Research protocol and recruitment redesign of a study of pregnant women and their infants during the COVID-19 pandemic: Childhood Allergy and the NeOnatal Environment (CANOE).

Background: Recruitment for research studies is a challenging endeavor that was further complicated by the coronavirus disease 2019 pandemic. We launched a new multicenter birth cohort, Childhood Allergy and the NeOnatal Environment (CANOE), supported by the National Institutes of Health in January 2020 across 4 sites. Although the pandemic temporarily halted clinical research, we restructured the study and instituted novel recruitment methods that we hypothesized would enable brisk enrollment when research activities resumed. Objective: We sought to develop protocol modifications and recruitment methods that promote successful recruitment of diverse populations in clinical research despite a global pandemic. Methods: Even though study activities were suspended, we modified recruitment strategies to limit in-person contact, shifting toward alternative HIPAA-compliant methods such as clinician referrals, institutional social media, and telemedicine screening and consent procedures. Protocol changes included reducing the frequency of in-person visits, leveraging clinical care visits to collect biospecimens, expanded self-collection of samples at home, and making study materials available online. Results: Remote methods, including targeted social media posts, mailed letters, and email, combined with in-clinic recruitment with modifications for social distancing led to successful recruitment at all sites. Rates of consent have been similar across recruitment sites, with the highest rates of enrollment of mother-infant dyads realized by sites that implemented multiple recruitment strategies. Conclusions: Study procedures that prioritize health and safety measures such as social distancing, study participant convenience, and use diverse recruitment strategies enable successful enrollment of pregnant women and their newborns into clinical research while adhering to public health restrictions during a global pandemic.

An Omicron-specific, self-amplifying mRNA booster vaccine for COVID-19: a phase 2/3 randomized trial.

Here we conducted a multicenter open-label, randomized phase 2 and 3 study to assess the safety and immunogenicity of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron-specific (BA.1/B.1.1.529), monovalent, thermostable, self-amplifying mRNA vaccine, GEMCOVAC-OM, when administered intradermally as a booster in healthy adults who had received two doses of BBV152 or ChAdOx1 nCoV-19. GEMCOVAC-OM was well tolerated with no related serious adverse events in both phase 2 and phase 3. In phase 2, the safety and immunogenicity of GEMCOVAC-OM was compared with our prototype mRNA vaccine GEMCOVAC-19 (D614G variant-specific) in 140 participants. At day 29 after vaccination, there was a significant rise in anti-spike (BA.1) IgG antibodies with GEMCOVAC-OM (P < 0.0001) and GEMCOVAC-19 (P < 0.0001). However, the IgG titers (primary endpoint) and seroconversion were higher with GEMCOVAC-OM (P < 0.0001). In phase 3, GEMCOVAC-OM was compared with ChAdOx1 nCoV-19 in 3,140 participants (safety cohort), which included an immunogenicity cohort of 420 participants. At day 29, neutralizing antibody titers against the BA.1 variant of SARS-CoV-2 were significantly higher than baseline in the GEMCOVAC-OM arm (P < 0.0001), but not in the ChAdOx1 nCoV-19 arm (P = 0.1490). GEMCOVAC-OM was noninferior (primary endpoint) and superior to ChAdOx1 nCoV-19 in terms of neutralizing antibody titers and seroconversion rate (lower bound 95% confidence interval of least square geometric mean ratio >1 and difference in seroconversion >0% for superiority). At day 29, anti-spike IgG antibodies and seroconversion (secondary endpoints) were significantly higher with GEMCOVAC-OM (P < 0.0001). These results demonstrate that GEMCOVAC-OM is safe and boosts immune responses against the B.1.1.529 variant. Clinical Trial Registry India identifier: CTRI/2022/10/046475 .

Broad de-regulated U2AF1 splicing is prognostic and augments leukemic transformation via protein arginine methyltransferase activation.

The role of splicing dysregulation in cancer is underscored by splicing factor mutations; however, its impact in the absence of such rare mutations is poorly understood. To reveal complex patient subtypes and putative regulators of pathogenic splicing in Acute Myeloid Leukemia (AML), we developed a new approach called OncoSplice. Among diverse new subtypes, OncoSplice identified a biphasic poor prognosis signature that partially phenocopies U2AF1-mutant splicing, impacting thousands of genes in over 40% of adult and pediatric AML cases. U2AF1-like splicing co-opted a healthy circadian splicing program, was stable over time and induced a leukemia stem cell (LSC) program. Pharmacological inhibition of the implicated U2AF1-like splicing regulator, PRMT5, rescued leukemia mis-splicing and inhibited leukemic cell growth. Genetic deletion of IRAK4, a common target of U2AF1-like and PRMT5 treated cells, blocked leukemia development in xenograft models and induced differentiation. These analyses reveal a new prognostic alternative-splicing mechanism in malignancy, independent of splicing-factor mutations.

Glucagon-Like Peptide-1 Receptors in the Gustatory Cortex Influence Food Intake.

The gustatory cortex (GC) region of the insular cortex processes taste information in manners important for taste-guided behaviors, including food intake itself. In addition to oral gustatory stimuli, GC activity is also influenced by physiological states including hunger. The specific cell types and molecular mechanisms that provide the GC with such abilities are unclear. Glucagon-like peptide 1 (GLP-1) is produced by neurons in the brain, where it can act on GLP-1 receptor-expressing (GLP-1R+) neurons found in several brain regions. In these brain regions, GLP-1R agonism suppresses homeostatic food intake and dampens the hedonic value of food. Here, we report in mice of both sexes that cells within the GC express Glp1r mRNA and further, by ex vivo brain slice recordings, that GC GLP-1R+ neurons are depolarized by the selective GLP-1R agonist, exendin-4. Next we found that chemogenetic stimulation of GLP-1R+ neurons, and also pharmacological stimulation of GC-GLP-1Rs themselves, both reduced homeostatic food intake. When mice were chronically maintained on diets with specific fat contents and then later offered foods with new fat contents, we also found that GLP-1R agonism reduced food intake toward foods with differing fat contents, indicating that GC GLP-1R influences may depend on palatability of the food. Together, these results provide evidence for a specific cell population in the GC that may hold roles in both homeostatic and hedonic food intake.SIGNIFICANCE STATEMENT The present study demonstrates that a population of neurons in the GC region of the insular cortex expresses receptors for GLP-1Rs, these neurons are depolarized by agonism of GLP-1Rs, and GC GLP-1Rs can influence food intake on their activation, including in manners depending on food palatability. This work is significant by adding to our understanding of the brain systems that mediate ingestive behavior, which holds implications for metabolic diseases.

Linking α-synuclein-induced synaptopathy and neural network dysfunction in early Parkinson's disease.

The prodromal phase of Parkinson's disease is characterized by aggregation of the misfolded pathogenic protein α-synuclein in select neural centres, co-occurring with non-motor symptoms including sensory and cognitive loss, and emotional disturbances. It is unclear whether neuronal loss is significant during the prodrome. Underlying these symptoms are synaptic impairments and aberrant neural network activity. However, the relationships between synaptic defects and network-level perturbations are not established. In experimental models, pathological α-synuclein not only impacts neurotransmission at the synaptic level, but also leads to changes in brain network-level oscillatory dynamics-both of which likely contribute to non-motor deficits observed in Parkinson's disease. Here we draw upon research from both human subjects and experimental models to propose a 'synapse to network prodrome cascade' wherein before overt cell death, pathological α-synuclein induces synaptic loss and contributes to aberrant network activity, which then gives rise to prodromal symptomology. As the disease progresses, abnormal patterns of neural activity ultimately lead to neuronal loss and clinical progression of disease. Finally, we outline goals and research needed to unravel the basis of functional impairments in Parkinson's disease and other α-synucleinopathies.

Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies.

Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.

Patient Satisfaction with Substance Use Disorder Rehabilitation Services: a Qualitative Study.

Despite its recognized value, there is a gap in the assessment of patient satisfaction among patients with substance use disorder (SUD) in rehabilitation. The study objective was to determine patient satisfaction dimensions relevant to individuals receiving residential rehabilitation for SUD. Semi-structured interviews were conducted with the following: (1) adult males enrolled in the program and (2) counseling staff involved in the care of these individuals. A literature review formed the basis for interviews, which were audio recorded and transcribed. Text data was analyzed using directed content analysis to identify dimensions relevant to patient satisfaction. Eighteen individuals participated, including 14 men with SUD and four staff. Content analysis of the interview transcripts resulted in five themes: (1) counselor (skill), (2) programmatic structure (adhering), (3) skill development (personal responsibility), (4) comparison to other programs, and (5) case management facilitation. These dimensions will be utilized to create a patient satisfaction tool specific to SUD rehabilitation.

Efficacy and safety of neural stem cell therapy for spinal cord injury: A systematic literature review.

AIMS: To summarize the evidence on the efficacy and safety of neural stem cell therapy (NSCT) for the treatment of spinal cord injury (SCI). METHODS: A systematic literature review of Medline®, EMBASE® and Cochrane library was performed to identify studies reporting efficacy and safety of NSCT in SCI. Articles were included if they reported efficacy and safety data of SCI patients who received NSCT. RESULTS: Overall, four studies of the 277 records met all the study eligibility criteria. Over the 1-year follow-up period, motor scores were significantly higher among patients who received NSCT compared with those who did not (American Spinal Injury Association [ASIA] motor scores (mean±standard deviation [SD]): 7.9±1.2 versus 3.9±0.6; upper extremity motor score: 7.8±2.1 versus 3.9±0.6, both P<0.05). Sensory scores (pinprick score: 4.8±1.3 versus 2.9±0.6; P=0.5; light touch score: 6.9±3.1 versus 2.3±0.5, P=0.3), ASIA impairment scale (26% versus 7%) or pain score (baseline: 2.4±0.6; 1-year: 3.4±0.4) were comparable in both NSCT and non-NSCT cohorts. Over the 1-year follow-up period, the graded redefined assessment of strength, sensibility, and prehension and international standards for neurological classification of SCI scores showed a mean improvement of 14.8 and 17.8 points respectively. Overall, treatment with NSCT showed favorable safety and tolerability profile. CONCLUSIONS: Due to the limited and poor-quality evidence, it is too early to make robust conclusions on the efficacy of NSCT in the treatment of SCI. However, based on the included studies, NSCT seems to be a potential option worth exploring among patients with SCI. Nonetheless, prospective, randomized trials in larger cohorts are needed to validate the efficacy and safety of NSCT in the treatment of SCI.

Perturbation of in vivo Neural Activity Following α-Synuclein Seeding in the Olfactory Bulb.

BACKGROUND: Parkinson's disease (PD) neuropathology is characterized by intraneuronal protein aggregates composed of misfolded α-Synuclein (α-Syn), as well as degeneration of substantia nigra dopamine neurons. Deficits in olfactory perception and aggregation of α-Syn in the olfactory bulb (OB) are observed during early stages of PD, and have been associated with the PD prodrome, before onset of the classic motor deficits. α-Syn fibrils injected into the OB of mice cause progressive propagation of α-Syn pathology throughout the olfactory system and are coupled to olfactory perceptual deficits. OBJECTIVE: We hypothesized that accumulation of pathogenic α-Syn in the OB impairs neural activity in the olfactory system. METHODS: To address this, we monitored spontaneous and odor-evoked local field potential dynamics in awake wild type mice simultaneously in the OB and piriform cortex (PCX) one, two, and three months following injection of pathogenic preformed α-Syn fibrils in the OB. RESULTS: We detected α-Syn pathology in both the OB and PCX. We also observed that α-Syn fibril injections influenced odor-evoked activity in the OB. In particular, α-Syn fibril-injected mice displayed aberrantly high odor-evoked power in the beta spectral range. A similar change in activity was not detected in the PCX, despite high levels of α-Syn pathology. CONCLUSION: Together, this work provides evidence that synucleinopathy impacts in vivo neural activity in the olfactory system at the network-level.

Publisher Correction: MBNL1 regulates essential alternative RNA splicing patterns in MLL-rearranged leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Hippo pathway oncoprotein YAP promotes melanoma cell invasion and spontaneous metastasis.

Melanoma is a deadly form of skin cancer that accounts for a disproportionally large proportion of cancer-related deaths in younger people. Compared with most other skin cancers, a feature of melanoma is its high metastatic capacity, although the mechanisms that confer this are not well understood. The Hippo pathway is a key regulator of organ growth and cell fate that is deregulated in many cancers. To analyse the Hippo pathway in cutaneous melanoma, we generated a transcriptional signature of melanoma cells that overexpressed YAP, the key downstream Hippo pathway oncoprotein. YAP-mediated transcriptional activity varied in melanoma cell lines but did not cluster with known genetic drivers of melanomagenesis such as BRAF and NRAS mutations. Instead, it correlated strongly with published gene expression profiles linked to melanoma cell invasiveness and varied throughout the metastatic cascade in melanoma patient tumours. Consistent with this, YAP was both necessary and sufficient for melanoma cell invasion in vitro. In vivo, YAP promoted spontaneous melanoma metastasis, whilst the growth of YAP-expressing primary tumours was impeded. Finally, we identified the YAP target genes AXL, THBS1 and CYR61 as key mediators of YAP-induced melanoma cell invasion. These data suggest that YAP is a critical regulator of melanoma metastasis.

The Hippo Pathway as a Driver of Select Human Cancers.

The Hippo pathway regulates myriad biological processes in diverse species and is a key cancer signaling network in humans. Although Hippo has been linked to multiple aspects of cancer, its role in this disease is incompletely understood. Large-scale pan-cancer analyses of core Hippo pathway genes reveal that the pathway is mutated at a high frequency only in select human cancers, including malignant mesothelioma and meningioma. Hippo pathway deregulation is also enriched in squamous epithelial cancers. We discuss cancer-related functions of the Hippo pathway and potential explanations for the cancer-restricted mutation profile of core Hippo pathway genes. Greater understanding of Hippo pathway deregulation in cancers will be essential to guide the imminent use of Hippo-targeted therapies.

MBNL1 regulates essential alternative RNA splicing patterns in MLL-rearranged leukemia.

Despite growing awareness of the biologic features underlying MLL-rearranged leukemia, targeted therapies for this leukemia have remained elusive and clinical outcomes remain dismal. MBNL1, a protein involved in alternative splicing, is consistently overexpressed in MLL-rearranged leukemias. We found that MBNL1 loss significantly impairs propagation of murine and human MLL-rearranged leukemia in vitro and in vivo. Through transcriptomic profiling of our experimental systems, we show that in leukemic cells, MBNL1 regulates alternative splicing (predominantly intron exclusion) of several genes including those essential for MLL-rearranged leukemogenesis, such as DOT1L and SETD1A. We finally show that selective leukemic cell death is achievable with a small molecule inhibitor of MBNL1. These findings provide the basis for a new therapeutic target in MLL-rearranged leukemia and act as further validation of a burgeoning paradigm in targeted therapy, namely the disruption of cancer-specific splicing programs through the targeting of selectively essential RNA binding proteins.

Breast Cancer Incidence and Mortality by Molecular Subtype: Statewide Age and Racial/Ethnic Disparities in New Jersey.

The objective of this study was to assess breast cancer incidence and mortality rates by molecular subtype for cases diagnosed in New Jersey. Data on all primary, histologically confirmed, invasive breast cancers diagnosed among women between January 1, 2008 and December 31, 2013 were retrieved from the New Jersey State Cancer Registry. Age-adjusted incidence rates were calculated for each subtype, by ageandrace/ethnicity. Logistic regression models, Cox proportional hazards models, and Kaplan Meier curves were used to describe the relative risks for breast cancer incidence, mortality, and survival, respectively. In this population-based sample of 32,770 breast cancer cases, non-Hispanic Blacks (NHBs) had the highest triple-negative breast cancer (TNBC) incidence rate (17.8 per 100,000, 95% CI 16.5-19.2) compared to other races/ethnicities. NHBs had also higher odds of TNBC (OR 2.1, 95% CI 1.95-2.36) and higher hazards of death when diagnosed with TNBC (HR 1.28, 95% CI 1.05-1.56), luminal A (HR 1.64, 95% CI 1.41-1.91), or luminal B (HR 1.54, 95% CI 1.10-2.15) than non-Hispanic Whites (NHWs). Younger women (20-39 years) had higher odds of TNBC (OR 1.77, 95% CI 1.54-2.02) and luminal B (OR 1.56, 95% CI 1.35-1.80) compared to women 50-64 years; minority women had higher odds of non-luminal HER2-expressing and lower odds of luminal A than NHWs. TNBC was associated with the poorest survival rates. These findings highlight a need for enhanced screening to promote earlier diagnosis and improve breast cancer outcomes, particularly in minorities and younger women, which will be essential for achieving health equity.

U2AF1 mutations induce oncogenic IRAK4 isoforms and activate innate immune pathways in myeloid malignancies.

Spliceosome mutations are common in myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML), but the oncogenic changes due to these mutations have not been identified. Here a global analysis of exon usage in AML samples revealed distinct molecular subsets containing alternative spliced isoforms of inflammatory and immune genes. Interleukin-1 receptor-associated kinase 4 (IRAK4) was the dominant alternatively spliced isoform in MDS and AML and is characterized by a longer isoform that retains exon 4, which encodes IRAK4-long (IRAK4-L), a protein that assembles with the myddosome, results in maximal activation of nuclear factor kappa-light-chain-enhancer of B cells (NF-κB) and is essential for leukaemic cell function. Expression of IRAK4-L is mediated by mutant U2 small nuclear RNA auxiliary factor 1 (U2AF1) and is associated with oncogenic signalling in MDS and AML. Inhibition of IRAK4-L abrogates leukaemic growth, particularly in AML cells with higher expression of the IRAK4-L isoform. Collectively, mutations in U2AF1 induce expression of therapeutically targetable 'active' IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signalling in MDS and AML.

Association of Medicaid enrollee characteristics and primary care utilization with cancer outcomes for the period spanning Medicaid expansion in New Jersey.

BACKGROUND: Cancer outcomes for Medicaid enrollees may be affected by patients' primary care (PC) utilization and complex Medicaid enrollment dynamics, which have recently changed for many states under the Affordable Care Act. METHODS: With New Jersey State Cancer Registry and linked Medicaid claims data, a retrospective cohort study was conducted for patients with incident breast, colorectal, or invasive cervical cancer (aged 21-64 years) diagnosed in 2012-2014. Associations of Medicaid enrollment factors and PC utilization with the stage at diagnosis and treatment delays were examined with multivariate logistic regression models. RESULTS: The study included 19,209 total cancer cases and 3253 linked Medicaid cases. Medicaid cases were more likely to be diagnosed at a late stage and to experience treatment delays in comparison with non-Medicaid cases. In adjusted analyses, Medicaid cases with 1 or more PC visits before the diagnosis had lower odds of a late-stage diagnosis (odds ratio, 0.47; 95% confidence interval, 0.33-0.67) in comparison with Medicaid cases with no outpatient visits. New enrollees (<6 months) and longer term enrollees in fee-for-service (FFS) Medicaid had greater odds of a late-stage diagnosis and treatment delays in comparison with those in Medicaid managed care. CONCLUSIONS: Medicaid patients with cancer diagnosed just before and in the initial year of eligibility expansion had worse outcomes than non-Medicaid cases. Poor outcomes were especially pronounced among new enrollees, those without outpatient visits before their diagnosis, and FFS enrollees. Targeted strategies to enhance care continuity, including access to PC providers before the diagnosis and a better understanding of pathways to cancer care upon Medicaid enrollment, are needed to improve outcomes in this population.

A Drosophila RNAi library modulates Hippo pathway-dependent tissue growth.

Libraries of transgenic Drosophila melanogaster carrying RNA interference (RNAi) constructs have been used extensively to perform large-scale functional genetic screens in vivo. For example, RNAi screens have facilitated the discovery of multiple components of the Hippo pathway, an evolutionarily conserved growth-regulatory network. Here we investigate an important technical limitation with the widely used VDRC KK RNAi collection. We find that approximately 25% of VDRC KK RNAi lines cause false-positive enhancement of the Hippo pathway, owing to ectopic expression of the Tiptop transcription factor. Of relevance to the broader Drosophila community, ectopic tiptop (tio) expression can also cause organ malformations and mask phenotypes such as organ overgrowth. To enhance the use of the VDRC KK RNAi library, we have generated a D. melanogaster strain that will allow researchers to test, in a single cross, whether their genetic screen of interest will be affected by ectopic tio expression.

PARP-14 binds specific DNA sequences to promote Th2 cell gene expression.

PARP-14, a member of the poly ADP-ribose polymerase super family, promotes T helper cell 2 (Th2) differentiation by regulating interleukin-4 (IL-4) and STAT6-dependent transcription. Yet, whether PARP-14 globally impacts gene regulation has not been determined. In this report, using an RNA pol II ChIP-seq approach, we identify genes in Th2 cells that are regulated by PARP-14, and either dependent or independent of ADP-ribosyltransferase catalytic activity. Our data demonstrate that PARP-14 enhances the expression of Th2 genes as it represses the expression of Th1-associated genes. Among the relevant targets are Signal Transducer and Activator of Transcription genes required for polarizing Th1 and Th2 cells. To define a mechanism for PARP-14 function, we use an informatics approach to identify putative PARP-14 DNA binding sites. Two putative PARP-14 binding motifs are identified in multiple Th2 cytokine genes, and we demonstrate that PARP-14 interacts with each motif using in vitro binding assays. Taken together our results indicate that PARP-14 is an important factor for T helper cell differentiation and it binds to specific DNA sequences to mediate its function.

Th9 cell development requires a BATF-regulated transcriptional network.

T helper 9 (Th9) cells are specialized for the production of IL-9, promote allergic inflammation in mice, and are associated with allergic disease in humans. It has not been determined whether Th9 cells express a characteristic transcriptional signature. In this study, we performed microarray analysis to identify genes enriched in Th9 cells compared with other Th subsets. This analysis defined a transcriptional regulatory network required for the expression of a subset of Th9-enriched genes. The activator protein 1 (AP1) family transcription factor BATF (B cell, activating transcription factor­like) was among the genes enriched in Th9 cells and was required for the expression of IL-9 and other Th9-associated genes in both human and mouse T cells. The expression of BATF was increased in Th9 cultures derived from atopic infants compared with Th9 cultures from control infants. T cells deficient in BATF expression had a diminished capacity to promote allergic inflammation compared with wild-type controls. Moreover, mouse Th9 cells ectopically expressing BATF were more efficient at promoting allergic inflammation than control transduced cells. These data indicate that BATF is a central regulator of the Th9 phenotype and contributes to the development of allergic inflammation.