Economic burden and secondary complications of influenza-related hospitalization among adults in the US: a retrospective cohort study.

OBJECTIVE: This study aims to describe the healthcare resource utilization (HCRU) and direct medical cost of influenza-related hospitalizations to illustrate the persistent economic burden of influenza among adults in the US. METHODS: A retrospective cohort study was conducted using the PINC AI Healthcare Database. Adults hospitalized with a diagnosis of influenza between August 1-May 31 from 2016-2023 were identified and stratified by age (18-49, 50-64 and ≥65 years). The index hospitalization was defined as the individual's first influenza-related hospitalization during each season. Patient demographics, comorbidities, and hospitalization characteristics were assessed during the index hospitalization. Index hospitalization length of stay (LOS), in-hospital mortality, intensive care unit (ICU) admissions, mechanical ventilation (MV) usage, and costs were evaluated overall and by MV usage, ICU admission, and secondary complication status. Pre-index influenza-related outpatient and emergency department (ED) visits (7 days prior) were also evaluated. RESULTS: Primarily initiated in the ED, the median LOS for influenza-related hospitalizations was 3-4 days. Inpatient mortality increased with age (2.2-4.4%). Combined mean hospitalization and initial ED visit costs were $12,556-$14,494 (2017/18; high severity season) and $11,384-$12,896 (2022/23; most recent season). Compared to other age groups, adults ≥65 years had higher proportions of hospitalization with no MV or ICU usage. Adults 18-49 years had the highest proportion of ICU admission only, whereas adults 50-64 years had the highest MV usage only and both MV and ICU admission. MV and/or ICU usage was associated with higher hospitalization costs. Increasing proportionally with age, the majority of influenza-related hospitalizations had a secondary complication diagnosis, which were associated with elevated costs. LIMITATIONS: Analysis of this hospital-based administrative database relied on coding accuracy. Only hospital system-associated outpatient/ED visits were captured; the full scope of HCRU was under-ascertained. CONCLUSIONS: The economic burden of influenza-related hospitalizations remains substantial, driven by underlying conditions, MV/ICU usage and secondary complications.

This study described the healthcare resource utilization (HCRU) and costs for US adults ≥18 years old hospitalized with influenza and associated secondary complications such as pneumonia, asthma exacerbation and malignant hypertension between 2016­2023. The researchers analyzed a hospital admission database and found that, for the healthcare system, average cost per influenza-related hospitalization ranged from $11,384 to $14,494, depending on the influenza season and age of the patient. Over 96% of patients admitted to a hospital initially presented at the emergency department, 20­30% of patients required mechanical ventilation (MV) or intensive care unit (ICU) admission, and the median hospital length of stay was 3­4 days. This study adds to the existing evidence by providing economic burden estimates for the 2022/23 influenza season, the most recent influenza season after the COVID-19 pandemic, and found slightly lower HCRU and cost for influenza hospitalizations relative to prior seasons. Also, the study comprehensively analyzed economic burden by patient age groups and found lower HCRU and costs among patients ≥65 years compared to adults 18­49 years and 50­64 years consistently for all seasons. Additionally, the study found that the proportion of patients with MV usage alone, with MV usage and an ICU admission, and average hospitalization costs were greatest among patients 50­64 years, highlighting the potential benefit of increasing rates of seasonal influenza vaccination among this age group. Finally, the study found higher costs among patients with complications related to their influenza infection compared to patients without complications. Overall, the study found that influenza-related hospitalization can contribute to substantial economic burden in the US in the most recent time period.

Estimated Effectiveness of Coadministration of the BNT162b2 BA.4/5 COVID-19 Vaccine With Influenza Vaccine.

Importance: No data comparing the estimated effectiveness of coadministering COVID-19 vaccines with seasonal influenza vaccine (SIV) in the community setting exist. Objective: To examine the comparative effectiveness associated with coadministering the BNT162b2 BA.4/5 bivalent mRNA COVID-19 vaccine (BNT162b2-biv [Pfizer BioNTech]) and SIV vs giving each vaccine alone. Design, Setting, and Participants: A retrospective comparative effectiveness study evaluated US adults aged 18 years or older enrolled in commercial health insurance or Medicare Advantage plans and vaccinated with BNT162b2-biv only, SIV only, or both on the same day between August 31, 2022, and January 30, 2023. Individuals with monovalent or another brand of mRNA bivalent COVID-19 vaccine were excluded. Exposure: Same-day coadministration of BNT162b2-biv and SIV; receipt of BNT162b2-biv only (for COVID-19-related outcomes) or SIV only (for influenza-related outcomes) were the comparator groups. For adults aged 65 years or older, only enhanced SIVs were included. Main Outcomes and Measures: COVID-19-related and influenza-related hospitalization, emergency department (ED) or urgent care (UC) encounters, and outpatient visits. Results: Overall, 3 442 996 individuals (57.0% female; mean [SD] age, 65 [16.7] years) were included. A total of 627 735 individuals had BNT162b2-biv and SIV vaccine coadministered, 369 423 had BNT162b2-biv alone, and 2 445 838 had SIV alone. Among those aged 65 years or older (n = 2 210 493; mean [SD] age, 75 [6.7] years; 57.9% female), the coadministration group had a similar incidence of COVID-19-related hospitalization (adjusted hazard ratio [AHR], 1.04; 95% CI, 0.87-1.24) and slightly higher incidence of emergency department or urgent care encounters (AHR, 1.12; 95% CI, 1.02-1.23) and outpatient visits (AHR, 1.06; 95% CI, 1.01-1.11) compared with the BNT162b2-biv-only group. Among individuals aged 18 to 64 years (n = 1 232 503; mean [SD] age, 47 [13.1] years; 55.4% female), the incidence of COVID-19-related outcomes was slightly higher among those who received both vaccines vs BNT162b2-biv alone (AHR point estimate range, 1.14-1.57); however, fewer events overall in this age group resulted in wider CIs. Overall, compared with those who received SIV alone, the coadministration group had a slightly lower incidence of most influenza-related end points (AHR point estimates 0.83-0.93 for those aged ≥65 years vs 0.76-1.08 for those aged 18-64 years). Negative control outcomes suggested residual bias and calibration of COVID-19-related and influenza-related outcomes with negative controls moved all estimates closer to the null, with most CIs crossing 1.00. Conclusions and Relevance: In this study, coadministration of BNT162b2-biv and SIV was associated with generally similar effectiveness in the community setting against COVID-19-related and SIV-related outcomes compared with giving each vaccine alone and may help improve uptake of both vaccines.

Risk of Pneumococcal Disease in US Adults by Age and Risk Profile.

Background: Older age and certain medical conditions are known to modify the risk of pneumococcal disease among adults. We quantified the risk of pneumococcal disease among adults with and without medical conditions in the United States between 2016 and 2019. Methods: This retrospective cohort study used administrative health claims data from Optum's de-identified Clinformatics Data Mart Database. Incidence rates of pneumococcal disease-all-cause pneumonia, invasive pneumococcal disease (IPD), and pneumococcal pneumonia-were estimated by age group, risk profile (healthy, chronic, other, and immunocompromising medical condition), and individual medical condition. Rate ratios and 95% CIs were calculated comparing adults with risk conditions with age-stratified healthy counterparts. Results: Among adults aged 18-49 years, 50-64 years, and ≥65 years, the rates of all-cause pneumonia per 100 000 patient-years were 953, 2679, and 6930, respectively. For the 3 age groups, the rate ratios of adults with any chronic medical condition vs healthy counterparts were 2.9 (95% CI, 2.8-2.9), 3.3 (95% CI, 3.2-3.3), and 3.2 (95% CI, 3.2-3.2), while the rate ratios of adults with any immunocompromising condition vs healthy counterparts were 4.2 (95% CI, 4.1-4.3), 5.8 (95% CI, 5.7-5.9), and 5.3 (95% CI, 5.3-5.4). Similar trends were observed for IPD and pneumococcal pneumonia. Persons with other medical conditions, such as obesity, obstructive sleep apnea, and neurologic disorders, were associated with increased risk of pneumococcal disease. Conclusions: The risk of pneumococcal disease was high among older adults and adults with certain risk conditions, particularly immunocompromising conditions.

Multi-Omic Analysis of Two Common P53 Mutations: Proteins Regulated by Mutated P53 as Potential Targets for Immunotherapy.

The p53 protein is mutated in more than 50% of human cancers. Mutated p53 proteins not only lose their normal function but often acquire novel oncogenic functions, a phenomenon termed mutant p53 gain-of-function. Mutant p53 has been shown to affect the transcription of a range of genes, as well as protein-protein interactions with transcription factors and other effectors; however, no one has intensively investigated and identified these proteins, or their MHC presented epitopes, from the viewpoint of their ability to act as targets for immunotherapeutic interventions. We investigated the molecular changes that occurred after the TP53 null osteosarcoma cells, SaOS-2, were transfected with one of two conformational p53-mutants, either R175H or R273H. We then examined the phenotypic and functional changes using macroscopic observations, proliferation, gene expression and proteomics alongside immunopeptidome profiling of peptide antigen presentation in the context of major histocompatibility complex (MHC) class I molecules. We identified several candidate proteins in both TP53 mutant cell lines with differential expression when compared to the TP53 null vector control, SaOS-V. Quantitative SWATH proteomics combined with immune-peptidome analysis of the class-I eluted peptides identified several epitopes presented on pMHC and in silico analysis shortlisted which antigens were expressed in a range of cancerous but not adjacent healthy tissues. Out of all the candidates, KLC1 and TOP2A showed high levels of expression in every tumor type examined. From these proteins, three A2 and four pan HLA-A epitopes were identified in both R175H and R273H from TOP2A. We have now provided a short list of future immunotherapy targets for the treatment of cancers harboring mutated TP53.

Cold Exposure Drives Weight Gain and Adiposity following Chronic Suppression of Brown Adipose Tissue.

Therapeutic activation of thermogenic brown adipose tissue (BAT) may be feasible to prevent, or treat, cardiometabolic disease. However, rodents are commonly housed below thermoneutrality (~20 °C) which can modulate their metabolism and physiology including the hyperactivation of brown (BAT) and beige white adipose tissue. We housed animals at thermoneutrality from weaning to chronically supress BAT, mimic human physiology and explore the efficacy of chronic, mild cold exposure (20 °C) and ß3-adrenoreceptor agonism (YM-178) under these conditions. Using metabolic phenotyping and exploratory proteomics we show that transfer from 28 °C to 20 °C drives weight gain and a 125% increase in subcutaneous fat mass, an effect not seen with YM-178 administration, thus suggesting a direct effect of a cool ambient temperature in promoting weight gain and further adiposity in obese rats. Following chronic suppression of BAT, uncoupling protein 1 mRNA was undetectable in the subcutaneous inguinal white adipose tissue (IWAT) in all groups. Using exploratory adipose tissue proteomics, we reveal novel gene ontology terms associated with cold-induced weight gain in BAT and IWAT whilst Reactome pathway analysis highlights the regulation of mitotic (i.e., G2/M transition) and metabolism of amino acids and derivatives pathways. Conversely, YM-178 had minimal metabolic-related effects but modified pathways involved in proteolysis (i.e., eukaryotic translation initiation) and RNA surveillance across both tissues. Taken together these findings are indicative of a novel mechanism whereby animals increase body weight and fat mass following chronic suppression of adaptive thermogenesis from weaning. In addition, treatment with a B3-adrenoreceptor agonist did not improve metabolic health in obese animals raised at thermoneutrality.

Aligned Poly-l-lactic Acid Nanofibers Induce Self-Assembly of Primary Cortical Neurons into 3D Cell Clusters.

Relative to two-dimensional (2D) culture, three-dimensional (3D) culture of primary neurons has yielded increasingly physiological responses from cells. Electrospun nanofiber scaffolds are frequently used as a 3D biomaterial support for primary neurons in neural tissue engineering, while hydrophobic surfaces typically induce aggregation of cells. Poly-l-lactic acid (PLLA) was electrospun as aligned PLLA nanofiber scaffolds to generate a structure with both qualities. Primary cortical neurons from E18 Sprague-Dawley rats cultured on aligned PLLA nanofibers generated 3D clusters of cells that extended highly aligned, fasciculated neurite bundles within 10 days. These clusters were viable for 28 days and responsive to AMPA and GABA. Relative to the 2D culture, the 3D cultures exhibited a more developed profile; mass spectrometry demonstrated an upregulation of proteins involved in cortical lamination, polarization, and axon fasciculation and a downregulation of immature neuronal markers. The use of artificial neural network inference suggests that the increased formation of synapses may drive the increase in development that is observed for the 3D cell clusters. This research suggests that aligned PLLA nanofibers may be highly useful for generating advanced 3D cell cultures for high-throughput systems.

MiR-423-5p prevents MALAT1-mediated proliferation and metastasis in prostate cancer.

BACKGROUND: The long non-coding RNA (lncRNA), MALAT1, plays a key role in the development of different cancers, and its expression is associated with worse prognosis in patients. However, its mechanism of action and its regulation are not well known in prostate cancer (PCa). A general mechanism of action of lncRNAs is their interaction with other epigenetic regulators including microRNAs (miRNAs). METHODS: Using lentiviral stable miRNA transfection together with cell biology functional assays and gene expression/target analysis, we investigated the interaction between MALAT1 and miR-423-5p, defined as a target with in silico prediction analysis, in PCa. RESULTS: Through bioinformatic analysis of data available from TCGA, we have found that MALAT1 expression correlates with high Gleason grade, metastasis occurrence, and reduced survival in PCa patients. These findings were validated on a TMA of PCa showing a significant correlation between MALAT1 expression with both stage and grading. We report that, in PCa cells, MALAT1 expression and activity is regulated by miR-423-5p that binds MALAT1, downregulates its expression and inhibits its activity in promoting proliferation, migration, and invasion. Using NanoString analysis, we unraveled downstream cell pathways that were affected by miR-423-5p expression and MALAT1 downregulation and identified several alterations in genes that are involved in metastatic response and angiogenic pathways. In addition, we showed that the overexpression of miR-423-5p increases survival and decreases metastases formation in a xenograft mouse model. CONCLUSIONS: We provide evidence on the role of MALAT1 in PCa tumorigenesis and progression. Also, we identify a direct interaction between miR-423-5p and MALAT1, which results in the suppression of MALAT1 action in PCa.

A Proteomic Approach Reveals That miR-423-5p Modulates Glucidic and Amino Acid Metabolism in Prostate Cancer Cells.

Recently, we have demonstrated that miR-423-5p modulates the growth and metastases of prostate cancer (PCa) cells both in vitro and in vivo. Here, we have studied the effects of miR-423-5p on the proteomic profile in order to identify its intracellular targets and the affected pathways. Applying a quantitative proteomic approach, we analyzed the effects on the protein expression profile of miR-423-5p-transduced PCa cells. Moreover, a computational analysis of predicted targets of miR-423-5p was carried out by using several target prediction tools. Proteomic analysis showed that 63 proteins were differentially expressed in miR-423-5-p-transfected LNCaP cells if compared to controls. Pathway enrichment analysis revealed that stable overexpression of miR-423-5p in LNCaP PCa cells induced inhibition of glycolysis and the metabolism of several amino acids and a parallel downregulation of proteins involved in transcription and hypoxia, the immune response through Th17-derived cytokines, inflammation via amphorin signaling, and ion transport. Moreover, upregulated proteins were related to the S phase of cell cycle, chromatin modifications, apoptosis, blood coagulation, and calcium transport. We identified seven proteins commonly represented in miR-423-5p targets and differentially expressed proteins (DEPs) and analyzed their expression and influence on the survival of PCa patients from publicly accessible datasets. Overall, our findings suggest that miR-423-5p induces alterations in glucose and amino acid metabolism in PCa cells paralleled by modulation of several tumor-associated processes.

Evaluation of COVID-19 vaccine breakthrough infections among immunocompromised patients fully vaccinated with BNT162b2.

OBJECTIVE: To evaluate COVID-19 vaccine breakthrough infections among immunocompromised (IC) individuals. METHODS: Individuals vaccinated with BNT162b2 were selected from the US HealthVerity database (10 December 2020 to 8  July 2021). COVID-19 vaccine breakthrough infections were examined in fully vaccinated (≥14 days after 2nd dose) IC individuals (IC cohort), 12 mutually exclusive IC condition groups, and a non-IC cohort. IC conditions were identified using an algorithm based on diagnosis codes and immunosuppressive (IS) medication usage. RESULTS: Of 1,277,747 individuals ≥16 years of age who received 2 BNT162b2 doses, 225,796 (17.7%) were identified as IC (median age: 58 years; 56.3% female). The most prevalent IC conditions were solid malignancy (32.0%), kidney disease (19.5%), and rheumatologic/inflammatory conditions (16.7%). Among the fully vaccinated IC and non-IC cohorts, a total of 978 breakthrough infections were observed during the study period; 124 (12.7%) resulted in hospitalization and 2 (0.2%) were inpatient deaths. IC individuals accounted for 38.2% (N = 374) of all breakthrough infections, 59.7% (N = 74) of all hospitalizations, and 100% (N = 2) of inpatient deaths. The proportion with breakthrough infections was 3 times higher in the IC cohort compared to the non-IC cohort (N = 374 [0.18%] vs. N = 604 [0.06%]; unadjusted incidence rates were 0.89 and 0.34 per 100 person-years, respectively. Organ transplant recipients had the highest incidence rate; those with >1 IC condition, antimetabolite usage, primary immunodeficiencies, and hematologic malignancies also had higher incidence rates compared to the overall IC cohort. Incidence rates in older (≥65 years old) IC individuals were generally higher versus younger IC individuals (<65). LIMITATIONS: This retrospective analysis relied on coding accuracy and had limited capture of COVID-19 vaccine receipt. CONCLUSIONS: COVID-19 vaccine breakthrough infections are rare but are more common and severe in IC individuals. The findings from this large study support the FDA authorization and CDC recommendations to offer a 3rd vaccine dose to increase protection among IC individuals.

Usher syndrome type 1-associated gene, pcdh15b, is required for photoreceptor structural integrity in zebrafish.

Blindness associated with Usher syndrome type 1 (USH1) is typically characterized as rod photoreceptor degeneration, followed by secondary loss of cones. The mechanisms leading to blindness are unknown because most genetic mouse models only recapitulate auditory defects. We generated zebrafish mutants for one of the USH1 genes, protocadherin-15b (pcdh15b), a putative cell adhesion molecule. Zebrafish Pcdh15 is expressed exclusively in photoreceptors within calyceal processes (CPs), at the base of the outer segment (OS) and within the synapse. In our mutants, rod and cone photoreceptor integrity is compromised, with early and progressively worsening abnormal OS disc growth and detachment, in part due to weakening CP contacts. These effects were attenuated or exacerbated by growth in dark and bright-light conditions, respectively. We also describe novel evidence for structural defects in synapses of pcdh15b mutant photoreceptors. Cell death does not accompany these defects at early stages, suggesting that photoreceptor structural defects, rather than overt cell loss, may underlie vision deficits. Thus, we present the first genetic animal model of a PCDH15-associated retinopathy that can be used to understand the aetiology of blindness in USH1. This article has an associated First Person interview with the first author of the paper.

Carnosine protects stimulus-secretion coupling through prevention of protein carbonyl adduction events in cells under metabolic stress.

Type 2 diabetes is characterised by failure to control glucose homeostasis, with numerous diabetic complications attributable to the resulting exposure of cells and tissues to chronic elevated concentrations of glucose and fatty acids. This, in part, results from formation of advanced glycation and advanced lipidation end-products that are able to modify protein, lipid, or DNA structure, and disrupt normal cellular function. Herein we used mass spectrometry to identify proteins modified by two such adduction events in serum of individuals with obesity, type 2 diabetes, and gestational diabetes, along with similar analyses of human and mouse skeletal muscle cells and mouse pancreatic islets exposed to glucolipotoxic stress. We also report that carnosine, a histidine containing dipeptide, prevented 65-90% of 4-hydroxynonenal and 3-nitrotyrosine adduction events, and that this in turn preserved mitochondrial function and protected stimulus-secretion coupling in cells exposed to metabolic stress. Carnosine therefore offers significant therapeutic potential against metabolic diseases.

Retinal Stem Cell 'Retirement Plans': Growth, Regulation and Species Adaptations in the Retinal Ciliary Marginal Zone.

The vertebrate retina develops from a specified group of precursor cells that adopt distinct identities and generate lineages of either the neural retina, retinal pigmented epithelium, or ciliary body. In some species, including teleost fish and amphibians, proliferative cells with stem-cell-like properties capable of continuously supplying new retinal cells post-embryonically have been characterized and extensively studied. This region, termed the ciliary or circumferential marginal zone (CMZ), possibly represents a conserved retinal stem cell niche. In this review, we highlight the research characterizing similar CMZ-like regions, or stem-like cells located at the peripheral margin, across multiple different species. We discuss the proliferative parameters, multipotency and growth mechanisms of these cells to understand how they behave in vivo and how different molecular factors and signalling networks converge at the CMZ niche to regulate their activity. The evidence suggests that the mature retina may have a conserved propensity for homeostatic growth and plasticity and that dysfunction in the regulation of CMZ activity may partially account for dystrophic eye growth diseases such as myopia and hyperopia. A better understanding of the properties of CMZ cells will enable important insight into how an endogenous generative tissue compartment can adapt to altered retinal physiology and potentially even restore vision loss caused by retinal degenerative conditions.

Homocitrullination of lysine residues mediated by myeloid-derived suppressor cells in the tumor environment is a target for cancer immunotherapy.

BACKGROUND: Homocitrullination is the post-translational modification of lysine that is recognized by T cells. METHODS: This study identified homocitrullinated peptides from aldolase, enolase, cytokeratin and binding immunoglobulin protein and used human leukocyte antigen (HLA) transgenic mice to assess immunogenicity by enzyme-linked immunosorbent spot assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma expressing constitutive or interferon γ (IFNγ)-inducible major histocompatibility complex class II (MHC-II) as represented by most human tumors. To determine the mechanism behind the therapy, immune cell infiltrates were analyzed using flow cytometry and therapy studies in the presence of myeloperoxidase (MPO) inhibitor and T-cell depletion performed. We assessed the T-cell repertoire to homocitrullinated peptides in patients with cancer and healthy donors using flow cytometry. RESULTS: Homocitrulline (Hcit) peptide vaccination stimulated strong CD4 T-cell responses and induced significant antitumor therapy in an established tumor model. The antitumor response was dependent on CD4 T cells and the effect was driven mainly via direct tumor recognition, as responses were only observed if the tumors were induced to express MHC-II. In vitro proliferation assays show that healthy donors and patients with cancer have an oligoclonal CD4 T-cell repertoire recognizing homocitrullinated peptides. Inhibition of cyanate generation, which mediates homocitrullination, by MPO inhibition reduced tumor therapy by the vaccine induced T cells (p=0.0018). Analysis of the tumor microenvironment (TME) suggested that myeloid-derived suppressor cells (MDSCs) were a potential source of MPO. The selected B16 melanoma model showed MDSC infiltration and was appropriate to see if the Hcit vaccine could overcome the immunosuppression associated with MDSCs. The vaccine was very effective (90% survival) as the induced CD4 T cells directly targeted the homocitrullinated tumor and likely reversed the immunosuppressive environment. CONCLUSION: We propose that MPO, potentially produced by MDSCs, catalyzes the buildup of cyanate in the TME which diffuses into tumor cells causing homocitrullination of cytoplasmic proteins which are degraded and, in the presence of IFNγ, presented by MHC-II for direct CD4 T-cell recognition. Homocitrullinated proteins are a new target for cancer vaccines and may be particularly effective against tumors containing high levels of MPO expressing MDSCs.

Rab27a co-ordinates actin-dependent transport by controlling organelle-associated motors and track assembly proteins.

Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that the motor protein myosin-Va works with dynamic actin tracks to drive long-range organelle dispersion in opposition to microtubules. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that co-operate with formin-1 to generate actin tracks required for myosin-Va-dependent transport in melanocytes. Thus, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIREs to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this, we suggest a model in which organelles and force generators (motors and track assemblers) are linked, forming an organelle-based, cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.

MKRN2 Physically Interacts with GLE1 to Regulate mRNA Export and Zebrafish Retinal Development.

The mammalian mRNA nuclear export process is thought to terminate at the cytoplasmic face of the nuclear pore complex through ribonucleoprotein remodeling. We conduct a stringent affinity-purification mass-spectrometry-based screen of the physical interactions of human RNA-binding E3 ubiquitin ligases. The resulting protein-interaction network reveals interactions between the RNA-binding E3 ubiquitin ligase MKRN2 and GLE1, a DEAD-box helicase activator implicated in mRNA export termination. We assess MKRN2 epistasis with GLE1 in a zebrafish model. Morpholino-mediated knockdown or CRISPR/Cas9-based knockout of MKRN2 partially rescue retinal developmental defects seen upon GLE1 depletion, consistent with a functional association between GLE1 and MKRN2. Using ribonomic approaches, we show that MKRN2 binds selectively to the 3' UTR of a diverse subset of mRNAs and that nuclear export of MKRN2-associated mRNAs is enhanced upon knockdown of MKRN2. Taken together, we suggest that MKRN2 interacts with GLE1 to selectively regulate mRNA nuclear export and retinal development.

Exercise Training in Obese Rats Does Not Induce Browning at Thermoneutrality and Induces a Muscle-Like Signature in Brown Adipose Tissue.

Aim: Exercise training elicits diverse effects on brown (BAT) and white adipose tissue (WAT) physiology in rodents housed below their thermoneutral zone (i.e., 28-32°C). In these conditions, BAT is chronically hyperactive and, unlike human residence, closer to thermoneutrality. Therefore, we set out to determine the effects of exercise training in obese animals at 28°C (i.e., thermoneutrality) on BAT and WAT in its basal (i.e., inactive) state. Methods: Sprague-Dawley rats (n = 12) were housed at thermoneutrality from 3 weeks of age and fed a high-fat diet. At 12 weeks of age half these animals were randomized to 4-weeks of swim-training (1 h/day, 5 days per week). Following a metabolic assessment interscapular and perivascular BAT and inguinal (I)WAT were taken for analysis of thermogenic genes and the proteome. Results: Exercise attenuated weight gain but did not affect total fat mass or thermogenic gene expression. Proteomics revealed an impact of exercise training on 2-oxoglutarate metabolic process, mitochondrial respiratory chain complex IV, carbon metabolism, and oxidative phosphorylation. This was accompanied by an upregulation of multiple proteins involved in skeletal muscle physiology in BAT and an upregulation of muscle specific markers (i.e., Myod1, CkM, Mb, and MyoG). UCP1 mRNA was undetectable in IWAT with proteomics highlighting changes to DNA binding, the positive regulation of apoptosis, HIF-1 signaling and cytokine-cytokine receptor interaction. Conclusion: Exercise training reduced weight gain in obese animals at thermoneutrality and is accompanied by an oxidative signature in BAT which is accompanied by a muscle-like signature rather than induction of thermogenic genes. This may represent a new, UCP1-independent pathway through which BAT physiology is regulated by exercise training.

ß2-Adrenergic Signalling Promotes Cell Migration by Upregulating Expression of the Metastasis-Associated Molecule LYPD3.

Metastasis is associated with poor prognosis in breast cancer. Although some studies suggest beta-blockers increase survival by delaying metastasis, others have been discordant. This study provides both insights into the anomalous findings and identifies potential biomarkers that may be treatment targets. Cell line models of basal-type and oestrogen receptor-positive breast cancer were profiled for basal levels of adrenoceptor gene/protein expression, and ß2-adrenoceptor mediated cell behaviour including migration, invasion, adhesion, and survival in response to adrenoceptor agonist/antagonist treatment. Protein profiling and histology identified biomarkers and drug targets. Baseline levels of adrenoceptor gene expression are higher in basal-type rather than oestrogen receptor-positive cancer cells. Norepinephrine (NE) treatment increased invasive capacity in all cell lines but did not increase proliferation/survival. Protein profiling revealed the upregulation of the pro-metastatic gene Ly6/PLAUR Domain-Containing Protein 3 (LYPD3) in norepinephrine-treated MDA-MB-468 cells. Histology confirmed selective LYPD3 expression in primary and metastatic breast tumour samples. These findings demonstrate that basal-type cancer cells show a more aggressive adrenoceptor-ß2-activated phenotype in the resting and stimulated state, which is attenuated by adrenoceptor-ß2 inhibition. This study also highlights the first association between ADRß2 signalling and LYPD3; its knockdown significantly reduced the basal and norepinephrine-induced activity of MCF-7 cells in vitro. The regulation of ADRß2 signalling by LYPD3 and its metastasis promoting activities, reveal LYPD3 as a promising therapeutic target in the treatment of breast and other cancers.

Interscapular and Perivascular Brown Adipose Tissue Respond Differently to a Short-Term High-Fat Diet.

Brown adipose tissue (BAT) function may depend on its anatomical location and developmental origin. Interscapular BAT (iBAT) regulates acute macronutrient metabolism, whilst perivascular BAT (PVAT) regulates vascular function. Although phenotypically similar, whether these depots respond differently to acute nutrient excess is unclear. Given their distinct anatomical locations and developmental origins and we hypothesised that iBAT and PVAT would respond differently to brief period of nutrient excess. Sprague-Dawley rats aged 12 weeks (n=12) were fed either a standard (10% fat, n=6) or high fat diet (HFD: 45% fat, n=6) for 72h and housed at thermoneutrality. Following an assessment of whole body physiology, fat was collected from both depots for analysis of gene expression and the proteome. HFD consumption for 72h induced rapid weight gain (c. 2.6%) and reduced serum non-esterified fatty acids (NEFA) with no change in either total adipose or depot mass. In iBAT, an upregulation of genes involved in insulin signalling and lipid metabolism was accompanied by enrichment of lipid-related processes and functions, plus glucagon and peroxisome proliferator-activated receptor (PPAR) signalling pathways. In PVAT, HFD induced a pronounced down-regulation of multiple metabolic pathways which was accompanied with increased abundance of proteins involved in apoptosis (e.g. Hdgf and Ywaq) and toll-like receptor signalling (Ube2n). There was also an enrichment of DNA-related processes and functions (e.g. nucleosome assembly and histone exchange) and RNA degradation and cell adhesion pathways. In conclusion, we show that iBAT and PVAT elicit divergent responses to short-term nutrient excess highlighting early adaptations in these depots before changes in fat mass.

PYK2 promotes HER2-positive breast cancer invasion.

BACKGROUND: Metformin, a biguanide, is one of the most commonly prescribed treatments for type 2 diabetes and has recently been recommended as a potential drug candidate for advanced cancer therapy. Although Metformin has antiproliferative and proapoptotic effects on breast cancer, the heterogenous nature of this disease affects the response to metformin leading to the activation of pro-invasive signalling pathways that are mediated by the focal adhesion kinase PYK2 in pure HER2 phenotype breast cancer. METHODS: The effect of metformin on different breast cancer cell lines, representing the molecular heterogenicity of the disease was investigated using in vitro proliferation and apoptosis assays. The activation of PYK2 by metformin in pure HER2 phenotype (HER2+/ER-/PR-) cell lines was investigated by microarrays, quantitative real time PCR and immunoblotting. Cell migration and invasion PYK2-mediated and in response to metformin were determined by wound healing and invasion assays using HER2+/ER-/PR- PYK2 knockdown cell lines. Proteomic analyses were used to determine the role of PYK2 in HER2+/ER-/PR- proliferative, migratory and invasive cellular pathways and in response to metformin. The association between PYK2 expression and HER2+/ER-/PR- patients' cancer-specific survival was investigated using bioinformatic analysis of PYK2 expression from patient gene expression profiles generated by the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) study. The effect of PYK2 and metformin on tumour initiation and invasion of HER2+/ER-/PR- breast cancer stem-like cells was performed using the in vitro stem cell proliferation and invasion assays. RESULTS: Our study showed for the first time that pure HER2 breast cancer cells are more resistant to metformin treatment when compared with the other breast cancer phenotypes. This drug resistance was associated with the activation of PTK2B/PYK2, a well-known mediator of signalling pathways involved in cell proliferation, migration and invasion. The role of PYK2 in promoting invasion of metformin resistant HER2 breast cancer cells was confirmed through investigating the effect of PYK2 knockdown and metformin on cell invasion and by proteomic analysis of associated cellular pathways. We also reveal a correlation between high level of expression of PYK2 and reduced survival in pure HER2 breast cancer patients. Moreover, we also report a role of PYK2 in tumour initiation and invasion-mediated by pure HER2 breast cancer stem-like cells. This was further confirmed by demonstrating a correlation between reduced survival in pure HER2 breast cancer patients and expression of PYK2 and the stem cell marker CD44. CONCLUSIONS: We provide evidence of a PYK2-driven pro-invasive potential of metformin in pure HER2 cancer therapy and propose that metformin-based therapy should consider the molecular heterogeneity of breast cancer to prevent complications associated with cancer chemoresistance, invasion and recurrence in treated patients.

Generation of In Vitro Model of Epithelial Mesenchymal Transition (EMT) Via the Expression of a Cytoplasmic Mutant Form of Promylocytic Leukemia Protein (PML).

Epithelial Mesenchymal Transition (EMT) is a key event in cancer progression. During this event, epithelial cancer cells undergo molecular and cellular changes leading to their trans-differentiation into mesenchymal cancer cells that are capable of migration, invasion, and metastasis to other tissues and organs. Here, we present a method for in vitro induction of EMT in prostate cancer cell lines using lentiviral expression of a PMLI isoform mutant construct.