Previous Knee Surgery, Anteromedial Portal Drilling, Quadriceps Tendon Autograft, and Meniscal Involvement Associated With Delayed Return to Sport After Anterior Cruciate Ligament Reconstruction in Amateur Athletes.

Purpose: To identify prognostic factors associated with a delayed return-to-sport (RTS) time in amateur athletes who return to full participation after a primary isolated anterior cruciate ligament (ACL) reconstruction. Methods: A retrospective review was performed among athletes who underwent ACL reconstruction between October 2014 and October 2021. Inclusion criteria were any amateur athletes with an ACL reconstruction who had a documented RTS and greater than 1-year follow-up. Nonathletes, those with multiligamentous knee injury, and those missing documented RTS timelines were excluded. RTS was defined as participation in athletics at a level equivalent to or greater than the preinjury level participation. Demographic and prognostic factors, including previous knee surgery, meniscal involvement, level of participation, surgical approach, and graft type, were recorded along with RTS time and analyzed via Poisson regression. Results: In total, 91 athletes, average age 18.8 (± 6.7) years, who underwent ACL reconstruction at a single institution from 2014 to 2021 were identified with an average follow-up time of 4.6 (± 2.5) years (range 1.1, 9.0). Meniscal involvement (1.11; 95% confidence interval [CI] 1.08-1.15, P < .001) and previous knee surgery (1.43; 95% CI 1.29-1.58; P < .001) were related to a delayed RTS. Quadriceps tendon and bone-patellar tendon-bone autografts, as well as allograft, showed a significant association with a longer RTS time when compared with hamstring autograft (1.16, 95% CI 1.13-1.20, P < .001; 1.04, 95% CI 1.01-1.07, P = .020; 1.11, 95% CI 1.03-1.19, P = .004, respectively), as did anteromedial portal drilling, when compared with the outside in approach for femoral drilling (1.19, 95% CI 1.16-1.23, P < .001). Conclusions: Previous knee surgery, anteromedial femoral drilling, quadriceps tendon autograft, and meniscus tear were most associated with a delayed timeline for RTS among young athletes who were able to return. Level of Evidence: Level IV, prognostic case series.

Assessment of a Pilot Peer Support Program for Suicide Prevention on a Medical School Campus: Impact on Awareness, Stigma, and Self-Efficacy for Outreach.

With the continued rise in mental health concerns, including suicide on college campuses nationwide, many academic institutions have developed peer-support programs. Correspondingly, the Medical College of Wisconsin developed and evaluated Seeking Peer Outreach* as its pioneer suicide prevention initiative. Seeking Peer Outreach* is an innovative approach to provide all medical students near-peer support and outreach encouraging engagement and conversations in effort to reduce the stigma and isolation often associated with mental health concerns in professional education. This study explores the effectiveness and efficacy of Seeking Peer Outreach* - a 3-tiered peer-support system. A survey of medical students, faculty, and staff demonstrated that the program increased knowledge on suicidal thoughts and behaviors and improved self-efficacy in talking about mental health with peers. It also showed that effective training helps individuals gain confidence with mental health interventions and suicide prevention.

The Impact of SIV-Induced Immunodeficiency on SARS-CoV-2 Disease, Viral Dynamics, and Antiviral Immune Response in a Nonhuman Primate Model of Coinfection.

The effects of immunodeficiency associated with chronic HIV infection on COVID-19 disease and viral persistence have not been directly addressed in a controlled setting. In this pilot study, we exposed two pigtail macaques (PTMs) chronically infected with SIVmac239, exhibiting from very low to no CD4 T cells across all compartments, to SARS-CoV-2. We monitored the disease progression, viral replication, and evolution, and compared these outcomes with SIV-naïve PTMs infected with SARS-CoV-2. No overt signs of COVID-19 disease were observed in either animal, and the SARS-CoV-2 viral kinetics and evolution in the SIVmac239 PTMs were indistinguishable from those in the SIV-naïve PTMs in all sampled mucosal sites. However, the single-cell RNA sequencing of bronchoalveolar lavage cells revealed an infiltration of functionally inert monocytes after SARS-CoV-2 infection. Critically, neither of the SIV-infected PTMs mounted detectable anti-SARS-CoV-2 T-cell responses nor anti-SARS-CoV-2 binding or neutralizing antibodies. Thus, HIV-induced immunodeficiency alone may not be sufficient to drive the emergence of novel viral variants but may remove the ability of infected individuals to mount adaptive immune responses against SARS-CoV-2.

Antibodies to Influenza A Virus in Lesser (Aytha affinis) and Greater Scaup (Aytha marila) in the USA.

Scaup, including both Lesser and Greater (Aythya affinis and Aythya marila, respectively), are a grouping of populous and widespread North American diving ducks. Few influenza type A viruses (IAV) have been reported from these species despite a high prevalence of antibodies to IAV being reported. Existing virologic and serologic data indicate that IAV infection routinely occurs in scaup, yet it is unknown which IAV subtypes are linked to these infections. In this study, we aimed to gain a more complete picture of IAV natural history in Lesser and Greater Scaup from two coastal flyways in North America in 2015-18 (302 samples from California in the Pacific Flyway and 471 samples from Maryland in the Atlantic Flyway). Low prevalence of active IAV infection was detected by real-time reverse-transcription PCR in Lesser Scaup sampled in Maryland and California (2.8% and 8.1%, respectively). A single IAV (H1N1) was isolated in embryonated chicken eggs from a bird sampled in California. Similarly low levels were observed in Greater Scaup in California (3.3%). Antibodies to the nucleoprotein as detected with a commercial blocking ELISA were observed in all species and flyway combinations. Antibody seroprevalence estimates were higher in adult Lesser Scaup than in juveniles at both the ≤0.5 (P<0.001, z=-3.582) and ≤0.7 serum-sample-to-negative-control absorbance thresholds (P=0.003, z=-2.996). Neutralizing antibodies to H1-H12, H14, and H15 were detected using a microtiter virus neutralization assay, with the highest prevalence of antibodies against H1 (38%), H6 (36%), and H11 (35%). The high prevalence of antibodies to IAV and evidence of previous exposure to numerous subtypes are consistent with a high level of population immunity and a low prevalence of infection. These results must be interpreted in the context of season (winter sampling), as results may vary with the annual influx of naïve juvenile birds.

Using an adaptive modeling framework to identify avian influenza spillover risk at the wild-domestic interface.

The wild to domestic bird interface is an important nexus for emergence and transmission of highly pathogenic avian influenza (HPAI) viruses. Although the recent incursion of HPAI H5N1 Clade 2.3.4.4b into North America calls for emergency response and planning given the unprecedented scale, readily available data-driven models are lacking. Here, we provide high resolution spatial and temporal transmission risk models for the contiguous United States. Considering virus host ecology, we included weekly species-level wild waterfowl (Anatidae) abundance and endemic low pathogenic avian influenza virus prevalence metrics in combination with number of poultry farms per commodity type and relative biosecurity risks at two spatial scales: 3 km and county-level. Spillover risk varied across the annual cycle of waterfowl migration and some locations exhibited persistent risk throughout the year given higher poultry production. Validation using wild bird introduction events identified by phylogenetic analysis from 2022 to 2023 HPAI poultry outbreaks indicate strong model performance. The modular nature of our approach lends itself to building upon updated datasets under evolving conditions, testing hypothetical scenarios, or customizing results with proprietary data. This research demonstrates an adaptive approach for developing models to inform preparedness and response as novel outbreaks occur, viruses evolve, and additional data become available.

Understanding the Phase Behavior of a Multistimuli-Responsive Elastin-like Polymer: Insights from Dynamic Light Scattering Analysis.

Elastin-like polymers are a class of stimuli-responsive protein polymers that hold immense promise in applications such as drug delivery, hydrogels, and biosensors. Yet, understanding the intricate interplay of factors influencing their stimuli-responsive behavior remains a challenging frontier. Using temperature-controlled dynamic light scattering and zeta potential measurements, we investigate the interactions between buffer, pH, salt, water, and protein using an elastin-like polymer containing ionizable lysine residues. We observed the elevation of transition temperature in the presence of the common buffering agent HEPES at low concentrations, suggesting a "salting-in" effect of HEPES as a cosolute through weak association with the protein. Our findings motivate a more comprehensive investigation of the influence of buffer and other cosolute molecules on elastin-like polymer behavior.

Isolation of Naegleria lustrarea n. sp. (Excavata, Discoba, Heterolobosea) from the feces of Ambystoma annulatum (Ringed Salamander) in Northwest Arkansas.

The salamander, Ambystoma annulatum, is considered a "species of special concern" in the state of Arkansas, USA, due to its limited geographic range, specialized habitat requirements and low population size. Although metazoan parasites have been documented in this salamander species, neither its native protists nor microbiome have yet been evaluated. This is likely due to the elusive nature and under-sampling of the animal. Here, we initiate the cataloguing of microbial associates with the identification of a new heterlobosean species, Naegleria lustrarea n. sp. (Excavata, Discoba, Heterolobosea), isolated from feces of an adult A. annulatum.

A Surgical Protocol for Establishing Spinal Cord Ischemia with Extended Lifespan and Low Complication Rates in Rats.

BACKGROUND: Experimental animal models of ischemic spinal cord injury (iSCI) are essential for studying its pathogenesis and for developing new therapeutic strategies to improve functional recovery in humans. Many existing models, however, exhibit high variability or early lethality. A reliable experimental iSCI model would significantly advance novel treatment approaches for these severe neurological disorders. To this end, we have established a rat model of persistent iSCI with an extended lifespan. METHODS: We have developed a novel iSCI model that induces localized ischemic lesions in the spinal cord of male Sprague-Dawley rats. This is achieved by cross clamping the descending aorta just rostral the azygos vein using an atraumatic bulldog clamp. RESULTS: The experimental iSCI model consistently demonstrated symptoms specific to spinal cord ischemia at the lumbar level. The procedure takes approximately 50 min and does not require specialized surgical equipment. It has a survival rate of 84%, a recovery rate of 40%, and a complication rate of 16%. CONCLUSIONS: We have successfully developed a rat model of persistent iSCI. This protocol proves to be highly reliable and holds promise for evaluating new therapeutic strategies aimed at promoting functional recovery in patients suffering from spinal cord ischemia.

Arthroscopic suture bridge fixation for acute bony Bankart with anterior glenohumeral instability: a case report and narrative review.

Background and Objective: Anterior shoulder dislocations can result in acute glenoid rim fractures that compromise the bony stability of the glenohumeral joint. Adequate fixation of these fractures is required to restore stability, decrease shoulder pain, and facilitate return to activity. The double-row suture bridge is a relatively novel fixation technique, first described in 2009, that accomplishes internal fixation with sufficient stability using an all-arthroscopic technique to restore the glenoid footprint. A 40-year-old female with recurrent anterior shoulder instability in the setting of seizure disorder was found to have a bony Bankart lesion of 25% to 30% with a concomitant superior labral tear. The patient was treated with a double-row bony Bankart bridge and labral repair. At six months follow-up, she has progressed to a full recovery with no recurrence. Methods: A search was conducted in May 2023 in PubMed, EMBASE, and CINAHL with the search terms bony Bankart, bone Bankart, osseous Bankart, acute, bridge, suture bridge, double row. Key Content and Findings: Double-row suture bridge repairs result in improvement in shoulder function as determined by ASES (93.5), QuickDASH (4.5), SANE (95.9), and SF-12 (55.6). The overall recurrence rate of anterior instability after a bony Bankart bridge repair is 8%. When examining the return to prior level of function, 81.4% of patients were able to do so with only 7.9% of patients reporting significant modifications to their activity level. In mid-term results, double row suture bridge demonstrates similar outcomes to other all-arthroscopic fixation methods of bony Bankart injuries. Importantly, bony Bankart bridge remains a viable option for critical glenoid lesions over the 20% cutoff used in other all arthroscopic techniques. Biomechanically, the double-row suture bridge offers distinct benefits over its single-row counterpart including increased compression, reduced displacement, and reduced step-off. Conclusions: Although there is limited data, the studies discussed and the demonstrative case show the potential benefit of all-arthroscopic double-row suture bridge fixation including increased compression, decreased displacement, and a lower complication rate in patients with large bony Bankart lesions traditionally requiring bony augmentation. However, more robust studies are necessary to determine the long-term success of the double-row suture bridge.

Critical Reflection to Investigate Medical Student Attitudes Toward Skin Tone in Their Preclinical Years.

INTRODUCTION: Implicit racial bias, defined as unreasoned judgement based solely on an individual's skin color, is a persistent barrier to quality medical care for people of color in the United States. Early, learner-centered intervention is crucial to establish cultural competence within health professional training programs. METHODS: Over 3 academic years, preclinical, second-year medical students were asked to submit an anonymous critical reflection regarding skin tone in medicine (n=794). Critical reflection is an instructional approach that encourages students to investigate their own thoughts and actions. Course credit was given based on the honor system. Reflection submission content and student feedback were analyzed quantitatively and qualitatively using constructivist thematic analysis. RESULTS: Most students completed the assignment (93.0%) and reported feeling comfortable expressing themselves honestly in the anonymous format (84.6%). Students' comfort level with honesty declined if they would have had to identify themselves (50.8%). Student comments indicated relief to have a place to process experiences and emphasized the importance of anonymity for value of this assignment. Thematic analysis identified 2 themes and 13 subthemes among student submissions. Submissions varied in format and typically contained multiple codes (4.08 ± 1.77 subthemes), indicating that students participated meaningfully in the assignment. CONCLUSIONS: Although some educators may hesitate to address these topics, students at our institution appreciated having a space to process their thoughts. This assignment structure is an effective way for educators to address a difficult, sensitive, and important topic in a meaningful way with students.

Thbs1 regulates skeletal muscle mass in a TGFß-Smad2/3-ATF4-dependent manner.

Loss of muscle mass is a feature of chronic illness and aging. Here, we report that skeletal muscle-specific thrombospondin-1 transgenic mice (Thbs1 Tg) have profound muscle atrophy with age-dependent decreases in exercise capacity and premature lethality. Mechanistically, Thbs1 activates transforming growth factor ß (TGFß)-Smad2/3 signaling, which also induces activating transcription factor 4 (ATF4) expression that together modulates the autophagy-lysosomal pathway (ALP) and ubiquitin-proteasome system (UPS) to facilitate muscle atrophy. Indeed, myofiber-specific inhibition of TGFß-receptor signaling represses the induction of ATF4, normalizes ALP and UPS, and partially restores muscle mass in Thbs1 Tg mice. Similarly, myofiber-specific deletion of Smad2 and Smad3 or the Atf4 gene antagonizes Thbs1-induced muscle atrophy. More importantly, Thbs1-/- mice show significantly reduced levels of denervation- and caloric restriction-mediated muscle atrophy, along with blunted TGFß-Smad3-ATF4 signaling. Thus, Thbs1-mediated TGFß-Smad3-ATF4 signaling in skeletal muscle regulates tissue rarefaction, suggesting a target for atrophy-based muscle diseases and sarcopenia with aging.

MCU genetically altered mice suggest how mitochondrial Ca2+ regulates metabolism.

Skeletal muscle has a major impact on total body metabolism and obesity, and is characterized by dynamic regulation of substrate utilization. While it is accepted that acute increases in mitochondrial matrix Ca2+ increase carbohydrate usage to augment ATP production, recent studies in mice with deleted genes for components of the mitochondrial Ca2+ uniporter (MCU) complex have suggested a more complicated regulatory scenario. Indeed, mice with a deleted Mcu gene in muscle, which lack acute mitochondrial Ca2+ uptake, have greater fatty acid oxidation (FAO) and less adiposity. By contrast, mice deleted for the inhibitory Mcub gene in skeletal muscle, which have greater acute mitochondrial Ca2+ uptake, antithetically display reduced FAO and progressive obesity. In this review we discuss the emerging concept that dynamic fluxing of mitochondrial matrix Ca2+ regulates metabolism.

Lactate transporter MCT1 in hepatic stellate cells promotes fibrotic collagen expression in nonalcoholic steatohepatitis.

Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline-deficient, high-fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.

MCU-independent Ca2+ uptake mediates mitochondrial Ca2+ overload and necrotic cell death in a mouse model of Duchenne muscular dystrophy.

Mitochondrial Ca2+ overload can mediate mitochondria-dependent cell death, a major contributor to several human diseases. Indeed, Duchenne muscular dystrophy (MD) is driven by dysfunctional Ca2+ influx across the sarcolemma that causes mitochondrial Ca2+ overload, organelle rupture, and muscle necrosis. The mitochondrial Ca2+ uniporter (MCU) complex is the primary characterized mechanism for acute mitochondrial Ca2+ uptake. One strategy for preventing mitochondrial Ca2+ overload is deletion of the Mcu gene, the pore forming subunit of the MCU-complex. Conversely, enhanced MCU-complex Ca2+ uptake is achieved by deleting the inhibitory Mcub gene. Here we show that myofiber-specific Mcu deletion was not protective in a mouse model of Duchenne MD. Specifically, Mcu gene deletion did not reduce muscle histopathology, did not improve muscle function, and did not prevent mitochondrial Ca2+ overload. Moreover, myofiber specific Mcub gene deletion did not augment Duchenne MD muscle pathology. Interestingly, we observed MCU-independent Ca2+ uptake in dystrophic mitochondria that was sufficient to drive mitochondrial permeability transition pore (MPTP) activation and skeletal muscle necrosis, and this same type of activity was observed in heart, liver, and brain mitochondria. These results demonstrate that mitochondria possess an uncharacterized MCU-independent Ca2+ uptake mechanism that is sufficient to drive MPTP-dependent necrosis in MD in vivo.

Identification of QTLs for symbiotic nitrogen fixation and related traits in a soybean recombinant inbred line population.

KEY MESSAGE: The genetic architecture of symbiotic N fixation and related traits was investigated in the field. QTLs were identified for percent N derived from the atmosphere, shoot [N] and C to N ratio. Soybean [Glycine max (L.) Merr.] is cultivated worldwide and is the most abundant source of plant-based protein. Symbiotic N2 fixation (SNF) in legumes such as soybean is of great importance; however, yields may still be limited by N in both high yielding and stressful environments. To better understand the genetic architecture of SNF and facilitate the development of high yielding cultivars and sustainable soybean production in stressful environments, a recombinant inbred line population consisting of 190 lines, developed from a cross between PI 442012A and PI 404199, was evaluated for N derived from the atmosphere (Ndfa), N concentration ([N]), and C to N ratio (C/N) in three environments. Significant genotype, environment and genotype × environment effects were observed for all three traits. A linkage map was constructed containing 3309 single nucleotide polymorphism (SNP) markers. QTL analysis was performed for additive effects of QTLs, QTL × environment interactions, and QTL × QTL interactions. Ten unique additive QTLs were identified across all traits and environments. Of these, two QTLs were detected for Ndfa and eight for C/N. Of the eight QTLs for C/N, four were also detected for [N]. Using QTL × environment analysis, six QTLs were detected, of which five were also identified in the additive QTL analysis. The QTL × QTL analysis identified four unique epistatic interactions. The results of this study may be used for genomic selection and introgression of favorable alleles for increased SNF, [N], and C/N via marker-assisted selection.

Batter's Shoulder: All-Knotless Posterior Labral Repair With Retensionable Anchors for Treatment of Batter's Shoulder.

Batter's Shoulder is a unique injury that may be associated with recurrent microtrauma followed by acute subluxation of the humeral head on the posterior glenoid edge, leading to posterior labral tears. Early identification of this injury is critical, as it may be treated with conservative nonsurgical treatments prior to labral tear onset. If conservative treatment fails and pain persists, surgical options include arthroscopic fixation to reapproximate the posterior labrum to the glenoid and restore capsular tension. Previous studies have shown the benefit of using knotless suture anchors in arthroscopic shoulder fixation. This technical note demonstrates that Batter's Shoulder is a unique injury associated with posterior labral tears of the shoulder and provides a contemporary method of arthroscopic fixation of a posterior labral tear using retensionable knotless all-suture anchors.

Evaluating the Need for Simultaneous Carpal Tunnel Release With Forearm Fasciotomy.

PURPOSE: The need to include simultaneous carpal tunnel release (sCTR) with forearm fasciotomy for acute compartment syndrome (ACS) or after vascular repair is unclear. We hypothesized that sCTR is more common when: 1) fasciotomies are performed by orthopedic or plastic surgeons, rather than general or vascular surgeons; 2) ACS occurred because of crush, blunt trauma, or fractures rather than vascular/reperfusion injuries; 3) elevated compartment pressures were documented. We also sought to determine the incidence of delayed CTR when not performed simultaneously. METHODS: Retrospective chart review identified patients who underwent forearm fasciotomy for ACS or vascular injury over a period of 10 years. Patient demographics, mechanism of ACS or indication for fasciotomy, surgeon subspecialty, compartment pressure measurements, inclusion of sCTR, complications, reoperations, and timing and method of definitive closure were analyzed. Logistic regression modeling was used to analyze predictors associated with delayed CTR. RESULTS: Fasciotomies were performed in 166 patients by orthopedic (63%), plastic (28%), and general/vascular (9%) surgeons. Orthopedic and plastic surgeons more frequently performed sCTR (67% and 63%, respectively). A total of 107 (65%) patients had sCTR. Fasciotomies for vascular/reperfusion injury were more likely to include sCTR (44%) compared with other mechanisms. If not performed simultaneously, 11 (19%) required delayed CTR at a median of 42 days. ACS secondary to fracture had the highest rate of delayed CTR (35%), and the necessity of late CTR for fractures was not supported by the logistic regression model. Residual hand paresthesias were less frequent in the sCTR group (6.5% vs 20%). Overall complication rates were similar in both groups (63% sCTR vs 70% without sCTR). CONCLUSION: When sCTR is excluded during forearm fasciotomy, 19% of patients required delayed CTR. This rate was higher (35%) when ACS was associated with fractures. Simultaneous CTR with forearm fasciotomy may decrease the incidence of residual hand paresthesias and the need for a delayed CTR. TYPE OF STUDY/LEVEL OF EVIDENCE: Prognosis IV.

MEK1-ERK1/2 signaling regulates the cardiomyocyte non-sarcomeric actin cytoskeletal network.

During select pathological conditions, the heart can hypertrophy and remodel in either a dilated or concentric ventricular geometry, which is associated with lengthening or widening of cardiomyocytes, respectively. The mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-related kinase 1 and 2 (ERK1/2) pathway has been implicated in these differential types of growth such that cardiac overexpression of activated MEK1 causes profound concentric hypertrophy and cardiomyocyte thickening, while genetic ablation of the genes encoding ERK1/2 in the mouse heart causes dilation and cardiomyocyte lengthening. However, the mechanisms by which this kinase signaling pathway controls cardiomyocyte directional growth as well as its downstream effectors are poorly understood. To investigate this, we conducted an unbiased phosphoproteomic screen in cultured neonatal rat ventricular myocytes treated with an activated MEK1 adenovirus, the MEK1 inhibitor U0126, or an eGFP adenovirus control. Bioinformatic analysis identified cytoskeletal-related proteins as the largest subset of differentially phosphorylated proteins. Phos-tag and traditional Western blotting were performed to confirm that many cytoskeletal proteins displayed changes in phosphorylation with manipulations in MEK1-ERK1/2 signaling. From this, we hypothesized that the actin cytoskeleton would be changed in vivo in the mouse heart. Indeed, we found that activated MEK1 transgenic mice and gene-deleted mice lacking ERK1/2 protein had enhanced non-sarcomeric actin expression in cardiomyocytes compared with wild-type control hearts. Consistent with these results, cytoplasmic ß- and γ-actin were increased at the subcortical intracellular regions of adult cardiomyocytes. Together, these data suggest that MEK1-ERK1/2 signaling influences the non-sarcomeric cytoskeletal actin network, which may be important for facilitating the growth of cardiomyocytes in length and/or width.NEW & NOTEWORTHY Here, we performed an unbiased analysis of the total phosphoproteome downstream of MEK1-ERK1/2 kinase signaling in cardiomyocytes. Pathway analysis suggested that proteins of the non-sarcomeric cytoskeleton were the most differentially affected. We showed that cytoplasmic ß-actin and γ-actin isoforms, regulated by MEK1-ERK1/2, are localized to the subcortical space at both lateral membranes and intercalated discs of adult cardiomyocytes suggesting how MEK1-ERK1/2 signaling might underlie directional growth of adult cardiomyocytes.

Rehabilitation facilitates functional improvement following intravenous infusion of mesenchymal stem cells in the chronic phase of cerebral ischemia in rats.

The primary objective of this study was to investigate the potential facilitating effects of daily rehabilitation for chronic cerebral ischemia following the intravenous infusion of mesenchymal stem cells (MSC) in rats. The middle cerebral artery (MCA) was occluded by intraluminal occlusion using a microfilament (MCAO). Eight weeks after MCAO induction, the rats were used as a chronic cerebral ischemia model. Four experimental groups were studied: Vehicle group (medium only, no cells); Rehab group (vehicle + rehabilitation), MSC group (MSC only); and Combined group (MSC + rehabilitation). Rat MSCs were intravenously infused eight weeks after MCAO induction, and the rats received daily rehabilitation through treadmill exercise for 20 min. Behavioral testing, lesion volume assessment using magnetic resonance imaging (MRI), and histological analysis were performed during the observation period until 16 weeks after MCAO induction. All treated animals showed functional improvement compared with the Vehicle group; however, the therapeutic efficacy was greatest in the Combined group. The combination therapy is associated with enhanced neural plasticity shown with histological analysis and MRI diffusion tensor imaging. These findings provide behavioral evidence for enhanced recovery by combined therapy with rehabilitation and intravenous infusion of MSCs, and may form the basis for the development of clinical protocols in the future.

Palmitoylation-dependent regulation of cardiomyocyte Rac1 signaling activity and minor effects on cardiac hypertrophy.

S-palmitoylation is a reversible lipid modification catalyzed by 23 S-acyltransferases with a conserved zinc finger aspartate-histidine-histidine-cysteine (zDHHC) domain that facilitates targeting of proteins to specific intracellular membranes. Here we performed a gain-of-function screen in the mouse and identified the Golgi-localized enzymes zDHHC3 and zDHHC7 as regulators of cardiac hypertrophy. Cardiomyocyte-specific transgenic mice overexpressing zDHHC3 show cardiac disease, and S-acyl proteomics identified the small GTPase Rac1 as a novel substrate of zDHHC3. Notably, cardiomyopathy and congestive heart failure in zDHHC3 transgenic mice is preceded by enhanced Rac1 S-palmitoylation, membrane localization, activity, downstream hypertrophic signaling, and concomitant induction of all Rho family small GTPases whereas mice overexpressing an enzymatically dead zDHHC3 mutant show no discernible effect. However, loss of Rac1 or other identified zDHHC3 targets Gαq/11 or galectin-1 does not diminish zDHHC3-induced cardiomyopathy, suggesting multiple effectors and pathways promoting decompensation with sustained zDHHC3 activity. Genetic deletion of Zdhhc3 in combination with Zdhhc7 reduces cardiac hypertrophy during the early response to pressure overload stimulation but not over longer time periods. Indeed, cardiac hypertrophy in response to 2 weeks of angiotensin-II infusion is not diminished by Zdhhc3/7 deletion, again suggesting other S-acyltransferases or signaling mechanisms compensate to promote hypertrophic signaling. Taken together, these data indicate that the activity of zDHHC3 and zDHHC7 at the cardiomyocyte Golgi promote Rac1 signaling and maladaptive cardiac remodeling, but redundant signaling effectors compensate to maintain cardiac hypertrophy with sustained pathological stimulation in the absence of zDHHC3/7.