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In single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of ZMWs in parallel without sophisticated equipment. Polystyrene 1 μm dia. microbeads self-assemble on a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead interstices. The width of those interstices (and subsequent posts) is adjusted within 100–300 nm by partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology avoiding the availability and expense of sophisticated clean-room environments and equipment. [ABSTRACT FROM AUTHOR]

Transforming growth factor‑β1 (TGF-β1) is a versatile cytokine. It has context-dependent pro- and anti-cell proliferation functions. Activation of latent TGF-β1 requires release of the growth factor from pro-complexes and is regulated through TGF-β binding proteins. Two types of TGF-β binding partners, latent TGF-β-binding proteins (LTBPs) and leucine-rich-repeat-containing protein 32 (LRRC32), have been identified and their expression are cell specific. TGF-β1 also plays important roles in acute myeloid leukemia (AML) cells. However, the expression of LTBPs and LRRC32 are lacking in myeloid lineage cells and the binding protein of TGF-β1 in these cells are unknown. Here we show that a novel leucine-rich-repeat-containing protein family member, LRRC33, with high mRNA level in AML cells, to be the binding and regulating protein of TGF-β1 in AML cells. Using two representative cell lines MV4-11 and AML193, we demonstrate that the protein expression of LRRC33 and TGF-β1 are correlated. LRRC33 co-localizes and forms complex with latent TGF-β1 protein on the cell surface and intracellularly in these cells. Similar as in other cell types, the activation of TGF-β1 in MV4-11 and AML193 cells are also integrin dependent. We anticipate our study to be a starting point of more comprehensive research on LRRC33 as novel TGF-β regulating protein and potential non-genomic based drug target for AML and other myeloid malignancy. [ABSTRACT FROM AUTHOR]

A detailed characterization of the chemical composition of complex substances, such as products of petroleum refining and environmental mixtures, is greatly needed in exposure assessment and manufacturing. The inherent complexity and variability in the composition of complex substances obfuscate the choices for their detailed analytical characterization. Yet, in lieu of exact chemical composition of complex substances, evaluation of the degree of similarity is a sensible path toward decision-making in environmental health regulations. Grouping of similar complex substances is a challenge that can be addressed via advanced analytical methods and streamlined data analysis and visualization techniques. Here, we propose a framework with unsupervised and supervised analyses to optimally group complex substances based on their analytical features. We test two data sets of complex oil-derived substances. The first data set is from gas chromatography-mass spectrometry (GC-MS) analysis of 20 Standard Reference Materials representing crude oils and oil refining products. The second data set consists of 15 samples of various gas oils analyzed using three analytical techniques: GC-MS, GC×GC-flame ionization detection (FID), and ion mobility spectrometry-mass spectrometry (IM-MS). We use hierarchical clustering using Pearson correlation as a similarity metric for the unsupervised analysis and build classification models using the Random Forest algorithm for the supervised analysis. We present a quantitative comparative assessment of clustering results via Fowlkes–Mallows index, and classification results via model accuracies in predicting the group of an unknown complex substance. We demonstrate the effect of (i) different grouping methodologies, (ii) data set size, and (iii) dimensionality reduction on the grouping quality, and (iv) different analytical techniques on the characterization of the complex substances. While the complexity and variability in chemical composition are an inherent feature of complex substances, we demonstrate how the choices of the data analysis and visualization methods can impact the communication of their characteristics to delineate sufficient similarity. [ABSTRACT FROM AUTHOR]

Background: Escherichia coli is currently unable to be reliably differentiated from Shigella species by routine matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. In the present study, a reliable and rapid identification method was established for Escherichia coli and Shigella species based on a short-term high-lactose culture using MALDI-TOF MS and artificial neural networks (ANN). Materials and methods: The Escherichia coli and Shigella species colonies, treated with (Condition 1)/without (Condition 2) a short-term culture with an in-house developed high-lactose fluid medium, were prepared for MALDI-TOF MS assays. The MS spectra were acquired in linear positive mode, with a mass range from 2000 to 12000 Da and were then compared to discover new biomarkers for identification. Finally, MS spectra data sets 1 and 2, extracted from the two conditions, were used for ANN training to investigate the benefit on bacterial classification produced by the new biomarkers. Results: Twenty-seven characteristic MS peaks from the Escherichia coli and Shigella species were summarized. Seven unreported MS peaks, with m/z 2330.745, m/z 2341.299, m/z 2371.581, m/z 2401.038, m/z 3794.851, m/z 3824.839 and m/z 3852.548, were discovered in only the spectra from the E. coli strains after a short-term high-lactose culture and were identified as belonging to acid shock protein. The prediction accuracies of the ANN models, based on data set 1 and 2, were 97.71±0.16% and 74.39±0.34% (n = 5), with an extremely remarkable difference (p < 0.001), and the areas under the curve of the receiver operating characteristic curve were 0.72 and 0.99, respectively. Conclusions: In summary, adding a short-term high-lactose culture approach before the analysis enabled a reliable and easy differentiation of Escherichia coli from the Shigella species using MALDI-TOF MS and ANN. [ABSTRACT FROM AUTHOR]

Guanylate binding proteins (GBPs) belong to the dynamin-related superfamily and exhibit various functions in the fight against infections. The functions of the human guanylate binding protein 1 (hGBP1) are tightly coupled to GTP hydrolysis and dimerization. Despite known crystal structures of the hGBP1 monomer and GTPase domain dimer, little is known about the dynamics of hGBP1. To gain a mechanistic understanding of hGBP1, we performed sub-millisecond multi-resolution molecular dynamics simulations of both the hGBP1 monomer and dimer. We found that hGBP1 is a highly flexible protein that undergoes a hinge motion similar to the movements observed for other dynamin-like proteins. Another large-scale motion was observed for the C-terminal helix α13, providing a molecular view for the α13–α13 distances previously reported for the hGBP1 dimer. Most of the loops of the GTPase domain were found to be flexible, revealing why GTP binding is needed for hGBP1 dimerization to occur. [ABSTRACT FROM AUTHOR]

Taste receptor type 1 (T1r) is responsible for the perception of essential nutrients, such as sugars and amino acids, and evoking sweet and umami (savory) taste sensations. T1r receptors recognize many of the taste substances at their extracellular ligand-binding domains (LBDs). In order to detect a wide array of taste substances in the environment, T1r receptors often possess broad ligand specificities. However, the entire ranges of chemical spaces and their binding characteristics to any T1rLBDs have not been extensively analyzed. In this study, we exploited the differential scanning fluorimetry (DSF) to medaka T1r2a/T1r3LBD, a current sole T1rLBD heterodimer amenable for recombinant preparation, and analyzed their thermal stabilization by adding various amino acids. The assay showed that the agonist amino acids induced thermal stabilization and shifted the melting temperatures (Tm) of the protein. An agreement between the DSF results and the previous biophysical assay was observed, suggesting that DSF can detect ligand binding at the orthosteric-binding site in T1r2a/T1r3LBD. The assay further demonstrated that most of the tested -amino acids, but no -amino acid, induced Tm shifts of T1r2a/T1r3LBD, indicating the broad -amino acid specificities of the proteins probably with several different manners of recognition. The Tm shifts by each amino acid also showed a fair correlation with the responses exhibited by the full-length receptor, verifying the broad amino-acid binding profiles at the orthosteric site in LBD observed by DSF. [ABSTRACT FROM AUTHOR]

Spleen stromal lines which support in vitro hematopoiesis are investigated for their lineage origin and hematopoietic support function in vivo. Marker expression and gene profiling identify a lineage relationship with mesenchymal stem cells and perivascular reticular cells described recently in bone marrow. Stromal lines commonly express Cxcl12, Pdgfra and Pdgfr typical of bone marrow derived perivascular reticular cells but reflect a unique cell type in terms of other gene and marker expression. Their classification as osteoprogenitors is confirmed through ability to undergo osteogenic, but not adipogenic or chondrogenic differentiation. Some stromal lines were shown to form ectopic niches for HSCs following engraftment under the kidney capsule of NOD/SCID mice. The presence of myeloid cells and a higher representation of a specific dendritic-like cell type over other myeloid cells within grafts was consistent with previous in vitro evidence of hematopoietic support capacity. These studies reinforce the role of perivascular/perisinusoidal reticular cells in hematopoiesis and implicate such cells as niches for hematopoiesis in spleen. [ABSTRACT FROM AUTHOR]

A novel polysaccharide STSP-I was isolated and purified from Sargassum thunbergii. Its structure and bioactivity were studied using gas chromatography (GC), fourier transform infrared spectroscopy (FTIR), periodate oxidation-smith degradation, partial acid hydrolysis, methylation-GC-MS, nuclear magnetic resonance (NMR), transmission electron microscopy (TEM), radicals scavenging assays and anti-inflammatory assays. STSP-I was consisted of fucose and galactose with a molar ratio of 1.2:1, and its mass was 373 kDa. The main structural components of STSP-I were →4)-α-D-Galp-(1→ and →3)-β-L-Fucp-(1→, STSP-I was a non-branched polysaccharide, and TEM further revealed the existence of entangled chains and linear forms. Compared with Vitamin C (Vc), STSP-I showed a higher scavenging effect of superoxide radical (EC50 = 0.22 mg/mL) and an equivalent scavenging effect of hydroxyl radical (EC50 = 0.88 mg/mL). STSP-I also exhibited good inhibitory effects of TNF-α, IL-6 and COX-2 mRNA expressions in LPS-stimulated RAW 264.7 mouse macrophage cells, and the inhibitory effects were more than 91% at the concentrations of 75 and 150 μg/ml. The results indicate that the polysaccharide STSP-I from S. thunbergii with the linear structure may serve as potential antioxidant and anti-inflammatory agents. [ABSTRACT FROM AUTHOR]

The spores of fungi come in a wide variety of forms and sizes, highly adapted to the route of dispersal and to survival under specific environmental conditions. The ascomycete Ashbya gossypii produces needle shaped spores with a length of 30 μm and a diameter of 1 μm. Formation of these spores relies on actin and actin regulatory proteins and is, therefore, distinct from the minor role that actin plays for spore formation in Saccharomyces cerevisiae. Using in vivo FRET-measurements of proteins labeled with fluorescent proteins, we investigate how the formin AgBnr2, a protein that promotes actin polymerization, integrates into the structure of the spindle pole body during sporulation. We also investigate the role of the A. gossypii homologs to the S. cerevisiae meiotic outer plaque proteins Spo74, Mpc54 and Ady4 for sporulation in A. gossypii. We found highest FRET of AgBnr2 with AgSpo74. Further experiments indicated that AgSpo74 is a main factor for targeting AgBnr2 to the spindle pole body. In agreement with these results, the Agspo74 deletion mutant produces no detectable spores, whereas deletion of Agmpc54 only has an effect on spore length and deletion of Agady4 has no detectable sporulation phenotype. Based on this study and in relation to previous results we suggest a model where AgBnr2 resides within an analogous structure to the meiotic outer plaque of S. cerevisiae. There it promotes formation of actin cables important for shaping the needle shaped spore structure. [ABSTRACT FROM AUTHOR]

Chromosome replication in Saccharomyces cerevisiae is initiated from ~300 origins that are regulated by DNA sequence and by the limited abundance of six trans-acting initiation proteins (Sld2, Sld3, Dpb11, Dbf4, Sld7 and Cdc45). We set out to determine how the levels of individual factors contribute to time of origin activation and/or origin efficiency using induced depletion of single factors and overexpression of sets of multiple factors. Depletion of Sld2 or Sld3 slows growth and S phase progression, decreases origin efficiency across the genome and impairs viability as a result of incomplete replication of the rDNA. We find that the most efficient early origins are relatively unaffected by depletion of either Sld2 or Sld3. However, Sld3 levels, and to a lesser extent Sld2 levels, are critical for firing of the less efficient early origins. Overexpression of Sld3 simultaneously with Sld2, Dpb11 and Dbf4 preserves the relative efficiency of origins. Only when Cdc45 and Sld7 are also overexpressed is origin efficiency equalized between early- and late-firing origins. Our data support a model in which Sld3 together with Cdc45 (and/or Sld7) is responsible for the differential efficiencies of origins across the yeast genome. [ABSTRACT FROM AUTHOR]

[Fam-] trastuzumab deruxtecan (DS-8201a) is a HER2 (ERBB2)-targeting antibody-drug conjugate, composed of a HER2-targeting antibody and a topoisomerase I inhibitor, exatecan derivative, that has antitumor effects in preclinical xenograft models and clinical trials. Recently, [fam-] trastuzumab deruxtecan was reported to enhance antitumor immunity and was beneficial in combination with an anti–PD-1 antibody in a mouse model. In this study, the antitumor effect of [fam-] trastuzumab deruxtecan in combination with an anti–CTLA-4 antibody was evaluated. [Fam-] trastuzumab deruxtecan monotherapy had antitumor activity in an immunocompetent mouse model with EMT6 human HER2-expressing mouse breast cancer cells (EMT6-hHER2). [Fam-] trastuzumab deruxtecan in combination with the anti–CTLA-4 antibody induced more potent antitumor activity than that by monotherapy with either agent. The combination therapy increased tumor-infiltrating CD4+ and CD8+ T cells in vivo. Mechanistically, cured mice with treatment of [fam-] trastuzumab deruxtecan and an anti–CTLA-4 antibody completely rejected EMT6-mock cells similar to EMT6-hHER2 cells, and splenocytes from the cured mice responded to both EMT6-hHER2 and EMT6-mock cells as measured by interferon-gamma release. Taken together, these results indicate that antitumor immunity is induced by [fam-] trastuzumab deruxtecan and is facilitated in combination with anti–CTLA-4 antibody. [ABSTRACT FROM AUTHOR]

The formation of the sea urchin spicule skeleton requires the participation of hydrogel-forming protein families that regulate mineral nucleation and nanoparticle assembly processes that give rise to the spicule. However, the structure and molecular behavior of these proteins is not well established, and thus our ability to understand this process is hampered. We embarked on a study of sea urchin spicule proteins using a combination of biophysical and bioinformatics techniques. Our biophysical findings indicate that recombinant variants of the two most studied spicule matrix proteins, SpSM50 and SpSM30B/C (S. purpuratus) have a conformational landscape that include a C-terminal random coil/intrinsically disordered MAPQG sequence coupled to a conserved, folded N-terminal C-type lectin-like (CTLL) domain, with SpSM50 > SpSM30B/C with regard to intrinsic disorder. Both proteins possess solvent-accessible unfolded MAQPG sequence regions where Asn, Gln, and Arg residues may be accessible for protein hydrogel interactions with water molecules. Our bioinformatics study included seven other spicule matrix proteins where we note similarities between these proteins and rare, unusual proteins that possess folded and unfolded traits. Moreover, spicule matrix proteins possess three types of sequences: intrinsically disordered, amyloid-like, and folded protein-protein interactive. Collectively these reactive domains would be capable of driving protein assembly and hydrogel formation. Interestingly, three types of global conformations are predicted for the nine member protein set, wherein we note variations in the arrangement of intrinsically disordered and interactive globular domains. These variations may reflect species-specific requirements for spiculogenesis. We conclude that the molecular landscape of spicule matrix protein families enables them to function as hydrogelators, nucleators, and assemblers of mineral nanoparticles. [ABSTRACT FROM AUTHOR]

To confirm that neoplastic monocyte-derived collagen- and fibronectin-producing fibrocytes induce bone marrow (BM) fibrosis in primary myelofibrosis (PMF), we injected PMF BM-derived fibrocyte-precursor CD14+/CD34- monocytes into the tail vein of NOD-SCID-γ (NSG) mice. PMF BM-derived CD14+/CD34- monocytes engrafted and induced a PMF-like phenotype with splenomegaly, myeloid hyperplasia with clusters of atypical megakaryocytes, persistence of the JAK2V617F mutation, and BM and spleen fibrosis. As control we used normal human BM-derived CD14+/CD34- monocytes. These monocytes also engrafted and gave rise to normal megakaryocytes that, like PMF CD14+/CD34--derived megakaryocytes, expressed HLA-ABC and human CD42b antigens. Using 2 clonogenic assays we confirmed that PMF and normal BM-derived CD14+/CD34- monocytes give rise to megakaryocyte colony-forming cells, suggesting that a subpopulation BM monocytes harbors megakaryocyte progenitor capacity. Taken together, our data suggest that PMF monocytes induce myelofibrosis-like phenotype in immunodeficient mice and that PMF and normal BM-derived CD14+/CD34- monocytes give rise to megakaryocyte progenitor cells. [ABSTRACT FROM AUTHOR]

Calreticulin (CRT) is a high-capacity Ca2+ protein whose expression is up-regulated during cellular transformation and is associated with disease progression in multiple types of malignancies. At the same time, CRT has been characterized as an important stress-response protein capable of inducing immunogenic cell death (ICD) when translocated to the cell surface. It remains unclear why CRT expression is preserved by malignant cells during the course of transformation despite its immunogenic properties. In this study, we identify a novel, critical function of CRT as a cell survival factor in multiple types of human solid-tissue malignancies. CRT knockdown activates p53, which mediates cell-death response independent of executioner caspase activity and accompanied full-length poly ADP ribose polymerase (PARP) cleavage. Mechanistically, we show that down-regulation of CRT results in mitochondrial Ca2+ overload and induction of mitochondria permeability transition pore (mPTP)-dependent cell death, which can be significantly rescued by the mPTP inhibitor, Cyclosporin A (CsA). The clinical importance of CRT expression was revealed in the analysis of the large cohort of cancer patients (N = 2,058) to demonstrate that high levels of CRT inversely correlates with patient survival. Our study identifies intracellular CRT as an important therapeutic target for tumors whose survival relies on its expression. [ABSTRACT FROM AUTHOR]

Objectives: Bloodstream infections in critically ill require a speeded-up microbiological diagnosis to improve clinical outcomes. In this pre-post intervention study, we evaluated how a molecular identification test directly performed on positive blood cultures of critically ill improves patient’s therapeutic management. Methods: All adult patients staying at the intensive care unit (ICU) at the time of positive blood culture detection were study-eligible. In the 8-month pre-intervention period (P0), standard positive blood culture management was performed. In the 10-month intervention period (P1), a BioFire® FilmArray® blood culture identification (FA-BCID) test (bioMérieux) was additionally performed 24/7 at detection. The evaluated clinical outcome was time to optimal antimicrobial treatment of the bloodstream infection. FA-BCID microbiological test performances were also analysed. Results: 163 positive blood culture episodes were allocated to P0 and 166 to P1. After the withdrawal of episodes in accordance with defined exclusion criteria, outcome analysis was performed on 110 bloodstream infections both in P0 and P1. Time to optimal antimicrobial treatment in P0 was 14h41 compared to 4h39 in P1. FA-BCID test results led to a treatment adjustment in 35/110 (31.8%) P1 episodes including 26 where the adjustment was the optimal antimicrobial treatment. FA-BCID testing identified 96.2% of the on-panel microorganisms thereby covering 85.2% of our ICU-strain epidemiology. Time to identification with FA-BCID testing was calculated at 1h35. Resistance detection was in complete concordance with routine results. Considering 150 FA-BCID tests were initially performed in P1, 4,3 tests were required to have 1 test leading to an improved therapeutic outcome. Conclusions: FA-BCID testing drastically reduced time to optimal antimicrobial treatment in critically ill with bloodstream infections. [ABSTRACT FROM AUTHOR]

The West Nile Virus (WNV) NS2B-NS3 protease is an attractive target for the development of therapeutics against this arboviral pathogen. In the present investigation, the screening of a small library of fifty-eight synthetic compounds against the NS2-NB3 protease of WNV is described. The following groups of compounds were evaluated: 3-(2-aryl-2-oxoethyl)isobenzofuran-1(3H)-ones; eugenol derivatives bearing 1,2,3-triazolic functionalities; and indan-1,3-diones with 1,2,3-triazolic functionalities. The most promising of these was a eugenol derivative, namely 4-(3-(4-allyl-2-methoxyphenoxy)-propyl)-1-(2-bromobenzyl)-1H-1,2,3-triazole (35), which inhibited the protease with IC50 of 6.86 μmol L-1. Enzyme kinetic assays showed that this derivative of eugenol presents competitive inhibition behaviour. Molecular docking calculations predicted a recognition pattern involving the residues His51 and Ser135, which are members of the catalytic triad of the WNV NS2B-NS3 protease. [ABSTRACT FROM AUTHOR]

Recent work has shown that weak protein-protein interactions are susceptible to the cellular milieu. One case in point is the binding of heat shock proteins (Hsps) to substrate proteins in cells under stress. Upregulation of the Hsp70 chaperone machinery at elevated temperature was discovered in the 1960s, and more recent studies have shown that ATPase activity in one Hsp70 domain is essential for control of substrate binding by the other Hsp70 domain. Although there are several denaturant-based assays of Hsp70 activity, reports of ATP-dependent binding of Hsp70 to a globular protein substrate under heat shock are scarce. Here we show that binding of heat-inducible Hsp70 to phosphoglycerate kinase (PGK) is remarkably different in vitro compared to in-cell. We use fluorescent-labeled mHsp70 and ePGK, and begin by showing that mHsp70 passes the standard β-galactosidase assay, and that it does not self-aggregate until 50°C in presence of ATP. Yet during denaturant refolding or during in vitro heat shock, mHsp70 shows only ATP-independent non-specific sticking to ePGK, as evidenced by nearly identical results with an ATPase activity-deficient K71M mutant of Hsp70 as a control. Addition of Hsp40 (co-factor) or Ficoll (crowder) does not reduce non-specific sticking, but cell lysate does. Therefore, Hsp70 does not act as an ATP-dependent chaperone on its substrate PGK in vitro. In contrast, we observe only specific ATP-dependent binding of mHsp70 to ePGK in mammalian cells, when compared to the inactive Hsp70 K71M mutant. We hypothesize that enhanced in-cell activity is not due to an unknown co-factor, but simply to a favorable shift in binding equilibrium caused by the combination of crowding and osmolyte/macromolecular interactions present in the cell. One candidate mechanism for such a favorable shift in binding equilibrium is the proven ability of Hsp70 to bind near-native states of substrate proteins in vitro. We show evidence for early onset of binding in-cell. Our results suggest that Hsp70 binds PGK preemptively, prior to its full unfolding transition, thus stabilizing it against further unfolding. We propose a “preemptive holdase” mechanism for Hsp70-substrate binding. Given our result for PGK, more proteins than one might think based on in vitro assays may be chaperoned by Hsp70 in vivo. The cellular environment thus plays an important role in maintaining proper Hsp70 function. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) is a seasonal pathogen with the potential to cause devastating pandemics. IAV infects multiple epithelial cell subsets in the respiratory tract, eliciting damage to the lungs. Clearance of IAV is primarily dependent on CD8+ T cells, which must balance control of the infection with immunopathology. Using a virus expressing Cre recombinase to permanently label infected cells in a Cre-inducible reporter mouse, we previously discovered infected club cells that survive both lytic virus replication and CD8+ T cell-mediated clearance. In this study, we demonstrate that ciliated epithelial cells, type I and type II alveolar cells can also become survivor cells. Survivor cells are stable in the lung long-term and demonstrate enhanced proliferation compared to uninfected cells. When we investigated how survivor cells evade CD8+ T cell killing we observed that survivor cells upregulated the inhibitory ligand PD-L1, but survivor cells did not use PD-L1 to evade CD8+ T cell killing. Instead our data suggest that survivor cells are not inherently resistant to CD8+ T cell killing, but instead no longer present IAV antigen and cannot be detected by CD8+ T cells. Finally, we evaluate the failure of CD8+ T cells to kill these previously infected cells. This work demonstrates that additional cell types can survive IAV infection and that these cells robustly proliferate and are stable long term. By sparing previously infected cells, the adaptive immune system may be minimizing pathology associated with IAV infection. [ABSTRACT FROM AUTHOR]

Ligand binding to death receptors activates apoptosis in cancer cells. Stimulation of death receptors results in the formation of intracellular multiprotein platforms that either activate the apoptotic initiator Caspase-8 to trigger cell death, or signal through kinases to initiate inflammatory and cell survival signalling. Two of these platforms, the Death-Inducing Signalling Complex (DISC) and the RIPoptosome, also initiate necroptosis by building filamentous scaffolds that lead to the activation of mixed lineage kinase domain-like pseudokinase. To explain cell decision making downstream of death receptor activation, we developed a semi-stochastic model of DISC/RIPoptosome formation. The model is a hybrid of a direct Gillespie stochastic simulation algorithm for slow assembly of the RIPoptosome and a deterministic model of downstream caspase activation. The model explains how alterations in the level of death receptor-ligand complexes, their clustering properties and intrinsic molecular fluctuations in RIPoptosome assembly drive heterogeneous dynamics of Caspase-8 activation. The model highlights how kinetic proofreading leads to heterogeneous cell responses and results in fractional cell killing at low levels of receptor stimulation. It reveals that the noise in Caspase-8 activation—exclusively caused by the stochastic molecular assembly of the DISC/RIPoptosome platform—has a key function in extrinsic apoptotic stimuli recognition. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, and is the most common type of cognitive impairment and dementia. There is a pressing need to improve the clinical efficacy and quality of life for AD patients, as limited treatments options for AD patients have been developed until now. In this study, we aim to investigate the protective effect of CH(II), a cerebroprotein hydrolysate consisted of abundant biological peptides, on preclinical model of AD. We found that CH(II) treatment effectively protects oxygen glucose deprivation (OGD)-induced N2A cell viability impairment and cell apoptosis. In addition, CH(II) significantly reduces H2O2-induced ROS accumulation and exhibits the protective activities against H2O2-induced oxidative injury. Intriguingly, we found that CH(II) treatment can effectively promote neurite outgrowth of N2A cells. Moreover, CH(II) obviously improve the cognitive and memorial function in scopolamine-induced amnesia mice model. Taken together, this study provides evidences of the neuroprotective activities of CH(II) and offers a potential therapeutic strategy for AD patients. [ABSTRACT FROM AUTHOR]

Objective: The immune rejection mediated by CD4+ T cell and antigen presenting macrophages is the leading cause of corneal transplantation failure. Bone marrow-derived mesenchymal stem cells (BM-MSCs) possess robust immunomodulatory potentials, and have been shown by us and others to promote corneal allograft survival. However, the immunological mechanism underlying the protective effects of BM-MSCs remains unclear. Therefore, in the current study, this mechanism was investigated in a BM-MSC-treated rat model of corneal allograft rejection, in the hope to facilitate the search for novel interventional targets to corneal allograft rejection. Methods: Lewis rats were subjected to corneal transplantation and then received subconjunctival injections of BM-MSCs (2×106 cells / 100 μl PBS) immediately and at day 3 post-transplantation. The control group received the injections of PBS with the same volume. The clinical parameters of the corneal allografts, including opacity, edema, and neovascularization, were regularly evaluated after transplantation. On day 10 post-transplantation, the corneal allografts were collected and subjected to flow cytometry and high-throughput RNA sequencing (RNA-seq). GO enrichment and KEGG pathways were analyzed. The quantitative realtime PCR (qPCR) and immunohistochemistry (IHC) were employed to validate the expression of the selected target genes at transcript and protein levels, respectively. Results: BM-MSC subconjunctival administration prolonged the corneal allograft survival, with reduced opacity, alleviated edema, and diminished neovascularization. Flow cytometry showed reduced CD4+ T cells and CD68+ macrophages as well as boosted regulatory T cells (Tregs) in the BM-MSC-treated corneal allografts as compared with the PBS-treated counterparts. Moreover, the RNA-seq and qPCR results demonstrated that the transcript abundance of Cytotoxic T-Lymphocyte Associated Protein 4 (Ctla4), Protein Tyrosine Phosphatase, Receptor Type C (Ptprc), and C-X-C Motif Chemokine Ligand 9 (Cxcl9) genes were increased in the allografts of BM-MSC group compared with PBS group; whereas the expression of Heat Shock Protein Family A (Hsp70) Member 8 (Hspa8) gene was downregulated. The expression of these genes was confirmed by IHC at protein level. Conclusion: Subconjunctival injections of BM-MSCs promoted corneal allograft survival, reduced CD4+ and CD68+ cell infiltration, and enriched Treg population in the allografts. The BM-MSC-induced upregulation of Ctla4, Ptprc, Cxcl9 genes and downregulation of Hspa8 gene might contribute to the protective effects of BM-MSCs and subserve the potential interventional targets to corneal allograft rejection. [ABSTRACT FROM AUTHOR]

Perfusion-related information is reportedly embedded in the low-frequency component of a blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) signal. The blood-propagation pattern through the cerebral vascular tree is detected as an interregional lag variation of spontaneous low-frequency oscillations (sLFOs). Mapping of this lag, or phase, has been implicitly treated as a projection of the vascular tree structure onto real space. While accumulating evidence supports the biological significance of this signal component, the physiological basis of the “perfusion lag structure,” a requirement for an integrative resting-state fMRI-signal model, is lacking. In this study, we conducted analyses furthering the hypothesis that the sLFO is not only largely of systemic origin, but also essentially intrinsic to blood, and hence behaves as a virtual tracer. By summing the small fluctuations of instantaneous phase differences between adjacent vascular regions, a velocity response to respiratory challenges was detected. Regarding the relationship to neurovascular coupling, the removal of the whole lag structure, which can be considered as an optimized global-signal regression, resulted in a reduction of inter-individual variance while preserving the fMRI response. Examination of the T2* and S0, or non-BOLD, components of the fMRI signal revealed that the lag structure is deoxyhemoglobin dependent, while paradoxically presenting a signal-magnitude reduction in the venous side of the cerebral vasculature. These findings provide insight into the origin of BOLD sLFOs, suggesting that they are highly intrinsic to the circulating blood. [ABSTRACT FROM AUTHOR]

High Content Analysis (HCA) has become a cornerstone of cellular analysis within the drug discovery industry. To expand the capabilities of HCA, we have applied the same analysis methods, validated in numerous mammalian cell models, to microbiology methodology. Image acquisition and analysis of various microbial samples, ranging from pure cultures to culture mixtures containing up to three different bacterial species, were quantified and identified using various machine learning processes. These HCA techniques allow for faster cell enumeration than standard agar-plating methods, identification of “viable but not plate culturable” microbe phenotype, classification of antibiotic treatment effects, and identification of individual microbial strains in mixed cultures. These methods greatly expand the utility of HCA methods and automate tedious and low-throughput standard microbiological methods. [ABSTRACT FROM AUTHOR]

Emission rates of diethylhexyl phthalate (DEHP) from building materials, such as vinyl floorings and wall paper, determined using a passive flux sampler (PFS) were constant over the week-long measurement period. Emission rates for vinyl floorings and wallpaper were linearly correlated to the inverse of diffusion distance, which corresponds to the internal depth of the PFS. Surface-air DEHP concentrations (y0) were estimated as 1.3–2.3 μg/m3 for materials having a boundary layer molecular diffusion rate-limiting step. The partition coefficient (Kmaterial-air) was estimated as 3.3–7.5 × 1010 for these materials. Additionally, emission rates of DEHP from same building materials determined using a micro-chamber were 4.5–6.1 μg/m2/h. Mass transfer coefficients in the micro-chamber (hm) were estimated by comparing the results using the PFS and micro-chamber, and these were 1.1–1.2 × 10−3 and 8.1 × 10−4 m/s for vinyl floorings (smooth surface) and wallpaper (rough surface), respectively. The thickness of boundary layer on the surface of building materials in the micro-chamber were estimated to be 2.5–2.6 and 3.7 mm for vinyl floorings and wallpaper, respectively. [ABSTRACT FROM AUTHOR]

A high work of breathing can compromise limb oxygen delivery during sustained high-intensity exercise. However, it is unclear if the same is true for intermittent sprint exercise. This project examined the effect of adding an inspiratory load on locomotor muscle tissue reoxygenation during repeated-sprint exercise. Ten healthy males completed three experiment sessions of ten 10-s sprints, separated by 30-s of passive rest on a cycle ergometer. The first two sessions were “all-out’ efforts performed without (CTRL) or with inspiratory loading (INSP) in a randomised and counterbalanced order. The third experiment session (MATCH) consisted of ten 10-s work-matched intervals. Tissue saturation index (TSI) and deoxy-haemoglobin (HHb) of the vastus lateralis and sixth intercostal space was monitored with near-infrared spectroscopy. Vastus lateralis reoxygenation (ΔReoxy) was calculated as the difference from peak HHb (sprint) to nadir HHb (recovery). Total mechanical work completed was similar between INSP and CTRL (effect size: -0.18, 90% confidence limit ±0.43), and differences in vastus lateralis TSI during the sprint (-0.01 ±0.33) and recovery (-0.08 ±0.50) phases were unclear. There was also no meaningful difference in ΔReoxy (0.21 ±0.37). Intercostal HHb was higher in the INSP session compared to CTRL (0.42 ±0.34), whilst the difference was unclear for TSI (-0.01 ±0.33). During MATCH exercise, differences in vastus lateralis TSI were unclear compared to INSP for both sprint (0.10 ±0.30) and recovery (-0.09 ±0.48) phases, and there was no meaningful difference in ΔReoxy (-0.25 ±0.55). Intercostal TSI was higher during MATCH compared to INSP (0.95 ±0.53), whereas HHb was lower (-1.09 ±0.33). The lack of difference in ΔReoxy between INSP and CTRL suggests that for intermittent sprint exercise, the metabolic O2 demands of both the respiratory and locomotor muscles can be met. Additionally, the similarity of the MATCH suggests that ΔReoxy was maximal in all exercise conditions. [ABSTRACT FROM AUTHOR]

Cotton gin trash (CGT), a waste product of cotton gins, make up about 10% of each bale of cotton bolls ginned. The current study investigates high value volatile compounds in CGT to add value to this by-product. The volatile compounds in CGT and different parts of the cotton plant were extracted using various methods, identified by gas chromatography-mass spectrometry (GC-MS) or nuclear magnetic resonance (NMR) spectroscopy, and then quantified by gas chromatography-flame ionisation detection (GC-FID) against available standards. Terpenoids including monoterpenoids and sesquiterpenoids were found to be the most abundant, making up 64.66% (area under peak) of total volatiles extracted by hydro-distillation. The major extractable terpenoids in CGT were α-pinene (13.69–23.05 μg/g), β-caryophyllene (3.99–74.32 μg/g), α-humulene (2.00–25.71 μg/g), caryophyllene oxide (41.50–102.08 μg/g) and β-bisabolol (40.05–137.32 μg/g). Recoveries varied between different extraction methods. The terpenoids were found to be more abundant in the calyx (659.12 μg/g) and leaves (627.72 μg/g) than in stalks (112.97 μg/g) and stems (24.24 μg/g) of the cotton plant, indicating the possible biological origin of CGT volatiles. This study is the first to identify and quantify the different terpenoids present in CGT and significantly, β-bisabolol, an abundant compound (sesquiterpene alcohol) which may have valuable biological prospects. These findings therefore contribute to identifying alternative management strategies and uses of CGT. [ABSTRACT FROM AUTHOR]

Determination and classification of the bruise degree for cherry can improve consumer satisfaction with cherry quality and enhance the industry’s competiveness and profitability. In this study, visible and near infrared (Vis-NIR) reflection spectroscopy was used for identifying bruise degree of cherry in 350–2500 nm. Sampling spectral data were extracted from normal, slight and severe bruise samples. Principal component analysis (PCA) was implemented to determine the first few principal components (PCs) for cluster analysis among samples. Optimal wavelengths were selected by loadings of PCs from PCA and successive projection algorithm (SPA) method, respectively. Afterwards, these optimal wavelengths were empolyed to establish the classification models as inputs of least square-support vector machine (LS-SVM). Better performance for qualitative discrimination of the bruise degree for cherry was emerged in LS-SVM model based on five optimal wavelengths (603, 633, 679, 1083, and 1803 nm) selected directly by SPA, which showed acceptable results with the classification accuracy of 93.3%. Confusion matrix illustrated misclassification generally occurred in normal and slight bruise samples. Furthermore, the latent relation between spectral property of cherries in varying bruise degree and its firmness and soluble solids content (SSC) was analyzed. The result showed both colour, firmness and SSC were consistent with the Vis-NIR reflectance of cherries. Overall, this study revealed that Vis-NIR reflection spectroscopy integrated with multivariate analysis can be used as a rapid, intact method to determine the bruise degree of cherry, laying a foundation for cherry sorting and postharvest quality control. [ABSTRACT FROM AUTHOR]

To escape CD8+ T-cell immunity, human cytomegalovirus (HCMV) US11 redirects MHC-I for rapid ER-associated proteolytic degradation (ERAD). In humans, classical MHC-I molecules are encoded by the highly polymorphic HLA-A, -B and -C gene loci. While HLA-C resists US11 degradation, the specificity for HLA-A and HLA-B products has not been systematically studied. In this study we analyzed the MHC-I peptide ligands in HCMV-infected cells. A US11-dependent loss of HLA-A ligands was observed, but not of HLA-B. We revealed a general ability of HLA-B to assemble with β2m and exit from the ER in the presence of US11. Surprisingly, a low-complexity region between the signal peptide sequence and the Ig-like domain of US11, was necessary to form a stable interaction with assembled MHC-I and, moreover, this region was also responsible for changing the pool of HLA-B ligands. Our data suggest a two-pronged strategy by US11 to escape CD8+ T-cell immunity, firstly, by degrading HLA-A molecules, and secondly, by manipulating the HLA-B ligandome. [ABSTRACT FROM AUTHOR]

Congenital hyperinsulinism (CHI) is characterised by inappropriate insulin secretion causing profound hypoglycaemia and brain damage if inadequately controlled. Pancreatic tissue isolated from patients with diffuse CHI shows abnormal proliferation rates, the mechanisms of which are not fully resolved. Understanding cell proliferation in CHI may lead to new therapeutic options, alongside opportunities to manipulate β-cell mass in patients with diabetes. We aimed to generate cell-lines from CHI pancreatic tissue to provide in vitro model systems for research. Three pancreatic mesenchymal stem cell-lines (CHIpMSC1-3) were derived from patients with CHI disease variants: focal, atypical and diffuse. All CHIpMSC lines demonstrated increased proliferation compared with control adult-derived pMSCs. Cell cycle alterations including increased CDK1 levels and decreased p27Kip1 nuclear localisation were observed in CHIpMSCs when compared to control pMSCs. In conclusion, CHIpMSCs are a useful in vitro model to further understand the cell cycle alterations leading to increased islet cell proliferation in CHI. [ABSTRACT FROM AUTHOR]

Nucleotide-binding, leucine-rich repeat containing X1 (NLRX1) is a mitochondria-located innate immune sensor that inhibits major pro-inflammatory pathways such as type I interferon and nuclear factor-κB signaling. We generated a novel, spontaneous, and rapidly progressing mouse model of multiple sclerosis (MS) by crossing myelin-specific T-cell receptor (TCR) transgenic mice with Nlrx1−/− mice. About half of the resulting progeny developed spontaneous experimental autoimmune encephalomyelitis (spEAE), which was associated with severe demyelination and inflammation in the central nervous system (CNS). Using lymphocyte-deficient mice and a series of adoptive transfer experiments, we demonstrate that genetic susceptibility to EAE lies within the innate immune compartment. We show that NLRX1 inhibits the subclinical stages of microglial activation and prevents the generation of neurotoxic astrocytes that induce neuronal and oligodendrocyte death in vitro. Moreover, we discovered several mutations within NLRX1 that run in MS-affected families. In summary, our findings highlight the importance of NLRX1 in controlling the early stages of CNS inflammation and preventing the onset of spontaneous autoimmunity. [ABSTRACT FROM AUTHOR]

Cytomegalovirus (CMV) is a ubiquitous β-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections. [ABSTRACT FROM AUTHOR]

Ten Hyptis suaveolens hairy root lines were established by infecting nodal explants with K599+pGus-GFP+ and ATCC15834+pTDT strains from Agrobacterium rhizogenes. Genetic transformation was confirmed by epifluorescence and plagiotropic hairy root growth in absence of growth regulators. Cytotoxicity was determined using the sulforhodamine B method, and the production of podophyllotoxin (PTOX) was measured by high performance thin layer chromatography scanning. Through these methodologies, HsTD10 was identified as the hairy root line with the highest cytotoxicity and PTOX production, which was corroborated by liquid chromatography-mass spectrometry and micrOTOF-Q II. A suspension culture of HsTD10 was established in which PTOX and carbohydrate consumption during growth kinetics were quantified by high-performance liquid chromatography. Procedures to increase the production and retrieval of PTOX in the HsTD10 line included selection of culture medium, addition of thiamine, and modification of the PTOX extraction method. The best combination of these variables was MS medium at 75% of its components with the addition of 2 mg L-1 of thiamine, extraction with methanol-dichloromethane, and sonication at 40 ± 5°C. During kinetics, growth-associated PTOX accumulation was observed. The specific growth rate (μ) was 0.11 d-1. The highest concentration of PTOX obtained with HsTD10 (5.6 mg g-1 DW) was 100 times higher than that reported for roots of wild plants and 56 times higher than that for in vitro nontransformed roots of H. suaveolens. [ABSTRACT FROM AUTHOR]

The inactivation of antibiotic resistant Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive) seeded in greywater by bimetallic bio-nanoparticles was optimized by using response surface methodology (RSM). The bimetallic nanoparticles (Cu/Zn NPs) were synthesized in secondary metabolite of a novel fungal strain identified as Aspergillus iizukae EAN605 grown in pumpkin medium. Cu/Zn NPs were very effective for inhibiting growth of E. coli and S. aureus. The maximum inactivation was optimized with 0.028 mg mL-1 of Cu/Zn NPs, at pH 6 and after 60 min, at which the reduction of E. coli and S. aureus was 5.6 vs. 5.3 and 5.2 vs. 5.4 log reduction for actual and predicted values, respectively. The inactivation mechanism was described based on the analysis of untreated and treated bacterial cells by Field emission scanning electron microscopy (FESEM), Energy Dispersive X-Ray Spectroscopy (EDS), Atomic Force Microscopy (AFM) revealed a damage in the cell wall structure due to the effect of Cu/Zn NPs. Moreover, the Raman Spectroscopy showed that the Cu/Zn NPs led to degradation of carbohydrates and amino structures on the bacteria cell wall. The Fourier transform infrared spectroscopy (FTIR) analysis confirmed that the destruction take place in the C-C bond of the functional groups available in the bacterial cell wall. The techno economic analysis revealed that the biosynthesis Cu/Zn NPs is economically feasible. These findings demonstrated that Cu/Zn NPs can effectively inhibit pathogenic bacteria in the greywater. [ABSTRACT FROM AUTHOR]

Multi-marker tests hold promise for identifying symptomatic women at risk of imminent preterm delivery (PTD, <37 week’s gestation). This study sought to determine the relationship of inflammatory mediators and metabolites in cervicovaginal fluid (CVF) with spontaneous PTD (sPTD) and delivery within 14 days of presentation with symptoms of preterm labour (PTL). CVF samples from 94 (preterm = 19, term = 75) singleton women with symptoms of PTL studied between 19+0–36+6 weeks’ gestation were analysed for cytokines/chemokines by multiplexed bead-based immunoassay, while metabolites were quantified by enzyme-based spectrophotometry in a subset of 61 women (preterm = 16, term = 45). Prevalence of targeted vaginal bacterial species was determined for 70 women (preterm = 14, term = 66) by PCR. Overall, 10 women delivered within 14 days of sampling. Predictive capacities of individual biomarkers and cytokine-metabolite combinations for sPTD and delivery within 14 days of sampling were analysed by logistic regression models and area under the receiver operating characteristic curve. Fusobacterium sp., Mubiluncus mulieris and Mycoplasma hominis were detected in more preterm-delivered than term women (P<0.0001), while, M. curtisii was found in more term-delivered than preterm women (P<0.0001). RANTES (0.91, 0.65–1.0), IL-6 (0.79, 0.67–0.88), and Acetate/Glutamate ratio (0.74, 0.61–0.85) were associated with delivery within 14 days of sampling (AUC, 95% CI). There were significant correlations between cytokines and metabolites, and several cytokine-metabolite combinations were associated with sPTD or delivery within 14 days of sampling (e.g. L/D-lactate ratio+Acetate/Glutamate ratio+IL-6: 0.84, 0.67–0.94). Symptomatic women destined to deliver preterm and within 14 days of sampling express significantly higher pro-inflammatory mediators at mid to late gestation. In this cohort, IL-6, Acetate/Glutamate ratio and RANTES were associated with delivery within 14 days of sampling, consistent with their roles in modulating infection-inflammation-associated preterm labour in women presenting with symptoms of preterm birth. Replication of these observations in larger cohorts of women could show potential clinical utility. [ABSTRACT FROM AUTHOR]

Fluorescent cytometry refers to the quantification of cell physical properties and surface biomarkers using fluorescently-tagged antibodies. The generally preferred techniques to perform such measurements are flow cytometry, which performs rapid single cell analysis by flowing cells one-by-one through a channel, and microscopy, which eliminates the complexity of the flow channel, offering multi-cell analysis at a lesser throughput. Low-magnification image-based cytometers, also called “cell astronomy” systems, hold promise of simultaneously achieving both instrumental simplicity and high throughput. In this magnification regime, a single cell is mapped to a handful of pixels in the image. While very attractive, this idea has, so far, not been proven to yield quantitative results of cell-labeling, mainly due to the poor signal-to-noise ratio present in those images and to partial volume effects. In this work we present a cell astronomy system that, when coupled with custom-developed algorithms, is able to quantify cell intensities and diameters reliably. We showcase the system using calibrated MESF beads and fluorescently stained leukocytes, achieving good population identification in both cases. The main contribution of the proposed system is in the development of a novel algorithm, H-EM, that enables inter-cluster separation at a very low magnification regime (2x). Such algorithm provides more accurate brightness estimates than DAOSTORM when compared to manual analysis, while fitting cell location, brightness, diameter, and background level concurrently. The algorithm first performs Fisher discriminant analysis to detect bright spots. From each spot an expectation-maximization algorithm is initialized over a heterogeneous mixture model (H-EM), this algorithm recovers both the cell fluorescence and diameter with sub-pixel accuracy while discriminating the background noise. Finally, a recursive splitting procedure is applied to discern individual cells in cell clusters. [ABSTRACT FROM AUTHOR]

Decidua is a transient uterine tissue shared by mammals with hemochorial placenta and is essential for pregnancy. The decidua is infiltrated by many immune cells promoting pregnancy. Adult bone marrow (BM)-derived cells (BMDCs) differentiate into rare populations of nonhematopoietic endometrial cells in the uterus. However, whether adult BMDCs become nonhematopoietic decidual cells and contribute functionally to pregnancy is unknown. Here, we show that pregnancy mobilizes mesenchymal stem cells (MSCs) to the circulation and that pregnancy induces considerable adult BMDCs recruitment to decidua, where some differentiate into nonhematopoietic prolactin-expressing decidual cells. To explore the functional importance of nonhematopoietic BMDCs to pregnancy, we used Homeobox a11 (Hoxa11)-deficient mice, having endometrial stromal-specific defects precluding decidualization and successful pregnancy. Hoxa11 expression in BM is restricted to nonhematopoietic cells. BM transplant (BMT) from wild-type (WT) to Hoxa11−/− mice results in stromal expansion, gland formation, and marked decidualization otherwise absent in Hoxa11−/− mice. Moreover, in Hoxa11+/− mice, which have increased pregnancy losses, BMT from WT donors leads to normalized uterine expression of numerous decidualization-related genes and rescue of pregnancy loss. Collectively, these findings reveal that adult BMDCs have a previously unrecognized nonhematopoietic physiologic contribution to decidual stroma, thereby playing important roles in decidualization and pregnancy. [ABSTRACT FROM AUTHOR]

The development of effective malaria vaccines remains a global health priority. Currently, the most advanced vaccine, known as RTS,S, has only shown modest efficacy in clinical trials. Thus, the development of more efficacious vaccines by improving the formulation of RTS,S for increased efficacy or to interrupt malaria transmission are urgently needed. The RTS,S vaccine is based on the presentation of a fragment of the sporozoite antigen on the surface of virus-like particles (VLPs) based on human hepatitis B virus (HBV). In this study, we have developed and evaluated a novel VLP platform based on duck HBV (known as Metavax) for malaria vaccine development. This platform can incorporate large and complex proteins into VLPs and is produced in a Hansenula cell line compatible with cGMP vaccine production. Here, we have established the expression of leading P. falciparum malaria vaccine candidates as VLPs. This includes Pfs230 and Pfs25, which are candidate transmission-blocking vaccine antigens. We demonstrated that the VLPs effectively induce antibodies to malaria vaccine candidates with minimal induction of antibodies to the duck-HBV scaffold antigen. Antibodies to Pfs230 also recognised native protein on the surface of gametocytes, and antibodies to both Pfs230 and Pfs25 demonstrated transmission-reducing activity in standard membrane feeding assays. These results establish the potential utility of this VLP platform for malaria vaccines, which may be suitable for the development of multi-component vaccines that achieve high vaccine efficacy and transmission-blocking immunity. [ABSTRACT FROM AUTHOR]

In Argentina, NDM metallo-β-lactamase was first reported in 2013. By now, it has disseminated throughout the country in diverse Gram negative bacteria. Here, we report the case of a paediatric patient that underwent a 1-year hospitalisation due to erythrodermic psoriasis in 2014 and received multiple antimicrobial treatments. During his stay, five isolates were obtained from rectal swabs (rs) or blood culture (bc) suspicious of carbapenemase production: a K. quasipneumoniae subsp. quasipneumoniae (rs), Citrobacter freundii (rs), Escherichia coli (bc), Enterobacter cloacae (rs), and a Serratia marcescens (bc). The isolates were studied with broth microdilution, biparental conjugation and plasmid and whole genome sequencing (Illumina). All isolates harboured an 138,998-bp type 1 IncC plasmid that carried blaNDM-1, bleMBL, blaCMY-6, rmtC, aac(6’)-Ib, and sul1 resistance genes. Additionally, the blaNDM-plasmids contained ISKpn8 an insertion sequence previously described as associated only to blaKPC. One isolate, a colistin-resistant E. coli, also carried a mcr-1-containing an IncI2 plasmid, which did not harbour additional resistance. The whole genome of K. quasipneumoniae subsp. quasipneumoniae isolate was fully sequenced. This isolate harboured, additionally to blaNDM, three plasmid-mediated quinolone resistance genes: qnrB4, qnrB52 and aac(6’)-Ib-cr1. The E. cloacae isolate also harboured qnrA1. These findings alert to the underestimated horizontal dissemination of multidrug-resistant plasmids limiting treatment options with last resort antimicrobials. [ABSTRACT FROM AUTHOR]

Melanin is a natural pigment that is produced by filamentous fungi. In this study, the endophytic species, Spissiomyces endophytica (strain SDBR-CMU319), produced a brown-black pigment in the mycelia. Consequently, the pigment was extracted from the dried fungal biomass. This was followed by pigment purification, characterization and identification. Physical and chemical characteristics of the pigment showed acid precipitation, alkali solubilization, decolorization with oxidizing agents, and insolubility in most organic solvents and water. The pigment was confirmed as melanin based on ultraviolet-visible spectroscopy, Fourier-transform infrared, and electron paramagnetic resonance spectra analyses. The analyses of the elemental composition indicated that the pigment possessed a low percentage of nitrogen, and therefore, was not 3,4-dihydroxyphenylalanine melanin. Inhibition studies involving specific inhibitors, both tricyclazole and phthalide, and suggest that fungal melanin could be synthesized through the 1,8-dihydroxynaphthalene pathway. The optimum conditions for fungal pigment production from this species were investigated. The highest fungal pigment yield was observed in glucose yeast extract peptone medium at an initial pH value of 6.0 and at 25°C over three weeks of cultivation. This is the first report on the production and characterization of melanin obtained from the genus Spissiomyces. [ABSTRACT FROM AUTHOR]

Background: Identification of blood sources of hematophagous arthropods is crucial for understanding the transmission cycles of vector-borne diseases. Many different approaches towards host determination were proposed, including precipitin test, ELISA, DNA- and mass spectrometry-based methods; yet all face certain complications and limitations, mostly related to blood degradation. This study presents a novel method for blood meal identification, peptide mass mapping (PMM) analysis of host-specific hemoglobin peptides using MALDI-TOF mass spectrometry. Methodology/Principal findings: To identify blood meal source, proteins from abdomens of engorged sand fly females were extracted, cleaved by trypsin and peptide fragments of host hemoglobin were sequenced using MALDI-TOF MS. The method provided correct host identification of 100% experimentally fed sand flies until 36h post blood meal (PBM) and for 80% samples even 48h PBM. In females fed on two hosts, both blood meal sources were correctly assigned for 60% of specimens until 36h PBM. In a validation study on field-collected females, the method yielded unambiguous host determination for 96% of specimens. The suitability of PMM-based MALDI-TOF MS was proven experimentally also on lab-reared Culex mosquitoes. Conclusions/Significance: PMM-based MALDI-TOF MS analysis targeting host specific hemoglobin peptides represents a sensitive and cost-effective method with a fast and simple preparation protocol. As demonstrated here on phlebotomine sand flies and mosquitoes, it allows reliable and rapid blood source determination even 48h PBM with minimal material input and provides more robust and specific results than other currently used methods. This approach was also successfully tested on field-caught engorged females and proved to be a promising useful tool for large-scale screening of host preferences studies. Unlike other methods including MALDI-TOF protein profiling, it allows correct identification of mixed blood meals as was demonstrated on both experimentally fed and field-collected sand flies. [ABSTRACT FROM AUTHOR]

The role of CD5 as a regulator of T cell signaling and tolerance is well recognized. Recent data show expression of CD5 on different subtypes of human dendritic cells, however its functional relevance in modulating DC mediated responses remains poorly understood. In this study, we show CD5 is expressed on CD11c+ DC from murine thymus, lymph node, spleen, skin and lung. Although the development of DC subpopulations in CD5-/- mice was normal, CD5-deficient DC produced significantly higher levels of IL-12 than wild type DC in response to LPS. CD5-/- DC, in comparison to CD5+/+ DC, enhanced the activation of CD4+ and CD8+ T cells in vitro and in vivo and induced significantly higher production of IL-2 and IFN-gamma by T cells. Consequently, CD5-/- DC were significantly more potent than wild type DC in the induction of anti-tumor immunity and contact hypersensitivity responses in mice. Restoration of CD5 expression in CD5-/- DC reduced IL-12 production and inhibited their capacity to stimulate T cells. Collectively, these data demonstrate that the specific expression of CD5 on DC inhibits the production of inflammatory cytokines and has a regulatory effect on their activity to stimulate T cells and induce immune responses. This study reveals a previously unrecognized regulatory role for CD5 on DC and provides novel insights into mechanisms for DC biology in immune responses. [ABSTRACT FROM AUTHOR]

Eosinophils are the prominent inflammatory cell involved in allergic asthma, atopic dermatitis, eosinophilic esophagitis, and hypereosinophilic syndrome and are found in high numbers in local tissue and/or circulating blood of affected patients. There is recent interest in a family of alarmins, including TSLP, IL-25 and IL-33, that are epithelial-derived and released upon stimulation of epithelial cells. Several genome wide association studies have found SNPs in genes encoding IL-33 to be risk factors for asthma. In two studies examining the direct role of IL-33 in eosinophils, there were differences in eosinophil responses. We sought to further characterize activation of eosinophils with IL-33 compared to activation by other cytokines and chemokines. We assessed IL-33 stimulated adhesion, degranulation, chemotaxis and cell surface protein expression in comparison to IL-3, IL-5, and eotaxin-1 on human eosinophils. Our results demonstrate that IL-33 can produce as potent eosinophil activation as IL-3, IL-5 and eotaxin-1. Thus, when considering specific cytokine targeting strategies, IL-33 will be important to consider for modulating eosinophil function. [ABSTRACT FROM AUTHOR]

The protein factor Glomulin (Glmn) is a regulator of the SCF (Skp1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex. Mutations of Glmn lead to glomuvenous malformations. Glmn has been reported to be associated with FK506-binding proteins (FKBP). Here we present in vitro binding analyses of the FKBP—Glmn interaction. Interestingly, the previously described interaction of Glmn and FKBP12 was found to be comparatively weak. Instead, the closely related FKBP12.6 and FKBP51 emerged as novel binding partners. We show different binding affinities of full length and truncated FKBP51 and FKBP52 mutants. Using FKBP51 as a model system, we show that two amino acids lining the FK506-binding site are essential for binding Glmn and that the FKBP51-Glmn interaction is blocked by FKBP ligands. This data suggest FKBP inhibition as a pharmacological approach to regulate Glmn and Glmn-controlled processes. [ABSTRACT FROM AUTHOR]

Vinculin (Vcn) is a ubiquitously expressed cytoskeletal protein that links transmembrane receptors to actin filaments, and plays a key role in regulating cell adhesion, motility, and force transmission. Metavinculin (MVcn) is a Vcn splice isoform that contains an additional exon encoding a 68-residue insert within the actin binding tail domain. MVcn is selectively expressed at sub-stoichiometic amounts relative to Vcn in smooth and cardiac muscle cells. Mutations in the MVcn insert are linked to various cardiomyopathies. In vitro analysis has previously shown that while both proteins can engage filamentous (F)-actin, only Vcn can promote F-actin bundling. Moreover, we and others have shown that MVcn can negatively regulate Vcn-mediated F-actin bundling in vitro. To investigate functional differences between MVcn and Vcn, we stably expressed either Vcn or MVcn in Vcn-null mouse embryonic fibroblasts. While both MVcn and Vcn were observed at FAs, MVcn-expressing cells had larger but fewer focal adhesions per cell compared to Vcn-expressing cells. MVcn-expressing cells migrated faster and exhibited greater persistence compared to Vcn-expressing cells, even though Vcn-containing FAs assembled and disassembled faster. Magnetic tweezer measurements on Vcn-expressing cells show a typical cell stiffening phenotype in response to externally applied force; however, this was absent in Vcn-null and MVcn-expressing cells. Our findings that MVcn expression leads to larger but fewer FAs per cell, in conjunction with the inability of MVcn to bundle F-actin in vitro and rescue the cell stiffening response, are consistent with our previous findings of actin bundling deficient Vcn variants, suggesting that deficient actin-bundling may account for some of the differences between Vcn and MVcn. [ABSTRACT FROM AUTHOR]

The dissemination of resistant pathogenic microbes has become one of the most challenging problems that modern medicine has faced. Developing novel drugs based on new molecular targets that previously were not targeted, is therefore the highest priority in antibiotics research. One approach that has been recently suggested is to inhibit copper transporters in prokaryotic systems. Copper is required for many biological pathways, but sometimes it can harm the cell. Pathogenic systems have a highly sophisticated copper-regulation network; therefore, a better understanding of how this network operates at the molecular level should assist in developing the next generation of antibiotics. The CusB protein is part of the CusCBA periplasmic Cu(I) efflux system in Gram-negative bacteria, and was recently reported to play a key role in the functioning of the whole CusCBA system, in which conformational changes as well as the assembly/disassembly process control the opening of the transporter. More knowledge of the underlying mechanism is needed to attain a full understanding of CusB functioning, which is associated with targeting specific and crucial residues in CusB. Here, we combine in-vitro structural measurements, which use EPR spectroscopy and UV-Vis measurements, with cell experiments to explore the role of the various methionine residues in CusB. We targeted two methionine residues (M227 and M241) that are essential for the proper functioning of CusB. [ABSTRACT FROM AUTHOR]

Exosomes represent a promising delivery tool for nucleic acid-based pharmaceuticals. They are highly suitable for transporting therapeutic miRNAs to tumor cells, due to their natural membrane components. Further, exosomes are capable of effectively protecting nucleic acids against ribonucleases and enable the delivery of their content through cell membranes. However, no suitable production host for miRNA containing exosomes of non-tumorigenic origin has yet been identified. In this study we engineered an immortalised human amniocyte cell line (CAP® cells), whose exosomes were enriched and characterised. The cell line modifications not only enabled the production of GFP-labelled but also pro-apoptotic miRNA containing exosomes without negative influence on host cell growth. Furthermore, we demonstrated that pro-apoptotic miRNA containing CAP exosomes are taken up by ovarian cancer cells. Strikingly, delivery of functional exosomal miRNA led to downregulation of several reported target genes in the treated tumor cells. In summary, we revealed CAP cells of non-tumorigenic origin as a novel and efficient exosome production host with the potential to produce functional miRNA-loaded exosomes. [ABSTRACT FROM AUTHOR]

IL-1β is a potent pro-inflammatory cytokine that promotes immunity and host defense, and its dysregulation is associated with immune pathology. Toxoplasma gondii infection of myeloid cells triggers the production and release of IL-1β; however, the mechanisms regulating this pathway, particularly in human immune cells, are incompletely understood. We have identified a novel pathway of T. gondii induction of IL-1β via a Syk-CARD9-NF-κB signaling axis in primary human peripheral blood monocytes. Syk was rapidly phosphorylated during T. gondii infection of primary monocytes, and inhibiting Syk with the pharmacological inhibitors R406 or entospletinib, or genetic ablation of Syk in THP-1 cells, reduced IL-1β release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced IL-1β transcripts and the production of pro-IL-1β. Furthermore, inhibition of PKCδ, CARD9/MALT-1 and IKK reduced p65 phosphorylation and pro-IL-1β production in T. gondii-infected primary monocytes, and genetic knockout of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1β protein levels and IL-1β release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1β transcriptional activation. IL-1β release from T. gondii-infected primary human monocytes required the NLRP3-caspase-1 inflammasome, but interestingly, was independent of gasdermin D (GSDMD) cleavage and pyroptosis. Moreover, GSDMD knockout THP-1 cells released comparable amounts of IL-1β to wild-type THP-1 cells after T. gondii infection. Taken together, our data indicate that T. gondii induces a Syk-CARD9/MALT-1-NF-κB signaling pathway and activation of the NLRP3 inflammasome for the release of IL-1β in a cell death- and GSDMD-independent manner. This research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection. [ABSTRACT FROM AUTHOR]

Understanding intrinsic and acquired resistance is crucial to overcoming cancer chemotherapy failure. While it is well-established that intratumor, subclonal genetic and phenotypic heterogeneity significantly contribute to resistance, it is not fully understood how tumor sub-clones interact with each other to withstand therapy pressure. Here, we report a previously unrecognized behavior in heterogeneous tumors: cooperative adaptation to therapy (CAT), in which cancer cells induce co-resistant phenotypes in neighboring cancer cells when exposed to cancer therapy. Using a CRISPR/Cas9 toolkit we engineered phenotypically diverse non-small cell lung cancer (NSCLC) cells by conferring mutations in Dicer1, a type III cytoplasmic endoribonuclease involved in small non-coding RNA genesis. We monitored three-dimensional growth dynamics of fluorescently-labeled mutant and/or wild-type cells individually or in co-culture using a substrate-free NanoCulture system under unstimulated or drug pressure conditions. By integrating mathematical modeling with flow cytometry, we characterized the growth patterns of mono- and co-cultures using a mathematical model of intra- and interspecies competition. Leveraging the flow cytometry data, we estimated the model’s parameters to reveal that the combination of WT and mutants in co-cultures allowed for beneficial growth in previously drug sensitive cells despite drug pressure via induction of cell state transitions described by a cooperative game theoretic change in the fitness values. Finally, we used an ex vivo human tumor model that predicts clinical response through drug sensitivity analyses and determined that cellular and morphologic heterogeneity correlates to prognostic failure of multiple clinically-approved and off-label drugs in individual NSCLC patient samples. Together, these findings present a new paradox in drug resistance implicating non-genetic cooperation among tumor cells to thwart drug pressure, suggesting that profiling for druggable targets (i.e. mutations) alone may be insufficient to assign effective therapy. [ABSTRACT FROM AUTHOR]

Expressed strongly by myeloid cells, damage-associated molecular pattern (DAMP) proteins S100A8 and S100A9 are found in the serum of patients with infectious and autoimmune diseases. Compared to S100A9, the role of S100A8 is controversial. We investigated its biological activity in collagen-induced arthritis using the first known viable and fertile S100a8-deficient (S100a8-/-) mouse. Although comparable to the wild type (WT) in terms of lymphocyte distribution in blood and in the primary and secondary lymphoid organs, S100a8-/- mice had increased numbers of neutrophils, monocytes and dendritic cells in the blood and bone marrow, and these all expressed myeloid markers such as CD11b, Ly6G and CD86 more strongly. Granulocyte-macrophage common precursors were increased in S100a8-/- bone marrow and yielded greater numbers of macrophages and dendritic cells in culture. The animals also developed more severe arthritic disease leading to aggravated osteoclast activity and bone destruction. These findings were correlated with increased inflammatory cell infiltration and cytokine secretion in the paws. This study suggests that S100A8 is an anti-inflammatory DAMP that regulates myeloid cell differentiation, thereby mitigating the development of experimental arthritis. [ABSTRACT FROM AUTHOR]