Activation of human STING by a molecular glue-like compound.

Stimulator of interferon genes (STING) is a dimeric transmembrane adapter protein that plays a key role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand, cGAMP, to the cytosolic ligand-binding domain. Here we report the discovery through functional screens of a class of compounds, named NVS-STGs, that activate human STING. Our cryo-EM structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models.

Health care administration and drug policy.

The American healthcare system faces a potential reorganization of the way in which services are provided and financed. We argue that healthcare administrators need to be increasingly aware of the ways in which our nation's illicit drug policy, commonly referred to as the 'War on Drugs', affects the provision of health services. A large and growing portion of the US population uses one or more of the currently illegal drugs and some of these persons suffer from an addiction or other substance use disorder. This is clearly demonstrated by the current opioid epidemic which is not yet being adequately controlled. Providing specialty treatment for drug abuse disorders will be increasingly important for healthcare administrators thanks to recent mental health parity legislation. At the same time, drug users and abusers will be increasingly encountered while providing care not specifically tied to drug use or abuse. The character of our current national drug policy has an important impact on how drug abuse disorders are treated and how the health delivery system responds to drug users who are increasingly often encountered in primary care, emergency care, specialty care, and long-term care settings.

Assessment of Perinatal Depression Risk among internally displaced Yazidi Women in Iraq: a descriptive cross-sectional study.

BACKGROUND: Yazidi survivors of a 2014 genocidal attack by the self-proclaimed Islamic State of Iraq and Syria (ISIS) have complex medical and mental health needs in the perinatal and postpartum period. Few studies have assessed perinatal mental health needs for this population of women who are living in camps for Internally Displaced Persons (IDP) in the Kurdistan Region of Iraq (KRI). METHODS: The specific aim of this formative cross-sectional study was to assess the prevalence of perinatal depressive symptoms, specifically the risk of perinatal depression symptoms, among a purposive sample of Yazidi women living in camps for internally displaced persons in the Kurdistan region of Iraq. One hundred twenty-two pregnant and recently postpartum (<1 year) Yazidi women completed a Kurdish-language version of the Edinburgh Postnatal Depression Scale (EPDS) questionnaire. Pregnant and postpartum participants' responses were analyzed together, in order to assess an overall combined risk of perinatal mental health issues for the study population. Logistic regression analyses were used to measure the association of participant characteristics with an elevated risk of perinatal depressive symptoms. RESULTS: Participants were 17-45 years of age (mean 32 years, SD 7.63) Among the 122 women, 67.2% (n=82) were pregnant and 32.8% (n=40) were <1 year postpartum. Overall, 78% (n=95) of participants were at an elevated risk of depression (EPDS >10), and 53% (n=65) of all participants were at risk of moderate to severe depression (EPDS >12). Thoughts of self-harm (EPDS item 10) were reported among 97% (n=118) of participants. Logistic regression analysis indicated that increased risk of perinatal depressive symptoms was significantly associated with reports of health problems during pregnancy (OR=3.22, 95% [CI]:1.08-9.61) and marital status (OR=16.00; 95% [CI]: 0.42-0.50). Age (OR= 0.84; 95% [CI]: 0.75-0.94) and level of education (OR=0.15; 95% [CI]: 0.42-0.50) had protective effects. CONCLUSIONS: Rates of perinatal depressive symptoms risk among internally displaced Yazid pregnant and postpartum women are higher than the general Kurdish-speaking population in Iraq (28.4%). Culturally responsive trauma informed perinatal and postpartum care services, which include both community-based and clinical strategies for perinatal depressive symptoms and suicide prevention for this population, are critically needed.

Focused Neck Ultrasound and Lymph Node Sampling by Respiratory Physicians in Suspected Lung Cancer.

BACKGROUND: Malignant cervical lymphadenopathy in the setting of lung cancer represents N3 disease, and neck ultrasound (NUS) with sampling is described in the Royal College of Radiologists ultrasound training curriculum for the non-radiologists. This study reviews the incorporation of NUS +/- biopsy in the routine practice of a lung cancer fast-track clinic in the UK. METHODS: We retrospectively assessed 29 months of activity of a lung cancer fast-track clinic. Systematic focused NUS was conducted in suspected thoracic malignancy, sampling nodes with a ≥5-mm short axis, under real-time US using a linear probe (5-12 Mhz). Fine-needle aspirations (FNAs) with or without 18 Ga core biopsies were taken. RESULTS: Between August 2017 and December 2019, of 152 peripheral lymph nodes (LNs)/deposits sampled, 98 (64.5%) were supraclavicular fossa LNs with median [IQR] size 12 [8-18] mm. Core biopsies were performed in 54/98 (55%) patients, while all patients had FNAs. No complications occurred. The representative yield was 90/95 (94.7%) in cases with suspected cancer. No difference was seen between FNA versus core biopsy (p = 0.44). Of the 5 non-diagnostic samples, one was FNA only. The commonest diagnosis was lung cancer in 66/98 (67.3%). PDL-1 was sufficient in 35/36 tested (97.2%). ALK-FISH was successful in 24/25 (96%) cases. EGFR mutation analysis was successful in 28/31 (90.3%) cases. Median time from clinic to initial diagnosis was 7 [5-10] days. Computed tomography (CT) scans reported no significant lymphadenopathy in 18/96 (18.7%) cases, yet 10/18 (55.5%) cases were positive for malignancy. CONCLUSION: Neck nodal sampling by respiratory physicians was safe, timely, with a high diagnostic yield and suitability for molecular testing. Neck US can provide a timely diagnosis in cases that may be missed by CT alone.

circSamd4 represses myogenic transcriptional activity of PUR proteins.

By interacting with proteins and nucleic acids, the vast family of mammalian circRNAs is proposed to influence many biological processes. Here, RNA sequencing analysis of circRNAs differentially expressed during myogenesis revealed that circSamd4 expression increased robustly in mouse C2C12 myoblasts differentiating into myotubes. Moreover, silencing circSamd4, which is conserved between human and mouse, delayed myogenesis and lowered the expression of myogenic markers in cultured myoblasts from both species. Affinity pulldown followed by mass spectrometry revealed that circSamd4 associated with PURA and PURB, two repressors of myogenesis that inhibit transcription of the myosin heavy chain (MHC) protein family. Supporting the hypothesis that circSamd4 might complex with PUR proteins and thereby prevent their interaction with DNA, silencing circSamd4 enhanced the association of PUR proteins with the Mhc promoter, while overexpressing circSamd4 interfered with the binding of PUR proteins to the Mhc promoter. These effects were abrogated when using a mutant circSamd4 lacking the PUR binding site. Our results indicate that the association of PUR proteins with circSamd4 enhances myogenesis by contributing to the derepression of MHC transcription.

Differential Metabotypes in Synovial Fibroblasts and Synovial Fluid in Hip Osteoarthritis Patients Support Inflammatory Responses.

Changes in cellular metabolism have been implicated in mediating the activated fibroblast phenotype in a number of chronic inflammatory disorders, including pulmonary fibrosis, renal disease and rheumatoid arthritis. The aim of this study was therefore to characterise the metabolic profile of synovial joint fluid and synovial fibroblasts under both basal and inflammatory conditions in a cohort of obese and normal-weight hip OA patients. Furthermore, we sought to ascertain whether modulation of a metabolic pathway in OA synovial fibroblasts could alter their inflammatory activity. Synovium and synovial fluid was obtained from hip OA patients, who were either of normal-weight or obese and were undergoing elective joint replacement surgery. The synovial fluid metabolome was determined by 1H NMR spectroscopy. The metabolic profile of isolated synovial fibroblasts in vitro was characterised by lactate secretion, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XF Analyser. The effects of a small molecule pharmacological inhibitor and siRNA targeted at glutaminase-1 (GLS1) were assessed to probe the role of glutamine metabolism in OA synovial fibroblast function. Obese OA patient synovial fluid (n = 5) exhibited a different metabotype, compared to normal-weight patient fluid (n = 6), with significantly increased levels of 1, 3-dimethylurate, N-Nitrosodimethylamine, succinate, tyrosine, pyruvate, glucose, glycine and lactate, and enrichment of the glutamine-glutamate metabolic pathway, which correlated with increasing adiposity. In vitro, isolated obese OA fibroblasts exhibited greater basal lactate secretion and aerobic glycolysis, and increased mitochondrial respiration when stimulated with pro-inflammatory cytokine TNFα, compared to fibroblasts from normal-weight patients. Inhibition of GLS1 attenuated the TNFα-induced expression and secretion of IL-6 in OA synovial fibroblasts. These findings suggest that altered cellular metabolism underpins the inflammatory phenotype of OA fibroblasts, and that targeted inhibition of glutamine-glutamate metabolism may provide a route to reducing the pathological effects of joint inflammation in OA patients who are obese.

Lysine-specific demethylase-2 is distinctively involved in brown and beige adipogenic differentiation.

Transcriptional and epigenetic regulation is fundamentally involved in initiating and maintaining progression of cellular differentiation. The 2 types of thermogenic adipocytes, brown and beige, are thought to be of different origins but share functionally similar phenotypes. Here, we report that lysine-specific demethylase 2 (LSD2) regulates the expression of genes associated with lineage identity during the differentiation of brown and beige adipogenic progenitors in mice. In HB2 mouse brown preadipocytes, short hairpin RNA-mediated knockdown (KD) of LSD2 impaired formation of lipid droplet-containing adipocytes and down-regulated brown adipogenesis-associated genes. Transcriptomic analysis revealed that myogenesis-associated genes were up-regulated in LSD2-KD cells under adipogenic induction. In addition, loss of LSD2 during later phases of differentiation had no obvious influence on adipogenic traits, suggesting that LSD2 functions during earlier phases of brown adipocyte differentiation. Using adipogenic cells from the brown adipose tissues of LSD2-knockout (KO) mice, we found reduced expression of brown adipogenesis genes, whereas myogenesis genes were not affected. In contrast, when LSD2-KO cells from inguinal white adipose tissues were subjected to beige induction, these cells showed a dramatic rise in myogenic gene expression. Collectively, these results suggest that LSD2 regulates distinct sets of genes during brown and beige adipocyte formation.-Takase, R., Hino, S., Nagaoka, K., Anan, K., Kohrogi, K., Araki, H., Hino, Y., Sakamoto, A., Nicholson, T. B., Chen, T., Nakao, M. Lysine-specific demethylase-2 is distinctively involved in brown and beige adipogenic differentiation.

Protein-carbohydrate ingestion alters Vps34 cellular localization independent of changes in kinase activity in human skeletal muscle.

NEW FINDINGS: What is the central question of the study? Is Vps34 a nutrient-sensitive activator of mTORC1 in human skeletal muscle? What is the main finding and its importance? We show that altering nutrient availability, via protein-carbohydrate feeding, does not increase Vps34 kinase activity in human skeletal muscle. Instead, feeding increased Vps34-mTORC1 co-localization in parallel to increased mTORC1 activity. These findings may have important implications in the understanding nutrient-induced mTORC1 activation in skeletal muscle via interaction with Vps34. ABSTRACT: The Class III PI3Kinase, Vps34, has recently been proposed as a nutrient sensor, essential for activation of the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). We therefore investigated the effects of increasing nutrient availability through protein-carbohydrate (PRO-CHO) feeding on Vps34 kinase activity and cellular localization in human skeletal muscle. Eight young, healthy males (21 ± 0.5 yrs, 77.7 ± 9.9 kg, 25.9 ± 2.7 kg/m2 , mean ± SD) ingested a PRO-CHO beverage containing 20/44/1 g PRO/CHO/FAT respectively, with skeletal muscle biopsies obtained at baseline and 1 h and 3 h post-feeding. PRO-CHO feeding did not alter Vps34 kinase activity, but did stimulate Vps34 translocation toward the cell periphery (PRE (mean ± SD) - 0.273 ± 0.040, 1 h - 0.348 ± 0.061, Pearson's Coefficient (r)) where it co-localized with mTOR (PRE - 0.312 ± 0.040, 1 h - 0.348 ± 0.069, Pearson's Coefficient (r)). These alterations occurred in parallel to an increase in S6K1 kinase activity (941 ± 466% of PRE at 1 h post-feeding). Subsequent in vitro experiments in C2C12 and human primary myotubes displayed no effect of the Vps34-specific inhibitor SAR405 on mTORC1 signalling responses to elevated nutrient availability. Therefore, in summary, PRO-CHO ingestion does not increase Vps34 activity in human skeletal muscle, whilst pharmacological inhibition of Vps34 does not prevent nutrient stimulation of mTORC1 in vitro. However, PRO-CHO ingestion promotes Vps34 translocation to the cell periphery, enabling Vps34 to associate with mTOR. Therefore, our data suggests that interaction between Vps34 and mTOR, rather than changes in Vps34 activity per se may be involved in PRO-CHO activation of mTORC1 in human skeletal muscle.

Binding of a glaucoma-associated myocilin variant to the αB-crystallin chaperone impedes protein clearance in trabecular meshwork cells.

Myocilin (MYOC) was discovered more than 20 years ago and is the gene whose mutations are most commonly observed in individuals with glaucoma. Despite extensive research efforts, the function of WT MYOC has remained elusive, and how mutant MYOC is linked to glaucoma is unclear. Mutant MYOC is believed to be misfolded within the endoplasmic reticulum, and under normal physiological conditions misfolded MYOC should be retro-translocated to the cytoplasm for degradation. To better understand mutant MYOC pathology, we CRISPR-engineered a rat to have a MYOC Y435H substitution that is the equivalent of the pathological human MYOC Y437H mutation. Using this engineered animal model, we discovered that the chaperone αB-crystallin (CRYAB) is a MYOC-binding partner and that co-expression of these two proteins increases protein aggregates. Our results suggest that the misfolded mutant MYOC aggregates with cytoplasmic CRYAB and thereby compromises protein clearance mechanisms in trabecular meshwork cells, and this process represents the primary mode of mutant MYOC pathology. We propose a model by which mutant MYOC causes glaucoma, and we propose that therapeutic treatment of patients having a MYOC mutation may focus on disrupting the MYOC-CRYAB complexes.

Bioinformatic evidence of widespread priming in type I and II CRISPR-Cas systems.

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against invading genetic elements, such as plasmids, bacteriophages and archaeal viruses. They consist of cas genes and CRISPR loci, which store genetic memories of previously encountered invaders as short sequences termed spacers. Spacers determine the specificity of CRISPR-Cas defence and immunity can be gained or updated by the addition of new spacers into CRISPR loci. There are two main routes to spacer acquisition, which are known as naïve and primed CRISPR adaptation. Naïve CRISPR adaptation involves the de novo formation of immunity, independent of pre-existing spacers. In contrast, primed CRISPR adaptation (priming) uses existing spacers to enhance the acquisition of new spacers. Priming typically results in spacer acquisition from locations near the site of target recognition by the existing (priming) spacer. Primed CRISPR adaptation has been observed in several type I CRISPR-Cas systems and it is potentially widespread. However, experimental evidence is unavailable for some subtypes, and for most systems, priming has only been shown in a small number of hosts. There is also no current evidence of priming by other CRISPR-Cas types. Here, we used a bioinformatic approach to search for evidence of priming in diverse CRISPR-Cas systems. By analysing the clustering of spacers acquired from phages, prophages and archaeal viruses, including strand and directional biases between subsequently acquired spacers, we demonstrate that two patterns of primed CRISPR adaptation dominate in type I systems. In addition, we find evidence of a priming-like pathway in type II CRISPR-Cas systems.

Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells.

DNA methylation at the 5 position of cytosine (5mC) in the mammalian genome is a key epigenetic event critical for various cellular processes. The ten-eleven translocation (Tet) family of 5mC-hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), offers a way for dynamic regulation of DNA methylation. Here we report that Tet1 binds to unmodified C or 5mC- or 5hmC-modified CpG-rich DNA through its CXXC domain. Genome-wide mapping of Tet1 and 5hmC reveals mechanisms by which Tet1 controls 5hmC and 5mC levels in mouse embryonic stem cells (mESCs). We also uncover a comprehensive gene network influenced by Tet1. Collectively, our data suggest that Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting 5mC to 5hmC through hydroxylase activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 targets, ultimately contributing to mESC differentiation and the onset of embryonic development.

Introduction of the Capsules environment to support further growth of the SBGrid structural biology software collection.

The expansive scientific software ecosystem, characterized by millions of titles across various platforms and formats, poses significant challenges in maintaining reproducibility and provenance in scientific research. The diversity of independently developed applications, evolving versions and heterogeneous components highlights the need for rigorous methodologies to navigate these complexities. In response to these challenges, the SBGrid team builds, installs and configures over 530 specialized software applications for use in the on-premises and cloud-based computing environments of SBGrid Consortium members. To address the intricacies of supporting this diverse application collection, the team has developed the Capsule Software Execution Environment, generally referred to as Capsules. Capsules rely on a collection of programmatically generated bash scripts that work together to isolate the runtime environment of one application from all other applications, thereby providing a transparent cross-platform solution without requiring specialized tools or elevated account privileges for researchers. Capsules facilitate modular, secure software distribution while maintaining a centralized, conflict-free environment. The SBGrid platform, which combines Capsules with the SBGrid collection of structural biology applications, aligns with FAIR goals by enhancing the findability, accessibility, interoperability and reusability of scientific software, ensuring seamless functionality across diverse computing environments. Its adaptability enables application beyond structural biology into other scientific fields.

Severe thermal and major traumatic injury results in elevated plasma concentrations of total heme that are associated with poor clinical outcomes and systemic immune suppression.

Background: Traumatic and thermal injuries result in a state of systemic immune suppression, yet the mechanisms that underlie its development are poorly understood. Released from injured muscle and lysed red blood cells, heme is a damage associated molecular pattern with potent immune modulatory properties. Here, we measured plasma concentrations of total heme in over 200 traumatic and thermally-injured patients in order to examine its relationship with clinical outcomes and post-injury immune suppression. Methods: Blood samples were collected from 98 burns (≥15% total body surface area) and 147 traumatically-injured (injury severity score ≥8) patients across the ultra-early (≤1 hour) and acute (4-72 hours) post-injury settings. Pro-inflammatory cytokine production by lipopolysaccharide (LPS) challenged whole blood leukocytes was studied, and plasma concentrations of total heme, and its scavengers haptoglobin, hemopexin and albumin measured, alongside the expression of heme-oxygenase-1 (HO-1) in peripheral blood mononuclear cells (PBMCs). LPS-induced tumour necrosis factor-alpha (TNF-α) production by THP-1 cells and monocytes following in vitro heme treatment was also examined. Results: Burns and traumatic injury resulted in significantly elevated plasma concentrations of heme, which coincided with reduced levels of hemopexin and albumin, and correlated positively with circulating levels of pro and anti-inflammatory cytokines. PBMCs isolated from trauma patients 4-12 and 48-72 hours post-injury exhibited increased HO-1 gene expression. Non-survivors of burn injury and patients who developed sepsis, presented on day 1 with significantly elevated heme levels, with a difference of 6.5 µM in heme concentrations corresponding to a relative 52% increase in the odds of post-burn mortality. On day 1 post-burn, heme levels were negatively associated with ex vivo LPS-induced TNF-α and interleukin-6 production by whole blood leukocytes. THP-1 cells and monocytes pre-treated with heme exhibited significantly reduced TNF-α production following LPS stimulation. This impairment was associated with decreased gene transcription, reduced activation of extracellular signal-regulated kinase 1/2 and an impaired glycolytic response. Conclusions: Major injury results in elevated plasma concentrations of total heme that may contribute to the development of endotoxin tolerance and increase the risk of poor clinical outcomes. Restoration of the heme scavenging system could be a therapeutic approach by which to improve immune function post-injury.

Oligonucleotide Therapeutics for Age-Related Musculoskeletal Disorders: Successes and Challenges.

Age-related disorders of the musculoskeletal system including sarcopenia, osteoporosis and arthritis represent some of the most common chronic conditions worldwide, for which there remains a great clinical need to develop safer and more efficacious pharmacological treatments. Collectively, these conditions involve multiple tissues, including skeletal muscle, bone, articular cartilage and the synovium within the joint lining. In this review, we discuss the potential for oligonucleotide therapies to combat the unmet clinical need in musculoskeletal disorders by evaluating the successes of oligonucleotides to modify candidate pathological gene targets and cellular processes in relevant tissues and cells of the musculoskeletal system. Further, we discuss the challenges that remain for the clinical development of oligonucleotides therapies for musculoskeletal disorders and evaluate some of the current approaches to overcome these.

Gastrointestinal Tolerability and Acute Glycemic Response of Oligosaccharides and Polysaccharides from Cellulose and Xylan in Healthy Adults: Two Double-Blinded, Randomized, Controlled, Cross-over Trials.

OBJECTIVE: The aim of this study was to investigate the gastrointestinal tolerability, glycemic and insulinemic responses of Plant Fiber Extract (PFE), a mixture comprising of oligosaccharides and polysaccharides derived from cellulose and xylan. METHODS: Two double-blind, randomized, controlled, cross-over trials were conducted in healthy adults. In the first trial, participants (n = 29) consumed either 25, 35 or 45 g per day of PFE or resistant maltodextrin (Control) for 14 days. The occurrence and severity of gastrointestinal (GI) symptoms, stool parameters, and safety outcomes were evaluated with a combination of surveys and blood analysis respectively. In the second trial (n = 20), the post-prandial glycemic and insulinemic responses after the ingestion of 20 g of PFE diluted in water or incorporated into chocolate chips was measured and then compared to that of glucose and regular chocolate, respectively. RESULTS: For all timepoints (0, 7 and 14 days), within any given dose group, there was no statistically significant difference in the GI symptoms score between PFE and Control. Further, for each test product (PFE or Control), no difference was observed in the same dose group from days 0 and 14. Stool consistency score and number of participants experiencing loose or watery stools was similar between products. No serious adverse events were reported and neither PFE nor Control significantly altered blood or urine safety parameters. The glycemic and insulinemic responses after PFE ingestion in comparison to glucose were 12% and 8% respectively. The glycemic and insulinemic responses after consuming chocolate containing PFE were 20% of that of regular chocolate. CONCLUSION: PFE was well-tolerated by healthy volunteers in doses up to 45 g/day and it elicited comparatively low glycemic and insulinemic responses when consumed alone or when incorporated into a food product.

Endobronchial Ultrasound-guided Sampling of Centrally Located Intrapulmonary Tumors Provides Suitable Material for Diagnostic and Molecular Testing.

BACKGROUND: Curvilinear endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) is a key diagnostic and staging procedure for patients with suspected lung cancer. However, sampling centrally located intrapulmonary tumors is feasible but less well established. METHODS: We retrospectively evaluated the diagnostic utility of EBUS-TBNA in patients who underwent sampling of centrally located intrapulmonary tumors. Diagnostic accuracy, sample suitability for molecular testing, and complications were assessed. RESULTS: Between January 2015 and April 2021, 102 EBUS-TBNA procedures sampled centrally located intrapulmonary tumors in 99 patients. The median age was 70 [interquartile range, 63 to 75] years and 51% (51/99) were male. The commonest site was the right upper lobe (n=42/99; 42%). The median tumor size was 29 [interquartile range, 21 to 35] mm. The diagnostic yield was 88/102 (86%) with a false negative rate of 14% (14/102). In addition to intrapulmonary tumor sampling, lymph nodes were sampled in 65/102 procedures and 30/65(46%) were positive for lung cancer. Cancer was diagnosed in 87/99 (88%) cases. When requested, molecular testing was adequate in ≥94% of samples. Complications included minor bleeding in 6/102 (6%) with 2 requiring cold saline instillation, desaturation in 1/102 (1%), and tachycardia in 1/102(1%). One procedure was abandoned due to patient tachycardia. Delayed complications occurred in 1 patient who was hospitalized ≤7 days with pneumonia. CONCLUSION: EBUS-TBNA sampling of centrally located intrapulmonary tumors provides similar diagnostic accuracy to lymph node sampling, provides suitable material for molecular testing, and has a low complication rate.

A detailed methodology for the long-term in vitro culture and analysis of three-dimensional, self-structuring bone models generated from cell lines or primary osteoblastic cell populations.

Background: There are insufficient in vitro bone models that accommodate long-term culture of osteoblasts and support their differentiation to osteocytes. The increased demand for effective therapies for bone diseases, and the ethical requirement to replace animals in research, warrants the development of such models.Here we present an in-depth protocol to prepare, create and maintain three-dimensional, in vitro, self-structuring bone models that support osteocytogenesis and long-term osteoblast survival (>1 year). Methods: Osteoblastic cells are seeded on a fibrin hydrogel, cast between two beta-tricalcium phosphate anchors. Analytical methods optimised for these self-structuring bone model (SSBM) constructs, including RT-qPCR, immunofluorescence staining and XRF, are described in detail. Results: Over time, the cells restructure and replace the initial matrix with a collagen-rich, mineralising one; and demonstrate differentiation towards osteocytes within 12 weeks of culture. Conclusions: Whilst optimised using a secondary human cell line (hFOB 1.19), this protocol readily accommodates osteoblasts from other species (rat and mouse) and origins (primary and secondary). This simple, straightforward method creates reproducible in vitro bone models that are responsive to exogenous stimuli, offering a versatile platform for conducting preclinical translatable research studies.

Identification of a novel functional specificity signal within the GPI anchor signal sequence of carcinoembryonic antigen.

Exchanging the glycophosphatidylinositol (GPI) anchor signal sequence of neural cell adhesion molecule (NCAM) for the signal sequence of carcinoembryonic antigen (CEA) generates a mature protein with NCAM external domains but CEA-like tumorigenic activity. We hypothesized that this resulted from the presence of a functional specificity signal within this sequence and generated CEA/NCAM chimeras to identify this signal. Replacing the residues (GLSAG) 6-10 amino acids downstream of the CEA anchor addition site with the corresponding NCAM residues resulted in GPI-anchored proteins lacking the CEA-like biological functions of integrin modulation and differentiation blockage. Transferring this region from CEA into NCAM in conjunction with the upstream proline (PGLSAG) was sufficient to specify the addition of the CEA anchor. Therefore, this study identifies a novel specificity signal consisting of six amino acids located within the GPI anchor attachment signal, which is necessary and sufficient to specify the addition of a particular functional GPI anchor and, thereby, the ultimate function of the mature protein.

e-Cigarette Vapour Condensate Reduces Viability and Impairs Function of Human Osteoblasts, in Part, via a Nicotine Dependent Mechanism.

Cigarette consumption negatively impacts bone quality and is a risk-factor for the development of multiple bone associated disorders, due to the highly vascularised structure of bone being exposed to systemic factors. However, the impact on bone to electronic cigarette (e-cigarette) use, which contains high doses of nicotine and other compounds including flavouring chemicals, metal particulates and carbonyls, is poorly understood. Here, we present the first evidence demonstrating the impact of e-cigarette vapour condensate (replicating changes in e-cigarette liquid chemical structure that occur upon device usage), on human primary osteoblast viability and function. 24 h exposure of osteoblasts to e-cigarette vapour condensate, generated from either second or third generation devices, significantly reduced osteoblast viability in a dose dependent manner, with condensate generated from the more powerful third generation device having greater toxicity. This effect was mediated in-part by nicotine, since exposure to nicotine-free condensate of an equal concentration had a less toxic effect. The detrimental effect of e-cigarette vapour condensate on osteoblast viability was rescued by co-treatment with the antioxidant N-Acetyl-L-cysteine (NAC), indicating toxicity may also be driven by reactive species generated upon device usage. Finally, non-toxic doses of either second or third generation condensate significantly blunted osteoblast osteoprotegerin secretion after 24 h, which was sustained for up to 7 days. In summary we demonstrate that e-cigarette vapour condensate, generated from commonly used second and third generation devices, can significantly reduce osteoblast viability and impair osteoblast function, at physiologically relevant doses. These data highlight the need for further investigation to inform users of the potential risks of e-cigarette use on bone health, including, accelerating bone associated disease progression, impacting skeletal development in younger users and to advise patients following orthopaedic surgery, dental surgery, or injury to maximise bone healing.