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Neurotrauma registries (NTR) collect data on traumatic brain injuries (TBI) to advance knowledge, shape policies, and improve outcomes. This study reviews global NTRs from High-Income (HICs) and Low- and Middle-Income countries (LMICs). A systematic review was conducted using PubMed, Google Scholar, Embase, and Web of Science following PRISMA guidelines to identify relevant NTRs. Twenty-six articles were included, revealing ten different NTRs from Europe, North America, Latin America, the Middle East, and Asia. North America had the most registries at four, followed by Europe and Asia with two each, and Latin America and the Middle East with one each. The median database size was 1,734 patients (Range: 65-25,000), with the largest registry from the United States (FITBIR DB) and the smallest from Iran (NSCIR-IR). The longest data collection period was 32 years, with a mean age of 43.1 years (Range: 9.07-60.0). Males comprised 70 % of patients. Sixty-six percent of articles emphasized outcomes such as functionality, length of stay, and mortality. Key challenges identified included issues with missing data and incomplete records (n = 4), lack of standardization in data collection procedures (n = 3), staffing shortages (n = 5), lack of IT infrastructure (n = 3), and problems with reproducibility, particularly in high-income countries (n = 4). Our review highlights the need for a large-scale global NTR, addressing LMIC barriers through private-public partnerships with organized neurosurgery members.

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Supported lipid bilayers (SLBs) are useful structures for mimicking cellular membranes, and they can be integrated with a variety of sensors. Although there are a variety of methods for forming SLBs, many of these methods come with limitations in terms of the lipid compositions that can be employed and the substrates upon which the SLBs can be deposited. Here we demonstrate the use of an all-aqueous chaotropic agent exchange process that can be used to form SLBs on two different substrate materials: SiO2, which is compatible with traditional SLB formation by vesicle fusion, and Al2O3, which is not compatible with vesicle fusion. When examined with a quartz crystal microbalance with dissipation monitoring, the SLBs generated by chaotropic agent exchange (CASLBs) have similar frequency and dissipation shifts to SLBs formed by the vesicle fusion technique. The CASLBs block nonspecific protein adsorption on the substrate and can be used to sense protein-lipid interactions. Fluorescence microscopy was used to examine the CASLBs, and we observed long-range lateral diffusion of fluorescent probes, which confirmed that the CASLBs were composed of a continuous, planar lipid bilayer. Our CASLB method provides another option for forming planar lipid bilayers on a variety of surfaces, including those that are not amenable to the widely used vesicle fusion method.

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Recent associations between Major Depressive Disorder (MDD) and measures of premature aging suggest accelerated biological aging as a potential biomarker for MDD susceptibility or MDD as a risk factor for age-related diseases. Statistical and machine learning regression models of biological age have been trained on various sources of high dimensional data to predict chronological age. Residuals or "gaps" between the predicted biological age and chronological age have been used for statistical inference, such as testing whether an increased age gap is associated with a given disease state. Recently, a gene expression-based model of biological age showed a higher age gap for individuals with MDD compared to healthy controls (HC). In the current study, we propose a machine learning approach that simplifies gene selection by using a least absolute shrinkage and selection operator (LASSO) penalty to construct an expression-based Gene Age Gap Estimate (GAGE) model. We construct the LASSO-GAGE (L-GAGE) model in an RNA-Seq study of 78 unmedicated individuals with MDD and 79 HC and then test for accelerated biological aging in MDD. When testing L-GAGE association with MDD, we account for factors such as sex and chronological age to mitigate regression to the mean effects. The L-GAGE shows higher biological aging in MDD subjects than HC, but the elevation is not statistically significant. However, when we dichotomize chronological age, the interaction between MDD status and age is significant in L-GAGE model. This effect remains statistically significant even after adjusting for chronological age and sex. We find cytomegalovirus (CMV) serostatus is associated with elevated L-GAGE. We also investigate feature selection methods Random Forest and nearest neighbor projected distance regression (NPDR) to characterize age related genes, and we find functional enrichment of infectious disease and SARS-COV pathways.

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BACKGROUND: Macrophage-based cell therapies have shown modest success in clinical trials, which can be attributed to their phenotypic plasticity, where transplanted macrophages get reprogrammed towards a pro-tumor phenotype. In most tumor types, including melanoma, the balance between antitumor M1-like and tumor-promoting M2-like macrophages is critical in defining the local immune response with a higher M1/M2 ratio favoring antitumor immunity. Therefore, designing novel strategies to increase the M1/M2 ratio in the TME has high clinical significance and benefits macrophage-based cell therapies. METHODS: In this study, we reprogrammed antitumor and proinflammatory macrophages ex-vivo with HDAC6 inhibitors (HDAC6i). We administered the reprogrammed macrophages intratumorally as an adoptive cell therapy (ACT) in the syngeneic SM1 murine melanoma model and patient-derived xenograft bearing NSG-SGM3 humanized mouse models. We phenotyped the tumor-infiltrated immune cells by flow cytometry and histological analysis of tumor sections for macrophage markers. We performed bulk RNA-seq profiling of murine bone marrow-derived macrophages treated with vehicle or HDAC6i and single-cell RNA-seq profiling of SM1 tumor-infiltrated immune cells to determine the effect of intratumor macrophage ACT on the tumor microenvironment (TME). We further analyzed the single-cell data to identify key cell-cell interactions and trajectory analysis to determine the fate of tumor-associated macrophages post-ACT. RESULTS: Macrophage ACT resulted in diminished tumor growth in both mouse models. We also demonstrated that HDAC6 inhibition in macrophages suppressed the polarization toward tumor-promoting phenotype by attenuating STAT3-mediated M2 reprogramming. Two weeks post-transplantation, ACT macrophages were viable, and inhibition of HDAC6 rendered intratumor transplanted M1 macrophages resistant to repolarization towards protumor M2 phenotype in-vivo. Further characterization of tumors by flow cytometry, single-cell transcriptomics, and single-cell secretome analyses revealed a significant enrichment of antitumor M1-like macrophages, resulting in increased M1/M2 ratio and infiltration of CD8 effector T-cells. Computational analysis of single-cell RNA-seq data for cell-cell interactions and trajectory analyses indicated activation of monocytes and T-cells in the TME. CONCLUSIONS: In summary, for the first time, we demonstrated the potential of reprogramming macrophages ex-vivo with HDAC6 inhibitors as a viable macrophage cell therapy to treat solid tumors.

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The determination of chemical mixture components is vital to a multitude of scientific fields. Oftentimes spectroscopic methods are employed to decipher the composition of these mixtures. However, the sheer density of spectral features present in spectroscopic databases can make unambiguous assignment to individual species challenging. Yet, components of a mixture are commonly chemically related due to environmental processes or shared precursor molecules. Therefore, analysis of the chemical relevance of a molecule is important when determining which species are present in a mixture. In this paper, we combine machine-learning molecular embedding methods with a graph-based ranking system to determine the likelihood of a molecule being present in a mixture based on the other known species and/or chemical priors. By incorporating this metric in a rotational spectroscopy mixture analysis algorithm, we demonstrate that the mixture components can be identified with extremely high accuracy (≥97%) in an efficient manner.

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STUDY DESIGN: Cross-sectional study. OBJECTIVE: To evaluate for areas of consensus and divergence of opinion within the spine community regarding the management of cervical spondylotic conditions and acute traumatic central cord syndrome (ATCCS) and the influence of the patient's age, disease severity, and myelomalacia. SUMMARY OF BACKGROUND DATA: There is ongoing disagreement regarding the indications for, and urgency of, operative intervention in patients with mild degenerative myelopathy, moderate to severe radiculopathy, isolated axial symptomatology with evidence of spinal cord compression, and ATCCS without myelomalacia. METHODS: A survey request was sent to 330 attendees of the Cervical Spine Research Society (CSRS) 2021 Annual Meeting to assess practice patterns regarding the treatment of cervical stenosis, myelopathy, radiculopathy, and ATCCS in 16 unique clinical vignettes with associated MRIs. Operative versus nonoperative treatment consensus was defined by a management option selected by >80% of survey participants. RESULTS: Overall, 116 meeting attendees completed the survey. Consensus supported nonoperative management for elderly patients with axial neck pain and adults with axial neck pain without myelomalacia. Operative management was indicated for adult patients with mild myelopathy and myelomalacia, adult patients with severe radiculopathy, elderly patients with severe radiculopathy and myelomalacia, and elderly ATCCS patients with pre-existing myelopathic symptoms. Treatment discrepancy in favor of nonoperative management was found for adult patients with isolated axial symptomatology and myelomalacia. Treatment discrepancy favored operative management for elderly patients with mild myelopathy, adult patients with mild myelopathy without myelomalacia, elderly patients with severe radiculopathy without myelomalacia, and elderly ATCCS patients without preceding symptoms. CONCLUSIONS: Although there is uncertainty regarding the treatment of mild myelopathy, operative intervention was favored for nonelderly patients with evidence of myelomalacia or radiculopathy and for elderly patients with ATCCS, especially if pre-injury myelopathic symptoms were present. LEVEL OF EVIDENCE: Level V.

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Radiolabeled small-molecule DOTA-haptens can be combined with antitumor/anti-DOTA bispecific antibodies (BsAbs) for pretargeted radioimmunotherapy (PRIT). For optimized delivery of the theranostic γ- and ß-emitting isotope 177Lu with DOTA-based PRIT (DOTA-PRIT), bivalent Gemini (DOTA-Bn-thiourea-PEG4-thiourea-Bn-DOTA, aka (3,6,9,12-tetraoxatetradecane-1,14-diyl)bis(DOTA-benzyl thiourea)) was developed. Methods: Gemini was synthesized by linking 2 S-2-(4-isothiocyanatobenzyl)-DOTA molecules together via a 1,14-diamino-PEG4 linker. [177Lu]Lu-Gemini was prepared with no-carrier-added 177LuCl3 to a molar-specific activity of 123 GBq/µmol and radiochemical purity of more than 99%. The specificity of BsAb-177Lu-Gemini was verified in vitro. Subsequently, we evaluated biodistribution and whole-body clearance for [177Lu]Lu-Gemini and, for comparison, our gold-standard monovalent [177Lu]Lu-S-2-(4-aminobenzyl)-DOTA ([177Lu]Lu-DOTA-Bn) in naïve (tumor-free) athymic nude mice. For our proof-of-concept system, a 3-step pretargeting approach was performed with an established DOTA-PRIT regimen (anti-GPA33/anti-DOTA IgG-scFv BsAb, a clearing agent, and [177Lu]Lu-Gemini) in mouse models. Results: Initial in vivo studies showed that [177Lu]Lu-Gemini behaved similarly to [177Lu]Lu-DOTA-Bn, with almost identical blood and whole-body clearance kinetics, as well as biodistribution and mouse kidney dosimetry. Pretargeting [177Lu]Lu-Gemini to GPA33-expressing SW1222 human colorectal xenografts was highly effective, leading to absorbed doses of [177Lu]Lu-Gemini for blood, tumor, liver, spleen, and kidneys of 3.99, 455, 6.93, 5.36, and 14.0 cGy/MBq, respectively. Tumor-to-normal tissue absorbed-dose ratios (i.e., therapeutic indices [TIs]) for the blood and kidneys were 114 and 33, respectively. In addition, we demonstrate that the use of bivalent [177Lu]Lu-Gemini in DOTA-PRIT leads to improved TIs and augmented [177Lu]Lu-Gemini tumor uptake and retention in comparison to monovalent [177Lu]Lu-DOTA-Bn. Finally, we established efficacy in SW1222 tumor-bearing mice, demonstrating that a single injection of anti-GPA33 DOTA-PRIT with 44 MBq (1.2 mCi) of [177Lu]Lu-Gemini (estimated tumor-absorbed dose, 200 Gy) induced complete responses in 5 of 5 animals and a histologic cure in 2 of 5 (40%) animals. Moreover, a significant increase in survival compared with nontreated controls was noted (maximum tolerated dose not reached). Conclusion: We have developed a bivalent DOTA-radiohapten, [177Lu]Lu-Gemini, that showed improved radiopharmacology for DOTA-PRIT application. The use of bivalent [177Lu]Lu-Gemini in DOTA-PRIT, as opposed to monovalent [177Lu]Lu-DOTA-Bn, allows curative treatments with considerably less administered 177Lu activity while still achieving high TIs for both the blood (>100) and the kidneys (>30).

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Epithelial and immune cells have long been appreciated for their contribution to the early immune response after injury; however, much less is known about the role of mesenchymal cells. Using single nuclei RNA-sequencing, we defined changes in gene expression associated with inflammation at 1-day post-wounding (dpw) in mouse skin. Compared to keratinocytes and myeloid cells, we detected enriched expression of pro-inflammatory genes in fibroblasts associated with deeper layers of the skin. In particular, SCA1+ fibroblasts were enriched for numerous chemokines, including CCL2, CCL7, and IL33 compared to SCA1- fibroblasts. Genetic deletion of Ccl2 in fibroblasts resulted in fewer wound bed macrophages and monocytes during injury-induced inflammation with reduced revascularization and re-epithelialization during the proliferation phase of healing. These findings highlight the important contribution of deep skin fibroblast-derived factors to injury-induced inflammation and the impact of immune cell dysregulation on subsequent tissue repair.

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STUDY DESIGN: Retrospective cohort study. OBJECTIVE: The impact of paraspinal sarcopenia following fusions that extend to the upper thoracic spine remain unknown. The purpose of the present study was to assess the impact of sarcopenia on the development of PJK and PJF following spine fusion surgery from the upper thoracic spine to the pelvis. METHODS: We performed a retrospective review of patients who underwent spine fusion surgery that extended caudally to the pelvis and terminated cranially between T1-6. The cohort was divided into 2 groups: (1) patients without PJK or PJF and (2) patients with PJK and/or PJF. Univariate and multivariate analyses were performed to determine risk factors for the development of proximal junctional complications. RESULTS: We identified 81 patients for inclusion in this study. Mean HU at the UIV was 186.1 ± 47.5 in the cohort of patients without PJK or PJF, which was substantially higher than values recorded in the PJK/PJF subgroup (142.4 ± 40.2) (P < 0.001). Severe multifidus sarcopenia was identified at a higher rate in the subgroup of patients who developed proximal junction pathology (66.7%) than in the subgroup of patients who developed neither PJK nor PJF (7.4%; P < 0.001). Multivariate analysis demonstrated both low HU at the UIV and moderate-severe multifidus sarcopenia to be risk factors for the development of PJK and PJF. CONCLUSIONS: Severe paraspinal sarcopenia and diminished bone density at the UIV impart an increased risk of developing PJK and PJF in following thoracolumbar fusions from the upper thoracic spine to the pelvis.

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Significance: Minimally invasive surgery (MIS) has shown vast improvement over open surgery by reducing post-operative stays, intraoperative blood loss, and infection rates. However, in spite of these improvements, there are still prevalent issues surrounding MIS that may be addressed through hyperspectral imaging (HSI). We present a laparoscopic HSI system to further advance the field of MIS. Aim: We present an imaging system that integrates high-speed HSI technology with a clinical laparoscopic setup and validate the system's accuracy and functionality. Different configurations that cover the visible (VIS) to near-infrared (NIR) range of electromagnetism are assessed by gauging the spectral fidelity and spatial resolution of each hyperspectral camera. Approach: Standard Spectralon reflectance tiles were used to provide ground truth spectral footprints to compare with those acquired by our system using the root mean squared error (RMSE). Demosaicing techniques were investigated and used to measure and improve spatial resolution, which was assessed with a USAF resolution test target. A perception-based image quality evaluator was used to assess the demosaicing techniques we developed. Two configurations of the system were developed for evaluation. The functionality of the system was investigated in a phantom study and by imaging ex vivo tissues. Results: Multiple configurations of our system were tested, each covering different spectral ranges, including VIS (460 to 600 nm), red/NIR (RNIR) (610 to 850 nm), and NIR (665 to 950 nm). Each configuration is capable of achieving real-time imaging speeds of up to 20 frames per second. RMSE values of 3.51 ± 2.03 % , 3.43 ± 0.84 % , and 3.47% were achieved for the VIS, RNIR, and NIR systems, respectively. We obtained sub-millimeter resolution using our demosaicing techniques. Conclusions: We developed and validated a high-speed hyperspectral laparoscopic imaging system. The HSI system can be used as an intraoperative imaging tool for tissue classification during laparoscopic surgery.

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BACKGROUND AND OBJECTIVES: Charcot-Marie-Tooth disease type 4J (CMT4J) is caused by autosomal recessive variants in the Factor-Induced Gene 4 (FIG4) gene. Recent preclinical work has demonstrated the feasibility of adeno-associated virus serotype 9-FIG4 gene therapy. This study aimed to further characterize the CMT4J phenotype and evaluate feasibility of validated CMT-related outcome measures for future clinical trials. METHODS: This cross-sectional study enrolled children and adults with genetically confirmed CMT4J, with 2 documented disease-causing variants in the FIG4 gene. Patients were recruited through the Inherited Neuropathy Consortium network. Disease severity was assessed using standardized CMT-specific outcome measures and exploratory biomarkers including muscle MRI fat fraction, electrophysiology, and neurofilament light chain levels. Descriptive statistics and correlation analyses were conducted to explore relationships between variables. RESULTS: We recruited a total of 19 patients, including 14 pediatric patients (mean age 10.9 ± 3.9 years) and 5 adults (mean age 40.0 ± 13.9 years). The most frequent symptoms were gross motor delay and distal more than proximal muscle weakness, which were observed in 14 of 19 patients. The most common non-neuromuscular symptoms were cognitive and respiratory deficits, each seen in 8 of 19 patients. We denoted asymmetric weakness in 2 patients and nonuniform slowing of conduction velocities in 6 patients. Charcot-Marie-Tooth Disease Pediatric Scale (CMTPedS), Pediatric Quality of Life Inventory, and Vineland Adaptive Behavior Scale scores were affected in most patients. We observed a significant positive correlation between neurofilament light chain levels and CMTPedS, but the study was underpowered to observe a correlation between CMTPedS and MRI fat fraction. DISCUSSION: We obtained baseline clinical and biomarker data in a broad cohort with CMT4J in pediatric and adult patients. Motor delay, muscle weakness, and respiratory and cognitive difficulties were the most common clinical manifestations of CMT4J. Many patients had nerve conduction studies with nonuniform slowing, and 2 had an asymmetric pattern of muscle weakness. We observed that the neurofilament light chain levels correlated with the CMTPedS in the pediatric population. This study showed feasibility of clinical outcomes including CMTPedS in assessment of disease severity in the pediatric patient population and provided baseline characteristics of exploratory biomarkers, neurofilament light chain levels, and muscle MRI fat fraction. The coronavirus disease 2019 pandemic affected some of the visits, resulting in a reduced number of some of the assessments.

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Surface grafting of polymer brushes drastically modifies surface properties, including wettability, compatibility, responsiveness, etc. A broad variety of functionalities can be introduced to the surface via different types of polymers. Bringing together properties of two or more types of polymer brushes to one surface opens up even more possibilities in brush-modified materials. However, while it is generally feasible to introduce several chemical compositions along the brushes via copolymerization, it is challenging to vary the types of polymer brushes along a surface. Although previous studies have demonstrated binary brushes via orthogonal polymerization techniques or partial deactivation/regrafting, they commonly limit the number of polymer types to two. Here, we propose a strategy to introduce dynamic covalent diketoenamine linkages at the root of polymer brushes. The grafting density could be precisely tuned during surface functionalization. The surface-anchored polymer brushes were cleaved by the addition of small molecule amines. New polymer brushes can be regrafted from the surface following refunctionalization of exposed sites. The maneuverability allows tuning of the types and densities of the polymer brushes, pointing the way to the preparation of a new generation of well-defined brush-modified materials with mixed grafts, with potential applications in the design of smart materials and surfaces.

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In vitro and in vivo tests for therapeutic agents are typically conducted in sterile environments, but many target areas for drug delivery are home to thousands of microbial species. Here, we examine the behaviour of lipidic nanomaterials after exposure to representative strains of four bacterial species found in the gastrointestinal tract and skin. Small angle X-ray scattering measurements show that the nanostructure of monoolein cubic and inverse hexagonal phases are transformed, respectively, into inverse hexagonal and inverse micellar cubic phases upon exposure to a strain of live Staphylococcus aureus often present on skin and mucosa. Further investigation demonstrates that enzymatic hydrolysis and cell membrane lipid transfer are both likely responsible for this effect. The structural responses to S. aureus are rapid and significantly reduce the rate of drug release from monoolein-based nanomaterials. These findings are the first to demonstrate how a key species in the live human microbiome can trigger changes in the structure and drug release properties of lipidic nanomaterials. The effect appears to be strain specific, varies from patient to patient and body region to body region, and is anticipated to affect the bioapplication of monoglyceride-based formulations.

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Academic medicine continues to characterize the experiences of Black and other minoritized faculty in medicine to enhance their careers and promote their advancement. An issue of discussion is tenure and its role in the advancement and retention of this group. Tenure is a sign of national presence, command of an area of study, and can demonstrate support from the institution in terms of permanent employment, eligibility to apply for awards, sit or vote on certain committees or qualify for certain leadership opportunities. Anecdotally there have been reports that tenure is a thing of the past that has lost relevance prompting some to end tenure in their institutions. Reasons for this are complex, however the literature does not include minoritized faculty as a reason for the need to revise or eliminate tenure and tenure earning tracks. The authors discuss 3 reasons why Black and other minoritized faculty should be afforded the opportunity to achieve permanent status in their academic health centers. They include histories of being denied freedom, having information concealed or being giving false information, and being denied permanent academic employment status.

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A comprehensive computational study on the underlying reactivity of iron tetra-NHC complexes for C2 + N1 aziridination catalysis is presented. A library of 18 unique iron tetra-NHC complexes was constructed, and a computational screening was performed on the reaction barriers associated with the rate-determining step (formation of an open chain radical intermediate). Thermodynamic barriers were computed along with a variety of steric and electronic properties, including the percentage of buried volume, orbital energies and ETS-NOCV analysis, which were used to identify key characteristics related to reactivity. The analysis performed in this study successfully identified key differences in tetracarbenes, such as linking groups (BMe2 or CH2) and the identity of the NHC groups (imidazole, imidazoline or benzimidazole) in terms of sterics, electronics and thermodynamics. Additionally, we have proposed two reaction pathways based on electronic structure arguments for the formation of the key open-chain radical intermediate. The first reaction pathway proceeds through a σ-hole channel where the Fe(IV)-imide intermediate evolves into Fe(III)-imidyl radical through electron donation into the antibonding σ* orbital, while the second involves a Fe(III)-imidyl radical formed through a π-hole channel (donation into π*). These pathways are consistent with the isoelectronic iron(IV)-oxo species for hydrogen atom abstraction mechanisms and they can be used as descriptors of the rate-determining step of the aziridination reaction.

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Major developmental events occurring in the hippocampus during the third trimester of human gestation and neonatally in altricial rodents include rapid and synchronized dendritic arborization and astrocyte proliferation and maturation. We tested the hypothesis that signals sent by developing astrocytes to developing neurons modulate dendritic development in vivo. We altered neuronal development by neonatal (third trimester-equivalent) ethanol exposure in mice; this treatment increased dendritic arborization in hippocampal pyramidal neurons. We next assessed concurrent changes in the mouse astrocyte translatome by translating ribosomal affinity purification (TRAP)-seq. We followed up on ethanol-inhibition of astrocyte Chpf2 and Chsy1 gene translation because these genes encode for biosynthetic enzymes of chondroitin sulfate glycosaminoglycan (CS-GAG) chains (extracellular matrix components that inhibit neuronal development and plasticity) and have not been explored before for their roles in dendritic arborization. We report that Chpf2 and Chsy1 are enriched in astrocytes and their translation is inhibited by ethanol, which also reduces the levels of CS-GAGs measured by Liquid Chromatography/Mass Spectrometry. Finally, astrocyte-conditioned medium derived from Chfp2-silenced astrocytes increased neurite branching of hippocampal neurons in vitro. These results demonstrate that CS-GAG biosynthetic enzymes in astrocytes regulates dendritic arborization in developing neurons.

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BACKGROUND: Eccentric resistance training elicits greater preservation of training-induced muscular adaptations compared with other training modalities, however the detraining profiles of different training dosages remain unknown. AIMS: To examine the detraining effects following once- or twice-weekly eccentric-specific resistance training in older adults. METHODS: Twenty-one older adults (age = 70.5 ± 6.0 year) completed a 12-week detraining period following the 12-week eccentric training programmes with neuromuscular function and muscle structure assessed six (mid-detraining) and 12 (post-detraining) weeks following training cessation. RESULTS: From post-training to post-detraining, no significant regression of the training-induced improvements (collapsed group data reported) occurred in power (0%), strength (eccentric = 0%, isometric = 39%), or explosive strength over numerous epochs (0-32%), resulting in values that remained significantly greater than at pre-training. However, significant regression in the improvements in muscle thickness (91%) and fascicle angle (100%) occurred, resulting in values that were not significantly greater than pre-training. DISCUSSION: The limited regression in neuromuscular function following a 12-week detraining period has important implications for supporting eccentric exercise prescription in older adults who often face periods of inactivity. However, further work is required to develop an effective maintenance dosage strategy that preserves improvements in muscle structure. CONCLUSIONS: Eccentric resistance training elicits improvements in the neuromuscular function of older adults, which are sustained for at least 12 weeks after eccentric training cessation.

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In orthopedic research, many studies have applied vitamin E as a protective antioxidant or used tert-butyl hydroperoxide to induce oxidative injury to chondrocytes. These studies often support the hypothesis that joint pathology causes oxidative stress and increased lipid peroxidation that might be prevented with lipid antioxidants to improve cell survival or function and joint health; however, lipid antioxidant supplementation was ineffective against osteoarthritis in clinical trials and animal data have been equivocal. Moreover, increased circulating vitamin E is associated with increased rates of osteoarthritis. This disconnect between benchtop and clinical results led us to hypothesize that oxidative stress-driven paradigms of chondrocyte redox function do not capture the metabolic and physiologic effects of lipid antioxidants and prooxidants on articular chondrocytes. We used ex vivo and in vivo cartilage models to investigate the effect of lipid antioxidants on healthy, primary, articular chondrocytes and applied immuno-spin trapping techniques to provide a broad indicator of high levels of oxidative stress independent of specific reactive oxygen species. Key findings demonstrate lipid antioxidants were pro-mitochondrial while lipid prooxidants decreased mitochondrial measures. In the absence of injury, radical formation was increased by lipid antioxidants; however, in the presence of injury, radical formation was decreased. In unstressed conditions, this relationship between chondrocyte mitochondria and redox regulation was reproduced in vivo with overexpression of glutathione peroxidase 4. In mice aged 18 months or more, overexpression of glutathione peroxidase 4 significantly decreased the presence of pro-mitochondrial peroxisome proliferation activated receptor gamma and deranged the relationship between mitochondria and the redox environment. This complex interaction suggests strategies targeting articular cartilage may benefit from adopting more nuanced paradigms of articular chondrocyte redox metabolism.

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Microcystins are hepatotoxic cyclic heptapeptides produced by some cyanobacterial species and usually contain the unusual ß-amino acid 3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyl-4E,6E-decadienoic acid (Adda) at position-5. The full microcystin gene cluster from Microcystis aeruginosa PCC 7806 has been expressed in Escherichia coli. In an earlier study, the engineered strain was shown to produce MC-LR and [d-Asp3]MC-LR, the main microcystins reported in cultures of M. aeruginosa PCC 7806. However, analysis of the engineered strain of E. coli using semitargeted liquid chromatography with high-resolution tandem mass spectrometry (LC-HRMS/MS) and thiol derivatization revealed the presence of 15 additional microcystin analogues, including four linear peptide variants and, in total, 12 variants with modifications to the Adda moiety. Four of the Adda-variants lacked the phenyl group at the Adda-terminus, a modification that has not previously been reported in cyanobacteria. Their HRMS/MS spectra contained the product-ion from Adda at m/z 135.1168, but the commonly observed product-ion at m/z 135.0804 from Adda-containing microcystins was almost completely absent. In contrast, three of the variants were missing a methyl group between C-2 and C-8 of the Adda moiety, and their LC-HRMS/MS spectra displayed the product-ion from Adda at m/z 135.0804. However, instead of the product-ion at m/z 135.1168, these three variants gave product-ions at m/z 121.1011. These observations, together with spectra from microcystin standards using in-source fragmentation, showed that the product-ion at m/z 135.1168 found in the HRMS/MS spectra of most microcystins originated from the C-2 to C-8 region of the Adda moiety. Identification of the fragmentation pathways for the Adda side chain will facilitate the detection of microcystins containing modifications in their Adda moieties that could otherwise easily be overlooked with standard LC-MS screening methods. Microcystin variants containing Abu at position-1 were also prominent components of the microcystin profile of the engineered bacterium. Microcystin variants with Abu1 or without the phenyl group on the Adda side chain were not detected in the original host cyanobacterium. This suggests not only that the microcystin synthase complex may be affected by substrate availability within its host organism but also that it possesses an unexpected degree of biosynthetic flexibility.

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The policymaking process is largely opaque, especially regarding the actual writing of the policy. To attempt to better understand this complex process, we utilized mixed methods in our evaluation of an intervention. However, the process of mixing methods can be messy, and thus may require recalibration during the evaluation itself. Yet, in comparison to reporting results, relatively little attention is paid to the effects of mixing methods on the evaluation process. In this article, we take a reflexive approach to reporting a mixed methods evaluation of an intervention on the use of research evidence in U.S. federal policymaking. We focus on the research process in a qualitative coding team, and the effects of mixing methods on that process. Additionally, we report in general terms how to interpret multinomial logistic regressions, an underused analysis type applicable to many evaluations. Thus, this reflexive piece contributes (1) findings from evaluation of the intervention on the policymaking process, (2) an example of mixing methods leading to unexpected findings and future directions, (3) a report about the evaluation process itself, and (4) a tutorial for those new to multinomial logistic regressions.

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