Protein liquid-liquid phase separation (LLPS), a pivotal phenomenon intricately linked to cellular processes, is regulated by various other proteins. However, there is still a lack of high-throughput methods for screening protein regulators of LLPS in target proteins. Here, we developed a CRISPR/Cas9-based screening method to identify protein phase separation regulators by integrating bimolecular fluorescence complementation (BiFC) and fluorescence-activated cell sorting (FACS). Using this newly developed method, we screened the RNA-binding proteins that regulate PABPN1 phase separation and identified the tumor suppressor QKI as a promoter of PABPN1 phase separation. Furthermore, QKI exhibits decreased expression levels and diminished nuclear localization in colorectal cancer cells, resulting in reduced PABPN1 phase separation, which, in turn, promotes alternative polyadenylation (APA), cell proliferation, and migration in colorectal cancer.
Humans ; Colorectal Neoplasms/genetics* ; RNA-Binding Proteins/genetics* ; Poly(A)-Binding Protein I/genetics* ; CRISPR-Cas Systems ; Flow Cytometry ; Cell Proliferation ; Cell Line, Tumor ; Cell Movement

Objective:To explore the deubiquitination modification of the deubiquitinase BRCC3 in rats with neuropathic pain (NPP) treated with meridian tuina of Zhuang medicine and its analgesic molecular mechanism.Methods:Rats were divided into normal group, sham-operation group, model group, sham-tuina group, and meridian tuina of Zhuang medicine group using a random number table method, with 9 rats in each group. Except for the normal and sham operation groups, spinal nerve ligation models were prepared in all other groups. On the first day after surgery, intervention was carried out on the meridian tuina of Zhuang medicine and sham-tuina groups for 14 days, while the other three groups were not intervened; the paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) of each group of rats were measured at 0 days before operation, 1, 7, and 14 days after operation. Western blot was used to detect the expressions of BRCC3 and NLRP3 proteins in spinal cord tissue, and ELISA was used to detect the level of IL-1β in serum.Results:On postoperative 7 and 14 d, compared with the model group, the meridian tuina of Zhuang medicine group showed an increase in PWMT and PWTL, a decrease in NLPR3 and BRCC3 expression in spinal cord tissue, and a decrease in serum IL-1β levels ( P<0.05). Compared with the sham-tuina group, the meridian tuina of Zhuang medicine group showed an increase in PWMT and PWTL, a decrease in NLRP3 protein expression in spinal cord tissue, and a decrease in IL-1β levels in serum ( P<0.05). Conclusion:Meridian tuina of Zhuang medicine can alleviate pain sensitivity in SNL model rats, and its mechanism is related to the inhibition of the expression of deubiquitinase BRCC3 by meridian massage of Zhuang medicine, which increases the ubiquitination level of NLRP3 and hinders its activation, thereby blocking the immune inflammatory response mediated by inflammatory factors.

Neuropathic pain(NPP)is a recurrent and intractable pain syndrome,and microglial polarization is closely related to it.Recent studies have shown that after cell polarization,M1 pro-inflammatory and M2 anti-inflammatory types could be formed,and M1 and M2 microglial cells are involved in NPP through the modulation of inflammatory responses.The two types of microglial cells have different roles in NPP,just like a double-edged sword.M1 pro-inflammatory type plays an injurious role in NPP through the release of pro-inflammatory factors and noxious substances,whereas M2 anti-inflammatory type plays a protective role by secreting anti-inflammatory factors.In this article,the role of microglia polarization on NPP are reviewed.

Objective:To explore the characteristics of nail fold microcirculation in hemodialysis patients and its correlation with cardiovascular events.Methods:The clinical data of 185 patients undergoing regular hemodialysis in Dongguan Donghua Hospital from January 2017 to December 2021 were retrospectively analyzed. Among them, there were 76 cases of cardiovascular events (cardiovascular events group) and 109 cases of no cardiovascular events (non-cardiovascular events group). The nail fold microcirculation detector was used to detect the nail fold microcirculation of the first row of capillaries in the nail fold dermal papilla of the left ring finger. The nail fold microcirculation indexes and morphology integral, flow integral, loop integral, total integral were recorded. The general information and laboratory indexes (peripheral venous blood) were recorded. The value of total integral of nail fold microcirculation in predicting the cardiovascular events in hemodialysis patients was evaluated using the receiver operating characteristics (ROC) curve.Results:The age, proportion of hypertension, proportion of diabetes, C-reactive protein (CRP), alkaline phosphatase and N-terminal pro-brain natriuretic peptide (NT-ProBNP) in cardiovascular events group were significantly higher than those in non-cardiovascular events group: 58 (44, 69) years vs. 49 (40, 63) years, 97.4% (74/76) vs. 83.5% (91/109), 43.4% (33/76) vs. 24.8% (27/109), 9.02 (2.73, 11.70) mg/L vs. 3.76 (1.28, 11.70) mg/L, 82 (75, 97) U/L vs. 72 (59, 82) U/L and 2 652 (1 020, 5 359) ng/L vs. 1 894 (780, 4 601) ng/L, the creatinine and triglyceride (TG) were significantly lower than those in non-cardiovascular events group: (961.95 ± 277.11) μmol/L vs. (1 058.93 ± 284.66) μmol/L and (1.73 ± 1.02) mmol/L vs. (2.27 ± 2.02) mmol/L, and there were statistical differences ( P<0.01 or <0.05). There were no statistical differences in gender composition, dialysis age, dialysis time, dialysis pathway, blood routine, serum iron, serum ferritin, total iron binding capacity, serum potassium, serum calcium, serum phosphorus, parathyroid hormone, urea nitrogen, albumin, total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol between two groups ( P>0.05). There was statistical difference in erythrocyte aggregation between two groups ( P<0.05); there were no statistical differences in the number of vascular loops, diameter of input branch, diameter of output branch, length of loop, diameter of loop top, blood flow rate, clarity, condition of cross loop, condition of malformed loop, shape of nipple and subpapillary venous plexus between two groups ( P>0.05). The morphology integral and total integral in cardiovascular events group were significantly higher than those in non-cardiovascular events group: 1.8 (1.1, 3.1) scores vs. 1.4 (0.8, 2.5) scores and 4.2 (2.4, 6.1) scores vs. 3.1 (1.8, 5.2) scores, and there were statistical differences ( P<0.05); there were no statistical differences in flow integral and loop integral between two groups ( P>0.05). ROC curve analysis result showed that the area under the curve of the total integral of nail fold microcirculation for predicting cardiovascular events in hemodialysis patients was 0.590 (95% CI 0.506 to 0.673), the best cut-off value was 2.85 scores, the sensitivity was 69.7% and the specificity was 45.9%. Conclusions:The nail fold microcirculation disorder is more serious in hemodialysis patients with cardiovascular events. The nail fold microcirculation detection may help to predict cardiovascular disease.