Cancer cell-derived exosomal circUHRF1 induces natural killer cell exhaustion and may cause resistance to anti-PD1 therapy in hepatocellular carcinoma.

OBJECTIVE: Natural killer (NK) cells play a critical role in the innate antitumor immune response. Recently, NK cell dysfunction has been verified in various malignant tumors, including hepatocellular carcinoma (HCC). However, the molecular biological mechanisms of NK cell dysfunction in human HCC are still obscure. METHODS: The expression of circular ubiquitin-like with PHD and ring finger domain 1 RNA (circUHRF1) in HCC tissues, exosomes, and cell lines was detected by qRT-PCR. Exosomes were isolated from the culture medium of HCC cells and plasma of HCC patients using an ultracentrifugation method and the ExoQuick Exosome Precipitation Solution kit and then characterized by transmission electronic microscopy, NanoSight and western blotting. The role of circUHRF1 in NK cell dysfunction was assessed by ELISA. In vivo circRNA precipitation, RNA immunoprecipitation, and luciferase reporter assays were performed to explore the molecular mechanisms of circUHRF1 in NK cells. In a retrospective study, the clinical characteristics and prognostic significance of circUHRF1 were determined in HCC tissues. RESULTS: Here, we report that the expression of circUHRF1 is higher in human HCC tissues than in matched adjacent nontumor tissues. Increased levels of circUHRF1 indicate poor clinical prognosis and NK cell dysfunction in patients with HCC. In HCC patient plasma, circUHRF1 is predominantly secreted by HCC cells in an exosomal manner, and circUHRF1 inhibits NK cell-derived IFN-γ and TNF-α secretion. A high level of plasma exosomal circUHRF1 is associated with a decreased NK cell proportion and decreased NK cell tumor infiltration. Moreover, circUHRF1 inhibits NK cell function by upregulating the expression of TIM-3 via degradation of miR-449c-5p. Finally, we show that circUHRF1 may drive resistance to anti-PD1 immunotherapy in HCC patients. CONCLUSIONS: Exosomal circUHRF1 is predominantly secreted by HCC cells and contributes to immunosuppression by inducing NK cell dysfunction in HCC. CircUHRF1 may drive resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for patients with HCC.

Germanicol induces selective growth inhibitory effects in human colon HCT-116 and HT29 cancer cells through induction of apoptosis, cell cycle arrest and inhibition of cell migration.

PURPOSE: The main aim of this research was to evaluate the anticancer and apoptotic effects of germanicol - a natural triterpene - in HCT-116 and HT29 human colon cancer cells and deciphering its mode of action by studying its effect on the cell cycle and cell migration. METHODS: Cell cytotoxicity was evaluated by MTT assay, while cell death was assessed by LDH assay. Fluorescence microscopy, using DAPI and acridine orange/ethidium bromide (AO-ETBR), was carried out to evaluate the effect of germanicol on cellular morphology and apoptosis induction. Apoptosis quantification was performed by Annexin V-FITC assay, while cell cycle analysis was performed by flow cytometry using propidium iodide (PI). RESULTS: The results revealed that germanicol showed selective, potent and dose-dependent cytotoxicity in HCT-116 and HT29 human colon cancer cells, while it showed lower cytotoxicity in normal colon cells (human colon fibroblast, CCD-18Co). LDH assay also showed that germanicol induced dose-dependent cell death in HCT-116 and HT29 cells. Fluorescence microscopy revealed that germanicol induced apoptosis via chromatin condensation and DNA damage in HCT-116 colon cancer cells. It also revealed that the percentage of cells with orange and red fluorescence increased when adding a germanicol dose, indicating apoptosis. Germanicol also inhibited cancer cell migration. CONCLUSION: The current findings reveal that germanicol exhibits selective antiproliferative activity against two human colon cancer cells. The normal cell line was less affected by the drug, as compared to the two cancer cell lines, indicating that germanicol will not target normal living cells. The antiproliferative effect was shown to be mediated through the induction of apoptosis and suppression of cell migration.

Population genetic structure and approximate Bayesian computation analyses reveal the southern origin and northward dispersal of the oriental fruit moth Grapholita molesta (Lepidoptera: Tortricidae) in its native range.

The oriental fruit moth (OFM) Grapholita molesta is one of the most destructive orchard pests. Assumed to be native to China, the moth is now distributed throughout the world. However, the evolutionary history of this moth in its native range remains unknown. In this study, we explored the population genetic structure, dispersal routes and demographic history of the OFM in China and South Korea based on mitochondrial genes and microsatellite loci. The Mantel test indicated a significant correlation between genetic distance and geographical distance in the populations. Bayesian analysis of population genetic structure (baps) identified four nested clusters, while the geneland analysis inferred five genetic groups with spatial discontinuities. Based on the approximate Bayesian computation approach, we found that the OFM was originated from southern China near the Shilin area of Yunnan Province. The early divergence and dispersal of this moth was dated to the Penultimate glaciation of Pleistocene. Further dispersal from southern to northern region of China occurred before the last glacial maximum, while the expansion of population size in the derived populations in northern region of China occurred after the last glacial maximum. Our results indicated that the current distribution and structure of the OFM were complicatedly influenced by climatic and geological events and human activities of cultivation and wide dissemination of peach in ancient China. We provide an example on revealing the origin and dispersal history of an agricultural pest insect in its native range as well as the underlying factors.

Macro CD5L+ deteriorates CD8+T cells exhaustion and impairs combination of Gemcitabine-Oxaliplatin-Lenvatinib-anti-PD1 therapy in intrahepatic cholangiocarcinoma.

Intratumoral immune status influences tumor therapeutic response, but it remains largely unclear how the status determines therapies for patients with intrahepatic cholangiocarcinoma. Here, we examine the single-cell transcriptional and TCR profiles of 18 tumor tissues pre- and post- therapy of gemcitabine plus oxaliplatin, in combination with lenvatinib and anti-PD1 antibody for intrahepatic cholangiocarcinoma. We find that high CD8 GZMB+ and CD8 proliferating proportions and a low Macro CD5L+ proportion predict good response to the therapy. In patients with a poor response, the CD8 GZMB+ and CD8 proliferating proportions are increased, but the CD8 GZMK+ proportion is decreased after the therapy. Transition of CD8 proliferating and CD8 GZMB+ to CD8 GZMK+ facilitates good response to the therapy, while Macro CD5L+-CD8 GZMB+ crosstalk impairs the response by increasing CTLA4 in CD8 GZMB+. Anti-CTLA4 antibody reverses resistance of the therapy in intrahepatic cholangiocarcinoma. Our data provide a resource for predicting response of the combination therapy and highlight the importance of CD8+T-cell status conversion and exhaustion induced by Macro CD5L+ in influencing the response, suggesting future avenues for cancer treatment optimization.

Effect of temperature and photoperiod on the development, reproduction, and predation of the predatory ladybird Cheilomenes sexmaculata (Coleoptera: Coccinellidae).

The polyphagous predatory ladybird Cheilomenes. sexmaculata (F.) (Coleoptera: Coccinellidae) is distributed throughout southern China and has been investigated as a potential biological control agent against herbivorous insects in various agroecosystems. In the current study, we evaluated the preimaginal development, eclosion rate, reproduction, fertility, adult longevity, and prey consumption of C. sexmaculata under five temperature and five photoperiod regimens. The results showed that preadult developmental duration decreased significantly with increasing temperature and amount of daylight. Adult eclosion rate was highest at 35 degrees C and under conditions of complete darkness. Higher temperatures shortened the duration of copulation and preoviposition, prolonged the duration of oviposition, and increased the level of fecundity. Hatchability was highest at 30 degrees C. By contrast, the shortest copulation and oviposition duration and lowest level of fecundity and hatchability occurred with a completely dark photoperiod. Temperature and the gender of C. sexmaculata influenced adult longevity. In addition, there was a significant interaction effect of photoperiod and gender on adult longevity. Furthermore, prey consumption by fourth instar larvae and adult females both increased with increasing temperature and photoperiod. Our results reveal the high thermal and light sensitivities of C. sexmaculata, which highlight the importance of environment regulation in the mass rearing of this natural enemy for application as a biological control in agroecosystems in China.

[Analysis of Irregular Blood Group Antibody Distribution and Blood Transfusion Efficacy in Patients with Malignant Tumor].

OBJECTIVE: To investigate the distribution of irregular blood group antibodies in patients with malignant tumors, and to analyze the relationship between it and efficacy of blood transfusion in patients. METHODS: 5 600 patients with malignant tumors treated in Shanxi Bethune Hospital from January 2019 to December 2021 were selected as the research subjects. All patients received blood transfusion, and cross matching test was conducted before blood transfusion, irregular antibody results of patients were tested; the irregular distribution of blood group antibodies was observed, and the relationship between it and efficacy of blood transfusion in patients was analyzed. RESULTS: Among 5 600 patients with malignant tumors, 96 cases were positive for irregular antibody, and the positive rate was 1.71%; the main blood group systems involved in the irregular antibody positive of 96 patients with malignant tumors were RH, MNSs and Duffy system, among which Rh blood group was the most common, and the proportion of anti-E was the highest; among the malignant tumor patients with positive blood group irregular antibody, the proportion of female was higher than that of male; the proportion of patients aged >60 years was the highest, followed by patients aged >40 and ≤50 years, and the proportion of patients aged 18-30 years was the lowest; the patients with positive blood group irregular antibody were mainly in blood system (including lymphoma), digestive system, reproductive and urinary system; the positive rate of irregular antibody of patients in the ineffective group was higher than that of patients in the effective group, the difference was statistically significant (P<0.05). Logistic regression analysis results showed that, irregular antibody positive was a risk factor for ineffective blood transfusion in patients with malignant tumor (OR>1, P<0.05). CONCLUSION: The irregular blood group antibody positive of patients with malignant tumor are mostly female, and the proportion of patients aged >60 is the highest, which is mainly distributed in malignant tumors of blood system, digestive system and urogenital system, and the positive blood group irregular antibody is related to the efficacy of blood transfusion in patients.

Rehabilitation Training after Spinal Cord Injury Affects Brain Structure and Function: From Mechanisms to Methods.

Spinal cord injury (SCI) is a serious neurological insult that disrupts the ascending and descending neural pathways between the peripheral nerves and the brain, leading to not only functional deficits in the injured area and below the level of the lesion but also morphological, structural, and functional reorganization of the brain. These changes introduce new challenges and uncertainties into the treatment of SCI. Rehabilitation training, a clinical intervention designed to promote functional recovery after spinal cord and brain injuries, has been reported to promote activation and functional reorganization of the cerebral cortex through multiple physiological mechanisms. In this review, we evaluate the potential mechanisms of exercise that affect the brain structure and function, as well as the rehabilitation training process for the brain after SCI. Additionally, we compare and discuss the principles, effects, and future directions of several rehabilitation training methods that facilitate cerebral cortex activation and recovery after SCI. Understanding the regulatory role of rehabilitation training at the supraspinal center is of great significance for clinicians to develop SCI treatment strategies and optimize rehabilitation plans.

Epidural combined optical and electrical stimulation induces high-specificity activation of target muscles in spinal cord injured rats.

Introduction: Epidural electrical stimulation (EES) has been shown to improve motor dysfunction after spinal cord injury (SCI) by activating residual locomotor neural networks. However, the stimulation current often spreads excessively, leading to activation of non-target muscles and reducing the accuracy of stimulation regulation. Objectives: Near-infrared nerve stimulation (nINS) was combined with EES to explore its regulatory effect on lower limb muscle activity in spinal-cord-transected rats. Methods: In this study, stimulation electrodes were implanted into the rats' L3-L6 spinal cord segment with T8 cord transected. Firstly, a series of EES parameters (0.2-0.6 mA and 20-60 Hz) were tested to determine those that specifically regulate the tibialis anterior (TA) and medial gastrocnemius (MG). Subsequently, to determine the effect of combined optical and electrical stimulation, near-infrared laser with a wavelength of 808 nm was used to irradiate the L3-L6 spinal cord segment while EES was performed. The amplitude of electromyography (EMG), the specific activation intensity of the target muscle, and the minimum stimulus current intensity to induce joint movement (motor threshold) under a series of optical stimulation parameters (power: 0.0-2.0 W; pulse width: 0-10 ms) were investigated and analyzed. Results: EES stimulation with 40 Hz at the L3 and L6 spinal cord segments specifically activated TA and MG, respectively. High stimulation intensity (>2 × motor threshold) activated non-target muscles, while low stimulation frequency (<20 Hz) produced intermittent contraction. Compared to electrical stimulation alone (0.577 ± 0.081 mV), the combined stimulation strategy could induce stronger EMG amplitude of MG (1.426 ± 0.365 mV) after spinal cord injury (p < 0.01). The combined application of nINS effectively decreased the EES-induced motor threshold of MG (from 0.237 ± 0.001 mA to 0.166 ± 0.028 mA, p < 0.001). Additionally, the pulse width (PW) of nINS had a slight impact on the regulation of muscle activity. The EMG amplitude of MG only increased by ~70% (from 3.978 ± 0.240 mV to 6.753 ± 0.263 mV) when the PW increased by 10-fold (from 1 to 10 ms). Conclusion: The study demonstrates the feasibility of epidural combined electrical and optical stimulation for highly specific regulation of muscle activity after SCI, and provides a new strategy for improving motor dysfunction caused by SCI.

Different macaque brain network remodeling after spinal cord injury and NT3 treatment.

Graph theory-based analysis describes the brain as a complex network. Only a few studies have examined modular composition and functional connectivity (FC) between modules in patients with spinal cord injury (SCI). Little is known about the longitudinal changes in hubs and topological properties at the modular level after SCI and treatment. We analyzed differences in FC and nodal metrics reflecting modular interaction to investigate brain reorganization after SCI-induced compensation and neurotrophin-3 (NT3)-chitosan-induced regeneration. Mean inter-modular FC and participation coefficient of areas related to motor coordination were significantly higher in the treatment animals than in the SCI-only ones at the late stage. The magnocellular part of the red nucleus may reflect the best difference in brain reorganization after SCI and therapy. Treatment can enhance information flows between regions and promote the integration of motor functions to return to normal. These findings may reveal the information processing of disrupted network modules.

Comparison of the conventional tube and erythrocyte-magnetized technology in titration of red blood cell alloantibodies.

BACKGROUND: Erythrocyte alloantibodies are mainly produced after immune stimulation, such as blood transfusion, pregnancy, and transplantation, and are the leading causes of severe hemolytic transfusion reactions and difficulty in blood grouping and matching. Therefore, antibody screening is critical to prevent and improve red cell alloantibodies. Routine tube assay is the primary detection method of antibody screening. Recently, erythrocyte-magnetized technology (EMT) has been increasingly used in clinical practice. This study intends to probe the application and efficacy of the conventional tube and EMT in red blood cell alloantibody titration to provide a reference for clinical blood transfusion. AIM: To investigate the application value of conventional tube and EMT in red blood cell alloantibody titration and enhance the safety of blood transfusion practice. METHODS: A total of 1298 blood samples were harvested from blood donors at the Department of Blood Transfusion of our hospital from March 2021 to December 2022. A 5 mL blood sample was collected in tubing, which was then cut, and the whole blood was put into a test tube for centrifugation to separate the serum. Different red blood cell blood group antibody titers were simultaneously detected using the tube polybrene test, tube antiglobulin test (AGT), and EMT screening irregular antibody methods to determine the best test method. RESULTS: Simultaneous detection was performed through the tube polybrene test, tube AGT and EMT screening irregular antibodies. It was discovered that the EMT screening irregular antibody method could detect all immunoglobulin G (IgG) and immunoglobulin M (IgM) irregular antibodies, and the results of manual tube AGT were satisfactory, but the operation time was lengthy, and the equipment had a large footprint. The EMT screening irregular antibody assay was also conducted to determine its activity against type O Rh (D) red blood cells, and the outcomes were satisfactory. Furthermore, compared to the conventional tube method, the EMT screening irregular antibody method was more cost-effective and had significantly higher detection efficiency. CONCLUSION: With a higher detection rate, the EMT screening irregular antibody method can detect both IgG and IgM irregular antibodies faster and more effectively than the conventional tube method.

Loss of 5-hydroxymethylcytosine induces chemotherapy resistance in hepatocellular carcinoma via the 5-hmC/PCAF/AKT axis.

Multidrug resistance is a major challenge in treating advanced hepatocellular carcinoma (HCC). Although recent studies have reported that the multidrug resistance phenotype is associated with abnormal DNA methylation in cancer cells, the epigenetic mechanism underlying multidrug resistance remains unknown. Here, we reported that the level of 5-hydroxymethylcytosine (5-hmC) in human HCC tissues was significantly lower than that in adjacent liver tissues, and reduced 5-hmC significantly correlated with malignant phenotypes, including poor differentiation and microvascular invasion; additionally, loss of 5-hmC was related to chemotherapy resistance in post-transplantation HCC patients. Further, the 5-hmC level was regulated by ten-eleven translocation 2 (TET2), and the reduction of TET2 in HCC contributes to chemotherapy resistance through histone acetyltransferase P300/CBP-associated factor (PCAF) inhibition and AKT signaling hyperactivation. In conclusion, loss of 5-hmC induces chemotherapy resistance through PCAF/AKT axis and is a promising chemosensitivity prediction biomarker and therapeutic target for HCC patients.

The clinical implications and molecular features of intrahepatic cholangiocarcinoma with perineural invasion.

BACKGROUND: Perineural invasion (PNI) is associated with metastasis in malignancies, including intrahepatic cholangiocarcinoma (ICC), and is correlated with poor prognosis. METHODS: The study included three large cohorts: ZS-ICC and TMA cohorts from our team, MSK cohort from a public database, and a small cohort named cohort 4. Prognostic implications of PNI were investigated in MSK cohort and TMA cohort. PNI-related genomic and transcriptomic profiles were analyzed in MSK and ZS-ICC cohorts. GO, KEGG, and ssGSEA analyses were performed. Immunohistochemistry was used to investigate the relationship between PNI and markers of neurons, hydrolases, and immune cells. The efficacy of adjuvant therapy in ICC patients with PNI was also assessed. RESULTS: A total of 30.6% and 20.7% ICC patients had PNI in MSK and TMA cohorts respectively. Patients with PNI presented with malignant phenotypes such as high CA19-9, the large bile duct type, lymph node invasion, and shortened overall survival (OS) and relapse-free survival (RFS). Nerves involved in PNI positively express tyrosine hydroxylase (TH), a marker of sympathetic nerves. Patients with PNI showed high mutation frequency of KRAS and an immune suppressive metastasis prone niche of decreased NK cell, increased neutrophil, and elevated PD-L1, CD80, and CD86 expression. Patients with PNI had an extended OS after adjuvant therapy with TEGIO, GEMOX, or capecitabine. CONCLUSION: Our study deciphered the genomic features and the immune suppressive metastasis-prone niche in ICC with PNI. Patients with PNI showed a poor prognosis after surgery but a good response to adjuvant chemotherapy.

[A study on the cerebral glucose metabolism in progressive supranuclear palsy].

OBJECTIVE: To study the regional cerebral glucose utilization with (18)F-fluorodeoxyglucose (FDG) PET and to investigate the correlation between cerebral glucose metabolism and the clinical characteristic of progressive supranuclear palsy (PSP). METHODS: A total of 13 patients with PSP and 30 matched healthy controls were performed (18)F-FDG PET imaging at rest state. Visual inspection and statistical parametric mapping (SPM) were used to investigate regional cerebral metabolic rate of glucose (rCMRglc). RESULTS: Based on the visual inspection, PET imaging in the PSP patients showed that the focal hypometabolic areas mainly included the bilateral frontal cortex, midbrain and subcortical structures. Compared to the controls, voxel-based analysis showed that the regional glucose metabolism decreased in bilateral superior, middle frontal gyrus, cingulate gyrus, midbrain and subcortical structures including basal ganglion and thalamus, which were consisted with the clinical characteristics, such as vertical gaze palsy, pseudobulbar palsy, postural instability, axial rigidity, dementia and so on. CONCLUSION: (18)F-FDG PET imaging is helpful for the early diagnosis of PSP.

NT3 treatment alters spinal cord injury-induced changes in the gray matter volume of rhesus monkey cortex.

Spinal cord injury (SCI) may cause structural alterations in brain due to pathophysiological processes, but the effects of SCI treatment on brain have rarely been reported. Here, voxel-based morphometry is employed to investigate the effects of SCI and neurotrophin-3 (NT3) coupled chitosan-induced regeneration on brain and spinal cord structures in rhesus monkeys. Possible association between brain and spinal cord structural alterations is explored. The pain sensitivity and stepping ability of animals are collected to evaluate sensorimotor functional alterations. Compared with SCI, the unique effects of NT3 treatment on brain structure appear in extensive regions which involved in motor control and neuropathic pain, such as right visual cortex, superior parietal lobule, left superior frontal gyrus (SFG), middle frontal gyrus, inferior frontal gyrus, insula, secondary somatosensory cortex, anterior cingulate cortex, and bilateral caudate nucleus. Particularly, the structure of insula is significantly correlated with the pain sensitivity. Regenerative treatment also shows a protective effect on spinal cord structure. The associations between brain and spinal cord structural alterations are observed in right primary somatosensory cortex, SFG, and other regions. These results help further elucidate secondary effects on brain of SCI and provide a basis for evaluating the effects of NT3 treatment on brain structure.

The impacts of fermented feed on laying performance, egg quality, immune function, intestinal morphology and microbiota of laying hens in the late laying cycle.

Fermented feed has the potential to improve poultry gastrointestinal microecological environment, health condition and production performance. Thus, the present study was undertaken to explore the effects of fermented feed on the laying performance, egg quality, immune function, intestinal morphology and microbiota of laying hens in the late laying cycle. A total of 360 healthy Hy-Line Brown laying hens aged 80 weeks were used to conduct a 56-day study. All hens were randomly separated into two treatment groups, with five replicates of 36 hens each as follows: basal diet containing 0.0% fermented feed (CON) and 20% fermented feed (FF). Subsequent analyses revealed that fermented feed supplementation was associated with significant increases in laying rates together with reduced broken egg rates and feed conversion ratio for hens in FF group (P < 0.05). There were additionally significant increases in both albumen height and Haugh unit values in hens following fermented feed supplementation (P < 0.05). Fermented feed was also associated with increases in duodenal, jejunal and ileac villus height (P < 0.05). Laying hens fed fermented feed had higher immune globulin (Ig)A, IgG, IgM levels (P < 0.01,) and higher interleukin 2, interleukin 6, tumour necrosis factor α and interferon γ (P < 0.05) concentrations than CON. Analysis of the microbiota in these laying hens revealed the alpha diversity was not significantly affected by fermented feed supplementation. Firmicutes abundance was reduced in caecal samples from FF hens relative to those from CON hens (30.61 vs 35.12%, P < 0.05). At the genus level, fermented feed was associated with improvements in relative Lactobacillus, Megasphaera and Peptococcus abundance and decreased Campylobacter abundance in laying hens. These results suggest that fermented feed supplementation may be beneficial to the laying performance, egg quality, immunological function, intestinal villus growth and caecal microecological environment of laying hens at the end of the laying cycle.

KSR2-14-3-3ζ complex serves as a biomarker and potential therapeutic target in sorafenib-resistant hepatocellular carcinoma.

BACKGROUND: Kinase suppressor of Ras 2 (KSR2) is a regulator of MAPK signaling that is overactivated in most hepatocellular carcinoma (HCC). We sought to determine the role of KSR2 in HCC pathogenesis. METHODS: We tested the level of KSR2 in HCC tissues and cell lines by tissue microarray, qPCR, and western blotting. Functionally, we determined the effects of KSR2 on the proliferation, migration, and invasion of HCC cells through colony formation assays, scratch assays, transwell migration assays, and xenograft tumor models. Co-immunoprecipitation (co-IP) experiments were used to assess the interaction of phospho-serine binding protein 14-3-3ζ and KSR2, and the effects of this interaction on growth and proliferation of human HCC cells were tested by co-overexpression and knockdown experiments. Additionally, we used flow cytometry to examine whether the KSR2 and 14-3-3ζ interaction conveys HCC resistance to sorafenib. RESULTS: KSR2 was significantly upregulated in HCC tissues and cell lines, and high KSR2 expression associated with poor prognosis in HCC patients. KSR2 knockdown significantly suppressed HCC cell proliferation, migration, and invasion in vitro and in vivo. Mechanistically, co-IP experiments identified that 14-3-3ζ complexed with KSR2, and elevated 14-3-3ζ increased KSR2 protein levels in HCC cells. Importantly, Kaplan-Meier survival analysis showed that patients with both high KSR2 and high 14-3-3ζ expression levels had the shortest survival times and poorest prognoses. Interestingly, HCC cells overexpressing both KSR2 and 14-3-3ζ, rather than either protein alone, showed hyperactivated MAPK signaling and resistance to sorafenib. CONCLUSIONS: Our results provide new insights into the pro-tumorigenic role of KSR2 and its regulation of the MAPK pathway in HCC. The KSR2-14-3-3ζ interaction may be a therapeutic target to enhance the sorafenib sensitivity of HCC.

The ribotoxin deoxynivalenol affects the viability and functions of glial cells.

Glial cells are responsible for maintaining brain homeostasis. Modification of the viability and functions of glial cells, including astrocytes and microglia, are associated with neuronal death and neurological diseases. Many toxins (heavy metals, pesticides, bacterial or viral toxins) are known to impact on brain cell viability and functions. Although recent publications suggest a potential link between environmental exposure of humans to mycotoxins and neurological diseases, data regarding the effects of fungal toxins on brain cells are scarce. In the present study, we looked at the impact of deoxynivalenol (DON), a fungal ribotoxin, on glial cells from animal and human origin. We found that DON decreased the viability of glial cells with a higher toxicity against microglial cells compared with astrocytes. In addition to cellular toxicity, DON affected key functions of glial cells. Thus, DON caused a biphasic effect on the neuroinflammatory response of microglia to lipopolysaccharide (LPS), while sublethal doses of DON increased the LPS-induced secretion of TNF-α and nitric oxide, toxic doses inhibited it. In addition to affecting microglial functions, sublethal doses of DON also suppressed the uptake of L-glutamate by astrocytes. This inhibition was associated with a modification of the expression of the glutamate transporters at the plasma membrane. Our results suggest that environmental ribotoxins such as DON could, at low doses, cause modifications of brain homeostasis and possibly participate in the etiology of neurological diseases in which alterations of the glia are involved.

Amplification of spatially isolated adenosine pathway by tumor-macrophage interaction induces anti-PD1 resistance in hepatocellular carcinoma.

BACKGROUND: Immune checkpoint blockade resistance narrows the efficacy of cancer immunotherapies, but the underlying mechanism remains elusive. Delineating the inherent mechanisms of anti-PD1 resistance is important to improve outcome of patients with advanced HCC. METHOD: The level of cricTMEM181 was measured in HCC patients with anti-PD1 therapy by RNA sequencing and then confirmed by qPCR and Sanger sequencing. Immune status in tumor microenvironment of HCC patients or mice models was evaluated by flow cytometry and IHC. Exosomes from HCC cell lines were isolated by ultracentrifugation, and their internalization by macrophage was confirmed by immunofluorescence. The underlying mechanism of HCC-derived exosomal circTMEM181 to macrophage was confirmed by SILAC, RNA FISH and RNA immunoprecipitation. The ATP-ADO pathway amplified by HCC-macrophage interaction was evaluated through ATP, AMP and ADO measurement and macrophage-specific CD39 knockout mice. The role of circTMEM181 in anti-PD1 therapy and its clinical significance were also determined in our retrospective HCC cohorts. RESULTS: Here, we found that circTMEM181 was elevated in hepatocellular carcinoma (HCC) patients responding poorly to anti-PD1 therapy and in HCC patients with a poor prognosis after operation. Moreover, we also found that high exosomal circTMEM181 favored the immunosuppressive microenvironment and endowed anti-PD1 resistance in HCC. Mechanistically, exosomal circTMEM181 sponged miR-488-3p and upregulated CD39 expression in macrophages. Using macrophage-specific CD39 knockout mice and pharmacologic approaches, we revealed a novel mode of anti-PD1 resistance in HCC. We discovered that cell-specific CD39 expression in macrophages and CD73 expression in HCC cells synergistically activated the eATP-adenosine pathway and produced more adenosine, thereby impairing CD8+ T cell function and driving anti-PD1 resistance. CONCLUSION: In summary, HCC-derived exosomal circTMEM181 contributes to immunosuppression and anti-PD1 resistance by elevating CD39 expression, and inhibiting the ATP-adenosine pathway by targeting CD39 on macrophages can rescue anti-PD1 therapy resistance in HCC.

CTLA-4 Synergizes With PD1/PD-L1 in the Inhibitory Tumor Microenvironment of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is highly invasive and carries high mortality due to limited therapeutic strategies. In other solid tumors, immune checkpoint inhibitors (ICIs) target cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD1), and the PD1 ligand PD-L1 has revolutionized treatment and improved outcomes. However, the relationship and clinical significance of CTLA-4 and PD-L1 expression in ICC remains to be addressed. Deciphering CTLA-4 and PD-L1 interactions in ICC enable targeted therapy for this disease. In this study, immunohistochemistry (IHC) was used to detect and quantify CTLA-4, forkhead box protein P3 (FOXP3), and PD-L1 in samples from 290 patients with ICC. The prognostic capabilities of CTLA-4, FOXP3, and PD-L1 expression in ICC were investigated with the Kaplan-Meier method. Independent risk factors related to ICC survival and recurrence were assessed by the Cox proportional hazards models. Here, we identified that CTLA-4+ lymphocyte density was elevated in ICC tumors compared with peritumoral hepatic tissues (P <.001), and patients with a high density of CTLA-4+ tumor-infiltrating lymphocytes (TILsCTLA-4 High) showed a reduced overall survival (OS) rate and increased cumulative recurrence rate compared with patients with TILsCTLA-4 Low (P <.001 and P = .024, respectively). Similarly, patients with high FOXP3+ TILs (TILsFOXP3 High) had poorer prognoses than patients with low FOXP3+ TILs (P = .021, P = .034, respectively), and the density of CTLA-4+ TILs was positively correlated with FOXP3+ TILs (Pearson r = .31, P <.001). Furthermore, patients with high PD-L1 expression in tumors (TumorPD-L1 High) and/or TILsCTLA-4 High presented worse OS and a higher recurrence rate than patients with TILsCTLA-4 LowTumorPD-L1 Low. Moreover, multiple tumors, lymph node metastasis, and high TumorPD-L1/TILsCTLA-4 were independent risk factors of cumulative recurrence and OS for patients after ICC tumor resection. Furthermore, among ICC patients, those with hepatolithiasis had a higher expression of CTLA-4 and worse OS compared with patients with HBV infection or undefined risk factors (P = .018). In conclusion, CTLA-4 is increased in TILs in ICC and has an expression profile distinct from PD1/PD-L1. TumorPD-L1/TILsCTLA-4 is a predictive factor of OS and ICC recurrence, suggesting that combined therapy targeting PD1/PD-L1 and CTLA-4 may be useful in treating patients with ICC.

Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation.

Membrane rafts are thought to be sphingolipid- and cholesterol-dependent lateral assemblies involved in diverse cellular functions. Their biological roles and even their existence, however, remain controversial. Using an original fluorescence correlation spectroscopy strategy that recently enabled us to identify nanoscale membrane organizations in live cells, we report here that highly dynamic nanodomains exist in both the outer and inner leaflets of the plasma membrane. Through specific inhibition of biosynthesis, we show that sphingolipids and cholesterol are essential and act in concert for formation of nanodomains, thus corroborating their raft nature. Moreover, we find that nanodomains play a crucial role in triggering the phosphatidylinositol-3 kinase/Akt signaling pathway, by facilitating Akt recruitment and activation upon phosphatidylinositol-3,4,5-triphosphate accumulation in the plasma membrane. Thus, through direct monitoring and controlled alterations of rafts in living cells, we demonstrate that rafts are critically involved in the activation of a signaling axis that is essential for cell physiology.