Abstract Introduction Surveillance of pertussis in Serbia is passive surveillance and every 3 to 4 years an increased number of positive cases are registered in the population. The last quarter of 2023 and the first quarter of 2024 are characterized by an increased number of pertussis cases. Methods Descriptive study of cases with confirmed pertussis in the territory of the Republic of Serbia based on the evidence by the Public Health Service System of the Institute of Public Health of Serbia Results According to data from the Public Health Service System in the Republic of Serbia, a total of 1348 confirmed cases of pertussis were registered by the end of April 2024 (incidence rate 20.2 per 100,000 inhabitants). Conclusions The largest number of cases was registered in the age groups of people older than 20 years, that is, aged 10 to 14 years, while the highest age-specific incidence rate was recorded in the age group of infants younger than 12 months and in children aged 10 to 14 years. Of the total number of reported cases, 120 (8.9%) were hospitalized, with the largest number of those hospitalized being in the age group younger than 12 months (85, ie 70.8% of hospitalized cases). As for the territorial distribution, the largest number of pertussis cases in relation to the place of residence was reported from the territory of two districts, Južnobački District and the City of Belgrade, where the highest incidence rates were also registered. The postponement of timely vaccination against pertussis and the impossibility of vaccinating newborns up to 2 months of age have influenced the increase in the number of infected persons. Also, advanced PCR diagnostics during Covid and the possibility of applying PCR diagnostics in pertussis helped the timely diagnosis of pertussis. The introduction of vaccination with another dose of acellular pertussis vaccine and vaccination of pregnant women would be a desirable public health intervention to prevent pertussis infection. Key messages • Protection of the target population before starting school and at the age of 14 will contribute to stopping the occurrence of new cases of pertussis. These are public health challenges. • PCR diagnosis of pertussis influenced the timely diagnosis of this infection and the possibility of its adequate and timely treatment.

Background: Approximately 2.3 million female breast cancer cases were identified globally in 2020, resulting in 685,000 fatalities among women. Serbia too experiences a high breast cancer burden. Effective reduction of breast cancer incidence and mortality necessitates strategic measures encompassing the implementation of cost-effective screening technology. However, various impediments to screening implementation persist. We aimed to estimate the impact of socioeconomic factors on breast cancer screening in Serbia. Methods: Data from the 2019 National Health Survey of the population of Serbia was. The research was a descriptive, crosssectional analytical study by design, on a representative sample of the population of Serbia. Data from women aged 15+ yr were used to examine the demographic and socioeconomic factors associated with breast cancer screening inequalities. Results: In Serbia the age group of women who predominantly participated in organized breast cancer screening (39.5%) were the ones aged 65+ yr. Women with a secondary education were 2.1x more likely to undergo a screening exam voluntarily (57.5%), compared to women with a higher education background (26.6%). When considering marital and financial circumstances, married/unmarried women from an affluent financial category exhibited a notably higher frequency of selfinitiating a mammography (73% and 48.5%) in comparison to those financially struggling (27.6%). Conclusion: Strong support is imperative for countries to establish prevention and early detection programs for cancer.

Abstract Background There was a registered increase in the number of confirmed pertussis cases in Central Serbia between June and December 2023. Among the possible causes of pertussis resurgence are increased awareness of pertussis among healthcare workers, availability of the sensitive RT PCR method in diagnostic, the limited duration of vaccine-induced immunity as well as decline of immunization coverage influenced by the COVID-19 pandemic. Methods The nasopharyngeal samples from clinics and primary care departments were transferred to Institute of Public Health of Serbia. DNA was extracted from each sample using the MagMAX Viral/Pathogen kit. The presence of B. pertussis was determined using two PCR assays, VIASURE Bordetella Real Time PCR Detection Kit and Bosphore B. pertussis/parapertussis Genotyping Kit, Anatolia Geneworks. Results RT-PCR confirmed a total of 338 pertussis cases in Institute of Public Health of Serbia during the observed period. Most of confirmed cases were among children 9-14 years old (49,7%) followed by infants younger than 12 months (21,9%). The more severe cases were among the unvaccinated and uncomplete vaccinated infants, 78,4% of which required hospitalization with two deaths in this age group. Conclusions Based on the analysis of the obtained results, we can highlight the importance of using sensitive methods in pathogen detection. Rapid diagnosis, laboratory confirmation and notification ensure early public health intervention to minimize the spread of disease. Despite long tradition of mandatory immunization against pertussis in the Republic of Serbia, wanning of vaccine induced immunity poses potential risk for the resurgence of pertussis. For pertussis control, in the future, it will be essential to achieve high immunization coverage as well as to introduce a booster dose of pertussis containing vaccine for children aged 14 and to implement vaccination of pregnant women. Key messages • For the better pertussis control vaccination of adolescents and adults, as well as vaccination of pregnant women should be considered in the future vaccination policies. • Clinicians should consider pertussis in the differential diagnosis of persistent cough illness in people of all ages, even those previously immunized.

Breast cancer (BC) remains the most common malignancy among women in Serbia, contributing significantly to both cancer morbidity and mortality annually. In December 2012, in response to the then-growing incidence of BC, the Republic of Serbia implemented a National Program for early BC detection, aligned with recommendations from the World Health Organization (WHO) and globally enforced practices. The program targets women aged 50 to 69 who are at higher risk for BC and is carried out in twoyear screening cycles designed to ensure comprehensive population coverage. All eligible women are encouraged to undergo mammographic examination - the diagnostic tool of choice - during each screening cycle. So far, five such cycles of organized BC screening have been completed, while the sixth cycle is currently underway (since the beginning of 2023) and will continue throughout 2024. As such, the program aims to reach at least 75% of the target population coverage. The screening process itself consists of blinded double reading with arbitration of yielding mammograms by two independent radiology specialists, and the introduction of a third in case of arbitration. Positive findings lead to further diagnostic procedures (ultrasound, targeted mammography) to confirm or rule out malignancy. Primary health centers, hospitals, and regional public health institutes support the existing decentralized screening infrastructure, to ensure a coordinated response for patients requiring follow-up care. Among the most significant advancements in the BC screening program in Serbia has been the integration of centralized electronic health record (EHR) systems. By integrating patient data from various healthcare providers, EHRs - as well as radiology platforms (RIS) - enable medical history and screening results to be made easily accessible, for better care coordination and timely follow-ups. These systems aim to allow standardized screening protocols, which will ensure consistent quality across healthcare facilities. Furthermore, once fully implemented, EHRs should create a digital space for public health officials to analyze screening rates, note trends, and optimize resource allocation accordingly. Patients stand to benefit from access to their health information, reminders for screenings, and educational resources, which should increase participation and improve overall health outcomes. BC management in Serbia stands to be further improved by other technology advancements in both diagnostics and treatment. Artificial intelligence (AI) and machine learning are expanding their roles in analyzing large language models, improving diagnostic precision, and optimizing treatment planning. AI-based algorithms promise to help clinicians identify minute patterns in medical data, resulting in more accurate diagnoses and more tailored, effective treatment strategies. Precision medicine, driven by genomic profiling, enables personalized treatment plans, improving patient outcomes by tailoring therapies to individual genetic markers. Additionally, cutting-edge radiation therapies, such as stereotactic body radiation therapy (SBRT) and proton therapy, reduce collateral damage to healthy tissues. The efforts to combat BC through organized screening, coupled with the integration of advanced technologies and treatment approaches in the future, are - and will continue - improving early detection rates, patient outcomes, and overall public health.

Abstract Introduction The association between Human Papiloma Virus (HPV) positive cervical swabs and the development of cervical cancer has lead to extensive research. Studies employing the RT-PCR method for typization in the young women demographic worldwide. Based on their proven oncogenic potential 14 HPV types have been classified as carcinogenic (HPV16,18,31,33,35,39,45,51,52,56, 58,59,66,68). The imporatnce of HPV diagnostics is a result of the existing link between viral infection and relative risk of cervical cancer, while the detection complexity reflects the large variety of HPV genotypes. Methods The samples analyzed in our study were obtained from cervical swabs of women aged 19 to 25 years between the months of October and December 2022. All collected samples were routinely screened for high risk genotype positivity (HPV16 and 18). There were no other inclusion criteria, the samples used in the study were selected at random. After DNA isolation, genotype identification was done by L2 amplification for HPV 16, the L1 for HPV 18, E6 for HPV 35, 56 and 58, and E7 for HPV 31,33,39,45,51,52,59,66 and 68 genotypes, with specific primers and flourescently labelled probes. Results The results demonstrated that among the DNA positive individuals HPV genotype distribution was as follows: 50% (31,39,56); 40,90% HPV(52,59,68); 34,10% HPV16; 22,70% HPV(35, 58, 66); 15,90% HPV 18. Individually, by type, the highest prevalence was noted for the high risk genotype HPV 16 (34,1%), followed by HPV18 (15,90%). As for group clusters, HPV(31,39,56) were most frequent, followed by HPV(52,59,68). Conclusions Given the forementioned results of our study, the most prevalent HPV genotype in the Republic of Serbia among the women of pre-set demographic is HPV 16, which is in conconcurrence with epidemiological results for Eastern Europe. By comparative analysis of the HPV16 and 31 distribution, the results imply a similarity in sensitivity to infection. Key messages • The susceptibility of young adults to HPV infection entails a well-developed preventive medicine program is paramount, and not limited to this demographic. • RT-PCR offers in-depth insight into the distribution of risk of cancer to the screening team.

Introduction: Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by inadequate glucose homeostasis. A common occurrence of T2DM is diabetic dyslipidemia. Given lipid droplets' role in intracellular lipid storage, these structures lie at the center of lipid and energy homeostasis. Lipolysosomes are cell organelles that have the structure of lipid droplets surrounded by a membrane. Lipophagy is a selective form of autophagy that enables lipid droplet degradation, thus representing an important mechanism in the regulation of lipid droplet homeostasis. Aim: The aim of our research was fractional volume analysis of lipid droplets, autophagic vesicles containing lipid droplets, and lipolysosomes in the lymphocytes of patients with T2DM and hyperlipidemia. Material and methods: Mononuclear cells were isolated from the peripheral blood of T2DM patients with hyperlipidemia and from healthy individuals. Cells were fixed in glutaraldehyde and postfixed in 1% osmium tetroxide. After contrasting with 4.7% uranyl acetate, the samples were embedded in epoxy resins and cut by an ultramicrotome. The ultrathin sections were then contrasted with uranyl acetate and lead citrate and analyzed using transmission electron microscopy. The fractional volume of lipid droplets, autophagic vesicles containing lipid droplets, and lipolysosomes was determined using the double "coherent point" grid with dots distributed at two different densities. Results: While there was no difference in the fractional volumes of lipid droplets and autophagic vesicles containing lipid droplets, the fractional volume of lipolysosomes was significantly higher in the lymphocytes of T2DM patients with hyperlipidemia compared to healthy individuals (p < 0.05). Conclusion: A higher fractional volume of lipolysosomes revealed in the lymphocytes of T2DM patients with hyperlipidemia can be due to an increase in the activity of these organelles, as well as an overall increase in cellular lipid metabolism in these patients.

Type 2 diabetes is a major health burden to the society. Macrophages and liver inflammation emerged as important factors in its development. We investigated ultrastructural changes in the liver, with a special emphasis on macrophages in high fat diet (HFD) fed C57BL/6 J mice treated with metformin or simvastatin, two drugs that are used frequently in diabetes. Both metformin and simvastatin reduced the liver damage in HFD fed animals, manifested as the prevention of nonalcoholic steatohepatitis development and reduced activation and number of macrophages in the liver, as well as the percentage of these cells with lipid droplets in the cytoplasm compared to untreated HFD animals. In contrast with untreated HFD-fed animals, lipid droplets were not observed in lysosomes of macrophages in HFD animals treated with metformin and simvastatin. These findings provide new insight into the effects of metformin and simvastatin on the liver in this experimental model of type 2 diabetes and provide further rationale for implementation of statins in the therapeutic regimens in this disease.

Abstract Introduction Mpox viral infection, previously known as an endemic zoonosis typical for West and South Africa, appeared in the Republic of Serbia in early summer among young and middle-aged men, particularly Belgrade's citizens. The aim of this study is to evaluate the epidemiological and clinical characteristics of Mpox cases registered in Serbia in 2022. Methods We conducted a national observational study using the national surveillance anonymized data of people who met case definition criteria on Mpox infection and confirmation by the polymerase chain reaction method. Clinical data were collected by standardized case investigation form used for epidemiological and clinical evaluation of cases. Results A total of 43 cases reported from the 17th of June to the 5th of October 2022 were men, aged between 20 and 54 (median age, 35 years), 74,4% living in Belgrade. Three-quarters of cases reported sex with other men as a potential disease transmission route. Out of the total, 15 (34,9%) Mpox cases had HIV co-infection. HIV-positive patients were older than negative cases (p = 0,023). The mean incubation period among patients who reported exposition to a probable or confirmed case was 9,3 (2-22) days. The most common systemic symptoms were fever (86%) and lymphadenopathy (44,2%). Three patients with common symptoms of fever were hospitalized. All cases presented a rash, mostly in pustule form (90,7%). The dominant localization of the skin lesions was the anogenital region (62,5%), whereas 9,3% of the patient's lower extremities were affected. Conclusions In Serbia, the outbreak of Mpox infection affected young and middle-aged men, mainly situated in the capital of Serbia. The disease had a polymorphic presentation, including a characteristical rash of the anogenital region implying the main transmission route. Key messages • Health promotion and community engagement regarding Mpox are very important in large urban settings. • Risk communication should be prioritized to promote safe sexual practices in vulnerable populations.

Discovery of the causal relationship between the human papilloma virus and cervical cancer formation increased the significance of the real-time PCR in HPV diagnostics. Based on evidence showing that they caused cervical cancer, 14 HPV types have been classified as carcinogenic (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68). This study analysed cervical smears taken from female patients, aged 19 to 25 years, using the Viasure diagnostic test for the detection of high-risk HPV genotypes and individual identification of HPV genotypes 16 and 18. A total of 110 cervical smears were analysed and 44 positive samples were detected (40%). DNA analysis of the positive samples found the following distribution of the HPV types: 27% HPV (31, 39, 56); 22% HPV (52, 59, 68); 18% HPV16; 13% HPV (33, 45, 51); 12% HPV (35, 58, 66); 8% HPV18. This study and the high positivity rate it found indicate that there is a lack of awareness among the youth on the measures of prevention, as well as a lack of understanding of HPV infection.

Numerous commercial tests for SARS-CoV-2 detection have been developed in response to the current COVID-19 pandemic. Our laboratory uses tests from two manufacturers, 2019-nCoV from Sansure and Xpert Xpress SARS-CoV-2 test from Cepheid. Protocols of different manufacturers list different predefined Ct (cycle threshold) values as cut-offs for a positive test. In this study, we focus on additional verification of Ct values for borderline positive samples, to confirm the results obtained. From May to November 2021, 88,708 tests were performed, of which 7,669 were positive and 224 were borderline positive. In retested borderline positive samples, positive results were obtained in 72.3% of the cases. Percentage of negative results upon retesting was 27.7%, which is almost a third of the total number of samples analysed. Based on these results, we can conclude that special attention must be paid when interpreting borderline positive results of RT-PCR tests. In the retesting process, if possible, it is particularly important to use a test with different properties.

The Republic of Serbia applied the booster dose of the following COVID-19 vaccines: BNT162b2 mRNA (Pfizer-BioNTech), Sinopharm BBIBP-CorV (Vero Cell®), Gam-COVID-Vac (Sputnik V) and ChAdOk1 nCoV-19 (AstraZeneca). We aimed to examine the immunogenicity and reactogenicity of the booster dose and identify factors related to immune response and adverse events. Panel study, conducted during August and September 2021, included 300 persons receiving the booster dose at the Institute of Public Health of Serbia. Blood samples were taken on the day of receiving the booster dose, and after 7 and 28 days. When applying homologous regimen, the average increase in anti-spike immunoglobulin G was 8782.2 (after 7 days), 1213.9 after 28 days, while 9179.5 (after 7 days) and 16,728.1 after 28 days of heterologous regimen. Sinopharm BBIBP-CorV (p < 0.001) and Sputnik V (p < 0.001), age 65 and over (p = 0.001) and currently smoking (p < 0.001) were independently associated with lower levels of anti-spike immunoglobulin G. Female sex (OR = 1.77; 95%CI = 1.01–3.12), previous COVID-19 infection (OR = 3.62; 95%CI = 1.13–11.63) and adverse events after the second dose (OR = 2.66; 95%CI = 1.33–5.32) were independently associated with intense systemic adverse events 7 days after. Booster dose significantly increased antibodies titers, especially 28 days after heterologous regimen, without a significant increase in reactogenicity.

Cervical cancer is the second most common type of cancer of the female reproductive organs i.e., the third most common malignant tumor among women globally. The significance of HPV genotyping, a method used to identify specific HPV genotypes, has been recognized in recent decades as an important tool for the early detection of cervical cancer risk. In recent years, great progress has been made in understanding HPV molecular biology, a large number of tests have been developed, and there is ongoing research on the association between their diagnostic and therapeutic use. In the conducted research, a diagnostic kit manufactured by Sansure Biotech was used for HPV genotyping. This test comprises pairs of specific primers and specific fluorescent probes for genotyping 15 high-risk HPV genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68). Out of a total of 41 cervical swabs analyzed, 17 were detected positive for 13 specific high-risk HPV genotypes. HPV 18 and HPV 52 genotypes were not detected in any analyzed sample, while HPV 16 (14%) and HPV 31 (17%) were detected with the highest frequency of occurrence. Although the results were obtained on a small number of samples, they have certainly indicated the importance of the application of the mentioned method for detecting oncogenic HPV variants that suggest an increased risk of cervical cancer in women in HPV screening programs in the wider population.

Abstract Objectives SUR2A is an ABC protein serving as a regulatory subunit of ATP-sensitive (KATP) channels. An increase in SUR2A levels is cardioprotective and it is a potential therapeutic strategy against ischaemic heart disease, heart failure and other diseases. However, whether overexpression of this protein has any adverse effects is yet to be fully understood. Here, we examined the heart rate and the heart rate diurnal variation in mice overexpressing SUR2A (SUR2A+) and their littermate controls (WT) using ECG telemetry that was continuously recorded for 14 days (days 8–23 post-radiotransmitter implantation). Methods Using SigmaPlot 14.0 and Microsoft Excel, Area Under the Curve (AUC) for each parameter was calculated and plotted in a graph. Results Both WT and SUR2A+ mice were more physically active during nights and there were no significant differences between two phenotypes. Physical activity was associated with increased heart rate in both phenotypes, but there were no differences in heart rate between phenotypes irrespective of physical activity or time of the day. A diurnal heart rate variation was preserved in the SUR2A+ mice. As area under the curve (AUC) analysis has the potential to reveal differences that are invisible with other statistical methods, we compared AUC of heart rate in SUR2A+ and WT mice. This analysis did not yield anything different from traditional analysis. Conclusions We conclude that increased SUR2A levels are not associated with changes in physical activity, heart rate and/or circadian rhythm influence on the heart rate. This lack of adverse effects supports a notion that manipulation with SUR2A levels is a promising cardioprotective strategy.

The purpose of this study was to determine whether increased expression of SUR2A, a regulatory subunit of sarcolemmal ATP-sensitive K+ (KATP) channels, improves adaptation to physical stress and regulates cardiac electrophysiology in physical stress. All experiments have been done on transgenic mice in which SUR2A expression was controlled by cytomegalovirus immediate-early (CMV) promoter (SUR2A) and their littermate wild-type controls (WT). The levels of mRNA in heart tissue were measured by real-time RT-PCR. Electrocardiogram (ECG) was monitored with telemetry. The physical adaptation to stress was elucidated using treadmill. We have found that SUR2A mice express 8.34 ± 0.20 times more myocardial SUR2A mRNA than WT (n = 8–18). The tolerated workload on exercise stress test was more than twofold higher in SUR2A than in WT (n = 5–7; P = 0.01). The pattern of Q-T interval from the beginning of the exercise test until drop point was as follows in the wild type: (1) increase in Q-T interval, (2) decrease in Q-T interval, (3) steady stage with a further decrease in Q-T interval, and (4) a sharp increase in Q-T interval. The pattern of Q-T interval was different in transgenic mice and the following stages have been observed: (1) increase in Q-T interval, (2) decrease in Q-T interval, and (3) prolonged steady-state stage with a slight decrease in Q-T interval. In SUR2A mice, no stage 4 (a sharp increase in Q-T interval) was observed. Based on the obtained results, we conclude that an increase in the expression of SUR2A improves adaptation to physical stress and physical endurance by increasing the number of sarcolemmal KATP channels and, by virtue of their channel activity, improving Ca2+ homeostasis in the heart.

Pyrazinamide is an anti-tubercular agent, used as a part of a three-drug regime (any three of the following: rifampicin, isoniazid, pyrazinamide, streptomycin or ethambutol) for the initial phase of treatment. One of the effects pyrazinamide has on mammalian cells is to regulate NAD + /NADH levels. We have recently found that changes in NAD + /NADH are associated with regulation of expression levels of SUR2A, a cardioprotective protein serving as a regulatory subunit of cardiac ATP-sensitive K + (K ATP ) channels. Here, we have tested whether pyrazinamide regulate expression of SUR2A/K ATP channel subunits and resistance to metabolic stress in embryonic heart-derived H9c2 cells. We have found that 24-h-long treatment with pyrazinamide (3 mcg/ml) increased mRNA levels of SUR2A, SUR2B and Kir6.1 without affecting mRNA levels of other K ATP channel subunits. This treatment with pyrazinamide (3 mcg/ml) protected H9c2 cells against stress induced by 10 mM 2,4-dinitrophenol (DNP). The survival rate of DNP-treated cells was 45.6 ± 2.3% ( n = 5) if not treated with pyrazinamide and 90.8 ± 2.3% ( n = 5; P < 0.001) if treated with pyrazinamide. We conclude that pyrazinamide increases resistance to metabolic stress in heart H9c2 cells probably by increasing SUR2A and SUR2B expression. Our results of this study indicate that pyrazinamide should be seriously considered as a drug of choice for patients with tuberculosis and ischaemic heart disease.

Bacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37°C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects.

It is generally accepted that activity of K+ channels maintain resting membrane potential and uterine quiescence during pregnancy, which is, at least in part, mediated by down-regulation of ATP-sensitive K+ (KATP) channels. Pregnancy-induced hypertension (PIH) is associated with pre-term and late pre-term labour. Here, we have used real time RT-PCR to compare mRNA levels of KATP channel subunits in PIH parturient and control parturient. We have found that Kir6.1, a pore forming, myometrial KATP channel subunit is down-regulated in PIH patients. This could perfectly explain increased rate of pre-term labour in patients suffering from PIH.

SPAK (Ste20/SPS1-related proline/alanine-rich kinase) has been recently identified as a protein kinase which targets the electroneutral cation-coupled chloride cotransporters and it stands out as a target for inhibition in novel anti-hypertensive agents. From this prospective, any information about physiological consequences of SPAK inhibition would be useful to better understand the efficacy and potential adverse effects of the SPAK-based anti-hypertensive therapy. Radiotelemetry was employed to continuously measure the parameters of blood pressure (mean arterial blood pressure, systolic blood pressure, and diastolic blood pressure), heart rate, and physical activity in SPAK knock-in (KI) mice and littermate controls for 24 h. For each parameter, the area under the curve (AUC) was calculated and compared between the SPAK KI mice and wild type mice. There was no statistically significant difference in the AUC of blood pressure parameters between SPAK KI and littermate mice. When mice were physically inactive, the AUCs for blood pressures were significantly lower in SPAK KI than in littermates. When physically active, blood pressures were significantly higher in SPAK KI than in littermates. The heart rate followed a similar pattern. The AUC of physical activity was significantly increased in SPAK KI mice when compared to littermates and the SPAK KI mice spent significantly less time in an inactive state and significantly more time in active states. Comparison between SPAK KI mice and unrelated wild type mice yielded similar results to the comparison between SPAK KI mice and littermates. We conclude that SPAK inhibition increases spontaneous locomotor activity, which has a significant effect on blood pressure.

Some recent studies associated insulin therapy with negative cardiovascular events and shorter lifespan. SUR2A, a K-ATP channel subunit, regulate cardioprotection and cardiac ageing. Here, we have tested whether glucose and insulin regulate expression of SUR2A/K-ATP channel subunits and resistance to metabolic stress in heart H9c2 cells. Absence of glucose in culture media decreased SUR2A mRNA, while mRNAs of Kir6.2, Kir6.1, SUR1 and IES SUR2B were increased. 2-deoxyglucose (50 mM) decreased mRNAs of SUR2A, SUR2B and SUR1, did not affect IES SUR2A and IES SUR2B mRNAs and increased Kir6.2 mRNA. No glucose and 2-deoxyglucose (50 mM) decreased resistance to an inhibitor of oxidative phosphorylation, DNP (10 mM). 50mM glucose did not alter K-ATP channel subunits nor cellular resistance to DNP (10 mM). Insulin (20 ng/ml) in both physiological and high glucose (50 mM) down-regulated SUR2A while upregulating Kir6.1 and Kir6.2 (in high glucose only). Insulin (20 ng/ml) in physiological and high glucose decreased cell survival in DNP (10 mM). As opposed to Kir6.2, infection with SUR2A resulted in titre-dependent cytoprotection. We conclude that insulin decreases resistance to metabolic stress in H9c2 cells by decreasing SUR2A expression. Lower cardiac SUR2A levels underlie increased myocardial susceptibility to metabolic stress and shorter lifespan.