Validation and clinical application of transactivation assays for RUNX1 variant classification

Familial platelet disorder with associated myeloid malignancies (RUNX1-FPD) is caused by heterozygous pathogenic germline variants of RUNX1. In the present study, we evaluate the applicability of transactivation assays to investigate RUNX1 variants in different regions of the protein. We studied 11 variants to independently validate transactivation assays supporting variant classification following the ClinGen Myeloid Malignancies variant curation expert panel guidelines. Variant classification is key for the translation of genetic findings. We showed that new assays need to be developed to assess C-terminal RUNX1 variants. Two variants of uncertain significance (VUS) were reclassified to likely pathogenic. Additionally, our analyses supported the (likely) pathogenic classification of two other variants. We demonstrated functionality of four VUS, but reclassification to (likely) benign was challenging and suggested the need to reevaluate current classification guidelines. Finally, clinical utility of our assays was illustrated in the context of seven families. Our data confirmed RUNX1-FPD suspicion in three families with RUNX1-FPD-specific family history. Whereas for three variants identified in non RUNX1-FPD-typical families, no functional defect was detected. Applying functional assays to support RUNX1 variant classification can be essential for adequate care of index patients and their relatives at risk. It facilitates translation of genetic data into personalized medicine.

The Effect of Isometric Hip External Rotation on Lower Extremity Muscles Activities During Pelvic Drop With Different Positions of the Hip Rotation in Subjects With and Without Genu Valgum: Controlled Laboratory Study

Background: Gluteus medius muscle (Gmed) dysfunction has been confirmed as a functional defect in subjects with Genu Valgum Deformity (GVD). In relation to these subjects, increase Gmed activity without synergist muscles dominance is considered as part of a specialized exercise program. Methods: A total of thirty female recreational athletes with (n=15) and without (n=15) GVD participated in this study. Surface electromyography measured Gmed, tensor fascia latae (TFL), and quadratus lumborum (QL) muscles activity when subjects performed pelvic drop (PD) in three different positions of hip rotations with and without applied isometric hip external rotation force. Results: There were differences in muscle activity between GVD and healthy subjects. The Gmed/TFL and Gmed/QL muscles activity ratio altered when placing the hip in different rotation positions and applying isometric load. Conclusions: The lower extremity muscles activity is affected by GVD, and changing the positions of the hip rotation in the PD task can be associated with altered muscle activity in both GVD and healthy Groups. However, applying isometric hip external rotation during PD can be suggested as an effective intervention to increase Gmed activity.

Platelet Defects in Acute Myeloid Leukemia—Potential for Hemorrhagic Events

Background and objectives: In acute myeloid leukemia (AML), extensive bleeding is one of the most frequent causes of death. Impaired activation and aggregation processes were identified in previous studies on platelet behaviour associated with this disease. This study’s aim was to examine platelet function in correlation with other haemorrhage risk factors (fever, sepsis, recent bleeding, uraemia, leucocytosis, haematocrit value, treatment). Design and methods: The analysis of platelet surface proteins (Glycoprotein Ib-IX (CD42b, CD42a), Glycoprotein IIb-IIIa (CD41, CD61), p-selectin (CD62P), granulophysin (CD63)) was conducted by flowcytometry from samples of whole blood in patients with acute myeloid leukaemia in different stages of diagnosis and therapy (n = 22) in comparison with healthy human controls (n = 10). Results and interpretations: Our results show a significant decrease in fluorescence level associated with platelet activation markers (CD63 (14.11% vs. 40.78 % p < 0.05); CD62P (15.26% vs. 28.23% p < 0.05)); adhesion markers (CD42b (69.08% vs. 84.41% p < 0.05)) and aggregation markers (CD61 (83.79% vs. 98.62% p < 0.001)) in patients compared to controls. The levels of CD41 (80.62% vs. 86.31%, p = 0.290) and CD42a (77.98% vs. 94.15%, p = 0.99) demonstrate no significant differences in the two groups. Conclusion: The AML patients present changes in adhesion receptors and activation markers, suggesting a functional defect or denatured intracellular signalling in platelets. The exposed data indicate that flow cytometry can effectively identify multiple functional platelet impairments in AML pathogenesis.

Kiss1/Gpr54 Prevents Bone Loss through Src Dephosphorylation by Dusp18 in Osteoclasts

Osteoclasts were over-activated as we age, which leads to bone loss. Src-deficient mice lead to only one phenotype -severe osteopetrosis due to functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCR) have been targets for ∼35% of approved drugs. However, how Src kinase activity is negatively regulated by GPCRs remains largely elusive. Herein we report that Src is dephosphorylated at Tyr 416 by Dusp18 upon GPR54 activation by its natural ligand Kp-10. Mechanically, both active Src and the Dusp18 phosphatase are recruited by GPR54 through the proline/arginine-rich motif (PR motif) in the C terminus, which is dependent on the Gαq signal pathway. As such, Kiss1, Gpr54, Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss. Accordingly, Kp-10 abrogated bone loss by suppressing osteoclasts activity in vivo. Therefore, Kiss1/Gpr54 is a promising therapeutic strategy governing bone resorption through Src dephosphorylation by Dusp18.

79 Platypnea–orthodeoxia syndrome as an uncommon cause of dyspnoea in a nonagenarian with aortic dissection

Methods and results Platypnea–Orthodeoxia syndrome (POS) is an uncommon but challenging clinical condition characterized by positional dyspnoea (platypnea) and arterial desaturation (orthodeoxia) in the upright position that improve in the supine position. POS can occur insidiously, as progressive unexplained oxygen desaturation for months or years, or as acute life-threatening unexplained hypoxaemia. The most common cause is the presence of an intracardiac shunt (patent foramen ovale and other interatrial defects) associated with a secondary anatomic or functional defect that serve as substrate for a right to left intracardiac shunt leading to oxygen desaturation. Conclusions Herein we present the case of a nonagenarian with a known history of ascending aortic aneurysm, that was admitted to the emergency department of our institution with a complaint of intermittent chest pain and dyspnoea. POS was then diagnosed, and the patient underwent a successful percutaneous closure with an Amplatzer™ device (25/25 mm) with minimal residual right-to-left shunt leading to a significant relief of the dyspnoea and improvement of arterial saturation. We also herein revise the clinical presentation, pathophysiology, diagnostic work-up, and management of patients with POS, aiming at increasing the awareness of this uncommon but often misdiagnosed treatable condition.

SEXUAL ACTIVITY: DEPENDENCE ON AGE, GENDER, VARIOUS CARDIOVASCULAR DISEASES AND CEREBRAL STROKE

Целью представленного исследования явилось определение места и роли сексуальных расстройств у представителей разного пола и возраста в развитии фоновой для церебрального инсульта патологии и формировании клинической картины острого периода инсульта и его последствий. Детально рассматривали вопросы интимной жизни 111 пациентов (58 мужчин и 53 женщины) в предшествовавшие инсульту периоды и определяли возможные связи с клиническими особенностями острого периода инсульта. У 77 из этих 111 пациентов изучали динамику сексуальных нарушений в связи с особенностями функционального дефекта, сформировавшегося спустя 3-24 мес после перенесенного инсульта. Выявлено, что снижение сексуальной активности до инсульта имеет, наряду с возрастом, отчетливое значение в развитии фоновой для инсульта патологии и в формировании последствий инсульта. У лиц пожилого возраста наиболее тяжелые функциональные последствия инсульта сформировались в 8,3 % при наличии нормальных сексуальных отношений в предшествовавший инсульту период и в 27,6 % при отсутствии таковых. Негативная динамика сексуальной активности после перенесенного инсульта выявлена в 46,8 % наблюдений, в основном у представителей пожилого возраста, и связана у женщин с наличием и выраженностью неврологического дефицита, у мужчин с присутствием церебральных атрофических изменений. The purpose of this study was to determine the place and role of sexual disorders in representatives of different sexes and ages in the development of background for cerebral stroke pathology and the formation of the clinical picture of acute stroke period and its consequences. Questions of intimate life of 111 patients (58 men and 53 women) in the periods preceding a stroke were considered in detail and possible connections with clinical features of an acute period of in-Soult were defined. In 77 of these 111 patients, we studied the dynamics of sexual disorders in connection with the features of a functional defect that formed 3-24 months after a stroke. It was revealed that the decrease in sexual activity before the stroke has a distinct significance along with age in the development of background for insulin pathology and in the formation of the consequences of stroke. In the elderly, the most severe functional consequences of stroke were formed in 8,3 % in the presence of normal sexual relations in the period preceding the stroke and in 27,6 % in the absence of such. Negative dynamics of sexual activity after a stroke was detected in 46,8 % of cases, mainly among the elderly, and is associated in women with the presence and severity of neurological deficits, in men-with the presence of cerebral atrophic changes.

Investigation of the Differences in Antithrombin to Heparin Binding among Antithrombin Budapest 3, Basel, and Padua Mutations by Biochemical and In Silico Methods

Antithrombin (AT) is a serine protease inhibitor, its activity is highly accelerated by heparin. Mutations at the heparin-binding region lead to functional defect, type II heparin-binding site (IIHBS) AT deficiency. The aim of this study was to investigate and compare the molecular background of AT Budapest 3 (p.Leu131Phe, ATBp3), AT Basel (p.Pro73Leu), and AT Padua (p.Arg79His) mutations. Advanced in silico methods and heparin-binding studies of recombinant AT proteins using surface plasmon resonance method were used. Crossed immunoelectrophoresis and Differential Scanning Fluorimetry (NanoDSF) were performed in plasma samples. Heparin affinity of AT Padua was the lowest (KD = 1.08 × 10−6 M) and had the most severe consequences affecting the allosteric pathways of activation, moreover significant destabilizing effects on AT were also observed. KD values for AT Basel, ATBp3 and wild-type AT were 7.64 × 10−7 M, 2.15 × 10−8 M and 6.4 × 10−10 M, respectively. Heparin-binding of AT Basel was slower, however once the complex was formed the mutation had only minor effect on the secondary and tertiary structures. Allosteric activation of ATBp3 was altered, moreover decreased thermostability in ATBp3 homozygous plasma and increased fluctuations in multiple regions of ATBp3 were observed by in silico methods suggesting the presence of a quantitative component in the pathogenicity of this mutation due to molecular instability.

Phosphoserine aminotransferase has conserved active site from microbes to higher eukaryotes with minor deviations

: Serine is ubiquitously synthesized in all living organisms from the glycolysis intermediate 3-phosphoglycerate (PGA) by phosphoserine biosynthetic pathway, consisting of three different enzymes, namely: 3-phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Any functional defect or mutation in these enzymes may cause deliberating conditions, such as colon cancer progression and chemoresistance in humans. Phosphoserine aminotransferase (PSAT) is the second enzyme in this pathway that converts phosphohydroxypyruvate (PHP) to O-phospho-L-serine (OPLS). Humans encode two isoforms of this enzyme: PSAT1 and PSAT2. PSAT1 exists as a functional dimer, where each protomer has a large and a small domain; each large domain contains a Lys residue that covalently binds PLP. The PLP-binding site of human PSAT1 and most of its active site residues are highly conserved in all known PSAT structures except for Cys-80. Interestingly, Two PSAT structures from different organisms show halide binding near their active site. While the human PSAT1 shows a water molecule at this site with different interacting residues, suggesting the inability of halide binding in the human enzyme. Analysis of the human PSAT1 structure showed a big patch of positive charge around the active site, in contrast to the bacterial PSATs. Compared to human PSAT1, the PSAT2 isoform lacks 46 residues at its C-terminal tail. This tail region is present at the opening of the active site as observed in the other PSAT structures. Further structural work on human PSAT2 may reveal the functional importance of these 46 residues.

CLINICAL AND STATISTICAL CHARACTERISTICS OF PATIENTS WITH CEREBRAL STROKE

A retrospective analysis of 1354 case histories of patients admitted to the multidisciplinary medical center of Bukhara with a diagnosis of "Acute cerebrovascular accident" for 2009-2019 was carried out. The study revealed that in the structure of hospitalizations, the largest number of patients with cerebral stroke (MI) lived in rural areas, and among them, females predominated. The ratio of ischemic and hemorrhagic strokes in Bukhara city and the Bukhara region was 4.9: 1. Among the patients with both IS and HI, females predominated. The totality of therevealed indicators, depending on the hemispheric lateralization of the focus, showed that the overall severity of the neurological and functional defect according to the NIHSS, Gusev-Skvortsova, and Bartel scales significantly predominates with the right-sided localization of the focus. Moreover, it was shown that in females with all IS forms, the indicators, according to the stated scales, are worse than inmales

Propagation of Crystal Defects during Directional Solidification of Silicon via Induction of Functional Defects

The introduction of directional solidified cast mono silicon promised a combination of the cheaper production via a casting process with monocrystalline material quality, but has been struggling with high concentration of structural defects. The SMART approach uses functional defects to maintain the monocrystalline structure with low dislocation densities. In this work, the feasibility of the SMART approach is shown for larger ingots. A G2 sized crystal with SMART and cast mono silicon parts has been analyzed regarding the structural defects via optical analysis, crystal orientation, and etch pit measurements. Photoluminescence measurements on passivated and processed samples were used for characterization of the electrical material quality. The SMART approach has successfully resulted in a crystal with mono area share over 90% and a confinement of dislocation structures in the functional defect region over the whole ingot height compared to a mono area share of down to 50% and extending dislocation tangles in the standard cast mono Si. Cellular structures in photoluminescence measurements could be attributed to cellular dislocation patterns. The SMART Si material showed very high and most homogeneous lifetime values enabling solar cell efficiencies up to 23.3%.