Circulating Variants of SARS-CoV-2 Among Macedonian COVID - 19 Patients in the First Year of Pandemic.

Genomic epidemiology has proven to be a useful tool for investigating pandemic outbreaks and tracking pathogen spread and evolution. This study describes the circulation of SARS-CoV-2 strains in N. Macedonia during a period of one year, encompassing three waves of the COVID-19 pandemic. A certain percentage (2-3%) of positive cases were continuously selected and analyzed by whole genome sequencing (WGS) technology. Using this approach, a total of 337 SARS-CoV-2 genomes were sequenced and 26 different lineages belonging to 7 clades were detected. During the first wave of the pandemic, the most dominant lineage was B.1.1, followed by B.1.1.70, which became the most dominant in the second wave. The B.1.1.7 lineage completely overpassed all other variants in the third wave. Our study strengthens the notion that the progression of COVID-19 pandemic is associated with emergence of new SARS-CoV-2 variants with increased virulence. The measure of the impact of this viral dynamic on the spread of the pandemic should be evaluated in association with other factors that might influence the transmission.

Inflammatory and oxidative stress markers in intracerebral hemorrhage: Relevance as prognostic markers for quantification of the edema volume.

We aimed to analyze the inflammatory and oxidative stress (OS) markers after intracerebral hemorrhage (ICH) and their temporal changes, interaction effects, and prognostic values as biomarkers for the prediction of the edema volume. Our prospective, longitudinal study included a cohort group of 73 conservatively treated patients with ICH, without hematoma expansion or intraventricular bleeding, which were initialized with the same treatment and provided with the same in-hospital care during the disease course. Study procedures included multilevel comprehensive analyses of clinical and neuroimaging data, aligned with the exploration of 19 inflammatory and five OS markers. White blood cells (WBC), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), neutrophilia, and lymphopenia peaked 3 days post-ICH, and they showed much stronger correlations with clinical and neuroimaging variables, when compared to the admission values. An intricate interplay among inflammatory (WBC, CRP, neutrophils, neutrophil-to-lymphocyte ratio [NLR], interleukin (IL)-6, and IL-10) and OS mechanisms (catalase activity and advanced oxidation protein products [AOPP]) was detected operating 3-days post-ICH, being assessed as relevant for prediction of the edema. The overall results suggested complex pathology of formation of post-ICH edema, via: (A) Not additive, but statistically significant synergistic interactions between CRP-ESR, neutrophils-CRP, and neutrophils-IL-6 as drivers for the edema formation; (B) Significant antagonistic effect of high protein oxidation on the CRP-edema dependence, suggesting a mechanism of potential OS-CRP negative feedback loop and redox inactivation of CRP. The final multiple regression model separated the third-day variables NLR, CRP × AOPP, and WBC, as significant prognostic biomarkers for the prediction of the edema volume, with NLR being associated with the highest effect size. Our developed mathematical equation with 3D modeling for prediction and quantification of the edema volume might be beneficial for taking timely adequate strategies for prevention of delayed neurological deteriorations.

A Comprehensive Molecular and Epidemiological Characterization of Influenza Viruses Circulating 2016-2020 in North Macedonia.

Influenza viruses know no boundaries, representing an example of rapid virus evolution combined with pressure exerted by the host's immune system. Seasonal influenza causes 4-50 million symptomatic cases in the EU/EEA each year, with a global death toll reaching 650,000 deaths. That being the case, in 2014 North Macedonia introduced the sentinel surveillance in addition to the existing influenza surveillance in order to obtain more precise data on the burden of disease, circulating viruses and to implement timely preventive measures. The aims of this study were to give a comprehensive virological and epidemiological overview of four influenza seasons (2016-2020), assess the frequency and distribution of influenza circulating in North Macedonia and to carry out molecular and phylogenetic analyses of the hemagglutinin (HA) and neuraminidase (NA) genes of influenza A(H1N1)pdm09, A(H3N2) from ILI and SARI patients. Our results showed that out of 1,632 tested samples, 46.4% were influenza positive, with influenza A(H1N1)pdm09 accounting for the majority of cases (44%), followed by influenza B (32%) and A(H3N2) (17%). By comparing the sentinel surveillance system to the routine surveillance system, we showed that the newly applied system works efficiently and gives great results in the selection of cases. Statistically significant differences (p = < 0.0000001) were observed when comparing the number of reported ILI cases among patients aged 0-4, 5-14, 15-29, and 30-64 years to the reference age group. The phylogenetic analysis of the HA sequences unveiled the resemblance of mutations circulating seasonally worldwide, with a vast majority of circulating viruses belonging to subclade 6B.1A. The PROVEAN analysis showed that the D187A substitution in the receptor binding site (RBS) of the A(H1N1)pdm09 HA has a deleterious effect on the its function. The A(H3N2) viruses fell into the 3C.2a and 3C.3a throughout the analyzed seasons. Molecular characterization revealed that various substitutions in the A(H3N2) viruses gradually replaced the parental variant in subsequent seasons before becoming the dominant variant. With the introduction of sentinel surveillance, accompanied by the advances made in whole-genome sequencing and vaccine therapeutics, public health officials can now modify their approach in disease management and intervene effectively and in a timely manner to prevent major morbidity and mortality from influenza.

Modeling prognostic factors for poor neurological outcome in conservatively treated patients with intracerebral hemorrhage: A focus on TNF-α.

OBJECTIVE: Prognostic models for Intracerebral hemorrhage (ICH), mainly based on clinical evaluation, have remained inherently confounded by subjective scoring assessments and limited accuracy. In this study, we aimed at assessing the risk for poor outcome after ICH based on peripheral biochemical markers (TNF-α, glutamate and glucose) and radiological variables (both at admission and five days after patient's care), for modeling purposes of prognostication. PATIENTS AND METHODS: The defined initial variables of fifty non-comatose conservatively treated ICH patients without severe complications during the hospitalization process (as intraventricular bleeding, or hematoma expansion) were aligned with the evaluated parameters during re-evaluation (3 months later). A comprehensive statistical approach has been applied by using different modeling strategies for prediction of their functional status and outcome. RESULTS: Higher blood plasma glutamate, TNF-α and initial ICH volume at admission, as well as higher volumes of ICH and perihematomal edema after five days of care were significantly more likely associated with the poor outcome. Nevertheless, in all of the constructed models, TNF-α was estimated as the only significant predictive risk factor, thus outperforming the capacity of the initial ICH volume and the radiological variables after 5 days, both in terms of prognostication of the functional status and the 3-month neurological outcome. The constructed canonical variable that has fairly marked off the different outcomes was also mainly weighed by the admission TNF-α levels. For the first time, we have carefully developed probability functions for the neurological outcome as a response to the admission TNF-α levels; TNF-α levels >110.35 pg/mL were assessed as an optimal cutoff point fairly identifying patients who will fall into the group with poor outcome. CONCLUSIONS: TNF-α based models and admission TNF-α screening might be appropriate as a key component that assists more objective prognostication and management of patient's care in clinical decision making, as rapid initial diagnosis and concentrated management are crucial for secondary prevention of further devastating neurological impairments after ICH.

SSRI-reduced platelet reactivity in non-responding patients with life-long Recurrent Depressive Disorder: Detection and involved mechanisms.

INTRODUCTION: Adverse effects with bleeding disorders are often associated with the administration of SSRI in depression, although the exact mechanisms remain contradicting. This study is aimed at detecting and exploring the mechanisms of SSRI-induced changes in platelet reactivity in non-responding patients with Recurrent Depressive Disorder (RDD) and life-long exposure to antidepressants. MATERIALS AND METHODS: Thirty-one patients and thirty-one healthy controls were included in the study. A comprehensive approach which includes evaluation of peripheral markers and microscopic analyses of platelet morphology changes has been used. RESULTS: RDD SSRI patients have shown blunted aggregatory responses towards collagen and epinephrine. Evident differences in the microscopic evaluation of platelet morphology were observed between the groups, with inherent absence of micro-aggregates and platelet shape changes within the patients; after quantification, the sensitivity and specificity of this method were assessed as high. The abnormalities were found in association with lower platelet serotonin content and high fluctuations of free plasma serotonin levels. Changes in the levels of CRP, fibrinogen and nitric oxide were not observed. Macroplatelets were also detected within RDD SSRI patients via increased MPV, PDW and P-LCR, which were associated with discoid shape and without procoagulant activity. CONCLUSIONS: The microscopic evaluation might be useful as a simple method for detection of SSRI-reduced platelet function for research purposes or systematic correlations with other biochemical parameters. The mechanisms involved in SSRI-reduced platelet function in non-responding RDD patients are complex, including combined effects of lower platelet serotonin content, high fluctuations in plasma serotonin concentration and abnormal α-AR function.

Evaluation of peripheral serotonin content and α2-adrenergic receptor function as potential markers for life-long recurrent depressive disorder by using methodological improvements.

OBJECTIVE: We aimed to evaluate the role and the relations between peripheral platelet serotonin content, blood plasma serotonin concentration and the function of platelet α2-adrenergic receptors (α2-AR) as potential state or trait biomarkers for recurrent depressive disorder (RDD). METHODS: 26 drug-free patients with life-long RDD and 31 healthy controls were included in the study. Several methodological improvements in blood collection and platelet isolation were implemented following the present standards in Haematology and Light transmission aggregometry. RESULTS: Our results have shown lower platelet serotonin content, higher plasma serotonin concentration and desensitization of platelet α2-AR in patients with RDD. The variables were found heterogeneous and mainly influenced by the clinical characteristics of the current episode. High amplitude of the α2-AR correlated with severe anxious symptoms and high platelet serotonin content (as well as low plasma serotonin levels) were associated with psychotic symptoms. CONCLUSIONS: The evaluated peripheral markers reflect only state (but not trait) abnormalities in patients with current severe episode of RDD. The observed peripheral α2-AR and serotonin abnormalities are mutually not related and they are probably triggered by different mechanisms.

In vivo effects of geranylgeraniol on the development of bisphosphonate-related osteonecrosis of the jaws.

BACKGROUND: Bisphosphonate-related osteonecrosis of the jaws (BRONJ) is a complication of the bisphosphonate (BP) treatment and its pathopysiology is still not fully understood. The existing preventive and treatment options require updates and more attention. Geranylgeraniol (GGOH) so far demonstrated an increased activity and viability of the cells previously treated with zoledronic acid (ZA). The aim of this study was to evaluate the in vivo effects of GGOH on the development of BRONJ. MATERIALS AND METHODS: A total of 30 male Wistar rats were included in the study, divided into three groups: two experimental groups (EG1 and EG2) and a control group (CG). Rats from EG1 and EG2 were treated with 0,06 mg/kg ZA ip weekly in a duration of five weeks, while CG received saline ip. On the third week all animals underwent extraction of the lower right first molars. The rats from EG2 received a local solution of GGOH in concentration of 5 mM in the socket every day after the tooth extraction. The analyses included clinical evaluation on the wound healing and pathohistological evaluation for presence and level of osteonecrosis. RESULTS: EG2 showed significantly improved wound healing and tissue proliferation, when compared to EG1. EG2 significantly differed from EG1 and CG (p<0,05) for the presence of microscopical osteonecrosis (80% vs 22,2% vs 0%). Regarding to the number of empty lacunes without osteocytes and the level of necrosis, all groups demonstrated significant differences. CONCLUSION: Geranylgeraniol in a form of local solution may be a promising option for prevention and treatment of BRONJ.

Peripheral glutamate and TNF-α levels in patients with intracerebral hemorrhage: Their prognostic values and interactions toward the formation of the edemal volume.

OBJECTIVE: We aimed to evaluate the prognostic values, contribution and interactions of the peripheral blood plasma glutamate and tumor-necrosis factor-α (TNF-α) levels toward the formation of the perifocal edema in patients with intracerebral hemorrhage (ICH). METHODS: Fifty patients with ICH and fifty healthy controls were included in the study. The peripheral markers were detected by high-sensitivity ELISA. RESULTS: A highly significant differences in plasma glutamate and TNF-α levels with good separation of their values was detected between patients and healthy controls. The two variables correlated with the severity of the symptoms and the initial volume of the ICH at admission. Both peripheral glutamate and TNF-α levels at admission were estimated as significant predictors for the formation of the perifocal edema five days after ICH; nevertheless, it was shown that they independently contribute to the development of the edema, without effects of interaction and regardless the localization of the ICH. CONCLUSIONS: Our results support the idea for the significance of glutamate and TNF-α as peripheral markers for excitotoxicity and inflammation in ICH patients. The developed multiple regression model for prediction of the development of the edema could be beneficial in decision making between conservative treatment and surgical intervention in the clinical practice.

The effect of low-level diode laser on COX-2 gene expression in chronic periodontitis patients.

Adjunctive treatments to scaling and root planing (SRP) such as lasers, have been utilized in the treatment of chronic periodontitis, mainly aiming to suppress and eliminate the bacteria, as well as enhancing the healing response. Eighty gingival papilla biopsy samples were obtained from 60 patients diagnosed with chronic advanced periodontitis; randomly assigned to three treatment groups (n = 20), as well as 20 subjects with no periodontal disease [group A]. Group B received SRP on a single quadrant/day for four consecutive days. On day 5, all quadrants were rescaled. Groups C and D received the same treatment as group B plus laser application with the low-level diode laser (630-670 nm, 1.875 J/cm2) for five and ten consecutive days, respectively. Papilla biopsies were obtained from subjects and evaluated by RT-PCR for expression of COX-2. The values in the control group were 0.028 0.014 and baseline values for the examined groups were 0.16 0.18. Significantly decreased level of COX-2 expression for groups C and D was found after treatment, while lowest average expression was found in the group that had the 10 laser treatments supplemental to SRP (0,035 0,014). The results of this study show suppression of COX-2 in gingival tissue after low-level laser treatment as adjunct to SRP.

Vitamin E supplementation in chronically hemodialyzed patients - influence on blood hemoglobin and plasma (anti)oxidant status.

Background: Disturbed oxidant/antioxidant status is involved in pathogenesis of anemia in end stage renal disease. There is evidence that vitamin E supplementation can increase blood hemoglobin in chronically hemodialyzed patients. However, the interindividual variation in response to the supplementation has not been fully addressed. Methods: 24 chronically hemodialyzed patients were supplemented with vitamin E (400 IU/day) in a period of two months. They had already been treated with erythropoiesis stimulating agents (ESA) and iron on a long-term basis, which was continued during the study period. A group of 20 healthy volunteers served as control subjects. Complete blood count, general biochemistry assays, the redox status by total thiols, oxidative stress by reactive oxygen metabolites, antioxidant status by biological antioxidant potential, and vitamin E (α- and γ- tocopherol) were measured before the start of supplementation, one month and two months later. Results: Overall, the vitamin E supplementation did not cause an increase of blood hemoglobin, hematocrit or red blood cells. However, 50 % of the patients with basal blood hemoglobin below 12.0 g/dL (N = 10) responded to the supplementation with its continuous increase. In addition, vitamin E exhibited a slight prooxidant effect only in the subgroup of patients with basal blood hemoglobin of ≥ 12.0 g/dL, two months after the start of supplementation (decreased total thiols: 300 ± 31 vs. 277 ± 36 µmol/L, p < 0.05; increased reactive oxygen metabolites: 183 ± 140 vs. 287 ± 112 CARR U, p > 0.05; decreased biological antioxidant potential: 2278 ± 150 vs. 2171 ± 126 µEq/L, p < 0.025), which coincided with their significantly increased serum α-tocopherol concentrations in comparison to the patients with basal blood hemoglobin below 12.0 g/dL (41.3 ± 7.2 vs. 59.9 ± 19.2 µmol/L, p < 0.025). Conclusions: When treated with ESA and iron on a long-term basis, the response to the vitamin E supplementation in chronically hemodialyzed patients is largely dependent on their basal blood hemoglobin and serum vitamin E concentrations.

L-citrulline supplementation reverses the impaired airway relaxation in neonatal rats exposed to hyperoxia.

BACKGROUND: Hyperoxia is shown to impair airway relaxation via limiting L-arginine bioavailability to nitric oxide synthase (NOS) and reducing NO production as a consequence. L-arginine can also be synthesized by L-citrulline recycling. The role of L-citrulline supplementation was investigated in the reversing of hyperoxia-induced impaired relaxation of rat tracheal smooth muscle (TSM). METHODS: Electrical field stimulation (EFS, 2-20 V)-induced relaxation was measured under in vitro conditions in preconstricted tracheal preparations obtained from 12 day old rat pups exposed to room air or hyperoxia (>95% oxygen) for 7 days supplemented with L-citrulline or saline (in vitro or in vivo). The role of the L-citrulline/L-arginine cycle under basal conditions was studied by incubation of preparations in the presence of argininosuccinate synthase (ASS) inhibitor [α-methyl-D, L-aspartate, 1 mM] or argininosuccinate lyase inhibitor (ASL) succinate (1 mM) and/or NOS inhibitor [Nω-nitro-L-arginine methyl ester; 100 µM] with respect to the presence or absence of L-citrulline (2 mM). RESULTS: Hyperoxia impaired the EFS-induced relaxation of TSM as compared to room air control (p < 0.001; 0.5 ± 0.1% at 2 V to 50.6 ± 5.7% at 20 V in hyperoxic group: 0.7 ± 0.2 at 2 V to 80.0 ± 5.6% at 20 V in room air group). Inhibition of ASS or ASL, and L-citrulline supplementation did not affect relaxation responses under basal conditions. However, inhibition of NOS significantly reduced relaxation responses (p < 0.001), which were restored to control level by L-citrulline. L-citrulline supplementation in vivo and in vitro also reversed the hyperoxia-impaired relaxation. The differences were significant (p <0.001; 0.8 ± 0.3% at 2 V to 47.1 ± 4.1% at 20 V without L-citrulline; 0.9 ± 0.3% at 2 V to 68.2 ± 4.8% at 20 V with L-citrulline). Inhibition of ASS or ASL prevented this effect of L-citrulline. CONCLUSION: The results indicate the presence of an L-citrulline/L-arginine cycle in the airways of rat pups. L-citrulline recycling does not play a major role under basal conditions in airways, but it has an important role under conditions of substrate limitations to NOS as a source of L-arginine, and L-citrulline supplementation reverses the impaired relaxation of airways under hyperoxic conditions.

Effect of laser on TNF-alpha expression in inflamed human gingival tissue.

This study sought to evaluate the effect of low-level laser treatment combined with scaling and root planing (SRP) on gingival tissue levels of TNF-alpha in subjects with periodontal disease. Eighty gingival papilla biopsy samples were obtained from 60 patients diagnosed with chronic advanced periodontitis; randomly assigned to three treatment groups (n = 20), as well as 20 subjects with no periodontal disease (group A). Group B received SRP on a single quadrant/day for four consecutive days. On day 5, all quadrants were rescaled. Groups C and D received the same treatment as group B plus laser application with the low-level diode laser (630-670 nm, 1.875 J/cm(2)) for five and ten consecutive days, respectively. Papilla biopsies were obtained from subjects and evaluated by ELISA for levels of TNF-alpha. The values in the control group were 5.2 ± 3.21 pg/mg and baseline values for the examined groups were 46.01 ± 16.69. Significantly decreased level of TNF-alpha for groups C and D was found after treatment, while group B demonstrated reduction of TNF-alpha of 31.34%. The results of this study show suppression of TNF-alpha in gingival tissue after low-level laser treatment as adjunct to SRP. Data may suggest beneficial anti-inflammatory effects of the laser treatment when used as adjunctive periodontal treatment.

Periodontal treatment with a low-level diode laser: clinical findings.

This study assessed the effects of low-level laser treatment in combination with scaling and root planing (SRP) in patients with periodontitis. Sixty subjects with chronic advanced periodontitis were assigned randomly to three treatment groups (n = 20) after collecting gingival clinical parameters. Group A received SRP on a single quadrant per day for four consecutive days; on the fifth day, all quadrants were rescaled. Group B received the same treatment as Group A, followed by laser application for five days. Group C received the same treatment as Group B but the laser treatment was administered for a total of 10 days. For Groups B and C, a low-level diode laser (630 to 670 nm) was used. The plaque index, gingival index, and sulcular bleeding index were recorded for all groups. For all clinical parameters, all three groups reported statistically significant differences (p < 0.005) compared to baseline data. Compared to Group A, Groups B and C showed statistically significant improvement for all clinical parameters. These findings suggest that a low-level diode laser can have a beneficial effect for treating inflammatory chronic advanced periodontitis.

Hepatoprotective effect of the ethyl acetate extract of Teucrium polium L. against carbontetrachloride-induced hepatic injury in rats.

Hepatoprotective activity of the ethyl acetate extract of Teucrium polium L. was investigated using rats with CCl4-induced liver damage. Specific biochemical parameters (glutathione peroxidase, superoxide dismutase, reduced glutathione and total antioxidative status) were estimated in blood and in liver homogenate. Lipid peroxidation in CCl4-intoxicated rats was evidenced by a marked increment in the levels of thiobarbituric acid reactive substances. Histopatological examinations of the liver were undertaken to monitor the liver status. Silymarin was used as a standard to compare the hepatoprotective activity of the extract. Some biochemical parameters in groups treated with the Teucrium polium extract at a dose of 25 mg kg(-1), showed significantly different values than that of the CCl4--treated group. The liver biopsy of all experimental rat groups treated with the Teucrium polium ethyl acetate extract showed significant restoration of the normal histomorphological pattern of liver cells. The study substantiates the potential hepatoprotective activity of the ethyl acetate extract of Teucrium polium L.

Redox kinetic measurements of glutathione at the mercury electrode by means of square-wave voltammetry. The role of copper, cadmium and zinc ions.

The electrode reaction of glutathione (GSH) at the hanging mercury drop electrode is studied by means of square-wave voltammetry (SWV). At potentials more positive than -0.350 V (vs. Ag/AgCl (3 mol/l KCl)) the oxidation of the mercury electrode in the presence of GSH leads to creation of a sparingly soluble mercury-GSH complex that deposits onto the electrode surface. Under cathodic potential scan, the deposited complex acts as a reducible reactant, giving raise to a well-defined cathodic stripping reversible SW voltammetric response. The electrode reaction can be described by the scheme: Hg(SG)(2(s))+e(-)+2H((aq))(+) = Hg((l))+2GSH((aq)). Thus, the electrode reaction provides information on both thermodynamics and kinetics of the chemical interactions of GSH with mercury. An experimental methodology for measuring the kinetics of the electrode reactions, based on the property known as "quasireversible maximum", is developed. The standard redox rate constant is 5.09, 5.75 and 5.22 cm s(-1) in a phosphate buffer at pH 5.6, 7.0 and 8.5, respectively, with a precision of +/-10%. The high rate of the electrode reaction reflects the strong affinity of GSH towards chemical interaction with mercury. The electrode reaction is particularly sensitive to the presence of heavy metal ions such as Cu(2+), Cd(2+), and Zn(2+.) The rate of the electrode reaction decreases significantly in the presence of these ions due to simultaneous interactions of GSH with the respective ion and mercury.