Hand-Jaw Coordination as Mice Handle Food Is Organized around Intrinsic Structure-Function Relationships.

Rodent jaws evolved structurally to support dual functionality, for either biting or chewing food. Rodent hands also function dually during food handling, for actively manipulating or statically holding food. How are these oral and manual functions coordinated? We combined electrophysiological recording of muscle activity and kilohertz kinematic tracking to analyze masseter and hand actions as mice of both sexes handled food. Masseter activity was organized into two modes synchronized to hand movement modes. In holding/chewing mode, mastication occurred as rhythmic (∼5 Hz) masseter activity while the hands held food below the mouth. In oromanual/ingestion mode, bites occurred as lower-amplitude aperiodic masseter events that were precisely timed to follow regrips (by ∼200 ms). Thus, jaw and hand movements are flexibly coordinated during food handling: uncoupled in holding/chewing mode and tightly coordinated in oromanual/ingestion mode as regrip-bite sequences. Key features of this coordination were captured in a simple model of hierarchically orchestrated mode-switching and intramode action sequencing. We serendipitously detected an additional masseter-related action, tooth sharpening, identified as bouts of higher-frequency (∼13 Hz) rhythmic masseter activity, which was accompanied by eye displacement, including rhythmic proptosis, attributable to masseter contractions. Collectively, the findings demonstrate how a natural, complex, and goal-oriented activity is organized as an assemblage of distinct modes and complex actions, adapted for the divisions of function arising from anatomical structure. These results reveal intricate, high-speed coordination of disparate effectors and show how natural forms of dexterity can serve as a model for understanding the behavioral neurobiology of multi-body-part coordination.

Axonal Barcode Analysis of Pyramidal Tract Projections from Mouse Forelimb M1 and M2.

Forelimb-related areas of the motor cortex communicate directly to downstream areas in the brainstem and spinal cord via axons that project to and through the pyramidal tract (PT). To better understand the diversity of the brainstem branching patterns of these pyramidal tract projections, we used MAPseq, a molecular barcode technique for population-scale sampling with single-axon resolution. In experiments using mice of both sexes, we first confirmed prior results demonstrating the basic efficacy of axonal barcode identification of primary motor cortex (M1) PT-type axons, including corticobulbar (CBULB) and corticospinal (CSPI) subclasses. We then used multiplexed MAPseq to analyze projections from M1 and M2 (caudal and rostral forelimb areas). The four basic axon subclasses comprising these projections (M1-CSPI, M1-CBULB, M2-CSPI, M2-CBULB) showed a complex mix of differences and similarities in their brainstem projection profiles. This included relatively abundant branching by all classes in the dorsal midbrain, by M2 subclasses in the pons, and by CSPI subclasses in the dorsal medulla. Cluster analysis showed graded distributions of the basic subclasses within the PT class. Clusters were of diversely mixed subclass composition and showed distinct rostrocaudal and/or dorsomedial projection biases. Exemplifying these patterns was a subcluster likely enriched in corticocuneate branches. Overall, the results indicate high yet systematic PT axon diversity at the level of brainstem branching patterns; projections of M1 and M2 appear qualitatively similar, yet with quantitative differences in subclasses and clusters.SIGNIFICANCE STATEMENT Axons of the PT class of cortical projection neurons, which includes corticospinal and corticobulbar neurons, anatomically link motor cortex to brainstem and spinal cord circuits. Both of these subclasses can form branches to brainstem destinations along the way, but the extent and diversity of these branching patterns is incompletely understood. Here, we used MAPseq to tag PT axons with individual molecular barcodes for high-throughput quantification of branching patterns across the brainstem. The results reveal diverse, complex, yet systematic branching patterns of corticospinal and corticobulbar neurons arising from two motor cortex areas, M1 and M2.

Targeting the P2Y13 Receptor Suppresses IL-33 and HMGB1 Release and Ameliorates Experimental Asthma.

Rationale: The alarmins IL-33 and HMGB1 (high mobility group box 1) contribute to type 2 inflammation and asthma pathogenesis. Objectives: To determine whether P2Y13-R (P2Y13 receptor), a purinergic GPCR (G protein-coupled receptor) and risk allele for asthma, regulates the release of IL-33 and HMGB1. Methods: Bronchial biopsy specimens were obtained from healthy subjects and subjects with asthma. Primary human airway epithelial cells (AECs), primary mouse AECs, or C57Bl/6 mice were inoculated with various aeroallergens or respiratory viruses, and the nuclear-to-cytoplasmic translocation and release of alarmins was measured by using immunohistochemistry and an ELISA. The role of P2Y13-R in AEC function and in the onset, progression, and exacerbation of experimental asthma was assessed by using pharmacological antagonists and mice with P2Y13-R gene deletion. Measurements and Main Results: Aeroallergen exposure induced the extracellular release of ADP and ATP, nucleotides that activate P2Y13-R. ATP, ADP, and aeroallergen (house dust mite, cockroach, or Alternaria antigen) or virus exposure induced the nuclear-to-cytoplasmic translocation and subsequent release of IL-33 and HMGB1, and this response was ablated by genetic deletion or pharmacological antagonism of P2Y13. In mice, prophylactic or therapeutic P2Y13-R blockade attenuated asthma onset and, critically, ablated the severity of a rhinovirus-associated exacerbation in a high-fidelity experimental model of chronic asthma. Moreover, P2Y13-R antagonism derepressed antiviral immunity, increasing IFN-λ production and decreasing viral copies in the lung. Conclusions: We identify P2Y13-R as a novel gatekeeper of the nuclear alarmins IL-33 and HMGB1 and demonstrate that the targeting of this GPCR via genetic deletion or treatment with a small-molecule antagonist protects against the onset and exacerbations of experimental asthma.

Assessment of Long-Term Macrolide Exposure on the Oropharyngeal Microbiome and Macrolide Resistance in Healthy Adults and Consequences for Onward Transmission of Resistance.

While the use of long-term macrolide therapy to prevent exacerbations in chronic respiratory diseases is widespread, its impact on the oropharyngeal microbiota and macrolide resistance, and the potential for onward transmission of resistance to close contacts are poorly understood. We determined the effects of long-term exposure to azithromycin or erythromycin on phenotypic and genotypic macrolide resistance within the oropharyngeal microbiome of healthy adults and their close contacts in a randomized, single-blinded, parallel-group trial of 4 weeks of twice-daily oral 400 mg erythromycin ethylsuccinate or twice-daily oral 125 mg azithromycin. Using oropharyngeal swabs collected from 20 index healthy adults and 20 paired close contacts, the oropharyngeal microbial composition and macrolide resistance in streptococci were assessed by 16S rRNA sequencing and antibiotic susceptibility testing of oropharyngeal cultures, respectively, at baseline and weeks 4 and 8 (washout). Targeted quantitative PCR of antibiotic resistance genes was performed to evaluate paired changes in resistance gene levels in index patients and close contacts and to relate the potential transmission of antibiotic resistance. Neither azithromycin nor erythromycin altered oropharyngeal microbiota characteristics significantly. Proportional macrolide resistance in oropharyngeal streptococci increased with both erythromycin and azithromycin, remaining above baseline levels for the azithromycin group at washout. Levels of resistance genes increased significantly with azithromycin[erm(B) and mef] and erythromycin (mef), returning to baseline levels at washout only for the erythromycin group. We found no evidence of onward transmission of resistance to close contacts, as indicated by the lack of concomitant changes in resistance gene levels detected in close contacts. (This study has been registered with the Australian and New Zealand Clinical Trials Registry under identifier ACTRN12617000278336.).

Airway Mucus Hyperconcentration in Non-Cystic Fibrosis Bronchiectasis.

Rationale: Non-cystic fibrosis bronchiectasis is characterized by airway mucus accumulation and sputum production, but the role of mucus concentration in the pathogenesis of these abnormalities has not been characterized.Objectives: This study was designed to: 1) measure mucus concentration and biophysical properties of bronchiectasis mucus; 2) identify the secreted mucins contained in bronchiectasis mucus; 3) relate mucus properties to airway epithelial mucin RNA/protein expression; and 4) explore relationships between mucus hyperconcentration and disease severity.Methods: Sputum samples were collected from subjects with bronchiectasis, with and without chronic erythromycin administration, and healthy control subjects. Sputum percent solid concentrations, total and individual mucin concentrations, osmotic pressures, rheological properties, and inflammatory mediators were measured. Intracellular mucins were measured in endobronchial biopsies by immunohistochemistry and gene expression. MUC5B (mucin 5B) polymorphisms were identified by quantitative PCR. In a replication bronchiectasis cohort, spontaneously expectorated and hypertonic saline-induced sputa were collected, and mucus/mucin concentrations were measured.Measurements and Main Results: Bronchiectasis sputum exhibited increased percent solids, total and individual (MUC5B and MUC5AC) mucin concentrations, osmotic pressure, and elastic and viscous moduli compared with healthy sputum. Within subjects with bronchiectasis, sputum percent solids correlated inversely with FEV1 and positively with bronchiectasis extent, as measured by high-resolution computed tomography, and inflammatory mediators. No difference was detected in MUC5B rs35705950 SNP allele frequency between bronchiectasis and healthy individuals. Hypertonic saline inhalation acutely reduced non-cystic fibrosis bronchiectasis mucus concentration by 5%.Conclusions: Hyperconcentrated airway mucus is characteristic of subjects with bronchiectasis, likely contributes to disease pathophysiology, and may be a target for pharmacotherapy.

A functional role for the cancer disparity-linked genes, CRYßB2 and CRYßB2P1, in the promotion of breast cancer.

BACKGROUND: In the USA, the breast cancer mortality rate is 41% higher for African-American women than non-Hispanic White women. While numerous gene expression studies have classified biological features that vary by race and may contribute to poorer outcomes, few studies have experimentally tested these associations. CRYßB2 gene expression has drawn particular interest because of its association with overall survival and African-American ethnicity in multiple cancers. Several reports indicate that overexpression of the CRYßB2 pseudogene, CRYßB2P1, and not CRYßB2 is linked with race and poor outcome. It remains unclear whether either or both genes are linked to breast cancer outcomes. This study investigates CRYßB2 and CRYßB2P1 expression in human breast cancers and breast cancer cell line models, with the goal of elucidating the mechanistic contribution of CRYßB2 and CRYßB2P1 to racial disparities. METHODS: Custom scripts for CRYßB2 or CRYßB2P1 were generated and used to identify reads that uniquely aligned to either gene. Gene expression according to race and tumor subtype were assessed using all available TCGA breast cancer RNA sequencing alignment samples (n = 1221). In addition, triple-negative breast cancer models engineered to have each gene overexpressed or knocked out were developed and evaluated by in vitro, biochemical, and in vivo assays to identify biological functions. RESULTS: We provide evidence that CRYßB2P1 is expressed at higher levels in breast tumors compared to CRYßB2, but only CRYßB2P1 is significantly increased in African-American tumors relative to White American tumors. We show that independent of CRYßB2, CRYßB2P1 enhances tumorigenesis in vivo via promoting cell proliferation. Our data also reveal that CRYßB2P1 may function as a non-coding RNA to regulate CRYßB2 expression. A key observation is that the combined overexpression of both genes was found to suppress cell growth. CRYßB2 overexpression in triple-negative breast cancers increases invasive cellular behaviors, tumor growth, IL6 production, immune cell chemoattraction, and the expression of metastasis-associated genes. These data underscore that both CRYßB2 and CRYßB2P1 promote tumor growth, but their mechanisms for tumor promotion are likely distinct. CONCLUSIONS: Our findings provide novel data emphasizing the need to distinguish and study the biological effects of both CRYßB2 and CRYßB2P1 as both genes independently promote tumor progression. Our data demonstrate novel molecular mechanisms of two understudied, disparity-linked molecules.

Silver nanoparticles alter epithelial basement membrane integrity, cell adhesion molecule expression, and TGF-ß1 secretion.

Silver nanoparticles (AgNPs) are widely used in consumer and pharmaceutical products due to their antipathogenic properties. However, safety concerns have been raised due to their bioactive properties. While reports have demonstrated AgNPs can embed within the extracellular matrix, their effects on basement membrane (BM) production, integrin engagement, and tissue-integrity are not well-defined. This study analyzed the effects of AgNPs on BM production, composition and integrin/focal adhesion interactions in representative lung, esophageal, breast and colorectal epithelia models. A multidisciplinary approach including focused proteomics, QPCR arrays, pathway analyses, and immune-based, structural and functional assays was used to identify molecular and physiological changes in cell adhesions and the BM induced by acute and chronic AgNP exposure. Dysregulated targets included CD44 and transforming growth factor-beta, two proteins frequently altered during pathogenesis. Results indicate AgNP exposure interferes with BM and cell adhesion dynamics, and provide insight into the mechanisms of AgNP-induced disruption of epithelial physiology.

Nitric Oxide-Releasing Macromolecule Exhibits Broad-Spectrum Antifungal Activity and Utility as a Topical Treatment for Superficial Fungal Infections.

Cutaneous and superficial fungal infections affecting the skin, nails, and hair of humans are caused primarily by dermatophytes of the genera Trichophyton and Epidermophyton or by yeasts of the genera Candida and Malassezia. Onychomycosis is a common fungal infection of the nail that frequently coexists with tinea pedis, the most prevalent mycotic skin infection. Efficacy rates for current topical onychomycosis therapies are hampered by low drug penetration across the nail plate, which is theoretically obviated with nitric oxide (NO)-based topical therapies. The Nitricil technology platform is comprised of polysiloxane-based macromolecules that stably release therapeutic levels of NO. In the reported studies, NVN1000, the lead candidate of the platform, was assessed for its spectrum of in vitro activity against a broad range of filamentous fungi and yeast species commonly associated with cutaneous fungal infections. Time-kill assays demonstrated that NVN1000 exhibited fungicidal activity as early as 4 h. Additionally, the penetration of several unique NVN1000 NO-releasing drug product formulations (gel, cream, and lacquer) was evaluated following a single topical application in an in vitro infected human nail assay, with all formulations showing similar inhibition of fungal growth. Repeated topical application in this model demonstrated that a lower-strength dose of NO could achieve the same efficacy as a higher-strength dose after 7 days. Together, these in vitro results demonstrate that NO-releasing treatments rapidly penetrate the nail plate and eradicate the fungal infection, representing promising novel topical therapies for the treatment of onychomycosis and other cutaneous fungal infections.

Chromosomal microarray testing identifies a 4p terminal region associated with seizures in Wolf-Hirschhorn syndrome.

BACKGROUND: Wolf-Hirschhorn syndrome (WHS) is a contiguous gene deletion syndrome involving variable size deletions of the 4p16.3 region. Seizures are frequently, but not always, associated with WHS. We hypothesised that the size and location of the deleted region may correlate with seizure presentation. METHODS: Using chromosomal microarray analysis, we finely mapped the breakpoints of copy number variants (CNVs) in 48 individuals with WHS. Seizure phenotype data were collected through parent-reported answers to a comprehensive questionnaire and supplemented with available medical records. RESULTS: We observed a significant correlation between the presence of an interstitial 4p deletion and lack of a seizure phenotype (Fisher's exact test p=3.59e-6). In our cohort, there were five individuals with interstitial deletions with a distal breakpoint at least 751 kbp proximal to the 4p terminus. Four of these individuals have never had an observable seizure, and the fifth individual had a single febrile seizure at the age of 1.5 years. All other individuals in our cohort whose deletions encompass the terminal 751 kbp region report having seizures typical of WHS. Additional examples from the literature corroborate these observations and further refine the candidate seizure susceptibility region to a region 197 kbp in size, starting 368 kbp from the terminus of chromosome 4. CONCLUSIONS: We identify a small terminal region of chromosome 4p that represents a seizure susceptibility region. Deletion of this region in the context of WHS is sufficient for seizure occurrence.

Mosaic deletion of 20pter due to rescue by somatic recombination.

We report on a unique case of a mosaic 20pter-p13 deletion due to a somatic repair event identified by allele differentiating single nucleotide polymorphism (SNP) probes on chromosomal microarray. Small terminal deletions of 20p have been reported in a few individuals and appear to result in a variable phenotype. This patient was a 24-month-old female who presented with failure to thrive and speech delay. Chromosomal microarray analysis (CMA) performed on peripheral blood showed a 1.6 Mb deletion involving the terminus of 20p (20pter-20p13). This deletion appeared mosaic by CMA and this suspicion was confirmed by fluorescence in situ hybridization (FISH) analysis. Additionally, the deletion interval at 20p was directly adjacent to 15 Mb of mosaic copy-neutral loss of heterozygosity (LOH). The pattern of SNP probes was highly suggestive of a somatic repair event that resulted in rescue of the deleted region using the non-deleted homologue as a template. Structural mosaicism is rare and most often believed to be due to a postzygotic mechanism. This case demonstrates the additional utility of allele patterns to help distinguish mechanisms and in this case identified the possibility of either a post-zygotic repair of a germline deletion or a post-zygotic deletion with somatic recombination repair in a single step.

Chromosomal Microarray Analysis of Consecutive Individuals with Autism Spectrum Disorders Using an Ultra-High Resolution Chromosomal Microarray Optimized for Neurodevelopmental Disorders.

Copy number variants (CNVs) detected by chromosomal microarray analysis (CMA) significantly contribute to understanding the etiology of autism spectrum disorder (ASD) and other related conditions. In recognition of the value of CMA testing and its impact on medical management, CMA is in medical guidelines as a first-tier test in the evaluation of children with these disorders. As CMA becomes adopted into routine care for these patients, it becomes increasingly important to report these clinical findings. This study summarizes the results of over 4 years of CMA testing by a CLIA-certified clinical testing laboratory. Using a 2.8 million probe microarray optimized for the detection of CNVs associated with neurodevelopmental disorders, we report an overall CNV detection rate of 28.1% in 10,351 consecutive patients, which rises to nearly 33% in cases without ASD, with only developmental delay/intellectual disability (DD/ID) and/or multiple congenital anomalies (MCA). The overall detection rate for individuals with ASD is also significant at 24.4%. The detection rate and pathogenic yield of CMA vary significantly with the indications for testing, age, and gender, as well as the specialty of the ordering doctor. We note discrete differences in the most common recurrent CNVs found in individuals with or without a diagnosis of ASD.

Perceptions of tissue storage in a dementia population among spouses and offspring.

Cognitively impaired patients with dementia often rely on health advocates or guardians, such as spouses or adult offspring, to consent for medical procedures. These family members may also decide whether an autopsy is performed after death or whether their family member donates tissues. However, spouses are not genetically related to the patient and may have different perspectives than genetically related family members when making medical decisions with genetic implications, such as participation in a tissue repository (biobank). Interviews were conducted with spouses and adult offspring of individuals with a progressive dementing disease. Both spouses and offspring were supportive of the patient with dementia to participate in tissue storage. The top perceived benefits of tissue storage in both offspring and spouses were future value for family members and advancement of medical knowledge. Concerns included misuse of the tissue and insurance discrimination. Although the personal genetic implications differ between spouses and offspring, they share similar attitudes about the importance of tissue banking for the individual with a dementing disease.

Clinical utility of chromosomal microarray analysis of DNA from buccal cells: detection of mosaicism in three patients.

Mosaic chromosomal abnormalities are relatively common. However, mosaicism may be missed due to multiple factors including failure to recognize clinical indications and order appropriate testing, technical limitations of diagnostic assays, or sampling tissue (s) in which mosaicism is either not present, or present at very low levels. Blood leukocytes have long been the "gold standard" sample for cytogenetic analysis; however, the culturing process for routine chromosome analysis can complicate detection of mosaicism since the normal cell line may have a growth advantage in culture, or may not be present in the cells that produce metaphases (the lymphocytes). Buccal cells are becoming increasingly utilized for clinical analyses and are proving to have many advantages. Buccal swabs allow for simple and noninvasive DNA collection. When coupled with a chromosomal microarray that contains single nucleotide polymorphic probes, analysis of buccal cells can maximize a clinician's opportunity to detect cytogenetic mosaicism. We present three cases of improved diagnosis of mosaic aberrations using buccal specimens for chromosomal microarray analysis. In each case, the aberration was either undetectable in blood or present at such a low level it likely could have gone undetected. These cases highlight the limitations of certain laboratory methodologies for identifying mosaicism. We also present practice implications for genetic counselors, including clinic workflow changes and counseling approaches based on increasing use of buccal samples.

The Efficiency of Phosphate Removal via Shallow Wastewater Injection into a Saline Carbonate Aquifer.

Wastewater-derived phosphate contributes to eutrophication if the phosphate is not efficiently removed before it is discharged to surface waters. In the Florida Keys (USA), shallow injection of treated wastewater into saline limestone aquifers is a common mode of wastewater disposal. We assessed the possibility of efficient and permanent phosphate removal following injection at a wastewater treatment facility in Marathon, Florida. The concentrations of nutrients, dissolved ions, and anthropogenic compounds in groundwater and nearshore waters were monitored over two years, as was the progression of a patch of fluorescent dye emplaced by the wastewater injection well. The density contrast between the wastewater effluent and saline groundwater caused the effluent plume to buoy to the shallow subsurface near the injection well. Soluble reactive phosphorus (SRP) and sucralose were both detected in nearshore waters, indicating incomplete removal of contaminants. However, ∼75% of the SRP is removed from the plume in the first 10 days of transit by adsorption followed by a slower removal mechanism, bringing the P removal efficiency above 90%. A positive relationship between excess calcium and phosphate removal efficiency, together with high levels of calcium phosphate mineral supersaturation, supports calcite dissolution followed by calcium phosphate mineralization as this slower removal process.

"Yeah, this is not going to work for me"-The impact of federal policy restrictions on methadone continuation upon release from jail or prison.

INTRODUCTION: Individuals impacted by the criminal-legal system face increased risk of opioid overdose. Medications for opioid use disorder (MOUD) provide a life-saving intervention. Multiple barriers prevent access to MOUD, including federal policies regulating opioid treatment programs (OTPs). This study aims to identify how federal policy affects anticipated barriers to methadone treatment access at a high-risk time for opioid mortality: community re-entry after incarceration. METHODS: The study used standard qualitative methods to conduct 40 in-depth-interviews with incarcerated individuals enrolled in the Rhode Island Department of Corrections MOUD treatment program. Semi-structured interviews took place between June and August 2018 and focused on participants' experiences with MOUD and anticipated treatment barriers upon re-entry. A deductive coding framework incorporating the SAMHSA "8-point" criteria for take-home methadone as the a priori codebook and additional identified barriers informed further inductive analysis. RESULTS: Four themes emerged: (1) logistical hurdles such as transportation and clinic location impeded clinic access; (2) punitive measures within clinics, like dose reduction for rule infractions, discouraged treatment continuation; (3) the environment of methadone clinics often tempted return to use; (4) while the structured nature of methadone treatment provided accountability, it also posed challenges. Federal policies, particularly around daily dosing and "take-home" regulations, exacerbated barriers for those re-entering the community. State and clinic level policies, however, were also identified as direct or exacerbating barriers to treatment access. CONCLUSION: Significant hurdles persist for methadone access among individuals released from incarceration. Though the federal 8-point criteria have now been replaced with more flexible take-home policies, our findings highlight critical treatment barriers for individuals during the high-risk period of community re-entry. State and clinic level policies also exacerbate many of the barrier-driven themes identified in this analysis. Future work can explore how to best implement the identified benefits of a structured program without forcing the punitive measures that discourage treatment retention. Additional policy reform can help mitigate the effects of other social determinants of health (including transportation access). Ultimately, the many barriers to community methadone treatment retention also apply to individuals involved in the criminal legal system; they can be exacerbated at the federal, state, and clinic policy level.

Clinical measures of disease in adult non-CF bronchiectasis correlate with airway microbiota composition.

RATIONALE: Despite the potentially important roles for infection in adult non-cystic fibrosis (CF) bronchiectasis disease progression, the bacterial species present in the lower airways of these patients is poorly characterised. OBJECTIVES: To provide a comprehensive cross-sectional analysis of bacterial content of lower airway samples from patients with non-CF bronchiectasis using culture-independent microbiology. METHODS: Paired induced sputum and bronchoalveolar lavage samples, obtained from 41 adult patients with non-CF bronchiectasis, were analysed by 16S ribosomal RNA gene pyrosequencing. Assessment of species distribution and dispersal allowed 'core' and 'satellite' bacterial populations to be defined for this patient group. Microbiota characteristics correlated with clinical markers of disease. MEASUREMENT AND MAIN RESULTS: 140 bacterial species were identified, including those associated with respiratory tract infections and opportunistic infections more generally. A group of core species, consisting of species detected frequently and in high abundance, was defined. Core species included those currently associated with infection in bronchiectasis, such as Pseudomonas aeruginosa, Haemophilus influenzae and Streptococcus pneumoniae, and many species that would be unlikely to be reported through standard diagnostic surveillance. These included members of the genera Veillonella, Prevotella and Neisseria. The comparative contribution of core and satellite groups suggested a low level of random species acquisition. Bacterial diversity was significantly positively correlated with forced expiratory volume in 1 s (FEV1) and bacterial community composition similarity correlated significantly with FEV1, neutrophil count and Leicester cough score. CONCLUSIONS: Characteristics of the lower airways microbiota of adult patients with non-CF bronchiectasis correlate significantly with clinical markers of disease severity.

Effect of long-term, low-dose erythromycin on pulmonary exacerbations among patients with non-cystic fibrosis bronchiectasis: the BLESS randomized controlled trial.

IMPORTANCE: Macrolide antibiotics such as erythromycin may improve clinical outcomes in non-cystic fibrosis (CF) bronchiectasis, although associated risks of macrolide resistance are poorly defined. OBJECTIVE: To evaluate the clinical efficacy and antimicrobial resistance cost of low-dose erythromycin given for 12 months to patients with non-CF bronchiectasis with a history of frequent pulmonary exacerbations. DESIGN, SETTING, AND PARTICIPANTS: Twelve-month, randomized (1:1), double-blind, placebo-controlled trial of erythromycin in currently nonsmoking, adult patients with non-CF bronchiectasis with a history of 2 or more infective exacerbations in the preceding year. This Australian study was undertaken between October 2008 and December 2011 in a university teaching hospital, with participants also recruited via respiratory physicians at other centers and from public radio advertisements. INTERVENTIONS: Twice-daily erythromycin ethylsuccinate (400 mg) or matching placebo. MAIN OUTCOME MEASURES: The primary outcome was the annualized mean rate of protocol-defined pulmonary exacerbations (PDPEs) per patient. Secondary outcomes included macrolide resistance in commensal oropharyngeal streptococci and lung function. RESULTS: Six-hundred seventy-nine patients were screened, 117 were randomized (58 placebo, 59 erythromycin), and 107 (91.5%) completed the study. Erythromycin significantly reduced PDPEs both overall (mean, 1.29 [95% CI, 0.93-1.65] vs 1.97 [95% CI, 1.45-2.48] per patient per year; incidence rate ratio [IRR], 0.57 [95% CI, 0.42-0.77]; P = .003), and in the prespecified subgroup with baseline Pseudomonas aeruginosa airway infection (mean difference, 1.32 [95% CI, 0.19-2.46]; P = .02). Erythromycin reduced 24-hour sputum production (median difference, 4.3 g [interquartile range [IQR], 1 to 7.8], P = .01) and attenuated lung function decline (mean absolute difference for change in postbronchodilator forced expiratory volume in the first second of expiration, 2.2 percent predicted [95% CI, 0.1% to 4.3%]; P = .04) compared with placebo. Erythromycin increased the proportion of macrolide-resistant oropharyngeal streptococci (median change, 27.7% [IQR, 0.04% to 41.1%] vs 0.04% [IQR, -1.6% to 1.5%]; difference, 25.5% [IQR,15.0% to 33.7%]; P < .001). CONCLUSION AND RELEVANCE: Among patients with non-CF bronchiectasis, the 12-month use of erythromycin compared with placebo resulted in a modest decrease in the rate of pulmonary exacerbations and an increased rate of macrolide resistance. TRIAL REGISTRATION: anzctr.org.au Identifier: ACTRN12609000578202.

Astrocyte reactivity and inflammation-induced depression-like behaviors are regulated by Orai1 calcium channels.

Astrocytes contribute to brain inflammation in neurological disorders but the molecular mechanisms controlling astrocyte reactivity and their relationship to neuroinflammatory endpoints are complex and poorly understood. In this study, we assessed the role of the calcium channel, Orai1, for astrocyte reactivity and inflammation-evoked depression behaviors in mice. Transcriptomics and metabolomics analysis indicated that deletion of Orai1 in astrocytes downregulates genes in inflammation and immunity, metabolism, and cell cycle pathways, and reduces cellular metabolites and ATP production. Systemic inflammation by peripheral lipopolysaccharide (LPS) increases hippocampal inflammatory markers in WT but not in astrocyte Orai1 knockout mice. Loss of Orai1 also blunts inflammation-induced astrocyte Ca2+ signaling and inhibitory neurotransmission in the hippocampus. In line with these cellular changes, Orai1 knockout mice showed amelioration of LPS-evoked depression-like behaviors including anhedonia and helplessness. These findings identify Orai1 as an important signaling hub controlling astrocyte reactivity and astrocyte-mediated brain inflammation that is commonly observed in many neurological disorders.

Linear and nonlinear rheology of liberase-treated breast cancer tumors.

Extracellular matrix (ECM) rigidity has been shown to increase the invasive properties of breast cancer cells, promoting transformation and metastasis through mechanotransduction. Reducing ECM stiffness via enzymatic digestion could be a promising approach to slowing breast cancer development by de-differentiation of breast cancer cells to less aggressive phenotypes and enhancing the effectiveness of existing chemotherapeutics via improved drug penetrance throughout the tumor. In this study, we examine the effects of injectable liberase (a blend of collagenase and thermolysin enzymes) treatments on the linear and nonlinear rheology of allograft 4T1 mouse mammary tumors. We perform two sets of in vivo mouse studies, in which either one or multiple treatment injections occur before the tumors are harvested for rheological analysis. The treatment groups in each study consist of a buffer control, free liberase enzyme in buffer, a thermoresponsive copolymer called LiquoGel (LQG) in buffer, and a combined, localized injection of LQG and liberase. All tumor samples exhibit gel-like linear rheological behavior with the elastic modulus significantly larger than the viscous modulus and both independent of frequency. Tumors that receive a single injection of localized liberase have significantly lower tumor volumes and lower tissue moduli at both the center and edge compared to buffer- and free liberase-injected control tumors, while tissue viscoelasticity remains relatively unaffected. Tumors injected multiple times with LQG and liberase also have lower tissue volumes but possess higher tissue moduli and lower viscoelasticities compared to the other treatment groups. We propose that a mechanotransductive mechanism could cause the formation of smaller but stiffer tumors after repeated, localized liberase injections. Large amplitude oscillatory shear (LAOS) experiments are also performed on tissues from the multiple injection study and the results are analyzed using MITlaos. LAOS analysis reveals that all 4T1 tumors from the multiple injection study exhibit nonlinear rheological behavior at high strains and strain rates. Examination of the Lissajous-Bowditch curves, Chebyshev coefficient ratios, elastic moduli, and dynamic viscosities demonstrate that the onset and type of nonlinear behavior is independent of treatment type and elastic modulus, suggesting that multiple liberase injections do not affect the nonlinear viscoelasticity of 4T1 tumors.

Regulation of neuropathic pain by microglial Orai1 channels.

Microglia are important mediators of neuroinflammation, which underlies neuropathic pain. However, the molecular checkpoints controlling microglial reactivity are not well-understood. Here, we investigated the role of Orai1 channels for microglia-mediated neuroinflammation following nerve injury and find that deletion of Orai1 in microglia attenuates Ca2+ signaling and the production of inflammatory cytokines by proalgesic agonists. Conditional deletion of Orai1 attenuated microglial proliferation in the dorsal horn, spinal cytokine levels, and potentiation of excitatory neurotransmission following peripheral nerve injury. These cellular effects were accompanied by mitigation of pain hyperalgesia in microglial Orai1 knockout mice. A small-molecule Orai1 inhibitor, CM4620, similarly mitigated allodynia in male mice. Unexpectedly, these protective effects were not seen in female mice, revealing sexual dimorphism in Orai1 regulation of microglial reactivity and hyperalgesia. Together, these findings indicate that Orai1 channels are key regulators of the sexually dimorphic role of microglia for the neuroinflammation that underlies neuropathic pain.