Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics.

CRISPR diagnostics based on nucleic acid amplification faces barriers to its commercial use, such as contamination risks and insufficient sensitivity. Here, we propose a robust solution involving optochemical control of CRISPR RNA (crRNA) activation in CRISPR detection. Based on this strategy, recombinase polymerase amplification (RPA) and CRISPR-Cas12a detection systems can be integrated into a completely closed test tube. crRNA can be designed to be temporarily inactivated so that RPA is not affected by Cas12a cleavage. After the RPA reaction is completed, the CRISPR-Cas12a detection system is activated under rapid light irradiation. This photocontrolled, fully closed CRISPR diagnostic system avoids contamination risks and exhibits a more than two orders of magnitude improvement in sensitivity compared with the conventional one-pot assay. This photocontrolled CRISPR method was applied to the clinical detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, achieving detection sensitivity and specificity comparable to those of PCR. Furthermore, a compact and automatic photocontrolled CRISPR detection device was constructed.

Light-Start CRISPR-Cas12a Reaction with Caged crRNA Enables Rapid and Sensitive Nucleic Acid Detection.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising platform for nucleic acid detection. Regulating the CRISPR reaction would be extremely useful to improve the detection efficiency and speed of CRISPR diagnostic applications. Here, we have developed a light-start CRISPR-Cas12a reaction by employing caged CRISPR RNA (crRNA). When combined with recombinase polymerase amplification, a robust photocontrolled one-pot assay is achieved. The photocontrolled one-pot assay is simpler and is 50-fold more sensitive than the conventional assay. This improved detection efficiency also facilitates the development of a faster CRISPR diagnostic method. The detection of clinical samples demonstrated that 10-20 min is sufficient for effective detection, which is much faster than the current gold-standard technique PCR. We expect this advance in CRISPR diagnostics to promote its widespread detection applications in biomedicine, agriculture, and food safety.

The clinical relevance of epithelial-mesenchymal transition and its correlations with tumorigenic immune infiltrates in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a cancer with extremely high mortality. Epithelial-mesenchymal transition (EMT) may play an important role in the occurrence, invasion and prognosis of HCC; however, its relationship with immunity in HCC has not yet been studied. Therefore, we investigated the diagnostic and prognostic values of EMT and explored its potential connections with tumorigenic immune infiltrates in HCC. We first proposed a quantitative metric of EMT activity, the EMT score. After applying this metric to 20 datasets from the Integrative Molecular Database of Hepatocellular Carcinoma, the Cancer Genome Atlas, and the Gene Expression Omnibus, we explored the ability of the EMT score to stratify across sample types. We then applied the EMT score for survival analysis and to differentiate patients with/without vascular invasion to test its prognostic value. We also collected and calculated data on the abundance of immune cells and immune cell markers in HCC and investigated their correlations with EMT scores. Finally, we synthesized and analyzed 20 datasets and constructed an EMT-gene-immune linkage network. The results showed higher EMT scores in HCC samples than in cirrhotic and normal livers. The cases with higher EMT scores also showed poorer performance in terms of prognostic factors such as vascular invasion and overall survival time. Our research demonstrated a broad correlation between EMT and the tumor immune microenvironment, and we uncovered multiple potential linkers associated with both EMT and immunity. Studying EMT has clinical relevance and high diagnostic and prognostic value for HCC.

Spatial maps of hepatocellular carcinoma transcriptomes highlight an unexplored landscape of heterogeneity and a novel gene signature for survival.

BACKGROUND: Hepatocellular carcinoma (HCC) often presents with satellite nodules, rendering current curative treatments ineffective in many patients. The heterogeneity of HCC is a major challenge in personalized medicine. The emergence of spatial transcriptomics (ST) provides a powerful strategy for delineating the complex molecular landscapes of tumours. METHODS: In this study, the heterogeneity of tissue-wide gene expression in tumour and adjacent nonneoplastic tissues using ST technology were investigated. The transcriptomes of nearly 10,820 tissue regions and identified the main gene expression clusters and their specific marker genes (differentially expressed genes, DEGs) in patients were analysed. The DEGs were analysed from two perspectives. First, two distinct gene profiles were identified to be associated with satellite nodules and conducted a more comprehensive analysis of both gene profiles. Their clinical relevance in human HCC was validated with Kaplan-Meier (KM) Plotter. Second, DEGs were screened with The Cancer Genome Atlas (TCGA) database to divide the HCC cohort into high- and low-risk groups according to Cox analysis. HCC patients from the International Cancer Genome Consortium (ICGC) cohort were used for validation. KM analysis was used to compare the overall survival (OS) between the high- and low-risk groups. Univariate and multivariate Cox analyses were applied to determine the independent predictors for OS. RESULTS: Novel markers for the prediction of satellite nodules were identified and a tumour clusters-specific marker gene signature model (6 genes) for HCC prognosis was constructed. CONCLUSION: The establishment of marker gene profiles may be an important step towards an unbiased view of HCC, and the 6-gene signature can be used for prognostic prediction in HCC. This analysis will help us to clarify one of the possible sources of HCC heterogeneity and uncover pathogenic mechanisms and novel antitumour drug targets.

TAZ promotes vasculogenic mimicry in gastric cancer through the upregulation of TEAD4.

BACKGROUND AND AIM: Vasculogenic mimicry (VM) is a unique blood supply pattern in malignant tumors that is closely associated with metastasis and poor prognosis. The Hippo signaling effector TAZ is upregulated in several cancers, promoting cancer proliferation and metastasis. This study aimed to identify the function of TAZ and its regulatory mechanism in promoting VM in gastric cancer (GC). METHODS: The expression of TAZ and TEAD4 and their correlations with overall survival and VM-related markers were analyzed in 228 cases of GC. The regulatory mechanism of TAZ and its interaction with TEAD4 in epithelial-mesenchymal transition (EMT) and VM were investigated in vitro and in vivo. RESULTS: TAZ was highly expressed in GC samples and was associated with shorter patient survival time. TAZ expression was positively correlated with TEAD4 and VM in patients with GC. TAZ enhanced the migration and invasion capacity of GC cells through EMT in vitro and upregulated the expression of VM-associated proteins, including VE-cadherin, MMP2, and MMP9, thus promoting VM formation. Overexpression of TAZ accelerated the growth of subcutaneous xenograft and promoted VM formation in vivo. Co-immunoprecipitation assays showed that TAZ can directly bind to TEAD4, and in vitro experiments showed that this binding mediates the function of TAZ in regulating EMT and VM formation in GC. CONCLUSIONS: TAZ promotes GC metastasis and VM by upregulating TEAD4 expression. Our findings expand the role of TAZ in VM and provide new theoretical support for the use of antiangiogenic therapy in the treatment of advanced GC.

Autophagy triggers endoplasmic reticulum stress and C/EBP homologous protein-mediated apoptosis in OGD/R-treated neurons in a caspase-12-independent manner.

We reported that a high level of autophagy was initiated by oxygen-glucose deprivation (OGD) and was maintained in neurons even after oxygen-glucose deprivation followed by reoxygenation (OGD/R), accompanied by neuronal apoptosis. This study focused on autophagy-induced apoptosis and its signaling network, especially the role of endoplasmic reticulum stress (ERS). Analysis of primary cultured cortical neurons from mice showed that the autophagy-induced apoptosis depended on caspase-8 and -9 but not on caspase-12. This finding did not mean that the endoplasmic reticulum did not participate in this process. Increases in the levels of endoplasmic reticulum (ER) biomarkers and binding immunoglobulin protein (BiP) were induced by autophagy in OGD/R-treated neurons. In addition, as an apoptotic transcription factor induced by ER stress, C/EBP homologous protein (CHOP) expression was significantly increased in neurons after OGD/R. This result suggested that the autophagy-BiP-CHOP-caspase (8 and 9)-dependent apoptotic signaling pathway at least partly participated in autophagy-induced apoptosis in primary cortical neurons. It revealed that ER induced apoptosis in neurons suffering from OGD/R injury in an ER stress-CHOP-dependent manner rather than a caspase-12-dependent manner. However, more research on signaling or cross-linking networks and intermediate links is needed. The realization of caspase-12-independent BiP-CHOP neuronal apoptosis pathway has expanded our understanding of the neuronal apoptosis network, which may eventually provide endogenous interventional strategies for OGD/R injury after stroke.NEW & NOTEWORTHY ER stress induced by autophagy mediates caspase-8- and caspase-9-dependent apoptosis pathways by regulating CHOP in neurons exposed to OGD/R. We hypothesized that the autophagy-BiP-CHOP-caspase (8 and 9)-dependent apoptotic signaling pathway at least partly participated in autophagy-induced apoptosis in primary cortical neurons.

Knockdown of plasmacytoma variant translocation 1 (PVT1) inhibits high glucose-induced proliferation and renal fibrosis in HRMCs by regulating miR-23b-3p/early growth response factor 1 (EGR1).

Long noncoding RNAs (lncRNAs) have been reported to play critical role in the development of diabetic nephropathy (DN). However, the effects and mechanism of plasmacytoma variant translocation 1 (PVT1) remain poorly understood. The expression of PVT1, miR-23b-3p, early growth response factor 1 (EGR1), Fibronectin (FN), Collagen IV (Col IV), alpha smooth muscle actin (α-SMA), E-cadherin, and vimentin, transforming growth factor (TGF)-ß1 was examined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was assessed by Cell Counting-8 (CCK-8) assay. Western blot assay was conducted to measure the protein levels of FN, Col IV, E-cadherin, α-SMA, vimentin, TGF-ß1, and EGR1. The interaction between miR-23b-3p and PVT1 or EGR1 was predicted by starBase or TargetScan and confirmed by the dual luciferase reporter assay. The oxidative stress factors were analyzed by corresponding kits. We found that the expression of PVT1 and EGR1 was increased and miR-23b-3p was decreased in serum samples of DN patients and HG-induced HRMCs. Knockdown of PVT1 significantly inhibited HG-induced proliferation, extracellular matrix (ECM) accumulation, epithelial-mesenchymal transition (EMT), and oxidative stress in HRMCs, while these effects were abated by inhibiting miR-23b-3p. In addition, EGR1 was confirmed as downstream target of miR-23b-3p and miR-23b-3p could specially bind to PVT1. Besides, downregulation of PVT1 inhibited the progression of DN partially via upregulating miR-23b-3p and downregulating EGR1. In conclusion, our results suggested that PVT1 knockdown suppressed DN progression though functioning as ceRNA of miR-23b-3p to regulate EGR1 expression in vitro, providing potential value for the treatment of DN.

Twist1 accelerates tumour vasculogenic mimicry by inhibiting Claudin15 expression in triple-negative breast cancer.

The up-regulation of EMT regulator Twist1 has been implicated in vasculogenic mimicry (VM) formation in human triple-negative breast cancer (TNBC). Twist1 targets the Claudin15 promoter in hepatocellular carcinoma cells. Claudin family members are related with TNBC. However, the relationship between Claudin15 and VM formation is not clear. In this study, we first found that Claudin15 expression was frequently down-regulated in human TNBC, and Claudin15 down-regulation was significantly associated with VM and Twist1 nuclear expression. Claudin15 down-regulation correlated with shorter survival compared with high levels. Claudin15 silence significantly enhanced cell motility, invasiveness and VM formation in the non-TNBC MCF-7 cells. Conversely, an up-regulation of Claudin15 remarkably reduced TNBC MDA-MB-231 cell migration, invasion and VM formation. We also showed that down-regulation of Claudin15 was Twist1-dependent, and Twist1 repressed Claudin15 promoter activity. Furthermore, GeneChip analyses of mammary glands of Claudin15-deficient mice indicated that Claudin18 and Jun might be downstream factors of Twist1-Claudin15. Our results suggest that Twist1 induced VM through Claudin15 suppression in TNBC, and Twist1 inhibition of Claudin15 might involve Claudin18 and Jun expression.

Supramolecular polymer assembly in aqueous solution arising from cyclodextrin host-guest complexation.

The employment of cyclodextrin host-guest complexation to construct supramolecular assemblies with an emphasis on polymer networks is reviewed. The main driving force for this supramolecular assembly is host-guest complexation between cyclodextrin hosts and guest groups either of which may be discrete molecular species or substituents on a polymer backbone. The effects of such complexation on properties at the molecular and macroscopic levels are discussed. It is shown that cyclodextrin complexation may be used to design functional polymer materials with tailorable properties, especially for photo-, pH-, thermo- and redox-responsiveness and self-healing.

Subpopulations of uPAR+ contribute to vasculogenic mimicry and metastasis in large cell lung cancer.

The urokinase plasminogen activator receptor (uPAR) is closely associated with poor prognosis in various aggressive cancers including large-cell lung cancer (LCLC). Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks involving the blood supply in early tumor formation. We demonstrate the statistically positive correlation of uPAR expression with VM formation, metastasis, and poor prognosis of LCLC patients. uPAR(+) cells sorted from the LCLC H460 cell line show higher invasion, migration capacity, and tube structure formation capability on Matrigel compared with uPAR(-) cells. uPAR(+) tumor cells highly expressed vimentin and VE-cadherin; the epithelial marker E-cadherin was low expressed. Higher EMT-regulated protein twist and snail expressions were also observed in these cells. uPAR(+) cells injected subcutaneously into nude mice markedly increased tumor growth, induced VM formation and liver metastasis; by contrast, uPAR(-) cells did not. The data suggest that uPAR expression may predict VM formation, tumor metastasis and poorer prognosis of LCLC patients. The uPAR gene may be used as a novel therapeutic target for inhibiting angiogenesis and metastasis in LCLC.

Hypoxia induced epithelial-mesenchymal transition and vasculogenic mimicry formation by promoting Bcl-2/Twist1 cooperation.

Hypoxia plays a pivotal role in tumor progression. The functions of hypoxia and subsequent Bcl-2/Twist1 activation in epithelial-mesenchymal transition (EMT) and vasculogenic mimicry (VM) formation are currently unclear. This study aimed to investigate the role of Bcl-2/Twist1 cooperation in hypoxia-induced EMT and VM formation. In in vitro experiments, we found that hypoxia resulted in co-overexpression of Bcl-2 and Twist1, facilitated Twist1 nuclear translocation and promoted EMT and VM formation. Co-overexpression of Bcl-2 and Twist1 under normoxia could also induce EMT and promote VM formation. Furthermore, blocking Bcl-2 or Twist1 attenuated the effects of hypoxia on EMT progress and VM formation in hepatocellular carcinoma cells. In in vivo experiments, the mechanism by which hypoxia promoted Bcl-2 and Twist1 co-overexpression and induced EMT process and VM formation was demonstrated using murine xenograft models. These results above suggest that hypoxia could activate the cooperation of Bcl-2 and Twist1, Bcl-2 plays an important role in assisting Twist1 nuclear translocation which could change the expression of a wide range of genes and lead to the induction of EMT and VM formation.

The U.S. FDA approved cardiovascular drugs from 2011 to 2023: A medicinal chemistry perspective.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. A total of 28 new molecular entities (NMEs) were approved by the U.S. Food and Drug Administration (FDA) for the treatment of cardiovascular diseases from 2011 to 2023. Approximately 25 % of the medications were sanctioned for the management of diverse vascular disorders. The other major therapeutic areas of focus included antilipemic agents (15 %), blood pressure disease (11 %), heart failure, hyperkalemia, and cardiomyopathy (7-8% each). Among all the approved drugs, there are a total of 22 new chemical entities (NCEs), including inhibitors, agonists, polymers, and inorganic compounds. In addition to NCEs, 6 biological agents (BLAs), including monoclonal antibodies, small interfering RNAs (siRNAs), and antisense oligonucleotides, have also obtained approval for the treatment of cardiovascular diseases. From this perspective, approved NCEs are itemized and discussed based on their disease, targets, chemical classes, major drug metabolites, and biochemical and pharmacological properties. Systematic analysis has been conducted to examine the binding modes of these approved drugs with their targets using cocrystal structure information or docking studies to provide valuable insights for designing next-generation agents. Furthermore, the synthetic approaches employed in the creation of these drug molecules have been emphasized, aiming to inspire the development of novel, efficient, and applicable synthetic methodologies. Generally, the primary objective of this review is to provide a comprehensive examination of the clinical applications, pharmacology, binding modes, and synthetic methodologies employed in small-molecule drugs approved for treating CVD. This will facilitate the development of more potent and innovative therapeutics for effectively managing cardiovascular diseases.

The CT-guided percutaneous drainage of pancreatic pseudocyst accompanied by pseudoaneurysm: A case report.

RATIONALE: This case report discusses the CT-guided percutaneous drainage of a pancreatic pseudocyst accompanied by a pseudoaneurysm. Pancreatic pseudocysts can erode the peripancreatic artery and produce pseudoaneurysms. This is rare, but it can be life-threatening. PATIENT CONCERNS: The case presented involves a 58-year-old female who was diagnosed with pancreatic cancer and underwent surgical treatment. She presented with hematochezia, dizziness, and hypodynamic findings with no obvious cause. Imaging revealed a pancreatic pseudocyst and small arterial aneurysms. To reduce the risk of aneurysm rupture, the patient underwent transcatheter arterial coil embolization. Three days later, CT-guided catheter drainage was performed to reduce the erosion of the arterial wall caused by pancreatic fluid. DIAGNOSES: The contrast-enhanced-CT imaging showed a round, slightly high-density lesion in the cyst, suggesting the presence of a pseudoaneurysm. INTERVENTIONS: The patient was sent for another transcatheter arterial embolization with coils and n-butyl-2-cyanoacrylate. OUTCOMES: After receiving the transcatheter arterial embolization, the patient had no serious bleeding or other complications. LESSONS: Early detection and accurate assessment of pseudoaneurysms are essential for appropriate management. This case shows that contrast-enhanced CT is necessary before CT-guided percutaneous drainage of pancreatic pseudocysts. It also shows that, due to the many complications that pancreatic pseudocysts may cause, appropriate treatment of pseudocysts complicated with pseudoaneurysm has important clinical significance.

Altered spontaneous brain activity in lumbar disc herniation patients: insights from an ALE meta-analysis of neuroimaging data.

Objective: To explore the characteristics of spontaneous brain activity changes in patients with lumbar disc herniation (LDH), and help reconcile the contradictory findings in the literature and enhance the understanding of LDH-related pain. Materials and methods: PubMed, Web of Science, Embase, Chinese National Knowledge Infrastructure (CNKI), SinoMed, and Wanfang databases were searched for literature that studies the changes of brain basal activity in patients with LDH using regional homogeneity (ReHo) and amplitude of low-frequency fluctuation/fraction amplitude of low-frequency fluctuation (ALFF/fALFF) analysis methods. Activation likelihood estimation (ALE) was used to perform a meta-analysis of the brain regions with spontaneous brain activity changes in LDH patients compared with healthy controls (HCs). Results: A total of 11 studies were included, including 7ALFF, 2fALFF, and 2ReHo studies, with a total of 269 LDH patients and 277 HCs. Combined with the data from the ALFF/fALFF and ReHo studies, the meta-analysis results showed that compared with HCs, LDH patients had increased spontaneous brain activity in the right middle frontal gyrus (MFG), left anterior cingulate cortex (ACC) and the right anterior lobe of the cerebellum, while they had decreased spontaneous brain activity in the left superior frontal gyrus (SFG). Meta-analysis using ALFF/fALFF data alone showed that compared with HCs, LDH patients had increased spontaneous brain activity in the right MFG and left ACC, but no decrease in spontaneous brain activity was found. Conclusion: In this paper, through the ALE Meta-analysis method, based on the data of reported rs-fMRI whole brain studies, we found that LDH patients had spontaneous brain activity changes in the right middle frontal gyrus, left anterior cingulate gyrus, right anterior cerebellar lobe and left superior frontal gyrus. However, it is still difficult to assess whether these results are specific and unique to patients with LDH. Further neuroimaging studies are needed to compare the effects of LDH and other chronic pain diseases on the spontaneous brain activity of patients. Furthermore, the lateralization results presented in our study also require further LDH-related pain side-specific grouping study to clarify this causation. Systematic review registration: PROSPERO, identifier CRD42022375513.

Mapping particulate organic carbon in lakes across China using OLCI/Sentinel-3 imagery.

Remote sensing monitoring of particulate organic carbon (POC) concentration is essential for understanding phytoplankton productivity, carbon storage, and water quality in global lakes. Some algorithms have been proposed, but only for regional eutrophic lakes. Based on in-situ data (N = 1269) in 49 lakes across China, we developed a blended POC algorithm by distinguishing Type-I and Type-II waters. Compared to Type-I, Type-II waters had higher reflectance peak around 560 nm (>0.0125 sr-1) and mean POC (4.65 ± 4.11 vs. 2.66 ± 3.37 mg/L). Furthermore, because POC was highly related to algal production (r = 0.85), a three-band index (R2 = 0.65) and the phytoplankton fluorescence peak height (R2 = 0.63) were adopted to estimate POC in Type-I and Type-II waters, respectively. The novel algorithm got a mean absolute percent difference (MAPD) of 35.93 % and outperformed three state-of-the-art formulas with MAPD values of 40.56-76.42 %. Then, the novel algorithm was applied to OLCI/Sentinel-3 imagery, and we first obtained a national map of POC in 450 Chinese lakes (> 20 km2), which presented an apparent spatial pattern of "low in the west and high in the east". In brief, water classification should be considered when remotely monitoring lake POC concentration over a large area. Moreover, a process-oriented method is required when calculating water column POC storage from satellite-derived POC concentrations in type-II waters. Our results contribute substantially to advancing the dynamic observation of the lake carbon cycle using satellite data.

Discovery of a Novel Series of Homo sapiens Caseinolytic Protease P Agonists for Colorectal Adenocarcinoma Treatment via ATF3-Dependent Integrated Stress Response.

Homo sapiens caseinolytic protease P (HsClpP) activation is a promising strategy for colon cancer treatment. In this study, CCG1423 was identified as a selective activator of HsClpP. After optimization, NCA029 emerged as the most potent compound, with an EC50 of 0.2 µM against HsClpP. Molecular dynamics revealed that the affinity of NCA029 for the YYW aromatic network is crucial for its selectivity toward HsClpP. Furthermore, NCA029 displayed favorable pharmacokinetics and safety profiles and significantly inhibited tumor growth in HCT116 xenografts, resulting in 83.6% tumor inhibition. Mechanistically, NCA029 targeted HsClpP, inducing mitochondrial dysfunction and activating the ATF3-dependent integrated stress response, ultimately causing cell death in colorectal adenocarcinoma. These findings highlight NCA029 as an effective HsClpP activator with potential for colon cancer therapy.

Chestnut-Inspired Hollow Hydroxyapatite 3D Printing Scaffolds Accelerate Bone Regeneration by Recruiting Calcium Ions and Regulating Inflammation.

This study aims to overcome the drawbacks associated with hydroxyapatite (HAP) dense structures after sintering, which often result in undesirable features such as large grain size, reduced porosity, high crystallinity, and low specific surface area. These characteristics hinder osseointegration and limit the clinical applicability of the material. To address these issues, a new method involving the preparation of hollow hydroxyapatite (hHAP) microspheres has been proposed. These microspheres exhibit distinctive traits including weak crystallization, high specific surface area, and increased porosity. The weak crystallization aligns more closely with early mineralization products found in the human body and animals. Moreover, the microspheres' high specific surface area and porosity offer advantages for protein loading and facilitating osteoblast attachment. This innovative approach not only mitigates the limitations of conventional HAP structures but also holds the potential for improving the effectiveness of hydroxyapatite in biomedical applications, particularly in enhancing osseointegration. Three-dimensional printed hHAP/chitosan (CS) scaffolds with different hHAP concentration gradients were manufactured, and the physical and biological properties of each group were systematically evaluated. In vitro and in vivo experiments show that the hHAP/CS scaffold has excellent performance in bone remodeling. Furthermore, in-scaffold components, hHAP and CS were cocultured with bone marrow mesenchymal stem cells to explore the regulatory role of hHAP and CS in the process of bone healing and to reveal the cell-level specific regulatory network activated by hHAP. Enrichment analysis showed that hHAP can promote bone regeneration and reconstruction by recruiting calcium ions and regulating inflammatory reactions.

Discovery and Optimization of Novel SaFabI Inhibitors as Specific Therapeutic Agents for MRSA Infection.

As the rate-limiting enzyme in fatty acid biosynthesis, Staphylococcus aureus enoyl-acyl carrier protein reductase (SaFabI) emerges as a compelling target for combating methicillin-resistant S. aureus (MRSA) infections. Herein, compound 1, featuring a 4-(1H-benzo[d]imidazol-2-yl)pyrrolidin-2-one scaffold, was identified as a potent SaFabI inhibitor (IC50 = 976.8 nM) from an in-house library. Subsequent optimization yielded compound n31, with improved inhibitory efficacy on enzymatic activity (IC50 = 174.2 nM) and selective potency against S. aureus (MIC = 1-2 µg/mL). Mechanistically, n31 directly inhibited SaFabI in cellular contexts. Moreover, n31 exhibited favorable safety and pharmacokinetic profiles, and dose-dependently treated MRSA-induced skin infections, outperforming the approved drug, linezolid. The chiral separation of n31 resulted in (S)-n31, with superior activities (IC50 = 94.0 nM, MIC = 0.25-1 µg/mL) and in vivo therapeutic efficacy. In brief, our research proposes (S)-n31 as a promising candidate for SaFabI-targeted therapy, offering specific anti-S. aureus efficacy and potential for further development.

[Preliminary study of influence of bone tissue from osteonecrosis of femoral head on the proliferation and differentiation of canine bone marrow mesenchymal stem cells].

OBJECTIVE: To examine the effects of bone tissue from osteonecrosis of femoral head on the proliferation and differentiation of canine bone marrow mesenchymal stem cells in vitro culture. METHODS: A canine model of femoral head osteonecrosis was induced by liquid nitrogen freezing. BMSC were isolated from dog ilium bone marrow by a combination of gradient centrifugation and adherent wall culture. Different bone tissues and BMSC were cultivated indirectly in vitro by co-cultured in Transwell plate. According to the culture media, 3 groups were established: blank group (10%FBS/DMEM), control group (10%FBS/DMEM+ bone tissue from natural femoral head) and experimental group (10%FBS/DMEM+ bone tissue from osteonecrosis of femoral head). Cell proliferation was measured by methyl-thiazol tetrazolium (MTT) method. Cell differentiation was examined by alkaline phosphatase (ALP) staining and its concentration examined. Alizarin red staining method was used to study the calcification effects and Oil red O staining method was used to detect if there was fat emergence. RESULTS: As compared with the blank group, the proliferation in the control and experiment groups were significantly promoted after culturing for Days 1, 3 and 5 (P < 0.05). The proliferation of the experiment group was higher than the control group at Day 5 and 7 day (P < 0.05). After a 7-day co-culturing, ALP staining was positive in the control and experiment groups. At Day 7 and 9, the ALP activity in culture fluid was in this order: control group > experiment group > blank group (P < 0.05). Alizarin red staining show control group had the most calcium nodules (12.17 ± 2.48, P < 0.05) and the number of calcium nodules in the experiment group was more than the blank group (P < 0.05). Oil red O staining show there was no fat emergence after 21 days in every group. CONCLUSION: Both natural and osteonecrotic bone tissue of femoral head could promoted the proliferation of canine BMSC and induces them osteogenic differentiation but not lipo differentiation, bone tissue from osteonecrosis of the femoral head had more competence in promoting proliferation but less capability in inducing osteogenic differentiation than natural bone tissue of the femoral head.

Targeting caseinolytic protease P and its AAA1 chaperone for tuberculosis treatment.

Caseinolytic protease P with its AAA1 chaperone, known as Mycobacterium tuberculosis (Mtb)ClpP1P2 proteolytic machinery, maintains protein homeostasis in Mtb cells and is essential for bacterial survival. It is regarded as an important biological target with the potential to address the increasingly serious issue of multidrug-resistant (MDR) TB. Over the past 10 years, many MtbClpP1P2-targeted modulators have been identified and characterized, some of which have shown potent anti-TB activity. In this review, we describe current understanding of the substrates, structure and function of MtbClpP1P2, classify the modulators of this important protein machine into several categories based on their binding subunits or pockets, and discuss their binding details; Such information provides insights for use in candidate drug research and development of TB treatments by targeting MtbClpP1P2 proteolytic machinery.