Multi-target potential of newly designed tacrine-derived cholinesterase inhibitors: Synthesis, computational and pharmacological study.

Simple and scalable synthetic approach was used for the preparation of thirteen novel tacrine derivatives consisting of tacrine and N-aryl-piperidine-4-carboxamide moiety connected by a five-methylene group linker. An anti-Alzheimer disease (AD) potential of newly designed tacrine derivatives was evaluated against two important AD targets, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). In vitro pharmacological evaluation showed strong ChE inhibitory activity of all compounds, with IC50 values ranging from 117.5 to 455 nM for AChE and 34 to 324 nM for BuChE. As a representative of the series with the best cytotoxicity / ChE inhibitory activity ratio, expressed as the selectivity index (SI), 2-chlorobenzoyl derivative demonstrated mixed-type inhibition on AChE and BuChE, suggesting binding to both CAS and PAS of the enzymes. It also exhibited antioxidant capacity and neuroprotective potential against amyloid-ß (Aß) toxicity in the culture of neuron-like cells. In-depth computational analysis corroborated well with in vitro ChE inhibition, illuminating that all compounds exhibit significant potential in targeting both enzymes. Molecular dynamics (MD) simulations revealed that 2-chlorobenzoyl derivative, created complexes with AChE and BuChE that demonstrated sufficient stability throughout the observed MD simulation. Computationally predicted ADME properties indicated that these compounds should have good blood-brain barrier (BBB) permeability, an important factor for CNS-targeting drugs. Overall, all tested compounds showed promising pharmacological behavior, highlighting the multi-target potential of 2-chlorobenzoyl derivative which should be further investigated as a new lead in the drug development process.

Prevalence of Carcinogenic Genotypes of HPV-Infected Women in a Ten-Year Period (2014-2023) in Vojvodina, Serbia.

Background and Objectives: Human papillomavirus (HPV) infection and its etiological role in the development of cervical cancer are well established. The cervical cancer mortality rate in Serbia is one of the highest among European countries, and this cancer is the second-leading cause of death in Serbian women aged from 15 to 44. Materials and Methods: This retrospective study was conducted at the Institute of Public Health of Vojvodina. A total of 10,062 cervical specimens from Serbian women were collected and HPV tested in ten years. The study patients were divided into five age groups. HPV genotype testing was performed using a commercial kit to detect 14 high-risk (HR) HPV genotypes. Additionally, cervix cytology data have been available for patients tested in 2022 and 2023. Results: An overall positive rate was found in 43.3% of patients (4356/10,062). A single HPV infection (62.1%) was the main infection pattern. The most frequent HR HPV genotypes were HPV 16, 31, 52, 56, 39, and 51, comprising 62.3% of the detected genotypes, including multiple infections. A significant difference was noted in the HPV prevalence across the different age groups, with a bimodal distribution of HPV infection. The highest prevalence was recorded in the age group ≤ 30 and those after 61 years. Women diagnosed with high-grade squamous intraepithelial lesions (HSIL) were significantly older compared to others. HR HPV is the most prevalent in patients with HSIL cytological findings (76.5%). The most common type, according to age-specific distribution and cytological findings, was HR HPV 16. Conclusions: This study provides comprehensive data on HR HPV distribution among Serbian women, which can serve as a basis for subsequent monitoring of genotypic distribution. It is particularly significant considering they are missing in the updated ICO/IARC Report for Serbia, and the cervical cancer mortality rate in Serbia is one of the highest among European countries.

Effects of early life overnutrition and hyperandrogenism on spatial learning and memory in a rat model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a complex disorder characterized by endocrine and metabolic abnormalities such as obesity and insulin resistance. PCOS is also associated with psychiatric disorders and cognitive impairment. The animal model of PCOS was induced by treating rats with 5α-dihydrotestosterone (5α-DHT) and additionally modified to induce adiposity by litter size reduction (LSR). Spatial learning and memory were assessed using the Barnes Maze test, and striatal markers of synaptic plasticity were analyzed. Striatal insulin signaling was estimated by the levels of insulin receptor substrate 1 (IRS1), its inhibitory phosphorylation at Ser307, and glycogen synthase kinase-3α/ß (GSK3α/ß) activity. Both LSR and DHT treatment significantly decreased striatal protein levels of IRS1, followed by increased GSK3α/ß activity in small litters. Results of the behavioral study showed that LSR had a negative effect on learning rate and memory retention, whereas DHT treatment did not induce impairment in memory formation. While protein levels of synaptophysin, GAP43, and postsynaptic density protein 95 (PSD-95) were not altered by the treatments, DHT treatment induced an increase in phosphorylation of PSD-95 at Ser295 in both normal and small litters. This study revealed that LSR and DHT treatment suppressed insulin signaling by downregulating IRS1 in the striatum. However, DHT treatment did not have an adverse effect on learning and memory, probably due to compensatory elevation in pPSD-95-Ser295, which had a positive effect on synaptic strength. This implies that hyperandrogenemia in this setting does not represent a threat to spatial learning and memory, opposite to the effect of overnutrition-related adiposity.

Human papillomavirus-specific antibody status among unvaccinated subjects in the region of Vojvodina, Serbia.

OBJECTIVES: The aim of the study was to evaluate the immune status of young people from the Vojvodina province, Serbia, through the detection of IgG antibodies specific for the L1 protein of HPV types 6, 11, 16, and 18 contained in quadrivalent vaccine. METHODS: The study enrolled 514 healthy persons of both genders, aged between 18 and 30 years. All potential participants were informed about the project's aims by trained interviewers before venous blood collection. Also, participants completed a specially designed anonymous questionnaire to identify socio-demographic characteristics and individual behaviours associated with HPV seroprevalence. VPL HPV L1-specific IgG antibodies were measured using a semi-quantitative HPV IgG ELISA kit (Dia.Pro, Italy). RESULTS: A total of 472 (91.8%) young subjects had no detectable antibodies against high- and low-risk HPV types covered by the quadrivalent vaccine. A slightly higher number of seropositive individuals were detected in the age group of 26-30 years compared to younger than 25. Multivariate analysis showed that the number of lifetime sexual partners was the most powerful predictor of HPV seropositivity (OR = 3.483, 95% CI: 1.294-9.379). CONCLUSIONS: Obtained data point out low levels of naturally induced HPV-specific serum antibodies among the target population in the Vojvodina province. The present work highlights the significance and potential benefits of HPV vaccination. Routine HPV vaccination should be the public health priority in our country and should be included in the national immunization programme as soon as possible.

Sulfur dioxide and exacerbation of allergic respiratory diseases: A time-stratified case-crossover study.

BACKGROUND: Strong epidemiological evidence suggests that air pollution plays a significant role in the exacerbation of allergic respiratory diseases. This study aimed to assess the potential relationship between daily levels of sulfur dioxide (SO2) and emergency department (ED) visits for allergic diseases. MATERIALS AND METHODS: Data regarding ED visits for allergic respiratory diseases were routinely collected from the EDs in the Zlatibor district, and the General Hospital, Uzice. The daily average concentrations of SO2 were obtained from the regional automatic air quality monitoring stations. All data were collected from June 2012 to July 2014. A time-stratified case-crossover design was used. Crude odds ratios (ORs) and ORs adjusted for weather conditions were calculated using conditional logistic regression. RESULTS: Statistically significant associations were seen between 0-day lagged exposure to SO2 and ED visits for all allergic diseases (OR = 1.62; 95% confidence interval [CI]: 1.05-2.48; P = 0.028) and between 2-day lagged exposure to SO2 and ED visits for asthma with allergic rhinitis (OR = 2.00; 95% CI: 1.03-3.88; P = 0.042). These results were adjusted for temperature, temperature2, and humidity. CONCLUSION: Our results suggest that short-term exposure to SO2 conferred an increased risk of ED visits for allergic respiratory diseases, particularly for asthma with concomitant allergic rhinitis.

Short-term effects of air pollution on exacerbations of allergic asthma in Uzice region, Serbia.

INTRODUCTION: Many time-series studies have shown a positive association between air pollution and asthma exacerbation. However, till now only one study in Serbia has examined this relationship. AIM: To examine the associations between air pollution and asthma emergency department (ED) visits in the Uzice region, Serbia. MATERIAL AND METHODS: A time-stratified case-crossover design was applied to 424 ED visits for asthma exacerbation that occurred in the Uzice region, Serbia, in 2012-2014. Data about ED visits were routinely collected in the Uzice Health Centre. The daily average concentrations of particulate matter (PM2.5 and PM10), sulphur dioxide (SO2), nitrogen dioxide (NO2), and black carbon (BC) were measured by automatic ambient air quality monitoring stations. Odds ratios and their corresponding 95% confidence intervals were estimated using conditional logistic regression adjusted for the potential confounding influence of weather variables (temperature, humidity and air pressure). RESULTS: Statistically significant associations were observed between ED visits for asthma and 3-day lagged exposure to BC (OR = 3.23; 95% CI: 1.05-9.95), and between ED visits for asthma with coexisting allergic rhinitis and 0-day lag exposure to NO2 (OR = 1.57; 95% CI: 0.94-2.65), 2-day lag exposure to SO2 (OR = 1.97; 95% CI: 1.02-3.80), and 3-day lag exposure to PM10 (OR = 2.38; 95% CI: 1.17-4.84). CONCLUSIONS: Exposure to ambient air pollution in the Uzice region increases the risk of ED visits for asthma, particularly during the heating season.

Prevalence of oncogenic Human papillomavirus and genetic diversity in the L1 gene of HPV16 HPV 18 HPV31 and HPV33 found in women from Vojvodina Province Serbia.

The main purpose of this paper is to estimate the pre-vaccination prevalence of 12 hrHPV types among 564 women from Vojvodina province (Serbia). The corrected contingency coefficient (Ccorr) was used to estimate the importance of association of examined HPV types and cytological diagnosis. The highest association with the abnormal cytology was observed for HPV 16 (Ccorr = 0.493) in all age groups of participants. The effect of HPV 16 was especially clear within the group of women older than 35 years (Ccorr = 0.691), compared with women younger than 35 (Ccorr = 0.333). The molecular characterization at the level of L1 gene of HPV 16, 18, 31 and 33 variants was for the first time assessed in our region. Nearly all HPV 16 isolates cluster with variant lineage A (96.4%) the remaining isolates clustering with variant lineage D. All of HPV 18 and HPV 33 isolates are clustering within the lineage A while isolates of HPV 31 group with lineages A and C. This contributes to understanding of intrinsic geographical and biological differences of examined HPV types and could be useful for development of cervical cancer screening strategies in Vojvodina (Serbia) and diagnosis of HPV related cervical cancer in general.

Evaluation of the Impact of Black Carbon on the Worsening of Allergic Respiratory Diseases in the Region of Western Serbia: A Time-Stratified Case-Crossover Study.

Background and Objectives: Many epidemiological studies have shown a positive association between black carbon (BC) and the exacerbation of allergic rhinitis and allergic asthma. However, none of the studies in Serbia examined this relationship yet. The aim of this study was to examine the associations between BC and emergency department (ED) visits for allergic rhinitis and allergic asthma in the Uzice region of Serbia. Materials and Methods: A time-stratified case-crossover design was applied to 523 ED visits for allergic rhinitis and asthma exacerbation that occurred in the Uzice region of Serbia between 2012-2014. Data regarding ED visits were routinely collected in the Health Center of Uzice. The daily average concentrations of BC were measured by automatic ambient air quality monitoring stations. Odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were estimated using conditional logistic regression adjusted for the potential confounding influence of weather variables (temperature, humidity, and air pressure). Results: Statistically significant associations were observed between ED visits for allergic rhinitis and 2-day lagged exposure to BC (OR = 3.20; CI = 1.00-10.18; p = 0.049) and allergic asthma and 3-day lagged exposure to BC (OR = 3.23; CI = 1.05-9.95; p = 0.041). Conclusion: Exposure to BC in the Uzice region increases the risk of ED visits for allergic rhinitis and asthma, particularly during the heating season.

West Nile virus 'circulation' in Vojvodina, Serbia: Mosquito, bird, horse and human surveillance.

Efforts to detect West Nile virus (WNV) in the Vojvodina province, northern Serbia, commenced with human and mosquito surveillance in 2005, followed by horse (2009) and wild bird (2012) surveillance. The knowledge obtained regarding WNV circulation, combined with the need for timely detection of virus activity and risk assessment resulted in the implementation of a national surveillance programme integrating mosquito, horse and bird surveillance in 2014. From 2013, the system showed highly satisfactory results in terms of area specificity (the capacity to indicate the spatial distribution of the risk for human cases of West Nile neuroinvasive disease - WNND) and sensitivity to detect virus circulation even at the enzootic level. A small number (n = 50) of Culex pipiens (pipiens and molestus biotypes, and their hybrids) females analysed per trap/night, combined with a high number of specimens in the sample, provided variable results in the early detection capacity at different administrative levels (NUTS2 versus NUTS3). The clustering of infected mosquitoes, horses, birds and human cases of WNND in 2014-2015 was highly significant, following the south-west to north-east direction in Vojvodina (NUTS2 administrative level). Human WNND cases grouped closest with infected mosquitoes in 2014, and with wild birds/mosquitoes in 2015. In 2014, sentinel horses showed better spatial correspondence with human WNND cases than sentinel chickens. Strong correlations were observed between the vector index values and the incidence of human WNND cases recorded at the NUTS2 and NUTS3 levels. From 2010, West Nile virus was detected in mosquitoes sampled at 43 different trap stations across Vojvodina. At 14 stations (32.56%), WNV was detected in two different (consecutive or alternate) years, at 2 stations in 3 different years, and in 1 station during 5 different years. Based on these results, integrated surveillance will be progressively improved to allow evidence-based adoption of preventive public health and mosquito control measures.

Tyramine-modified pectins via periodate oxidation for soybean hull peroxidase induced hydrogel formation and immobilization.

Pectin was modified by oxidation with sodium periodate at molar ratios of 2.5, 5, 10, 15 and 20 mol% and reductive amination with tyramine and sodium cyanoborohydride afterwards. Concentration of tyramine groups within modified pectin ranged from 54.5 to 538 µmol/g of dry pectin while concentration of ionizable groups ranged from 3.0 to 4.0 mmol/g of dry polymer compared to 1.5 mmol/g before modification due to the introduction of amino group. All tyramine-pectins showed exceptional gelling properties and could form hydrogel both by cross-linking of carboxyl groups with calcium or by cross-linking phenol groups with peroxidase in the presence of hydrogen peroxide. These hydrogels were tested as carriers for soybean hull peroxidase (SHP) immobilization within microbeads formed in an emulsion based enzymatic polymerization reaction. SHP immobilized within tyramine-pectin microbeads had an increased thermal and organic solvent stability compared to the soluble enzyme. Immobilized SHP was more active in acidic pH region and had slightly decreased K m value of 2.61 mM compared to the soluble enzyme. After 7 cycles of repeated use in batch reactor for pyrogallol oxidation microbeads, immobilized SHP retained half of the initial activity.

Liver phospholipids fatty acids composition in response to different types of diets in rats of both sexes.

BACKGROUND: Dietary intake influence changes in fatty acids (FA) profiles in liver which plays a central role in fatty acid metabolism, triacylglycerol synthesis and energy homeostasis. We investigated the effects of 4-weeks treatment with milk- and fish-based diet, on plasma biochemical parameters and FA composition of liver phospholipids (PL) in rats of both sexes. METHODS: Adult, 4 months old, Wistar rats of both sexes, were fed with different types of diets: standard, milk-based and fish-based, during 4 weeks. Analytical characterization of different foods was done. Biochemical parameters in plasma were determined. Fatty acid composition was analyzed by gas-chromatography. Statistical significance of FA levels was tested with two-way analysis of variance (ANOVA) using the sex of animals and treatment (type of diet) as factors on logarithmic or trigonometric transformed data. RESULTS: Our results showed that both, milk- and fish-based diet, changed the composition and ratio of rat liver phospholipids FA, in gender-specific manner. Initially present sex differences appear to be dietary modulated. Although, applied diets changed the ratio of total saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA), and effects were gender specific. Milk-based diet lowered SFA and elevated MUFA in males and increased PUFA in females vs. standard diet. The same diet decreased n-3, increased n-6 and n-6/n-3 ratio in males. Fish-based diet increased n-3, decreased n-6 and n-6/n-3 ratio vs. standard and milk-based diet in females. However, the ratio of individual FA in liver PL was also dietary-influenced, but with gender specific manner. While in females fish-based diet decreased AA (arachidonic acid) increased level of EPA (eicosapentaenoic acid), DPA (docosapentaenoic acid) and DHA (docosahexaenoic acid), the same diet elevated only DHA levels in males. CONCLUSION: Gender related variations in FA composition of rat liver PL were observed, and results have shown that those initial differences could be significantly modulated by the type of diet. Furthermore, the modulatory effects of milk- and fish-based diets on liver phospholipids FA profiles appeared to be sex-specific.

Testing for viral material in water of public bathing areas of the Danube during summer, Vojvodina, Serbia, 2014.

From August to September 2014 a water quality study was conducted on five popular public Danube beaches in Vojvodina, Serbia. To assess the safety of Danube water for bathing, physical, chemical, bacteriological tests were performed. While many parameters for monitoring the quality of water are regulated by law, there are neither national nor international legislations addressing the presence of viruses in recreational waters. In this study, we performed analyses that surpassed national requirements, and investigated if adenovirus, enterovirus or rotavirus genetic material was present in samples of recreational water collected for quality monitoring. Of 90 water samples obtained during the study, enterovirus material was not found in any sample, but adenovirus and rotavirus genetic materials were respectively detected in 60 and 31 samples. Statistical analyses showed a significant correlation between adenovirus DNA and total coliforms in the water. Even when water samples were adequate for recreational use, adenoviruses were detected in 75% (57/76) of such samples. Our results indicate that implementation of viral indicators in recreational water might be helpful to better assess public health safety. This might be particularly relevant in areas where urban wastewater treatment is insufficient and surface waters affected by wastewater are used for recreation.

Coordinated activation of AMP-activated protein kinase, extracellular signal-regulated kinase, and autophagy regulates phorbol myristate acetate-induced differentiation of SH-SY5Y neuroblastoma cells.

We explored the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)-induced neuronal differentiation of SH-SY5Y human neuroblastoma cells. PMA-triggered expression of neuronal markers (dopamine transporter, microtubule-associated protein 2, ß-tubulin) was associated with an autophagic response, measured by the conversion of microtubule-associated protein light chain 3 (LC3)-I to autophagosome-bound LC3-II, increase in autophagic flux, and expression of autophagy-related (Atg) proteins Atg7 and beclin-1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference-mediated silencing of AMPK suppressed PMA-induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA-induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA-induced differentiation of SH-SY5Y cells. Therefore, PMA-induced neuronal differentiation of SH-SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response. Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule-associated protein 2, and ß-tubulin, and subsequent neuronal differentiation of SH-SY5Y neuroblastoma cells through AMP-activated protein kinase (AMPK)-dependent activation of extracellular signal-regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin-1 and Atg7, and increases LC3 conversion, thereby triggering the autophagic response that counteracts differentiation process.

Seroepidemiology of Human Parvovirus B19 Infection among the Population of Vojvodina, Serbia, over a 16-Year Period (2008-2023).

This study aimed to estimate the serological status and dynamic changes in the prevalence of Parvovirus B19 (PVB19) antibodies within the general population residing in the northern part of the Republic of Serbia (Province of Vojvodina) during a 16-year period. Serum samples were analyzed for Human PVB19-specific IgM and IgG antibodies using enzyme-linked immunosorbent assay (ELISA). Throughout the study period, the overall seroprevalence was 49.51%. Approximately 10% of patients exhibited a serologic profile positive for PVB19 IgM antibodies. Notably, seroprevalence varied significantly, ranging from 9.12% in the pediatric cohort (ages 1-4 years) to 65.50% in the adult demographic (40-59 years old). Seroprevalence was higher (51.88%) among women compared to men (42.50%). Immunologically naive pregnant women in the age groups 26-36 and 36-45 years had 45% (OR = 0.55, 95% CI: 0.31-1.00) and 52% (OR = 0.48; 95% CI: 0.24-0.94) lower odds of having negative IgM and IgG compared to those in age group 16-25 years old. Improved knowledge of the epidemiology of PVB19 may assist clinicians in the differential diagnosis of PVB19 clinical manifestations. The PVB19 detection is particularly important for monitoring individuals in risk groups such as women of reproductive age, medical staff, patients with hematological disorders, and those with immunodeficiency.

Main features and disease outcome of congenital myotonic dystrophy - experience from a single tertiary center.

Congenital myotonic dystrophy type 1 (CDM1) is a rare neuromuscular disease. The aim of our study was to evaluate clinical variability of CDM1 and factors that may influence survival in CDM1. Research included 24 pediatric patients with CDM1. Most of our patients had some form of hypoxic ischemic encephalopathy (HIE) (74 %), from mild to severe. Prolonged and complicated deliveries (75 %), high percentage of children resuscitated at birth (57 %) and respiratory insufficiency (46 %) with consequent hypoxia were the main reasons that could explain high percentage of HIE. Therapeutic hypothermia was applied in three children with poor outcome. Median survival of all CDM1 was 14.2 ± 1.5 years. Six patients had a fatal outcome (25 %). Their mean age of death was 3.0 ± 2.8 years. Poor prognostic factors for the survival of our CDM1 patients were: preterm delivery, resuscitation at birth, severe HIE, hypothermia treatment and permanent mechanical ventilation. Respiratory insufficiency was the main life-threatening factor. Our data clearly indicates the need to develop natural history studies in CDM1 in order to enhance the standards of care and to develop clinical trials investigating causative therapies in pediatric patients with CDM1.

Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases.

Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had kcat values of 33.3 and 61.3s(-1) and Km values for glucose of 33.4 and 27.9mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.

Negative trend in seroprevalence of anti-toxoplasma gondii IgG antibodies among the general population of the province of Vojvodina, Serbia, 2008-2021.

This study aimed to estimate dynamic changes in seroprevalence of Toxoplasma gondii within the general population living in the northern part of the Republic of Serbia (Province of Vojvodina) during a 14-year period. The differences in prevalence of anti-toxoplasma antibodies were analyzed in correlation with age, gender, residential area (rural/urban) and meteorological factors. In this cohort retrospective study, 24,440 subjects between 1 and 88 years old were enrolled. To determine the presence of T. gondii-specific IgM and IgG antibodies in serum samples, commercially available ELISA kits were used (Euroimmun, Luebeck, Germany). During the study period, the overall T. gondii seroprevalence was 23.5%. The seroprevalence continuously decreased over time from 31.7% in 2008 to 20.4% in 2021 (0.81% per year, p < 0.001). Approximately 2% of patients had a serologic profile positive for both anti-Toxoplasma IgG and IgM antibodies. The seroprevalence was higher (28.87%) among men compared to women (24.28%), while urban residents (24.94%) had lower seroprevalence than the rural population (28.17%). A statistically significant negative correlation (r = -0.559) was found between serologic profile of patients positive for both T. gondii IgG and IgM antibodies and the annual mean air temperature. No significant association was observed between seropositivity to T. gondii infection and examined meteorological factors. These data could be useful to national and regional health authorities to create an optimal health policy to reduce rate of T. gondii infections.

Diagnostic value of urine qRT-PCR for the diagnosis of West Nile virus neuroinvasive disease.

Short and low-level viremia and virorachia, antibody cross-reactivity, IgM persistence, and inaccessibility of neutralization test, make laboratory diagnosis of West Nile virus (WNV) infection difficult. Recent investigations imply that WNV is excreted in urine longer and at higher concentrations compared to blood. The detection of WNV nucleic acid in cerebrospinal fluid (CSF), serum, and urine samples collected from 41 patients with suspected WNV neuroinvasive disease, was done by real-time RT-PCR assay. CSF and serum samples were also serologically tested using anti-WNV IgM/IgG ELISA kits. WNV infection was confirmed in 46.3% of patients by positive WNV RNA results in serum and/or CSF samples. The WNV RNA testing of urine allowed confirmation of 31.7% more cases. No association between WNV RNA urine positivity and age, gender, or the day of sample collection was found. The urine qRT-PCR can be a valuable diagnostic test for confirmation of probable cases of WNV neuroinvasive disease.

Titrating Avidity of Yeast-Displayed Proteins Using a Transcriptional Regulator.

Yeast surface display is a valuable tool for protein engineering and directed evolution; however, significant variability in the copy number (i.e., avidity) of displayed variants on the yeast cell wall complicates screening and selection campaigns. Here, we report an engineered titratable display platform that modulates the avidity of Aga2-fusion proteins on the yeast cell wall dependent on the concentration of the anhydrotetracycline (aTc) inducer. Our design is based on a genomic Aga1 gene copy and an episomal Aga2-fusion construct both under the control of an aTc-dependent transcriptional regulator that enables stoichiometric and titratable expression, secretion, and display of Aga2-fusion proteins. We demonstrate tunable display levels over 2-3 orders of magnitude for various model proteins, including glucose oxidase enzyme variants, mechanostable dockerin-binding domains, and anti-PDL1 affibody domains. By regulating the copy number of displayed proteins, we demonstrate the effects of titratable avidity levels on several specific phenotypic activities, including enzyme activity and cell adhesion to surfaces under shear flow. Finally, we show that titrating down the display level allows yeast-based binding affinity measurements to be performed in a regime that avoids ligand depletion effects while maintaining small sample volumes, avoiding a well-known artifact in yeast-based binding assays. The ability to titrate the multivalency of proteins on the yeast cell wall through simple inducer control will benefit protein engineering and directed evolution methodology relying on yeast display for broad classes of therapeutic and diagnostic proteins of interest.