Clinical characteristics and outcome of children with acute cryptorchid testicular torsion: A single-center, retrospective case series study.

BACKGROUND: Cryptorchidism and testicular torsion (TT) are relatively common conditions in clinical practice; however, sparse information about cryptorchid TT is available in the current literature. METHODS: We retrospectively reviewed the clinical characteristics, treatment modalities, and long-term outcomes of pediatric patients treated for acute cryptorchid TT. RESULTS: We found eight patients with unilateral acute cryptorchid TT with a prevalence of 8.9% (8/90) among all TT cases. The left testis was affected in six patients. The median age of patients at the time of the surgery was 65 months (interquartile range (IQR) 4-136 months). The median duration of symptoms was 16 h (IQR 9-25 h), while the median time to treatment was 60 min (IQR 59-63 min). The most common symptoms were pain (abdominal and inguinal) and inguinal mass with no palpable testis in the ipsilateral hemiscrotum. Preoperative color Doppler ultrasonography revealed absent or decreased testicular blood flow in the affected testes in 7/8 of patients. Various degrees of testicular torsion (median 540°, min 360°, max 1260°) were found during surgery. A necrotic testis that led to orchidectomy was found in 4/8 of patients. The median follow-up period was 42.6 months (IQR 12.5-71.2 months), revealing only one patient with testicular atrophy. The final testicular salvage rate was 35%. CONCLUSIONS: Greater awareness among caregivers and primary care physicians about acute cryptorchid TT is required to improve their timely diagnosis and treatment. A physical examination of the external genitalia and inguinal regions should be mandatory to attain a proper diagnosis and treatment without delay.

Association of ACE2 and TMPRSS2 genes variants with disease severity and most important biomarkers in COVID-19 patients in Bosnia and Herzegovina.

AIM: To assess the association of single nucleotide polymorphisms (SNPs) in the ACE2 and TMPRSS2 genes with COVID-19 severity and key biomarkers. METHODS: The study involved 750 COVID-19 patients from Bosnia and Herzegovina, divided into three groups: mild, moderate, and severe cases. Genetic variations within the ACE2 (rs2285666) and TMPRSS2 (rs2070788) genes were examined with real-time polymerase chain reaction. Biochemical markers were determined with standard procedures. RESULTS: There was a significant difference in the rs2070788 genotype distribution between patients with mild and moderate symptoms, but not between other groups. For the rs2285666 polymorphism, no significant difference in genotype distribution was found. In patients with mild symptoms, carriers of the GG genotype of rs2070788 had significantly higher total bilirubin levels than carriers of the AA genotype. Similarly, carriers of the TT genotype of rs2285666 had significantly higher activated partial thromboplastin time and international normalized ratio, and lower lactate dehydrogenase levels compared with the CC genotype. Among patients with severe symptoms, carriers of the GG genotype showed significantly higher potassium levels than carriers of the AA genotype, while carriers of the TT genotype showed significantly higher erythrocyte count as well as hemoglobin and hematocrit levels compared with the CC genotype. CONCLUSION: This study highlights the role of genetic factors, particularly SNPs in the ACE2 and TMPRSS2 genes, in determining COVID-19 severity, aiding patient risk assessment and prognosis.

The Predictive Value of Plasma Sodium and Other Laboratory Parameters in Determining Complicating Appendicitis in Children.

OBJECTIVE: Finding a reliable preoperative predictor of complicated acute appendicitis (AA) has been a challenging diagnostic problem. The present study aimed to identify potential factors that may predict complicated AA in the pediatric emergency department (ED) based on routine, widely available laboratory tests on admission to the ED, including plasma sodium concentration. METHODS: We retrospectively reviewed clinical and laboratory data of pediatric patients with AA who underwent emergency surgery at our department between January 2020 and December 2022. The patients were divided into two groups: histopathologically proven complicated AA (n = 80), and non-complicated AA (n = 155). RESULTS: Complicated AA was associated with reduced plasma sodium and chloride concentrations (p < 0.001, both), decreased values of lymphocytes (p = 0.002), elevated C-reactive protein (CRP) ( p < 0.001), elevated values of white blood cells (WBC) and neutrophils (p = 0.012 and 0.001, respectively). In binomial logistic regression, increased levels of CRP and WBC, and decreased levels of sodium were predictors of complicated AA. The area under the ROC curve was 0.825 (95% CI 0.764, 0.886). CONCLUSION: We identified mild hyponatremia and elevated CRP and WBC values as potential markers for distinguishing complicated from uncomplicated pediatric AA with implications for surgical approaches for treating complicated AA and conservative approaches for treating uncomplicated AA.

Therapy adherence, safety and efficacy of rivaroxaban in prevention of venous thromboembolism in patients with hip or knee endoprosthesis.

PURPOSE: Rivaroxaban is a potent, selective direct inhibitor of factor Xa. The aim of this study was to evaluate the therapy adherence, safety and efficacy of rivaroxaban therapy in reducing the risk of venous thromboembolism in patients undergoing elective hip or knee replacement. METHODS: The prospective, post-marketing clinical trial was conducted on adult patients after knee or hip endoprosthesis. Data were collected at the baseline and three control visits (five days, a month and three months after the baseline). Morisky Medication Adherence Scale (MMAS-8) was used for evaluation of therapy adherence. RESULTS: The study included 60 patients who received rivaroxaban therapy in a dose of 10 mg once a day. A low adherence to the drug was observed in 15% patients. All patients had an average MMAS-8 score in the range of high adherence 0.65 ± 0.90. Symptomatic venous thromboembolism was observed in two patients with numerous risk factors. No major bleeding was recorded during entire follow-up period. During the five-day postoperative in-hospital follow-up, signs of wound complications were recorded in 8 (13.3%) patients, and 4 (6.7%) of them underwent surgical revision of the wound. CONCLUSION: Generally, there was high adherence to rivaroxaban therapy, but low adherence was present in 15% of patients. Rivaroxaban showed good safety and efficacy. However, high proportion of wound complications and patients needing surgical revision of the wound should be further evaluated through larger studies.

The predictors of perforated appendicitis in the pediatric emergency department: A retrospective observational cohort study.

OBJECTIVE: Appendiceal perforation has significant effects on perioperative morbidity and postoperative outcome. The present study aimed to identify possible predictive factors associated with perforated appendicitis (PA) in children at admission in the emergency department (ED). METHODS: In this retrospective observational cohort study, consecutive medical records of children <18 years old with surgically and histopathologically confirmed acute appendicitis (AA) over three years (2013-2015) were analyzed. Patients were divided into two groups: PA and non-perforated appendicitis (NPA). The differences between the two groups and potential predictors of PA were explored using univariate and multivariate analyses. RESULTS: During the study period, 295 patients underwent an appendectomy and had confirmatory AA diagnoses. Ninety-two patients had a PA (31.2%). In the univariate analysis, male gender, vomiting, diarrhea, fever, elevated white blood cell count (WBC) levels, and high C-reactive protein (CRP) were identified as predictors of PA. In the multivariate analysis, male gender (odds ratio [OR]: 3.133; 95% confidence interval [CI]: 1.610-6.096); vomiting (OR: 2.346; 95% CI: 1.141-4.822); diarrhea (OR: 4.549; 95% CI: 1.850-11.181); fever (OR: 3.429; 95% CI: 1.765-6.663); elevated WBC (OR: 2.962; 95% CI: 1.491-5.884) and elevated CRP (OR: 3.061; 95% CI: 1.267-7.396) were variables that predicted the PA in children. CONCLUSION: Our data indicate that several clinical and biochemical parameters can reliably distinguish between pediatric PA and NPA at admission in the emergency department.

Adverse drug reaction reporting and development of pharmacovigilance systems in Bosnia and Herzegovina, Croatia, Serbia, and Montenegro: a retrospective pharmacoepidemiological study.

AIM: To compare individual case safety reports (ICSR) rates and characteristics between Croatia, Serbia, Montenegro, and Bosnia and Herzegovina (B&H). METHODS: This retrospective pharmacoepidemiological study used the data from ICSR received by the Agency for Medicines and Medical Devices in B&H in 2011-2016. The number, characteristics, and sources of reports, suspected drugs, and patient characteristics were analyzed. The results were compared with the publicly available data from Croatia, Serbia, and Montenegro. RESULTS: The number of reported adverse drug reactions per one million of inhabitants was lowest in B&H and highest in Croatia. There were significant differences in reporter characteristics, sources of reports, and the percentage of missing data in ICSR, while the Anatomical Therapeutic Chemical product classes, patient's sex, and adverse drug reaction System Organ Classes were similar. CONCLUSION: Despite the historical and geographical vicinity of B&H and its neighboring countries, there were significant differences in indicators of pharmacovigilance development.

Synthesis, Biological Evaluation and Docking Studies of Benzoxazoles Derived from Thymoquinone.

Thymoquinone (TQ), a natural compound with antimicrobial and antitumor activity, was used as the starting molecule for the preparation of 3-aminothymoquinone (ATQ) from which ten novel benzoxazole derivatives were prepared and characterized by elemental analysis, IR spectroscopy, mass spectrometry and NMR (¹H, 13C) spectroscopy in solution. The crystal structure of 4-methyl-2-phenyl-7-isopropyl-1,3-benzoxazole-5-ol (1a) has been determined by X-ray diffraction. All compounds were tested for their antibacterial, antifungal and antitumor activities. TQ and ATQ showed better antibacterial activity against tested Gram-positive and Gram-negative bacterial strains than benzoxazoles. ATQ had the most potent antifungal effect against Candida albicans, Saccharomyces cerevisiae and Aspergillus brasiliensis. Three benzoxazole derivatives and ATQ showed the highest antitumor activities. The most potent was 2-(4-fluorophenyl)-4-methyl-7-isopropyl-1,3-benzoxazole-5-ol (1f). Western blot analyses have shown that this compound inhibited phosphorylation of protein kinase B (Akt) and Insulin-like Growth Factor-1 Receptor (IGF1R ß) in HeLa and HepG2 cells. The least toxic compound against normal fibroblast cells, which maintains similar antitumor activities as TQ, was 2-(4-chlorophenyl)-4-methyl-7-isopropyl-1,3-benzoxazole-5-ol (1e). Docking studies indicated that 1e and 1f have significant effects against selected receptors playing important roles in tumour survival.

The correlation between ureteric orifice morphology and primary vesicoureteral reflux grade and the impact on the effectiveness of endoscopic reflux correction.

INTRODUCTION: Early diagnosis and treatment of primary vesicoureteral reflux (VUR) are essential for preserving renal function. OBJECTIVES: The study explored whether preoperative cystoscopic grading of refluxing ureteric orifices (UO) correlated with their shape in an institution with non-performance of hydrodistention of the UO in the diagnosis and grading of VUR. We also assessed the relationship between the UO shape and VUR grade with the effectiveness of endoscopic correction of primary VUR in children. METHODS: This retrospective study included consecutive patients ≤15 years treated for primary VUR. The reflux grade was based on the results of preoperative voiding cystourethrography as mild, moderate, or severe. RESULTS: Fifty-one patients with 77 renal refluxing units (RRU) underwent endoscopic treatment with Deflux®. VUR was bilateral in 51 % of patients. VUR was mild in 13 %, moderate in 53 %, and severe in 34 % of cases. The patients with mild and moderate VUR had stadium-shaped UOs in 60 % and 54 % RRUs, respectively. Horseshoe-shaped UOs constituted 42 % of UOs in patients with severe VUR, followed by 31 % of golf-hole UOs. The reflux resolution rate after the first endoscopic injection was 84 %. The preoperative VUR grade correlated with UOs shape (p < 0.001). No significant correlation between UOs configuration and the outcome of endoscopic treatment was seen (p = 0.452). The preoperative VUR grade negatively correlated with a favorable endoscopic treatment (p = 0.043). DISCUSSION AND CONCLUSION: Our data indicate ureteral orifice shapes are closely related to preoperative VUR grade. There was no correlation between the UO configuration and the success rate of endoscopic treatment of VUR, in contrast to the significant negative correlation between the VUR grade and the success rate of endoscopic treatment.

The Frequency, Severity, and Risk Factors of Hickman CatheterRelated Complications in Pediatric Cancer Patients: A Single-Center Experience from Bosnia and Herzegovina.

OBJECTIVE: This study aimed to identify the frequency, severity, and risk factors associated with Hickman catheter-related complications in children with hemato-oncological malignancies at the largest pediatric tertiary care unit in Bosnia and Herzegovina. MATERIALS AND METHODS: A cross-sectional study was conducted on a cohort of pediatric cancer patients who underwent Hickman central venous catheters (CVCs) between January 2019 and December 2022. Mechanical, infectious, and thrombotic Hickman catheter-related complications were evaluated and analyzed. We also investigated possible risk factors associated with these complications. RESULTS: Seventy-one Hickman CVCs were inserted in 68 children (44 boys and 24 girls) at a mean age of 6.9 ± 4.6. Forty (58.8%) children had hematological malignancies and 28 (41.2%) solid cancers. The median follow-up after Hickman CVC insertion was 190 days (95% CI [160-212]) for 12 644 catheter days. During follow-up, 10 (14.1%) mechanical, 7 (9.9%) infectious, and 1 (1.4%) thrombotic complications were recorded (0.8, 0.48, and 0.08 for mechanical, infectious, and thrombotic complications per 1000 catheter days, respectively). A slightly higher incidence of complications was recorded in children with hematological malignancies (1.59 per 1000 catheter days) compared with children with solid cancers (1.22 complications per 1000 catheter days). CONCLUSION: Using Hickman CVCs for long-term venous access in infusional chemotherapy for pediatric cancer patients is safe but is associated with significant morbidity.

Role of Lactate Dehydrogenase as a Biomarker of Early Cardiac Remodeling: A Cross-Sectional Study.

BACKGROUND: Lactate dehydrogenase (LDH) isoenzyme assay was used widely in the past to diagnose myocardial infarction (MI). Recent studies show that lactate dehydrogenase seems to be a promising biomarker of adverse left ventricular remodeling. OBJECTIVES: Higher levels of these biomarkers were associated with lower odds for favorable reverse remodeling in patients with MI. METHODS: The study was performed on patients with the first occurrence of acute myocardial infarction (ST-elevation myocardial infarction (STEMI) or non-ST-elevation myocardial infarction (NSTEMI)), aged 34 to 80 years who underwent catheterization at the admission or during their hospital stay depending on indications. In this study, we compared peak levels of lactate dehydrogenase (LDH) and left ventricular ejection fraction (LVEF). Peak values of LDH were used from the second to the fourth day of hospitalization. Echocardiography has been done in the first 72 hours, which represents an early phase of cardiac remodeling. The ejection fraction was evaluated using the Simpson method. RESULTS: Spearman's rank test showed a negative, statistically significant correlation between LDH and ejection fraction ρ(80)=-0.543, p<0.001. Weighted least squares regression model included LDH concentration, age, and type of myocardial infarction (STEMI/NSTEMI), and the slope coefficient for the LDH level was -0.010 (95% confidence interval (CI): -0.013 to -0.006). With each unit of LDH increase, there was a decrease of 0.01% in left ventricular ejection fraction when age and type of myocardial infarction were held constant. CONCLUSION: The increased LDH level could be a new predictor for early myocardial remodeling after the first occurrence of myocardial infarction independent of age and type of myocardial infarction.

HPLC method for the determination of thymoquinone in growth cell medium.

BACKGROUND: Preclinical drug testing requires in vitro and in vivo assessments that are vital for studying drug pharmacokinetics and toxicity. Distinct factors that play an important role in drug screening, such as hydrophobicity, solubility of the substance and serum protein binding can be challenging by inducing result inconsistencies. Hence, establishing accurate methods to quantify drug concentrations in cell cultures becomes pivotal for reliable and reproducible results important for in vivo dosing predictions. OBJECTIVE: This research focuses on developing an optimized analytical approach via high-pressure liquid chromatography (HPLC) to determine thymoquinone (TQ) levels in monolayer cell cultures. METHODS: The method's validation adheres to the International Council for Harmonisation (ICH) guideline M10, ensuring its acceptance and applicability. Using an HPLC system with a Diode Array Detector (DAD), the study fine-tuned various parameters to achieve an efficient separation of TQ. Validation covered specificity, sensitivity, matrix effects, linearity, precision, and accuracy, alongside assessing TQ stability in RPMI-1640 medium. RESULTS: The HPLC method exhibited remarkable TQ specificity, free from interfering peaks at the analyte retention. Sensitivity analysis at the lower limit of quantification (LLOQ) revealed 5.68% %CV and 98.37% % mean accuracy. Matrix effect evaluation showcased accuracy within 85-115%. Linearity spanned in the concentration range of 2-10 µM with a correlation coefficient (r2) of 0.9993. Precision and accuracy were aligned with acceptance criteria. The proposed method was found to be greener in terms of usage of persistent, bioaccumulative, and toxic chemicals and solvents, corrosive samples, and waste production. CONCLUSION: The developed HPLC-DAD method emerges as specific, accurate, sensitive, and reliable for TQ determination in cell cultures. It ensures robust TQ quantification, enhancing precise in vitro assessments and dependable dosing predictions for in vivo studies. Further research is advocated to investigate TQ's stability across diverse environmental conditions.

Polyphenol stability and bioavailability in cell culture medium: Challenges, limitations and future directions.

Polyphenols are abundant natural plant micronutrients that commonly contribute to human health due to their anti-inflammatory, antioxidant, antiviral, anti-carcinogenic, anti-aging, anti-allergic, and other biological activities. Their therapeutic benefits mainly depend on the structure, stability, chemical interactions, and absorption, which ultimately affect the bioavailability of these compounds. The bioactivity of polyphenols is evaluated by in vitro and in vivo studies, sometimes yielding inconsistent results due to numerous differences between used models. Among the main differences is the production of reactive oxygen species (ROS) in cultured cell models, potentially leading to misinterpretation of the effects of polyphenolic compounds. Little attention is paid to the polyphenol stability in cell culture medium and the potential generation of artifacts due to their chemical instability. Stability tests of polyphenols are strongly advised to be performed in parallel with cell culture, to help avoid misleading conclusions. This review highlights the existing challenges with cell-based research, focusing on polyphenols' stability in the cell culture media. We also emphasize that new methods analyzing the molecular interactions of compounds with cell culture media supplements are essential to provide a comprehensive understanding of the polyphenols in in vitro models.

Clinical Significance and Expression Pattern of RIP5 and VGLL4 in Clear Cell Renal Cell Carcinoma Patients Treated with Sunitinib.

While clear cell renal cell carcinoma (ccRCC) is curable, advanced metastatic (mRCC) remains a clinical challenge. We analyzed clinical, pathohistological, and molecular data (Receptor Interacting Protein 5-RIP5 and Vestigial Like Family Member 4-VGLL4 expression) of 55 mRCC patients treated with first-line treatment with sunitinib. The trend of linear increase in the protein expression of RIP5 was observed with the progression of tumor grade. Overall, 80% of RIP5-positive cells were in the control kidneys and high-grade mRCC. On the contrary, RIP5 displayed low expression in grade 2 mRCC (5.63%). The trend of linear decrease in the expression of VGLL4 was observed with the progression of tumor grade. The highest protein expression of VGLL4 was observed in grade 2 (87.82%) in comparison to grade 3 and 4 and control. High expression of RIP5 mRNA was associated with longer first-line overall survival and longer progression-free survival in mRCC. In addition, a high VGLL4 mRNA expression showed better overall survival in patients with ccRCC. In conclusion, high mRNA expression of RIP5 and VGLL4 are important markers of better survival rates in mRCC patients.

Molecular Mechanisms Related to Responses to Oxidative Stress and Antioxidative Therapies in COVID-19: A Systematic Review.

The coronavirus disease (COVID-19) pandemic is a leading global health and economic challenge. What defines the disease's progression is not entirely understood, but there are strong indications that oxidative stress and the defense against reactive oxygen species are crucial players. A big influx of immune cells to the site of infection is marked by the increase in reactive oxygen and nitrogen species. Our article aims to highlight the critical role of oxidative stress in the emergence and severity of COVID-19 and, more importantly, to shed light on the underlying molecular and genetic mechanisms. We have reviewed the available literature and clinical trials to extract the relevant genetic variants within the oxidative stress pathway associated with COVID-19 and the anti-oxidative therapies currently evaluated in the clinical trials for COVID-19 treatment, in particular clinical trials on glutathione and N-acetylcysteine.

Viral Agents as Potential Drivers of Diffuse Large B-Cell Lymphoma Tumorigenesis.

Among numerous causative agents recognized as oncogenic drivers, 13% of total cancer cases occur as a result of viral infections. The intricacy and diversity of carcinogenic processes, however, raise significant concerns about the mechanistic function of viruses in cancer. All tumor-associated viruses have been shown to encode viral oncogenes with a potential for cell transformation and the development of malignancies, including diffuse large B-cell lymphoma (DLBCL). Given the difficulties in identifying single mechanistic explanations, it is necessary to combine ideas from systems biology and viral evolution to comprehend the processes driving viral cancer. The potential for more efficient and acceptable therapies lies in targeted medicines that aim at viral proteins or trigger immune responses to either avoid infection or eliminate infected or cancerous cells. In this review, we aim to describe the role of viral infections and their mechanistic approaches in DLBCL tumorigenesis. To the best of our knowledge, this is the first review summarizing the oncogenic potential of numerous viral agents in DLBCL development.

Curcumin Decreases Viability and Inhibits Proliferation of Imatinib-Sensitive and Imatinib-Resistant Chronic Myeloid Leukemia Cell Lines.

Chronic myeloid leukemia (CML) is a myeloproliferative haematological malignancy characterized by constitutive activation of BCR-ABL1 tyrosine kinase in the majority of patients. BCR-ABL1 expression activates signaling pathways involved in cell proliferation and survival. Current treatment options for CML include tyrosine kinase inhibitors (TKI) with resistance as a major issue. Various treatment options for overcoming resistance are being investigated. Among them, phytochemical curcumin could play an important role. Curcumin has been found to exhibit anti-cancerous effects in various models, including CML, through regulation of multiple molecular signaling pathways contributing to tumorigenesis. We have evaluated curcumin's effects on imatinib-sensitive LAMA84S and K562, as well as imatinib-resistant LAMA84R cell lines. Our results indicate a significant dose-dependent decrease in cell viability and proliferation of imatinib-sensitive and imatinib-resistant cell lines after curcumin treatment. Suppression of key signaling molecules regulating metabolic and proliferative events, such as Akt, P70S6K and NF-kB, was observed. Increased expression of caspase-3 suggests the potential pro-apoptotic effect of curcumin in the imatinib-resistant CML model. Additional in silico molecular docking studies revealed binding modes and affinities of curcumin with different targets and the results are in accordance with in vitro findings. Altogether, these results indicate the potential role of curcumin in the treatment of CML.

Nifuroxazide Has Better Efficacy Than Probiotic Treatment in Adult Patients with Acute Diarrheal Syndrome.

Background: Acute gastroenteritis remains an extremely common problem among the general population. In Western countries, an average person will probably face one or two episodes of gastrointestinal infections every year. Objective: The aim of this study was to compare the efficacy of nifuroxazide and probiotic preparation containing lactic acid bacteria in the treatment of acute diarrheal syndrome. Methods: The study was prospective, comparative study. Patients who suffered from acute infective diarrhoea for ≤72 hours and had ≥3 unformed stools per day, with no administration of antibiotics during 10 days before enrolment were divided into two groups: nifuroxazide group and the lactic acid probiotic group. All patients received therapies four times a day for three days. Data was collected at the baseline visit (before the initiation of the treatment) and two follow-up examinations on the third and seventh day from the treatment start. Results: The study included 61 patients, 36 in nifuroxazide group and 25 in probiotic group. Nifuroxazide group compared to probiotic group showed faster improvement of patients' condition with lower number of stools three and seven days after therapy start (p=0.001 and p<0.001 respectively) and faster stool consistency normalization. On the seventh day from therapy start medium mushy stool consistency was observed in the majority of patients in nifuroxazide group (n=31, 86%) and only in small number of patients in probiotic group (n=5, 20%). Patients were feeling better and there was a trend of reporting better therapy efficacy in nifuroxazide group. Subjective assessment of therapy tolerability was also better in nifuroxazide group. Compliance to therapy and recommended dietary regime was similar between groups and there were no significant differences between groups regarding age, gender, elevated body temperature, abdominal pain, cramps, nausea and vomiting. Conclusion: Although probiotics are sometimes used in the treatment of acute diarrheal syndrome, nifuroxazide has better efficacy and greater patients' satisfaction. Nifuroxazide can be recommended as the first choice empirical treatment in adult patients with the acute diarrheal syndrome.

Crystallography and DFT Studies of Synthesized Tetraketones.

Two tetraketone derivatives, one previously reported and one novel, were synthesized, whose structures have been confirmed by elemental analyses, NMR, HPLC-MS, and IR spectroscopy. The crystal structures of synthesized tetraketones were determined using X-ray single-crystal diffraction. To analyze the molecular geometry and compare with experimentally obtained X-ray crystal data of synthesized compounds 1 (2,2'-((4-nitrophenyl)methylene)bis(5,5-dimethylcyclohexane-1,3-dione)) and 2 (2,2'-((4-hydroxy-3-methoxy-5-nitrophenyl)methylene)bis(5,5-dimethylcyclohexane-1,3-dione)), DFT calculations were performed with the standard 6-31G*(d), 6-31G**, and 6-31+G* basis sets. The calculated HOMO-LUMO energy gap for compound 1 was 4.60 eV and this value indicated that compound 1 is chemically more stable compared to compound 2 whose energy gap was 3.73 eV. Both compounds' calculated bond lengths and bond angles were in very good accordance to experimental values determined by X-ray single-crystal diffraction.

Metformin and Thymoquinone Synergistically Inhibit Proliferation of Imatinib-Resistant Human Leukemic Cells.

Chemotherapy resistance is one of the major challenges in cancer treatment, including leukemia. A massive array of research is evaluating combinations of drugs directed against different intracellular signaling molecules to overcome cancer resistance, increase therapy effectiveness, and decrease its adverse effects. Combining chemicals with proven safety profiles, such as drugs already used in therapy and active substances isolated from natural sources, could potentially have superior effects compared to monotherapies. In this study, we evaluated the effects of metformin and thymoquinone (TQ) as monotherapy and combinatorial treatments in chronic myeloid leukemia (CML) cell lines sensitive and resistant to imatinib therapy. The effects were also evaluated in primary monocytic acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) cells. Both compounds induced a dose- and time-dependent decrease of viability and proliferation in tested cells. Metformin had similar IC50 values in imatinib-sensitive and imatinib-resistant cell lines. IC50 values of TQ were significantly higher in imatinib-resistant cells, but with a limited resistance index (2.4). Synergistic effects of combinatorial treatments were observed in all tested cell lines, as well as in primary cells. The strongest synergistic effects were observed in the inhibition of imatinib-resistant cell line proliferation. Metformin and TQ inhibited the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and induced apoptosis in tested cell lines and primary cells. The enhanced effects of combinatorial treatments on the induction of apoptosis were more dominant in imatinib-resistant compared to imatinib-sensitive CML cells. Primary cells were more sensitive to combinatorial treatments compared to cell lines. A combination of 1.25 mM metformin and 0.625 µM TQ increased the levels of cleaved poly (ADP-ribose) polymerase (PARP), decreased the levels of proliferation regulatory proteins, and inhibited protein kinase B (Akt) and NF-κB signaling in primary CLL cells. This study demonstrates that combinatorial treatments of imatinib-resistant malignant clones with metformin and TQ by complementary intracellular multi-targeting represents a promising approach in future studies.