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Vibrio is a genus of halophilic, gram-negative bacteria found in estuaries around the globe. Integral parts of coastal cultures often involve contact with vectors of pathogenic Vibrio spp. (e.g., consuming raw shellfish). High rates of mortality from certain Vibrio spp. infections demonstrate the need for an improved understanding of Vibrio spp. dynamics in estuarine regions. Our study assessed meteorological, hydrographic, and biological correlates of Vibrio parahaemolyticus and V. vulnificus at 10 sites in the Eastern Mississippi Sound System (EMSS) from April to October 2019. During the sampling period, median abundances of V. parahaemolyticus and V. vulnificus were 2.31 log MPN/L and 2.90 log MPN/L, respectively. Vibrio spp. dynamics were largely driven by site-based variation, with sites closest to freshwater inputs having the highest abundances. The E-W wind scalar, which affects Ekman transport, was a novel Vibrio spp. correlate observed. A potential salinity effect on bacterial-particle associations was identified, where V. vulnificus was associated with larger particles in conditions outside of their optimal salinity. Additionally, V. vulnificus abundances were correlated to those of harmful algal species that did not dominate community chlorophyll. Correlates from this study may be used to inform the next iteration of regionally predictive Vibrio models and may lend additional insight to Vibrio spp. ecology in similar systems., Importance: Vibrio spp. are bacteria found in estuaries worldwide; some species can cause illness and infections in humans. Relationships between Vibrio spp. abundance, salinity, and temperature are well documented, but correlations to other environmental parameters are less understood. This study identifies unique correlates (e.g., E-W wind scalar and harmful algal species) that could potentially inform the next iteration of predictive Vibrio models for the EMSS region. Additionally, these correlates may allow existing environmental monitoring efforts to be leveraged in providing data inputs for future Vibrio risk models. An observed correlation between salinity and V. vulnificus /particle-size associations suggests that predicted environmental changes may affect the abundance of Vibrio spp. in certain reservoirs, which may alter which vectors present the greatest vibrio risk., Competing Interests: The authors declare no conflict of interest.

The global spread of the novel coronavirus first reported in December 2019 led to drastic changes in the social and economic dynamics of everyday life. Nationwide, racial, gender, and geographic disparities in symptom severity, mortality, and access to health care evolved, which impacted stress and anxiety surrounding COVID-19. On university campuses, drastic shifts in learning environments occurred as universities shifted to remote instruction, which further impacted student mental health and anxiety. Our study aimed to understand how students from diverse backgrounds differ in their worry and stress surrounding COVID-19 upon return to hybrid or in-person classes during the Fall of 2020. Specifically, we addressed the differences in COVID-19 worry, stress response, and COVID-19-related food insecurity related to race/ethnicity (Indigenous American, Asian/Asian American, black/African American, Latinx/Hispanic, white, or multiple races), gender (male, female, and gender expressive), and geographic origin (ranging from rural to large metropolitan areas) of undergraduate students attending a regional-serving R2 university, in the southeastern U.S. Overall, we found significance in worry, food insecurity, and stress responses with females and gender expressive individuals, along with Hispanic/Latinx, Asian/Asian American, and black/African American students. Additionally, students from large urban areas were more worried about contracting the virus compared to students from rural locations. However, we found fewer differences in self-reported COVID-related stress responses within these students. Our findings can highlight the disparities among students' worry based on gender, racial differences, and geographic origins, with potential implications for mental health of university students from diverse backgrounds. Our results support the inclusion of diverse voices in university decisioning making around the transition through the COVID-19 pandemic., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Correia et al.)

Inositol hexakisphosphate kinase 2 (IP6K2), a member of the inositol hexakisphosphate kinase family, functions as a growth suppressive and apoptosis-enhancing kinase during cell stress. We created mice with a targeted deletion of IP6K2; these mice display normal embryogenesis, development, growth and fertility. Chronic exposure to the carcinogen 4-nitroquinoline 1-oxide (4-NQO, a UV-mimetic compound) in drinking water resulted in fourfold increased incidence of invasive squamous cell carcinoma (SCC) formation in the oral cavity and esophagus of the knockout (KO) mice compared to the wild-type (WT) littermates. Paradoxically, KO mice displayed relative resistance to ionizing radiation and exhibit enhanced survival following 8-10 Gy total body irradiation. Primary KO fibroblasts displayed resistance to antiproliferative effects of interferon-beta and increased colony forming units following ionizing radiation. Radioresistance of KO fibroblasts was associated with accelerated DNA repair measured by comet assay. Direct microinjection of 5-PP-Ins(1,2,3,4,6)P(5) (the enzymatic product of IP6K2), but not InsP(6) (the substrate of IP6K2) induced cell death in SCC22A squamous carcinoma cells.

Background: Nitrosylcobalamin (NO-Cbl) is a chemotherapeutic pro-drug derived from vitamin B12 that preferentially delivers nitric oxide (NO) to tumor cells, based upon increased receptor expression. NO-Cbl induces Apo2L/TRAIL-mediated apoptosis and inhibits survival signaling in a variety of malignant cell lines. Chemotherapeutic agents often simultaneously induce an apoptotic signal and activation of NF-kappaB, which has the undesired effect of promoting cell survival. The specific aims of this study were to 1) measure the anti-tumor effects of NO-Cbl alone and in combination with conventional chemotherapeutic agents, and to 2) examine the mechanism of action of NO-Cbl as a single agent and in combination therapy., Methodology: Using anti-proliferative assays, electrophoretic mobility shift assay (EMSA), immunoblot analysis and kinase assays, we demonstrate an increase in the effectiveness of chemotherapeutic agents in combination with NO-Cbl as a result of suppressed NF-kappaB activation., Results: Eighteen chemotherapeutic agents were tested in combination with NO-Cbl, in thirteen malignant cell lines, resulting in a synergistic anti-proliferative effect in 78% of the combinations tested. NO-Cbl pre-treatment resulted in decreased NF-kappaB DNA binding activity, inhibition of IkappaB kinase (IKK) enzymatic activity, decreased AKT activation, increased caspase-8 and PARP cleavage, and decreased cellular XIAP protein levels., Conclusion: The use of NO-Cbl to inhibit survival signaling may enhance drug efficacy by preventing concomitant activation of NF-kappaB or AKT.

We previously showed that inositol hexakisphosphate kinase 2 (IHPK2) functions as a growth-suppressive and apoptosis-enhancing kinase during cell stress. Overexpression of IHPK2 sensitized ovarian carcinoma cell lines to the growth-suppressive and apoptotic effects of interferon beta (IFN-beta), IFN-alpha2, and gamma-irradiation. Expression of a kinase-dead mutant abrogated 50% of the apoptosis induced by IFN-beta. Because the kinase-dead mutant retained significant response to cell stressors, we hypothesized that a portion of the death-promoting function of IHPK2 was independent of its kinase activity. We now demonstrate that IHPK2 binds to tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2 and interferes with phosphorylation of transforming growth factor beta-activated kinase 1 (TAK1), thereby inhibiting NF-kappaB signaling. IHPK2 contains two sites required for TRAF2 binding, Ser-347 and Ser-359. Compared with wild type IHPK2-transfected cells, cells expressing S347A and S359A mutations displayed 3.5-fold greater TAK1 activation following TNF-alpha. This mutant demonstrated a 6-10-fold increase in NF-kappaB DNA binding following TNF-alpha compared with wild type IHPK2-expressing cells in which NF-kappaB DNA binding was inhibited. Cells transfected with wild type IHPK2 or IHPK2 mutants that lacked S347A and S359A mutations displayed enhanced terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining following TNF-alpha. We believe that IHPK2-TRAF2 binding leads to attenuation of TAK1- and NF-kappaB-mediated signaling and is partially responsible for the apoptotic activity of IHPK2.

Previously, we have reported that overexpression of IHPK2 (inositol hexakisphosphate kinase 2) sensitized NIH-OVCAR-3 ovarian carcinoma cell lines to the growth-suppressive and apoptotic effects of IFN-beta (interferon-beta) treatment and gamma-irradiation. In the present study, we demonstrate that Apo2L/TRAIL (Apo2L/tumour-necrosis-factor-related apoptosis-inducing ligand) is a critical mediator of IFN-induced apoptosis in these cells. Compared with IFN-alpha2, IFN-beta is a more potent inducer of Apo2L/TRAIL and IHPK2 activity. Overexpression of IHPK2 converts IFN-alpha2-resistant cells into cells that readily undergo apoptosis in response to IFN-alpha2. In untreated cells transfected with IHPK2-eGFP (where eGFP stands for enhanced green fluorescent protein), the fusion protein is localized to the cytoplasm and perinuclear region. After treatment with IFN-beta, IHPK2-eGFP translocated to the nucleus. In cells transfected with mutant IHPK2-NLS-eGFP (where NLS stands for nuclear localization sequence), containing point mutations in the NLS, the fusion protein remained trapped in the cytoplasm, even after IFN-beta treatment. Cells expressing mutant NLS mutation were more resistant to IFN-beta. The IC50 value of IHPK2-expressing cells was 2-3-fold lower than vector control. The IC50 value of NLS-mutant-expressing cells was 3-fold higher than vector control. Blocking antibodies to Apo2L/TRAIL or transfection with a dominant negative Apo2L/TRAIL receptor (DR5Delta) inhibited the antiproliferative effects of IFN-beta. Thus overexpression of IHPK2 enhanced apoptotic effects of IFN-beta, and expression of the NLS mutant conferred resistance to IFN-beta. Apo2L/TRAIL expression and nuclear localization of IHPK2 are both required for the induction of apoptosis by IFN-beta in ovarian carcinoma.

We have previously demonstrated the anti-tumor activity of nitrosylcobalamin (NO-Cbl), an analog of vitamin B12 that delivers nitric oxide (NO) and increases the expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. The specific aim of this study was to examine whether NO-Cbl could sensitize drug-resistant melanomas to Apo2L/TRAIL. Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibroblast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by TUNEL and confirmed by examining levels and activity of key mediators of apoptosis. The activation status of NF-kappa B was established by assaying DNA binding, luciferase reporter activity, the phosphorylation status of I kappa B alpha, and in vitro IKK activity. NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. Treatment with NO-Cbl followed by Apo2L/TRAIL induced apoptosis in Apo2L/TRAIL-resistant tumor cells, characterized by cleavage of caspase-3, caspase-8, and PARP. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of I kappa B alpha and inhibition of NF-kappa B DNA binding activity. NO-Cbl suppressed Apo2L/TRAIL- and TNF-alpha-mediated activation of a transfected NF-kappa B-driven luciferase reporter. XIAP, an inhibitor of apoptosis, was inactivated by NO-Cbl. NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of NO-Cbl and Apo2L/TRAIL capitalizes on the tumor-specific properties of both agents and represents a promising anti-cancer combination.

Angiogenesis is an absolute requirement for tumor growth and metastasis. The purpose of this study was to evaluate the antiangiogenic activity of interferon-alpha2b (IFN-alpha2b) and thalidomide, as single agents and in combination. The murine dermis model was used to assess tumor-induced angiogenesis in nude mice. Human ACHN (renal), NIH-OVCAR-3 (ovarian), LNCaP (prostate), and SK-Mel-1 (melanoma) tumor cells were inoculated intradermally into the flanks of nude mice. IFN-alpha2b and thalidomide, administered daily, were effective inhibitors of angiogenesis induced by all four tumor types. The combination of IFN-alpha2b and thalidomide caused a synergistic decrease in mean vessel count in tumors that were resistant to the antiproliferative effects of IFN-alpha2b and thalidomide in vitro. This enhanced suppression of angiogenesis translated into synergistic antitumor activity in a xenograft model. Pegylated IFN-alpha (PEG-IFN-alpha2b) (10(6) U) administered once in 10 days was as effective as daily IFN-alpha2b treatment (10(6) U x 10 days). IFN-alpha2b and thalidomide have potentiated antiangiogenic activity when used in combination. A single dose of PEG-IFN-alpha2b (10(6) U) was as effective at suppressing vessel growth as an equivalent dose of IFN-alpha2b given daily for 10 days.

Background: The ubiquitous plasma membrane transcobalamin II receptor (TC II-R) mediates uptake of cobalamin (Cbl; vitamin B12), an essential micronutrient. Tumors often require more Cbl than normal tissue, and increased Cbl uptake may result from increased TC II-R expression. To examine whether Cbl could therefore be used as a carrier molecule to target a chemotherapy drug, we tested an analogue of Cbl with nitric oxide as a ligand, nitrosylcobalamin (NO-Cbl). Because interferon beta (IFN-beta) has antitumor effects and increases expression of some membrane receptors, we examined whether it may enhance the effects of NO-Cbl., Methods: Antiproliferative effects of NO-Cbl were assessed in 24 normal and cancer cell lines. Xenograft tumors of human ovarian cancer NIH-OVCAR-3 cells were established in athymic nude mice, and tumor growth was monitored after treatment with NO-Cbl and IFN-beta, both individually and concomitantly. TC II-R expression and apoptosis was monitored in vitro and in vivo. RNA protection assays and mitochondrial membrane potential assays were used to distinguish the extrinsic and intrinsic apoptotic pathways, respectively., Results: Cancer cell lines were more sensitive to NO-Cbl (with ID(50)s [the dose that inhibits growth by 50%] as low as 2 microM) than normal cell lines (with ID(50)s of 85-135 microM). Single-agent NO-Cbl and IFN-beta treatment of NIH-OVCAR-3 xenografts induced tumor regression, whereas combination treatment induced tumor eradication. IFN-beta treatment increased TC II-R expression in vitro and uptake of [(57)Co]cobalamin in vivo. Compared with NIH-OVCAR-3 cells treated with NO-Cbl, cells treated with NO-Cbl and IFN-beta were more apoptotic and expressed higher mRNA levels of various apoptosis-associated genes. No changes in mitochondrial membrane potential were observed in cells treated with NO-Cbl., Conclusion: NO-Cbl inhibited tumor growth in vivo by activating the extrinsic apoptotic pathway. The increased expression of TC II-R induced by IFN-beta resulted in enhanced antitumor effects with NO-Cbl both in vitro and in vivo.

We recently identified inositol hexakisphosphate kinase 2 (IP6K2) as a positive regulator of apoptosis. Overexpression of IP6K2 enhances apoptosis induced by interferon-beta (IFN-beta) and cytotoxic agents in NIH-OVCAR-3 ovarian carcinoma cells. In this study, we contrast and compare IFN-beta and radiation-induced death, and show that IP6K2 expression sensitizes tumor cells. Unirradiated NIH-OVCAR-3 cells transfected with IP6K2 formed fewer colonies compared to unirradiated vector-expressing cells. IP6K2 overexpression caused increased radiosensitivity, evidenced by decreased colony forming units (CFU). Both IFN-beta and radiation induced caspase 8. IFN-beta, but not gamma-irradiation, induced TRAIL in NIH-OVCAR-3 cells. Gamma irradiation, but not IFN-beta, induced DR4 mRNA. Apoptotic effects of IFN-beta or gamma-irradiation were blocked by expression of a dominant negative mutant death receptor 5 (DR5Delta) or by Bcl-2. Caspase-8 mRNA induction was more pronounced in IP6K2-expressing cells compared to vector-expressing cells. These data suggest that overexpression of IP6K2 enhances sensitivity of some ovarian carcinomas to radiation and IFN-beta. IP6K2 may function to enhance the expression and/or function of caspase 8 and DR4 following cell injury. Both IFN-beta and gamma-irradiation induce apoptosis through the extrinsic, receptor-mediated pathway, IFN-beta through TRAIL, radiation through DR4, and both through caspase 8. The function of both death inducers is positively regulated by IP6K2.

Interferons (IFNs) regulate the expression of genes that mediate their antiviral, antitumor, and immunomodulatory actions. We have previously shown that IFN-beta suppresses growth of human ovarian carcinoma xenografts in vivo and induces apoptosis of ovarian carcinoma cells in vitro. To investigate mechanisms of IFN-beta-induced apoptosis we employed an antisense technical knockout approach to identify gene products that mediate cell death and have isolated several regulators of interferon-induced death (RIDs). In this investigation, we have characterized one of the RIDs, RID-2. Sequence analysis revealed that RID-2 was identical to human inositol hexakisphosphate kinase 2 (IP6K2). IP6K2 is post-transcriptionally induced by IFN-beta in ovarian carcinoma cells. A mutant IP6K2 with substitutions in the putative inositol phosphate binding domain abrogates IFN-beta-induced apoptosis. These studies identify a novel function for IP6K2 in cell growth regulation and apoptosis.

Objectives: We have previously described that bioactive lysophospholipids-lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), and sphingosylphosphorylcholine (SPC)-are present in ascitic fluids from patients with ovarian cancer. To understand the role of these lipids in ovarian cancer, we investigated the effects of these lipids on interleukin-8 (IL-8) production in ovarian cancer cells. IL-8 is a proinflammatory and proangiogenic factor, which is potentially involved in ovarian cancer development., Methods: The Clontech PCR-Select cDNA subtraction method (Clontech Laboratories, Inc., Palo Alto, CA) was used to identify genes potentially regulated by LPA in HEY and OCC1 ovarian cancer cell lines. Northern blot analysis was used to confirm and examine IL-8 mRNA regulation by lysolipids. Enzyme-linked immunosorbent assay (ELISA) was used for detecting secreted IL-8., Results: We describe here that LPA, S1P, and SPC increased mRNA levels (2- to 7-fold) and protein secretion (2- to 12-fold) of IL-8 from ovarian cancer cells (HEY, OCC1, and SKOV3) in vitro. These regulations were both dose- and time-dependent. All three lipids increased the stability IL-8 mRNA in HEY cells. In contrast to malignant ovarian cancer cells, immortalized human ovarian epithelial cells did not respond to any of these lipids to increase the secretion of IL-8, although these cells secreted similar basal levels of IL-8 (310 pg/ml/10,000 cells). Two breast cancer cell lines (MCF7 and T47D) secreted lower basal levels of IL-8 (48-80 pg/ml/10,000 cells), compared with ovarian cancer cells (200-500 pg/ml/10,000 cells). MCF7 cells responded to LPA, but not S1P and SPC, by increasing the secretion of IL-8. T47D and MCF10A, an immortalized breast cell line, did not respond to LPA, S1P, or SPC to increase IL-8 secretion., Conclusions: LPA, S1P, and SPC regulate the mRNA and protein levels of the proinflammatory and proangiogenic factor IL-8 in ovarian cancer cells. The pathological significance of these regulations in ovarian cancer remains to be further investigated., (Copyright 2001 Academic Press.)

In this paper, we discuss the value of scientific conferences to both the discipline area and the individual participants and trace the growth in grant support of biomedical meetings by the National Institutes of Health and, in particular, the National Cancer Institute.