Three-Dimensional Mechanical Microenvironment Rescued the Decline of Osteogenic Differentiation of Old Human Jaw Bone Marrow Mesenchymal Stem Cells.

Resorption and atrophy of the alveolar bone, as two consequences of osteoporosis that remarkably complicate the orthodontic and prosthodontic treatments, contribute to the differentiated biological features and force-induced response of jaw bone marrow-derived mesenchymal stem cells (JBMSCs) in elderly patients. We isolated and cultured JBMSCs from adolescent and adult patients and then simulated the loading of orthodontic tension stress by constructing an in vitro three-dimensional (3D) stress loading model. The decline in osteogenic differentiation of aged JBMSCs was reversed by tensile stress stimulation. It is interesting to note that tension stimulation had a stronger effect on the osteogenic differentiation of elderly JBMSCs compared to the young ones, indicating a possible mechanism of aging rescue. High-throughput sequencing of microRNA (miRNAs) was subsequently performed before and after tension stimulation in all JBMSCs, followed by the comprehensive comparison of mechanically responsive miRNAs in the 3D strain microenvironment. The results suggested a significant reduction in the expression of miR-210-3p and miR-214-3p triggered by the 3D strain microenvironment in old-JBMSCs. Bioinformatic analysis indicated that both miRNAs participate in the regulation of critical pathways of aging and cellular senescence. Taken together, this study demonstrated that the 3D strain microenvironment efficiently rescued the cellular senescence of old-JBMSCs via modulating specific miRNAs, which provides a novel strategy for coordinating periodontal bone loss and regeneration of the elderly.

Unilateral biportal endoscopy vs. open decompression for lumbar epidural lipomatosis-cohort study using a prospective registry.

Objective: This study aimed to compare the outcomes of unilateral biportal endoscopy, unilateral laminectomy bilateral decompression (UBE-ULBD), and open lumbar decompression (OLD) in patients with lumbar epidural lipomatosis (LEL). Methods: This prospective observational study was conducted from March 2019 to May 2022 and encompassed 33 patients with LEL who underwent lumbar decompression. The study included 15 cases of UBE-ULBD decompression and 18 cases of open decompression, which were followed up for 1 year. The baseline characteristics, initial clinical manifestations, and surgical details [including estimated blood loss (EBL) and preoperative complications] of all patients were recorded. Radiographic evaluation included the cross-sectional area (CSA) of the thecal sac and paraspinal muscles on MRI. Clinical results were analyzed using the Short Form-36 Score (SF-36), the Numeric Pain Rating Scale (NRS) for lumbar and leg pain, creatine kinase, the Roland and Morris Disability Questionnaire (RMDQ), and the Oswestry Disability Index (ODI). Results: The dural sac CSA increased considerably at the 1-year postoperative follow-up in both groups (p < 0.001). The operative duration in the OLD group (48.2 ± 7.2 min) was shorter than that in the UBE-ULBD group (67.7 ± 6.3 min, p < 0.001). The OLD group (97.2 ± 19.8 mL) was associated with more EBL than the UBE-ULBD group (40.6 ± 13.6 mL, p < 0.001). The duration of hospitalization in the OLD group (5.4 ± 1.3 days) was significantly longer compared with the UBE-ULBD group (3.5 ± 1.2 days, p < 0.01). The SF-36, NRS, RMDQ, and ODI scores improved in both groups postoperatively (p < 0.001). Serum creatine kinase values in the UBE-ULBD group (101.7 ± 15.5) were significantly lower than those in the OLD group (330.8 ± 28.1 U/L) 1 day after surgery (p < 0.001). The degree of paraspinal muscle atrophy in the UBE-ULBD group (4.81 ± 1.94) was significantly lower than that in the OLD group (12.15 ± 6.99) at 1 year (p < 0.001). Conclusion: UBE-ULBD and OLD demonstrated comparable clinical outcomes in treating LEL. However, UBE-ULBD surgery was associated with shorter hospital stays, lower rates of incision infection, lighter paravertebral muscle injury, and lower EBL than OLD surgery. Consequently, UBE-ULBD can be recommended in patients with LEL if conservative treatment fails.

In vitro and in vivo anticancer activity of novel Rh(III) and Pd(II) complexes with pyrazolopyrimidine derivatives.

Six pyrazolopyrimidine rhodium(III) or palladium(II) complexes, [Rh(L1)(H2O)Cl3] (1), [Rh(L2)(CH3OH)Cl3] (2), [Rh(L3)(H2O)Cl3] (3), [Rh2(L4)Cl6]·CH3OH (4), [Rh(L5)(CH3CN)Cl3]·0.5CH3CN (5), and [Pd(L5)Cl2] (6), were synthesized and characterized. These complexes showed high cytotoxicity against six tested cancer cell lines. Most of the complexes showed higher cytotoxicity to T-24 cells in vitro than cisplatin. Mechanism studies indicated that complexes 5 and 6 induced G2/M phase cell cycle arrest through DNA damage, and induced apoptosis via endoplasmic reticulum stress response. In addition, complex 5 also induced cell apoptosis via mitochondrial dysfunction. Complexes 5 and 6 showed low in vivo toxicity and high tumor growth inhibitory activity in mouse tumor models. The inhibitory effect of rhodium complex 5 on tumor growth in vivo was more pronounced than that of palladium complex 6.

CTLA-4 blockade induces a microglia-Th1 cell partnership that stimulates microglia phagocytosis and anti-tumor function in glioblastoma.

The limited efficacy of immunotherapies against glioblastoma underscores the urgency of better understanding immunity in the central nervous system. We found that treatment with αCTLA-4, but not αPD-1, prolonged survival in a mouse model of mesenchymal-like glioblastoma. This effect was lost upon the depletion of CD4+ T cells but not CD8+ T cells. αCTLA-4 treatment increased frequencies of intratumoral IFNγ-producing CD4+ T cells, and IFNγ blockade negated the therapeutic impact of αCTLA-4. The anti-tumor activity of CD4+ T cells did not require tumor-intrinsic MHC-II expression but rather required conventional dendritic cells as well as MHC-II expression on microglia. CD4+ T cells interacted directly with microglia, promoting IFNγ-dependent microglia activation and phagocytosis via the AXL/MER tyrosine kinase receptors, which were necessary for tumor suppression. Thus, αCTLA-4 blockade in mesenchymal-like glioblastoma promotes a CD4+ T cell-microglia circuit wherein IFNγ triggers microglia activation and phagocytosis and microglia in turn act as antigen-presenting cells fueling the CD4+ T cell response.

Rhodium(III)-Picolinamide Complexes Act as Anticancer and Antimetastasis Agents via Inducing Apoptosis and Autophagy.

As a continuation of our endeavors in discovering metal-based drugs with cytotoxic and antimetastatic activities, herein, we reported the syntheses of 11 new rhodium(III)-picolinamide complexes and the exploration of their potential anticancer activities. These Rh(III) complexes showed high antiproliferative activity against the tested cancer cell lines in vitro. The mechanism study indicated that Rh1 ([Rh(3a)(CH3CN)Cl2]) and Rh2 ([Rh(3b)(CH3CN)Cl2]) inhibited cell proliferation by multiple modes of action via cell cycle arrest, apoptosis, and autophagy and inhibited cell metastasis via FAK-regulated integrin ß1-mediated suppression of EGFR expression. Furthermore, Rh1 and Rh2 significantly inhibited bladder cancer growth and breast cancer metastasis in a xenograft model. These rhodium(III) complexes could be developed as potential anticancer agents with antitumor growth and antimetastasis activity.

Low Glucose plus ß-Hydroxybutyrate Induces an Enhanced Inflammatory Response in Yak Alveolar Macrophages via Activating the GPR109A/NF-κB Signaling Pathway.

Yaks are often subject to long-term starvation and a high prevalence of respiratory diseases and mortality in the withered season, yet the mechanisms that cause this remain unclear. Research has demonstrated that ß-hydroxybutyrate (BHB) plays a significant role in regulating the immune system. Hence, we hypothesize that the low glucose and high BHB condition induced by severe starvation might have an effect on the pro-inflammatory response of the alveolar macrophages (AMs) in yaks. To validate our hypothesis, we isolated and identified primary AMs from freshly slaughtered yaks and cultured them in a medium with 5.5 mM of glucose or 2.8 mM of glucose plus 1-4 mM of BHB. Utilizing a real-time quantitative polymerase chain reaction (RT-qPCR), immunoblot assay, and enzyme-linked immunosorbent assay (ELISA), we evaluated the gene and protein expression levels of GPR109A (G-protein-coupled receptor 109A), NF-κB p65, p38, and PPARγ and the concentrations of pro-inflammatory cytokines interleukin (IL)-1ß and IL-6 and tumor necrosis factor (TNF)-α in the supernatant. The results demonstrated that AMs exposed to low glucose plus BHB had significantly higher levels of IL-1ß, IL-6, and TNF-α (p < 0.05) and higher activity of the GPR109A/NF-κB signaling pathway. A pretreatment of either pertussis toxin (PTX, inhibitor of GPR109A) or pyrrolidinedithiocarbamic (PDTC, inhibitor of NF-κB p65) was effective in preventing the elevated secretion of pro-inflammatory cytokines induced by low glucose plus BHB (p < 0.05). These results indicated that the low glucose plus BHB condition would induce an enhanced pro-inflammatory response through the activation of the GPR109A/NF-κB signaling pathway in primary yak AMs, which is probably the reason why yaks experience a higher rate of respiratory diseases and mortality. This study will offer new insight into the prevention and treatment of bovine respiratory diseases.

Decompression via unilateral biportal endoscopy for severe degenerative lumbar spinal stenosis: A comparative study with decompression via open discectomy.

Background: Previous studies have shown that the Unilateral Biportal Endoscopy is an effective and safety surgery for sufficient decompression of degenerative lumbar spinal stenosis. However, data are lacking in terms of its benefits when compared with conventional open lumbar discectomy (OLD), especially in patients with severe degenerative lumbar spinal stenosis (DLSS). Aim: To compare the clini cal outcomes of two types decompressive surgery: unilateral biportal endoscopy-unilateral laminectomy bilateral decompression (UBE-ULBD) and conventional open lumbar discectomy (OLD) in severe degenerative lumbar spinal stenosis (DLSS). Methods: We retrospectively analyzed patients who underwent UBE-ULBD (n = 50, operated at 50 levels; UBE-ULBD group) and conventional open lumbar discectomy (n = 59, operated at 47 levels; OLD group) between February 2019 and July 2021. All patients were diagnosed with severe stenosis based on the Schizas classification (Grade C or D) on MRI. We compared radiographic and clinical outcome scores [including the visual analog scale (VAS), Oswestry Disability Index (ODI), and Zurich Claudication Questionnaire (ZCQ)] between the 2 groups at 1 year of follow-up. The radiographic evaluation included the cross-sectional area (CSA) of the thecal sac and paraspinal muscles on MRI. Fasting blood was drawn before and 1 and 7 days after the operation to detect creatine kinase (CK). Surgical data perioperative complications were also investigated. Results: The baseline demographic data of the 2 groups were comparable, including VAS, ODI and ZCQ scores, the cross-sectional area of the thecal sac and paraspinal muscles and creatine kinase levels. The dural sac CSA significantly increased post -operatively in both groups, which confirmed they benefited from comparable decompressive effects. The operative duration in the OLD group was less than the UBE-ULBD group (43.9 ± 5.6 min vs. 74.2 ± 9.3 min, p < 0.05). The OLD group was associated with more estimated blood loss than the UBE-ULBD group (111.2 ± 25.0 ml vs. 41.5 ± 22.2 ml, P < 0.05). The length of hospital stay (HS) was significantly longer in the OLD group than in the UBE-ULBD group (6.8 ± 1.6 vs. 4.0 ± 1.4 days, P < 0.05). The VAS, ODI, and ZCQ scores improved in both groups after the operation. Serum creatine kinase values in the UBE-ULBD group were significantly lower than in the OLD group at 1 day after surgery (108. 1 ± 11.9 vs. 347.0 ± 19.5 U/L, P < 0.05). The degree of paraspinal muscle atrophy in the UBE-ULBD group was significantly lower than in the OLD group at 1 year (4.50 ± 0.60 vs. 11.42 ± 0.87, P < 0.05). Conclusions: UBE-ULBD and conventional OLD demonstrate comparable short-term clinical outcomes in treating severe DLSS. However, UBE-ULBD surgery was associated with a shorter hospital stay, less EBL and paravertebral muscle injury than OLD surgery.

Digital Subtraction Angiography-Guided Percutaneous Kyphoplasty in Treatment of Multi-Segmental Osteoporotic Vertebral Compression Fracture: A retrospective single-Center study.

Purpose: This study aimed to explore the effectiveness and safety of digital subtractionangiography (DSA)-guided percutaneous kyphoplasty (PKP) in treating multi-segmental osteoporotic vertebral compression fracture (OVCF). Methods: We retrospectively reviewed 68 patients with multi-segmental OVCF who had unilateral PKP surgeries using DSA and C arm guiding at our hospital between October 2016 and June 2020 and were followed for at least two years. All patients were divided into two groups: DSA guidance (n = 31) and C-arm guidance (n=37). In addition, we collected the clinical and radiological evaluation results during postoperative and last follow-up periods. Results: Our findings revealed that the DSA guidance group required lesser time for channel establishment and surgery than the C-arm guidance group at P < 0.05. The incidences of bone cement leakage, fluoroscopy times, and radiation dose of the DSA guidance group were significantly lesser than the C-arm guidance group (P < 0.05). Compared to the C-arm guidance group, the deviation of puncture in the DSA guidance group was significantly lower, the puncture angle in the DSA guidance group was significantly larger, and better bone cement distribution was obtained (P < 0.05). Compared to preoperative data, the VAS score, median vertebral height, and Cobb angle were significantly improved one day after surgery and the final follow-up in both groups (P < 0.05). However, the VAS score, the median vertebral height, average length of stay, and Cobb angle were not significantly different between the two groups (P > 0.05). Conclusion: DSA-guided PKP in treating multi-segmental OVCF can shorten the operation time, improve puncture accuracy, reduce the times and dose of fluoroscopy, reduce the leakage of bone cement, and achieve better cement distribution.

Multiomics atlas-assisted discovery of transcription factors enables specific cell state programming.

The same types of cells can assume diverse states with varying functionalities. Effective cell therapy can be achieved by specifically driving a desirable cell state, which requires the elucidation of key transcription factors (TFs). Here, we integrated epigenomic and transcriptomic data at the systems level to identify TFs that define different CD8 + T cell states in an unbiased manner. These TF profiles can be used for cell state programming that aims to maximize the therapeutic potential of T cells. For example, T cells can be programmed to avoid a terminal exhaustion state (Tex Term ), a dysfunctional T cell state that is often found in tumors or chronic infections. However, Tex Term exhibits high similarity with the beneficial tissue-resident memory T states (T RM ) in terms of their locations and transcription profiles. Our bioinformatic analysis predicted Zscan20 , a novel TF, to be uniquely active in Tex Term . Consistently, Zscan20 knock-out thwarted the differentiation of Tex Term in vivo , but not that of T RM . Furthermore, perturbation of Zscan20 programs T cells into an effector-like state that confers superior tumor and virus control and synergizes with immune checkpoint therapy. We also identified Jdp2 and Nfil3 as powerful Tex Term drivers. In short, our multiomics-based approach discovered novel TFs that enhance anti-tumor immunity, and enable highly effective cell state programming. One sentence summary: Multiomics atlas enables the systematic identification of cell-state specifying transcription factors for therapeutic cell state programming.

Identification and validation of a novel signature as a diagnostic and prognostic biomarker in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignant neoplasms worldwide. Although marker genes associated with CRC have been identified previously, only a few have fulfilled the therapeutic demand. Therefore, based on differentially expressed genes (DEGs), this study aimed to establish a promising and valuable signature model to diagnose CRC and predict patient's prognosis. METHODS: The key genes were screened from DEGs to establish a multiscale embedded gene co-expression network, protein-protein interaction network, and survival analysis. A support vector machine (SVM) diagnostic model was constructed by a supervised classification algorithm. Univariate Cox analysis was performed to construct two prognostic signatures for overall survival and disease-free survival by Kaplan-Meier analysis, respectively. Independent clinical prognostic indicators were identified, followed by univariable and multivariable Cox analysis. GSEA was used to evaluate the gene enrichment analysis and CIBERSORT was used to estimate the immune cell infiltration. Finally, key genes were validated by qPCR and IHC. RESULTS: In this study, four key genes (DKC1, FLNA, CSE1L and NSUN5) were screened. The SVM diagnostic model, consisting of 4-gene signature, showed a good performance for the diagnostic (AUC = 0.9956). Meanwhile, the four-gene signature was also used to construct a risk score prognostic model for disease-free survival (DFS) and overall survival (OS), and the results indicated that the prognostic model performed best in predicting the DFS and OS of CRC patients. The risk score was validated as an independent prognostic factor to exhibit the accurate survival prediction for OS according to the independent prognostic value. Furthermore, immune cell infiltration analysis demonstrated that the high-risk group had a higher proportion of macrophages M0, and T cells CD4 memory resting was significantly higher in the low-risk group than in the high-risk group. In addition, functional analysis indicated that WNT and other four cancer-related signaling pathways were the most significantly enriched pathways in the high-risk group. Finally, qRT-PCR and IHC results demonstrated that the high expression of DKC1, CSE1L and NSUN5, and the low expression of FLNA were risk factors of CRC patients with a poor prognosis. CONCLUSION: In this study, diagnosis and prognosis models were constructed based on the screened genes of DKC1, FLNA, CSE1L and NSUN5. The four-gene signature exhibited an excellent ability in CRC diagnosis and prognostic prediction. Our study supported and highlighted that the four-gene signature is conducive to better prognostic risk stratification and potential therapeutic targets for CRC patients.

Molecular characterization and functional exploration of GPR84 in Chinese Giant Salamander (Andrias davidianus).

The G protein-coupled receptor 84 (GPR84) is a putative medium-chain fatty acids (MCFAs) receptor involved in immune regulation and other metabolic processes. Most available studies focused on the GPR84 characterization from mammals, neglecting vital information that could be obtained from other levels of life, such as amphibians, necessary for an apt evolutionary understanding of the orphan GPR84. Hence, this study molecularly characterized and functionally explored the GPR84 from the Chinese Giant Salamander (Andrias davidianus). Therefore, we report that the Chinese Giant Salamander (CGS), one of the world's largest amphibians, expresses a GPR84 protein having 376 amino acids, with about 70% homologous to other amphibians and around 50% to human GPR84. Investigating the relative localized expression of gpr84 mRNA in CGS using quantitative PCR revealed the highest expression in the kidney and liver. Furthermore, four medium-chain fatty acids (MCFAs) at micromolar levels activated CGS-GPR84 transfected and expressed in HEK293 cells. In HEK293 cells, four different concentrations of MCFAs inhibited forskolin-induced cAMP accumulation and resulted in a dose-dependent increase in extracellular signal-regulated kinases 1 and 2 (ERK1/2). Interestingly, MCFAs activation of GPR84 concomitantly led to the upregulation of inflammatory mediators such as Nuclear Factor Kappa B (NF-κB) and IL-6. Conclusively, this study successfully elucidated the intriguing molecular and functional properties of CGS GPR84, particularly as an immune modulator, and has positioned the findings within the existing body of knowledge for a better overall understanding of GPR84, especially in amphibians.

Role of the mucin-like glycoprotein FCGBP in mucosal immunity and cancer.

IgGFc-binding protein (FCGBP) is a mucin first detected in the intestinal epithelium. It plays an important role in innate mucosal epithelial defense, tumor metastasis, and tumor immunity. FCGBP forms disulfide-linked heterodimers with mucin-2 and members of the trefoil factor family. These formed complexes inhibit bacterial attachment to mucosal surfaces, affect the motility of pathogens, and support their clearance. Altered FCGBP expression levels may be important in the pathologic processes of Crohn's disease and ulcerative colitis. FCGBP is also involved in regulating the infiltration of immune cells into tumor microenvironments. Thus, the molecule is a valuable marker of tumor prognosis. This review summarizes the functional relevance and role of FCGBP in immune responses and disease development, and highlights the potential role in diagnosis and predicting tumor prognosis.

Arene-Ruthenium(II)/Osmium(II) Complexes Potentiate the Anticancer Efficacy of Metformin via Glucose Metabolism Reprogramming.

Targeting metabolic reprogramming to treat cancer could increase overall survival and reduce side effects. Here, we put forward a strategy using arene-ruthenium(II)/osmium(II) complexes to potentiate the anticancer effect of metformin (Met.) via glucose metabolism reprogramming. Complexes 1-6 with oxoglaucine derivatives as ligands were synthesized and their anti-tumor activities were tested under hypoglycemia. Results indicated that 2 and 5 potentiated the anticancer effects of Met. under hypoglycemia, exhibiting lower toxicity, slower blood glucose decline and inhibition of early tumor liver metastasis. Combination of 5 with Met. could be used as a new strategy to treat cancer under hypoglycemia through glucose metabolism reprogramming.

Effects of iodoacetic acid drinking water disinfection byproduct on the gut microbiota and its metabolism in rats.

Iodoacetic acid (IAA) is an unregulated disinfection byproduct in drinking water and has been shown to exert cytotoxicity, genotoxicity, tumorigenicity, and reproductive and developmental toxicity. However, the effects of IAA on gut microbiota and its metabolism are still unknown, especially the association between gut microbiota and the metabolism and toxicity of IAA. In this study, female and male Sprague-Dawley rats were exposed to IAA at 0 and 16 mg/kg bw/day daily for 8 weeks by oral gavage. Results of 16S rRNA gene sequencing showed that IAA could alter the diversity, relative abundance and function of gut microbiota in female and male rats. IAA also increased the abundance of genes related to steroid hormone biosynthesis in the gut microbiota of male rats. Moreover, metabolomics profiling revealed that IAA could significantly disturb 6 and 13 metabolites in the feces of female and male rats, respectively. In female rats, the level of androstanediol increased in the IAA treatment group. These results were consistent with our previous findings, where IAA was identified as an androgen disruptor. Additionally, the perturbed gut microbiota and altered metabolites were correlated with each other. The results of this study indicated that IAA could disturb gut microbiota and its metabolism. These changes in gut microbiota and its metabolism were associated with the reproductive and developmental toxicity of IAA.

Inhibition of Streptococcus mutans Biofilm Formation by the Joint Action of Oxyresveratrol and Lactobacillus casei.

Microbial dysbiosis in dental plaque contributes to the occurrence of dental caries, to which Streptococcus mutans is a major contributor. Lactobacillus casei can be used as probiotic therapy to treat caries by replacing S. mutans within the dental plaque. However, the effects of probiotic treatment are not always stable. Oxyresveratrol (ORV), a plant-derived polyphenol, displays opposite effects in that it inhibits cariogenic and promotes commensal bacteria. Thus, the objectives of this study are to investigate the effects of ORV on bacterial proportions in S. mutans-L. casei biofilm and to elucidate how ORV weakens the competitiveness of S. mutans. Quantitative real-time PCR confirms a decreased S. mutans-L. casei ratio in dual-species biofilm by action of ORV. The culture supernatant of L. casei after being incubated with ORV (ORVLC) is prepared to explore the joint action of ORV and L. casei. ORVLC displays the strongest anti-biofilm effect against S. mutans when compared with the effects of L. casei supernatant or ORV alone. As a result of this treatment, both exopolysaccharides and bacteria contents in the biofilm are greatly reduced. The biofilm is transformed from water-insoluble glucan-dominant to water-soluble glucan-dominant by ORVLC through the modulation of the glycometabolism-related genes of S. mutans. As for the interactions between ORV and L. casei, ORV promotes L. casei to produce acetic acid, which provides L. casei with a competitive advantage against S. mutans. Taken together, ORV may be very suitable as an adjuvant medicine for probiotic therapy in the control of dental caries. IMPORTANCE The homeostatic imbalance in dental plaque associated with a sharp increase in the number of cariogenic bacteria such as Streptococcus mutans is critical for the occurrence and development of caries. Probiotic therapy can restore ecological balance by replacing cariogenic pathogens with probiotics. The current study innovatively finds that oxyresveratrol, a natural polyphenol, can provide probiotic Lactobacillus casei with competitive dominance in its dual-species biofilm with S. mutans. The joint action of oxyresveratrol and L. casei strongly inhibits the biofilm formation of S. mutans. Additionally, oxyresveratrol promotes L. casei to produce acetic acid, which facilitates L. casei to compete with S. mutans. Through the effects of these two mechanisms, oxyresveratrol leads to a significantly decreased S. mutans-L. casei ratio in their dual-species biofilm. Thus, oxyresveratrol is speculated to be an ideal medicine for the prevention and treatment of caries by regulating oral flora balance.

LONP-1 and ATFS-1 sustain deleterious heteroplasmy by promoting mtDNA replication in dysfunctional mitochondria.

The accumulation of deleterious mitochondrial DNA (∆mtDNA) causes inherited mitochondrial diseases and ageing-associated decline in mitochondrial functions such as oxidative phosphorylation. Following mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcriptional programme to restore mitochondrial function. Paradoxically, ATFS-1 is also required to maintain ∆mtDNAs in heteroplasmic worms. The mechanism by which ATFS-1 promotes ∆mtDNA accumulation relative to wild-type mtDNAs is unclear. Here we show that ATFS-1 accumulates in dysfunctional mitochondria. ATFS-1 is absent in healthy mitochondria owing to degradation by the mtDNA-bound protease LONP-1, which results in the nearly exclusive association between ATFS-1 and ∆mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the binding of the mtDNA replicative polymerase (POLG) to ∆mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wild-type mtDNAs. LONP-1 inhibition in Caenorhabditis elegans and human cybrid cells improved the heteroplasmy ratio and restored oxidative phosphorylation. Our findings suggest that ATFS-1 promotes mtDNA replication in dysfunctional mitochondria by promoting POLG-mtDNA binding, which is antagonized by LONP-1.

Ru(III) complexes with pyrazolopyrimidines as anticancer agents: bioactivities and the underlying mechanisms.

Three ruthenium(III) complexes with pyrazolopyrimidine [Ru(Ln)(H2O)Cl3] (1-3, n = 1-3) were prepared and characterized. These Ru(III) compounds show strong cytotoxicity against six cancer cell lines and low toxicity to normal human liver cells. Particularly, they exhibited stronger cytotoxicity to SK-OV-3 cells than cisplatin. Mechanism studies revealed that complex 1 inhibited tumor cell invasion and suppressed cell proliferation, induced apoptosis by elevating the levels of intracellular ROS (reactive oxygen species) and free calcium (Ca2+), and reduced mitochondrial membrane potential (ΔΨ). It also activated the caspase cascade, accompanied with upregulation of cytochrome c, Bax, p53, Apaf-1 and downregulation of Bcl-2. Moreover, complex 1 caused cell cycle arrest at S phase by inhibiting the expression of CDC 25, cyclin A2 and CDK 2 proteins, and induced DNA damage by interacting with DNA and inhibiting the topoisomerase I enzyme. Complex 1 exhibited efficient in vivo anticancer activity in a model of SK-OV-3 tumor xenograft.

Genome-wide detection of CRISPR editing in vivo using GUIDE-tag.

Analysis of off-target editing is an important aspect of the development of safe nuclease-based genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. Herein we describe GUIDE-tag, which enables one-step, off-target genome editing analysis in mouse liver and lung. The GUIDE-tag system utilizes tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. GUIDE-tag enables detection of off-target sites where editing rates are ≥ 0.2%. UDiTaS analysis utilizing the same tagmented genomic DNA detects low frequency translocation events with off-target sites and large deletions in vivo. The SpyCas9-mSA and biotin-dsDNA system provides a method to capture DSB loci in vivo in a variety of tissues with a workflow that is amenable to analysis of gross genomic alterations that are associated with genome editing.

One-pot synthesis of oxoaporphines as potent antitumor agents and investigation of their mechanisms of actions.

An efficient one-pot reaction for the synthesis of oxoaporphine alkaloids has been developed. Twenty-three compounds of oxoaporphine alkaloids were prepared and assessed for their antitumor activities. Most compounds inhibited the growth of T-24 tumor cells in vitro. Particularly, 4B displayed the most potent activity with an IC50 value of 0.5 µM, which was 19-fold more potent than the parent compound 4. The substitution at C3-position of oxoaporphine core by -NO2 significantly enhanced the anticancer activity. Mechanism studies indicated that 4 and 4B induced cell cycle arrest at G2/M phase; in contrast, 4V induced cell cycle arrest at the S phase. Increase of mitochondrial ROS/Ca2+ and decrease of MMP, accompanied by activation of caspase-3/9, were observed in T-24 cells after exposure to compounds 4, 4B and 4V, suggesting that the mitochondrial pathway was involved in the induced apoptosis. Moreover, compound 4B effectively inhibited tumor growth in a mouse xenograft model bearing T-24.

Transcription Elongation Machinery Is a Druggable Dependency and Potentiates Immunotherapy in Glioblastoma Stem Cells.

Glioblastoma (GBM) is the most lethal primary brain cancer characterized by therapeutic resistance, which is promoted by GBM stem cells (GSC). Here, we interrogated gene expression and whole-genome CRISPR/Cas9 screening in a large panel of patient-derived GSCs, differentiated GBM cells (DGC), and neural stem cells (NSC) to identify master regulators of GSC stemness, revealing an essential transcription state with increased RNA polymerase II-mediated transcription. The YY1 and transcriptional CDK9 complex was essential for GSC survival and maintenance in vitro and in vivo. YY1 interacted with CDK9 to regulate transcription elongation in GSCs. Genetic or pharmacologic targeting of the YY1-CDK9 complex elicited RNA m6A modification-dependent interferon responses, reduced regulatory T-cell infiltration, and augmented efficacy of immune checkpoint therapy in GBM. Collectively, these results suggest that YY1-CDK9 transcription elongation complex defines a targetable cell state with active transcription, suppressed interferon responses, and immunotherapy resistance in GBM. SIGNIFICANCE: Effective strategies to rewire immunosuppressive microenvironment and enhance immunotherapy response are still lacking in GBM. YY1-driven transcriptional elongation machinery represents a druggable target to activate interferon response and enhance anti-PD-1 response through regulating the m6A modification program, linking epigenetic regulation to immunomodulatory function in GBM.This article is highlighted in the In This Issue feature, p. 275.