Impact of repeated blast exposure on active-duty United States Special Operations Forces.

United States (US) Special Operations Forces (SOF) are frequently exposed to explosive blasts in training and combat, but the effects of repeated blast exposure (RBE) on SOF brain health are incompletely understood. Furthermore, there is no diagnostic test to detect brain injury from RBE. As a result, SOF personnel may experience cognitive, physical, and psychological symptoms for which the cause is never identified, and they may return to training or combat during a period of brain vulnerability. In 30 active-duty US SOF, we assessed the relationship between cumulative blast exposure and cognitive performance, psychological health, physical symptoms, blood proteomics, and neuroimaging measures (Connectome structural and diffusion MRI, 7 Tesla functional MRI, [11C]PBR28 translocator protein [TSPO] positron emission tomography [PET]-MRI, and [18F]MK6240 tau PET-MRI), adjusting for age, combat exposure, and blunt head trauma. Higher blast exposure was associated with increased cortical thickness in the left rostral anterior cingulate cortex (rACC), a finding that remained significant after multiple comparison correction. In uncorrected analyses, higher blast exposure was associated with worse health-related quality of life, decreased functional connectivity in the executive control network, decreased TSPO signal in the right rACC, and increased cortical thickness in the right rACC, right insula, and right medial orbitofrontal cortex-nodes of the executive control, salience, and default mode networks. These observations suggest that the rACC may be susceptible to blast overpressure and that a multimodal, network-based diagnostic approach has the potential to detect brain injury associated with RBE in active-duty SOF.

Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue.

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. Here we develop and validate a method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and use solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise a mixture of single protofilament and two protofilament fibrils with very low twist. The protofilament fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural characterization of LBD Asyn fibrils and approaches for studying disease mechanisms, imaging agents and therapeutics targeting Asyn.

Mineralization of palm oil mill effluent by advanced oxidation processes: A review on current trends and the way forward.

Palm oil mill effluent (POME) is regarded as deleterious to the environment, primarily owing to the substantial volume of waste it produces during palm oil extraction. In terms of contaminant composition, POME surpasses the pollutant content typically found in standard municipal sewage, therefore releasing it without treatment into water bodies would do irreparable damage to the environment. Main palm oil mills are normally located in the proximity of natural rivers in order to take advantage of the cheap and abundant water source. The same rivers are also used as a water source for many villages situated along the river banks. As such, it is imperative to degrade POME before its disposal into the water bodies for obvious reasons. The treatment methods used so far include the biological processes such as open ponding/land application, which consist of aerobic as well as anaerobic ponds, physicochemical treatment including membrane technology, adsorption and coagulation are successful for the mitigation of contaminants. As the above methods require large working area and it takes more time for contaminant degradation, and in consideration of the strict environmental policies as well as palm oil being the most sort of vegetable oil in several countries, numerous researchers have concentrated on the emerging technologies such as advanced oxidation processes (AOPs) to remediate POME. Methods such as the photocatalysis, Fenton process, sonocatalysis, sonophotocatalysis, ozonation have attained special importance for the degradation of POME because of their efficiency in complete mineralization of organic pollutants in situ. This review outlines the AOP technologies currently available for the mineralization of POME with importance given to sonophotocatalysis and ozonation as these treatment process removes the need to transfer the pollutant while possibly degrading the organic matter sufficiently to be used in other industry like fertilizer manufacturing.

A comparison of pre- and post-clinical education learning preferences among medical students who elected to dissect compared to those who did not during the COVID-19 pandemic.

Anatomy instructional methods varied widely during the COVID-19 pandemic and programs are assessing innovations for retention. Learning preferences were assessed among medical students dichotomized as elective dissectors (ED) or non-dissectors (ND) during the COVID-19 partial re-opening in 2020 (preclinical) and again in 2022 after clinical exposure (post-clinical) to assess the viability of elective dissection post-pandemic. A mixed-method approach was used for the assessment of test scores, learning preference surveys, learning activities rankings, and thematic analyses. No significant differences occurred in anatomy examination scores. Dissection was considered useful by both preclinical groups but significantly more so by ED, while the presence of an instructor was significantly preferred by ED although a majority of ND agreed. Elective dissection was significantly preferred by ND but also by a large minority of ED students. Pre- and post-clinical ND believed that elective dissection offered more academic flexibility, did not hinder clinical learning, and did not negatively impact medical education. The corresponding ED stated that confidence improved, clinical experiences were enhanced, and dissection was irreplaceable. Preclinical ND preferred self-learning, while ED students preferred online learning, but these differences largely disappeared post-clinically. Learning activity rankings were not significantly different among all groups (ND, ED, preclinical, and post-clinical). A hybrid laboratory with a virtual learning environment ranked highest across groups and preferences increased over time suggesting that students benefited from this instructional method during clinical exposure. The absence of laboratory experience ranked lowest, and preference decreased over time suggesting that anatomy dissection is valued.

Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal.

Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.

13C and 15N resonance assignments of alpha synuclein fibrils amplified from Lewy Body Dementia tissue.

Fibrils of the protein α-synuclein (Asyn) are implicated in the pathogenesis of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. Numerous forms of Asyn fibrils have been studied by solid-state NMR and resonance assignments have been reported. Here, we report a new set of 13C, 15N assignments that are unique to fibrils obtained by amplification from postmortem brain tissue of a patient diagnosed with Lewy Body Dementia.

Optimizing Brain Health of United States Special Operations Forces.

United States Special Operations Forces (SOF) personnel are frequently exposed to explosive blasts in training and combat. However, the effects of repeated blast exposure on the human brain are incompletely understood. Moreover, there is currently no diagnostic test to detect repeated blast brain injury (rBBI). In this "Human Performance Optimization" article, we discuss how the development and implementation of a reliable diagnostic test for rBBI has the potential to promote SOF brain health, combat readiness, and quality of life.

Segmental Dynamics of Membranous Cholesterol are Coupled.

Cholesterol promotes the structural integrity of the fluid cell membrane and interacts dynamically with many membrane proteins to regulate function. Understanding site-resolved cholesterol structural dynamics is thus important. This long-standing challenge has thus far been addressed, in part, by selective isotopic labeling approaches. Here we present a new 3D solid-state NMR (SSNMR) experiment utilizing scalar 13C-13C polarization transfer and recoupling of the 1H-13C interactions in order to determine average dipolar couplings for all 1H-13C vectors in uniformly 13C-enriched cholesterol. The experimentally determined order parameters (OP) agree exceptionally well with molecular dynamics (MD) trajectories and reveal coupling among several conformational degrees of freedom in cholesterol molecules. Quantum chemistry shielding calculations further support this conclusion and specifically demonstrate that ring tilt and rotation are coupled to changes in tail conformation and that these coupled segmental dynamics dictate the orientation of cholesterol. These findings advance our understanding of physiologically relevant dynamics of cholesterol, and the methods that revealed them have broader potential to characterize how structural dynamics of other small molecules impact their biological functions.

Aerosolized, Gas-Phase, Intravitreal Methotrexate Reduces Proliferative Vitreoretinopathy in a Randomized Trial in a Porcine Model.

Purpose: To determine the effectiveness of aerosol-delivered methotrexate (AD-MTx) in a large-animal (porcine) model of proliferative vitreoretinopathy (PVR). Design: Prospective, randomized, interventional, double-masked, controlled, large-animal study with predetermined clinical and histopathologic outcome criteria. Controls: Half of the pigs were randomly assigned to receive an identical volume of aerosol-delivered normal saline (AD-NS) using identical delivery systems and treatment intervals. Methods: Proliferative vitreoretinopathy was surgically induced in 16 pigs (8 males and 8 females), randomly assigned to receive 2 doses (group A) or 3 doses (group B) of either AD-MTx (1.6 mg/0.4 ml) or normal saline (AD-NS). Group A pigs were euthanized at week 2 (n = 8), and group B pigs were euthanized at week 3 (n = 8). Masked clinical PVR scores (0-6) by a vitreoretinal surgeon and histopathology PVR scores (0-8) by a masked ophthalmic pathologist were used to determine outcomes. Main Outcome Measures: The mean, combined clinical and histopathology scores (both anterior and posterior) were used to determine the overall treatment effect between the groups. Results: The mean masked score (± standard deviation) when all grading end points (clinical + histopathology) were combined was a mean of 8.0 ± 2.3 in the AD-MTx group versus a higher 9.9 ± 2.0 in the AD-NS control group (P = 0.05). The clinical score was 3.88 ± 1.2 in the AD-MTx group versus 4.63 ± 1.6 in the AD-NS group (P = 0.16). The histopathology score for anterior PVR was 2.5 ± 0.8 in the AD-MTx group versus 2.5 ± 0.5 in the AD-NS group (P = 0.50), and the posterior PVR was 1.63 ± 1.6 in the AD-MTx group versus 2.75 ± 1.3 in the AD-NS group (P = 0.07). When the frequency of methotrexate dosing in group A (2 doses) was compared with that in group B (3 doses), the mean score was 8.75 versus 9.13 (P = 0.38), respectively, suggesting an insignificant difference. Conclusions: After surgical induction of PVR in an aggressive, high-risk, large-animal model, AD-MTx reduced posterior PVR formation compared with AD-NS. Additional dosing at week 3 did not improve the outcomes. No difference in anterior PVR formation was noted with intervention. This novel drug delivery system has implications for PVR reduction and warrants further investigation. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Rescue of glutaric aciduria type I in mice by liver-directed therapies.

Glutaric aciduria type I (GA-1) is an inborn error of metabolism with a severe neurological phenotype caused by the deficiency of glutaryl-coenzyme A dehydrogenase (GCDH), the last enzyme of lysine catabolism. Current literature suggests that toxic catabolites in the brain are produced locally and do not cross the blood-brain barrier. In a series of experiments using knockout mice of the lysine catabolic pathway and liver cell transplantation, we uncovered that toxic GA-1 catabolites in the brain originated from the liver. Moreover, the characteristic brain and lethal phenotype of the GA-1 mouse model was rescued by two different liver-directed gene therapy approaches: Using an adeno-associated virus, we replaced the defective Gcdh gene or we prevented flux through the lysine degradation pathway by CRISPR deletion of the aminoadipate-semialdehyde synthase (Aass) gene. Our findings question the current pathophysiological understanding of GA-1 and reveal a targeted therapy for this devastating disorder.

13C and 15N Resonance Assignments of Alpha Synuclein Fibrils Amplified from Lewy Body Dementia Tissue.

Fibrils of the protein α-synuclein (Asyn) are implicated in the pathogenesis of Parkinson Disease, Lewy Body Dementia, and Multiple System Atrophy. Numerous forms of Asyn fibrils have been studied by solid-state NMR and resonance assignments have been reported. Here, we report a new set of 13C, 15N assignments that are unique to fibrils obtained by amplification from postmortem brain tissue of a patient diagnosed with Lewy Body Dementia.

Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue.

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. We developed and validated a novel method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and used solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise two protofilaments with pseudo-21 helical screw symmetry, very low twist and an interface formed by antiparallel beta strands of residues 85-93. The fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural landscape of LBD Asyn fibrils and inform further studies of disease mechanisms, imaging agents and therapeutics targeting Asyn.

Search for an Excess of Electron Neutrino Interactions in MicroBooNE Using Multiple Final-State Topologies.

We present a measurement of ν_{e} interactions from the Fermilab Booster Neutrino Beam using the MicroBooNE liquid argon time projection chamber to address the nature of the excess of low energy interactions observed by the MiniBooNE Collaboration. Three independent ν_{e} searches are performed across multiple single electron final states, including an exclusive search for two-body scattering events with a single proton, a semi-inclusive search for pionless events, and a fully inclusive search for events containing all hadronic final states. With differing signal topologies, statistics, backgrounds, reconstruction algorithms, and analysis approaches, the results are found to be either consistent with or modestly lower than the nominal ν_{e} rate expectations from the Booster Neutrino Beam and no excess of ν_{e} events is observed.

2H,13C-Cholesterol for Dynamics and Structural Studies of Biological Membranes.

We present a cost-effective means of 2H and 13C enrichment of cholesterol. This method exploits the metabolism of 2H,13C-acetate into acetyl-CoA, the first substrate in the mevalonate pathway. We show that growing the cholesterol producing strain RH6827 of Saccharomyces cerevisiae in 2H,13C-acetate-enriched minimal media produces a skip-labeled pattern of deuteration. We characterize this cholesterol labeling pattern by mass spectrometry and solid-state nuclear magnetic resonance spectroscopy. It is confirmed that most 2H nuclei retain their original 2H-13C bonds from acetate throughout the biosynthetic pathway. We then quantify the changes in 13C chemical shifts brought by deuteration and the impact upon 13C-13C spin diffusion. Finally, using adiabatic rotor echo short pulse irradiation cross-polarization (RESPIRATIONCP), we acquire the 2H-13C correlation spectra to site specifically quantify cholesterol dynamics in two model membranes as a function of temperature. These measurements show that cholesterol acyl chains at physiological temperatures in mixtures of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingomyelin, and cholesterol are more dynamic than cholesterol in POPC. However, this overall change in motion is not uniform across the cholesterol molecule. This result establishes that this cholesterol labeling pattern will have great utility in reporting on cholesterol dynamics and orientation in a variety of environments and with different membrane bilayer components, as well as monitoring the mevalonate pathway product interactions within the bilayer. Finally, the flexibility and universality of acetate labeling will allow this technique to be widely applied to a large range of lipids and other natural products.

Soluble ECM promotes organotypic formation in lung alveolar model.

Micropatterned suspension culture creates consistently sized and shaped cell aggregates but has not produced organotypic structures from stable cells, thus restricting its use in accurate disease modeling. Here, we show that organotypic structure is achieved in hybrid suspension culture via supplementation of soluble extracellular matrix (ECM). We created a viable lung organoid from epithelial, endothelial, and fibroblast human stable cell lines in suspension culture. We demonstrate the importance of soluble ECM in organotypic patterning with the emergence of lumen-like structures with airspace showing feasible gas exchange units, formation of branching, perfusable vasculature, and long-term 70-day maintenance of lumen structure. Our results show a dependent relationship between enhanced fibronectin fibril assembly and the incorporation of ECM in the organoid. We successfully applied this technology in modeling lung fibrosis via bleomycin induction and test a potential antifibrotic drug in vitro while maintaining fundamental cell-cell interactions in lung tissue. Our human fluorescent lung organoid (hFLO) model represents features of pulmonary fibrosis which were ameliorated by fasudil treatment. We also demonstrate a 3D culture method with potential of creating organoids from mature cells, thus opening avenues for disease modeling and regenerative medicine, enhancing understanding of lung cell biology in health and lung disease.

Spectroscopic signatures of bilayer ordering in native biological membranes.

Membrane proteins and polycyclic lipids like cholesterol and hopanoids coordinate phospholipid bilayer ordering. This phenomenon manifests as partitioning of the liquid crystalline phase into liquid-ordered (Lo) and liquid-disordered (Ld) regions. In Eukaryotes, microdomains are rich in cholesterol and sphingolipids and serve as signal transduction scaffolds. In Prokaryotes, Lo microdomains increase pathogenicity and antimicrobial resistance. Previously, we identified spectroscopically distinct chemical shift signatures for all-trans (AT) and trans-gauche (TG) acyl chain conformations, cyclopropyl ring lipids (CPR), and hopanoids in prokaryotic lipid extracts and used Polarization Transfer (PT) SSNMR to investigate bilayer ordering. To investigate how these findings relate to native bilayer organization, we interrogate whole cell and whole membrane extract samples of Burkholderia thailendensis to investigate bilayer ordering in situ. In 13C-13C 2D SSNMR spectra, we assigned chemical shifts for lipid species in both samples, showing conservation of lipids of interest in our native membrane sample. A one-dimensional temperature series of PT SSNMR and transverse relaxation measurements of AT versus TG acyl conformations in the membrane sample confirm bilayer ordering and a broadened phase transition centered at a lower-than-expected temperature. Bulk protein backbone Cα dynamics and correlations consistent with lipid-protein contacts within are further indicative of microdomain formation and lipid ordering. In aggregate, these findings provide evidence for microdomain formation in vivo and provide insight into phase separation and transition mechanics in biological membranes.

A Cholesterol Dimer Stabilizes the Inactivated State of an Inward-Rectifier Potassium Channel.

Cholesterol oligomers reside in multiple membrane protein X-ray crystal structures. Yet, there is no direct link between these oligomers and a biological function. Here we present the structural and functional details of a cholesterol dimer that stabilizes the inactivated state of an inward-rectifier potassium channel KirBac1.1. K+ efflux assays confirm that high cholesterol concentration reduces K+ conductance. We then determine the structure of the cholesterol-KirBac1.1 complex using Xplor-NIH simulated annealing calculations driven by solid-state NMR distance measurements. These calculations identified an α-α cholesterol dimer docked to a cleft formed by adjacent subunits of the homotetrameric protein. We compare these results to coarse grain molecular dynamics simulations. This is one of the first examples of a cholesterol oligomer performing a distinct biological function and structural characterization of a conserved promiscuous lipid binding region.

Sarcomatoid Dedifferentiated Melanoma: The Diagnostic Role of Next-Generation Sequencing.

ABSTRACT: Sarcomatoid dedifferentiated melanoma (SDDM) represents a diagnostic challenge as this cutaneous spindle cell melanoma lacks expression of classic melanocytic markers including S100, SOX10, Melan-A, HMB45, and MITF. The expression of the emerging melanoma marker preferentially expressed antigen in melanoma (PRAME) in SDDM is largely unknown. In this article, a case of SDDM arising in association with a nodular melanoma is highlighted. A 65-year-old man presented with a several week history of an ulcerated lesion on the right medial knee. A shave biopsy of the lesion revealed a biphasic neoplasm, which consisted of a centrally located poorly differentiated spindle cell component and an adjacent nodular component consisting of atypical melanocytes arranged in nests and fascicles. While the nodular component stained for S100, SOX10, and Melan-A, the spindle cell component failed to stain for these conventional melanocytic markers, only staining diffusely for CD10 and faintly for CD68. Both components stained for PRAME diffusely albeit less intensely within the spindle cell component. Next-generation DNA sequencing assay of the microdissected biphasic components revealed a shared mutation of NRAS. The results of the PRAME immunohistochemical stain and next-generation DNA sequencing assay facilitated in establishing the diagnosis of SDDM in association with nodular melanoma.

Water Accessibility Refinement of the Extended Structure of KirBac1.1 in the Closed State.

NMR structures of membrane proteins are often hampered by poor chemical shift dispersion and internal dynamics which limit resolved distance restraints. However, the ordering and topology of these systems can be defined with site-specific water or lipid proximity. Membrane protein water accessibility surface area is often investigated as a topological function via solid-state NMR. Here we leverage water-edited solid-state NMR measurements in simulated annealing calculations to refine a membrane protein structure. This is demonstrated on the inward rectifier K+ channel KirBac1.1 found in Burkholderia pseudomallei. KirBac1.1 is homologous to human Kir channels, sharing a nearly identical fold. Like many existing Kir channel crystal structures, the 1p7b crystal structure is incomplete, missing 85 out of 333 residues, including the N-terminus and C-terminus. We measure solid-state NMR water proximity information and use this for refinement of KirBac1.1 using the Xplor-NIH structure determination program. Along with predicted dihedral angles and sparse intra- and inter-subunit distances, we refined the residues 1-300 to atomic resolution. All structural quality metrics indicate these restraints are a powerful way forward to solve high quality structures of membrane proteins using NMR.