Factor Activity Research Articles

Mitochondria and Alcohol-Associated Liver Disease: Pathogenic Role and Target for Therapy.

Alcohol-associated liver disease (ALD) is one of the leading causes of chronic liver disease and a major cause of liver-related death. ALD is a multifactorial disease triggered by the oxidative metabolism of alcohol which leads to the activation of multiple factors that promote the progression from steatosis to more advanced stages like alcohol-associated steatohepatitis (AH) that culminate in alcohol-associated cirrhosis and hepatocellular carcinoma. Poor understanding of the complex heterogeneous pathology of ALD has limited drug development for this disease. Alterations in mitochondrial performance are considered a crucial event in paving the progression of ALD due to the crucial role of mitochondria in energy production, intermediate metabolism, calcium homeostasis, and cell fate decisions. Therefore, understanding the role of mitochondria in eliciting steatosis and progression toward AH may open the door to new opportunities for treatment. In this review, we will cover the physiological function of mitochondria, its contribution to ALD in experimental models and human disease, and explore whether targeting mitochondria may represent a game changer in the treatment of ALD.

Hypoxia drives estrogen receptor β-mediated cell growth via transcription activation in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a highly malignant tumor with a poor prognosis. Hypoxia conditions affect multiple cellular processes promoting the adaptation and progression of cancer cells via the activation of hypoxia-inducible factors (HIF) and subsequent transcription activation of their target genes. Preliminary studies have suggested that estrogen receptor β (ERβ) might play a promoting role in the progression of NSCLC. However, the precise mechanisms, particularly its connection to HIF-1α-mediated modulation under hypoxia, remain unclear. Our findings demonstrated that the overexpression of ERβ, not ERα, increased cell proliferation and inhibition of apoptosis in NSCLC cells and xenografts. Tissue microarray staining revealed a strong correlation between the protein expression of HIF-1α and ERβ. HIF-1α induced ERβ gene transcription and protein expression in CoCl2-induced hypoxia, 1% O2 incubation, or HIF-1α overexpressing cells. ChIP identified HIF-1α binding to a hypoxia response element in the ESR2 promoter. The suppression of HIF-1α and ERβ both in vitro and in vivo effectively reduced the tumor growth, thus emphasizing the promising prospects of targeting HIF-1α and ERβ as a therapeutic approach for the treatment of NSCLC. KEY MESSAGES: ERβ, not ERα, increases cell proliferation and inhibition of apoptosis in NSCLC cells and xenografts. A strong correlation exists between the protein expression of HIF-1α and ERβ. HIF-1α induced ERβ gene transcription and protein expression in hypoxic cells via binding to HRE in the ESR2 promoter. The suppression of HIF-1α and ERβ both in vitro and in vivo effectively reduced the NSCLC tumor growth.

Activity Assessment of Factor Replacement Therapies to Guide Infusion Rate Protocols in Patients with Bleeding Disorders

Abstract Introduction The development of institutional protocols guiding administration of clotting factor replacements in patients with bleeding disorders requires an understanding of product stability over infusion duration. Inclusion of new factor products on formulary at our institution raised consideration for optimal administration routes, including whether a bolus intravenous push versus infusion over 4-12h provided acceptable factor activities. Here we assessed the activity of two clotting factor replacement therapies over a 24h period to guide clinical care protocols. Method This study was performed in the Special Coagulation Lab as a collaboration between pharmacy, hematology, and laboratory medicine. Two vials each of Novoeight, a recombinant antihemophilic factor used in patients with factor VIII deficiency, and Vonvendi, a recombinant von Willebrand Factor (VWF) replacement approved for prophylaxis in adults with Type 3 von Willebrand Disease, were reconstituted to clinically relevant potencies (13 IU/ml, Novoeight; 27 IU/ml, Vonvendi). Products were spiked separately into factor VIII-deficient plasma (Novoeight) or assay diluent (Vonvendi), given inability to obtain VWF-deficient plasma, and tested for activity at various timepoints. Novoeight samples were assessed by traditional one-stage FVIII assays, while Vonvendi samples were assessed for VWF antigen, ristocetin cofactor activity (RCA), and glycoprotein Ib binding activity (GP1bM). Results Novoeight samples maintained similar FVIII activities at all timepoints tested, with differences falling within the coefficient of variation of the assay. Specifically, the two samples had FVIII levels of 108% and 95% at baseline and levels of 98% and 91%, respectively, at 12h, which was the timepoint of greatest interest given plans to allow 12h infusion rates. For Vonvendi samples, measurement of VWF antigen remained fairly stable throughout the 24h period. However, VWF activity decreased in both RCA and GP1bM assays over time, including by 4h. RCA values were 193% and 190% at baseline, 113% and 95% at 4h, and 63% and 63% at 12h. GP1bM was 140% and 133% at baseline, 92% and 91% at 4h, and 56% and 70% at 12h. Together, these results supported implementation of protocols allowing for extended (up to 12h) infusion of Novoeight and short (3-5min) bolus infusion of Vonvendi. Conclusion Our study demonstrated maintained Novoeight activity over the study period, confirming infusion rates of 12h should be efficacious. Conversely, Vonvendi yielded significantly decreased activity levels in as little as 4h, indicating the need for faster infusion rates. Based on our results, current practices at our institution were adapted to include extended infusion for Novoeight only. Drug activity studies may not directly reflect patient outcomes, and additional studies of factor activities following product infusion into patients and clinical hemostasis correlates are necessary. Nevertheless, the clinical lab should continue to play a primary role in testing, monitoring, and developing patient care protocols for therapeutic drugs through multidisciplinary collaboration.

Comprehensive Transcriptomic Analysis Reveals Defense-Related Genes and Pathways of Rice Plants in Response to Fall Armyworm (Spodoptera frugiperda) Infestation.

Rice (Oryza sativa L.) serves as a substitute for bread and is a staple food for half of the world's population, but it is heavily affected by insect pests. The fall armyworm (Spodoptera frugiperda) is a highly destructive pest, threatening rice and other crops in tropical regions. Despite its significance, little is known about the molecular mechanisms underlying rice's response to fall armyworm infestation. In this study, we used transcriptome analysis to explore the global changes in gene expression in rice leaves during a 1 h and 12 h fall armyworm feeding. The results reveal 2695 and 6264 differentially expressed genes (DEGs) at 1 and 12 h post-infestation, respectively. Gene Ontology (GO) and KEGG enrichment analyses provide insights into biological processes and pathways affected by fall armyworm feeding. Key genes associated with hormone regulation, defense metabolic pathways, and antioxidant and detoxification processes were upregulated, suggesting the involvement of jasmonic acid (JA) signaling, salicylic acid biosynthesis pathways, auxin response, and heat shock proteins in defense during 1 h and 12 h after fall armyworm infestation. Similarly, key genes involved in transcriptional regulation and defense mechanisms reveal the activation of calmodulins, transcription factors (TFs), and genes related to secondary metabolite biosynthesis. Additionally, MYB, WRKY, and ethylene-responsive factors (ERFs) are identified as crucial TF families in rice's defense response. This study provides a comprehensive understanding of the molecular dynamics in rice responding to fall armyworm infestation, offering valuable insights for developing pest-resistant rice varieties and enhancing global food security. The identified genes and pathways provide an extensive array of genomic resources that can be used for further genetic investigation into rice herbivore resistance. This also suggests that rice plants may have evolved strategies against herbivorous insects. It also lays the groundwork for novel pest-resistance techniques for rice.

Modulating Microbial Resistance: Transcription Factor Regulation of Antibiotic Resistance in Escherichia coli

Abstract Introduction/Objective Antibiotic resistance is a global health emergency, with resistance detected to all antibiotics currently in clinical use and only a few novel drugs in the development pipeline. Antibiotics target key biological processes in bacteria, such as protein synthesis, cell wall synthesis, DNA replication and protein translation. Cells tightly control their internal states in different environments by regulating their transcriptional program through the activity of transcription factors (TFs) that control the expression of multiple downstream effector genes. We hypothesized that efficacy of specific antibiotics can be adjusted by altering the transcriptional states of bacteria prior to treatment, using TF knockout (KO) and over- expression (O-E) strains. Methods/Case Report Aim: Calculate the IC50 of 9 TF KO strains and 9 TF O-E strains across across a panel of representative antibiotics. A previous barcode screen study had identified TF KOs which were associated with antibiotic resistance/susceptibility. A serial antibiotic dilution was prepared in M9 media. E. coli TF KO and O-E strains were cultured for 24 hours in M9 media+Antibiotic (37°C, shaking). Each strain/antibiotic combination contained a technical replicate. Growth was measured by OD, allowing for calculation of growth curves, drug response curves, IC50, and EC50. Strain-specific IC50 and EC50 were compared to wild-type (WT) IC50 and EC50 as a measure of drug resistance/susceptibility. Results (if a Case Study enter NA) Experiments demonstrated consistent, TF-specific patterns of drug susceptibility. Compared to WT, TF KO strains demonstrated altered antibiotic IC50s (drug susceptibility). Compared to WT, TF O-E strains also demonstrated altered antibiotic IC50s (drug susceptibility). Conclusion Hypothesis Weakly Supported – TF states affect IC50s (Abx resistance). The relationships outlined in initial screen were consistently represented in experimental trials. Strain resistance/susceptibility is directly related to TF system and drug mechanism of action. For example, drugs that inhibited ribosomal function demonstrated greater effectiveness against KO/O-E strains that relied more heavily on protein synthesis pathways. Drug resistance is not binary but is shaped by a number of internal cellular factors, including gene regulation by TFs.

A Closer Look at the Newer GP1bM Assay Sheds Light on Unusual Findings

Abstract Von Willibrand disease (VWD) is the most common inherited bleeding disorder. The 2021 Guidelines on VWD diagnosis recommend newer assays measuring the platelet-binding activity of von Willebrand factor (VWF), such as VWF: GPIbM and VWF: GPIbR, over the traditional ristocetin cofactor assay (VWF: RCo). Among these newer assays, the GP1bM INNOVANCE VWF Ac assay obtained de novo FDA approval in October 2022. Its ristocetin-independent activity distinguishes it from other assays. Our laboratory conducted a comprehensive validation on the Stago StaR-Max platform, utilizing Siemens Standard Plasma for calibration. The VWF activity measured by the GP1bM assay was compared with the existing VWF: Ab assay (IL, HemosIL® von Willebrand Factor Activity). The validation studies revealed a good correlation between the two assays. Since implementing the GP1bM assay in October 2023, our attention has been drawn to at least four cases exhibiting unexpectedly high activity levels compared to VWF antigen levels. On average, the activity-to-antigen ratio was 1.9. Notably, all these cases showed the presence of all multimers (low, intermediate, and high). Despite extensive investigation, encompassing considerations like heterophile antibody interference using scantibodies, retesting with different lot numbers of Innovance VWF Ac reagent, and rerunning tests at an outside laboratory, the cause of this anomaly remains elusive. A notable variable lies in the platform disparity between Siemens and Stago. Given the recent introduction of this assay, it has yet to gain widespread adoption in laboratories, resulting in a limited understanding of its practical variations. While recent studies affirm the overall superior performance of newer assays compared to the ristocetin cofactor assay, intricate details of each assay remain less explored. The novelty of the GP1bM assay and its limited usage in laboratories underscore the potential emergence of stable outliers as it becomes more widely adopted. The peculiar cases in our study pose an enigma as to why the reported activity is approximately double the antigen level. As laboratories increasingly embrace and integrate the GP1bM assay, a more complete understanding of its potential variations is expected to unfold. This may elucidate the mysteries surrounding these peculiar cases and ensure the assay’s robust interpretation in clinical settings.

OsPUB75-OsHDA716 mediates deactivation and degradation of OsbZIP46 to negatively regulate drought tolerance in rice.

Histone deacetylases (HDACs) play crucial roles in plant stress responses via modification of histone as well as non-histone proteins; however, how HDAC-mediated deacetylation of non-histone substrates affects protein functions remains elusive. Here, we report that the Reduced Potassium Dependency3/Histone Deacetylase1 (RPD3/HDA1)-type histone deacetylase OsHDA716 and plant U-box (PUB) E3 ubiquitin ligase OsPUB75 form a complex to regulate rice drought response via deactivation and degradation of basic leucine zipper (bZIP) transcription factor OsbZIP46 in rice (Oryza sativa). OsHDA716 decreases ABA-induced drought tolerance, and mechanistic investigations showed that OsHDA716 interacts with and deacetylates OsbZIP46, a key regulator in ABA signaling and drought response, thus inhibiting its transcriptional activity. Furthermore, OsHDA716 recruits OsPUB75 to facilitate ubiquitination and degradation of deacetylated OsbZIP46. Therefore, the OsPUB75-OsHDA716 complex exerts double restrictions on the transcriptional activity and protein stability of OsbZIP46, leading to repression of downstream drought-responsive gene expression and consequently resulting in reduced drought tolerance. Conversely, OsbZIP46 acts as an upstream repressor to repress OsHDA716 expression, and therefore OsHDA716 and OsbZIP46 form an antagonistic pair to reciprocally inhibit each other. Genetic evidence showed that OsHDA716 works with OsbZIP46 in a common pathway to antagonistically regulate rice drought response, revealing that plants can fine-tune stress responses by the complex interplay between chromatin regulators and transcription factors. Our findings unveil an acetylation-dependent regulatory mechanism governing protein functions and shed light on the precise coordination of activity and stability of key transcription factors through a combination of different post-translational modifications.

WS6 and 5-iodotubercidin small molecules and growth factors; TGF, HGF, and EGF synergistically enhance proliferation of β-like human induced pluripotent stem cells (iPSCs)

Abstract Objectives It has been shown that growth factors and small molecules play an essential role in the proliferation of β cells and insulin production. In this study, we investigated the effects of small molecules (WS6 and 5-iodotubercidin) and growth factors (TGFβ, HGF, and EGF) on the proliferation of β-like human ipSCs. Methods iPSCs derived β cells were treated with small molecules and growth factors. Cytotoxic activity of small molecules and growth factors was determined using MTT assay. Insulin gene expression and secretion were measured by qPCR and ELISA, respectively. The protein expression of insulin was evaluated by western blot as well. Results Simltananeous addition of WS6 and Harmine into the culture media increased insulin gene expression compared to treatment by each molecule alone (p < 0.05). It was found that the simultaneous recruitment of EGH, HGF, and TGF-β increased insulin expression compared to treatment by each molecule alone (p < 0.05). Results showed that EGF, HGF, TGF-β growth factors increased insulin gene expression, eventually leading to insulin secretion from β cells (p < 0.05). Conclusions Growth factors and small molecules synergistically enhanced the proliferation of β cells and insulin production.

LABORATORY DIAGNOSIS OF VON WILLEBRAND DISEASE

The aim of the study. To determine the frequency of Willebrand disease (VWD) in patients which turned to the SI IBPTM of the NAMS of Ukraine with hemorrhagic manifestations. Research metods. Object and research methods. The object of the study were 298 people with hemorrhagic syndrome aged from 7 months to 66 years old who have turned to the specialists of the institution since 01.01.2021, of which 198 (66,4%) came in for the first time. Coagulation hemostasis was investigated by methods that meet international standards and recommendations. Ristocetin-cofactor activity of factor Willebrand (VWF: RCo), content of the antigen VWF (VWF: AG), and coagulant activity of factor VIII (FVIII: C) were performed for the diagnosis of VWD. Results and Conclusions. VWD were found in 29 patients (9,7%) of the 298 people with hemorrhagic syndrome. VWD was revealed in 7,6% of patients from the 198 patients who were first investigate. The 15 (51,7%) VWD patients was diagnosed for the first time. In 48 (24,2%) was first diagnosed with hereditary or caused by various factors of disaggregational thrombocytopathy. Von Willebrand disease is the most common type of congenital hemostasis disorders (0,6-1,3% of the population), the type of inheritance is autosomal recessive/dominant. An algorithmic approach should be applied to diagnosis. Laboratory diagnosis consists of screening coagulological tests and three levels special tests. To establish the diagnosis, the necessary tests are determined by VWF: RCo, VWF: Ag and FVIII: C.

Sex-specific and developmental effects of early life adversity on stress reactivity are rescued by postnatal knockdown of 5-HT1A autoreceptors.

Early Life Adversity (ELA) predisposes to stress hypersensitivity in adulthood, but neurobiological mechanisms that protect from the enduring effects of ELA are poorly understood. Serotonin 1A (5HT1A) autoreceptors in the raphé nuclei regulate adult stress vulnerability, but whether 5HT1A could be targeted to prevent ELA effects on susceptibility to future stressors is unknown. Here, we exposed mice with postnatal knockdown of 5HT1A autoreceptors to the limited bedding and nesting model of ELA from postnatal day (P)3-10 and tested behavioral, neuroendocrine, neurogenic, and neuroinflammatory responses to an acute swim stress in male and female mice in adolescence (P35) and in adulthood (P56). In females, ELA decreased raphé 5HT neuron activity in adulthood and increased passive coping with the acute swim stress, corticosterone levels, neuronal activity, and corticotropin-releasing factor (CRF) levels in the paraventricular nucleus (PVN) of the hypothalamus. ELA also reduced neurogenesis in the ventral dentate gyrus (vDG) of the hippocampus, an important mediator of individual differences in stress susceptibility, and increased microglia activation in the PVN and vDG. These effects of ELA were specific to females and manifested predominantly in adulthood, but not earlier on in adolescence. Postnatal knockdown of 5HT1A autoreceptors prevented these effects of ELA on 5HT neuron activity, stress reactivity, neurogenesis, and neuroinflammation in adult female mice. Our findings demonstrate that ELA induces long-lasting and sex-specific impairments in the serotonin system, stress reactivity, and vDG function, and identify 5HT1A autoreceptors as potential targets to prevent these enduring effects of ELA.

Shade-induced ROS/NO reinforce COP1-mediated diffuse cell growth

Canopy shade enhances the activity of PHYTOCHROME INTERACTING FACTORs (PIFs) to boost auxin synthesis in the cotyledons. Auxin, together with local PIFs and their positive regulator CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), promotes hypocotyl growth to facilitate access to light. Whether shade alters the cellular redox status thereby affecting growth responses, remains unexplored. Here, we show that, under shade, high auxin levels increased reactive oxygen species and nitric oxide accumulation in the hypocotyl of Arabidopsis. This nitroxidative environment favored the promotion of hypocotyl growth by COP1 under shade. We demonstrate that COP1 is S-nitrosylated, particularly under shade. Impairing this redox regulation enhanced COP1 degradation by the proteasome and diminished the capacity of COP1 to interact with target proteins and to promote hypocotyl growth. Disabling this regulation also generated transversal asymmetries in hypocotyl growth, indicating poor coordination among different cells, which resulted in random hypocotyl bending and predictably low ability to compete with neighbors. These findings highlight the significance of redox signaling in the control of diffuse growth during shade avoidance.

Identification of oxylipins and lipid mediators in pulmonary embolism

BackgroundThis study aimed to investigate the role of oxylipins and lipid mediators in Pulmonary Embolism (PE), a serious cardiovascular condition associated with high morbidity and mortality rates.MethodsA total of 6,365 hospitalized patients with thrombosis and 200 healthy individuals were recruited as the control group from 2015 to 2023. Thrombus type, coagulation, and lipid-related parameters were statistically analysed. Additionally, lipidomic characteristics of serum samples from the PE and control groups were examined via LC-MS/MS for the first time.ResultsAmong the 6,365 hospitalized patients with thrombosis, 72.1% (4,587/6,365) had venous thromboembolism (VTE). Within the VTE group, the incidence of PE was 12.1% (555/4,587). In comparison to the healthy control (HC) group, the PE group exhibited significant elevations in coagulation-related parameters, such as factor VIII (F VIII) and von Willebrand factor (vWF) activities, while antithrombin III (AT III) and factor XII (F XII) activities were notably reduced. Lipid-related parameters, including serum cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein A (apoA), were significant reductions in PE patients (P < 0.0001), with areas under the curve (AUCs) exceeding 0.9. LC-MS/MS analysis of serum samples revealed 118 oxidized lipid metabolites. Compared to the HC group, the PE group exhibited 10 upregulated oxidized lipid metabolites, with the most significant difference observed in 20-hydroxyPGF2α derived from arachidonic acid (ARA). The study identified upregulated oxidized lipid metabolites primarily linked to the ARA metabolism signalling pathway.ConclusionThis research indicates a notable correlation between lipid metabolism and the occurrence and development of PE. Specifically, upregulation of the arachidonic acid metabolism signalling pathway may be an important pathogenic factor for PE, and 20-hydroxyPGF2α derived from ARA has potential as a biomarker for PE disease.

Seasonal influence on miRNA expression dynamics of extracellular vesicles in equine follicular fluid.

Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting follicle development, ovulation, and oocyte quality. Cells in the follicles release tiny particles called extracellular vesicles (EVs) containing vital regulatory molecules, such as microRNAs (miRNAs). These miRNAs are pivotal in facilitating communication within the follicles through diverse signaling and information transfer forms. EV-coupled miRNA signaling is implicated to be associated with ovarian function, follicle and oocyte growth and response to various environmental insults. Herein, we investigated how seasonal variations directly influence the ovulatory and anovulatory states of ovarian follicles and how are they associated with follicular fluid EV-coupled miRNA dynamics in horses. Ultrasonographic monitoring and follicular fluid aspiration of preovulatory follicles in horses during the anovulatory (spring: non-breeding) and ovulatory (spring, summer, and fall: breeding) seasons and subsequent EV isolation and miRNA profiling identified significant variation in EV-miRNA cargo content. We identified 97 miRNAs with differential expression among the groups and specific clusters of miRNAs involved in the spring transition (miR-149, -200b, -206, -221, -328, and -615) and peak breeding period (including miR-143, -192, -451, -302b, -100, and let-7c). Bioinformatic analyses showed enrichments in various biological functions, e.g., transcription factor activity, transcription and transcription regulation, nucleic acid binding, sequence-specific DNA binding, p53 signaling, and post-translational modifications. Cluster analyses revealed distinct sets of significantly up- and down-regulated miRNAs associated with spring anovulatory (Cluster 1) and summer ovulation-the peak breeding season (Clusters 4 and 6). The findings from the current study shed light on the dynamics of FF-EV-coupled miRNAs in relation to equine ovulatory and anovulatory seasons, and their roles in understanding the mechanisms involved in seasonal shifts and ovulation during the breeding season warrant further investigation.

Retinol-binding protein 4 is a potential biomarker of changes in lean mass in postmenopausal women.

Identifying biomarkers can help in the early detection of muscle loss and drive the development of new therapies. Research suggests a potential link between retinol-binding protein 4 (RBP4) and muscle mass, particularly in postmenopausal women. This study aimed to examine the association between baseline RBP4 levels and changes in appendicular lean mass (ALM), an indicator of muscle mass, in postmenopausal women. A 12-month follow-up period (n=153) included baseline and 12-month ALM assessments using DXA. ALM was normalized to squared height (ALMI). Baseline evaluations encompassed insulin resistance via HOMA-IR and immunoassay magnetic bead panel measurements of RPB4, IL-6, TNF-α, and IL-10. Postmenopausal women were categorized into higher (n=77) and lower (n=76) RPB4 groups based on baseline RPB4 values. Their changes in ALMI were compared using Mann-Whitney tests. General linear model was employed to evaluate the predictive power of baseline RBP4 for ALMI changes, adjusting for confounding variables: age, physical activity, smoking status, body fat, HOMA-IR, inflammatory markers (TNF-α and IL-6), and anti-inflammatory factor (IL-10). The higher RBP4 group exhibited a more pronounced reduction in ALMI compared to the lower RBP4 group (Higher RBP4 = -0.39kg/m2, 95% CI: -0.48 to -0.31kg/m2vs. Lower RBP4 = -0.24kg/m2, 95% CI: -0.32 to -0.15kg/m2, P=0.011). After adjusting for confounding factors, the association between baseline RBP4 changes and ALMI remained (b = -0.008, SE=0.002, P<0.001), indicating higher baseline RBP4 values linked to greater ALMI reduction. Our findings support RBP4 as a potential biomarker for changes in muscle mass in postmenopausal women.

UV-induced reactive oxygen species and transcriptional control of 3-deoxyanthocyanidin biosynthesis in black sorghum pericarp.

Black pericarp sorghum has notable value due to the biosynthesis of 3-deoxyanthocyanidins (3-DOAs), a rare class of bioactive polyphenols valued as antioxidant food additives and as bioactive compounds with cytotoxicity to human cancer cells. A metabolic and transcriptomic study was conducted to ascertain the cellular events leading to the activation of 3-DOA biosynthesis in black sorghum pericarp. Prolonged exposure of pericarp during grain maturation to high-fluence ultraviolet (UV) light resulted in elevated levels of reactive oxygen species (ROS) and the activation of 3-DOA biosynthesis in pericarp tissues. In conjunction with 3-DOA biosynthesis was the transcriptional activation of specific family members of early and late flavonoid biosynthesis pathway genes as well as the downstream activation of defense-related pathways. Promoter analysis of genes highly correlated with 3-DOA biosynthesis in black pericarp were enriched in MYB and HHO5/ARR-B motifs. Light microscopy studies of black pericarp tissues suggest that 3-DOAs are predominantly localized in the epicarp and are associated with the cell wall. A working model of UV-induced 3-DOA biosynthesis in black pericarp is proposed that shares features of plant immunity associated with pathogen attack or mechanical wounding. The present model depicts ROS accumulation, the transcriptional activation of receptor kinases and transcription factors (TFs) including NAC, WRKY, bHLH, AP2, and C2H2 Zinc finger domain. This study identified key biosynthetic and regulatory genes of 3-DOA accumulation in black pericarp and provided a deeper understanding of the gene networks and cellular events controlling this tissue-and genotype-specific trait.

Medical Rehabilitation and Health Resort Treatment at the Resort “Sernovodsk-Kavkazsky”

INTRODUCTION. Medical rehabilitation and health resort treatment are important parts of the health care system of the Chechen Republic, which are actively developing with high potential and competitiveness. Studies on the treatment of patients with chronic non-communicable diseases show the effectiveness of natural therapeutic resources and other innovative non-medication methods. AIM. To analyze and identify the most promising for the development of the Resort “Sernovodsk-Kavkazsky” directions of treatment of patients with chronic non-infectious diseases. MATERIALS AND METHODS. Data from information-analytical systems of the sanatorium-resort industry of the Russian Federation and the data of our own research were used for the analysis. RESULTS. The analysis and the main results confirming the effectiveness of treatment of patients with chronic non-infectious diseases with the use of natural healing resources and other innovative non-medication methods realized in the Resort “Sernovodsk-Kavkazsky” are presented. The most promising ways of development of health resort treatment and medical rehabilitation are determined. CONCLUSION. The increasing activity of unfavorable environmental factors, which negatively affect the functional state of the organism and the level of health of the population as a whole, require from the health care system continuous improvement, including in the sanatorium and resort industry.

8141 A Rare Case of Follicular Adenoma with Osseous Metaplasia and Mature Bone Formation- Is Surveillance Important?

Abstract Disclosure: K. Madani: None. Y. Al-Khazraji: None. D.H. Sacoto: None. G.K. Rai: None. R. Belokovskaya: None. A.A. Franco-Akel, MD: None. Osseous metaplasia and mature bone formation (MBF) are common in malignant thyroid and parathyroid tumors. It is very unusual to have MBF in a benign tumor-like follicular adenoma, as in the case we describe. We found only twelve such cases reported in the literature, and none addressed the cancer risk or need for surveillance for the development of potential malignancy. Thus, further studies are required to investigate the need for surveillance in patients not undergoing excision and in those with remnant thyroid tissue post-removal of the adenoma for recurrence. A 43-year-old female with history of hyperlipidemia and hypothyroidism had an incidental finding of midline neck mass during the evaluation of a left upper extremity lipoma. Ultrasound of the neck showed two nodules in the Left (L) thyroid lobe: a complex nodule measured 2.2 x 1.2 x 2.1 cm and a calcified nodule measuring 1.5 x 0.8 x 0.6 cm. MRI of the neck revealed a 1.9 cm heterogeneously enhancing nodule in the mid to lower pole of the L thyroid lobe. The patient had symptoms suggestive of compression, such as dysphagia, odynophagia, and hoarseness; thus, left hemothyroidectomy was performed. Tissue biopsy was notable for follicular adenoma with osseous metaplasia and mature bone formation. The patient remained clinically and biochemically euthyroid, with only mildly elevated thyrotropin level, and treatment with levothyroxine 25 mcg once daily was initiated. On post-surgical follow-up, there was a lack of data to support close monitoring modality and strategy for the remnant thyroid lobe and neck nodal compartments. We concluded with the patient to follow up in a clinical visit for a physical exam of the neck and to obtain a follow-up sonogram at an interval of six months, in addition to monitoring TSH levels to guide hypothyroidism therapy. Osseous metaplasia has not been established as a premalignant condition, but in certain tissues like the esophagus, metaplasia is a risk factor for malignant conversion, for instance, in Barrett’s esophagus leading to esophageal adenocarcinoma. Moreover, MBF in thyroid tissue has been attributed to the activity of basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2), which are linked with mitogenesis and tumorigenesis. Thus, the question arises if post-excision surveillance is required in patients with a history of osseous metaplasia in remnant thyroid tissue post-lobectomy or partial thyroidectomy. At this point, more epidemiological data on similar cases may help us understand the true risk of this clinical scenario, in order to develop a reasonable approach for surveillance. Presentation: 6/3/2024

8385 Coexistence of Parathyroid Adenoma &amp; Papillary Thyroid Cancer: A Rare Case Report

Abstract Disclosure: K. Dash: None. S. Verma: None. U. Ayyagari: None. M. Roy: None. S. Rathore: None. Prevalence of primary hyperparathyroidism (pHPT) ranges from 0.1- 0.4% in general population. Nodular thyroid disease is an increasingly common incidental diagnosis due to widespread use of ultrasound. Papillary thyroid cancer (PTC) is the most common cancer of thyroid gland. Here we report a 53 year old post-menopausal woman presented with weight loss, generalised body aches, arthralgia, myalgia, and proximal muscle weakness. She was hypertensive and clinically euthyroid. Physical examination revealed generalized tenderness all over the body but no thyroid or parathyroid nodules on palpation. Biochemical evaluation showed hypercalcemia 12.1mg/dl (8.4-10.2mg/dl), PTH&amp;gt;2000pg/ml (14-72pg/ml), ALP-1855 U/L (40–125 U/L), decreased phosphorous of 2.4mg/dl (25-4.6 mg/dl), and vitamin-D of 46 nmol/L ( 75-250 nmol/L). USG neck revealed a heterogeneous lesion at the lower pole of right lobe of thyroid gland &amp; two small hypoechoic nodules seen in the right lobe of thyroid gland. FNAC from nodules was inconclusive. USG abdomen revealed nephrocalcinosis. DEXA scan showed severe osteoporosis. Tc99mSestamibi SPECT-CT showed uptake at lower pole of right lobe of thyroid. On exploration, two nodules were palpated in the right lobe of the thyroid and simultaneous right hemi-thyroidectomy was performed. Histopathologic report confirmed parathyroid adenoma (figure 1) as well as presence of carcinoma in the two nodules: one was a papillary thyroid cancer (Classic variant) and the other a follicular variant of papillary thyroid carcinoma (figure 2). Margins were negative for malignancy. She was treated with levothyroxine to maintain TSH &amp;lt;0.1U/L and followed up with annual ultrasound screening. At 2 year follow up, patient was symptom free with improved T score on DEXA scan and no nodule over residual thyroid gland. Discussion: Co-existence of pHPT and PTC ranges from 2.3-4.3%. The use of ultrasound neck as a routine imaging modality for localisation of parathyroid adenoma allows detection of thyroid nodules. Benign and/or malignant thyroid nodules are commonly seen in patients with pHPT and this can range from 2-15%. Women have higher risk for co-existence. The relationship between the two diseases is thought to be incidental. Some authors have suggested possible role of shared genes, embryological factors, transcription factors, vitamin-D deficiency and hyperparathyroidism that could lead to activation of angiogenic factors and carcinogenesis. Fig. 1. Parathyroid adenoma. (a)Tc99m sestamibi scan showing right inferior parathyroid adenoma (b) Parathyroid adenoma (400x high power) comprising predominantly of Chief cells with mild pleomorphism. Fig. 2. Histopathology of PTC. (a)Papillary Carcinoma thyroid. 10xPhotomicrograph showing areas of calcification. (b) Papillary Carcinoma thyroid. Photomicrograph showing thyroid with papillary pattern with areas of infiltration. Presentation: 6/1/2024

7615 Genomic Consequences of GRHL2 Overexpression in ER+ Breast Cancer Cells

Abstract Disclosure: K.O. Allen: None. C. Zheng: None. N. Solodin: None. M.S. Ozers: Owner/Co-Owner; Self; Proteovista, LLC. C.L. Warren: None. P.E. Messa: None. D.T. Vogt: None. E.T. Alarid: None. Breast cancer progression is characterized by the presence and prevalence of metastatic disease. At a genomic level, metastasis can be facilitated via rearrangement of the chromatin landscape to expose oncogenic genes for transcription. Grainyhead like protein 2 (GRHL2) is a nuclear transcription factor implicated in epithelial cell differentiation. The GRHL2 motif has been found near activated estrogen receptor (ER) binding sites, and elevated levels of GRHL2 have been linked to worse prognosis in hormone receptor positive breast cancer patients. In its capacity to contribute to a more severe disease state when overexpressed, GRHL2 may function as either a transcription or pioneer factor to aid in the expression of metastasis-associated genes. Our lab previously showed that GRHL2 genomic binding precedes ER binding at co-regulated binding sites. Also, overexpression of GRHL2 in MCF7 cells for 24 hours did not appreciably alter gene expression by RNA-Seq. These data taken together indicate that GRHL2 may be exhibiting pioneer factor activity. However, the specific role of GRHL2 overexpression in hormone positive breast cancer progression remains unknown. In order to examine the mechanism of GRHL2 action, GRHL2 genomic binding was investigated using a tetracycline-inducible MCF7 model that allows controlled overexpression of GRHL2. ChIP studies were performed to determine GRHL2 binding intensity at specific and genome wide GRHL2 binding sites. Specific focus was given to established GRHL2 binding sites near oncogenic genes responsible for epithelial to mesenchymal transition (EMT), dormancy, and proto-oncogenic behavior. A similar analysis examined H3K27 acetylation to validate active gene transcription. An orthogonal technology, Specificity and Affinity for Protein (SNAP) microarrays, which characterizes direct GRHL2 binding to tiled genomic regions identified in ChIP-Seq analyses from multiple labs, was utilized to discriminate direct and indirect GRHL2 binding sites. The requirement for GRHL2 transactivation function in regulation of EMT related genes was further interrogated using transactivation GRHL2 dominant negative mutants. This work aims to distinguish the transcriptional and non-transcriptional activities of GRHL2 in breast cancer cells on a genome wide level and thereby, provide a deeper understanding of how overexpressed GRHL2 contributes to hormone positive breast cancer. Presentation: 6/3/2024

A20 intrinsically influences human effector T-cell survival and function by regulating both NF-κB and JNK signaling.

A20 is a dual-function ubiquitin-editing enzyme that maintains immune homeostasis by restraining inflammation. Although A20 serves a similar negative feedback function for T-cell receptor (TCR) signaling, the molecular mechanisms utilized and their ultimate impact on human T-cell function remain unclear. TCR engagement triggers the assembly of the CARD11-BCL10-MALT1 (CBM) protein complex, a signaling platform that governs the activation of downstream transcription factors including NF-κB and c-Jun/AP-1. Utilizing WT and A20 knockout Jurkat T cells, we found that A20 is required to negatively regulate NF-κB and JNK. Utilizing a novel set of A20 mutants in NF-κB and AP-1-driven reporter systems, we discovered the ZnF7 domain is crucial for negative regulatory capacity, while deubiquitinase activity is dispensable. Successful inactivation of A20 in human primary effector T cells congruently conferred sustained NF-κB and JNK signaling, including enhanced upregulation of activation markers, and increased secretion of several cytokines including IL-9. Finally, loss of A20 in primary human T cells resulted in decreased sensitivity to restimulation-induced cell death and increased sensitivity to cytokine withdrawal-induced death. These findings demonstrate the importance of A20 in maintaining T-cell homeostasis via negative regulation of both NF-κB and JNK signaling.