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Background: Chronic pain from peripheral neuromas is difficult to manage and often requires surgical excision, though intraoperative identification of neuromas can be challenging due to anatomical ambiguity. Mechanical manipulation of the neuroma during surgery can elicit a characteristic "startle sign", which can help guide surgical management. However, it is unknown how anesthetic management affects detection of the startle sign., Methods: We performed a retrospective cohort study of 73 neuroma excision surgeries performed recently at Massachusetts General Hospital. Physiological changes in the anesthetic record were analyzed to identify associations with a startle sign event. Anesthesia type and doses of pharmacological agents were analyzed between startle sign and no-startle sign groups., Results: Of the 64 neuroma resection surgeries included, 13 had a startle sign. Combined intravenous and inhalation anesthesia (CIVIA) was more frequently used in the startle sign group vs. no-startle sign group (54% vs. 8%), while regional blockade with monitored anesthetic care was not associated with the startle sign group (12% vs. 0%), p = 0.001 for anesthesia type. Other factors, such as neuromuscular blocking agents, ketamine infusion, remifentanil infusion, and intravenous morphine equivalents showed no differences between groups., Conclusions: Here, we identified hypothesis-generating descriptive differences in anesthetic management associated with the detection of the neuroma startle sign during neuroma excision surgery, suggesting ways to deliver anesthesia facilitating detection of this phenomenon. Prospective trials are needed to further validate the hypotheses generated., (© 2024. The Author(s).)

Although combination antiretroviral therapy (ART) has been a landmark achievement for the treatment of human immunodeficiency virus (HIV), an HIV cure has remained elusive. Elimination of latent HIV reservoirs that persist throughout HIV infection is the most challenging barrier to an HIV cure. The progressive HIV infection is marked by the increasing size and diversity of latent HIV reservoirs until an effective immune response is mobilized, which can control but not eliminate HIV infection. The stalemate between HIV replication and the immune response is manifested by the establishment of a viral set point. ART initiation during the early stage limits HIV reservoir development, preserves immune function, improves the quality of life, and may lead to ART-free viral remission in a few people living with HIV (PLWH). However, for the overwhelming majority of PLWH, early ART initiation alone does not cure HIV, and lifelong ART is needed to sustain viral suppression. A critical area of research is focused on determining whether HIV could be functionally cured if additional treatments are provided alongside early ART. Several HIV interventions including Block and Lock, Shock and Kill, broadly neutralizing antibody (bNAb) therapy, adoptive CD8+ T cell therapy, and gene therapy have demonstrated delayed viral rebound and/or viral remission in animal models and/or some PLWH. Whether or not their application during early infection can improve the success of HIV remission is less studied. Herein, we review the current state of clinical and investigative HIV interventions and discuss their potential to improve the likelihood of post-treatment remission if initiated during early infection.

The mission of NIH-sponsored institutional training programs such as the T32 is to provide strong research and career training for early career scientists. One of the main avenues to pursuing health-related research is becoming research faculty at an academic institution. It is therefore important to know whether these programs are succeeding in this mission, or, if barriers exist that prevent trainees from pursuing these careers. Our institution currently trains ~ 2400 post-doctoral scholars per year, approximately 5% of whom are enrolled in one of our 33 T32 programs. In this study, we 1) compare the proximal professional career trajectories of T32 trainees with non-T32 trainees at our institution, 2) compare proximal career trajectories of trainees in a subset of cardiovascular T32 programs based on their previous training backgrounds, and 3) survey past and current T32 trainees in a subset of cardiovascular T32 programs about the barriers and enablers they experienced to pursuing research-oriented careers. We find that former T32 trainees are significantly more likely to attain appointments as primarily research faculty members, compared to other trainees. Trainees report a perceived lack of stability, the paucity of open positions, and the 'publish or perish' mentality of academia as the top barriers to pursuing careers in academia. However, they were still more likely to choose research over clinical careers after participating in a dedicated T32 program. Our results support the conclusion that structured training programs strengthen the pipeline of young scientists pursuing careers in academic research, including those from underrepresented backgrounds. However, T32 postdoctoral researchers are held back from pursuing academic careers by a perceived lack of stability and high competition for faculty positions., Competing Interests: Author Adrienne Mueller manages and authors Philip Tsao and Nicholas Leeper codirect a T32 program at Stanford University. All authors declare no additional competing interests. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Mueller et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Xenografts of human skeletal muscle generated in mice can be used to study muscle pathology and to test drugs designed to treat myopathies and muscular dystrophies for their efficacy and specificity in human tissue. We previously developed methods to generate mature human skeletal muscles in immunocompromised mice starting with human myogenic precursor cells (hMPCs) from healthy individuals and individuals with facioscapulohumeral muscular dystrophy (FSHD). Here, we examine a series of alternative treatments at each stage in order to optimize engraftment. We show that (i) X-irradiation at 25Gy is optimal in preventing regeneration of murine muscle while supporting robust engraftment and the formation of human fibers without significant murine contamination; (ii) hMPC lines differ in their capacity to engraft; (iii) some hMPC lines yield grafts that respond better to intermittent neuromuscular electrical stimulation (iNMES) than others; (iv) some lines engraft better in male than in female mice; (v) coinjection of hMPCs with laminin, gelatin, Matrigel, or Growdex does not improve engraftment; (vi) BaCl 2 is an acceptable replacement for cardiotoxin, but other snake venom preparations and toxins, including the major component of cardiotoxin, cytotoxin 5, are not; and (vii) generating grafts in both hindlimbs followed by iNMES of each limb yields more robust grafts than housing mice in cages with running wheels. Our results suggest that replacing cardiotoxin with BaCl 2 and engrafting both tibialis anterior muscles generates robust grafts of adult human muscle tissue in mice., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Background: Bedside electrical impedance tomography could be useful to visualize evolving pulmonary perfusion distributions when acute respiratory distress syndrome worsens or in response to ventilatory and positional therapies. In experimental acute respiratory distress syndrome, this study evaluated the agreement of electrical impedance tomography and dynamic contrast-enhanced computed tomography perfusion distributions at two injury time points and in response to increased positive end-expiratory pressure (PEEP) and prone position., Methods: Eleven mechanically ventilated (VT 8 ml · kg-1) Yorkshire pigs (five male, six female) received bronchial hydrochloric acid (3.5 ml · kg-1) to invoke lung injury. Electrical impedance tomography and computed tomography perfusion images were obtained at 2 h (early injury) and 24 h (late injury) after injury in supine position with PEEP 5 and 10 cm H2O. In eight animals, electrical impedance tomography and computed tomography perfusion imaging were also conducted in the prone position. Electrical impedance tomography perfusion (QEIT) and computed tomography perfusion (QCT) values (as percentages of image total) were compared in eight vertical regions across injury stages, levels of PEEP, and body positions using mixed-effects linear regression. The primary outcome was agreement between QEIT and QCT, defined using limits of agreement and Pearson correlation coefficient., Results: Pao2/Fio2 decreased over the course of the experiment (healthy to early injury, -253 [95% CI, -317 to -189]; early to late injury, -88 [95% CI, -151 to -24]). The limits of agreement between QEIT and QCT were -4.66% and 4.73% for the middle 50% quantile of average regional perfusion, and the correlation coefficient was 0.88 (95% CI, 0.86 to 0.90]; P < 0.001). Electrical impedance tomography and computed tomography showed similar perfusion redistributions over injury stages and in response to increased PEEP. QEIT redistributions after positional therapy underestimated QCT in ventral regions and overestimated QCT in dorsal regions., Conclusions: Electrical impedance tomography closely approximated computed tomography perfusion measures in experimental acute respiratory distress syndrome, in the supine position, over injury progression and with increased PEEP. Further validation is needed to determine the accuracy of electrical impedance tomography in measuring perfusion redistributions after positional changes., (Copyright © 2023 American Society of Anesthesiologists. All Rights Reserved.)

Cys 2 His 2 zinc finger (ZF) domains engineered to bind specific target sequences in the genome provide an effective strategy for programmable regulation of gene expression, with many potential therapeutic applications. However, the structurally intricate engagement of ZF domains with DNA has made their design challenging. Here we describe the screening of 49 billion protein-DNA interactions and the development of a deep-learning model, ZFDesign, that solves ZF design for any genomic target. ZFDesign is a modern machine learning method that models global and target-specific differences induced by a range of library environments and specifically takes into account compatibility of neighboring fingers using a novel hierarchical transformer architecture. We demonstrate the versatility of designed ZFs as nucleases as well as activators and repressors by seamless reprogramming of human transcription factors. These factors could be used to upregulate an allele of haploinsufficiency, downregulate a gain-of-function mutation or test the consequence of regulation of a single gene as opposed to the many genes that a transcription factor would normally influence., (© 2023. The Author(s).)

Background: Despite the growing contributions of critical care anesthesiologists to clinical practice, research, and administrative leadership of intensive care units (ICUs), relatively little is known about the subspecialty-specific clinical practice environment. An understanding of contemporary clinical practice is essential to recognize the opportunities and challenges facing critical care anesthesia, optimize staffing patterns, assess sustainability and satisfaction, and strategically plan for future activity, scope, and training. This study surveyed intensivists who are members of the Society of Critical Care Anesthesiologists (SOCCA) to evaluate practice patterns of critical care anesthesiologists, including compensation, types of ICUs covered, models of overnight ICU coverage, and relationships between these factors. We hypothesized that variability in compensation and practice patterns would be observed between individuals., Methods: Board-certified critical care anesthesiologists practicing in the United States were identified using the SOCCA membership distribution list and invited to take a voluntary online survey between May and June 2021. Multiple-choice questions with both single- and multiple-select options were used for answers with categorical data, and adaptive questioning was used to clarify stem-based responses. Respondents were asked to describe practice patterns at their respective institutions and provide information about their demographics, salaries, effort in ICUs, as well as other activities., Results: A total of 490 participants were invited to take this survey, and 157 (response rate 32%) surveys were completed and analyzed. The majority of respondents were White (73%), male (69%), and younger than 50 years of age (82%). The cardiothoracic/cardiovascular ICU was the most common practice setting, with 69.5% of respondents reporting time working in this unit. Significant variability was observed in ICU practice patterns. Respondents reported spending an equal proportion of their time in clinical practice in the operating rooms and ICUs (median, 40%; interquartile range [IQR], 20%-50%), whereas a smaller proportion-primarily those who completed their training before 2009-reported administrative or research activities. Female respondents reported salaries that were $36,739 less than male respondents; however, this difference was not statistically different, and after adjusting for age and practice type, these differences were less pronounced (-$27,479.79; 95% confidence interval [CI], -$57,232.61 to $2273.03; P = .07)., Conclusions: These survey data provide a current snapshot of anesthesiology critical care clinical practice patterns in the United States. Our findings may inform decision-making around the initiation and expansion of critical care services and optimal staffing patterns, as well as provide a basis for further work that focuses on intensivist satisfaction and burnout., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 International Anesthesia Research Society.)

Background: Post-laparoscopic cholecystectomy (LC) pain control is still an issue postoperatively., Objectives: We investigated the effectiveness of the unilateral right-side ultrasound-guided erector spinae plane block (ESPB) on post-LC pain intensity and opioid consumption., Methods: This is a parallel-arm randomized control trial on 62 adult patients with an American Society of Anesthesiologists (ASA) physical status ≤ 2 who underwent LC. The patients were randomized into 2 groups (the block group [BG] and the control group [CG]; n = 31 per group). BG received a single-shot right-sided T7 ESPB with 20 mL of 0.2% ropivacaine at arrival time in the post-anesthesia care unit (PACU). CG) received no regional anesthesia. Both groups received patient-controlled intravenous fentanyl and rescue meperidine for analgesia. The primary outcome was the pain intensity determined using a Numerical Rating Scale (NRS) in the first 24 hours after surgery. Secondary outcomes included total fentanyl and meperidine consumption within 24 hours., Results: Median pain scores were significantly higher in CG at rest and with coughing up to 12 hours after surgery compared with BG. Pain scores were higher in CG with a cough at 24 hours compared with BG (median 1 [interquartile range (IQR) 1, 2] vs. 1 [1, 0]; P = 0.0005). Total fentanyl consumption and meperidine consumption within 24 hours were significantly lower in BG compared with CG (median 60 µg [IQR 60, 90] vs 250 µg [90, 300]; P < 0.0001 and median 20 µg [IQR 10, 20] vs 25 [20, 25]; P = 0.002, respectively)., Conclusions: A single-shot, right-sided, unilateral ESPB decreases post-LC opioid consumption and pain., Competing Interests: Conflict of Interests: Professors FI, PR and SHRF are members of Board of Trustees of the journal and they have no financial interests related to this study. The other authors have no conflict of interest to declare., (Copyright © 2022, Author(s).)

Breakdown of the blood-central nervous system barrier (BCNSB) is a hallmark of many neuroinflammatory disorders, such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we show that endothelial-to-mesenchymal transition (EndoMT) occurs in the CNS before the onset of clinical symptoms and plays a major role in the breakdown of BCNSB function. EndoMT can be induced by an IL-1β-stimulated signaling pathway in which activation of the small GTPase ADP ribosylation factor 6 (ARF6) leads to crosstalk with the activin receptor-like kinase (ALK)-SMAD1/5 pathway. Inhibiting the activation of ARF6 both prevents and reverses EndoMT, stabilizes BCNSB function, reduces demyelination, and attenuates symptoms even after the establishment of severe EAE, without immunocompromising the host. Pan-inhibition of ALKs also reduces disease severity in the EAE model. Therefore, multiple components of the IL-1β-ARF6-ALK-SMAD1/5 pathway could be targeted for the treatment of a variety of neuroinflammatory disorders., Competing Interests: Declaration of interests The University of Utah has filed intellectual property concerning ARF6 pathways. The authors declare competing financial interests: The University of Utah has licensed technology to Navigen, a biotechnology company owned in part by the University of Utah Research Foundation. A.L.M. is an employee of Navigen. Correspondence and requests for materials should be addressed to sodelber@genetics.utah.edu or weiquan.zhu@u2m2.utah.edu., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Common anesthesia practice for hand surgery combines a preoperative regional anesthetic and intraoperative monitored anesthesia care (MAC). Despite adequate regional anesthesia, patients may receive doses of intraoperative sedatives which can result in oversedation and potentially avoidable complications. VR could prove to be a valuable tool for patients and providers by distracting the mind from processing noxious stimuli resulting in minimized sedative use and reduced risk of oversedation without negatively impacting patient satisfaction. Our hypothesis was that intraoperative VR use reduces sedative dosing during elective hand surgery without detracting from patient satisfaction as compared to a usual care control., Methods: Forty adults undergoing hand surgery were randomized to receive either intraoperative VR in addition to MAC, or usual MAC. Patients in both groups received preoperative regional anesthesia at provider discretion. Intraoperatively, the VR group viewed programming of their choice via a head-mounted display. The primary outcome was intraoperative propofol dose per hour (mg · hr-1). Secondary outcomes included patient reported pain and anxiety, overall satisfaction, functional outcome, and post anesthesia care unit (PACU) length of stay (LOS)., Results: Of the 40 enrolled patients, 34 completed the perioperative portion of the trial. VR group patients received significantly less propofol per hour than the control group (Mean (±SD): 125.3 (±296.0) vs 750.6 (±334.6) mg · hr-1, p<0.001). There were no significant differences between groups in patient reported overall satisfaction, (0-100 scale, Median (IQR) 92 (77-100) vs 100 (100-100), VR vs control, p = 0.087). There were no significant differences between groups in PACU pain scores, perioperative opioid analgesic dose, or in postoperative functional outcome. PACU LOS was significantly decreased in the VR group (53.0 (43.0-72.0) vs 75.0 (57.5-89.0) min, p = 0.018)., Conclusion: VR immersion during hand surgery led to significant reductions in intraoperative propofol dose and PACU LOS without negatively impacting key patient reported outcomes., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: BOG is a consultant for Sedana Medical. GK has commercial affiliation with Spectrum Healthcare Partners as an employee. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Tumor microenvironment (TME) is one of the most important factors in tumor aggressiveness, with an active exchange between tumor and other TME-associated cells that promotes metastasis. The tumor-inhibitory effect of resveratrol on colorectal cancer (CRC) cells has been frequently reported. However, whether resveratrol can specifically suppress TME-induced CRC invasion via β1-integrin receptors has not been fully elucidated yet. Methods: Two CRC cell lines (HCT116, RKO) were cultured in multicellular, pro-inflammatory 3D-alginate TME cultures (containing fibroblasts, T-lymphocytes) to investigate the role of β1-integrin receptors in the anti-invasive and anti-metastatic effect of resveratrol by antisense oligonucleotides (ASO). Results: Our results show that resveratrol dose-dependently suppressed the migration-promoting adhesion adapter protein paxillin and simultaneously enhanced the expression of E-cadherin associated with the phenotype change of CRC cells, and their invasion. Moreover, resveratrol blocked TME-induced phosphorylation and nuclear translocation of p65-NF-κB, which was associated with changes in the expression pattern of epithelial-mesenchymal-transition-related biomarkers (slug, vimentin, E-cadherin), metastasis-related factors (CXCR4, MMP-9, FAK), and apoptosis (caspase-3). Finally, transient transfection of β1-integrin, in contrast to knockdown of NF-κB, abrogated most anti-invasive, anti-metastatic effects as well as downstream signaling of resveratrol, resulting in a concomitant increase in CRC cell invasion, indicating a central role of β1-integrin receptors in the anti-invasive function of resveratrol. Conclusion: These results demonstrate for the first time that silencing β1-integrins may suppress, at least in part the inhibitory effects of resveratrol on invasion and migration of CRC cells, underscoring the crucial homeostatic role of β1-integrin receptors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Brockmueller, Mueller, Shayan and Shakibaei.)

Inactivated influenza vaccines induce greater antibody responses in females than males among both humans and mice. To test the breadth of protection, we used recombinant mouse-adapted A/California/2009 (maA/Cal/09) H1N1 viruses containing mutations at one (1M), two (2M), or three (3M) antigenic sites, in addition to a virus containing the 1M mutation and a substitution of the Ca2 antigenic site (Sub) with one derived from an H5 hemagglutinin (HA) to challenge mice of both sexes. Following maA/Cal/09 vaccination, females produced greater virus-specific, class-switched total IgG and IgG2c antibodies against the vaccine and all mutant viruses, and antibodies from females recognized a greater number of unique, linear HA epitopes than did antibodies from males. While females had greater neutralizing antibody titers against the vaccine virus, both sexes showed a lower neutralization capacity against mutant viruses. After virus challenge, vaccinated females had lower pulmonary virus titers and reduced morbidity than males for the 1M and 2M viruses, but not the Sub virus. Females generated greater numbers of germinal center (GC) B cells containing superior somatic hypermutation (SHM) frequencies than vaccinated males. Deletion of activation-induced cytidine deaminase ( Aicda ) eliminated female-biased immunity and protection against the 2M virus. Harnessing methods to improve GC B cell responses and frequencies of SHM, especially in males, should be considered in the development of universal influenza vaccines. IMPORTANCE Adult females develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection would be dependent on the extent of virus diversity as well as molecular mechanisms in B cells which constrain the breadth of epitope recognition. We developed a panel of mouse-adapted (ma) A/Cal/09 viruses that had mutations in the immunodominant hemagglutinin. Following vaccination against maA/Cal/09, females were better able to neutralize maA/Cal/09 than males, but neutralization of mutant maA/Cal/09 viruses was equally poor in both sexes, despite vaccinated females being better protected against these viruses. Vaccinated females benefited from the greater production of class-switched, somatically hypermutated antibodies generated in germinal center B cells, which increased recognition of more diverse maA/Cal/09 hemagglutinin antigen epitopes. Female-biased protection against influenza infection and disease after vaccination is driven by differential mechanisms in males versus females and should be considered in the design of novel vaccine platforms.

The prevalence and severity of cancers in non-reproductive tissues are greater in males than females, but the sex-specific factors contributing to this remain ill described. In this issue of Immunity, Yang et al. (2022) uncover a mechanism of androgen signaling leading to an exhausted, terminally differentiated CD8 + T cell phenotype in male mice., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Tendinitis (tendinopathy) is a pro-inflammatory and painful tendon disease commonly linked with mechanical overuse and associated injuries, drug abuse, and lifestyle factors (including poor diet and physical inactivity) that causes significant healthcare expenditures due to its high incidence. Nuclear factor kappa B (NF-κB) is one of the major pro-inflammatory transcription factors, along with other inflammation signaling pathways, triggered by a variety of stimuli, including cytokines, endotoxins, physical and chemical stressors, hypoxia, and other pro-inflammatory factors. Their activation is known to regulate the expression of a multitude of genes involved in inflammation, degradation, and cell death. The pathogenesis of tendinitis is still poorly understood, whereas efficient and sustainable treatment is missing. Targeting drug suppression of the key inflammatory regulators represents an effective strategy for tendinitis therapy, but requires a comprehensive understanding of their principles of action. Conventional monotherapies are often ineffective and associated with severe side effects in patients. Therefore, agents that modulate multiple cellular targets represent therapeutic treatment potential. Plant-derived nutraceuticals have been shown to act as multi-targeting agents against tendinitis via various anti-oxidant and anti-inflammatory mechanisms, whereat they were able to specifically modulate numerous signaling pathways, including NF-κB, p38/MAPK, JNK/STAT3, and PI3K/Akt, thus down-regulating inflammatory processes. This review discusses the utility of herbal nutraceuticals that have demonstrated safety and tolerability as anti-inflammatory agents for the prevention and treatment of tendinitis through the suppression of catabolic signaling pathways. Limitations associated with the use of nutraceuticals are also described.

Background To support diversity in biomedical science, the American Heart Association launched the Supporting Undergraduate Research Experiences for undergraduate students from underrepresented backgrounds to provide mentorship and high-level exposure at 5 leading medical institutions. Here we describe the initial formation of the partnership and the alteration made in response to the program to accommodate COVID-19 safety precautions. Methods and Results We outline how programming shifted from local, in-person programming in the summer of 2019 to a collaborative, mainly virtual curriculum in 2020 using students' self-reported before and after surveys from both 2019 (n=33) and 2020 (n=42). Students from both in-person (2019) and virtual programs (2020) self-reported significant gains in scientific proficiency. A qualitative-directed content analysis of student open-response questions was performed. Students reported extensive benefits from the 2020 virtual training, including Personal Gains, Research Skills, Thinking and Working Like a Scientist, and Attitudes and Behaviors. Notedly, we observed increases in the Attitudes and Behaviors category. We outline the pros and cons of in-person and virtual programming and make recommendations moving forward in a postpandemic world with hybrid work and learning systems. Conclusions Our effort informs the development of future undergraduate research training programs, significantly maximizing a hybrid training modality. The American Heart Association Supporting Undergraduate Research Experiences serves as a model for building multi-institutional partnerships and providing research experiences that overcome institutional barriers and support students' interests, commitment, and ability to persist in science, technology, engineering, and math fields.

Colorectal cancer (CRC) is one of the most common tumors worldwide, with a higher rate of distant metastases than other malignancies and with regular occurrence of drug resistance. Therefore, scientists are forced to further develop novel and innovative therapeutic treatment strategies, whereby it has been discovered microorganisms, albeit linked to CRC pathogenesis, are able to act as highly selective CRC treatment agents. Consequently, researchers are increasingly focusing on bacteriotherapy as a novel therapeutic strategy with less or no side effects compared to standard cancer treatment methods. With multiple successful trials making use of various bacteria-associated mechanisms, bacteriotherapy in cancer treatment is on its way to become a promising tool in CRC targeting therapy. In this study, we describe the anti-cancer effects of bacterial therapy focusing on the treatment of CRC as well as diverse modulatory mechanisms and techniques that bacteriotherapy offers such as bacterial-related biotherapeutics including peptides, toxins, bacteriocins or the use of bacterial carriers and underlying molecular processes to target colorectal tumors.

Calebin A (CA) is one of the active constituents of turmeric and has anti-inflammatory and antioxidant effects. Excessive inflammation and cell apoptosis are the main causes of tendinitis and tendinopathies. However, the role of CA in tendinitis is still unclear and needs to be studied in detail. Tenocytes in monolayer or 3D-alginate cultures in the multicellular tendinitis microenvironment (fibroblast cells) with T-lymphocytes (TN-ME) or with TNF-α or TNF-β, were kept without treatment or treated with CA to study their range of actions in inflammation. We determined that CA blocked TNF-β-, similar to TNF-α-induced adhesiveness of T-lymphocytes to tenocytes. Moreover, immunofluorescence and immunoblotting showed that CA, similar to BMS-345541 (specific IKK-inhibitor), suppressed T-lymphocytes, or the TNF-α- or TNF-β-induced down-regulation of Collagen I, Tenomodulin, tenocyte-specific transcription factor (Scleraxis) and the up-regulation of NF-κB phosphorylation; thus, its translocation to the nucleus as well as various NF-κB-regulated proteins was implicated in inflammatory and degradative processes. Furthermore, CA significantly suppressed T-lymphocyte-induced signaling, similar to TNF-β-induced signaling, and NF-κB activation by inhibiting the phosphorylation and degradation of IκBα (an NF-κB inhibitor) and IκB-kinase activity. Finally, inflammatory TN-ME induced the functional linkage between NF-κB and Scleraxis, proposing that a synergistic interaction between the two transcription factors is required for the initiation of tendinitis, whereas CA strongly attenuated this linkage and subsequent inflammation. For the first time, we suggest that CA modulates TN-ME-promoted inflammation in tenocytes, at least in part, via NF-κB/Scleraxis signaling. Thus, CA seems to be a potential bioactive compound for the prevention and treatment of tendinitis.

Background: High-dose (≥ 80 ppm) inhaled nitric oxide (INO) has antimicrobial effects. We designed a trial to test the preventive effects of high-dose NO on coronavirus disease 2019 (COVID-19) in health care providers working with patients with COVID-19. The study was interrupted prematurely due to the introduction of COVID-19 vaccines for health care professionals. We thereby present data on safety and feasibility of breathing 160 ppm NO using 2 different NO sources, namely pressurized nitrogen/NO cylinders (INO) and electric NO (eNO) generators., Methods: NO gas was inhaled at 160 ppm in air for 15 min twice daily, before and after each work shift, over 14 d by health care providers (NCT04312243). During NO administration, vital signs were continuously monitored. Safety was assessed by measuring transcutaneous methemoglobinemia (SpMet) and the inhaled nitrogen dioxide (NO 2 ) concentration., Results: Twelve healthy health care professionals received a collective total of 185 administrations of high-dose NO (160 ppm) for 15 min twice daily. One-hundred and seventy-one doses were delivered by INO and 14 doses by eNO. During NO administration, SpMet increased similarly in both groups ( P = .82). Methemoglobin decreased in all subjects at 5 min after discontinuing NO administration. Inhaled NO 2 concentrations remained between 0.70 ppm (0.63-0.79) and 0.75 ppm (0.67-0.83) in the INO group and between 0.74 ppm (0.68-0.78) and 0.88 ppm (0.70-0.93) in the eNO group. During NO administration, peripheral oxygen saturation and heart rate did not change. No adverse events occurred., Conclusions: This pilot study testing high-dose INO (160 ppm) for 15 min twice daily using eNO seems feasible and similarly safe when compared with INO., Competing Interests: Drs Zapol and Yu disclose a relationship with Third Pole. Dr Carroll discloses a relationship with UNITAID. Dr Berra discloses relationships with iNO Therapeutics, Praxair, Masimo, the National Institutes of Health, and Fast Grant. The remaining authors have disclosed no conflicts of interest., (Copyright © 2022 by Daedalus Enterprises.)

Rheumatoid arthritis (RA) is considered a chronic systemic, multi-factorial, inflammatory, and progressive autoimmune disease affecting many people worldwide. While patients show very individual courses of disease, with RA focusing on the musculoskeletal system, joints are often severely affected, leading to local inflammation, cartilage destruction, and bone erosion. To prevent joint damage and physical disability as one of many symptoms of RA, early diagnosis is critical. Auto-antibodies play a pivotal clinical role in patients with systemic RA. As biomarkers, they could help to make a more efficient diagnosis, prognosis, and treatment decision. Besides auto-antibodies, several other factors are involved in the progression of RA, such as epigenetic alterations, post-translational modifications, glycosylation, autophagy, and T-cells. Understanding the interplay between these factors would contribute to a deeper insight into the causes, mechanisms, progression, and treatment of the disease. In this review, the latest RA research findings are discussed to better understand the pathogenesis, and finally, treatment strategies for RA therapy are presented, including both conventional approaches and new methods that have been developed in recent years or are currently under investigation.

Inflammation has a fundamental impact on the pathophysiology of osteoarthritis (OA), a common form of degenerative arthritis. It has previously been established that curcumin, a component of turmeric ( Curcuma longa ), has anti-inflammatory properties. This research evaluates the potentials of curcumin on the pathophysiology of OA in vitro. To explore the anti-inflammatory efficacy of curcumin in an inflamed joint, an osteoarthritic environment (OA-EN) model consisting of fibroblasts, T-lymphocytes, 3D-chondrocytes is constructed and co-incubated with TNF-α, antisense oligonucleotides targeting NF-kB (ASO-NF-kB), or an IkB-kinase (IKK) inhibitor (BMS-345541). Our results show that OA-EN, similar to TNF-α, suppresses chondrocyte viability, which is accompanied by a significant decrease in cartilage-specific proteins (collagen II, CSPG, Sox9) and an increase in NF-kB-driven gene proteins participating in inflammation, apoptosis, and breakdown (NF-kB, MMP-9, Cox-2, Caspase-3). Conversely, similar to knockdown of NF-kB at the mRNA level or at the IKK level, curcumin suppresses NF-kB activation, NF-kB-promotes gene proteins derived from the OA-EN, and stimulates collagen II, CSPG, and Sox9 expression. Furthermore, co-immunoprecipitation assay shows that curcumin reduces OA-EN-mediated inflammation and chondrocyte apoptosis, with concomitant chondroprotective effects, due to modulation of Sox-9/NF-kB signaling axis. Finally, curcumin selectively hinders the interaction of p-NF-kB-p65 directly with DNA-this association is disrupted through DTT. These results suggest that curcumin suppresses inflammation in OA-EN via modulating NF-kB-Sox9 coupling and is essential for maintaining homeostasis in OA by balancing chondrocyte survival and inflammatory responses. This may contribute to the alternative treatment of OA with respect to the efficacy of curcumin.

Background: Despite evidence suggesting detrimental effects of perioperative hyperoxia, hyperoxygenation remains commonplace in cardiac surgery. Hyperoxygenation may increase oxidative damage and neuronal injury leading to potential differences in postoperative neurocognition. Therefore, this study tested the primary hypothesis that intraoperative normoxia, as compared to hyperoxia, reduces postoperative cognitive dysfunction in older patients having cardiac surgery., Methods: A randomized double-blind trial was conducted in patients aged 65 yr or older having coronary artery bypass graft surgery with cardiopulmonary bypass. A total of 100 patients were randomized to one of two intraoperative oxygen delivery strategies. Normoxic patients (n = 50) received a minimum fraction of inspired oxygen of 0.35 to maintain a Pao2 above 70 mmHg before and after cardiopulmonary bypass and between 100 and 150 mmHg during cardiopulmonary bypass. Hyperoxic patients (n = 50) received a fraction of inspired oxygen of 1.0 throughout surgery, irrespective of Pao2 levels. The primary outcome was neurocognitive function measured on postoperative day 2 using the Telephonic Montreal Cognitive Assessment. Secondary outcomes included neurocognitive function at 1, 3, and 6 months, as well as postoperative delirium, mortality, and durations of mechanical ventilation, intensive care unit stay, and hospital stay., Results: The median age was 71 yr (interquartile range, 68 to 75), and the median baseline neurocognitive score was 17 (16 to 19). The median intraoperative Pao2 was 309 (285 to 352) mmHg in the hyperoxia group and 153 (133 to 168) mmHg in the normoxia group (P < 0.001). The median Telephonic Montreal Cognitive Assessment score on postoperative day 2 was 18 (16 to 20) in the hyperoxia group and 18 (14 to 20) in the normoxia group (P = 0.42). Neurocognitive function at 1, 3, and 6 months, as well as secondary outcomes, were not statistically different between groups., Conclusions: In this randomized controlled trial, intraoperative normoxia did not reduce postoperative cognitive dysfunction when compared to intraoperative hyperoxia in older patients having cardiac surgery. Although the optimal intraoperative oxygenation strategy remains uncertain, the results indicate that intraoperative hyperoxia does not worsen postoperative cognition after cardiac surgery., (Copyright © 2020, the American Society of Anesthesiologists, Inc. All Rights Reserved.)

The rise in multidrug-resistant (MDR) organisms portends a serious global threat to the health care system with nearly untreatable infectious diseases, including pneumonia and its often fatal sequelae, acute respiratory distress syndrome (ARDS) and sepsis. Gram-negative bacteria (GNB), including Acinetobacter baumannii , Pseudomonas aeruginosa , and carbapenemase-producing Klebsiella pneumoniae (CPKP), are among the World Health Organization's and National Institutes of Health's high-priority MDR pathogens for targeted development of new therapies. Here, we show that stabilizing the host's vasculature by genetic deletion or pharmacological inhibition of the small GTPase ADP-ribosylation factor 6 (ARF6) increases survival rates of mice infected with A. baumannii , P. aeruginosa , and CPKP. We show that the pharmacological inhibition of ARF6-GTP phenocopies endothelium-specific Arf6 disruption in enhancing the survival of mice with A. baumannii pneumonia, suggesting that inhibition is on target. Finally, we show that the mechanism of protection elicited by these small-molecule inhibitors acts by the restoration of vascular integrity disrupted by GNB lipopolysaccharide (LPS) activation of the TLR4/MyD88/ARNO/ARF6 pathway. By targeting the host's vasculature with small-molecule inhibitors of ARF6 activation, we circumvent microbial drug resistance and provide a potential alternative/adjunctive treatment for emerging and reemerging pathogens., (Copyright © 2020 American Society for Microbiology.)

The Cys2His2 zinc finger is the most common DNA-binding domain expanding in metazoans since the fungi human split. A proposed catalyst for this expansion is an arms race to silence transposable elements yet it remains poorly understood how this domain is able to evolve the required specificities. Likewise, models of its DNA binding specificity remain error prone due to a lack of understanding of how adjacent fingers influence each other's binding specificity. Here, we use a synthetic approach to exhaustively investigate binding geometry, one of the dominant influences on adjacent finger function. By screening over 28 billion protein-DNA interactions in various geometric contexts we find the plasticity of the most common natural geometry enables more functional amino acid combinations across all targets. Further, residues that define this geometry are enriched in genomes where zinc fingers are prevalent and specificity transitions would be limited in alternative geometries. Finally, these results demonstrate an exhaustive synthetic screen can produce an accurate model of domain function while providing mechanistic insight that may have assisted in the domains expansion., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Traditional approaches to clinical risk assessment utilize age as a marker of increased vulnerability to stress. Relatively recent advancements in the study of aging have led to the concept of the frailty syndrome, which represents a multidimensional state of depleted physiologic and psychosocial reserve and clinical vulnerability that is related to but variably present with advancing age. The frailty syndrome is now a well-established clinical entity that serves as both a guide for clinical intervention and a predictor of poor outcomes in the primary and acute care settings. The biological aspects of the syndrome broadly represent a network of interrelated perturbations involving the age-related accumulation of molecular, cellular, and tissue damage that leads to multisystem dysregulation, functional decline, and disproportionately poor response to physiologic stress. Given the complexity of the underlying biologic processes, several well-validated approaches to define frailty clinically have been developed, each with distinct and reasonable considerations. Stemming from this background, the past several years have seen a number of observational studies conducted in intensive care units that have established that the determination of frailty is both feasible and prognostically useful in the critical care setting. Specifically, frailty as determined by several different frailty measurement tools appears associated with mortality, increased health care utilization, and disability, and has the potential to improve risk stratification of intensive care patients. While substantial variability in the implementation of frailty measurement likely limits the generalizability of specific findings, the overall prognostic trends may offer some assistance in guiding management decisions with patients and their families. Although no trials have assessed interventions to improve the outcomes of critically ill older people living with frailty, the particular vulnerability of this population offers a promising target for intervention in the future.

The severity and outcome of coronavirus disease 2019 (COVID-19) largely depends on a patient's age. Adults over 65 years of age represent 80% of hospitalizations and have a 23-fold greater risk of death than those under 65. In the clinic, COVID-19 patients most commonly present with fever, cough and dyspnea, and from there the disease can progress to acute respiratory distress syndrome, lung consolidation, cytokine release syndrome, endotheliitis, coagulopathy, multiple organ failure and death. Comorbidities such as cardiovascular disease, diabetes and obesity increase the chances of fatal disease, but they alone do not explain why age is an independent risk factor. Here, we present the molecular differences between young, middle-aged and older people that may explain why COVID-19 is a mild illness in some but life-threatening in others. We also discuss several biological age clocks that could be used in conjunction with genetic tests to identify both the mechanisms of the disease and individuals most at risk. Finally, based on these mechanisms, we discuss treatments that could increase the survival of older people, not simply by inhibiting the virus, but by restoring patients' ability to clear the infection and effectively regulate immune responses.

Objective: To study the association between hypertensive diseases of pregnancy and immediate postpartum development of heart failure in a large national database., Patients and Methods: Using the 2013 to 2014 National Readmissions Database, which covered admissions from January 1 through September 30 in years 2013 and 2014, we examined 90-day readmission rates in parturients with a diagnosis of hypertensive disease of pregnancy who were discharged after delivery. The primary outcome was the association between the presence of hypertensive disease of pregnancy and readmission with heart failure within 90 days of delivery discharge. Secondary outcomes included readmission mortality, time between delivery discharge and readmission, length of stay, and costs of readmission., Results: Women with hypertensive disease of pregnancy were more likely to be readmitted with heart failure (1809 of 25,908 readmissions (7.0%) vs 2622 of 89,660 readmissions (2.9%); P<.001). This difference persisted after adjustment for potential cofounders (6.3% vs 3.1%; odds ratio, 2.15; 95% CI, 1.92-2.40; P<.001). Women with a diagnosis of heart failure at readmission were readmitted sooner (11 days vs 23 days; P<.001) and had a longer length of stay (4 days vs 3 days; P<.001) and higher costs of readmission ($10,361 vs $6977; P<.001) than did women without a diagnosis of heart failure., Conclusion: Parturients with hypertensive disease of pregnancy were more likely to be readmitted with heart failure within 90 days of delivery. Most patients readmitted with heart failure were readmitted within 2 weeks of discharge after delivery. Patients readmitted with heart failure had substantial health care expenditures., (Copyright © 2018 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

Feeding dried distillers grains with solubles (DDGS) to lactating dairy cows has been implicated as a cause of late blowing defects in the production of Swiss-style cheeses. Our objectives were (1) to test the effect of feeding reduced-fat DDGS (RF-DDGS; ∼6% fat) to lactating dairy cows on the composition of milk and on the suitability of the milk for production of baby Swiss cheese and (2) to evaluate the effect of diet on cow lactation performance. Lactating Holstein dairy cows were fed both dietary treatments in a 2 × 2 crossover design. Cows were housed in a 48-cow freestall pen equipped with individual feeding gates to record feed intake. The control diet was a corn, corn silage, and alfalfa hay diet supplemented with mechanically expelled soybean meal. The experimental diet was the same base ration, but 20% (dry matter basis) RF-DDGS were included in place of the expelled soybean meal. The RF-DDGS diet was additionally supplemented with rumen-protected lysine; diets were formulated to be isoenergetic and isonitrogenous. Cows were allowed ad libitum access to feed and water, fed twice daily, and milked 3 times daily. For cheese production, milk was collected and pooled 6 times for each dietary treatment. There was no treatment effect on milk yield (35.66 and 35.39 kg/d), milk fat production (1.27 and 1.25 kg/d), milk fat percentage (3.65 and 3.61%), milk protein production (1.05 and 1.08 kg/d), lactose percentage (4.62 and 4.64%), milk total solids (12.19 and 12.28%), and somatic cell count (232.57 and 287.22 × 10 3 cells/mL) for control and RF-DDGS, respectively. However, dry matter intake was increased by treatment, which implied a reduction in feed efficiency. Milk protein percentage also increased (3.01 and 3.11%), whereas milk urea nitrogen decreased (14.18 and 12.99 mg/dL), indicating that protein utilization may be more efficient when cows are fed RF-DDGS. No differences in cheese were observed by a trained panel except cheese appearance; control cheese eyes were significantly, but not practically, larger than the RF-DDGS cheese. These results indicate that RF-DDGS can be effectively used in the rations of lactating Holstein cows with no deleterious effects on milk production and composition and metrics of the physiology of the cow (i.e., blood glucose and nonesterified fatty acids); however, feeding RF-DDGS increased dry matter intake, which decreased feed efficiency. Finally, feeding RF-DDGS did not negatively influence quality and suitability of milk for production of baby Swiss cheese., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

B6.A-Dysf prmd /GeneJ (BLAJ) mice model human limb-girdle muscular dystrophy 2B (LGMD2B), which is linked to mutations in the dysferlin (DYSF) gene. We tested the hypothesis that, the calcium ion (Ca 2+ ) channel blocker diltiazem (DTZ), reduces contraction-induced skeletal muscle damage, in BLAJ mice. We randomly assigned mice (N = 12; 3-4 month old males) to one of two groups - DTZ (N = 6) or vehicle (VEH, distilled water, N = 6). We conditioned mice with either DTZ or VEH for 1 week, after which, their tibialis anterior (TA) muscles were tested for contractile torque and susceptibility to injury from forced eccentric contractions. We continued dosing with DTZ or VEH for 3 days following eccentric contractions, and then studied torque recovery and muscle damage. We analyzed contractile torque before eccentric contractions, immediately after eccentric contractions, and at 3 days after eccentric contractions; and counted damaged fibers in the injured and uninjured TA muscles. We found that DTZ improved contractile torque before and immediately after forced eccentric contractions, but did not reduce delayed-onset muscle damage that was observed at 3 days after eccentric contractions., (© 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs - ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy.

Activating mutations in Gαq proteins, which form the α subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic Gαq signaling to induce all of these downstream pathways as well as β-catenin signaling. ARF6 activates these diverse pathways through a common mechanism: the trafficking of GNAQ and β-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small-molecule inhibitor reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for Gα-mediated diseases., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Studies of the pathogenic mechanisms underlying human myopathies and muscular dystrophies often require animal models, but models of some human diseases are not yet available. Methods to promote the engraftment and development of myogenic cells from individuals with such diseases in mice would accelerate such studies and also provide a useful tool for testing therapeutics. Here, we investigate the ability of immortalized human myogenic precursor cells (hMPCs) to form mature human myofibers following implantation into the hindlimbs of non-obese diabetic-Rag1 (null) IL2rγ (null) (NOD-Rag)-immunodeficient mice., Results: We report that hindlimbs of NOD-Rag mice that are X-irradiated, treated with cardiotoxin, and then injected with immortalized control hMPCs or hMPCs from an individual with facioscapulohumeral muscular dystrophy (FSHD) develop mature human myofibers. Furthermore, intermittent neuromuscular electrical stimulation (iNMES) of the peroneal nerve of the engrafted limb enhances the development of mature fibers in the grafts formed by both immortal cell lines. With control cells, iNMES increases the number and size of the human myofibers that form and promotes closer fiber-to-fiber packing. The human myofibers in the graft are innervated, fully differentiated, and minimally contaminated with murine myonuclei., Conclusions: Our results indicate that control and FSHD human myofibers can form in mice engrafted with hMPCs and that iNMES enhances engraftment and subsequent development of mature human muscle.

Perineuronal nets (PNNs) are extracellular molecules that form around neurons near the end of critical periods during development. They surround neuronal cell bodies and proximal dendrites. PNNs inhibit the formation of new connections and may concentrate around rapidly firing inhibitory interneurons. Previous work characterized the important role of perineuronal nets in plasticity in the visual system, amygdala, and spinal cord of rats. In this study, we use immunohistochemistry to survey the distribution of perineuronal nets in representative areas of the primate brain. We also document changes in PNN prevalence in these areas in animals of different ages. We found that PNNs are most prevalent in the cerebellar nuclei, surrounding >90% of the neurons there. They are much less prevalent in cerebral cortex, surrounding less than 10% of neurons in every area that we examined. The incidence of perineuronal nets around parvalbumin-positive neurons (putative fast-spiking interneurons) varies considerably between different areas in the brain. Our survey indicates that the presence of PNNs may not have a simple relationship with neural plasticity and may serve multiple functions in the central nervous system.

Mutations in the dysferlin gene (DYSF) lead to human muscular dystrophies known as dysferlinopathies. The dysferlin-deficient A/J mouse develops a mild myopathy after 6 months of age, and when younger models the subclinical phase of the human disease. We subjected the tibialis anterior muscle of 3- to 4-month-old A/J mice to in vivo large-strain injury (LSI) from lengthening contractions and studied the progression of torque loss, myofiber damage, and inflammation afterward. We report that myofiber damage in A/J mice occurs before inflammatory cell infiltration. Peak edema and inflammation, monitored by magnetic resonance imaging and by immunofluorescence labeling of neutrophils and macrophages, respectively, develop 24 to 72 hours after LSI, well after the appearance of damaged myofibers. Cytokine profiles 72 hours after injury are consistent with extensive macrophage infiltration. Dysferlin-sufficient A/WySnJ mice show much less myofiber damage and inflammation and lesser cytokine levels after LSI than do A/J mice. Partial suppression of macrophage infiltration by systemic administration of clodronate-incorporated liposomes fails to suppress LSI-induced damage or to accelerate torque recovery in A/J mice. The findings from our studies suggest that, although macrophage infiltration is prominent in dysferlin-deficient A/J muscle after LSI, it is the consequence and not the cause of progressive myofiber damage., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Dysferlinopathies, most commonly limb girdle muscular dystrophy 2B and Miyoshi myopathy, are degenerative myopathies caused by mutations in the DYSF gene encoding the protein dysferlin. Studies of dysferlin have focused on its role in the repair of the sarcolemma of skeletal muscle, but dysferlin's association with calcium (Ca(2+)) signaling proteins in the transverse (t-) tubules suggests additional roles. Here, we reveal that dysferlin is enriched in the t-tubule membrane of mature skeletal muscle fibers. Following experimental membrane stress in vitro, dysferlin-deficient muscle fibers undergo extensive functional and structural disruption of the t-tubules that is ameliorated by reducing external [Ca(2+)] or blocking L-type Ca(2+) channels with diltiazem. Furthermore, we demonstrate that diltiazem treatment of dysferlin-deficient mice significantly reduces eccentric contraction-induced t-tubule damage, inflammation, and necrosis, which resulted in a concomitant increase in postinjury functional recovery. Our discovery of dysferlin as a t-tubule protein that stabilizes stress-induced Ca(2+) signaling offers a therapeutic avenue for limb girdle muscular dystrophy 2B and Miyoshi myopathy patients.

NPS Pharmaceuticals, Inc. (NPS) has synthesized a series of open-channel blockers with varying potencies at the NMDA receptor. NPS 1506 (Fig. 1) is a moderate affinity antagonist that inhibits NMDA/glycine-induced increases in cytosolic calcium in cultured rat cerebellar granule cells (IC50 = 476nM) and displaces the binding of [3H]MK-801 to rat cortical membranes (IC50 = 664nM).

Early in development, murine B-lineage progenitor cells express two classes of IgG Fc receptors (FcgammaR) designated as FcgammaRII (CD32) and FcgammaRIII (CD16), but mature B lymphocytes only express FcgammaRII (CD32), which functions as an inhibitor of B-cell activation when it is induced to associate with mIgM. The functions of CD16 and CD32 on B-lineage precursor cells have not previously been investigated. To search for FcgammaR functions on developing B-lineage cells, normal murine bone marrow cells were cultured in the presence of 2.4G2, a rat monoclonal antibody that binds to CD16 and CD32, or in the presence of control normal rat IgG, and then the B-lineage compartment was analyzed for effects. Cultures that contained 2.4G2 showed enhanced growth and differentiation of B-lineage cells compared with control cultures. The enhancing effect of 2.4G2 also occurred when fluorescence-activated cell-sorted B-cell precursors (B220(+), sIgM-, HSAhigh, FcgammaR+) from normal bone marrow were cocultured with BMS2, a bone marrow stromal cell line, but not when they were cultured in BMS2-conditioned media. The enhancement of B-lineage development induced by 2.4G2 was CD16-dependent and CD32-dependent, because 2.4G2 did not effect B-lineage growth or differentiation in cultures of bone marrow from mice in which either the gene encoding CD16 or CD32 had been disrupted. Analysis of fresh bone marrow from the CD16 gene-disrupted mice showed normal numbers and distribution of cells within the B-cell compartment, but in CD32 gene-disrupted mice, the B-cell compartment was significantly enlarged. These experiments provide several lines of evidence that the FcgammaR expressed on murine B-cell precursors can influence their growth and differentiation., (Copyright 1998 by The American Society of Hematology.)

Murine granulocytes and precursors express low-affinity IgG Fc receptors (Fc gammaR). We investigated the effects of FcyR ligation on the development of eosinophils in cultures of normal murine bone marrow. Eosinophilopoiesis was induced by culture of bone marrow cells in the presence of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF], interleukin-3 [IL-3], and IL-5). Addition to the cultures of 2.4G2, a rat monoclonal antibody (mAb) that reacts with Fc gammaRII (CD32) and Fc gammaRIII (CD16), induced granulocyte apoptosis within 24 hours. Granulocytes in cultures that contained 2.4G2 showed chromatin condensation, binding of Annexin-V, and fas induction, and by electron microscopy, apoptosis was most commonly observed in cells of the eosinophil lineage. Since murine granulocytes can express both Fc gammaRII (CD32) and Fc gammaRIII (CD16), we investigated the effect of 2.4G2 on cultures of bone marrow obtained from Fc gammaRIII (CD16) gene-disrupted mice and found that the apoptosis induced with 2.4G2 was CD16-independent. Studies with bone marrow cultures from B6MLR-lpr/lpr and C3H/HEJ-gld/gld mice established that the Fc gammaRII (CD32)-triggered apoptosis was fas-fasL-dependent. When mature eosinophils isolated from hepatic granulomas of Schistosoma mansoni-infected mice were cultured in cytokines in the presence of 2.4G2, the eosinophils underwent apoptosis within 24 hours. These findings identify a previously unknown linkage between Fc gammaR on eosinophils and fas-mediated apoptosis, a connection that could be relevant to mechanisms by which eosinophils mediate tissue injury and antibody-dependent cellular cytotoxicity reactions.

Chronic infection with Schistosoma mansoni induces in humans and mice a Th2-dominant immune response in which eosinophils and IgE are conspicuously elevated. Human eosinophils express IgE receptors that participate in an IgE-dependent eosinophil-mediated ADCC reaction against Schistosomula larvae in vitro. To investigate the expression of IgE receptors on murine eosinophils, they were purified (>95% pure by Giemsa-stained cytospin preparations) from liver granulomas of Schistosoma-infected mice. Flow cytometric analysis showed the absence of the low-affinity IgE receptor Fc-epsilon RII (CD23) and Mac-2 and the absence of binding of murine IgE. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of granuloma eosinophil mRNA did not detect transcripts for Fc-epsilon RII or the alpha-chain of the high-affinity IgE receptor Fc-epsilon RI, but did detect transcripts that encode Mac-2 and the low-affinity IgG receptors Fc-gamma RIIb2, Fc-gamma RIII, and the FcR-associated gamma-chain. In vitro stimulation of granuloma eosinophils with interleukin-4 (IL-4) did not induce IgE binding, surface expression of Mac-2, or the transcription of Fc-epsilon receptors (Fc-epsilon RI, Fc-epsilon RII/CD23). To investigate normal murine eosinophils, we cultured normal mouse bone marrow cells with recombinant IL-3, recombinant IL-5, and recombinant granulocyte-macrophage colony-stimulating factor, conditions that promote eosinophil differentiation. Flow cytometric analysis of bone marrow-derived eosinophils failed to detect IgE binding or cell surface expression of Fc-epsilon RII and Mac-2, and RT-PCR analysis of fluorescence-activated cell sorted bone marrow-derived eosinophils failed to detect transcripts that encode Fc-epsilon RI or Fc-epsilon RII. These findings show that, in contrast to human eosinophils, murine eosinophils do not express cell surface receptors that bind IgE. However, because IgG receptors (Fc-gamma RIIb2, Fc-gamma RII) were present on eosinophils purified from granulomas, we investigated whether they might be involved in eosinophil activation. We found that an oxidative burst in eosinophils could be triggered through their IgG receptors.

Fetal pre-T cells express low-affinity receptors for IgG (Fc gamma R) at a developmental stage prior to the rearrangement and expression of immunoglobulin genes. The present studies investigated the possible functional significance of Fc gamma R on fetal pre-T cells. Between 13 and 17 days of fetal development a subpopulation of T-cell receptor-, Thy-1+ thymocytes express for gamma R. The same cells contain mRNA for several forms of Fc gamma R (Fc gamma RII beta 1, beta 2, and Fc gamma RIII). Concurrently, a Pgp-1-, Thy-1-, surface-immunoglobulin- fetal thymic cell binds recombinant soluble Fc gamma R. In principle this cell can interact with the pre-T cells through this counter-receptor. To test this possibility anti-Fc gamma RII/III antibody (2.4G2) was injected into pregnant mice and then into their offspring for 6 wk postpartum. The injected antibody induced a slight increase in the proportion of CD4 or CD8 single-positive, alpha/beta T cells in the thymus. However, in fetal thymic cultures in the presence of 2.4G2 or the recombinant soluble Fc gamma R there was an accelerated differentiation of thymocytes to single-positive, CD3-bright, heat-stable antigen-dull, alpha/beta T cells. These experiments show that Fc gamma Rs are present on pre-T cells during early fetal thymic development, and that a non-IgG ligand of the Fc gamma R is expressed concurrently on Thy- fetal thymocytes. Furthermore, the presumed interaction of Fc gamma R and the alternative ligand(s) influences T-cell development. IgG binding could be an adapted function of Fc gamma Rs, and, as shown for many members of the Ig super family, these receptors may have originally served as cell-cell recognition/interaction molecules required for hematopoietic development.

The venom of the North American funnel-web spider Agelenopsis aperta contains a variety of arylamine toxins (the alpha-agatoxins) that paralyze insects by blocking glutamatergic neuromuscular transmission. We have tested six synthetic alpha-agatoxins for their ability to antagonize glutamate receptor function in mammalian brain. These compounds produce, at submicromolar concentrations, noncompetitive inhibition of N-methyl-D-aspartate (NMDA) receptor-mediated elevations in the concentration of cytosolic free calcium in cultured rat cerebellar granule neurons. In contrast, the alpha-agatoxins are relatively weak antagonists of elevations in the cytosolic free calcium concentration induced by non-NMDA receptor agonists. The alpha-agatoxins also produce reversible suppression of the NMDA receptor-mediated excitatory postsynaptic potential in rat hippocampal slices at concentrations that have little effect on the non-NMDA receptor-mediated population spike. We conclude that the alpha-agatoxins are selective and reversible noncompetitive antagonists at NMDA receptors in mammalian brain.

Addition of a disulfonic stilbene, 4-acetamido 4'-isothiocyano-2,2'-disulfonic stilbene (SITS), to the serosal side of the turtle bladder blocks the efflux of bicarbonate ions from the acidifying cells and thereby inhibits hydrogen ion secretion into the luminal solution. Because SITS has little effect on other transport systems, we used it to define the relationship between hydrogen ion secretion (JH) and the different cell types facing the luminal surface. Cells were identified by scanning and transmission electron microscopy (SEM and TEM) and by histochemical localization of carbonic anhydrase (CA). SEM revealed that SITS caused marked alterations in luminal surface characteristics of a cell population with prominent microplicae. Two hours after the serosal addition of SITS, cells with identifiable microplicae had decreased from 12.7 to 0.5% of total cells. TEM studies and CA histochemistry showed that the number of cells rich in CA (CA cells) remained the same, whereas the individual luminal surface areas of a subpopulation of CA cells had decreased markedly. A comparison of the distribution of individual surface areas of cells with microplicae and CA cells revealed that the CA cells with large surface areas corresponded to the cells with microplicae and that both were affected by the serosal addition of SITS. Acetazolamide, which also inhibits JH, caused similar changes. The luminal addition of SITS and an inactive analogue of acetazolamide, which have no effect on JH, did not alter surface morphology, These results indicate that the CA cell with microplicae represents the active state of the hydrogen ion secreting cell.

The electrophysiological actions of somatostatin (somatotropin release inhibiting factor; SRIF) were investigated in the in vitro hippocampal slice preparation. Intracellular recordings were obtained from pyramidal neurons in area CA1 in slices of hippocampus from guinea pigs and rabbits. Somatostatin, applied via micropressure ejection to CA1 pyramidal-cell somata, was primarily excitatory. The effects, however, were quite variable, with nearly all cells displaying pronounced tachyphylaxis. A majority of cells was depolarized by SRIF, but hyperpolarizations or biphasic depolarization/hyperpolarization responses were also recorded. Only minimal conductance changes were associated with the SRIF-induced voltage changes. Depletion of SRIF, by injection of the intact animal with cysteamine several hours before preparing slices, resulted in no obvious abnormalities in hippocampal slice electrophysiology. Our results obtained with application of exogenous SRIF are consistent with the concept that SRIF acts as an excitatory neurotransmitter/neuromodulator in hippocampus. However, our attempts to demonstrate endogenous SRIF action have thus far been unsuccessful.

The ability of l-norepinephrine to influence epileptiform activity was examined in an in vitro rat hippocampal slice preparation. Exogenously applied norepinephrine (NE) had anticonvulsant properties in that it slowed or stopped spontaneous interictal discharges which had been initiated by superfusion of slices with medium containing penicillin and elevated levels of potassium. This anticonvulsant property of NE was shared by the alpha receptor agonists 6-fluoro-norepinephrine, l-alpha-methyl-norepinephrine, and clonidine, but not by d-alpha-methyl-norepinephrine or l-phenylephrine. The beta receptor agonists 2-fluoro-norepinephrine and l-isoproterenol, on the other hand, were proconvulsant in that they increased interictal discharge rate. The alpha receptor antagonist phentolamine selectively blocked anticonvulsant responses, whereas the beta receptor antagonist timolol selectively blocked proconvulsant activity. These results suggest that activation of alpha or beta receptors has opposing inhibitory or excitatory effects respectively on epileptiform discharges in the rat hippocampus.