CYLD alleviates NLRP3 inflammasome-mediated pyroptosis in osteoporosis by deubiquitinating WNK1.

BACKGROUND: Osteoporosis (OP) is the result of bone mass reduction and bone structure disorder. Bone marrow mesenchymal stem cells (BMSCs) are the main source of osteogenic precursor cells involved in adult bone remodeling. The involvement of the deubiquitinating enzyme CYLD in OP has recently been discovered. However, the detailed role and mechanism of CYLD remain unknown. METHODS: The OP mouse model was established by performing ovariectomy (OVX) on mice. Hematoxylin and eosin staining, Masson and Immunohistochemical staining were used to assess pathologic changes. Real-time quantitative PCR, Western blot, and immunofluorescence were employed to assess the expression levels of CYLD, WNK1, NLRP3 and osteogenesis-related molecules. The binding relationship between CYLD and WNK1 was validated through a co-immunoprecipitation assay. The osteogenic capacity of BMSCs was determined using Alkaline phosphatase (ALP) and alizarin red staining (ARS). Protein ubiquitination was evaluated by a ubiquitination assay. RESULTS: The levels of both CYLD and WNK1 were decreased in bone tissues and BMSCs of OVX mice. Overexpression of CYLD or WNK1 induced osteogenic differentiation in BMSCs. Additionally, NLRP3 inflammation was activated in OVX mice, but its activation was attenuated upon overexpression of CYLD or WNK1. CYLD was observed to reduce the ubiquitination of WNK1, thereby enhancing its protein stability and leading to the inactivation of NLRP3 inflammation. However, the protective effects of CYLD on osteogenic differentiation and NLRP3 inflammation inactivation were diminished upon silencing of WNK1. CONCLUSION: CYLD mitigates NLRP3 inflammasome-triggered pyroptosis in osteoporosis through its deubiquitination of WNK1.

Approach to difficult-to-treat asthma in childhood: a narrative review.

ABSTRACT: Asthma is a major chronic disease affecting children, and children with difficult-to-treat asthma account for a disproportionate share of resource utilisation and healthcare costs. This review presents a comprehensive and up-to-date overview of the treatment strategies in difficult-to-treat paediatric asthma. Mimickers of asthma must first be ruled out, and the diagnosis confirmed with objective tests whenever possible. The effect of comorbid conditions such as obesity, smoking, other atopic conditions and psychosocial factors on asthma control and severity should be considered. Treatment can then be optimised by implementing personalised strategies, including the use of appropriate drug delivery devices and adherence monitoring. Biologics can be an alternative treatment option for selected patients but should not be a substitute for addressing poor adherence. Many patients with difficult-to-treat asthma may not have severe asthma, and the physician should work with patients and families to achieve good asthma control via an individualised approach.

Innexin-Mediated Adhesion between Glia Is Required for Axon Ensheathment in the Peripheral Nervous System.

Glia are essential to protecting and enabling nervous system function and a key glial function is the formation of the glial sheath around peripheral axons. Each peripheral nerve in the Drosophila larva is ensheathed by three glial layers, which structurally support and insulate the peripheral axons. How peripheral glia communicate with each other and between layers is not well established and we investigated the role of Innexins in mediating glial function in the Drosophila periphery. Of the eight Drosophila Innexins, we found two (Inx1 and Inx2) are important for peripheral glia development. In particular loss of Inx1 and Inx2 resulted in defects in the wrapping glia leading to disruption of the glia wrap. Of interest loss of Inx2 in the subperineurial glia also resulted in defects in the neighboring wrapping glia. Inx plaques were observed between the subperineurial glia and the wrapping glia suggesting that gap junctions link these two glial cell types. We found Inx2 is key to Ca2+ pulses in the peripheral subperineurial glia but not in the wrapping glia, and we found no evidence of gap junction communication between subperineurial and wrapping glia. Rather we have clear evidence that Inx2 plays an adhesive and channel-independent role between the subperineurial and wrapping glia to ensure the integrity of the glial wrap.SIGNIFICANCE STATEMENT Gap junctions are critical for glia communication and formation of myelin in myelinating glia. However, the role of gap junctions in non-myelinating glia is not well studied, yet non-myelinating glia are critical for peripheral nerve function. We found the Innexin gap junction proteins are present between different classes of peripheral glia in Drosophila. Here Innexins form junctions to facilitate adhesion between the different glia but do so in a channel-independent manner. Loss of adhesion leads to disruption of the glial wrap around axons and leads to fragmentation of the wrapping glia membranes. Our work points to an important role for gap junction proteins in mediating insulation by non-myelinating glia.

Using genotype to assist clinical surveillance: a retrospective study of Chinese familial adenomatous polyposis patients.

Without treatment, familial adenomatous polyposis (FAP) patients will inevitably develop colorectal cancer (CRC) during lifetime. Yet, surgical trauma is a high risk of desmoid tumor (DT), one of the main causes of death in FAP patients. So far, the timing for colectomy is primarily based on the clinician's experience and the patient's preference; most patients undergo surgery at mid-20's. In this study, we analyzed the germline mutation distribution in 35 FAP patients from different families, 16 of them diagnosed with DTs. We also investigated the association between the molecular alterations and the clinicopathological features. Capture-based targeted sequencing using a panel of 520 genes was performed on tumor tissue and paired normal mucosa or white blood cells from 18 FAP probands who were initially diagnosed with CRC. Of all 35 FAP patients, 30 (85.7%) of them harbored germline APC mutations scattered from codon 161 to 1578. The mutations in the 16 DT patients scattered from codon 457 to 1578. All three patients with the mutation at the 3' of 1444 codon were diagnosed with DT. The percentage of high-risk DT (stage III or IV) harboring mutations at the 5' of 1062 or 1062-1578 was 14.3% and 77.8%, respectively, and all three patients with 3' of 1399 codon mutation had high risk. In addition, by using public database, we compared 140 FAP patients with DT to all 1880 FAP patients on the Leiden Open Variation Database and found that the odd ratio of DT in codon 159 to 495 was 0.34, while in codon 1310 to 2011 was 2.36. Compared to sporadic CRCs, the somatic spectrum of FAP CRCs was similar to the early onset CRCs, with higher TP53 (94.1%) and lower somatic APC mutations (65.7%), but the KRAS mutation rate was the highest (58.5%). One of the 18 FAP CRCs was identified as microsatellite instability-high (MSI-H), with tumor mutation burden (TMB) of 115.65 mut/Mb. Given that no TP53 mutations were detected in the low- and high-grade adenomas, ctDNA TP53 sequencing might be used for the close monitoring before FAP colectomy. In conclusion, except mutations at the 5' end of APC (5' to 495), all FAP patients need to consider the risk of DT after colectomy. The chance of life-threating DTs was higher in patients with 3' 1062 codon mutation and peaked in patients with 3' 1399 codon mutation. Scheduled monitoring of TP53 ctDNA is proposed to be a novel tool for optimizing the operation time.

The Formation Mechanism of Nanocrystals after Martensitic Transformation.

Understanding the ultrafine substructure in freshly formed Fe-C martensite is the key point to reveal the real martensitic transformation mechanism. As-quenched martensite, whose transformation temperature is close to room temperature, has been investigated in detail by means of transmission electron microscopy (TEM) in this study. The observation results revealed that the freshly formed martensite after quenching is actually composed of ultrafine crystallites with a grain size of 1−2 nm. The present observation result matches well with the suggestion based on X-ray studies carried out one hundred years ago. Such nanocrystals are distributed throughout the entire martensite. The whole martensite shows a uniform contrast under both bright and dark field observation modes, irrespective of what observation directions are chosen. No defect contrast can be observed inside each nanocrystal. However, a body-centered cubic {112}<111>-type twinning relationship exists among the ultrafine α-Fe grains. Such ultrafine α-Fe grains or crystallites are the root cause of the fine microstructure formed in martensitic steels and high hardness after martensitic transformation. The formation mechanism of the ultrafine α-Fe grains in the freshly formed martensite will be discussed based on a new γ → α phase transformation mechanism.

Liquiritigenin decreases tumorigenesis by inhibiting DNMT activity and increasing BRCA1 transcriptional activity in triple-negative breast cancer.

As a selective estrogen receptor ß agonist, the natural flavonoid liquiritigenin reportedly inhibits invasiveness of breast cancer cells, but its specific role and mechanism remain largely unclear. In this study, cells from the triple negative breast cancer lines MDA-MB-231 and BT549 were incubated with different concentrations of liquiritigenin. The results indicated that low concentrations had no significant cytotoxic effect, whereas high concentrations decreased viability of both MDA-MB-231 and BT549 cells. Liquiritigenin treatment also resulted in increased apoptosis and enhanced Caspase3 activity. After liquiritigenin treatment, we observed decreased invasive and migratory capacities of cells, as well as upregulated E-cadherin and downregulated N-cadherin, vimentin, and MMP9. Interestingly, liquiritigenin increased the mRNA and protein expression of breast cancer 1 (BRCA1). It also increased p21 and growth arrest and DNA-damage-inducible 45 alpha (GADD45A) levels, accompanied by decreased cellular DNA methyltransferase (DNMT) activity and downregulation of DNMT1, DNMT3a, and DNMT3b. These findings suggest that liquiritigenin can inhibit malignant behavior of triple negative breast cancer cells by inhibiting DNMT activity and increasing BRCA1 expression and its transcriptional activity. Liquiritigenin thus may be a promising candidate for the treatment of breast cancer.

Ablation of LGR4 signaling enhances radiation sensitivity of prostate cancer cells.

AIM: Our previous study has shown that leucine-rich repeat containing GPCR-4 (LGR4, or GPR48) LGR4 plays a role in cell migration, invasion, proliferation and apoptosis of prostate cancer (PCa). In this study, we aimed to explore whether LGR4 would affect radiation response in PCa. MATERIALS AND METHODS: LGR4 expression was silenced by shRNA transfection. qRT-PCR was employed to determine mRNA expression of LGR4 and DNA damage repair genes. Western blot was used to evaluate protein expression of LGR4, RSPO1-4, androgen receptor (AR), cyclic AMP response-element binding protein (CREB1), γH2A.X, and H2A.X. Cell proliferation was detected by CCK-8 assay and apoptosis was assayed by flow cytometry. Additionally, a xenograft model was also established to validate the role of LGR4 in PCa cells after radiation. KEY FINDINGS: LGR4 expression was enhanced in PCa cells by radiation treatment in dose- and time-dependent means. RSPO1-4 were also upregulated post-radiation. Furthermore, LGR4 knockdown exacerbated apoptosis, reduced cell viabilities and strengthened nuclear γH2A.X staining in AR positive PCa cells but not in AR negative cells in the presence of radiation. Likewise, LGR4 ablation diminished AR and CREB1 expression induced by radiation. In contrast, RSPO1 stimulation augmented cell viabilities, promoted AR and CREB1 expression, and upregulated DNA repair gene expression, which could be reversed by enzalutamide, except for AR expression. Additionally, LGR4 knockdown further suppressed tumor growth and AR/CREB1 expression but enhanced γH2A.X expression in xenografts. SIGNIFICANCE: In all, our study suggested that LGR4 might serve as an important regulator of radiation sensitivity in PCa.

Prevalence of Myocardial Fibrosis in Intensive Endurance Training Athletes: A Systematic Review and Meta-Analysis.

Objective: To review the published literature reporting on the incidence of myocardial fibrosis (MF) in high-intensity endurance athletes measured by late gadolinium enhancement (LGE) with cardiac magnetic resonance imaging (CMR). Methods: Five databases (PubMed, Cochrane Controlled Trials Register, EMBASE, Web of Science, and SPORTDiscus) were searched to obtain case cohort studies published before November 10, 2019. From 96 abstracts or reports extracted, 18 full-text articles were reviewed. The incidence of LGE was reported as outcome measures. Subgroup analysis was performed by age (under or above 50 years). Pooled estimates were obtained using a fixed-effects model. Results: After a full-text assessment, 12 studies involving 1,359 participants were included for analysis. Among them, 163/772 participants in the endurance athletes group showed LGE positive, compared with 19/587 participants in the comparison group. The results of the meta-analysis suggested that the prevalence of LGE was higher in the athletes group with long-term endurance exercise (OR 7.20;95%CI: 4.51-11.49). In addition, the same conclusion was drawn after the stratification of age. Conclusions: The available evidence demonstrates that high-intensity endurance athletes is associated with an increased incidence of LGE positive.

MicroRNA­137 regulates hypoxia­mediated migration and epithelial­mesenchymal transition in prostate cancer by targeting LGR4 via the EGFR/ERK signaling pathway.

MicroRNAs (miRs) serve an integral role in prostate cancer. The present study aimed to investigate the effects and mechanisms of miR­137 in hypoxia­mediated migration and epithelial­mesenchymal transition (EMT). PC3 and DU145 prostate cancer cells were exposed to hypoxia for 24 h, after which the expression of miR­137 was determined by reverse transcription­quantitative PCR (RT­qPCR). The cells were transfected with a miR­137 mimic or inhibitor, followed by hypoxia exposure. The results demonstrated that hypoxia reduced miR­137 expression. Further results from the Cell Counting Kit­8, Cell Death Detection ELISA plus kit, Transwell assay, RT­qPCR and western blotting assays revealed that the miR­137 mimic prevented cell proliferation, facilitated apoptosis and repressed cell migration, invasiveness, and expression of N­cadherin, vimentin and matrix metalloproteinase 2; the miR­137 inhibitor exerted the opposite effects. A dual­-luciferase reporter assay determined that miR­137 directly targeted leucine­rich repeat­containing G protein­coupled receptor 4 (LGR4). Additionally, miR­137 negatively regulated the epidermal growth factor receptor/extracellular signal­-regulated kinase (EGFR/ERK) signaling pathway by targeting LGR4. LGR4 silencing or EGFR/ERK inhibition abolished the effects of miR­137 inhibitor on cell migration and EMT. In conclusion, by targeting LGR4 via the EGFR/ERK signaling pathway, miR­137 inhibited prostate cancer cell migration and EMT.

Cardiac arrhythmias and cardiac arrest related to mushroom poisoning: A case report.

BACKGROUND: Mushroom exposure is a global health issue. The manifestations of mushroom poisoning (MP) may vary. Some species have been reported as rhabdomyolytic, hallucinogenic, or gastrointestinal poisons. Critical or even fatal MPs are mostly attributable to Amanita phalloides, with the development of severe liver or renal failure. Myocardial injury and even cases mimicking ST-segment elevation myocardial infarction (STEMI) have been previously reported, while cardiac arrhythmia or cardiac arrest is not commonly seen. CASE SUMMARY: We report a 68-year-old woman with MP who suffered from delirium, seizure, long QT syndrome on electrocardiogram (ECG), severe cardiac arrhythmias of multiple origins, and cardiac arrest. She was intubated and put on blood perfusion. Her kidney and liver functions were intact; creatine kinase-MB was mildly elevated, and then fell within normal range during her hospital stay. We sent the mushrooms she left for translation elongation factor subunit 1α, ribosomal RNA gene sequence, and internal transcribed spacer sequence analyses. There were four kinds of mushrooms identified, two of which were found to be toxic. CONCLUSION: This is the first time that we found cardiac toxicity caused by Panaeolus subbalteatus and Conocybe lactea, which were believed to be toxic to the liver, kidney, and brain. We suggest that intensive monitoring and ECG follow-up are essential to diagnose prolonged QT interval and different forms of tachycardia in MP patients, even without the development of severe liver or renal failure. The mechanisms need to be further investigated and clarified based on animal experiments and molecular signal pathways.

Juvenile-Onset Immunodeficiency Secondary to Anti-Interferon-Gamma Autoantibodies.

Immunodeficiency secondary to anti-interferon-gamma (anti-IFN-γ) autoantibodies was first described in 2004 as an acquired defect in the IFN-γ pathway leading to susceptibility to multiple opportunistic infections, including dimorphic fungi, parasites, and bacteria, especially tuberculosis and non-tuberculous mycobacterium (NTM) species. It has so far only been described in adult patients. We present 2 cases of disseminated NTM infections in otherwise immunocompetent children. A 16-year-old girl with Sweet's syndrome-like neutrophilic dermatosis developed recurrent fever and cervical lymphadenitis secondary to Mycobacterium abscessus. A 10-year-old boy with a history of prolonged fever, aseptic meningitis, aortitis, and arteritis in multiple blood vessels developed thoracic vertebral osteomyelitis secondary to Mycobacterium avium complex. Both patients were found to have positive serum neutralizing anti-IFNγ autoantibodies. Testing for anti-IFNγ autoantibodies should be considered in otherwise healthy immunocompetent hosts with recurrent or disseminated NTM infection. This represents a phenocopy of primary immunodeficiency which has been recently described only in adults. We report the first two cases of this phenomenon to affect children.

TROP2 promotes cell proliferation and migration in osteosarcoma through PI3K/AKT signaling.

Human trophoblast cell surface antigen 2 (TROP2) has been noted to serve an important role in the proliferation and migration of various types of human cancers. However, the potential role and the molecular mechanisms of TROP2 in osteosarcoma (OS) remain largely unclear. In the present study, high expression of TROP2 in human OS tissues and cell lines was observed. Overexpression of TROP2 promoted the proliferation and migration of OS cell lines, while TROP2 knockdown markedly decreased cell growth and migration. Furthermore, it was revealed that TROP2 overexpression significantly activated the phosphoinositide 3­kinase/protein kinase B (PI3K/AKT) signaling pathway. Collectively, these results suggested that TROP2 may promote OS cell proliferation and migration via PI3K/AKT signaling and may serve as a novel treatment target for OS.

Hepatitis B virus upregulates the expression of kinesin family member 4A.

Hepatitis B virus (HBV) infection is one of the major causes of hepatocellular carcinoma (HCC). Kinesin family member 4A (KIF4A) is a microtubule­based motor protein, which is upregulated in cervical and lung cancer. However, the expression of KIF4A in HBV­associated HCC, and the effect of HBV on the expression of KIF4A remain to be elucidated. In the present study, the expression profiles of KIF4A were examined in cancerous tissues and paracancerous tissues from patients with HCC, who presented with histories of chronic HBV infection, and the role of HBV in the induction of the expression of KIF4A was investigated. HepG2 cells were transfected with the pHBV1.3, HBV infectious clone and a construct, which contained the luciferase gene under the control of the KIF4A gene promoter. The results demonstrated that the expression of KIF4A was significantly higher in the HCC tissues than in the paracancerous tissues. HBV activated the KIF4A gene promoter and upregulated the mRNA and protein expression of KIF4A. Furthermore, activation of the gene expression of KIF4A increased in a pHBV1.3 concentration­dependent manner. These results provide novel insights into the understanding of HCC oncogenesis caused by HBV.

Enchanced levels of apolipoprotein M during HBV infection feedback suppresses HBV replication.

BACKGROUND: Chronic liver diseases can interfere with hepatic metabolism of lipoproteins, apolipoproteins. Hepatitis B virus (HBV) is a major etiological agent causing acute and chronic liver diseases. Apolipoprotein M (ApoM) is a high-density lipoprotein (HDL) apolipoprotein and exclusively expressed in the liver parenchyma cells and in the tubular cells of the kidney. This study was to determine the correlation between HBV infection and ApoM expression. MATERIALS AND METHODS: Serum ApoM levels in patients with HBV infection and in healthy individuals were measured by ELISA, ApoM mRNA expression were determined by RT-PCR, and the expression of S and E proteins of HBV, as well as the synthesis of viral DNA were measured by ELISA and real-time PCR. RESULTS: The levels of serum ApoM was significantly elevated in patients as compared to healthy individuals (P < 0.001), ApoM promoter activity, mRNA and protein expression were all stimulated in cells transfected with infectious HBV clone. In addition, ApoM decreases the expression of S and E proteins of HBV and the synthesis of viral DNA. CONCLUSION: Raised ApoM levels in HBV infection may in turn suppress HBV replication, one of the protective mechanisms of nature.