Cell Variant Research Articles

Electrification pathways for light-duty logistics vehicles based on perceived cost of ownership in Northern China

Urban decarbonization and environmental mitigation necessitate the electrification of light-duty logistics vehicles (LDLVs), including battery electric, plug-in hybrid, and hydrogen fuel cell variants. Although the market uptake of electric LDLVs is ecologically imperative, it is impeded by range anxiety and charging infrastructure limitations, particularly pronounced in Northern China’s cold climates. This paper employs a system dynamics model to assess the Perceived Cost of Ownership of electric LDLVs, integrating both direct expenses - initial investment and energy costs - and indirect factors like energy replenishment, vehicle substitution, and lifecycle carbon emissions. This analysis reveals that, notwithstanding higher upfront costs, electric LDLVs offer substantial economic and environmental advantages, with significant energy and maintenance savings projected by 2030 under various electrification scenarios. This paper predicts that policy incentives, electricity pricing, and technological progress will significantly influence the market dynamics and industry output of new energy vehicles in Northern China. Notably, the findings indicate that by 2030, electric LDLVs could achieve substantial cost savings and environmental benefits, with market penetration and industry output contingent on the interplay of policy support and technological advancements. The baseline scenario forecasts a 48.17% market share and CNY 60.015 billion in industry output, whereas the high-speed electrification scenario projects the most optimistic outcomes, with a 75.29% market share and CNY 306.087 billion in output.

Hyperlipidemia interferes with the behavior of variant NKT cells

Abstract Background NKT cells significantly influence atherosclerosis development. However, inconsistent findings on the function of the NKT cells and the invariant subset in atherosclerosis remains a challenge. Moreover, there is no clarity on the role and phenotype of the variant (vNKT) subset in atherosclerosis. Purpose To understand the role of the vNKT subsets in HFD-induced atherosclerosis. Methods We extracted the data for meta-analysis to understand the role of the whole NKT and iNKT cells in atherosclerosis. PRISMA guidelines were followed for the meta-analysis. In addition, C57BL/6 mice (WT) were fed with HFD for 20 weeks from 6-8 weeks of age. vNKTs were identified as CD3+NK1.1+CD1d-tetramer- within the CD3+NK1.1+ NKT cell population in both liver and spleen by flow cytometry. Intracellular cyotkines staining was performed. Statistical differences inthe means of two groups are calculated by two-tailed Student’s t-test with nonparametric Mann-Whitney tests. P-value<0.05 was considered significant. All data represented as mean±SEM. Results The NKT cell numbers increased significantly in the spleen, lymph nodes, blood, and liver in the HFD-fed mice fed from 1.5 to 24 weeks in Apoe-/-, Ldlr-/- in comparison to wild-type mice (SMD, 2.51 [95% CI, 1.42, 3.61]). Contrary to this, the iNKT cell numbers and the iNKT TCR gene expression levels, decreased significantly in the HFD-fed mice (SMD, -2.04 [95% CI, -3.34, -0.75]). The reduction in iNKT cell numbers is also supported by the clinical data from the AMI patients when compared to healthy controls [SMD = -1.81, 95% CI = -2.89, -0.74] (Figure 1). In HFD-fed C57BL/6 hyperlipidemic mice, significantly increased levels of total cholesterol (HFD vs Chow; 226.5±29.66 vs 118.5±10.56 mg/dL, n=4) and LDLc (44.56±11.96 vs 16.69±1.13 mg%, n=4) was observed. The number of vNKTs in the liver was significantly increased by 46% (HFD vs Chow 28.35±2.28 vs 19.41±1.36, n=6-7) (Figure 2A). However, the CD8+ vNKTs were significantly decreased by 68.6% in the liver (4.72±0.99 vs 15.06±0.96) and the CD4+ vNKTs decreased more than two times in the HFD-fed WT liver (7.39±0.76 vs 18.28±0.36) (Figure 2B). Also, the intracellular IFN-γ+ vNKT cells doubled in their numbers in the HFD-fed spleen (14.31±1.39 vs 6.367±0.87) compared to the chow-fed mice (Figure 2C). Conclusion(s) Meta-analysis revealed dual nature of the NKT cells and its invariant subsets in atherosclerosis, since the whole NKTs increase whereas the iNKTs decrease in number. The vNKT subset actively participates in immune modulation in the HFD-induced hyperlipidemia by increasing their number, as well as by differentially regulating CD8 and CD4 co-receptors between chow and HFD. HFD induced a pro-inflammatory nature in the splenic vNKTs but not in liver vNKTs, that in turn may aggravate other immune cells. This data confirms the dual nature of the NKT subsets and reveals a crucial systemic involvement of vNKT cells in the progression of dyslipidemia.

Micro-capture-C in vessel wall cell types identifies mechanisms underlying coronary artery disease susceptibility loci

Abstract Genetics is estimated to account for 50% of Coronary Artery Disease (CAD) risk. Genome-Wide Association Studies (GWAS) have revealed 100s of genomic loci which are associated with CAD (1). However, moving from GWAS signals to causal genes has proved challenging, slowing progress in developing interventions for CAD and other diseases. The greatest challenge in interpreting GWAS data is that only 10% of variants are in protein-coding regions and 90% are in "non-coding" genomic regions. Many of these non-coding variants are found in regulatory elements such as enhancers. Enhancers are distal regulatory elements that bind transcription factors and regulate genes up to 1 million base pairs away. This is achieved by DNA looping bringing enhancers into physical proximity with target genes. However, it is not possible to predict which gene(s) a given enhancer will regulate based on genomic location alone. We aimed to develop a generalisable workflow to solve CAD GWAS signals accurately and sensitively, by identifying causal enhancer SNPs and linking them mechanistically to output genes in a cell-type specific manner. We combined cell-specific epigenomic mapping with machine learning to prioritise 21,959 SNPs from 861 candidate CAD loci, in endothelial cells (HUVECs) and smooth muscle cells (HCASMCs). Micro-Capture-C (MCC) was used to resolve enhancer-promoter interactions at base-pair resolution (2). MNase footprinting determined differential transcription factor binding at specific enhancer SNPs. In HCASMCs, 20 putative enhancers containing CAD SNPs were found to interact with 32 genes, and in HUVECs 25 enhancers interacted with 48 genes. These genes were enriched for pathways including TGFb signalling, RUNX3 activity, and MAPK6/MAPK4 signalling. To prove causality at specific loci, heterozygous SNPs were identified in the individual primary cell lines. The interaction frequency at these elements was statistically compared between risk and non-risk alleles. For example, at the 9p21.3 locus, an enhancer containing a non-risk T allele was found to interact more strongly with the CDKN2B gene, compared to the risk G allele. Interestingly, this imbalance between allele interaction was potentiated by stimulation with TGFb, a key mediator in atherosclerosis. Furthermore, the transcription factor footprint at the SNP location was altered by the risk allele, indicating the mechanistic cause of the CAD-associated change in CDKN2B expression. We demonstrate a combined computational and experimental approach to identify causal non-coding variants in CAD risk loci, at scale. Among the key outputs of this study is the identification of the risk variant (rs1537373), output gene (CDKN2B), and specific cell type (HCASMC) underlying the 9p21.3 locus association with CAD risk. Using this approach to translate GWAS signals into cell-type specific outputs genes, will lead to a better understanding of the pathogenesis of CAD, and new therapeutic opportunities.

Epithelioid solitary fibrous tumors from CNS and soft tissues: an unusual morphologic variant

BackgroundSolitary fibroous tumors (SFTs) are distinctive soft tissue tumors characterized by rearrangements of NAB2-STAT6 gene, which are associated with thin-walled, branching, “staghorn”-shaped vessels. SFTs are originally classified as a type of hemangiopericytoma (HPC). Classical SFTs are composed of spindle to ovoid cells arranged haphazardly or in fascicles. Rarely, SFTs exhibit unusual morphological variants such as fat formation, giant cells, dedifferentiation, or epithelioid variant. The epithelioid cell variant, which is composed almost entirely of epithelioid cells and arranged in solid or nest patterns, is extremely rare and frequently malignant.Case presentationIn this study, we reported three cases of epithelioid SFTs (ESFTs) located in extrathoracic sites (right lateral ventricle, right lumbar, left pelvis). All the subjects in this study were elderly, with a predominance of female patients, accounting for two out of the three cases, and only one case involved a male patient. The tumor cells were entirely composed of epithelioid cells and exhibited positive for CD34 and STAT-6 markers. Ultimately, the majority of cases (two out of three) were diagnosed as malignant SFTs.ConclusionThis study aims to enhance the awareness of ESFTs. In these cases, irrespective of the onset location, the arrangement patterns of tumor cells, such as papillary structures and the morphology of epithelial-like cells, conspicuously lack the hallmark histological characteristics of Solitary Fibrous Tumors (SFTs). Consequently, it requires differential diagnosis from a plethora of malignant neoplasms. Moreover, the elevated malignancy level of this cohort of cases poses substantial diagnostic challenges to pathologists, compounding the complexity of accurate interpretation.

Multiplexed profiling of intracellular protein abundance, activity, interactions and druggability with LABEL-seq.

Here we describe labeling with barcodes and enrichment for biochemical analysis by sequencing (LABEL-seq), an assay for massively parallel profiling of pooled protein variants in human cells. By leveraging the intracellular self-assembly of an RNA-binding domain (RBD) with a stable, variant-encoding RNA barcode, LABEL-seq facilitates the direct measurement of protein properties and functions using simple affinity enrichments of RBD protein fusions, followed by high-throughput sequencing of co-enriched barcodes. Measurement of ~20,000 variant effects for ~1,600 BRaf variants revealed that variation at positions frequently mutated in cancer minimally impacted intracellular abundance but could dramatically alter activity, protein-protein interactions and druggability. Integrative analysis identified networks of positions with similar biochemical roles and enabled modeling of variant effects on cell proliferation and small molecule-promoted degradation. Thus, LABEL-seq enables direct measurement of multiple biochemical properties in a native cellular context, providing insights into protein function, disease mechanisms and druggability.

Distinct chikungunya virus polymerase palm subdomains contribute to viral protein accumulation and virion production.

Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus has established the importance of the nsP4 residue cysteine 483 in replication. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the coxsackievirus B3 3D polymerase. We rescued each variant in mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to host-specific replication. In C6/36 cells, we found that while the nsP4 variants had replicase function similar to that of wild-type CHIKV, many variants presented changes in protein accumulation and virion production even when viral nonstructural and structural proteins were produced. Finally, we found that WT CHIKV and nsP4 variant replication and protein production could be enhanced in mammalian cells at 28°C, yet growing virus under these conditions led to changes in virus infectivity. Taken together, these studies highlight that distinct nsP4 subdomains are required for proper RNA transcription and translation, having major effects on virion production.

Role of PNPLA3 in Hepatic Stellate Cells and Hepatic Cellular Crosstalk.

Since its discovery, the patatin-like phospholipase domain containing 3 (PNPLA3) (rs738409 C>G p.I148M) variant has been studied extensively to unravel its molecular function. Although several studies proved a causal relationship between the PNPLA3 I148M variant and MASLD development and particularly fibrosis, the pathological mechanisms promoting this phenotype have not yet been fully clarified. We summarise the latest data regarding the PNPLA3 I148M variant in hepatic stellate cells (HSCs) activation and macrophage biology or the path to inflammation-induced fibrosis. Elegant but contradictory studies have ascribed PNPLA3 a hydrolase or an acyltransferase function. The PNPLA3 I148M results in hepatic lipid accumulation, which predisposes the hepatocyte to lipotoxicity and lipo-apoptosis, producing DAMPs, cytokines and chemokines leading to recruitment and activation of macrophages and HSCs, propagating fibrosis. Recent studies showed that the PNPLA3 I148M variant alters HSCs biology via attenuation of PPARγ, AP-1, LXRα and TGFβ activity and signalling. The advent of refined techniques in isolating HSCs has made PNPLA3's direct role in HSCs for liver fibrosis development more apparent. However, many other mechanisms still need detailed investigations.

Polyoxazolines with Cholesterol Lipid Anchor for Fast Intracellular Delivery.

Due to the increasing challenges posed by the growing immunity to poly(ethylene glycol) (PEG), there is growing interest in innovative polymer-based materials as viable alternatives. In this study, the advantages of lipids and polymers are combined to allow efficient and rapid cytoplasmic drug delivery. Specifically, poly(2-methyl-2-oxazoline) is modified with a cholesteryl hemisuccinate group as a lipid anchor (CHEMSPOx). The CHEMSPOx is additionally functionalized with a coumarin group (CHEMSPOx-coumarin). Both polymers self-assembled in water into vesicles of ≈100nm and are successfully loaded with a hydrophobic model drug. The loaded vesicles reveal high cellular internalization across variant cell lines within 1 h at 37°C as well as 4°C, albeit to a lesser extent. A kinetic study confirms the fast internalization within 5 min after the sample's addition. Therefore, different internalization pathways are involved, e.g., active uptake but also nonenergy dependent mechanisms. CHEMSPOx and CHEMSPOx-coumarin further demonstrate excellent cyto-, hemo-, and membrane compatibility, as well as a membrane-protecting effect, which underlines their good safety profile for potential biological intravenous application. Overall, CHEMSPOx, as a lipopolyoxazoline, holds great potential for versatile biological applications such as fast and direct intracellular delivery or cellular lysis protection.

8788 Implications of NTRK gene fusion in Papillary thyroid cancer

Abstract Disclosure: W. Medina-Torres: None. L. El Musa Penna: None. J. Segarra-Villafane: None. L.R. Sepulveda-Garcia: None. Z. Maisonet -Feliciano: None. I.C. Arroyo: None. M. Ramirez: None. M. Alvarado: None. L.A. Gonzalez-Rodriguez: None. V.J. Carlo: None. Thyroid cancer (TC) is the most common endocrine cancer. Papillary thyroid carcinoma (PTC) is the predominant contributor of this occurrence, comprising approximately 80% of all thyroid carcinomas. PTC has a good prognosis with a 5-year survival rate of more than 90% after resection. Nonetheless, factors such as tumor size, multifocality, nodal and distant metastases, characterize more aggressive cases including tall cell variant, columnar cell variant, and hobnail variant Aside from staging parameters, PTC behavior is mostly determined by morphologic variant. This is the case of 31 y/o female without known past medical history who was evaluated at endocrinology clinic due to a fine needle thyroid aspiration (FNA) positive for PTC, tall cell variant. Total thyroidectomy with central compartment dissection was performed. Pathology report presented a main tumoral mass of 3 cm but permeating multinodular pattern involving the entire gland with focal extrathyroidal extension, positive margins and six level VI positive lymph nodes. As per report, the tumor presented both papillary and follicular patterns of growth and other findings including oncocytic change, foci of clear cell change, low mitotic rate, and most notably "reverse polarization”. This pattern has the pathological features suggestive of the novel NTRK gene fusion mutation. The patient was treated with radioactive iodine (RAI) and one year post treatment there was evidence of elevated Thyroglobulin level at 904 ng/mLand thyroglobulin Ab at 2.48 IU/mL. A WBS +SPECT was done resulting in small uptake at left posterior thyroid bed and pulmonary intake. A chest CT revealed multiple bilateral sub centimeter nodules. Based on this result, a second course of RAI was given. Follow up neck ultrasound and chest CT presented persistent left level VI lymph nodes, increased amount of bilateral sub-centimeter pulmonary nodules and new thoracic(T11) lesion. FNA of left neck lymph nodes were positive for metastatic PTC for which left neck dissection was performed. Evaluation for actionable somatic mutations was requested based on resistance behavior, remarkable for TRIM33-NTRK1 chromosomal rearrangement. Patient was evaluated by Oncology service who started Larotrectinib treatment since it was approved for NTRK driven malignancies. Six-month post therapy patient achieved clinical and biochemical improvement. In the thyroid, NTRK-driven malignancies are rare and aggressive but treatable, in the evolving genomic era. As reviewed in the literature, NTRK fusion-positive TC exhibits distinctive clinicopathological features that could aid in identifying potential patients. Thus, in such patients who have PTC with non-classic pathologic patterns, specific molecular profile of the main tumor may have significant prognostic value that must be integrated into stratification system in order to provide target therapy on time. Presentation: 6/3/2024

8549 A Pre-ablative Stimulated Thyroglobulin Value That Predicts ATA High Risk Patients and Their Treatment Response

Abstract Disclosure: H. Gondaliya: None. N. Nangadda: None. D. Bhat: None. K.S. Khadilkar: None. S. Kumbenahalli Siddegowda: None. B.G. Sooragonda: None. A. Shetty: None. S. Kannan: None. In ATA risk stratification system for differentiated thyroid cancer (DTC), one of the variables placing patients in ATA high risk category is thyroglobulin (Tg) suggestive of distant metastases. However there is no defined cut-off Tg level for guiding the clinicians. We aimed to determine pre-ablative stimulated Tg (psTg) value that predicts ATA high risk patients (based on non-Tg parameters) and their treatment response on follow up. This was an ambispective observational study of 285 patients with DTC treated at our centre from April 2017 to November 2022. psTg was measured in all patients 3-4 weeks after surgery and they were subjected to RAIA depending on clinician’s discretion and followed up using the ATA dynamic risk stratification every 6 months. The cohort was divided into two groups: ATA high risk and ATA non-high risk (Low and intermediate risk). Patients among high risk group were divided based on response to treatment into structural incomplete group and non-structural incomplete group (Excellent, Indeterminate and Biochemically incomplete response). The mean age of the study population was 41±15 years and 68% (n=194) were female and 87% (n=249) were papillary thyroid cancer (PTC) and 13% (n=36) were Follicular Thyroid Cancer (FTC). Median duration of follow up was 2.95 years (IQR: 1.42 - 5.12). Among those with PTC, 75% were Classic, 15% Follicular variant and 10% aggressive variants that included tall cell, hobnail, diffuse sclerosing and columnar variant. On ATA risk stratification, 81.75% (n=233) patients were in non-high risk group with median psTg 3.4 ng/ml (IQR 0.5-12.2) and 18.25% (n=52) were in high-risk group with median psTg 150.5 ng/ml (IQR 7.15-439.5). On ROC analysis of all the patients, a psTg value >53.3 ng/ml was predictive of ATA high risk group with PPV of 64%; NPV of 93.5% and AUC 0.81. In ATA high-risk group, there were 73% (n=38) of patients with structural incomplete response on follow up and their median psTg was 287 ng/ml (IQR 95-500) and there were 27% (n=14) with non-structural incomplete response and their median psTg level of 6.2 ng/ml (IQR 2.3-110). A psTg value >53 ng/ml discriminated structural incomplete from non-structural incomplete response with PPV of 77%; NPV 87% and AUC 0.83. A psTg value >53 ng/ml not only predicts ATA high risk patients but also indicates those who are likely to remain with structural residual disease at follow up of 3 years. Presentation: 6/1/2024

7568 Sickle Cell Variant Promotes Glycemic Dysregulation In A Mouse Model Of Human Sickle Cell Trait And Sickle Cell Disease

Abstract Disclosure: J.L. Zapater: None. B.T. Layden: None. Since cases of type 2 diabetes mellitus (T2DM) are rising due to environmental and societal factors, it is estimated that in the coming decades that T2DM will affect twenty percent of the United States population and that mortality from diabetic complications will continue to rise. It is therefore critical to accurately diagnose and treat T2DM early, however this proves difficult in certain populations. Sickle cell anemia is a common hemoglobinopathy in which changes in red blood cell turnover lead to inaccurate measurement of hemoglobin A1c and incongruencies between this and other glucose measures, making it difficult to establish a diagnosis of T2DM. The sickle cell variant has been linked to higher hemoglobin A1c, and with 1-in-10 African Americans having the sickle cell trait (SCT), an increasing prevalence of sickle cell disease (SCD), and a higher risk of diabetic complications in those with SCT or SCD, it is critical that we elucidate the precise mechanisms by which the sickle cell variant affects glycemic homeostasis. In this work, we utilized a mouse model of human sickle cell anemia to begin to determine the biochemical mechanisms governing glucose handling in the presence of the sickle cell variant by examining glycemic homeostasis under normal and obesogenic conditions. Townes sickle cell mice containing human hemoglobin with either two normal β-globin chains (littermate controls) or one normal and one sickle β-globin chain (SCT) were placed on either a normal chow diet or on a high fat diet (HFD) until 20 weeks of age to induce an obesogenic state. Although no overt glycemic phenotype was noted, 20-week-old HFD-fed SCT mice exhibited an increased glucose excursion following an oral glucose load compared to littermate controls. Though serum insulin levels were comparable, HFD-fed SCT mice also exhibited a reduced whole-body insulin sensitivity during insulin tolerance testing compared to controls, correlating with the noted increased glycemic excursion. With these findings, we have initiated work with Townes mice containing two normal α-chains and two sickle β-chains (SCD), which is found in human sickle cell anemia. Early results show that chow-fed SCD mice exhibit greater random and fasting glucose levels at 8 and 12 weeks of age, despite similar fasting insulin levels. These findings suggest that the presence of one sickle β-globin chain, as in SCT, promotes hyperglycemia in the obesogenic state through reduction of whole-body insulin sensitivity. Furthermore, the presence of two sickle β-globin chains, as in SCD, is sufficient to dysregulate glycemic control under normal conditions. In total, these findings demonstrate that the presence of the sickle cell variant promotes glucose dysregulation and hyperglycemia, meaning that it is essential to understand the glycemic regulation in patients with sickle cell trait and sickle cell anemia. Presentation: 6/1/2024

7962 Transformation of Poorly Differentiated Papillary Thyroid Carcinoma to Anaplastic Thyroid Carcinoma, an Atypical Course!

Abstract Disclosure: T. Altaweel: None. S. Makadsi: None. M. Hussein: None. J. Samantray: None. Introduction: Anaplastic thyroid cancers are undifferentiated, rare and aggressive. The transformation of papillary thyroid cancer to anaplastic thyroid cancer is documented in literature, however, rare. In this report, we present a case of Anaplastic thyroid carcinoma likely arising from a co-existing poorly differentiated Papillary carcinoma. Case Presentation: A 74-year-old male with past medical history of tobacco smoking who presented with the onset of voice hoarseness for 6 months. He has also noticed a left neck mass that is in the mid supraclavicular region. A Neck CT neck was performed at the time of presentation and was significant for a 4.6x3.4x5.9 cm left thyroid mass with displacement of the trachea. FNA biopsy showed papillary carcinoma with focal features of Tall Cell Variant. Poorly differentiated components with fragments of stroma and muscle were seen and suggested invasion. These findings raised the possibility of anaplastic thyroid carcinoma in the setting of papillary carcinoma and poorly differentiated carcinoma. BRAF V600E pathogenic variant mutation along with TERT mutation were found. Patient underwent direct laryngoscopy, bronchoscopy with biopsies, and tracheostomy with surgical pathology confirming papillary thyroid cancer with muscles invasion. Combination dabrafenib and trametinib were started. No surgical resection of thyroid tissue was planned. A follow up CT showed a significant decrease in the low-density soft tissue involving the medial thyroid gland bilaterally, consistent with interim positive response to therapy. Three months later, CT neck showed near complete resolution of low-density soft tissue involving the medial thyroid and parathyroid glands and subglottic trachea consistent with further interim positive response to therapy. Six months post initiation of therapy, CT Neck showed focal low density in the mid and inferior right thyroid gland which may represent residual and/or recurrent thyroid carcinoma. That density was further demonstrated on a repeat CT neck as a 1.8 cm hypodense nodule in the inferior right thyroid gland and concerning for recurrent thyroid tumor with possible infiltration of the trachea. Conclusion: ATC prognosis is affected by many factors such as metastasis, age, treatment strategies, socioeconomic status and the presence of BRAF V600E and TERT mutations. Diagnosis can be challenging. Our patient presented months after his symptoms started and this is an atypical course of Anaplastic Thyroid Carcinoma as it usually progresses aggressively and rapidly. Given the pathology reports and the more slow presentation of this patient’s carcinoma, it can be suggested that this is a case of papillary carcinoma with a later transformation to ATC. In these cases, surgical resection Presentation: 6/2/2024

Molecular mechanisms of Coxiella burnetii formalin-fixed cellular vaccine reactogenicity.

Local and systemic reactogenic responses to Q-VAX have prevented licensing of this vaccine outside of Australia. These reactogenic responses occur in previously sensitized individuals and have not been well defined at the cellular level, in part because many studies have been done in guinea pigs that have limited molecular tools. We previously characterized a mouse model of reactogenicity where local reaction sites showed an influx of CD8+ and IFNγ-expressing IL17a+ CD4+ T cells consistent with a Th1 delayed-type hypersensitivity. In this study, we determined, using depletion and adoptive transfer experiments, that both anti-Coxiella antibodies and CD4+ T cells were essential for localized reactions at the site of vaccination. Furthermore, IFNγ depletion showed significant histological changes at the local reaction sites demonstrating the essential nature of this cytokine to reactogenicity. In addition to the cells and cytokines required for this response, we determined that whole cell vaccine (WCV) material remained at the site of vaccination for at least 26 weeks post-injection. Transmission electron microscopy (TEM) of these sites demonstrated intact rod-shaped bacteria at 2 weeks post-injection and partially degraded bacteria within macrophages at 26 weeks post-injection. Finally, because small cell variants (SCVs) are an environmentally stable form, we determined that local reactions were more severe when the WCV material was prepared with higher levels of SCVs compared to typical WCV or with higher levels of large cell variant (LCV). These studies support the hypothesis that antigen persistence at the site of injection contributes to this reactogenicity and that anti-Coxiella antibodies, CD4+ T cells, and IFNγ each contribute to this process.

De novo design of mini-protein binders broadly neutralizing Clostridioides difficile toxin B variants

Clostridioides difficile toxin B (TcdB) is the key virulence factor accounting for C. difficile infection-associated symptoms. Effectively neutralizing different TcdB variants with a universal solution poses a significant challenge. Here we present the de novo design and characterization of pan-specific mini-protein binders against major TcdB subtypes. Our design successfully binds to the first receptor binding interface (RBI-1) of the varied TcdB subtypes, exhibiting affinities ranging from 20 pM to 10 nM. The cryo-electron microscopy (cryo-EM) structures of the mini protein binder in complex with TcdB1 and TcdB4 are consistent with the computational design models. The engineered and evolved variants of the mini-protein binder and chondroitin sulfate proteoglycan 4 (CSPG4), another natural receptor that binds to the second RBI (RBI-2) of TcdB, better neutralize major TcdB variants both in cells and in vivo, as demonstrated by the colon-loop assay using female mice. Our findings provide valuable starting points for the development of therapeutics targeting C. difficile infections (CDI).

Epithelioid Fibrous Histiocytoma Is on a Continuum With Superficial ALK-rearranged Myxoid Spindle Cell Neoplasm: A Clinicopathologic Series of 35 Cases Including Alternate RET and NTRK3 Fusions.

Anaplastic lymphoma kinase (ALK) rearrangements drive most examples of epithelioid fibrous histiocytoma (EFH) and have been reported in an emerging family of receptor tyrosine kinase (RTK) fusion-positive mesenchymal neoplasms, including superficial ones described under the rubric of "superficial ALK-rearranged myxoid spindle cell neoplasm" (SAMS). Here, we describe 35 superficial tumors with SAMS morphology, which occurred in 18 females (51%) and 17 males at a median age at presentation of 39 years (range: 6 to 82y). Most tumors occurred on the lower extremity (25 tumors; 71%), followed by upper extremity (5; 14%), trunk (3; 9%), and face (2; 6%). Nine tumors were reported to have grown slowly before presentation, including >10 years in 2 cases. Tumors occurred primarily in the dermis (32 tumors; 91%) or subcutis (3; 9%); 8 dermal tumors extended into the subcutis. Median tumor size was 1.3cm (range: 0.5 to 8.0cm). Clinical follow-up was available for 12 patients (34%; range: 2mo to 21y; median: 2.7y), none of whom experienced metastasis. One incompletely resected tumor recurred locally at 19 months, and no other patients experienced recurrence. Histologically, tumors were characterized by bland spindle-to-ovoid cells showing whorled growth and myxoid-to-collagenous stroma. Recurrent features included an epidermal collarette (19/30; 63%), perivascular hyalinization (20/35; 57%), amianthoid collagen (14/35; 40%), and metaplastic ossification (2/35; 6%). Immunohistochemistry (IHC) demonstrated expression of ALK (24/31; 77%), CD34 (15/21; 71%), EMA (17/28; 61%), and S-100 (9/32; 28%). Eleven tumors showed hybrid morphologic features between EFH and SAMS; 9 of them (82%) showed cytomorphology typical of EFH but with whorled growth, myxoid stroma, and/or regions of spindle cell morphology. Two hybrid tumors showed sharp transitions between a region characteristic of EFH and a region characteristic of SAMS, with a concomitant sharp transition in EMA, CD34, and S-100 expression by IHC. Sequencing revealed ALK fusions in 15 of 19 tumors: 2 each with fusion partners FLNA, SQSTM1, and VCL, and 1 each with COL1A2, DCTN1, EML4, FXR1, MPRIP, PLEKHH2, PRKAR1A, SPECC1L, and TLN2. Thirteen of 14 ALK-rearranged tumors expressed ALK by IHC. Three tumors negative for ALK fusions instead harbored alternate RTK fusions (NCOA4::RET, TRIM27::RET, and VIM::NTRK3), and 1 tumor was negative for RTK alterations. CDKN2A/B deletions were found in 2 tumors with ALK fusions and both tumors with RET fusions. SAMS is on a morphologic and molecular genetic spectrum with EFH, with a similar body site distribution, frequent clinical presentation as an exophytic skin tumor, and invariably benign outcomes; we conclude that SAMS should be considered a histologic variant of EFH. Some morphologically typical examples harbor alternate RET and NTRK3 fusions, such that SAMS is not an appropriate designation for this morphologic class; instead, to highlight the clinicopathologic similarities to EFH, we propose the diagnostic term "myxoid spindle cell variant of epithelioid fibrous histiocytoma."

The effect of sodium deoxyribonucleate with iron complex on the expression of surface markers of MT-4 cells infected with human immunodeficiency virus type 1 (HIV-1) (Retroviridae: Primate lentivirus group)

The persistence of immune dysfunction during therapy has serious consequences for the health of HIV-infected people. Therefore, an important direction is the search for drugs that can reduce the inflammatory potential of the immune system and serve as an additional component of antiviral therapy. Aim ‒ to study the effect of the immunomodulatory drug Sodium deoxyribonucleate with iron complex (DNA-Na-Fe) on the expression of activation markers in MT-4 cells infected with HIV-1. Expression levels of CD4, CD28, CD38, CD62L and HLA-DR proteins on the plasma membrane were measured in cells. To assess viral activity, the p24 protein was quantified by ELISA. The two cell variants with different replicative activity were analyzed. Control cells, cells with DNA-Na-Fe, infected cells and infected cells with DNA-Na-Fe were tested. Based on the results obtained, it can be concluded that antiviral activity of the drug in MT-4 cells infected with HIV-1 is associated with immunomodulatory activity that enhances the expression of membrane proteins CD4, CD28, CD38 and CD62L. Diversity in the effect of DNA-Na-Fe on the studied surface proteins expression in two cell lines indicates that they depend on the characteristics of the combined molecular biological processes occurring in cells. And the increased effects observed in a system with changes in replicative activity assumes its active participation in virus replication at the stages of virus penetration and budding. Studies have shown that DNA-Na-Fe has antiviral and immunomodulatory activity.

Selective Elimination of Genetic Variants of Human Embryonic Stem Cells from High Vulnerability to Ferroptosis.

Selective Elimination of Genetic Variants of Human Embryonic Stem Cells from High Vulnerability to Ferroptosis.

Directed evolution of an orthogonal transcription engine for programmable gene expression in eukaryotes.

T7 RNA polymerase has enabled orthogonal control of gene expression and recombinant protein production across diverse prokaryotic host chassis organisms for decades. However, the absence of 5' methyl guanosine caps on T7 RNAP derived transcripts has severely limited its utility and widespread adoption in eukaryotic systems. To address this shortcoming, we evolved a fusion enzyme combining T7 RNAP with the single subunit capping enzyme from African swine fever virus using Saccharomyces cerevisiae. We isolated highly active variants of this fusion enzyme, which exhibited roughly two orders of magnitude higher protein expression compared to the wild-type enzyme. We demonstrate the programmable control of gene expression using T7 RNAP-based genetic circuits in yeast and validate enhanced performance of these engineered variants in mammalian cells. This study presents a robust, orthogonal gene regulatory system applicable across diverse eukaryotic hosts, enhancing the versatility and efficiency of synthetic biology applications.

Sacsin levels in PBMCs: A diagnostic assay for SACS variants in peripheral blood cells - A PROSPAX study.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a common recessive ataxia that is still underdiagnosed worldwide. An easily accessible diagnostic biomarker might help to diagnostically confirm patients presenting SACS variants of unknown significance (VUS) or atypical phenotypes. To detect sacsin in peripheral blood mononuclear cells (PBMCs) and to validate its diagnostic biomarker quality to discriminate biallelic SACS patients (including patients with VUS and/or atypical phenotypes) against healthy controls, non-ARSACS spastic ataxia patients, and heterozygous SACS carriers. Sacsin protein levels in PBMCs were assessed in patients versus controls and validated in skin-derived fibroblasts. Patients with biallelic SACS variants - including patients with VUS and/or atypical phenotypes - showed loss of sacsin in PBMCs, with discriminative performance against healthy, heterozygous, and non-ARSACS controls. This included all investigated SACS missense variants. Also, C-terminal variants escaping nonsense-mediated decay, while not differing from controls in expression level, showed lower molecular weight in this assay. Assessing sacsin levels using PBMCs offers an easy, peripherally accessible diagnostic biomarker for ARSACS, with PBMCs being much less invasive and easier to handle than fibroblasts. Additionally, this might be a potential target-engagement blood biomarker for sacsin-increasing therapies. © 2024 International Parkinson and Movement Disorder Society.

Abstract C084: Genetic polymorphism rs3820282 and WNT4-mediated metabolic plasticity contributes to differential therapy response in ovarian cancer clear carcinoma

Abstract Purpose of research: Ovarian clear cell carcinoma (OCCC) is a rare and notoriously chemoresistant gynecologic malignancy that is more likely to affect patients of Asian race. Unlike the more common tubo-ovarian high-grade serous carcinoma, OCCC is rising in incidence. Previous work highlights the increased risk of reproductive pathology associated with single nucleotide polymorphism (SNP) rs3820282, a genetic variant that is also overrepresented in people of Asian race. In particular, rs3820282 results in increased WNT4 expression and is associated with metabolic reprogramming and elevated phospho-AMPK as demonstrated in murine models and primary human tumors. We hypothesize the WNT4/rs3820282 regulatory axis in OCCC mediates the response to chemotherapy via metabolic plasticity. Methods: Using SNP-variant genotype (OVTOKO) and wild-type (TOV-21G) OCCC cell lines, we manipulated the expression of WNT4 via short interfering RNA knockdown and lentiviral overexpression. We evaluated the proliferation and survival of cells in nutrient deprivation conditions. We used Agilent Seahorse to evaluate metabolic outputs of oxidative phosphorylation in the presence of cytotoxic stress conditions (platinum chemotherapy [cisplatin]), antimetabolic agents [metformin and dimethyl malonate], lipid- and glucose-deprived media, and fatty acid oxidation and glycolysis inhibition). We used immunoblotting to measure total AMPK and phospho-AMPK as signals of cellular response to metabolic stress. To evaluate the effect of WNT4 on response to chemotherapy, we measured changes in the half-maximal inhibitory concentration (IC50) of cisplatin. Results: Knockdown and overexpression of WNT4 in variant and wild-type OCCC cells altered cellular proliferation and survival in nutrient deprivation conditions. Seahorse cellular energetics conducted in variant OCCC cells demonstrated that WNT4 knockdown mitigates the metabolic detriment of cytotoxic and metabolic stress conditions with the opposite trend observed in wild-type WNT4 overexpressing OCCC cells. The variant phenotype is consistent with models of high-grade serous carcinoma. Over five experiments, WNT4 knockdown reduced the IC50 of cisplatin in variant cells (mean log fold change -0.29, range -0.91 to 0.07). While WNT4 knockdown led to a modest increase in total AMPK protein expression, in lipid- and glucose-deprived conditions WNT4 loss significantly attenuated phospho-AMPK levels. Conclusions: Genetically variant OCCC cells exposed to metabolic and antineoplastic stressors demonstrate metabolic plasticity which is regulated by WNT4. Knockdown of WNT4 induces metabolic rigidity that may convey increased susceptibility to chemotherapy, as indicated by the WNT4-induced reduction in cisplatin IC50. The SNP is highly prevalent in people of Asian race, and further investigation of this axis of metabolic reprogramming represents an opportunity to further understand an established cancer disparity and improve chemotherapeutic response in ovarian clear cell carcinoma. Citation Format: Julia Dexter, Madeleine Shackleford, Sydney Robinson, Matthew Sikora, Benjamine Bitler. Genetic polymorphism rs3820282 and WNT4-mediated metabolic plasticity contributes to differential therapy response in ovarian cancer clear carcinoma [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C084.