly divided into GBR intervention group (n = 56) and control group (n = 56). Except those who lost follow-up and withdrew, final GBR group and control group consisted of 42 and 43 patients, respectively. Participants in GBR group were asked to consume 100 g/d GBR instead of equal refined grain (RG) for 3 months, while control group maintain their usual eating habits. A structured questionnaire was used for demographic information at baseline, and basic indicators were measured both at the beginning and end of the trail to evaluate plasma glucose and lipids levels. RESULTS: In GBR group, mean dietary inflammation index (DII) decreased, indicating GBR intervention retarded patient inflammation. Besides, glycolipid related parameters, including FBG, HbA1c, TC and HDL, were all significantly lower than those in control group. Excitingly, fatty acid composition was changed by intake of GBR, especially n-3 PUFA and n-3/n-6 PUFA rate were significantly increased. Moreover, subjects in GBR group had higher levels of n-3 metabolites, such as RVE, MaR1 and PD1, reducing inflammatory effect. In contrast, n-6 metabolites, like LTB4 and PGE2 which could promote inflammatory effect, were lower in GBR group. CONCLUSION: We confirmed that diet with 100 g/d GBR for 3 months could really improve T2DM to some extent. This beneficial effect may be related to n-3 metabolites, namely inflammation changes. TRIAL REGISTRATION: ChiCRT-IOR-17013999, www.chictr.org.cn.

3-Deoxysappanchalcone isolated from Caesalpinia sinensis shows anticancer effects on HeLa and PC3 cell lines: invasion, migration, cell cycle arrest, and signaling pathway.

To study the antitumor activity of compound 3-desoxysulforaphane (3-DSC) isolated from Caesalpinia sinensis, SRB assay, clone formation assay, flow cytometric cell cycle assay, scratch assay, transwell assay, and molecular docking were used to investigate the inhibitory effect of 3-DSC on HeLa and PC3 cells. The results showed that 3-DSC inhibited the cell migration and invasion by down-regulating expression of N-cadherin, Vimentin, MMP-2, and MMP-9 in HeLa and PC3 cells; It also inhibits cell proliferation by promoting the expression of CDK1 (cyclin-dependent kinases 1) and CDK2 (cyclin-dependent kinases 2), which arrests the tumor cell cycle at G2 phase. 3-DSC inhibits phosphorylation of AKT and ERK and upregulates the expression of the tumor suppressor gene p53. Molecular docking results confirmed that 3-DSC could bind firmly to AKT. In conclusion, 3-DSC inhibited the proliferation, migration and invasion of HeLa and PC3 cells.

Study on the chemical composition of Caesalpinia sinensis.

Five compounds were isolated from the methanolic extract of Caesalpinia sinensis stems and leaves including a new cassane-type butenolide norditerpenoid compound (1) and a new type of biphenyl compound (2); the compounds were identified as Norcaesalpin-one (1), 4'-hexyl 3-methyl 6-methoxy-[1,1'-biphenyl]-3,4'-dicarboxylate (2), rhapontigenin (3), 3-deoxysappanchalcone (4), isoliquiritigenin (5). Compounds 1-5 were first isolated from C. sinensis. Their structures were elucidaded on the basis of MS, IR, NMR spectroscopic, X-ray diffraction data analyses. The NGF-induced PC12 differentiation assay was performed on compound 1, and the results showed that compound 1 had a promotive effect on PC12 cell differentiation, with a differentiation rate of 12.32%. In addition, compounds 1-5 were evaluated for their cytotoxic activities against four human cancer cell lines (including A-549, BGC-823, MDA-MB-231, HepG2), and the results showed that compounds 3-5 showed inhibitory activity against these cancer cell lines with IC50 values ranging from 22.96 to 74.92 µmol/L, compound 4 showing the best activity against human malignant melanoma cells A375 with an IC50 value of 22.96 µmol/L.

Buffalo-Origin Seneca Valley Virus in China: First Report, Isolation, Genome Characterization, and Evolution Analysis.

Pigs are the main host of Seneca Valley virus (SVV), previously known as Senecavirus A (SVA). Pigs affected by SVV have vesicles in the nose, hooves, and limp and may cause death in some severe cases. Occasionally, SVV has also been detected in mice, houseflies, environmental equipment, and corridors in pig farms. Moreover, it was successfully isolated from mouse tissue samples. In this study, an SVV strain (SVA/GD/China/2018) was isolated from a buffalo with mouth ulcers in the Guangdong province of China using seven mammalian cell lines (including BHK-21, NA, PK-15, ST, Vero, Marc-145, and MDBK). The genome of SVA/GD/China/2018 consists of 7,276 nucleotides. Multiple-sequence alignment showed that SVA/GD/China/2018 shared the highest nucleotide similarity (99.1%) with one wild boar-origin SVV strain (Sichuan HS-01) from the Sichuan province of China. Genetic analysis revealed that SVA/GD/China/2018 clustered with those porcine-origin SVV strains. To the best of our knowledge, this is the first report of SVV infection in buffalo, which might expand the host range of the virus. Surveillance should be expanded, and clinical significance of SVV needs to be further evaluated in cattle.

Genome Characterization and Phylogenetic Analysis of the First Bovine Rhinitis B Virus Isolate in China.

Bovine rhinitis B virus (BRBV) is an emerging viral species in the genus Aphthovirus, family Picornaviridae. Studies suggested that BRBV was considered a potential etiological agent of bovine respiratory disease complex (BRDC). BRBV has been reported in the United States, Sweden, Canada, Japan, and Mexico. However, little information of BRBV was available in China. In this study, we performed viral metagenomic analysis in a calf with respiratory disease. The results showed high abundance (3.85) of BRBV nucleotide and 248 mapped reads in calf samples. Online BLASTn analysis showed that three contigs of those had the highest nucleotide similarity (95%) with one Swedish BRBV isolate (BRBV_SWE1, GenBank accession no. KY432299). To identify the genome characterization of the Chinese BRBV isolate (designated CHN1), six couples of overlapping RT-PCR primers were designed according to genome sequences of BRBV_SWE1. Through gene cloning and splicing, we obtained the genome information of CHN1, possessing 7,465 nucleotides (46.6% G+C). Although CHN1 had the highest nucleotide similarity (95.1%) with BRBV_SWE1, one 11-nucleotide (ACATTTGTTGT) deletion occurred in the 5' untranslated region compared to SWE1. Phylogenetic analysis showed that CHN1 clustered together with BRBV_SWE1, and far from other BRBV isolates. This study recorded the first discovery of BRBV infection in China. Further investigation should be made in order to evaluate the infection status and epidemiological significance of BRBV in China.

Development of a Real-Time Quantitative RT-PCR Assay for Detection of Bovine Rhinitis B Virus.

Bovine rhinitis B virus (BRBV) has been frequently identified in cattle diagnosed with bovine respiratory disease complex (BRDC) in recent years, suggesting its potential contribution to BRDC. The goal of this study was to develop a TaqMan-based real-time quantitative RT-PCR assay for efficient BRBV detection. A pair of primers and a probe were designed based on the 3D gene of the BRBV genome. The assay was specific for BRBV and able to exclude bovine rhinitis A virus, foot-and-mouth disease virus and Senecavirus A. The limit of detection of the assay was 4.46 copies per reaction. A standard curve was plotted, with a coefficient of determination of 0.999 in the concentration range of 100-108 copies/µl. The reproducibility of the assay was acceptable, with the standard deviations of cycle threshold values lower than 1.00 in both intra- and inter-assay. Of 200 samples collected from 150 head of cattle in recent years in China, 11% (22/200) of the samples tested positive in the assay, i.e., 4.6% (7/150) of the cattle were BRBV positive. This study provides an efficient diagnostic tool for the epidemiological investigations of BRBV.

Reservoirs of Porcine Circoviruses: A Mini Review.

Porcine circovirus (PCV) is one of the smallest known DNA viruses in mammals. At present, PCVs are divided into three species, PCV1, PCV2, and PCV3. PCV1 and PCV2 were found in the 1970s and the 1990s, respectively, whereas PCV3 was discovered recently in 2016. PCV1 does not cause diseases in pigs. However, PCV3, similar to PCV2, is reported to be associated with several swine diseases, including porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure. PCVs are very common in domestic pigs as well as wild boars. However, PCVs have been occasionally isolated from non-porcine animals, including ruminants (such as cattle, goats, wild chamois, and roe deers), rodents (such as NMRI mice, BALB/c mice, Black C57 mice, ICR mice, Mus musculus, and Rattus rattus), canines (such as dogs, minks, foxes, and raccoon dogs), insects (such as flies, mosquitoes, and ticks), and shellfish. Moreover, PCVs are frequently reported in biological products, including human vaccines, animal vaccines, porcine-derived commercial pepsin products, and many cell lines. PCVs are also abundant in the environment, including water samples and air samples. Interestingly, PCV1 and/or PCV2 antibody or antigen has also been detected in sera, stool samples and respiratory swab samples of human, revealing zoonotic potential of PCVs. Thus, PCVs inhabit many types of reservoirs. In this review, we summarize the reservoirs of PCVs, and this information would be helpful in understanding the natural circulating status and possible cross-species transmission of PCVs.

A Novel Enzootic Nasal Tumor Virus Circulating in Goats from Southern China.

Enzootic nasal tumor virus (ENTV) has two types, ENTV-1 in sheep and ENTV-2 in goats, respectively. In China, the incidence of ENTV-2 related diseases has increased year by year. In this study, we reported an outbreak of ENTV-2 in a commercial goat farm in Qingyuan city, Guangdong province, southern China. A full-length genome of ENTV-2 (designated GDQY2017), with 7479 base pairs, was sequenced. Although GDQY2017 shared the highest nucleotide identity with a Chinese ENTV-2 isolate (ENTV-2CHN4, GenBank accession number KU258873), it possesses distinct genome characteristics undescribed, including a non-continuous 21-nucleotide insertion in the gag gene and a non-continuous 12-nucleotide deletion in the env gene. Notably, most of these indel nucleotide sequences were originated from a Chinese jaagsiekte sheep retrovirus (JSRV) isolate (GenBank accession number DQ838494). In the gag and env genes, GDQY2017 was phylogenetically related to those Chinese ENTV-2 isolates and a Chinese JSRV isolate (DQ838494). For GDQY2017-like viruses, more surveillance work should be made to explain their pathogenicity in goat herds. To our knowledge, this study represents the first to demonstrate the circulating pattern of ENTV-2 in Guangdong province, China, which will help to better understand the epidemiology and genetic diversity of ENTV-2.

Scutellarein selectively targets multiple myeloma cells by increasing mitochondrial superoxide production and activating intrinsic apoptosis pathway.

OBJECTIVES: Scutellarein is a flavonoid monomer found in traditional Chinese medicine such as Scutellaria barbata. This study aimed to investigate the cytotoxic effect of scutellarein treatment on multiple myeloma (MM) cells. METHODS: circulating B lymphocytes (CBL) isolated from healthy donors' peripheral blood served as control for MM.1R and IM-9 MM cells. CLB and MM cells were treated with various concentrations of scutellarein before their cell viability and apoptosis being evaluated. Nude mice burdened with MM xenograft tumor were intravenously injected with different concentrations of scutellarein, and their tumor burden change were monitored. Apoptosis of MM cells or CBL after scutellarein treatment was assayed by measuring caspase-3, -8 and -9 activities. FADD or APAF1 gene knockdown in MM cells was achieved by lentiviral transfection. Amount of Cytochrome C in cytosol or mitochondria as well as that of Bax and Bcl-2 protein were evaluated by Western blot. Mitochondria-induced apoptosis was assayed by measuring mitochondrial membrane potential change. Production of general reactive oxygen species and mitochondrial superoxide in MM or CBL was detected after scutellarein treatment, which was reduced by MitoTEMPO or apocynin treatment, respectively. RESULTS: Scutellarein treatment showed potent cytotoxicity on MM cells but not on viable CBL, and intravenous injection of scutellarein significantly reduced MM xenograft tumor burden in nude mice. Scutellarein treatment in MM cells activated the mitochondrial-mediated intrinsic apoptosis pathway by increasing the production of mitochondrial superoxide, which was reduced to ROS by NADPH, but this effect was weakened in healthy CBL. Co-treatment with scutellarein synergized with bortezomib in inducing apoptosis in MM cells in vitro and in reducing tumor volume in MM xenografted nude mice. CONCLUSIONS: Scutellarein induced mitochondrial-mediated intrinsic apoptosis selectively on malignant cells comparing to healthy cells.

Phylogeographic analysis of porcine reproductive and respiratory syndrome virus 1 in Guangdong province, Southern China.

Porcine reproductive and respiratory syndrome virus (PRRSV) is considered an important economic pathogen for the international swine industry. At present, both PRRSV-1 and PRRSV-2 have been confirmed to be co-circulating in China. However, there is little available information about the prevalence or distribution of PRRSV-1 in Guangdong province, southern China. In this study, we performed molecular detection of PRRSV-1 in 750 samples collected from 50 farms in 15 major pig farming regions in this province. After RT-PCR testing, 64% (32/50) of farms were confirmed as PRRSV-1-positive. Surprisingly, PRRSV-1 was circulating on at least one pig farm in all 15 regions; of the 750 samples, 186 samples (24.8%) were positive for PRRSV-1. Furthermore, 15 representative PRRSV-1 ORF5 sequences (606 bp) (n = 1 per region) were obtained from those PRRSV-1-positive regions. Sequence alignment analysis indicated that they shared 81.8% ~ 100% nucleotide and 81.2% ~ 100% amino acid similarity with each other. Although all current PRRSV-1 sequences were divided into pandemic subtype 1, most of them had unique glycoprotein-5 amino acid sequences that are significantly different from other known PRRSV-1 isolates. To conclude, the present findings revealed wide geographical distribution of PRRSV-1 in Guangdong province, southern China. This study further extends the epidemiological significance of PRRSV-1 in China.

Preliminary serological evidence for Schmallenberg virus infection in China.

Schmallenberg virus (SBV) is an emerging and rampant arbovirus in Europe, and even Africa and West Asia. Investigating whether SBV existed in new regions or countries, it was very helpful for the early warning and control of SBV. In this study, we collected 317 serum samples (n = 242 for dairy cattle, n = 13 for yellow cattle, n = 21 for buffalo, and n = 41 for goats) from Guangdong province of southern China, which is located in a subtropical region and is an important distribution area for arboviral diseases. A commercial competitive enzyme-linked immunosorbent assay (cELISA) kit and a previously established real-time PCR were used to detect SBV antibody and RNA in those serum samples. Via testing, serological evidence of SBV was confirmed, with total positive rates (57.4, 15.4, 19, and 9.8%) in dairy cattle, yellow cattle, buffalo, and goats, respectively, while no positive signal for SBV RNA was found. To summarize, this study for the first time provided preliminary serological evidence of SBV infection in China, East Asia. Further investigations on molecular evidence, origin, and pathogenesis of SBV in ruminants needed to be studied in China.

Comparative epidemiology of porcine circovirus type 3 in pigs with different clinical presentations.

BACKGROUND: Porcine circovirus type 3 (PCV3), as an emerging circovirus species, was reported to be widely circulating in the United States, China, South Korea and Poland. Previous studies revealed that PCV3 was mainly concentrated in sick animals with respiratory disease, skin disease, reproductive disorders and so on. However, the circulating status of PCV3 in pigs with other clinical presentations (especilly asymptomatic or diarrhea) was not well established. FINDINGS: In this study, to conduct a comparative epidemiological survey of PCV3, 80 weaned pig serum samples with severe respiratory disease (SRD), 175 weaned pig serum samples with mild respiratory disease (MRD), 216 asymptomatic weaned pig serum samples, 35 diarrheal weaned pig samples and 35 non-diarrheal weaned pig samples were collected from eight provinces of China. Via qPCR testing, PCV3 was circulating in all sampling provinces, with total positive rates varying from 1.04% to 100%. Interestingly, the PCV3-positive rate was significantly higher in weaned pigs with SRD (63.75%, 51/80) than in those weaned pigs with MRD (13.14%, 23/175) and asymptomatic pigs (1.85%, 4/216) (P < 0.01). Similarly, the PCV3-positive rate was significantly higher in diarrheal weaned pigs (17.14%, 6/35) than in non-diarrheal weaned pigs (2.86%, 1/35) (P < 0.05). Moreover, the lower Ct values of qPCR were frequently found in those weaned pigs or fattening pigs with respiratory disease and diarrhea rather than that in asymptomatic pigs. Sequence analysis showed that low genetic diversity existed among those PCV3 sequences collected from pigs with different clinical presentations. CONCLUSIONS: The present study further extends evidence that newly described PCV3 widely circulates in six additional provinces of Southern and Northern China and has high similarity to previously reported isolates. As an emerging virus of swine, although the present case-control study reveals that PCV3 has a potential association with swine respiratory disease and diarrhea, further investigations into the pathogenesis are needed to ascertain the role of PCV3 in swine health.

Influenza D Virus in Animal Species in Guangdong Province, Southern China.

Molecular tests revealed influenza D viruses of D/OK lineage widely circulating in farmed animal species in Guangdong Province, southern China. In particular, we found high levels of influenza D virus infection in goats and pigs. We also detected viral RNA in serum specimens and feces of animals with certain severe diseases.