IGSF8 is an innate immune checkpoint and cancer immunotherapy target.

Antigen presentation defects in tumors are prevalent mechanisms of adaptive immune evasion and resistance to cancer immunotherapy, whereas how tumors evade innate immunity is less clear. Using CRISPR screens, we discovered that IGSF8 expressed on tumors suppresses NK cell function by interacting with human KIR3DL2 and mouse Klra9 receptors on NK cells. IGSF8 is normally expressed in neuronal tissues and is not required for cell survival in vitro or in vivo. It is overexpressed and associated with low antigen presentation, low immune infiltration, and worse clinical outcomes in many tumors. An antibody that blocks IGSF8-NK receptor interaction enhances NK cell killing of malignant cells in vitro and upregulates antigen presentation, NK cell-mediated cytotoxicity, and T cell signaling in vivo. In syngeneic tumor models, anti-IGSF8 alone, or in combination with anti-PD1, inhibits tumor growth. Our results indicate that IGSF8 is an innate immune checkpoint that could be exploited as a therapeutic target.

Glia-derived adenosine in the ventral hippocampus drives pain-related anxiodepression in a mouse model resembling trigeminal neuralgia.

Glial activation and dysregulation of adenosine triphosphate (ATP)/adenosine are involved in the neuropathology of several neuropsychiatric illnesses. The ventral hippocampus (vHPC) has attracted considerable attention in relation to its role in emotional regulation. However, it is not yet clear how vHPC glia and their derived adenosine regulate the anxiodepressive-like consequences of chronic pain. Here, we report that chronic cheek pain elevates vHPC extracellular ATP/adenosine in a mouse model resembling trigeminal neuralgia (rTN), which mediates pain-related anxiodepression, through a mechanism that involves synergistic effects of astrocytes and microglia. We found that rTN resulted in robust activation of astrocytes and microglia in the CA1 area of the vHPC (vCA1). Genetic or pharmacological inhibition of astrocytes and connexin 43, a hemichannel mainly distributed in astrocytes, completely attenuated rTN-induced extracellular ATP/adenosine elevation and anxiodepressive-like behaviors. Moreover, inhibiting microglia and CD39, an enzyme primarily expressed in microglia that degrades ATP into adenosine, significantly suppressed the increase in extracellular adenosine and anxiodepressive-like behaviors. Blockade of the adenosine A2A receptor (A2AR) alleviated rTN-induced anxiodepressive-like behaviors. Furthermore, interleukin (IL)-17A, a pro-inflammatory cytokine probably released by activated microglia, markedly increased intracellular calcium in vCA1 astrocytes and triggered ATP/adenosine release. The astrocytic metabolic inhibitor fluorocitrate and the CD39 inhibitor ARL 67156, attenuated IL-17A-induced increases in extracellular ATP and adenosine, respectively. In addition, astrocytes, microglia, CD39, and A2AR inhibitors all reversed rTN-induced hyperexcitability of pyramidal neurons in the vCA1. Taken together, these findings suggest that activation of astrocytes and microglia in the vCA1 increases extracellular adenosine, which leads to pain-related anxiodepression via A2AR activation. Approaches targeting astrocytes, microglia, and adenosine signaling may serve as novel therapies for pain-related anxiety and depression.

Development and validation of a prediction model for in-hospital death in patients with heart failure and atrial fibrillation.

BACKGROUND: To develop a prediction model for in-hospital mortality of patients with heart failure (HF) and atrial fibrillation (AF). METHODS: This cohort study extracted the data of 10,236 patients with HF and AF upon intensive care unit (ICU) from the Medical Information Mart for Intensive Care (MIMIC). The subjects from MIMIC-IV were divided into the training set to construct the prediction model, and the testing set to verify the performance of the model. The samples from MIMIC-III database and eICU-CRD were included as the internal and external validation set to further validate the predictive value of the model, respectively. Univariate and multivariable Logistic regression analyses were used to explore predictors for in-hospital death in patients with HF and AF. The receiver operator characteristic (ROC), calibration curves and the decision curve analysis (DCA) curves were plotted to evaluate the predictive values of the model. RESULTS: The mean survival time of participants from MIMIC-III was 11.29 ± 10.05 days and the mean survival time of participants from MIMIC-IV was 10.56 ± 9.19 days. Simplified acute physiology score (SAPSII), red blood cell distribution width (RDW), beta-blocker, race, respiratory rate, urine output, coronary artery bypass grafting (CABG), Charlson comorbidity index, renal replacement therapies (RRT), antiarrhythmic, age, and anticoagulation were predictors finally included in the prediction model. The AUC of our prediction model was 0.810 (95%CI: 0.791-0.828) in the training set, 0.757 (95%CI: 0.729-0.786) in the testing set, 0.792 (95%CI: 0.774-0.810) in the internal validation set, and 0.724 (95%CI: 0.687-0.762) in the external validation set. The calibration curves of revealed that the predictive probabilities of our model for the in-hospital death in patients with HF and AF deviated slightly from the ideal model. The DCA curves revealed that the use of our prediction model increased the net benefit than use no model. CONCLUSION: The prediction model had good discriminative ability, and might provide a tool to timely identify patients with HF complicated with AF who were at high risk of in-hospital mortality.

Long Noncoding RNA LNC_000898 Alleviates Cardiomyocyte Apoptosis and Promotes Cardiac Repair After Myocardial Infarction Through Modulating the miR-375/PDK1 Axis.

Increasing evidence has confirmed that both long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) exert key roles in the pathogenesis of myocardial infarction (MI). Previous microarray assay results revealed that lncRNA LNC_000898 expression was significantly downregulated in acute MI. However, the specific function of LNC_000898 on MI is still unclear. Our study was aimed to explore the role of LNC_000898 on cardiac MI injury and investigate its underlying mechanism. The male C57BL/6 mouse was used as cardiac MI injury animal models, and neonatal mouse ventricular cardiomyocytes (NMCMs) exposed to hypoxia were used as an in vitro model. Quantitative real-time polymerase chain reaction analysis, Western blot analysis, Tunel immunofluorescence staining assay, and cardiac echocardiography measurement were conducted to detect corresponding indicators. The results indicated that LNC_000898 expression was downregulated in marginal tissue of MI and in NMCMs exposed to hypoxia. Overexpression of LNC_000898 decreased cardiomyocyte apoptosis both in vivo and in vitro. In addition, we elaborated that LNC_000898 exerts its inhibitory effect on apoptosis after MI through the miR-375/PDK1 axis. Through miR-375 overexpression or silencing PDK1, the biological effects of LNC_000898 on hypoxia-induced NMCM injury were partially reversed. These data not only demonstrate that LNC_000898 could protect the heart against MI injury by regulating miR-375/PDK1 but also provide a new understanding to better protection of MI injury through the LNC_000898/miR-375/PDK1 axis.

Regulation of TERRA on telomeric and mitochondrial functions in IPF pathogenesis.

BACKGROUND: Aging is a known risk factor of idiopathic pulmonary fibrosis (IPF). However, the pathogenic mechanisms underlying the effects of advanced aging remain largely unknown. Telomeric repeat-containing RNA (TERRA) represents a type of long noncoding RNA. In this study, the regulatory roles of TERRA on human telomeres and mitochondria and IPF epithelial injury model were identified. METHODS: Blood samples were collected from patients with IPF (n = 24) and matched control individuals (n = 24). The significance of clinical research on the TERRA expression correlated with pulmonary fibrosis was assessed. The expression levels of TERRA in vivo and in vitro were determined through quantitative real-time polymerase chain reaction analysis. Telomerase activity was observed using a fluorescent quantitative TRAP assay kit. The functions of telomeres, mitochondria, and associated genes were analyzed through RNA interference on TERRA. RESULTS: TERRA expression levels significantly increased in the peripheral blood mononuclear cells of IPF patients. The expression levels also exhibited a direct and significantly inverse correlation with the percentage of predicted force vital capacity, which is a physiological indicator of fibrogenesis during IPF progression. This finding was confirmed in the epithelial injury model of IPF in vitro. RNA interference on TERRA expression can ameliorate the functions of telomeres; mitochondria; associated genes; components associated with telomeres, such as telomerase reverse transcriptase, telomerase, and cell nuclear antigen, cyclin D1; and mitochondria-associated cyclin E genes, including the MMP and Bcl-2 family. The RNA interference on TERRA expression can also improve the functions of oxidative-stress-associated genes, such as reactive oxygen species, superoxide dismutase, and catalase, and apoptosis-related genes, such as cytochrome c, caspase-9, and caspase-3. CONCLUSIONS: In this study, the regulation of TERRA expression on telomeres and mitochondria during IPF pathogenesis was identified for the first time. The results may provide valuable insights for the discovery of a novel biomarker or therapeutic approach for IPF treatment.

Safety and efficacy of a novel abluminal groove-filled biodegradable polymer sirolimus-eluting stent.

Late stent thrombosis (LST) following drug-eluting stent (DES) implantation in patients with coronary artery disease (CAD) is often associated with delayed vascular healing, resulting from vascular inflammation and hypersensitivity to durable polymers and drugs. Therefore, DES design, materials, and coatings have been technologically revolutionized. Herein, we designed a novel abluminal groove-filled biodegradable polymer sirolimus-eluting stent (AGF-BP-SES), with a sirolimus content of only about one-third of traditional DES. The mechanical performances of AGF-BP-SES during compression and expansion were investigated. The pharmacokinetic (PK) profile of sirolimus was studied in the swine model. The in vivo efficacy of AGF-BP-SES was compared with that of Xience PRIME® stent. The results showed that AGF-BP-SES exhibited mechanical properties similar to traditional DES, including the rebound ratio of radial contraction/direction, rebound ratio of axial contraction/direction, and inhomogeneity of compression/expansion. Despite utilizing a reduced dose of sirolimus, AGF-BP-SES delivered sirolimus to the coronary artery in a controlled and efficient manner. The stent maintained a safe and effective local drug concentration without local or systemic risks. In the swine model, histopathological indicators predicted safety and biocompatibility of AGF-BP-SES. In conclusion, AGF-BP-SES maintained similar mechanical properties as other stents while reducing the drug-loading capacity, and showed a favorable safety and efficacy profile of the targeted DES.

miR-30a as Potential Therapeutics by Targeting TET1 through Regulation of Drp-1 Promoter Hydroxymethylation in Idiopathic Pulmonary Fibrosis.

Several recent studies have indicated that miR-30a plays critical roles in various biological processes and diseases. However, the mechanism of miR-30a participation in idiopathic pulmonary fibrosis (IPF) regulation is ambiguous. Our previous study demonstrated that miR-30a may function as a novel therapeutic target for lung fibrosis by blocking mitochondrial fission, which is dependent on dynamin-related protein1 (Drp-1). However, the regulatory mechanism between miR-30a and Drp-1 is yet to be investigated. Additionally, whether miR-30a can act as a potential therapeutic has not been verified in vivo. In this study, the miR-30a expression in IPF patients was evaluated. Computational analysis and a dual-luciferase reporter assay system were used to identify the target gene of miR-30a, and cell transfection was utilized to confirm this relationship. Ten-eleven translocation 1 (TET1) was validated as a direct target of miR-30a, and miR-30a mimic and inhibitor transfection significantly reduced and increased the TET1 protein expression, respectively. Further experimentation verified that the TET1 siRNA interference could inhibit Drp-1 promoter hydroxymethylation. Finally, miR-30a agomir was designed and applied to identify and validate the therapeutic effect of miR-30a in vivo. Our study demonstrated that miR-30a could inhibit TET1 expression through base pairing with complementary sites in the 3'untranslated region to regulate Drp-1 promoter hydroxymethylation. Furthermore, miR-30a could act as a potential therapeutic target for IPF.

[Effect of P2X7 receptor knock-out on bone cancer pain in mice].

Cancer pain is one of the most common symptoms in patients with late stage cancer. Lung, breast and prostate carcinoma are the most common causes of pain from osseous metastasis. P2X7 receptor (P2X7R) is one of the subtypes of ATP-gated purinergic ion channel family, predominately distributed in microglia in the spinal cord. Activation of P2X7Rs in the spinal dorsal horn has been associated with release of proinflammatory cytokines from glial cells, causing increased neuronal excitability and exaggerated nociception. Mounting evidence implies a critical role of P2X7R in inflammatory and neuropathic pain. However, whether P2X7R is involved in cancer pain remains controversial. Here we established a bone cancer pain model by injecting the Lewis lung carcinoma cells into the femur bone marrow cavity of C57BL/6J wild-type mice (C57 WT mice) and P2X7R knockout mice (P2rx7(-/-) mice) to explore the role of P2X7R in bone cancer pain. Following intrafemur carcinoma inoculation, robust mechanical allodynia and thermal hyperalgesia in C57 WT mice were developed on day 7 and 14, respectively, and persisted for at least 28 days in the ipsilateral hindpaw of the affected limb. CatWalk gait analysis showed significant decreases in the print area and stand phase, and a significant increase in swing phase in the ipsilateral hindpaw on day 21 and 28 after carcinoma cells inoculation. Histopathological sections (hematoxylin and eosin stain) showed that the bone marrow of the affected femur was largely replaced by invading tumor cells, and the femur displayed medullary bone loss and bone destruction on day 28 after inoculation. Unexpectedly, no significant changes in bone cancer-induced hypersensitivity of pain behaviors were found in P2rx7(-/-) mice, and the changes of pain-related values in CatWalk gait analysis even occurred earlier in P2rx7(-/-) mice, as compared with C57 WT mice. Together with our previous study in rats that blockade of P2X7R significantly alleviated bone cancer pain, it is implied that P2X7R may play different roles in bone cancer pain in different species (e.g. rat vs mouse). These results implicated a huge difference between the pathophysiology discovered in the experimental animal models and that of human disease.

DKVMN&MRI: A new deep knowledge tracing model based on DKVMN incorporating multi-relational information.

Knowledge tracing is a technology that models students' changing knowledge state over learning time based on their historical answer records, thus predicting their learning ability. It is the core module that supports the intelligent education system. To address the problems of sparse input data, lack of interpretability and weak capacity to capture the relationship between exercises in the existing models, this paper build a deep knowledge tracing model DKVMN&MRI based on the Dynamic Key-Value Memory Network (DKVMN) that incorporates multiple relationship information including exercise-knowledge point relations, exercise-exercise relations, and learning-forgetting relations. In the model, firstly, the Q-matrix is utilized to map the link between knowledge points and exercises to the input layer; secondly, improved DKVMN and LSTM are used to model the learning process of learners, then the Ebbinghaus forgetting curve function is introduced to simulate the process of memory forgetting in learners, and finally, the prediction strategies of Item Response Theory (IRT) and attention mechanism are used to combine the similarity relationship between learners' knowledge state and exercises to calculate the probability that learners would correctly respond during the subsequent time step. Through extensive experiments on three real-world datasets, we demonstrate that DKVMN&MRI has significant improvements in both AUC and ACC metrics contrast with the latest models. Furthermore, the study provides explanations at both the exercise level and learner knowledge state level, demonstrating the interpretability and efficacy of the proposed model.

HOTTIP is upregulated in esophageal cancer and triggers the drug resistance.

PURPOSE: To explore the role of HOTTIP in the development of esophageal cancer and the drug resistance. METHODS: Serum level of HOTTIP in esophageal cancer patients was detected by RT-PCR. After treatment with different concentrations of Adriamycin (ADM) in Eca109 cells for 24 h, IC50 was measured by MTT assay. Subsequently, Eca109 cells were treated with 0, 0.2, 0.4 or 0.8 µg/ml ADM, respectively, followed by extraction of extracellular vesicles (EVs). HOTTIP level in EVs was detected. In addition, Eca109 cells were pre-treated with EVs for 48 h, and different concentrations of ADM for another 24 h. IC50, cell cycle determination and relative levels of HOTTIP and ABCG2 were examined. Pearson correlation test was conducted to assess the correlation between levels of HOTTIP and ABCG2. RESULTS: Serum level of HOTTIP was higher in esophageal cancer patients. IC50 in ADM-treated Eca109 cells for 24 h was 0.43±0.02 µg/ml. EVs1-4 were respectively isolated from Eca109 cells treated with 0, 0.2, 0.4 or 0.8 µg/ml ADM for 24 h, respectively. HOTTIP remained the highest level in EVs4 than the other three groups. Notably, IC50 was much higher in Eca109 cells incubated with EVs4 than those treated with EVs1-3. EVs4 intervention in Eca109 cells for 48 h markedly increased the proliferation index (PI), and relative levels of HOTTIP and ABCG2 than the other three groups. HOTTIP displayed a positive correlation with ABCG2 in Eca109 cells. CONCLUSIONS: HOTTIP is highly expressed in serum of esophageal cancer patients. EVs-containing HOTTIP regulates drug resistance in esophageal cancer by positively activating ABCG2.

Inhibition of TRPV1 by SHP-1 in nociceptive primary sensory neurons is critical in PD-L1 analgesia.

Recently programmed death-ligand 1 (PD-L1) receptor PD-1 was found in dorsal root ganglion (DRG) neurons, and PD-L1 activates PD-1 to inhibit inflammatory and neuropathic pain by modulating neuronal excitability. However, the downstream signaling of PD-1 in sensory neurons remains unclear. Here, we show that PD-L1 activated Src homology 2 domain-containing tyrosine phosphatase-1 (SHP-1) to downregulate transient receptor potential vanilloid 1 (TRPV1) in DRG neurons and inhibit bone cancer pain in mice. Local injection of PD-L1 produced analgesia. PD-1 in DRG neurons colocalized with TRPV1 and SHP-1. PD-L1 induced the phosphorylation of SHP-1 in DRG TRPV1 neurons and inhibited TRPV1 currents. Loss of TRPV1 in mice abolished bone cancer-induced thermal hyperalgesia and PD-L1 analgesia. Conditioned deletion of SHP-1 in NaV1.8+ neurons aggravated bone cancer pain and diminished the inhibition of PD-L1 on TRPV1 currents and pain. Together, our findings suggest that PD-L1/PD-1 signaling suppresses bone cancer pain via inhibition of TRPV1 activity. Our results also suggest that SHP-1 in sensory neurons is an endogenous pain inhibitor and delays the development of bone cancer pain via suppressing TRPV1 function.

Interleukin-17 Regulates Neuron-Glial Communications, Synaptic Transmission, and Neuropathic Pain after Chemotherapy.

The proinflammatory cytokine interleukin-17 (IL-17) is implicated in pain regulation. However, the synaptic mechanisms by which IL-17 regulates pain transmission are unknown. Here, we report that glia-produced IL-17 suppresses inhibitory synaptic transmission in the spinal cord pain circuit and drives chemotherapy-induced neuropathic pain. We find that IL-17 not only enhances excitatory postsynaptic currents (EPSCs) but also suppresses inhibitory postsynaptic synaptic currents (IPSCs) and GABA-induced currents in lamina IIo somatostatin-expressing neurons in mouse spinal cord slices. IL-17 mainly expresses in spinal cord astrocytes, and its receptor IL-17R is detected in somatostatin-expressing neurons. Selective knockdown of IL-17R in spinal somatostatin-expressing interneurons reduces paclitaxel-induced hypersensitivity. Overexpression of IL-17 in spinal astrocytes is sufficient to induce mechanical allodynia in naive animals. In dorsal root ganglia, IL-17R expression in nociceptive sensory neurons is sufficient and required for inducing neuronal hyperexcitability after paclitaxel. Together, our data show that IL-17/IL-17R mediate neuron-glial interactions and neuronal hyperexcitability in chemotherapy-induced peripheral neuropathy.

A shape-memory and spiral light-emitting device for precise multisite stimulation of nerve bundles.

We previously demonstrated that for long-term spastic limb paralysis, transferring the seventh cervical nerve (C7) from the nonparalyzed side to the paralyzed side results in increase of 17.7 in Fugl-Meyer score. One strategy for further improvement in voluntary arm movement is selective activation of five target muscles innervated by C7 during recovery process. In this study, we develop an implantable multisite optogenetic stimulation device (MOSD) based on shape-memory polymer. Two-site stimulation of sciatic nerve bundles by MOSD induces precise extension or flexion movements of the ankle joint, while eight-site stimulation of C7 nerve bundles induce selective limb movement. Long-term implant of MOSD to mice with severed and anastomosed C7 nerve is proven to be both safe and effective. Our work opens up the possibility for multisite nerve bundle stimulation to induce highly-selective activations of limb muscles, which could inspire further applications in neurosurgery and neuroscience research.

Interaction network of coexpressed mRNA, miRNA, and lncRNA activated by TGF­ß1 regulates EMT in human pulmonary epithelial cell.

Noncoding RNAs (ncRNAs), such as microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), play increasingly important roles in pathological processes involved in disease development. However, whether mRNAs interact with miRNAs and lncRNAs to form an interacting regulatory network in diseases remains unknown. In this study, the interaction of coexpressed mRNAs, miRNAs and lncRNAs during tumor growth factor­ß1­activated (TGF­ß1) epithelial­mesenchymal transition (EMT) was systematically analyzed in human alveolar epithelial cells. For EMT regulation, 24 mRNAs, 11 miRNAs and 33 lncRNAs were coexpressed, and interacted with one another. The interaction among coexpressed mRNAs, miRNAs and lncRNAs were further analyzed, and the results showed the lack of competing endogenous RNAs (ceRNAs) among them. The mutual regulation may be correlated with other modes, such as histone modification and transcription factor recruitment. However, the possibility of ceRNA existence cannot be ignored because of the generally low abundance of lncRNAs and frequent promiscuity of protein­RNA interactions. Thus, conclusions need further experimental identification and validation. In this context, disrupting many altered disease pathways remains one of the challenges in obtaining effective pathway­based therapy. The reason being that one specific mRNA, miRNA or lncRNA may target multiple genes that are potentially implicated in a disease. Nevertheless, the results of the present study provide basic mechanistic information, possible biomarkers and novel treatment strategies for diseases, particularly pulmonary tumor and fibrosis.

A novel lnc-PCF promotes the proliferation of TGF-ß1-activated epithelial cells by targeting miR-344a-5p to regulate map3k11 in pulmonary fibrosis.

Emerging evidence suggests that microRNA (miRNA) and long noncoding RNA (lncRNA) play important roles in disease development. However, the mechanism underlying mRNA interaction with miRNA and lncRNA in idiopathic pulmonary fibrosis (IPF) remains unknown. This study presents a novel lnc-PCF that promotes the proliferation of TGF-ß1-activated epithelial cells through the regulation of map3k11 by directly targeting miR-344a-5p during pulmonary fibrogenesis. Bioinformatics and in vitro translation assay were performed to confirm whether or not lnc-PCF is an actual lncRNA. RNA fluorescent in situ hybridization (FISH) and nucleocytoplasmic separation showed that lnc-PCF is mainly expressed in the cytoplasm. Knockdown and knockin of lnc-PCF indicated that lnc-PCF could promote fibrogenesis by regulating the proliferation of epithelial cells activated by TGF-ß1 according to the results of xCELLigence real-time cell analysis system, flow cytometry, and western blot analysis. Computational analysis and a dual-luciferase reporter system were used to identify the target gene of miR-344a-5p, whereas RNA pull down, anti-AGO2 RNA immunoprecipitation, and rescue experiments were conducted to confirm the identity of this direct target. Further experiments verified that lnc-PCF promotes the proliferation of activated epithelial cells that were dependent on miR-344a-5p, which exerted its regulatory functions through its target gene map3k11. Finally, adenovirus packaging sh-lnc-PCF was sprayed into rat lung tissues to evaluate the therapeutic effect of lnc-PCF. These findings revealed that lnc-PCF can accelerate pulmonary fibrogenesis by directly targeting miR-344a-5p to regulate map3k11, which may be a potential therapeutic target in IPF.

Transport of heterologous proteins to the periplasmic space of Pseudomonas fluorescens using a variety of native signal sequences.

Bacterial expression of recombinant proteins containing disulfide bonds is facilitated by transport of the proteins to the periplasmic space. Several Pseudomonas fluorescens signal sequences have been identified that efficiently direct proteins to the periplasm and provide solubility and yield advantages over the production of proteins fused to the PelB signal sequence in E. coli. For a single chain antibody fragment, the final yield varied from about 1 g/l to 10 g/l when expression in P. fluorescens involved fusion to various P. fluorescens signal sequences.