Bone morphogenetic protein 9 is a candidate prognostic biomarker and host-directed therapy target for sepsis.

Defining next-generation immune therapeutics for the treatment of sepsis will involve biomarker-based therapeutic decision-making. Bone morphogenetic protein 9 (BMP9) is a cytokine in the transforming growth factor-ß superfamily. Here, circulating BMP9 concentrations were quantified in two independent cohorts of patients with sepsis. Decreased concentrations of serum BMP9 were observed in the patients with sepsis at the time of admission as compared with healthy controls. Concentrations of BMP9 at the time of admission were also associated with 28-day mortality, because patients with sepsis at a higher risk of death had lower BMP9 concentrations. The mechanism driving the contribution of BMP9 to host immunity was further investigated using in vivo murine sepsis models and in vitro cell models. We found that BMP9 treatment improved outcome in mice with experimental sepsis. BMP9-treated mice exhibited increased macrophage influx into the peritoneal cavity and more efficient bacterial clearance than untreated mice. In vitro, BMP9 promoted macrophage recruitment, phagocytosis, and subsequent bacterial killing. We further found that deletion of the type 1 BMP receptor ALK1 in macrophages abolished BMP9-mediated protection against polymicrobial sepsis in vivo. Further experiments indicated that the regulation of macrophage activation by the BMP9-ALK1 axis was mainly mediated through the suppressor of mother against decapentaplegic 1/5 signaling pathway. Together, these results suggest that BMP9 can both serve as a biomarker for patient stratification with an independent prognostic value and be developed as a host-directed therapy for sepsis.

Absence of toll-like receptor 7 ameliorates survival and reduces intestinal injury in mice after Clostridium difficile infection.

Clostridioides difficile (CD) is a major cause of antibiotic-associated diarrhea and pseudomembranous enteritis. C. difficile infection (CDI) is increasingly present in the community and represents a significant burden on the healthcare system. Identification of novel immune-based therapeutic targets from a better understanding of their molecular pathogenesis is urgently required. Toll-like receptor 7 (TLR7) is an important pattern recognition receptor and function as an immune sensor that can trigger host defenses against pathogens, but the relationship between TLR7 and CDI remains unknown. Here, we reported that the expression levels of TLR7 increased significantly in patients and mice with CDI. Absence of TLR7 in mice with CDI demonstrated enhanced bacterial clearance of intestinal contents and reduced intestinal inflammation, edema, injury and prolonged the survival. TLR7 loss decreased the concentrations of tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IFN-α1 in the intestine and improved tissue damage and inflammation. Flow cytometry and immunofluorescence results indicated that TLR7 enhanced leukocyte recruitment in the infected intestine. In-vitro results have shown that TLR7 impairs the phagocytosis and killing ability of macrophages to CD, prompts reactive oxygen species (ROS) production and accelerates apoptosis. To our knowledge, our study first identified TLR7 as a critical factor that contributes to the immunopathology of CDI, suggesting that targeting TLR7 might serve as a potential treatment for CDI.

miR-486-5p Attenuates Steroid-Induced Adipogenesis and Osteonecrosis of the Femoral Head Via TBX2/P21 Axis.

Enhanced adipogenic differentiation of mesenchymal stem cells (MSCs) is considered as a major risk factor for steroid-induced osteonecrosis of the femoral head (SOFNH). The role of microRNAs during this process has sparked interest. miR-486-5p expression was down-regulated significantly in femoral head bone tissues of both SONFH patients and rat models. The purpose of this study was to reveal the role of miR-486-5p on MSCs adipogenesis and SONFH progression. The present study showed that miR-486-5p could significantly inhibit adipogenesis of 3T3-L1 cells by suppressing mitotic clonal expansion (MCE). And upregulated expression of P21, which was caused by miR-486-5p mediated TBX2 decrease, was responsible for inhibited MCE. Further, miR-486-5p was demonstrated to effectively inhibit steroid-induced fat formation in the femoral head and prevented SONFH progression in a rat model. Considering the potent effects of miR-486-5p on attenuating adipogenesis, it seems to be a promising target for the treatment of SONFH.

Progranulin aggravates lethal Candida albicans sepsis by regulating inflammatory response and antifungal immunity.

Candida albicans is the most frequent pathogen of fungal sepsis associated with substantial mortality in critically ill patients and those who are immunocompromised. Identification of novel immune-based therapeutic targets from a better understanding of its molecular pathogenesis is required. Here, we reported that the production of progranulin (PGRN) levels was significantly increased in mice after invasive C.albicans infection. Mice that lacked PGRN exhibited attenuated kidney injury and increased survival upon a lethal systemic infection with C. albicans. In mice, PGRN deficiency protected against systemic candidiasis by decreasing aberrant inflammatory reactions that led to renal immune cell apoptosis and kidney injury, and by enhancing antifungal capacity of macrophages and neutrophils that limited fungal burden in the kidneys. PGRN in hematopoietic cell compartment was important for this effect. Moreover, anti-PGRN antibody treatment limited renal inflammation and fungal burden and prolonged survival after invasive C. albicans infection. In vitro, PGRN loss increased phagocytosis, phagosome formation, reactive oxygen species production, neutrophil extracellular traps release, and killing activity in macrophages or neutrophils. Mechanistic studies demonstrated that PGRN loss up-regulated Dectin-2 expression, and enhanced spleen tyrosine kinase phosphorylation and extracellular signal-regulated kinase activation in macrophages and neutrophils. In summary, we identified PGRN as a critical factor that contributes to the immunopathology of invasive C.albicans infection, suggesting that targeting PGRN might serve as a novel treatment for fungal infection.

Intranuclear cardiac troponin I plays a functional role in regulating Atp2a2 expression in cardiomyocytes.

In the past studies, it is shown that cardiac troponin I (cTnI, encoded by TNNI3), as a cytoplasmic protein, is an inhibitory subunit in troponin complex, and involves in cardiomyocyte diastolic regulation. Here, we assessed a novel role of cTnI as a nucleoprotein. Firstly, the nuclear translocation of cTnI was found in mouse, human fetuses and rat heart tissues. In addition, there were differences in percentage of intranuclear cTnI in different conditions. Based on weighted gene co-expression network analyses (WGCNA) and verification in cell experiments, a strong expression correlation was found between TNNI3 and Atp2a2, which encodes sarco-endoplasmic reticulum Ca2+ ATPase isoform 2a (SERCA2a), and involves in ATP hydrolysis and Ca2+ transient. TNNI3 gain and loss caused Atpa2a2 increase/decrease in a dose-dependent manner both in mRNA and protein levels, in vivo and in vitro. By using ChIP-sequence we demonstrated specific binding DNA sequences of cTnI were enriched in ATP2a2 promoter -239∼-889 region and the specific binding sequence motif of cTnI was analyzed by software as "CCAT", which has been reported to be required for YY1 binding to the promoter region of YY1-related genes. Moreover, it was further verified that pcDNA3.1 (-)-TNNI3 could express cTnI proteins and increase the promoter activity of Atp2a2 through luciferase report assay. In the end, we evaluated beat frequencies, total ATP contents, Ca2+ transients in TNNI3-siRNA myocardial cells. These findings indicated, for the first time, cTnI may regulate Atp2a2 in cardiomyocytes as a co-regulatory factor and participate in the regulation of intracellular Ca ions.

CXCL14 Protects against Polymicrobial Sepsis by Enhancing Antibacterial Functions of Macrophages.

Rapid and effective control of bacterial infection is critical for the treatment of bacterial sepsis. CXCL14 (CXC motif ligand 14) is an important chemokine involved in infection and immunity, which can bind to CXCR4. However, the contribution of the CXCL14/CXCR4 chemokine axis to bacterial clearance in sepsis remains unknown. Here, the impact of CXCL14/CXCR4 blockade or CXCL14 administration on sepsis was assessed using murine and cell models, as well as human samples. CXCL14 protein concentrations were elevated in mice after cecal ligation and puncture (CLP)-induced sepsis. In vivo, CXCL14 blockade using anti-CXCL14 antibody or CXCL14 knockdown by adeno-associated virus carrying-CXCL14 shRNA significantly increased mortality and bacterial burden, which was paralleled by significantly decreased macrophage influx and M2 macrophage polarization at the site of infection after CLP. Therapeutic administration of CXCL14 improved mortality and bacterial clearance after CLP in a CXCR4-dependent manner, and macrophages, but not neutrophils, were important for the protective effect of CXCL14 in sepsis. In vitro, CXCL14 directly enhanced bacterial phagocytosis and killing of macrophages, and it also increased phagosome formation and reactive oxygen species production in macrophages. Furthermore, inhibiting the activation of PI3K/Akt and NF-κB signaling pathways, but not STAT1 (signal transducer and activator of transcription 1), abrogated the enhanced antibacterial effects of CXCL14 on macrophages. Finally, circulating CXCL14 concentrations were significantly upregulated in patients with sepsis. CXCL14 could enhance bacterial phagocytosis and killing in human monocyte-derived macrophages, which was dependent on CXCR4. Therefore, our results indicate a previously undescribed role of the CXCL14/CXCR4 axis and suggest CXCL14 as a potential adjunct therapy in bacterial sepsis.

[miR-100-5p is involved in non-traumatic osteonecrosis of the femoral head by inhibiting the migration and osteogenic differentiation of BMSCs].

Objective To investigate the role of miR-100-5p in the pathogenesis of non-traumatic osteonecrosis of the femoral head (NONFH). Methods The miRNA expression in patients with NONFH was detected by real-time quantitative PCR, the high expression of miR-100-5p in femoral head tissues of the patients determined. Rat bone marrow mesenchymal stem cells (rBMSCs) were cultured and divided into 5 groups: blank control group, dexamethasone treatment group (treated with dexamethasone for 3 days), miR-NC group (transfected with miR-NC), agomiR-100-5p group (overexpression of miR-100-5p), and antagomiR-100-5p group (transfected with miR-100-5p antagonist). The mRNA expression levels of miR-100-5p, alkaline phosphatase (ALP), Runt-associated transcription factor 2 (RUNX2), and collagen type I (Col1) were detected by real-time quantitative PCR. The protein expressions of ALP, RUNX2, Col1, and bone morphogenetic protein receptor 2 (BMPR2) were detected by Western blotting. The effect of miR-100-5p on the migration ability of rBMSCs was evaluated by scratch healing assay. And the effect of miR-100-5p on osteogenic differentiation ability of rBMSCs was investigated by alizarin red staining. Results miR-100-5p was significantly upregulated in the femoral head bone tissue of NONFH patients compared with normal femoral head bone tissue. Compared with those in the normal rBMSCs, the expression of miR-100-5p in rBMSCs treated with 20 µmol/L of dexamethasone was up-regulated. The upregulation of miR-100-5p in rBMSCs reduced the expressions of ALP, RUNX2, Col1, and BMPR2, and inhibited the osteogenic differentiation and migration abilities of rBMSCs. Conclusion The expression of miR-100-5p is elevated in bone tissues of NONFH patients and in rBMSCs treated with 20 µmol/L of dexamethasone. The up-regulated miR-100-5p may be involved in the pathogenesis of NONFH by inhibiting the migration and osteogenic differentiation of rBMSCs.

Regulating the synthesis rate and yield of bio-assembled FeS nanoparticles for efficient cancer therapy.

Biosynthesis has gained growing interest due to its energy efficiency and environmentally benign nature. Recently, biogenic iron sulfide nanoparticles (FeS NPs) have exhibited excellent performance in environmental remediation and energy recovery applications. However, their biosynthesis regulation strategy and application prospects in the biomedical field remain to be explored. Herein, biogenic FeS NPs are controllably synthesized by Shewanella oneidensis MR-1 and applied for cancer therapy. Tuning the synthesis rate and yield of biogenic FeS NPs is realized by altering the initial iron precursor dosage. Notably, increasing the precursor concentration decreases and delays FeS NP biosynthesis. The biogenic FeS NPs (30 nm) are homogeneously anchored on the cell surface of S. oneidensis MR-1. Moreover, the good hydrophilic nature and outstanding Fenton properties of the as-prepared FeS NPs endow them with good cancer therapy performance. The intracellular location of the FeS NPs taken up is visualized with a soft X-ray microscope (SXM). Highly efficient cancer cell killing can be achieved at extremely low concentrations (<12 µg mL-1), lower than those in reported works. Such good performance is attributed to the Fe2+ release, elevated ROS, reduced glutathione (GSH) consumption, and lipid hydroperoxide (LPO) generation. The resulting FeS NPs show excellent in vivo therapeutic performance. This work provides a facile, eco-friendly, and scalable approach to produce nanomedicine, demonstrating the potential of biogenic nanoparticles for use in cancer therapy.

Exosomal miR-100-5p inhibits osteogenesis of hBMSCs and angiogenesis of HUVECs by suppressing the BMPR2/Smad1/5/9 signalling pathway.

BACKGROUND: Nontraumatic osteonecrosis of the femoral head (NONFH) is a common, progressive, and refractory orthopaedic disease. Decreased osteogenesis and angiogenesis are considered the main factors in the pathogenesis of NONFH. We aimed to figure out whether exosomes and exosomal miRNA from necrotic bone tissues of patients with NONFH are involved in the pathogenesis of NONFH and reveal the underlying mechanisms. METHODS: RT-PCR and western blotting (WB) were used to detect the expression of osteogenic, adipogenic, and angiogenic markers. ALP staining and Alizarin Red S (ARS) staining were used to evaluate osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Oil Red O staining was performed to assess the adipocyte deposition. A tube formation assay was used to study angiogenesis of human umbilical vascular endothelial cells (HUVECs). H&E staining and immunohistochemistry (IHC) staining were used to detect the effect of the NONFH exosomes in vivo. MicroRNA sequencing was conducted to identify potential regulators in the NONFH exosomes. The target relationship between miR-100-5p and BMPR2 was predicted and confirmed by a dual luciferase reporter assay and WB. RESULTS: The NONFH exosomes reduced the osteogenic differentiation of hBMSCs and angiogenesis of HUVECs. In addition, the injection of the NONFH exosomes caused thinning and disruption of bone trabeculae in the femoral heads of rats. MiR-100-5p expression was upregulated in the NONFH exosomes and inhibited the osteogenesis of hBMSCs and angiogenesis of HUVECs by targeting BMPR2 and suppressing the BMPR2/SMAD1/5/9 signalling pathway. Silencing miR-100-5p expression rescued the reduction in osteogenesis and angiogenesis caused by the NONFH exosomes by activating the BMPR2/SMAD1/5/9 signalling pathway. CONCLUSION: The NONFH exosomal miR-100-5p can lead to NONFH-like damage by targeting BMPR2 and suppressing the BMPR2/SMAD1/5/9 signalling pathway, which may be involved in the pathophysiological mechanisms of nontraumatic osteonecrosis of the femoral head (NONFH).

Precise correlative method of Cryo-SXT and Cryo-FM for organelle identification.

Cryogenic soft X-ray tomography (Cryo-SXT) is ideally suitable to image the 3D sub-cellular architecture and organization of cells with high resolution in the near-native preservation state. Cryogenic fluorescence microscopy (Cryo-FM) can determine the location of a molecule of interest that has been labeled with a fluorescent tag, thus revealing the function of the cells. To understand the relations between the sub-cellular architecture and the function of cells, correlative Cryo-SXT and Cryo-FM was applied. This method required the matching of images of different modalities, and the accuracy of the matching is important. Here, a precise correlative method of Cryo-SXT and Cryo-FM is introduced. The capability of matching images of different modalities with high resolution was verified by simulations and practical experiments, and the method was used to identify vacuoles and mitochondria.

Gene expression profiling of the bone trabecula in patients with osteonecrosis of the femoral head by RNA sequencing.

Early diagnosis and treatment of osteonecrosis of the femoral head (ONFH) is challenging. Bone trabecula play a vital role in the severity and progression of ONFH. In the present study, the investigators used gene expression profiling of bone trabecula to investigate gene alterations in ONFH patients. Osteonecrotic bone trabecula (ONBT) such as necrosis, fibrosis, and lacuna were confirmed by histological examination in the patients. The adjacent 'normal' bone trabecula (ANBT) did not show any pathological changes. Gene sequencing data revealed that although ANBT showed no significant histological changes, alteration of mRNA profiling in ANBT was observed, similar to that in ONBT. Our results indicated that the alteration of mRNA profiling in ANBT may cause normal bone tissue to develop into necrotic bone. RNA-seq data indicated that 2,297 differentially abundant mRNAs were found in the ONBT group (1,032 upregulated and 1,265 downregulated) and 1,523 differentially abundant mRNAs in the ANBT group (744 upregulated and 799 downregulated) compared with the healthy control group. Gene ontology (GO) enrichment analysis suggested that fatty acid metabolism and degradation were the main zones enriched with differentially expressed genes (DEG). Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis indicated that peroxisome proliferator-activated receptor γ (PPAR-γ) pathway was the most significantly regulated pathway. Lipocalin-2 (LCN2), an osteoblast-enriched secreted protein, was significantly decreased in ONBT suggesting that downregulation of LCN2 might affect lipid metabolism and lead to hyperlipidemia, and thus promote pathogenesis of ONFH.

A modified discrete tomography for improving the reconstruction of unknown multi-gray-level material in the `missing wedge' situation.

Full angular rotational projections cannot always be acquired in tomographic reconstructions because of the limited space in the experimental setup, leading to the `missing wedge' situation. In this paper, a recovering `missing wedge' discrete algebraic reconstruction technique algorithm (rmwDART) has been proposed to solve the `missing wedge' problem and improve the quality of the three-dimensional reconstruction without prior knowledge of the material component's number or the material's values. By using oversegmentation, boundary extraction and mathematical morphological operations, `missing wedge' artifact areas can be located. Then, in the iteration process, by updating the located areas and regions, high-quality reconstructions can be obtained from the simulations, and the reconstructed images based on the rmwDART algorithm can be obtained from soft X-ray nano-computed tomography experiments. The results showed that there is the potential for discrete tomography.

Controllable long focal length microlens based on thermal expansion.

A novel method to fabricate a microlens array with a long focal length has been developed in this paper. It is based on the fabricating and heating of a microlens consisting of two materials with a great difference in coefficient of thermal expansion. A thermal expansion process leads to considerable deformation of the microlens surface and significant increase in focal length, which could be controlled by altering the processing temperature. Cylindrical polymeric microlens arrays with different focal lengths were successfully fabricated. By measuring the focal length and temperature dependence of the cylindrical microlens geometry, the formation mechanism was analyzed and validated. While the temperature is ranged from 20°C to 50°C, the focal length of the cylindrical microlens has been extended by 38.2% and the longest focal length was obtained up to 6.6 mm for the microlens with a linewidth of 240 µm.