Chitin, Chitinase Responses, and Invasive Fungal Infections

The human immune system is capable of recognizing and degrading chitin, an important cell wall component of pathogenic fungi. In the context of host-immune responses to fungal infections, herein we review the particular contributions and interplay of fungus and chitin recognition, and chitin-degrading enzymes, known as chitinases. The mechanisms of host chitinase responses may have implications for diagnostic assays as well as novel therapeutic approaches for patients that are at risk of contracting fatal fungal infections

Detection of secreted peptides by using hypothesis-driven multistage mass spectrometry

A method is presented for the rapid detection and characterization of trace amounts of peptides secreted from microorganisms, including pheromones, virulence factors, and quorum-sensing peptides. The procedure, based on targeted multistage MS, uses a novel matrix-assisted laser desorptionionization-ion trap mass spectrometer to overcome limitations of current MS methods (limited dynamic range, signal suppression effects, and chemical noise) that impair observation of low abundance peptides from complex biological matrixes. Here, secreted peptides that are hypothesized to be present in the supernatant, but that may not be sufficiently abundant to be observed in single-stage mass spectra, are subjected to multistage MS. Highly specific fragmentation signatures enable unambiguous identification of the peptides of interest and differentiation of the signals from the background. As examples, we demonstrate the rapid (<1 min) determination of the mating type of cells in colonies of Saccharomyces cerevisiae and the elucidation of autoinducing peptides (AIPs) from supernatants of pathogenic Staphylococci. We confirm the primary structures of the agrD encoded cyclic AIPs of Staphylococcus aureus for groups I, II, and IV and provide direct evidence that the native group-III AIP is a heptapeptide (INCDFLL). We also show that the homologous peptide from Staphylococcus intermedius is a nonapeptide (RIPTSTGFF) with a lactone ring formed through condensation of the serine side chain with the C terminus of the peptide. This is the first demonstration of cyclization in a staphylococcal AIP that occurs via lactone formation. These examples demonstrate the analytical power of the present procedure for characterizing secreted peptides and its potential utility for identifying microorganisms

Addition of platinum derivatives to neoadjuvant single-agent fluoropyrimidine chemoradiotherapy in patients with stage II/III rectal cancer: protocol for a systematic review and meta-analysis (PROSPERO CRD42017073064)

Background Neoadjuvant (chemo-)radiation has proven to improve local control compared to surgery alone, but this improvement did not translate into better overall or disease-specific survival. The addition of oxaliplatin to fluoropyrimidine-based neoadjuvant chemoradiotherapy holds the potential of positively affecting survival in this context since it has been proven effective in the palliative and adjuvant setting of colorectal cancer. Thus, the objective of this systematic review is to assess the efficacy, safety, and quality of life resulting from adding a platinum derivative to neoadjuvant single-agent fluoropyrimidine-based chemoradiotherapy in patients with Union for International Cancer Control stage II and III rectal cancer. Methods: MEDLINE, Web of Science, and Cochrane Central Register of Controlled Trials will be systematically searched to identify all randomized controlled trials comparing single-agent fluoropyrimidine-based chemoradiotherapy to combined neoadjuvant therapy including a platinum derivative. Predefined data on trial design, quality, patient characteristics, and endpoints will be extracted. Quality of included trials will be assessed according to the Cochrane Risk of Bias Tool, and the GRADE recommendations will be applied to judge the quality of the resulting evidence. The main outcome parameter will be survival, but also treatment toxicity, perioperative morbidity, and quality of life will be assessed. Discussion: The findings of this systematic review and meta-analysis will provide novel insights into the efficacy and safety of combined neoadjuvant chemoradiotherapy including a platinum derivative and may form a basis for future clinical decision-making, guideline evaluation, and research prioritization. Systematic review registration PROSPERO CRD4201707306

Prothymosin α fragmentation in apoptosis

AbstractWe observed fragmentation of an essential proliferation-related human nuclear protein prothymosin α in the course of apoptosis induced by various stimuli. Prothymosin α cleavage occurred at the DDVD99 motif. In vitro, prothymosin α could be cleaved at D99 by caspase-3 and -7. Caspase hydrolysis disrupted the nuclear localization signal of prothymosin α and abrogated the ability of the truncated protein to accumulate inside the nucleus. Prothymosin α fragmentation may therefore be proposed to disable intranuclear proliferation-related function of prothymosin α in two ways: by cleaving off a short peptide containing important determinants, and by preventing active nuclear uptake of the truncated protein

Effects of Acyclovir and IVIG on Behavioral Outcomes after HSV1 CNS Infection

Herpes simplex virus 1 (HSV) encephalitis (HSE) has serious neurological complications, involving behavioral and cognitive impairments that cause significant morbidity and a reduced quality of life. We showed that HSE results from dysregulated central nervous system (CNS) inflammatory responses. We hypothesized that CNS inflammation is casually involved in behavioral abnormalities after HSE and that treatment with ACV and pooled human immunoglobulin (IVIG), an immunomodulatory drug, would improve outcomes compared to mice treated with phosphate buffered saline (PBS) or ACV alone. Anxiety levels were high in HSV-infected PBS and ACV-treated mice compared to mice treated with ACV + IVIG, consistent with reports implicating inflammation in anxiety induced by lipopolysaccharide (LPS) or stress. Female, but not male, PBS-treated mice were cognitively impaired, and unexpectedly, ACV was protective, while the inclusion of IVIG surprisingly antagonized ACV’s beneficial effects. Distinct serum proteomic profiles were observed for male and female mice, and the antagonistic effects of ACV and IVIG on behavior were paralleled by similar changes in the serum proteome of ACV- and ACV + IVIG-treated mice. We conclude that inflammation and other factors mediate HSV-induced behavioral impairments and that the effects of ACV and IVIG on behavior involve novel mechanisms

Quantitative Proteomic Analysis of Bacillus pumilus from Spores that Survived in Outer Space Conditions

The hardy spores of Bacillus species are well known for their resistance to unfavorable conditions such as UV and gamma radiation, hydrogen peroxide desiccation, chemical disinfection, or starvation. In particular, Bacillus pumilus strain SAFR-032 that was originally recovered from the Jet Propulsion Lab Spacecraft Assembly Facility has shown to exhibit unusually high resistance to UV radiation and peroxide treatment compared to other Bacillus species. To further understand the resistance of bacterial endospores to relevant outer space environments, spores of B. pumilus SAFR-032 were exposed for 1.5 years to selected parameters of space on board of the International Space Station (ISS). Here we are using a quantitative proteomics approach to gain insights into the resistance mechanism of B. pumilus

Proteomic and Metabolomic Characteristics of Extremophilic Fungi Under Simulated Mars Conditions

Filamentous fungi have been associated with extreme habitats, including nuclear power plant accident sites and the International Space Station (ISS). Due to their immense adaptation and phenotypic plasticity capacities, fungi may thrive in what seems like uninhabitable niches. This study is the first report of fungal survival after exposure of monolayers of conidia to simulated Mars conditions (SMC). Conidia of several Chernobyl nuclear accident-associated and ISS-isolated strains were tested for UV-C and SMC sensitivity, which resulted in strain-dependent survival. Strains surviving exposure to SMC for 30 min, ISSFT-021-30 and IMV 00236-30, were further characterized for proteomic, and metabolomic changes. Differential expression of proteins involved in ribosome biogenesis, translation, and carbohydrate metabolic processes was observed. No significant metabolome alterations were revealed. Lastly, ISSFT-021-30 conidia re-exposed to UV-C exhibited enhanced UV-C resistance when compared to the conidia of unexposed ISSFT-021

Long non-coding RNA lncMGC mediates the expression of TGF-β-induced genes in renal cells via nucleosome remodelers

Background: MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) play key roles in diabetic kidney disease (DKD). The miR-379 megacluster of miRNAs and its host transcript lnc-megacluster (lncMGC) are regulated by transforming growth factor-β (TGF-β), increased in the glomeruli of diabetic mice, and promote features of early DKD. However, biochemical functions of lncMGC are unknown. Here, we identified lncMGC-interacting proteins by in vitro-transcribed lncMGC RNA pull down followed by mass spectrometry. We also created lncMGC-knockout (KO) mice by CRISPR-Cas9 editing and used primary mouse mesangial cells (MMCs) from the KO mice to examine the effects of lncMGC on the gene expression related to DKD, changes in promoter histone modifications, and chromatin remodeling.Methods:In vitro-transcribed lncMGC RNA was mixed with lysates from HK2 cells (human kidney cell line). lncMGC-interacting proteins were identified by mass spectrometry. Candidate proteins were confirmed by RNA immunoprecipitation followed by qPCR. Cas9 and guide RNAs were injected into mouse eggs to create lncMGC-KO mice. Wild-type (WT) and lncMGC-KO MMCs were treated with TGF-β, and RNA expression (by RNA-seq and qPCR) and histone modifications (by chromatin immunoprecipitation) and chromatin remodeling/open chromatin (by Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq) were examined.Results: Several nucleosome remodeling factors including SMARCA5 and SMARCC2 were identified as lncMGC-interacting proteins by mass spectrometry, and confirmed by RNA immunoprecipitation–qPCR. MMCs from lncMGC-KO mice showed no basal or TGF-β-induced expression of lncMGC. Enrichment of histone H3K27 acetylation and SMARCA5 at the lncMGC promoter was increased in TGF-β-treated WT MMCs but significantly reduced in lncMGC-KO MMCs. ATAC peaks at the lncMGC promoter region and many other DKD-related loci including Col4a3 and Col4a4 were significantly lower in lncMGC-KO MMCs compared to WT MMCs in the TGF-β-treated condition. Zinc finger (ZF), ARID, and SMAD motifs were enriched in ATAC peaks. ZF and ARID sites were also found in the lncMGC gene.Conclusion: lncMGC RNA interacts with several nucleosome remodeling factors to promote chromatin relaxation and enhance the expression of lncMGC itself and other genes including pro-fibrotic genes. The lncMGC/nucleosome remodeler complex promotes site-specific chromatin accessibility to enhance DKD-related genes in target kidney cells

Attomolar Detection of Botulinum Toxin Type A in Complex Biological Matrices

BACKGROUND: A highly sensitive, rapid and cost efficient method that can detect active botulinum neurotoxin (BoNT) in complex biological samples such as foods or serum is desired in order to 1) counter the potential bioterrorist threat 2) enhance food safety 3) enable future pharmacokinetic studies in medical applications that utilize BoNTs. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a botulinum neurotoxin serotype A assay with a large immuno-sorbent surface area (BoNT/A ALISSA) that captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate. In direct comparison with the "gold standard" mouse bioassay, the ALISSA is four to five orders of magnitudes more sensitive and considerably faster. Our method reaches attomolar sensitivities in serum, milk, carrot juice, and in the diluent fluid used in the mouse assay. ALISSA has high specificity for the targeted type A toxin when tested against alternative proteases including other BoNT serotypes and trypsin, and it detects the holotoxin as well as the multi-protein complex form of BoNT/A. The assay was optimized for temperature, substrate concentration, size and volume proportions of the immuno-sorbent matrix, enrichment and reaction times. Finally, a kinetic model is presented that is consistent with the observed improvement in sensitivity. CONCLUSIONS/SIGNIFICANCE: The sensitivity, specificity, speed and simplicity of the BoNT ALISSA should make this method attractive for diagnostic, biodefense and pharmacological applications

Histamine Derived from Probiotic Lactobacillus reuteri Suppresses TNF via Modulation of PKA and ERK Signaling

Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s) produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC) separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA), histidine/histamine antiporter (hdcP), and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2)-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H2 receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA) and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases