Development of a Cell-Based Reporter Potency Assay for Live Virus Vaccines.

The rapid development of potency assays is critical in the development of life-saving vaccines. The traditional plaque assay or fifty percent tissue culture infectious dose (TCID50) assay used to measure the potency of live virus vaccines is time consuming, labor intensive, low throughput and with high variability. Described here is the development and qualification of a cell-based reporter potency assay for two vaccines for respiratory viral infection, one based on the recombinant vesicular stomatitis virus (rVSV) backbone, termed Vaccine 1 in this paper, and the other based on the measles virus vector, termed Vaccine 2. The reporter potency assay used a Vero E6 cell line engineered to constitutively express NanuLuc® luciferase, termed the VeroE6-NLuc or JM-1 cell line. Infection of JM-1 cells by a live virus, such as rVSV or measles virus, causes a cytopathic effect (CPE) and release of NanuLuc® from the cytoplasm into the supernatant, the amount of which reflects the intensity of the viral infection. The relative potency was calculated by comparison to a reference standard using parallel line analysis (PLA) in a log-log linear model. The reporter assay demonstrated good linearity, accuracy, and precision, and is therefore suitable for a vaccine potency assay. Further evaluation of the Vaccine 1 reporter assay demonstrated the robustness to a range of deliberate variation of the selected assay parameters and correlation with the plaque assay. In conclusion, we have demonstrated that the reporter assay using the JM-1 cell line could be used as a potency assay to support the manufacturing and release of multiple live virus vaccines.

Pulsed electric field effect on acrylamide reduction and quality attributes of continuous-style Lamoka potato chips.

Potato chips are a popular snack, well-liked because of their texture-flavor combination. Potato chips are made by frying slices of potato in vegetable oil to achieve a crispy texture. Frying potato slices initiates the Maillard reaction, resulting in chemical changes that enhance taste, color, and texture, but also undesired acrylamides, which are suspected carcinogens. The application of pulsed electric field (PEF) technology is commonly used in French fry processing operations to prolong cutting blade sharpness and reduce waste, energy consumption, and water usage. Despite these attributes, PEF systems have not yet gained widespread adoption by potato chip producers. In the current study, Lamoka potatoes were PEF-treated prior to continuous frying into potato chips. The effect of specific energy at 0.75 kJ/kg (Low-PEF) and 1.5 kJ/kg (High-PEF) and electric field strength of 1 kV/cm, frequency of 24 kV, and pulse width of 6 µs versus untreated (control) samples was studied, then batches of 250 g of slices were fried at 170 °C or 185 °C for two frying times to obtain potato chips with acrylamide levels below the California Proposition 65 limit (275 ng/g). The Lamoka potato chip product quality metrics that were assessed include moisture, fat, reducing sugars, asparagine, acrylamide, chip color, and texture. PEF treatment of Lamoka potatoes resulted in chips fried in 10 % less time, lower oil content by 8 %, and a decrease of reducing sugars by 19.2 %, asparagine by 42.0 %, and acrylamide by 28.9 %. The PEF fried chips were lighter in color but maintained textural attributes compared to continuous frying cooking. The process of frying potato slices at 170 °C for 150 s with High-PEF yielded potato chips with acrylamide content below the California Proposition 65 limit; which speaks to the health implications for consumers and the quality and safety of these chips.

Large-scale next generation sequencing based analysis of SLCO1B1 pharmacogenetics variants in the Saudi population.

BACKGROUND: SLCO1B1 plays an important role in mediating hepatic clearance of many different drugs including statins, angiotensin-converting enzyme inhibitors, chemotherapeutic agents and antibiotics. Several variants in SLCO1B1 have been shown to have a clinically significant impact, in relation to efficacy of these medications. This study provides a comprehensive overview of SLCO1B1 variation in Saudi individuals, one of the largest Arab populations in the Middle East. METHODS: The dataset of 11,889 (9,961 exomes and 1,928 pharmacogenetic gene panel) Saudi nationals, was used to determine the presence and frequencies of SLCO1B1 variants, as described by the Clinical Pharmacogenetic Implementation Consortium (CPIC). RESULTS: We identified 141 previously described SNPs, of which rs2306283 (50%) and rs4149056 (28%), were the most common. In addition, we observed six alleles [*15 (24.7%) followed by *20 (8.04%), *14 (5.86%), *5 (3.84%), *31 (0.21%) and *9 (0.03%)] predicted to be clinically actionable. Allele diplotype to phenotype conversion revealed 41 OATP1B1 diplotypes. We estimated the burden of rare, and novel predicted deleterious variants, resulting from 17 such alterations. CONCLUSIONS: The data we present, from one of the largest Arab cohorts studied to date, provides the most comprehensive overview of SLCO1B1 variants, and the subsequent OATP1B1 activity of this ethnic group, which thus far remains relatively underrepresented in available international genomic databases. We believe that the presented data provides a basis for further clinical investigations and the application of personalized statin drug therapy guidance in Arabs.

Improving predictions of compound amenability for liquid chromatography-mass spectrometry to enhance non-targeted analysis.

Mass-spectrometry-based non-targeted analysis (NTA), in which mass spectrometric signals are assigned chemical identities based on a systematic collation of evidence, is a growing area of interest for toxicological risk assessment. Successful NTA results in better identification of potentially hazardous pollutants within the environment, facilitating the development of targeted analytical strategies to best characterize risks to human and ecological health. A supporting component of the NTA process involves assessing whether suspected chemicals are amenable to the mass spectrometric method, which is necessary in order to assign an observed signal to the chemical structure. Prior work from this group involved the development of a random forest model for predicting the amenability of 5517 unique chemical structures to liquid chromatography-mass spectrometry (LC-MS). This work improves the interpretability of the group's prior model of the same endpoint, as well as integrating 1348 more data points across negative and positive ionization modes. We enhance interpretability by feature engineering, a machine learning practice that reduces the input dimensionality while attempting to preserve performance statistics. We emphasize the importance of interpretable machine learning models within the context of building confidence in NTA identification. The novel data were curated by the labeling of compounds as amenable or unamenable by expert curators, resulting in an enhanced set of chemical compounds to expand the applicability domain of the prior model. The balanced accuracy benchmark of the newly developed model is comparable to performance previously reported (mean CV BA is 0.84 vs. 0.82 in positive mode, and 0.85 vs. 0.82 in negative mode), while on a novel external set, derived from this work's data, the Matthews correlation coefficients (MCC) for the novel models are 0.66 and 0.68 for positive and negative mode, respectively. Our group's prior published models scored MCC of 0.55 and 0.54 on the same external sets. This demonstrates appreciable improvement over the chemical space captured by the expanded dataset. This work forms part of our ongoing efforts to develop models with higher interpretability and higher performance to support NTA efforts.

Suspect Screening Analysis of Pooled Human Serum Samples Using GC × GC/TOF-MS.

Humans interact with thousands of chemicals. This study aims to identify substances of emerging concern and in need of human health risk evaluations. Sixteen pooled human serum samples were constructed from 25 individual samples each from the National Institute of Environmental Health Sciences' Clinical Research Unit. Samples were analyzed using gas chromatography (GC) × GC/time-of-flight (TOF)-mass spectrometry (MS) in a suspect screening analysis, with follow-up confirmation analysis of 19 substances. A standard reference material blood sample was also analyzed through the confirmation process for comparison. The pools were stratified by sex (female and male) and by age (≤45 and >45). Publicly available information on potential exposure sources was aggregated to annotate presence in serum as either endogenous, food/nutrient, drug, commerce, or contaminant. Of the 544 unique substances tentatively identified by spectral matching, 472 were identified in females, while only 271 were identified in males. Surprisingly, 273 of the identified substances were found only in females. It is known that behavior and near-field environments can drive exposures, and this work demonstrates the existence of exposure sources uniquely relevant to females.

Evaluation of luciferase and prefusion-stabilized F protein from respiratory syncytial virus mRNA/LNPs in pre-clinical models using jet delivery compared to needle and syringe.

Described here is the evaluation of a luciferase (luc) and respiratory syncytial virus (RSV) messenger RNA / lipid nanoparticle (mRNA/LNP) vaccine using a Needle-free Injection System, Tropis®, from PharmaJet® (Golden, Colorado USA). Needle-free jet delivery offers an alternative to needle/syringe. To perform this assessment, compatibility studies with Tropis were first performed with a luc mRNA/LNP and compared to needle/syringe. Although minor changes in particle size and encapsulation efficiency were observed when using Tropis on the benchtop, in vitro luciferase activity remained the same. Next, the luc mRNA/LNP was administered to rats intramuscularly using Tropis or needle/syringe and tracking of the injection and distribution was performed. Lastly, an mRNA encoding a prefusion-stabilized F protein from RSV was delivered intramuscularly using both Tropis and needle/syringe at 1 and 5 mcg mRNA. An equivalent IgG response was observed using both Tropis and needle/syringe. The cell mediated immune (CMI) response was also evaluated, and responses to RSV-F were detected from animals immunized with needle/syringe at all dose levels, and from the animals immunized with Tropis in the 5 and 25 ug groups. These results indicated that delivery of mRNA/LNPs with Tropis is a potential means of administration and an alternative to needle/syringe.

Novel Homozygous Variants of SLC13A5 Expand the Functional Heterogeneity of a Homogeneous Syndrome of Early Infantile Epileptic Encephalopathy.

BACKGROUND: Early infantile epileptic encephalopathy 25 (EIEE25) is a distinct type of neonatal epileptic encephalopathy caused by autosomal recessive mutations in the SLC13A5 gene. SLC13A5 encodes a transmembrane sodium/citrate cotransporter required for regulating citrate entry into cells. METHODS: Four families with recessively inherited epileptic encephalopathy were sequenced by clinically accredited laboratories using commercially available epilepsy gene panels. Patients were examined by a neurologist and were clinically diagnosed with infantile epileptic encephalopathy. RESULTS: We present four families with global developmental delay, intellectual disability, and defective tooth development with four novel homozygous mutations in SLC13A5. The neurological examination showed spastic quadriplegia with increased deep tendon reflexes. Brain magnetic resonance imaging showed nonspecific signal abnormality of the bilateral hemispheric white matter. Despite similar clinical features, the conditions were based on different molecular mechanisms acting on SLC13A5 (abnormal splicing, large-scale deletions, and tandem-residue insertion). CONCLUSIONS: Our results extend the landscape of autosomal recessive inherited homozygous mutations in SLC13A5 that cause a distinctive syndrome of severe neonatal epileptic encephalopathy. Our observations confirm the homogeneity of epileptic encephalopathy and dental abnormalities as a distinct clinical marker for EIEE25 despite the heterogeneous functional and mutational background.

Wild-type S100A3 and S100A13 restore calcium homeostasis and mitigate mitochondrial dysregulation in pulmonary fibrosis patient-derived cells.

Patients with digenic S100A3 and S100A13 mutations exhibited an atypical and progressive interstitial pulmonary fibrosis, with impaired intracellular calcium homeostasis and mitochondrial dysfunction. Here we provide direct evidence of a causative effect of the mutation on receptor mediated calcium signaling and calcium store responses in control cells transfected with mutant S100A3 and mutant S100A13. We demonstrate that the mutations lead to increased mitochondrial mass and hyperpolarization, both of which were reversed by transfecting patient-derived cells with the wild type S100A3 and S100A13, or extracellular treatment with the recombinant proteins. In addition, we demonstrate increased secretion of inflammatory mediators in patient-derived cells and in control cells transfected with the mutant-encoding constructs. These findings indicate that treatment of patients' cells with recombinant S100A3 and S100A13 proteins is sufficient to normalize most of cellular responses, and may therefore suggest the use of these recombinant proteins in the treatment of this devastating disease.

ß-catenin attenuation leads to up-regulation of activating NKG2D ligands and tumor regression in BrafV600E-driven thyroid cancer cells.

Introduction: BRAFV600E mutations frequently occur in papillary thyroid cancer (PTC). ß-catenin, encoded by CTNNB1, is a key downstream component of the canonical Wnt signaling pathway and is often overexpressed in PTC. BRAFV600E-driven PTC tumors rely on Wnt/ß-catenin signaling to sustain growth and progression. Methods: In the present study, we investigated the tumorigenicity of thyroid cancer cells derived from BRAFV600E PTC mice following Ctnnb1 ablation (BVE-Ctnnb1null). Results: Remarkably, the tumorigenic potential of BVE-Ctnnb1null tumor cells was lost in nude mice. Global gene expression analysis of BVE-Ctnnb1null tumor cells showed up-regulation of NKG2D receptor activating ligands (H60a, H60b, H60c, Raet1a, Raet1b, Raet1c, Raet1d, Raet1e, and Ulbp1) and down-regulation of inhibitory MHC class I molecules H-2L and H-2K2 in BVE-Ctnnb1null tumor cells. In vitro cytotoxicity assay demonstrated that BVE-Ctnnb1wt tumor cells were resistant to NK cell-mediated cytotoxicity, whereas BVE-Ctnnb1null tumor cells were sensitive to NK cell-mediated killing. Furthermore, the overexpression of any one of these NKG2D ligands in the BVE-Ctnnb1wt cell line resulted in a significant reduction of tumor growth in nude mice. Conclusions: Our results indicate that active ß-catenin signaling inhibits NK cell-mediated immune responses against thyroid cancer cells. Targeting the ß-catenin signaling pathway may have significant therapeutic benefits for BRAF-mutant thyroid cancer by not only inhibiting tumor growth but also enhancing host immune surveillance.

The clinical utility of rapid exome sequencing in a consanguineous population.

BACKGROUND: The clinical utility of exome sequencing is now well documented. Rapid exome sequencing (RES) is more resource-intensive than regular exome sequencing and is typically employed in specialized clinical settings wherein urgent molecular diagnosis is thought to influence acute management. Studies on the clinical utility of RES have been largely limited to outbred populations. METHODS: Here, we describe our experience with rapid exome sequencing (RES) in a highly consanguineous population. Clinical settings included intensive care units, prenatal cases approaching the legal cutoff for termination, and urgent transplant decisions. RESULTS: A positive molecular finding (a pathogenic or likely pathogenic variant that explains the phenotype) was observed in 80 of 189 cases (42%), while 15 (8%) and 94 (50%) received ambiguous (variant of uncertain significance (VUS)) and negative results, respectively. The consanguineous nature of the study population gave us an opportunity to observe highly unusual and severe phenotypic expressions of previously reported genes. Clinical utility was observed in nearly all (79/80) cases with positive molecular findings and included management decisions, prognostication, and reproductive counseling. Reproductive counseling is a particularly important utility in this population where the overwhelming majority (86%) of identified variants are autosomal recessive, which are more actionable in this regard than the de novo variants typically reported by RES elsewhere. Indeed, our cost-effectiveness analysis shows compelling cost savings in the study population. CONCLUSIONS: This work expands the diversity of environments in which RES has a demonstrable clinical utility.

Wikipedia on the CompTox Chemicals Dashboard: Connecting Resources to Enrich Public Chemical Data.

The online encyclopedia Wikipedia aggregates a large amount of data on chemistry, encompassing well over 20,000 individual Wikipedia pages and serves the general public as well as the chemistry community. Many other chemical databases and services utilize these data, and previous projects have focused on methods to index, search, and extract it for review and use. We present a comprehensive effort that combines bulk automated data extraction over tens of thousands of pages, semiautomated data extraction over hundreds of pages, and fine-grained manual extraction of individual lists and compounds of interest. We then correlate these data with the existing contents of the U.S. Environmental Protection Agency's (EPA) Distributed Structure-Searchable Toxicity (DSSTox) database. This was performed with a number of intentions including ensuring as complete a mapping as possible between the Dashboard and Wikipedia so that relevant snippets of the article are loaded for the user to review. Conflicts between Dashboard content and Wikipedia in terms of, for example, identifiers such as chemical registry numbers, names, and InChIs and structure-based collisions such as SMILES were identified and used as the basis of curation of both DSSTox and Wikipedia. This work also allowed us to evaluate available data for sets of chemicals of interest to the Agency, such as synthetic cannabinoids, and expand the content in DSSTox as appropriate. This work also led to improved bidirectional linkage of the detailed chemistry and usage information from Wikipedia with expert-curated structure and identifier data from DSSTox for a new list of nearly 20,000 chemicals. All of this work ultimately enhances the data mappings that allow for the display of the introduction of the Wikipedia article in the community-accessible web-based EPA Comptox Chemicals Dashboard, enhancing the user experience for the thousands of users per day accessing the resource.

Comparing the performance and coverage of selected in silico (liver) metabolism tools relative to reported studies in the literature to inform analogue selection in read-across: A case study.

Changes in the regulatory landscape of chemical safety assessment call for the use of New Approach Methodologies (NAMs) including read-across to fill data gaps. One critical aspect of analogue evaluation is the extent to which target and source analogues are metabolically similar. In this study, a set of 37 structurally diverse chemicals were compiled from the EPA ToxCast inventory to compare and contrast a selection of metabolism in silico tools, in terms of their coverage and performance relative to metabolism information reported in the literature. The aim was to build understanding of the scope and capabilities of these tools and how they could be utilised in a read-across assessment. The tools were Systematic Generation of Metabolites (SyGMa), Meteor Nexus, BioTransformer, Tissue Metabolism Simulator (TIMES), OECD Toolbox, and Chemical Transformation Simulator (CTS). Performance was characterised by sensitivity and precision determined by comparing predictions against literature reported metabolites (from 44 publications). A coverage score was derived to provide a relative quantitative comparison between the tools. Meteor, TIMES, Toolbox, and CTS predictions were run in batch mode, using default settings. SyGMa and BioTransformer were run with user-defined settings, (two passes of phase I and one pass of phase II). Hierarchical clustering revealed high similarity between TIMES and Toolbox. SyGMa had the highest coverage, matching an average of 38.63% of predictions generated by the other tools though was prone to significant overprediction. It generated 5,125 metabolites, which represented 54.67% of all predictions. Precision and sensitivity values ranged from 1.1-29% and 14.7-28.3% respectively. The Toolbox had the highest performance overall. A case study was presented for 3,4-Toluenediamine (3,4-TDA), assessed for the derivation of screening-level Provisional Peer Reviewed Toxicity Values (PPRTVs), was used to demonstrate the practical role in silico metabolism information can play in analogue evaluation as part of a read-across approach.

Clinical, Neurophysiological, Radiological, Pathological, and Genetic Features of Dysferlinopathy in Saudi Arabia.

Background: To characterize the phenotypic, neurophysiological, radiological, pathological, and genetic profile of 33 Saudi Arabian families with dysferlinopathy. Methods: A descriptive observational study was done on a cohort of 112 Saudi Arabian families with LGMD. Screening for the Dysferlin (DYSF) gene was done in a tertiary care referral hospital in Saudi Arabia. Clinical, Neurophysiological, Radiological, Pathological, and Genetic findings in subjects with dysferlin mutation were the primary outcome variables. Statistical analysis was done by Epi-info. Results: 33 out of 112 families (29.46%) registered in the LGMD cohort had Dysferlinopathy. 53 subjects (28 males, 52.83%) from 33 families were followed up for various periods ranging from 1 to 28 years. The mean age of onset was 17.79 ± 3.48 years (Range 10 to 25 years). Miyoshi Myopathy phenotype was observed in 50.94% (27 out of 53), LGMDR2 phenotype in 30.19% (16 out of 53), and proximodistal phenotype in 15.09% (8 out of 53) of the subjects. Loss of ambulation was observed in 39.62% (21 out of 53 subjects). Electrophysiological, Radiological, and histopathological changes were compatible with the diagnosis. Mean serum Creatinine Kinase was 6,464.45 ± 4,149.24 with a range from 302 to 21,483 IU/L. In addition, 13 dysferlin mutations were identified two of them were compound heterozygous. One founder mutation was observed c.164_165insA in 19 unrelated families. Conclusion: The prevalence of Dysferlinopathy was 29.46% in the native Saudi LGMD cohort. It is the most prevalent subtype seconded by calpainopathy. The clinical course varied among the study subjects and was consistent with those reported from different ethnic groups. One founder mutation was identified. Initial screening of the founder mutations in new families is highly recommended.

Identification of pharmacogenetic variants from large scale next generation sequencing data in the Saudi population.

It is well documented that drug responses are related to Absorption, Distribution, Metabolism, and Excretion (ADME) characteristics of individual patients. Several studies have identified genetic variability in pharmacogenes, that are either directly responsible for or are associated with ADME, giving rise to individualized treatments. Our objective was to provide a comprehensive overview of pharmacogenetic variation in the Saudi population. We mined next generation sequencing (NGS) data from 11,889 unrelated Saudi nationals, to determine the presence and frequencies of known functional SNP variants in 8 clinically relevant pharmacogenes (CYP2C9, CYP2C19, CYP3A5, CYP4F2, VKORC1, DPYD, TPMT and NUDT15), recommended by the Clinical Pharmacogenetics Implementation Consortium (CPIC), and collectively identified 82 such star alleles. Functionally significant pharmacogenetic variants were prevalent especially in CYP genes (excluding CYP3A5), with 10-44.4% of variants predicted to be inactive or to have decreased activity. In CYP3A5, inactive alleles (87.5%) were the most common. Only 1.8%, 0.7% and 0.7% of NUDT15, TPMT and DPYD variants respectively, were predicted to affect gene activity. In contrast, VKORC1 was found functionally, to be highly polymorphic with 53.7% of Saudi individuals harboring variants predicted to result in decreased activity and 31.3% having variants leading to increased metabolic activity. Furthermore, among the 8 pharmacogenes studied, we detected six rare variants with an aggregated frequency of 1.1%, that among several other ethnicities, were uniquely found in Saudi population. Similarly, within our cohort, the 8 pharmacogenes yielded forty-six novel variants predicted to be deleterious. Based upon our findings, 99.2% of individuals from the Saudi population carry at least one actionable pharmacogenetic variant.

Insights of Noncanonical Splice-site Variants on RNA Splicing in Patients With Congenital Hypothyroidism.

CONTEXT: Congenital hypothyroidism (CH) is caused by mutations in the genes for thyroid hormone synthesis. In our previous investigation of CH patients, approximately 53% of patients had mutations in either coding exons or canonical splice sites of causative genes. Noncanonical splice-site variants in the intron were detected but their pathogenic significance was not known. OBJECTIVE: This work aims to evaluate noncanonical splice-site variants on pre-messenger RNA (pre-mRNA) splicing of CH-causing genes. METHODS: Next-generation sequencing data of 55 CH cases in 47 families were analyzed to identify rare intron variants. The effects of variants on pre-mRNA splicing were investigated by minigene RNA-splicing assay. RESULTS: Four intron variants were found in 3 patients: solute carrier family 26 member 4 (SLC26A4) c.1544+9C>T and c.1707+94C>T in one patient, and solute carrier family 5 member 5 (SLC5A5) c.970-48G>C and c.1652-97A>C in 2 other patients. The c.1707+94C>T and c.970-48G>C caused exons 15 and 16 skipping, and exon 8 skipping, respectively. The remaining variants had no effect on RNA splicing. Furthermore, we analyzed 28 previously reported noncanonical splice-site variants (4 in TG and 24 in SLC26A4). Among them, 15 variants (~ 54%) resulted in aberrant splicing and 13 variants had no effect on RNA splicing. These data were compared with 3 variant-prediction programs (FATHMM-XF, FATHMM-MKL, and CADD). Among 32 variants, FATHMM-XF, FATHMM-MKL, and CADD correctly predicted 20 (63%), 17 (53%), and 26 (81%) variants, respectively. CONCLUSION: Two novel deep intron mutations have been identified in SLC26A4 and SLC5A5, bringing the total number of solved families with disease-causing mutations to approximately 45% in our cohort. Approximately 46% (13/28) of reported noncanonical splice-site mutations do not disrupt pre-mRNA splicing. CADD provides highest prediction accuracy of noncanonical splice-site variants.

Transcending self therapy: Four-session individual integrative cognitive-behavioral treatment: A case report.

Effective treatments for Substance Use Disorders (SUDs) are of critical importance, particularly among veterans. We present a successful application of Transcending Self Therapy: Four-Session Individual Integrative Cognitive Behavioral Treatment (Individual TST-I-CBT), that helped a male combat veteran with Alcohol Use Disorder, Severe, and PTSD enter recovery and reduce depressive symptoms. Session 1 focused on problem solving and behaviors; Session 2 centered on checking and changing thoughts; Session 3 emphasized behaviors, thoughts, and coping; and Session 4 consisted of review and finalizing the recovery plan. After the second Individual TST-I-CBT session, he was abstinent from alcohol use and remained abstinent throughout the remainder of treatment. At the end of treatment, his depressive symptoms declined substantially from pretreatment (from severe to low), his desire to stop using alcohol was 10/10, and his confidence in his ability to stop using alcohol was 10/10. He demonstrated personal growth and accomplishments throughout the course of treatment, such as improving his self-concept and relationships; living in accordance with his values; developing the passionate pursuit of being a productive member of society by holding a job; looking for a job; and taking steps to continue his education. Accordingly, he accomplished all of his identified treatment goals. This case suggests Individual TST-I-CBT is a potentially effective adjunctive treatment for SUD. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Established and candidate transthyretin amyloidosis variants identified in the Saudi population by data mining.

BACKGROUND: Familial transthyretin (TTR) amyloidosis (ATTR) is an autosomal dominant disease with significant phenotypic heterogeneity. Its prevalence in Saudi Arabia has not previously been investigated. An existing exome variant database of Saudi individuals, sequenced to globally investigate rare diseases in the population, was mined for TTR variants and filtered for missense mutations resulting in single amino acid changes. A total of 13,906 Saudi exomes from unrelated individuals were analyzed blindly. RESULTS: Three TTR variants known to be associated with ATTR amyloidosis were identified. Additionally, three novel TTR mutations were identified. Structural analysis of the three novel variants suggests that at least two could be amyloidogenic. The most common variant associated with amyloidosis was p.Val142Ile (allele frequency 0.001). Further investigation of these variants and their translation to clinical practice may help to diagnose, monitor, and manage patients with ATTR amyloidosis. CONCLUSION: Multiple TTR variants potentially associated with systemic ATTR amyloidosis were identified in the Saudi population. Early diagnosis and intervention, facilitated by familial genetic testing of patients with ATTR amyloidosis, may benefit in the management of this disease. Early diagnosis could be enhanced through inclusion of ATTR variants in existing population-based screening programs.

Frontiers in assessing septic systems vulnerability in coastal Georgia, USA: Modeling approach and management implications.

Threats to public health and environmental quality from septic systems are more prevalent in areas with poorly draining soils, high water tables, or frequent flooding. Significant research gaps exist in assessing these systems' vulnerability and evaluating factors associated with higher rates of septic systems replacement and repair. We developed a novel GIS-based framework for assessing septic system vulnerability using a database of known septic system specifications and a modified Soil Topographic Index (STI) that incorporates seasonal high groundwater elevation to assess risks posed to septic systems in coastal Georgia. We tested the hypothesis that both the modified STI and septic system specifications such as tank capacity per bedroom and drainfield type would explain most of the variance in septic system repair and replacement using classification inference tree and generalized logistic regression models. Our modeling results indicate that drainfield type (level vs. mounded) is the most significant variable (p-value < 0.001) in predicting septic systems functionality followed by septic tank capacity per bedroom (p-value < 0.01). These show the importance of septic system design regulations such as a minimum requirement for horizontal separation distance between the bottom of trenches and seasonal water table, and adequate tank capacity design. However, for septic systems with a mounded drainfield and a larger tank capacity per bedroom, the modified STI representing hydrological characteristics of septic system location is a significant predictor of a high septic system repair and replacement rate. The methodology developed in this study can have important implications for managing existing septic systems and planning for future development in coastal areas, especially in an environment of rapid climatic change.

ß-Catenin Attenuation Inhibits Tumor Growth and Promotes Differentiation in a BRAFV600E-Driven Thyroid Cancer Animal Model.

BRAFV600E mutation is the most frequent genetic alteration in papillary thyroid cancer (PTC). ß-Catenin (Ctnnb1) is a key downstream component of canonical Wnt signaling pathway and is frequently overexpressed in PTC. BRAF V600E-driven tumors have been speculated to rely on Wnt/ß-catenin signaling to sustain its growth, although many details remain to be elucidated. In this study, we investigated the role of ß-catenin in BrafV600E -driven thyroid cancer in a transgenic mouse model. In Braf V600E mice with wild-type (WT) Ctnnb1 (BVE-Ctnnb1WT or BVE), overexpression of ß-catenin was observed in thyroid tumors. In Braf V600E mice with Ctnnb1 knockout (BVE-Ctnnb1null), thyroid tumor growth was slowed with significant reduction in papillary architecture. This was associated with increased expression of genes involved in thyroid hormone synthesis, elevated 124iodine uptake, and serum T4. The survival of BVE-Ctnnb1null mice was increased by more than 50% during 14-month observation. Mechanistically, downregulation of MAPK, PI3K/Akt, and TGFß pathways and loss of epithelial-mesenchymal transition (EMT) were demonstrated in the BVE-Ctnnb1null tumors. Treatment with dual ß-catenin/KDM4A inhibitor PKF118-310 dramatically improved the sensitivity of BVE-Ctnnb1WT tumor cells to BRAFV600E inhibitor PLX4720, resulting in significant growth arrest and apoptosis in vitro, and tumor regression and differentiation in vivo These findings indicate that ß-catenin signaling plays an important role in thyroid cancer growth and resistance to BRAFV600E inhibitors. Simultaneously targeting both Wnt/ß-catenin and MAPK signaling pathways may achieve better therapeutic outcome in BRAFV600E inhibitor-resistant and/or radioiodine-refractory thyroid cancer.