Cas1 mediates the interference stage in a phage-encoded CRISPR-Cas system.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are prokaryotic adaptive immune systems against invading phages and other mobile genetic elements. Notably, some phages, including the Vibrio cholerae-infecting ICP1 (International Center for Diarrheal Disease Research, Bangladesh cholera phage 1), harbor CRISPR-Cas systems to counteract host defenses. Nevertheless, ICP1 Cas8f lacks the helical bundle domain essential for recruitment of helicase-nuclease Cas2/3 during target DNA cleavage and how this system accomplishes the interference stage remains unknown. Here, we found that Cas1, a highly conserved component known to exclusively work in the adaptation stage, also mediates the interference stage through connecting Cas2/3 to the DNA-bound CRISPR-associated complex for antiviral defense (Cascade; CRISPR system yersinia, Csy) of the ICP1 CRISPR-Cas system. A series of structures of Csy, Csy-dsDNA (double-stranded DNA), Cas1-Cas2/3 and Csy-dsDNA-Cas1-Cas2/3 complexes reveal the whole process of Cas1-mediated target DNA cleavage by the ICP1 CRISPR-Cas system. Together, these data support an unprecedented model in which Cas1 mediates the interference stage in a phage-encoded CRISPR-Cas system and the study also sheds light on a unique model of primed adaptation.

Liver-specific mitochondrial amidoxime-reducing component 1 (Mtarc1) knockdown protects the liver from diet-induced MASH in multiple mouse models.

BACKGROUND: Human genetic studies have identified several mitochondrial amidoxime-reducing component 1 (MTARC1) variants as protective against metabolic dysfunction-associated steatotic liver disease. The MTARC1 variants are associated with decreased plasma lipids and liver enzymes and reduced liver-related mortality. However, the role of mARC1 in fatty liver disease is still unclear. METHODS: Given that mARC1 is mainly expressed in hepatocytes, we developed an N-acetylgalactosamine-conjugated mouse Mtarc1 siRNA, applying it in multiple in vivo models to investigate the role of mARC1 using multiomic techniques. RESULTS: In ob/ob mice, knockdown of Mtarc1 in mouse hepatocytes resulted in decreased serum liver enzymes, LDL-cholesterol, and liver triglycerides. Reduction of mARC1 also reduced liver weight, improved lipid profiles, and attenuated liver pathological changes in 2 diet-induced metabolic dysfunction-associated steatohepatitis mouse models. A comprehensive analysis of mARC1-deficient liver from a metabolic dysfunction-associated steatohepatitis mouse model by metabolomics, proteomics, and lipidomics showed that Mtarc1 knockdown partially restored metabolites and lipids altered by diet. CONCLUSIONS: Taken together, reducing mARC1 expression in hepatocytes protects against metabolic dysfunction-associated steatohepatitis in multiple murine models, suggesting a potential therapeutic approach for this chronic liver disease.

Suppression of MALAT1 promotes human synovial mesenchymal stem cells enhance chondrogenic differentiation and prevent osteoarthritis of the knee in a rat model via regulating miR-212-5p/MyD88 axis.

Osteoarthritis (OA) is one of the most common diseases of the skeleton. Long non-coding RNAs (lncRNAs) are emerging as key players in OA pathogenesis. This work sets out to determine the function of lncRNA MALAT1 in OA and the mechanisms by which it does so. Mesenchymal stem cells isolated from the human synovial membrane are called hSMSCs. The hSMSCs' surface markers were studied using flow cytometry. To determine whether or not hSMSC might differentiate, researchers used a number of different culture settings and labeling techniques. The expression levels of associated genes and proteins were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), and immunostaining. A dual luciferase reporter experiment and RNA immunoprecipitation (RIP) test demonstrated the direct association between miR-212-5p and MALAT1 or MyD88. MALAT1 was downregulated during the chondrogenic differentiation of hSMSCs, and underexpression of MALAT1 promotes chondrogenesis in hSMSCs. Using dual luciferase reporter and RIP assays facilitated the identification of MALAT1 as a competitive endogenous RNA (ceRNA) that sequesters miR-212-5p. Additionally, the expression of MYD88 was regulated by MALAT1 through direct binding with miR-212-5p. Significantly, the effects of MALAT1 on the chondrogenic differentiation of hSMSCs were counteracted by miR-212-5p/MYD88. Furthermore, our in vivo investigation revealed that the inhibition of MALAT1 mitigated osteoarthritis progression in rat models. In conclusion, the promotion of chondrogenic differentiation in hSMSCs and the protective effect on cartilage tissue in OA can be achieved by suppressing MALAT1, which regulates the miR-212-5p/MyD88 axis.

Bacteriophage strategies for overcoming host antiviral immunity.

Phages and their bacterial hosts together constitute a vast and diverse ecosystem. Facing the infection of phages, prokaryotes have evolved a wide range of antiviral mechanisms, and phages in turn have adopted multiple tactics to circumvent or subvert these mechanisms to survive. An in-depth investigation into the interaction between phages and bacteria not only provides new insight into the ancient coevolutionary conflict between them but also produces precision biotechnological tools based on anti-phage systems. Moreover, a more complete understanding of their interaction is also critical for the phage-based antibacterial measures. Compared to the bacterial antiviral mechanisms, studies into counter-defense strategies adopted by phages have been a little slow, but have also achieved important advances in recent years. In this review, we highlight the numerous intracellular immune systems of bacteria as well as the countermeasures employed by phages, with an emphasis on the bacteriophage strategies in response to host antiviral immunity.

A Cadaveric Limb Analysis of the Posterior Tibial Musculotendinous Junction to determine the feasibility of Interosseous Membrane Tendon Transfer.

The transfer of the posterior tibial tendon through the interosseous membrane is potentially an effective treatment to correct the deformity of the foot and ankle. Our study aimed to evaluate the anatomical feasibility of anterior transfer of the posterior tibial tendon through the interosseous membrane route using the musculotendinous junction (MTJ). Eighteen adult cadavers were used. The width and thickness of the tibial posterior MTJ, width of the interosseous membrane at the corresponding level, and the window size of the interosseous membrane were measured. Additionally, the distance between the distal end of the MTJ and the tip of the medial malleolus were recorded. The mean length of the posterior tibial tendon was 83.60 mm, the mean distance of the posterior tibial MTJ to medial malleolus tip was 45.48 mm and the mean length of MTJ was 31.74 mm. The mean width of distal end of MTJ was 7.76 mm, thickness of distal end of MTJ was 4.07 mm and the mean width of the interosseous membrane at the distal end of MTJ was 4.76 mm. We found the mean width of the proximal end of MTJ was 20.68 mm, the mean thickness of proximal end of MTJ was 5.52 mm, and mean width of interosseous membrane at the proximal end of MTJ was 8.76 mm. Our study has demonstrated that a 31 mm length incision made at approximately 45 mm from the proximal end of the medial malleolus can safely reach the MTJ. We recommend an opening length of the interosseous membrane of at least 20 mm.

Anti-CRISPR protein AcrIF4 inhibits the type I-F CRISPR-Cas surveillance complex by blocking nuclease recruitment and DNA cleavage.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system provides prokaryotes with protection against mobile genetic elements such as phages. In turn, phages deploy anti-CRISPR (Acr) proteins to evade this immunity. AcrIF4, an Acr targeting the type I-F CRISPR-Cas system, has been reported to bind the crRNA-guided surveillance (Csy) complex. However, it remains controversial whether AcrIF4 inhibits target DNA binding to the Csy complex. Here, we present structural and mechanistic studies into AcrIF4, exploring its unique anti-CRISPR mechanism. While the Csy-AcrIF4 complex displays decreased affinity for target DNA, it is still able to bind the DNA. Our structural and functional analyses of the Csy-AcrIF4-dsDNA complex revealed that AcrIF4 binding prevents rotation of the helical bundle of the Cas8f subunit induced by dsDNA binding, therefore resulting in failure of nuclease Cas2/3 recruitment and DNA cleavage. Overall, our study provides an interesting example of attack on the nuclease recruitment event by an Acr, but not conventional mechanisms of blocking binding of target DNA.

Target RNA activates the protease activity of Craspase to confer antiviral defense.

In the type III-E CRISPR-Cas system, a Cas effector (gRAMP) is associated with a TPR-CHAT to form Craspase (CRISPR-guided caspase). However, both the structural features of gRAMP and the immunity mechanism remain unknown for this system. Here, we report structures of gRAMP-crRNA and gRAMP:cRNA:target RNA as well as structures of Craspase and Craspase complexed with cognate target RNA (CTR) or non-cognate target RNA (NTR). Importantly, the 3' anti-tag region of NTR and CTR binds at two distinct channels in Craspase, and CTR with a non-complementary 3' anti-tag induces a marked conformational change of the TPR-CHAT, which allosterically activates its protease activity to cleave an ancillary protein Csx30. This cleavage then triggers an abortive infection as the antiviral strategy of the type III-E system. Together, our study provides crucial insights into both the catalytic mechanism of the gRAMP and the immunity mechanism of the type III-E system.

Can noninvasive Brain Stimulation Improve Pain and Depressive Symptoms in Patients With Neuropathic Pain? A Systematic Review and Meta-Analysis.

CONTEXT: Noninvasive brain stimulations (NIBS) have been increasingly applied to the patients with neuropathic pain (NP), while the effectiveness of NIBS in the management of NP is still conflicting. OBJECTIVES: To examine the effectiveness of NIBS on pain and depression symptoms of patients with NP. METHODS: A comprehensive literature retrieval was performed on MEDLINE, Embase, PsycINFO, PEDro, and CENTRAL from the establishment of the databases to June 2021. Randomized controlled trials comparing NIBS with sham stimulation were included. RESULTS: A total of thirteen trials comprising 498 participants met the inclusion criteria. The pooled analysis found a significant effect on the improvement of pain scores at post-treatment, favoring NIBS over sham stimulation (SMD = -0.60; 95% CI: -1.00 to -0.20; P = 0.004). Subgroup analysis showed that only transcranial direct current stimulation (tDCS) (SMD = -0.38; 95% CI: -0.71 to -0.04; P = 0.030) and high-frequency repetitive transcranial magnetic stimulation (H-rTMS) (SMD = -0.95; 95% CI: -1.85 to -0.04; P = 0.040) had positive effects on pain reduction among all types of NIBS. The favorable effects of NIBS remained significant at follow-up visit (SMD = -0.51; 95% CI: -0.79 to -0.23; P = 0.000), while only H-rTMS was found in subgroup analyses to significantly improve pain scales of the patients (SMD = -0.54; 95% CI: -0.85 to -0.24; P = 0.000). Additionally, overall NIBS showed no beneficial effect over sham stimulation in reducing depression symptoms of NP patients either at post-treatment (SMD = -0.19; 95% CI: -0.39 to 0.01; P = 0.061) or at follow-up visit (SMD = -0.18; 95% CI: -0.45 to 0.10; P = 0.202). CONCLUSION: This meta-analysis revealed the analgesic effect of NIBS on patients with NP, while no beneficial effect was observed on reducing concomitant depression symptoms. The findings recommended the clinical application of NIBS in patients with NP.

AcrIF5 specifically targets DNA-bound CRISPR-Cas surveillance complex for inhibition.

CRISPR-Cas systems are prokaryotic antiviral systems, and phages use anti-CRISPR proteins (Acrs) to inactivate these systems. Here we present structural and functional analyses of AcrIF5, exploring its unique anti-CRISPR mechanism. AcrIF5 shows binding specificity only for the target DNA-bound form of the crRNA-guided surveillance (Csy) complex, but not the apo Csy complex from the type I-F CRISPR-Cas system. We solved the structure of the Csy-dsDNA-AcrIF5 complex, revealing that the conformational changes of the Csy complex caused by dsDNA binding dictate the binding specificity for the Csy-dsDNA complex by AcrIF5. Mechanistically, five AcrIF5 molecules bind one Csy-dsDNA complex, which destabilizes the helical bundle domain of Cas8f, thus preventing subsequent Cas2/3 recruitment. AcrIF5 exists in symbiosis with AcrIF3, which blocks Cas2/3 recruitment. This attack on the recruitment event stands in contrast to the conventional mechanisms of blocking binding of target DNA. Overall, our study reveals an unprecedented mechanism of CRISPR-Cas inhibition by AcrIF5.

Location of motor branches of tibialis posterior muscle and its relation in treatment of spastic equinovarus foot: a cadaveric study.

BACKGROUND AND OBJECTIVES: Nerve block or neurolysis is an important approach in the treatment of spastic equinovarus foot. To illustrate the accurate location of the nerve branch to the tibialis posterior muscle (TP) in clinical practice, 21 adult cadavers were dissected and 14 complete both lower limb specimens were obtained. A total of 28 lower limbs were included. METHODS: We measured the length of the motor branch nerve (LM) of the tibialis posterior muscle, the length of the fibula (LF), the vertical distance (D1) from the midpoint of LM to the fibula tip as well as the horizontal distance (D2) from the midpoint of LM to the inner edge of the fibula. RESULTS: The LM was higher (35.74 ± 7.28 mm) in male than in female (30.40 ± 6.88 mm) specimens but there was no significant correlation between LM and gender (p > 0.05). Additionally, among male specimens, the LM on the right side was longer than that on the left (p ≤ 0.05) while among female specimens, the D1 on the left side was longer than that on the right (p ≤ 0.05). The LF in male specimen was significantly longer than that in female (p ≤ 0.05). The midpoint of the nerve to the motor branch of the tibialis posterior muscle was about 50 mm distal to the fibular head and 10 mm at the inner edge of the fibula. CONCLUSION: Using this coordinate, the midpoint of the nerve branch to the TP could be accurately located.

Antibody-mediated inhibition of GDF15-GFRAL activity reverses cancer cachexia in mice.

Cancer cachexia is a highly prevalent condition associated with poor quality of life and reduced survival1. Tumor-induced perturbations in the endocrine, immune and nervous systems drive anorexia and catabolic changes in adipose tissue and skeletal muscle, hallmarks of cancer cachexia2-4. However, the molecular mechanisms driving cachexia remain poorly defined, and there are currently no approved drugs for the condition. Elevation in circulating growth differentiation factor 15 (GDF15) correlates with cachexia and reduced survival in patients with cancer5-8, and a GDNF family receptor alpha like (GFRAL)-Ret proto-oncogene (RET) signaling complex in brainstem neurons that mediates GDF15-induced weight loss in mice has recently been described9-12. Here we report a therapeutic antagonistic monoclonal antibody, 3P10, that targets GFRAL and inhibits RET signaling by preventing the GDF15-driven interaction of RET with GFRAL on the cell surface. Treatment with 3P10 reverses excessive lipid oxidation in tumor-bearing mice and prevents cancer cachexia, even under calorie-restricted conditions. Mechanistically, activation of the GFRAL-RET pathway induces expression of genes involved in lipid metabolism in adipose tissues, and both peripheral chemical sympathectomy and loss of adipose triglyceride lipase protect mice from GDF15-induced weight loss. These data uncover a peripheral sympathetic axis by which GDF15 elicits a lipolytic response in adipose tissue independently of anorexia, leading to reduced adipose and muscle mass and function in tumor-bearing mice.

Posterior Tibial Tendinopathy and Osteopenia as Primary Symptoms of Celiac Disease: A Case Report.

This case report describes posterior tibial tendon (PTT) tendinopathy, valgus deformity with tenosynovitis, and osteopenia at the medial malleolus as the primary symptoms of a young patient with celiac disease (CD) without gastrointestinal symptoms. CD is an autoimmune condition that is a chronic inflammatory disorder of the small intestine triggered by ingestion of gluten in individuals with a particular genetic background. Without typical gastrointestinal symptoms, CD patients are often misdiagnosed or undiagnosed. The patient was diagnosed with CD by duodenal biopsy. He underwent a surgical procedure, including medial displacement calcaneal osteotomy, tenosynovectomy of the PTT and flexor digitorum longus (FDL), FDL transfer to the navicular for a pes planovalgus deformity, and drilling of the medial malleolus for a stress reaction. The mechanism of the PTT tear and associated heel valgus deformity was assumed to be related to the fact that his heel alignment on the affected side changed gradually from normal to valgus and pes planus owing to CD and mechanical stress, because his normal-side heel alignment was neutral before surgery and at final follow-up. His operated ankle was pain-free, with full range of motion, 1.5 years after surgery. The patient was able to restart running and exercise gradually. Foot and ankle specialists should consider the possibility of CD in patients presenting with a PTT tear without injury or trauma and osteopenia with no obvious reason.

The self-reduction synthesis and luminescent properties of Eu2+ /Eu3+ activated BaZrx Si3 O7+2x phosphors with white light emission for white light-emitting diodes.

The BaZrx Si3 O7+2x :Eu2+ /Eu3+ (2%, molar ratio) phosphors were prepared with adding different amounts of zirconium dioxide (ZrO2 ) (x = 0, 0.2, 0.4, 0.6, 0.8, and 1) by a self-reduction method under air conditions. X-ray diffraction, photoluminescence properties, thermal quenching properties and luminescent lifetimes of the prepared samples were systematically analyzed. In BaZrx Si3 O7+2x :Eu2+ /Eu3+ , both the 4f â†’ 5d transitions of Eu2+ and sharp emissions assigned to 5 D0 → 7 FJ (J = 0, 1, 2, 3, 4) transitions of Eu3+ were observed. In addition, the results also reveal that the amount of ZrO2 affects the crystal structure, the self-reduction process and further influences the relative intensity of Eu2+ /Eu3+ in all samples. Therefore, the diverse emission colors can be obtained and modulated by controlling the content of ZrO2 . The highlight of this work is that the white light emission is observed, when x is 0.4, 0.6, 0.8 and 1, respectively.

Anatomical variations in the first extensor compartment: a cadaver study.

BACKGROUND: Anatomical variations in the first extensor compartment are associated with the pathogenesis of de Quervain's disease. Here, we report two novel anatomical variations of the first extensor compartment. METHODS: The wrists of two adult cadavers were dissected to reveal the anatomical variations in the first extensor compartment. RESULTS: In one of the cadavers, no septum was present in the first extensor compartment. However, the extensor pollicis brevis tendon and its proximal muscle belly were absent. The abductor pollicis longus (APL) tendon had multiple slips, and one of them inserted on the dorsal base of the proximal thumb phalanx. In another cadaver, a septum was present in the first extensor compartment. One of the multiple APL tendon slips ran into the septum alongside the extensor pollicis brevis tendon for 4 mm, which then exited the septum and inserted into the base of the first phalanx together with the APL tendon. CONCLUSION: Our findings may help to improve the awareness of the anatomical variations in the first extensor compartment.

COL5A1 may contribute the metastasis of lung adenocarcinoma.

BACKGROUND: Lung cancer leads to the largest number of cancer-related deaths worldwide and is usually accompanied with metastasis which is the primary cause of those death and correlated with poor prognosis. However, the mechanism of lung cancer metastasis is still lack of definition. METHODS: We compared the primary lung adenocarcinoma (AD) and its metastasis tissues induced by overexpression of KrasG12D and inactivation of P53 in mouse lungs by analyzing GSE40222 about the differentially expressed genes (DEGs), pathways and hub genes. And human lung AD databases are used to verify the conversed changes of identified key gene and then followed functional studies are performed to explore the functions of key gene. RESULTS: We identified 165 genes differentially expressed in lung AD metastasis compared to primary AD. Pathway analysis identified 649 GO biological processes and 8 KEGG pathways, such as ECM-receptor interaction. Biological network interaction identified the hub genes during lung adenocarcinoma metastasis, such as the up-regulated COL5A1, a novel gene in AD metastasis. We found it's also increased in human AD and advanced stage. Knockdown of COL5A1 in human AD metastatic cells inhibited cell growth and invasion, and induced cell apoptosis. Notably, higher expression of COL5A1 was observed in the lung AD patients with recurrence and short survive. CONCLUSION: By analyzing mouse lung AD and its metastases, we identified the potential key genes and pathways regulating lung AD metastasis, such as COL5A1. The following analysis of COL5A1 in human AD database and cells explores its functions, holding the implications of target therapy in AD metastasis and also providing more clues for future studies.

Micrometer-Sized Titanium Particles Induce Aseptic Loosening in Rabbit Knee.

Wear debris induced aseptic loosening is the leading cause of total knee arthroplasty (TKA) failure. The complex mechanism of aseptic loosening has been a major issue for introducing effective prevention and treatment methods, so a simplified yet efficient rabbit model was established to address this concern with the use of micrometer-sized titanium particles. 20 New Zealand white rabbits were selected and divided into two groups (control = 10, study = 10). A TKA surgery was then performed for each of them, with implantation of a titanium rod prosthesis which was coated evenly with micrometer-sized titanium in the study group and nothing in the control group, into right femoral medullary cavity. After 12 weeks, all the animals were euthanized and X-ray analyses, H&E staining, Goldner Masson trichrome staining, Von Kossa staining, PCR, and Western blotting of some specific mRNAs and proteins in the interface membrane tissues around the prosthesis were carried out. The implantation of a titanium rod prosthesis coated with 20 µm titanium particles into the femoral medullary cavity of rabbits caused continuous titanium particle stimulation around the prosthesis, effectively inducing osteolysis and aseptic loosening. Titanium particle-induced macrophages produce multiple inflammatory factors able to activate osteoclast differentiation through the OPG/RANKL/RANK signaling pathway, resulting in osteolysis while suppressing the function of osteoblasts and reducing bone ingrowth around the prosthesis. This model simulated the implantation and loosening process of an artificial prosthesis, which is an ideal etiological model to study the aseptic prosthetic loosening.

Autophagy Strengthens Intestinal Mucosal Barrier by Attenuating Oxidative Stress in Severe Acute Pancreatitis.

BACKGROUND: Intestinal mucosal barrier dysfunction can be caused by severe acute pancreatitis (SAP). It is normally associated with changes to mucosal autophagy and oxidative stress. OBJECTIVE: The aim of this study was to investigate the correlation between autophagy and oxidative stress on the intestinal mucosal barrier of SAP rat model. METHODS: SAP was induced by retrograde injection of sodium taurocholate (5%) into the biliopancreatic duct. Bacterial translocation (BT) was detected by 16S rDNA sequencing analysis. Morphological alterations in the pancreas and gut were determined by hematoxylin-eosin staining. Oxidative stress status was determined by measuring the level of intestinal malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx). Western blot, RT-PCR, and immunofluorescent staining were preformed to analyze the expression of tight junction and autophagy proteins. RESULTS: According to the sequencing analysis, rats in SAP group were divided into BT (+) group (n = 9) and BT (-) group (n = 8). Pancreatic and intestinal injuries in SAP group were significantly higher than sham operation group. The content of MDA was clearly elevated, and SOD as well as GPx activities were decreased in BT (+) group as compared with BT (-) group. The expression of LC3II and Beclin1 in BT (-) group was higher than that observed in BT (+). In contrast, BT (+) group had a higher level of claudin-2 and a lower level of zonula occluden-1, occludin, and claudin-1. CONCLUSION: These results suggest that activated autophagy may attenuate intestinal mucosal barrier dysfunction by preventing and reducing the oxidative stress in SAP.

Erratum: Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15.

This corrects the article DOI: 10.1038/nature24042.

A novel classification of the anatomical variations of the first extensor compartment.

The presence of a septum in the first extensor compartment is closely associated with the pathophysiology of de Quervain disease, and affects the efficacy of corticosteroid injection and surgical release. This study aimed to examine the incidence and length of the first extensor compartment septum.Forty sides of the wrists in 20 cadavers were used. The presence of a septum in the first extensor compartment was examined. The septum length was recorded with the radial styloid process as the reference point.The anatomical variations of the first extensor compartment were classified into 3 types. Type I compartment was found in 7 sides in males (29.2%) versus 6 sides in females (37.5%, P = .733), type II was found in 6 sides in males (25%) versus 1 side in females (6.25%, P = .21), and type III was found in 11 sides in males (45.8%) versus 9 sides in females (56.25%, P = .56). There was no significant difference in the septum length between males and females (5.3 ±â€Š2.3 vs 4.8 ±â€Š1.1 mm, P = .54).The incidence of a septum in the first extensor compartment is approximately 50%. The mean septum length is 5 mm. Injection at 5 mm proximal to the radial styloid process has a great chance of delivering the steroids into both subcompartments. Exposure to 5 mm proximal to the radial styloid process can avoid the overlook of subcompartment and achieve adequate decompression of the first extensor compartment.