Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage.

Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR-Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium MA2020 (Lb2) and the Lb2Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2Cas12a assumes a unique, elongated conformation, whereas the Lb2Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2Cas12a is dynamic and can exist in both elongated and compact forms. Residues from Met493 to Leu523 of the WED domain undergo major conformational changes to facilitate the required structural rearrangements. The REC lobe of Lb2Cas12a rotates 103° concomitant with rearrangement of the hinge region close to the WED and RuvC II domains to position the RNA-DNA duplex near the catalytic site. Our findings provide insight into crRNA recognition and the mechanism of target DNA cleavage.

Autophagy-related LncRNA PRDM10-DT responds to UVB radiation in keratinocytes.

Ultraviolet (UV)-B radiation is a major environmental risk factor that is responsible for the development and progression of many skin disorders. Autophagy is the process of degradation and recycling of damaged cytoplasmic organelles, macromolecular aggregates, and long-lived proteins. Previously, we found that the autophagy inducer apigenin restored UVB-impaired autophagy and the cellular response by downregulating the expression of autophagy-related genes such as ATG5. To explore long noncoding RNAs (lncRNAs) involved in regulating these autophagy-related genes, in this study, we assessed the expression profiles of lncRNAs and mRNAs using a microarray in human epidermal keratinocytes (HEKs) treated with or without apigenin after UVB radiation. The expression levels of 80 selected autophagy-related genes and related lncRNAs were confirmed by quantitative real-time polymerase chain reaction (qRT‒PCR). The lncRNA PRDM10-DT was proposed to regulate IRGM based on the ceRNA and coexpression pattern and was demonstrated to be involved in autophagy regulation, proliferation and migration of HEKs by qRT‒PCR, Western blotting, colony formation and scratch wound assays, respectively. These findings suggest an autophagy-related lncRNA in response to UVB radiation that promotes the proliferation and migration of HEKs through inducing autophagy by competing microRNAs for IRGM.

Effect of low-magnitude different-frequency whole-body vibration on subchondral trabecular bone microarchitecture, cartilage degradation, bone/cartilage turnover, and joint pain in rabbits with knee osteoarthritis.

BACKGROUD: Whole-body vibration(WBV) has been suggested for the prevention of subchondral bone loss of knee osteoarthritis (OA) . This study examined the effects of different frequency of whole-body vibration on subchondral trabecular bone microarchitecture, cartilage degradation and metabolism of the tibia and femoral condyle bone, and joint pain in an anterior cruciate ligament transection (ACLT)-induced knee osteoarthritisrabbit model. METHOD: Ninety adult rabbits were divided into six groups: all groups received unilateral ACLT; Group 1, ACLT only; Group 2, 5 Hz WBV; Group 3, 10 Hz WBV; Group 4, 20 Hz WBV; Group 5, 30 Hz WBV; and Group 6, 40 Hz WBV. Pain was tested via weight-bearing asymmetry. Subchondral trabecular bone microarchitecture was examined using in vivo micro-computed tomography. Knee joint cartilage was evaluated by gross morphology, histology, and ECM gene expression level (aggrecan and type II collagen [CTX-II]). Serum bone-specific alkaline phosphatase, N-mid OC, cartilage oligometric protein, CPII, type I collagen, PIIANP, G1/G2 aggrecan levels, and urinary CTX-II were analyzed. RESULTS: After 8 weeks of low-magnitude WBV, the lower frequency (10 Hz and 20 Hz) WBV treatment decreased joint pain and cartilage resorption, accelerated cartilage formation, delayed cartilage degradation especially at the 20 Hz regimen. However, the higher frequencies (30 Hz and 40 Hz) had worse effects, with worse limb function and cartilage volume as well as higher histological scores and cartilage resorption. In contrast, both prevented loss of trabeculae and increased bone turnover. No significant change was observed in the 5 Hz WBV group. CONCLUSION: Our data demonstrate that the lower frequencies (10 Hz and 20 Hz) of low-magnitude WBV increased bone turnover, delayed cartilage degeneration, and caused a significant functional change of the OA-affected limb in ACLT-induced OA rabbit model but did not reverse OA progression after 8 weeks of treatment.

Correlation between slow transit constipation and spleen deficiency, and gut microbiota: a pilot study.

OBJECTIVE: To investigate the effect of slow transit constipation (STC) and spleen deficiency on gut microbiota, and the mechanism underlying the action that the positive drug Maren Runchang (MR) alleviates STC. METHODS: STC was induced, using the cathartic method of Senna and the hunger-fullness disorder method, in ICR mice; one group of model mice was treated with MR (6.24 g/kg). The changes in the general condition, fecal parameters, D-xylose content in the serum, intestinal propulsion rate, and histopathology of the colon were assessed after STC induction in the control, model, and MR groups. Fecal microbiota transplantation (FMT) was performed from STC mice into pseudo germ-free mice. Changes in the contents of substance P (SP), vasoactive intestinal peptide (VIP), and gut microbiota in STC mice and pseudo germ-free mice were assessed after FMT. RESULTS: Compared with the control group, the model mice showed the following results: the time of the first black stool was significantly longer ( 0.01), the number and weight of black stools were significantly reduced within 6 h ( 0.05), the D-xylose content in the serum was significantly reduced ( < 0.05), the intestinal propulsion rate decreased ( < 0.01), the content of VIP in colon tissue significantly increased ( < 0.05), and SP content in the colon tissue significantly decreased ( < 0.01); moreover, the colon showed significant inflame-mation and injury. Furthermore, the abundance of Firmicutes was increased, the abundance of Bacteroides decreased, and the abundance of decreased, while the abundance of the conditional pathogenic bacteria and Klebsiella increased. However, after treatment with MR, the time of the first black stool decreased (0.01), the number of black stools within 6 h increased, and the intestinal propulsion rate increased ( < 0.05). Moreover, the content of D-xylose in the serum and the content of VIP in colon tissue significantly decreased ( < 0.05), the content of SP in colon tissue significantly increased ( < 0.01), and colon inflammation significantly improved. Additionally, the abundance of Firmicutes decreased, and the abundance of Bacteroides increased. The abundance of increased, and the abundance of decreased. In the model + FMT group, compared with control + FMT group, the content of VIP in colon tissue decreased ( < 0.05), the content of SP in colon tissue significantly increased ( < 0.01), and the abundance of probiotics, such as , decreased. In the MR + FMT group, compared with the model + FMT group, the content of VIP in colon tissue increased, the content of SP in colon tissue significantly decreased ( < 0.01), and the abundance of probiotics increased. CONCLUSIONS: STC mice with spleen deficiency show a decreased abundance of beneficial bacteria, such as , and an increased abundance of the conditional pathogenic bacteria . Furthermore, the mechanism of action of MR in treating STC may involve the regulation of intestinal movement, reduction of intestinal inflammation, elevation of intestinal absorption, and regulation of gut microbiota.