Mutation of SIVA, a candidate metastasis gene identified from clonally related bilateral breast cancers, promotes breast cancer cell spread in vitro and in vivo.

Metastasis is the most dreaded outcome after a breast cancer diagnosis, and little is known regarding what triggers or promotes breast cancer to spread distally, or how to prevent or eradicate metastasis effectively. Bilateral breast cancers are an uncommon form of breast cancers. In our study, a percentage of bilateral breast cancers were clonally related based on copy number variation profiling. Whole exome sequencing and comparative sequence analysis revealed that a limited number of somatic mutations were acquired in this "breast-to-breast" metastasis that might promote breast cancer distant spread. One somatic mutation acquired was SIVA-D160N that displayed pro-metastatic phenotypes in vivo and in vitro. Over-expression of SIVA-D160N promoted migration and invasion of human MB-MDA-231 breast cancer cells in vitro, consistent with a dominant negative interfering function. When introduced via tail vein injection, 231 cells over-expressing SIVA-D160N displayed enhanced distant spread on IVIS imaging. Over-expression of SIVA-D160N promoted invasion and anchorage independent growth of mouse 4T1 breast cancer cells in vitro. When introduced orthotopically via mammary fat pad injection in syngeneic Balb/c mice, over-expression of SIVA-D160N in 4T1 cells increased orthotopically implanted mammary gland tumor growth as well as liver metastasis. Clonally related bilateral breast cancers represented a novel system to investigate metastasis and revealed a role of SIVA-D160N in breast cancer metastasis. Further characterization and understanding of SIVA function, and that of its interacting proteins, may elucidate mechanisms of breast cancer metastasis, providing clinically useful biomarkers and therapeutic targets.

Grb7 Ablation in Mice Improved Glycemic Control, Enhanced Insulin Signaling, and Increased Abdominal fat Mass in Females.

OBJECTIVES: Growth factor receptor bound protein 7 (GRB7) is a multidomain signaling adaptor. Members of the Grb7/10/14 family, specifically Gbrb10/14, have important roles in metabolism. We ablated the Grb7 gene in mice to examine its metabolic function. METHODS: Global ablation of Grb7 in FVB/NJ mice was generated. Growth, organ weight, food intake, and glucose homeostasis were measured. Insulin signaling was examined by Western blotting. Fat and lean body mass was measured by nuclear magnetic resonance, and body composition after fasting or high-fat diet was assessed. Energy expenditure was measured by indirect calorimetry. Expression of adiposity and lipid metabolism genes was measured by quantitative PCR. RESULTS: Grb7-null mice were viable, fertile, and without obvious phenotype. Grb7 ablation improved glycemic control and displayed sensitization to insulin signaling in the liver. Grb7-null females but not males had increased gonadal white adipose tissue mass. Following a 12-week high-fat diet, Grb7-null female mice gained fat body mass and developed relative insulin resistance. With fasting, there was less decrease in fat body mass in Grb7-null female mice. Female mice with Grb7 ablation had increased baseline food intake, less energy expenditure, and displayed a decrease in the expression of lipolysis and adipose browning genes in gonadal white adipose tissue by transcript and protein analysis. CONCLUSION: Our study suggests that Grb7 is a negative regulator of glycemic control. Our results reveal a role for Grb7 in female mice in the regulation of the visceral adipose tissue mass, a powerful predictor of metabolic dysfunction in obesity.

Five Years of Extreme Risk Protection Orders in Oregon: A Descriptive Analysis.

Extreme Risk Protection Order (ERPO) laws have received increasing attention as a tool to prevent firearm suicide and homicide, including mass shootings. However, important gaps remain in our understanding of ERPO usage and implementation. Using the Oregon Judicial Case Information Network database, we abstracted data from all ERPO petitions filed in Oregon from 2018 to 2022, the first five years after the law took effect (N = 649). ERPO petitions were filed in 29 of 36 counties (81%, range 0-105 per county, median 11), against respondents 17-96 years of age (median: 42). Of ERPOs filed, 78% were initially granted. While only 22% of respondents in initially-granted ERPOs requested a hearing, when a hearing was held, nearly half (44%) of ERPOs were dismissed. The majority of ERPO petitions were motivated by threats of harm to respondents and others (n = 327, 50%), followed by threats of harm to others-only (n = 220, 34%) or respondents-only (n = 81, 12%). During the 5-year period, 72 (11%) ERPO petitions cited threats of mass violence as a motivating factor, including 24 (4%) petitions citing threats to schools or college campuses. The majority of ERPOs were filed by law enforcement officers (60%), and these petitions were significantly more often granted than those filed by family/household members (96% vs. 67%, p < .0001). We also found evidence of important gaps in documentation, including of respondent race (unavailable for 191 respondents, 29%) and of weapon removal or disposition after the ERPO was granted (unavailable in 350 cases, 69%). This study of long-term patterns of ERPO petitions highlights trends in usage and suggests areas where improvement may be possible, with implications for other states that have adopted or are considering similar ERPO laws.

Mayaro virus pathogenesis and immunity in rhesus macaques.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes debilitating and persistent arthritogenic disease. While MAYV was previously reported to infect non-human primates (NHP), characterization of MAYV pathogenesis is currently lacking. Therefore, in this study we characterized MAYV infection and immunity in rhesus macaques. To inform the selection of a viral strain for NHP experiments, we evaluated five MAYV strains in C57BL/6 mice and showed that MAYV strain BeAr505411 induced robust tissue dissemination and disease. Three male rhesus macaques were subcutaneously challenged with 105 plaque-forming units of this strain into the arms. Peak plasma viremia occurred at 2 days post-infection (dpi). NHPs were taken to necropsy at 10 dpi to assess viral dissemination, which included the muscles and joints, lymphoid tissues, major organs, male reproductive tissues, as well as peripheral and central nervous system tissues. Histological examination demonstrated that MAYV infection was associated with appendicular joint and muscle inflammation as well as presence of perivascular inflammation in a wide variety of tissues. One animal developed a maculopapular rash and two NHP had viral RNA detected in upper torso skin samples, which was associated with the presence of perivascular and perifollicular lymphocytic aggregation. Analysis of longitudinal peripheral blood samples indicated a robust innate and adaptive immune activation, including the presence of anti-MAYV neutralizing antibodies with activity against related Una virus and chikungunya virus. Inflammatory cytokines and monocyte activation also peaked coincident with viremia, which was well supported by our transcriptomic analysis highlighting enrichment of interferon signaling and other antiviral processes at 2 days post MAYV infection. The rhesus macaque model of MAYV infection recapitulates many of the aspects of human infection and is poised to facilitate the evaluation of novel therapies and vaccines targeting this re-emerging virus.

The association between preconception cannabis use and depression and anxiety during pregnancy.

OBJECTIVE: Cannabis use among individuals of reproductive age has increased with cannabis legalization and heightened stress during the COVID-19 pandemic. Our study provides data on preconception cannabis use and cannabis use disorder (CUD) during the pandemic and models the association between preconception cannabis use and depression and anxiety during pregnancy. METHODS: Data on substance use and depression and anxiety symptoms were collected from questionnaires and the Structured Clinical Interview for DSM-5 (SCID-5) from pregnant individuals in Oregon in 2019-2022. Linear regression was used to model the association between the frequency of preconception cannabis use and scores on the Center for Epidemiological Studies of Depression-Revised (CESD-R) and Beck Anxiety Inventory (BAI). RESULTS: The prevalence of preconception cannabis use was 27.8% among 227 study participants. CUD was diagnosed in 19% of cannabis users, or 5.3% of the overall sample. Daily cannabis use, compared to rare/never use, was associated with increases in CESD-R (ß = 6.22, p 0.029) and BAI (ß = 4.71, p 0.045) scores. CONCLUSIONS: Cannabis use and CUD are common among individuals of reproductive age. Given the association between preconception cannabis use and depression and anxiety during pregnancy, more attention is needed on screening and counseling of cannabis use among people of reproductive age.

PdeB, a cyclic Di-GMP-specific phosphodiesterase that regulates Shewanella oneidensis MR-1 motility and biofilm formation.

Shewanella oneidensis MR-1, a gammaproteobacterium with respiratory versatility, forms biofilms on mineral surfaces through a process controlled by the cyclic dinucleotide messenger c-di-GMP. Cellular concentrations of c-di-GMP are maintained by proteins containing GGDEF and EAL domains, which encode diguanylate cyclases for c-di-GMP synthesis and phosphodiesterases for c-di-GMP hydrolysis, respectively. The S. oneidensis MR-1 genome encodes several GGDEF and EAL domain proteins (50 and 31, respectively), with a significant fraction (∼10) predicted to be multidomain (e.g., GGDEF-EAL) enzymes containing an additional Per-Arnt-Sim (PAS) sensor domain. However, the biochemical activities and physiological functions of these multidomain enzymes remain largely unknown. Here, we present genetic and biochemical analyses of a predicted PAS-GGDEF-EAL domain-containing protein, SO0437, here named PdeB. A pdeB deletion mutant exhibited decreased swimming motility and increased biofilm formation under rich growth medium conditions, which was consistent with an increase in intracellular c-di-GMP. A mutation inactivating the EAL domain also produced similar swimming and biofilm phenotypes, indicating that the increase in c-di-GMP was likely due to a loss in phosphodiesterase activity. Therefore, we also examined the enzymatic activity of purified PdeB and found that the protein exhibited phosphodiesterase activity via the EAL domain. No diguanylate cyclase activity was observed. In addition to the motility and biofilm phenotypes, transcriptional profiling by DNA microarray analysis of biofilms of pdeB (in-frame deletion and EAL) mutant cells revealed that expression of genes involved in sulfate uptake and assimilation were repressed. Addition of sulfate to the growth medium resulted in significantly less motile pdeB mutants. Together, these results indicate a link between c-di-GMP metabolism, S. oneidensis MR-1 biofilm development, and sulfate uptake/assimilation.

Energy-dependent stability of Shewanella oneidensis MR-1 biofilms.

Stability and resistance to dissolution are key features of microbial biofilms. How these macroscopic properties are determined by the physiological state of individual biofilm cells in their local physical-chemical and cellular environment is largely unknown. In order to obtain molecular and energetic insight into biofilm stability, we investigated whether maintenance of biofilm stability is an energy-dependent process and whether transcription and/or translation is required for biofilm dissolution. We found that in 12-hour-old Shewanella oneidensis MR-1 biofilms, a reduction in cellular ATP concentration, induced either by oxygen deprivation or by addition of the inhibitor of oxidative phosphorylation carbonyl cyanide m-chlorophenylhydrazone (CCCP), dinitrophenol (DNP), or CN(-), resulted in massive dissolution. In 60-hour-old biofilms, the extent of uncoupler-induced cell loss was strongly attenuated, indicating that the integrity of older biofilms is maintained by means other than those operating in younger biofilms. In experiments with 12-hour-old biofilms, the transcriptional and translational inhibitors rifampin, tetracycline, and erythromycin were found to be ineffective in preventing energy starvation-induced detachment, suggesting that neither transcription nor translation is required for this process. Biofilms of Vibrio cholerae were also induced to dissolve upon CCCP addition to an extent similar to that in S. oneidensis. However, Pseudomonas aeruginosa and P. putida biofilms remained insensitive to CCCP addition. Collectively, our data show that metabolic energy is directly or indirectly required for maintaining cell attachment, and this may represent a common but not ubiquitous mechanism for stability of microbial biofilms.

Indirect modulation of the intracellular c-Di-GMP level in Shewanella oneidensis MR-1 by MxdA.

The GGDEF domain protein MxdA, which is important for biofilm formation in Shewanella oneidensis MR-1, was hypothesized to possess diguanylate cyclase activity. Here, we demonstrate that while MxdA controls the cellular level of c-di-GMP in S. oneidensis, it modulates the c-di-GMP pool indirectly.