N1-methylation of adenosine (m1A) in ND5 mRNA leads to complex I dysfunction in Alzheimer's disease.

One mechanism of particular interest to regulate mRNA fate post-transcriptionally is mRNA modification. Especially the extent of m1A mRNA methylation is highly discussed due to methodological differences. However, one single m1A site in mitochondrial ND5 mRNA was unanimously reported by different groups. ND5 is a subunit of complex I of the respiratory chain. It is considered essential for the coupling of oxidation and proton transport. Here we demonstrate that this m1A site might be involved in the pathophysiology of Alzheimer's disease (AD). One of the pathological hallmarks of this neurodegenerative disease is mitochondrial dysfunction, mainly induced by Amyloid ß (Aß). Aß mainly disturbs functions of complex I and IV of the respiratory chain. However, the molecular mechanism of complex I dysfunction is still not fully understood. We found enhanced m1A methylation of ND5 mRNA in an AD cell model as well as in AD patients. Formation of this m1A methylation is catalyzed by increased TRMT10C protein levels, leading to translation repression of ND5. As a consequence, here demonstrated for the first time, TRMT10C induced m1A methylation of ND5 mRNA leads to mitochondrial dysfunction. Our findings suggest that this newly identified mechanism might be involved in Aß-induced mitochondrial dysfunction.

Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development.

SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.

NMR-based Fragment Screening in a Minimum Sample but Maximum Automation Mode.

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Exploring the Druggability of Conserved RNA Regulatory Elements in the SARS-CoV-2 Genome.

SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1 H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.

1H, 13C, and 15N backbone chemical shift assignments of coronavirus-2 non-structural protein Nsp10.

The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.

19 F NMR-Based Fragment Screening for 14 Different Biologically Active RNAs and 10 DNA and Protein Counter-Screens.

We report here the nuclear magnetic resonance 19 F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess the druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter-screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low-micromolar binding affinity without losing binding specificity between two different terminator structures.

Leptin-mediated changes in hepatic mitochondrial metabolism, structure, and protein levels.

Leptin reduces body weight in ob/ob mice by decreasing food intake and increasing energy expenditure; however, the mechanisms by which it does the latter are not known. Here we report that 30% of the weight loss induced by leptin treatment of ob/ob mice is due to changes in energy expenditure. In assessing leptin's effects on specific tissues, we found that hepatic basal metabolic rate was paradoxically decreased 1.7-fold with leptin treatment, which was the result of a 1.6-fold reduction in mitochondrial volume density and altered substrate oxidation kinetics. The altered kinetics were associated with a decrease in protein levels of 2 mitochondrial respiratory chain components--cytochrome c oxidase subunit VIa and cytochrome c oxidase subunit IV. In addition to reduced hepatic metabolism, there was reduced long chain fatty acid production and a 2.5-fold increase in hepatic lipid export, both of which explain the reduced steatosis in leptin-treated animals. These data help clarify the role of the liver in leptin-mediated weight loss and define the mechanisms by which leptin alters hepatic metabolism and corrects steatosis.

Identification of malignancy factors by analyzing cystic tumors of the pancreas.

AIM: The diversity in the aggressiveness of cystic tumors of the pancreas - ranging from the usually benign serous cystadenoma to lesions of variable degrees of malignancy - was utilized for the identification of molecular factors that are involved in the occurrence of malignancy. METHODS: We analyzed the transcript profiles of different cystic tumor types. The results were confirmed at the protein level by immunohistochemistry. Also, functional studies with siRNA silencing were performed. RESULTS: Expression variations at the RNA and protein level were identified that are closely correlated with the degree of malignancy. Besides, all tumors could be classified effectively by this means. Many of the identified factors had not previously been known to be associated with malignant cystic lesions. siRNA silencing of the gene with the most prominent variation - the anti-apoptotic factor FASTK (Fas-activated serine/threonine kinase) - revealed a regulative effect on several genes known to be relevant to the development of tumors. CONCLUSION: By a molecular analysis of rare types of pancreatic cancer, which are less frequent in terms of disease, variations could be identified that could be critical for the regulation of malignancy and thus relevant to the treatment of also the majority of pancreatic tumors.

Still any role for transduodenal local excision in tumors of the papilla of Vater?

Tumors of the papilla and ampulla of Vater are rare neoplasms which are usually detected at an early stage due to their symptoms. The accurate preoperative histological diagnosis and staging of ampullary tumors is often difficult and inconclusive, leading to controversy over the adequate treatment of these lesions. Three procedures are currently being used to treat such tumors. Pancreatoduodenectomy (PD) is a procedure with low morbidity and mortality at experienced centers, and is considered the treatment of choice for invasive carcinoma and large benign ampullary lesions with suspicion of malignancy. Transduodenal local excision (TDE) of ampullary tumors is a relatively simple procedure with operative morbidity and mortality rates comparable to PD. TDE is challenged at endoscopic centers by endoscopic snare excision (ESE). Due to technical advances, the safety and outcomes of ESE for ampullary tumors have improved in recent years. ESE and TDE represent adequate methods for treatment of benign tumors and also for small malignant tumors detected at an early stage if the diagnosis and stage have been accurately established preoperatively. Due to the safety of PD and the technical advances of ESE, TDE is reserved for selected patients. Randomized controlled studies are needed to establish the correct indications for PD, TDE, and ESE.

Moxifloxacin penetration into human gastrointestinal tissues.

OBJECTIVE: Moxifloxacin is a recently developed fourth-generation methoxyquinolone with a broad spectrum of activity against both Gram-positive and Gram-negative aerobic bacteria and anaerobes. The aim of the present study was to assess the penetration of moxifloxacin into gastrointestinal (GI) mucosal tissues to evaluate its potential role as an antimicrobial drug in bacterial infections of the GI tract. PATIENTS AND METHODS: Twenty-eight patients undergoing GI-tract surgery received 400 mg of moxifloxacin twice pre-operatively [eight patients orally (po) and 20 patients intravenously (iv)], of whom 22 completed the study. Mucosal tissues (three stomach, three small bowel and 16 colon) and serum samples were collected and moxifloxacin concentrations were measured by HPLC. RESULTS: The highest tissue concentrations were detected in the mucosa of the stomach (10.9 +/- 5.1 mg/kg), followed by colon mucosa (7.8 +/- 7.1 mg/kg after iv; 6.6 +/- 3.6 mg/kg after po) and small bowel mucosa (5.4 +/- 0.5 mg/kg). The tissue-to-serum ratio of moxifloxacin was 2.0 +/- 1.6 in the small bowel mucosa, 5.8 +/- 3.4 and 6.8 +/- 3.9 in the colon mucosa after po and iv administration, respectively, and 9.7 +/- 5.7 in the stomach mucosa. CONCLUSION: Moxifloxacin penetrates into and accumulates in the mucosa of the stomach, small bowel and colon. The clinical applicability of moxifloxacin administration for bacterial GI-tract infections should be investigated in controlled trials.

Up-regulation of p75 neurotrophin receptor (p75NTR) is associated with apoptosis in chronic pancreatitis.

Activation of apoptosis in chronic pancreatitis has been demonstrated. The low-affinity neurotrophin receptor p75 (p75NTR) mediates apoptosis in many cell types in vivo and in vitro. The aim of this study was to examine whether p75NTR is involved in the apoptotic process in chronic pancreatitis. The quantity and localization of the receptor was evaluated using northern blot analysis, in situ hybridization, immunohistochemistry, and western blot analysis. Apoptosis was determined by TUNEL assay. By northern blot analysis, p75NTR mRNA levels were increased 40-fold in chronic pancreatitis compared with normal pancreas (P < 0.01). In situ hybridization revealed weak p75NTR mRNA expression in some ductal cells in the normal pancreas. In contrast in chronic pancreatitis moderate p75NTR expression was present in acinar cells next to fibrosis, ductal cells, and cells of ductular structures as well as in some islet cells. Immunostaining of p75NTR in normal pancreas and chronic pancreatitis tissue samples showed a similar intensity and distribution pattern as found by in situ hybridization. Higher p75NTR protein levels could be confirmed by western blot analysis, which revealed an 8.6-fold increase of p75NTR in chronic pancreatitis. TUNEL staining showed, in chronic pancreatitis samples, positivity in some acinar cells next to fibrosis, some ductal cells, and cells of ductular structures. Also some islet cells were positive by TUNEL staining. The presence of p75NTR immunoreactivity was positively correlated (P < 0.05) with the apoptotic index in the exocrine and endocrine pancreas. In conclusion, p75NTR, the low-affinity receptor of neurotrophins which mediates apoptosis, is up-regulated in CP and is involved in the apoptotic process of the exocrine and endocrine pancreas.

Surgical treatment and long-term follow-up in chronic pancreatitis.

In the past two decades our knowledge of the pathophysiology and surgical treatment options in chronic pancreatitis have improved substantially. Surgical treatment in chronic pancreatitis has evolved from radical to organ-preserving procedures. The classic Whipple resection is no longer indicated in chronic pancreatitis, and operations like the duodenum-preserving pancreatic head resection and the pylorus-preserving Whipple have replaced it as surgical standards. These procedures allow the preservation of exocrine and endocrine pancreatic function, provide pain relief in up to 90% of patients, and contribute to an improvement in the quality of life.

Glypican-3 expression is markedly decreased in human gastric cancer but not in esophageal cancer.

BACKGROUND: Deregulation of the expression of glypican-3, a heparan sulfate proteoglycan, has been demonstrated in several human cancers. METHODS: In the present study, glypican-3 mRNA expression was analyzed by Northern blotting and in situ hybridization in 20 normal and 41 cancerous esophageal specimens as well as in 15 normal and 32 cancerous gastric tissues. RESULTS: Glypican-3 mRNA was expressed in both normal and esophageal cancer tissues without a significant difference between normal and cancerous tissues, and without a correlation with histological type, tumor stage, tumor grade, or patient survival. Moderate to strong glypican-3 mRNA signals were found in the cytoplasm of squamous epithelial cells of the normal esophagus. In both squamous and adenocarcinomas of the esophagus glypican-3 mRNA signals were also moderately to strongly present in the cytoplasm of the cancer cells. In gastric tissues, glypican-3 mRNA was present in 53% of normal gastric tissue samples, but was below the detection level in all examined gastric cancer samples. Glypican-3 mRNA signals were moderately to strongly present in the cytoplasm of gastric mucosal epithelial cells, but were only very faintly present in some cancer cells. CONCLUSIONS: Glypican-3 may be involved in the growth control of normal esophageal and gastric epithelial cells. Furthermore, our results suggest that glypican-3 may play a tumor suppressor role in gastric but not in esophageal cancer.