Differential impact of sex on regulation of skeletal muscle mitochondrial function and protein homeostasis by hypoxia-inducible factor-1α in normoxia.

Hypoxia-inducible factor (HIF)-1α is continuously synthesized and degraded in normoxia. During hypoxia, HIF1α stabilization restricts cellular/mitochondrial oxygen utilization. Cellular stressors can stabilize HIF1α even during normoxia. However, less is known about HIF1α function(s) and sex-specific effects during normoxia in the basal state. Since skeletal muscle is the largest protein store in mammals and protein homeostasis has high energy demands, we determined HIF1α function at baseline during normoxia in skeletal muscle. Untargeted multiomics data analyses were followed by experimental validation in differentiated murine myotubes with loss/gain of function and skeletal muscle from mice without/with post-natal muscle-specific Hif1a deletion (Hif1amsd). Mitochondrial oxygen consumption studies using substrate, uncoupler, inhibitor, titration protocols; targeted metabolite quantification by gas chromatography-mass spectrometry; and post-mitotic senescence markers using biochemical assays were performed. Multiomics analyses showed enrichment in mitochondrial and cell cycle regulatory pathways in Hif1a deleted cells/tissue. Experimentally, mitochondrial oxidative functions and ATP content were higher with less mitochondrial free radical generation with Hif1a deletion. Deletion of Hif1a also resulted in higher concentrations of TCA cycle intermediates and HIF2α proteins in myotubes. Overall responses to Hif1amsd were similar in male and female mice, but changes in complex II function, maximum respiration, Sirt3 and HIF1ß protein expression and muscle fibre diameter were sex-dependent. Adaptive responses to hypoxia are mediated by stabilization of constantly synthesized HIF1α. Despite rapid degradation, the presence of HIF1α during normoxia contributes to lower mitochondrial oxidative efficiency and greater post-mitotic senescence in skeletal muscle. In vivo responses to HIF1α in skeletal muscle were differentially impacted by sex. KEY POINTS: Hypoxia-inducible factor -1α (HIF1α), a critical transcription factor, undergoes continuous synthesis and proteolysis, enabling rapid adaptive responses to hypoxia by reducing mitochondrial oxygen consumption. In mammals, skeletal muscle is the largest protein store which is determined by a balance between protein synthesis and breakdown and is sensitive to mitochondrial oxidative function. To investigate the functional consequences of transient HIF1α expression during normoxia in the basal state, myotubes and skeletal muscle from male and female mice with HIF1α knockout were studied using complementary multiomics, biochemical and metabolite assays. HIF1α knockout altered the electron transport chain, mitochondrial oxidative function, signalling molecules for protein homeostasis, and post-mitotic senescence markers, some of which were differentially impacted by sex. The cost of rapid adaptive responses mediated by HIF1α is lower mitochondrial oxidative efficiency and post-mitotic senescence during normoxia.

Pain and its Perceived Relatedness to the Onset, Maintenance, and Relapse of Opioid use Disorder: A Descriptive Study of Non-Treatment-Seeking Individuals.

Background: Previous research has found chronic pain to be prevalent among individuals with opioid use disorder (OUD). The perception that pain is related to OUD onset, maintenance, relapse, and treatment delay has been noted in this population. However, prior works primarily involved treatment-engaged populations. Scant research describes such perceptions among non-treatment-seeking individuals. Aims: This study describes pain burden and perceptions regarding the role of pain in OUD onset, maintenance, relapse, and addiction treatment delay in a sample of individuals with untreated OUD. Methods: This cross-sectional study surveyed syringe exchange participants (n = 141). Participants responded to a survey including Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition OUD criteria, pain survey scales, demographic characteristics, and questions regarding pain and its perceived relatedness to aspects of OUD. Results: Most participants reported pain within the past 4 weeks (127, 91.4%). Data displayed a skew toward more intense pain ratings, with 120 reporting their pain as greater than mild (86.3%). A majority of participants agreed that pain was responsible for their OUD onset (79, 56.4%), maintenance (76, 54.3%), past relapse experience (82, 57.9%), and treatment delay (81, 57.9%). Correlative analyses revealed that pain severity and interference measures displayed moderate and statistically significant associations with extent of perceived relatedness of pain to these aspects of OUD. Conclusions: Among this sample of individuals with untreated OUD, pain and pain interference were prevalent. Pain was perceived to be related to OUD onset, maintenance, relapse, and treatment delay by a majority of the sample. These findings are in accordance with and expand upon prior works. Abbreviations: OUD: opioid use disorder; DSM-5: Diagnostic and Statistical Manual 5; BPI: Brief Pain Inventory; NIDA: National Institute on Drug Abuse; IASP: International Association for the Study of Pain; MOUD: Medications for Opioid Use Disorder; IQR: Interquartile Range.

Contexte: Des recherches antérieures ont montré que la douleur chronique est prévalente chez les personnes souffrant d'un trouble lié à l'utilisation d'opioïdes (TUO). La perception que la douleur est liée à l'apparition, au maintien, à la rechute et au retard dans le début du traitement a été constatée au sein de cette population. Toutefois, les travaux antérieurs portaient principalement sur les populations engagées dans un traitement. Peu de recherches décrivent ces perceptions chez les personnes qui ne sont pas à la recherche d'un traitement.Objectifs: Cette étude décrit le fardeau de la douleur et les perceptions concernant le rôle de la douleur dans l'apparition, le maintien, la rechute et le retard dans le début du traitement du trouble lié à l'utilisation d'opioïdes chez un échantillon de personnes souffrant d'un trouble lié à l'utilisation d'opioïdes non traité.Méthodes: Cette étude transversale a interrogé des participants pratiquant l'échange de seringues (n = 141). Les participants ont répondu à un questionnaire comprenant les critères de la cinquième édition du Manuel diagnostique et statistique des troubles mentaux pour le trouble lié à l'utilisation d'opioïdes, des échelles d'évaluation de la douleur, leurs caractéristiques démographiques, ainsi que des questions concernant la douleur et son lien perçu avec différents aspects du trouble lié à l'utilisation d'opioïdes.Résultats: La plupart des participants ont déclaré avoir ressenti de la douleur au cours des quatre dernières semaines (127, 91,4 %). Les données ont montré une tendance à évaluer la douleur de façon plus intense, 120 participants ayant déclaré que leur douleur était plus que légère (86,3 %). La majorité des participants ont reconnu que la douleur était responsable de l'apparition (79, 56,4 %), du maintien (76, 54,3 %), des rechutes antérieures (82, 57,9 %) et du retard dans le début du traitement (81, 57,9 %) du trouble lié à l'utilisation des opioïdes. Les analyses corrélatives ont révélé que les mesures de l'intensité et de l'interférence de la douleur présentaient des associations modérées et statistiquement significatives avec l'étendue du lien perçu entre la douleur et ces aspects du trouble lié à l'utilisation des opioïdes.Conclusions: Dans cet échantillon de personnes atteintes d'un trouble lié à l'utilisation des opioïdes non traité, la douleur et l'interférence de la douleur étaient prévalentes. La douleur était perçue comme étant liée à l'apparition, au maintien, à la rechute et au retard dans le début du traitement du trouble lié à l'utilisation des opioïdes par la majeure partie de l'échantillon. Ces résultats sont conformes aux travaux antérieurs et les étayent.Abréviations: TUO : trouble lié à l'utilisation d'opioïdes; DSM-5 : Manuel diagnostique et statistique 5; BPI : Questionnaire concis sur la douleur; NIDA : National Institute on Drug Abuse; IASP : Association internationale pour l'étude de la douleur; MTUO : Médicaments pour le trouble lié à l'utilisation des opioïdes; EIQ : Écart interquartile.

Multivariate investigation of aging in mouse models expressing the Alzheimer's protective APOE2 allele: integrating cognitive metrics, brain imaging, and blood transcriptomics.

APOE allelic variation is critical in brain aging and Alzheimer's disease (AD). The APOE2 allele associated with cognitive resilience and neuroprotection against AD remains understudied. We employed a multipronged approach to characterize the transition from middle to old age in mice with APOE2 allele, using behavioral assessments, image-derived morphometry and diffusion metrics, structural connectomics, and blood transcriptomics. We used sparse multiple canonical correlation analyses (SMCCA) for integrative modeling, and graph neural network predictions. Our results revealed brain sub-networks associated with biological traits, cognitive markers, and gene expression. The cingulate cortex emerged as a critical region, demonstrating age-associated atrophy and diffusion changes, with higher fractional anisotropy in males and middle-aged subjects. Somatosensory and olfactory regions were consistently highlighted, indicating age-related atrophy and sex differences. The hippocampus exhibited significant volumetric changes with age, with differences between males and females in CA3 and CA1 regions. SMCCA underscored changes in the cingulate cortex, somatosensory cortex, olfactory regions, and hippocampus in relation to cognition and blood-based gene expression. Our integrative modeling in aging APOE2 carriers revealed a central role for changes in gene pathways involved in localization and the negative regulation of cellular processes. Our results support an important role of the immune system and response to stress. This integrative approach offers novel insights into the complex interplay among brain connectivity, aging, and sex. Our study provides a foundation for understanding the impact of APOE2 allele on brain aging, the potential for detecting associated changes in blood markers, and revealing novel therapeutic intervention targets.

Injectable pH and Thermo-Responsive Hydrogel Scaffold with Enhanced Osteogenic Differentiation of Preosteoblasts for Bone Regeneration.

Bone fractures are common in the geriatric population and pose a great economic burden worldwide. While traditional methods for repairing bone defects have primarily been autografts, there are several drawbacks limiting its use. Bone graft substitutes have been used as alternative strategies to improve bone healing. However, there remain several impediments to achieving the desired healing outcomes. Injectable hydrogels have become attractive scaffold materials for bone regeneration, given their high performance in filling irregularly sized bone defects and their ability to encapsulate cells and bioactive molecules and mimic the native ECM of bone. We investigated the use of an injectable chitosan-based hydrogel scaffold to promote the differentiation of preosteoblasts in vitro. The hydrogels were characterized by evaluating cell homogeneity, cell viability, rheological and mechanical properties, and differentiation ability of preosteoblasts in hydrogel scaffolds. Cell-laden hydrogel scaffolds exhibited shear thinning behavior and the ability to maintain shape fidelity after injection. The CNC-CS hydrogels exhibited higher mechanical strength and significantly upregulated the osteogenic activity and differentiation of preosteoblasts, as shown by ALP activity assays and histological analysis of hydrogel scaffolds. These results suggest that this injectable hydrogel is suitable for cell survival, can promote osteogenic differentiation of preosteoblasts, and structurally support new bone growth.

Clonal evolution and stereotyped sequences of human IgE lineages in aeroallergen-specific immunotherapy.

BACKGROUND: Allergic disease reflects specific inflammatory processes initiated by interaction between allergen and allergen-specific IgE. Specific immunotherapy (SIT) is an effective long-term treatment option, but the mechanisms by which SIT provides desensitization are not well understood. OBJECTIVE: Our aim was to characterize IgE sequences expressed by allergen-specific B cells over a 3-year longitudinal study of patients with aeroallergies who were undergoing SIT. METHODS: Allergen-specific IgE-expressing clones were identified by using combinatorial single-chain variable fragment libraries and tracked in PBMCs and nasal biopsy samples over a 3-year period with antibody gene repertoire sequencing. The characteristics of private IgE-expressing clones were compared with those of stereotyped or "public" IgE responses to the grass pollen allergen Phleum pratense (Phl p) 2. RESULT: Members of the same allergen-specific IgE lineages were observed in nasal biopsy samples and blood, and lineages detected at baseline persisted in blood and nasal biopsy samples after 3 years of SIT, including B cells that express IgE. Evidence of progressive class switch recombination to IgG subclasses was observed after 3 years of SIT. A common stereotyped Phl p 2-specific antibody heavy chain sequence was detected in multiple donors. The amino acid residues enriched in IgE-stereotyped sequences from seropositive donors were analyzed with machine learning and k-mer motif discovery. Stereotyped IgE sequences had lower overall rates of somatic hypermutation and antigen selection than did single-chain variable fragment-derived allergen-specific sequences or IgE sequences of unknown specificity. CONCLUSION: Longitudinal tracking of rare circulating and tissue-resident allergen-specific IgE+ clones demonstrates persistence of allergen-specific IgE+ clones, progressive class switch recombination to IgG subtypes, and distinct maturation of a stereotyped Phl p 2 clonotype.

Adaptive exhaustion during prolonged intermittent hypoxia causes dysregulated skeletal muscle protein homeostasis.

Nocturnal hypoxaemia, which is common in chronic obstructive pulmonary disease (COPD) patients, is associated with skeletal muscle loss or sarcopenia, which contributes to adverse clinical outcomes. In COPD, we have defined this as prolonged intermittent hypoxia (PIH) because the duration of hypoxia in skeletal muscle occurs through the duration of sleep followed by normoxia during the day, in contrast to recurrent brief hypoxic episodes during obstructive sleep apnoea (OSA). Adaptive cellular responses to PIH are not known. Responses to PIH induced by three cycles of 8 h hypoxia followed by 16 h normoxia were compared to those during chronic hypoxia (CH) or normoxia for 72 h in murine C2C12 and human inducible pluripotent stem cell-derived differentiated myotubes. RNA sequencing followed by downstream analyses were complemented by experimental validation of responses that included both unique and shared perturbations in ribosomal and mitochondrial function during PIH and CH. A sarcopenic phenotype characterized by decreased myotube diameter and protein synthesis, and increased phosphorylation of eIF2α (Ser51) by eIF2α kinase, and of GCN-2 (general controlled non-derepressed-2), occurred during both PIH and CH. Mitochondrial oxidative dysfunction, disrupted supercomplex assembly, lower activity of Complexes I, III, IV and V, and reduced intermediary metabolite concentrations occurred during PIH and CH. Decreased mitochondrial fission occurred during CH. Physiological relevance was established in skeletal muscle of mice with COPD that had increased phosphorylation of eIF2α, lower protein synthesis and mitochondrial oxidative dysfunction. Molecular and metabolic responses with PIH suggest an adaptive exhaustion with failure to restore homeostasis during normoxia. KEY POINTS: Sarcopenia or skeletal muscle loss is one of the most frequent complications that contributes to mortality and morbidity in patients with chronic obstructive pulmonary disease (COPD). Unlike chronic hypoxia, prolonged intermittent hypoxia is a frequent, underappreciated and clinically relevant model of hypoxia in patients with COPD. We developed a novel, in vitro myotube model of prolonged intermittent hypoxia with molecular and metabolic perturbations, mitochondrial oxidative dysfunction, and consequent sarcopenic phenotype. In vivo studies in skeletal muscle from a mouse model of COPD shared responses with our myotube model, establishing the pathophysiological relevance of our studies. These data lay the foundation for translational studies in human COPD to target prolonged, nocturnal hypoxaemia to prevent sarcopenia in these patients.

Injectable pH Thermo-Responsive Hydrogel Scaffold for Tumoricidal Neural Stem Cell Therapy for Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults and despite recent advances in treatment modalities, GBM remains incurable. Injectable hydrogel scaffolds are a versatile delivery system that can improve delivery of drug and cell therapeutics for GBM. In this report, we investigated an injectable nanocellulose/chitosan-based hydrogel scaffold for neural stem cell encapsulation and delivery. Hydrogels were prepared using thermogelling beta-glycerophosphate (BGP) and hydroxyethyl cellulose (HEC), chitosan (CS), and cellulose nanocrystals (CNCs). We evaluated the impact of neural stem cells on hydrogel gelation kinetics, microstructures, and degradation. Furthermore, we investigated the biomaterial effects on cell viability and functionality. We demonstrated that the incorporation of cells at densities of 1, 5 and 10 million does not significantly impact rheological and physical properties CS scaffolds. However, addition of CNCs significantly prolonged hydrogel degradation when cells were seeded at 5 and 10 million per 1 mL hydrogel. In vitro cell studies demonstrated high cell viability, release of TRAIL at therapeutic concentrations, and effective tumor cell killing within 72 h. The ability of these hydrogel scaffolds to support stem cell encapsulation and viability and maintain stem cell functionality makes them an attractive cell delivery system for local treatment of post-surgical cancers.

A phenomenological study identifying facilitators and barriers to Black and Latinx youth's engagement in hospital-based violence intervention programs.

Black and Latinx youth are disproportionately affected by violence in the United States. Hospital-based violence intervention programs (HVIPs) have emerged as an effective response to this epidemic; however, participation rates remain low. This study aimed to identify facilitators and barriers to recruitment and engagement amongst black and Latinx youth from the perspective of HVIP staff. Employing a phenomenological approach, a purposive sample of key informants was recruited. Focus groups and semi-structured interviews lasting approximately 90 min were conducted with representatives (N = 12) from five HVIPs in U.S. cities across the Midwest and Northeast, making up 15% of all HVIPs in the United States. Each interview was recorded and transcribed verbatim. The research team employed rigorous content analysis of the data. Three themes and subsequent categories resulted from the analysis: (1) Interpersonal/Relational Facilitators (building rapport; connecting with youth; enhancing the teachable moment; building relational health); (2) Structural/Systemic Barriers (lack of reinforcement; difficulties connecting after discharge from the hospital; hospital workflow; institutional challenges); (3) Structural/Systemic Facilitators (embedding the HVIP; trauma-informed practices and policies). Given the limited research on black and Latinx youth and the disproportionate rate of violent injuries amongst these groups, an evidence-based systematic approach to engage youth is essential to promote health equity. The findings from this study suggest that there are several steps that HVIPs and hospitals can take to enhance their recruitment and engagement of youth and their families.

Effects of Injection Volume and Route of Administration on Dolutegravir In Situ Forming Implant Pharmacokinetics.

Due to the versatility of the in situ forming implant (ISFI) drug delivery system, it is crucial to understand the effects of formulation parameters for clinical translation. We utilized ultrasound imaging and pharmacokinetics (PK) in mice to understand the impact of administration route, injection volume, and drug loading on ISFI formation, degradation, and drug release in mice. Placebo ISFIs injected subcutaneously (SQ) with smaller volumes (40 µL) exhibited complete degradation within 30-45 days, compared to larger volumes (80 µL), which completely degraded within 45-60 days. However, all dolutegravir (DTG)-loaded ISFIs along the range of injection volumes tested (20-80 µL) were present at 90 days post-injection, suggesting that DTG can prolong ISFI degradation. Ultrasound imaging showed that intramuscular (IM) ISFIs flattened rapidly post administration compared to SQ, which coincides with the earlier Tmax for drug-loaded IM ISFIs. All mice exhibited DTG plasma concentrations above four times the protein-adjusted 90% inhibitory concentration (PA-IC90) throughout the entire 90 days of the study. ISFI release kinetics best fit to zero order or diffusion-controlled models. When total administered dose was held constant, there was no statistical difference in drug exposure regardless of the route of administration or number of injections.

Investigating crosstalk between H3K27 acetylation and H3K4 trimethylation in CRISPR/dCas-based epigenome editing and gene activation.

Epigenome editing methods enable the precise manipulation of epigenetic modifications, such as histone posttranscriptional modifications (PTMs), for uncovering their biological functions. While histone PTMs have been correlated with certain gene expression status, the causalities remain elusive. Histone H3 Lysine 27 acetylation (H3K27ac) and histone H3 Lysine 4 trimethylation (H3K4me3) are both associated with active genes, and located at active promoters and enhancers or around transcriptional start sites (TSSs). Although crosstalk between histone lysine acetylation and H3K4me3 has been reported, relationships between specific epigenetic marks during transcriptional activation remain largely unclear. Here, using clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-based epigenome editing methods, we discovered that the ectopic introduction of H3K27ac in the promoter region lead to H3K4me3 enrichment around TSS and transcriptional activation, while H3K4me3 installation at the promoter cannot induce H3K27ac increase and failed to activate gene expression. Blocking the reading of H3K27ac by BRD proteins using inhibitor JQ1 abolished H3K27ac-induced H3K4me3 installation and downstream gene activation. Furthermore, we uncovered that BRD2, not BRD4, mediated H3K4me3 installation and gene activation upon H3K27ac writing. Our studies revealed the relationships between H3K27ac and H3K4me3 in gene activation process and demonstrated the application of CRISPR/dCas-based epigenome editing methods in elucidating the crosstalk between epigenetic mechanisms.

Glioblastoma Multiforme-A Look at the Past and a Glance at the Future.

Gliomas are the most common type of brain tumor that occur in adults and children. Glioblastoma multiforme (GBM) is the most common, aggressive form of brain cancer in adults and is universally fatal. The current standard-of-care options for GBM include surgical resection, radiotherapy, and concomitant and/or adjuvant chemotherapy. One of the major challenges that impedes success of chemotherapy is the presence of the blood-brain barrier (BBB). Because of the tightly regulated BBB, immune surveillance in the central nervous system (CNS) is poor, contributing to unregulated glioma cell growth. This review gives a comprehensive overview of the latest advances in treatment of GBM with emphasis on the significant advances in immunotherapy and novel therapeutic delivery strategies to enhance treatment for GBM.

Mental health services in the Dominican Republic from the perspective of health care providers.

This study examines mental health services in five different regions of the Dominican Republic (DR) from the perspectives of health care providers. The purpose of this research was to (1) examine existing mental health care services; (2) identify barriers to treatment and mental health services delivery; and (3) explore potential strategies to improve mental health services delivery. Thirty-seven health care workers including physicians, nurses, psychologists, governmental administrators, and non-governmental community health workers were part of five focus groups and subsequent follow-up interviews. Transcripts were coded and analysed to obtain the most parsimonious categories of themes. Results indicated that there is insufficient funding allocated to mental health. The unreliable distribution of psychiatric medications precludes care for patients with severe chronic mental illness. Stigmatising attitudes among health care providers influences the quality of care. The prevalence of domestic violence is a significant public health problem contributing to mental illness. In conclusion, our study findings call for a re-examination of priority public health foci, with special attention to mental health and domestic violence in the DR. From a policy perspective, mental health care should be integrated into primary care and coupled with provider and patient education to reduce stigma. A social determinants approach could ameliorate systemic factors contributing to mental illness.

Preparing Global Trauma Nurses for Leadership Roles in Global Trauma Systems.

Trauma leads to 5.7 million annual deaths globally, accounting for 25%-33% of global unintentional deaths and 90% of the global trauma burden in low- and middle-income countries. The Lancet Commission on Global Surgery and the World Health Organization assert that emergent and essential surgical capacity building and trauma system improvement are essential to address the global burden of trauma. In response, the Rutgers Global Surgery program, the School of Nursing and Medicine, and the Robert Wood Johnson University Hospital faculty collaborated in the first Interprofessional Models in Global Injury Care and Education Symposium in June 2016. This 2-week symposium combined lectures, high-fidelity simulation, small group workshops, site visits to Level I trauma centers, and a 1-day training course from the Panamerican Trauma Society. The aim was to introduce global trauma nurses to trauma leadership and trauma system development. After completing the symposium, 10 nurses from China, Colombia, Kenya, Puerto Rico, and Uruguay were surveyed. Overall, 88.8% of participants reported high levels of satisfaction with the program and 100% stated being very satisfied with trauma lectures. Symposia, such as that developed and offered by Rutgers University, prepare nurses to address trauma within system-based care and facilitate trauma nursing leadership in their respective countries.

Eastern Association for the Surgery of Trauma and Society of Trauma Nurses advanced practitioner position paper: Optimizing the integration of advanced practitioners in trauma and critical care.

Nurse practitioners and physician assistants, collectively known as advanced practitioners (APs), enhance the provision of care for the acutely injured patient. Despite their prevalence, institutions employ, train, and utilize these providers with significant variability. The Eastern Association for the Surgery of Trauma, the Society of Trauma Nurses, and the American Association of Surgical Physician Assistants acknowledge the value of APs and support their utilization in the management of injured and critically ill patients. This position paper offers insight into the history of, scope of practice for, and opportunities for optimal utilization of APs in trauma, critical care, and acute care surgery services.

Persistence and evolution of allergen-specific IgE repertoires during subcutaneous specific immunotherapy.

BACKGROUND: Specific immunotherapy (SIT) is the only treatment with proved long-term curative potential in patients with allergic disease. Allergen-specific IgE is the causative agent of allergic disease, and antibodies contribute to SIT, but the effects of SIT on aeroallergen-specific B-cell repertoires are not well understood. OBJECTIVE: We sought to characterize the IgE sequences expressed by allergen-specific B cells and track the fate of these B-cell clones during SIT. METHODS: We used high-throughput antibody gene sequencing and identification of allergen-specific IgE with combinatorial antibody fragment library technology to analyze immunoglobulin repertoires of blood and the nasal mucosa from aeroallergen-sensitized subjects before and during the first year of subcutaneous SIT. RESULTS: Of 52 distinct allergen-specific IgE heavy chains from 8 allergic donors, 37 were also detected by using high-throughput antibody gene sequencing of blood samples, nasal mucosal samples, or both. The allergen-specific clones had increased persistence, higher likelihood of belonging to clones expressing other switched isotypes, and possibly larger clone size than the rest of the IgE repertoire. Clone members in nasal tissue showed close mutational relationships. CONCLUSION: In the future, combining functional binding studies, deep antibody repertoire sequencing, and information on clinical outcomes in larger studies might aid assessment of SIT mechanisms and efficacy.

Human B-cell isotype switching origins of IgE.

BACKGROUND: B cells expressing IgE contribute to immunity against parasites and venoms and are the source of antigen specificity in allergic patients, yet the developmental pathways producing these B cells in human subjects remain a subject of debate. Much of our knowledge of IgE lineage development derives from model studies in mice rather than from human subjects. OBJECTIVE: We evaluate models for isotype switching to IgE in human subjects using immunoglobulin heavy chain (IGH) mutational lineage data. METHODS: We analyzed IGH repertoires in 9 allergic and 24 healthy adults using high-throughput DNA sequencing of 15,843,270 IGH rearrangements to identify clonal lineages of B cells containing members expressing IgE. Somatic mutations in IGH inherited from common ancestors within the clonal lineage are used to infer the relationships between B cells. RESULTS: Data from 613,641 multi-isotype B-cell clonal lineages, of which 592 include an IgE member, are consistent with indirect switching to IgE from IgG- or IgA-expressing lineage members in human subjects. We also find that these inferred isotype switching frequencies are similar in healthy and allergic subjects. CONCLUSIONS: We found evidence that secondary isotype switching of mutated IgG1-expressing B cells is the primary source of IgE in human subjects, with lesser contributions from precursors expressing other switched isotypes and rarely IgM or IgD, suggesting that IgE is derived from previously antigen-experienced B cells rather than naive B cells that typically express low-affinity unmutated antibodies. These data provide a basis from which to evaluate allergen-specific human antibody repertoires in healthy and diseased subjects.

Single B-cell deconvolution of peanut-specific antibody responses in allergic patients.

BACKGROUND: The frequencies, cellular phenotypes, epitope specificity, and clonal diversity of allergen-specific B cells in patients with food allergy are not fully understood but are of major pathogenic and therapeutic significance. OBJECTIVE: We sought to characterize peanut allergen-specific B-cell populations and the sequences and binding activities of their antibodies before and during immunotherapy. METHODS: B cells binding fluorescently labeled Ara h 1 or Ara h 2 were phenotyped and isolated by means of flow cytometric sorting from 18 patients at baseline and 13 patients during therapy. Fifty-seven mAbs derived from allergen-binding single B cells were evaluated by using ELISA, Western blotting, and peptide epitope mapping. Deep sequencing of the B-cell repertoires identified additional members of the allergen-specific B-cell clones. RESULTS: Median allergen-binding B-cell frequencies were 0.0097% (Ara h 1) or 0.029% (Ara h 2) of B cells in baseline blood from allergic patients and approximately 3-fold higher during immunotherapy. Five of 57 allergen-specific cells belonged to clones containing IgE-expressing members. Almost all allergen-specific antibodies were mutated, and binding to both conformational and linear allergen epitopes was detected. Increasing somatic mutation of IgG4 members of a clone was seen in immunotherapy, whereas IgE mutation levels in the clone did not increase. CONCLUSION: Most peanut allergen-binding B cells isolated by means of antigen-specific flow sorting express mutated and isotype-switched antibodies. Immunotherapy increases their frequency in the blood, and even narrowly defined allergen epitopes are recognized by numerous distinct B-cell clones in a patient. The results also suggest that oral immunotherapy can stimulate somatic mutation of allergen-specific IgG4.

Comprehensive whole-genome sequencing of an early-stage primary myelofibrosis patient defines low mutational burden and non-recurrent candidate genes.

In order to identify novel somatic mutations associated with classic BCR/ABL1-negative myeloproliferative neoplasms, we performed high-coverage genome sequencing of DNA from peripheral blood granulocytes and cultured skin fibroblasts from a patient with MPL W515K-positive primary myelofibrosis. The primary myelofibrosis genome had a low somatic mutation rate, consistent with that observed in similar hematopoietic tumor genomes. Interfacing of whole-genome DNA sequence data with RNA expression data identified three somatic mutations of potential functional significance: i) a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; ii) a 19-base pair deletion involving a potential regulatory region in the 5'-untranslated region of BRD2, implicated in transcriptional regulation and cell cycle control; and iii) a non-synonymous point mutation in KIAA0355, an uncharacterized protein. Additional mutations in three genes (CAP2, SOX30, and MFRP) were also evident, albeit with no support for expression at the RNA level. Re-sequencing of these six genes in 178 patients with polycythemia vera, essential thrombocythemia, and myelofibrosis did not identify recurrent somatic mutations in these genes. Finally, we describe methods for reducing false-positive variant calls in the analysis of hematologic malignancies with a low somatic mutation rate. This trial is registered with ClinicalTrials.gov (NCT01108159).

The ELF4-ELF3-LUX complex links the circadian clock to diurnal control of hypocotyl growth.

The circadian clock is required for adaptive responses to daily and seasonal changes in environmental conditions. Light and the circadian clock interact to consolidate the phase of hypocotyl cell elongation to peak at dawn under diurnal cycles in Arabidopsis thaliana. Here we identify a protein complex (called the evening complex)--composed of the proteins encoded by EARLY FLOWERING 3 (ELF3), ELF4 and the transcription-factor-encoding gene LUX ARRHYTHMO (LUX; also known as PHYTOCLOCK 1)--that directly regulates plant growth. ELF3 is both necessary and sufficient to form a complex between ELF4 and LUX, and the complex is diurnally regulated, peaking at dusk. ELF3, ELF4 and LUX are required for the proper expression of the growth-promoting transcription factors encoded by PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and PIF5 (also known as PHYTOCHROME INTERACTING FACTOR 3-LIKE 6) under diurnal conditions. LUX targets the complex to the promoters of PIF4 and PIF5 in vivo. Mutations in PIF4 and/or PIF5 are epistatic to the loss of the ELF4-ELF3-LUX complex, suggesting that regulation of PIF4 and PIF5 is a crucial function of the complex. Therefore, the evening complex underlies the molecular basis for circadian gating of hypocotyl growth in the early evening.