Beyond dopamine: Novel strategies for schizophrenia treatment.

Despite extensive research efforts aimed at discovering novel antipsychotic compounds, a satisfactory pharmacological strategy for schizophrenia treatment remains elusive. All the currently available drugs act by modulating dopaminergic neurotransmission, leading to insufficient management of the negative and cognitive symptoms of the disorder. Due to these challenges, several attempts have been made to design agents with innovative, non-dopaminergic mechanisms of action. Consequently, a number of promising compounds are currently progressing through phases 2 and 3 of clinical trials. This review aims to examine the rationale behind the most promising of these strategies while simultaneously providing a comprehensive survey of study results. We describe the versatility behind the cholinergic neurotransmission modulation through the activation of M1 and M4 receptors, exemplified by the prospective drug candidate KarXT. Our discussion extends to the innovative approach of activating TAAR1 receptors via ulotaront, along with the promising outcomes of iclepertin, a GlyT-1 inhibitor with the potential to become the first treatment option for cognitive impairment associated with schizophrenia. Finally, we evaluate the 5-HT2A antagonist paradigm, assessing two recently developed serotonergic agents, pimavanserin and roluperidone. We present the latest advancements in developing novel solutions to the complex challenges posed by schizophrenia, offering an additional perspective on the diverse investigated drug candidates.

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.

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.

Expression of Alternative Splice Variants of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase-4 in Normoxic and Hypoxic Melanoma Cells.

Cancer-specific isoenzyme of phosphofructokinase II (PFKFB4), as our previous research has shown, may be one of the most important enzymes contributing to the intensification of glycolysis in hypoxic malignant melanoma cells. Although the PFKFB4 gene seems to play a crucial role in the progression of melanoma, so far there are no complete data on the expression of PFKFB4 at the isoform level and the influence of hypoxia on alternative splicing. Using RT-qPCR and semi-quantitative RT-PCR, we presented the PFKFB4 gene expression profile at the level of six isoforms described in the OMIM NCBI database in normoxic and hypoxic melanoma cells. Additionally, using VMD software, we analyzed the structure of isoforms at the protein level, concluding about the catalytic activity of individual isoforms. Our research has shown that five isoforms of PFKFB4 are expressed in melanoma cells, of which the D and F isoforms are highly constitutive, while the canonical B isoform seems to be the main isoform induced in hypoxia. Our results also indicate that the expression profile at the level of the PFKFB4 gene does not reflect the expression at the level of individual isoforms. Our work clearly indicates that the PFKFB4 gene expression profile should be definitely analyzed at the level of individual isoforms. Moreover, the analysis at the protein level allowed the selection of those isoforms whose functional validation should be performed to fully understand the importance of PFKFB4 expression in the metabolic adaptation of malignant melanoma cells.

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.

Evaluation of Cysteine Metabolism in the Rat Liver and Kidney Following Intravenous Cocaine Administration and Abstinence.

Many toxic effects of cocaine are attributed to reactive oxygen species (ROS) generated during its metabolism. Recently, it has been suggested that the biological action of ROS is often confused with endogenously generated reactive sulfur species (RSS). The aim of this study was to evaluate the impact of cocaine on thiols and RSS in the rat liver and kidney in the drug self-administration (SA) paradigm and the cocaine yoked delivery model (YC) followed by drug abstinence with extinction training. The level of thiols as well as RSS formed during anaerobic metabolism of cysteine and sulfate were assayed. In addition, the activity of enzymes involved in RSS formation and glutathione metabolism were determined. In the liver, following direct cocaine administration (SA and YC), the RSS levels decreased, while in the kidneys, cocaine increased the RSS contents in both groups. These changes were maintained in these tissues during drug abstinence. The level of sulfates was changed by cocaine only in the liver. In the kidney, cocaine shifted cysteine metabolism towards an anaerobic pathway. Our study demonstrates for the first time the changes in cysteine metabolism and thiol levels in the liver and kidney of rats after cocaine self-administration and abstinence.

Influence of metformin on HIF-1 pathway in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is defined as plasma cells malignancy, developing in the bone marrow. At the beginning of the disease, the malignant plasma cells are dependent on bone marrow microenvironment, providing growth and survival factors. Importantly, the recent studies pointed hypoxia as an important factor promoting progression of MM. In particular, hypoxia-triggered HIF-1 signaling was shown to promote chemoresistance, angiogenesis, invasiveness and induction of immature phenotype, suggesting that strategies targeting HIF-1 may contribute to improvement of anti-myeloma therapies. METHODS: The Western Blot and RT-PCR techniques were applied to analyze the influence of metformin on HIF-1 pathway in MM cells. To evaluate the effect of metformin on the growth of MM cell lines in normoxic and hypoxic conditions the MTT assay was used. The apoptosis induction in metformin treated hypoxic and normoxic cells was verified by Annexin V/PI staining followed by FACS analysis. RESULTS: Our results showed, for the first time, that metformin inhibits HIF-1 signaling in MM cells. Moreover, we demonstrated the effect of metformin to be mainly oxygen dependent, since the HIF-1 pathway was not significantly affected by metformin in anoxic conditions as well as after application of hypoxic mimicking compound, CoCl2. Our data also revealed that metformin triggers the growth arrest without inducing apoptosis in either normoxic or hypoxic conditions. CONCLUSIONS: Taken together, our study indicates metformin as a promising candidate for developing new treatment strategies exploiting HIF-1 signaling inhibition to enhance the overall anti-MM effect of currently used therapies, that may considerably benefit MM patients.

Aberrant promoter methylation may be responsible for the control of CD146 (MCAM) gene expression during breast cancer progression.

The CD146 (also known as MCAM, MUC-18, Mel-CAM) was initially reported in 1987, as a protein crucial for the invasiveness of malignant melanoma. Recently, it has been confirmed that CD146 has been involved in progression and poor overall survival of many cancers including breast cancer. Importantly, in independent studies, CD146 was reported to be a trigger of epithelial to mesenchymal transition in breast cancer cells. The goal of our current study was to verify the potential involvement of epigenetic mechanism behind the regulation of CD146 expression in breast cancer cells, as it has been previously reported in prostate cancer. First, we analysed the response of breast cancer cell lines, differing in the initial CD146 mRNA and protein content, to epigenetic modifier, 5-aza-2-deoxycytidine, and subsequently the methylation status of CD146 gene promoter was investigated, using direct bisulfite sequencing. We observed that treatment with demethylating agent led to induction of CD146 expression in all analysed breast cancer cell lines, both at mRNA and protein level, what was accompanied by increased expression of selected mesenchymal markers. Importantly, CD146 gene promoter analysis showed aberrant CpG island methylation in 2 out of 3 studied breast cancer cells lines, indicating epigenetic regulation of CD146 gene expression. In conclusion, our study revealed, for the first time, that aberrant methylation maybe involved in expression control of CD146, a very potent EMT inducer in breast cancer cells. Altogether, the data obtained may provide the basis for novel therapies as well as diagnostic approaches enabling sensitive and very accurate detection of breast cancer cells.

The Epigenetic Modifier 5-Aza-2-deoxycytidine Triggers the Expression of CD146 Gene in Prostate Cancer Cells.

BACKGROUND/AIM: During cancer progression cells undergo epithelial-to-mesenchymal transition (EMT). Although EMT is a complex process, recently, it has been reported that CD146 overexpression in prostate cancer cells is sufficient to induce mesenchymal phenotype. The following study aimed to investigate whether the expression of CD146 is altered by an epigenetic modifier in prostate cancer cells, in vitro. MATERIALS AND METHODS: Three human prostate cancer cell lines were treated with 5-aza-2-deoxycytidine; the expression of CD146 and EMT-related factors was analyzed by RT-PCR and western Blot. The methylation status of the CD146 promoter area was assessed using bisulfite sequencing. RESULTS: Our data showed that, the expression of CD146 was evidently increased in all three studied cell lines in response to a demethylating agent, both at the mRNA and protein level, suggesting epigenetic regulation of the analyzed gene. However, there was no methylation in the studied CpG island in CD146 gene promoter. Moreover, the demethylating agent induced the expression of EMT-related transcription factors (SNAI1, SNAI2, TWIST1 and ZEB1), the pattern of which differed among the cell lines, as well as alterations in cell morphology; altogether accounting for the mesenchymal phenotype. CONCLUSION: The demethylating agent 5-aza-2-deoxycytidine triggers the expression of CD146 in prostate cancer cells independently on the methylation status of the analyzed CpG island fragment in CD146 gene promoter. Moreover, demethylation treatment induces a mesenchymal profile in prostate cancer cells.

Application of case study to introduce medical students to molecular biology techniques used in HIV diagnostics.

Diagnostic molecular biology is a fast developing discipline of laboratory medicine widely used in numerous medical branches such as oncology, hematology, immunology, internal medicine, or infectious diseases, which will certainly have a major impact on clinical medicine in the near future. Nowadays, educational process is forced to face the quickly growing overflow of easily accessible data and properly guide the students not to be lead astray in the information chaos. Hence, in view of the foregoing, it appears obvious that modern medical education should put particular stress on selective acquiring, interpreting, and applying integrated multidisciplinary knowledge rather than on just absorbing and memorizing huge amount of scattered information. The presented case study aims at familiarizing the students with basic molecular biology techniques such as enzyme-linked immunosorbent assay, Western blot, and quantitative reverse transcription-polymerase chain reaction. Importantly, it is not limited only to discussing and learning the principles of the assays mentioned earlier, but it also shows their practical application in a particular diagnostic process and give the guidelines on how to explain and interpret exemplary results. In parallel, the way the case study is constructed allows a tutor to lead students into discussion on clinical aspects related to HIV infection what should eventually create complete picture of a HIV diagnostic process, thereby integrating basic knowledge of molecular biology laboratory techniques, HIV biology, and immunological response. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):355-360, 2019.

Effects of kinetin riboside on proliferation and proapoptotic activities in human normal and cancer cell lines.

Kinetin riboside (KR) is a N6-substituted derivative of adenosine. It is a natural compound which occurs in the milk of coconuts on the nanomole level. KR was initially shown to selectively inhibit proliferation of cancer cells and induce their apoptosis. We observed that KR inhibited growth (20-80%) of not only human cancer, but also normal cells and that this effect strongly depended on the type of cells. The anti-apoptotic Bcl-2 protein was downregulated, while proapoptotic Bax was upregulated in normal as well as in cancer cell lines, upon exposure to KR. Cytochrome c level increased in the cytosol upon treatment of cells with KR. The activity of caspases (ApoFluor®Green Caspase Activity Assay), as well as caspase-3 (caspase-3 activation assay) were increased mainly in cancer cells. The expression of procaspase 9 and its active form in the nucleus as well as in cytosol of KR-treated cells was elevated. In contrast, no effect of KR on caspase 8 expression was noted. The results indicated that non-malignant cells were less sensitive to KR then their cancer analogs and that KR most likely stimulated apoptosis mechanism of cancer cells through the intrinsic pathway.

Kinetic intermediates of unfolding of dimeric prostatic phosphatase.

Kinetics of guanidine hydrochloride (GdnHCl)-induced unfolding of human prostatic acid phosphatase (hPAP), a homodimer of 50 kDa subunit molecular mass was investigated with enzyme activity measurements, capacity for binding an external hydrophobic probe, 1-anilinonaphtalene-8-sulfonate (ANS), accessibility of thiols to reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and 2-(4'-maleimidylanilino)naphthalene-6-sulfonate (MIANS) and ability to bind Congo red dye. Kinetic analysis was performed to describe a possible mechanism of hPAP unfolding and dissociation that leads to generation of an inactive monomeric intermediate that resembles, in solution of 1.25 M GdnHCl pH 7.5, at 20 degrees C, in equilibrium, a molten globule state. The reaction of hPAP inactivation in 1.25 M GdnHCl followed first order kinetics with the reaction rate constant 0.0715 +/- 0.0024 min(-1) . The rate constants of similar range were found for the pseudo-first-order reactions of ANS and Congo red binding: 0.0366 +/- 0.0018 min(-1) and 0.0409 +/- 0.0052 min(-1), respectively. Free thiol groups, inaccessible in the native protein, were gradually becoming, with the progress of unfolding, exposed for the reactions with DTNB and MIANS, with the pseudo-first-order reaction rate constants 0.327 +/- 0.014 min(-1) and 0.216 +/- 0.010 min(-1), respectively. The data indicated that in the course of hPAP denaturation exposure of thiol groups to reagents took place faster than the enzyme inactivation and exposure of the protein hydrophobic surface. This suggested the existence of a catalytically active, partially unfolded, but probably dimeric kinetic intermediate in the process of hPAP unfolding. On the other hand, the protein inactivation was accompanied by exposure of a hydrophobic, ANS-binding surface, and with an increased capacity to bind Congo red. Together with previous studies these results suggest that the stability of the catalytically active conformation of the enzyme depends mainly on the dimeric structure of the native hPAP.

[Glutatione transferases--structure and functions. beta-lyase-dependent bioactivation of cysteine S-conjugates]. / Tranferazy glutationowe--bioaktywacja S-koniugatów glutationu z udzialem beta-liazy.

Glutathione transferases (GSTs) catalyze nucleophilic attack of glutathione on electrophilic center of the second substrate, hydrophobic in character. It leads to the formation of glutathione S-conjugates (thioethers), which are subsequently eliminated from the organism as mercapturic acids. However, in some reactions, glutathione can also fulfills the role of a cofactor, facilitating transformation of a hydrophobic substrate molecule, and released after the structure has been changed. Glutathione transferases participate in the processes of conjugation, reduction, isomerization, synthesis of sex hormones, prostaglangins and leukotrienes, degradation of aromatic compounds and signal transduction. The role of these enzymes consists principally in increasing glutathione nucleophilicity by its appropriate positioning and binding in active center, and its following activation by catalytic amino acid residues. There are also so-called ligandins, i.e. glutathione transferases which can bind hydrophobic, non-substrate ligands, thereby contributing to their sequestration. GSTs play a dominating role in detoxification of xenobiotics eliminated from the body in the form of thioethers, which however, under certain conditions, can be bioactivated in beta-liase-catalyzed reaction to form compounds capable of forming tissue adducts. Inhibition of S-transferase activity can have therapeutic significance both when thioethers are activated by beta-liase and during carcinogenesis, which is often accompanied by overexpression of GSTs.