Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Quantification of ARID1A in Tissue Lysates

ARID1A is a subunit of the mammalian SWI/SNF complex, which is thought to regulate gene expression through restructuring chromatin structures. Its gene ARID1A is frequently mutated and ARID1A levels are lowered in several human cancers, especially gynecologic ones. A functional ARID1A loss may have prognostic or predictive value in terms of therapeutic strategies but has not been proposed based on a quantitative method. Hardly any literature is available on ARID1A levels in tumor samples. We developed an indirect enzyme-linked immunosorbent assay (ELISA) for ARID1A based on the current EMA and FDA criteria. We demonstrated that our ELISA provides the objective, accurate, and precise quantification of ARID1A concentrations in recombinant protein solutions, cell culture standards, and tissue lysates of tumors. A standard curve analysis yielded a ‘goodness of fit’ of R2 = 0.99. Standards measured on several plates and days achieved an inter-assay accuracy of 90.26% and an inter-assay precision with a coefficient of variation of 4.53%. When tumor lysates were prepared and measured multiple times, our method had an inter-assay precision with a coefficient of variation of 11.78%. We believe that our suggested method ensures a high reproducibility and can be used for a high sample throughput to determine the ARID1A concentration in different tumor entities. The application of our ELISA on various tumor and control tissues will allow us to explore whether quantitative ARID1A measurements in tumor samples are of predictive value

Intracranial Intracerebral Schwannoma: a Case Report and Review of the Literature

Intracranial schwannomas are relatively uncommon, accounting for approximately 8% of all intracranial tumors, while intracerebral schwannomas represent an even rarer entity, responsible for roughly 1% of all intracranial schwannomas. After reviewing the relevant literature, we discussed the clinical journey of a 74-year-old woman who presented with a 3-week history of dizziness and nausea. Magnetic resonance imaging revealed a right temporal mass lesion with perifocal edema. The initial suspicion was the diagnosis of a glioblastoma or metastasis, prompting surgical intervention. During the surgery, a gross total resection of a noninvasive tumor was successfully performed. The patient’s postoperative recovery was uneventful. Histopathological examination and confrmatory immunohistochemistry played a crucial role in reaching the fnal diagnosis of an intracerebral temporal schwannoma, highlighting the diagnostic challenges posed by radiologically indistinguishable features from metastasis and gliomas. Despite these challenges, complete surgical removal remains the most preferred treatment option, resulting in a favorable long-term prognosis without the need for adjuvant or neoadjuvant chemotherapy. Intracerebral schwannomas are exceedingly rare brain tumors, often found on the brain’s surface or adjacent ventricles. Early and accurate diagnosis can be challenging due to radiological features overlapping with other intracranial pathologies. Nonetheless, histopathological examination and immunohistochemistry remain indispensable tools in establishing a defnitive diagnosis and guiding efective treatment strategies. With complete surgical excision, patients with intracerebral schwannomas can expect a positive outcome and a promising long-term prognosis. Further research and case studies are warranted to enhance our understanding of these rare tumors and improve patient outcomes

Bisulfite profiling of the MGMT promoter and comparison with routine testing in glioblastoma diagnostics

Background: Promoter methylation of the DNA repair gene O6 -methylguanine-DNA methyltransferase (MGMT) is an acknowledged predictive epigenetic marker in glioblastoma multiforme and anaplastic astrocytoma. Patients with methylated CpGs in the MGMT promoter beneft from treatment with alkylating agents, such as temozolomide, and show an improved overall survival and progression-free interval. A precise determination of MGMT promoter methyla‑ tion is of importance for diagnostic decisions. We experienced that diferent methods show partially divergent results in a daily routine. For an integrated neuropathological diagnosis of malignant gliomas, we therefore currently apply a combination of methylation-specifc PCR assays and pyrosequencing. Results: To better rationalize the variation across assays, we compared these standard techniques and assays to deep bisulfte sequencing results in a cohort of 80 malignant astrocytomas. Our deep analysis covers 49 CpG sites of the expanded MGMT promoter, including exon 1, parts of intron 1 and a region upstream of the transcription start site (TSS). We observed that deep sequencing data are in general in agreement with CpG-specifc pyrosequencing, while the most widely used MSP assays published by Esteller et al. (N Engl J Med 343(19):1350–1354, 2000. https://doi.org/ 10.1056/NEJM200011093431901) and Felsberg et al. (Clin Cancer Res 15(21):6683–6693, 2009. https://doi.org/10.1158/ 1078-0432.CCR-08-2801) resulted in partially discordant results in 22 tumors (27.5%). Local deep bisulfte sequencing (LDBS) revealed that CpGs located in exon 1 are suited best to discriminate methylated from unmethylated samples. Based on LDBS data, we propose an optimized MSP primer pair with 83% and 85% concordance to pyrosequencing and LDBS data. A hitherto neglected region upstream of the TSS, with an overall higher methylation compared to exon 1 and intron 1 of MGMT, is also able to discriminate the methylation status. Conclusion: Our integrated analysis allows to evaluate and redefne co-methylation domains within the MGMT pro‑ moter and to rationalize the practical impact on assays used in daily routine diagnostics

Molecular communication: An acid tale of prion formation

Some bacteria use lactic acid to communicate with yeast cells

Study of Metabolite Repair in Eukaryotic Cells: Metabolic origin and fate of D-2-hydroxyglutarate in yeast and effect of NAD(P)HX repair deficiency on yeast and human cells

Abnormal metabolites, which are useless and can even be toxic, are constantly generated inside the cell by unwanted chemical reactions or by enzymatic side reactions. Metabolite repair enzymes clean the metabolite pool from these molecules. The proportion of proteins annotated as metabolite repair enzymes is currently very small but accumulating evidence suggests that a bigger part might be hidden among proteins of unknown function. The aim of this thesis was to study two of these metabolite repair systems and their physiological relevance in more detail as their importance is well illustrated through implication in disease processes. D-2-hydroxyglutaric aciduria, a severe human neurometabolic disorder, can be caused by a deficiency in the metabolite repair enzyme D-2-hydroxyglutarate (D-2HG) dehydrogenase. Higher levels of D-2HG have also been observed in cancerous cells with a mutated form of isocitrate dehydrogenase. Strikingly, in the model organism Saccharomyces cerevisiae, 2-hydroxyglutarate metabolism had remained completely unexplored. We elucidated the metabolic pathways involved in D-2HG formation and degradation in yeast using bioinformatics, metabolomics, yeast genetics, and classical biochemical tools. We discovered that Dld3, currently annotated as a D-lactate dehydrogenase, actually degrades D-2HG to α-ketoglutarate while reducing pyruvate to D-lactate, thereby acting as a transhydrogenase. We also demonstrated that the yeast phosphoglycerate dehydrogenases Ser3 and Ser33 are major sources for D-2HG formation. These findings paved the way to integrate 2HG and its associated genes into the yeast metabolic network and might help, on the long-term, to better understand underlying mechanisms in human disease as well. Other recently identified metabolite repair enzymes, NAD(P)HX dehydratase and NAD(P)HX epimerase (encoded in yeast by the YKL151C and YNL200C genes, respectively), specifically act on NADHX and NADPHX, hydrated and inactive forms of the central NADH and NADPH cofactors. Although extensively biochemically characterized, the physiological importance of these two enzymes still remains largely unclear. Only very recently, case reports were published indicating a correlation between NAD(P)HX repair deficiency and severe neuropathological symptoms starting in early childhood upon events of febrile illnesses and rapidly leading to a fatal outcome. We systematically analyzed extracts of NAD(P)HX repair deficient yeast and human cells using HPLC and LC-MS/MS methods. This enabled us to demonstrate that NADHX and NADPHX can be formed intracellularly. In the yeast system, NADHX accumulation, which could be modulated by the cultivation temperature, was accompanied by a decrease in intracellular NAD+ levels. Furthermore, we showed that NADHX interferes with serine metabolism by inhibiting the first step of the main synthesis pathway of this amino acid. In the human cell system, NAD(P)HX dehydratase deficiency led, as in yeast, to intracellular NADHX accumulation, but also to a marked decrease in cell viability after prolonged cultivation times. This is, to our knowledge, the first report about the effect of NADHX accumulation on cellular metabolism. Expanding our experimental strategy of combined transcriptomics and metabolomics approaches to the human cell model might ultimately lead to the discovery of the disease-causing cellular process. The findings in both projects led to an unexpected connection between NAD(P)HX and 2HG metabolism via the yeast homologues of 3-phoshpoglycerate dehydrogenase, Ser3 and Ser33. Both proteins catalyze the oxidation of 3-phosphoglycerate to 3-phosphohydroxypyruvate in the initial step of de novo serine biosynthesis with a concomitant reduction of α-ketoglutarate to D-2-hydroxyglutarate. By acting as transhydrogenases, they substantially, even though not exclusively, contribute to D-2HG formation in yeast. The very same enzymes were strongly inhibited in vitro and, as suggested by our findings, also in vivo by the presence of NADHX, leading to serine depletion in NAD(P)HX repair deficient cells

Saccharomyces cerevisiae Forms D-2-Hydroxyglutarate and Couples its Degradation to D-Lactate Formation via a Cytosolic Transhydrogenase.

The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders and high D-2HG levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here we show that S. cerevisiae actively forms the D enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human D-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3Delta knockout strain accumulates millimolar levels of D-2HG, while a dld2Delta knockout strain displayed only very moderate increases in D-2HG. Recombinant Dld2 and Dld3, both currently annotated as D-lactate dehydrogenases, efficiently oxidized D-2HG to alpha-ketoglutarate. Depletion of D-lactate levels in the dld3Delta, but not in the dld2Delta mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert D-2HG to alpha-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce alpha-ketoglutarate to D-2HG using NADH and represent major intracellular sources of D-2HG in yeast. Based on our observations, we propose that D-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples D-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the D-lactate dehydrogenase Dld1

3‑Phosphoglycerate Transhydrogenation Instead of Dehydrogenation Alleviates the Redox State Dependency of Yeast de Novo L‑Serine Synthesis

The enzymatic mechanism of 3-phosphoglycerate to 3-phosphohydroxypyruvate oxidation, which forms the first step of the main conserved de novo serine synthesis pathway, has been revisited recently in certain microorganisms. While this step is classically considered to be catalyzed by an NAD-dependent dehydrogenase (e.g., PHGDH in mammals), evidence has shown that in Pseudomonas, Escherichia coli, and Saccharomyces cerevisiae, the PHGDH homologues act as transhydrogenases. As such, they use α-ketoglutarate, rather than NAD+, as the final electron acceptor, thereby producing D-2-hydroxyglutarate in addition to 3-phosphohydroxypyruvate during 3-phosphoglycerate oxidation. Here, we provide a detailed biochemical and sequence−structure relationship characterization of the yeast PHGDH homologues, encoded by the paralogous SER3 and SER33 genes, in comparison to the human and other PHGDH enzymes. Using in vitro assays with purified recombinant enzymes as well as in vivo growth phenotyping and metabolome analyses of yeast strains engineered to depend on either Ser3, Ser33, or human PHGDH for serine synthesis, we confirmed that both yeast enzymes act as transhydrogenases, while the human enzyme is a dehydrogenase. In addition, we show that the yeast paralogs differ from the human enzyme in their sensitivity to inhibition by serine as well as hydrated NADH derivatives. Importantly, our in vivo data support the idea that a 3PGA transhydrogenase instead of dehydrogenase activity confers a growth advantage under conditions where the NAD+:NADH ratio is low. The results will help to elucidate why different species evolved different reaction mechanisms to carry out a widely conserved metabolic step in central carbon metabolism

NAD(P)HX repair deficiency causes central metabolic perturbations in yeast and human cells

NADHX and NADPHX are hydrated and redox inactive forms of the NADH and NADPH cofactors, known to inhibit several dehydrogenases in vitro. A metabolite repair system that is conserved in all domains of life and that comprises the two enzymes NAD(P)HX dehydratase and NAD(P)HX epimerase, allows reconversion of both the S- and R-epimers of NADHX and NADPHX to the normal cofactors. An inherited deficiency in this system has recently been shown to cause severe neurometabolic disease in children. Although evidence for the presence of NAD(P)HX has been obtained in plant and human cells, little is known about the mechanism of formation of these derivatives in vivo and their potential effects on cell metabolism. Here, we show that NAD(P)HX dehydratase deficiency in yeast leads to an important, temperature-dependent NADHX accumulation in quiescent cells with a concomitant depletion of intracellular NAD+ and serine pools. We demonstrate that NADHX potently inhibits the first step of the serine synthesis pathway in yeast. Human cells deficient in the NAD(P)HX dehydratase also accumulated NADHX and showed decreased viability. In addition, those cells consumed more glucose and produced more lactate, potentially indicating impaired mitochondrial function. Our results provide first insights into how NADHX accumulation affects cellular functions and pave the way for a better understanding of the mechanism(s) underlying the rapid and severe neurodegeneration leading to early death in NADHX repair deficient children

Bisulfite profiling of the MGMT promoter and comparison with routine testing in glioblastoma diagnostics

Background: Promoter methylation of the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT) is an acknowledged predictive epigenetic marker in glioblastoma multiforme and anaplastic astrocytoma. Patients with methylated CpGs in the MGMT promoter benefit from treatment with alkylating agents, such as temozolomide, and show an improved overall survival and progression-free interval. A precise determination of MGMT promoter methylation is of importance for diagnostic decisions. We experienced that different methods show partially divergent results in a daily routine. For an integrated neuropathological diagnosis of malignant gliomas, we therefore currently apply a combination of methylation-specific PCR assays and pyrosequencing. Results: To better rationalize the variation across assays, we compared these standard techniques and assays to deep bisulfite sequencing results in a cohort of 80 malignant astrocytomas. Our deep analysis covers 49 CpG sites of the expanded MGMT promoter, including exon 1, parts of intron 1 and a region upstream of the transcription start site (TSS). We observed that deep sequencing data are in general in agreement with CpG-specific pyrosequencing, while the most widely used MSP assays published by Esteller et al. (N Engl J Med 343(19):1350-1354, 2000. https://doi.org/10.1056/NEJM200011093431901 ) and Felsberg et al. (Clin Cancer Res 15(21):6683-6693, 2009. https://doi.org/10.1158/1078-0432.CCR-08-2801 ) resulted in partially discordant results in 22 tumors (27.5%). Local deep bisulfite sequencing (LDBS) revealed that CpGs located in exon 1 are suited best to discriminate methylated from unmethylated samples. Based on LDBS data, we propose an optimized MSP primer pair with 83% and 85% concordance to pyrosequencing and LDBS data. A hitherto neglected region upstream of the TSS, with an overall higher methylation compared to exon 1 and intron 1 of MGMT, is also able to discriminate the methylation status. Conclusion: Our integrated analysis allows to evaluate and redefine co-methylation domains within the MGMT promoter and to rationalize the practical impact on assays used in daily routine diagnostics.Funding: Open Access funding enabled and organized by Projekt DEAL. This work was supported by the German Epigenome Programme (DEEP) of the Federal Ministry of Education and Research in Germany (BMBF) (01KU1216F). MS is supported by the BMBF project de.NBI-epi (031L0101D) and the EU H2020 project SYSCID (733100