Knockout of butyrophilin subfamily 1 member A1(BTN1A1) alters lipid droplet formation and phospholipid composition in bovine mammary epithelial cells

Background: Milk lipids originate from cytoplasmic lipid droplets(LD) that are synthesized and secreted from mammary epithelial cells by a unique membrane-envelopment process. Butyrophilin 1 A1(BTN1 A1) is one of the membrane proteins that surrounds LD, but its role in bovine mammary lipid droplet synthesis and secretion is not well known.Methods: The objective was to knockout BTN1 A1 in bovine mammary epithelial cells(BMEC) via the CRISPR/Cas9 system and evaluate LD formation, abundance of lipogenic enzymes, and content of cell membrane phospholipid(PL) species. Average LD diameter was determined via Oil Red O staining, and profiling of cell membrane phospholipid species via liquid chromatography-tandem mass spectrometry(LC-MS/MS).Results: Lentivirus-mediated infection of the Cas9/sg RNA expression vector into BMEC resulted in production of a homozygous clone BTN1 A1^((-/-)). The LD size and content decreased following BTN1 A1 gene knockout. The m RNA abundance of fatty acid synthase(FASN) and peroxisome proliferator-activated receptor-gamma(PPARG) was downregulated in the BTN1 A1^((-/-))clone. Subcellular analyses indicated that BTN1 A1 and LD were co-localized in the cytoplasm. BTN1 A1 gene knockout increased the percentage of phosphatidylethanolamine(PE) and decreased phosphatidylcholine(PC), which resulted in a lower PC/PE ratio.Conclusions: Results suggest that BTN1 A1 plays an important role in regulating LD synthesis via a mechanism involving membrane phospholipid composition.

Genomic mutation signatures in primary breast cancer and their axillary metastatic lymph nodes

Breast cancer is one of the most common malignant tumors in women all over the world.Metastasis represents a major adverse progression of breast cancer,resulting in poor survival duration.Axillary lymph node metastasis is often the first step of systemic metastasis process of breast cancer.However,themechanismof lymph node metastasis and the genomic signatures of primary breast tumors and lymph node metastasis are still under exploration.Whole exome sequencing was applied to primary breast cancer,axillary metastatic lymph nodes,andwhite blood cells from10Chinesewomen patients in our study.Single nucleotide variants(SNVs)and copynumber variants(CNVs)were compared between primary tumors and lymph nodes for individual patients.There are somatic SNVs(average 5.58±2.56 per megabase)in primary breast cancers and somatic SNVs(average 5.46±2.66 per megabase)in axillary metastatic lymph nodes were identified,which is corresponding to a semblable mutation burden in two malignant sites(P=0.81).No difference was found in CNVs(P=0.33).In primary breast cancer,somatic SNVs(48.12±13.80%)and CNVs(61.72±35.00%)were overlapping with somatic SNVs(49.43±12.30%)and CNVs(72.01±24.31%)in axillary metastatic lymph nodes.Nine genes were screened for significant specificmutations in primary tumors,and 15 genes were significantly mutated in metastatic lymph nodes.Using MutSigCV screening,it was found that HRNR and AHNAK2 are lymph node metastasis-specific genes.In our study,primary breast tumors are directly related to axillary lymph node metastases together and there are most SNVs and CNVs which were overlapping in primary andmetastatic sites.These variants which are overlapping is closely related to themetastatic process of tumor invasion with early genetic variability.This is the first timeto prove the concept of polyclonalmetastaticmodel and in thismodelmore than one clonemigrates establish the metastases to axillary lymph nodes.This study was approved by the institutional review board(IRB)of the Cancer Hospital,Chinese Academy of Medical Sciences,and Peking Union Medical College,China(approval No.NCC2016G-030)on March 3,2016.

Degradation of extracellular genomic, plasmid DNA and specific antibiotic resistance genes by chlorination

There is a need to improve understanding of the effect of chlorine disinfection on antibiotic resistance genes (ARGs) in order to advance relevant drinking water, wastewater, and reuse treatments. However, few studies have explicitly assessed the physical effects on the DNA. Here we examined the effects of free chlorine (1-20 mg CI2/L) on extracellular genomic, plasmid DNA and select ARGs. Chlorination was found to decrease the fluorometric signal of extracellular genomic and plasmid DNA (ranging from 0.005 to 0.05 μg/mL) by 70%, relative to a no-chlorine control. Resulting DNA was further subject to a fragment analysis using a Bioanalyzer, indicating that chlorination resulted in fragmentation. Moreover, chlorine also effectively deactivated both chromosomal- and plasmidborne ARGs, mecK and tetA., respectively. For concentrations >2 mg CI2//L× 30 min, chlorine efficiently reduced the qPCR signal when the initial concentration of ARGs was 10^5 copies/μL or less. Notably, genomic DNA and mecA gene signals were more readily reduced by chlorine than the plasmid-bome tetK gene (by ~2 fold). Based on the results of qPCR with short (~200 bps) and long amplicons (-1200 bps), chlorination could destroy the integrity of ARGs, which likely reduces the possibility of natural transformation. Overall, our findings strongly illustrate that chlorination could be an effective method for inactivating extracellular chromosomal- and plasmid-bome DNA and ARGs.

Salinity-oriented environmental flows for keystone species in the Modaomen Estuary, China

Rapid development and urbanization in recent years have contributed to a reduction in freshwater discharge and intensified saltwater intrusion in the Pearl River Delta. This comprises a significant threat to potable water supplies and overall estuary ecosystem health. In this study, the environmental flows of the Modaomen Estuary, one of the estuaries of the Pear/River Delta in China, were determined based on the salinity demand of keystone species and the linear relationship between river discharge and estuarine salinity. The estimated minimum and optimal annual environmental flows in the Modaomen Estuary were 116.8 ~ I09m3 and 273.8 ~ 109m3, respectively, representing 59.3% and 139.0% of the natural runoff. Water quality assessments in recent years indicate that the environmental flows have not been satisfied most of the time, particularly the optimal environmental flow, despite implementation of various water regulations since 2005. Therefore, water regulations and wetland network recov- eries based on rational environmental flows should be implemented to alleviate saltwater intrusion and for the creation of an ideal estuarine habitat.

Paget's disease of the nipple in males:two case reports and literature review

Male breast cancer acounts for less than 0.5%of all breast cancer,and Paget's disease of the breast in males is extremely rare.Here,we report 2 cases of Paget's disease of the nipple areola complex with invasive ductal carcinoma as typical examples of male PDB.Case 1 was a 64-year old man with an altered appearance of the left nipple,itching and redness,without a palpable breast mass at first.Case 2 was a 55-year-old man with a palpable mass in the left breast and histologically confirmed Paget'sdisease of the nipple with invasive ductal carcinoma.Both patients underwent modified radical mastectomy but with different adjuvant therapies and remained well during follow-up with no recurrence.Furthermore,we reviewed all the sporadic cases of male PDB from the literature.This may help contribute to the development of diagnostic strategies and appropriate interventions for male Paget's disease of the breast.

Control of slope-pattern on the deposition of fan-delta systems:a case study of the Upper Karamay Formation,Junggar Basin

The Mesozoic fan deltas in the north-west margin of the Junggar Basin,as important petroleum reservoirs,exhibited complex facies change and internal structures with strong heterogeneity which were controlled by the transformation of slope-patterns,bringing great challenges to the study of sedimentary characteristics.The Upper Karamay Formation at north-west margin of the Junggar Basin was the objective in this paper which attempts to clarify the mechanism of sedimentary response and sand-body distribution of fan delta systems under the control of slope-pattern change.Based on a data set of cores,well logs and seismic,two types of slope-pattern were identified in the study area,which include steep-to-gentle in the south and gentle-to-steep in the north.The control of difference slope-patterns on the sand-body distribution was clarified based on the analysis of the sedimentary dynamics,facies characteristics,and depositional evolution of the fan deltas.The study shows that the transport mechanism of sediments on the steep-slope was dominated by debris flows,developing coarse-grained,thick-bedded lobes with poor structural maturity of clasts.On the gentle-slope,the deposition was dominated by hyperconcentrated-traction currents,forming relatively fine-grained,thin-bedded lobes with increased sandy matrix.The sand-bodies show frequent bar-channel transformation and channel down-cutting under the steep slope setting,which exhibit migration of isolated river channels on the gentle slopes.Under the steep-to-gentle pattern,the coase-grained sediments were mainly accumulated at slope toe,generally developed equiaxial lobes.However,the coarse-grained clasts were preserved both at proximal and distal lobes on the gentle-to-steep slopes,showing obvious lateral extension of the fan delta.The slope patterns controlled sedimentary respond rates of the fan deltas during lake level change.By comparing the modern cases of fan systems worldwide,the control of slope patterns on deposition of coarse-grained fans was clarified,providing insight into hydrocarbon exploration on basin margins.