Overcoming Lot-to-Lot Variability in Protein Activity Using Epitope-Specific Calibration-Free Concentration Analysis.

Concentration determination is a fundamental hallmark of protein reagent characterization, providing a means to ensure reproducibility and unify measurements from various assays. However, lot-to-lot differences in protein activity often still occur, leading to uncertainty in the accuracy of downstream measurements. Here, we postulate that those differences are caused by a misrepresentation of the protein concentration as measured by traditional total protein techniques, which can include multiple types of inactive protein species. To overcome this, we developed a standardized method to quantify a protein's active concentration via calibration-free concentration analysis (CFCA). As a pilot study, we compare the biophysical and immunoassay responses from three batches of recombinant soluble lymphocyte-activation gene 3 (sLAG3), as defined by either their total or active concentrations. Defining the sLAG3 reagents by their assay-specific concentration improved consistency in reported kinetic binding parameters and decreased immunoassay lot-to-lot coefficients of variation (CVs) by over 600% compared to the total protein concentration. These findings suggest that the total concentration of a protein reagent may not be the ideal metric to correlate in-assay signals between lots, and by instead quantifying the concentrations of a reagent's assay-specific epitopes, CFCA may prove a useful tool in overcoming lot-to-lot variability.

Biallelic variants in GTF3C5, a regulator of RNA polymerase III-mediated transcription, cause a multisystem developmental disorder.

General transcription factor IIIC subunit 5 (GTF3C5) encodes transcription factor IIIC63 (TFIIIC63). It binds to DNA to recruit another transcription factor, TFIIIB, and RNA polymerase III (Pol III) to mediate the transcription of small noncoding RNAs, such as tRNAs. Here, we report four individuals from three families presenting with a multisystem developmental disorder phenotype with biallelic variants in GTF3C5. The overlapping features include growth retardation, developmental delay, intellectual disability, dental anomalies, cerebellar malformations, delayed bone age, skeletal anomalies, and facial dysmorphism. Using lymphoblastoid cell lines (LCLs) from two affected individuals, we observed a reduction in TFIIIC63 protein levels compared to control LCLs. Genome binding of TFIIIC63 protein is also reduced in LCL from one of the affected individuals. Additionally, approximately 40% of Pol III binding regions exhibited reduction in the level of Pol III occupancy in the mutant genome relative to the control, while approximately 54% of target regions showed comparable levels of Pol III occupancy between the two, indicating partial impairment of Pol III occupancy in the mutant genome. Yeasts with subject-specific variants showed temperature sensitivity and impaired growth, supporting the notion that the identified variants have deleterious effects. gtf3c5 mutant zebrafish showed developmental defects, including a smaller body, head, and eyes. Taken together, our data show that GTF3C5 plays an important role in embryonic development, and that biallelic variants in this gene cause a multisystem developmental disorder. Our study adds GTF3C5-related disorder to the growing list of genetic disorders associated with Pol III transcription machinery.

A framework for estimating soil water characteristic curve and hydraulic conductivity function of permeable reactive media.

The unsaturated behavior of permeable reactive barriers (PRB) is a critical component in predicting the removal efficiency through the adsorption of contaminants. This study investigates the framework to estimate the soil water characteristic curve (SWCC) and hydraulic conductivity function (HCF) for iron oxide-coated sand (IOCS) and zeolite, which are common materials used in PRBs. A multistep outflow (MSO) experiment was performed and the results of the MSO experiment were used to optimize associated parameters in Kosugi's SWCC and HCF. In addition, three scenarios of optimization analysis were investigated to evaluate the best-fitting model for estimating SWCC and HCF. The low root mean square error (RMSE) of fitted parameters indicates the Kosugi model well described the observed suction profiles in MSO experiments. In addition, the lowest RMSE and coefficient of variation suggested the inclusion of the additional parameter ß provided the best estimation of the three materials (clean sand, IOCS, and zeolite). The physically reasonable estimation of SWCC and HCF of the three materials from the optimized parameters suggests the proposed framework is a reasonable model for the unsaturated behavior of PRBs.

Recurrent missense variant identified in two unrelated families with MPZL2-related hearing loss, expanding the variant spectrum associated with DFNB111.

MPZL2-related hearing loss is a rare form of autosomal recessive hearing loss characterized by progressive, mild sloping to severe sensorineural hearing loss. Thirty-five previously reported patients had biallelic truncating variants in MPZL2, with the exception of one patient with a missense variant of uncertain significance and a truncating variant. Here, we describe the clinical characteristics and genotypes of five patients from four families with confirmed MPZL2-related hearing loss. A rare missense likely pathogenic variant [NM_005797.4(MPZL2):c.280C>T,p.(Arg94Trp)] located in exon 3 was confirmed to be in trans with a recurrent pathogenic truncating variant that segregated with hearing loss in three of the patients from two unrelated families. This is the first recurrent likely pathogenic missense variant identified in MPZL2. Apparently milder or later-onset hearing loss associated with rare missense variants in MPZL2 indicates that some missense variants in this gene may cause a milder phenotype than that resulting from homozygous or compound heterozygous truncating variants. This study, along with the identification of truncating loss of function and missense MPZL2 variants in several diverse populations, suggests that MPZL2-related hearing loss may be more common than previously appreciated and demonstrates the need for MPZL2 inclusion in hearing loss testing panels.

Diagnostic Yield of Exome Sequencing in Pediatric Cardiomyopathy.

OBJECTIVE: To assess the diagnostic yield of exome sequencing (ES) in pediatric cardiomyopathy. STUDY DESIGN: A single-institution, retrospective chart review of 91 patients with pediatric cardiomyopathy was performed. While pediatric cardiomyopathy is often genetic in nature, no genetic test is recommended as standard of care. All our patients were diagnosed with cardiomyopathy and evaluated by a medical geneticist between January 2010 through September 2022. Demographic information and clinical data were abstracted. RESULTS: Of 91 patients with pediatric cardiomyopathy, 36 (39.6%) received a diagnosis by ES. Twenty-two (61.1%) of these diagnoses would have been missed on cardiac multigene panel testing. The diagnostic yield for cardiomyopathy presenting under 1 year of age was 38.3%, while the yield for patients over 1 year of age was 41.9%. CONCLUSIONS: ES has a high diagnostic yield in pediatric cardiomyopathy compared with a gene panel. Over 60% of patients with diagnosis by ES would not have received their molecular genetic diagnosis if only multigene panel testing was sent. Diagnostic yield did not vary significantly between the subtypes of cardiomyopathy and patient age groups, highlighting the likely clinical utility of ES for all pediatric cardiomyopathy patients.

Unconventional Rocky Mountain Spotted Fever Presentation From Kentucky: A Compelling Case Report and Literature Review.

Rocky Mountain spotted fever (RMSF) is a potentially lethal tick-borne disease caused by Rickettsia rickettsii, known for its tropism for vascular endothelial cells. Its classic symptoms include fever, headaches, and a rash, but atypical presentations can challenge diagnosis. We present the case of a 71-year-old male with fever, weakness, and hiccups, evolving into confusion. Laboratory findings showed severe hyponatremia, leukocytosis, and abnormal blood parameters. Initial management addressed sepsis and hyponatremia, leading to symptom improvement. Later, a fever of 106.5°F prompted ICU transfer, broad-spectrum antibiotics, and testing for tick-borne diseases. The patient reported tick exposure and received prophylactic doxycycline. Follow-up confirmed the RMSF diagnosis based on serological testing and clinical symptoms. This case highlights the diagnostic challenges posed by atypical RMSF presentations and underscores the importance of early detection and treatment to prevent complications.

An electrokinetic-biocementation study for clay stabilisation using carbonic anhydrase-producing bacteria.

This study investigates the feasibility of biocementing clay soil underneath a railway embankment of the UK rail network via carbonic anhydrase (CA) biocementation, implementing the treatments electrokinetically. Compared to previous biocementation studies using the ureolytic route, the CA pathway is attractive as CA-producing bacteria can sequester CO2 to produce biocement. Clay soil samples were treated electrokinetically using biostimulation and bioaugmentation conditions to induce biocementation. The effects of the treatment were assessed in terms of undrained shear strength using the cone penetration test, moisture content, and calcium carbonate content measurements. Scanning electron microscopy (SEM) analyses were also conducted on soil samples before and after treatment to evaluate the reaction products. The results showed that upon biostimulation, the undrained shear strength of the soil increased uniformly throughout the soil, from 17.6 kPa (in the natural untreated state) to 106.6 kPa. SEM micrographs also showed a clear change in the soil structure upon biostimulation. Unlike biostimulation, bioaugmentation did not have the same performance, although a high amount of CaCO3 precipitates was detected, and bacteria were observed to have entered the soil. The prospects are exciting, as it was shown that it is possible to achieve a considerable strength increase by the biostimulation of native bacteria capturing CO2 while improving the soil strength, thus having the potential to contribute both to the resilience of existing railway infrastructure and to climate change mitigation.

Caryophyllene Sesquiterpenes from a Chaetomium globosum Endophyte of the Canadian Medicinal Plant Empetrum nigrum.

Punctaporonins T (1) and U (2), new caryophyllene sesquiterpenes, were isolated with three known punctaporonins, A (3), B (4), and C (5), from the endophytic fungus Chaetomium globosum (TC2-041). The structures and relative configurations of punctaporonins T and U were elucidated based on a combination of HRESIMS, 1D/2D NMR spectroscopic analysis, and X-ray diffraction analysis, while their absolute configuration is presumed to be consistent with the co-isolated 3-5 on biogenetic arguments. Compound 1 showed weak inhibitory activity against both Mycobacterium tuberculosis and Staphylococcus aureus.

Tolypocaibols: Antibacterial Lipopeptaibols from a Tolypocladium sp. Endophyte of the Marine Macroalga Spongomorpha arcta.

Two new lipopeptaibols, tolypocaibols A (1) and B (2), and the mixed NRPS-polyketide-shikimate natural product maximiscin [(P/M)-3)] were isolated from a Tolypocladium sp. fungal endophyte of the marine alga Spongomorpha arcta. Analysis of NMR and mass spectrometry data revealed the amino acid sequences of the lipopeptaibols, which both comprise 11 residues with a valinol C-terminus and a decanoyl acyl chain at the N-terminus. The configuration of the amino acids was determined by Marfey's analysis. Tolypocaibols A (1) and B (2) showed moderate, selective inhibition against Gram-positive and acid-fast bacterial strains, while maximiscin [(P/M)-3)] showed moderate, broad-spectrum antibiotic activity.

Ventricular Tachycardia and ICD Therapy Burden With Catheter Ablation Versus Escalated Antiarrhythmic Drug Therapy.

BACKGROUND: Catheter ablation improves ventricular tachycardia (VT) event-free (time to event) survival in patients with antiarrhythmic drug (AAD)-refractory VT and previous myocardial infarction (MI). The effects of ablation on recurrent VT and implantable cardioverter-defibrillator (ICD) therapy (burden) have yet to be investigated. OBJECTIVES: This study sought to compare the VT and ICD therapy burden following treatment with either ablation or escalated AAD therapy among patients with VT and previous MI in the VANISH (Ventricular tachycardia AblatioN versus escalated antiarrhythmic drug therapy in ISchemic Heart disease) trial. METHODS: The VANISH trial randomized patients with previous MI and VT despite initial AAD therapy to either escalated AAD treatment or catheter ablation. VT burden was defined as the total number of VT events treated with ≥1 appropriate ICD therapy. Appropriate ICD therapy burden was defined as the total number of appropriate shocks or antitachycardia pacing therapies (ATPs) delivered. The Anderson-Gill recurrent event model was used to compare burden between the treatment arms. RESULTS: Of the 259 enrolled patients (median age, 69.8 years; 7.0% women), 132 patients were randomized to ablation and 129 patients were randomized to escalated AAD therapy. Over 23.4 months of follow-up, ablation-treated patients had a 40% lower shock-treated VT event burden and a 39% lower appropriate shock burden compared with patients who received escalated AAD therapy (P <0.05 for all). A reduction in VT burden, ATP-treated VT event burden, and appropriate ATP burden among ablation patients was only demonstrated in the stratum of patients with amiodarone-refractory VT (P <0.05 for all). CONCLUSIONS: Among patients with AAD-refractory VT and a previous MI, catheter ablation reduced shock-treated VT event burden and appropriate shock burden compared with escalated AAD therapy. There was also lower VT burden, ATP-treated VT event burden, and appropriate ATP burden among ablation-treated patients; however, the effect was limited to patients with amiodarone-refractory VT.

Monoallelic loss-of-function BMP2 variants result in BMP2-related skeletal dysplasia spectrum.

PURPOSE: Bone morphogenic proteins (BMPs) regulate gene expression that is related to many critical developmental processes, including osteogenesis for which they are named. In addition, BMP2 is widely expressed in cells of mesenchymal origin, including bone, cartilage, skeletal and cardiac muscle, and adipose tissue. It also participates in neurodevelopment by inducing differentiation of neural stem cells. In humans, BMP2 variants result in a multiple congenital anomaly syndrome through a haploinsufficiency mechanism. We sought to expand the phenotypic spectrum and highlight phenotypes of patients harboring monoallelic missense variants in BMP2. METHODS: We used retrospective chart review to examine phenotypes from an international cohort of 18 individuals and compared these with published cases. Patient-derived missense variants were modeled in zebrafish to examine their effect on the ability of bmp2b to promote embryonic ventralization. RESULTS: The presented cases recapitulated existing descriptions of BMP2-related disorders, including craniofacial, cardiac, and skeletal anomalies and exhibit a wide phenotypic spectrum. We also identified patients with neural tube defects, structural brain anomalies, and endocrinopathies. Missense variants modeled in zebrafish resulted in loss of protein function. CONCLUSION: We use this expansion of reported phenotypes to suggest multidisciplinary medical monitoring and management of patients with BMP2-related skeletal dysplasia spectrum.

Retrospective identification of patients with SRRM2-related neurodevelopmental disorder in a single tertiary children's hospital.

SRRM2-related neurodevelopmental disorder is a recently described genetic diagnosis caused by loss-of-function variants in SRRM2. In order to understand the clinical spectrum of SRRM2-related neurodevelopmental disorder, we performed a retrospective exome data and clinical chart review at a single tertiary children's hospital, Children's Hospital of Philadelphia (CHOP). Among approximately 3100 clinical exome sequencing cases performed at CHOP, we identified three patients with SRRM2 loss-of-function pathogenic variants, in addition to one patient previously described in the literature. Common clinical features include developmental delay, attention deficit hyperactivity disorder, macrocephaly, hypotonia, gastroesophageal reflux, overweight/obesity, and autism. While developmental disabilities are commonly seen in all individuals with SRRM2 variants, the degree of developmental delay and intellectual disability is variable. Our data suggest that SRRM2-related neurodevelopmental disorder can be identified in 0.3% of individuals with developmental disabilities receiving exome sequencing.

Human germline heterozygous gain-of-function STAT6 variants cause severe allergic disease.

STAT6 (signal transducer and activator of transcription 6) is a transcription factor that plays a central role in the pathophysiology of allergic inflammation. We have identified 16 patients from 10 families spanning three continents with a profound phenotype of early-life onset allergic immune dysregulation, widespread treatment-resistant atopic dermatitis, hypereosinophilia with esosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and anaphylaxis. The cases were either sporadic (seven kindreds) or followed an autosomal dominant inheritance pattern (three kindreds). All patients carried monoallelic rare variants in STAT6 and functional studies established their gain-of-function (GOF) phenotype with sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. Precision treatment with the anti-IL-4Rα antibody, dupilumab, was highly effective improving both clinical manifestations and immunological biomarkers. This study identifies heterozygous GOF variants in STAT6 as a novel autosomal dominant allergic disorder. We anticipate that our discovery of multiple kindreds with germline STAT6 GOF variants will facilitate the recognition of more affected individuals and the full definition of this new primary atopic disorder.

Annotation of natural product compound families using molecular networking topology and structural similarity fingerprinting.

Spectral matching of MS2 fragmentation spectra has become a popular method for characterizing natural products libraries but identification remains challenging due to differences in MS2 fragmentation properties between instruments and the low coverage of current spectral reference libraries. To address this bottleneck we present Structural similarity Network Annotation Platform for Mass Spectrometry (SNAP-MS) which matches chemical similarity grouping in the Natural Products Atlas to grouping of mass spectrometry features from molecular networking. This approach assigns compound families to molecular networking subnetworks without the need for experimental or calculated reference spectra. We demonstrate SNAP-MS can accurately annotate subnetworks built from both reference spectra and an in-house microbial extract library, and correctly predict compound families from published molecular networks acquired on a range of MS instrumentation. Compound family annotations for the microbial extract library are validated by co-injection of standards or isolation and spectroscopic analysis. SNAP-MS is freely available at www.npatlas.org/discover/snapms .

Genomic and phenotypic characterization of 404 individuals with neurodevelopmental disorders caused by CTNNB1 variants.

PURPOSE: Germline loss-of-function variants in CTNNB1 cause neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV; OMIM 615075) and are the most frequent, recurrent monogenic cause of cerebral palsy (CP). We investigated the range of clinical phenotypes owing to disruptions of CTNNB1 to determine the association between NEDSDV and CP. METHODS: Genetic information from 404 individuals with collectively 392 pathogenic CTNNB1 variants were ascertained for the study. From these, detailed phenotypes for 52 previously unpublished individuals were collected and combined with 68 previously published individuals with comparable clinical information. The functional effects of selected CTNNB1 missense variants were assessed using TOPFlash assay. RESULTS: The phenotypes associated with pathogenic CTNNB1 variants were similar. A diagnosis of CP was not significantly associated with any set of traits that defined a specific phenotypic subgroup, indicating that CP is not additional to NEDSDV. Two CTNNB1 missense variants were dominant negative regulators of WNT signaling, highlighting the utility of the TOPFlash assay to functionally assess variants. CONCLUSION: NEDSDV is a clinically homogeneous disorder irrespective of initial clinical diagnoses, including CP, or entry points for genetic testing.

MYH7 variants cause complex congenital heart disease.

MYH7, encoding the myosin heavy chain sarcomeric ß-myosin heavy chain, is a common cause of both hypertrophic and dilated cardiomyopathy. Additionally, families with left ventricular noncompaction cardiomyopathy (LVNC) and congenital heart disease (CHD), typically septal defects or Ebstein anomaly, have been identified to have heterozygous pathogenic variants in MHY7. One previous case of single ventricle CHD with heart failure due to a MYH7 variant has been identified. Herein, we present a single center's experience of complex CHD due to MYH7 variants. Three probands with a history of CHD, LVNC, and/or arrhythmias were identified to have MYH7 variants through multigene panel testing or exome sequencing. These three patients collectively had 12 affected family members, four with a history of Ebstein anomaly and seven with a history of LVNC. These findings suggest a wider phenotypic spectrum in MYH7-related CHD than previously understood. Further investigation into the possible role of MYH7 in CHD and mechanism of disease is necessary to fully delineate the phenotypic spectrum of MYH7-related cardiac disease. MYH7 should be considered for families with multiple individuals with complex CHD in the setting of a family history of LVNC or arrhythmias.

Ventricular tachycardia characteristics and outcomes with catheter ablation vs. antiarrhythmic therapy: insights from the VANISH trial.

AIMS: Catheter ablation is superior to escalated antiarrhythmic drugs among patients with ventricular tachycardia (VT) and prior myocardial infarction (MI). However, it is uncertain whether clinical VT characteristics, should influence choice of therapy. The purpose of this study was to evaluate whether presentation with electrical storm and the clinical VT cycle length predicted response to ablation vs. escalated antiarrhythmic therapy. METHODS AND RESULTS: All patients enrolled in the Ventricular Tachycardia Ablation vs. Escalated Antiarrhythmic Drug Therapy in Ischaemic Heart Disease (VANISH) trial were included. The association between VT cycle length and presentation with electrical storm and the primary outcome of death, subsequent VT storm or appropriate ICD shock was evaluated. Among the study population of 259 patients, escalated antiarrhythmic drug therapy had worse outcomes for those presenting with a VT cycle length >400 ms [<150 b.p.m., 89/259, hazard ratio (HR) 1.7 (1.02-3.13)]. This effect was more pronounced among those taking amiodarone at baseline [HR of 2.22 (1.19-4.16)]. Presentation with VT storm (32/259) did not affect the primary outcome between groups. However, those presenting with VT storm on amiodarone had a trend towards worse outcomes with escalated antiarrhythmic therapy [HR 4.31 (0.55-33.93)]. CONCLUSION: The VT cycle length can influence response to either ablation or escalated drug therapy in patients with VT and prior MI. Those with slow VT had improved outcomes with ablation. Patients presenting with electrical storm demonstrated similar outcomes to the overall trial population, with a trend to benefit of catheter ablation, particularly in those on amiodarone.

Disruption of RFX family transcription factors causes autism, attention-deficit/hyperactivity disorder, intellectual disability, and dysregulated behavior.

PURPOSE: We describe a novel neurobehavioral phenotype of autism spectrum disorder (ASD), intellectual disability, and/or attention-deficit/hyperactivity disorder (ADHD) associated with de novo or inherited deleterious variants in members of the RFX family of genes. RFX genes are evolutionarily conserved transcription factors that act as master regulators of central nervous system development and ciliogenesis. METHODS: We assembled a cohort of 38 individuals (from 33 unrelated families) with de novo variants in RFX3, RFX4, and RFX7. We describe their common clinical phenotypes and present bioinformatic analyses of expression patterns and downstream targets of these genes as they relate to other neurodevelopmental risk genes. RESULTS: These individuals share neurobehavioral features including ASD, intellectual disability, and/or ADHD; other frequent features include hypersensitivity to sensory stimuli and sleep problems. RFX3, RFX4, and RFX7 are strongly expressed in developing and adult human brain, and X-box binding motifs as well as RFX ChIP-seq peaks are enriched in the cis-regulatory regions of known ASD risk genes. CONCLUSION: These results establish a likely role of deleterious variation in RFX3, RFX4, and RFX7 in cases of monogenic intellectual disability, ADHD and ASD, and position these genes as potentially critical transcriptional regulators of neurobiological pathways associated with neurodevelopmental disease pathogenesis.

Laboratory Scale Slow Cook-Off Testing of Rocket Propellants: The Combustion Rate Analysis of a Slowly Heated Propellant (CRASH-P) Test.

Solid rocket propellants are widely used for propulsion applications by military and space agencies. Although highly effective, they can be dangerous to personnel and equipment under certain conditions, with slow heating in confined conditions being a particular danger. This paper describes a more affordable laboratory test that is easier to set up and was developed for screening rocket propellant ingredients. Rocket propellants are cast into sample holders that have been designed to have the same confinement as standard rocket motors (propellant volume to total volume in the container) and ensure that the propellant is not easily vented. Reaction violence is quantified by the time it takes to reach 90% of the maximum pressure after autoignition, which is analogous to blast overpressure gauges used to measure violence in a full-scale test. A positive correlation was observed between the speed and pressure produced from the reaction and the power produced by the rocket propellant during the reaction.

Double-blind, randomized, placebo-controlled crossover trial of alpha-lipoic acid for the treatment of fibromyalgia pain: the IMPALA trial.

ABSTRACT: Fibromyalgia is a common and challenging chronic pain disorder with few, if any, highly effective and well-tolerated treatments. Alpha-lipoic acid (ALA) is a nonsedating antioxidant with evidence of efficacy in the treatment of symptomatic diabetic neuropathy that has not been evaluated in the setting of fibromyalgia treatment. Thus, we conducted a single-centre, proof-of-concept, randomized, placebo-controlled, crossover trial of ALA for the treatment of fibromyalgia. Twenty-seven participants were recruited, and 24 participants completed both treatment periods of the trial. The median maximal tolerated dose of ALA in this trial was 1663 mg/day. Treatment-emergent adverse events with ALA were infrequent and not statistically different from placebo. For the primary outcome of pain intensity, and for several other validated secondary outcomes, there were no statistically significant differences between placebo and ALA. A post hoc exploratory subgroup analysis showed a significant interaction between gender and treatment with a significant favourable placebo-ALA difference in pain for men, but not for women. Overall, the results of this trial do not provide any evidence to suggest promise for ALA as an effective treatment for fibromyalgia, which is predominantly prevalent in women. This negative clinical trial represents an important step in a collective strategy to identify new, better tolerated and more effective treatments for fibromyalgia.