Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal.

Regulatory myeloid immune cells, such as myeloid-derived suppressor cells (MDSCs), populate inflamed or cancerous tissue and block immune cell effector functions. The lack of mechanistic insight into MDSC suppressive activity and a marker for their identification has hampered attempts to overcome T cell inhibition and unleash anti-cancer immunity. Here, we report that human MDSCs were characterized by strongly reduced metabolism and conferred this compromised metabolic state to CD8+ T cells, thereby paralyzing their effector functions. We identified accumulation of the dicarbonyl radical methylglyoxal, generated by semicarbazide-sensitive amine oxidase, to cause the metabolic phenotype of MDSCs and MDSC-mediated paralysis of CD8+ T cells. In a murine cancer model, neutralization of dicarbonyl activity overcame MDSC-mediated T cell suppression and, together with checkpoint inhibition, improved the efficacy of cancer immune therapy. Our results identify the dicarbonyl methylglyoxal as a marker metabolite for MDSCs that mediates T cell paralysis and can serve as a target to improve cancer immune therapy.

Base editing of haematopoietic stem cells rescues sickle cell disease in mice.

Sickle cell disease (SCD) is caused by a mutation in the ß-globin gene HBB1. We used a custom adenine base editor (ABE8e-NRCH)2,3 to convert the SCD allele (HBBS) into Makassar ß-globin (HBBG), a non-pathogenic variant4,5. Ex vivo delivery of mRNA encoding the base editor with a targeting guide RNA into haematopoietic stem and progenitor cells (HSPCs) from patients with SCD resulted in 80% conversion of HBBS to HBBG. Sixteen weeks after transplantation of edited human HSPCs into immunodeficient mice, the frequency of HBBG was 68% and hypoxia-induced sickling of bone marrow reticulocytes had decreased fivefold, indicating durable gene editing. To assess the physiological effects of HBBS base editing, we delivered ABE8e-NRCH and guide RNA into HSPCs from a humanized SCD mouse6 and then transplanted these cells into irradiated mice. After sixteen weeks, Makassar ß-globin represented 79% of ß-globin protein in blood, and hypoxia-induced sickling was reduced threefold. Mice that received base-edited HSPCs showed near-normal haematological parameters and reduced splenic pathology compared to mice that received unedited cells. Secondary transplantation of edited bone marrow confirmed that the gene editing was durable in long-term haematopoietic stem cells and showed that HBBS-to-HBBG editing of 20% or more is sufficient for phenotypic rescue. Base editing of human HSPCs avoided the p53 activation and larger deletions that have been observed following Cas9 nuclease treatment. These findings point towards a one-time autologous treatment for SCD that eliminates pathogenic HBBS, generates benign HBBG, and minimizes the undesired consequences of double-strand DNA breaks.

DNA Methylation Clocks in Aging: Categories, Causes, and Consequences.

Age-associated changes to the mammalian DNA methylome are well documented and thought to promote diseases of aging, such as cancer. Recent studies have identified collections of individual methylation sites whose aggregate methylation status measures chronological age, referred to as the DNA methylation clock. DNA methylation may also have value as a biomarker of healthy versus unhealthy aging and disease risk; in other words, a biological clock. Here we consider the relationship between the chronological and biological clocks, their underlying mechanisms, potential consequences, and their utility as biomarkers and as targets for intervention to promote healthy aging and longevity.

Comprehensive safety assessment of serendipity berry sweet protein produced from Komagataella phaffii.

Serendipity berry plant (Dioscoreophyllum cumminsii (Stapf) Diels) is the source of a naturally sweet protein referred to as monellin. The safety of serendipity berry sweet protein (SBSP) containing single polypeptide monellin (MON) expressed in Komagataella phaffii (formerly Pichia pastoris) and produced via precision fermentation was examined comprehensively through assessments of in vitro and in silico protein digestion, in silico allergenicity, in vitro genotoxicity (reverse mutation and mammalian micronucleus assays), and 14-day and 90-day oral (dietary) toxicity studies in rats. There was no indication of allergenicity for SBSP in the in silico analyses. Results from both in vitro and in silico protein digestibility assessments indicated that SBSP is digested upon ingestion and would therefore be unlikely to pose a toxigenic or allergenic risk to consumers. SBSP was non-genotoxic in in vitro assays and showed no adverse effects in the 14-day or 90-day toxicity studies up to the highest dose tested. The 90-day toxicity study supports a NOAEL for SBSP of 1954 mg/kg bw/day, which corresponds to a NOAEL for MON of 408 mg/kg bw/day.

Development of a potent high-affinity human therapeutic antibody via novel application of recombination signal sequence-based affinity maturation.

Therapeutic antibody development requires discovery of an antibody molecule with desired specificities and drug-like properties. For toxicological studies, a therapeutic antibody must bind the ortholog antigen with a similar affinity to the human target to enable relevant dosing regimens, and antibodies falling short of this affinity design goal may not progress as therapeutic leads. Herein, we report the novel use of mammalian recombination signal sequence (RSS)-directed recombination for complementarity-determining region-targeted protein engineering combined with mammalian display to close the species affinity gap of human interleukin (IL)-13 antibody 731. This fully human antibody has not progressed as a therapeutic in part because of a 400-fold species affinity gap. Using this nonhypothesis-driven affinity maturation method, we generated multiple antibody variants with improved IL-13 affinity, including the highest affinity antibody reported to date (34 fM). Resolution of a cocrystal structure of the optimized antibody with the cynomolgus monkey (or nonhuman primate) IL-13 protein revealed that the RSS-derived mutations introduced multiple successive amino-acid substitutions resulting in a de novo formation of a π-π stacking-based protein-protein interaction between the affinity-matured antibody heavy chain and helix C on IL-13, as well as an introduction of an interface-distant residue, which enhanced the light chain-binding affinity to target. These mutations synergized binding of heavy and light chains to the target protein, resulting in a remarkably tight interaction, and providing a proof of concept for a new method of protein engineering, based on synergizing a mammalian display platform with novel RSS-mediated library generation.

Assessing the effects of smoking status on outcomes of elective minimally invasive paraesophageal hernia repair.

BACKGROUND: Patients are often advised on smoking cessation prior to elective surgical interventions, but the impact of active smoking on paraesophageal hernia repair (PEHR) outcomes is unclear. The objective of this cohort study was to evaluate the impact of active smoking on short-term outcomes following PEHR. METHODS: Patients who underwent elective PEHR at an academic institution between 2011 and 2022 were retrospectively reviewed. The National Surgical Quality Improvement Program (NSQIP) database from 2010 to 2021 was queried for PEHR. Patient demographics, comorbidities, and 30-day post-operative data were collected and maintained in an IRB-approved database. Cohorts were stratified by active smoking status. Primary outcomes included rates of death or serious morbidity (DSM) and radiographically identified recurrence. Bivariate and multivariable regressions were performed, and p value < 0.05 was considered statistically significant. RESULTS: 538 patients underwent elective PEHR in the single-institution cohort, of whom 5.8% (n = 31) were smokers. 77.7% (n = 394) were female, median age was 67 [IQR 59, 74] years, and median follow-up was 25.3 [IQR 3.2, 53.6] months. Rates of DSM (non-smoker 4.5% vs smoker 6.5%, p = 0.62) and hernia recurrence (33.3% vs 48.4%, p = 0.09) did not differ significantly. On multivariable analysis, smoking status was not associated with any outcome (p > 0.2). On NSQIP analysis, 38,284 PEHRs were identified, of whom 8.6% (n = 3584) were smokers. Increased DSM was observed among smokers (non-smoker 5.1%, smoker 6.2%, p = 0.004). Smoking status was independently associated with increased risk of DSM (OR 1.36, p < 0.001), respiratory complications (OR 1.94, p < 0.001), 30-day readmission (OR 1.21, p = 0.01), and discharge to higher level of care (OR 1.59, p = 0.01). No difference was seen in 30-day mortality or wound complications. CONCLUSION: Smoking status confers a small increased risk of short-term morbidity following elective PEHR without increased risk of mortality or hernia recurrence. While smoking cessation should be encouraged for all active smokers, minimally invasive PEHR in symptomatic patients should not be delayed on account of patient smoking status.

The effect of frailty and age on outcomes in elective paraesophageal hernia repair.

INTRODUCTION: Paraesophageal hernia repair (PEHR) is a safe and effective operation. Previous studies have described risk factors for poor peri-operative outcomes such as emergent operations or advanced patient age, and pre-operative frailty is a known risk factor in other major surgery. The goal of this retrospective cohort study was to determine if markers of frailty were predictive of poor peri-operative outcomes in elective paraesophageal hernia repair. METHODS: Patients who underwent elective PEHR between 1/2011 and 6/2022 at a single university-based institution were identified. Patient demographics, modified frailty index (mFI), and post-operative outcomes were recorded. A composite peri-operative morbidity outcome indicating the incidence of any of the following: prolonged length of stay (≥ 3 days), increased discharge level of care, and 30-day complications or readmissions was utilized for statistical analysis. Descriptive statistics and logistic regression were used to analyze the data. RESULTS: Of 547 patients who underwent elective PEHR, the mean age was 66.0 ± 12.3, and 77.1% (n = 422) were female. Median length of stay was 1 [IQR 1, 2]. ASA was 3-4 in 65.8% (n = 360) of patients. The composite outcome occurred in 32.4% (n = 177) of patients. On multivariate analysis, increasing age (OR 1.021, p = 0.02), high frailty (OR 2.02, p < 0.01), ASA 3-4 (OR 1.544, p = 0.05), and redo-PEHR (OR 1.72, p = 0.02) were each independently associated with the incidence of the composite outcome. On a regression of age for the composite outcome, a cutoff point of increased risk is identified at age 72 years old (OR 2.25, p < 0.01). CONCLUSION: High frailty and age over 72 years old each independently confer double the odds of a composite morbidity outcome that includes prolonged post-operative stay, peri-operative complications, the need for a higher level of care after elective paraesophageal hernia repair, and 30-day readmission. This provides additional information to counsel patients pre-operatively, as well as a potential opportunity for targeted pre-habilitation.

COVID-19 Vaccination Rates and Vaccine Hesitancy Among Spanish-Speaking Free Clinic Patients.

We sought to assess COVID-19 vaccination rates, as well as attitudes and beliefs towards the vaccine, of patients in a Spanish-speaking student-run free clinic in Columbus, Ohio. A cross-sectional study was performed. Surveys were distributed to all individuals over 18 years who presented to La Clínica Latina between July, 2022 and September, 2022. A convenience sample was used: patients in the waiting room and their accompanying family members or friends were invited to participate. Subjects were excluded if under the age of 18 or over the age of 75, or if non-Spanish speaking. Of the 158 individuals who agreed to participate in our study, 146 responded to the question regarding vaccination status, revealing 90.4% of respondents had received a COVID-19 vaccination. Most respondents learned about the vaccine from social media (26.4%) or television (22.7%). The majority of participants sought answers to questions surrounding the vaccine by asking their doctor (49.1%). The most common reason among unvaccinated participants for not undergoing vaccination was fear of an adverse reaction to the vaccine (n = 11). We found that a large proportion (90.4%) of individuals seeking care at a Spanish-speaking free clinic were vaccinated against COVID-19. Our study also provides perspective on the means of health knowledge acquisition and behaviors in this predominantly Latinx patient population in central Ohio. We can utilize our results to optimize and tailor clinic services and initiatives for COVID-19 boosters to meet the needs of this community.

Enzyme directed evolution using genetically encodable biosensors.

Directed evolution has been remarkably successful in identifying enzyme variants with new or improved properties, such as altered substrate scope or novel reactivity. Genetically encodable biosensors (GEBs), which convert the concentration of a small molecule ligand into an easily detectable output signal, have seen increasing application to enzyme directed evolution in the last decade. GEBs enable the use of high-throughput methods to assess enzyme activity of very large libraries, which can accelerate the search for variants with desirable activity. Here, we review different classes of GEBs and their properties in the context of enzyme evolution, how GEBs have been integrated into directed evolution workflows, and recent examples of enzyme evolution efforts utilizing GEBs. Finally, we discuss the advantages, challenges, and opportunities for using GEBs in the directed evolution of enzymes.

Reductions in Gray Matter Linked to Epigenetic HIV-Associated Accelerated Aging.

A growing literature suggests a relationship between HIV-infection and a molecular profile of age acceleration. However, despite the widely known high prevalence of HIV-related brain atrophy and HIV-associated neurocognitive disorder (HAND), epigenetic age acceleration has not been linked to HIV-related changes in structural MRI. We applied morphological MRI methods to study the brain structure of 110 virally suppressed participants with HIV infection and 122 uninfected controls age 22-72. All participants were assessed for cognitive impairment, and blood samples were collected from a subset of 86 participants with HIV and 83 controls to estimate epigenetic age. We examined the group-level interactive effects of HIV and chronological age and then used individual estimations of epigenetic age to understand the relationship between age acceleration and brain structure. Finally, we studied the effects of HAND. HIV-infection was related to gray matter reductions, independent of age. However, using epigenetic age as a biomarker for age acceleration, individual HIV-related age acceleration was associated with reductions in total gray matter. HAND was associated with decreases in thalamic and hippocampal gray matter. In conclusion, despite viral suppression, accentuated gray matter loss is evident with HIV-infection, and greater biological age acceleration specifically relates to such gray matter loss.

The serum protein transthyretin as a platform for dimerization and tetramerization of antibodies and Fab fragments to enable target clustering.

Transthyretin (TTR) is an abundant homotetrameric serum protein and was selected here for engineering higher-valency molecules because of its compact size, simple structure, and natural propensity to tetramerize. To demonstrate this utility, we fused TTR to the C terminus of conatumumab, an antibody that targets tumor necrosis factor-related apoptosis-inducing ligand receptor 2, as heavy chains to form antibody dimers and Fab heavy chains to form Fab tetramers. Moreover, we used constant heavy domain 3 heterodimerization substitutions to create TTR-mediated conatumumab tetramers. The conatumumab-TTR fusions displayed substantially enhanced potency in cell-based assays, as well as in murine tumor xenograft models. We conclude that antibody-TTR fusions may provide a powerful platform for multimerizing antibody and Fab fragments to enhance the capabilities of human therapeutics that benefit from target clustering and higher-order antigen-binding valency.

Fascial thickness and stiffness in hypermobile Ehlers-Danlos syndrome.

There is a high prevalence of myofascial pain in people with hypermobile Ehlers-Danlos Syndrome (hEDS). The fascial origin of pain may correspond to changes in the extracellular matrix. The objective of this study was to investigate structural changes in fascia in hEDS. A series of 65 patients were examined prospectively-26 with hEDS, and 39 subjects with chronic neck, knee, or back pain without hEDS. The deep fascia of the sternocleidomastoid, iliotibial tract, and iliac fascia were examined with B-mode ultrasound and strain elastography, and the thicknesses were measured. Stiffness (strain index) was measured semi-quantitatively using elastography comparing fascia to muscle. Differences between groups were compared using one-way analysis of variance. hEDS subjects had a higher mean thickness in the deep fascia of the sternocleidomastoid compared with non-hEDS subjects. There was no significant difference in thickness of the iliac fascia and iliotibial tract between groups. Non-hEDS subjects with pain had a higher strain index (more softening of the fascia with relative stiffening of the muscle) compared with hEDS subjects and non-hEDS subjects without back or knee pain. In myofascial pain, softening of the fascia may occur from increase in extracellular matrix content and relative increase in stiffness of the muscle; this change is not as pronounced in hEDS.

Epigenetic Markers of Aging Predict the Neural Oscillations Serving Selective Attention.

Chronological age remains an imperfect measure of accumulated physiological stress. Biological measures of aging may provide key advantages, allowing scientists focusing on age-related functional changes to use metrics derived from epigenetic factors like DNA methylation (DNAm), which could provide greater precision. Here we investigated the relationship between methylation-based age and an essential cognitive function that is known to exhibit age-related decline: selective attention. We found that DNAm-age predicted selective attention abilities and fully mediated the relationship between selective attention and chronological age. Using neuroimaging with magnetoencephalography, we found that gamma activity in the anterior cingulate was robustly predicted by DNAm-derived biological age, revealing the neural dynamics underlying this DNAm age-related cognitive decline. Anterior cingulate gamma activity also significantly predicted behavior on the selective attention task, indicating its functional relevance. These findings suggest that DNAm age may be a better predictor of cognitive and brain aging than more traditional chronological metrics.

Methylglyoxal-derived posttranslational arginine modifications are abundant histone marks.

Histone posttranslational modifications (PTMs) regulate chromatin dynamics, DNA accessibility, and transcription to expand the genetic code. Many of these PTMs are produced through cellular metabolism to offer both feedback and feedforward regulation. Herein we describe the existence of Lys and Arg modifications on histones by a glycolytic by-product, methylglyoxal (MGO). Our data demonstrate that adduction of histones by MGO is an abundant modification, present at the same order of magnitude as Arg methylation. These modifications were detected on all four core histones at critical residues involved in both nucleosome stability and reader domain binding. In addition, MGO treatment of cells lacking the major detoxifying enzyme, glyoxalase 1, results in marked disruption of H2B acetylation and ubiquitylation without affecting H2A, H3, and H4 modifications. Using RNA sequencing, we show that MGO is capable of altering gene transcription, most notably in cells lacking GLO1. Finally, we show that the deglycase DJ-1 protects histones from adduction by MGO. Collectively, our findings demonstrate the existence of a previously undetected histone modification derived from glycolysis, which may have far-reaching implications for the control of gene expression and protein transcription linked to metabolism.

Continuous directed evolution of proteins with improved soluble expression.

We report the development of soluble expression phage-assisted continuous evolution (SE-PACE), a system for rapidly evolving proteins with increased soluble expression. Through use of a PACE-compatible AND gate that uses a split-intein pIII, SE-PACE enables two simultaneous positive selections to evolve proteins with improved expression while maintaining their desired activities. In as little as three days, SE-PACE evolved several antibody fragments with >5-fold improvement in expression yield while retaining binding activity. We also developed an activity-independent form of SE-PACE to correct folding-defective variants of maltose-binding protein (MBP) and to evolve variants of the eukaryotic cytidine deaminase APOBEC1 with improved expression properties. These evolved APOBEC1 variants were found to improve the expression and apparent activity of Cas9-derived base editors when used in place of the wild-type cytidine deaminase. Together, these results suggest that SE-PACE can be applied to a wide variety of proteins to rapidly improve their soluble expression.

Low persistence to rivaroxaban or warfarin among patients with new venous thromboembolism at a safety net academic medical center.

Recent guidelines recommend direct acting oral anticoagulants (DOAC) over vitamin-k antagonist (VKA) for acute venous thromboembolism (VTE). Non-adherence to anticoagulation has been associated with increased frequency of VTE or stroke. This study evaluated 90 day persistence among patients prescribed rivaroxaban or warfarin for the treatment of acute VTE at an academic safety net hospital. We conducted a single center, retrospective cohort study of 314 consecutive patients newly prescribed rivaroxaban or warfarin for acute VTE between January 2016 and July 2017. Primary outcome was 90 day persistence, and secondary outcomes included 90 day readmission and/or ED visit, time to 90 m day readmission and/or ED visits, and attendance of direct oral anticoagulant education class. Of 314 patients, 78 were prescribed warfarin and 236 rivaroxaban. Patients had a mean age of 52 years, 62% were men, and 96% were diagnosed with deep vein thrombosis and/or pulmonary embolism. Persistence at 90 days was 52.6% among patients prescribed warfarin compared to 45.3% for patients prescribed rivaroxaban (p = 0.2678). Persistencewas associated with decreased 90 day hospital or ED readmission. Among patients prescribed rivaroxaban, attending a pharmacist led educational class was associated with a 2.5 fold increase in persistence (p < 0.0001). Among patients with new onset venous thromboembolism, 90 day persistence with anticoagulation was similarly low with either rivaroxaban or warfarin therapy. Participation in a pharmacist led DOAC class was associated with a 2.5-fold increase in persistence on rivaroxaban.

Acute myeloid leukemia with complex cytogenetic abnormalities associated with long-term use of oral colloidal silver as nutritional supplement - Case report and review of literature.

We report a case of acute myeloid leukemia with complex cytogenetic abnormalities suggestive of preexisting myelodysplastic syndrome in a patient with habitual ingestion of colloidal silver as nutritional supplement for over 10 years and the medical literature is reviewed.

Nitric oxide homeostasis is maintained during acute in vitro hypoxia and following reoxygenation in naked mole-rat but not mouse cortical neurons.

Reactive nitrogen species (RNS), including nitric oxide (NO), are important cellular messengers when tightly regulated, but unregulated production of RNS during hypoxia or ischemia and reoxygenation is deleterious to hypoxia-intolerant brain. Therefore, maintaining NO homeostasis during hypoxia/ischemia and reoxygenation may be a hallmark of hypoxia-tolerant brain. Unlike most mammals, naked mole-rats (NMRs; Heterocephalus glaber) are tolerant of repeated bouts of hypoxia in vivo. Although there is some evidence that NMR brain is tolerant of hypoxia/ischemia, little is known about the underlying neuroprotective mechanism(s), and their tolerance to reoxygenation injury has not been examined. We hypothesized that NMR brain would maintain NO homeostasis better than hypoxia-intolerant mouse brain during hypoxic/ischemic stresses and following reoxygenation. To test this, we exposed adult NMR and mouse cortical slices to transitions from normoxia (21% O2) to hypoxia (< 1% O2) or ischemia (oxygen glucose deprivation, OGD), followed by reoxygenation, while measuring neuronal NO production. We report that NMR cortical neurons maintain NO homeostasis during hypoxia/OGD and avoid bursts of NO upon reoxygenation. Conversely, mouse cortical neurons maintain NO homeostasis in OGD but not hypoxia and exhibit a burst of NO upon reperfusion. This suggests that maintenance of NO homeostasis during fluctuating O2 availability may be a contributing neuroprotective mechanism against hypoxia/ischemia and reoxygenation injury in hypoxia-tolerant NMR brain.

Arthroscopic Vastus Elevation (AVE) for Arthrofibrosis of the Knee: Surgical Technique and Literature Review.

Arthrofibrosis of the knee continues to challenge Orthopaedic surgeons. With a wide etiology, lack of knee motion can be debilitating. Its surgical management has several complications. The purpose of this study is to describe a modification of previously described techniques to aid in the management of knee arthrofibrosis. Arthroscopic vastus elevation in conjunction with adjuvant hemostatic agents allows for a controlled quadriceps elevation in the setting of arthrofibrosis. In addition to a thorough intra-articular lysis of adhesions, this appears to improve motion, while minimizing postoperative complications. Minimized postoperative complications include extensor lag, skin necrosis, and bleeding complications. (Journal of Surgical Orthopaedic Advances 29(2):117-120, 2020).

Myofascial Injection Using Fascial Layer-Specific Hydromanipulation Technique (FLuSH) and the Delineation of Multifactorial Myofascial Pain.

Background and objectives: The aims of this study were to delineate the contribution of specific fascial layers of the myofascial unit to myofascial pain and introduce the use of ultrasound-guided fascial layer-specific hydromanipulation (FLuSH) as a novel technique in the treatment of myofascial pain. Materials and Methods: The clinical data of 20 consecutive adult patients who underwent myofascial injections using FLuSH technique for the treatment of myofascial pain were reviewed. The FLuSH technique involved measuring the pain pressure threshold using an analog algometer initially and after each ultrasound guided injection of normal saline into the specific layers of the myofascial unit (superficial fascia, deep fascia, or muscle) in myofascial points corresponding with Centers of Coordination/Fusion (Fascial Manipulation®). The outcome measured was the change in pain pressure threshold after injection of each specific fascial layer. Results: Deep fascia was involved in 73%, superficial fascia in 55%, and muscle in 43% of points. A non-response to treatment of all three layers occurred in 10% of all injected points. The most common combinations of fascial layer involvement were deep fascia alone in 23%, deep fascia and superficial fascia in 22%, and deep fascia and muscle in 18% of injected points. Each individual had on average of 3.0 ± 1.2 different combinations of fascial layers contributing to myofascial pain. Conclusions: The data support the hypothesis that multiple fascial layers are responsible for myofascial pain. In particular, for a given patient, pain may develop from discrete combinations of fascial layers unique to each myofascial point. Non-response to treatment of the myofascial unit may represent a centralized pain process. Adequate treatment of myofascial pain may require treatment of each point as a distinct pathologic entity rather than uniformly in a given patient or across patients.