Reconciling the Structural Attributes of Avian Antibodies

Antibodies are high value therapeutic, diagnostic, biotechnological, and research tools. Combinatorial approaches to antibody discovery have facilitated access to unique antibodies by surpassing the diversity limitations of the natural repertoire, exploitation of immune repertoires from multiple species, and tailoring selections to isolate antibodies with desirable biophysical attributes. The V-gene repertoire of the chicken does not utilize highly diverse sequence and structures, which is in stark contrast to the mechanism employed by humans, mice, and primates. Recent exploitation of the avian immune system has generated high quality, high affinity antibodies to a wide range of antigens for a number of therapeutic, diagnostic and biotechnological applications. Furthermore, extensive examination of the amino acid characteristics of the chicken repertoire has provided significant insight into mechanisms employed by the avian immune system. A paucity of avian antibody crystal structures has limited our understanding of the structural consequences of these uniquely chicken features. This paper presents the crystal structure of two chicken single chain fragment variable (scFv) antibodies generated from large libraries by phage display against important human antigen targets, which capture two unique CDRL1 canonical classes in the presence and absence of a non-canonical disulfide constrained CDRH3. These structures cast light on the unique structural features of chicken antibodies and contribute further to our collective understanding of the unique mechanisms of diversity and biochemical attributes that render the chicken repertoire of particular value for antibody generation.     

Animal models of gastrointestinal inflammation and cancer

Inflammation and cancer are the two major disorders in the gastrointestinal tract. They are causally related in their pathogenesis. It is important to study animal models' causal relationship and, in particular, to discover new therapeutic agents for such diseases. There are several criteria for these models in order to make them useful in better understanding the etiology and treatment of the said diseases in humans. In this regard, animal models should be similar as possible to human diseases and also be easy to produce and reproducible and also economic to allow a continuous replication in different laboratories. In this review, we summarize the various animal models for inflammatory and cancerous disorders in the upper and lower gastrointestinal tract. Experimental approaches are as simple as by giving a single oral dose of alcohol or other noxious agents or by injections of multiple dosages of ulcer inducing agents or by parenteral administration or in drinking water of carcinogens or by modifying the genetic makeups of animals to produce relatively long-term pathological changes in particular organs. With these methods they could induce consistent inflammatory responses or tumorigenesis in the gastrointestinal mucosa. These animal models are widely used in laboratories in understanding the pathogenesis as well as the mechanisms of action for therapeutic agents in the treatment of gastrointestinal inflammation and cancer.     

Vibrio fischeri and its host: it takes two to tango

The association of Vibrio fischeri and Euprymna scolopes provides insights into traits essential for symbiosis, and the signals and pathways of bacteria-induced host development. Recent studies have identified important bacterial colonization factors, including those involved in motility, bioluminescence and biofilm formation. Surprising links between symbiosis and pathogenesis have been revealed through discoveries that nitric oxide is a component of the host defense, and that V. fischeri uses a cytotoxin-like molecule to induce host development. Technological advances in this system include the genome sequence of V. fischeri, an expressed sequence tagged library for E. scolopes and the availability of dual-fluorescence markers and confocal microscopy to probe symbiotic structures and the dynamics of colonization.     

Efficient microwave enhanced regioselective synthesis of a series of benzimidazolyl/triazolyl spiro [indole-thiazolidinones] as potent antifungal agents and crystal structure of spiro[3H-indole-3,2'-thiazolidine]-3'(1,2,4-triazol-3-yl)-2,4'(1H)-dione

A microwave-assisted three-component, regioselective one-pot cyclocondensation method has been developed for the synthesis of a series of novel spiro[indole-thiazolidinones] (6a-l) using an environmentally benign procedure at atmospheric pressure in open vessel. This rapid method produces pure products in high yields within few minutes in comparison to a conventional two-step procedure. The crystal structure of one representative compound has been determined by X-ray diffraction. The synthesized compounds have been screened 'in vitro' for antifungal activity against Rhizoctonia solani, Fusarium oxysporum and Collectotrichum capsici. All compounds have shown good activity against these pathogens.     

Hypoxia dysregulates the production of adiponectin and plasminogen activator inhibitor-1 independent of reactive oxygen species in adipocytes

Low plasma levels of adiponectin (hypoadiponectinemia) and elevated circulating concentrations of plasminogen activator inhibitor (PAI)-1 are causally associated with obesity-related insulin resistance and cardiovascular disease. However, the mechanism that mediates the aberrant production of these two adipokines in obesity remains poorly understood. In this study, we investigated the effects of hypoxia and reactive oxygen species (ROS) on production of adiponectin and PAI-1 in 3T3-L1 adipocytes. Quantitative PCR and immunoassays showed that ambient hypoxia markedly suppressed adiponectin mRNA expression and its protein secretion, and increased PAI-1 production in mature adipocytes. Dimethyloxallyl glycine, a stabilizer of hypoxia-inducible factor 1alpha (HIF-1alpha), mimicked the hypoxia-mediated modulations of these two adipokines. Hypoxia caused a modest elevation of ROS in adipocytes. However, ablation of intracellular ROS by antioxidants failed to alleviate hypoxia-induced aberrant production of adiponectin and PAI-1. On the other hand, the antioxidants could reverse hydrogen peroxide (H2O2)-induced dysregulation of adiponectin and PAI-1 production. H2O2 treatment decreased the expression levels of peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer binding protein (C/EBPalpha), but had no effect on HIF-1alpha, whereas hypoxia stabilized HIF-1alpha and decreased expression of C/EBPalpha, but not PPARgamma. Taken together, these data suggest that hypoxia and ROS decrease adiponectin production and augment PAI-1 expression in adipocytes via distinct signaling pathways. These effects may contribute to hypoadiponectinemia and elevated PAI-1 levels in obesity, type 2 diabetes, and cardiovascular diseases.     

A Preliminary Proteomic Investigation of Circulating Exosomes and Discovery of Biomarkers Associated with the Progression of Osteosarcoma in a Clinical Model of Spontaneous Disease

Circulating cancer exosomes are microvesicles which originate from malignant cells and other organs influenced by the disease and can be found in blood. The exosomal proteomic cargo can often be traced to the cells from which they originated, reflecting the physiological status of these cells. The similarities between cancer exosomes and the tumor cells they originate from exhibit the potential of these vesicles as an invaluable target for liquid biopsies. Exosomes were isolated from the serum of eight osteosarcoma-bearing dogs, five healthy dogs, and five dogs with traumatic fractures. We also characterized exosomes which were collected longitudinally from patients with osteosarcoma prior and 2 weeks after amputation, and eventually upon detection of lung metastasis. Exosomal proteins fraction were analyzed by label-free mass spectrometry proteomics and were validated with immunoblots of selected proteins. Ten exosomal proteins were found that collectively discriminate serum of osteosarcoma patients from serum healthy or fractured dogs with an accuracy of 85%. Additionally, serum from different disease stages could be distinguished with an accuracy of 77% based on exosomal proteomic composition. The most discriminating protein changes for both sample group comparisons were related to complement regulation, suggesting an immune evasion mechanism in early stages of osteosarcoma as well as in advanced disease.     

Attachment of diaminopimelic acid to bdelloplast peptidoglycan during intraperiplasmic growth of Bdellovibrio bacteriovorus 109J.

An early event in the predatory lifestyle of Bdellovibrio bacteriovorus 109J is the attachment of diaminopimelic acid (DAP) to the peptidoglycan of its prey. Attachment occurs over the first 60 min of the growth cycle and is mediated by an extracellular activity(s) produced by the bdellovibrio. Some 40,000 DAP residues are incorporated into the Escherichia coli bdelloplast wall, amounting to ca. 2 to 3% of the total initial DAP content of its prey cells. Incorporation of DAP occurs when E. coli, Pseudomonas putida, or Spirillum serpens are the prey organisms. The structurally similar compounds lysine, ornithine, citrulline, and 2,4-diaminobutyric acid are not attached. The attachment process is not affected by heat-killing the prey nor by the addition of inhibitors of either energy generation (cyanide, azide, or arsenate), protein or RNA synthesis (chloramphenicol and rifamycin), or de novo synthesis of cell wall (penicillin or vancomycin). Approximately one-third of the incorporated DAP is exchangeable with exogenously added unlabeled DAP, whereas the remaining incorporated DPA is solubilized only during the lysis of the bdelloplast wall. Examination of DAP incorporation at low prey cell densities suggests that bdellovibrios closely couple the incorporation to an independent, enzymatic solubilization of DAP by a peptidase. The data indicate that DAP incorporation is a novel process, representing the second example of the ability of the bdellovibrio to biosynthetically modify the wall of its prey.