Optimizing Morbidity in Unplanned Soft Tissue Sarcoma Excisions: Should We Skip the Reconstructive Ladder?

INTRODUCTION: Soft tissue sarcomas (STSs) are rare and diverse primary malignant tumors that comprise approximately 1% of all malignancies. Misdiagnoses and unplanned excisions of STSs are common due to the tumor's rarity, leading to secondary tumor bed excisions (TBEs). Reconstructive outcomes for TBEs remain poorly understood, prompting this study to address the knowledge gap and inform preoperative discussions. METHODS: This was a retrospective cohort study of patients who underwent STS excisions at a quaternary cancer center. Patients were categorized into mass excision (ME) and TBE groups. Reconstructive approaches were divided into simple (primary closure, complex repair, skin grafts, local flaps) and advanced (pedicled or free flaps). The groups were compared for postoperative outcomes, including complications, recurrence, and death. RESULTS: When simple reconstructive techniques were used, TBEs exhibited higher rates of overall and major complications, whereas MEs had higher rates of overall and minor complications. Intergroup analysis revealed that with simple reconstruction, rates of overall and major complications were higher in TBEs than in MEs, and rates of minor complications were higher in MEs than in TBEs. Regression analyses revealed that simple reconstruction of TBEs had 90% and 180% higher odds of major complications and reoperation compared to simple reconstruction of MEs (P < 0.05). CONCLUSION: TBEs, despite their smaller size, exhibited a heightened susceptibility to overall and major complications, challenging the notion that simpler techniques suffice in these cases. Our findings encourage the consideration of advanced reconstructive techniques for TBEs that may seem amenable to simple reconstructive techniques.

Microglia regulate motor neuron plasticity via reciprocal fractalkine/adenosine signaling.

Microglia are innate CNS immune cells that play key roles in supporting key CNS functions including brain plasticity. We now report a previously unknown role for microglia in regulating neuroplasticity within spinal phrenic motor neurons, the neurons driving diaphragm contractions and breathing. We demonstrate that microglia regulate phrenic long-term facilitation (pLTF), a form of respiratory memory lasting hours after repetitive exposures to brief periods of low oxygen (acute intermittent hypoxia; AIH) via neuronal/microglial fractalkine signaling. AIH-induced pLTF is regulated by the balance between competing intracellular signaling cascades initiated by serotonin vs adenosine, respectively. Although brainstem raphe neurons release the relevant serotonin, the cellular source of adenosine is unknown. We tested a model in which hypoxia initiates fractalkine signaling between phrenic motor neurons and nearby microglia that triggers extracellular adenosine accumulation. With moderate AIH, phrenic motor neuron adenosine 2A receptor activation undermines serotonin-dominant pLTF; in contrast, severe AIH drives pLTF by a unique, adenosine-dominant mechanism. Phrenic motor neuron fractalkine knockdown, cervical spinal fractalkine receptor inhibition on nearby microglia, and microglial depletion enhance serotonin-dominant pLTF with moderate AIH but suppress adenosine-dominant pLTF with severe AIH. Thus, microglia play novel functions in the healthy spinal cord, regulating hypoxia-induced neuroplasticity within the motor neurons responsible for breathing.

Invasive Oral Squamous Cell Carcinoma Mimicking a Dentoalveolar Abscess: Report of a Case in a 14-Year-Old Patient.

INTRODUCTION: A diagnosis of oral squamous cell carcinoma in adolescent patients is extremely rare. When an oral squamous cell carcinoma lesion arises near the teeth and/or periodontium, it can be easily misdiagnosed as an inflammatory condition of endodontic or periodontal origin. METHODS: This is a case report of an otherwise healthy 14-year-old patient who was referred for endodontic evaluation and treatment of a soft-tissue swelling in the anterior maxilla. RESULTS: The unexpected definitive diagnosis of invasive oral squamous cell carcinoma underscores the importance of proper diagnostic testing. CONCLUSIONS: Accurate interpretation of pulp testing results, periapical and cone beam computed tomography imaging, timely biopsy, and prompt definitive treatment are critical when a lesion of nonodontogenic origin is suspected.

Prolonged intermittent hypoxia differentially regulates phrenic motor neuron serotonin receptor expression in rats following chronic cervical spinal cord injury.

Low-dose (< 2 h/day), acute intermittent hypoxia (AIH) elicits multiple forms of serotonin-dependent phrenic motor plasticity and is emerging as a promising therapeutic strategy to restore respiratory and non-respiratory motor function after spinal cord injury (SCI). In contrast, high-dose (> 8 h/day), chronic intermittent hypoxia (CIH) undermines some forms of serotonin-dependent phrenic motor plasticity and elicits pathology. CIH is a hallmark of sleep disordered breathing, which is highly prevalent in individuals with cervical SCI. Interestingly, AIH and CIH preconditioning differentially impact phrenic motor plasticity. Although mechanisms of AIH-induced plasticity in the phrenic motor system are well-described in naïve rats, we know little concerning how these mechanisms are affected by chronic SCI or intermittent hypoxia preconditioning. Thus, in a rat model of chronic, incomplete cervical SCI (lateral spinal hemisection at C2 (C2Hx), we assessed serotonin type 2A, 2B and 7 receptor expression in and near phrenic motor neurons and compared: 1) intact vs. chronically injured rats; and 2) the impact of preconditioning with varied "doses" of intermittent hypoxia (IH). While there were no effects of chronic injury or intermittent hypoxia alone, CIH affected multiple receptors in rats with chronic C2Hx. Specifically, CIH preconditioning (8 h/day; 28 days) increased serotonin 2A and 7 receptor expression exclusively in rats with chronic C2Hx. Understanding the complex, context-specific interactions between chronic SCI and CIH and how this ultimately impacts phrenic motor plasticity is important as we leverage AIH-induced motor plasticity to restore breathing and other non-respiratory motor functions in people with chronic SCI.

Fibro-adipogenic progenitors in physiological adipogenesis and intermuscular adipose tissue remodeling.

Excessive accumulation of intermuscular adipose tissue (IMAT) is a common pathological feature in various metabolic and health conditions and can cause muscle atrophy, reduced function, inflammation, insulin resistance, cardiovascular issues, and unhealthy aging. Although IMAT results from fat accumulation in muscle, the mechanisms underlying its onset, development, cellular components, and functions remain unclear. IMAT levels are influenced by several factors, such as changes in the tissue environment, muscle type and origin, extent and duration of trauma, and persistent activation of fibro-adipogenic progenitors (FAPs). FAPs are a diverse and transcriptionally heterogeneous population of stromal cells essential for tissue maintenance, neuromuscular stability, and tissue regeneration. However, in cases of chronic inflammation and pathological conditions, FAPs expand and differentiate into adipocytes, resulting in the development of abnormal and ectopic IMAT. This review discusses the role of FAPs in adipogenesis and how they remodel IMAT. It highlights evidence supporting FAPs and FAP-derived adipocytes as constituents of IMAT, emphasizing their significance in adipose tissue maintenance and development, as well as their involvement in metabolic disorders, chronic pathologies and diseases. We also investigated the intricate molecular pathways and cell interactions governing FAP behavior, adipogenesis, and IMAT accumulation in chronic diseases and muscle deconditioning. Finally, we hypothesize that impaired cellular metabolic flexibility in dysfunctional muscles impacts FAPs, leading to IMAT. A deeper understanding of the biology of IMAT accumulation and the mechanisms regulating FAP behavior and fate are essential for the development of new therapeutic strategies for several debilitating conditions.

Inactivity-induced phrenic motor facilitation requires PKCζ activity within phrenic motor neurons.

Prolonged inhibition of respiratory neural activity elicits a long-lasting increase in phrenic nerve amplitude once respiratory neural activity is restored. Such long-lasting facilitation represents a form of respiratory motor plasticity known as inactivity-induced phrenic motor facilitation (iPMF). Although facilitation also occurs in inspiratory intercostal nerve activity after diminished respiratory neural activity (iIMF), it is of shorter duration. Atypical PKC activity in the cervical spinal cord is necessary for iPMF and iIMF, but the site and specific isoform of the relevant atypical PKC are unknown. Here, we used RNA interference to test the hypothesis that the zeta atypical PKC isoform (PKCζ) within phrenic motor neurons is necessary for iPMF but PKCζ within intercostal motor neurons is unnecessary for transient iIMF. Intrapleural injections of siRNAs targeting PKCζ (siPKCζ) to knock down PKCζ mRNA within phrenic and intercostal motor neurons were made in rats. Control rats received a nontargeting siRNA (NTsi) or an active siRNA pool targeting a novel PKC isoform, PKCθ (siPKCθ), which is required for other forms of respiratory motor plasticity. Phrenic nerve burst amplitude and external intercostal (T2) electromyographic (EMG) activity were measured in anesthetized and mechanically ventilated rats exposed to 30 min of respiratory neural inactivity (i.e., neural apnea) created by modest hypocapnia (20 min) or a similar recording duration without neural apnea (time control). Phrenic burst amplitude was increased in rats treated with NTsi (68 ± 10% baseline) and siPKCθ (57 ± 8% baseline) 60 min after neural apnea vs. time control rats (-3 ± 3% baseline), demonstrating iPMF. In contrast, intrapleural siPKCζ virtually abolished iPMF (5 ± 4% baseline). iIMF was transient in all groups exposed to neural apnea; however, intrapleural siPKCζ attenuated iIMF 5 min after neural apnea (50 ± 21% baseline) vs. NTsi (97 ± 22% baseline) and siPKCθ (103 ± 20% baseline). Neural inactivity elevated the phrenic, but not intercostal, responses to hypercapnia, an effect that was blocked by siPKCζ. We conclude that PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient iIMF.NEW & NOTEWORTHY We report important new findings concerning the mechanisms regulating a form of spinal neuroplasticity elicited by prolonged inhibition of respiratory neural activity, inactivity-induced phrenic motor facilitation (iPMF). We demonstrate that the atypical PKC isoform PKCζ within phrenic motor neurons is necessary for long-lasting iPMF, whereas intercostal motor neuron PKCζ contributes to, but is not necessary for, transient inspiratory intercostal facilitation. Our findings are novel and advance our understanding of mechanisms contributing to phrenic motor plasticity.

Labeling of methyl groups: a streamlined protocol and guidance for the selection of 2H precursors based on molecular weight.

A streamlined one-day protocol is described to produce isotopically methyl-labeled protein with high levels of deuterium for NMR studies. Using this protocol, the D2O and 2H-glucose content of the media and protonation level of ILV labeling precursors (ketobutyrate and ketovalerate) were varied. The relaxation rate of the multiple-quantum (MQ) state that is present during the HMQC-TROSY pulse sequence was measured for different labeling schemes and this rate was used to predict upper limits of molecular weights for various labeling schemes. The use of deuterated solvents (D2O) or deuterated glucose is not required to obtain 1H-13C correlated NMR spectra of a 50 kDa homodimeric protein that are suitable for assignment by mutagenesis. High quality spectra of 100-150 kDa proteins, suitable for most applications, can be obtained without the use of deuterated glucose. The proton on the ß-position of ketovalerate appears to undergo partial exchange with deuterium under the growth conditions used in this study.

Self-brushing for nanopatterning: achieving perpendicular domain orientation in block copolymer thin films.

The self-assembly of thin films of block copolymers (BCPs) with perpendicular domain orientation offers a promising approach for nanopatterning on a variety of substrates, which is required by advanced applications such as ultrasmall transistors in integrated circuits, nanopatterned materials for tissue engineering, and electrocatalysts for fuel cell applications. In this study, we created BCPs with an A-b-(B-r-C) architecture that have blocks with equal surface energy (γair) and that can bind to the substrate, effectively creating a non-preferential substrate coating via self-brushing that enables the formation of through-film perpendicular domains in thin films of BCPs. We employed a thiol-epoxy click reaction to functionalize polystyrene-block-poly(glycidyl methacrylate) with a pair of thiols to generate an A-b-(B-r-C) BCP and tune γair of the B-r-C block. The secondary hydroxyl and thiol ether functionality generated by the click reaction was utilized to bind the BCP to the substrates. Scanning electron microscopy revealed that perpendicular orientation was achieved by simply annealing a thin film of the BCP on the bare substrate without the usual extra step of coating a random copolymer brush on the substrate. The self-brushing capability of the BCP was also examined on gold, platinum, titanium, aluminum nitride, and silicon nitride surfaces. These results demonstrate that self-brushing is a promising approach for achieving perpendicular domain orientation in thin films of BCP for nanopatterning on a variety of useful surfaces.

Poor association between 13-valent pneumococcal conjugate vaccine-induced serum and mucosal antibody responses with experimental Streptococcus pneumoniae serotype 6B colonisation.

BACKGROUND: Pneumococcal carriage is the primary reservoir for transmissionand a prerequisite for invasive pneumococcal disease. Pneumococcal Conjugate Vaccine 13 (PCV13) showed a 62% efficacy in protection against experimental Streptococcus pneumoniae serotype 6B (Spn6B) carriage in a controlled human infection model (CHIM) of healthy Malawian adults. We, therefore, measured humoral responses to experimental challenge and PCV-13 vaccination and determined the association with protection against pneumococcal carriage. METHODS: We vaccinated 204 young, healthy Malawian adults with PCV13 or placebo and nasally inoculated them with Spn6B at least four weeks post-vaccination to establish carriage. We collected peripheral blood and nasal lining fluid at baseline, 4 weeks post-vaccination (7 days pre-inoculation), 2, 7, 14 and > 1 year post-inoculation. We measured the concentration of anti-serotype 6B Capsular Polysaccharide (CPS) Immunoglobulin G (IgG) and IgA antibodies in serum and nasal lining fluid using the World Health Organization (WHO) standardised enzyme-linked immunosorbent assay (ELISA). RESULTS: PCV13-vaccinated adults had higher serum IgG and nasal IgG/IgA anti-Spn6B CPS-specific binding antibodies than placebo recipients 4 to 6 weeks post-vaccination, which persisted for at least a year after vaccination. Nasal challenge with Spn6B did not significantly alter serum or nasal anti-CPS IgG binding antibody titers with or without experimental pneumococcal carriage. Pre-challenge titers of PCV13-induced serum IgG and nasal IgG/IgA anti-Spn6B CPS binding antibodies did not significantly differ between those that got experimentally colonised by Spn6B compared to those that did not. CONCLUSION: This study demonstrates that despite high PCV13 efficacy against experimental Spn6B carriage in young, healthy Malawian adults, robust vaccine-induced systemic and mucosal anti-Spn6B CPS binding antibodies did not directly relate to protection.

The BALB/c.mdx62 mouse exhibits a dystrophic muscle pathology and is a model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a devastating monogenic skeletal muscle-wasting disorder. Although many pharmacological and genetic interventions have been reported in preclinical studies, few have progressed to clinical trials with meaningful benefit. Identifying therapeutic potential can be limited by availability of suitable preclinical mouse models. More rigorous testing across models with varied background strains and mutations can identify treatments for clinical success. Here, we report the generation of a DMD mouse model with a CRISPR-induced deletion within exon 62 of the dystrophin gene (Dmd) and the first generated in BALB/c mice. Analysis of mice at 3, 6 and 12 months of age confirmed loss of expression of the dystrophin protein isoform Dp427 and resultant dystrophic pathology in limb muscles and the diaphragm, with evidence of centrally nucleated fibers, increased inflammatory markers and fibrosis, progressive decline in muscle function, and compromised trabecular bone development. The BALB/c.mdx62 mouse is a novel model of DMD with associated variations in the immune response and muscle phenotype, compared with those of existing models. It represents an important addition to the preclinical model toolbox for developing therapeutic strategies.

Mitochondrial fusion and altered beta-oxidation drive muscle wasting in a Drosophila cachexia model.

Cancer cachexia is a tumour-induced wasting syndrome, characterised by extreme loss of skeletal muscle. Defective mitochondria can contribute to muscle wasting; however, the underlying mechanisms remain unclear. Using a Drosophila larval model of cancer cachexia, we observed enlarged and dysfunctional muscle mitochondria. Morphological changes were accompanied by upregulation of beta-oxidation proteins and depletion of muscle glycogen and lipid stores. Muscle lipid stores were also decreased in Colon-26 adenocarcinoma mouse muscle samples, and expression of the beta-oxidation gene CPT1A was negatively associated with muscle quality in cachectic patients. Mechanistically, mitochondrial defects result from reduced muscle insulin signalling, downstream of tumour-secreted insulin growth factor binding protein (IGFBP) homologue ImpL2. Strikingly, muscle-specific inhibition of Forkhead box O (FOXO), mitochondrial fusion, or beta-oxidation in tumour-bearing animals preserved muscle integrity. Finally, dietary supplementation with nicotinamide or lipids, improved muscle health in tumour-bearing animals. Overall, our work demonstrates that muscle FOXO, mitochondria dynamics/beta-oxidation and lipid utilisation are key regulators of muscle wasting in cancer cachexia.

Atrogin-1 promotes muscle homeostasis by regulating levels of endoplasmic reticulum chaperone BiP.

Skeletal muscle wasting results from numerous pathological conditions affecting both the musculoskeletal and nervous systems. A unifying feature of these pathologies is the upregulation of members of the E3 ubiquitin ligase family, resulting in increased proteolytic degradation of target proteins. Despite the critical role of E3 ubiquitin ligases in regulating muscle mass, the specific proteins they target for degradation and the mechanisms by which they regulate skeletal muscle homeostasis remain ill-defined. Here, using zebrafish loss-of-function models combined with in vivo cell biology and proteomic approaches, we reveal a role of atrogin-1 in regulating the levels of the endoplasmic reticulum chaperone BiP. Loss of atrogin-1 resulted in an accumulation of BiP, leading to impaired mitochondrial dynamics and a subsequent loss in muscle fiber integrity. We further implicated a disruption in atrogin-1-mediated BiP regulation in the pathogenesis of Duchenne muscular dystrophy. We revealed that BiP was not only upregulated in Duchenne muscular dystrophy, but its inhibition using pharmacological strategies, or by upregulating atrogin-1, significantly ameliorated pathology in a zebrafish model of Duchenne muscular dystrophy. Collectively, our data implicate atrogin-1 and BiP in the pathogenesis of Duchenne muscular dystrophy and highlight atrogin-1's essential role in maintaining muscle homeostasis.

Plasma beta-hydroxy-beta-methylbutyrate availability after enteral administration during critical illness after trauma: An exploratory study.

BACKGROUND: During critical illness skeletal muscle wasting occurs rapidly. Although beta-hydroxy-beta-methylbutyrate (HMB) is a potential treatment to attenuate this process, the plasma appearance and muscle concentration is uncertain. METHODS: This was an exploratory study nested within a blinded, parallel group, randomized clinical trial in which critically ill patients after trauma received enteral HMB (3 g daily) or placebo. Plasma samples were collected at 0, 60, and 180 min after study supplement administration on day 1. Needle biopsies of the vastus lateralis muscle were collected (baseline and day 7 of the HMB treatment intervention period). An external standard curve was used to calculate HMB concentrations in plasma and muscle. RESULTS: Data were available for 16 participants (male n = 12 (75%), median [interquartile range] age 50 [29-58] years) who received placebo and 18 participants (male n = 14 (78%), age 49 [34-55] years) who received HMB. Plasma HMB concentrations were similar at baseline but increased after HMB (T = 60 min: placebo 0.60 [0.44-1.31] µM; intervention 51.65 [22.76-64.72] µM). Paired muscle biopsies were collected from 11 participants (placebo n = 7, HMB n = 4). Muscle HMB concentrations were similar at baseline between groups (2.35 [2.17-2.95]; 2.07 [1.78-2.31] µM). For participants in the intervention group who had the repeat biopsy within 4 h of HMB administration, concentrations were greater (7.2 and 12.3 µM) than those who had the repeat biopsy >4 h after HMB (2.7 and 2.1 µM). CONCLUSION: In this exploratory study, enteral HMB administration increased plasma HMB availability. The small sample size limits interpretation of the muscle HMB findings.

Trigonelline is an NAD+ precursor that improves muscle function during ageing and is reduced in human sarcopenia.

Mitochondrial dysfunction and low nicotinamide adenine dinucleotide (NAD+) levels are hallmarks of skeletal muscle ageing and sarcopenia1-3, but it is unclear whether these defects result from local changes or can be mediated by systemic or dietary cues. Here we report a functional link between circulating levels of the natural alkaloid trigonelline, which is structurally related to nicotinic acid4, NAD+ levels and muscle health in multiple species. In humans, serum trigonelline levels are reduced with sarcopenia and correlate positively with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle. Using naturally occurring and isotopically labelled trigonelline, we demonstrate that trigonelline incorporates into the NAD+ pool and increases NAD+ levels in Caenorhabditis elegans, mice and primary myotubes from healthy individuals and individuals with sarcopenia. Mechanistically, trigonelline does not activate GPR109A but is metabolized via the nicotinate phosphoribosyltransferase/Preiss-Handler pathway5,6 across models. In C. elegans, trigonelline improves mitochondrial respiration and biogenesis, reduces age-related muscle wasting and increases lifespan and mobility through an NAD+-dependent mechanism requiring sirtuin. Dietary trigonelline supplementation in male mice enhances muscle strength and prevents fatigue during ageing. Collectively, we identify nutritional supplementation of trigonelline as an NAD+-boosting strategy with therapeutic potential for age-associated muscle decline.

Inaugural Review Prize 2023: The exercise hyperpnoea dilemma: A 21st-century perspective.

During mild or moderate exercise, alveolar ventilation increases in direct proportion to metabolic rate, regulating arterial CO2 pressure near resting levels. Mechanisms giving rise to the hyperpnoea of exercise are unsettled despite over a century of investigation. In the past three decades, neuroscience has advanced tremendously, raising optimism that the 'exercise hyperpnoea dilemma' can finally be solved. In this review, new perspectives are offered in the hope of stimulating original ideas based on modern neuroscience methods and current understanding. We first describe the ventilatory control system and the challenge exercise places upon blood-gas regulation. We highlight relevant system properties, including feedforward, feedback and adaptive (i.e., plasticity) control of breathing. We then elaborate a seldom explored hypothesis that the exercise ventilatory response continuously adapts (learns and relearns) throughout life and ponder if the memory 'engram' encoding the feedforward exercise ventilatory stimulus could reside within the cerebellum. Our hypotheses are based on accumulating evidence supporting the cerebellum's role in motor learning and the numerous direct and indirect projections from deep cerebellar nuclei to brainstem respiratory neurons. We propose that cerebellar learning may be obligatory for the accurate and adjustable exercise hyperpnoea capable of tracking changes in life conditions/experiences, and that learning arises from specific cerebellar microcircuits that can be interrogated using powerful techniques such as optogenetics and chemogenetics. Although this review is speculative, we consider it essential to reframe our perspective if we are to solve the till-now intractable exercise hyperpnoea dilemma.

Enhanced SARS-CoV-2 case prediction using public health data and machine learning models.

Objectives: The goal of this study is to propose and test a scalable framework for machine learning (ML) algorithms to predict near-term severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases by incorporating and evaluating the impact of real-time dynamic public health data. Materials and Methods: Data used in this study include patient-level results, procurement, and location information of all SARS-CoV-2 tests reported in West Virginia as part of their mandatory reporting system from January 2021 to March 2022. We propose a method for incorporating and comparing widely available public health metrics inside of a ML framework, specifically a long-short-term memory network, to forecast SARS-CoV-2 cases across various feature sets. Results: Our approach provides better prediction of localized case counts and indicates the impact of the dynamic elements of the pandemic on predictions, such as the influence of the mixture of viral variants in the population and variable testing and vaccination rates during various eras of the pandemic. Discussion: Utilizing real-time public health metrics, including estimated Rt from multiple SARS-CoV-2 variants, vaccination rates, and testing information, provided a significant increase in the accuracy of the model during the Omicron and Delta period, thus providing more precise forecasting of daily case counts at the county level. This work provides insights on the influence of various features on predictive performance in rural and non-rural areas. Conclusion: Our proposed framework incorporates available public health metrics with operational data on the impact of testing, vaccination, and current viral variant mixtures in the population to provide a foundation for combining dynamic public health metrics and ML models to deliver forecasting and insights in healthcare domains. It also shows the importance of developing and deploying ML frameworks in rural settings.