Enzymatic Tests Research Articles

Effect of different carbon sources and concentrations on in vitro propagation of chestnut

The in vitro propagation of chestnut is a crucial approach to address the limitations of traditional propagation methods. It encounters issues like insufficient rooting and multiplication. This research investigated the impact of sucrose, glucose, maltose and concentrations (20 g/L and 30 g/L) on the growth and biochemical profiles of the hybrid chestnut cultivar ‘Marsol’ (C. sativa × C. crenata). Essential parameters encompassed plant height, leaf area, callus size, chlorophyll content, enzymatic activity, and secondary metabolite composition determined using GC-MS analysis. For each carbon source and concentration, thirty-six biological replicates were analyzed. The findings underlined the essential function of sucrose, especially at 30 g/L, in enhancing plant height (5.30 ± 0.04 cm), shoot multiplication (4.50 ± 0.50 shoots per explant), and the coloration of leaves (minimum L value of 35.78 ± 0.27, maximum SPAD value of 13.21 ± 1.27). Glucose similarly promoted growth but to a slightly lower degree, whereas maltose continuously exhibited inferior performance. The callus’s diameters, a crucial indicator of regeneration potential, were greatest with 30 g/L sucrose (1.664 ± 0.26 cm width), highlighting its effectiveness in cellular differentiation. GC-MS analysis demonstrated various biochemical profiles affected by the carbon source. Sucrose treatments showed elevated concentrations of pyrogallol and hexadecanoic acid derivatives associated with stress tolerance and metabolic control. Furthermore, enzymatic tests revealed that the carbon source and concentration influenced phenol oxidase (PO) and catalase (CAT) activity, with sucrose producing ideal equilibria between oxidative stress and antioxidant responses. The findings provide critical insights for optimizing the nutritional medium to improve propagation efficiency for important chestnut cultivars/genotypes.

Exploiting unique NP1 interface: Oriented immobilization via electrostatic and affinity interactions in a tailored PDA/PEI microenvironment enhanced by concanavalin A.

Enzyme immobilization techniques are crucial for enhancing enzyme stability and catalytic efficiency. Traditional methods such as physical adsorption and simple covalent binding often fail to maintain enzyme activity and stability. In this study, an innovative multi-level immobilization strategy was proposed to achieve efficient targeted immobilization of nuclease P1 (NP1) by fine-tuning the surface microenvironment. Molecular simulation results revealed that the distinctive electrostatic distribution and the specific placement of basic amino acids, such as lysine, on the NP1 surface caused dopamine to preferentially adsorb on areas away from NP1's active site. This selective adsorption facilitated the directed immobilization of NP1, while the positively charged environment generated by the co-deposited surface further enhanced NP1's adsorption capacity. This multilevel modification was found to significantly optimize the physicochemical environment of the immobilized surface through surface characterization and enzymatic testing. This strategy greatly improves enzyme activity (3590.0 U/mg), stability, and reusability (70% after 10 cycles). In particular, NP1 on this surface exhibited an optimal Michaelis constant (Km) of 34.0mM and a maximum reaction rate of 5.5mMmin-1, demonstrating the remarkable effect of the modification strategy in enhancing the enzyme catalytic performance. The present study provides an efficient and stable immobilization platform for enzyme catalytic applications by precisely modulating the surface microenvironment and the oriented immobilization strategy, which has an important potential for practical applications. This stable and reusable NP1 platform allows for efficient DNA/RNA cleavage, facilitating its application in industrial biocatalysis, biomedical enzyme-based processes, and biosensors.

Relevance of muscle biopsies in the neonatal and early infantile period: a 52 years retrospective study in the gene-sequencing era

Neuromuscular disorders (NMD) with neonatal or early infantile onset are usually severe and differ in symptoms, complications, and treatment options. The establishment of a diagnosis relies on the combination of clinical examination, morphological analyses of muscle biopsies, and genetic investigations. Here, we re-evaluated and classified a unique collection of 535 muscle biopsies from NMD infants aged 0–6 months examined over a period of 52 years. We aimed to assess the importance and contribution of morphological muscle biopsy analyses for the establishment of a precise and accurate molecular diagnosis. Altogether, 82% of the biopsies showed typical structural myofiber anomalies highly suggestive of specific NMD classes (congenital myopathies, metabolic myopathies, lower motor neuron (LMN) and neuromuscular junction (NMJ) disorders, muscular dystrophies, inflammatory myopathies), while the remaining 18% showed no or only non-specific histological abnormalities. The diagnostic success rate differed among the NMD classes and was particularly high for congenital myopathies as illustrated by the identification of causative genes in 61% of cases. This is essentially due to the presence of characteristic histopathological hallmarks on biopsies visible by light or electron microscopy often pointing to specific genes. In contrast, metabolic myopathies commonly displayed non-specific features on muscle sections, led to the identification of causative genes in only 19% of the patients, and typically required additional enzymatic tests to establish a more precise diagnosis. The evolution of sequencing technologies fundamentally improved molecular diagnosis and also shifted the relevance of muscle biopsies within the diagnostic process. Depending on the clinical presentation of the patients, direct gene or panel sequencing may be the preferred method nowadays. However, histological and ultrastructural examinations of muscle sections are still frequently useful and can constitute an elemental step in the diagnostic process—either by directing purposeful gene sequencing or pointing to genes and pathogenic variants identified by next-generation sequencing (NGS), or by complementing clinical findings and biochemical analysis methods.

Oxidative damage and mitochondrial dysfunction in cystathionine beta-synthase deficiency

Cystathionine beta-synthase deficiency (CBSD) is the most prevalent inherited disorder of homocysteine metabolism in the transsulphuration pathway. Research have suggested oxidative stress and inflammation as candidate pathogenic mechanisms in CBSD. This study aims to evaluate mitochondrial dysfunction and oxidative stress biomarkers in cystathionine beta-synthase deficiency (CBSD) patients, which may aid in understanding the pathogenesis of CBSD and improving treatment. The study group comprised 23 patients with a diagnosis of CBSD and healthy controls. We analysed serum levels of NAD+ and NADH by fluorometric assay, FGF-21 and GDF-15 by ELISA, mitochondrial DAMPs by real time qRT-PCR, total homocysteine levels in plasma by enzymatic test and compared the results in CBSD group with healthy controls. In patient group, a positive correlation was found between the total homocysteine level and both GDF-15 and NAD+/NADH levels. Furthermore, there was a negative correlation between total homocysteine levels and both total NAD++NADH and NADH levels. The alterations in NAD+, FGF-21, GDF-15 levels, and NAD+/NADH ratio in patients suggest that oxidative damage coexists with mitochondrial dysfunction in CBSD. Assessment of oxidative damage and addition of anti-oxidant therapy together with mitochondrial support may have additional benefits in reducing long-term morbidity in CBSD patients.

Multivariate statistical analysis as a tool for monitoring drinking water sources in an Atlantic Rainforest Conservation Unit.

Water quality monitoring is paramount in identifying and mitigating pollution sources, protecting aquatic ecosystems, and ensuring safe water for human and wildlife consumption. This study is aimed at evaluating the quality of drinking water sources in three communities located in a Sustainable Use Conservation Unit in the municipality of Mangaratiba, Rio de Janeiro, Brazil, employing a multivariate statistical analysis. A total of 161 water samples were collected from January to December 2022, encompassing 32 surface water and 129 tap water samples. Physicochemical parameters were determined in situ employing a Horiba U50 multiparameter probe. The samples were stored and transported at 4°C to the laboratory for microbiological analyses concerning total coliforms and Escherichia coli using a commercial enzymatic test. All samples contained coliforms, while E. coli were detected in 87% of the samples. The multivariate analysis indicated that the microbiological water quality in sampling region R2 was influenced by rainy periods and that, in general, the water quality within R3 was the most affected by the transport of solids to the water sources. The statistical methods applied herein aided in characterizing the study areas and detecting points of attention regarding physicochemical and microbiological parameters that significantly influence the water quality of each sampling point. Representative points for each study region were identified and may be employed for future monitoring and prevention actions.

Unexpected increase in microalgal removal of doxylamine induced by bicarbonate addition: synergistic chem-/bio-degradation mechanisms

Microalgae-based approaches serve as promising methods for the remediation of pharmaceutical contaminants (PCs) compared to conventional wastewater treatment processes. However, how to decrease hydraulic retention times of the microalgal system currently has been one of the main bottlenecks. This study constructed an unexpected synergistic extra-chemical/intra-biological degradation system by adding 5.95 mM bicarbonate to the microalgal system, which achieved complete removal (100%) of a representative PC, doxylamine (DOX) in 96 h, compared to that 192 h in the control. Removal capacities and mass balance analyses demonstrated that biodegradation rate per unit microalgal density was significantly increased by 207%. Further analyses using transcriptomic, enzymatic inhibiting tests, and high-resolution mass spectrometry revealed that after addition of bicarbonate for metabolism of DOX, a hydrolase (CYP97C1) and a primary amine oxidase (TynA) can transform DOX into doxylamine N-oxide and an intermediate (C15H17NO2) with a m/z of 244.1335. Meanwhile, bicarbonate reacted with microalgae-excreted hydrogen peroxide to form more oxidative radicals such as superoxide and hydroxyl radicals extracellularly, which promised the extracellular degradation of DOX according to the oxidative radical inhibiting tests. Further investigation showed addiing bicarbonate to the microalgal system improved the removal rate of 17 PCs by up to 500.8%. Therefore, this study not only developed an approach to enhance treatment efficiencies of diverse PCs by microalgae within a shorter time, but also carried unique mechanistic insights into the underlying principles.

In vitro study of the gel cohesivity and persistence to hyaluronidase degradation of a novel stabilized composition of 26 mg/mL of high molecular weight HA

Aim: Hyaluronic acid (HA) is extensively used in injectable skin quality products due to its documented role in skin rejuvenation. The rapid in vivo degradation of HA by the enzyme hyaluronidase necessitates the development of advanced formulations to ensure the efficacy and longevity of the treatments. In this context, a novel 2.6% high molecular weight HA (H-HA)/3.2% sorbitol composition has been introduced, featuring stabilization through hydrogen bonds rather than traditional crosslinking. Methods: The stabilized composition was evaluated through two in vitro enzymatic degradation tests. In the first test, the efficiency on the gel degradation was followed by rheology and compared with two crosslinked HA products available on the market. In the second test, the effect on the gel structure of a less diluted hyaluronidase dose was followed by rheological and cohesivity measurements. Results: In vitro study demonstrates that, before its complete degradation into a liquid-like state, the stabilized composition exhibits high elasticity and cohesivity during the enzymatic degradation process, surpassing traditional crosslinked HA products. Conclusion: The stabilization provided by the sorbitol in the stabilized composition effectively enhances product properties and protects them during gel degradation. These attributes indicate significant potential for improved clinical outcomes in skin quality treatments.

Serum selenium, selenoprotein P and glutathione peroxidase 3 in rheumatoid, psoriatic, juvenile idiopathic arthritis, and osteoarthritis

Selenoprotein P (SELENOP) controls selenium (Se) transport, and glutathione peroxidase 3 (GPx3) elicits antioxidant activity in blood. Inflammation associates with Se deficiency, but knowledge concerning selenoproteins in inflammatory rheumatic musculoskeletal diseases (iRMD) is limited. We compared three Se biomarkers in patients with rheumatoid (RA), psoriatic (PsA), and juvenile idiopathic arthritis (JIA) in comparison to osteoarthritis (OA) and healthy subjects, to improve the data base on selenoprotein expression in iRMD.The cross-sectional study enrolled n=272 patients with RA (n=131), PsA (n=67), JIA (n=22) and OA (n=52). Serum Se was quantified by total reflection X-ray fluorescence, SELENOP by ELISA and GPx3 by an enzymatic test. Data from the EPIC trial served as reference. Impairment of daily life was assessed by the Functional Ability Questionnaire (FfbH).Serum SELENOP and Se concentrations correlated linearly in all groups and were below the average measured in EPIC. Se concentration was not different between the patient groups. Compared to controls, SELENOP levels were low in iRMD patients. GPx3 activity was particularly low in JIA and PsA. Seropositive but not seronegative RA patients displayed a disrupted interaction between GPx3 and Se or SELENOP. SELENOP associated with the functional status measured by the FfbH, most pronounced in OA (R=0.76, P < .01).The data indicate selenoprotein deficiency in the majority of patients with iRMD, and a positive relation of SELENOP with functional status in OA. Since increased Se supply improves selenoprotein biosynthesis, a personalized correction of diagnosed deficiency merits consideration to improve Se transport and ameliorate disease burden.

Salivary cysteine levels as a potential biochemical indicator of oral cancer risk in tobacco consumers.

Aim: Oral cancer is the leading cause of mortality, with a survival rate of less than 5 years, and is predominantly influenced by tobacco mutagens. Invasive diagnostic methods hinder early detection of oral cancer biomarkers. The present study performed salivary biochemical analysis for early oral cancer screening in tobacco consumers.Materials & methods: Three study groups included healthy controls (n=25), tobacco users (n=25) and oral cancer patients (n=25). Salivary total protein, amylase, TNF-α and amino acid levels were evaluated using enzymatic tests, Enzyme linked Immunosorbent Assay (ELISA) and High-Performance Liquid Chromatography (HPLC).Results: Compared with healthy controls, salivary total protein and TNF-αlevels were significantly (p=0.04) higher in oral cancer patients. Salivary amylase levels were significantly lower in tobacco smokers (p=0.02) and higher in oral cancer patients (p=0.01). Interestingly, the amino acid cysteine concentration was significantly higher (p=0.02) in tobacco consumers (62.5±10) than in healthy controls (116.1±28).Conclusion: In high-risk populations, such as tobacco users, salivary biochemical analysis can serve as a promising noninvasive diagnostic method for early oral cancer screening. As a salivary biomarker, the amino acid cysteine exhibits potential as a means of detecting the progression of oral cancer in individuals who consume tobacco.

Whole genome sequencing and characterization of Corynebacterium isolated from the healthy and dry eye ocular surface

BackgroundThe purpose of this study was to characterize Corynebacterium isolated from the ocular surface of dry eye disease patients and healthy controls. We aimed to investigate the pathogenic potential of these isolates in relation to ocular surface health. To this end, we performed whole genome sequencing in combination with biochemical, enzymatic, and antibiotic susceptibility tests. In addition, we employed deferred growth inhibition assays to examine how Corynebacterium isolates may impact the growth of potentially competing microorganisms including the ocular pathogens Pseudomonas aeruginosa and Staphylococcus aureus, as well as other Corynebacterium present on the eye.ResultsThe 23 isolates were found to belong to 8 different species of Corynebacterium with genomes ranging from 2.12 mega base pairs in a novel Corynebacterium sp. to 2.65 mega base pairs in C. bovis. Whole genome sequencing revealed the presence of a range of antimicrobial targets present in all isolates. Pangenome analysis showed the presence of 516 core genes and that the pangenome is open. Phenotypic characterization showed variously urease, lipase, mucinase, protease and DNase activity in some isolates. Attention was particularly drawn to a potentially new or novel Corynebacterium species which had the smallest genome, and which produced a range of hydrolytic enzymes. Strikingly the isolate inhibited in vitro the growth of a range of possible pathogenic bacteria as well as other Corynebacterium isolates. The majority of Corynebacterium species included in this study did not seem to possess canonical pathogenic activity.ConclusionsThis study is the first reported genomic and biochemical characterization of ocular Corynebacterium. A number of potential virulence factors were identified which may have direct relevance for ocular health and contribute to the finding of our previous report on the ocular microbiome, where it was shown that DNA libraries were often dominated by members of this genus. Particularly interesting in this regard was the observation that some Corynebacterium, particularly new or novel Corynebacterium sp. can inhibit the growth of other ocular Corynebacterium as well as known pathogens of the eye.

Correlation between diet, body mass index, and lipid profile in Kosovar women treated for primary infertility

The increasing prevalence of female fertility requires the identification of potentially modifiable factors and non-pharmacological treatments. There is strong evidence that healthy preconception dietary patterns of women of reproductive age have a beneficial effect on fertility. In accordance with the hypothesis, the aim of the study is to evaluate the influence of diet on overweight and obesity in infertile women as well as the correlation of serum lipid concentration by making a comparison between three observed groups with different body mass index (BMI). A total of 107 women who were treated for infertility were investigated. Diet and food pattern survey, BMI, and lipidogram determination from venous blood samples were carried out. The determination of lipidogram parameters was carried out by enzymatic colorimetric test with the COBAS INTEGRA 400 apparatus. From the total number of patients included in the study (107), infertile women with abnormal BMI accounted for 26.16%, while fertile women with normal BMI accounted for 73.83% (p&lt;0.001). The total cholesterol value for groups with BMI&gt;25 kg/m2 was 6.78 mmol/L (p=0.031). High triglycerides with increased BMI were observed (2.27 mmol/L versus 2.68 mmol/L) (p=0.028). Low-density lipoprotein-cholesterol had higher concentrations in obese women (n=28), and high-density lipoprotein-cholesterol had lower concentrations in women with BMI&gt;25 km2 (p&lt;0.05). Western dietary patterns of nutrition results in obesity and increased BMI values in infertile women. Increased BMI has a positive correlation with lipid profile in obese infertile women.

High-risk screening for late-onset Pompe disease in China: An expanded multicenter study.

Late-onset Pompe disease (LOPD) is caused by a genetic deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA), leading to progressive limb-girdle weakness and respiratory impairment. The insidious onset of non-specific early symptoms often prohibits timely diagnosis. This study aimed to validate the high-risk screening criteria for LOPD in the Chinese population. A total of 726 patients were included, including 96 patients under 14 years of age. Dried blood spots (DBS) and tandem mass spectrometry (MS/MS) were employed to evaluate serum GAA activity. Forty-four patients exhibited a decreased GAA activity, 16 (2.2%) of which were confirmed as LOPD by genetic testing. Three previously unreported GAA mutations were also identified. The median diagnostic delay was shortened to 3 years, which excelled the previous retrospective studies. At diagnosis, most patients exhibited impaired respiratory function and/or limb-girdle weakness. Elevated serum creatine kinase (CK) levels were more frequently observed in patients who manifested before age 16. Overall, high-risk screening is a feasible and efficient method to identify LOPD patients at an early stage. Patients over 1 year of age with either weakness in axial and/or proximal limb muscles, or unexplained respiratory distress shall be subject to GAA enzymatic test, while CK levels above 2 times the upper normal limit shall be an additional criterion for patients under 16. This modified high-risk screening criteria for LOPD requires further validation in larger Chinese cohorts.

Fabrication of small-diameter in situ tissue engineered vascular grafts with core/shell fibrous structure and a one-year evaluation via rat abdominal vessel replacement model

A high vascular patency was realized in the bulk or surface heparinized small-diameter in situ tissue-engineered vascular grafts (TEVGs) via a rabbit carotid artery replacement model in our previous studies. Those surface heparinized TEVGs could reduce the occurrence of aneurysms, but with a low level of the remodeled elastin, whereas those bulk heparinized TEVGs displayed a faster degradation and an increasing occurrence of aneurysms, but with a high level of the regenerated elastin. To combine the advantages of the bulk and surface graft heparinization to boost the remodeling of elastin and defer the occurrence of aneurysms, a coaxial electro-spinning technique was used to fabricate a kind of small-diameter core/shell fibrous structural in situ TEVGs with a faster degradable poly(lactic-co-glycolic acid) (PLGA) as a core layer and a relatively lower degradable poly(ε-caprolactone) (PCL) as a shell layer followed by the surface heparinization. The in vitro mechanical performance and enzymatic degradation tests revealed the resulting PLGA@PCL-Hep in situ TEVGs possessing not only a faster degradation rate, but also the mechanical properties comparable to those of human saphenous veins. After implanted in the rat abdominal aorta for 12 months, the good endothelialization, low inflammation, and no calcification were evidenced. Furthermore, the neointima layer of regenerated new blood vessels was basically constructed with a well-organized arrangement of elastin and collagen proteins. The results showed the great potential of these in situ TEVGs to be used as a novel type of long-term small-diameter vascular grafts.

Improvement of bioanalytical parameters through automation: suitability of a hand-like robotic system.

Commercial automation systems for small- and medium-sized laboratories, including research environments, are often complex to use. For liquid handling systems (LHS), development is required not only for the robot's movements but also for adapting the bioanalytical method to the automated system. This study investigates whether a more human-like automation strategy-using a robotic system (RS)-is more suitable for research laboratories than a professional automation approach utilizing a commercial automated LHS. We conducted a series of measurements for protein determination using a Bradford assay manually, with a fully automated LHS, and with our human-like RS. Although the hand-like RS approach requires more than twice the time of the LHS, it achieved the best standard deviation in this setup (RS = 0.5, manual = 0.71, LHS = 0.86). Due to the low limit of detection (LOD) and limit of quantification (LOQ), most protein samples could be quantified with the RS (samples below LOQ = 9.7%, LOD = 0.23; LOQ = 0.25) compared to manual (samples below LOQ = 28.8%, LOD = 0.24; LOQ = 0.26) and the LHS (samples below LOQ = 36.1%, LOD = 0.27; LOQ = 0.31). In another time-dependent enzymatic assay test, the RS achieved results comparable to the manual method and the LHS, although the required time could be a constraint for short incubation times. Our results demonstrate that a more hand-like automation system closely models the manual process, leading easier to accurate bioanalytical results. We conclude that such a system could be more suitable for typical research environments than a complex LHS.

Effect of the crystal orientation on the enzymatic degradation rate of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) fibers

In this study, the relationship between the crystal orientation and enzymatic degradation rate of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) was investigated. The enzymatic degradation rates of melt-spun PHBH fibers were quantified via PHB depolymerase from Ralstonia pickettii T1, and the degradation rate decreased with increasing drawing ratio. The solid structure was investigated via differential scanning calorimetry and small-angle X-ray scattering, and kinetic analysis was performed, indicating that the degradation rate was affected not only by crystallinity and lamellar thickness but also by highly ordered structure. The highly ordered structure was examined via wide-angle X-ray diffraction. The enzymatic degradation rate from the fiber cross-sections was then quantified for the first time, and it increased with drawing ratio and was larger than that from the fiber sides. This result showed that the degradation rate varied depending on the crystal orientation. Furthermore, an enzymatic degradation test of PHBH ring-banded spherulites was conducted, and it was found that the degradation rate of flat-on lamellar crystals was greater than that of edge-on lamellar crystals. This is the first study to quantitatively evaluate the relationship between the crystal orientation and the enzymatic degradation rate.

A new strategy for the preparation of polylactic acid composites with UV resistance, light conversion, and antibacterial properties

A novel rare earth complex, Eu(IAA)2(phen)2 (EuIP), was synthesized by solution-based synthesis method. Then, EuIP and polylactic acid (PLA) were melt-blended at 190 °C to obtain a multifunctional PLA/EuIP composite. The incorporation of EuIP provided PLA/EuIP composites with good light conversion ability. Under UV irradiation, PLA/EuIP composites converted the absorbed UV light into red light. Moreover, the PLA/1.0EuIP composite exhibited excellent light transmittance of 88 % in the visible region and showed strong red emission under UV light. After UV irradiation for 96 h, the molecular weights and mechanical properties of neat PLA decreased dramatically. Interestingly, the molecular weights and mechanical properties of PLA/EuIP composites did not deteriorate after 96 h of UV irradiation. The reason was that EuIP could absorb UV light and utilize the absorbed energy to emit red fluorescence. Furthermore, PLA/EuIP composites showed good antibacterial activities against E. coli and S. aureus. In addition, in vitro cell experiments showed that PLA/EuIP composites was suitable for the growth of murine breast cancer (4 T1) cells. Besides, enzymatic degradation testing also proved that PLA/EuIP composites had good biodegradability. This work provides an ingenious design strategy for the preparation of PLA/EuIP composites possessing light conversion ability, UV resistance, and antibacterial properties.

Serum ceruloplasmin oxidase activity: A neglected diagnostic biomarker for Wilson disease

BackgroundLow serum ceruloplasmin concentration is considered robust marker for Wilson disease (WD) screening, measuring serum ceruloplasmin oxidase activity might be an even more valuable diagnostic tool, but it has not been sufficiently studied. MethodsAll patients who were assessed for serum ceruloplasmin oxidase activity between January 1, 2016, and September 2, 2019, were enrolled in this study. The diagnostic performance of serum ceruloplasmin oxidase activity was analyzed using receiver operating characteristic curve analysis (ROC), Spearman's rank correlation, and Mann-Whitney U test. ResultsSerum ceruloplasmin oxidase activity was significantly decreased in WD patients (0.87 U/L, IQR 0.61–1.54). The optimal cut-off of serum ceruloplasmin oxidase activity to identified WD is 7 U/L, with sensitivity and specificity of 97.03 % and 98.19 %, respectively. Furthermore, this study revealed a positive correlation between enzymatic and immunoreactive serum ceruloplasmin tests. As primary diagnostic methods, serum ceruloplasmin levels below the diagnostic cut-offs for either the enzymatic or immunoreactive tests were observed in 818 out of 842 WD patients (97.15 %). Compared with the presence of K-F rings in asymptomatic patients, the accuracy of serum ceruloplasmin tests was significantly higher (56.12 % VS 95.08 %). Moreover, the positive rate of cranial MRI in neurological patients was similar to the tests of serum ceruloplasmin (92.91 % VS 97.40 %). Moreover, 71 patients had ambiguous genetic results, complicating the diagnosis. However, serum ceruloplasmin tests successfully identified 65 out of these 71 patients (91.55 %). ConclusionSerum ceruloplasmin oxidase activity has excellent performance in diagnosing WD, which should be widely used as preferred test in WD patients.

Analytical and operational considerations of measuring glucose 6-phosphate dehydrogenase (G6PD) activity using a fully automated assay.

This study determined the performance characteristics of a quantitative glucose-6-phosphate dehydrogenase (G6PD) assay with automated lysis and evaluated the robustness of the operational workflow following implementation in a hospital laboratory. The G6PD activity was measured in whole blood using an enzymatic quantitative test on a Roche cobas c501 analyzer with onboard lysis configuration and normalized to hemoglobin (Hb). The performance characteristics of the method and stability of G6PD in whole blood collected in EDTA-containing tubes were evaluated, and the reference interval was established on a population of healthy individuals (n = 279). The robustness of this automated workflow for sample lysis was evaluated during validation and after implementation for routine clinical use for 18 months and in 2,181 patients. The G6PD assay was linear from 0.7 to 16.5 U/g Hb. Inter- and intra-assay precision using control and patient samples was below 12%. The G6PD results correlated well with a reference laboratory method (r = 0.96, y = 0.9615x - 1.222). The reference interval in our population was 9.8 to 15.5 U/g Hb. There were no interferences by lipemia and icteria, although grossly hemolyzed specimens may be affected. The testing workflow requires analyzing samples within minutes from mixing and loading into the instrument to avoid sample sedimentation. Measures to repeat samples with Hb 8.0g/dL or less identified sedimented samples. In our patient population, 10.6% and 5.8% of the total males and females tested were G6PD deficient, respectively. The G6PD assay with automated lysis is acceptable for patient testing. Several measures ensured the robustness of this workflow in a hospital laboratory.

Quantification of Ions in Human Urine-A Review for Clinical Laboratories.

Urine is an organic fluid produced by the kidney, and its analysis is one of the most requested laboratory tests by clinicians. The ionic composition of urine has been shown to be a good health indicator: it is useful for the diagnosis of several diseases, as well as monitoring therapeutics. This review considers laboratorial techniques that have been used throughout time for the quantification of ions in urine, and also considers some methodologies that can potentially be used in clinical laboratories for this kind of analysis. Those methods include gravimetry, titration, flame emission spectrophotometry (flame photometry), fluorimetry, potentiometry (ion selective electrodes), ion chromatography, electrophoresis, kinetic colorimetric tests, enzymatic colorimetric tests, flow cytometry, atomic absorption, plasma atomic emission spectrometry, and paper-based devices. Sodium, potassium, chloride, calcium, and magnesium are among the most important physiological ions, and their determination is frequently requested in hospitals. There have been many advances regarding the analysis of these ions in 24 h urine. However, there is still some way to go concerning the importance of intracellular ions in this type of sample as well as the use of occasional urine for monitoring these parameters.

Phenotypic and Genotypic Characterization of Resistance and Virulence Markers in Candida spp. Isolated from Community-Acquired Infections in Bucharest, and the Impact of AgNPs on the Highly Resistant Isolates.

This study aimed to determine, at the phenotypic and molecular levels, resistance and virulence markers in Candida spp. isolated from community-acquired infections in Bucharest outpatients during 2021, and to demonstrate the efficiency of alternative solutions against them based on silver nanoparticles (AgNPs). A total of 62 Candida spp. strains were isolated from dermatomycoses and identified using chromogenic culture media and MALDI-TOF MS, and then investigated for their antimicrobial resistance and virulence markers (VMs), as well as for metabolic enzymes using enzymatic tests for the expression of soluble virulence factors, their biofilm formation and adherence capacity on HeLa cells, and PCR assays for the detection of virulence markers and the antimicrobial activity of alternative solutions based on AgNPs. Of the total of 62 strains, 45.16% were Candida parapsilosis; 29.03% Candida albicans; 9.67% Candida guilliermondii; 3.22% Candida lusitaniae, Candia pararugosa, and Candida tropicalis; and 1.66% Candida kefyr, Candida famata, Candida haemulonii, and Candida metapsilosis. Aesculin hydrolysis, caseinase, and amylase production were detected in the analyzed strains. The strains exhibited different indices of adherence to HeLa cells and were positive in decreasing frequency order for the LIP1, HWP1, and ALS1,3 genes (C. tropicalis/C. albicans). An inhibitory effect on microbial growth, adherence capacity, and on the production of virulence factors was obtained using AgNPs. The obtained results in C. albicans and Candida non-albicans circulating in Bucharest outpatients were characterized by moderate-to-high potential to produce VMs, necessitating epidemiological surveillance measures to minimize the chances of severe invasive infections.