Highly unstable heterogeneous representations in VIP interneurons of the anterior cingulate cortex.

A hallmark of the anterior cingulate cortex (ACC) is its functional heterogeneity. Functional and imaging studies revealed its importance in the encoding of anxiety-related and social stimuli, but it is unknown how microcircuits within the ACC encode these distinct stimuli. One type of inhibitory interneuron, which is positive for vasoactive intestinal peptide (VIP), is known to modulate the activity of pyramidal cells in local microcircuits, but it is unknown whether VIP cells in the ACC (VIPACC) are engaged by particular contexts or stimuli. Additionally, recent studies demonstrated that neuronal representations in other cortical areas can change over time at the level of the individual neuron. However, it is not known whether stimulus representations in the ACC remain stable over time. Using in vivo Ca2+ imaging and miniscopes in freely behaving mice to monitor neuronal activity with cellular resolution, we identified individual VIPACC that preferentially activated to distinct stimuli across diverse tasks. Importantly, although the population-level activity of the VIPACC remained stable across trials, the stimulus-selectivity of individual interneurons changed rapidly. These findings demonstrate marked functional heterogeneity and instability within interneuron populations in the ACC. This work contributes to our understanding of how the cortex encodes information across diverse contexts and provides insight into the complexity of neural processes involved in anxiety and social behavior.

A Window Into the Tired Brain: Neurophysiological Dynamics of Visuospatial Working Memory Under Fatigue.

OBJECTIVE: We examine the spatiotemporal dynamics of neural activity and its correlates in heart rate and its variability (HR/HRV) during a fatiguing visuospatial working memory task. BACKGROUND: The neural and physiological drivers of fatigue are complex, coupled, and poorly understood. Investigations that combine the fidelity of neural indices and the field-readiness of physiological measures can facilitate measurements of fatigue states in operational settings. METHOD: Sixteen healthy adults, balanced by sex, completed a 60-minute fatiguing visuospatial working memory task. Changes in task performance, subjective measures of effort and fatigue, cerebral hemodynamics, and HR/HRV were analyzed. Peak brain activation, functional and effective connections within relevant brain networks were contrasted against spectral and temporal features of HR/HRV. RESULTS: Task performance elicited increased neural activation in regions responsible for maintaining working memory capacity. With the onset of time-on-task effects, resource utilization was seen to increase beyond task-relevant networks. Over time, functional connections in the prefrontal cortex were seen to weaken, with changes in the causal relationships between key regions known to drive working memory. HR/HRV indices were seen to closely follow activity in the prefrontal cortex. CONCLUSION: This investigation provided a window into the neurophysiological underpinnings of working memory under the time-on-task effect. HR/HRV was largely shown to mirror changes in cortical networks responsible for working memory, therefore supporting the possibility of unobtrusive state recognition under ecologically valid conditions. APPLICATIONS: Findings here can inform the development of a fieldable index for cognitive fatigue.

Tracking POPs in Global Air from the First 10 Years of the GAPS Network (2005 to 2014).

The Global Atmospheric Passive Sampling (GAPS) network, initiated in 2005 across 55 global sites, supports the global monitoring plan (GMP) of the Stockholm Convention on Persistent Organic Pollutants (POPs) by providing information on POP concentrations in air on a global scale. These data inform assessments of the long-range transport potential of POPs and the effectiveness evaluation of chemical regulation efforts, by observing changes in concentrations over time. Currently, measurements spanning 5-10 sampling years are available for 40 sites from the GAPS Network. This study was the first time that POP concentrations in air were reported on a global scale for an extended time period and the first to evaluate worldwide trends with an internally consistent sample set. For consistency between sampling years, site- and sample specific sampling rates were calculated with a new, public online model, which accounts for the effects of wind speed variability. Concentrations for legacy POPs in air between 2005 and 2014 show different trends for different organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs). The POPs discussed in this study were chosen due to being the most frequently detected, with detection at the majority of sites. PCB, endosulfan, and hexachlorocyclohexane (HCH) concentrations in air are decreasing at most sites. The global trends reflect global sources and recycling of HCH, ongoing emissions from old stockpiles for PCBs, and recent use restrictions for endosulfan. These chlorinated OCPs continue to present exposure threat to humans and ecosystems worldwide. Concentrations of other OCPs, such as chlordanes, heptachlor and dieldrin, are steady and/or declining slowly at the majority of sites, reflecting a transition from primary to secondary sources (i.e., re-emission from reservoirs where these POPs have accumulated historically) which now control ambient air burdens.

Characterization of Phenolic Plant Exudates by Nuclear Magnetic Resonance Spectroscopy.

The class of plant exudates that contain the phenol functionality, termed phenolics, is defined, surveyed, and characterized by solid-state 13C NMR spectroscopy and by solution-state 1H NMR spectroscopy. Materials in this group are identified by the phenolic 13C resonance (from the ipso carbon of ArOH) at δ 145-160 (δ 160-167 for ArOR). The resonance patterns define several subclasses based on the collective similarity of their 13C spectra, specifically, aloetics from the genus Aloe, guaiacs from the genus Guaiacum and other eurosid and conifer genera, xanthics from the genus Garcinia, and kinos from the genus Eucalyptus and many other genera. Phenolic exudates often are mixed with terpenoid materials (the building block of exudates known as resins) and carbohydrates (the building block of exudates known as gums) to form hybrid subgroups such as guaiac gums, guaiac resins, and kino resins. There are numerous phenolic exudates not affiliated with any of these groups, both as pure phenolics and as hybrids (phenolic resins, phenolic gum resins, and phenolic waxes).

Hospital Volumes of Inpatient Pediatric Surgery in the United States.

BACKGROUND: Perioperative outcomes of children depend on the skill and expertise in managing pediatric patients, as well as integration of surgical, anesthesiology, and medical teams. We compared the types of pediatric patients and inpatient surgical procedures performed in low- versus higher-volume hospitals throughout the United States. METHODS: Retrospective analysis of 323,258 hospitalizations with an operation for children age 0 to 17 years in 2857 hospitals included in the Agency for Healthcare Research and Quality (AHRQ) Kids' Inpatient Database (KID) 2016. Hospitals were categorized by their volume of annual inpatient surgical procedures. Specific surgeries were distinguished with the AHRQ Clinical Classification System. We assessed complex chronic conditions (CCCs) using Feudtner and Colleagues' system. RESULTS: The median annual volume of pediatric inpatient surgeries across US hospitals was 8 (interquartile range [IQR], 3-29). The median volume of inpatient surgeries for children with a CCC was 4 (IQR, 1-13). Low-volume hospitals performed significantly fewer types of surgeries (median 2 vs 131 types of surgeries in hospitals with 1-24 vs ≥2000 volumes). Appendectomy and fixation of bone fracture were among the most common surgeries in low-volume hospitals. As the volume of surgical procedures increased from 1 to 24 to ≥2000, the percentage of older children ages 11 to 17 years decreased (70.9%-32.0% [P < .001]) and the percentage of children with a CCC increased (11.2%-60.0% [P < .001]). CONCLUSIONS: Thousands of US hospitals performed inpatient surgeries on few pediatric patients, including those with CCCs who have the highest risk of perioperative morbidity and mortality. Evaluation of perioperative decision making, workflows, and pediatric clinicians in low- and higher-volume hospitals is warranted.

One Size Does Not Fit All: A Perspective on the American Society of Anesthesiologists Physical Status Classification for Pediatric Patients.

BACKGROUND: The American Society of Anesthesiologists physical status (ASA-PS) classification system is used worldwide to classify patients based on comorbid conditions before general anesthesia. Despite its popularity, the ASA-PS classification system has been shown to have poor interrater reliability due to its subjective definitions, especially when applied to the pediatric population. We hypothesized that the clarification of ASA-PS definitions to better reflect pediatric conditions would improve the accuracy of ASA-PS applied to this population. METHODS: A stratified, randomized sample of 120 pediatric surgical cases was collected from a tertiary-care pediatric hospital. A team of senior anesthesiologists reclassified ASA-PS within this patient sample using the suggested pediatric-specific ASA-PS definitions. Interrater reliability was measured using intraclass correlation (ICC) and Fleiss κ statistic. In addition, a qualitative study component using small focus groups of senior anesthesiologists identified areas of ambiguity within the ASA-PS system. RESULTS: Among the 90 reclassifications within each ASA-PS group, 42.2% (n = 38) of ASA-PS I were upgraded to ASA-PS II, and 36.7% (n = 33) of ASA-PS II were upgraded to ASA-PS III. In addition, 28.9% (n = 26) of ASA-PS III were upgraded to ASA-PS IV, and 24.4% (n = 22) of ASA-PS IV were downgraded to III. ICC across the reclassified ASA-PS categories was 0.77 (95% confidence interval [CI], 0.71-0.83; P < .001) demonstrating strong overall agreement. Fleiss κ statistic was lowest in ASA-PS II and III patients (κ = 0.41 and κ = 0.30, respectively) indicating lower agreement beyond chance within these subgroups. Focus groups revealed common themes such as active sequelae of disease, active versus well-controlled presence of comorbidities, and the possible inclusion of functional limitations as important considerations. CONCLUSIONS: The ASA-PS classification system has several benefits including ease-of-use, simplicity, and flexibility. However, revising the ASA-PS system to provide better guidance for pediatric patients could be valuable. While this study demonstrates good interrater reliability with the included ASA-PS pediatric definitions, further work is needed to clarify accurate assignment of ASA-PS within the midrange of the scale (ASA-PS II and III) and explore its implementation in other institutions.

Implementing a Pediatric Perioperative Surgical Home Integrated Care Coordination Pathway for Laryngeal Cleft Repair.

BACKGROUND: The Pediatric Perioperative Surgical Home (PPSH) model is an integrative care model designed to provide better patient care and value by shifting focus from the patient encounter level to the overarching surgical episode of care. So far, no PPSH model has targeted a complex airway disorder. It was hypothesized that the development of a PPSH for laryngeal cleft repair would reduce the high rates of postoperative resource utilization observed in this population. METHODS: Institutional review board approval was obtained for the purpose of data collection and analysis. A multidisciplinary team of anesthesiologists, surgeons, nursing staff, information technology specialists, and finance administrators was gathered during the PPSH development phase. Standardized perioperative (preoperative, intraoperative, and postoperative) protocols were developed, with a focus on preoperative risk stratification. Patients presenting before surgery with ≥1 predefined medical comorbidity were triaged to the intensive care unit (ICU) postoperatively, while patients without severe systemic disease were triaged to a lower-acuity floor for overnight observation. The success of the PPSH protocol was defined by quality outcome and value measurements. RESULTS: The PPSH initiative included 120 patients, and the pre-PPSH period included 115 patients who underwent laryngeal cleft repair before implementation of the new process. Patients in the pre-PPSH period were reviewed and classified as ICU candidates or lower acuity floor candidates had they presented in the post-PPSH period. Among the 79 patients in the pre-PPSH period who were identified as candidates for the lower-acuity floor transfer, 70 patients (89%) were transferred to the ICU (P < .001). Retrospective analysis concluded that 143 ICU bedded days could have been avoided in the pre-PPSH group by using PPSH risk stratification. Surgery duration (P = .034) and hospital length of stay (P = .015) were found to be slightly longer in the group of pre-PPSH observation unit candidates. Rates of 30-day unplanned readmissions to the hospital were not associated with the new PPSH initiative (P = .093). No patients in either group experienced emergent postoperative intubation or other expected complications. Total hospital costs were not lower for PPSH observation unit patients as compared to pre-PPSH observation unit candidates (difference = 8%; 95% confidence interval, -7% to 23%). CONCLUSIONS: A well-defined preoperative screening protocol for patients undergoing laryngeal cleft repair can reduce postoperative ICU utilization without affecting patient safety. Further research is needed to see if these findings are applicable to other complex airway surgeries.

Novel Peptidomic Approach for Identification of Low and High Molecular Weight Tauopathy Peptides Following Calpain Digestion, and Primary Culture Neurotoxic Challenges.

Tauopathy is a class of a neurodegenerative disorder linked with tau hyperphosphorylation, proteolysis, and aggregation. Tau can be subjected to proteolysis upon calpain activation in Alzheimer disease (AD), and traumatic brain injury (TBI). We and others have extensively researched calpain-mediated tau breakdown products (Tau-BDP; 45K, 35K, and 17K). Tau proteolysis might also generate low molecular weight (LMW ≤10K) proteolytic peptides after neurodegenerative damage. In this study, we have subjected purified tau protein (phospho and non-phospho) and mouse brain lysate to calpain-1 digestion to characterize the LMW generated by nano-liquid chromatography coupled to electrospray ionization to tandem mass spectrometry (nano-LC-ESI-MS/MS). We have also challenged differentiated primary cerebrocortical neuronal cultures (CTX) with neurotoxic agents (calcium ionophore calcimycin (A23187), staurosporine (STS), N-methyl-D-aspartate (NMDA), and Maitotoxin (MTX)) that mimic neurodegeneration to investigate the peptidome released into the conditioned cell media. We used a simple workflow in which we fractionate LMW calpain-mediated tau peptides by ultrafiltration (molecular weight cut-off value (MWCO) of 10K) and subject filtrate fractions to nano-LC-MS/MS analysis. The high molecular weight (HMW) peptides and intact proteins retained on the filter were analyzed separately by western blotting using total and phospho-specific tau antibodies. We have identified several novel proteolytic tau peptides (phosphorylated and non-phosphorylated) that are only present in samples treated with calpain or cell-based calpain activation model (particularly N- and C-terminal peptides). Our findings can help in developing future research strategies emphasizing on the suppression of tau proteolysis as a target.

Coronary artery calcification is often not reported in pulmonary CT angiography in patients with suspected pulmonary embolism: an opportunity to improve diagnosis of acute coronary syndrome.

OBJECTIVE: In patients with suspected pulmonary thromboembolism (PTE), coronary artery calcification (CAC) can be an incidental finding on pulmonary CT angiography. We evaluated the frequency of CAC not being reported and its association with a diagnosis of acute coronary syndrome (ACS). MATERIALS AND METHODS: Data of 469 consecutive patients who were referred to the emergency radiology department for pulmonary CT angiography of suspected PTE were reviewed. Radiology reports were rechecked and positive CAC findings were recorded. All pulmonary CT angiograms were reevaluated by one radiologist and CAC findings were recorded. The rates of ACS and PTE as final diagnoses for that hospital admission were calculated. The association between CAC and ACS diagnosis was assessed in different subgroups of patients. RESULTS: Approximately 11.1% of patients had PTE and 43.8% had CAC. The incidence of CAC was significantly higher in patients with an ACS diagnosis than in those without ACS (56.2% vs 40.4%, respectively; odds ratio [OR] = 1.9). There was a strong positive association (OR = 3.5) between CAC and ACS in younger patients (men ≤ 45 years and women ≤ 55 years), patients without PTE (OR = 2.2), and those without cardiometabolic risk factors (OR = 3.8). CAC was not reported in 45% of patients (n = 98) with positive CAC findings on imaging. ACS was the final diagnosis in 31.6% of patients with unreported CAC. There was a significant association between CAC and ACS in patients with unreported CAC (OR = 2.2). This association was more prominent in the subgroups described. CONCLUSION: CAC is often not reported in pulmonary CT angiography studies. CAC is a significant predictor of ACS particularly in younger patients, patients without PTE, and those without cardiometabolic risk factors. Especially in these subgroups, radiologists should assess CAC findings.

Hallux Valgus Bunion Splint: Surgical Dressing Technique.

BACKGROUND: Hallux valgus is a common condition with a complex etiology resulting in numerous treatment options. Recurrence of the deformity can occur following correction. Surgical technique and possibly also postoperative care play a role in reducing recurrence rates. This article highlights a postoperative surgical dressing technique which allows for semirigid support during the immediate postoperative period. METHODS: A wooden tongue depressor placed along the medial border of the hallux comprises the primary support for the dressing. The rigidity of the tongue depressor allows for the hallux to be drawn toward the depressor, encouraging neutral alignment of the hallux. Dressings are removed 2 weeks postoperatively, with new dressings applied in similar fashion and maintained in place until 6 weeks postoperatively. RESULTS: Based upon our observations, our surgical dressing technique provides sufficient support following hallux valgus correction surgery while being straightforward to replicate without the need for frequent dressing changes. The dressing materials are of negligible cost and are typically readily available. No associated wound complications have been observed. CONCLUSIONS: We present an easily replicable and affordable option for postoperative hallux valgus correction surgical dressings. LEVELS OF EVIDENCE: Level V: Expert Opinion.

Intraduodenal nutrient infusion differentially alters intestinal nutrient sensing, appetite, and satiety responses in lean and obese subjects.

BACKGROUND: Intestinal nutrient sensing regulates food intake and energy metabolism by acting locally and relaying nutritional status to the brain. It is unclear whether these mechanisms are altered in obese humans. OBJECTIVES: We aimed to investigate differences in duodenal nutrient sensing in humans with or without obesity and the effects of transiently blocking vagal transmission on nutrient sensing, hunger, and appetite. METHODS: In a single-blinded, randomized, cross-over design, subjects with or without obesity (n = 14 and n = 11, respectively) were infused intraduodenally with saline or a combination of glucose and oleic acid for 90 min (glucose load: 22.5 g, 1 kcal/min; oleic acid load: 10 g, 1 kcal/min) in the presence or absence of local anesthetic (benzocaine). Blood was sampled at 10-min intervals (120-240 min) and 15-min intervals until termination of the study for measurements of gut hormones, insulin, leptin, and C-peptide. Hunger and satiety sensations were scored using the visual analog scale, and hepatic glucose production and glucose oxidation rates were measured. RESULTS: Duodenal nutrient infusion in lean subjects led to a 65% drop in acyl ghrelin release and robustly increased cholecystokinin 8 (CCK-8) release (65%; P = 0.023); benzocaine infusion delayed this response (2-factor repeated-measures analysis of variance, P = 0.0065). In contrast, subjects with obesity had significantly blunted response to nutrient infusion, and no further effects were observed with benzocaine. Additionally, significant delays were observed in peptide YY (3-36), pancreatic polypeptide, glucose inhibitory peptide, and glucagon-like peptide 1 (7-36) response. No significant interactions were found between body mass index (BMI) or baseline hormone levels and areas under the curve for hormones except CCK-8 (BMI, P = 0.018; baseline CCK, P = 0.013). Nutrient-induced hunger and satiety sensations were impeded by benzocaine only in the lean cohort. Hunger and satiety sensations in subjects with obesity were not responsive to nutrient entry into the duodenum, and no additional effects were observed by blocking neural signaling. CONCLUSION: Nutrient-induced gut hormone release and response to transient vagal blockade are significantly blunted in subjects with obesity. This trial was registered at clinicaltrials.org as NCT02537314.

Neurobiochemical, Peptidomic, and Bioinformatic Approaches to Characterize Tauopathy Peptidome Biomarker Candidates in Experimental Mouse Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) is a multidimensional damage, and currently, no FDA-approved medicine is available. Multiple pathways in the cell are triggered through a head injury (e.g., calpain and caspase activation), which truncate tau and generate variable fragment sizes (MW 400-45,000 K). In this study, we used an open-head TBI mouse model generated by controlled cortical impact (CCI) and collected ipsilateral (IC) and contralateral (CC) mice htau brain cortices at one (D1) three (D3), and seven (D7) days post-injury. We implemented immunological (antibody-based detection) and peptidomic approaches (nano-reversed-phase liquid chromatography/tandem mass spectrometry) to investigate proteolytic tau peptidome (low molecular weight (LMW) < 10 K)) and pathological phosphorylation sites (high-molecular-weight (HMW); > 10 K) derived from CCI-TBI animal models. Our immunoblotting analysis verified tau hyperphosphorylation, HMW, and HMW breakdown products (HMW-BDP) formation of tau (e.g., pSer202, pThr181, pThr231, pSer396, and pSer404), following CCI-TBI. Peptidomic data revealed unique sequences of injury-dependent proteolytic peptides generated from human tau protein. Among the N-terminal tau peptides, EIPEGTTAEEAGIGDTPSLEDEAAGHVTQA (a.a. 96-125) and AQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARM (a.a. 91-127). Examples of tau C-terminal peptides identified include NVSSTGSIDMVDSPQLATLADEVSASLAKQGL (a.a. 410-441) and QLATLADEVSASLAKQGL (a.a. 424-441). Our peptidomic bioinformatic tools showed the association of proteases, such as CAPN1, CAPN2, and CTSL; CASP1, MMP7, and MMP9; and ELANE, GZMA, and MEP1A, in CCI-TBI tau peptidome. In clinical trials for novel TBI treatments, it might be useful to monitor a subset of tau peptidome as targets for biomarker utility and use them for a "theranostic" approach.

Hedgehog signaling via its ligand DHH acts as cell fate determinant during skeletal muscle regeneration.

Successful muscle regeneration relies on the interplay of multiple cell populations. However, the signals required for this coordinated intercellular crosstalk remain largely unknown. Here, we describe how the Hedgehog (Hh) signaling pathway controls the fate of fibro/adipogenic progenitors (FAPs), the cellular origin of intramuscular fat (IMAT) and fibrotic scar tissue. Using conditional mutagenesis and pharmacological Hh modulators in vivo and in vitro, we identify DHH as the key ligand that acts as a potent adipogenic brake by preventing the adipogenic differentiation of FAPs. Hh signaling also impacts muscle regeneration, albeit indirectly through induction of myogenic factors in FAPs. Our results also indicate that ectopic and sustained Hh activation forces FAPs to adopt a fibrogenic fate resulting in widespread fibrosis. In this work, we reveal crucial post-developmental functions of Hh signaling in balancing tissue regeneration and fatty fibrosis. Moreover, they provide the exciting possibility that mis-regulation of the Hh pathway with age and disease could be a major driver of pathological IMAT formation.

C4 induces pathological synaptic loss by impairing AMPAR trafficking.

During development, activation of the complement pathway, an extracellular proteolytic cascade, results in microglia-dependent synaptic elimination via complement receptor 3 (CR3). Here, we report that decreased connectivity caused by overexpression of C4 (C4-OE), a schizophrenia-associated gene, is CR3 independent. Instead, C4-OE triggers GluR1 degradation through an intracellular mechanism involving endosomal trafficking protein SNX27, resulting in pathological synaptic loss. Moreover, the connectivity deficits associated with C4-OE were rescued by increasing levels of SNX27, linking excessive complement activity to an intracellular endolysosomal recycling pathway affecting synapses.

ZNF683 marks a CD8+ T cell population associated with anti-tumor immunity following anti-PD-1 therapy for Richter syndrome.

Unlike many other hematologic malignancies, Richter syndrome (RS), an aggressive B cell lymphoma originating from indolent chronic lymphocytic leukemia, is responsive to PD-1 blockade. To discover the determinants of response, we analyze single-cell transcriptome data generated from 17 bone marrow samples longitudinally collected from 6 patients with RS. Response is associated with intermediate exhausted CD8 effector/effector memory T cells marked by high expression of the transcription factor ZNF683, determined to be evolving from stem-like memory cells and divergent from terminally exhausted cells. This signature overlaps with that of tumor-infiltrating populations from anti-PD-1 responsive solid tumors. ZNF683 is found to directly target key T cell genes (TCF7, LMO2, CD69) and impact pathways of T cell cytotoxicity and activation. Analysis of pre-treatment peripheral blood from 10 independent patients with RS treated with anti-PD-1, as well as patients with solid tumors treated with anti-PD-1, supports an association of ZNF683high T cells with response.

Evolutionary history of transformation from chronic lymphocytic leukemia to Richter syndrome.

Richter syndrome (RS) arising from chronic lymphocytic leukemia (CLL) exemplifies an aggressive malignancy that develops from an indolent neoplasm. To decipher the genetics underlying this transformation, we computationally deconvoluted admixtures of CLL and RS cells from 52 patients with RS, evaluating paired CLL-RS whole-exome sequencing data. We discovered RS-specific somatic driver mutations (including IRF2BP2, SRSF1, B2M, DNMT3A and CCND3), recurrent copy-number alterations beyond del(9p21)(CDKN2A/B), whole-genome duplication and chromothripsis, which were confirmed in 45 independent RS cases and in an external set of RS whole genomes. Through unsupervised clustering, clonally related RS was largely distinct from diffuse large B cell lymphoma. We distinguished pathways that were dysregulated in RS versus CLL, and detected clonal evolution of transformation at single-cell resolution, identifying intermediate cell states. Our study defines distinct molecular subtypes of RS and highlights cell-free DNA analysis as a potential tool for early diagnosis and monitoring.

A Guide to Examining Intramuscular Fat Formation and its Cellular Origin in Skeletal Muscle.

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build and maintain the extracellular matrix that acts as a molecular myofiber scaffold. In addition, FAPs are indispensable for myofiber regeneration as they secrete a multitude of beneficial factors sensed by the muscle stem cells (MuSCs). In diseased states, however, FAPs are the cellular origin of intramuscular fat and fibrotic scar tissue. This fatty fibrosis is a hallmark of sarcopenia and neuromuscular diseases, such as Duchenne Muscular Dystrophy. One significant barrier in determining why and how FAPs differentiate into intramuscular fat is effective preservation and subsequent visualization of adipocytes, especially in frozen tissue sections. Conventional methods of skeletal muscle tissue processing, such as snap-freezing, do not properly preserve the morphology of individual adipocytes, thereby preventing accurate visualization and quantification. To overcome this hurdle, a rigorous protocol was developed that preserves adipocyte morphology in skeletal muscle sections allowing visualization, imaging, and quantification of intramuscular fat. The protocol also outlines how to process a portion of muscle tissue for RT-qPCR, enabling users to confirm observed changes in fat formation by viewing differences in the expression of adipogenic genes. Additionally, it can be adapted to visualize adipocytes by whole-mount immunofluorescence of muscle samples. Finally, this protocol outlines how to perform genetic lineage tracing of Pdgfrα-expressing FAPs to study the adipogenic conversion of FAPs. This protocol consistently yields high-resolution and morphologically accurate immunofluorescent images of adipocytes, along with confirmation by RT-qPCR, allowing for robust, rigorous, and reproducible visualization and quantification of intramuscular fat. Together, the analysis pipeline described here is the first step to improving our understanding of how FAPs differentiate into intramuscular fat, and provides a framework to validate novel interventions to prevent fat formation.

Botulinum Injection Into the Proximal Intestinal Wall of Diet-Induced Obese Mice Leads to Weight Loss and Improves Glucose and Fat Tolerance.

Botulinum neurotoxin (available commercially as BOTOX) has been used successfully for treatment of several neuromuscular disorders, including blepharospasm, dystonia, spasticity, and cerebral palsy in children. Our data demonstrate that injection of Botox into the proximal intestinal wall of diet-induced obese (DIO) mice induces weight loss and reduces food intake. This was associated with amelioration of hyperglycemia, hyperlipidemia, and significant improvement of glucose tolerance without alteration of energy expenditure. We also observed accelerated gastrointestinal transit and significant reductions in glucose and lipid absorption, which may account, at least in part, for the observed weight loss and robust metabolic benefits, although possible systemic effects occurring as a consequence of central and/or peripheral signaling cannot be ignored. The observed metabolic benefits were found to be largely independent of weight loss, as demonstrated by pair-feeding experiments. Effects lasted ∼8 weeks, for as long as the half-life of Botox as reported in prior rodent studies. These results have valuable clinical implications. If the observed effects are translatable in humans, this approach could lay the foundation for therapeutic approaches geared toward robust and sustained weight loss, mimicking some of the benefits of bariatric operations without its cost and complications.

Preoperative hematocrit and platelet count are associated with blood loss during spinal fusion for children with neuromuscular scoliosis.

AIM: To assess the relationship of preoperative hematology laboratory results with intraoperative estimated blood loss and transfusion volumes during posterior spinal fusion for pediatric neuromuscular scoliosis. METHODS: Retrospective chart review of 179 children with neuromuscular scoliosis undergoing spinal fusion at a tertiary children's hospital between 2012 and 2017. The main outcome measure was estimated blood loss. Secondary outcomes were volumes of packed red blood cells, fresh frozen plasma, and platelets transfused intraoperatively. Independent variables were preoperative blood counts, coagulation studies, and demographic and surgical characteristics. Relationships between estimated blood loss, transfusion volumes, and independent variables were assessed using bivariable analyses. Classification and Regression Trees were used to identify variables most strongly correlated with outcomes. RESULTS: In bivariable analyses, increased estimated blood loss was significantly associated with higher preoperative hematocrit and lower preoperative platelet count but not with abnormal coagulation studies. Preoperative laboratory results were not associated with intraoperative transfusion volumes. In Classification and Regression Trees analysis, binary splits associated with the largest increase in estimated blood loss were hematocrit ≥44% vs. <44% and platelets ≥308 vs. <308 × 109/L. CONCLUSIONS: Preoperative blood counts may identify patients at risk of increased bleeding, though do not predict intraoperative transfusion requirements. Abnormal coagulation studies often prompted preoperative intervention but were not associated with increased intraoperative bleeding or transfusion needs.