Silica Nanoparticles Disclose a Detailed Neurodegeneration Profile throughout the Life Span of a Model Organism.

The incidence of age-related neurodegenerative diseases is rising globally. However, the temporal sequence of neurodegeneration throughout adult life is poorly understood. To identify the starting points and schedule of neurodegenerative events, serotonergic and dopaminergic neurons were monitored in the model organism C. elegans, which has a life span of 2-3 weeks. Neural morphology was examined from young to old nematodes that were exposed to silica nanoparticles. Young nematodes showed phenotypes such as dendritic beading of serotonergic and dopaminergic neurons that are normally not seen until late life. During aging, neurodegeneration spreads from specifically susceptible ADF and PDE neurons in young C. elegans to other more resilient neurons, such as dopaminergic CEP in middle-aged worms. Investigation of neurodegenerative hallmarks and animal behavior revealed a temporal correlation with the acceleration of neuromuscular defects, such as internal hatch in 2-day-old C. elegans. Transcriptomics and proteomics of young worms exposed to nano silica showed a change in gene expression concerning the gene ontology groups serotonergic and dopaminergic signaling as well as neuropeptide signaling. Consistent with this, reporter strains for nlp-3, nlp-14 and nlp-21 confirmed premature degeneration of the serotonergic neuron HSN and other neurons in young C. elegans. The results identify young nematodes as a vulnerable age group for nano silica-induced neural defects with a significantly reduced health span. Neurodegeneration of specific neurons impairs signaling by classical neurotransmitters as well as neuropeptides and compromises related neuromuscular behaviors in critical phases of life, such as the reproductive phase.

Stromal alterations in patients with MGUS, smoldering myeloma and multiple myeloma.

The hallmark of multiple myeloma (MM) is a clonal plasma cell infiltration in the bone marrow accompanied by myelosuppression and osteolysis. Premalignant stages like monoclonal gammopathy of undetermined significance (MGUS) and asymptomatic stages like smoldering myeloma (SMM) can progress to multiple myeloma (MM). Mesenchymal stromal cells (MSC) are an integral component of the bone marrow microenvironment and play an important role for osteoblast differentiation and hematopoietic support. While stromal alterations have been reported in MM contributing to hematopoietic insufficiency and osteolysis, it is not clear whether alterations in MSC already occur in MGUS or SMM. In this study we analyzed MSC from MGUS, SMM and MM towards their properties and functionality and performed mRNA sequencing to find underlying molecular signatures in different disease stages. A high number of senescent cells and a reduced osteogenic differentiation capacity and hematopoietic support was already present in MGUS MSC. As shown by RNA sequencing there was a broad spectrum of differentially expressed genes including genes of the BMP/TGF-signaling pathway, detected already in MGUS and that clearly increases in SMM and MM patients. Our data may help to block these signaling pathways in the future to hinder progression to multiple myeloma.

Affordable, accurate and unbiased RNA sequencing by manual library miniaturization: A case study in barley.

We present an easy-to-reproduce manual miniaturized full-length RNA sequencing (RNAseq) library preparation workflow that does not require the upfront investment in expensive lab equipment or long setup times. With minimal adjustments to an established commercial protocol, we were able to manually miniaturize the RNAseq library preparation by a factor of up to 1:8. This led to cost savings for miniaturized library preparation of up to 86.1% compared to the gold standard. The resulting data were the basis of a rigorous quality control analysis that inspected: sequencing quality metrics, gene body coverage, raw read duplications, alignment statistics, read pair duplications, detected transcripts and sequence variants. We also included a deep dive data analysis identifying rRNA contamination and suggested ways to circumvent these. In the end, we could not find any indication of biases or inaccuracies caused by the RNAseq library miniaturization. The variance in detected transcripts was minimal and not influenced by the miniaturization level. Our results suggest that the workflow is highly reproducible and the sequence data suitable for downstream analyses such as differential gene expression analysis or variant calling.

Positive allosteric GABAA receptor modulation counteracts lipotoxicity-induced gene expression changes in hepatocytes in vitro.

Introduction: We have previously shown that the novel positive allosteric modulator of the GABAA receptor, HK4, exerts hepatoprotective effects against lipotoxicity-induced apoptosis, DNA damage, inflammation and ER stress in vitro. This might be mediated by downregulated phosphorylation of the transcription factors NF-κB and STAT3. The current study aimed to investigate the effect of HK4 on lipotoxicity-induced hepatocyte injury at the transcriptional level. Methods: HepG2 cells were treated with palmitate (200 µM) in the presence or absence of HK4 (10 µM) for 7 h. Total RNA was isolated and the expression profiles of mRNAs were assessed. Differentially expressed genes were identified and subjected to the DAVID database and Ingenuity Pathway Analysis software for functional and pathway analysis, all under appropriate statistical testing. Results: Transcriptomic analysis showed substantial modifications in gene expression in response to palmitate as lipotoxic stimulus with 1,457 differentially expressed genes affecting lipid metabolism, oxidative phosphorylation, apoptosis, oxidative and ER stress among others. HK4 preincubation resulted in the prevention of palmitate-induced dysregulation by restoring initial gene expression pattern of untreated hepatocytes comprising 456 genes. Out of the 456 genes, 342 genes were upregulated and 114 downregulated by HK4. Enriched pathways analysis of those genes by Ingenuity Pathway Analysis, pointed towards oxidative phosphorylation, mitochondrial dysregulation, protein ubiquitination, apoptosis, and cell cycle regulation as affected pathways. These pathways are regulated by the key upstream regulators TP53, KDM5B, DDX5, CAB39 L and SYVN1, which orchestrate the metabolic and oxidative stress responses including modulation of DNA repair and degradation of ER stress-induced misfolded proteins in the presence or absence of HK4. Discussion: We conclude that HK4 specifically targets mitochondrial respiration, protein ubiquitination, apoptosis and cell cycle. This not only helps to counteract lipotoxic hepatocellular injury through modification of gene expression, but - by targeting transcription factors responsible for DNA repair, cell cycle progression and ER stress - might even prevent lipotoxic mechanisms. These findings suggest that HK4 has a great potential for the treatment of non-alcoholic fatty liver disease (NAFLD).

Complete sequences of six major histocompatibility complex haplotypes, including all the major MHC class II structures.

Accurate and comprehensive immunogenetic reference panels are key to the successful implementation of population-scale immunogenomics. The 5Mbp Major Histocompatibility Complex (MHC) is the most polymorphic region of the human genome and associated with multiple immune-mediated diseases, transplant matching and therapy responses. Analysis of MHC genetic variation is severely complicated by complex patterns of sequence variation, linkage disequilibrium and a lack of fully resolved MHC reference haplotypes, increasing the risk of spurious findings on analyzing this medically important region. Integrating Illumina, ultra-long Nanopore, and PacBio HiFi sequencing as well as bespoke bioinformatics, we completed five of the alternative MHC reference haplotypes of the current (GRCh38/hg38) build of the human reference genome and added one other. The six assembled MHC haplotypes encompass the DR1 and DR4 haplotype structures in addition to the previously completed DR2 and DR3, as well as six distinct classes of the structurally variable C4 region. Analysis of the assembled haplotypes showed that MHC class II sequence structures, including repeat element positions, are generally conserved within the DR haplotype supergroups, and that sequence diversity peaks in three regions around HLA-A, HLA-B+C, and the HLA class II genes. Demonstrating the potential for improved short-read analysis, the number of proper read pairs recruited to the MHC was found to be increased by 0.06%-0.49% in a 1000 Genomes Project read remapping experiment with seven diverse samples. Furthermore, the assembled haplotypes can serve as references for the community and provide the basis of a structurally accurate genotyping graph of the complete MHC region.

Transcriptome analysis of newly established carboplatin-resistant ovarian cancer cell model reveals genes shared by drug resistance and drug-induced EMT.

BACKGROUND: In ovarian cancer (OC) therapy, even initially responsive patients develop drug resistance. METHODS: Here, we present an OC cell model composed of variants with differing degrees of acquired resistance to carboplatin (CBP), cross-resistance to paclitaxel, and CBP-induced metastatic properties (migration and invasion). Transcriptome data were analysed by two approaches identifying differentially expressed genes and CBP sensitivity-correlating genes. The impact of selected genes and signalling pathways on drug resistance and metastatic potential, along with their clinical relevance, was examined by in vitro and in silico approaches. RESULTS: TMEM200A and PRKAR1B were recognised as potentially involved in both phenomena, also having high predictive and prognostic values for OC patients. CBP-resistant MES-OV CBP8 cells were more sensitive to PI3K/Akt/mTOR pathway inhibitors Rapamycin, Wortmannin, SB216763, and transcription inhibitor Triptolide compared with parental MES-OV cells. When combined with CBP, Rapamycin decreased the sensitivity of parental cells while Triptolide sensitised drug-resistant cells to CBP. Four PI3K/Akt/mTOR inhibitors reduced migration in both cell lines. CONCLUSIONS: A newly established research model and two distinct transcriptome analysis approaches identified novel candidate genes enrolled in CBP resistance development and/or CBP-induced EMT and implied that one-gene targeting could be a better approach than signalling pathway inhibition for influencing both phenomena.

The Mycotoxin Beauvericin Exhibits Immunostimulatory Effects on Dendritic Cells via Activating the TLR4 Signaling Pathway.

Beauvericin (BEA), a mycotoxin of the enniatin family produced by various toxigenic fungi, has been attributed multiple biological activities such as anti-cancer, anti-inflammatory, and anti-microbial functions. However, effects of BEA on dendritic cells remain unknown so far. Here, we identified effects of BEA on murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-cultured bone marrow derived dendritic cells (BMDCs) and the underlying molecular mechanisms. BEA potently activates BMDCs as signified by elevated IL-12 and CD86 expression. Multiplex immunoassays performed on myeloid differentiation primary response 88 (MyD88) and toll/interleukin-1 receptor (TIR) domain containing adaptor inducing interferon beta (TRIF) single or double deficient BMDCs indicate that BEA induces inflammatory cytokine and chemokine production in a MyD88/TRIF dependent manner. Furthermore, we found that BEA was not able to induce IL-12 or IFNß production in Toll-like receptor 4 (Tlr4)-deficient BMDCs, whereas induction of these cytokines was not compromised in Tlr3/7/9 deficient BMDCs. This suggests that TLR4 might be the functional target of BEA on BMDCs. Consistently, in luciferase reporter assays BEA stimulation significantly promotes NF-κB activation in mTLR4/CD14/MD2 overexpressing but not control HEK-293 cells. RNA-sequencing analyses further confirmed that BEA induces transcriptional changes associated with the TLR4 signaling pathway. Together, these results identify TLR4 as a cellular BEA sensor and define BEA as a potent activator of BMDCs, implying that this compound can be exploited as a promising candidate structure for vaccine adjuvants or cancer immunotherapies.

Single particle adsorbing transfer system.

Here we present a novel approach for horizontal transfer of single particles after laser microdissection. The developed technique is a single particle adsorbing system for highly selective and gentle horizontal transfer of microdissected fixed and living material. As mediated via low-pressure technology, the transfer process can be precisely controlled, thus facilitating horizontal particle transfer of any isolated material, e.g. tissue material, single cells or chromosomes, in addition to precise positioning for sample release. This collection method allows one to predefine target positions and enables material transfer without contamination to any planar microchip device. This contamination free transfer is indispensable for novel lab-on-a-chip systems performing nanoscale polymerase chain reaction analyses. Using virtual reaction chamber microdevices, small amounts of microdissected material--as little as one single cell--can be directly transmitted and immediately used for single cell analysis.