Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways.

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

Comparison of 3 Different Surgical Techniques for Rotator Cuff Repair in a Rabbit Model: Direct Suture, Inlay Suture, and Polyether Ether Ketone (PEEK) Suture Anchor.

BACKGROUND: Rotator cuff tears have been repaired using the transosseous method for decades. The direct suture (DS) technique has been widely used for rotator cuff tears; however, the retear rate is relatively high. Suture anchors are now used frequently for rotator cuff repair (RCR) in accordance with recent developments in materials. However, polyether ether ketone (PEEK) may still cause complications such as the formation of cysts and osteophytes. Some studies have developed the inlay suture (IS) technique for RCR. PURPOSE/HYPOTHESIS: To compare how 3 different surgical techniques-namely, the DS, IS, and PEEK suture anchor (PSA)-affect tendon-bone healing after RCR. We hypothesized that the IS technique would lead to better tendon-to-bone healing and that the repaired structure would be similar to the normal enthesis. STUDY DESIGN: Controlled laboratory study. METHODS: Acute infraspinatus tendon tears were created in 36 six-month-old male rabbits, which were divided into 3 groups based on the technique used for RCR: DS, IS, and PSA. Animals were euthanized at 6 and 12 weeks postoperatively and underwent a histological assessment and imaging. The expression of related proteins was demonstrated by immunohistochemistry and immunofluorescence staining. Mechanical properties were evaluated by biomechanical testing. RESULTS: At 12 weeks, regeneration of the enthesis was observed in the 3 groups. However, the DS group showed a lower type I collagen content than the PSA and IS groups, which was similar to the results for scleraxis. The DS group displayed a significantly inferior type II collagen expression and proteoglycan deposition after safranin O/fast green and sirius red staining. With regard to runt-related transcription factor 2 and alkaline phosphatase, the IS group showed upregulated expression levels compared with the other 2 groups. CONCLUSION: Compared with the DS technique, the PSA and IS techniques contributed to the improved maturation of tendons and fibrocartilage regeneration, while the IS technique particularly promoted osteogenesis at the enthesis. CLINICAL RELEVANCE: The IS and PSA techniques may be more beneficial for tendon-bone healing after RCR.

Environmental-related doses of afidopyropen induced toxicity effects in earthworms (Eisenia fetida).

Afidopyropen has high activity against pests. However, it poses potential risks to the soil ecology after entering the environment. The toxicity of afidopyropen to earthworms (Eisenia fetida) was studied for the first time in this study. The results showed that afidopyropen had low level of acute toxicity to E. fetida. Under the stimulation of chronic toxicity, the increase of reactive oxygen species (ROS) level activated the antioxidant and detoxification system, which led to the increase of superoxide dismutase (SOD) and glutathione S-transferase (GST) activities. Lipid peroxidation and DNA damage were characterized by the increase of malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) contents. Meanwhile, the functional genes SOD, CAT, GST, heat shock protein 70 (HSP70), transcriptionally controlled tumor protein (TCTP), and annetocin (ANN) played a synergistic role in antioxidant defense. However, the comprehensive toxicity of high concentration still increased on the 28th day. In addition, strong histopathological damage in the body wall and intestine was observed, accompanied by weight loss, which indicated that afidopyropen inhibited the growth of E. fetida. The molecular docking revealed that afidopyrene combined with the surface structure of SOD and GST proteins, which made SOD and GST become sensitive biomarkers reflecting the toxicity of afidopyropen to E. fetida. Summing up, afidopyropen destroys the homeostasis of E. fetida through chronic toxic. These results provide theoretical data for evaluating the environmental risk of afidopyropen to soil ecosystem.

Blockage of mechanosensitive Piezo1 channel alleviates the severity of experimental malaria-associated acute lung injury.

BACKGROUND: Malaria-associated acute lung injury (MA-ALI) is a well-recognized clinical complication of severe, complicated malaria that is partly driven by sequestrations of infected red blood cells (iRBCs) on lung postcapillary induced impaired blood flow. In earlier studies the mechanosensitive Piezo1 channel emerged as a regulator of mechanical stimuli, but the function and underlying mechanism of Piezo1 impacting MA-ALI severity via sensing the impaired pulmonary blood flow are still not fully elucidated. Thus, the present study aimed to explore the role of Piezo1 in the severity of murine MA-ALI. METHODS: Here, we utilized a widely accepted murine model of MA-ALI using C57BL/6 mice with Plasmodium berghei ANKA infection and then added a Piezo1 inhibitor (GsMTx4) to the model. The iRBC-stimulated Raw264.7 macrophages in vitro were also targeted with GsMTx4 to further explore the potential mechanism. RESULTS: Our data showed an elevation in the expression of Piezo1 and number of Piezo1+-CD68+ macrophages in lung tissues of the experimental MA-ALI mice. Compared to the infected control mice, the blockage of Piezo1 with GsMTx4 dramatically improved the survival rate but decreased body weight loss, peripheral blood parasitemia/lung parasite burden, experimental cerebral malaria incidence, total protein concentrations in bronchoalveolar lavage fluid, lung wet/dry weight ratio, vascular leakage, pathological damage, apoptosis and number of CD68+ and CD86+ macrophages in lung tissues. This was accompanied by a dramatic increase in the number of CD206+ macrophages (M2-like subtype), upregulation of anti-inflammatory cytokines (e.g. IL-4 and IL-10) and downregulation of pro-inflammatory cytokines (e.g. TNF-α and IL-1ß). In addition, GsMTx4 treatment remarkably decreased pulmonary intracellular iron accumulation, protein level of 4-HNE (an activator of ferroptosis) and the number of CD68+-Piezo1+ and CD68+-4-HNE+ macrophages but significantly increased protein levels of GPX4 (an inhibitor of ferroptosis) in experimental MA-ALI mice. Similarly, in vitro study showed that the administration of GsMTx4 led to a remarkable elevation in the mRNA levels of CD206, IL-4, IL-10 and GPX-4 but to a substantial decline in CD86, TNF-α, IL-1ß and 4-HNE in the iRBC-stimulated Raw264.7 cells. CONCLUSIONS: Our findings indicated that blockage of Piezo1 with GsMTx4 alleviated the severity of experimental MA-ALI in mice partly by triggering pulmonary macrophage M2 polarization and subsequent anti-inflammatory responses but inhibited apoptosis and ferroptosis in lung tissue. Our data suggested that targeting Piezo1 in macrophages could be a promising therapeutic strategy for treating MA-ALI.

Single-cell multi-omics in the study of digestive system cancers.

Digestive system cancers are prevalent diseases with a high mortality rate, posing a significant threat to public health and economic burden. The diagnosis and treatment of digestive system cancer confront conventional cancer problems, such as tumor heterogeneity and drug resistance. Single-cell sequencing (SCS) emerged at times required and has developed from single-cell RNA-seq (scRNA-seq) to the single-cell multi-omics era represented by single-cell spatial transcriptomics (ST). This article comprehensively reviews the advances of single-cell omics technology in the study of digestive system tumors. While analyzing and summarizing the research cases, vital details on the sequencing platform, sample information, sampling method, and key findings are provided. Meanwhile, we summarize the commonly used SCS platforms and their features, as well as the advantages of multi-omics technologies in combination. Finally, the development trends and prospects of the application of single-cell multi-omics technology in digestive system cancer research are prospected.

Ultrasound-Controlled Delivery of Growth Factor-Loaded Cerasomes Combined with Polycaprolactone Scaffolds Seeded with Bone Marrow Mesenchymal Stem Cells for Biomimetic Tendon-to-Bone Interface Engineering.

Rotator cuff tear (RCT) is a prevalent shoulder injury that poses challenges for achieving continuous and functional regeneration of the tendon-to-bone interface (TBI). In this study, we controlled the delivery of growth factors (GFs) from liposomal nanohybrid cerasomes by ultrasound and implanted three-dimensional printed polycaprolactone (PCL) scaffolds modified with polydopamine loaded with bone marrow mesenchymal stem cells (BMSCs) to repair tears of the infraspinatus tendon in a lapine model. Direct suturing (control, CTL) was used as a control. The PCL/BMSC/cerasome (PBC) devices are sutured with the enthesis of the infraspinatus tendon. The cerasomes and PCL scaffolds are highly stable with excellent biocompatibility. The roles of GFs BMP2, TGFß1, and FGF2 in tissue-specific differentiation are validated. Compared with the CTL group, the PBC group had significantly greater proteoglycan deposition (P = 0.0218), collagen volume fraction (P = 0.0078), and proportions of collagen I (P = 0.0085) and collagen III (P = 0.0048). Biotin-labeled in situ hybridization revealed a high rate of survival for transplanted BMSCs. Collagen type co-staining at the TBI is consistent with multiple collagen regeneration. Our studies demonstrate the validity of biomimetic scaffolds of TBI with BMSC-seeded PCL scaffolds and GF-loaded cerasomes to enhance the treatment outcomes for RCTs.

Anthracyclines inhibit SARS-CoV-2 infection.

Vaccines and drugs are two effective medical interventions to mitigate SARS-CoV-2 infection. Three SARS-CoV-2 inhibitors, remdesivir, paxlovid, and molnupiravir, have been approved for treating COVID-19 patients, but more are needed, because each drug has its limitation of usage and SARS-CoV-2 constantly develops drug resistance mutations. In addition, SARS-CoV-2 drugs have the potential to be repurposed to inhibit new human coronaviruses, thus help to prepare for future coronavirus outbreaks. We have screened a library of microbial metabolites to discover new SARS-CoV-2 inhibitors. To facilitate this screening effort, we generated a recombinant SARS-CoV-2 Delta variant carrying the nano luciferase as a reporter for measuring viral infection. Six compounds were found to inhibit SARS-CoV-2 at the half maximal inhibitory concentration (IC50) below 1 µM, including the anthracycline drug aclarubicin that markedly reduced viral RNA-dependent RNA polymerase (RdRp)-mediated gene expression, whereas other anthracyclines inhibited SARS-CoV-2 by activating the expression of interferon and antiviral genes. As the most commonly prescribed anti-cancer drugs, anthracyclines hold the promise of becoming new SARS-CoV-2 inhibitors.

Malaria-derived exosomes exacerbate liver injury during blood stage of Plasmodium berghei infection.

Liver injury is a common clinical feature of Plasmodium spp. infection and contributes to multi-organ failure of severe malaria. Malaria-derived exosomes (MD-Exos) have recently engaged as key mediators in parasite-host interactions, modulating the subsequent pathogenic process. However, the role of MD-Exos in malaria-related liver injury and the underlying mechanisms remain unclear. Herein, exosomes from C57BL/6 mice infected with or without P. berghei ANKA serum (namely inf-Exos or un-Exos) were isolated and characterized by transmission electron microscopy, western blotting, and nanoparticle tracking analysis. The miRNAs profiling between inf-Exos and un-Exos were generated using RNA-seq and qPCR. The functions of inf-Exos on liver injury were investigated after two types of exosomes injected into mice intravenously (i.v.), by examining histopathological and apoptotic changes, macrophage polarization, and pro-inflammatory response. The infected red blood cells-stimulated mouse Raw264.7 macrophage cells targeted by inf-Exos or un-Exos were cultured for further study and verification the potential mechanisms. We found that both inf-Exos and un-Exos displayed a typical cup-shaped structure with a diameter of 60-200 nm, and had a positive expression of exosomal markers (e.g., CD9, CD63, and CD81). Compared with infected control mice, the treatment of inf-Exos but not un-Exos dramatically enhanced peripheral blood parasitemia and ECM incidence, exacerbated liver histopathological damage, elevated numbers of liver apoptotic cells, CD68+and CD86+ macrophages. The CD68+-TREM-1+ macrophages in liver tissues and the mRNA levels of pro-inflammatory cytokines (e.g., iNOS, TNF-α, IL-1ß, and IL-6) were increased by inf-Exos treatment in vivo. Meanwhile, the treatment of inf-Exos resulted in a substantial increase of the mRNA levels of CD86, iNOS, TNF-α, IL-1ß, and IL-6, but led to a remarkable decrease of Bcl-6 and SOCS-1 in Raw264.7 cells stimulated with iRBC in vitro. Notably, compared to un-Exos, five types of miRNAs (including miR-10a-5p, miR-10b-5p, miR-155-5p, miR-205-5p, and miR-21a-5p), that were previously reported to target Bcl-6 or SOCS-1, present higher abundance on inf-Exos, as demonstrated by RNA-seq and qPCR. Collectively, our data suggest that inf-Exos exacerbate malaria-induced liver pathology via triggering excessive pro-inflammatory response and promoting macrophage M1 polarization. Our findings will provide new insights into the roles of inf-Exos in malaria parasite-host interaction and pathogenesis of liver injury.

A newly identified interaction between nucleolar NPM1/B23 and the HTLV-I basic leucine zipper factor in HTLV-1 infected cells.

Human T-cell leukemia virus type 1 is the causative agent of HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T-cell leukemia-lymphoma (ATL). The HTLV-1 basic leucine zipper factor (HBZ) has been associated to the cancer-inducing properties of this virus, although the exact mechanism is unknown. In this study, we identified nucleophosmin (NPM1/B23) as a new interaction partner of HBZ. We show that sHBZ and the less abundant uHBZ isoform interact with nucleolar NPM1/B23 in infected cells and HTLV-1 positive patient cells, unlike equivalent antisense proteins of related non-leukemogenic HTLV-2, -3 and-4 viruses. We further demonstrate that sHBZ association to NPM1/B23 is sensitive to RNase. Interestingly, sHBZ was shown to interact with its own RNA. Through siRNA and overexpression experiments, we further provide evidence that NPM1/B23 acts negatively on viral gene expression with potential impact on cell transformation. Our results hence provide a new insight over HBZ-binding partners in relation to cellular localization and potential function on cell proliferation and should lead to a better understanding of the link between HBZ and ATL development.

Schlafen 5 suppresses human immunodeficiency virus type 1 transcription by commandeering cellular epigenetic machinery.

Schlafen-5 (SLFN5) is an interferon-induced protein of the Schlafen family, which are involved in immune responses and oncogenesis. To date, little is known regarding its anti-HIV-1 function. Here, the authors report that overexpression of SLFN5 inhibits HIV-1 replication and reduces viral mRNA levels, whereas depletion of endogenous SLFN5 promotes HIV-1 replication. Moreover, they show that SLFN5 markedly decreases the transcriptional activity of HIV-1 long terminal repeat (LTR) via binding to two sequences in the U5-R region, which consequently represses the recruitment of RNA polymerase II to the transcription initiation site. Mutagenesis studies show the importance of nuclear localization and the N-terminal 1-570 amino acids fragment in the inhibition of HIV-1. Further mechanistic studies demonstrate that SLFN5 interacts with components of the PRC2 complex, G9a and Histone H3, thereby promoting H3K27me2 and H3K27me3 modification leading to silencing HIV-1 transcription. In concert with this, they find that SLFN5 blocks the activation of latent HIV-1. Altogether, their findings demonstrate that SLFN5 is a transcriptional repressor of HIV-1 through epigenetic modulation and a potential determinant of HIV-1 latency.

5-Iodotubercidin inhibits SARS-CoV-2 RNA synthesis.

Coronavirus disease 2019 (COVID-19) is a newly emerged infectious disease caused by a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid global emergence of SARS-CoV-2 highlights the importance and urgency for potential drugs to control the pandemic. The functional importance of RNA-dependent RNA polymerase (RdRp) in the viral life cycle, combined with structural conservation and absence of closely related homologs in humans, makes it an attractive target for designing antiviral drugs. Nucleos(t)ide analogs (NAs) are still the most promising broad-spectrum class of viral RdRp inhibitors. In this study, using our previously developed cell-based SARS-CoV-2 RdRp report system, we screened 134 compounds in the Selleckchemicals NAs library. Four candidate compounds, Fludarabine Phosphate, Fludarabine, 6-Thio-20-Deoxyguanosine (6-Thio-dG), and 5-Iodotubercidin, exhibit remarkable potency in inhibiting SARS-CoV-2 RdRp. Among these four compounds, 5-Iodotubercidin exhibited the strongest inhibition upon SARS-CoV-2 RdRp, and was resistant to viral exoribonuclease activity, thus presenting the best antiviral activity against coronavirus from a different genus. Further study showed that the RdRp inhibitory activity of 5-Iodotubercidin is closely related to its capacity to inhibit adenosine kinase (ADK).

A cell-based assay to discover inhibitors of SARS-CoV-2 RNA dependent RNA polymerase.

Antiviral therapeutics is one effective avenue to control and end this devastating COVID-19 pandemic. The viral RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 has been recognized as a valuable target of antivirals. However, the cell-free SARS-CoV-2 RdRp biochemical assay requires the conversion of nucleotide prodrugs into the active triphosphate forms, which regularly occurs in cells yet is a complicated multiple-step chemical process in vitro, and thus hinders the utility of this cell-free assay in the rapid discovery of RdRp inhibitors. In addition, SARS-CoV-2 exoribonuclease provides the proof-reading capacity to viral RdRp, thus creates relatively high resistance threshold of viral RdRp to nucleotide analog inhibitors, which must be examined and evaluated in the development of this class of antivirals. Here, we report a cell-based assay to evaluate the efficacy of nucleotide analog compounds against SARS-CoV-2 RdRp and assess their tolerance to viral exoribonuclease-mediated proof-reading. By testing seven commonly used nucleotide analog viral polymerase inhibitors, Remdesivir, Molnupiravir, Ribavirin, Favipiravir, Penciclovir, Entecavir and Tenofovir, we found that both Molnupiravir and Remdesivir showed the strong inhibition of SARS-CoV-2 RdRp, with EC50 value of 0.22 µM and 0.67 µM, respectively. Moreover, our results suggested that exoribonuclease nsp14 increases resistance of SARS-CoV-2 RdRp to nucleotide analog inhibitors. We also determined that Remdesivir presented the highest resistance to viral exoribonuclease activity in cells. Therefore, we have developed a cell-based SARS-CoV-2 RdRp assay which can be deployed to discover SARS-CoV-2 RdRp inhibitors that are urgently needed to treat COVID-19 patients.

CD38 Deficiency Downregulates the Onset and Pathogenesis of Collagen-Induced Arthritis through the NF-κB Pathway.

AIM: The RelB gene plays an important role in guiding the progression of arthritis. We have previously demonstrated that the expression of the RelB gene is decreased significantly in bone marrow DCs of CD38-/- mice. In this study, we demonstrate that the cluster of the differentiation (CD38) gene could be a potentially therapeutic target for autoimmune arthritis. METHOD: Collagen-induced arthritis (CIA) models were generated with both the wild-type (WT) C57BL/6 and CD38-/- mice. The expression of the RelB gene and maturation of bone marrow-derived dendritic cells (DCs) from the WT and CD38-/- mice were detected. Antigen-specific T cell responses, joint damage, and expression of proinflammatory cytokines were assessed. The effects of the Nuclear Factor Kappa B (NF-κB) transcription factor and its mechanisms were characterized. RESULTS: We demonstrated that in CD38-/- mice, the expression of the RelB gene and major histocompatibility complex II (MHC II) was decreased, accompanied with the inhibited T cell reaction in a mixed lymphocyte reaction (MLR) in bone marrow-derived DCs. Compared to the serious degeneration of the cartilage and the enlarged gap of the cavum articular in WT CIA mice, joint pathological changes of the CD38-/- CIA mice revealed marked attenuation, while the joint structures were well preserved. The preserved effects were observed by the inhibition of proinflammatory cytokines and promotion of anti-inflammatory cytokines. Furthermore, decreased phosphorylation of NF-κB was also observed in CD38-/- CIA mice. CONCLUSION: We demonstrate that CD38 could regulate CIA through NF-κB and this regulatory molecule could be a novel target for the treatment of autoimmune inflammatory joint disease.

Anatomical single bundle anterior cruciate ligament reconstruction with rounded rectangle tibial tunnel and oval femoral tunnel: a prospective comparative study versus conventional surgery.

Conventional anatomical single bundle anterior cruciate ligament (ACL) reconstruction technique with round tunnels could not simulate morphology of native insertion, while studies about ACL reconstruction technique with modified tunnels based on morphology of anatomical insertion are rare. The purpose of this study was to demonstrate an ACL reconstruction technique with rounded rectangle tibial tunnel and oval femoral tunnel and compare clinical outcomes with conventional technique. A prospective comparative study was performed in 80 consecutive subjects who underwent ACL reconstruction with the conventional round tunnels (RT-Group, n=40) or modified tunnels (MT-Group, n=40). For the modified surgery, the tunnel was modified with a bone file based on the anatomical direction and area of the remnant insertion fibers. Graft maturity were evaluated by MR images at 12 months postoperatively and patients were examined for functional scores, physical examinations at 2-year follow-up. The primary variable was the pivot-shift test. No serious complications were experienced in either group. Seventy patients (87.5%) were examined at 2-year follow-up, significant improvements were seen in both groups compared with the preoperative values in terms of all clinical assessments. Tegner scores, pivot-shift test results and SNQ value in the MT-Group were significantly better than RT-Group (P=0.04, P=0.03 and P=0.001, respectively). There were no significant differences in Lysholm scores, IKDC scores, KT-2000 measurements and Lachman tests. We successfully developed the ACL reconstruction technique with rounded rectangle tibial tunnel and oval femoral tunnel, which was superior to conventional technique in terms of postoperative Tegner scores, pivot-shift tests and early graft maturity.

Increased TLR4 Expression Aggravates Sepsis by Promoting IFN-γ Expression in CD38-/- Mice.

Gram-negative bacterial sepsis accounts for up to 50% worldwide sepsis that causes hospital mortality. Acute kidney injury (AKI), a common complication of Gram-negative bacterial sepsis, is caused by Toll-like receptor 4 (TLR4) activation. Lipopolysaccharide (LPS) is an endotoxin in Gram-negative bacteria and is recognized specifically by TLR4, which initiates innate immune response. Also, TLR4 signaling pathway activation is essential in response to LPS infection. CD38 is one of the well-known regulators of innate immunity, whose dysregulation contributes to sepsis. Many studies have proven that an attenuated Gram-positive bacterium induces sepsis in a CD38-blocking model. However, the pathogenesis of Gram-negative bacteria-induced sepsis in a CD38-/- mouse model remains unclear. The aim of this study is to investigate whether kidney injury is still attenuated in a LPS-induced CD38-/- sepsis model and identify the potential mechanism. We assess the severity of kidney injury related to proinflammatory cytokine expressions (IFN-γ, TNF-α, IL-1ß, and IL-6) in WT and CD38-/- mice. Our results showed more aggravated kidney damage in CD38-/- mice than in WT mice, accompanied with an increase of proinflammatory cytokine expression. In addition, compared with CD38-/-TLR4mut mice, we found an increase of TLR4 expression and mRNA expression of these cytokines in the kidney of CD38-/- mice, although only increased IFN-γ level was detected in the serum. Taken together, these results demonstrated that an increased TLR4 expression in CD38-/- mice could contribute to the aggravation of AKI through boosting of the production of IFN-γ.

HIV-1 Antisense Protein of Different Clades Induces Autophagy and Associates with the Autophagy Factor p62.

The existence of the antisense transcript-encoded HIV-1 antisense protein (ASP) was recently reinforced by in silico analyses providing evidence for recent appearance of this gene in the viral genome. Our previous studies led to the detection of ASP in various cell lines by Western blotting, flow cytometry, and confocal microscopy analyses and reported that it induced autophagy, potentially through multimer formation. Here, our goals were to assess autophagy induction by ASP from different clades and to identify the implicated autophagy factors. We first demonstrated that ASP formed multimers, partly through its amino-terminal region and cysteine residues. Removal of this region was further associated with lower induction of autophagy, as assessed by autophagosome formation. ASPs from different clades (A, B, C, D, and G) were tested next and were detected in monomeric and multimeric forms at various levels, and all induced autophagy (clade A ASP was less efficient), as determined by LC3-II and p62 (SQSTM1) levels. Furthermore, CRISPR-based knockout of ATG5, ATG7, and p62 genes led to increased ASP levels. Confocal microscopy analyses showed that ASP colocalized with p62 and LC3-II in autophagosome-like structures. Coimmunoprecipitation experiments further demonstrated that p62 associated with ASP through its PB1 domain. Interestingly, immunoprecipitation experiments supported the idea that ASP is ubiquitinated and that ubiquitination was modulating its stability. We are thus suggesting that ASP induces autophagy through p62 interaction and that its abundance is controlled by autophagy, in which ubiquitin plays an important role. Understanding the mechanisms underlying ASP degradation is essential to better assess its function.IMPORTANCE In the present study, we provide the first evidence that a new HIV-1 protein termed ASP derived from different clades acts similarly in inducing autophagy, an important cellular process implicated in the degradation of excess or defective cellular material. We have gained further knowledge on the mechanism mediating the activation of autophagy. Our studies have important ramifications in the understanding of viral replication and the pathogenesis associated with HIV-1 in infected individuals. Indeed, autophagy is implicated in antigen presentation during immune response and could thus be rendered inefficient in infected cells, such as dendritic cells. Furthermore, a possible link with HIV-1-associated neurological disorder (HAND) might also be a possible association with the capacity of ASP to induce autophagy. Our studies hence demonstrate the importance in conducting further studies on this protein as it could represent a new interesting target for antiretroviral therapies and vaccine design.

Clinical study of anatomical ACL reconstruction with adjustable oval shaped bone tunnels: a CT evaluation.

The purpose of this article was to demonstrate an adjustable oval bone tunnel ACLR technique. Aim of this technique was to fit the direction and shape of the footprint and tendon-bone healing passage (TBHP) which was defined as the passage of the normal ACL insertion embedded in the bone as closely as possible. 30 fresh-frozen human cadaveric knees were used to do the ACL anatomical insertions research. 20 patients underwent adjustable oval bone tunnel surgery and 20 patients were in round tunnel group. The tunnel of the presented technique was first drilled with a small diameter round drill bit. Then according to the direction and area of the remnant insersion fibers, the major axes of oval tunnels were expanded to theoretical value with a bone file. Major and minor axes, positions of bone apertures, and areas were evaluated on CT scans. These results were compared with cadaveric and theoretical values. The distance of major axis of oval femoral and tibial tunnel apertures were 10.42 ± 0.55 mm and 12.63 ± 0.5 mm respectively. There're no statistical significance compared with theoretical distance (femoral: P = 0.068, tibial: P = 0.058). The distance of minor axis of oval femoral and tibial tunnel apertures were 6.79 ± 0.28 mm and 6.02 ± 0.29 mm respectively. Both of them were longer than theoretical values (P < 0.001). Compared with the round femoral tunnel, the major/minor axis ratio of oval tunnel (1.53) was more close to the cadaveric results (1.83, P < 0.001). The areas of femoral and tibial apertures were 53.12 ± 1.87 mm2 and 54.22 ± 3.21 mm2 respectively. Both of them were smaller than the round tunnel area and lager than theoretical areas (P < 0.001). We successfully developed the adjustable single oval bone tunnel ACLR technique, which mimic the direction and shape of the tibial and femoral footprints together with the BTHP better than single round tunnel.

Microfracture combined with functional pig peritoneum-derived acellular matrix for cartilage repair in rabbit models.

Due to avascular and hypocellular nature of cartilage, repair of articular cartilage defects within synovial joints still poses a significant clinical challenge. To promote neocartilage properties, we established a functional scaffold named APM-E7 by conjugating a bone marrow-derived mesenchymal stem cell (BM-MSC) affinity peptide (E7) onto the acellular peritoneum matrix (APM). During in vitro culture, the APM-E7 scaffold can support better proliferation as well as better differentiation into chondrocytes of BM-MSCs. After implanting into cartilage defects in rabbits for 24weeks, compared with microfracture and APM groups, the APM-E7 scaffolds exhibited superior quality of neocartilage without transplant rejection, according to general observations, histological assessment, synovial fluid analysis, magnetic resonance imaging (MRI) and nanomechanical properties. This APM-E7 scaffold provided a scaffold for cell attachment, which was crucial for cartilage regeneration. Overall, the APM-E7 is a promising biomaterial with low immunogenicity for one-step cartilage repair by promoting autologous connective tissue progenitor (CTP) attachment. STATEMENT OF SIGNIFICANCE: We report the one-step transplantation of functional acellular peritoneum matrix (APM-E7) with specific mesenchymal stem cell recruitment to repair rabbit cartilage injury. The experimental results illustrated that the APM-E7 scaffold was successfully fabricated, which could specifically recruit MSCs and fill the cartilage defects in the femoral trochlear of rabbits at 24weeks post-surgery. The repaired tissue was hyaline cartilage, which exhibited ideal mechanical stability. The APM-E7 biomaterial could provide scaffold for MSCs and improve cell homing, which are two key factors required for cartilage tissue engineering, thereby providing new insights into cartilage tissue engineering.

A novel rabbit model of early osteoarthritis exhibits gradual cartilage degeneration after medial collateral ligament transection outside the joint capsule.

Though many surgical animal models have been used to induce osteoarthritis (OA) of the knee joint, they always open the capsule of the joint. Any surgical procedures that incises the capsule may cause inflammation, pain, and possibly altered gait. One common disadvantage of these surgically induced animal models is that they may affect the initial structures and synovial fluid in joint. These animal models may not be suitable for research into synovial fluid changes during early OA. This study aimed to create an animal model of early OA by resecting the medial collateral ligament (MCL) outside of the capsule. At 1, 2, 3, 4, 5 and 6 weeks after surgery, eight knees from each group were harvested. The joint gap was measured on posteroanterior radiographs after MCL-transection (MCLT). Gross examination and histological analysis were performed to evaluate cartilage damage to the medial femoral condyles, and knee joints were scanned using a Micro-CT system. The MCLT group experienced early stage OA from 3 to 6 weeks according to the histological scores. IL-6, MMP-1 and MMP-13 content in the synovial fluid were higher after MCLT than anterior cruciate ligament transection (ACLT) at 1 and 2 weeks.

Inhibition of transforming growth factor ß-activated kinase 1 prevents inflammation-related cartilage degradation in osteoarthritis.

Osteoarthritis (OA) is a common debilitating joint disorder, there's still no available disease-modifying drug for OA currently. This study aims to explore the role of TAK1 in OA pathogenesis and therapeutic efficiency of TAK1 inhibition for OA. The contribution of TAK1 to OA pathogenesis was investigated by intra-articular injection of TAK1-encoding adenovirus in rats. TAK1 inhibitor 5Z-7-induced expression changes of extracellular matrix (ECM)-related genes were detected by real-time PCR. The protective effect of 5Z-7 against OA progression was evaluated in a post-traumatic OA rat model. Our results showed that intra-articular injection of Ad-Tak1 induced cartilage destruction and OA-related cytokine secretion in rat joints. TAK1 inhibition by 5Z-7 efficiently blocked NF-κB, JNK and p38 pathways activation in OA chondrocytes and synoviocytes, Meanwhile, 5Z-7 significantly decreased the expression of matrix-degrading enzymes and pro-inflammatory cytokine, while increased ECM protein expression, which are all crucial components in OA. 5Z-7 also ameliorated ECM loss in OA cartilage explants. More importantly, 5Z-7 significantly protected against cartilage destruction in a rat model of OA. In conclusion, our findings provide the first in vivo evidence that TAK1 contributes to OA by disrupting cartilage homeostasis, thus represents an ideal target for OA treatment, with 5Z-7 as a candidate therapeutic.