Targeting ATP-binding site of WRN Helicase: Identification of novel inhibitors through pocket analysis and Molecular Dynamics-Enhanced virtual screening.

WRN helicase is a critical protein involved in maintaining genomic stability, utilizing ATP hydrolysis to dissolve DNA secondary structures. It has been identified as a promising synthetic lethal target for microsatellite instable (MSI) cancers. However, few WRN helicase inhibitors have been discovered, and their potential binding sites remain unexplored. In this study, we analyzed potential binding sites for WRN inhibitors and focused on the ATP-binding site for screening new inhibitors. Through molecular dynamics-enhanced virtual screening, we identified two compounds, h6 and h15, which effectively inhibited WRN's helicase and ATPase activity in vitro. Importantly, these compounds selectively targeted WRN's ATPase activity, setting them apart from other non-homologous proteins with ATPase activity. In comparison to the homologous protein BLM, h6 exhibits some degree of selectivity towards WRN. We also investigated the binding mode of these compounds to WRN's ATP-binding sites. These findings offer a promising strategy for discovering new WRN inhibitors and present two novel scaffolds, which might be potential for the development of MSI cancer treatment.

Ethylene-MPK8-ERF.C1-PR module confers resistance against Botrytis cinerea in tomato fruit without compromising ripening.

The plant hormone ethylene plays a critical role in fruit defense against Botrytis cinerea attack, but the underlying mechanisms remain poorly understood. Here, we showed that ethylene response factor SlERF.C1 acts as a key regulator to trigger the ethylene-mediated defense against B. cinerea in tomato fruits without compromising ripening. Knockout of SlERF.C1 increased fruit susceptibility to B. cinerea with no effect on ripening process, while overexpression enhanced resistance. RNA-Seq, transactivation assays, EMSA and ChIP-qPCR results indicated that SlERF.C1 activated the transcription of PR genes by binding to their promoters. Moreover, SlERF.C1 interacted with the mitogen-activated protein kinase SlMPK8 which allowed SlMPK8 to phosphorylate SlERF.C1 at the Ser174 residue and increases its transcriptional activity. Knocking out of SlMPK8 increased fruit susceptibility to B. cinerea, whereas overexpression enhanced resistance without affecting ripening. Furthermore, genetic crosses between SlMPK8-KO and SlERF.C1-OE lines reduced the resistance to B. cinerea attack in SlERF.C1-OE fruits. In addition, B. cinerea infection induced ethylene production which in turn triggered SlMPK8 transcription and enhanced the phosphorylation of SlERF.C1. Overall, our findings reveal the regulatory mechanism of the 'Ethylene-MPK8-ERF.C1-PR' module in resistance against B. cinerea and provide new insight into the manipulation of gray mold disease in fruits.

Discovery of Novel Coumarin-quinolinium Derivatives as Pan-KRAS Translation Inhibitors by Targeting 5'-UTR RNA G-Quadruplexes.

Hyperactivated KRAS mutations fuel tumorigenesis and represent attractive targets for cancer treatment. While covalent inhibitors have shown clinical benefits against the KRASG12C mutant, advancements for non-G12C mutants remain limited, highlighting the urgent demand for pan-KRAS inhibitors. RNA G-quadruplexes (rG4s) in the 5'-untranslated region of KRAS mRNA can regulate KRAS translation, making them promising targets for pan-KRAS inhibitor development. Herein, we designed and synthesized 50 novel coumarin-quinolinium derivatives, leveraging our previously developed rG4-specific ligand, QUMA-1. Notably, several compounds exhibited potent antiproliferative activity against cancer cells as pan-KRAS translation inhibitors. Among them, 15a displayed exceptional capability in stabilizing KRAS rG4s, suppressing KRAS translation, and consequently modulating MAPK and PI3K-AKT pathways. 15a induced cell cycle arrest, prompted apoptosis in KRAS-driven cancer cells, and effectively inhibited tumor growth in a KRAS mutant xenograft model. These findings underscore the potential of 15a as a pan-KRAS translation inhibitor, offering a novel and promising approach to target various KRAS-driven cancers.

Knockdown of NADPH oxidase 4 reduces mitochondrial oxidative stress and neuronal pyroptosis following intracerebral hemorrhage.

Intracerebral hemorrhage is often accompanied by oxidative stress induced by reactive oxygen species, which causes abnormal mitochondrial function and secondary reactive oxygen species generation. This creates a vicious cycle leading to reactive oxygen species accumulation, resulting in progression of the pathological process. Therefore, breaking the cycle to inhibit reactive oxygen species accumulation is critical for reducing neuronal death after intracerebral hemorrhage. Our previous study found that increased expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (NADPH oxidase 4, NOX4) led to neuronal apoptosis and damage to the blood-brain barrier after intracerebral hemorrhage. The purpose of this study was to investigate the role of NOX4 in the circle involving the neuronal tolerance to oxidative stress, mitochondrial reactive oxygen species and modes of neuronal death other than apoptosis after intracerebral hemorrhage. We found that NOX4 knockdown by adeno-associated virus (AAV-NOX4) in rats enhanced neuronal tolerance to oxidative stress, enabling them to better resist the oxidative stress caused by intracerebral hemorrhage. Knockdown of NOX4 also reduced the production of reactive oxygen species in the mitochondria, relieved mitochondrial damage, prevented secondary reactive oxygen species accumulation, reduced neuronal pyroptosis and contributed to relieving secondary brain injury after intracerebral hemorrhage in rats. Finally, we used a mitochondria-targeted superoxide dismutase mimetic to explore the relationship between reactive oxygen species and NOX4. The mitochondria-targeted superoxide dismutase mimetic inhibited the expression of NOX4 and neuronal pyroptosis, which is similar to the effect of AAV-NOX4. This indicates that NOX4 is likely to be an important target for inhibiting mitochondrial reactive oxygen species production, and NOX4 inhibitors can be used to alleviate oxidative stress response induced by intracerebral hemorrhage.

Effects of cadmium stress on the morphology, physiology, cellular ultrastructure, and BvHIPP24 gene expression of sugar beet (Beta vulgaris L.).

To clarify the mechanism of the response of sugar beet (Beta vulgaris L.) to cadmium (Cd) stress, this study investigated changes in the phenotype, physiological indexes, and subcellular structure of B. vulgaris under Cd treatment and the transcriptional pattern of the BvHIPP24 gene (a heavy metal-associated isoprenylated plant protein involved in heavy metal detoxification). The plant height and shoot and root growth of B. vulgaris seedlings were inhibited to some extent under 0.5 and 1 mM Cd, with gradually wilting and yellowing of leaves and dark brown roots. When the Cd concentration was increased, malondialdehyde content and the activities of peroxidase, superoxide dismutase, and glutathione S-transferase increased differentially. qPCR indicated that the expression of BvHIPP24 was induced by different concentrations of Cd. Although transmission electron microscopy revealed damage to nuclei, mitochondria, and chloroplasts, B. vulgaris exhibited strong adaptability to 0.5 mM Cd according to a comprehensive analysis using the membership function. The results showed that B. vulgaris may reduce cell damage and improve its Cd tolerance by regulating functional gene expression and antioxidant enzymes. This study increases our understanding of the Cd-tolerance mechanism of B. vulgaris and provides insights into the use of B. vulgaris in Cd bioremediation.

Sugar beet is a novel energy crop with superior characteristics for both heavy metal phytoremediation and biomass energy development. This work is the first to investigate both the morphological, physiological, and ultrastructural response of sugar beet to cadmium stress and the induction of a functional metallochaperone gene by cadmium. This study explains the cadmium tolerance mechanism of sugar beet based on a comprehensive evaluation and provides an important theoretical basis for further application of beet in heavy metal bioremediation.

Lysionotin Induces Ferroptosis to Suppress Development of Colorectal Cancer via Promoting Nrf2 Degradation.

Extensive use of substances derived from natural sources has been documented in the treatment of colorectal cancer (CRC). Lysionotin (Lys) is a flavonoid present in the flowers and leaves of Gesneriaceae family plants. Despite its various pharmacological properties, which include neuroprotective, pro, antimalarial, and anticancer effects, the therapeutic advantages of Lys for CRC remain uncertain. In this present study, we demonstrated that Lys treatment successfully inhibited cell proliferation, migration, and invasion in HCT116 and SW480 CRC cells in vitro. Intriguingly, significant ferroptosis and reactive oxygen species (ROS) accumulation in CRC cells were induced by Lys treatment, whereas antagonism of ferroptosis by Liproxstatin-1 (Lip1) pretreatment retarded the anti-CRC effects of Lys. In addition, Lys reduced the amount of Nrf2 protein in CRC cells by increasing the rate at which it is degraded. Overexpression of Nrf2 rescued Lys reduced ferroptosis, suggesting the Nrf2 signaling is a crucial determinant of whether Lys induces ferroptosis in CRC cells. We also revealed that Lys suppressed tumor growth in vivo without obvious adverse effects on the main organs of mice. In conclusion, our results discovered that Lys treatment induced ferroptosis to exert antitumor effects in HCT116 and SW480 CRC cells by modulating Nrf2 signaling, providing a potential therapeutic approach for the prevention of colorectal cancer.

Fabrication of Nano Iron Oxide-Modified Biochar from Co-Hydrothermal Carbonization of Microalgae and Fe(II) Salt for Efficient Removal of Rhodamine B.

Dye adsorption by magnetic modified biochar has now received growing interest due to its excellent adsorption performance and facile separation for recycling. In this study, nano iron oxide-modified biochar was fabricated via the successive hydrothermal-pyrolyzing method using Chlorella vulgaris (Cv) and FeSO4·7H2O as raw materials, and its adsorption on Rhodamine B (RhB) in aqueous solution was studied. Multiple techniques such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Brunauer-Emmett-Teller (BET), vibrating sample magnetometry (VSM) and X-ray photoelectron spectroscopy (XPS) were employed to comprehensively characterize the structure, morphology and physicochemical properties of the adsorbent. The as-synthesized nano iron oxide-modified biochar (CBC-Fe(II)) exhibited a large surface area (527.6 m2/g) and high magnetic saturation value (13.7 emu/g) to facilitate magnetic separation. Compared with CBC and CBC-Fe(III), CBC-Fe(II) exhibited superior adsorption ability towards RhB in aqueous solution, with a maximum adsorption capacity of 286.4 mg/g. The adsorption process of RhB onto CBC-Fe(II) was well described by the pseudo-second-order kinetic model and Langmuir isotherm model, indicating monolayer chemisorption behaviors for the adsorption system. Facile preparation, great adsorption performance and magnetic recovery properties endow CBC-Fe(II) to be a promising adsorbent for dye removal.

Dysregulation of MiR-144-5p/RNF187 Axis Contributes To the Progression of Colorectal Cancer.

Background and Objectives: RING finger protein 187 (RNF187) belongs to RING domain-containing E3 ligases family, which was recently reported to be involved in oncogenesis and development of several cancers. This research aims to clarify the role of RNF187 in colorectal cancer (CRC) development. Methods: The expression of RNF187 and miR-144-4p were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The levels of RNF187 protein were assessed by western blot analysis. Cell Counting Kit-8 (CCK8) assay, clonogenic assay, cell scratch test and transwell assay were used to determine the proliferation, migration and invasion of CRC cells in vitro. The binding of miR-144-5p and RNF197 mRNA was validated by luciferase reporter assays. Tumor-bearing nude mice were used to determine CRC cells growth in vivo. Results: RNF187 expression significantly increased in CRC specimens and cell lines compared to normal colon tissues and normal colonic mucosa cell line, respectively. Upregulation of RNF187 expression was inversely correlated to poor prognosis in CRC patients. In addition, knockdown of RNF187 expression inhibited the proliferation, migration, and invasion but promoted the apoptosis of CRC lines Caco-2 and SW480 cells. Further studies validated that RNF187 was the direct target of miR-144-5p. The expression of miR-144-5p was downregulated in CRC tissues, which was negatively correlated to the expression of RNF187. Restoration of miR-144-5p significantly inhibited the progression of CRC cells and its anti-tumor effects could be abrogated by overexpression of RNF187. Conclusion: Our findings demonstrate the deregulation of miR-144-5p/ RNF187 axis in CRC, as well as its role in regulation of the tumor progression, thus providing a novel therapeutic strategy for CRC treatment.

Comparison of Minimally Invasive Fixation of Mid-Shaft Clavicular Fractures with Threaded Elastic Intramedullary Nail (TEIN) and Elastic Locking Intramedullary Nail (ELIN).

OBJECTIVE: To compare the effectiveness of threaded elastic intramedullary nail and elastic locking intradullary nail (ELIN) for mid-shaft clavicular fractures. METHODS: The clinical data of 47 patients with middle clavicle fracture treated by TEIN and ELIN from August 2017 to March 2019 were analyzed retrospectively. Twenty-three patients received intramedullary fixation treatment with ELIN, nine males and 14 females, AO/OTA fracture classification type 2A (n = 17) and 2B (n = 6). Twenty-four patients received intramedullary fixation treatment with TEIN, including nine males and 15 females, AO/OTA classification: type 2A (n = 18) and 2B (n = 6). All patients were anesthetized with ipsilateral cervical plexus block. After internal fixation was removed, the clinical outcomes were assessed and evaluated. The Constant-Murley score and disabilities of the arm, shoulder and hand questionnaire (DASH) score were compared between the two groups to evaluate the functional status of all patients. The study was done accordingly to the guidelines provided by the ethics committee. RESULTS: All patients in the two groups completed the operation successfully and were followed up. In the ELIN group, the operation time was 20.78 ± 7.71 min, intra-operative blood loss was 13.26 ± 9.72 mL, incision length was 1.60 ± 0.92 cm, internal fixation removal time was 12.86 ± 2.24 weeks, Constant-Murley score was 99.30 ± 1.36 points and DASH score was 1.43 ± 3.00 points. In the TEIN group, the operation time, intra-operative blood loss, incision length, internal fixation removal time, Constant-Murley and DASH scores were 22.83 ± 8.17 min, 22.08 ± 11.22 mL, 2.48 ± 0.84 cm, 15.66 ± 5.58 weeks, 95.79 ± 7.38 point and 6.69 ± 11.55 point respectively. In the ELIN group, four cases developed skin irritation, and the symptoms were relieved after removal of internal fixation. In the TEIN group, one patient's internal fixation broke and had an obvious scar at the incision, but there was no fracture after replacement of internal fixation; withdrawal of TEIN occurred in four patients, the nail did not shift again until the last follow-up; skin irritation and temporary bursitis occurred in six patients, and the symptoms were relieved after internal fixation was removed. No other conditions were found in the patients, and bony healing was achieved in all patients. CONCLUSION: ELIN prevents shortening and malunion of the clavicle, reduces secondary damage to related tissues, and leads to restoration of clavicle length and faster osseous healing.

Identification of Key Candidate Genes and Chemical Perturbagens in Diabetic Kidney Disease Using Integrated Bioinformatics Analysis.

Globally, nearly 40 percent of all diabetic patients develop serious diabetic kidney disease (DKD). The identification of the potential early-stage biomarkers and elucidation of their underlying molecular mechanisms in DKD are required. In this study, we performed integrated bioinformatics analysis on the expression profiles GSE111154, GSE30528 and GSE30529 associated with early diabetic nephropathy (EDN), glomerular DKD (GDKD) and tubular DKD (TDKD), respectively. A total of 1,241, 318 and 280 differentially expressed genes (DEGs) were identified for GSE30258, GSE30529, and GSE111154 respectively. Subsequently, 280 upregulated and 27 downregulated DEGs shared between the three GSE datasets were identified. Further analysis of the gene expression levels conducted on the hub genes revealed SPARC (Secreted Protein Acidic And Cysteine Rich), POSTN (periostin), LUM (Lumican), KNG1 (Kininogen 1), FN1 (Fibronectin 1), VCAN (Versican) and PTPRO (Protein Tyrosine Phosphatase Receptor Type O) having potential roles in DKD progression. FN1, LUM and VCAN were identified as upregulated genes for GDKD whereas the downregulation of PTPRO was associated with all three diseases. Both POSTN and SPARC were identified as the overexpressed putative biomarkers whereas KNG1 was found as downregulated in TDKD. Additionally, we also identified two drugs, namely pidorubicine, a topoisomerase inhibitor (LINCS ID- BRD-K04548931) and Polo-like kinase inhibitor (LINCS ID- BRD-K41652870) having the validated role in reversing the differential gene expression patterns observed in the three GSE datasets used. Collectively, this study aids in the understanding of the molecular drivers, critical genes and pathways that underlie DKD initiation and progression.

HNRNPC impedes m6A-dependent anti-metastatic alternative splicing events in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a malignancy with poor prognosis due to early metastasis. The aberrant N6-methyladenosine (m6A) RNA modification has emerged as an important mechanism in cancer progression and metastasis, but its role in PDAC remained largely unknown. Here, we demonstrated that an m6A regulator, heterogeneous nuclear ribonucleoprotein C (HNRNPC), modulated alternative splicing events to promote PDAC metastasis. In clinical PDAC tissues, high expression of HNRNPC was correlated with metastasis, resulting in poor prognosis in PDAC patients. Knockdown of HNRNPC significantly reduced PDAC cell invasion in vitro and metastasis in vivo. In contrast, overexpression of HNRNPC provoked malignant phenotypes of PDAC cells. Mechanistically, HNRNPC antagonized the anti-metastatic isoform of TAF8 (TAF8L) but increased the pro-metastatic alternative splicing isoform of TAF8 (TAF8S). Mutation of the m6A-site of TAF8 attenuated the interaction between HNRNPC and TAF8 transcript, leading to the decrease of TAF8S. Furthermore, experimental manipulation of the anti-metastasis splicing isoform TAF8L revealed that splice isoform switching of TAF8 is crucial for PDAC metastasis. In conclusion, our findings demonstrate the essentiality of HNRNPC-mediated alternative splicing events that impinges on metastatic PDAC.

[Exhaustion of CD8+T Lymphocytes Plays a Critical Role in the Pathogenesis of Secondary Hemophagocytic Lymphohistiocytosis].

OBJECTIVE: To investigate the changes in function of CD8+ T cell subsets, and explore its significance in pathogenesis of secondary hemophagocytic lymphohistiocytosis (sHLH). METHODS: Flow cytometry was used to detect the expressions of PD-1, TIM-3, and LAG-3, which were the markers of exhausted CD8+ T cell, as well as the secretion levels of interferon γ (IFN-γ) and expression of ΔCD107a after the stimulation; the numbers of effector-memory CD8+ T cells, regulatory T cells and double negative T cells were also detected. RESULTS: The expressions of inhibitory receptors (PD-1, TIM3 and LAG-3) on CD8+ T cells of sHLH patients were 40.73±22.64, 15.97±14.45 and 0.73 (0-37.41), respectively, which were significantly higher than those of the control group (P<0.001). In contrast, the expression of ΔCD107a (4.49±2.71 vs 6.07±2.14, P=0.035) and secretion level of IFN-γ (37.30±24.46 vs 55.17±22.23, P=0.034) were significantly lower. The number of effector-memory CD8+ T cells in sHLH patients was also lower as compared with that in control group (14.35±10.37 vs 22.92±11.12, P=0.016). But there was no significant difference in the number of regulatory T cell and double negative T cell. In addition, the expressions of PD-1, TIM3 and LAG-3 in active stage of sHLH were 38.09±21.87, 14.35±13.70 and 0.82 (0-13.22), respectively, which were significant higher than those in remission stage [24.27±17.23 (P=0.03), 8.64±5.60 (P=0.014) and 0.13 (0-3.69)]. CONCLUSION: The exhausted CD8+ T lymphocytes may play a critical role in the development of sHLH.

Paeoniflorin elicits the anti-proliferative effects on glioma cell via targeting translocator protein 18 KDa.

As a natural compound isolated from Paeoniae radix, Paeoniflorin (PF) has been shown the antitumor effects in various types of human cancers including glioma, which is one of the serious tumors in central nervous system. Translocator protein 18 KDa (TSPO) has been shown to be relevant to the glioma aetiology. However, the regulation of PF in TSPO and neurosteriods biosynthesis on glioma is still unclear. In the present study, the glioma cell (U87 and U251) were cultured and used to quantify the bindings of PF on TSPO. Results indicated that there was not significant different between IC50 of PF and TSPO ligand PK11195. Moreover, PF exerted the anti-proliferative effects in glioma cell with a dose dependent inhibition from 12.5 to 100 µM in vitro. Consistent with the effects of PK11195, lowered levels on progesterone, allopregnanolone, as well as TSPO mRNA were induced by PF (25 and 50 µM). Furthermore, a xenograft mouse model with U87 cell-derived was significant inhibited by PF treatment, as well as the PK11195 administration. These results demonstrate that PF exerts its antitumor effects associated with the TSPO and neurosteroids biosynthesis in glioma cells could be a promising therapeutic agent for glioma therapy.

[Establishment of Secondary HLH Mouse Model and Effect of Ruxolitinib on Disease Manifestations of Model Mide].

OBJECTIVE: To establish a secondary hemophagocytic lymphohistiocytosis(HLH) mouse model, and to investigate the effect of ruxolitinib on the disease manifestation of model mice. METHODS: Wild type C57BL/6 mice were randomly divided into 4 groups: two groups of mice were intraperitoneally injected with CpG oligodeoxynucleotide 1826 (CpG-ODN1826) every other day to induce HLH, and other two groups were control groups. One group of the CpG-ODN1826 groups and one of the control groups were given ruxolitinib, and other two groups were given the same amount of PBS. Blood samples, serum ferritin and hepatic/spleen weights of experimental mice were detected and serum cytokine levels were measured by ELISA. RESULTS: Compared with the control groups, the levels of white blood cells, hemoglobin and platelets in the CpG-ODN1826 groups were significantly lower (P＜0.05); and liver/body weight, spleen/body weight, serum ferritin, sCD25, IL-10, IL-1ß, IFN-Ƴ, IL-12p70, GM-CSF, TNF-α and IL-18 levels significantly increased (P＜0.05). There was no significant difference in the levels of IL-2, IL-4, IL-5, IL-6, IL-22, IL-13, IL-27 and IL-23 between the two groups (P＞0.05). The spleen in CpG group had disordered internal structure, expanding red pulp and hyperplastic nucleated cells. The liver had severe perivascular inflammations. The spleen/weight of the ruxolitinib-treated mice in the CpG-ODN1826 group was significantly smaller than that of the unapplied ruxolitinib (P＜0.05). CONCLUSION: The CpG-ODN1826 can induce secondary HLH symptoms in wild type C57BL/6 mice. Ruxolitinib can alleviate the symptoms of splenomegaly in HLH model mice.

Genotype characteristics and immunological indicator evaluation of 311 hemophagocytic lymphohistiocytosis cases in China.

BACKGROUND: Primary hemophagocytic lymphohistiocytosis (pHLH) is a genetic disorder that is classically diagnosed by genetic testing. Secondary HLH (sHLH) is usually caused by infections, malignancies, or autoimmune disorders, but may display some mutations or polymorphisms. Rapid immunological assays examining natural killer (NK) cell activity, degranulation function (CD107a), and protein expression related to genetic deficiencies have been recommended for early pHLH identification. METHODS: A retrospective analysis of 311 HLH patients from a Chinese population was performed to evaluate the potential correlations between genetic testing and rapid immunological assays; genotyping characteristics, age of onset, and etiology were examined. RESULTS: Among the 128 (128/311) patients who were positive in the genetic screening, the most frequently detected mutant gene was UNC13D (29%), followed by LYST (21%), PRF1 (17%), and STXBP2 (10%). Among pHLH patients (n = 39), the majority (67%) had PRF1 and UNC13D defects. FHL-2 was predominant (12/27, 44%) in patients aged under 18, while FHL-3 was the most common (6/12, 50%) in adults. Differences in genetic variant types and etiological components were noted in HLH patients based on the age of onset. NK cell activity and CD107a were observed to show a consistent trend (Ptrend < 0.001) when grouping patients according to the severity of the genetic variant type. Moreover, NK cell activity was generally consistent within a certain range of ΔCD107a values (Ptrend < 0.001). The PPV for bi-allelic degranulation gene mutations in patients with CD107a < 5% was 38.9% (7/18), while the PPV in patients with CD107a ≤10% was 16.7% (13/78). The PPV for pHLH was 41.4% (29/70) with NK cell activity ≤13%. To further evaluate the diagnostic efficacy of NK cell activity assay in pHLH, a receiver operating characteristic (ROC) curve was generated and showed an area under the curve (AUC) of 0.872, and the optimal cutoff value was determined to be 13.425% with a sensitivity of 84.21% and specificity of 80.67% when the corresponding Youden index was maximized. Flow cytometry screening for deficient proteins, including perforin, SAP, and XIAP, showed a relatively high sensitivity (83.33-93.33%). The positive predictive values (PPVs) of perforin and XIAP were relatively low (20.83-26.92%), but the negative predictive values (NPVs) for all three were excellent (all > 98%). CONCLUSIONS: Various immunological indicators have different clinical prediction and application values for the diagnosis of pHLH. The degree of reduction of immunological indicators also needs attention, and choosing appropriate cutoff value may be of important significance in guiding clinical judgment for pHLH.

The role of allogeneic hematopoietic stem cell transplantation and Epstein-Barr virus infection on the treatment for child primary hemophagocytic lymphohistiocytosis patients with X-linked lymphoproliferative disease: A rare case report and family survey study.

XLP-2 is known as a rare primary immunodeficiency disease, which is characterized by the susceptibility to EBV infection and potential development into the pHLH. The existing studies believe that the dysfunction of XIAP represents one of the most significant pathogenic mechanisms of XLP-2, and allo-HSCT is regarded as a crucial treatment for the long-term survival in XLP-2 patients. In our present study, a 2-year-old male patient was diagnosed with XLP-2. After receiving chemotherapy by using HLH-2004 without allo-HSCT, he reached a complete remission, and his EBV load was brought under control. Our family survey revealed a novel frameshift mutation in the XIAP gene in this patient, as well as in his cousin and grandfather. Until now, the patient has been followed up for 22 months with no recurrence reported yet. Based on these findings, it is believed that for child pHLH patients with XLP-2, the treatment by controlling symptoms alone without allo-HSCT and with regular monitoring of EBV load could be conducive to a long-term survival.

MFAP5 facilitates the aggressiveness of intrahepatic Cholangiocarcinoma by activating the Notch1 signaling pathway.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer. The dismal outcome of ICC patients is due to lack of early diagnosis, the aggressive biological behavior of ICC and the lack of effective therapeutic options. Early diagnosis and prognosis of ICC by non-invasive methods would be helpful in providing valuable information and developing effective treatment strategies. METHODS: Expression of microfibrillar-associated protein 5 (MFAP5) in the serum of ICC patients was detected by ELISA. Human ICC specimens were immunostained by MFAP5 antibodies. The growth rate of human ICC cell lines treated with MFAP5 or MFAP5 shRNAs was examined by CCK8 and colony formation assays. Cell cycle analysis was performed with PI staining. The effect of MFAP5 inhibition was assessed by xenograft models in nude mice. RNA-seq and ATAC-seq analyses were used to dissect the molecular mechanism by which MFAP5 promoted ICC aggressiveness. RESULTS: We identified MFAP5 as a biomarker for the diagnosis and prognosis of ICC. Upregulated MFAP5 is a common feature in aggressive ICC patients' tissues. Importantly, MFAP5 level in the serum of ICC patients and healthy individuals showed significant differential expression profiles. Furthermore, we showed that MFAP5 promoted ICC cell growth and G1 to S-phase transition. Using RNA-seq expression and ATAC-seq chromatin accessibility profiling of ICC cells with suppressed MFAP5 secretion, we showed that MFAP5 regulated the expression of genes involved in the Notch1 signaling pathway. Furthermore, FLI-06, a Notch signaling inhibitor, completely abolished the MFAP5-dependent transcriptional programs. CONCLUSIONS: Raised MFAP5 serum level is useful for differentiating ICC patients from healthy individuals, and could be helpful in ICC diagnosis, prognosis and therapies.

The inhibitory receptors on NK cells and CTLs are upregulated in adult and adolescent patients with secondary hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) includes primary HLH (pHLH) and secondary HLH (sHLH). Mutations that cause abnormal functions in natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) are frequently identified in pHLH. However, why NK cells and CTLs exhibit abnormal functions in sHLH remains unclear. Here, we demonstrated that NK cells in sHLH exhibited a high expression of inhibitory receptor NKG2A and a low expression of activating receptor NKG2D. Besides, the expression of HLA-E on lymphocyte, the adaptor of NKG2A on NK cells, was elevated in sHLH. Moreover, CTLs in sHLH patients expressed a higher level of functional exhaustion markers PD-1, TIM-3 and LAG-3 as well as a lower secretion of IFN-γ and CD107a upon stimulation. In addition, the expression of MHC-I on lymphocytes was decreased. Taken together, our study indicates a potentially pathological mechanism of sHLH and may open up new avenues for the development of immunotherapies against sHLH.

Reactive oxygen species are responsible for the cell aggregation and production of pro-inflammatory mediators in phorbol ester (PMA)-treated U937 cells on gelatin-coated dishes through upregulation of autophagy.

Purpose: Our previous studies indicate that phorbol 12-myristate 13-acetate (PMA)-treated U937 cells cultured on collagen I-coated dishes express lowered production of pro-inflammatory mediators in parallel through reduced reactive oxygen species (ROS) levels. By contrast, PMA-treated U937 cells on gelatin, the denatured collagen, show enhanced production of pro-inflammatory mediators, mediated by up-regulating autophagy levels. The present study is aimed to investigate the effect of ROS levels in PMA-treated U937 cells cultured on gelatin-coated surface. Material and methods: MTT assay, flow cytometric analysis of ROS and autophagy, biochemical detection of antioxidant levels, enzyme-linked immunosorbent assay, and western blot were used. Results: Gelatin-coating increased ROS levels in PMA-treated U937 cells. Increased ROS levels are involved in the regulation of cell aggregation and the release of pro-inflammatory mediators in gelatin-coated culture. These results lead to the query about the crosstalk between the two positive regulators, the autophagy and ROS. Autophagy induction is attenuated by N-acetyl-L-cysteine treatment, but the treatment with autophagy inhibitor, 3-methyladenine, does not affect ROS levels, suggesting ROS are upstream of autophagy in the regulation axis of differentiated U937 cells on gelatin-coated surface. Further study confirmed that upregulation of autophagy was responsible for ROS-induced cell aggregation and production of pro-inflammatory mediators. Conclusion: The results suggest that gelatin-coating promotes the aggregation of PMA-treated U937 cells and the production of pro-inflammatory mediators by ROS-autophagy signaling pathway.