Genetic drift and purifying selection shape within-host influenza A virus populations during natural swine infections.

Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype swine IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intrahost Single Nucleotide Variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics.

The influence of needleless connectors and inserted catheters on flow rates through vascular introducer sheaths.

SummaryA vascular introducer sheath is often used for rapid volume replacement. However, common manipulations such as the addition of needleless connectors to infusion ports and the insertion of catheters or other devices through the introducer sheath may impede flow. In this study we utilised a rapid infuser to deliver room-temperature normal saline through two introducer sheath configurations with and without the addition of needleless connectors and the placement of catheters through the introducer sheaths. The maximal flow rate delivered by the rapid infuser was 1000 mL/min, which was observed with both introducer sheath sizes tested without additional resistive elements. However, with the addition of a needleless connector, flow rates through the introducer sheaths were substantially lower (64 (standard deviation (SD) 6) mL/min and 61 (SD 7) mL/min for the 8.5 Fr and 9 Fr introducers, respectively). Flow rates were also reduced when catheters were placed within the sheaths (298 (SD 9) mL/min with the 7 Fr catheter and 74 (SD 9) mL/min with the 8 Fr catheter placed in an 8.5 Fr sheath; 649 (SD 6) mL/min with the 7 Fr catheter and 356 (SD 14) mL/min with the 8 Fr catheter placed in the 9 Fr sheath). These findings indicated that both needleless connectors and the placement of catheters through vascular introducer sheaths substantially reduced potential flow rates. Even 'large' vascular introducer sheaths capable of delivering high flow rates could be rendered minimally effective for rapid fluid administration when used in this way. Clinicians should consider these impediments to flow when rapid fluid administration is required, and obtain alternative vascular access if necessary.

Malignant transformation and subsequent leptomeningeal carcinomatosis of a gastric polyp in a dog.

Progressive carcinogenesis of a gastric polyp with transformation to gastric adenocarcinoma and subsequent development of leptomeningeal carcinomatosis is described in an adult male Scottish terrier. Presenting clinical signs consisted of vomiting with intermittent hematemesis. Surgical biopsies over the course of 14 months documented the progression from gastric polyp to minimally invasive gastric carcinoma to invasive gastric adenocarcinoma, a pathogenesis not previously documented in veterinary oncology. The patient ultimately developed neurologic pathology and was euthanized, and necropsy evaluation identified widespread carcinomatosis with accompanying leptomeningeal metastasis. As in humans, gastric polyps in dogs rarely have malignant potential.

The Paraventricular Thalamic Nucleus and Its Projections in Regulating Reward and Context Associations.

The paraventricular thalamic nucleus (PVT) is a brain region that mediates aversive and reward-related behaviors as shown in animals exposed to fear conditioning, natural rewards, or drugs of abuse. However, it is unknown whether manipulations of the PVT, in the absence of external factors or stimuli (e.g., fear, natural rewards, or drugs of abuse), are sufficient to drive reward-related behaviors. Additionally, it is unknown whether drugs of abuse administered directly into the PVT are sufficient to drive reward-related behaviors. Here, using behavioral as well as pathway and cell-type specific approaches, we manipulate PVT activity as well as the PVT-to-nucleus accumbens shell (NAcSh) neurocircuit to explore reward phenotypes. First, we show that bath perfusion of morphine (10 µM) caused hyperpolarization of the resting membrane potential, increased rheobase, and decreased intrinsic membrane excitability in PVT neurons that project to the NAcSh. Additionally, we found that direct injections of morphine (50 ng) in the PVT of mice were sufficient to generate conditioned place preference (CPP) for the morphine-paired chamber. Mimicking the inhibitory effect of morphine, we employed a chemogenetic approach to inhibit PVT neurons that projected to the NAcSh and found that pairing the inhibition of these PVT neurons with a specific context evoked the acquisition of CPP. Lastly, using brain slice electrophysiology, we found that bath-perfused morphine (10 µM) significantly reduced PVT excitatory synaptic transmission on both dopamine D1 and D2 receptor-expressing medium spiny neurons in the NAcSh, but that inhibiting PVT afferents in the NAcSh was not sufficient to evoke CPP.

Evolution of influenza A viruses in exhibition swine and transmission to humans, 2013-2015.

AIMS: Swine are a mixing vessel for the emergence of novel reassortant influenza A viruses (IAV). Interspecies transmission of swine-origin IAV poses a public health and pandemic risk. In the United States, the majority of zoonotic IAV transmission events have occurred in association with swine exposure at agricultural fairs. Accordingly, this human-animal interface necessitates mitigation strategies informed by understanding of interspecies transmission mechanisms in exhibition swine. Likewise, the diversity of IAV in swine can be a source for novel reassortant or mutated viruses that pose a risk to both swine and human health. METHODS AND RESULTS: In an effort to better understand those risks, here we investigated the epidemiology of IAV in exhibition swine and subsequent transmission to humans by performing phylogenetic analyses using full genome sequences from 272 IAV isolates collected from exhibition swine and 23 A(H3N2)v viruses from human hosts during 2013-2015. Sixty-seven fairs (24.2%) had at least one pig test positive for IAV with an overall estimated prevalence of 8.9% (95% CI: 8.3-9.6, Clopper-Pearson). Of the 19 genotypes found in swine, 5 were also identified in humans. There was a positive correlation between the number of human cases of a genotype and its prevalence in exhibition swine. Additionally, we demonstrated that A(H3N2)v viruses clustered tightly with exhibition swine viruses that were prevalent in the same year. CONCLUSIONS: These data indicate that multiple genotypes of swine-lineage IAV have infected humans, and highly prevalent IAV genotypes in exhibition swine during a given year are also the strains detected most frequently in human cases of variant IAV. Continued surveillance and rapid characterization of IAVs in exhibition swine can facilitate timely phenotypic evaluation and matching of candidate vaccine strains to those viruses present at the human-animal interface which are most likely to spillover into humans.

Genetic drift and purifying selection shape within-host influenza A virus populations during natural swine infections.

Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intra-host single nucleotide variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics.

Accelerated evolution of SARS-CoV-2 in free-ranging white-tailed deer.

The zoonotic origin of the COVID-19 pandemic virus highlights the need to fill the vast gaps in our knowledge of SARS-CoV-2 ecology and evolution in non-human hosts. Here, we detected that SARS-CoV-2 was introduced from humans into white-tailed deer more than 30 times in Ohio, USA during November 2021-March 2022. Subsequently, deer-to-deer transmission persisted for 2-8 months, disseminating across hundreds of kilometers. Newly developed Bayesian phylogenetic methods quantified how SARS-CoV-2 evolution is not only three-times faster in white-tailed deer compared to the rate observed in humans but also driven by different mutational biases and selection pressures. The long-term effect of this accelerated evolutionary rate remains to be seen as no critical phenotypic changes were observed in our animal models using white-tailed deer origin viruses. Still, SARS-CoV-2 has transmitted in white-tailed deer populations for a relatively short duration, and the risk of future changes may have serious consequences for humans and livestock.

Lack of SARS-CoV-2 Viral RNA Detection among a Convenience Sampling of Ohio Wildlife, Companion, and Agricultural Animals, 2020-2021.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in humans in late 2019 and spread rapidly, becoming a global pandemic. A zoonotic spillover event from animal to human was identified as the presumed origin. Subsequently, reports began emerging regarding spillback events resulting in SARS-CoV-2 infections in multiple animal species. These events highlighted critical links between animal and human health while also raising concerns about the development of new reservoir hosts and potential viral mutations that could alter the virulence and transmission or evade immune responses. Characterizing susceptibility, prevalence, and transmission between animal species became a priority to help protect animal and human health. In this study, we coalesced a large team of investigators and community partners to surveil for SARS-CoV-2 in domestic and free-ranging animals around Ohio between May 2020 and August 2021. We focused on species with known or predicted susceptibility to SARS-CoV-2 infection, highly congregated or medically compromised animals (e.g., shelters, barns, veterinary hospitals), and animals that had frequent contact with humans (e.g., pets, agricultural animals, zoo animals, or animals in wildlife hospitals). This included free-ranging deer (n = 76 individuals), free-ranging mink (n = 57), multiple species of bats (n = 59), and other wildlife in addition to domestic cats (n = 275) and pigs (n = 184). In total, we tested 792 individual animals (34 species) via rRT-PCR for SARS-CoV-2 RNA. SARS-CoV-2 viral RNA was not detected in any of the tested animals despite a major peak in human SARS-CoV-2 cases that occurred in Ohio subsequent to the peak of animal samplings. Importantly, we did not test for SARS-CoV-2 antibodies in this study, which limited our ability to assess exposure. While the results of this study were negative, the surveillance effort was critical and remains key to understanding, predicting, and preventing the re-emergence of SARS-CoV-2 in humans or animals.

Multivariable model of postoperative delirium in cardiac surgery patients: proteomic and demographic contributions.

Background: Delirium following cardiac surgery is common, morbid, and costly, but may be prevented with risk stratification and targeted intervention. Preoperative protein signatures may identify patients at increased risk for worse postoperative outcomes, including delirium. In this study, we aimed to identify plasma protein biomarkers and develop a predictive model for postoperative delirium in older patients undergoing cardiac surgery, while also uncovering possible pathophysiological mechanisms. Methods: SOMAscan analysis of 1,305 proteins in the plasma from 57 older adults undergoing cardiac surgery requiring cardiopulmonary bypass was conducted to define delirium-specific protein signatures at baseline (PREOP) and postoperative day 2 (POD2). Selected proteins were validated in 115 patients using the ELLA multiplex immunoassay platform. Proteins were combined with clinical and demographic variables to build multivariable models that estimate the risk of postoperative delirium and bring light to the underlying pathophysiology. Results: A total of 115 and 85 proteins from SOMAscan analyses were found altered in delirious patients at PREOP and POD2, respectively (p<0.05). Using four criteria including associations with surgery, delirium, and biological plausibility, 12 biomarker candidates (Tukey's fold change (|tFC|)>1.4, Benjamini-Hochberg (BH)-p<0.01) were selected for ELLA multiplex validation. Eight proteins were significantly altered at PREOP, and seven proteins at POD2 (p<0.05), in patients who developed postoperative delirium compared to non-delirious patients. Statistical analyses of model fit resulted in the selection of a combination of age, sex, and three proteins (angiopoietin-2 (ANGPT2); C-C motif chemokine 5 (CCL5); and metalloproteinase inhibitor 1 (TIMP1); AUC=0.829) as the best performing predictive model for delirium at PREOP. The delirium-associated proteins identified as biomarker candidates are involved with inflammation, glial dysfunction, vascularization, and hemostasis, highlighting the multifactorial pathophysiology of delirium. Conclusion: Our study proposes a model of postoperative delirium that includes a combination of older age, female sex, and altered levels of three proteins. Our results support the identification of patients at higher risk of developing postoperative delirium after cardiac surgery and provide insights on the underlying pathophysiology. ClinicalTrials.gov ( NCT02546765 ).

Shortening Duration of Swine Exhibitions to Reduce Risk for Zoonotic Transmission of Influenza A Virus.

Reducing zoonotic influenza A virus (IAV) risk in the United States necessitates mitigation of IAV in exhibition swine. We evaluated the effectiveness of shortening swine exhibitions to <72 hours to reduce IAV risk. We longitudinally sampled every pig daily for the full duration of 16 county fairs during 2014-2015 (39,768 nasal wipes from 6,768 pigs). In addition, we estimated IAV prevalence at 195 fairs during 2018-2019 to test the hypothesis that <72-hour swine exhibitions would have lower IAV prevalence. In both studies, we found that shortening duration drastically reduces IAV prevalence in exhibition swine at county fairs. Reduction of viral load in the barn within a county fair is critical to reduce the risk for interspecies IAV transmission and pandemic potential. Therefore, we encourage fair organizers to shorten swine shows to protect the health of both animals and humans.

Anterior cingulate cortex and its projections to the ventral tegmental area regulate opioid withdrawal, the formation of opioid context associations and context-induced drug seeking.

Clinical evidence suggests that there are correlations between activity within the anterior cingulate cortex (ACC) following re-exposure to drug-associated contexts and drug craving. However, there are limited data contributing to our understanding of ACC function at the cellular level during re-exposure to drug-context associations as well as whether the ACC is directly related to context-induced drug seeking. Here, we addressed this issue by employing our novel behavioral procedure capable of measuring the formation of drug-context associations as well as context-induced drug-seeking behavior in male mice (8-12 weeks of age) that orally self-administered oxycodone. We found that mice escalated oxycodone intake during the long-access training sessions and that conditioning with oxycodone was sufficient to evoke conditioned place preference (CPP) and drug-seeking behaviors. Additionally, we found that thick-tufted, but not thin-tufted pyramidal neurons (PyNs) in the ACC as well as ventral tegmental area (VTA)-projecting ACC neurons had increased intrinsic membrane excitability in mice that self-administered oxycodone compared to controls. Moreover, we found that global inhibition of the ACC or inhibition of VTA-projecting ACC neurons was sufficient to significantly reduce oxycodone-induced CPP, drug seeking, and spontaneous opioid withdrawal. These results demonstrate a direct role of ACC activity in mediating context-induced opioid seeking among other behaviors, including withdrawal, that are associated with the DSM-V criteria of opioid use disorder.

The RAL Enigma: Distinct Roles of RALA and RALB in Cancer.

RALA and RALB are highly homologous small G proteins belonging to the RAS superfamily. Like other small GTPases, the RALs are molecular switches that can be toggled between inactive GDP-bound and active GTP-bound states to regulate diverse and critical cellular functions such as vesicle trafficking, filopodia formation, mitochondrial fission, and cytokinesis. The RAL paralogs are activated and inactivated by a shared set of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) and utilize similar sets of downstream effectors. In addition to their important roles in normal cell biology, the RALs are known to be critical mediators of cancer cell survival, invasion, migration, and metastasis. However, despite their substantial similarities, the RALs often display striking functional disparities in cancer. RALA and RALB can have redundant, unique, or even antagonistic functions depending on cancer type. The molecular basis for these discrepancies remains an important unanswered question in the field of cancer biology. In this review we examine the functions of the RAL paralogs in normal cellular physiology and cancer biology with special consideration provided to situations where the roles of RALA and RALB are non-redundant.

SARS-CoV-2 infection in free-ranging white-tailed deer.

Humans have infected a wide range of animals with SARS-CoV-21-5, but the establishment of a new natural animal reservoir has not been observed. Here we document that free-ranging white-tailed deer (Odocoileus virginianus) are highly susceptible to infection with SARS-CoV-2, are exposed to multiple SARS-CoV-2 variants from humans and are capable of sustaining transmission in nature. Using real-time PCR with reverse transcription, we detected SARS-CoV-2 in more than one-third (129 out of 360, 35.8%) of nasal swabs obtained from O. virginianus in northeast Ohio in the USA during January to March 2021. Deer in six locations were infected with three SARS-CoV-2 lineages (B.1.2, B.1.582 and B.1.596). The B.1.2 viruses, dominant in humans in Ohio at the time, infected deer in four locations. We detected probable deer-to-deer transmission of B.1.2, B.1.582 and B.1.596 viruses, enabling the virus to acquire amino acid substitutions in the spike protein (including the receptor-binding domain) and ORF1 that are observed infrequently in humans. No spillback to humans was observed, but these findings demonstrate that SARS-CoV-2 viruses have been transmitted in wildlife in the USA, potentially opening new pathways for evolution. There is an urgent need to establish comprehensive 'One Health' programmes to monitor the environment, deer and other wildlife hosts globally.

Cotesiacassina sp. nov. from southwestern Colombia: a new gregarious microgastrine wasp (Hymenoptera, Braconidae) reared from the pest species Opsiphanescassina Felder & Felder (Lepidoptera, Nymphalidae) feeding on Elaeis oil palm trees (Arecaceae).

A new species of microgastrine wasp, Cotesiacassina Salgado-Neto, Vásquez & Whitfield, sp. nov., is described from southwestern Colombia in Tumaco, Nariño. This species is a koinobiont gregarious larval endoparasitoid, and spins a common mass of cocoons underneath the host caterpillars of Opsiphanescassina (Felder & Felder) (Lepidoptera, Nymphalidae), feeding on oil palm trees (interspecific hybrid Elaeisoleifera × E.guineensis) (Arecaceae). While superficially similar, both morphologically and biologically, to C.invirae Salgado-Neto & Whitfield from southern Brazil, the two species are distinct based on DNA barcodes, host species, geographical range and morphological characters.

SARS-CoV-2 infection in free-ranging white-tailed deer ( Odocoileus virginianus ).

Human-to-animal spillover of SARS-CoV-2 virus has occurred in a wide range of animals, but thus far, the establishment of a new natural animal reservoir has not been detected. Here, we detected SARS-CoV-2 virus using rRT-PCR in 129 out of 360 (35.8%) free-ranging white-tailed deer ( Odocoileus virginianus ) from northeast Ohio (USA) sampled between January-March 2021. Deer in 6 locations were infected with at least 3 lineages of SARS-CoV-2 (B.1.2, B.1.596, B.1.582). The B.1.2 viruses, dominant in Ohio at the time, spilled over multiple times into deer populations in different locations. Deer-to-deer transmission may have occurred in three locations. The establishment of a natural reservoir of SARS-CoV-2 in white-tailed deer could facilitate divergent evolutionary trajectories and future spillback to humans, further complicating long-term COVID-19 control strategies. ONE-SENTENCE SUMMARY: A significant proportion of SARS-CoV-2 infection in free-ranging US white-tailed deer reveals a potential new reservoir.

SARS-CoV-2 infection in free-ranging white-tailed deer (Odocoileus virginianus)

Human-to-animal spillover of SARS-CoV-2 virus has occurred in a wide range of animals, but thus far, the establishment of a new natural animal reservoir has not been detected. Here, we detected SARS-CoV-2 virus using rRT-PCR in 129 out of 360 (35.8%) free-ranging white-tailed deer (Odocoileus virginianus) from northeast Ohio (USA) sampled between January-March 2021. Deer in 6 locations were infected with at least 3 lineages of SARS-CoV-2 (B.1.2, B.1.596, B.1.582). The B.1.2 viruses, dominant in Ohio at the time, spilled over multiple times into deer populations in different locations. Deer-to-deer transmission may have occurred in three locations. The establishment of a natural reservoir of SARS-CoV-2 in white-tailed deer could facilitate divergent evolutionary trajectories and future spillback to humans, further complicating long-term COVID-19 control strategies. One-Sentence SummaryA significant proportion of SARS-CoV-2 infection in free-ranging US white-tailed deer reveals a potential new reservoir.

ATP Synthase and Mitochondrial Bioenergetics Dysfunction in Alzheimer's Disease.

Alzheimer's Disease (AD) is the most common neurodegenerative disorder in our society, as the population ages, its incidence is expected to increase in the coming decades. The etiopathology of this disease still remains largely unclear, probably because of the highly complex and multifactorial nature of AD. However, the presence of mitochondrial dysfunction has been broadly described in AD neurons and other cellular populations within the brain, in a wide variety of models and organisms, including post-mortem humans. Mitochondria are complex organelles that play a crucial role in a wide range of cellular processes, including bioenergetics. In fact, in mammals, including humans, the main source of cellular ATP is the oxidative phosphorylation (OXPHOS), a process that occurs in the mitochondrial electron transfer chain (ETC). The last enzyme of the ETC, and therefore the ulterior generator of ATP, is the ATP synthase. Interestingly, in mammalian cells, the ATP synthase can also degrade ATP under certain conditions (ATPase), which further illustrates the crucial role of this enzyme in the regulation of cellular bioenergetics and metabolism. In this collaborative review, we aim to summarize the knowledge of the presence of dysregulated ATP synthase, and of other components of mammalian mitochondrial bioenergetics, as an early event in AD. This dysregulation can act as a trigger of the dysfunction of the organelle, which is a clear component in the etiopathology of AD. Consequently, the pharmacological modulation of the ATP synthase could be a potential strategy to prevent mitochondrial dysfunction in AD.

Anterior cingulate cortex is necessary for spontaneous opioid withdrawal and withdrawal-induced hyperalgesia in male mice.

The anterior cingulate cortex (ACC) is implicated in many pathologies, including depression, anxiety, substance-use disorders, and pain. There is also evidence from brain imaging that the ACC is hyperactive during periods of opioid withdrawal. However, there are limited data contributing to our understanding of ACC function at the cellular level during opioid withdrawal. Here, we address this issue by performing ex vivo electrophysiological analysis of thick-tufted, putative dopamine D2 receptor expressing, layer V pyramidal neurons in the ACC (ACC L5 PyNs) in a mouse model of spontaneous opioid withdrawal. We found that escalating doses of morphine (20, 40, 60, 80, and 100 mg/kg, i.p. on days 1-5, respectively) injected twice daily into male C57BL/6 mice evoked withdrawal behaviors and an associated withdrawal-induced mechanical hypersensitivity. Brain slices prepared 24 h following the last morphine injection showed increases in ACC L5 thick-tufted PyN-intrinsic membrane excitability, increases in membrane resistance, reductions in the rheobase, and reductions in HCN channel-mediated currents (IH). We did not observe changes in intrinsic or synaptic properties on thin-tufted, dopamine D1-receptor-expressing ACC L5 PyNs recorded from male Drd1a-tdTomato transgenic mice. In addition, we found that chemogenetic inhibition of the ACC blocked opioid-induced withdrawal and withdrawal-induced mechanical hypersensitivity. These results demonstrate that spontaneous opioid withdrawal alters neuronal properties within the ACC and that ACC activity is necessary to control behaviors associated with opioid withdrawal and withdrawal-induced mechanical hypersensitivity. The ability of the ACC to regulate both withdrawal behaviors and withdrawal-induced mechanical hypersensitivity suggests overlapping mechanisms between two seemingly distinguishable behaviors. This commonality potentially suggests that the ACC is a locus for multiple withdrawal symptoms.

The small G-protein RalA promotes progression and metastasis of triple-negative breast cancer.

BACKGROUND: Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-associated mortality in women. In particular, triple-negative BC (TNBC) has the highest rate of mortality due in large part to the lack of targeted treatment options for this subtype. Thus, there is an urgent need to identify new molecular targets for TNBC treatment. RALA and RALB are small GTPases implicated in growth and metastasis of a variety of cancers, although little is known of their roles in BC. METHODS: The necessity of RALA and RALB for TNBC tumor growth and metastasis were evaluated in vivo using orthotopic and tail-vein models. In vitro, 2D and 3D cell culture methods were used to evaluate the contributions of RALA and RALB during TNBC cell migration, invasion, and viability. The association between TNBC patient outcome and RALA and RALB expression was examined using publicly available gene expression data and patient tissue microarrays. Finally, small molecule inhibition of RALA and RALB was evaluated as a potential treatment strategy for TNBC in cell line and patient-derived xenograft (PDX) models. RESULTS: Knockout or depletion of RALA inhibited orthotopic primary tumor growth, spontaneous metastasis, and experimental metastasis of TNBC cells in vivo. Conversely, knockout of RALB increased TNBC growth and metastasis. In vitro, RALA and RALB had antagonistic effects on TNBC migration, invasion, and viability with RALA generally supporting and RALB opposing these processes. In BC patient populations, elevated RALA but not RALB expression is significantly associated with poor outcome across all BC subtypes and specifically within TNBC patient cohorts. Immunohistochemical staining for RALA in patient cohorts confirmed the prognostic significance of RALA within the general BC population and the TNBC population specifically. BQU57, a small molecule inhibitor of RALA and RALB, decreased TNBC cell line viability, sensitized cells to paclitaxel in vitro and decreased tumor growth and metastasis in TNBC cell line and PDX models in vivo. CONCLUSIONS: Together, these data demonstrate important but paradoxical roles for RALA and RALB in the pathogenesis of TNBC and advocate further investigation of RALA as a target for the precise treatment of metastatic TNBC.

Influenza Vaccination of Swine Reduces Public Health Risk at the Swine-Human Interface.

Influenza A viruses (IAV) in swine (IAV-S) pose serious risk to public health through spillover at the human-animal interface. Continued zoonotic transmission increases the likelihood novel IAV-S capable of causing the next influenza pandemic will emerge from this animal reservoir. Because current mitigation strategies are insufficient to prevent IAV zoonosis, we investigated the ability of swine vaccination to decrease IAV-S zoonotic transmission risk. We assessed postchallenge viral shedding in market-age swine vaccinated with either live-attenuated influenza virus (LAIV), killed influenza virus (KV), or sham vaccine (NV). We also assessed postchallenge transmission by exposing naive ferrets to pigs with contact types reflective of those experienced by humans in a field setting. LAIV and KV swine groups exhibited a nearly 100-fold reduction in peak nasal titer (LAIV mean, 4.55 log 50% tissue culture infectious dose [TCID50]/ml; KV mean, 4.53 log TCID50/ml) compared to NV swine (mean, 6.40 log TCID50/ml). Air sampling during the postchallenge period revealed decreased cumulative IAV in LAIV and KV study room air (LAIV, area under the concentration-time curve [AUC] of 57.55; KV, AUC = 24.29) compared to the NV study room (AUC = 86.92). Pairwise survival analysis revealed a significant delay in onset of infection among ferrets exposed to LAIV pigs versus NV pigs (rate ratio, 0.66; P = 0.028). Ferrets exposed to vaccinated pigs had lower cumulative virus titers in nasal wash samples (LAIV versus NV, P < 0.0001; KV versus NV, P= 0.3490) and experienced reduced clinical signs during infection. Our findings support the implementation of preexhibition influenza vaccination of swine to reduce the public health risk posed by IAV-S at agricultural exhibitions. IMPORTANCE Swine exhibited at agricultural fairs in North America have been the source of repeated zoonotic influenza A virus transmission, which creates a pathway for influenza pandemic emergence. We investigated the effect of using either live-attenuated influenza virus or killed influenza virus vaccines as prefair influenza vaccination of swine on zoonotic influenza transmission risk. Ferrets were exposed to the pigs in order to simulate human exposure in a field setting. We observed reductions in influenza A virus shedding in both groups of vaccinated pigs as well as the corresponding ferret exposure groups, indicating vaccination improved outcomes on both sides of the interface. There was also significant delay in onset of infection among ferrets that were exposed to live-attenuated virus-vaccinated pigs, which might be beneficial during longer fairs. Our findings indicate that policies mandating influenza vaccination of swine before fairs, while not currently common, would reduce the public health risk posed by influenza zoonosis.