Differences in nonoxidative sulfur metabolism between normal human breast MCF-12A and adenocarcinoma MCF-7 cell lines.

Recent studies have revealed the role of endogenous hydrogen sulfide (H2S) in the development of breast cancer. The capacity of cells to generate H2S and the activity and expression of the main enzymes (cystathionine beta synthase; CBS, cystathionase γ-lyase; CGL, 3-mercaptopyruvate sulfurtransferase; MPST and thiosulfate sulfurtransferase; TST) involved in H2S metabolism were analyzed using an in vitro model of a non-tumourigenic breast cell line (MCF-12A) and a human breast adenocarcinoma cell line (MCF-7). In both cell lines, MPST, CGL, and TST expression was confirmed at the mRNA (RT-PCR) and the protein (Western Blot) level, while CBS expression was detected only in MCF-7 cells. Elevated levels of GSH, sulfane sulfur and increased CBS and TST activity were presented in the MCF-7 compared to the MCF-12A cells. It appears that cysteine might be mainly a substrate for GSH synthesis in breast adenocarcinoma. Increased capacity of the cells to generate H2S was shown for MCF-12A compared to MCF-7 cell line. Results suggest an important function of CBS in H2S metabolism in breast adenocarcinoma. The presented work may contribute to further research on new therapeutic possibilities for breast cancer - one of the most frequently diagnosed types of cancer among women.

Molecular biology techniques in real life - case study introducing medical students to QF-PCR technique used in medical diagnostics.

QF-PCR is a widely used molecular biology method. To name just a few of its uses, it is considered to be useful in paternity tests, identification tests or prenatal diagnostics. Therefore, there is a good chance that medical faculty students would come into contact with this technology - directly or indirectly - during their professional work. The following article proposes a teaching classes scenario conducted in the problem-based learning manner, which aims to familiarize students with the QF-PCR technique. In addition, other modern methods of molecular genetics are among topics that students can learn during the problem-based learning modules. The classes are divided into three parts. In the first part, students learn about the possible usage of QF-PCR in paternity tests. The second part focuses on learning about the advantages and limitations of QF-PCR in prenatal diagnosis. Learning activities in the last part are designed to show the limitations of the diagnostic properties of the method - students analyze the case study, in which QF-PCR must be replaced by other modern methods of molecular genetics. By analyzing three independent stories, students learn about usage, advantages and limitations of QF-PCR, and additionally gain knowledge in basic, pre-clinical and clinical sciences. This course is designated as an elective course for final year medical students who have completed either: a basic genetics course, a molecular genetics course, a biochemistry course or a molecular biology course. The focus of the classes is to draw students' attention to the possible application and rapid development of molecular biology techniques, which is the base for modern therapeutic and diagnostic strategies.

Analysis of biochemical parameters in an experimental model of chronic pancreatitis in rats.

BACKGROUND: Despite advanced research and great progress in understanding the chronic pancreatitis (CP) pathogenesis, no current causal treatment for the condition is available. For preclinical studies, the existence of a well-characterized CP animal model is essential. The aim of the study was to assess the impact of chronic pancreatitis on the antioxidant enzymes activity in rat blood serum and on the level of glutathione (intracellular antioxidant) in rat pancreas. METHODS: The experiments were carried out on the Wistar Kyoto rats in two groups: control and study group (CP), in which chemical induction of pancreatitis with dibutyl dichloride was performed. Serum enzyme activities of amylase, lipase, catalase and superoxide dismutase were analyzed. The levels of the following biochemical parameters were also investigated: total protein, albumin, calcium, magnesium, and triglycerides. Levels of low-molecular-weight thiols: reduced (GSH) and oxidized (GSSG) glutathione, were determined in pancreatic homogenates. RESULTS: Histopathological imaging of rat pancreatic parenchyma with induced inflammation confirmed focal lymphocytic interstitial chronic pancreatitis with fibrosis features and mild parenchymal atrophy, as well as pancreatic islets degeneration. In the CP group, we observed a statistically significant decrease in serum amylase and lipase activities and in total protein/albumin levels. Also, the elevated catalase activity was registered. In CP rats' tissues, we observed a 15-fold reduction in GSH levels. The other examined parameters remained unchanged. Clinically relevant are hypoalbuminemia and a moderate decrease in lipase activity. The described changes are most probably indicative of the impaired exocrine pancreas function, however without organ failure features.

S-Allyl-L-Cysteine Affects Cell Proliferation and Expression of H2S-Synthetizing Enzymes in MCF-7 and MDA-MB-231 Adenocarcinoma Cell Lines.

S-allyl-L-cysteine (SAC) is a sulfur compound present in fresh garlic. The reference literature describes its anticancer, antioxidant and neuroprotective effects. Breast cancer is infamously known as one of the most commonly diagnosed malignancies among women worldwide. Its morbidity and mortality make it reasonable to complete and expand knowledge on this cancer's characteristics. Hydrogen sulfide (H2S) and its naturally occurring donors are well-known investigation subjects for diverse therapeutic purposes. This study was conducted to investigate the SAC antiproliferative potential and effect on three enzymes involved in H2S metabolism: 3-mercaptopyruvate sulfurtransferase (MPST), cystathionine γ-lyase (CTH), and cystathionine ß-synthase (CBS). We chose the in vitro cellular model of human breast adenocarcinomas: MCF-7 and MDA-MB-231. The expression of enzymes after 2, 4, 6, 8, and 24 h incubation with 2.24 mM, 3.37 mM, and 4.50 mM SAC concentrations was examined. The number of living cells was determined by the MTS assay. Changes in cellular plasma membrane integrity were measured by the LDH test. Expression changes at the protein level were analyzed using Western blot. A significant decrease in viable cells was registered for MCF-7 cells after all incubation times upon 4.50 mM SAC exposure, and after 6 and 24 h only in MDA-MB-231 upon 4.50 mM SAC. In both cell lines, the MPST gene expression significantly increased after the 24 h incubation with 4.50 mM SAC. S-allyl-L-cysteine had opposite effects on changes in CTH and CBS expression in both cell lines. In our research model, we confirmed the antiproliferative potential of SAC and concluded that our studies provided current information about the increase in MPST gene expression mediated by S-allyl-L-cysteine in the adenocarcinoma in vitro cellular model for the MCF-7 and MDA-MB-231 cell lines. Further investigation of this in vitro model can bring useful information regarding sulfur enzyme metabolism of breast adenocarcinoma and regulating its activity and expression (gene silencing) in anticancer therapy.

Teaching "Biochemistry with Elements of Chemistry" in the COVID-19 pandemic.

The purpose of the presented article is to evaluate the students' perception of the online teaching educational model as a part of the "Biochemistry with Elements of Chemistry" course conducted during the COVID-19 pandemic at the Faculty of Medicine of the Jagiellonian University Medical College. The first part of the article reflects upon the pandemic impact on the transition from the in-person (standard format) to a complete remote learning format. The next part is based on the responses of the students to a questionnaire and presents an analysis of the students' preferences and perceptions regarding synchronous and asynchronous teaching methods. Students answered questions about the advantages and disadvantages of distance learning programs. They indicated the most suitable learning mode in terms of gaining knowledge and enhancing their motivation to learn. They listed factors that facilitated remote learning as well as those which made it difficult and posed a challenge to adjust to the new system, in all types of classes, that is, lectures, seminars, and laboratories. The last part of this paper presents the results of the students' performance in the pandemic-enforced system and compares them with the results from the previous class of students from the past year, when teaching was conducted mainly in the standard format. The conclusions from the given analysis may enable beneficial changes for the "Biochemistry with Elements of Chemistry" course for remote teaching in the future. It may also provide valuable insights for such types of courses conducted at other Universities and promote deliberations to continuously improve learning outcomes.

Does the absence of SARS-CoV-2 specific genes always exclude the infection? How to interpret RT-PCR results?-The scenario of interactive online workshop.

The aim of this online workshop is to familiarize biomedical faculties students with the principle of RT-PCR method. The following assumption is made, students participating in the workshop: are already familiar with the principle of PCR reaction, can distinguish PCR from RT-PCR, know the basic possibilities of using the above techniques. During the online workshop participants are supposed to learn the interpretation of PCR and RT-PCR results and to understand the crucial importance of controlling the reaction conditions. The workshop involves active students' learning, critical analysis of the data, group discussion, brainstorming method, involvement of e-tools such as pool everywhere or e-learning platforms, as well as interpreting the real-life example results that allows putting the topic in the proper future work-related tasks. The final part of the workshop focuses on the analysis of the RT-PCR results performed in order to confirm or exclude the presence of the SARS-CoV-2 genome in potentially infected individuals. The students are expected to see the practical/work-related part of the knowledge gained during the workshop.