Septin 7 interacts with Numb to preserve sarcomere structural organization and muscle contractile function.

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

Use of levetiracetam for the successful treatment of suspected myoclonic seizures: five dogs (2016-2022).

OBJECTIVES: Myoclonic seizures are considered a type of generalised seizure characterised by brief, jerking movements of the body. The aim of this study is to describe cases of suspected canine myoclonic seizure of idiopathic aetiology and to discuss the successful use of the anticonvulsant levetiracetam as treatment in each of these cases. MATERIALS AND METHODS: Dogs with epileptic myoclonus suspected to be idiopathic in aetiology were considered for inclusion. Medical records were reviewed for physical and neurologic examination findings, clinicopathologic results, and diagnostic imaging results. All included dogs were treated with levetiracetam, and their response was reported. RESULTS: Five dogs were included, all of which had suspected myoclonic seizures either observed in-person or on video recording by a board-certified veterinary neurologist. The duration of myoclonic seizures preceding treatment ranged from one day to one year. One dog also experienced a generalised tonic-clonic seizure. All dogs were treated with levetiracetam. Two dogs experienced long-term myoclonic seizure freedom (duration seizure-free of at least 1 year), and two dogs experienced marked decreased myoclonic seizure frequency. One dog experienced immediate abatement of myoclonic seizures, although levetiracetam was only utilised for 1 month following onset of myoclonic seizures in this patient. CLINICAL SIGNIFICANCE: Myoclonic seizures can be idiopathic in aetiology. Levetiracetam can be used effectively to rapidly stop myoclonic seizures and to decrease the frequency of myoclonic seizures.

Data-Independent Acquisition and Label-Free Quantification for Quantitative Proteomics Analysis of Human Cerebrospinal Fluid.

This article presents a practical guide to mass spectrometry-based data-independent acquisition and label-free quantification for proteomics analysis applied to cerebrospinal fluid, offering a robust and scalable approach to probing the proteomic composition of the central nervous system. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Cerebrospinal fluid sample collection and preparation for mass spectrometry analysis Basic Protocol 2: Mass spectrometry sample analysis with data-independent acquisition Support Protocol: Data-dependent mass spectrometry and spectral library construction Basic Protocol 3: Analysis of mass spectrometry data.

The complement system in neurodegenerative diseases.

Complement is an important component of innate immune defence against pathogens and crucial for efficient immune complex disposal. These core protective activities are dependent in large part on properly regulated complement-mediated inflammation. Dysregulated complement activation, often driven by persistence of activating triggers, is a cause of pathological inflammation in numerous diseases, including neurological diseases. Increasingly, this has become apparent not only in well-recognized neuroinflammatory diseases like multiple sclerosis but also in neurodegenerative and neuropsychiatric diseases where inflammation was previously either ignored or dismissed as a secondary event. There is now a large and rapidly growing body of evidence implicating complement in neurological diseases that cannot be comprehensively addressed in a brief review. Here, we will focus on neurodegenerative diseases, including not only the 'classical' neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, but also two other neurological diseases where neurodegeneration is a neglected feature and complement is implicated, namely, schizophrenia, a neurodevelopmental disorder with many mechanistic features of neurodegeneration, and multiple sclerosis, a demyelinating disorder where neurodegeneration is a major cause of progressive decline. We will discuss the evidence implicating complement as a driver of pathology in these diverse diseases and address briefly the potential and pitfalls of anti-complement drug therapy for neurodegenerative diseases.

Electrodiagnostic testing in dogs with disorders of the spinal cord or cauda equina.

Electrodiagnostic (EDX) testing is uncommonly utilized in dogs other than for investigation of disorders of the neuromuscular system. In dogs with diseases affecting the spinal cord or cauda equina, EDX testing can provide functional data complementary to imaging information that together can guide therapeutic and management approaches. Additionally, in some clinical scenarios, EDX testing prior to advanced imaging is integral to identifying if there is spinal cord or cauda equina involvement and can aid in determining the appropriate diagnostic path. This review will outline EDX testing methods that have been reported in dogs relating to the diagnosis, monitoring or prognosis of various conditions affecting the spinal cord and cauda equina. The various tests will be briefly outlined regarding how they are performed and what information is provided. The main focus will be on clinical applications including highlighting situations where EDX testing is useful for differentiating between neurologic and non-neurologic presentations. Additional ways these EDX techniques could be incorporated in the management of diseases of the spinal cord and cauda equina in dogs will be presented.

Supervised exercise training improves cardiorespiratory fitness and reduces perioperative risk in peripheral artery disease patients with intermittent claudication.

INTRODUCTION: This study examined to what extent supervised aerobic and resistance exercise combined with continued unsupervised exercise training improves cardiorespiratory fitness and corresponding perioperative risk in peripheral artery disease (PAD) patients with intermittent claudication. METHODS: A total of 106 patients (77% male) were enrolled into the study, alongside 155 healthy non-PAD control participants. Patients completed supervised exercise therapy (aerobic and resistance exercises of the upper and lower limbs) twice a week for 10 weeks. Thereafter, 52 patients completed 12 weeks of an unsupervised tailored home-based exercise. Pain-free walking distance (PWD), maximum walking distance (MWD), peak oxygen uptake ([Formula: see text]) and perioperative risk were assessed before and after both exercise interventions. RESULTS: Patients were highly unconditioned relative to healthy controls ([Formula: see text]=11.9 vs 24.2ml/kg/min, p=<0.001) with 91% classified as high perioperative risk (peak oxygen uptake <15ml/kg/min). Supervised exercise increased PWD (+44±81m, p=<0.001), MWD (+44±71m, p=<0.001) and [Formula: see text] (+1.01±1.63ml/kg/min, p=<0.001) and lowered perioperative risk (91% to 85%, p=<0.001). When compared with supervised exercise, the improvements in PWD were maintained following unsupervised exercise (+11±91m vs supervised exercise, p=0.572); however, MWD and [Formula: see text] decreased (-15±48m, p=0.030 and -0.34±1.11ml/kg/min, p=0.030, respectively) and perioperative risk increased (+3%, p=<0.001) although still below baseline (p=<0.001). CONCLUSIONS: Supervised aerobic and resistance exercise training and, to a lesser extent, unsupervised tailored exercise improves walking capacity and cardiorespiratory fitness and reduces perioperative risk in PAD patients with intermittent claudication.

Pericellular Matrix Formation and Atomic Force Microscopy of Single Primary Human Chondrocytes Cultured in Alginate Microgels.

One of the main components of articular cartilage is the chondrocyte's pericellular matrix (PCM), which is critical for regulating mechanotransduction, biochemical cues, and healthy cartilage development. Here, individual primary human chondrocytes (PHC) are encapsulated and cultured in 50 µm diameter alginate microgels using drop-based microfluidics. This unique culturing method enables PCM formation and manipulation of individual cells. Over ten days, matrix formation is observed using autofluorescence imaging, and the elastic moduli of isolated cells are measured using AFM. Matrix production and elastic modulus increase are observed for the chondrons cultured in microgels. Furthermore, the elastic modulus of cells grown in microgels increases ≈ten-fold over ten days, nearly reaching the elastic modulus of in vivo PCM. The AFM data is further analyzed using a Gaussian mixture model and shows that the population of PHCs grown in microgels exhibit two distinct populations with elastic moduli averaging 9.0 and 38.0 kPa. Overall, this work shows that microgels provide an excellent culture platform for the growth and isolation of PHCs, enabling PCM formation that is mechanically similar to native PCM. The microgel culture platform presented here has the potential to revolutionize cartilage regeneration procedures through the inclusion of in vitro developed PCM.

Complex Autism Spectrum Disorder in a Patient with a Novel De Novo Heterozygous MYT1L Variant.

Autism spectrum disorder (ASD) comprises a group of complex neurodevelopmental features seen in many different forms due to variable causes. Highly impactful ASD-susceptibility genes are involved in pathways associated with brain development, chromatin remodeling, and transcription regulation. In this study, we investigate a proband with complex ASD. Whole genome sequencing revealed a novel de novo missense mutation of a highly conserved amino acid residue (NP_001289981.1:p.His516Gln; chr2:1917275; hg38) in the MYT1L neural transcription factor gene. In combination with in silico analysis on gene effect and pathogenicity, we described the proband's phenotype and made comparisons with previously reported cases to explore the spectrum of clinical features in MYT1L single nucleotide variant (SNV) cases. The phenotype-genotype correlation showed a high degree of clinical similarity with previously reported cases of missense variants in MYT1L, indicating MYT1L as the causal gene for the observed phenotype in our proband. The variant was also predicted to be damaging according to multiple in silico pathogenicity predicting tools. This study expands the clinical description of SNVs on the MYT1L gene and provides insight into its contribution to ASD.

Debridement, antibiotics and implant retention (DAIR) following hip and knee arthroplasty: results and findings of a multidisciplinary approach from a non-specialist prosthetic infection centre.

INTRODUCTION: Prosthetic joint infection (PJI) is a catastrophic complication following arthroplasty surgery. Recently a debridement, antibiotics and implant retention (DAIR) procedure has gained popularity for PJI where a thorough debridement, irrigation and modular component exchange is undertaken. METHOD: We present the outcome for DAIR, data collected prospectively, in a busy orthopaedic unit but not one specialising in PJI. All patients with PJI were included without loss of data or patients from 2012 to 2018 with a minimum follow-up of 5 years. RESULTS: Four total knee replacements, 17 total hip replacements, one revision total hip replacement and three hip hemiarthroplasties are included with an average duration from onset of symptoms to the DAIR procedure of 11 days (range 1-22 days). Staphylococcus aureus (24%) and Staphylococcus epidermidis (32%) were the most common causative organisms, and the most common antibiotic regimens included intravenous teicoplanin and flucloxacillin. Average follow-up was 67 months (range 9-104 months). Only four patients went on to require revision surgery. An analysis of midterm patient outcome measures for 6 of the total hip replacement (THR) DAIR patients were compared with a database of 792 THRs (with a minimum two-year follow-up) carried out by the same surgeon revealed no significant difference in Oxford hip scores at one-year post-surgery (OHS DAIR 36.2 vs 39 for control group). CONCLUSION: This study includes 25 consecutive patients treated with DAIR with only one reinfection, with a mean follow-up period of 5 years. Using a strict protocol, DAIR appears to offer a successful treatment strategy for the management of early PJI.

Adaptation of land treadmill scoring system for underwater treadmill in dogs with thoracolumbar intervertebral disc extrusion.

The underwater treadmill (UWTM) is utilized in dogs recovering from thoracolumbar intervertebral disc extrusion (TL-IVDE). Gait scoring is validated for dogs with TL-IVDE walking on the land treadmill (LT) but has not been reported for the UWTM. Our objective was to investigate if LT gait analysis could be applied to the UWTM and if non-ambulatory dogs walking unassisted on the UWTM, at a standardized water level, would be more likely to generate gait scores compared to on the LT. This was a prospective, observational study in dogs with TL-IVDE managed surigcally. At 0, 2, 4, 8 and 12 weeks post-operatively, paired video footage of dogs walking on the LT and UWTM (water level at the greater trochanter) was used to generate 0-100 stepping (SS) and coordination (regularity index, RI) scores. Scores were compared between treadmill type and over time. Twenty dogs were enrolled and seventy-eight paired recordings were available for review. Median gait scores increased over time but did not differ by treadmill type (P = 0.262 for SS, P = 0.533 for RI). Combining SS and RI, more recordings received scores of 0 for the LT (n = 58/156; 37.2 %) compared to the UWTM (n = 44/156; 28.2 %; P = 0.043). Scores of 0, at visits when there was at least movement present at multiple joints, was more common on the LT (n = 11/108; 10.2 %) compared to the UWTM (n = 2/108, 1.9 %; P = 0.026). In dogs recovering from TL-IVDE, LT-based gait scoring was feasible in dogs walking on the UWTM and might complement other gait analysis methods, especially for non-ambulatory dogs.

Complement C3 Reduces Apoptosis via Interaction with the Intrinsic Apoptotic Pathway.

Myocardial ischemia/reperfusion (I/R) elicits an acute inflammatory response involving complement factors. Recently, we reported that myocardial necrosis was decreased in complement C3-/- mice after heart I/R. The current study used the same heart model to test the effect of C3 on myocardial apoptosis and investigated if C3 regulation of apoptosis occurred in human cardiomyocytes. Comparative proteomics analyses found that cytochrome c was present in the myocardial C3 complex of WT mice following I/R. Incubation of exogenous human C3 reduced apoptosis in a cell culture system of human cardiomyocytes that did not inherently express C3. In addition, human C3 inhibited the intrinsic apoptosis pathway in a cell-free apoptosis system. Finally, human pro-C3 was found to bind with an apoptotic factor, pro-caspase 3, in a cell-free system. Thus, we present firsthand evidence showing that C3 readily reduces myocardial apoptosis via interaction with the intrinsic apoptotic pathway.

Fourier Transform Infrared microspectroscopy identifies single cancer cells in blood. A feasibility study towards liquid biopsy.

The management of cancer patients has markedly improved with the advent of personalised medicine where treatments are given based on tumour antigen expression amongst other. Within this remit, liquid biopsies will no doubt improve this personalised cancer management. Identifying circulating tumour cells in blood allows a better assessment for tumour screening, staging, response to treatment and follow up. However, methods to identify/capture these circulating tumour cells using cancer cells' antigen expression or their physical properties are not robust enough. Thus, a methodology that can identify these circulating tumour cells in blood regardless of the type of tumour is highly needed. Fourier Transform Infrared (FTIR) microspectroscopy, which can separate cells based on their biochemical composition, could be such technique. In this feasibility study, we studied lung cancer cells (squamous cell carcinoma and adenocarcinoma) mixed with peripheral blood mononuclear cells (PBMC). The data obtained shows, for the first time, that FTIR microspectroscopy together with Random Forest classifier is able to identify a single lung cancer cell in blood. This separation was easier when the region of the IR spectra containing lipids and the amide A (2700 to 3500 cm-1) was used. Furthermore, this work was carried out using glass coverslips as substrates that are widely used in pathology departments. This allows further histopathological cell analysis (staining, immunohistochemistry, …) after FTIR spectra are obtained. Hence, although further work is needed using blood samples from patients with cancer, FTIR microspectroscopy could become another tool to be used in liquid biopsies for the identification of circulating tumour cells, and in the personalised management of cancer.

Dimerization of European Robin Cryptochrome 4a.

Homo-dimer formation is important for the function of many proteins. Although dimeric forms of cryptochromes (Cry) have been found by crystallography and were recently observed in vitro for European robin Cry4a, little is known about the dimerization of avian Crys and the role it could play in the mechanism of magnetic sensing in migratory birds. Here, we present a combined experimental and computational investigation of the dimerization of robin Cry4a resulting from covalent and non-covalent interactions. Experimental studies using native mass spectrometry, mass spectrometric analysis of disulfide bonds, chemical cross-linking, and photometric measurements show that disulfide-linked dimers are routinely formed, that their formation is promoted by exposure to blue light, and that the most likely cysteines are C317 and C412. Computational modeling and molecular dynamics simulations were used to generate and assess a number of possible dimer structures. The relevance of these findings to the proposed role of Cry4a in avian magnetoreception is discussed.

The checkpoint inhibitor PD-1H/VISTA controls osteoclast-mediated multiple myeloma bone disease.

Multiple myeloma bone disease is characterized by the development of osteolytic bone lesions. Recent work identified matrix metalloproteinase 13 as a myeloma-derived fusogen that induces osteoclast activation independent of its proteolytic activity. We now identify programmed death-1 homolog, PD-1H, as the bona fide MMP-13 receptor on osteoclasts. Silencing PD-1H or using Pd-1h-/- bone marrow cells abrogates the MMP-13-enhanced osteoclast fusion and bone-resorptive activity. Further, PD-1H interacts with the actin cytoskeleton and plays a necessary role in supporting c-Src activation and sealing zone formation. The critical role of PD-1H in myeloma lytic bone lesions was confirmed using a Pd-1h-/- myeloma bone disease mouse model wherein myeloma cells injected into Pd-1h-/-Rag2-/- results in attenuated bone destruction. Our findings identify a role of PD-1H in bone biology independent of its known immunoregulatory functions and suggest that targeting the MMP-13/PD-1H axis may represent a potential approach for the treatment of myeloma associated osteolysis.

SEPTIN 7 INTERACTS WITH NUMB TO PRESERVE SARCOMERE STRUCTURAL ORGANIZATION AND MUSCLE CONTRACTILE FUNCTION.

Here, we investigated mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development including asymmetric cell division, cell-type specification and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (NumbL) in mouse myofibers caused weakness, disorganization of sarcomeres and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, NumbL knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb, that Septin 7 is a potential Numb binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

The impact of preoperative sarcopenia on postoperative ileus following colorectal cancer surgery.

PURPOSE: Sarcopenia is associated with poor short- and long-term patient outcomes following colorectal surgery. Despite postoperative ileus (POI) being a major complication following colorectal surgery, the predictive value of sarcopenia for POI is unclear. We assessed the association between sarcopenia and POI in patients with colorectal cancer. METHODS: Elective colorectal cancer surgery patients were retrospectively included (2018-2022). The cross-sectional psoas area was calculated using preoperative staging imaging at the level of the 3rd lumbar vertebrae. Sarcopenia was determined using gender-specific cut-offs. The primary outcome POI was defined as not achieving GI-2 by day 4. Demographics, operative characteristics, and complications were compared via univariate and multivariate analyses. RESULTS: Of 297 patients, 67 (22.6%) were sarcopenic. Patients with sarcopenia were older (median 74 (IQR 67-82) vs. 69 (58-76) years, p < 0.001) and had lower body mass index (median 24.4 (IQR 22.2-28.6) vs. 28.8 (24.9-31.9) kg/m2, p < 0.001). POI was significantly more prevalent in patients with sarcopenia (41.8% vs. 26.5%, p = 0.016). Overall rate of complications (85.1% vs. 68.3%, p = 0.007), Calvien-Dindo grade > 3 (13.4% vs. 10.0%, p = 0.026) and length of stay were increased in patients with sarcopenia (median 7 (IQR 5-12) vs. 6 (4-8) days, p = 0.013). Anastomotic leak rate was higher in patients with sarcopenia although the difference was not statistically significant (7.5% vs. 2.6%, p = 0.064). Multivariate analysis demonstrated sarcopenia (OR 2.0, 95% CI 1.1-3.8), male sex (OR 1.9, 95% CI 1.0-3.5), postoperative hypokalemia (OR 3.2, 95% CI 1.6-6.5) and increased opioid use (OR 2.4, 95% CI 1.3-4.3) were predictive of POI. CONCLUSION: Sarcopenia demonstrates an association with POI. Future research towards truly identifying the predictive value of sarcopenia for postoperative complications could improve informed consent and operative planning for surgical patients.

Complement activation and increased anaphylatoxin receptor expression are associated with cortical grey matter lesions and the compartmentalised inflammatory response of multiple sclerosis.

Background: The extent of cortical pathology is an important determinant of multiple sclerosis (MS) severity. Cortical demyelination and neurodegeneration are related to inflammation of the overlying leptomeninges, a more inflammatory CSF milieu and with parenchymal microglia and astroglia activation. These are all components of the compartmentalised inflammatory response. Compartmentalised inflammation is a feature of progressive MS, which is not targeted by disease modifying therapies. Complement is differentially expressed in the MS CSF and complement, and complement receptors, are associated with demyelination and neurodegeneration. Methods: To better understand if complement activation in the leptomeninges is associated with underlying cortical demyelination, inflammation, and microglial activation, we performed a neuropathological study of progressive MS (n = 22, 14 females), neuroinflammatory (n = 8), and non-neurological disease controls (n = 10). We then quantified the relative extent of demyelination, connective tissue inflammation, complement, and complement receptor positive microglia/macrophages. Results: Complement was elevated at the leptomeninges, subpial, and within and around vessels of the cortical grey matter. The extent of complement C1q immunoreactivity correlated with connective tissue infiltrates, whilst activation products C4d, Bb, and C3b associated with grey matter demyelination, and C3a receptor 1+ and C5a receptor 1+ microglia/macrophages closely apposed C3b labelled cells. The density of C3a receptor 1+ and C5a receptor 1+ cells was increased at the expanding edge of subpial and leukocortical lesions. C5a receptor 1+ cells expressed TNFα, iNOS and contained puncta immunoreactive for proteolipid protein, neurofilament and synaptophysin, suggesting their involvement in grey matter lesion expansion. Interpretation: The presence of products of complement activation at the brain surfaces, their association with the extent of underlying pathology and increased complement anaphylatoxin receptor positive microglia/macrophages at expanding cortical grey matter lesions, could represent a target to modify compartmentalised inflammation and cortical demyelination.

Effect of Geometry and Orientation on the Tensile Properties and Failure Mechanisms of Compliant Suture Joints.

Compliant sutures surrounded by stiff matrices are present in biological armors and carapaces, providing enhanced mechanical performance. Understanding the mechanisms through which these sutured composites achieve outstanding properties is key to developing engineering materials with improved strength and toughness. This article studies the impact of suture geometry and load direction on the performance of suture joints using a two-stage reactive polymer resin that enables facile photopatterning of mechanical heterogeneity within a single polymer network. Compliant sinusoidal sutures with varying geometries are photopatterned into stiff matrices, generating a modulus contrast of 2 orders of magnitude. Empirical relationships are developed connecting suture wavelength and amplitude to composite performance under parallel and perpendicular loading conditions. Results indicate that a greater suture interdigitation broadly improves composite performance when loading is applied perpendicular to suture joints but has deleterious effects when loading is applied parallel to the joint. Investigations into the failure mechanisms under perpendicular loading highlight the interplay between suture geometry and crack growth stability after damage initiation occurs. Our findings could enable a framework for engineering composites and bio-inspired structures in the future.

Tobacco-Derived Nicotine Pouch Brands and Marketing Messages on Internet and Traditional Media: Content Analysis.

BACKGROUND: Nicotine pouches and lozenges are increasingly available in the United States, and sales are growing. The brands of nicotine pouch products with the largest market share are produced by tobacco companies. OBJECTIVE: The aim of this study is to examine the marketing of 5 oral nicotine products sold by tobacco companies. METHODS: Internet, radio, television, print, and web-based display advertisements between January 2019 and March 2020 for 6 brands of nicotine pouches and lozenges were identified through commercially available marketing surveillance systems supplemented by a manual search of trade press and a review of brand websites. A total of 711 advertisements (122 unique) were analyzed to identify characteristics, themes, marketing strategies, and target audiences, and qualitatively compared by brand. All 5 brand websites were also analyzed. Coders examined the entirety of each advertisement or website for products, marketing claims, and features and recorded the presence or absence of 27 marketing claims and lifestyle elements. RESULTS: All 6 brands of nicotine pouch products spent a total of US $11.2 million on advertising in 2019, with the most (US $10.7 million) spent by the brand Velo, and 86.1% (n=105) of the unique advertisements were web-based. Of the 711 total nicotine pouch advertisements run in 2019, the 2 brands Velo (n=407, 57%) and ZYN (n=303, 42%) dominated. These brands also made the greatest number of advertising claims in general. These claims focused on novelty, modernity, and use in a variety of contexts, including urban contexts, workplaces, transportation, and leisure activities. Of the 122 unique advertisements, ZYN's most common claims were to be "tobacco-free," featuring many flavors or varieties, and modern. Velo was the only brand to include urban contexts (n=14, 38.9% of advertisements) or freedom (n=8, 22.2%); Velo advertisements portrayed use in the workplace (n=15, 41.7%), bars or clubs (n=5, 13.9%), leisure activities (n=4, 11.1%), transportation (n=4, 11.1%), sports (n=3, 8.3%), cooking (n=2, 5.6%), and with alcohol (n=1, 2.8%). Velo and ZYN also included most of the images of people, including women and people of color. The 36 Velo ads included people in advertising in 77.8% (n=28) of advertisements, and of those advertisements with identifiable people, 40% (n=4) were young adults and 50% (n=5) were middle-aged. About one-third (n=11, 35.5%) of the 31 unique ZYN advertisements included people, and most identifiable models appeared to be young adults. Brands such as Rogue, Revel, Dryft, and on! focused mainly on product features. All nicotine pouch products made either tobacco-free, smoke-free, spit-free, or vape-free claims. The most common claim overall was "tobacco-free," found in advertisements from Rogue (1/1, 100%), ZYN (30/31, 96.8%), Velo (19/36, 52.8%), and Dryft (1/3, 33.3%), but not Revel. CONCLUSIONS: Nicotine pouches and lozenges may expand the nicotine market as tobacco-free claims alleviate concerns about health harms and advertising features a greater diversity of people and contexts than typical smokeless tobacco advertising. The market leaders and highest-spending brands, ZYN and Velo, included more lifestyle claims. Surveillance of nicotine pouch marketing and uptake, including influence on tobacco use behaviors, is necessary.

The matricellular protein Drosophila Cellular Communication Network Factor is required for synaptic transmission and female fertility.

Within the extracellular matrix, matricellular proteins are dynamically expressed nonstructural proteins that interact with cell surface receptors, growth factors, and proteases, as well as with structural matrix proteins. The cellular communication network factors family of matricellular proteins serve regulatory roles to regulate cell function and are defined by their conserved multimodular organization. Here, we characterize the expression and neuronal requirement for the Drosophila cellular communication network factor family member. Drosophila cellular communication network factor is expressed in the nervous system throughout development including in subsets of monoamine-expressing neurons. Drosophila cellular communication network factor-expressing abdominal ganglion neurons innervate the ovaries and uterus and the loss of Drosophila cellular communication network factor results in reduced female fertility. In addition, Drosophila cellular communication network factor accumulates at the synaptic cleft and is required for neurotransmission at the larval neuromuscular junction. Analyzing the function of the single Drosophila cellular communication network factor family member will enhance our potential to understand how the microenvironment impacts neurotransmitter release in distinct cellular contexts and in response to activity.