Fluorescence-detection size-exclusion chromatography specifically detects autoantibodies targeting the ganglionic acetylcholine receptor in patients with autoimmune autonomic ganglionopathy.

Autoimmune autonomic ganglionopathy (AAG) is a rare disease wherein autoantibodies target the ganglionic acetylcholine receptor (gAChR). Current diagnosis in the United States depends upon clinical symptoms and positive autoantibody detection using a radioimmunoprecipitation assay (RIA). Here we offer a proof-of-principle study on an alternative method, fluorescence-detection size-exclusion-chromatography (FSEC). We show FSEC can detect autoantibodies against gAChR from patient sera but not healthy controls or samples from other autoimmune diseases. We compare FSEC to RIA and find good correlation. We discuss potential advantages of using FSEC as an alternative or as a first-step diagnostic prior to pursuing existing methodologies.

Unmasking Fluorinated Moieties on the Surface of Hydride-Terminated Silicon Nanoparticles.

Despite the widespread use of hydrofluoric acid (HF) in the preparation of silicon surfaces, the true nature of fluorinated surface species remains unclear. Here, we employ an array of characterization techniques led by solid-state nuclear magnetic resonance spectroscopy to uncover the nature of fluorinated moieties on the surface of hydride-terminated silicon nanoparticles (H-SiNPs). A structural model that explains the observed trends in 19F and 29Si magnetic shielding is proposed and further supported by quantum chemical computations. Fluorine is incorporated into local oxidation domains on the surface and clustered at the interface of the oxide and surrounding hydride-terminated surface. Silicon sites capped by a single fluorine are also identified by their distinct 19F and 29Si chemical shifts, providing insight into how fluorine termination influences the electronic structure. The extent of fluorine passivation and the effects of fluorine on the optical properties of SiNPs are also discussed. Finally, challenges associated with Teflon contamination are highlighted that future explorations of nanomaterials may have to contend with.

B cell and aquaporin-4 antibody relationships with neuromyelitis optica spectrum disorder activity.

This post hoc analysis of the randomized, placebo-controlled N-MOmentum study (NCT02200770) of inebilizumab in neuromyelitis optica spectrum disorder (NMOSD) evaluated relationships between circulating B-cell subsets and aquaporin-4 immunoglobulin G (AQP4-lgG) titers and attacks. Among participants receiving placebo, CD20+ and CD27+ B-cell counts were modestly increased from the pre-attack visit to attack; plasmablast/plasma cell gene signature was increased from baseline to the pre-attack visit (p = 0.016) and from baseline to attack (p = 0.009). With inebilizumab treatment, B-cell subset counts decreased and did not increase with attacks. No difference in change of AQP4-IgG titers from baseline to time of attack was observed.

CleanUpRNAseq: An R/Bioconductor Package for Detecting and Correcting DNA Contamination in RNA-Seq Data.

RNA sequencing (RNA-seq) has become a standard method for profiling gene expression, yet genomic DNA (gDNA) contamination carried over to the sequencing library poses a significant challenge to data integrity. Detecting and correcting this contamination is vital for accurate downstream analyses. Particularly, when RNA samples are scarce and invaluable, it becomes essential not only to identify but also to correct gDNA contamination to maximize the data's utility. However, existing tools capable of correcting gDNA contamination are limited and lack thorough evaluation. To fill the gap, we developed CleanUpRNAseq, which offers a comprehensive set of functionalities for identifying and correcting gDNA-contaminated RNA-seq data. Our package offers three correction methods for unstranded RNA-seq data and a dedicated approach for stranded data. Through rigorous validation on published RNA-seq datasets with known levels of gDNA contamination and real-world RNA-seq data, we demonstrate CleanUpRNAseq's efficacy in detecting and correcting detrimental levels of gDNA contamination across diverse library protocols. CleanUpRNAseq thus serves as a valuable tool for post-alignment quality assessment of RNA-seq data and should be integrated into routine workflows for RNA-seq data analysis. Its incorporation into OneStopRNAseq should significantly bolster the accuracy of gene expression quantification and differential expression analysis of RNA-seq data.

Hide-&-Go-PhiP-Seq: Finding an elusive predictive MS biomarker.

Whole-proteome autoantibody profiling reveals an immunological signature that predates the clinical onset of multiple sclerosis.

Airlift bioreactor-based strategies for prolonged semi-continuous cultivation of edible Agaricomycetes.

Submerged cultivation of edible filamentous fungi (Agaricomycetes) in bioreactors enables maximum mass transfer of nutrients and has the potential to increase the volumetric productivity of fungal biomass compared to solid state cultivation. These aspects are paramount if one wants to increase the range of bioactives (e.g. glucans) in convenient time frames. In this study, Trametes versicolor (M9911) outperformed four other Agaricomycetes tested strains (during batch cultivations in an airlift bioreactor). This strain was therefore further tested in semi-continuous cultivation. Continuous and semi-continuous cultivations (driven by the dilution rate, D) are the preferred bioprocess strategies for biomass production. We examined the semi-continuous cultivation of T. versicolor at dilution rates between 0.02 and 0.1 h-1. A maximum volumetric productivity of 0.87 g/L/h was obtained with a D of 0.1 h-1 but with a lower total biomass production (cell dry weight, CDW 8.7 g/L) than the one obtained at lower dilution rates (12.3 g/L at D of 0.04 and vs 13.4 g/L, at a D of 0.02 h-1). However, growth at a D of 0.1 h-1 resulted in a very short fermentation (18 h) which terminated due to washout (the specific D exceeded the maximum growth rate of the fungal biomass). At a D of 0.04 h-1, a CDW of 12.3 g/L was achieved without compromising the total residence time (184 h) of the fermentation. While the D of 0.04 h-1 and 0.07 h-1 achieved comparable volumetric productivities (0.5 g/L/h), the total duration of the fermentation at D of 0.07 h-1 was only 85 h. The highest glucan content of cells (27.8 as percentage of CDW) was obtained at a D of 0.07 h-1, while the lowest glucan content was observed in T. versicolor cells grown at a D of 0.02 h-1. KEY POINTS: • The highest reported volumetric productivity for fungal biomass was 0.87 g/L/h. • Semi-continuous fermentation at D of 0.02 h-1 resulted in 13.4 g/L of fungal biomass. • Semi-continuous fermentation at D of 0.07 h-1 resulted in fungal biomass with 28% of total glucans.

Unveiling the proteome-wide autoreactome enables enhanced evaluation of emerging CAR T cell therapies in autoimmunity.

Given the global surge in autoimmune diseases, it is critical to evaluate emerging therapeutic interventions. Despite numerous new targeted immunomodulatory therapies, comprehensive approaches to apply and evaluate the effects of these treatments longitudinally are lacking. Here, we leveraged advances in programmable-phage immunoprecipitation methodology to explore the modulation, or lack thereof, of autoantibody profiles, proteome-wide, in both health and disease. Using a custom set of over 730,000 human-derived peptides, we demonstrated that each individual, regardless of disease state, possesses a distinct and complex constellation of autoreactive antibodies. For each individual, the set of resulting autoreactivites constituted a unique immunological fingerprint, or "autoreactome," that was remarkably stable over years. Using the autoreactome as a primary output, we evaluated the relative effectiveness of various immunomodulatory therapies in altering autoantibody repertoires. We found that therapies targeting B cell maturation antigen (BCMA) profoundly altered an individual's autoreactome, while anti-CD19 and anti-CD20 therapies had minimal effects. These data both confirm that the autoreactome comprises autoantibodies secreted by plasma cells and strongly suggest that BCMA or other plasma cell-targeting therapies may be highly effective in treating currently refractory autoantibody-mediated diseases.

Comprehensive Proteomics Analysis of Polyhydroxyalkanoate (PHA) Biology in Pseudomonas putida KT2440: The Outer Membrane Lipoprotein OprL is a Newly Identified Phasin.

Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress.

The impact of size on middle-ear sound transmission in elephants, the largest terrestrial mammal.

Elephants have a unique auditory system that is larger than any other terrestrial mammal. To quantify the impact of larger middle ear (ME) structures, we measured 3D ossicular motion and ME sound transmission in cadaveric temporal bones from both African and Asian elephants in response to air-conducted (AC) tonal pressure stimuli presented in the ear canal (PEC). Results were compared to similar measurements in humans. Velocities of the umbo (VU) and stapes (VST) were measured using a 3D laser Doppler vibrometer in the 7-13,000 Hz frequency range, stapes velocity serving as a measure of energy entering the cochlea-a proxy for hearing sensitivity. Below the elephant ME resonance frequency of about 300 Hz, the magnitude of VU/PEC was an order of magnitude greater than in human, and the magnitude of VST/PEC was 5x greater. Phase of VST/PEC above ME resonance indicated that the group delay in elephant was approximately double that of human, which may be related to the unexpectedly high magnitudes at high frequencies. A boost in sound transmission across the incus long process and stapes near 9 kHz was also observed. We discuss factors that contribute to differences in sound transmission between these two large mammals.

The role of quiescent thymic progenitors in TAL/LMO2-induced T-ALL chemotolerance.

Relapse in T-cell acute lymphoblastic leukemia (T-ALL) may signify the persistence of leukemia-initiating cells (L-ICs). Ectopic TAL1/LMO expression defines the largest subset of T-ALL, but its role in leukemic transformation and its impact on relapse-driving L-ICs remain poorly understood. In TAL1/LMO mouse models, double negative-3 (DN3; CD4-CD8-CD25+CD44-) thymic progenitors harbored L-ICs. However, only a subset of DN3 leukemic cells exhibited L-IC activity, and studies linking L-ICs and chemotolerance are needed. To investigate L-IC heterogeneity, we used mouse models and applied single-cell RNA-sequencing and nucleosome labeling techniques in vivo. We identified a DN3 subpopulation with a cell cycle-restricted profile and heightened TAL1/LMO2 activity, that expressed genes associated with stemness and quiescence. This dormant DN3 subset progressively expanded throughout leukemogenesis, displaying intrinsic chemotolerance and enrichment in genes linked to minimal residual disease. Examination of TAL/LMO patient samples revealed a similar pattern in CD7+CD1a- thymic progenitors, previously recognized for their L-IC activity, demonstrating cell cycle restriction and chemotolerance. Our findings substantiate the emergence of dormant, chemotolerant L-ICs during leukemogenesis, and demonstrate that Tal1 and Lmo2 cooperate to promote DN3 quiescence during the transformation process. This study provides a deeper understanding of TAL1/LMO-induced T-ALL and its clinical implications in therapy failure.

Dietary Hydroxytyrosol Supplementation on Growth Performance, Gut Morphometry, and Oxidative and Inflammatory Status in LPS-Challenged Broilers.

This study assessed the effects of hydroxytyrosol (HT) on 8- to 20-day-old broilers challenged with lipopolysaccharide (LPS); 180 Cobb500™ male chicks were randomly assigned to 3 treatment groups, each comprising 10 replicates with 6 birds per replicate. Treatments included a control diet (CON), CON with LPS administration, and CON + LPS supplemented with 10 mg of HT/kg of feed. LPS was administered intraperitoneally on days 14, 16, 18, and 20. Body weight (BW), body weight gain (BWG), and the feed conversion ratio (FCR) were measured. On day 20, ten birds per treatment were slaughtered for analysis. Bursa, spleen, and liver were collected, and their respective relative weight was determined. The jejunum was destined for morphological analyses of villus height (VH), crypt depth (CD), and their ratio (VH:CD), and for mRNA expression of nuclear factor kappa B (NF-κB), catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and interleukins 10 (IL-10), 1 beta (IL-1ß), and 8 (IL-8). HT improved BW, BWG, and FCR, and reduced crypt depth (CD) while increasing the VH:CD ratio in the jejunum. Moreover, HT downregulated mRNA expression of CAT, GPx, IL-10, and IL-1ß. In conclusion, HT enhances broiler growth performance, mitigates jejunal mucosa damage from LPS, and modulates antioxidant and immune responses.

Synthesis of high-entropy germanides and investigation of their formation process.

High-entropy alloys and compounds have emerged as an attractive research area in part because of their distinctive solid-solution structure and multi-element compositions that provide near-limitless tailorability. A diverse array of reports describing high-entropy compounds, including carbides, nitrides, sulfides, oxides, fluorides, silicides, and borides, has resulted. Strikingly, exploration of high-entropy germanides (HEGs) has remained relatively limited. In this study, we present a detailed investigation into the synthesis of HEGs, specifically AuAgCuPdPtGe and FeCoNiCrVGe, via a rapid thermal annealing. The structural, compositional, and morphological characteristics of the synthesized HEGs were assessed using laboratory X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and transmission electron microscopy (TEM). Complementing these post-synthesis analyses, we interrogated the formation and growth mechanisms using in situ heating XRD and TEM and determined that HEG formation involved initial decomposition of germanane (GeNSs) during the annealing, followed by gradual grain growth via atom diffusion at temperatures below 600 °C, and finally a rapid grain growth process at elevated temperatures.

A Noncanonical CD56dimCD16dim/- NK Cell Subset Indicative of Prior Cytotoxic Activity Is Elevated in Patients with Autoantibody-Mediated Neurologic Diseases.

Neuromyelitis optica spectrum disorder (NMOSD), myelin oligodendrocyte glycoprotein Ab disease, and autoimmune myasthenia gravis (MG) are autoantibody-mediated neurologic conditions where autoantibodies can induce Ab-dependent cellular cytotoxicity (ADCC), a NK cell-mediated effector function. However, whether ADCC is a pathogenic mechanism in patients with these conditions has not been confirmed. We sought to characterize circulatory NK cells using functional assays, phenotyping, and transcriptomics to elucidate their role in pathology. NK cells from NMOSD patients and MG patients with elevated disease burden exhibited reduced ADCC and CD56dimCD16hi NK cells, along with an elevated frequency of CD56dimCD16dim/- NK cells. We determined that ADCC induces a similar phenotypic shift in vitro. Bulk RNA sequencing distinguished the CD56dimCD16dim/- population from the canonical CD56dimCD16hi cytotoxic and CD56hiCD16- immunomodulatory subsets, as well as CD56hiCD16+ NK cells. Multiparameter immunophenotyping of NK cell markers, functional proteins, and receptors similarly showed that the CD56dimCD16dim/- subset exhibits a unique profile while still maintaining expression of characteristic NK markers CD56, CD94, and NKp44. Notably, expression of perforin and granzyme is reduced in comparison with CD56dimCD16hi NK cells. Moreover, they exhibit elevated trogocytosis capability, HLA-DR expression, and many chemokine receptors, including CCR7. In contrast with NMOSD and MG, myelin oligodendrocyte glycoprotein Ab disease NK cells did not exhibit functional, phenotypic, or transcriptomic perturbations. In summary, CD56dimCD16dim/- NK cells are a distinct peripheral blood immune cell population in humans elevated upon prior cytotoxic activity by the CD56dimCD16hi NK cell subset. The elevation of this subset in NMOSD and MG patients suggests prior ADCC activity.

Not all silicon quantum dots are equal: photostability of silicon quantum dots with and without a thick amorphous shell.

Luminescent colloidal silicon quantum dots (SiQDs) are sustainable alternatives to metal-based QDs for various optical applications. While the materials are reliant on their photoluminescence efficiency, the relationship between the structure and photostability of SiQDs is yet to be well studied. An amorphous silicon (a-Si) shell was recently discovered in SiQDs prepared by thermally-processed silicon oxides. As a-Si is known as a source of defects upon UV irradiation, the disordered shell could potentially have an adverse effect on the optical properties of nanoparticles. Herein, the photostability of ∼5 nm diameter SiQDs with an amorphous shell was compared with that of over-etched SiQDs of equivalent dimensions that bore an a-Si shell of negligible thickness. An UV-induced degradation study was conducted by subjecting toluene solutions of SiQDs to 365 nm light-emitting diodes (LEDs) under an inert atmosphere for predetermined times up to 72 hours. The structure, composition, and optical responses of the exposed SiQDs were evaluated.

Impact of pre-bypass ultrafiltration on prime values and clinical outcomes in neonatal and infant cardiopulmonary bypass.

BACKGROUND: A standard blood prime for cardiopulmonary bypass (CPB) in congenital cardiac surgery may possess non-physiologic values for electrolytes, glucose, and lactate. Pre-bypass Ultrafiltration (PBUF) can make these values more physiologic and standardized prior to bypass initiation. We aimed to determine if using PBUF on blood primes including packed red blood cells and thawed plasma would make prime values more predictable and physiologic. Additionally, we aimed to evaluate whether the addition of PBUF had an impact on outcome measures. METHODS: Retrospective review of consecutive patients ≤ 1 year of age undergoing an index cardiac operation on CPB between 8/2017 and 9/2021. As PBUF was performed at the perfusionists' discretion, a natural grouping of patients that received PBUF vs. those that did not occur. Differences in electrolytes, glucose, and lactate were compared at specific time points using Fisher's exact test for categorical variables and the Wilcoxon rank sum test for continuous variables. Clinical outcomes were also assessed. RESULTS: In both cohorts, the median age at surgery was 3 months and 47% of patients were female; 308/704 (44%) of the PBUF group and 163/414 (39%) of the standard prime group had at least one preoperative risk factor. The proportion of PBUF circuits which demonstrated more physiologic values for glucose (318 [45%]), sodium (434, [62%]), potassium (688 [98%]), lactate (612 [87%]) and osmolality (595 [92%]) was significantly higher when compared to standard prime circuit levels for glucose (8 [2%]), sodium (13 [3%], potassium (150 [36%]), lactate (56 [13%]) and osmolality (23 [6%]) prior to CPB initiation. There were no differences in clinical outcomes or rates of major adverse events between the two cohorts. CONCLUSIONS: PBUF creates standardized and more physiologic values for electrolytes, glucose, and lactate before the initiation of bypass without significant impacts on in-hospital outcomes.

Sulfasalazine-Induced Delayed Hypersensitivity Reaction Presenting as Fever, Aseptic Meningitis, and Mesenteric Panniculitis in a Patient with Seronegative Arthritis.

BACKGROUND An 82-year-old woman presented with acute pyrexial illness and mesenteric panniculitis and developed biochemical aseptic meningitis (cerebrospinal fluid pleocytosis with no identifiable pathogen). Investigation determined her illness was likely a delayed hypersensitivity reaction caused by sulfasalazine. Sulfasalazine-induced aseptic meningitis is a rare condition often diagnosed late in a patient's admission owing to initial non-specific illness symptomatology requiring the exclusion of more common "red flag" etiologies, such as infection and malignancy. CASE REPORT An 82-year-old woman with a history of recurrent urinary tract infections and seronegative arthritis presented with a 3-day history of fatigue, headache, dyspnea, and lassitude. On admission, she was treated as presumed sepsis of uncertain source owing to pyrexia and tachycardia. Brain computer tomography (CT) revealed no acute intracranial abnormality. Furthermore, CT of the chest, abdomen, and pelvis did not reveal any source of sepsis or features of malignancy. After excluding infective etiologies with serological and cerebrospinal fluid testing, sulfasalazine-induced aseptic meningitis (SIAM) was diagnosed. The patient was then commenced on intravenous steroids, resulting in immediate defervescence and symptom resolution. CONCLUSIONS SIAM remains a diagnostic challenge since patients present with non-specific signs and symptoms, such as pyrexia, headaches, and lassitude. These patients require a thorough investigative battery starting with anamnesis, physical examination, biochemical testing, and radiologic imaging. This case illustrates the need for a high suspicion index of drug-induced hypersensitivity reaction in a rheumatological patient with pyrexial illness where infective etiologies have been confidently excluded. Prompt initiation of intravenous steroids in SIAM provides a dramatic recovery and resolution of symptoms.

Pilot-scale production of mcl-PHA by Pseudomonas citronellolis using acetic acid as the sole carbon source.

Medium-chain-length polyhydroxyalkanoates (mcl-PHA) are biobased materials with promising properties for environmentally friendly applications. Due to high production costs, which are related to the cost of the carbon sources combined with conversion insufficiencies, currently only small quantities are produced. This results in a lack of reliable data on properties and application potential for the variety of polymers from different types of production strains. This study investigated the potential for the production of mcl-PHA from volatile fatty acids (VFA) at a larger scale, given their potential as low-cost and sustainable raw material within a carboxylate-platform based biorefinery. Pseudomonas citronellolis (DSMZ 50332) was chosen as the production strain, and acetic acid was selected as the main carbon and energy source. Nitrogen was limited to trigger polymer production, and a fed-batch process using a pH-stat feeding regime with concentrated acid was established. We report successful production, extraction, and characterization of mcl PHA, obtaining a total of 1.76 kg from two 500-litre scale fermentations. The produced polymer was identified as a copolymer of 3-hydroxydecanoate (60.7%), 3-hydroxyoctanoate (37.3%), and 3-hydroxyhexanoate (2.0%) with a weight average molecular weight (Mw) of 536 kDa. NMR analysis indicates the presence of unsaturated side chains, which may offer additional possibilities for modification. The results confirm that there is a potential to produce significant amounts of mcl-PHA with interesting rubber-like properties from waste-derived VFA.

The impact of size on middle-ear sound transmission in elephants, the largest terrestrial mammal.

Elephants have a unique auditory system that is larger than any other terrestrial mammal. To quantify the impact of larger middle ear (ME) structures, we measured 3D ossicular motion and ME sound transmission in cadaveric temporal bones from both African and Asian elephants in response to air-conducted (AC) tonal pressure stimuli presented in the ear canal (P EC ). Results were compared to similar measurements in humans. Velocities of the umbo (V U ) and stapes (V ST ) were measured using a 3D laser Doppler vibrometer in the 7-13,000 Hz frequency range, stapes velocity serving as a measure of energy entering the cochlea-a proxy for hearing sensitivity. Below the elephant ME resonance frequency of about 300 Hz, the magnitude of V U /P EC was an order of magnitude greater than in human, and the magnitude of V ST /P EC was 5x greater. Phase of V ST /P EC above ME resonance indicated that the group delay in elephant was approximately double that of human, which may be related to the unexpectedly high magnitudes at high frequencies. A boost in sound transmission across the incus long process and stapes near 9 kHz was also observed. We discuss factors that contribute to differences in sound transmission between these two large mammals.

Understanding the Formation Mechanisms of Silicon Particles from the Thermal Disproportionation of Hydrogen Silsesquioxane.

Crystalline silicon particles sustaining Mie resonances are readily obtained from the thermal processing of hydrogen silsesquioxane (HSQ). Here, the mechanisms involved in silicon particle formation and growth from HSQ are investigated through real-time in situ analysis using an environmental transmission electron microscope and X-ray diffractometer. The nucleation of Si nanodomains is observed starting around 1000 °C. For the first time, a highly mobile intermediate phase is experimentally observed, thus demonstrating a previously unknown growth mechanism. At least two growth processes occur simultaneously: the coalescence of small particles into larger particles and growth mode by particle displacement through the matrix toward the HSQ grain surface. Postsynthetic characterization by scanning electron microscopy further supports the latter growth mechanism. The gaseous environment employed during synthesis impacts particle formation and growth under both in situ and ex situ conditions, impacting the particle yield and structural homogeneity. Understanding the formation mechanisms of particles provides promising pathways for reducing the energy cost of this synthetic route.