ChatGPT (GPT-3.5) as an assistant tool in microbial pathogenesis studies in Sweden: a cross-sectional comparative study

ChatGPT (GPT-3.5) has entered higher education and there is a need to determine how to use it effectively. This descriptive study compared the ability of GPT-3.5 and teachers to answer questions from dental students and construct detailed intended learning outcomes. When analyzed according to a Likert scale, we found that GPT-3.5 answered the questions from dental students in a similar or even more elaborate way compared to the answers that had previously been provided by a teacher. GPT-3.5 was also asked to construct detailed intended learning outcomes for a course in microbial pathogenesis, and when these were analyzed according to a Likert scale they were, to a large degree, found irrelevant. Since students are using GPT-3.5, it is important that instructors learn how to make the best use of it both to be able to advise students and to benefit from its potential

Thymidine Kinase 2 Deficiency-Induced mtDNA Depletion in Mouse Liver Leads to Defect beta-Oxidation

Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2(-/-)) that progressively loses its mtDNA. The TK2(-/-) mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2(-/-) mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2(-/-) mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2(-/-) mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial beta-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2(-/-) mice causes impaired mitochondrial function that leads to defect beta-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies

Human mitochondrial pyrophosphatase: cDNA cloning and analysis of the gene in patients with mtDNA depletion syndromes

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Nucleoside analog phosphorylation and mitochondrial enzymes : Studies on molecular targets of the anti-leukemic compound 9-beta-D-arabinofuranosylguanine

Nucleoside analogs are commonly used in treatment of cancer and viral infections. A way to improve the therapies would be to minimize the acquirement of resistance and side effects such as delayed cytotoxicity. Nucleoside analogs are phosphorylated and thereby activated by cellular kinases and to understand more about their phosphorylation by mitochondrial enzymes we have studied the molecular targets of the guanosine analog 9-beta-D-arabinofuranosylguanine (araG). This analog is a substrate of both the mitochondrial deoxyguanosine kinase (dGK) and the cytosolic deoxycytidine kinase (dCK). The prodrug of the biologically active araG, nelarabine, has proven highly efficient in particular in patients with T-cell acute lymphoblastic leukemia. Although the mechanism of action of araG is not fully understood, the accumulation of araG tripbosphates (araGTP) has been correlated to cytotoxicity both in vitro and in vivo. AraGTP acts as a structural analog of deoxyguanosine triphosphate (dGTP) and is thereby incorporated into DNA. The accumulation of araGTP is independent of the cell cycle, which is not surprising since both dCK and dGK are expressed throughout the cell cycle. Incorporation of araGMP into nuclear DNA has been suggested as a critical event for cytotoxicity. A recent study has suggested a role of mitochondria in the cell specific toxicity of dGTP with intra-mitochondrial accumulation of dGTP and inhibition of mtDNA repair. The doselimiting toxicity in the clinical trials with nelarabine has been neurotoxicity, but less pronounced adverse effects include other symptoms similar of drugs causing mitochondrial toxicity. We have shown that araG can be incorporated into mtDNA but the mtDNA incorporation does not, however, cause the acute cytotoxicity of araG and we do presently not know to what extent it contributes to the cytotoxic action of the analog. It cannot be excluded that long-term exposure to araG may cause mtDNA alterations with subsequent delayed mitochondrial toxicity. Several studies on mechanisms of resistance to araG have been performed. These studies have shown partly conflicting results as to the molecular mechanism of resistance. In our studies we found that araG resistance can occur by two separate molecular mechanisms that can occur sequentially. The first mechanism is associated with a decrease of araG incorporation into mtDNA and the second event is associated with loss of dCK activity, whereas the dGK activity remained at the same level as in the control cells. We do not yet know how the decreased incorporation of araG into mtDNA contributes to the resistant phenotype, but we know that araG does not cause mtDNA depletion or altered translation of mtDNAencoded genes. To study differences in gene expression in the araG resistant cells we have initiated microarray analysis. In the search for enzymes that could contribute to the activation of nucleoside analogs in the mitochondria we found a UMP-CMP kinase from Drosophila melanogaster that localized to the mitochondria. The recombinant enzyme accepted pyrimidine nucleoside monophosphates as substrates. The enzyme contained an N-terminal signal targeting the enzyme to the mitochondria. The identification of a functional mitochondrial import signal in the Dm.UMP-CMP kinase suggests that this enzyme and its homologues in other species may be involved in the mitochondrial phosphorylation of pyrimidine nucleoside monophosphates. However, the mitochondrial homologue in human cells remains to be identified. It was recently shown that mutations in the genes coding for dGK and the mitochondrial thymidine kinase 2 (TK2) are associated with mtDNA depletion in patients. However, for the majority of patients with mtDNA depletion syndromes (MDS) the genetic defect causing the syndrome remains to be identified. It is known that the yeast mitochondrial pyrophosphatase is necessary for maintained mtDNA content in the yeast cells, and the human mitochondrial pyrophosphatase would be a candidate gene for MDS. Biochemical properties of mammalian mitochondrial pyrophosphatases have been studied on enzyme purified from tissues, but cloning of the gene encoding the enzyme has not been reported previously. Based on sequence similarity to other pyrophosphatases we identified the cDNA encoding the human mitochondrial pyrophosphatase. We cloned the enzyme and showed that it encoded a functional N-terminal mitochondrial targeting signal. The recombinant enzyme was active and ubiquitously expressed with highest levels in tissues rich in mitochondria such as muscle, liver and kidney. The ubiquitous expression suggests that the mitochondrial pyrophosphatase, like the cytosolic pyrophosphatase, is involved in "house-keeping" hydrolyzing of pyrophosphate which is generated by different metabolic processes in the cells. To test if the human mitochondrial pyrophosphatase is required for normal mtDNA copy number, material from more than 50 patients in the USA with unknown cause of MDS is presently being screened for alterations in the gene encoding the mitochondrial pyrophosphate

Students’ performance of and perspective on an objective structured practical examination for the assessment of preclinical and practical skills in biomedical laboratory science students in Sweden: a 5-year longitudinal study

Purpose It aims to find students’ performance of and perspectives on an objective structured practical examination (OSPE) for assessment of laboratory and preclinical skills in biomedical laboratory science (BLS). It also aims to investigate the perception, acceptability, and usefulness of OSPE from the students’ and examiners’ point of view. Methods This was a longitudinal study to implement an OSPE in BLS. The student group consisted of 198 BLS students enrolled in semester 4, 2015–2019 at Karolinska University Hospital Huddinge, Sweden. Fourteen teachers evaluated the performance by completing a checklist and global rating scales. A student survey questionnaire was administered to the participants to evaluate the student perspective. To assess quality, 4 independent observers were included to monitor the examiners. Results Almost 50% of the students passed the initial OSPE. During the repeat OSPE, 73% of the students passed the OSPE. There was a statistically significant difference between the first and the second repeat OSPE (P<0.01) but not between the first and the third attempt (P=0.09). The student survey questionnaire was completed by 99 of the 198 students (50%) and only 63 students responded to the free-text questions (32%). According to these responses, some stations were perceived as more difficult, albeit they considered the assessment to be valid. The observers found the assessment protocols and examiner’s instructions assured the objectivity of the examination. Conclusion The introduction of an OSPE in the education of biomedical laboratory scientists was a reliable, and useful examination of practical skills

Long term survival and abnormal liver fat accumulation in mice with specific thymidine kinase 2 deficiency in liver tissue.

Deficiency in thymidine kinase 2 (TK2) causes mitochondrial DNA depletion. Liver mitochondria are severely affected in Tk2 complete knockout models and have been suggested to play a role in the pathogenesis of the Tk2 knockout phenotype, characterized by loss of hypodermal fat tissue, growth retardation and reduced life span. Here we report a liver specific Tk2 knockout (KO) model to further study mechanisms contributing to the phenotypic changes associated with Tk2 deficiency. Interestingly, the liver specific Tk2 KO mice had a normal life span despite a much lower mtDNA level in liver tissue. Mitochondrial DNA encoded peptide COXI did not differ between the Tk2 KO and control mice. However, the relative liver weight was significantly increased in the male Tk2 KO mouse model. Histology analysis indicated an increased lipid accumulation. We conclude that other enzyme activities can partly compensate Tk2 deficiency to maintain mtDNA at a low but stable level throughout the life span of the liver specific Tk2 KO mice. The lower level of mtDNA was sufficient for survival but led to an abnormal lipid accumulation in liver tissue

Methodological aspects of ELISA analysis of thioredoxin 1 in human plasma and cerebrospinal fluid.

Thioredoxin-1 (Trx1) is a protein antioxidant involved in major cellular processes. Increased plasma levels of Trx1 have been associated with human diseases suggesting that Trx1 is a marker for oxidative stress with putative clinical use. However, the reported mean levels of Trx1 in the control cohorts vary a hundred-fold between studies (0.8-87 ng/ml), possibly due to methodological differences between the capture ELISA used in the different studies. The aim of this study was to investigate methodological aspects related to the ELISA measurement of Trx1. ELISAs utilizing different capture and detection combinations of antibodies to Trx1 and as well as recombinant human (rh) Trx1 standards from two sources were characterized. The different ELISAs were subsequently used to measure Trx1 in human plasma and cerebrospinal fluid samples (CSF) from healthy donors and from patients with various neurological diagnoses. The Trx1 standards differed in their content of monomeric and oligomeric Trx1, which affected the ELISAs composed of different antibody combinations. Thus, the levels of Trx1 determined in human plasma and CSF samples varied depending on the antibody used in the ELISAs and on the rhTrx1 standard. Furthermore, the relevance of preventing interference by heterophilic antibodies (HA) in human plasma and CSF was investigated. The addition of a HA blocking buffer to human samples drastically reduced the ELISA signals in many samples showing that HA are likely to cause false positive results unless they are blocked. In conclusion, the study shows that the design of a Trx1 ELISA in regards to antibodies and standards used has an impact on the measured Trx1 levels. Importantly, analyses of human plasma and CSF without preventing HA interference may obscure the obtained data. Overall, the results of this study are crucial for the improvement of future studies on the association of Trx1 levels with various diseases

Clozapine protects adult neural stem cells from ketamine-induced cell death in correlation with decreased apoptosis and autophagy

Adult neurogenesis, the production of newborn neurons from neural stem cells (NSCs) has been suggested to be decreased in patients with schizophrenia. A similar finding was observed in an animal model of schizophrenia, as indicated by decreased bromodeoxyuridine (BrdU) labelling cells in response to a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist. The antipsychotic drug clozapine was shown to counteract the observed decrease in BrdU-labelled cells in hippocampal dentate gyrus (DG). However, phenotypic determination by immunohistochemistry analysis could not reveal whether BrdU-positive cells were indeed NSCs. Using a previously established cell model for analysing NSC protection in vitro, we investigated a protective effect of clozapine on NSCs. Primary NSCs were isolated from the mouse subventricular zone (SVZ), we show that clozapine had a NSC protective activity alone, as evident by employing an ATP cell viability assay. In contrast, haloperidol did not show any NSC protective properties. Subsequently, cells were exposed to the non-competitive NMDA-receptor antagonist ketamine. Clozapine, but not haloperidol, had a NSC protective/anti-apoptotic activity against ketamine-induced cytotoxicity. The observed NSC protective activity of clozapine was associated with increased expression of the anti-apoptotic marker Bcl-2, decreased expression of the pro-apoptotic cleaved form of caspase-3 and associated with decreased expression of the autophagosome marker 1A/1B-light chain 3 (LC3-II). Collectively, our findings suggest that clozapine may have a protective/anti-apoptotic effect on NSCs, supporting previous in vivo observations, indicating a neurogenesis-promoting activity for clozapine. If the data are further confirmed in vivo, the results may encourage an expanded use of clozapine to restore impaired neurogenesis in schizophrenia

Additional file 2: of Inhibition of glutamate oxaloacetate transaminase 1 in cancer cell lines results in altered metabolism with increased dependency of glucose

Figure S2. Rescue of GOT1 down-regulated and GOT1-null cells by oxaloacetate. Relative cell viabilities after 8 h (a) and 24 h (b) in wild type 143B cells and 8 h (c) and 24 h (d) in wild type A549 cells. Relative cell viabilities after 8 h (e) and 24 h (f) in GOT1 siRNA knock-down A549 cells. Rescue of GOT1-null 143B cells with OAA at different concentrations upon glucose deprivation (g). Mean ± s.d. from 3 independent experiments. One-way ANOVA test was performed. *** p < 0.001; ** p < 0.01;* p < 0.05. NS: not significant. (TIF 230 kb