A comparative study of binding interaction between Safranin O (SO) and Neutral Red (NR) with lysozyme (Lyz) has been reported using several spectroscopic methods along with computational approaches. Steady-state fluorescence measurements revealed static quenching as the major quenching mechanism in Lyz-SO and Lyz-NR interaction, which is further supported by time-resolved fluorescence and UV-vis measurements. Additionally, binding and thermodynamic parameters of these interactions are calculated from temperature dependent fluorescence data. Moreover, conformational changes of protein upon binding with SO and NR are provided by synchronous and circular dichroism (CD) measurements. Molecular docking study provided the exact binding location of SO and NR in lysozyme. Along with this study, molecular dynamics simulation is carried out to measure the stability of Lyz, Lyz-SO, and Lyz-NR complex. The present study revealed the strong binding affinity of dyes with lysozyme, and this study would be helpful toward medical and environmental science.

The toxic effects of paraquat (PQ) on the transcription of immunoglobulin M (IgM), complement C3 (C3), and lysozyme (LYZ) and the histopathological alteration of the liver, kidney, and spleen of common carp were evaluated by subacute exposure to 1.596 or 3.192 mg/L of PQ for 7 days. The results demonstrated that PQ exposure altered the transcription of IgM, C3, and LYZ. For example, IgM and C3 expression was generally downregulated, but LYZ was either down- or upregulated, suggesting that PQ may disturb the function of the fish immune system. The results of the histopathological examination revealed that the liver, kidney, and spleen of PQ-treated fish were injured. These injuries included cellular swelling, intracytoplasmic vacuolization, nucleus distortion, and pycnosis in the liver and spleen, and vacuolization of the renal parenchyma and intumescence of the renal tubule in the kidney. This finding indicates that PQ causes toxicity in common carp.

The contribution of colostrum to passive immunity transfer and intestinal protection is well known; however, the effects of colostrum intake on the expression of antimicrobial peptides (AP) and Fc receptors in the intestine of neonatal calves are unclear. Our aim was to investigate changes in the expression of AP and Fc receptor in the small intestine of calves in the first 36 h postpartum. Twenty-four Holstein bull calves were used in this study, of which 18 calves were administered 3.2 L of pooled colostrum for each calf per meal via an esophageal tube. Calves were slaughtered at 8 h (1 meal at 1-2 h), 24 h (2 meals at 1-2 h and 10-12 h), and 36 h (3 meals at 1-2 h, 10-12 h, and 22-24 h) postpartum. The remaining 6 calves without any milk administration were slaughtered at 2 h postpartum. Samples of blood and jejunum digesta were collected to determine immunoglobulin concentration using ELISA. Samples of the duodenum, jejunum, and ileum tissues after slaughter were collected to determine AP and Fc receptor expression using quantitative real-time PCR. In calves administered colostrum, IgG concentration in jejunum digesta rapidly decreased in an age-dependent manner (33.41, 9.47, and 0.34 mg/mL at 8, 24, and 36 h, respectively), whereas serum IgG concentration increased significantly, from 0.25 μg/mL at 2 h to 21.72 mg/mL at 24 h. Cathelicidin-4, β-defensin (DEFB)-7, and enteric β-defensin expression was upregulated at 8 h postpartum in the duodenum and jejunum compared with that at 2 h, but progressive recovery was detected from 24 h onward. Higher expression of cathelicidin-4, regenerating family member 3γ, lysozyme (<em>LYZ</em>), <em>LYZ</em>1, and <em>LYZ</em>2 and lower expression of DEFB, DEFB1, DEFB7, DEFB10, and enteric β-defensin were observed in the duodenum and jejunum compared with the ileum. Differences in AP expression between intestinal regions suggested that the innate immune defense mechanism varied significantly among the duodenum, jejunum, and ileum. No difference in the expression of Fc fragment of the IgG receptor was observed either among ages or small intestinal regions. The Fcγ receptor (FcγR)Ia and FcγRIIb expression was the highest at 8 h compared with that at 2, 24, and 36 h, and expression of FcγRIa, FcγRIIb, and FcγRIIIa was higher in the duodenum and jejunum than in the ileum. These results indicated that AP and Fcγ receptors might play important roles in intestinal defense during the passive immunity period.

Keywords: Fc receptor; antimicrobial peptide; neonatal calf; small intestine.

Here we report on the excited-state behavior in terms of the excited-state proton-transfer (ESPT) reaction as well as the ground-state acid-base property of pyranine [8-hydroxypyrene-1,3,6-trisulfonate (HPTS)] in the presence of an enzymatic protein, human lysozyme (LYZ). HPTS forms a 1:1 ground-state complex with LYZ having the binding constant KBH = (1.4 ± 0.05) × 10(4) M(-1), and its acid-base equilibrium gets shifted toward the deprotonated conjugate base (RO(-)), resulting in a downward shift in pKa. This suggests that the conjugate base (RO(-)) is thermodynamically more favored over the protonated (ROH) species inside the lysozyme matrix, resulting in an increased population of the deprotonated form. However, for the release of the proton from the excited photoacid, interestingly, the rate of proton transfer gets slowed down due to the "slow" acceptor biological water molecules present in the immediate vicinity of the fluorophore binding region inside the protein. The observed ESPT time constants, ∼140 and ∼750 ps, of protein-bound pyranine are slower than in bulk aqueous media (∼100 ps, single exponential). The molecular docking study predicts that the most probable binding location of the fluorophore is in a region near to the active site of the protein. Here we also report on the effect of external electrolyte (NaCl) on the reverse modulation of ground-state prototropy as well as the ESPT process of the protein-bound pyranine. It is found that there is a dominant role of electrostatic forces in the HPTS-LYZ interaction process, because an increase in ionic strength by the addition of NaCl dislodges the fluorophore from the protein pocket to the bulk again. The study shows a considerably different perspective of the perturbation offered by the model macromolecular host used, unlike the available literature reports on the concerned photoacid.

A combination strategy of moderate self-polymerization and assembly technique was proposed to fabricate superparamagnetic surface imprinted nanocomposites (SSINs) for efficient protein recognition. Homogeneous Fe3O4/Poly (methyl methacrylate) (PMMA)/Poly (dihydroxyphenylacetic acid) (PDOPA) SSINs were obtained via self-polymerization of DOPA on the surface of Fe3O4/PMMA nanospheres in the presence of lysozyme (Lyz) as a template. The Lyz-imprinted Fe3O4/PMMA/PDOPA SSINs possessed average diameters of 200nm, high magnetic content, high saturation magnetization, as well as excellent specific recognition capacity toward Lyz template, exhibiting their great potential for biomacromolecular recognition.

<AbstractText>Lactobacilli in rabbit intestine is rare and its function on rabbit gut health is not fully understood. The present study aimed to evaluate in vivo the probiotic potential of Lactobacillus casei for suckling rabbits.</AbstractText><AbstractText>Two healthy 5-day-old suckling rabbits with similar weights from each of 12 New Zealand White litters were selected and disturbed to control group and treatment group. All rabbits were artificially fed. The treatment group had been supplemented with live Lactobacillus casei in the milk from the beginning of the trial to 13 days of age. At 15 days of age, healthy paired rabbits were slaughtered to collect intestinal samples.</AbstractText><AbstractText>1) Oral administration of Lactobacillus casei significantly increased the proportion of Lactobacilli in the total intestinal bacteria (P < 0.01) and obviously reduced that of Escherichia-Shigella (P < 0.01); 2) treatment increased the length of vermiform appendix (P < 0.05); 3) a higher percentage of degranulated paneth cells was observed in the duodenum and jejunum when rabbits administered with Lactobacillus casei (P < 0.01); and 4) the expression of toll-like receptor 9 (TLR9), Lysozyme (<em>LYZ</em>) and defensin-7-like (DEFEN) in the duodenum and jejunum was stimulated by supplemented Lactobacillus casei (P < 0.05).</AbstractText><AbstractText>orally administered Lactobacillus casei could increase the abundance of intestinal Lactobacilli, decrease the relative abundance of intestinal Escherichia-Shigella, promote the growth of appendix vermiform, stimulate the degranulation of paneth cells and induce the expression of defensin-7-like and Lysozyme. The results of the present study implied that Lactobacillus casei exhibited probiotic potential for suckling rabbit.</AbstractText>

Monocytes and macrophages play a key role in defending pathogens, removing the dead cells or cell debris, and wound healing. The mammalian target of rapamycin (mTOR) inhibitor rapamycin (RPM) is widely used in clinics to treat patients with organ transplantation or tumors. The role of mTOR in monocyte/macrophage development remains to be clarified. Here we found that mTOR intrinsically controls monocyte/macrophage development, as evidenced by the decreased percentages and cell numbers of CD11b+F4/80+ cells resulting from mTOR inhibition in SCID mice, mTOR-deficient mice, and mixed chimera mice, and the in vitro colony formation and monocyte/macrophage induction assays. However, Lyzs-mTOR knockout mice displayed normal levels of monocytes/macrophages, indicating that mTOR is not essential for the survival and maturation of monocytes/macrophages. Further studies showed that mTOR deficiency significantly reduced macrophage colony-stimulating factor receptor CD115 expression at the transcriptional and translational levels. The molecular mechanism studies indicate that the impaired monocyte/macrophage development caused by mTOR deficiency is mainly a result of the overactivated STAT5 and subsequent downregulation of IRF8, but not the altered cell metabolism and autophagy. Therefore, our work identifies that mTOR is an intrinsic master for monocyte/macrophage development at the early stages through regulating STAT5-IRF8-dependent CD115-expressing pathway. Long-term usage of RPM may cause a defect of myeloid progenitors in bone marrow.

BACKGROUND

Graves' orbitopathy (GO) is an autoimmune inflammatory ocular complication and one of the most frequent manifestations of Graves' disease (GD). Clinical judgment of GO is subjective sometimes leading to clinical and therapeutic challenges. Better tools to diagnose this severe complication are warranted.

METHODS

The aim of the present study was to evaluate tear levels of LYZ, LACRT and AZGP1 in GD patients with or without GO, as possible biomarkers for GO. Tear samples were collected from GD patients with moderate-to-severe GO (n = 21) and no clinical signs of GO (n = 21). Additionally, 18 GD patients with mild GO and 9 patients without GO were included in a further part of the study.

RESULTS

Tear levels of LYZ (p < 0.001), LACRT (p = 0.004) and AZGP1 (p = 0.001) were significantly elevated in GD patients with moderate-to-severe GO compared to GD patients without GO. The discriminatory power of the three biomarkers, combined in a panel was confirmed by ROC plot analysis, with an AUC value of 0.93 (sensitivity of 95%; specificity of 80%). Since LYZ showed the best performance in discriminating between GD patients with (moderate-to-severe) and without GO (in combination with limited sample volume available), LYZ levels were also measured in tears from GD patients with mild GO and without GO. Significantly higher levels of LYZ were measured in GD patients with mild GO compared to those without GO (p = 0.003).

CONCLUSIONS

We have established a novel three-protein biomarker panel that is able to discriminate between GD patients with and without GO, which might aid in diagnostic evaluation of GO as well as an indicator for disease activity.

Aquatic animals are frequently threated by bacterial pathogens. The most economic and efficient protection against bacterial infection are through vaccine immunization. The various serotypes of the pathogens, such as Vibrios, hurdle the development of the vaccines, especially polyvalent vaccines. Here, we demonstrate that recombinant bacterial ghost is a good candidate for multivalent vaccine. By expressing PhiX174 gene E alone or co-expressing the gene E with two genes encoding outer membrane proteins (VP1667 and VP2369) in V. parahaemolyticus, we generated the recombinant V. parahaemolyticus ghosts VPG and rVPGs respectively. Fish immunized with either VPG or rVPG showed increased survival against the infection by either V. parahaemolyticus or V. alginolyticus, with a better protective effect by immunization with rVPG. Our furthermore studies show that rVPG stimulates stronger innate immune responses by increasing the expression of tnfα, il1β, il6, il8 and il10 as well as that of c3b, lyz, and tlr5, the key players linking the innate and adaptive immune responses upon microbial stimulation. In summary, VPG and rVPG can protect zebrafish against the infection from at least two Vibrio species, suggesting its potential value for further aquaculture vaccines development.

Keywords: Bacterial ghosts; Immunoprotection; Polyvalent vaccine; Vibrio parahaemolyticus.

Lysozymes are an ancient group of antimicrobial enzymes of the innate immune system. Here we provide a comparative analysis of the evolution and function of lysozymes during early development in fish, the most speciose vertebrate group. In fishes, lineage and species-specific evolution of both C-type (chicken or conventional) and G-type (goose type) genes occurred. Phylogenetic analysis revealed that the teleost lysozyme G-type members group with the tetrapod homologues but the teleost C-type form three different clusters with the tetrapods. Most of the teleost C-type cluster with tetrapod Lyz but there are some that group with the mammalian Lyzl1/2 and LALBA. This suggests that early in gnathostome evolution these genes already existed and that lyzl1/2 and lalba genes are present in fish and tetrapods. Gene synteny analysis to confirm sequence orthologies failed to identify conserved genome regions between teleosts and other vertebrates lysozyme gene regions suggesting that in the ancestral bony fish genome lyz, lyzl1/2, lalba and lyg precursor genes were transposed to different chromosome regions. The homologue of the mammalian lactalbumin (LALBA) gene was identified for the first time in teleosts and was expressed in skin and during egg and larval development. Lysozyme activity was detected in teleost eggs and varied between species and in the gilthead sea bream lyg and lalba transcript abundance differed in eggs and larvae from different brood stock suggesting differences exist in maternal innate immune protection.

Keywords: evolution; fish; innate immunity; lysozymes; ontogeny.

Nanoparticulate and water-soluble formulations of ionic polyphosphazenes and protein cargo - lysozyme (LYZ) were prepared by their self-assembly in aqueous solutions at near physiological pH (pH 7.4) in the presence and absence of an ionic cross-linker - spermine tetrahydrochloride. Efficiency of LYZ encapsulation, physico-chemical characteristics of formulations, and the effect of reaction parameters were investigated using asymmetric flow field flow fractionation (AF4) and dynamic light scattering (DLS) methods. The effect of both polymer formulations on encapsulated LYZ was evaluated using soluble oligosaccharide substrate, whereas their ability to present the protein to cellular surfaces was assessed by measuring enzymatic activity of encapsulated LYZ against Micrococcus lysodeikticus cells. It was found that both soluble and cross-linked polymer matrices reduce lysis of bacterial cells by LYZ, whereas activity of encapsulated protein against oligosaccharide substrate remained practically unchanged indicating no adverse effect of polyphosphazene on protein integrity. Moreover, nanoparticulate formulations display distinctly different behavior in cellular assays when compared to their soluble counterparts. LYZ encapsulated in polyphosphazene nanoparticles shows approximately 2.5-fold higher activity in its ability to lyse cells as compared with water-soluble LYZ-PCPP formulations. A new approach to PEGylation of polyphosphazene nanoparticles was also developed. The method utilizes a new ionic polyphosphazene derivative, which contains graft (polyethylene glycol) chains. PEGylation allows for an improved control over the size of nanoparticles and broader modulation of their cross-linking density, while still permitting for protein presentation to cellular substrates.

Background: Defining epithelial cell contributions to inflammatory bowel disease (IBD) is essential for the development of much needed therapies for barrier repair. Children with very early onset (VEO)-IBD have more extensive, severe, and refractory disease than older children and adults with IBD and, in some cases, have defective barrier function. We therefore evaluated functional and transcriptomic differences between pediatric IBD (VEO and older onset) and non-IBD epithelium using 3-dimensional, biopsy-derived organoids.

Methods: We measured growth efficiency relative to histopathological and clinical parameters in patient enteroid (ileum) and colonoid (colon) lines. We performed RNA-sequencing on patient colonoids and subsequent flow cytometry after multiple passages to evaluate changes that persisted in culture.

Results: Enteroids and colonoids from pediatric patients with IBD exhibited decreased growth associated with histological inflammation compared with non-IBD controls. We observed increased LYZ expression in colonoids from pediatric IBD patients, which has been reported previously in adult patients with IBD. We also observed upregulation of antigen presentation genes HLA-DRB1 and HLA-DRA, which persisted after prolonged passaging in patients with pediatric IBD.

Conclusions: We present the first functional evaluation of enteroids and colonoids from patients with VEO-IBD and older onset pediatric IBD, a subset of which exhibits poor growth. Enhanced, persistent epithelial antigen presentation gene expression in patient colonoids supports the notion that epithelial cell-intrinsic differences may contribute to IBD pathogenesis.

Keywords: VEO-IBD; colonoids; enteroids; epithelial cells; inflammatory bowel disease.

In this work, we clearly focus on the comparative cytotoxicity investigations of several protein-stabilized gold nanoclusters (Au NCs) towards lymphocytes B (COLO-720 L) and lymphocytes T (HUT-78) cells. For synthesis, the one-pot template-assisted method was carried out using lysozyme (LYZ), human (HSA) and bovine (BSA) serum albumins, and gamma globulin (γG) as stabilizing agents. Regardless of the type of proteins, all synthesized Au NCs possess intense red emission (λ_em ∼ 650 nm) and have similar size of a metal core (ca. 1.4 nm) with negative surface charge at pH = 7.4. During the treatment of cells with clusters, changes in mitochondrial activity, membrane integrity, secretion of inflammatory and apoptosis mediators of the lymphocytes were studied to determine the potential effect of protein layers on the toxicity of clusters. It was found that γG-Au NCs induced the highest disorders in mitochondrial activity, but the influence of other NCs on the cell viability was minor. Besides, all Au NCs caused oxidative stress by peroxidation of membrane lipids. The secretion of malonic dialdehyde (MDA) was enhanced by LYZ- and γG-Au NCs. Apart from LYZ-Au NCs, the clusters did not exhibit strong proinflammatory and apoptotic properties. The enhanced secretion of tumor necrosis factor (TNF-α) by lymphocytes B, in comparison to control, was independent of the clusters type. Despite the lack of significant influence of the Au NCs on the viability of the lymphocytes, they can stimulate undesirable cellular processes, which clearly depends on the stabilizing proteins.

Keywords: Cytotoxicity; Gold nanoclusters; Lymphocytes; Proteins.

In susceptible hosts, protection from Leptospira infection is mediated by the innate immune response at the point of entry and humoral immunity. Thus, identifying and segregating the initial host response at the representative host-pathogen interface is needed to understand the typical outcomes of Leptospira infection, clearance, persistence, or disease. An in vitro whole blood culture system to study the overall immune response using pathogenic and non-pathogenic Leptospira strains was explored in this study. Using an ELISA, increased IL-8, TNF alpha, and IL-1 in blood samples stimulated with pathogenic and nonpathogenic Leptospira compared to unstimulated controls were detected. In RT² Profiler PCR Array assays, consistent upregulation of 22 genes and downregulation of 25 genes were observed. Few of the notable upregulated genes included BPI, CCL3, CXCL2, IL-6, IL-8, TLR1, TLR2, TLR6, and TNF and downregulated genes included, LBP, LYZ, MPO, MYD88. IFNβ was upregulated in samples treated with pathogenic Leptospira and IL-1β was upregulated in samples treated with nonpathogenic Leptospira. Toll- like Receptor signaling and expression of pattern recognition receptors were two of the five prominent canonical pathways observed. Individual deconvolution of each of the specific and significant pathways observed in this study may improve the understanding of the pathogenesis of this important zoonotic agent. The use of this system in conjunction with whole transcriptome analysis in a larger population, may unveil the robust nature of host/Leptospira interaction.

Keywords: Canine; Innate immune response; Leptospira; Whole blood culture.

Soft tissue sarcomas (STSs) are rare, heterogeneous mesenchymal neoplasias. Understanding the tumor microenvironment (TME) and identifying potential biomarkers for prognosis associated with the TME of STS might provide effective clues for immune therapy. We evaluated the immune scores and stromal scores of STS patients by using the RNA sequencing dataset from The Cancer Genome Atlas (TCGA) database and the ESTIMATE algorithm. Then, the differentially expressed mRNAs (DEGs), miRNAs (DEMs) and lncRNAs (DELs) were identified after comparing the high- and low-score groups. Next, we established a competing endogenous RNA (ceRNA) network and explored the prognostic values of biomarkers involved in the network with the help of bioinformatics analysis. High immune score was significantly associated with favorable overall survival in STS patients. A total of 328 DEGs, 18 DEMs and 67 DELs commonly regulated in the immune and stromal score groups were obtained. A ceRNA network and protein-protein interaction (PPI) network identified some hub nodes with considerable importance in the network. Kaplan-Meier survival analysis demonstrated that nine mRNAs, two miRNAs and three lncRNAs were closely associated with overall survival of STS patients. Gene set enrichment analysis (GSEA) suggested that these three lncRNAs were mainly involved in immune response-associated pathways in STS patients. Finally, the expression levels of five mRNAs (APOL1, EFEMP1, LYZ, RARRES1 and TNFAIP2) were verified, which were consistent with the results of the TCGA cohort. The results of our study confirmed the prognostic value of immune scores for STS patients. We also identified several TME-related biomarkers that might contribute to prognostic prediction and immune therapy.

Keywords: Soft tissue sarcomas; ceRNA; estimate; prognosis; tumor microenvironment.

In contrast to mammals, zebrafish (Danio rerio) has the ability to regenerate injured sites such as different tissues present in the fin. It is known that cells of the innate immune system play essential roles in regeneration, however, some aspects of the molecular mechanisms by which these cells orchestrate regeneration remain unknown. This study aimed to evaluate the infiltration dynamics of neutrophils and macrophages in the regenerative process of fin fold in regards to the influence of the redox environment and oxidative pathways. Fin fold amputation was performed on transgenic larvae for macrophage-expressed gene 1 (mpeg1), lysozyme (lyz), myeloperoxidase (mpo), and tumor necrosis factor alpha (TNFα) at 3 days post-fertilization, followed by confocal microscopy imaging and measurement of the activities of oxidant and antioxidant enzymes. We observed initially an increase in the number of neutrophils (lyz:DsRed+/mpx:GFP+) and then macrophages (mpeg1+) in the injury site followed by a decrease in neutrophils at 7 days post-amputation (dpa). Moreover, macrophages switch from a pro-inflammatory to an anti-inflammatory profile throughout the process, while the activity of superoxide dismutase (SOD) increased at 1 dpa and catalase (CAT) at 5 dpa. Higher levels of lipid peroxidation were also detected during regeneration. Despite oxidative stress, there is, therefore, an antioxidant response throughout the regeneration of the caudal fin. The present work can contribute to future studies on the development of cell therapies, achieving greater effectiveness in the treatment of diseases related to the formation of fibrotic tissue.

Keywords: Zebrafish; macrophages; redox.

The interactions of levofloxacin (LEV) with lysozyme (LYZ), trypsin and bovine hemoglobin (BHb) were investigated, respectively, by using multi-spectral techniques and molecular docking in vitro. Fluorescence studies showed that LEV quenched LYZ/trypsin fluorescence in a combined quenching ways and BHb fluorescence in a static quenching with binding constants of .14, .51 and .20 × 105 L mol-1 at 298 K, respectively. The thermodynamic parameters demonstrated that hydrophobic forces, hydrogen bonds, and van der Waals forces played the major role in the binding process. The binding distances between LEV and the inner tryptophan residues of LYZ, trypsin, and BHb were calculated to be 4.04, 3.38, and 4.52 nm, respectively. Furthermore, the results of circular dichroism spectra (CD), UV-vis, and three-dimensional fluorescence spectra indicated that the secondary structures of LYZ, trypsin, and BHb were partially changed by LEV with the α-helix percentage of LYZ-LEV system increased while that of BHb-LEV system was decreased, the β-sheet percentage of trypsin-LEV system increased from 41.3 to 42.9%. UV-vis spectral results showed that the binding interactions could cause conformational and some micro-environmental changes of LYZ, trypsin, and BHb. The results of molecular docking revealed that in LYZ and trypsin systems, LEV bound to the active sites residues GLU 35 and ASP 52 of LYZ and trypsin at the active site SER 195, and in BHb system, LEV was located in the central cavity, which was consistent with the results of synchronous fluorescence experiment. Besides, LEV made the activity of LYZ decrease while the activity of trypsin increased.

The paper presents and analyzes the results of experiments on the Escherichia coli-Salmonella relationships in combined infection of Ornithodoros papillipes ticks. The findings have led to the following conclusions. The body of hungry adult O. papillipes ticks can retain the pathogen of Salmonella infection for a month. This model object is also favourable for avirulent E. coli strain persistent in the body's cavity both alone and in combination with Salmonella typhimurium strain in a 1:1 ratio. Binary carriage in hungry Ornithodoros papillipes ticks revealed that the ticks associated with Salmonella typhimurium and E. coli 083 mcl + lyz No 225, microcine and lysozyme producers, are freed of pathogenic bacteria during 24 hours. The initial bacteria E. coli 083 mcl also suppress salmonellae; however, their elimination occurs much later--in 5 days. Calculating the correlation between the pairs (S. typhi and E. coli) has revealed a linear functional relationship of the rate of E. coli growth to S. typhimurium suppression.

OBJECTIVE

Antimicrobial peptides (AMPs) are one of the most active components of innate immunity and have characteristics that could place them at the heart of the pathogenesis of periodontal disease. This study investigated differences in the expression of AMP coding genes obtained using a simple harvesting technique, gingival smear, between two groups of patients: chronic periodontitis subjects versus healthy ones.

METHODS

Twenty-three patients were enrolled in two groups: 12 were diagnosed with moderate or severe generalized chronic periodontitis, and 11 were diagnosed as clinically healthy. Gingival smears were retrieved and studied using reverse transcription-quantitative PCR (RT-qPCR) after mRNA purification.

RESULTS

Fifteen gene expressions were obtained using real-time RT-qPCR. Three AMP genes, histatin 3 (HTN3), α-defensin 4 (DEFA4) and lysozyme C (LYZ), presented different expression levels in periodontitis patients compared with healthy subjects. The relative expression level of DEFA4 appeared to be a protective factor against periodontitis.

CONCLUSIONS

Gingival smears studied by RT-qPCR may be used to assess the expression of AMPs coding genes. A lack of expression of DEFA4 could be a potential indicator of periodontitis status.

In foregut-fermenting mammals (e.g., colobine monkeys, artiodactyl ruminants) the enzymes pancreatic ribonuclease (RNASE1) and lysozyme C (LYZ), originally involved in immune defense, have evolved new digestive functions. Howler monkeys are folivorous non-colobine primates that lack the multi-chambered stomachs of colobines and instead digest leaves using fermentation in the caeco-colic region. We present data on the RNASE1 and LYZ genes of four species of howler monkey (Alouatta spp.). We find that howler monkey LYZ is conserved and does not share the substitutions found in colobine and cow sequences, whereas RNASE1 was duplicated in the common ancestor of A. palliata, A. seniculus, A. sara, and A. pigra. While the parent gene (RNASE1) is conserved, the daughter gene (RNASE1B) has multiple amino acid substitutions that are parallel to those found in RNASE1B genes of colobines. The duplicated RNase in Alouatta has biochemical changes similar to those in colobines, suggesting a novel, possibly digestive function. These findings suggest that pancreatic ribonuclease has, in parallel, evolved a new role for digesting the products of microbial fermentation in both foregut- and hindgut-fermenting folivorous primates. This may be a vital digestive enzyme adaptation allowing howler monkeys to survive on leaves during periods of low fruit availability.

Serine protease inhibitors (SPIs) are an important group of protease inhibitors involved in a variety of biological processes. In the present study, a Kazal-type serine protease inhibitor homolog gene (designated as CsKPI) was identified from a Cyclina sinensis cDNA library. The open reading frame consists of 456 bp and encodes a protein of 151 amino acid residues with a theoretical molecular mass of 16.85 kDa and an isoelectric point of 5.74. Furthermore, using quantitative real-time PCR, we focused on the expression patterns of CsKPI found in tissues and on the stimulation of this gene's expression by bacteria. The results show that a higher-level mRNA expression of CsKPI was detected in hemocytes (P < 0.05) and was significantly upregulated at 3 h (P < 0.01) upon receiving bacterial challenges with Vibrio anguillarum. In addition, after the CsKPI gene was silenced by RNA interference, the expression of the CsTLR2 and CsMyD88 genes was extremely significantly decreased (P < 0.01) in C. sinensis. Finally, the recombinant CsKPI (rCsKPI) protein was purified and shown to exhibit less inhibitory activity than C-lyz against V. anguillarum in vitro. Hence, we propose that CsKPI plays an important role in the innate immunity and mediates TLR2 and MyD88-dependent pathway initiation in C. sinensis.

Interaction of an anticancer drug, vandetanib (VDB) with a ligand transporter, lysozyme (LYZ) was explored using multispectroscopic techniques, such as fluorescence, absorption and circular dichroism along with computational analysis. Fluorescence data and absorption results confirmed VDB-LYZ complexation. VDB-induced quenching was characterized as static quenching based on inverse correlation of KSV with temperature as well as kq values. The complex was characterized by the weak binding constant (Ka=4.96-3.14×103M-1). Thermodynamic data (ΔS=+12.82Jmol-1K-1; ΔH=-16.73kJmol-1) of VDB-LYZ interaction revealed participation of hydrophobic and van der Waals forces along with hydrogen bonds in VDB-LYZ complexation. Microenvironmental perturbations around tryptophan and tyrosine residues as well as secondary and tertiary structural alterations in LYZ upon addition of VDB were evident from the 3-D fluorescence, far- and near-UV CD spectral analyses, respectively. Interestingly, addition of VDB to LYZ significantly increased protein's thermostability. Molecular docking results suggested the location of VDB binding site near the LYZ active site while molecular dynamics simulation results suggested stability of VDB-LYZ complex. Presence of Mg2+, Ba2+ and Zn2+ was found to interfere with VDB-LYZ interaction.

Biofloc technology is increasingly becoming the most promising aquaculture tool especially in places where water is scarce and the land is very expensive. The dynamics of water quality, as well as plankton and microbial abundance, are collectively necessary for successful fish farming. The prospective use of jaggery as a potential carbon source and its influence on water quality, growth performance, innate immunity, serum bactericidal capacity, and disease resistance to Aeromonas hydrophila was investigated in Oreochromis niloticus. A completely randomized design was used in triplicates, where the control group was reared in a water system with no carbon source, while T1, T2, and T3 groups were raised in biofloc systems at C:N ratios of C:N12, C:N15, and C:N20, respectively. Water specimens were collected daily and fortnightly, while blood, serum, and head kidneys were collected at 75 days of experimental period for further analysis. TAN, nitrite, and ammonia values were considerably reduced, while the TSS values elevated significantly in all treated groups compared to the control. Jaggery-based biofloc system (JB-BFT) has a pronounced effect on hematological and growth performance parameters rather than control. Similarly, serum antioxidants, lysozyme, protease, antiprotease and bactericidal capacity were significantly increased (p < 0.05) in the treated groups in a dose-dependent manner. LYZ, TNF-α, and IL-1β genes were upregulated in proportion to C:N ratios with the highest fold in C:N20. Furthermore, fish treated with JB-BFT presented lower cumulative mortalities and better relative levels of production (RLP) after experimental challenge with A. hydrophila compared to control. In conclusion, JB-BFT has a robust influence on Nile tilapia (O. niloticus) innate immunity through favorable innovation of various immune-cells and enzymes as well as upregulating the expression levels of immune-related genes. This study offers jaggery as a new carbon source with unique properties that satisfy all considerations of biofloc technology in an eco-friendly manner.

Keywords: Biofloc; Experimental challenge; Innate immunity; Jaggery powder; Nile tilapia.

Hazardous contaminants, such as nitrite and microcystin-leucine arginine (MC-LR), are released into water bodies during cyanobacterial blooms and may adversely influence the normal physiological function of hydrobiontes. The combined effects of nitrite and MC-LR on the antioxidant defense and innate immunity were evaluated through an orthogonal experimental design (nitrite: 0, 29, 290 μM; MC-LR: 0, 3, 30 nM). Remarkable increases in malondialdehyde (MDA) levels have suggested that nitrite and/or MC-LR exposures induce oxidative stress in fish spleen, which were indirectly confirmed by significant downregulations of total antioxidant capacity (T-AOC), glutathione (GSH) contents, as well as transcriptional levels of antioxidant enzyme genes cat1, sod1 and gpx1a. Simultaneously, nitrite and MC-LR significantly decreased serum complement C3 levels as well as the transcriptional levels of splenic c3b, lyz, il1β, ifnγ and tnfα, and indicated that they could jointly impact the innate immunity of fish. The severity and extent of splenic lesions were aggravated by increased concentration of nitrite or MC-LR and became more serious in combined groups. The damages of mitochondria and pseudopodia in splenic macrophages suggest that oxidative stress exerted by nitrite and MC-LR aimed at the membrane structure of immune cells and ultimately disrupted immune function. Our results clearly demonstrate that nitrite and MC-LR exert synergistic suppressive effects on fish innate immunity via interfering antioxidant responses, and their joint toxicity should not be underestimated in eutrophic lakes.