A Dual Inhibitory Mechanism Sufficient to Maintain Cell-Cycle-Restricted CENP-A Assembly.

Chromatin featuring the H3 variant CENP-A at the centromere is critical for its mitotic function and epigenetic maintenance. Assembly of centromeric chromatin is restricted to G1 phase through inhibitory action of Cdk1/2 kinases in other phases of the cell cycle. Here, we identify the two key targets sufficient to maintain cell-cycle control of CENP-A assembly. We uncovered a single phosphorylation site in the licensing factor M18BP1 and a cyclin A binding site in the CENP-A chaperone, HJURP, that mediated specific inhibitory phosphorylation. Simultaneous expression of mutant proteins lacking these residues results in complete uncoupling from the cell cycle. Consequently, CENP-A assembly is fully recapitulated under high Cdk activities, indistinguishable from G1 assembly. We find that Cdk-mediated inhibition is exerted by sequestering active factors away from the centromere. Finally, we show that displacement of M18BP1 from the centromere is critical for the assembly mechanism of CENP-A.

Endovascular Treatment of "Donut-Shaped" Aneurysm-A Case Series.

Background and Objectives: Partially thrombosed aneurysms represent a subset primarily found within large and giant aneurysms. The presence of an intraluminal thrombus can cause an aneurysm to present in different shapes upon angiographic examination. We present a series of five cases of "donut-shaped" aneurysms observed over the past decade at the Clinic for Neurosurgery in the University Clinical Centre of Serbia. Materials and Methods: The management of "donut-shaped" aneurysms was accomplished through endovascular interventions, employing techniques such as the deployment of flow-diverting stents or a combination of stent placement and coil embolization. Results: Four out of five patients underwent endovascular treatment, yielding positive outcomes with complete thrombosis of the aneurysms during follow-up. The fifth patient was successfully diagnosed; however, due to their deteriorating condition, treatment was not feasible. Conclusions: Given the potential life-threatening complications associated with this entity, accurate diagnosis and appropriate management are crucial. In our cohort, endovascular interventions demonstrated efficacy in the majority of cases, underscoring the significance of this approach in treating "donut-shaped" aneurysms. Nevertheless, considering the rarity of this condition, further research is justified to refine diagnostic and therapeutic strategies for these complex intracranial vascular anomalies.

Quantitative Microscopy Reveals Centromeric Chromatin Stability, Size, and Cell Cycle Mechanisms to Maintain Centromere Homeostasis.

Centromeres are chromatin domains specified by nucleosomes containing the histone H3 variant, CENP-A. This unique centromeric structure is at the heart of a strong self-templating epigenetic mechanism that renders centromeres heritable. We review how specific quantitative microscopy approaches have contributed to the determination of the copy number, architecture, size, and dynamics of centromeric chromatin and its associated centromere complex and kinetochore. These efforts revealed that the key to long-term centromere maintenance is the slow turnover of CENP-A nucleosomes, a critical size of the chromatin domain and its cell cycle-coupled replication. These features come together to maintain homeostasis of a chromatin locus that directs its own epigenetic inheritance and facilitates the assembly of the mitotic kinetochore.

Improving quality in the preanalytical phase through innovation, on behalf of the European Federation for Clinical Chemistry and Laboratory Medicine (EFLM) Working Group for Preanalytical Phase (WG-PRE).

It is now undeniable that laboratory testing is vital for the diagnosis, prognostication and therapeutic monitoring of human disease. Despite the many advances made for achieving a high degree of quality and safety in the analytical part of diagnostic testing, many hurdles in the total testing process remain, especially in the preanalytical phase ranging from test ordering to obtaining and managing the biological specimens. The Working Group for the Preanalytical Phase (WG-PRE) of the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) has planned many activities aimed at mitigating the vulnerability of the preanalytical phase, including the organization of three European meetings in the past 7 years. Hence, this collective article follows the previous three opinion papers that were published by the EFLM WGPRE on the same topic, and brings together the summaries of the presentations that will be given at the 4th EFLM-BD meeting "Improving quality in the preanalytical phase through innovation" in Amsterdam, 24-25 March, 2017.

Acthar gel in the treatment of nephrotic syndrome: a multicenter retrospective case series.

BACKGROUND: Current first-line anti-proteinuric treatments for nephrotic syndrome (NS) do not produce an effective response in all patients and are not tolerated by some patients. Additional effective and tolerable treatment options in NS are strongly needed. This retrospective case series is the largest to date to examine Acthar gel (adrenocorticotropic hormone, ACTH) in patients with varied-etiology NS. METHODS: This multicenter retrospective case series included adult patients with NS (N = 44) treated with Acthar gel at 6 clinical practices. NS etiologies included idiopathic focal segmental glomerulosclerosis (FSGS, 15), idiopathic membranous nephropathy (iMN, 11), IgA nephropathy (IgAN, 5), diabetic nephropathy (DN, 4), systemic lupus erythematosus class V membranous lupus nephritis (MLN, 2), minimal change disease (MCD, 2), membranoproliferative glomerulonephritis (MPGN, 1), fibrillary glomerulonephritis (FGN, 1), and unbiopsied NS (3). Proteinuria response was assessed as percent reduction from baseline and percent of patients meeting complete remission (final proteinuria <500 mg/d), partial remission (≥50 % reduction in proteinuria from baseline and final proteinuria 500-3500 mg/d), clinical response (≥30 % reduction in proteinuria from baseline that did not meet criteria for complete or partial remission), and no response (failed to meet remission or clinical response criteria) following Acthar gel therapy. Safety and tolerability were examined using adverse event (AE) frequency reported by patients or treating nephrologists and frequency of early discontinuation of treatment due to AEs. RESULTS: 68.2 % (30/44) of patients had received prior NS treatment with immunosuppressive or cytotoxic therapies. Thirty-seven patients completed Acthar gel treatment. Seven patients (15.9 %) had early termination due to AEs, including weight gain (2), hypertension (2), edema (1), fatigue (1), seizures (1) and for reasons not stated (2). Proteinuria reduction ≥30 % was shown in 81.1 % (30/37) of patients and 62.2 % (23/37) showed ≥50 % proteinuria reduction. Proteinuria responses were greatest in MCD (n = 2/2 complete remission), MLN (n = 2/2 partial remission), MPGN (n = 1/1 partial remission), FSGS (n = 12/15 [80.0 %] partial remission or clinical response), and iMN (n = 8/11 [72.7 %] complete remission, partial remission, or clinical response). CONCLUSIONS: Acthar gel may meet an important treatment need in patients with treatment-resistant NS in response to first-line therapies, patients unable to tolerate first-line therapies, and in patients with advanced disease.

Intracranial arterial variations: a comprehensive evaluation using CT angiography.

Background Intracranial arterial variations are a frequent finding in the general population. Knowledge of these vascular variations has significant clinical impact because some of them predispose patients to development of an aneurysm or cerebrovascular ischemic disease. The purpose of this study was to evaluate the frequency of intracranial vascular variations and associated vascular lesions on computed tomography angiography (CTA) examinations. Material and Methods CTA examinations performed by 16-detector computed tomography were prospectively reviewed in 455 patients for the presence of fenestrations, duplications, hypoplasia, aplasia, aneurysms, and other vascular lesions. Results Arterial fenestrations were found in 2.4% of patients, with the vertebrobasilar system as the most common location. The remaining fenestrations were located on the middle cerebral artery M1 segment (0.2%), anterior communicating artery (0.4%), and anterior cerebral artery A1 segment (0.6%). No associated aneurysms were noted in these patients. The prevalence of an azygos anterior cerebral artery was 1.5%. Bihemispheric anterior cerebral artery was found in 0.9%, hypoplastic A1 segment in 17.6%, and congenital absence of A1 segment in 0.4% of patients. Fetal origin of the posterior cerebral artery was found in 37% of cases. Hypoplastic vertebral artery terminating as posterior inferior cerebellar artery was observed in 9 patients, while transversal anastomosis between vertebral arteries was seen in only 1 patient. Conclusions CTA precisely demonstrates the diversity of intracranial arterial variations, whose overall frequency in this study is similar to previous radiological reports. Furthermore, our results do not show significant association between the frequency of aneurysms and cerebral arterial anomalies.

Case report: Rapidly progressive neurocognitive disorder with a fatal outcome in a patient with PU.1 mutated agammaglobulinemia.

Introduction: PU.1-mutated agammaglobulinemia (PU.MA) represents a recently described autosomal-dominant form of agammaglobulinemia caused by mutation of the SPI1 gene. This gene codes for PU.1 pioneer transcription factor important for the maturation of monocytes, B lymphocytes, and conventional dendritic cells. Only six cases with PU.MA, presenting with chronic sinopulmonary and systemic enteroviral infections, have been previously described. Accumulating literature evidence suggests a possible relationship between SPI1 mutation, microglial phagocytic dysfunction, and the development of Alzheimer's disease (AD). Case description: We present a Caucasian female patient born from a non-consanguineous marriage, who was diagnosed with agammaglobulinemia at the age of 15 years when the immunoglobulin replacement therapy was started. During the following seventeen years, she was treated for recurrent respiratory and intestinal infections. At the age of 33 years, the diagnosis of celiac-like disease was established. Five years later progressive cognitive deterioration, unstable gait, speech disturbances, and behavioral changes developed. Comprehensive microbiological investigations were negative, excluding possible infective etiology. Brain MRI, 18FDG-PET-CT, and neuropsychological testing were suggestive for a diagnosis of a frontal variant of AD. Clinical exome sequencing revealed the presence of a novel frameshift heterozygous variant c.441dup in exon 4 of the SPI1 gene. Despite intensive therapy, the patient passed away a few months after the onset of the first neurological symptoms. Conclusion: We describe the first case of PU.MA patient presenting with a rapidly progressive neurocognitive deterioration. The possible role of microglial dysfunction in patients with SPI1 mutation could explain their susceptibility to neurodegenerative diseases thus highlighting the importance of genetic testing in patients with inborn errors of immunity. Since PU.MA represents a newly described form of agammaglobulinemia, our case expands the spectrum of manifestations associated with SPI1 mutation.

Preanalytic Competency Assessment: A Q-Probes Study Involving 46 Health Care Institutions, 447 Blood Collectors/Phlebotomists, and 2212 Individual Assessments.

CONTEXT.­: Health care organizations face a challenge of assessing preanalytic competency of blood collectors/phlebotomists (BC/Ps). OBJECTIVE.­: To pilot a novel methodology for BC/P preanalytic competency assessment and identify potential areas for improvement. DESIGN.­: Study participants identified preanalytic errors present in 5 blood collection video vignettes. Submitted error descriptions were categorized and then consolidated into a list of standardized required errors for evaluation. RESULTS.­: The correct identification of required error rates across all videos viewed by 447 BC/Ps from 46 institutions ranged from 0.7% to 91.9%. The median phlebotomist score across all 5 videos was 55.9% for 440 eligible blood collectors and ranged between 38.2% (10th percentile) and 70.6% (90th percentile). The median institutional score from 42 eligible institutions was 55.9% (range, 43.3%-65.3% for the 10th to 90th percentiles). There were no significant associations between any laboratory practice characteristics and the institutional average overall phlebotomist scores. The following phlebotomist characteristics were significantly associated with overall phlebotomist scores: level of education (P = .01), having phlebotomy technician (American Society for Clinical Pathology) certification compared with no or other certifications (P = .002), years of experience in collecting blood specimens (P = .01), and higher average number of venipuncture specimens collected per shift (P = .001). CONCLUSIONS.­: Improvement of the awareness and knowledge of correct blood collection practices is needed, because the best performers (90th percentile) did not recognize approximately one-third of the errors. Using hypothetical blood collection scenarios that incorporate performance errors may be a way to assess preanalytic competency of BC/Ps and create opportunities for continuous improvement.

Enhancement of ZnO@RuO2 bifunctional photo-electro catalytic activity toward water splitting.

Catalytic materials are the greatest challenge for the commercial application of water electrolysis (WEs) and fuel cells (FCs) as clean energy technologies. There is a need to find an alternative to expensive and unavailable platinum group metal (PGM) catalysts. This study aimed to reduce the cost of PGM materials by replacing Ru with RuO2 and lowering the amount of RuO2 by adding abundant and multifunctional ZnO. A ZnO@RuO2 composite in a 10:1 molar ratio was synthesized by microwave processing of a precipitate as a green, low-cost, and fast method, and then annealed at 300°C and 600°C to improve the catalytic properties. The physicochemical properties of the ZnO@RuO2 composites were investigated by X-ray powder diffraction (XRD), Raman and Fourier transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FESEM), UV-Vis diffuse reflectance spectroscopy (DRS), and photoluminescence (PL) spectroscopy. The electrochemical activity of the samples was investigated by linear sweep voltammetry in acidic and alkaline electrolytes. We observed good bifunctional catalytic activity of the ZnO@RuO2 composites toward HER and OER in both electrolytes. The improved bifunctional catalytic activity of the ZnO@RuO2 composite by annealing was discussed and attributed to the reduced number of bulk oxygen vacancies and the increased number of established heterojunctions.

Garlic Ecotypes Utilise Different Morphological, Physiological and Biochemical Mechanisms to Cope with Drought Stress.

Drought negatively affects plants by altering morphological, physiological and metabolic processes and ultimately reducing yields. Garlic (Allium sativum L.), an important member of the Alliaceae family, is also sensitive to drought and maximizing the yield of garlic bulbs is largely dependent on water availability. The objective of this study was to determine the effects of drought stress on morphological and physiological characteristics, as well as on phenolic, sugar, inulin and free amino acid content and antioxidant activity in two Croatian garlic ecotypes, 'Istarski crveni' (IC) and Istarski bijeli (IB). Drought was induced by using polyethylene glycol 8000 (PEG) solution (-0.6 MPa) starting 21 days after clove planting and lasted for 20 days. Drought reduced plant height, number of leaves and plant weight, but increased root length in both ecotypes compared to the control treatment. Among the physiological parameters, significant differences were observed between the two ecotypes studied in the spectral characteristics of the leaves, namely reflection in red, green and blue, VAL, values of the vegetation indices related to the chlorophyll content (CHI, GI), and the anthocyanin content (ARI). Ecotype IC showed higher antioxidant activity in the control treatment due to higher total phenolic content (TPC), but under drought conditions higher DPPH radical scavenging activity was determined in ecotype IB and higher values of FRAP in IC. Sucrose and glucose generally decreased under drought, while inulin increased in IB but decreased in IC. Total free amino acid content increased under drought in both ecotypes. In conclusion, drought tolerance of IB might be associated with increased accumulation of inulin and higher levels of amino acids, especially those shown to contribute to drought resistance. In IC, drought tolerance is associated with an increase in some amino acid compounds and better root growth in depth, probably due to a more efficient translocation of sucrose to the underground part of the plant.

Virtual screening, drug-likeness analysis, and molecular docking study of potential severe acute respiratory syndrome coronavirus 2 main protease inhibitors.

Due to the length of time required to develop specific antiviral agents, the World Health Organization adopted the strategy of repurposing existing medications to treat Coronavirus disease 2019 infection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease is possible biological target for potential antiviral drugs. We selected various compounds from PubChem database based on the structure of main protease inhibitors in Protein Data Bank database. Ten compounds showed nontumorigenic and nonmutagenic potential and met Egan's and Lipinski's rules. Molecular docking analysis was performed using AutoDock Vina software. Based on number and type of key binding interactions, as well as docking scores, we selected compounds 6, 8, and 17 that demonstrated the highest binding affinity for the target protein. Molecular dynamics simulations were then carried out on the protein-top docked ligand complexes which were subjected to molecular mechanics/generalized Born and surface area calculations. The molecular dynamics simulation results indicated that protein-top docked ligand complexes showed good conformational stability. Among analyzed molecules, compound 17 emerged as the best in silico hit based on the docking score, MM/GBSA binding energy and MD results.

Benchmarking challenging small variants with linked and long reads.

Genome in a Bottle benchmarks are widely used to help validate clinical sequencing pipelines and develop variant calling and sequencing methods. Here we use accurate linked and long reads to expand benchmarks in 7 samples to include difficult-to-map regions and segmental duplications that are challenging for short reads. These benchmarks add more than 300,000 SNVs and 50,000 insertions or deletions (indels) and include 16% more exonic variants, many in challenging, clinically relevant genes not covered previously, such as PMS2. For HG002, we include 92% of the autosomal GRCh38 assembly while excluding regions problematic for benchmarking small variants, such as copy number variants, that should not have been in the previous version, which included 85% of GRCh38. It identifies eight times more false negatives in a short read variant call set relative to our previous benchmark. We demonstrate that this benchmark reliably identifies false positives and false negatives across technologies, enabling ongoing methods development.

Preanalytical quality improvement: from dream to reality.

Abstract Laboratory diagnostics (i.e., the total testing process) develops conventionally through a virtual loop, originally referred to as "the brain to brain cycle" by George Lundberg. Throughout this complex cycle, there is an inherent possibility that a mistake might occur. According to reliable data, preanalytical errors still account for nearly 60%-70% of all problems occurring in laboratory diagnostics, most of them attributable to mishandling procedures during collection, handling, preparing or storing the specimens. Although most of these would be "intercepted" before inappropriate reactions are taken, in nearly one fifth of the cases they can produce inappropriate investigations and unjustifiable increase in costs, while generating inappropriate clinical decisions and causing some unfortunate circumstances. Several steps have already been undertaken to increase awareness and establish a governance of this frequently overlooked aspect of the total testing process. Standardization and monitoring preanalytical variables is of foremost importance and is associated with the most efficient and well-organized laboratories, resulting in reduced operational costs and increased revenues. As such, this article is aimed at providing readers with significant updates on the total quality management of the preanalytical phase to endeavour further improvement for patient safety throughout this phase of the total testing process.

Quality improvements in the preanalytical phase: focus on urine specimen workflow.

In the past, laboratories have addressed issues of preanalytical variability in an opportunistic way, addressing discrete parts of the preanalytical process, such as patient identification, specimen rejection, and blood/urine culture contamination. To obtain needed quality improvements and error reduction, it is necessary to look at the preanalytical process as a whole - from test ordering to the moment the specimen is processed by the analyzer and apply process improvement methodologies, such as LEAN and Six Sigma. To achieve this, laboratories should map the preanalytical phase in its entirety, identify steps that are potential causes of unnecessary variability that lead can to laboratory errors, and find ways either to remove them or error proof them. At the same time, by using this approach it is possible to reduce unnecessary waste and obtain needed process efficiencies.

Synthesis, characterization and cytotoxic activity of binuclear copper(II)-complexes with some S-isoalkyl derivatives of thiosalicylic acid. Crystal structure of the binuclear copper(II)-complex with S-isopropyl derivative of thiosalicylic acid.

Isoalkyl (isoalkyl = isopropyl-(L1), isobutyl-(L2) and isoamyl-(L3)) derivatives of thiosalicylic acid (TSA) were prepared by alkylation of TSA with corresponding isoalkyl-chlorides in the alkaline water-ethanol solution. The new free copper(II)-complexes with corresponding S-isoalkyl derivatives of TSA (C1-copper(II)-complex with S-isopropyl derivative of thiosalicylic acid, C2-copper(II)-complex with S-isobutyl derivative of thiosalicylic acid and C3-copper(II)-complex with S-isoamyl derivative of thiosalicylic acid) have been synthesized by direct reaction of copper(II)-nitrate with ligand precursor and then characterized by microanalysis, infrared spectra (IR) and EPR (electron paramagnetic resonance) spectra. The spectroscopically predicted structure of the obtained binuclear copper(II)-complex with S-isopropyl derivative of thiosalicylic acid was confirmed by X-ray analysis. Single crystals suitable for X-ray measurements were obtained by slow crystallization from a water solution. Newly synthesized precursors S-isoalkyl derivatives of thiosalicylic acid and corresponding copper(II)-complexes moderately reduced viability of human and murine lung cancer cells, they showed similar cytotoxic effect on human colorectal cancer cells as cisplatin and lower cytotoxic effect than cisplatin toward normal fibroblasts, evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric technique. All new complexes exhibited apoptotic effect toward lung cancer cells, stronger than cisplatin, whereas only C3 induced significant apoptosis of colorectal cancer cells. Complex C1 showed significant antiproliferative effect against murine lung cancer cells, LLC1, while C2 reduced expression of Ki67 in human colorectal cancer cells. All tested complexes induced cell cycle arrest of HCT116 cells in G2/M phase.

A Multicenter Evaluation of a Nongel Mechanical Separator Plasma Blood Collection Tube for Testing of Selected Therapeutic Drugs.

BACKGROUND: Some therapeutic drugs are unstable during sample storage in gel tubes. BD Vacutainer® Barricor™ Plasma Blood Collection Tube with nongel separator was compared with plasma gel tubes, BD Vacutainer PST™, PST II, and BD Vacutainer Serum Tube for acetaminophen, salicylate, digoxin, carbamazepine, phenytoin, valproic acid, and vancomycin during sample storage for up to 7 days. METHODS: Seven hospital sites enrolled 705 participants who were taking at least one selected drug. The study tubes were collected and tested at initial time (0 h), after 48 h of storage at room temperature and on day 7 (after additional 5 days of refrigerated storage). The performance of BD Barricor tube was evaluated for each drug by comparing BD Barricor samples with samples from the other tubes at 0 h from the same participant; stability was evaluated by comparing test results from the same tube at 0 h, 48 h, and 7 days. RESULTS: At 0 h, BD Barricor showed clinically equivalent results for selected therapeutic drugs compared with the other tubes, except phenytoin in BD PST. Phenytoin samples ≥20 µg/mL in BD PST had 10-12% lower values than samples in BD Barricor. During sample storage, all selected drugs remained stable for 7 days in BD Barricor and in serum aliquots. In BD PST, all drugs remained stable except phenytoin and carbamazepine and in BD PST II except for phenytoin. CONCLUSION: The BD Barricor Tube is effective for the collection and storage of plasma blood samples for therapeutic drug monitoring without sample aliquoting.

Quality improvements in the preanalytical phase: focus on urine specimen workflow.

Despite advances in the performance of analytic systems, there still remains a high level of variability in the total testing process. Most laboratory errors currently occur in the preanalytical phase, which is complex because it consists of numerous steps. To reduce the number of errors in the preanalytical phase and achieve targeted quality improvements, particular attention should focus on process improvement and reduction of unnecessary steps. Using the example of urine specimen collection and processing, major causes of preanalytical variability are identified, as are potential areas of process improvement that significantly impact quality of urine testing. Means of achieving them through application of various Lean techniques are also discussed.

Evaluation of lancets for pain perception and capillary blood volume for glucose monitoring.

OBJECTIVE: The purpose of this study was to assess patient pain perception and capillary blood volume of four currently marketed lancets [BD Microtainer Contact-Activated Lancet, Low Flow (Contact-Activated Lancet); LifeScan OneTouch SureSoft Gentle (OneTouch SureSoft Gentle); BD Genie Blue; SurgiLance Safety] in a diabetic population following routine finger-puncture procedures and glucose monitoring. METHODS: Data were collected from adult subjects diagnosed with type I or type II diabetes mellitus at a 300-bed US hospital following finger-puncture procedures for glucose monitoring. Based on quantitative and qualitative measurements, each blood collection device was evaluated for pain perception and calculated total capillary blood volume. RESULTS: A total of 80 subjects received four skin punctures in an alternating finger and hand sequence using each lancet. The ten clinicians (nurses and phlebotomists) conducted the study, collected and then calculated total capillary blood volume. It was determined that the Contact-Activated Lancet produced less perceived pain and bleeding, while obtaining an adequate capillary blood volume for glucose monitoring. CONCLUSION: This study demonstrated that the Contact-Activated Lancet provided an adequate sample volume required for blood glucose monitoring. In addition, less perceived pain was elicited with this lancet when compared with the other lancets evaluated in the study.