NADH elevation during chronic hypoxia leads to VHL-mediated HIF-1α degradation via SIRT1 inhibition.

BACKGROUND: Under conditions of hypoxia, cancer cells with hypoxia inducible factor-1α (HIF-1α) from heterogeneous tumor cells show greater aggression and progression in an effort to compensate for harsh environmental conditions. Extensive study on the stability of HIF-1α under conditions of acute hypoxia in cancer progression has been conducted, however, understanding of its involvement during the chronic phase is limited. METHODS: In this study, we investigated the effect of SIRT1 on HIF1 stability in a typical chronic hypoxic conditon that maintains cells for 24 h under hypoxia using Western blotting, co-IP, measurement of intracellular NAD + and NADH levels, semi-quantitative RT-PCR analysis, invasion assay, gene knockdown. RESULTS: Here we demonstrated that the high concentration of pyruvate in the medium, which can be easily overlooked, has an effect on the stability of HIF-1α. We also demonstrated that NADH functions as a signal for conveyance of HIF-1α degradation via the SIRT1 and VHL signaling pathway under conditions of chronic hypoxia, which in turn leads to attenuation of hypoxically strengthened invasion and angiogenic activities. A steep increase in the level of NADH occurs during chronic hypoxia, leading to upregulation of acetylation and degradation of HIF-1α via inactivation of SIRT1. Of particular interest, p300-mediated acetylation at lysine 709 of HIF-1α is recogonized by VHL, which leads to degradation of HIF-1α via ubiquitin/proteasome machinary under conditions of chronic hypoxia. In addition, we demonstrated that NADH-elevation-induced acetylation and subsequent degradation of HIF-1α was independent of proline hydroxylation. CONCLUSIONS: Our findings suggest a critical role of SIRT1 as a metabolic sensor in coordination of hypoxic status via regulation of HIF-1α stability. These results also demonstrate the involvement of VHL in degradation of HIF-1α through recognition of PHD-mediated hydroxylation in normoxia and p300-mediated HIF-1α acetylation in hypoxia.

Struggling, helping and adapting: Crowdfunding motivations and outcomes during the early US COVID-19 pandemic.

During the early months of COVID-19, many people in the US turned to charitable crowdfunding to seek and provide assistance. Little is known about the needs, hopes or experiences that motivated US pandemic crowdfunding and how these were correlated with campaign success. This study uses a mixed-methods data analysis of a randomised cluster sample of 919 US GoFundMe campaigns during the first 7 months of the pandemic. Overall, most campaigns performed poorly, and 38% got no donations at all. The largest proportion of campaigns aimed to address individual, acute financial struggles, often arising from considerable challenges accessing or qualifying for government assistance. These campaigns, as well as those involving campaigners and beneficiaries of colour, tended to be least successful. Qualitative thematic analysis revealed three key crowdfunding motivations that reflect individualistic, agentive responses to the pandemic: struggling, helping and adapting. These motivations reveal a shift away from social suffering and collective mobilisation and towards largely individualised efforts of survival as digital crowdfunding becomes a key domain of crisis response. Crowdfunding platforms are playing an increasingly important role in mediating and influencing individual and collective responses to crisis, which has important political ramifications for how societies perceive and address health emergencies.

Crowdfunding as a response to COVID-19: Increasing inequities at a time of crisis.

During the first seven months of the COVID-19 pandemic, more than 175,000 crowdfunding campaigns were established in the US for coronavirus-related needs using the platform GoFundMe. Though charitable crowdfunding has been popular in recent years, the widespread creation of COVID-19 related campaigns points to potential shifts in how the platform is being used, and the volume of needs users have brought to the site during a profound economic, social, and epidemiological crisis. This study offers a systematic examination of the scope and impacts of COVID-19 related crowdfunding in the early months of the pandemic and assesses how existing social and health inequities shaped crowdfunding use and outcomes. Using data collected from all US-based GoFundMe campaigns mentioning COVID or coronavirus, we used descriptive analysis and a series of negative binomial and linear models to assess the contributions of demographic factors and COVID-19 impacts to campaign creation and outcome. We find significant evidence of growing inequalities in outcomes for campaigners. We find that crowdfunding provides substantially higher benefits in wealthier counties with higher levels of education. People from these areas are more likely to initiate campaigns in response to adverse health and economic impacts of COVID-19, and they also receive more funding compared to people living in areas with lower income and education. Modeling also indicates differential outcomes based on the racial and ethnic composition of county population, though without more detail about who is creating and funding campaigns we cannot explain causality. A targeted qualitative analysis of the top earning COVID-19 campaigns offers further evidence of how user privilege and corporate practices contribute to highly unequal outcomes. Taken together, these findings demonstrate how a market-oriented digital technology used to respond to large-scale crisis can exacerbate inequalities and further benefit already privileged groups.

Usefulness of the Alcohol Use Disorders Identification Test-Korean Revised Version in Screening for Diagnostic and Statistical Manual of Mental Disorders 5th Edition Alcohol Use Disorder among College Students.

BACKGROUND: There is a distinction in alcohol consumption behavior between adults and college students. This study aims to verify the usability and the optimal cutoff point of Alcohol Use Disorders Identification Test-Korean revised version (AUDIT-KR) for screening alcohol use disorder in college students when the diagnostic and statistical manual of mental disorders (DSM), 5th edition diagnostic criteria is applied. METHODS: A total of 922 college students living in Daejeon were enrolled and divided into two groups based on how many items they corresponded to among DSM-5 alcohol use disorder diagnostic criteria: those who corresponded to ≥2 of the 11 items were classified into the patient group (107 males, 89 females) while the others into the control group (311 males, 415 females). The participants were evaluated using AUDIT-KR to find the optimal cutoff point for screening alcohol use disorder, sensitivity, and specificity. RESULTS: The mean±standard deviation scores in the AUDIT-KR were 12.76±7.27, 10.72±4.62 for males and females, respectively, in the patient group. In contrast, in the control group the scores were 6.26±5.23 and 3.95±3.59 in males and females, respectively. The area under the receiver operating characteristic curve (95% confidence interval) regarding alcohol use disorder screening by AUDIT-KR was 0.768 (0.715-0.821) and 0.883 (0.848-0.919) for males and females, respectively. The optimal cutoff point of alcohol use disorder for males was >9, sensitivity 64.49%, and specificity 76.85%. The optimal cutoff point for females was >6, sensitivity 82.02%, and specificity 80.48%. CONCLUSION: This study suggested that AUDIT-KR can be used as a screening tool for alcohol use disorder in groups of college students when DSM-5 diagnosis criteria are applied.

SIRT1 deacetylates and stabilizes hypoxia-inducible factor-1α (HIF-1α) via direct interactions during hypoxia.

Upon shift to a hypoxic environment, cellular HIF-1α protein is stabilized, with a rapid decline in oxygen-sensitive hydroxylation. Several additional post-translational modifications of HIF-1α are critical in controlling protein stability during hypoxia. In the present study, we showed that SIRT1 stabilizes HIF-1α via direct binding and deacetylation during hypoxia. SIRT1 depletion or inactivation led to reduced hypoxic HIF-1α accumulation, accompanied by an increase in HIF-1α acetylation. Impaired HIF-1α accumulation was recovered upon inhibition of 26S proteasome activity, indicating that SIRT1 is essential for HIF-1α stabilization during hypoxia. Consistently, HIF-1α accumulation was enhanced upon overexpression of wild-type SIRT1, but not its dominant-negative form. SIRT1-mediated accumulation of HIF-1α protein led to increased expression of HIF-1α target genes, including VEGF, GLUT1 and MMP2, and ultimate promotion of cancer cell invasion. These findings collectively imply that hypoxic HIF-1α stabilization requires SIRT1 activation. Furthermore, SIRT1 protection of HIF-1α from acetylation may be a prerequisite for stabilization and consequent enhancement of cell invasion.

How far?: Using geographical information systems (GIS) to examine maternity care access for expectant mothers in a rural state.

Various factors have made rural access to maternity care a significant problem for rural women. The geographic distance between a mother's county of residence and the county in which she gave birth was examined in a rural state. Analyzing North Dakota county-level data using geographic information system (GIS) software, women from over half of the state's counties, making up nearly 18% of all births, were found to be over 40 miles to the hospital in which they gave birth. These findings suggest that rural women may experience significant geographic barriers as they receive health services in the prenatal, delivery, and postpartum periods of their pregnancy. We highlight the value of GIS, particularly geovisualization power, and note models of care that may be effective for rural women.

Hepatitis B virus X protein overcomes all-trans retinoic acid-induced cellular senescence by downregulating levels of p16 and p21 via DNA methylation.

Despite current molecular evidence suggesting that hepatitis B virus (HBV) X protein (HBx) plays an important role during HBV-mediated hepatocarcinogenesis, the detailed mechanism is still controversial. Here, it was shown that HBx overcomes cellular senescence provoked by all-trans retinoic acid (ATRA) in HepG2 cells, as demonstrated by the impaired induction of irreversible G(1) arrest and senescence-associated ß-galactosidase activity by ATRA in the presence of HBx. The anti-senescence effect of HBx was also observed in another human hepatoma cell line, Hep3B, but not in Huh-7 cells in which the p16 and p21 proteins are absent. In addition, HBx suppressed ATRA-mediated induction of p16 and p21 in HepG2 cells via promoter hypermethylation, resulting in inactivation of retinoblastoma protein. Furthermore, the ability of HBx to overcome ATRA-induced cellular senescence almost completely disappeared when the levels of p16 and p21 in the HBx-expressing cells became similar to those in the control cells by complementation in the former by exogenous expression, knockdown of their expression in the latter using specific small interfering RNA or treatment with a DNA methylation inhibitor, 5-Aza-2'-deoxycytidine. These results suggest that HBx executes its potential by downregulating levels of p16 and p21 via DNA methylation. As cellular senescence is a tumour-suppression process, the present study provides a new strategy by which HBV promotes hepatocarcinogenesis.

Hepatitis B virus X protein overcomes stress-induced premature senescence by repressing p16(INK4a) expression via DNA methylation.

Cellular senescence is an important tumor suppression process under diverse oncogenic conditions, entering a state of irreversible growth arrest to prevent damaged cells from undergoing aberrant proliferation. Developing a means of evading senescence thus seems to be a fundamental task that all cancer cells should solve early on. Here, we show that an oncogenic X protein of hepatitis B virus (HBx) overcomes cellular senescence provoked by a universal premature senescence inducer, H(2)O(2), in human hepatoma cells, as demonstrated by impaired induction of senescence-associated biomarkers, including morphological change, G(1) arrest, and beta-galactosidase activity, in the presence of HBx. HBx induced DNA hypermethylation of p16(INK4a) promoter and subsequently interfered action of transcription factors like Ets1 and Ets2 activated by H(2)O(2) through the p38(MAPK) pathway, resulting in inhibition of its transcription. Down-regulation of p16(INK4a) expression by HBx subsequently led to activation of G(1)-CDKs, phosphorylation of Rb, activation of E2F1, and finally evasion from G(1) arrest induced by H(2)O(2). Levels of another senescence regulator, p21(waf1), however, were not affected by HBx under our senescence-inducing conditions. In addition, the potentials of HBx to inactivate Rb and subsequently inhibit cellular senescence almost completely disappeared when levels of p16(INK4a) were recovered either by exogenous complementation or inhibition of the promoter hypermethylation. To our knowledge, our present study represents the first report that an oncogenic virus evades cellular senescence through epigenetic down-regulation of p16(INK4a) expression.

Adalimumab treatment for life threatening pulmonary artery aneurysm in Behçet disease: a case report.

Behçet's disease (BD) is a multisystem disorder characterized by vasculitis. Pulmonary vascular problems such as pulmonary artery aneurysms (PAA) are reported to indicate poor prognosis and high mortality. We describe a 43-year-old man who presented with life threatening bilateral PAA and thromboembolic disease due to BD. He was treated with prednisone and pulse cyclophosphamide and was poorly responsive to the conventional immunosuppression. The introduction of adalimumab therapy stabilized his PAA. We report that the inhibition of TNF-alpha using the neutralizing monoclonal antibody adalimumab has the potential to induce rapid, complete, and long-lasting remission in a life-threatening manifestation of BD.

Hepatitis B virus X protein overcomes the growth-inhibitory potential of retinoic acid by downregulating retinoic acid receptor-beta2 expression via DNA methylation.

Aberrant promoter methylation of retinoic acid receptor-beta(2) (RAR-beta(2)) is frequently detected in hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC); however, the mechanism of methylation and its biological significance are unknown. This study showed that HBx, the principal oncogene product of HBV, induced promoter hypermethylation of RAR-beta(2) via upregulation of DNA methyltransferases 1 and 3a, resulting in downregulation of its expression in human HCC cells. In addition, HBx abolished the potential of retinoic acid (RA) to downregulate levels of G(1)-checkpoint regulators including p16, p21 and p27, resulting in activation of E2F1 in the presence of RA. As a consequence, HBx-expressing cells were less susceptible to RA-induced cell growth inhibition compared with control cells. These effects almost completely disappeared when levels of RAR-beta(2) in HBx-expressing cells were restored by treatment with a universal DNA methylation inhibitor, 5-aza-2'-deoxycytidine. As RAR-beta(2) is a major executor of the anti-tumour potential of RA, its epigenetic downregulation by HBx is likely to be an important step during HBV-mediated tumorigenesis.

Hepatitis C virus core protein downregulates E-cadherin expression via activation of DNA methyltransferase 1 and 3b.

E-cadherin is a major cell adhesion molecule implicated as a potent tumor suppressor, which is frequently altered in human tumors including hepatocellular carcinoma. Here, we report that hepatitis C virus Core downregulates E-cadherin expression at the transcription level. This effect was abolished after treatment of 5'-Aza-2'dC, a specific inhibitor of DNA methyltransferase (DNMT). In addition, this repression was strongly correlated with hypermethylation of CpG islands of E-cadherin promoter via concerted action of both DNMT1 and 3b in Core-expressing cells. The decreased E-cadherin expression results in dramatic morphological changes in Core-expressing cells. In addition, Core-expressing cells aggregate poorly in suspension culture, reflecting their altered cell-cell interactions. The biological significance was further demonstrated by the increased collagen invasion ability of Core-expressing cells. Therefore, our finding suggests that Core plays a role in hepatocellular carcinogenesis by favoring cell detachment from the surrounding cells and migration outside of the primary tumor site.

Hepatitis B virus X protein differentially affects the ubiquitin-mediated proteasomal degradation of beta-catenin depending on the status of cellular p53.

Abnormal accumulation of beta-catenin is considered to be a strong driving force in hepatocellular carcinogenesis; however, the mechanism of beta-catenin accumulation in tumours is unclear. Here, it was demonstrated that hepatitis B virus X protein (HBx) differentially regulates the level of beta-catenin through two ubiquitin-dependent proteasome pathways depending on p53 status. In the presence of p53, HBx downregulated beta-catenin through the activation of a p53-Siah-1 proteasome pathway. For this purpose, HBx upregulated Siah-1 expression at the transcriptional level via activation of p53. In the absence of p53, however, HBx stabilized beta-catenin through the inhibition of a glycogen synthase kinase-3beta-dependent pathway. Interestingly, HBx variants with a Pro-101 to Ser substitution were unable to activate p53 and thus could stabilize beta-catenin irrespective of p53 status. Based on these findings, a model of beta-catenin regulation by HBx is proposed whereby the balance between the two opposite activities of HBx determines the overall expression level of beta-catenin. Differential regulation of beta-catenin by HBx depending on host (p53 status) and viral factors (HBx sequence variation) helps not only to explain the observation that cancers accumulating beta-catenin also exhibit a high frequency of p53 mutations but also to understand the contradictory reports on the roles of HBx during hepatocellular carcinogenesis.

Expression of DNA methyltransferase 1 is activated by hepatitis B virus X protein via a regulatory circuit involving the p16INK4a-cyclin D1-CDK 4/6-pRb-E2F1 pathway.

DNA methyltransferase 1 (DNMT1) is responsible for copying DNA methylation patterns to the daughter strands during DNA replication. Its expression is frequently up-regulated in human tumors, including hepatocellular carcinoma, but the mechanism of overexpression and its biological significance remain unclear. Here, we show that hepatitis B virus X protein (HBx) activates DNMT1 expression via a regulatory circuit involving the p16(INK4a)-cyclin D1-cyclin-dependent kinase (CDK) 4/6-retinoblastoma protein (pRb)-E2F1 pathway. HBx induced DNA hypermethylation of p16(INK4a) promoter to repress its expression, which subsequently led to activation of G1-CDKs, phosphorylation of pRb, activation of E2F1, and finally transcriptional activation of DNMT1. Inhibition of DNMT1 activity by either treatment with 5'-Aza-2'dC or introduction of DNMT1 small interfering RNA not only abolished the DNA methylation-mediated p16(INK4a) repression but also impaired DNMT1 expression itself, suggesting a cross-talk between DNMT1 and p16(INK4a). The up-regulation of cyclin D1 by HBx is likely to serve as an initiative impulse for the circuit because it was absolutely required for the activation of DNMT1 expression. We also observed that accumulated DNMT1 via this pathway inactivates E-cadherin expression through promoter hypermethylation. Considering that the pRb-E2F1 pathway is commonly activated in human tumors, activation of this circuit might be widespread and a potential therapeutic target.

Hepatitis B virus X protein represses E-cadherin expression via activation of DNA methyltransferase 1.

E-cadherin is a key cell adhesion molecule implicated as a tumor suppressor, which is frequently altered in hepatocellular carcinoma, especially in hepatitis B virus (HBV)-related tumors. Here, we report that HBV X protein (HBx) represses E-cadherin expression at the transcription level. Based on the differential effects of HBx natural variants, we determined that Lys-130 in the transactivation domain of HBx is critical for the E-cadherin repression. The repression effect of HBx was abolished after treatment with DNA methyltransferase inhibitor, 5'-Aza-2'dC. In addition, methylation-specific PCR analysis revealed that the CpG island 1 of E-cadherin promoter is hypermethylated by HBx. Furthermore, HBx induces DNA methyltransferase 1 expression by stimulating its transcription. Therefore, we conclude that HBx represses E-cadherin expression by inducing methylation-mediated promoter inactivation. The reduced E-cadherin expression results in dramatic morphological changes of the HBx-expressing cells. In addition, HBx-expressing cells aggregate poorly in suspension culture, reflecting their altered intercellular interactions. The biological significance was further demonstrated by the increased collagen invasion ability of HBx-expressing cells. Therefore, the present study suggests that HBx plays a role during hepatocellular carcinogenesis by favoring cell detachment from the surrounding cells and migration outside of the primary tumor site.