Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target.

Drugs that target histone deacetylase (HDAC) entered the pharmacopoeia in the 2000s. However, some enigmatic phenotypes suggest off-target engagement. Here, we developed a quantitative chemical proteomics assay using immobilized HDAC inhibitors and mass spectrometry that we deployed to establish the target landscape of 53 drugs. The assay covers 9 of the 11 human zinc-dependent HDACs, questions the reported selectivity of some widely-used molecules (notably for HDAC6) and delineates how the composition of HDAC complexes influences drug potency. Unexpectedly, metallo-ß-lactamase domain-containing protein 2 (MBLAC2) featured as a frequent off-target of hydroxamate drugs. This poorly characterized palmitoyl-CoA hydrolase is inhibited by 24 HDAC inhibitors at low nanomolar potency. MBLAC2 enzymatic inhibition and knockdown led to the accumulation of extracellular vesicles. Given the importance of extracellular vesicle biology in neurological diseases and cancer, this HDAC-independent drug effect may qualify MBLAC2 as a target for drug discovery.

Transmating: conjugative transfer of a new broad host range expression vector to various Bacillus species using a single protocol.

BACKGROUND: The genus Bacillus includes a great variety of species with potential applications in biotechnology. While species such as B. subtilis or B. licheniformis are well-known and used to provide various products at industrial scale, other Bacillus species are less characterized and are not yet used in commercial processes. One reason for this is the fact that genetic manipulation of new isolates is usually complicated with conventional techniques which have to be adapted to each new strain. Even in well-established strains, the available transformation protocols often suffer from low efficiencies. RESULTS: In this paper, we provide a new broad host range E. coli/Bacillus shuttle vector, named pBACOV (Bacillus conjugation vector), that can be efficiently transferred to various Bacillus species using a single protocol. A variant of pBACOV carrying the sfGFP gene was successfully transferred to eight different species from the genus Bacillus and to one Paenibacillus species using triparental conjugation ("transmating"). This was achieved using a single protocol and worked for nine out of eleven tested acceptor species. The transmating procedure was used to test expression of the heterologous reporter gene sfGFP under control of the PaprE-promoter from B. subtilis in several Bacillus species in parallel. Expression of sfGFP was found in eight out of nine transmates. For several of the tested species, this is the first report of a method for genetic modification and heterologous gene expression. The expression level, analyzed by measuring the relative sfGFP-fluorescence normalized to the cell density of the cultures, was highest in B. mojavensis. CONCLUSIONS: The new shuttle vector pBACOV can be transferred to many different Bacillus and Paenibacillus species using a simple and efficient transmating protocol. It is a versatile tool facilitating the application of recombinant DNA technology in new as well as established strains, or selection of an ideal host for heterologous gene expression from a multitude of strains. This paves the way for the genetic modification and biotechnological exploitation of the broad diversity of species of Bacillus and related genera as well as different strains from these species.

A pipeline for the development and analysis of extracellular vesicle-based transcriptomic biomarkers in molecular diagnostics.

Extracellular vesicles are shed by every cell type and can be found in any biofluid. They contain different molecules that can be utilized as biomarkers, including several RNA species which they protect from degradation. Here, we present a pipeline for the development and analysis of extracellular vesicle-associated transcriptomic biomarkers that our group has successfully applied multiple times. We highlight the key steps of the pipeline and give particular emphasis to the necessary quality control checkpoints, which are linked to numerous available guidelines that should be considered along the workflow. Our pipeline starts with patient recruitment and continues with blood sampling and processing. The purification and characterization of extracellular vesicles is explained in detail, as well as the isolation and quality control of extracellular vesicle-associated RNA. We point out the possible pitfalls during library preparation and RNA sequencing and present multiple bioinformatic tools to pinpoint biomarker signature candidates from the sequencing data. Finally, considerations and pitfalls during the validation of the biomarker signature using RT-qPCR will be elaborated.

Extracellular vesicle-mediated trafficking of molecular cues during human brain development.

Cellular crosstalk is an essential process influenced by numerous factors, including secreted vesicles that transfer nucleic acids, lipids, and proteins between cells. Extracellular vesicles (EVs) have been the center of many studies focusing on neurodegenerative disorders, but whether EVs display cell-type-specific features for cellular crosstalk during neurodevelopment is unknown. Here, using human-induced pluripotent stem cell-derived cerebral organoids, neural progenitors, neurons, and astrocytes, we identify heterogeneity in EV protein content and dynamics in a cell-type-specific and time-dependent manner. Our results support the trafficking of key molecules via EVs in neurodevelopment, such as the transcription factor YAP1, and their localization to differing cell compartments depending on the EV recipient cell type. This study sheds new light on the biology of EVs during human brain development.

Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems.

Background: Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Methods: Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. Results: We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Conclusion: Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

Characterization of the Staphylococcus xylosus methylome reveals a new variant of type I restriction modification system in staphylococci.

Restriction modification (RM) systems are known to provide a strong barrier to the exchange of DNA between and within bacterial species. Likewise, DNA methylation is known to have an important function in bacterial epigenetics regulating essential pathways such as DNA replication and the phase variable expression of prokaryotic phenotypes. To date, research on staphylococcal DNA methylation focused mainly on the two species Staphylococcus aureus and S. epidermidis. Less is known about other members of the genus such as S. xylosus, a coagulase-negative commensal of mammalian skin. The species is commonly used as starter organism in food fermentations but is also increasingly considered to have an as yet elusive function in bovine mastitis infections. We analyzed the methylomes of 14 S. xylosus strains using single-molecular, real-time (SMRT) sequencing. Subsequent in silico sequence analysis allowed identification of the RM systems and assignment of the respective enzymes to the discovered modification patterns. Hereby the presence of type I, II, III and IV RM systems in varying numbers and combinations among the different strains was revealed, clearly distinguishing the species from what is known for other members of the genus so far. In addition, the study characterizes a newly discovered type I RM system, encoded by S. xylosus but also by a variety of other staphylococcal species, with a hitherto unknown gene arrangement that involves two specificity units instead of one (hsdRSMS). Expression of different versions of the operon in E. coli showed proper base modification only when genes encoding both hsdS subunits were present. This study provides new insights into the general understanding of the versatility and function of RM systems as well as the distribution and variations in the genus Staphylococcus.

Obtaining Reliable RT-qPCR Results in Molecular Diagnostics-MIQE Goals and Pitfalls for Transcriptional Biomarker Discovery.

In this review, we discuss the development pipeline for transcriptional biomarkers in molecular diagnostics and stress the importance of a reliable gene transcript quantification strategy. Hence, a further focus is put on the MIQE guidelines and how to adapt them for biomarker discovery, from signature validation up to routine diagnostic applications. First, the advantages and pitfalls of the holistic RNA sequencing for biomarker development will be described to establish a candidate biomarker signature. Sequentially, the RT-qPCR confirmation process will be discussed to validate the discovered biomarker signature. Examples for the successful application of RT-qPCR as a fast and reproducible quantification method in routinemolecular diagnostics are provided. Based on the MIQE guidelines, the importance of "key steps" in RT-qPCR is accurately described, e.g., reverse transcription, proper reference gene selection and, finally, the application of automated RT-qPCR data analysis software. In conclusion, RT-qPCR proves to be a valuable tool in the establishment of a disease-specific transcriptional biomarker signature and will have a great future in molecular diagnostics or personalized medicine.

Non-invasive and high-throughput interrogation of exon-specific isoform expression.

Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.