The rumen-derived Lact. mucosae LLK-XR1 exhibited greater free gossypol degradation capacity during solid-state fermentation of cottonseed meal and probiotic potential.

BACKGROUND: This study aimed to isolate the rumen-derived bacteria with the ability to degrade free gossypol (FG), and to evaluate the probiotic potential in vitro for ensuring safe utilization. METHODS: The strains were anaerobically isolated from fresh rumen fluid of sheep with long-term fed cottonseed meal (CSM) with the screening agar medium containing gossypol as the sole carbon source. Afterwards, the isolated strain incubated with CSM was subjected to the determination of the FG degradation and in vitro evaluation of probiotic characteristics. RESULTS: The target strain labeled Lact. mucosae LLK-XR1 [Accession number: OQ652016.1] was obtained, and its growth on MRS Liquid medium exhibited degradation efficiency of FG up to 69.5% which was significantly greater than its growth on Man-Rogosa-Sharpe medium with glucose free for 24 h (p < 0.01). Meanwhile, LLK-XR1 showed 40.652% degradation rate of FG for unautoclaved, non-pulverized, and no additional nutrients supplementation CSM. Furthermore, LLK-XR1 presented good survivability at pH 3.0 (above 88.6%), and 0.3% bile (78.5%). LLK-XR1 showed sensitivity to broad-spectrum antibiotics except Sulfamethoxazole, Ciprofloxacin and Gentamycin and significantly inhibited E. coli CICC 10,899, Staph. aureus CICC 21,600, and Salmonella. Typhimurium CICC 21,483. LLK-XR1 demonstrated good cell surface hydrophobicity and auto-aggregation ability. CONCLUSIONS: Taken together, this study for the first time noted that rumen-originated Lact. mucosae LLK-XR1 with probiotic properties exhibited substantial FG degradation capacity when it was applied to the solid-state fermentation of CSM.

Risk factors for the development of severe breast cancer-related lymphedema: a retrospective cohort study.

BACKGROUND: Severe lymphedema presents a challenge in terms of treatment due to the significant formation of scar tissue that accompanies it. The aim of this study was to identify intraoperative and preoperative risk factors of severe lymphedema and to develop a nomogram for estimating the risk of severe lymphedema within 3 years of surgery. METHOD: Data was collected from a retrospective cohort of 326 patients with BCRL at the Zhejiang Cancer Hospital from November 2015 to November 2018. Univariate and multivariate logistic regression analysis was conducted to identify predictive indicators of severe lymphedema. A nomogram was developed to further improve the clinical applicability. RESULTS: In the retrospective cohort, the ratio of severe/non-severe lymphedema within 3 years of surgery was 1:3. Independent risk factors for severe lymphedema were determined to be age, positive lymph nodes, interpectoral (Rotter's) lymph nodes (IPNs) dissection, and educational level. IPNs dissection was found to contribute greatly to the development of severe lymphedema with a higher odds ratio (7.76; 95% CI: 3.87-15.54) than other risk factors. A nomogram was developed by integrating age, positive lymph nodes, IPNs dissection, and educational level, which yielded a C-index of 0.810 and 0.681 in the training and validation cohort, respectively. This suggested a moderate performance of the nomogram in predicting the risk of severe lymphedema within 3 years of surgery. The cut-off values of the low-, medium- and high-risk probabilities were 0.0876 and 0.3498, and the severe lymphedema exhibited a significantly higher risk probability as compared with the non-severe lymphedema. CONCLUSION: This study identified the risk factors of severe lymphedema and highlighted the substantial contribution of IPNs dissection to the severity of lymphedema.

Complementary hepatic metabolomics and proteomics reveal the adaptive mechanisms of dairy cows to the transition period.

The transition period from late pregnancy to early lactation is a vital time of the lifecycle of dairy cows due to the marked metabolic challenges. Besides, the liver is the pivot point of metabolism in cattle. Nevertheless, the hepatic physiological molecular adaptation during the transition period has not been elucidated, especially from the metabolomics and proteomics view. Therefore, the present study aims to investigate the hepatic metabolic alterations in transition cows by using integrative metabolomics and proteomics methods. Gas chromatography quadrupole-time-of-flight mass spectrometry-based metabolomics and data-independent acquisition-based quantitative proteomics methods were used to analyze liver tissues collected from 8 healthy multiparous Holstein dairy cows 21 d before and after calving. In total, 44 metabolites and 250 proteins were identified as differentially expressed from 233 metabolites and 3,539 proteins detected from the liver biopsies during the transition period. Complementary functional analysis of different metabolites and proteins indicated the upregulated gluconeogenesis, tricarboxylic acid cycles, AA degradation, fatty acid oxidation, AMP-activated protein kinase signaling pathway, peroxisome proliferator-activated receptor signaling pathway, and ribosome proteins in postpartum dairy cows. In terms of the metabolites and proteins, glucose-6-phosphate, fructose-6-phosphate, carnitine palmitoyltransferase 1A, and phosphoenolpyruvate carboxykinase played a significant role in these pathways. The upregulated oxidative status may be accompanied by the pathways mentioned above. In addition, the upregulated glucagon and insulin signaling pathways also indicated the significant requirement for glucose in postpartum dairy cows. These outcomes, from the view of global metabolites and proteins, may present a better comprehension of the biology of the transition period, which can be helpful in further developing nutritional regulation strategies targeting the liver to help cows overcome this metabolically challenging time.

Growth performance, blood metabolites, ruminal fermentation, and bacterial community in preweaning dairy calves fed corn silage-included starter and total mixed ration.

The objective of this study was to evaluate the effects of the inclusion of whole-plant corn silage (WPCS) in a starter or total mixed ration (TMR) on growth, blood metabolites, ruminal fermentation, and microbial community in preweaning dairy calves. A total of 45 healthy dairy calves were blocked by date of birth and randomly assigned to 1 of 3 treatments: 100% calf starter (CONS), a mix of 85% calf starter and 15% WPCS [dry matter (DM) basis; CSCS], or 100% WPCS-based lactation TMR (CTMR). Pasteurized normal milk was fed to all the animals under the same regimen. The experiment ran from when the calves were 2 d old to weaning at 63 d. Milk and feed intakes were recorded daily. Growth performance data and blood samples were collected on wk 3, 5, 7, and 9 of the experiment. Rumen fluid was sampled at 40 and 60 d. The 3 treatments had different particle size fractions. The CSCS group had greater medium fraction (<19 mm, >8 mm) and particles retained on 8-mm sieves than the other 2 groups, whereas the CTMR group had the greatest long (>19 mm) and fine (<4 mm) fractions and physically effective neutral detergent fiber (NDF) on 8- and 4-mm sieves, but had the smallest short fraction (<8 mm, >4 mm) and particles retained on 4-mm sieves. The 24-h in vitro digestibility of DM, crude protein (CP), NDF, and acid detergent fiber (ADF) were decreased in order by the CONS, CSCS, and CTMR groups. Compared with the CONS group, the digestibility of ether extract (EE) was lower in the CSCS and CTMR groups, whereas the digestibility of starch was similar among treatments. During the experimental period, the DM, CP, and metabolizable energy intakes from milk, solid feed, and total feed were not affected by treatments. The NDF, ADF, and EE intakes and potentially digestible intakes were greater in the CTMR group than in the other 2 groups. With the exception that body barrel was greater for calves fed CSCS, growth parameters and blood metabolites were similar among treatments. Compared with the CSCS group, the CTMR group had greater rumen pH and total volatile fatty acids, propionate, and isovalerate concentrations, but a lower acetate:propionate ratio. The CTMR group had greater relative abundances of some cellulolytic bacteria (Rikenellaceae RC9 gut group, Christensenellaceae R7, Ruminococcaceae NK4A214, Ruminococcaceae UCG, Ruminococcus, and Erysipelotrichaceae UCG) in the rumen, which may be beneficial for the early acquisition of specific adult-associated microorganisms. In summary, a WPCS-based lactation TMR, but not the WPCS-included starter, had the potential to be an alternative starter in preweaning calves without having significant adverse effects. These findings provide theoretical and practical implications for the rational application of TMR in the early life of dairy calves.

Neoadjuvant pyrotinib, trastuzumab, and docetaxel for HER2-positive breast cancer (PHEDRA): a double-blind, randomized phase 3 trial.

BACKGROUND: Pyrotinib (an irreversible pan-ErbB inhibitor) plus capecitabine has survival benefits and acceptable tolerability in patients with HER2-positive metastatic breast cancer. We further assessed addition of pyrotinib to trastuzumab and docetaxel in the neoadjuvant setting. METHODS: In this multicenter, double-blind, phase 3 study (PHEDRA), treatment-naive women with HER2-positive early or locally advanced breast cancer were randomly assigned (1:1) to receive four neoadjuvant cycles of oral pyrotinib or placebo (400 mg) once daily, plus intravenous trastuzumab (8 mg/kg loading dose, followed by 6 mg/kg) and docetaxel (100 mg/m2) every 3 weeks. The primary endpoint was the total pathological complete response (tpCR; ypT0/is and ypN0) rate per independent central review. RESULTS: Between Jul 23, 2018, and Jan 8, 2021, 355 patients were randomly assigned, 178 to the pyrotinib group and 177 to the placebo group. The majority of patients completed four cycles of neoadjuvant treatment as planned (92.7% and 97.7% in the pyrotinib and placebo groups, respectively). The tpCR rate was 41.0% (95% CI 34.0 to 48.4) in the pyrotinib group compared with 22.0% (95% CI 16.6 to 28.7) in the placebo group (difference, 19.0% [95% CI 9.5 to 28.4]; one-sided P < 0.0001). The objective response rate per investigator was 91.6% (95% CI 86.6 to 94.8) in the pyrotinib group and 81.9% (95% CI 75.6 to 86.9) in the placebo group after the neoadjuvant treatment, resulting in an increase of 9.7% (95% CI 2.7 to 16.6). The most common grade 3 or worse adverse events were diarrhea (79 [44.4%] in the pyrotinib group and nine [5.1%] in the placebo group), neutropenia (33 [18.5%] and 36 [20.3%]), and decreased white blood cell count (29 [16.3%] and 24 [13.6%]). No deaths were reported during neoadjuvant treatment. CONCLUSIONS: The primary endpoint of the study was met. Neoadjuvant pyrotinib, trastuzumab, and docetaxel significantly improved the tpCR rate compared with placebo, trastuzumab, and docetaxel, with manageable toxicity, providing a new option for HER2-positive early or locally advanced breast cancer. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03588091.

Cobalt-Catalyzed 2-(1-Methylhydrazinyl)pyridine-Assisted C-H Alkylation/Annulation: Mechanistic Insights and Rapid Access to Cyclopenta[c]isoquinolinone Derivatives.

We have developed cobalt-catalyzed, bidentate 2-(1-methylhydrazinyl)pyridine (MHP)-directed C(sp2)-H alkylation/annulation of benzoic hydrazides with various alkenes. Notably, diverse cyclopenta[c]isoquinolinones and dihydroisoquinolinones were obtained via this functional group-tolerant protocol. The reaction can be performed on a gram scale while maintaining an excellent yield, and the directing group can be removed efficiently under mild conditions. Furthermore, density-functional theory (DFT) calculations provide an incisive understanding of the observed regioselectivities for different olefins.

Synthesis of 5H-Chromeno[2,3-d]pyrimidin-5-one Derivatives via Microwave-Promoted Multicomponent Reaction.

A microwave-promoted multicomponent reaction of 3-formylchromones, amines, and paraformaldehyde was achieved under catalyst-free and solvent-free conditions, delivering 5H-chromeno[2,3-d]pyrimidin-5-one derivatives in good to excellent yields via an unexpected annulation pathway, which further expanded the synthetic application of paraformaldehyde as a C1 building block.

A Prospective Validation Cohort Study of a Prediction Model on Non-sentinel Lymph Node Involvement in Early Breast Cancer.

BACKGROUND: Early breast cancer with one or two sentinel lymph nodes (SLNs) may omit axillary lymph node dissection (ALND) if followed by radiotherapy. However, only less than one-third of the patients have positive non-SLNs and can truly benefit from radiotherapy. Before any regional treatment decision, the risk of non-SLN metastasis must be identified. The authors previously developed a predictive model for non-SLN involvement using CK19 mRNA and contrast-enhanced ultrasound (CEUS) score in a training set. They designed a further study to evaluate the predictive effect using the model prospectively in a validation set of one or two involved SLNs. METHODS: This study identified early breast cancer patients at Zhejiang Cancer Hospital from July 2017 to June 2018. The CK19 mRNA tested by quantitative real-time polymerase chain reaction and CEUS scores were collected before surgery. Patients with one or two involved SLNs were enrolled and underwent ALND. The estimated percentage of non-SLN involvement was calculated by the authors' model formula and the Memorial Sloan Kettering Cancer Center (MSKCC) nomogram. The false-negative rates, predictive accuracy, and area under curve (AUC) were compared between two predictive models. RESULTS: The study enrolled 235 patients, and 35.36% (83/235) of them had non-SLN involvement. The authors' model had a false-negative rate of 6% and an accuracy of 94.9%. The AUC was 0.952 (95% confidence interval [CI] 0.922-0.982), which was significantly higher than that of the MSKCC model at all three cutoff value levels. CONCLUSION: The authors' model, using CK19 mRNA and the CEUS score, showed the potential predictive value of non-SLNs before surgery for early breast cancer patients. CLINICALTRIALS REGISTRY: NCT02992067, NCT03280134.

Effect of dietary fiber fermentation on short-chain fatty acid production and microbial composition in vitro.

BACKGROUND: The efficient utilization of fiber-rich co-products is important for optimizing feed resource utilization and animal health. This study was conducted to evaluate the fermentation characteristics of fiber-rich co-products, which had equal quantities of total dietary fiber (TDF), at different time points using batch in vitro methods. It considered their gas production, short-chain fatty acid (SCFA) production, and microbial composition. RESULTS: The fermentation of wheat bran (WB) and oat bran (OB) showed higher and faster (P < 0.05) gas and SCFA production than corn bran (CB), sugar beet pulp (SBP), and soybean hulls (SH). The α-diversity was higher in the CB, SBP, and SH groups than in the WB and OB groups (P < 0.05). At the phylum level, OB and WB fermentation showed lower (P < 0.05) relative abundance of Actinobacteria than the CB, SBP, and SH groups. At the genus level, OB and WB fermentation increased the Enterococcus population in comparison with the CB, SBP, and SH groups, whereas CB and SBP fermentation improved the relative abundance of the Christensenellaceae R-7 group more than the WB, OB, and SH groups (P < 0.05). CONCLUSION: Overall, WB and OB were rapidly fermented by fecal microbiota, in contrast with SBP, SH, and CB. Fermentation of different fiber-rich co-products with an equal TDF content gives different responses in terms of microbial composition and SCFA production due to variations in their physicochemical properties and molecular structure. © 2020 Society of Chemical Industry.

Long noncoding RNA TALNEC2 plays an oncogenic role in breast cancer by binding to EZH2 to target p57KIP2 and involving in p-p38 MAPK and NF-κB pathways.

We aimed to investigate the potential role and regulatory mechanism of long noncoding RNA tumor-associated lncRNA expressed in chromosome 2 (TALNEC2) in breast cancer. The expression of TALNEC2 in breast cancer tissues and cells were investigated. MCF-7 and MDA-MB-231 cells were transfected with small interfering RNA (siRNA) duplexes for targeting TALNEC2 (si-TALNEC2), enhancer of zeste homolog 2 (EZH2; si-EZH2) and p57KIP2 (si-p57 KIP2 ), and their corresponding controls (si-NC). The viability, colony forming ability, cell cycle, apoptosis, and autophagy of transfected cells were assessed. The expressions of p-p38 mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathway-related proteins were investigated. The results showed that TALNEC2 was highly expressed in breast cancer tissues and cells. Knockdown of TALNEC2 significantly inhibited the malignant behaviors of MCF-7 and MDA-MB-231 cells, including inhibiting cell viability and colony forming, arresting cell cycle at G0/G1 phase, inducing cell apoptosis, and promoting cell autophagy. EZH2 was a TALNEC2 binding protein, which was upregulated in breast cancer tissues and cells and could negatively regulate p57 KIP2 . Effects of TALNEC2 knockdown on malignant behaviors of MCF-7 cells were reversed by p57 KIP2 knockdown. The expressions of p-p38, RelA, and RelB in MCF-7 cells were decreased after knockdown of TALNEC2 or EZH2, which were reversed by knockdown of p57 KIP2 concurrently. In conclusion, TALNEC2 may play an oncogenic role in breast cancer by binding to EZH2 to target p57 KIP2 . Activation of p-p38 MAPK and NF-κB pathways may be key mechanisms mediating the oncogenic role of TALNEC2 in breast cancer. TALNEC2 may serve as a promising target in the therapy of breast cancer.

The Dysregulated Expression of KCNQ1OT1 and Its Interaction with Downstream Factors miR-145/CCNE2 in Breast Cancer Cells.

BACKGROUND/AIMS: Next-generation sequencing (NGS) has revealed abundant long noncoding RNAs (lncRNAs) that have been characterized as critical components of cancer biology in humans. The present study aims to investigate the role of the lncRNA KCNQ1OT1 in breast cancer (BRCA) as well as the underlying molecular mechanisms and functions of KCNQ1OT1 involved in the progression of BRCA. METHODS: The Cancer Genome Atlas (TCGA) and StarBase v2.0 were used to obtain the required gene data. Dual luciferase reporter gene assays were conducted to verify the relevant intermolecular target relationships. QRT-PCR and Western blot were performed to measure the expression levels of different molecules. Cell proliferation was detected by using the MTT and colony formation assays, while cell migration and invasion were examined by transwell assay. Variations in cell apoptosis and cell cycle were determined through flow cytometry. A tumor xenograft model was applied to assess tumor growth in vivo. RESULTS: KCNQ1OT1 was found to be remarkably highly expressed in BRCA tissues and cells. KCNQ1OT1 modulated CCNE2 through sponging miR-145 in BRCA. KCNQ1OT1 promoted tumor growth in vivo by regulating miR-145/CCNE2. CONCLUSION: The KCNQ1OT1/miR-145/CCNE2 axis plays a critical regulatory role in BRCA, potentially giving rise to BRCA tumorigenesis and progression. These findings provide valuable evidence for improving the diagnosis and treatment of BRCA in the future.

The Expanding Utility of Rhodium-Iminocarbenes: Recent Advances in the Synthesis of Natural Products and Related Scaffolds.

Rhodium-iminocarbenes that are derived from N-sulfonyl-1,2,3-triazoles have become an important class of reactive species and useful intermediates in organic synthesis. Over the last several years, many practical and versatile approaches involving rhodium-iminocarbene intermediates to synthetically challenging molecules (scaffolds) have been developed. This Minireview mainly summarizes the recent advance of rhodium-iminocarbene involved reactions in the synthesis of natural products and their related scaffolds by the end of 2017. Several applications in important pharmaceuticals are documented as well.

Digital gene expression profiling analysis and its application in the identification of genes associated with improved response to neoadjuvant chemotherapy in breast cancer.

BACKGROUND: This study aimed to screen sensitive biomarkers for the efficacy evaluation of neoadjuvant chemotherapy in breast cancer. METHODS: In this study, Illumina digital gene expression sequencing technology was applied and differentially expressed genes (DEGs) between patients presenting pathological complete response (pCR) and non-pathological complete response (NpCR) were identified. Further, gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were then performed. The genes in significant enriched pathways were finally quantified by quantitative real-time PCR (qRT-PCR) to confirm that they were differentially expressed. Additionally, GSE23988 from Gene Expression Omnibus database was used as the validation dataset to confirm the DEGs. RESULTS: After removing the low-quality reads, 715 DEGs were finally detected. After mapping to KEGG pathways, 10 DEGs belonging to the ubiquitin proteasome pathway (HECTD3, PSMB10, UBD, UBE2C, and UBE2S) and cytokine-cytokine receptor interactions (CCL2, CCR1, CXCL10, CXCL11, and IL2RG) were selected for further analysis. These 10 genes were finally quantified by qRT-PCR to confirm that they were differentially expressed (the log2 fold changes of selected genes were - 5.34, 7.81, 6.88, 5.74, 3.11, 19.58, 8.73, 8.88, 7.42, and 34.61 for HECTD3, PSMB10, UBD, UBE2C, UBE2S, CCL2, CCR1, CXCL10, CXCL11, and IL2RG, respectively). Moreover, 53 common genes were confirmed by the validation dataset, including downregulated UBE2C and UBE2S. CONCLUSION: Our results suggested that these 10 genes belonging to these two pathways might be useful as sensitive biomarkers for the efficacy evaluation of neoadjuvant chemotherapy in breast cancer.

Rhodium-Catalyzed Denitrogenative [3+2] Cycloaddition: Access to Functionalized Hydroindolones and the Framework of Montanine-Type Amaryllidaceae Alkaloids.

Rhodium-catalyzed denitrogenative [3+2] cycloaddition of 1-sulfonyl-1,2,3-triazoles with cyclic silyl dienol ethers has been developed for the synthesis of functionalized hydroindolones or their corresponding silyl ethers. The present method has been employed to construct synthetically valuable bicyclo[3.3.1]alkenone derivatives and pyrrolidine-ring-containing bicyclic indole compounds. As a further synthetic application, a stereoselective synthesis of 5,11-methanomorphanthridin-3-one, which shares a key skeleton with montanine-type Amaryllidaceae alkaloids has been achieved by using this chemistry.

RNA Sequencing Uncovers Molecular Mechanisms Underlying Pathological Complete Response to Chemotherapy in Patients with Operable Breast Cancer.

BACKGROUND This study aimed to identify key genes contributing to pathological complete response (pCR) to chemotherapy by mRNA sequencing (RNA-seq). MATERIAL AND METHODS RNA was extracted from the frozen biopsy tissue of patients with pathological complete response and patients with non-pathological complete response. Sequencing was performed on the HiSeq2000 platform. Differentially expressed genes (DEGs) were identified between the pCR group and non-pCR (NpCR) group. Pathway enrichment analysis of DEGs was performed. A protein-protein interaction network was constructed, then module analysis was performed to identify a subnetwork. Finally, transcription factors were predicted. RESULTS A total of 673 DEGs were identified, including 419 upregulated ones and 254 downregulated ones. The PPI network constructed consisted of 276 proteins forming 471 PPI pairs, and a subnetwork containing 18 protein nodes was obtained. Pathway enrichment analysis revealed that PLCB4 and ADCY6 were enriched in pathways renin secretion, gastric acid secretion, gap junction, inflammatory mediator regulation of TRP channels, retrograde endocannabinoid signaling, melanogenesis, cGMP-PKG signaling pathway, calcium signaling pathway, chemokine signaling pathway, cAMP signaling pathway, and rap1 signaling pathway. CNR1 was enriched in the neuroactive ligand-receptor interaction pathway, retrograde endocannabinoid signaling pathway, and rap1 signaling pathway. The transcription factor-gene network consists of 15 transcription factors and 16 targeted genes, of which 5 were downregulated and 10 were upregulated. CONCLUSIONS We found key genes that may contribute to pCR to chemotherapy, such as PLCB4, ADCY6, and CNR1, as well as some transcription factors.

Metabolic responses and "omics" technologies for elucidating the effects of heat stress in dairy cows.

Heat stress (HS) negatively affects various industries that rely on animal husbandry, particularly the dairy industry. A better understanding of metabolic responses in HS dairy cows is necessary to elucidate the physiological mechanisms of HS and offer a new perspective for future research. In this paper, we review the current knowledge of responses of body metabolism (lipid, carbohydrate, and protein), endocrine profiles, and bovine mammary epithelial cells during HS. Furthermore, we summarize the metabolomics and proteomics data that have revealed the metabolite profiles and differentially expressed proteins that are a feature of HS in dairy cows. Analysis of metabolic changes and "omics" data demonstrated that HS is characterized by reduced lipolysis, increased glycolysis, and catabolism of amino acids in dairy cows. Here, analysis of the impairment of immune function during HS and of the inflammation that arises after long-term HS might suggest new strategies to ameliorate the effects of HS in dairy production.

Long-term heat stress induces the inflammatory response in dairy cows revealed by plasma proteome analysis.

In this work we employed a comparative proteomic approach to evaluate seasonal heat stress and investigate proteomic alterations in plasma of dairy cows. Twelve lactating Holstein dairy cows were used and the treatments were: heat stress (n = 6) in hot summer (at the beginning of the moderate heat stress) and no heat stress (n = 6) in spring natural ambient environment, respectively. Subsequently, heat stress treatment lasted 23 days (at the end of the moderate heat stress) to investigate the alterations of plasma proteins, which might be employed as long-term moderate heat stress response in dairy cows. Changes in plasma proteins were analyzed by two-dimensional electrophoresis (2-DE) combined with mass spectrometry. Analysis of the properties of the identified proteins revealed that the alterations of plasma proteins were related to inflammation in long-term moderate heat stress. Furthermore, the increase in plasma tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) directly demonstrated that long-term moderate heat stress caused an inflammatory response in dairy cows.

Novel germline mutations and unclassified variants of BRCA1 and BRCA2 genes in Chinese women with familial breast/ovarian cancer.

BACKGROUND: Germline mutations in the BRCA1 and BRCA2 genes greatly increase a woman's risk of developing breast and/or ovarian cancer. The prevalence and distribution of such mutations differ across races/ethnicities. Several studies have investigated Chinese women with high-risk breast cancer, but the full spectrum of the mutations in these two genes remains unclear. METHODS: In this study, 133 unrelated Chinese women with familial breast/ovarian cancer living in Zhejiang, eastern China, were enrolled between the years 2008 and 2014. The complete coding regions and exon-intron boundaries of BRCA1 and BRCA2 were screened by PCR-sequencing assay. Haplotype analysis was performed to confirm BRCA1 and BRCA2 founder mutations. In silico predictions were performed to identify the non-synonymous amino acid changes that were likely to disrupt the functions of BRCA1 and BRCA2. RESULTS: A total of 23 deleterious mutations were detected in the two genes in 31 familial breast/ovarian cancer patients with a total mutation frequency of 23.3% (31/133). The highest frequency of 50.0% (8/16) was found in breast cancer patients with a history of ovarian cancer. The frequencies of BRCA1 and BRCA2 mutations were 13.5 % (18/133) and 9.8% (13/133), respectively. We identified five novel deleterious mutations (c.3295delC, c.3780_3781delAG, c.4063_4066delAATC, c.5161 > T and c.5173insA) in BRCA1 and seven (c.1-40delGA, c.4487delC, c.469_473delAAGTC, c.5495delC, c.6141T > A, c.6359C > G and c.7588C > T) in BRCA2, which accounted for 52.2% (12/23) of the total mutations. Six recurrent mutations were found, including four (c.3780_3781delAG, c.5154G > A, c.5468-1del8 and c.5470_5477del8) in BRCA1 and two (c.3109C > T and c.5682C > G) in BRCA2. Two recurrent BRCA1 mutations (c.5154G > A and c.5468-1del8) were identified as putative founder mutations. We also found 11 unclassified variants, and nine of these are novel. The possibility was that each of the non-synonymous amino acid changes would disrupt the function of BRCA1 and BRCA2 varied according to the different algorithms used. CONCLUSIONS: BRCA1 and BRCA2 mutations accounted for a considerable proportion of hereditary breast/ovarian cancer patients from eastern China and the spectrum of the mutations of these two genes exhibited some unique features. The two BRCA1 putative founder mutations may provide a cost-effective option to screen Chinese population, while founder effects of the two mutations should be investigated in a lager sample size of patients.

Drug response to HER2 gatekeeper T798M mutation in HER2-positive breast cancer.

The gatekeeper T798M mutation in HER2 kinase domain has been observed to considerably shift drug sensitivity to HER2 in breast cancer therapy. Here, drug response of clinical tyrosine kinase inhibitors (TKIs) to the mutation was profiled using a synthetic biology protocol. It was found that TKIs can be grouped into three classes in terms of their response behavior to T798M mutation: class I inhibitors exhibit drug resistance upon the mutation, such as lapatinib, TAK-285 and AEE788; class II inhibitors are insensitive to the mutation, such as erlotinib and gefitinib; and class III inhibitors can be sensitized by the mutation, such as staurosporine. However, kinetic study indicated that the mutation has only a modest effect on the binding of substrate ATP to HER2. Binding free energy analysis revealed that the drug response is primarily determined by direct interaction between the kinase and inhibitors, but not by indirect kinase interaction with competitive ATP. This is different to the molecular mechanism of "generic" drug resistance conferring from EGFR gatekeeper T790M mutation, which is caused by increased ATP affinity upon the mutation. Structural analysis of kinase-inhibitor complexes unraveled that HER2 T798M mutation induces significant steric hindrance to class I inhibitors, but can establish additional nonbonded interactions for class III inhibitors.

Fiber degradation potential of natural co-cultures of Neocallimastix frontalis and Methanobrevibacter ruminantium isolated from yaks (Bos grunniens) grazing on the Qinghai Tibetan Plateau.

Several natural anaerobic fungus-methanogen co-cultures have been isolated from rumen and feces source of herbivores with strong fiber degrading ability. In this study, we isolated 7 Neocallimastix with methanogen co-cultures from the rumen of yaks grazing on the Qinghai Tibetan Plateau. Based on morphological characteristics and internal transcribed spacer 1 sequences (ITS1), all the fungi were identified as Neocallimastix frontalis. The co-cultures were confirmed as the one fungus - one methanogen pattern by the PCR-denatured gradient gel electrophoresis (DGGE) assay. All the methanogens were identified as Methanobrevibacter ruminantium by 16s rRNA gene sequencing. We investigated the biodegrading capacity of the co-culture (N. frontalis + M. ruminantium) Yaktz1 on wheat straw, corn stalk and rice straw in a 7 days-incubation. The in vitro dry matter digestibility (IVDMD), acid detergent fiber digestibility (ADFD) and neural detergent fiber digestibility (NDFD) values of the substrates in the co-culture were significantly higher than those in the mono-culture N. frontalis Yaktz1. The co-culture exhibited high polysaccharide hydrolase (xylanase and FPase) and esterase activities. The xylanase in the co-culture reached the highest activity of 12500 mU/ml on wheat straw at the day 3 of the incubation. At the end of the incubation, 3.00 mmol-3.29 mmol/g dry matter of methane were produced by the co-culture. The co-culture also produced high level of acetate (40.00 mM-45.98 mM) as the end-product during the biodegradation. Interestingly, the N. frontalis Yaktz1 mono-culture produced large amount of lactate (8.27 mM-11.60 mM) and ethanol (163.11 mM-242.14 mM), many times more than those recorded in the previously reported anaerobic fungi. Our data suggests that the (N. frontalis + M. ruminantium) Yaktz1 co-culture and the N. frontalis Yaktz1 mono-culture both have great potentials for different industrial use.