Demonstrating drug treatment efficacies by monitoring superoxide dynamics in human lung cancer cells with time-lapse fluorescence microscopy.

Metformin hydrochloride, an antihyperglycemic agent, and sulindac, a nonsteroidal anti-inflammatory drug, are FDA-approved drugs known to exert anticancer effects. Previous studies demonstrated sulindac and metformin's anticancer properties through mitochondrial dysfunction and inhibition of mitochondrial electron transport chain complex I and key signaling pathways. In this study, various drugs were administered to A549 lung cancer cells, and results revealed that a combination of sulindac and metformin enhanced cell death compared to the administration of the drugs separately. To measure superoxide production over time, we employed a time-lapse fluorescence imaging technique using mitochondrial-targeted hydroethidine. Fluorescence microscopy data showed the most significant increases in superoxide production in the combination treatment of metformin and sulindac. Results showed significant differences between the combined drug treatment and control groups and between the positive control and control groups. This approach can be utilized to quantify the anticancer efficacy of drugs, creating possibilities for additional therapeutic options.

The Molecular Basis and Clinical Consequences of Chronic Inflammation in Prostatic Diseases: Prostatitis, Benign Prostatic Hyperplasia, and Prostate Cancer.

Chronic inflammation is now recognized as one of the major risk factors and molecular hallmarks of chronic prostatitis, benign prostatic hyperplasia (BPH), and prostate tumorigenesis. However, the molecular mechanisms by which chronic inflammation signaling contributes to the pathogenesis of these prostate diseases are poorly understood. Previous efforts to therapeutically target the upstream (e.g., TLRs and IL1-Rs) and downstream (e.g., NF-κB subunits and cytokines) inflammatory signaling molecules in people with these conditions have been clinically ambiguous and unsatisfactory, hence fostering the recent paradigm shift towards unraveling and understanding the functional roles and clinical significance of the novel and relatively underexplored inflammatory molecules and pathways that could become potential therapeutic targets in managing prostatic diseases. In this review article, we exclusively discuss the causal and molecular drivers of prostatitis, BPH, and prostate tumorigenesis, as well as the potential impacts of microbiome dysbiosis and chronic inflammation in promoting prostate pathologies. We specifically focus on the importance of some of the underexplored druggable inflammatory molecules, by discussing how their aberrant signaling could promote prostate cancer (PCa) stemness, neuroendocrine differentiation, castration resistance, metabolic reprogramming, and immunosuppression. The potential contribution of the IL1R-TLR-IRAK-NF-κBs signaling molecules and NLR/inflammasomes in prostate pathologies, as well as the prospective benefits of selectively targeting the midstream molecules in the various inflammatory cascades, are also discussed. Though this review concentrates more on PCa, we envision that the information could be applied to other prostate diseases. In conclusion, we have underlined the molecular mechanisms and signaling pathways that may need to be targeted and/or further investigated to better understand the association between chronic inflammation and prostate diseases.

Predicting residues involved in anti-DNA autoantibodies with limited neural networks.

Computer-aided rational vaccine design (RVD) and synthetic pharmacology are rapidly developing fields that leverage existing datasets for developing compounds of interest. Computational proteomics utilizes algorithms and models to probe proteins for functional prediction. A potentially strong target for computational approach is autoimmune antibodies, which are the result of broken tolerance in the immune system where it cannot distinguish "self" from "non-self" resulting in attack of its own structures (proteins and DNA, mainly). The information on structure, function, and pathogenicity of autoantibodies may assist in engineering RVD against autoimmune diseases. Current computational approaches exploit large datasets curated with extensive domain knowledge, most of which include the need for many resources and have been applied indirectly to problems of interest for DNA, RNA, and monomer protein binding. We present a novel method for discovering potential binding sites. We employed long short-term memory (LSTM) models trained on FASTA primary sequences to predict protein binding in DNA-binding hydrolytic antibodies (abzymes). We also employed CNN models applied to the same dataset for comparison with LSTM. While the CNN model outperformed the LSTM on the primary task of binding prediction, analysis of internal model representations of both models showed that the LSTM models recovered sub-sequences that were strongly correlated with sites known to be involved in binding. These results demonstrate that analysis of internal processes of LSTM models may serve as a powerful tool for primary sequence analysis.

Influence of Ile655Val polymorphism on trastuzumab-induced cardiotoxicity in early-stage HER2 positive breast cancer.

Trastuzumab has improved the prognosis of HER2 positive breast cancer, but cardiotoxicity remains a concern. We aimed to identify risk factors for trastuzumab-induced cardiotoxicity, with an emphasis on the HER2 Ile655Val single nucleotide polymorphism. This single-center case-control study included 1056 patients with early-stage HER2 positive breast cancer that received adjuvant trastuzumab. Cardiotoxicity was defined as a decline in left ventricular ejection fraction (LVEF) > 15% in patients without previous cardiomyopathy, or > 10% in patients with baseline LVEF of < 50%. Patient characteristics and cardiac parameters were compared in 78 (7.38%) cases and 99 randomly assigned controls, and the polymorphism was genotyped using real-time polymerase chain reaction. Cardiotoxicity was independently associated with advanced age (P = 0.024), lower body mass index (P = 0.023), left breast involvement (P = 0.001), N3 status (P = 0.004), diabetes (P = 0.016), and a family history of coronary artery disease (P = 0.019). Genotype distribution was as follows: A/A (Ile/Ile) was found in 111 (62.7%) patients, A/G (Ile/Val) in 60 (33.9%) patients, and G/G (Val/Val) in 6 (3.4%) patients. The genotype was not associated with cardiotoxicity or the severity of heart failure, reversibility, and recovery time. We found no association between the HER2 Ile655Val polymorphism and trastuzumab-induced cardiotoxicity; therefore, we do not recommend routine cardiotoxicity-risk stratification using this polymorphism.

CHARACTERISTICS AND PROGNOSIS OF TRIPLE-NEGATIVE BREAST CANCER PATIENTS: A CROATIAN SINGLE INstitution RETROSPECTIVE COHORT STUDY.

Triple-negative breast cancer (TNBC) occurs in around one-sixth of all breast cancer (BC) patients, with the most aggressive behavior and worst prognosis of all BC subtypes. It is a heterogeneous disease, with specific molecular characteristics and natural dynamics of early recurrence and fast progression. Due to the lack of biomarkers or any valid treatment targets, it can only be treated with classic cytotoxic chemotherapy. We analyzed a cohort of 152 patients, median age 58 years, diagnosed with and treated for early stage TNBC at the University Hospital for Tumors, Sestre milosrdnice University Hospital Centre, Zagreb, Croatia, during the 2009-2012 period. Patients were treated with primary surgical approach, adjuvant chemotherapy and adjuvant irradiation. We observed a relatively large proportion of locally advanced TNBC at diagnosis, with large tumor size and nodal involvement, with high grade and high proliferation index Ki67. Patient age, tumor size and lymph node involvement, as expected, were significant and clinically most important prognostic factors for 5-year disease-free survival (67%; 95% CI 60%-75%) and overall absolute survival rate (74%; 95% CI 66%-81%).

Coronavirus epidemic in Croatia: case fatality decline during summer?

AIM: To describe the SARS-CoV-2 epidemic pattern in Croatia during February-September 2020 and compare the case fatality ratio (CFR) between spring and summer. METHODS: National data were used to calculate the weekly and monthly CFRs, stratified by three age groups: 0-64, 65-79, and 80+ years. We also calculated the standardized mortality ratios (SMR) to offset the differences in age composition. RESULTS: The epidemic consisted of the initial wave, a trough in June, and two conjoined summer waves, yielding 17206 coronavirus disease 2019 cases and 290 deaths. While the number of confirmed cases nearly quadrupled during summer, case fatality estimates decreased; CFR in spring was 4.81 (95% confidence interval 3.91-5.71), compared with 1.24 (1.06-1.42) in summer. The SMR for summer was 0.45 (0.37-0.55), suggesting that the case fatality risk halved compared with spring. Cardiovascular comorbidity was an important risk factor for case fatality (SMR 2.63 [2.20-3.13] during spring and 1.28 [1.02-1.59] during summer). The risk of death in ventilated patients remained unchanged (SMR 0.98 [0.77-1.24]). CONCLUSIONS: The epidemic dynamics suggests summer decline in case fatality, except in ventilated patients. While the effect of comorbidity also decreased, cardiovascular comorbidity remained an important risk factor for death even during summer. A plethora of possible confounders and an ever-changing landscape of SARS-CoV-2 epidemic in Croatia require constant monitoring and evaluation, with an aim to prevent the uncontrolled spread of the virus and a disruption of health care functioning.

OXALIPLATIN INDUCED PULMONARY FIBROSIS: A CASE REPORT.

Oxaliplatin is part of the standard chemotherapy regimens for treating colorectal carcinoma. Pulmonary fibrosis is a serious but rare side effect of oxaliplatin treatment, which resulted in patient death in more than half of the reported cases. The precise pathophysiological mechanism of this phenomenon has not been clarified yet. Analysis of the reported cases strongly suggests that early diagnosis and immediate corticosteroid treatment are crucial for better prognosis. Here we report a case of pulmonary fibrosis related to the FOLFOX regimen in a patient with early colorectal carcinoma.

Rapid hair depigmentation in patient treated with pazopanib.

Pazopanib is multitargeted tyrosine kinase inhibitor used for the treatment of metastatic renal cell carcinoma. Hair colour change is a common side effect of pazopanib therapy which usually develops gradually during few months of therapy. We report a case of the patient who developed multiple pazopanib side effects followed by rapid overnight hair and eyebrow depigmentation after only few weeks of therapy. In our research, we found no literature data of rapid loss of hair pigment due to therapy with any of listed multitargeted tyrosine kinase inhibitors. To the best of our knowledge, this is the first such case being reported. We presume that summation of different mechanisms probably led to rapid hair depigmentation. Considering the fact that pazopanib treatment was very effective in our patient, this side effect could be a good predictor of therapy success, although it presents very stressful event for patient and his family.

Circulating tumor cell isolation, culture, and downstream molecular analysis.

Circulating tumor cells (CTCs) are a major contributor of cancer metastases and hold a promising prognostic significance in cancer detection. Performing functional and molecular characterization of CTCs provides an in-depth knowledge about this lethal disease. Researchers are making efforts to design devices and develop assays for enumeration of CTCs with a high capture and detection efficiency from whole blood of cancer patients. The existing and on-going research on CTC isolation methods has revealed cell characteristics which are helpful in cancer monitoring and designing of targeted cancer treatments. In this review paper, a brief summary of existing CTC isolation methods is presented. We also discuss methods of detaching CTC from functionalized surfaces (functional assays/devices) and their further use for ex-vivo culturing that aid in studies regarding molecular properties that encourage metastatic seeding. In the clinical applications section, we discuss a number of cases that CTCs can play a key role for monitoring metastases, drug treatment response, and heterogeneity profiling regarding biomarkers and gene expression studies that bring treatment design further towards personalized medicine.

UNCOMMON SIDE EFFECTS OF SUNITINIB THERAPY IN A PATIENT WITH METASTATIC RENAL CELL CANCER: CASE REPORT.

- Sunitinib is an orally administered multikinase inhibitor. This therapy can provoke uncommon side effects such as pancytopenia, tumor lysis syndrome, cardiac disorders, thromboembolic incidents, intestinal perforation, pancreatitis, acute renal failure, etc. We report a case of a 63-year-old female admitted to the hospital due to abdominal pain, nausea, vomiting and elevated blood pressure. One month earlier, sunitinib therapy for metastatic renal cell carcinoma was initiated. During the first cycle of therapy, after three weeks of sunitinib 50 mg daily, symptoms started and she stopped taking the drug. At admission, laboratory tests revealed elevated serum and urine amylase, C-reactive protein, urea and creatinine, and lowered platelet and leukocyte counts and hemoglobin value. Urine test showed proteinuria, erythrocyturia, leukocyturia and granulated cylinder. The patient was diagnosed with acute pancreatitis grade III, acute renal failure grade II, pancytopenia and urinary infection, and was hospitalized for five days. She was treated symptomatically and with antibiotic therapy because of persistently elevated C-reactive protein and pathologic urinary sediment, which led to subjective and clinical improvement. Acute pancreatitis, renal insufficiency and pancytopenia are rarely described side effects of sunitinib therapy, and clear connection between these conditions and drug activity is not yet determined. Medical specialists who prescribe and treat patients with sunitinib should be aware of the possible occurrence of these conditions and perform regular checkups of sunitinib treated patients.

Strategies in Ebola virus disease (EVD) diagnostics at the point of care.

Ebola virus disease (EVD) is a devastating, highly infectious illness with a high mortality rate. The disease is endemic to regions of Central and West Africa, where there is limited laboratory infrastructure and trained staff. The recent 2014 West African EVD outbreak has been unprecedented in case numbers and fatalities, and has proven that such regional outbreaks can become a potential threat to global public health, as it became the source for the subsequent transmission events in Spain and the USA. The urgent need for rapid and affordable means of detecting Ebola is crucial to control the spread of EVD and prevent devastating fatalities. Current diagnostic techniques include molecular diagnostics and other serological and antigen detection assays; which can be time-consuming, laboratory-based, often require trained personnel and specialized equipment. In this review, we discuss the various Ebola detection techniques currently in use, and highlight the potential future directions pertinent to the development and adoption of novel point-of-care diagnostic tools. Finally, a case is made for the need to develop novel microfluidic technologies and versatile rapid detection platforms for early detection of EVD.

Software tools for simultaneous data visualization and T cell epitopes and disorder prediction in proteins.

We have developed EpDis and MassPred, extendable open source software tools that support bioinformatic research and enable parallel use of different methods for the prediction of T cell epitopes, disorder and disordered binding regions and hydropathy calculation. These tools offer a semi-automated installation of chosen sets of external predictors and an interface allowing for easy application of the prediction methods, which can be applied either to individual proteins or to datasets of a large number of proteins. In addition to access to prediction methods, the tools also provide visualization of the obtained results, calculation of consensus from results of different methods, as well as import of experimental data and their comparison with results obtained with different predictors. The tools also offer a graphical user interface and the possibility to store data and the results obtained using all of the integrated methods in the relational database or flat file for further analysis. The MassPred part enables a massive parallel application of all integrated predictors to the set of proteins. Both tools can be downloaded from http://bioinfo.matf.bg.ac.rs/home/downloads.wafl?cat=Software. Appendix A includes the technical description of the created tools and a list of supported predictors.

[CANCER PATIENTS FOLLOW-UP ­ CROATIAN SOCIETY OF MEDICAL ONCOLOGY CLINICAL GUIDELINES Part III: neuroendocrine neoplasms, hepatocellular carcinoma, pancreatic cancer, cancer of the bile ducts]. / PRACENJE ONKOLOSKIH BOLESNIKA ­ KLINICKE PREPORUKE HRVATSKOG DRUSTVA ZA INTERNISTICKU ONKOLOGIJU HLZ-a III. dio: neuroendokrine neoplazme, hepatocelularni karcinom, rak gusterace, rak zucnih vodova.

The treatment of oncological patients must be based upon multidisciplinary approach, and takes place in specialized oncological centers. By the end of a specific oncological treatment further follow-up is being managed mostly by the oncologists, but the role of the general practitioners becomes more important every day and therefore should be precisely defined. Nowadays, most of the existing follow-up guidelines are not based on prospective studies, but on the experts opinion of individual oncological centers or specialists. The aim of the Croatian Society of Medical Oncology (CSMO) with these recommendations is to standardize and rationalize the diagnostic procedures algorithm in the follow-up of oncological patients after primary treatment, in patients with neuroendocrine neoplasms, hepatocellular carcinoma, pancreatic cancer and cancer of the bile ducts.

A cohesive analysis of DNA/RNA sequences via entropy, energetics and spectral-domain methods to assess genomic features across single viral diversity.

In virology context, a particular virus may prevail in different forms of serotypes (as in the case of dengue 1-4 viral strains) with common and distinct genomic features. Finding such genomic details of a serogroup is useful in knowing related information for unique vaccine designs compatible for immunity across the viral diversity. For robust comparison of genomes of serovars of a virus in order to decide on their common and differential genomic details, proposed here is a set of sequence analyses exercised side-by-side via entropy, energetic and spectral-domain methods. Results obtained thereof with dengue viral serotypes, namely DEN1, DEN2, DEN3 and DEN4, are presented. Hence, inferences on distinct as well as common features extracted are annotated and indicated for possible vaccine design applications.

Epitope distribution in ordered and disordered protein regions. Part B - Ordered regions and disordered binding sites are targets of T- and B-cell immunity.

Intrinsically disordered proteins exist in highly flexible conformational states linked to different protein functions. In this work, we have presented evidence that HLA class-I- and class-II-binding T-cell epitopes, experimentally verified in several tumor-associated antigens and nuclear systemic autoantigens, are predominantly located in ordered protein regions or at disorder/order borderlines, defined by the majority of analyzed publicly available disorder predictors. We have also observed the overlapping of secondary structural elements and prevalently hydrophobic regions with T-cell epitopes in Epstein Barr Virus (EBV) nuclear antigen 1 (EBNA-1), cancer/testis antigen MAGE-A4, and Sm-B/B', U1 snRNPA (U1A) and U1-70kDa autoantigens. The results are in accordance with the clustering of the predicted HLA class-I and class-II epitopes in protein parts which encompass the consensus of ordered regions, determined by individual disorder predictors. Some HLA class-II epitopes and linear B-cell epitopes were located near the segments predicted to have elevated crystallographic B factor in EBNA-1, Sm-B/B' and U1 snRNP A proteins, suggesting that protein flexibility could influence the structural availability of epitopes. Naturally processed T-cell epitopes and linear B-cell epitopes could also be found within putative disordered binding sites, determined by "dips" in the prevalently disordered parts of prediction profiles of the majority of disorder predictors, and peaks in ANCHOR-prediction profile. Two minor antigenic regions within EBNA-1, mapped to the residues 58-85 and 398-458, encompassing putative disordered binding sites, contain epitopes connected with anti-Ro 60kDa and anti-Sm B/B' autoimmunity in systemic lupus erythematosus. One of these regions overlaps residues 395-450, identified as the binding site of USP7 (HAUSP), which regulates the EBNA-1 replication function. In Sm-B/B', one of the putative disordered binding sites (residues 114-165) encompasses the T-cell epitope 136-153, while another, residues 200-216, flanks two proline-rich B-cell epitopes (residues 190-198 and 216-222), overlapping the preferred CD2BP2-GYF-binding motif (R/K/G)XXPPGX(R/K), characteristic of splicosomal proteins. We have noticed that the same motif (residues 397-403) is mimicked in EBNA-1 and overlaps epitope 398-404, involved in anti-Sm B/B' autoimmunity. The majority of recognized T- and B-cell epitopes in analyzed autoantigens or tumor-associated antigens appertain to the ordered or transient protein structures. The congruence between certain B- and T-cell epitopes and predicted disordered binding sites or protein-binding eukaryotic motifs in the antigens participating in molecular complexes might influence the capture of antigens, their processing and subsequent presentation and immunodominance.