RAP80 phase separation at DNA double-strand break promotes BRCA1 recruitment.

RAP80 has been characterized as a component of the BRCA1-A complex and is responsible for the recruitment of BRCA1 to DNA double-strand breaks (DSBs). However, we and others found that the recruitment of RAP80 and BRCA1 were not absolutely temporally synchronized, indicating that other mechanisms, apart from physical interaction, might be implicated. Recently, liquid-liquid phase separation (LLPS) has been characterized as a novel mechanism for the organization of key signaling molecules to drive their particular cellular functions. Here, we characterized that RAP80 LLPS at DSB was required for RAP80-mediated BRCA1 recruitment. Both cellular and in vitro experiments showed that RAP80 phase separated at DSB, which was ascribed to a highly disordered region (IDR) at its N-terminal. Meanwhile, the Lys63-linked poly-ubiquitin chains that quickly formed after DSBs occur, strongly enhanced RAP80 phase separation and were responsible for the induction of RAP80 condensation at the DSB site. Most importantly, abolishing the condensation of RAP80 significantly suppressed the formation of BRCA1 foci, encovering a pivotal role of RAP80 condensates in BRCA1 recruitment and radiosensitivity. Together, our study disclosed a new mechanism underlying RAP80-mediated BRCA1 recruitment, which provided new insight into the role of phase separation in DSB repair.

Phase separation of α-crystallin-GFP protein and its implication in cataract disease.

Cataract, the leading cause of blindness worldwide, is caused by crystallin protein aggregation within the protected lens environment. Phase separation has been implicated as an important mechanism of protein aggregation diseases, such as neurodegeneration. Similarly, cataract has been proposed to be a protein condensation disease in the last century. However, whether crystallin proteins aggregate via a phase separation mechanism and which crystallin protein initiates the aggregation remain unclear. Here, we showed that all types of crystallin-GFP proteins remain soluble under physiological conditions, including protein concentrations, ion strength, and crowding environments. However, in age or disease-induced aberrant conditions, α-crystallin-GFP, including αA- and αB-crystallin-GFP, but not other crystallin-GFP proteins, undergo phase separation in vivo and in vitro. We found that aging-related changes, including higher crystallin concentrations, increased Na+, and decreased K+ concentrations, induced the aggregation of α-crystallin-GFP. Furthermore, H2O2, glucose, and sorbitol, the well-known risk factors for cataract, significantly enhanced the aggregation of αB-crystallin-GFP. Taken together, our results revealed that α-crystallin-GFP forms aggregates via a phase transition process, which may play roles in cataract disease. Opposite to the previously reported function of enhancing the solubility of other crystallin, α-crystallin may be the major aggregated crystallin in the lens of cataract patients.

Screening of key biomarkers of tendinopathy based on bioinformatics and machine learning algorithms.

Tendinopathy is a complex multifaceted tendinopathy often associated with overuse and with its high prevalence resulting in significant health care costs. At present, the pathogenesis and effective treatment of tendinopathy are still not sufficiently elucidated. The purpose of this research is to intensely explore the genes, functional pathways, and immune infiltration characteristics of the occurrence and development of tendinopathy. The gene expression profile of GSE106292, GSE26051 and GSE167226 are downloaded from GEO (NCBI comprehensive gene expression database) and analyzed by WGCNA software bag using R software, GSE26051, GSE167226 data set is combined to screen the differential gene analysis. We subsequently performed gene enrichment analysis of Gene Ontology (GO) and "Kyoto Encyclopedia of Genes and Genomes" (KEGG), and immune cell infiltration analysis. By constructing the LASSO regression model, Support vector machine (SVM-REF) and Gaussian mixture model (GMMs) algorithms are used to screen, to identify early diagnostic genes. We have obtained a total of 171 DEGs through WGCNA analysis and differentially expressed genes (DEGs) screening. By GO and KEGG enrichment analysis, it is found that these dysregulated genes were related to mTOR, HIF-1, MAPK, NF-κB and VEGF signaling pathways. Immune infiltration analysis showed that M1 macrophages, activated mast cells and activated NK cells had infiltration significance. After analysis of THE LASSO SVM-REF and GMMs algorithms, we found that the gene MACROD1 may be a gene for early diagnosis. We identified the potential of tendon disease early diagnosis way and immune gene regulation MACROD1 key infiltration characteristics based on comprehensive bioinformatics analysis. These hub genes and functional pathways may as early biomarkers of tendon injuries and molecular therapy level target is used to guide drug and basic research.

Local environment in biopsy better predict the pathological response to neoadjuvant chemoradiotherapy in rectal cancer.

Neoadjuvant chemoradiotherapy (nCRT) followed by surgery is the standard treatment for locally advanced rectal cancer. Here, we analyzed the impact of local and systemic environments on the tumor response to preoperative chemoradiotherapy in rectal cancer. We recruited 141 patients with rectal cancer treated with nCRT. We evaluated the local tumor environment, including tumor-infiltrating lymphocytes (TILs), intratumor budding (ITB), and the systemic inflammatory environment, including the neutrophil-to-lymphocyte ratio (NLR) and C-reactive protein (CRP) level. Our finding revealed that tumor regression was significantly associated with the density of CD8+ TILs in the intraepithelial, the presence of ITB, the combination of NLR and CRP (NLR-CRP) value, and the combination of CD8+ intraepithelial TIL (iTIL) density and ITB presence. Moreover, multivariate analysis showed that only the combination of CD8+ iTILs and ITB was an independent predictive factor for the pathological response to nCRT in rectal cancer. Our finding demonstrate that the local tumor environment was a better predictor of the tumor response than the systemic environment and thus provided new insight into screening for patients who are more likely to benefit from cancer treatment.

Prognostic and predictive value of interstitial cells of Cajal populations following stapled transanal rectal resection (STARR) in patients with obstructed defecation syndrome.

OBJECTIVE: The present study was designed to evaluate the functional outcome of stapled transanal rectal resection (STARR) and to examine the relationship between the population density of the interstitial cells of Cajal (ICC) and the efficacy of the STARR operation in the management of obstructed defecation syndrome (ODS) patients. METHODS: Full-thickness rectal samples were obtained from 50 ODS patients who underwent STARR. Samples were analysed using ICC immunohistochemistry. Clinical and functional parameters obtained with defecography and anorectal manometry were compared with 20 controls. RESULTS: ICCs were significantly decreased in patients in the submucosal (SM), intramuscular (IM) and myenteric (MY) regions when compared with the control group (P < 0.05). The mean pre-operative Cleveland Constipation Score (CCS) was 24.2 ± 4.1, whilst the CCS at 1, 2, 3, 4 and 5 years post-operatively decreased significantly (P < 0.05). At 3 post-operative years, 58.3% (28/48) of the patients reported a favorable outcome (CCS ≤ 10). On univariate analysis, the functional results were worse in those with pre-operative digitation (P = 0.017), a decreased ICC-MY cell population (P = 0.067), a higher resting anal canal pressure (P = 0.039) and a higher rectal sensory threshold (P = 0.073). Multivariate analysis showed the decreased ICC-MY cell population was an independent predictor for low unfavorable functional outcome (odds ratio = 0.097, 95% confidence interval: 0.012-0.766). CONCLUSIONS: STARR achieved acceptable results at the cost of a slight deterioration over a more prolonged follow-up. Patients with a decreased ICC number in the rectal specimen showed an unfavorable functional outcome where pre-operative histological assessment of a full-thickness rectal sample might predict for the functional outcome following STARR.

Improvement of endothelial dysfunction in atherosclerotic rabbit aortas by ex vivo gene transferring of dimethylarginine dimethylaminohydrolase-2.

BACKGROUND: Elevated endogenous asymmetric dimethylarginine (ADMA) is an independent risk factor for atherosclerosis, and dimethylarginine dimethylaminohydrolase (DDAH) is the key enzyme responsible for the metabolism of ADMA. This study was to determine whether reduced vascular DDAH activity was implicated in endothelial dysfunction of atherosclerosis and whether ex vivo gene transferring of DDAH2 could upregulate vascular DDAH activity and improve endothelial dysfunction associated with atherosclerosis. METHODS: Recombinant adenovirus encoding human DDAH2 gene driven by a cytomegalovirus (CMV) promoter was constructed and used to infect thoracic aortic rings from hyperlipidemic rabbits. Vascular hDDAH2 transcription, DDAH activity and endothelium-dependent relaxation were measured in thoracic aortas of hyperlipidemic and control rabbits. RESULTS: Vascular DDAH activity was distinctly reduced (0.048±0.002 vs 0.095 ± 0.007U/g protein, n=5, P<0.01) in atherosclerotic aortas in accompany with impaired endothelium-dependent relaxation, whereas serum ADMA levels were markedly elevated in hyperlipidemic rabbits (2.24 ± 0.12 vs 1.22 ± 0.12µmol/L, n=5, P<0.01) compared to control rabbits. Ex vivo gene transferring of hDDAH2 to atherosclerotic aortas not only increased the functional expression of hDDAH2 as shown by presenting hDDAH2 mRNA and enhancing DDAH activity (0.112 ± 0.008 U/g protein, n=5) but also markedly improved the impaired endothelium-dependent relaxation in atherosclerotic arteries. CONCLUSIONS: Reduced vascular DDAH activity contributes to endothelial dysfunction in hyperlipidemic rabbits. Ex vivo gene transferring of hDDAH2 can improve the endothelial dysfunction and inhibited DDAH activity, indicating that targeted modulation of DDAH2 gene in vascular endothelium may be a novel approach for treatment of endothelial dysfunction in atherosclerosis.