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Wooden breast (WB) is a myopathy that occurs in pectoralis major (PM) muscles, predominately affecting large, fast-growing broilers. Severe myodegeneration, increased hypoxia, reduced blood flow, and increased collagen deposition are hallmark characteristics of WB that culminate in unsatisfactory fresh meat quality attributes, such as poor water-holding capacity, tenderness, and processing characteristics. Therefore, WB meat is often downgraded resulting in economic losses for the United States poultry industry. Although WB has been well characterized, its etiology remains undefined. As the scientific community continues to resolve mechanisms responsible for WB onset, understanding biochemical changes associated with WB may facilitate solutions to negate its poor meat quality attributes. Given changes in metabolism of living muscle can alter biochemical processes during the conversion of muscle to meat, this review aims to summarize and discuss the current knowledge of WB muscle and meat biochemistry. For example, it appears metabolic pathways that support combating stress are upregulated in WB muscle at the expense of glycolytic flux, which presumably contributes to the high ultimate pH of WB meat. Further, perturbed function of WB mitochondria, such as altered calcium handling, impacts aspects of postmortem metabolism and proteolysis. Collectively, metabolic dysfunction of WB muscle alters the biochemical processes that occur during the conversion of muscle to meat, and thus contributes to the poor WB meat quality.

Abstract Temporal community stability, here defined as temporal mean divided by temporal SD, plays an important role in predicting certain ecosystem services. However, temporal stability can change with invasion, with greater abundances of invasive species potentially having greater impacts on native community stability. The exact consequences of invasion for temporal stability are unclear and, in part, depend on the particular metric of stability measured. In rangeland ecosystems, predicable forage is important for livestock production but can be threatened by invasion. Therefore, using an observational field study conducted over three years in Wyoming, we assessed which metrics of plant community stability were altered by invasion and whether those effects were mediated by two environmental variables (light and soil moisture). Bromus arvensis and Bromus tectorum are two invasive annual weeds found across US rangelands, including the northern mixed‐grass prairies of Wyoming. We established plots along natural invasion blocks of B. arvensis and B. tectorum abundance and collected plant species composition data over three growing seasons. We tested associations between seven different metrics of plant community stability and invasion by B. arvensis and B. tectorum. We found that species turnover increases with invasion by both species, while stability of forb (both brome species), C4 grass (B. arvensis only), and C3 grass (B. tectorum only) cover decreases with invasion. All metrics of stability associated with invasion supported the hypothesis of a destabilizing effect of invasion on the native plant community. Further, we found that light and soil moisture did mediate some associations between stability and invasion. Overall, our results align with previous work suggesting that invasive annual bromes can lead to decreased native plant stability, which has important implications for forage production and, thus, food security.

Metabolic programming underpins inflammation and liver macrophage activation in the setting of chronic liver disease. Here, we sought to identify the role of an important metabolic regulator, AMP-activated protein kinase (AMPK), specifically within myeloid cells during the progression of non-alcoholic steatohepatitis (NASH) and whether treatment with metformin, a firstline therapy for diabetes and activator of AMPK could stem disease progression. Male and female Prkaa1 fl/fl/Prkaa2fl/fl (Flox) control and Flox-LysM-Cre+ (MacKO) mice were fed a low-fat control or a choline-deficient, amino acid defined 45% Kcal high-fat diet (CDAHFD) for 8 weeks, where metformin was introduced in the drinking water (50 or 250 mg/kg/day) for the last 4 weeks. Hepatic steatosis and fibrosis were dramatically increased in response to CDAHFD-feeding compared to low-fat control. While myeloid AMPK signaling had no effect on markers of hepatic steatosis or circulating markers, fibrosis as measured by total liver collagen was significantly elevated in livers from MacKO mice, independent of sex. Although treatment with 50 mg/kg/day metformin had no effect on any parameter, intervention with 250 mg/kg/day metformin completely ameliorated hepatic steatosis and fibrosis in both male and female mice. While the protective effect of metformin was associated with lower final body weight, and decreased expression of lipogenic and Col1a1 transcripts, it was independent of myeloid AMPK signaling. These results suggest that endogenous AMPK signaling in myeloid cells, both liver-resident and infiltrating, acts to restrict fibrogenesis during CDAHFD-induced NASH progression but is not the mechanism by which metformin improves markers of NASH.

Background: Mitochondria are considered the powerhouse of cells, and skeletal muscle cells are no exception. However, information regarding muscle mitochondria from different species is limited. Methods: Different muscles from cattle, pigs and chickens were analyzed for mitochondrial DNA (mtDNA), protein and oxygen consumption. Results: Bovine oxidative muscle mitochondria contain greater mtDNA (p < 0.05), protein (succinate dehydrogenase, SDHA, p < 0.01; citrate synthase, CS, p < 0.01; complex I, CI, p < 0.05), and oxygen consumption (p < 0.01) than their glycolytic counterpart. Likewise, porcine oxidative muscle contains greater mtDNA (p < 0.01), mitochondrial proteins (SDHA, p < 0.05; CS, p < 0.001; CI, p < 0.01) and oxidative phosphorylation capacity (OXPHOS, p < 0.05) in comparison to glycolytic muscle. However, avian oxidative skeletal muscle showed no differences in absolute mtDNA, SDHA, CI, complex II, lactate dehydrogenase, or glyceraldehyde 3 phosphate dehydrogenase compared to their glycolytic counterpart. Even so, avian mitochondria isolated from oxidative muscles had greater OXPHOS capacity (p < 0.05) than glycolytic muscle. Conclusions: These data show avian mitochondria function is independent of absolute mtDNA content and protein abundance, and argue that multiple levels of inquiry are warranted to determine the wholistic role of mitochondria in skeletal muscle.

Combining information from multiple senses is essential to object recognition, core to the ability to learn concepts, make new inferences, and generalize across distinct entities. Yet how the mind combines sensory input into coherent crossmodal representations – the crossmodal binding problem – remains poorly understood. Here, we applied multi-echo fMRI across a 4-day paradigm, in which participants learned three-dimensional crossmodal representations created from well-characterized unimodal visual shape and sound features. Our novel paradigm decoupled the learned crossmodal object representations from their baseline unimodal shapes and sounds, thus allowing us to track the emergence of crossmodal object representations as they were learned by healthy adults. Critically, we found that two anterior temporal lobe structures – temporal pole and perirhinal cortex – differentiated learned from non-learned crossmodal objects, even when controlling for the unimodal features that composed those objects. These results provide evidence for integrated crossmodal object representations in the anterior temporal lobes that were different from the representations for the unimodal features. Furthermore, we found that perirhinal cortex representations were by default biased toward visual shape, but this initial visual bias was attenuated by crossmodal learning. Thus, crossmodal learning transformed perirhinal representations such that they were no longer predominantly grounded in the visual modality, which may be a mechanism by which object concepts gain their abstraction.

Background: Symptomatic genu recurvatum is defined as greater than 5° of knee hyperextension and can be caused by osseous deformity, soft tissue laxity, or a combination. Common symptoms include pain, weakness, instability, decreased range of motion, leg length discrepancy, and stretching of the posterior capsuloligamentous structures of the knee. In instances where the genu recurvatum is caused by reverse tibial slope, literature supports the use of anterior opening-wedge proximal tibial osteotomy (OW PTO) to treat genu recurvatum by increasing tibial slope. Correction of anterior slope to a more anatomic, posterior orientation allows any stressed ligaments to return to their normal tension and restores the native biomechanics of the knee. Indications: The primary indication for OW PTO is genu recurvatum that is nonresponsive to physical therapy or genu recurvatum with concurrent ligamentous injury. The heel-height test provides an objective assessment for the identification and measurement of knee hyperextension. Technique Description: 2 guide pins are placed parallel to the tibial plateau, engaging the posterior cortex. A small micro sagittal saw is used to cut the anterior cortex. Osteotomes are used to complete the osteotomy, preserving a posterior hinge. An opening spreader device is placed and opened slowly while keeping the posterior cortex intact. The new slope is maintained by use of an opening wedge osteotomy plate and screws. Allograft bone graft is packed thoroughly into the osteotomy site. Fluoroscopy is used throughout the case to assess appropriate orientation and depth of the osteotomy, as well as the final opening width. Results: A review of 5 studies demonstrated adequate reduction in hyperextension, with a mean knee hyperextension ranging from 17° to 32° preoperatively and 0° to 7° postoperatively. Patients had significantly improved postoperative clinical outcomes compared with the preoperative state. Discussion/Conclusion: Anterior OW PTO has been shown to be a safe method of accurately correcting tibial plateau slope for the treatment of genu recurvatum. Patients can expect correction of knee hyperextension, restoration of anatomic posterior tibial slope, decreased posterior tibial translation, and increased subjective outcome scores. Patient Consent Disclosure Statement: The author(s) attest that consent has been obtained from any patient(s) appearing in this publication. If the individual may be identifiable, the author(s) has included a statement of release or other written form of approval from the patient(s) with this submission for publication.

Abstract Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in infants, the immunocompromised, and the elderly. RSV infects the airway epithelium via the apical membrane and almost exclusively sheds progeny virions back into the airway mucus (AM), making RSV difficult to target by systemically administered therapies. An inhalable “muco‐trapping” variant of motavizumab (Mota‐MT), a potent neutralizing mAb against RSV F is engineered. Mota‐MT traps RSV in AM via polyvalent Fc‐mucin bonds, reducing the fraction of fast‐moving RSV particles in both fresh pediatric and adult AM by ≈20–30‐fold in a Fc‐glycan dependent manner, and facilitates clearance from the airways of mice within minutes. Intranasal dosing of Mota‐MT eliminated viral load in cotton rats within 2 days. Daily nebulized delivery of Mota‐MT to RSV‐infected neonatal lambs, beginning 3 days after infection when viral load is at its maximum, led to a 10 000‐fold and 100 000‐fold reduction in viral load in bronchoalveolar lavage and lung tissues relative to placebo control, respectively. Mota‐MT‐treated lambs exhibited reduced bronchiolitis, neutrophil infiltration, and airway remodeling than lambs receiving placebo or intramuscular palivizumab. The findings underscore inhaled delivery of muco‐trapping mAbs as a promising strategy for the treatment of RSV and other acute respiratory infections.

Objective: Chronic exposure to persistent organic pollutants (POPs) is associated with increased incidence of type 2 diabetes, hyperglycemia, and poor insulin secretion in humans. Dioxins and dioxin-like compounds are a broad class of POPs that exert cellular toxicity through activation of the aryl hydrocarbon receptor (AhR). We previously showed that a single high-dose injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, aka dioxin; 20 μg/kg) in vivo reduced fasted and glucose-stimulated plasma insulin levels for up to 6 weeks in male and female mice. TCDD-exposed male mice were also modestly hypoglycemic and had increased insulin sensitivity, whereas TCDD-exposed females were transiently glucose intolerant. Whether these effects are driven by AhR activation in β-cells requires investigation. Methods: We exposed female and male β-cell specific Ahr knockout (βAhrKO) mice and littermate Ins1-Cre genotype controls (βAhrWT) to a single high dose of 20 μg/kg TCDD and tracked the mice for 6 weeks. Results: Under baseline conditions, deleting AhR from β-cells caused hypoglycemia in female mice, increased insulin secretion ex vivo in female mouse islets, and promoted modest weight gain in male mice. Importantly, high-dose TCDD exposure impaired glucose homeostasis and β-cell function in βAhrWT mice, but these phenotypes were largely abolished in TCDD-exposed βAhrKO mice. Conclusion: Our study demonstrates that AhR signaling in β-cells is important for regulating baseline β-cell function in female mice and energy homeostasis in male mice. We also show that β-cell AhR signaling largely mediates the effects of TCDD on glucose homeostasis in both sexes, suggesting that the effects of TCDD on β-cell function and health are driving metabolic phenotypes in peripheral tissues.

Skeletal muscle metabolism has implications for swine feed efficiency (FE); however, it remains unclear if the metabolic profile of skeletal muscle changes during postnatal growth. To assess the metabolic changes, samples were collected from the longissimus dorsi (LD, glycolytic muscle), latissimus dorsi (LAT, mixed muscle), and masseter (MS, oxidative muscle) at 20, 53, 87, 120, and 180 days of age from barrows. Muscles were assessed to determine the abundance of several metabolic enzymes. Lactate dehydrogenase (LDHα) decreased in all muscles from 20 to 87 d (p < 0.01), which may be attributed to the muscles being more glycolytic at weaning from a milk-based diet. Pyruvate carboxylase (PC) increased in all muscles at 53 d compared to the other time points (p < 0.01), while pyruvate dehydrogenase α 1 (PDHα1) increased at 87 and 180 d in MS compared to LD (p < 0.05), indicating that potential changes occur in pyruvate entry into the tricarboxylic acid (TCA) cycle during growth. Isolated mitochondria from each muscle were incubated with 13C-labeled metabolites to assess isotopomer enrichment patterns of TCA intermediates. Citrate M + 2 and M + 4 derived from [13C3]-pyruvate increased at 87 d in LAT and MS mitochondria compared to LD mitochondria (p < 0.05). Regardless of the muscle, citrate M+3 increased at 87 d compared to 20, 53, and 120 d, while 180 d showed intermediate values (p < 0.01). These data support the notion that pyruvate metabolism is dynamic during growth. Our findings establish a metabolic fingerprint associated with postnatal muscle hypertrophy.

Background: The popliteus tendon is a primary stabilizer of external knee rotation and has been described as the “fifth ligament of the knee.” Injuries involving the posterolateral corner of the knee commonly involve the popliteus tendon; isolated injury to the popliteus tendon is relatively rare and usually involves a rotatory injury mechanism and symptoms of instability and pain. In this patient, there was a concomitant injury to the proximal tibiofibular joint. Indications: Popliteus tendon reconstruction is indicated for patients with abnormal external rotation on examination and symptoms of pain and instability. Proximal tibiofibular joint reconstruction is indicated for patients with instability of the tibiofibular joint. Technique Description: Preoperative examination under anesthesia is essential to determine the extent of external rotation and assess for additional posterolateral pathology. The popliteus tendon procedure begins with a meticulous common peroneal nerve neurolysis to minimize nerve irritation and decrease the risk of a postoperative foot drop. Semitendinosus and gracilis graft harvest is performed with at least 16 cm of graft length followed by tunnel reaming of the popliteus tendon femoral attachment and tibia tunnel identification and reaming. The femoral attachment of the popliteus tendon is secured, and the semitendinosus graft is anatomically oriented along the native path and passed from posterior to anterior through the femoral tunnel. The gracilis graft for anatomic proximal tibiofibular joint reconstruction requires an anterior to posterior tunnel through the fibular head. Finally, both grafts are secured in the tibial tunnel with the application of an anterior traction force. Results: A cadaveric study by LaPrade et al reported that an anatomic popliteus tendon reconstruction with a semitendinosus autograft significantly reduced external rotation laxity compared with the sectioned state. In addition, popliteus tendon reconstruction resulted in significantly decreased varus laxity, anterior tibial translation, and internal rotation. Discussion: This is a technique that effectively restores external rotation knee stability utilizing an anatomically oriented reconstruction of the popliteus tendon and tibiofibular joint. The popliteus tendon serves as a primary stabilizer to external rotation and facilitates static function of the knee. The proximal tibiofibular joint reconstruction restores the function of the posterior proximal tibiofibular joint ligaments. Patient Consent Disclosure Statement: The author(s) attests that consent has been obtained from any patient(s) appearing in this publication. If the individual may be identifiable, the author(s) has included a statement of release or other written form of approval from the patient(s) with this submission for publication.

Background: Fibular collateral ligament (FCL) injuries commonly present in a multiligament knee injury pattern. These injuries are associated with significant instability leading to altered tibiofemoral biomechanics and therefore require surgical intervention. Similarly, grade 3 posterior cruciate ligament (PCL) injuries may disrupt normal tibiofemoral and patellofemoral biomechanics and increase the risk of secondary osteoarthritis. Therefore, concomitant reconstruction of the FCL and PCL should be performed to decrease knee laxity and optimize functional outcomes. Indications: Early operative treatment is indicated for patients with combined grade 3 FCL injuries and complete PCL tears. Contraindications to this procedure include patients who have significant osteoarthritis, open knee dislocations, or medical comorbidities making them unfit for surgery. Technique Description: The fundamental idea behind this technique is a stepwise treatment starting with open aspects of the procedure and followed by arthroscopic work. The technique is initiated with a lateral approach, common peroneal neurolysis, fibular and femoral FCL reconstruction tunnel preparation, and a gracilis or semitendinosus tendon autograft harvest. After that, focus shifts to intra-articular work such as associated meniscal assessment and treatment, PCL femoral and tibial tunnel preparation, graft passage, and PCL femoral tunnel fixation. Final graft fixation order is as follows: anterolateral bundle of PCL, posteromedial bundle of PCL, and finally FCL. Results: Multiple studies have reported that an anatomic FCL reconstruction in the setting of multiligament injury results in improved patient outcomes. In a prospective study of 20 patients, LaPrade et al reported −0.4 mm difference in side-to-side lateral compartment gapping and significant postoperative improvement of symptom and functional scores at a minimum 2 year postoperative follow-up after anatomic reconstruction of the FCL. Similarly, Moulton et al reported significant improvement in the average Western Ontario and Lysholm scores at 2.7 years follow-up. LaPrade et al also reported significant improvement in function and objective outcome scores at 3 years’ follow-up from anatomic double-bundle PCL reconstruction. Discussion: Anatomic FCL and PCL reconstructions successfully restore near native knee objective stability and provide superior clinical outcomes when compared to nonanatomic-based FCL reconstructions that continue to be performed. Patient Consent Disclosure Statement: The author(s) attests that consent has been obtained from any patient(s) appearing in this publication. If the individual may be identifiable, the author(s) has included a statement of release or other written form of approval from the patient(s) with this submission for publication.

Abstract mRNA‐based molecular subtypes have implications for bladder cancer prognosis and clinical benefit from certain therapies. Whether small extracellular vesicles (sEVs) can reflect bladder cancer molecular subtypes is unknown. We performed whole transcriptome RNA sequencing for formalin fixed paraffin embedded (FFPE) tumour tissues and sEVs separated from matched tissue explants, urine and plasma in patients with bladder cancer. sEVs were separated using size‐exclusion chromatography, and characterized by transmission electron microscopy, nano flow cytometry and western blots, respectively. High yield of sEVs were obtained using approximately 1 g of tissue, incubated with media for 30 min. FFPE tumour tissue and tumour tissue‐derived sEVs demonstrated good concordance in molecular subtype classification. All urinary sEVs were classified as luminal subtype, while all plasma sEVs were classified as Ba/Sq subtype, regardless of the molecular subtypes indicated by their matched FFPE tumour tissue. The comparison within urine sEVs, which may exclude the sample type specific background, could pick up the different biology between NMIBC and MIBC, as well as the signature genes related to molecular subtypes. Four candidate sEV‐related bladder cancer‐specific mRNA biomarkers, FAM71E2, OR4K5, FAM138F and KRTAP26‐1, were identified by analysing matched urine sEVs, tumour tissue derived sEVs, and adjacent normal tissue derived sEVs. Compared to sEVs separated from biofluids, tissue‐derived sEVs may reflect more tissue‐ or disease‐specific biological features. Urine sEVs are promising biomarkers to be used for liquid biopsy‐based molecular subtype classification, but the current algorithm needs to be modified/adjusted. Future work is needed to validate the four new bladder cancer‐specific biomarkers in large cohorts.

Background: Complete meniscal root tears disrupt dispersion of axial loading forces through hoop stresses. This increases point-loading on tibiofemoral cartilage and leads to chondromalacia and accelerated osteoarthritis (OA). Posterior root tears may be treated successfully with a transtibial pullout repair. Varus malalignment also leads to increased medial compartment pressures, increasing the risk of early OA and putting increased stress on the meniscus and stabilizing knee ligaments. In particular, revision medial meniscal root repairs without correction of varus malalignment are at increased risk of failure. Genu varum may be corrected with a medial opening-wedge proximal tibial osteotomy (OW PTO). Indications: Meniscal root repairs are indicated for acute or chronic tears in active patients with healthy cartilage. OW PTO is indicated for varus malalignment in ambulatory patients with healthy cartilage, or who are at risk for failure of meniscal or ligamentous procedures. Technique Description: After exposure of the osteotomy site, arthroscopy is performed through the incision and the revision posterior meniscus root repair is performed via a double-tunnel transtibial pullout technique. The positioning of these tunnels is modified superiorly so as to not cross the planned osteotomy site. The osteotomy is then performed by drilling 2 guide pins under fluoroscopy to delineate the plane of the cut. An OW plate is placed, and the root repair is tensioned last. Results: Double-tunnel transtibial pullout repairs increase meniscal fixation contact surface and have been shown to be biomechanically superior to all-inside fixation techniques. Medial OW PTO restores knee alignment and reduces supra-anatomic stresses in the medial compartment, additionally decreasing the risk of a revision medial meniscus repair failure. Discussion: Biomechanical studies have shown that meniscal root tears are functionally equivalent to complete meniscectomy. Varus malalignment increases the risk of medial meniscal tears, and reduces the risk of a successful long-term repair. We describe a technique for a revision transosseous posterior root repair with concomitant proximal tibial osteotomy, with discussion of surgical pearls and pitfalls. This technique restores anatomic position and native function of the medial meniscus while correcting tibiofemoral malalignment that could jeopardize the repair. Patient Consent Disclosure Statement: The author(s) attests that consent has been obtained from any patient(s) appearing in this publication. If the individual may be identifiable, the author(s) has included a statement of release or other written form.

OBJECTIVES/GOALS: Scaffolding aids learning by gradually removing assistance to encourage independence (Bickhard, 2013; Gross, 2015). Mightier is a commercially available biofeedback game that fosters emotion regulation (ER) skill practice through play. This study aims to evaluate the ability of Mightier to scaffold the learning of ER and reduce irritability. METHODS/STUDY POPULATION: Data were collected via online caregiver report. Inclusion criteria were age of child (

Summary: Atherosclerotic cardiovascular disease is characterized by both chronic low-grade inflammation and dyslipidemia. The AMP-activated protein kinase (AMPK) inhibits cholesterol synthesis and dampens inflammation but whether pharmacological activation reduces atherosclerosis is equivocal. In the current study, we found that the orally bioavailable and highly selective activator of AMPKβ1 complexes, PF-06409577, reduced atherosclerosis in two mouse models in a myeloid-derived AMPKβ1 dependent manner, suggesting a critical role for macrophages. In bone marrow-derived macrophages (BMDMs), PF-06409577 dose dependently activated AMPK as indicated by increased phosphorylation of downstream substrates ULK1 and acetyl-CoA carboxylase (ACC), which are important for autophagy and fatty acid oxidation/de novo lipogenesis, respectively. Treatment of BMDMs with PF-06409577 suppressed fatty acid and cholesterol synthesis and transcripts related to the inflammatory response while increasing transcripts important for autophagy through AMPKβ1. These data indicate that pharmacologically targeting macrophage AMPKβ1 may be a promising strategy for reducing atherosclerosis.

Type 2 cytokines like IL-4 are hallmarks of helminth infection and activate macrophages to limit immunopathology and mediate helminth clearance. In addition to cytokines, nutrients and metabolites critically influence macrophage polarization. Choline is an essential nutrient known to support normal macrophage responses to lipopolysaccharide; however, its function in macrophages polarized by type 2 cytokines is unknown. Using murine IL-4-polarized macrophages, targeted lipidomics revealed significantly elevated levels of phosphatidylcholine, with select changes to other choline-containing lipid species. These changes were supported by the coordinated up-regulation of choline transport compared to naïve macrophages. Pharmacological inhibition of choline metabolism significantly suppressed several mitochondrial transcripts and dramatically inhibited select IL-4-responsive transcripts, most notably, Retnla. We further confirmed that blocking choline metabolism diminished IL-4-induced RELMα (encoded by Retnla) protein content and secretion and caused a dramatic reprogramming toward glycolytic metabolism. To better understand the physiological implications of these observations, naïve or mice infected with the intestinal helminth Heligmosomoides polygyrus were treated with the choline kinase α inhibitor, RSM-932A, to limit choline metabolism in vivo. Pharmacological inhibition of choline metabolism lowered RELMα expression across cell-types and tissues and led to the disappearance of peritoneal macrophages and B-1 lymphocytes and an influx of infiltrating monocytes. The impaired macrophage activation was associated with some loss in optimal immunity to H. polygyrus, with increased egg burden. Together, these data demonstrate that choline metabolism is required for macrophage RELMα induction, metabolic programming, and peritoneal immune homeostasis, which could have important implications in the context of other models of infection or cancer immunity.

Fibrosis, or excessive scarring, is characterized by the emergence of alpha-smooth muscle actin (αSMA)-expressing myofibroblasts and the excessive accumulation of fibrotic extracellular matrix (ECM). Currently, there is a lack of effective treatment options for fibrosis, highlighting an unmet need to identify new therapeutic targets. The acquisition of a fibrotic phenotype is associated with changes in chromatin structure, a key determinant of gene transcription activation and repression. The major repressive histone mark, H3K27me3, has been linked to dynamic changes in gene expression in fibrosis through alterations in chromatin structure. H3K27-specific homologous histone methylase (HMT) enzymes, Enhancer of zeste 1 and 2 (EZH1, EZH2), which are the alternative subunits of the Polycomb Repressive Complex 2 (PRC2) and demethylase (KDM) enzymes, Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), and Lysine demethylase 6B (KDM6B), are responsible for regulating methylation status of H3K27me3. In this review, we explore how these key enzymes regulate chromatin structure to alter gene expression in fibrosis, highlighting them as attractive targets for the treatment of fibrosis.

Background: Chronic posterior cruciate ligament (PCL) insufficiency leads to instability of the knee, resulting in increased loading of the medial and patellofemoral compartments. Chronic PCL insufficiency progressively damages the menisci and chondral surfaces and can accentuate and worsen varus malalignment. Biplanar proximal tibial opening-wedge osteotomy (BPTO) is a treatment option for chronic PCL tears with medial compartment arthritis which concurrently treats sagittal (flat slope) and varus coronal alignment. Indications: Opening-wedge BPTO is indicated in active patients who present with varus malalignment and flat slope with concomitant degenerative medial compartment osteoarthritis and PCL insufficiency. In general, BPTO is contraindicated in patients with a significantly elevated body mass index (BMI), smokers, or in those who have advanced tricompartmental osteoarthritis. Technique Description: Preoperative long-leg radiographs are essential to preoperative evaluation. Ideal coronal alignment following correction would have the mechanical axis pass through the apex of the lateral tibial eminence to properly off-load the medial compartment. Magnetic resonance imaging (MRI) studies are also useful for determining the presence and extent of concomitant soft tissue and chondral injury, especially in the lateral compartment. This video demonstrates the senior author’s preferred technique for a biplanar proximal tibial opening (PTO) with concomitant chondroplasty and partial medial meniscectomy (PMM). The senior author’s preferred order of operations is also displayed and involves first performing an arthroscopy, then proceeding with the open portions of the case, including the medial approach and subsequent osteotomy. Results: Past studies have supported PTO as a reliable treatment option for varus malalignment. Bode et al reported a 5-year survival rate of 96% of patients with an overall complication rate of 8.6%. The authors also reported that Lysholm scores were also significantly improved at 60-month follow-up compared with preoperative scores. Discussion: This is a technique for the correction of a varus malalignment with concomitant flat slope and PCL insufficiency with biplanar open-wedge PTO. This operation corrects the mechanical axis and sagittal slope of the knee and restores proper load distribution across the tibiofemoral articulation. In conclusion, a biplanar open-wedge PTO is a useful alternative to joint arthroplasty for active patients with a chronic PCL tear, medial compartment arthritis, and varus malalignment. Patient Consent Disclosure Statement: The author(s) attests that consent has been obtained from any patient(s) appearing in this publication. If the individual may be identifiable, the author(s) has included a statement of release or other written form of approval from the patient(s) with this submission for publication.

Abstract Introduction Better transparency of research results and participant engagement may help address poor participant accrual in paediatric clinical research. We conducted formative research to assess the acceptability of lay summaries and thank you notes, as well as to refine and expand guidance on participant and family engagement in Pediatric Trials Network's (PTN) pragmatic paediatric clinical research. Methods Informed by draft PTN guidance, we conducted in‐depth qualitative interviews with adolescent clinical trial participants and caregivers of paediatric participants in four trials conducted by PTN across eight sites. Participants were shown multiple versions of mock lay summaries and thank you notes and asked questions on their preferences for content and layout, and on trial communications. We used applied thematic analysis to analyse the data. Results We interviewed 27 individuals engaged in PTN research: 24 caregivers and 3 adolescents. During a trial, participants want regular updates on study progress, reminders of the study purpose and reassurances of data confidentiality. After the trial, participants want to learn the aggregated results, particularly medication effectiveness. Participants reported that lay summaries should include a review of the study's purpose, methods and length, and that they expect to learn individual‐level results. Participants stated that thank you notes must be of sufficient length to be meaningful. Conclusions This is the first study to describe stakeholder preferences for thank you note content and layout. Using these findings, we finalized PTN's trial communication guidance for use in future PTN trials. Research is needed to determine the effect of lay summaries and thank you notes on improving public transparency regarding clinical trials and paediatric trial recruitment and completion. Patient or Public Contribution By design, stakeholders (adolescent trial participants and caregivers of pediatric trial participants) contributed to PTN's guidance on the content and layout of lay summaries and thank you notes through their participation in the in‐depth interviews.

IntroductionWhile increased time spent on social media can be negatively related to one’s overall mental health, social media research often fails to account for what behaviors users are actually engaging in while they are online. The present research helps to address this gap by measuring participants’ active and passive social media behavioral styles and investigates whether and how these two social media behavioral styles are related to depression, anxiety, and stress, and the mediating role of emotion recognition ability in this relationship.MethodsA pre-study (N = 128) tested whether various social media behaviors reliably grouped into active and passive behavioral styles, and a main study (N = 139) tested the relationships between social media use style, emotion recognition, and mental health.ResultsWhile we did not find evidence of a mediating relationship between these variables, results supported that more active social media use was related to more severe anxiety and stress as well as poorer emotion recognition skill, while passive social media use was unrelated to these outcomes.DiscussionThese findings highlight that, beyond objective time spent on social media, future research must consider how users are spending their time online.

Fermentable oligo-, di-, monosaccharides and polyols (FODMAPs) have emerged as key contributors to digestive discomfort and intolerance to certain vegetables, fruits, and plant-based foods. Although strategies exist to minimize FODMAP consumption and exposure, exogenous enzyme supplementation targeting the fructan-type FODMAPs has been underexploited. The objective of this study was to test the hydrolytic efficacy of a food-grade, non-genetically engineered microbial inulinase preparation toward inulin-type fructans in the INFOGEST in vitro static simulation of gastrointestinal (GI) digestion. Purified inulin was shown to undergo acid-mediated hydrolysis at high gastric acidity as well as predominantly inulinase-mediated hydrolysis at lower gastric acidity. Inulinase dose-response simulations of inulin, garlic, and high-fructan meal digestion in the gastric phase suggest that as little as 50 inulinase units (INU) and up to 800 INU per serving promote fructan hydrolysis better than the control simulations without inulinase. Liquid chromatography-mass spectrometry (LC-MS) profiling of fructo-oligosaccharides (FOS) in the gastric digestas following inulinase treatment confirms the fructolytic activity of inulinase under simulated digestive conditions. Altogether, these in vitro digestion data support the use of microbial inulinase as an exogenous enzyme supplement for reducing dietary fructan-type FODMAP exposure.

Background: Injuries to the fibular collateral ligament (FCL) seldom occur in isolation and may present with a concomitant injury to the biceps femoris tendon and anterior cruciate ligament (ACL). Injuries to structures of the posterolateral corner (PLC) lead to varus and rotational instability of the knee, subjecting the cruciate ligaments to increased forces that may result in graft failure. Therefore, reconstruction of these structures should be performed concurrently with the ACL. Indications: Grade III FCL injuries heal poorly without operative treatment and often result in residual varus instability of the knee that increases medial knee compartment forces, and forces on both the native ACL and the graft status post ACL reconstruction. Therefore, preservation of biomechanical stability and long-term health of the knee are reliant on addressing injuries to the PLC surgically. Technique Description: A key concept of this surgical technique is a meticulous peroneal nerve neurolysis in the setting of altered biceps femoris anatomy, and the proper order of the surgical steps for tunnel creation, graft passage, and fixation and suture anchor insertion to achieve optimal patient outcomes. The described technique involves a lateral surgical approach, peroneal neurolysis, and preparation of fibular and femoral FCL tunnels, followed by a Bone-patellar tendon-bone graft (BTB) graft harvest. Attention is then turned to intra-articular work including the diagnostic arthroscopy, femoral and tibial tunnel preparation, passage of the ACL graft, and fixation of the grafts in femoral tunnels. Last, fixation is achieved in the following order: FCL graft on fibula, ACL graft on tibia, and biceps femoris tendon to fibular head. Results: Compared with the preoperative state, Moulton et al reported significant improvements in the average Lysholm and Western Ontario scores at 2.7 years postoperatively following anatomic FCL reconstrution. Furthermore, Thompson et al reported on primary suture anchor repair of distal biceps femoris in 22 elite athletes and reported that all patients had returned to their preinjury level of sporting activity at 2-year follow-up. Discussion: Anatomic reconstructions of the FCL and ACL, such as the one described in our technique, effectively restore near native knee biomechanics and offer superior clinical outcomes compared with nonanatomic-based FCL reconstructions. Patient Consent Disclosure Statement: The author(s) attests that consent has been obtained from any patient(s) appearing in this publication. If the individual may be identifiable, the author(s) has included a statement of release or other written form of approval from the patient(s) with this submission for publication.

Background: Valgus malalignment leads to excessive loading of the lateral compartment and has been associated with lateral compartment meniscal injuries and early-onset knee osteoarthritis. Distal femoral osteotomy (DFO) is a treatment option that allows correction of the mechanical access to offload the lateral compartment. Lateral opening-wedge distal femoral osteotomy and medial meniscal root repairs are both aimed to address the pathologic biomechanics of the tibiofemoral joint and when successful can delay or even prevent progression to end-stage osteoarthritic changes in the lateral and medial compartment, respectively. Indications: Distal femoral osteotomy is indicated for patients with normal metabolic bone health and valgus malalignment who may present with concurrent lateral meniscal injury, symptomatic lateral compartment arthritis, or a chronic medial collateral ligament tear/laxity. Technique Description: Long-leg alignment radiographs and preoperative magnetic resonance images are required to evaluate the degree of mechanical access deviation and to evaluate for soft tissue injury, as there is often concomitant lateral meniscal or cartilage pathology present. This technique demonstrates the optimal order of surgical steps for DFO with a concurrent medial meniscal root repair. The senior author’s preferred order is to first perform the open lateral approach and expose the lateral cortex for the placement of plate prior to arthroscopic work and excess fluid extravasation. Intra-articular work is then performed with a diagnostic arthroscopy followed by medial meniscal root repair. Finally, the osteotomy and subsequent internal fixation are performed. Results: The literature on opening-wedge DFO outcomes is heterogeneous, with variable rates of successful outcomes and complications. Saithna et al reported that International Knee Documentation Committee and the pain subdomain of Knee injury and Osteoarthritis Outcome Scores significantly improved postoperatively following lateral opening-wedge DFOs in patients with lateral compartment osteoarthritis. The 10-year success rate of varus-producing DFO has been reported to be 64% to 85%. Discussion: We describe a technique for correction of a valgus deformity with opening-wedge DFO and concurrent meniscal root repair. This surgical technique effectively restores the native femoral and tibial axis with appropriate load distribution and also concurrently restores the appropriate function of the medial meniscus which allows axial force dispersion via hoop stresses for the medial compartment. Patient Consent Disclosure Statement: The author(s) attests that consent has been obtained from any patient(s) appearing in this publication. If the individual may be identifiable, the author(s) has included a statement of release or other written form of approval from the patient(s) with this submission for publication.

Summary: Mice systemically lacking dipeptidyl peptidase-4 (DPP4) have improved islet health, glucoregulation, and reduced obesity with high-fat diet (HFD) feeding compared to wild-type mice. Some, but not all, of this improvement can be linked to the loss of DPP4 in endothelial cells (ECs), pointing to the contribution of non-EC types. The importance of intra-islet signaling mediated by α to β cell communication is becoming increasingly clear; thus, our objective was to determine if β cell DPP4 regulates insulin secretion and glucose tolerance in HFD-fed mice by regulating the local concentrations of insulinotropic peptides. Using β cell double incretin receptor knockout mice, β cell- and pancreas-specific Dpp4−/− mice, we reveal that β cell incretin receptors are necessary for DPP4 inhibitor effects. However, although β cell DPP4 modestly contributes to high glucose (16.7 mM)-stimulated insulin secretion in isolated islets, it does not regulate whole-body glucose homeostasis.

Summary: Cell reprogramming to a myofibroblast responsible for the pathological accumulation of extracellular matrix is fundamental to the onset of fibrosis. Here, we explored how condensed chromatin structure marked by H3K72me3 becomes modified to allow for activation of repressed genes to drive emergence of myofibroblasts. In the early stages of myofibroblast precursor cell differentiation, we discovered that H3K27me3 demethylase enzymes UTX/KDM6B creates a delay in the accumulation of H3K27me3 on nascent DNA revealing a period of decondensed chromatin structure. This period of decondensed nascent chromatin structure allows for binding of pro-fibrotic transcription factor, Myocardin-related transcription factor A (MRTF-A) to nascent DNA. Inhibition of UTX/KDM6B enzymatic activity condenses chromatin structure, prevents MRTF-A binding, blocks activation of the pro-fibrotic transcriptome, and results in an inhibition of fibrosis in lens and lung fibrosis models. Our work reveals UTX/KDM6B as central coordinators of fibrosis, highlighting the potential to target its demethylase activity to prevent organ fibrosis.

Background: Anterior cruciate ligament (ACL) reconstructions in skeletally immature patients have an increased risk of graft failure and a decreased rate of successful return to sports. Anterior cruciate ligament reconstructions (ACLRs) combined with lateral extra-articular tenodesis (LET) procedures are reported to limit anterolateral rotary instability of the knee, decreasing tension on the ACL graft. However, the open physes found in pediatric patients present unique challenges for ACLR. This video demonstrates a technique to avoid the femoral physis and necessary precautions for performing an ACLR with LET in a skeletally immature patient. Indications: The indications for combined ACLR and LET, as opposed to ACLR alone, mainly center on the risk factors for graft failure. These include the following: posterior tibial slope >12°, revision ACLR, high-grade pivot shift on physical examination, meniscal deficiency, generalized ligamentous hyperlaxity, and—especially pertinent to this procedure—skeletally immature patients. Technique Description: Standard plain radiographs and long-leg alignment films, along with a magnetic resonance imaging (MRI) study, are necessary to assess for malalignment, bone age, and concomitant meniscal and chondral pathology. This technique describes the senior author’s preferred technique for pediatric ACLR with LET. The senior author’s preferred order of operations includes performing the autologous hamstring graft harvest and LET before performing the diagnostic arthroscopy. This is followed by the intra-articular work including the ACLR. It is also the senior author’s preference to perform physeal-sparing femoral tunnels, using fluoroscopy for tunnel placement. Results: Getgood et al reported that an ACLR with an LET in young patients with a high risk of graft failure had a significant reduction in graft rupture compared to an ACLR alone. Furthermore, Noyes et al reported a significantly lower rate of graft failure and a higher rate of return to sports in those who received LET in addition to ACLR compared with those who received ACLR alone. Discussion: An ACLR combined with an LET has been reported to reduce graft failure rates and improve clinical outcomes in high-risk patients. An ACLR with LET may also be considered in skeletally immature patients with appropriate indications and extra precautions to avoid growth arrest or deformity. Patient Consent Disclosure Statement: The author(s) attests that consent has been obtained from any patient(s) appearing in this publication. If the individual may be identifiable, the author(s) has included a statement of release or other written form of approval from the patient(s) with this submission for publication.

Effective second-line treatment options for patients with recurrent ovarian clear cell carcinoma (OCCC) are limited. This case series sought to report tumor characteristics and oncologic outcomes in a small group of patients treated with combination lenvatinib and pembrolizumab.A retrospective analysis of patients with ovarian clear cell carcinoma treated with combination lenvatinib and pembrolizumab at a single institution was performed. Patient and tumor characteristics were collected including demographics and germline/somatic testing. Clinical outcomes were also evaluated and reported.Three patients with recurrent OCCC were included in the study. The median age of patients was 48 years old. All patients had platinum-resistant disease and had received 1–3 prior lines of therapy. The overall response rate was 100% (3/3). Progression-free survival ranged from 10 months to not-yet-reached. One patient remains on treatment, while the other two died of disease with overall survival of 14 and 27 months.Combination lenvatinib-pembrolizumab demonstrated favorable clinical response in these patients with platinum-resistant, recurrent, ovarian clear cell carcinoma.

Background: Lateral meniscal root tears often occur in the acute setting in concert with anterior cruciate ligament (ACL) tears. Severe changes in tibiofemoral biomechanics and joint degeneration are encountered when these injuries are either unrecognized or are treated with meniscectomy; therefore, meniscal root surgical repair is now preferred for longer-term benefits. The most common repair techniques include side-to-side and transtibial pull-out repair, which may be performed via single or double transtibial tunnel techniques. Indications: The primary indication for lateral meniscal root repair with double transtibial tunnels is suspicion for a lateral meniscal root tear based upon injury mechanism, physical examination findings, magnetic resonance imaging findings, and confirmation with diagnostic arthroscopy. Technique Description: The root attachment site is decorticated. Adhesions that cause lateral meniscal root retraction are arthroscopically released. Two separate transtibial tunnels are drilled 5 mm apart from the anterolateral tibia, entering the anterolateral tibia distal to Gerdy’s tubercle and entering the joint at the decorticated lateral root attachment site. Two suture tapes are passed through the torn lateral meniscal root in a vertical mattress configuration and shuttled with a passing suture through the tibial tunnels. The suture tapes are tied over a surgical button on the anterolateral tibia with the knee flexed to approximately 90° while the repair is viewed arthroscopically. Results: Lateral meniscal root repairs are safe and have very low reoperation rates. It has been reported that the transtibial pullout repair technique of the lateral meniscus significantly decreases lateral meniscus extrusion compared with other repair techniques in patients with combined ACL reconstruction. Zhuo et al also reported significantly improved postoperative clinical outcomes compared with the preoperative state in patients who underwent pullout repair for posterior lateral meniscal root avulsion tears. Discussion/Conclusion: The biomechanical and clinical evidence supports concomitant lateral meniscal root repair in the setting of concurrent ACL injury, and no studies to date have demonstrated a clear harm associated with this procedure. In addition, failure to repair the lateral meniscal root places supraphysiologic loads on the ACL graft and may increase the risk of graft failure. Patient Consent Disclosure Statement: The author(s) attests that consent has been obtained from any patient(s) appearing in this publication. If the individual may be identifiable, the author(s) has included a statement of release or other written form of approval from the patient(s) with this submission for publication.

Recent interest has uncovered the importance of understanding meniscal root anatomy, its effect on biomechanics of the knee joint, and appropriate treatment modalities. Meniscal root tears may account for up to one-fifth of all meniscal tears and have downstream consequences that can be as severe as total meniscectomy due to the unique force-dispersing properties of the meniscus. Meniscal biomechanics rely heavily upon the integrity of root attachments, and this historically overlooked etiology of knee pain and early onset osteoarthritis plays a vital role in maintaining the longevity of the knee joint. As meniscal root pathology becomes more commonly known and clinically assessed, an understanding of classifications, repair methods, and indications is important for surgeons.

Pannexin-1 ion channels on endothelial cells regulate vascular inflammation and remodeling to mediate aortic aneurysm formation. Pharmacological blockade of Pannexin-1 channels may offer translational therapeutic mitigation of aneurysmal pathology.

While the lens is an avascular tissue with an immune-privileged status, studies have now revealed that there are immune responses specifically linked to the lens. The response to lens injury, such as following cataract surgery, has been shown to involve the activation of the resident immune cell population of the lens and the induction of immunomodulatory factors by the wounded epithelium. However, there has been limited investigation into the immediate response of the lens to wounding, particularly those induced factors that are intrinsic to the lens and its associated resident immune cells. Using an established chick embryo ex vivo cataract surgery model has made it possible to determine the early immune responses of this tissue to injury, including its resident immune cells, through a transcriptome analysis. RNA-seq studies were performed to determine the gene expression profile at 1 h post wounding compared to time 0. The results provided evidence that, as occurs in other tissues, the resident immune cells of the lens rapidly acquired a molecular signature consistent with their activation. These studies also identified the expression of many inflammatory factors by the injured lens that are associated with both the induction and regulation of the immune response.

Elevated circulating dipeptidyl peptidase-4 (DPP4) is a biomarker for liver disease, but its involvement in gluconeogenesis and metabolic associated fatty liver disease progression remains unclear. Here, we identified that DPP4 in hepatocytes but not TEK receptor tyrosine kinase–positive endothelial cells regulates the local bioactivity of incretin hormones and gluconeogenesis. However, the complete absence of DPP4 (Dpp4–/–) in aged mice with metabolic syndrome accelerates liver fibrosis without altering dyslipidemia and steatosis. Analysis of transcripts from the livers of Dpp4–/– mice displayed enrichment for inflammasome, p53, and senescence programs compared with littermate controls. High-fat, high-cholesterol feeding decreased Dpp4 expression in F4/80+ cells, with only minor changes in immune signaling. Moreover, in a lean mouse model of severe nonalcoholic fatty liver disease, phosphatidylethanolamine N-methyltransferase mice, we observed a 4-fold increase in circulating DPP4, in contrast with previous findings connecting DPP4 release and obesity. Last, we evaluated DPP4 levels in patients with hepatitis C infection with dysglycemia (Homeostatic Model Assessment of Insulin Resistance > 2) who underwent direct antiviral treatment (with/without ribavirin). DPP4 protein levels decreased with viral clearance; DPP4 activity levels were reduced at long-term follow-up in ribavirin-treated patients; but metabolic factors did not improve. These data suggest elevations in DPP4 during hepatitis C infection are not primarily regulated by metabolic disturbances.

Abstract The respiratory tract represents the key target for antiviral delivery in early interventions to prevent severe COVID‐19. While neutralizing monoclonal antibodies (mAb) possess considerable efficacy, their current reliance on parenteral dosing necessitates very large doses and places a substantial burden on the healthcare system. In contrast, direct inhaled delivery of mAb therapeutics offers the convenience of self‐dosing at home, as well as much more efficient mAb delivery to the respiratory tract. Here, building on our previous discovery of Fc‐mucin interactions crosslinking viruses to mucins, we showed that regdanvimab, a potent neutralizing mAb already approved for COVID‐19 in several countries, can effectively trap SARS‐CoV‐2 virus‐like particles in fresh human airway mucus. IN‐006, a reformulation of regdanvimab, was stably nebulized across a wide range of concentrations, with no loss of activity and no formation of aggregates. Finally, nebulized delivery of IN‐006 resulted in 100‐fold greater mAb levels in the lungs of rats compared to serum, in marked contrast to intravenously dosed mAbs. These results not only support our current efforts to evaluate the safety and efficacy of IN‐006 in clinical trials, but more broadly substantiate nebulized delivery of human antiviral mAbs as a new paradigm in treating SARS‐CoV‐2 and other respiratory pathologies.

Abstract Marine‐derived resource subsidies can generate intrapopulation variation in the behaviors and diets of terrestrial consumers. How omnivores respond, given their multiple trophic interactions, is not well understood. We sampled mice (Peromyscus keeni) and their food sources at five sites on three islands of the Central Coast of British Columbia, Canada, to test predictions regarding variation in the spatial behavior and consumption of marine‐subsidized foods among individuals. About 50% of detections (n = 27 recaptures) occurred at traps closest to shoreline (25 m), with capture frequencies declining significantly inland (up to 200 m). Stable isotope signatures (δ13C and δ15N), particularly δ15N, in plant foods, forest arthropod prey, and mouse feces were significantly enriched near shorelines compared with inland, while δ13C patterns were more variable. Bayesian isotope mixing models applied to isotope values in mouse hair indicated that over one‐third (35–37%) of diet was comprised of beach‐dwelling arthropods, a marine‐derived food source. Males were more abundant near the shoreline than females and consumed more marine‐derived prey, regardless of reproductive status or availability of other food sources. Our results identify how multiple pathways of marine nutrient transfer can subsidize terrestrial omnivores and how subsets of recipient populations can show variation in spatial and dietary response.

Swine heat production (HP) data are an essential element of numerous aspects affecting swine production sustainability, such as, housing environmental control design, energetics and thermoregulation modeling, as well as understanding of feed energy partitioning. Accurate HP values that reflect the continuous advances in growth, nutrition, health, and reproduction are needed to update outdated models and data; hence, this review of swine HP values is a critical contribution. This review updates the last previous review conducted in 2004, by reviewing literature from growing and breeding pigs from 2003 to 2020. In total, 33 references were identified that provided relevant HP data and from these references, 192 records were identified for pigs ranging in weight from 12.5 to 283 kg and exposed to temperatures between 12.0°C and 35.5°C. For growing pigs at thermoneutral conditions, a 4.7% average increase in HP was observed compared to HP data summarized from 1988 to 2004. Only five records were identified for gestating sows and the 43 records for lactating sows plus litter. This sow data shows high variability and inconsistent trends with temperature, most likely attributed to variation in experimental protocols, management, and limited reported information. There is still a lack of data on growing pigs greater than 105 kg, gilts and gestating sows housed in different systems (stall, pen, mixed, etc.), and latent HP values that reflect different housing systems. Further, there is a need to standardize reporting of HP values (with an example provided) across different disciplines to drive documentation of increased swine production efficiency, environmental control design, and energetics modeling.

The application of circulating tumor cells (CTCs) in both clinical practice and research has been continuously limited by the rare number of targets that can be found in a tube of peripheral blood. Diagnostic leukapheresis (DLA) was used to increase the sampling volume. AdnaTest was used to process the whole leukopak, and the RNAs of captured CTCs was then profiled by NanoString nCounter platform. Spike-in experiments and leukopaks from patients with metastatic prostate cancer were used to validate this new strategy. The whole leukopak was further concentrated five times to reduce the total volume from 150 ​mL to 30 ​mL, which enabled it to be processed by 3 separate AdnaTest kits. The spike-in experiment demonstrated a reliable capture when there were more than 100 cancer cells/10 ​mL of concentrated leukopak. In 1 out of 5 real patient samples, CTCs were only detected in the leukopak, but not in peripheral blood. The RNA profiling of DLA CTCs indicated a more aggressive phenotype of CTCs occurred when the patient was experiencing a disease relapse, even when the serum prostate specific antigen (PSA) level was still relatively low and CTCs in peripheral blood were not detectable. We established a new protocol, integrating DLA, AdnaTest and NanoString nCounter technology, to profile RNAs from CTCs captured from a large blood screening volume. The new protocol can process the whole leukopak with sensitive CTC capture. The RNA profiling of CTCs can provide valuable information for disease monitoring.

Abstract Environmental DNA (eDNA) has been increasingly utilized by academic, industry, and government groups for environmental monitoring due to its efficiency in regards to both time and cost, as well as non‐invasiveness to target organisms, and reduced dependency on trained biologists for sample collection. The methods typically employ quantitative real‐time polymerase chain reaction (qPCR) to detect the presence of a given organism's DNA in a sample. Currently, there is a drive to use qPCR data to infer biomass or abundance by quantitating the copy number or concentration of a given target gene fragment in a sample, which is often very dilute. Before eDNA can be fully accepted as an environmental decision‐making tool, however, certain aspects of the methods require standardization, including the quantification of target DNA in low copy number samples. Models that are not able to properly make use of data from highly dilute samples are severely hampered in their definitions of the limits of detection and quantification at the lower end of the detection curve. We propose a statistical model for a standard curve that relates the number of qPCR‐detected technical replicates to the copy number in the case of low copy number samples. Likelihood methods are used to estimate the parameters of the model and we derive inverse regression estimates together with their standard errors. Limits of copy number detection and quantification, and their confidence intervals are derived using a well‐accepted statistical approach thus providing a more broadly applicable and robust method for reporting eDNA abundance into the low copy number range. The method is illustrated using experimental results from multiple laboratories.

Variations in postmortem metabolism in muscle impact pork quality development. Curiously, some genetic lines are more refractile to adverse pork quality development than others and may regulate energy metabolism differently. The aim of this study was to challenge pork carcasses from different genetic populations with electrical stimulation (ES) to determine how postmortem metabolism varies with genetic line and explore control points that reside in glycolysis in dying muscle. Three genetic populations (GP) were subjected to ES (100 V or 200 V, 13 pulses, 2 s on/2 s off) at 15- or 25-min post-exsanguination, or no stimulation (NS). Genetic population affected relative muscle relative abundance of different myosin heavy chains, glycogen, G6P, and lactate concentrations. Genetic lines responded similarly to ES, but a comparison of ES treatment groups revealed a trend for an interaction between voltage, time of ES, and time postmortem. Higher voltage accelerated pH decline at 20 min up to 60 min postmortem. Trends in color and firmness scores and L* values were consistent with pH and metabolite data. These data show that genetic populations respond differently to postmortem perturbation by altering glycolytic flux and suggest differences in postmortem glycolysis may be partially responsible for differences in meat quality between genetic populations, though not entirely.

Solanum section Leptostemonum is an ideal lineage to test the theoretical framework regarding proposed evolutionary benefits of outcrossing sexual systems in comparison to cosexuality. Theoretically, non-cosexual taxa should support more genetic diversity within populations, experience less inbreeding, and have less genetic structure due to a restricted ability to self-fertilize. However, many confounding factors present challenges for a confident inference that inherent differences in sexual systems influence observed genetic patterns among populations. This study provides a foundational baseline of the population genetics of several species of different sexual systems with the aim of generating hypotheses of any factor—including sexual system—that influences genetic patterns. Importantly, results indicate that dioecious S. asymmetriphyllum maintains less genetic structure and greater admixture among populations than cosexual S. raphiotes at the same three locations where they co-occur. This suggests that when certain conditions are met, the evolution of dioecy may have proceeded as a means to avoid genetic consequences of self-compatibility and may support hypotheses of benefits gained through differential resource allocation partitioned across sexes. Arguably, the most significant finding of this study is that all taxa are strongly inbred, possibly reflective of a shared response to recent climate shifts, such as the increased frequency and intensity of the region’s fire regime.

Objectives: Dysregulation of cholesterol metabolism in the liver and hematopoietic stem and progenitor cells (HSPCs) promotes atherosclerosis development. Previously, it has been shown that HMG-CoA-Reductase (HMGCR), the rate-limiting enzyme in the mevalonate pathway, can be phosphorylated and inactivated by the metabolic stress sensor AMP-activated protein kinase (AMPK). However, the physiological significance of AMPK regulation of HMGCR to atherogenesis has yet to be elucidated. The aim of this study was to determine the role of AMPK/HMGCR axis in the development of atherosclerosis. Methods: We have generated a novel atherosclerotic-prone mouse model with defects in the AMPK regulation of HMGCR (Apoe−/−/Hmgcr KI mice). Atherosclerotic lesion size, plaque composition, immune cell and lipid profiles were assessed in Apoe−/− and Apoe−/−/Hmgcr KI mice. Results: In this study, we showed that both male and female atherosclerotic-prone mice with a disruption of HMGCR regulation by AMPK (Apoe−/−/Hmgcr KI mice) display increased aortic lesion size concomitant with an increase in plaque-associated macrophages and lipid accumulation. Consistent with this, Apoe−/−/Hmgcr KI mice exhibited an increase in total circulating cholesterol and atherogenic monocytes, Ly6-Chi subset. Mechanistically, increased circulating atherogenic monocytes in Apoe−/−/Hmgcr KI mice was associated with enhanced egress of bone marrow HSPCs and extramedullary myelopoiesis, driven by a combination of elevated circulating 27-hydroxycholesterol and intracellular cholesterol in HSPCs. Conclusions: Our results uncovered a novel signalling pathway involving AMPK-HMGCR axis in the regulation of cholesterol homeostasis in HSPCs, and that inhibition of this regulatory mechanism accelerates the development and progression of atherosclerosis. These findings provide a molecular basis to support the use of AMPK activators that currently undergoing Phase II clinical trial such as O–3O4 and PXL 770 for reducing atherosclerotic cardiovascular disease risks.

Abstract While atypical sensory processing is one of the more ubiquitous symptoms in autism spectrum disorder, the exact nature of these sensory issues remains unclear, with different studies showing either enhanced or deficient sensory processing. Using a well-established continuous cued-recall task that assesses visual working memory, the current study provides novel evidence reconciling these apparently discrepant findings. Autistic children exhibited perceptual advantages in both likelihood of recall and recall precision relative to their typically-developed peers. When autistic children did make errors, however, they showed a higher probability of erroneously binding a given colour with the incorrect spatial location. These data align with neural-architecture models for feature binding in visual working memory, suggesting that atypical population-level neural noise in the report dimension (colour) and cue dimension (spatial location) may drive both the increase in probability of recall and precision of colour recall as well as the increase in proportion of binding errors when making an error, respectively. These changes are likely to impact core symptomatology associated with autism, as perceptual binding and working memory play significant roles in higher-order tasks, such as communication.

Resilience of power systems is already a key issue that is getting frequent attention all over the world. It is useful to analyze resilience issues not only for bulk supply, but at all levels including at a customer level. This is because distributed energy resources can play a prominent role in enhancing resilience. Although the literature on planning models, tools and data for bulk supply and distribution systems have expanded in recent years, customer-centric planning, e.g., for an individual household, is yet to receive adequate attention. Although solar PV and battery storage at a household level have been analyzed, how these resources can be optimally combined, together with grid supply, from a resilience perspective is the focus of this study. The study demonstrates how a conceptual framework can be developed to show the trade-off between system costs and resilience including its dimensions such as duration, depth and frequency of service outages. A planning model is developed that incorporates multiple facets of resilience and individual customer preferences. The model considers power system resilience explicitly as a constraint. The model is implemented for a household level case study in Miami, Florida. The results show there are complex trade-offs among different dimensions of resilience. The study demonstrates how combined resilience metrics can be formulated and evaluated using the proposed least-cost planning model at a household level to optimize grid supply together with solar, battery storage and diesel generators. The model allows a planner to directly embed a resilience standard to drive the optimal supply mix. These concepts and the modeling construct can also be applied at other levels of planning, including community level and bulk supply system planning.

The dysregulation of myeloid-derived cell metabolism can drive atherosclerosis. AMP-activated protein kinase (AMPK) controls various aspects of macrophage dynamics and lipid homeostasis, which are important during atherogenesis. Using LysM-Cre to drive the deletion of both the α1 and α2 catalytic subunits (MacKO), we aimed to clarify the role of myeloid-specific AMPK signaling in male and female mice made acutely atherosclerotic by injection of AAV vector encoding a gain-of-function mutant PCSK9 (PCSK9-AAV) and WD feeding. After 6 weeks of WD feeding, mice received a daily injection of either the AMPK activator A-769662 or a vehicle control for an additional 6 weeks. Following this (12 weeks total), we assessed myeloid cell populations and differences between genotype or sex were not observed. Similarly, aortic sinus plaque size, lipid staining, and necrotic area did not differ in male and female MacKO mice compared with their littermate floxed controls. Moreover, therapeutic intervention with A-769662 showed no treatment effect. There were also no observable differences in the amount of circulating total cholesterol or triglyceride, and only minor differences in the levels of inflammatory cytokines between groups. Finally, CD68+ area and markers of autophagy showed no effect of either lacking AMPK signaling or AMPK activation. Our data suggest that while defined roles for each catalytic AMPK subunit have been identified, complete deletion of myeloid AMPK signaling does not significantly impact atherosclerosis. Additionally, these findings suggest that intervention with the first-generation AMPK activator A-769662 is not able to stem the progression of atherosclerosis.

Early stages of Alzheimer's disease (AD) are associated with volume reductions in specific subregions of the medial temporal lobe (MTL). Using a manual segmentation method-the Olsen-Amaral-Palombo (OAP) protocol-previous work in healthy older adults showed that reductions in grey matter volumes in MTL subregions were associated with lower scores on the Montreal Cognitive Assessment (MoCA), suggesting atrophy may occur prior to diagnosis of mild cognitive impairment, a condition that often progresses to AD. However, current "gold standard" manual segmentation methods are labour intensive and time consuming. Here, we examined the utility of Automatic Segmentation of Hippocampal Subfields (ASHS) to detect volumetric differences in MTL subregions of healthy older adults who varied in cognitive status as determined by the MoCA. We trained ASHS on the OAP protocol to create the ASHS-OAP atlas and then examined how well automated segmentation replicated manual segmentation. Volumetric measures obtained from the ASHS-OAP atlas were also contrasted against those from the ASHS-PMC atlas, a widely used atlas provided by the ASHS team. The pattern of volumetric results was similar between the ASHS-OAP atlas and manual segmentation for anterolateral entorhinal cortex and perirhinal cortex, suggesting that ASHS-OAP is a viable alternative to current manual segmentation methods for detecting group differences based on cognitive status. Although ASHS-OAP and ASHS-PMC produced varying volumes for most regions of interest, they both identified early signs of neurodegeneration in CA2/CA3/DG and identified marginal differences in entorhinal cortex. Our findings highlight the utility of automated segmentation methods but still underscore the need for a unified and harmonized MTL segmentation atlas.

Choline is an essential nutrient and a critical component of the membrane phospholipid phosphatidylcholine (PC), the neurotransmitter acetylcholine, while also contributing to the methylation pathway. In the liver specifically, PC is the major membrane constituent and can be synthesized by the cytidine diphosphate–choline or the phosphatidylethanolamine N‐methyltransferase pathway. With the continuing global rise in the rates of obesity and nonalcoholic fatty liver disease, we sought to explore how excess fatty acids on primary hepatocytes and diet‐induced obesity affect choline uptake and metabolism. Our results demonstrate that hepatocytes chronically treated with palmitate, but not oleate or a mixture, had decreased choline uptake, which was associated with lower choline incorporation into PC and lower expression of choline transport proteins. Interestingly, a reduction in the rate of degradation spared PC levels in response to palmitate when compared with control. The effects of palmitate treatment were independent of endoplasmic reticulum stress, which counterintuitively augmented choline transport and transporter expression. In a model of obesity‐induced hepatic steatosis, male mice fed a 60% high‐fat diet for 10 weeks had significantly diminished hepatic choline uptake compared with lean mice fed a control diet. Although the transcript and protein expression of various choline metabolic enzymes fluctuated slightly, we observed reduced protein expression of choline transporter‐like 1 (CTL1) in the liver of mice fed a high‐fat diet. Polysome profile analyses revealed that in livers of obese mice, the CTL1 transcript, despite being more abundant, was translated to a lesser extent compared with lean controls. Finally, human liver cells demonstrated a similar response to palmitate treatment. Conclusion: Our results suggest that the altered fatty acid milieu seen in obesity‐induced fatty liver disease progression may adversely affect choline metabolism, potentially through CTL1, but that compensatory mechanisms work to maintain phospholipid homeostasis.

Obesity, dyslipidemia, and insulin resistance, the increasingly common metabolic syndrome, are risk factors for CVD and type 2 diabetes that warrant novel therapeutic interventions. The flavonoid nobiletin displays potent lipid-lowering and insulin-sensitizing properties in mice with metabolic dysfunction. However, the mechanisms by which nobiletin mediates metabolic protection are not clearly established. The central role of AMP-activated protein kinase (AMPK) as an energy sensor suggests that AMPK is a target of nobiletin. We tested the hypothesis that metabolic protection by nobiletin required phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in mouse hepatocytes, in mice deficient in hepatic AMPK (Ampkβ1−/−), in mice incapable of inhibitory phosphorylation of ACC (AccDKI), and in mice with adipocyte-specific AMPK deficiency (iβ1β2AKO). We fed mice a high-fat/high-cholesterol diet with or without nobiletin. Nobiletin increased phosphorylation of AMPK and ACC in primary mouse hepatocytes, which was associated with increased FA oxidation and attenuated FA synthesis. Despite loss of ACC phosphorylation in Ampkβ1−/− hepatocytes, nobiletin suppressed FA synthesis and enhanced FA oxidation. Acute injection of nobiletin into mice did not increase phosphorylation of either AMPK or ACC in liver. In mice fed a high-fat diet, nobiletin robustly prevented obesity, hepatic steatosis, dyslipidemia, and insulin resistance, and it improved energy expenditure in Ampkβ1−/−, AccDKI, and iβ1β2AKO mice to the same extent as in WT controls. Thus, the beneficial metabolic effects of nobiletin in vivo are conferred independently of hepatic or adipocyte AMPK activation. These studies further underscore the therapeutic potential of nobiletin and begin to clarify possible mechanisms.

Small-scale battery energy storage systems (BESS), especially for behind-the-meter applications, are still relatively expensive, but we show that it can be a potent solution to render homes resilient to storm related power outages. We present a stochastic programming model formulation to optimize PV/BESS explicitly accounting for resilience benefits these investments entail, over and above their ability to reduce cost of supply. The stochastic optimization considers uncertainties around storm related grid supply failures as well as variability of solar PV. The model includes an embedded Monte Carlo simulation module that considers storm related outage risks using climate model reanalysis data. It is a least-cost planning framework that optimizes selection of BESS, solar PV, grid supply, and diesel generator, from a home-owner's perspective. We present two case studies with low and high storm risks that demonstrate how different risk exposures can impact on the selection of alternative options to build resilience. Duals, or shadow prices, of demand-supply constraints from the model for both normal days and for storm related contingencies, provide interesting insights into the marginal cost of supply that can inform innovative pricing schemes to promote customer level resilience measures. The case study results reveal significant merits of BESS, in combination with PV, to enhance resilience. We find that in low-risk areas like Bethesda, MD, incremental PV and BESS required for a more resilient system can add $79 (4%) to annual electricity costs for a typical household, and a considerably higher $208 (10.6%) in Miami, FL which is at a much greater risk. These options are, however, 27% (in Bethesda) and 20% (in Florida) less expensive than the conventional solution of installing a diesel generator. These results provide insights into the value of BESS as part of a resilient and clean energy solution for households.

This paper presents the design and results of a model that uses household smart meter data, electric vehicle (EV) travel load and charging options, and multiple solar resource profiles, to make decisions on optimal combinations of photovoltaics (PV), battery energy storage systems (BESS) and EV charging strategies. The least-cost planning model is formulated as a stochastic mixed integer programming (MIP) problem that makes first stage decisions on PV/BESS investments, and recourse decisions on purchase/sell from/to the grid to minimize expected household electricity costs. The model undertakes a customer-centric optimization taking into consideration net metering policy, time-of-use grid pricing, and uncertainties around inter-annual variability of solar irradiance. The model adds to the existing literature in terms of stochastic representation of inter-annual variability of solar irradiance, together with BESS capacity optimization, and EV charging mode selection. Three case studies are presented: two for a residential house with and without EV load, and a third for a larger community facility. Results from the model for the first residential house case study are compared with commercially available software to show the impacts of an accurate load profile and different policy parameters. The stochastic feature of the model proves useful in understanding the impact of variability in solar resource profiles on PV sizing. Finally, simulations of alternative EV travel patterns and tariff policies that discourage charging during the evening peak show the efficacy of `super off-peak' pricing being introduced in the state of Maryland.

Mining and mineral processing continues to be a source of lasting environmental problems in many developing economies. Phytoremediation has proven to be a viable strategy to remediate contaminated lands and limit environmental damage, but it has not been widely implemented partially due to social and economic challenges. However, by encouraging phytoremediation with a focus on phytoextraction, it may be possible to rehabilitate contaminated lands while simultaneously providing economic support to local communities. This can be achieved by the sale of phytoextracted metals to fund large-scale phytoremediation, particularly in Sub-Saharan Africa. To this end, this paper provides a conceptual approach for phytoremediation-based mineral recovery and explores the social and economic challenges related to large-scale deployment. The viability of the approach is explored and future work on phytoremediation implementation is defined with the goal of advancing research and collaboration.

Objective: Salsalate is a prodrug of salicylate that lowers blood glucose in people with type 2 diabetes. AMP-activated protein kinase (AMPK) is an αβγ heterotrimer which inhibits macrophage inflammation and the synthesis of fatty acids and cholesterol in the liver through phosphorylation of acetyl-CoA carboxylase (ACC) and HMG-CoA reductase (HMGCR), respectively. Salicylate binds to and activates AMPKβ1-containing heterotrimers that are highly expressed in both macrophages and liver, but the potential importance of AMPK and ability of salsalate to reduce atherosclerosis have not been evaluated. Methods: ApoE−/− and LDLr−/− mice with or without (−/−) germline or bone marrow AMPKβ1, respectively, were treated with salsalate, and atherosclerotic plaque size was evaluated in serial sections of the aortic root. Studies examining the effects of salicylate on markers of inflammation, fatty acid and cholesterol synthesis and proliferation were conducted in bone marrow–derived macrophages (BMDMs) from wild-type mice or mice lacking AMPKβ1 or the key AMPK-inhibitory phosphorylation sites on ACC (ACC knock-in (KI)-ACC KI) or HMGCR (HMGCR-KI). Results: Salsalate reduced atherosclerotic plaques in the aortic roots of ApoE−/− mice, but not ApoE−/− AMPKβ1−/− mice. Similarly, salsalate reduced atherosclerosis in LDLr−/− mice receiving wild-type but not AMPKβ1−/− bone marrow. Reductions in atherosclerosis by salsalate were associated with reduced macrophage proliferation, reduced plaque lipid content and reduced serum cholesterol. In BMDMs, this suppression of proliferation by salicylate required phosphorylation of HMGCR and the suppression of cholesterol synthesis. Conclusions: These data indicate that salsalate suppresses macrophage proliferation and atherosclerosis through an AMPKβ1-dependent pathway, which may involve HMGCR phosphorylation and cholesterol synthesis. Since rapidly-proliferating macrophages are a hallmark of atherosclerosis, these data indicate further evaluation of salsalate as a potential therapeutic agent for treating atherosclerotic cardiovascular disease.