Defect In Synthesis Research Articles

Anti-Müllerian hormone, testicular descent and cryptorchidism.

Anti-Müllerian hormone (AMH) is a Sertoli cell-secreted glycoprotein involved in male fetal sex differentiation: it provokes the regression of Müllerian ducts, which otherwise give rise to the Fallopian tubes, the uterus and the upper part of the vagina. In the first trimester of fetal life, AMH is expressed independently of gonadotropins, whereas from the second trimester onwards AMH testicular production is stimulated by FSH and oestrogens; at puberty, AMH expression is inhibited by androgens. AMH has also been suggested to participate in testicular descent during fetal life, but its role remains unclear. Serum AMH is a well-recognized biomarker of testicular function from birth to the first stages of puberty. Especially in boys with nonpalpable gonads, serum AMH is the most useful marker of the existence of testicular tissue. In boys with cryptorchidism, serum AMH levels reflect the mass of functional Sertoli cells: they are lower in patients with bilateral than in those with unilateral cryptorchidism. Interestingly, serum AMH increases after testis relocation to the scrotum, suggesting that the ectopic position result in testicular dysfunction, which may be at least partially reversible. In boys with cryptorchidism associated with micropenis, low AMH and FSH are indicative of central hypogonadism, and serum AMH is a good marker of effective FSH treatment. In patients with cryptorchidism in the context of disorders of sex development, low serum AMH is suggestive of gonadal dysgenesis, whereas normal or high AMH is found in patients with isolated androgen synthesis defects or with androgen insensitivity. In syndromic disorders, assessment of serum AMH has shown that Sertoli cell function is preserved in boys with Klinefelter syndrome until mid-puberty, while it is affected in patients with Noonan, Prader-Willi or Down syndromes.

Serum visfatin level in β-thalassemia and its correlation with disease severity.

Thalassemia is a group of genetic hematological conditions characterized by the defective synthesis of one or more hemoglobin chains. This genetic anomaly alters globin chain balance, causing hemolysis, ineffective erythropoiesis, and chronic inflammatory diseases. The proinflammatory adipocytokine visfatin is predominantly produced in visceral adipose tissue. Its evaluation in individuals with thalassemia may provide valuable insights into the assessment of disease severity. The aim of this study was to investigate the potential role of visfatin in the development of β-thalassemia and its association with the severity of the illness. The study included 40 patients with β-thalassemia and ten healthy individuals matched by age and sex. Serum visfatin level was measured using ELISA. We found that individuals with β-thalassemia major had significantly higher levels of serum visfatin than those with β-thalassemia minor and the control group (P < 0.001). A receiver operating characteristic curve revealed that serum visfatin levels were different in the three groups. Our results suggest that the serum level of visfatin is significantly correlated with the severity of β-thalassemia.

Vcp overexpression and leucine supplementation extend lifespan and ameliorate neuromuscular junction phenotypes of a SOD1G93A-ALS mouse model.

Many genes with distinct molecular functions have been linked to genetically heterogeneous amyotrophic lateral sclerosis (ALS), including SuperOxide Dismutase 1 (SOD1) and Valosin-Containing Protein (VCP). SOD1 converts superoxide to oxygen and hydrogen peroxide. VCP acts as a chaperon to regulate protein degradation and synthesis and various other cellular responses. Although the functions of these two genes differ, in the current report we show that overexpression of wild-type VCP in mice enhances lifespan and maintains the size of neuromuscular junctions (NMJs) of both male and female SOD1G93A mice, a well-known ALS mouse model. Although VCP exerts multiple functions, its regulation of ER formation and consequent protein synthesis has been shown to play the most important role in controlling dendritic spine formation and social and memory behaviors. Given that SOD1 mutation results in protein accumulation and aggregation, it may direct VCP to the protein degradation pathway, thereby impairing protein synthesis. Since we previously showed that the protein synthesis defects caused by Vcp deficiency can be improved by leucine supplementation, to confirm the role of the VCP-protein synthesis pathway in SOD1-linked ALS, we applied leucine supplementation to SOD1G93A mice and, similar to Vcp overexpression, we found that it extends SOD1G93A mouse lifespan. In addition, the phenotypes of reduced muscle strength and fewer NMJs of SOD1G93A mice are also improved by leucine supplementation. These results support the existence of crosstalk between SOD1 and VCP and suggest a critical role for protein synthesis in ASL. Our study also implies a potential therapeutic treatment for ALS.

Update on cirrhotic cardiomyopathy: from etiopathogenesis to treatment.

Cirrhotic cardiomyopathy represents a syndrome of cardiac dysfunction associated with advanced liver disease. It is the result of complex pathophysiological processes that complicate the course of the disease, and is generally associated with a poor prognosis. Pathophysiologically, portal hypertension is the key factor leading to hyperdynamic circulation, via over-activation of the neurohumoral axis. Intestinal obstruction, subclinical inflammation and hepatocellular insufficiency, with defective synthesis or metabolism of several vasoactive mediators, are essential components of this process. Since it is usually unapparent at rest and only unmasked by an inadequate cardiac response to hemodynamic stress, the diagnosis of cirrhotic cardiomyopathy is challenging and demands a multimodal approach. There is currently no specific therapy, but there are prognostically effective drugs available to treat heart failure. Therefore, it is crucial to identify patients with chronic liver disease and heart failure in order to ameliorate their outcome. This article attempts to highlight the most important aspects of cirrhotic cardiomyopathy and draws attention to this condition.

Bilateral dead bag syndrome in skeletal dysplasia: A case report and review of literature

Abstract: Posterior chamber intraocular lens (IOL) dislocation is a rare yet grave complication of cataract surgery. Late in-the-bag IOL dislocation is usually spontaneous and occurs due to progressive zonular weakness. Aging, high myopia, pseudoexfoliation, vitrectomized eye, and connective tissue disorder are the predisposing factors. A floppy capsular bag without any proliferative deposits has been termed a “dead bag.” Bilateral in-the-bag IOL dislocation or dead bag syndrome is very infrequent. We present such a case in a 67-year-old male with skeletal dysplasia. The patient had undergone uneventful cataract surgery in both eyes 11 years and 6 years ago respectively. Skeletal dysplasia may cause zonulopathy and associated defective collagen synthesis can affect the anchorage points of the zonules. In these patients, cataract surgery should be accomplished by experienced surgeons after detailed counseling of such complications.

Morc2a variants cause hydroxyl radical-mediated neuropathy and are rescued by restoring GHKL ATPase.

Mutations in the Microrchidia CW-type zinc finger 2 (MORC2) GHKL ATPase module cause a broad range of neuropathies, such as Charcot-Marie-Tooth disease type 2Z; however, the aetiology and therapeutic strategy are not fully understood. Previously, we reported that the Morc2a p.S87L mouse model exhibited neuropathy and muscular dysfunction through DNA damage accumulation. In the present study, we analysed the gene expression of Morc2a p.S87L mice and designated the primary causing factor. We investigated the pathological pathway using Morc2a p.S87L mouse embryonic fibroblasts and human fibroblasts harbouring MORC2 p.R252W. We subsequently assessed the therapeutic effect of gene therapy administered to Morc2a p.S87L mice. This study revealed that Morc2a p.S87L causes a protein synthesis defect, resulting in the loss of function of Morc2a and high cellular apoptosis induced by high hydroxyl radical levels. We considered the Morc2a GHKL ATPase domain as a therapeutic target because it simultaneously complements hydroxyl radical scavenging and ATPase activity. We used the adeno-associated virus (AAV)-PHP.eB serotype, which has a high CNS transduction efficiency, to express Morc2a or Morc2a GHKL ATPase domain protein in vivo. Notably, AAV gene therapy ameliorated neuropathy and muscular dysfunction with a single treatment. Loss-of-function characteristics due to protein synthesis defects in Morc2a p.S87L were also noted in human MORC2 p.S87L or p.R252W variants, indicating the correlation between mouse and human pathogenesis. In summary, CMT2Z is known as an incurable genetic disorder, but the present study demonstrated its mechanisms and treatments based on established animal models. This study demonstrates that the Morc2a p.S87L variant causes hydroxyl radical-mediated neuropathy, which can be rescued through AAV-based gene therapy.

Iron homeostasis and post-hemorrhagic hydrocephalus: a review.

Iron physiology is regulated by a complex interplay of extracellular transport systems, coordinated transcriptional responses, and iron efflux mechanisms. Dysregulation of iron metabolism can result in defects in myelination, neurotransmitter synthesis, and neuronal maturation. In neonates, germinal matrix-intraventricular hemorrhage (GMH-IVH) causes iron overload as a result of blood breakdown in the ventricles and brain parenchyma which can lead to post-hemorrhagic hydrocephalus (PHH). However, the precise mechanisms by which GMH-IVH results in PHH remain elusive. Understanding the molecular determinants of iron homeostasis in the developing brain may lead to improved therapies. This manuscript reviews the various roles iron has in brain development, characterizes our understanding of iron transport in the developing brain, and describes potential mechanisms by which iron overload may cause PHH and brain injury. We also review novel preclinical treatments for IVH that specifically target iron. Understanding iron handling within the brain and central nervous system may provide a basis for preventative, targeted treatments for iron-mediated pathogenesis of GMH-IVH and PHH.

An examination of relationships between vitamin B12 status and functional measures of peripheral neuropathy in young adult vegetarians.

Prevalence rates for vitamin B12 deficiency in U.S. adult vegetarians may exceed 30%, which is concerning given the role for this vitamin in numerous nervous system functions, including the synthesis of myelin sheaths. Defective myelin synthesis and repair are directly linked to peripheral neuropathy; yet, few investigations have examined how physical indicators of peripheral neuropathy (e.g., hand dexterity, vibration sensitivity and balance) are impacted in individuals adhering to vegetarian diets. This feasibility research explored the relationships between peripheral neuropathy and vitamin B12 status using a cross-sectional study design. In addition, a small pilot trial was conducted for limited-efficacy testing of vitamin B12 supplementation for reducing peripheral neuropathy. Healthy, able-bodied adults (n = 38; 19-40 years of age) reported exclusive adherence to a vegetarian or vegan diet for 3 years. Peripheral neuropathy was measured using a force plate for assessing balance, and a vibration sensitivity tester and pegboard tests to assess hand dexterity. Serum vitamin B12 and folate were measured using standard radioimmunoassay techniques. Twenty-six percent of the sample displayed deficient or marginal vitamin B12 status (serum vitamin B12 <221 pmol/L). Participants with adequate vitamin B12 status scored 10% higher on the Purdue pegboard assembly test and 20% higher on the left hand adjusted functional dexterity test in comparison to participants with marginal-to-deficient vitamin B12 status (p < 0.05). These data provide preliminary evidence that peripheral neuropathy can be detected in individuals with marginal-to-deficient vitamin B12 status.

Molybdenum - a scoping review for Nordic Nutrition Recommendations 2023.

Molybdenum is an essential element in the form of the molybdenum cofactor (Moco). In humans, Moco is required for four enzymes: xanthine oxidase (XO), aldehyde oxidase, sulfite oxidase (SO), and mitochondrial amidoxime-reducing component (mARC). The enzymes are involved in the oxidation of purines to uric acid, metabolism of aromatic aldehydes and heterocyclic compounds, and in the catabolism of sulfur amino acids. Molybdenum cofactor deficiency is a rare autosomal recessive syndrome due to a defective synthesis of Moco, resulting in a deficiency of all the molybdoenzymes. There are no reports on clinical signs of dietary molybdenum deficiency in otherwise healthy humans. Water-soluble molybdate is efficiently absorbed from the digestive tract. The body retention is regulated by urinary excretion. Plasma molybdenum reflects long-term intake and 24-h urinary excretion is related to recent intake. There are no biochemical markers of molybdenum status. Cereal products are the main contributors to molybdenum dietary intake, estimated to 100-170 μg/day in Nordic studies. Little data are available on molybdenum toxicity in humans. A tolerable upper intake level of molybdenum has been based on reproductive toxicity in rats, but the effects have not been reproduced in more recent studies. The U.S. Institute of Medicine (IOM, present National Academy of Sciences, Engineering, and Medicine; NASEM) established a Recommended Dietary Allowance of 45 μg/day in adult men and women in 2001, based on a small study reporting urinary excretion in balance with intake at 22 μg/day. The European Food Safety Authority (EFSA) considered in 2013 the evidence to be insufficient to derive an Average Requirement and a Population Reference Intake, but proposed an Adequate Intake of 65 μg/day for adults.

Highly Reliable Van Der Waals Memory Boosted by a Single 2D Charge Trap Medium.

Charge trap materials that can store carriers efficiently and controllably are desired for memory applications. 2D materials are promising for highly compacted and reliable memory mainly due to their ease of constructing atomically uniform interfaces, however, remain unexplored as being charge trap media. Here it is discovered that 2D semiconducting PbI2 is an excellent charge trap material for nonvolatile memory and artificial synapses. It is simple to construct PbI2 -based charge trap devices since no complicated synthesis or additional defect manufacturing are required. As a demonstration, MoS2 /PbI2 device exhibits a large memory window of 120V, fast write speed of 5 µs, high on-off ratio around 106 , multilevel memory of over 8 distinct states, high reliability with endurance up to 104 cycles and retention over 1.2 × 104 s. It is envisioned that PbI2 with ionic activity caused by the natively formed iodine vacancies is unique to combine with unlimited 2D materials for versatile van der Waals devices with high-integration and multifunctionality.

The ATF4-RPS19BP1 Axis Modulates Ribosome Biogenesis to Promote Erythropoiesis

The function of hematopoietic stem cells (HSCs) and the process of hematopoiesis are intricately governed by a complex interplay of intrinsic programs and extrinsic signals from the microenvironment (Crane et al., Nat Rev Immunol, 2017). Previous work reported by our group and others has demonstrated that ATF4, a basic region-leucine zipper transcription factor, plays pivotal roles in fetal liver hematopoiesis, HSC maintenance, bone formation and tumorigenesis (Zhao et al., Blood, 2015; Sun et al., Sci Adv, 2021; Yang et al., Cell, 2004). However, the precise function of ATF4 in the bone marrow niche and how ATF4 regulates adult hematopoiesis remain largely unknown. Here, we employ four cell-type-specific mouse Cre lines to conditionally knock out Atf4 in Cdh5 + endothelial cells (ECs), Prx1 + bone marrow stromal cells (BMSCs), Osx + osteo-progenitor cells, and Mx1 + hematopoietic cells, and uncover the role of Atf4 in niche cells and hematopoiesis. We found that Atf4 deletion in BMSCs reduced BMSC numbers and impaired their CFU-F activity as well as differentiation toward osteoblasts, but spares hematopoiesis and HSC function in adult mice. Furthermore, Atf4 depletion in the ECs or osteo-progenitors of adult mice has a very mild effect on the BM hematopoiesis and HSC maintenance. Intriguingly, Atf4 depletion in the hematopoietic system resulted in severe anemia. More than 70% of the Mx1cre; Atf4 fl/fl (Δ/Δ) mice gradually died from anemia. Atf4 deficiency markedly reduced the frequency of erythroid progenitor cells (EPCs) (including MEP, PreCFU-E, and CFU-E cells) and impaired their CFU-E and BFU-E colony-forming ability. Atf4 depletion severely impaired the repopulation ability of BM cells and the self-renewal capacity of HSCs. Single cell RNA-seq (scRNA-seq) analysis of BM Lin -cKit + cells confirmed the defects of erythropoiesis after Atf4 depletion. The absence of ATF4 compelled early erythroid progenitors to enter the S phase, which led to replication stress and in turn activated both the DNA damage response and apoptosis. Subsequently, we conducted an integrative analysis of the genes that were identified as downregulated in MEPs by RNA-seq, ATAC-seq, and H3K4me3 Cut&amp;Tag and genes that were identified as downregulated in erythroid progenitors (i.e., PreMegE, Ery1, and Ery2) by scRNA-seq, which predicted Rps19bp1 as the top target gene of Atf4. Luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays confirmed that ATF4 was a transcriptional activator of Rps19bp1. Downregulation of Rps19bp1 caused by Atf4 deletion led to decreased assembles of 40S proteins, such as RPS3, RPS6 and RPS19. O-propargyl-puromycin (OP-Puro) incorporation and SUnSET assay further confirmed the global protein synthesis defect in Atf4-deficient MEP cells. The protein synthesis defects and impaired erythropoiesis were rescued upon RPS19BP1 overexpression in Atf4 knockdown MEL cell line or BM cKit + cells from Atf4-deficient mice. Ribosome profiling of CMP cells from Δ/Δ and fl/fl mice further demonstrated that the expression of proteins involved in ribosome biogenesis was significantly lower in Atf4-depleted than in wildtype CMP cells. The expression of gene sets linked to PreCFU-E signature was also lower in Atf4-depleted than in wildtype CMPs, suggesting that the reduction in ribosome biogenesis significantly affected the translation efficiency of erythroid-related pathway components, thereby impeding erythroid lineage commitment. Finally, we demonstrate that under conditions of 5-fluorouracil-induced stress, Atf4 depletion impedes the recovery of hematopoietic lineages, which requires efficient ribosome biogenesis. Taken together, we demonstrated that, unlike in the fetal liver, ATF4 governs adult HSC function and erythropoiesis in a cell-intrinsic manner. We revealed a novel role for ATF4 in erythropoiesis, which links it to RPS19BP1, ribosome biogenesis and protein translation. Our findings have highlighted the crucial importance of protein synthesis regulation during erythroid lineage commitment. This discovery will likely have extended implications for understanding and treating ribosomopathy-associated erythroid failure.

Predicting Thalassemia Using Feature Selection Techniques: A Comparative Analysis.

Thalassemia represents one of the most common genetic disorders worldwide, characterized by defects in hemoglobin synthesis. The affected individuals suffer from malfunctioning of one or more of the four globin genes, leading to chronic hemolytic anemia, an imbalance in the hemoglobin chain ratio, iron overload, and ineffective erythropoiesis. Despite the challenges posed by this condition, recent years have witnessed significant advancements in diagnosis, therapy, and transfusion support, significantly improving the prognosis for thalassemia patients. This research empirically evaluates the efficacy of models constructed using classification methods and explores the effectiveness of relevant features that are derived using various machine-learning techniques. Five feature selection approaches, namely Chi-Square (χ2), Exploratory Factor Score (EFS), tree-based Recursive Feature Elimination (RFE), gradient-based RFE, and Linear Regression Coefficient, were employed to determine the optimal feature set. Nine classifiers, namely K-Nearest Neighbors (KNN), Decision Trees (DT), Gradient Boosting Classifier (GBC), Linear Regression (LR), AdaBoost, Extreme Gradient Boosting (XGB), Random Forest (RF), Light Gradient Boosting Machine (LGBM), and Support Vector Machine (SVM), were utilized to evaluate the performance. The χ2 method achieved accuracy, registering 91.56% precision, 91.04% recall, and 92.65% f-score when aligned with the LR classifier. Moreover, the results underscore that amalgamating over-sampling with Synthetic Minority Over-sampling Technique (SMOTE), RFE, and 10-fold cross-validation markedly elevates the detection accuracy for αT patients. Notably, the Gradient Boosting Classifier (GBC) achieves 93.46% accuracy, 93.89% recall, and 92.72% F1 score.

PSVIII-11 Antinutritional Factors Are Largely Mitigated By Extrusion and Did Not Negatively Impact Digestibility of Canine Grain-Free and Grain-Inclusive Foods

Abstract Ingredients commonly used in companion animal foods, such as whole grains and legumes, naturally contain antinutritional factors (ANF). Some ANF are known contributors to reduced nutrient digestibility and bioavailability, potentially resulting in defective protein synthesis, which is one hypothesis of potential root cause(s) for dilated cardiomyopathy. Yet some ANF, such as trypsin inhibitor (TI) and isoflavones, may act as beneficial bioactives at lower inclusion levels. While various processing methods can impact the presence or activity of ANF, the purpose of this investigation was to assess the presence of ANF in ingredients commonly used in canine foods, the effect of extrusion on ANF, and the subsequent digestibility of foods formulated using different ingredient strategies. Four test foods were formulated to meet requirements for adult dogs at maintenance: 1) grain-inclusive without peas or potatoes, 2) grain-inclusive with peas and pea fiber, 3) grain-free with peas and potatoes, and 4) grain-free with potatoes (Table 1). Grains, legumes, plant proteins, and finished test foods were analyzed for 41 ANF. Percent destruction of ANF was calculated from the inclusion levels of ingredients in the formulations and the tested levels in finished foods. Digestibility studies were then conducted to assess macronutrient digestibility of the test foods. Cereal grains and soybean meal were the primary sources of ANF in raw ingredients. Phytic acid, TI, and isoflavones (daidzin and genistin) were the most common ANF measured in ingredients. Lectin was not detectable in any ingredients or test foods; tannins were detected only in soybean meal. Except for phytic acid and TI, ANF were effectively (&amp;gt;99%) destroyed by this extrusion process. Trypsin inhibitor was detectable at levels slightly above the detection limit (1000 TIU/g) in all finished test foods (Table 2; 1,200 to 1,700 TIU/g). Phytic acid was detectable (0.49 to 0.76%) in all finished test foods except for the grain-free with potatoes formulation. This was unsurprising as both are known to be resistant to inactivation by heat, pressure and steam. Despite their presence in finished test foods, apparent DM, carbohydrate (NFE), and energy digestibility were high (&amp;gt;80%) for all foods; apparent fat and true protein digestibility were &amp;gt;90% (Figure 1). These data demonstrate the effectiveness of this specific extrusion process to minimize or mitigate the majority of ANF in canine foods when formulated with a variety of common ingredients. Despite the presence of these ANF, test foods remained highly digestible, demonstrating the importance and effectiveness of balanced nutritional formulation in mitigating their effects. We assert that the presence or absence of ANF in ingredients should not, by itself, be the reason for use or avoidance of an ingredient. Additional factors such as the quality of the raw ingredient, processing methods, and overall nutritional design must be considered.

Possible Protective Effect of Low Dose of Papaverine on ANIT Induce Cholestasis in Rat

Abstract&#x0D; Intrahepatic cholestasis is clinical syndrome which cause either by defect in synthesis or bile acid flow, the pathophysiology of cholestasis is complicated by a number of variables, including oxidative stress, inflammatory response, and dysregulation of bile acid transporter . Rats, mice, and guinea pigs were utilized as experimental animals, and ANIT was administered to them in order to create a model that closely resembled intrahepatic cholestasis in human. This study examined the protective effects of papaverine, a non-narcotic opium alkaloid derived from papaver somniferum and discovered as an FXR agonist, on cholestasis in rats induced by alpha-naphthylisothiocyanate (ANIT). Rats utilized in this study divided into 3 groups (10 rats per each groups), group I (control) or vehicle group rat administered corn oil (1ml/kg) once daily 48 hour before sacrifice group II rats orally administered alpha-naphthylisothiocyanate (ANIT) 100mg/kg single dose 48hour before sacrifice group III rats administered 100mg/kg papaverine orally for 7 consecutive days and at day 5 rat administered alpha-naphthylisothiocyanate (ANIT) The results showed that papaverine treatment decreased alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), total bilirubin, total bile acid, as well as increased antioxidant enzyme GPX and decreased MDA and inflammatory mediators tumor necrosis factor TNF- and interleukin IL1-β. In conclusion, papaverine may have a protective effect to alleviate ANIT-induced cholestasis and may be a therapeutic target to treat cholestasis.

Identifying Novel Regulators of γ-Globin Expression Using a Genome-Scale CRISPR Activation Screen

β-hemoglobinopathies are the most common monogenic disorders worldwide, and are defined based on whether patients have quantitative (β-thalassemia) or qualitative (Sickle Cell Disease (SCD)) defects in β-globin synthesis. Unfortunately, there are limited treatment options for β-hemoglobinopathies, and the majority of patients continue to develop life-threatening complications from their diseases. An alternative β-like subunit, γ-globin, has been shown to inhibit pathogenic hemoglobin polymerization in SCD and to functionally replace β-globin in β-thalassemia. The discovery of novel strategies to upregulate γ-globin expression may lead to effective therapies for β-hemoglobinopathies. To identify novel regulators of γ-globin expression, we performed a genome-wide pooled CRISPR activation (CRISPRa) screen, using the Synergistic Activation Mediator (SAM) system. The screen was performed in the HUDEP-2 cell line, which expresses adult β-globin. We generated a clonal HUDEP-2-MPH cell line, which stably expresses the transcriptional activator complex MPH (MS2-P65-HSF1). This cell line was transduced with a viral library that delivers a VP64 activation domain fused to a catalytically dead Cas9 (dCas9-VP64), a blasticidin resistance cassette, and one of 3 unique sgRNAs targeting virtually every coding gene in the human genome (the library consists of 70,290 sgRNAs). At day 8 of erythroid differentiation, the top and bottom 10% γ-expressing cells were sorted, and integrated sgRNAs were sequenced using NextGen sequencing. As expected, sgRNAs targeting BCL11A and ZBTB7A (known negative regulators of γ-globin) were enriched in the bottom 10% γ-expressing cells, while sgRNAs targeting HIF1A (known positive regulator of γ-globin) were enriched in the top 10% γ-expressing cells. Notably, this screen identified several novel candidate positive regulators of γ-globin expression, including the transcriptional repressor Hypermethylated in Cancer 1 (HIC1). In preliminary validation studies, we used 2 independent sgRNAs to activate HIC1 in HUDEP-2-MPH cells and measured the percentage of γ-globin expressing cells (F-cells) by flow cytometry. Compared to cells transduced with control sgRNAs, increased HIC1 transcription (&amp;gt;200-fold) resulted in a profound increase in the proportion of F-cells, from a baseline of ~6% up to ~76%. Similarly, HIC1 overexpression in HUDEP-2 cells resulted in a ~13-fold increase in γ-globin mRNA levels and reduction in BCL11A mRNA level to ~30% of normal. These results suggest that the increased γ-globin expression resulting from HIC1 overexpression may result, at least in part, from reduced BCL11A levels (which we are currently validating). Recently, increased expression of HIC2, a paralogous protein for HIC1, was reported to result in increased γ-globin expression. To exclude the possibility that HIC1 targeting sgRNAs may also target HIC2, we measured the HIC2 mRNA level in HUDEP-2 cells targeted with sgRNAs aimed at increasing HIC1 expression. HIC2 mRNA was not increased in the latter cells. We next overexpressed HIC1 cDNA in erythroid cells differentiated from primary human CD34+ hematopoietic stem and progenitor cells (HSPCs). In early preliminary results, we found that HIC1 overexpression resulted in increased γ-globin expression, validating the HUDEP-2 data. Additional studies are ongoing to define the role of HIC1 in the regulation of γ-globin expression, and to define the impact of HIC1 overexpression on erythroid differentiation. In summary, our screen uncovered HIC1, and other potential novel regulators of γ-globin expression, which we are currently validating. These findings may result in future therapeutic approaches for β-hemoglobinopathies.

Borealin/CDCA8 deficiency alters thyroid development and results in papillary tumor-like structures.

BOREALIN/CDCA8 mutations are associated with congenital hypothyroidism and thyroid dysgenesis. Borealin is involved in mitosis as part of the Chromosomal Passenger Complex. Although BOREALIN mutations decrease thyrocyte adhesion and migration, little is known about the specific role of Borealin in the thyroid. We characterized thyroid development and function in Borealin-deficient (Borealin +/-) mice using histology, transcriptomic analysis, and quantitative PCR. Thyroid development was impaired with a hyperplastic anlage on embryonic day E9.5 followed by thyroid hypoplasia from E11.5 onward. Adult Borealin +/- mice exhibited euthyroid goiter and defect in thyroid hormone synthesis. Borealin +/- aged mice had disorganized follicles and papillary-like structures in thyroids due to ERK pathway activation and a strong increase of Braf-like genes described by The Cancer Genome Atlas (TCGA) network of papillary thyroid carcinoma. Moreover, Borealin +/- thyroids exhibited structural and transcriptomic similarities with papillary thyroid carcinoma tissue from a human patient harboring a BOREALIN mutation, suggesting a role in thyroid tumor susceptibility. These findings demonstrate Borealin involvement in critical steps of thyroid structural development and function throughout life. They support a role for Borealin in thyroid dysgenesis with congenital hypothyroidism. Close monitoring for thyroid cancer seems warranted in patients carrying BOREALIN mutations.

A case-control study of early-stage radiological markers of endothelial dysfunction and cardiovascular findings in patients with osteogenesis imperfecta: genotype-phenotype correlations.

Osteogenesis imperfecta (OI) is a disease caused by defective collagen synthesis. Collagen type 1 is found in many structures in the cardiovascular system. Endothelial dysfunction, which develops prior to the emergence of structural and clinical signs of atherosclerosis, isbelieved to play a key role in atherogenesis. Endothelial dysfunction may be detected presymptomatically by non-invasive radiologic methods, such as flow-mediated dilatation (FMD) and carotid intima-media thickness (CIMT). These modalities may provide early indicators of endothelial dysfunction. This cross-sectional comparative study aimed to investigate early-stage radiological markers of endothelial dysfunction and cardiovascular diseases in OI patients and healthy controls and to investigate the correlation of findings with OI genotype. Thirty patients diagnosed with OI were paired with thirty healthy age- and gender-matched controls and echocardiogram findings were compared. None of the patients had known underlying cardiovascular disease. The mean age was 13.18±2.91 years. According to Sillence classification, 15 patients had type 1 OI, 10 had type III, and 5 had type IV. Mean CIMT in the OI group was higher in the control group (OI group: 0.42±0.06 vs. healthy controls: 0.34±0.04mm, p<0.01), and mean FMD percent was lower in the patient group (p<0.01). Left ventricular ejection fraction was 78.97±10.32 vs. 77.56±8.50 %, (OI group: 7.00±3.06 vs. healthy controls: 12.14±1.99, p=0.56), and fractional shortening was 42.68±11.94 vs. 40.23±7.99 %, (p=0.35), in OI patients and controls, respectively. Pediatric patients with OI without clinical signs of cardiovascular abnormality had significantly worse CIMT and FMD findings than healthy controls. However, no difference was determined when comparing left ventricular ejection fraction or fractional shortening. OI patients may need to be screened for cardiovascular system complications starting from an early age.

The Saccharomyces cerevisiae SR protein Npl3 interacts with hyperphosphorylated CTD of RNA Polymerase II

The catalytic subunit of RNA Polymerase II contains a highly conserved carboxy terminal domain (CTD) composed of multiple tandem heptad sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. The non-proline residues in CTD undergo posttranslational modifications, with Ser5 phosphorylation (Ser5P) predominating at the start of the transcription cycle and Ser2P at the end, while other phosphorylation levels are high all throughout. The differentially phosphorylated CTD is recognized by regulatory proteins, helpful during mRNA transcription and export. One such protein Npl3 is composed of two RNA binding domains and a C-terminus RGG/SR domain. The Ser411 of Npl3 is reported to make direct contact with Ser2P of CTD for its recruitment and function, while the Npl3 lacking of C-terminal 25 amino acids (Npl3Δ389–414) showed no apparent defects in mRNA synthesis. Here, we report that the RNA binding domains of Npl3 are separate folding units and interact also with the CTD. The interaction between Npl3 and CTD appears to involve not just Ser2P, but also the Ser5P and Ser7P. The Arg126 of the first RNA binding domain interacts with Ser2P whereas the Arg235 of the second RNA binding domain interacts with either Ser7P or Ser5P of another heptad. The finding provides new insight of Npl3 function for mRNA transcription.

THU455 Sclerostin Inhibitor Use In The Medical Management Of Osteogenesis Imperfecta Type III

Abstract Disclosure: M.D. Lundholm: None. A. Derkyi: None. S. Talvacchio: None. J.C. Marini: Other; Self; Ultragenyx, Amgen Inc. L.Z. Khan: None. Background: Osteogenesis imperfecta (OI) is a rare collagen-related hereditary skeletal disorder with an increased fracture risk from minimal trauma, short stature, and bone deformity. OI is associated with defects in collagen structure or synthesis, high bone turnover, and paradoxical bone hypermineralization. The optimal medical therapies for bone health in these patients are unknown. Clinical Case: A 27-year-old woman with OI presented to clinic for bone health. She was diagnosed with OI type III by phenotype, collagen biochemistry, and genetic analyses (heterozygous for a COL1A1 Gly187Ala substitution) following fractures at age 4 mos. She experienced dozens of fractures, has had orthopedic hardware in bilateral femora, left tibia, humerus, and radius, and right tibia, as well as spine fusion at age 7 yrs. She had not fractured in over 3 yrs which she attributed to the stability of her automatic wheelchair. She had no family history of OI and had been in studies at the NIH; from ages 6-9 yrs she received pamidronate (1 mg/kg/cycle q3m) with BMD improvement. Presently, her vitals were normal, with height of 3’6” (25-50th percentile for type III OI females) and weight of 64 lbs (BMI 25.5 kg/m2). On exam she had blue sclera, midface hypoplasia, dentinogenesis imperfecta, severe kyphosis and scoliosis, and a barrel chest. She had normal calcium, PTH, and 25-OH vitamin D levels. Skull CT showed 1 mm increase in basilar invagination (16 to 17 mm). DEXA demonstrated progressive decline in radial BMD from 0.671 g/cm2 to 0.625 g/cm2 over 3 yrs (z-score -0.4 to -1.1) into the osteopenic range. For worsening bone disease, she is starting romosozumab 210 mg monthly. Discussion: While several agents increase BMD in OI, there are no placebo-controlled studies to show an effect on fracture rate. Bisphosphonates including pamidronate are used most commonly, with increased areal BMD, decreased bone turnover, but also increased mineralization of already brittle bone. Overall, two Cochran reviews failed to support decreased fracture rates from bisphosphonates, and there is risk of adynamic bone with chronic use. There is even less evidence for use of denosumab which is additionally associated with a rebound effect upon discontinuation. Sclerostin inhibitors are the newest agents with both anabolic and antiresorptive effects and good results in OI mouse models. Phase 2 data with sestrusumab showed increased BMD and decreased bone turnover. Ongoing studies are looking at sclerostin inhibitor use in adults and adolescents. From a practical standpoint, romosozumab has a shorter effect with quicker offset than bisphosphonates if needed to stop for fracture healing. Conclusion: Sclerostin inhibitors are an option for the medical treatment of OI. Further investigation is needed to determine long-term safety, optimal duration, and use of sclerostin inhibitors in combination with antiresorptive agents in OI. Presentation: Thursday, June 15, 2023

Investigation of Antidiabetic Activity of Isolated Molecule from the Bark of Olax scandens

Hyperglycemia caused by defects in insulin synthesis and/or activity characterizes diabetes mellitus (DM), which is categorized as a metabolic condition. Some patients may develop side effects or drug interactions when treating these disorders with synthetic drugs. Due to this, there is an increased interest in researching the medical properties of plants and herbal substances while also considering synthetic medications’ interactions and negative effects. To isolate and identify the flavonoid 5-hydroxy-apigenin-7-O-Dglucopyranoside, the bark of Olax scandens was extracted with methanol in a soxhlet system (5HAG). The current study set out to examine 5HAG’s potential ability to treat albino wistar rats with STZ-induced diabetes. The chemical component significantly lowered rats’ blood glucose levels and improved lipid markers. Hepatic enzymes improved significantly, including SGOT, SGPT, ALP, plasma insulin, and total proteins. Overall, this study exposes new avenues for researching how 5HAG reduces blood sugar levels so drastically, but according to the aforementioned study, the flavonoid possesses antidiabetic properties because of its antioxidant activity. In addition, the isolated flavonoid may significantly impact the reduction of blood sugar and its complications by acting as a lead molecule in the identification of novel antidiabetic drug candidates.