Staphylococcus aureus is a major human pathogen causing multiple pathologies, from cutaneous lesions to life-threatening sepsis. Although neutrophils contribute to immunity against S. aureus, multiple lines of evidence suggest that these phagocytes can provide an intracellular niche for staphylococcal dissemination. However, the mechanism of neutrophil subversion by intracellular S. aureus remains unknown. Targeting of intracellular pathogens by macroautophagy/autophagy is recognized as an important component of host innate immunity, but whether autophagy is beneficial or detrimental to S. aureus-infected hosts remains controversial. Here, using larval zebrafish, we showed that the autophagy marker Lc3 rapidly decorates S. aureus following engulfment by macrophages and neutrophils. Upon phagocytosis by neutrophils, Lc3-positive, non-acidified spacious phagosomes are formed. This response is dependent on phagocyte NADPH oxidase as both cyba/p22phox knockdown and diphenyleneiodonium (DPI) treatment inhibited Lc3 decoration of phagosomes. Importantly, NADPH oxidase inhibition diverted neutrophil S. aureus processing into tight acidified vesicles, which resulted in increased host resistance to the infection. Some intracellular bacteria within neutrophils were also tagged by Sqstm1/p62-GFP fusion protein and loss of Sqstm1 impaired host defense. Together, we have shown that intracellular handling of S. aureus by neutrophils is best explained by Lc3-associated phagocytosis (LAP), which appears to provide an intracellular niche for bacterial pathogenesis, while the selective autophagy receptor Sqstm1 is host-protective. The antagonistic roles of LAP and Sqstm1-mediated pathways in S. aureus-infected neutrophils may explain the conflicting reports relating to anti-staphylococcal autophagy and provide new insights for therapeutic strategies against antimicrobial-resistant Staphylococci.Abbreviations: ATG: autophagy related; CFU: colony-forming units; CMV: cytomegalovirus; Cyba/P22phox: cytochrome b-245, alpha polypeptide; DMSO: dimethyl sulfoxide; DPI: diphenyleneiodonium; EGFP: enhanced green fluorescent protein; GFP: green fluorescent protein; hpf: hours post-fertilization; hpi: hours post-infection; Irf8: interferon regulatory factor 8; LAP: LC3-associated phagocytosis; lyz: lysozyme; LWT: london wild type; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NADPH oxidase: nicotinamide adenine dinucleotide phosphate oxidase; RFP: red fluorescent protein; ROS: reactive oxygen species; RT-PCR: reverse transcriptase polymerase chain reaction; Sqstm1/p62: sequestosome 1; Tg: transgenic; TSA: tyramide signal amplification.

We cloned zebrafish runx3/aml2/cbfa3 and examined its expression and function during embryogenesis. In the developing embryo, runx3 is dynamically expressed in hematopoietic, neuronal, and cartilaginous tissues. Hematopoietic expression of runx3 commences late in embryogenesis in the ventral tail intermediate cell mass and later colocalizes with spi1 and lyz in circulating blood cells. In the cloche mutant, hematopoietic expression was absent, suggesting that Runx3 functions downstream of cloche in a hematopoietic pathway. Neuronal tissues expressing runx3 include the trigeminal ganglia and Rohon-Beard neurons. Runx3 appears to contribute to normal development of primitive and definitive hematopoietic cells. When Runx3 function was compromised using morpholino oligonucleotides, a reduction in the number of mature blood cells was observed. Furthermore, Runx3 depletion decreased runx1 expression in the ventral wall of the dorsal aorta and reduced the number of spi1- and lyz-containing blood cells. Conversely, ubiquitous overexpression of runx3 led to an increase in primitive blood cell numbers, together with an increase in runx1-expressing cells in the ventral wall of the dorsal aorta. We propose a role for Runx3 in the regulation of blood cell numbers.

This work examines physico-chemical properties influencing protein adsorption to anionic PLG microparticles and demonstrates the ability to bind and release vaccine antigens over a range of loads, pH values, and ionic strengths. Poly(lactide-co-glycolide) microparticles were synthesized by a w/o/w emulsification method in the presence of the anionic surfactant DSS (dioctyl sodium sulfosuccinate). Ovalbumin (OVA), carbonic anhydrase (CAN), lysozyme (LYZ), lactic acid dehydrogenase, bovine serum albumin (BSA), an HIV envelope glyocoprotein, and a Neisseria meningitidis B protein were adsorbed to the PLG microparticles, with binding efficiency, initial release and zeta potentials measured. Protein (antigen) binding to PLG microparticles was influenced by both electrostatic interaction and other mechanisms such as van der Waals forces. The protein binding capacity was directly proportional to the available surface area and may have a practical upper limit imposed by the formation of a complete protein monolayer as suggested by AFM images. The protein affinity for the PLG surface depended strongly on the isoelectric point (pI) and electrostatic forces, but also showed contributions from nonCoulombic interactions. Protein antigens were adsorbed on anionic PLG microparticles with varying degrees of efficiency under different conditions such as pH and ionic strength. Observable changes in zeta potentials and morphology suggest the formation of a surface monolayer. Antigen binding and release occur through a combination of electrostatic and van der Waals interactions occurring at the polymer-solution interface.

The preventive effect of Thea sinensis melanin (TSM) against cisplatin-induced nephrotoxicity was studied on ICR mice. Animals were given 20mg/kg i.p. of cisplatin, and TSM was injected i.p. in doses 10-40 mg/kg 2h before intoxication. The protective effects were evidenced by a complete inhibition of the cisplatin-induced elevation of serum Blood Urea nitrogen (BUN), prevention of oxidative stress, and complete blockade of cisplatin-induced elevation of serum creatinine. TSM by itself, however, did not affect the renal functional parameters, including serum BUN and creatinine. Real-time RT-PCR was applied to quantify mRNA levels of cisplatin-treated mouse kidney compared to normal mouse kidney for selected marker genes. Cisplatin treatment increases mRNA levels 40-fold for glutathione-S-transferases (Gstp2), 15-fold for soluble epoxide hydrolase (Ephx1), 15-fold for lipocalin 2 (Lcn2), 9-fold for lysozyme (Lyz), 5-fold for UDP glycosyltransferase 2 (Utg2b), 30-fold for survival motor neuron (Smn1), 30-fold for guanidinoacetate methyltransferase (Gamt), 80-fold for urine retinol binding protein (Rbp4), 60-fold for aminopeptidase N (Apn), 60-fold for cytochrome P450 (Cyp2d18), and 100-fold for ornithine aminotransferase (Oat). Pre-administration of TSM restored normal expression of marker genes for cisplatin-treated mouse kidneys. TSM by itself, however, did not affect the transcription for marker genes. Results obtained demonstrate that TSM pre-administration can prevent the renal toxic effects of cisplatin.

Despite the common use of immunohistochemistry in autopsy tissues, the stability of most proteins over extended time periods is unknown. The robustness of signal for 16 proteins (MMP1, MMP2, MMP3, MMP9, TIMP1, TIMP2, TIMP3, AGER, MSR, SCARB1, OLR1, CD36, LTF, LGALS3, LYZ, and DDOST) and two measures of advanced glycation end products (AGE, CML) was evaluated. Two formalin-fixed, paraffin-embedded human tissue arrays containing 16 tissues each were created to evaluate 48 hr of autolysis in a warm or cold environment. For these classes of proteins, matrix metalloproteinases and their inhibitors, scavenger receptors, and advanced glycation end product receptors, we saw no systematic diminution of signal intensity during a period of 24 hr. Analysis was performed by two independent observers and confirmed for a subset of proteins by digital analysis and Western blotting. We conclude that these classes of proteins degrade slowly and faithfully maintain their immunohistochemistry characteristics over at least a 24-hr time interval in devitalized tissues. This study supports the use of autopsy tissues with short postmortem intervals for immunohistochemical studies for diseases such as diabetic vascular disease, cancer, Alzheimer's disease, atherosclerosis, and other pathological states. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Many applications of nanobiomaterials rely on or are enhanced by specific, protein-mediated interactions with biological systems. These interactions can be engineered by chemically modifying the surface of the material to affect protein adsorption, or by altering the topography of the nanoscale surface. The covalent attachment or adsorption of proteins onto materials can greatly affect their structure and function, giving rise to either beneficial effects or to unpredictable and potentially undesirable effects. Thus, it is essential to develop a detailed understanding of how nanostructured surface characteristics, such as atomic-scale topography, surface energy, and chemical structure may affect protein adsorption, structure, function, and stability. Herein we observe that nanoparticle morphology and protein surface coverage affect the structure, activity, and stability of adsorbed lysozyme (Lyz) and α-chymotrypsin (ChT) in a manner that is protein specific. Wet chemical methods were used to synthesize gold nanocubes (AuNC) with {100} facets and gold nanooctahedra (AuNO) with {111} facets. Differences in adsorption on AuNC and AuNO are observed, which may be attributed to the atomic topography of the material. Nanoparticles, as well as the final form of the resulting protein conjugates, were thoroughly characterized through various physical, microscopic, and spectroscopic techniques. As a result, additional insight into the influence of nanoscale surface properties was obtained, which will enhance our fundamental understanding of how such properties affect protein structure and function, and will hence assist us in strategically engineering protein-nanomaterial conjugates for a variety of biomedical applications.

The immobilization of protein molecules on self-assembled monolayers (SAM) by physical interactions and chemical bonding has been studied using atomic force microscopy (AFM). The proteins used for our investigation are bovine serum albumin (BSA), lysozyme (LYZ), and normal rabbit immunoglobulin G (IgG). The surfaces are methyl-, hydroxyl-, carboxylic acid- and aldehyde-terminated SAMs. We found that BSA and LYZ can be readily immobilized on SAMs at their isoelectric point (IEP). The detailed surface morphology of adsorbed proteins varies with the functionality of the SAMs. The strong hydrophobic interaction at the IEP is attributed to immobilization. If the solution pH is deviated from the IEP, proteins may be attached onto the surface via electrostatic interactions. Covalent binding between the aldehyde-terminated SAM and the H2N-groups in the protein results in immobilization of all three proteins. The individual proteins and their orientations on SAMs are clearly resolved from high-resolution AFM images. The stability and bioactivity of these immobilized proteins are also studied.

In the present study, we analyzed the oxidative stress related indices and immune related gene expression of zebrafish embryos after a short-term exposure to various concentrations of di-n-butyl phthalate (DBP), diethyl phthalate (DEP) and their mixture (DBP-DEP) from 4h post-fertilization (hpf) to 96hpf. Exposure to the chemicals was found to enhance the production of reactive oxygen species (ROS) and lipid peroxidation (LPO) in a concentration-dependent manner. Simultaneously, adaptive responses to DBP/DEP-induced oxidative stress were observed. The activity of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were all increased in a concentration-dependent manner. The transcription of innate immune related genes including interferon γ (IFNγ), interleukin-1β (IL1β), Myxovirus resistance (Mx), tumor necrosis factor α (TNFα), CC-chemokine, CXCL-clc, lysozyme (Lyz) and complement factor C3B (C3) were up-regulated upon DBP, DEP and their mixture exposure, suggesting the induction of immune response. In addition, co-exposure to DBP-DEP also induced antioxidant defense and immune response in zebrafish embryo. The results demonstrat that DBP/DEP exposure could induce the antioxidant and immune responses in zebrafish embryos.

The function of Toll pathway defense against bacterial infection has been well established in shrimp, however how this pathway responds to viral infection is still largely unknown. In this study, we report the Toll4-Dorsal-AMPs cascade restricts the white spot syndrome virus (WSSV) infection of shrimp. A total of nine Tolls from Litopenaeus vannamei namely Toll1-9 are identified, and RNAi screening in vivo reveals the Toll4 is important for shrimp to oppose WSSV infection. Knockdown of Toll4 results in elevated viral loads and renders shrimp more susceptible to WSSV. Furthermore, Toll4 could be a one of upstream pattern recognition receptor (PRR) to detect WSSV, and thereby leading to nuclear translocation and phosphorylation of Dorsal, the known NF-κB transcription factor of the canonical Toll pathway. More importantly, silencing of Toll4 and Dorsal contributes to impaired expression of a specific set of antimicrobial peptides (AMPs) such as anti-LPS-factor (ALF) and lysozyme (<em>LYZ</em>) family, which exert potent anti-WSSV activity. Two AMPs of ALF1 and <em>LYZ</em>1 as representatives are demonstrated to have the ability to interact with several WSSV structural proteins to inhibit viral infection. Taken together, we therefore identify that the Toll4-Dorsal pathway mediates strong resistance to WSSV infection by inducing some specific AMPs.

A drug delivery system was developed by combining composite scaffolds made up of collagen and hydroxyapatite (Col/HA) with bisphosphonate (BP)-derivatized liposomes. The Col/HA scaffold was prepared by a freeze-drying method to yield a porous scaffold. The liposomes were composed of distearoylphosphocholine, cholesterol, distearoylphosphoethanolamine-poly(ethylene glycol) (DSPE-PEG), and a bone-binding bisphosphonate (BP) attached to the DSPE-PEG (DSPE-PEG-BP). By taking advantage of the specific interaction between the liposomal BP and the HA incorporated into the scaffold, the BP-decorated liposomes (BP-liposomes) were shown to display a strong affinity to Col/HA scaffolds. Three different model drugs, carboxyfluorescein (CF), doxorubicin (DOX), and lysozyme (LYZ) were entrapped in liposomes; there were no differences in drug release from the liposomes whether the liposomes were BP decorated or not. Whereas unencapsulated drugs and drugs encapsulated in PEG-liposomes displayed rapid release from the scaffolds, the drugs entrapped in BP-liposomes showed a slower release from the Col/HA scaffolds. We conclude that the proposed system can prolong the in situ residence of model drugs and has the potential to provide a sustained drug release platform in bone regeneration and repair.

Novel biomarker verification assays are urgently required to improve the efficiency of biomarker development. Benefitting from lower development costs, multiple reaction monitoring (MRM) has been used for biomarker verification as an alternative to immunoassay. However, in general MRM analysis, only one sample can be quantified in a single experiment, which restricts its application. Here, a Hyperplex-MRM quantification approach, which combined mTRAQ for absolute quantification and iTRAQ for relative quantification, was developed to increase the throughput of biomarker verification. In this strategy, equal amounts of internal standard peptides were labeled with mTRAQ reagents Δ0 and Δ8, respectively, as double references, while 4-plex iTRAQ reagents were used to label four different samples as an alternative to mTRAQ Δ4. From the MRM trace and MS/MS spectrum, total amounts and relative ratios of target proteins/peptides of four samples could be acquired simultaneously. Accordingly, absolute amounts of target proteins/peptides in four different samples could be achieved in a single run. In addition, double references were used to increase the reliability of the quantification results. Using this approach, three biomarker candidates, ademosylhomocysteinase (AHCY), cathepsin D (CTSD), and lysozyme C (LYZ), were successfully quantified in colorectal cancer (CRC) tissue specimens of different stages with high accuracy, sensitivity, and reproducibility. To summarize, we demonstrated a promising quantification method for high-throughput verification of biomarker candidates.

Langerhans cell histiocytosis (LCH) is a neoplastic disorder that results in clonal proliferation of cells with a Langerhans cell (LC) phenotype. The pathogenesis of LCH is still poorly understood. In the present study, serial analysis of gene expression (SAGE) was applied to LCs generated from umbilical cord blood CD34+ progenitor cells to identify LC-specific genes and the expression of these genes in LCH was investigated. Besides the expression of several genes known to be highly expressed in LCs and LCH such as CD1a, LYZ, and CD207, high expression of genes not previously reported to be expressed in LCs, such as GSN, MMP12, CCL17, and CCL22, was also identified. Further analysis of these genes by quantitative RT-PCR revealed high expression of FSCN1 and GSN in all 12 LCH cases analysed; of CD207, MMP12, CCL22, and CD1a in the majority of these cases; and CCL17 in three of the 12 cases. Immunohistochemistry confirmed protein expression in the majority of cases. The expression of MMP12 was most abundant in multi-system LCH, which is the LCH type with the worst prognosis. This suggests that expression of MMP12 may play a role in the progression of LCH. These data reveal new insight into the pathology of LCH and provide new starting points for further investigation of this clonal proliferative disorder.

OBJECTIVE

This study aimed to identify rheumatoid arthritis (RA) related genes based on microarray data using the WGCNA (weighted gene co-expression network analysis) method.

METHODS

Two gene expression profile datasets GSE55235 (10 RA samples and 10 healthy controls) and GSE77298 (16 RA samples and seven healthy controls) were downloaded from Gene Expression Omnibus database. Characteristic genes were identified using metaDE package. WGCNA was used to find disease-related networks based on gene expression correlation coefficients, and module significance was defined as the average gene significance of all genes used to assess the correlation between the module and RA status. Genes in the disease-related gene co-expression network were subject to functional annotation and pathway enrichment analysis using Database for Annotation Visualization and Integrated Discovery. Characteristic genes were also mapped to the Connectivity Map to screen small molecules.

RESULTS

A total of 599 characteristic genes were identified. For each dataset, characteristic genes in the green, red and turquoise modules were most closely associated with RA, with gene numbers of 54, 43 and 79, respectively. These genes were enriched in totally enriched in 17 Gene Ontology terms, mainly related to immune response (CD97, FYB, CXCL1, IKBKE, CCR1, etc.), inflammatory response (CD97, CXCL1, C3AR1, CCR1, LYZ, etc.) and homeostasis (C3AR1, CCR1, PLN, CCL19, PPT1, etc.). Two small-molecule drugs sanguinarine and papaverine were predicted to have a therapeutic effect against RA.

CONCLUSIONS

Genes related to immune response, inflammatory response and homeostasis presumably have critical roles in RA pathogenesis. Sanguinarine and papaverine have a potential therapeutic effect against RA.

OBJECTIVE

Current culture schema for human intestinal stem cells (hISCs) frequently rely on a 3D culture system using Matrigel™, a laminin-rich matrix derived from murine sarcoma that is not suitable for clinical use. We have developed a novel 2D culture system for the in vitro expansion of hISCs as an intestinal epithelial monolayer without the use of Matrigel.

METHODS

Cadaveric duodenal samples were processed to isolate intestinal crypts from the mucosa. Crypts were cultured on a thin coat of type I collagen or laminin. Intestinal epithelial monolayers were supported with growth factors to promote self-renewal or differentiation of the hISCs. Proliferating monolayers were sub-cultured every 4-5days.

RESULTS

Intestinal epithelial monolayers were capable of long-term cell renewal. Less differentiated monolayers expressed high levels of gene marker LGR5, while more differentiated monolayers had higher expressions of CDX2, MUC2, LYZ, DEF5, and CHGA. Furthermore, monolayers were capable of passaging into a 3D culture system to generate spheroids and enteroids.

CONCLUSIONS

This 2D system is an important step to expand hISCs for further experimental studies and for clinical cell transplantation.

METHODS

1 Experimental.

In the present study zebrafish (Danio rerio) has been used as model organism to establish the effects of dietary supplementation of Gracilaria gracilis powder (GP) on mucosal and innate immune parameters, antioxidant enzymes, and growth. In order to establish these features, zebrafish were fed for eight weeks with experimental diets containing different levels of Red algae, 0.25, 0.5 and 1% of GP; also, a group was fed with control diet. At the end of the experimental period the antioxidant superoxide dismutase and catalase (SOD, CAT) genes expression, interleukin 1 beta (il-1β), lysozyme (LYZ), tumor necrosis factor alpha (TNF-α) for immune-related genes expression, total immunoglobulin (Ig), total protein, alkaline phosphatase (ALP) activity for innate immune parameters, and growth performance have been established. The GP dietary supplementation showed differences in SOD and CAT expression in zebrafish whole body respect to the control group. Non-signifcant differences were noticed among the different groups in case of TNF-α, LYZ and il-1expression (P>> 0.05). The skin mucus total Ig and total protein in the group fed on 1% of GP were significantly higher respect to control group (P < 0.05). 0.25 and 0.5% of GP dietary supplementation significantly enhanced skin mucus ALP activity levels (P < 0.05). No significant differences were recorded for growth performances among groups (P>> 0.05). The results obtained in the present study revealed that G. gracilis could be takes in account as fishes diet supplementation for its immune system stimulants effects.

The insulin receptor (IR) regulates nutrient uptake and utilization in multiple organs, but its role in the intestinal epithelium is not defined. This study developed a mouse model with villin-Cre (VC) recombinase-mediated intestinal epithelial cell (IEC)-specific IR deletion (VC-IR(Δ/Δ)) and littermate controls with floxed, but intact, IR (IR(fl/fl)) to define in vivo roles of IEC-IR in mice fed chow or high-fat diet (HFD). We hypothesized that loss of IEC-IR would alter intestinal growth, biomarkers of intestinal epithelial stem cells (IESC) or other lineages, body weight, adiposity, and glucose or lipid handling. In lean, chow-fed mice, IEC-IR deletion did not affect body or fat mass, plasma glucose, or IEC proliferation. In chow-fed VC-IR(Δ/Δ) mice, mRNA levels of the Paneth cell marker lysozyme (Lyz) were decreased, but markers of other differentiated lineages were unchanged. During HFD-induced obesity, IR(fl/fl) and VC-IR(Δ/Δ) mice exhibited similar increases in body and fat mass, plasma insulin, mRNAs encoding several lipid-handling proteins, a decrease in Paneth cell number, and impaired glucose tolerance. In IR(fl/fl) mice, HFD-induced obesity increased circulating cholesterol; numbers of chromogranin A (CHGA)-positive enteroendocrine cells (EEC); and mRNAs encoding Chga, glucose-dependent insulinotrophic peptide (Gip), glucagon (Gcg), Lyz, IESC biomarkers, and the enterocyte cholesterol transporter Scarb1. All these effects were attenuated or lost in VC-IR(Δ/Δ) mice. These results demonstrate that IEC-IR is not required for normal growth of the intestinal epithelium in lean adult mice. However, our findings provide novel evidence that, during HFD-induced obesity, IEC-IR contributes to increases in EEC, plasma cholesterol, and increased expression of Scarb1 or IESC-, EEC-, and Paneth cell-derived mRNAs.

Rickettsiae can cause life-threatening infections in humans. Macrophages are one of the initial targets for rickettsiae after inoculation by ticks. However, it remains poorly understood how rickettsiae remain free in macrophages prior to establishing their infection in microvascular endothelial cells. Here, we demonstrated that the concentration of Rickettsia australis was significantly greater in infected tissues of Atg5flox/flox mice compared to the counterparts of Atg5flox/flox Lyz-Cre mice, in association with a reduced level of IL-1β in serum. The greater concentration of R. australis in Atg5 flox/flox bone marrow-derived macrophages (BMMs) compared to Atg5 flox/flox Lyz-Cre BMMs in vitro was abolished by exogenous treatment with recombinant IL-1β. Rickettsia australis induced significantly increased levels of LC3-II and LC3 puncta in Atg5-competent BMMs, but not in Atg5- deficient BMMs, while no p62 turnover was observed. Further analysis found that the co-localization of LC3 with a small portion of R. australis and Rickettsia-containing double-membrane-bound vacuoles in BMMs of B6 mice. Moreover, treatment with rapamycin significantly increased the concentrations of R. australis in B6 BMMs compared to the untreated controls. Taken together, our results demonstrated that Atg5 favors R. australis infection in mouse macrophages in association with a suppressed production level of IL-1β but not active autophagy flux. These data highlight the contribution of Atg5 in macrophages to the pathogenesis of rickettsial diseases.

Four genes, VTN, KERA, LYZ, and a non-annotated EST (Affymetrix probe set ID: Ssc.25503.1.S1_at), whose candidacy for traits related to water-holding capacity of meat arises from their trait-dependent differential expression, were selected for candidate gene analysis. Based on in silico analysis SNPs were detected, confirmed by sequencing and used to genotype animals of 4 pig populations including 3 commercial herds of Pietrain (PI), Pietrain x (German Large White x German Landrace) (PIF1), German Landrace (DL) and 1 experimental F2 population Duroc x Pietrain (DUPI). Comparative and genetic mapping established the location of VTN on SSC12, of LYZ and KERA on SSC5 and of UN on SSC7, coinciding with QTL regions for meat quality traits. VTN showed association with pH1, pH24 and drip loss. LYZ revealed association with conductivity 24, pH1 and drip loss. KERA was associated with pH. UN showed association with pH24 and drip loss, respectively. However, none of the candidate genes showed significant associations for a particular trait across all populations. This may be due to breed specific effects that are related to the differences in meat quality of theses pig breeds. The studies revealed statistic evidence for a link of genetic variation at these loci or close to them and promoted those four candidate genes as functional and/or positional candidate genes for meat quality traits.

Antimicrobial peptides (AMPs) have a crucial role in the fish innate immune response, being considered a fundamental component of the first line of defence against pathogens. Moreover, AMPs have not been studied in the fish gonad since this is used by some pathogens as a vehicle or a reservoir to be transmitted to the progeny, as occurs with nodavirus (VNNV), which shows vertical transmission through the gonad and/or gonadal fluids, but no study has looked into the gonad of infected fish. In this framework, we have characterized the antimicrobial response triggered by VNNV in the testis of European sea bass, a very susceptible species of the virus, and in the gilthead seabream, which acts as a reservoir, both in vivo and in vitro, and compared with that present in the serum and brain (target tissue of VNNV). First, our data show a great antiviral response in the brain of gilthead seabream and in the gonad of European sea bass. In addition, for the first time, our results demonstrate that the antimicrobial activities (complement, lysozyme and bactericidal) and the expression of AMP genes such as complement factor 3 (c3), lysozyme (lyz), hepcidin (hamp), dicentracin (dic), piscidin (pis) or β-defensin (bdef) in the gonad of both species are very different, but generally activated in the European sea bass, probably related with the differences of susceptibility upon VNNV infection, and even differs to the brain response. Furthermore, the in vitro data suggest that some AMPs are locally regulated playing a local immune response in the gonad, while others are more dependent of the systemic immune system. Data are discussed in the light to ascertain their potential role in viral clearance by the gonad to avoid vertical transmission.

lysozyme C (lyz), a glycoside hydrolase expressed exclusively in myeloid cells, is involved in the host defense against bacterial infection. We isolated a 2.4kb zebrafish lyz promoter region and established transgenic lines that drive enhanced green fluorescent protein (EGFP) to examine how lyz expression is regulated during myelopoiesis. We found that the 2.4kb lyz promoter is sufficient to drive myeloid-specific expression of EGFP in zebrafish. We identified potential transcriptional regulatory elements including a Runx element (TGTGGT at -1.70kb) and a C/ebp element (TTTGGCAAT at -1.46kb) in the lyz promoter, and showed that they are required for myeloid-specific expression of EGFP. We found that the myeloid-lineage transcription factors C/ebp1, Runx1 and Pu.1 can bind to the 2.4kb lyz promoter. Forced expression of runx1, c/ebp1 and pu.1 together induced ectopic lyz expression in the intermediated cell mass (ICM). Thus, we propose that c/ebp1 and runx1 presumably cooperated with pu.1 in the transcriptional regulation of lyz during zebrafish myelopoiesis.

Magnetite nanoparticles have been widely used in biomedical applications, especially as contrast agents in magnetic resonance imaging. In this work, the antifouling property of polyampholyte-coating (poly(acrylic acid) (PAA)-co-3-(diethylamino)-propylamine (DEAPA)) is systematically demonstrated. Polyampholyte-coated magnetite nanoparticles (NP1) and PAA-coated magnetite nanoparticles (NP2) were synthesized to investigate their interactions with BSA and lysozyme (LYZ) by high-resolution turbidimetric titration, dynamic light scattering (DLS), and isothermal titration calorimetry (ITC) in phosphate buffer saline (PBS) buffer with pH 7.4. The abundant carboxyl groups of NP2 and polyampholyte coating of NP1 were well proven by TGA, ζ-potential, and titration methods. Turbidity change shows that NP1 have no interaction with both proteins other than NP2 having adsorption with LYZ, which was further confirmed by DLS. Besides, ITC gives the exact enthalpy change and unveils the binding stoichiometry for each interaction. All characterizations demonstrate the antifouling property of NP1 to both negatively charged protein BSA and positively charged protein LYZ. The polyampholyte-coated magnetite nanoparticles were shown to be a promising material to eliminate the strong interaction with proteins in complex medium, for example, when it is applied for MRA contrast agents with long in vivo circulation time.

Fat deposition is a widely studied trait in pigs because of its implications with animal growth efficiency, technological and nutritional characteristics of meat products, but the global framework of the biological and molecular processes regulating fat deposition in pigs is still incomplete. This study describes the backfat tissue transcription profile in Italian Large White pigs and reports genes differentially expressed between fat and lean animals according to RNA-seq data. The backfat transcription profile was characterised by the expression of 23 483 genes, of which 54.1% were represented by known genes. Of 63 418 expressed transcripts, about 80% were non-previously annotated isoforms. By comparing the expression level of fat vs. lean pigs, we detected 86 robust differentially expressed transcripts, 72 more highly expressed (e.g. ACP5, BCL2A1, CCR1, CD163, CD1A, EGR2, ENPP1, GPNMB, INHBB, LYZ, MSR1, OLR1, PIK3AP1, PLIN2, SPP1, SLC11A1, STC1) and 14 lower expressed (e.g. ADSSL1, CDO1, DNAJB1, HSPA1A, HSPA1B, HSPA2, HSPB8, IGFBP5, OLFML3) in fat pigs. The main functional categories enriched in differentially expressed genes were immune system process, response to stimulus, cell activation and skeletal system development, for the overexpressed genes, and unfolded protein binding and stress response, for the underexpressed genes, which included five heat shock proteins. Adipose tissue alterations and impaired stress response are linked to inflammation and, in turn, to adipose tissue secretory activity, similar to what is observed in human obesity. Our results provide the opportunity to identify biomarkers of carcass fat traits to improve the pig production chain and to identify genetic factors that regulate the observed differential expression.

Acute myeloid leukemia (AML) is the most common and severe form of acute leukemia diagnosed in adults. Owing to its heterogeneity, AML is divided into classes associated with different treatment outcomes and specific gene expression profiles. Based on previous studies on AML, in this study, we designed and generated an AML-array containing 900 oligonucleotide probes complementary to human genes implicated in hematopoietic cell differentiation and maturation, proliferation, apoptosis and leukemic transformation. The AML-array was used to hybridize 118 samples from 33 patients with AML of the M1 and M2 subtypes of the French-American‑British (FAB) classification and 15 healthy volunteers (HV). Rigorous analysis of the microarray data revealed that 83 genes were differentially expressed between the patients with AML and the HV, including genes not yet discussed in the context of AML pathogenesis. The most overexpressed genes in AML were STMN1, KITLG, CDK6, MCM5, KRAS, CEBPA, MYC, ANGPT1, SRGN, RPLP0, ENO1 and SET, whereas the most underexpressed genes were IFITM1, LTB, FCN1, BIRC3, LYZ, ADD3, S100A9, FCER1G, PTRPE, CD74 and TMSB4X. The overexpression of the CPA3 gene was specific for AML with mutated NPM1 and FLT3. Although the microarray-based method was insufficient to differentiate between any other AML subgroups, quantitative PCR approaches enabled us to identify 3 genes (ANXA3, S100A9 and WT1) whose expression can be used to discriminate between the 2 studied AML FAB subtypes. The expression levels of the ANXA3 and S100A9 genes were increased, whereas those of WT1 were decreased in the AML-M2 compared to the AML-M1 group. We also examined the association between the STMN1, CAT and ABL1 genes, and the FLT3 and NPM1 mutation status. FLT3+/NPM1- AML was associated with the highest expression of STMN1, and ABL1 was upregulated in FLT3+ AML and CAT in FLT3- AML, irrespectively of the NPM1 mutation status. Moreover, our results indicated that CAT and WT1 gene expression levels correlated with the response to therapy. CAT expression was highest in patients who remained longer under complete remission, whereas WT1 expression increased with treatment resistance. On the whole, this study demonstrates that the AML-array can potentially serve as a first-line screening tool, and may be helpful for the diagnosis of AML, whereas the differentiation between AML subgroups can be more successfully performed with PCR-based analysis of a few marker genes.

BACKGROUND

Chinese traditional herbal medicine Fuzhengkangai (FZKA) formulation combination with gefitinib can overcome drug resistance and improve the prognosis of lung adenocarcinoma patients. However, the pharmacological and molecular mechanisms underlying the active ingredients, potential targets, and overcome drug resistance of the drug are still unclear. Therefore, it is necessary to explore the molecular mechanism of FZKA.

METHODS

A systems pharmacology and bioinformatics-based approach was employed to investigate the molecular pathogenesis of EGFR-TKI resistance with clinically effective herb formula. The differential gene expressions between EGFR-TKI sensitive and resistance cell lines were calculated and used to find overlap from targets as core targets. The prognosis of core targets was validated from the cancer genome atlas (TCGA) database by Cox regression. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment is applied to analysis core targets for revealing mechanism in biology.

RESULTS

The results showed that 35 active compounds of FZKA can interact with eight core targets proteins (ADRB2, BCL2, CDKN1A, HTR2C, KCNMA1, PLA2G4A, PRKCA and LYZ). The risk score of them were associated with overall survival and relapse free time (HR = 6.604, 95% CI: 2.314-18.850; HR = 5.132, 95% CI: 1.531-17.220). The pathway enrichment suggested that they involved in EGFR-TKI resistance and non-small cell lung cancer pathways, which directly affect EGFR-TKI resistance. The molecular docking showed that licochalcone a and beta-sitosterol can closely bind two targets (BCL2 and PRKCA) that involved in EGFR-TKI resistance pathway.

CONCLUSIONS

This study provided a workflow for understanding mechanism of CHM for against drug resistance.