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DDX3 is a DEAD‐box RNA helicase that plays multiple roles in RNA metabolism, including translation. We previously reported that DDX3 is required for translation of PACT, a binding partner of Dicer, suggesting a role for DDX3 in microRNA (miRNA) biogenesis and RNA interference (RNAi). Emerging evidence suggests that DDX3 plays a vital role in tumorigenesis and cancer progression, however, its underlying mechanism is still not fully understood. Here, we showed that the control of PACT by DDX3 is conserved in human cells and Caenorhabditis elegans. Using a miRNA microarray, we found that DDX3 regulates the expression of a small subset of cancer‐related miRNAs. These oncogenic miRNAs were down‐regulated by knockdown of DDX3 or PACT and up‐regulated by overexpression of DDX3 or PACT in HEK293T cells. Similar results were obtained in human cancer HCT116 and HeLa cells. Dual luciferase reporter assay showed that DDX3 and PACT are required for short hairpin RNA (shRNA)‐induced RNAi. We also performed co‐immunoprecipitation to confirm the interaction between DDX3 and AGO2, a significant component of the RNA‐induced silencing complex, supporting a role for DDX3 in the RNAi pathway. We further examined the effects of DDX3 and PACT on cell proliferation, and stable overexpression of DDX3 in HEK293 cells results in loss of contact inhibition of cell growth. Hence, we propose that DDX3 may participate in cancer development by regulating the RNAi pathway. [ABSTRACT FROM AUTHOR]

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1β secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, and only the phosphorylated ASC could participate in speck formation and trigger IL-1β secretion. Moreover, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 reduced monosodium urate-mediated peritonitis, a disease model used for studying the consequences of NLRP3 activation. Our results suggest that although Pyk2 and FAK are involved in inflammasome activation, only Pyk2 directly phosphorylates ASC and brings ASC into an oligomerization-competent state by allowing Tyr146 phosphorylation to participate ASC speck formation and subsequent NLRP3 inflammation.

Various vaccine platforms were developed and deployed against the COVID‐19 disease. The Fc‐mediated functions of IgG antibodies are essential in the adaptive immune response elicited by vaccines. However, the long‐term changes of protein subunit vaccines and their combinations with messenger RNA (mRNA) vaccines are unknown. A total of 272 serum and plasma samples were collected from individuals who received first to third doses of the protein subunit Medigen, the mRNA (BNT, Moderna), or the adenovector AstraZeneca vaccines. The IgG subclass level was measured using enzyme‐linked immunosorbent assay, and Fc‐N glycosylation was measured using liquid chromatography coupled to tandem mass spectrometry. Antibody‐dependent‐cellular‐phagocytosis (ADCP) and complement deposition (ADCD) of anti‐spike (S) IgG antibodies were measured by flow cytometry. IgG1 and 3 reached the highest anti‐S IgG subclass level. IgG1, 2, and 4 subclass levels significantly increased in mRNA‐ and Medigen‐vaccinated individuals. Fc‐glycosylation was stable, except in female BNT vaccinees, who showed increased bisection and decreased galactosylation. Female BNT vaccinees had a higher anti‐S IgG titer than that of males. ADCP declined in all groups. ADCD was significantly lower in AstraZeneca‐vaccinated individuals. Each vaccine produced specific long‐term changes in Fc structure and function. This finding is critical when selecting a vaccine platform or combination to achieve the desired immune response. [ABSTRACT FROM AUTHOR]

JMJD5, a Jumonji C domain-containing dioxygenase, is important for embryonic development and cancer growth. Here, we show that JMJD5 is up-regulated by hypoxia and is crucial for hypoxia-induced cell proliferation. JMJD5 interacts directly with pyruvate kinase muscle isozyme (PKM)2 to modulate metabolic flux in cancer cells. The JMJD5-PKM2 interaction resides at the intersubunit interface region of PKM2, which hinders PKM2 tetramerization and blocks pyruvate kinase activity. This interaction also influences translocation of PKM2 into the nucleus and promotes hypoxia-inducible factor (HIF)-1α-mediated transactivation. JMJD5 knockdown inhibits the transcription of the PKM2-HIF-1α target genes involved in glucose metabolism, resulting in a reduction of glucose uptake and lactate secretion in cancer cells. JMJD5, along with PKM2 and HIF-1α, is recruited to the hypoxia response element site in the lactate dehydrogenase A and PKM2 loci and mediates the recruitment of the latter two proteins. Our data uncover a mechanism whereby PKM2 can be regulated by factor-binding-induced homo/heterooligomeric restructuring, paving the way to cell metabolic reprogram.

Cullin 4A (Cul4A) is important in cell survival, development, growth and the cell cycle, but its role in mesothelioma has not been studied. For the first time, we identified amplification of the Cul4A gene in four of five mesothelioma cell lines. Consistent with increased Cul4A gene copy number, we found that Cul4A protein was overexpressed in mesothelioma cells as well. Cul4A protein was also overexpressed in 64% of primary malignant pleural mesothelioma (MPM) tumours. Furthermore, knockdown of Cul4A with shRNA in mesothelioma cells resulted in up-regulation of p21 and p27 tumour suppressor proteins in a p53-independent manner in H290, H28 and MS-1 mesothelioma cell lines. Knockdown of Cul4A also resulted in G0/G1 cell cycle arrest and decreased colony formation in H290, H28 and MS-1 mesothelioma cell lines. Moreover, G0/G1 cell cycle arrest was partially reversed by siRNA down-regulation of p21 and/or p27 in Cul4A knockdown H290 cell line. In the contrary, overexpression of Cul4A resulted in down-regulation of p21 and p27 proteins and increased colony formation in H28 mesothelioma cell line. Both p21 and p27 showed faster degradation rates in Cul4A overexpressed H28 cell line and slower degradation rates in Cul4A knockdown H28 cell line. Our study indicates that Cul4A amplification and overexpression play an oncogenic role in the pathogenesis of mesothelioma. Thus, Cul4A may be a potential therapeutic target for MPM.

Vascular ectasia observed in port wine stain (PWS) birthmarks might be secondary to localized reduction of neural innervation and associated loss of autonomic stimulation. Our objective was to investigate this theory and evaluate nerve density, blood vessel density, and average blood vessel size in untreated and pulsed dye laser with cryogen spray cooling (PDL-CSC) treated PWS skin.Biopsy skin specimens were taken from 14 adults with a PWS, categorized by: uninvolved skin; untreated PWS skin; PWS skin with a history of good blanching, and PWS skin with a history of poor blanching both in response to PDL treatment. Seven specimens of normal, unaffected skin were used as the experimental control group. Indirect immunohistochemistry was performed on all specimens followed by confocal microscopy imaging with computer analysis to determine nerve density, blood vessel density, and average blood vessel size.Nerve density was significantly decreased in all PWS sites as compared to uninvolved skin (p < 0.01). Average blood vessel diameter was larger in untreated as compared to treated PWS sites and varied between different sites within a single PWS.Nerve density was decreased in all evaluated PWS sites, and this may be a factor in lesion pathogenesis. PWS blood vessel size correlated with the PDL blanching response and may prove to be a useful prognostic indicator of therapeutic outcome.