The relevance of tissue thiol histochemistry to diagnostic hematopathology

Expression analyses suggest that alterations of the antioxidant state of some diffuse large B-cell lymphomas can assist prognosis; reversibly oxidized thiols may serve as a surrogate marker for identifying such cases. Little is known about the distribution of free thiols and reversibly oxidized thiols in human tissues. We developed a staining technique that enables visualization of tissue thiols in situ using bright field microscopy and validated it using gastrointestinal tissue specimens. We used our thiol staining technique to assess benign tonsillectomy and diffuse large B-cell lymphoma specimens. The gastrointestinal series revealed the presence of free thiols within epithelial cells and cells of the lamina propria. Staining for reversibly oxidized thiols was robust in gastric foveolar cells, intestinal goblet cells and the mucus they produce. Tonsillectomy specimens exhibited diffuse presence of free thiols. Staining for reversibly oxidized thiols was confined to germinal center macrophages and sinus histiocytes. Among the diffuse large B-cell lymphoma specimens, we observed strong staining for free thiols within malignant cells. By contrast to benign B-cells, the malignant cells demonstrated pronounced and diffuse staining for reversibly oxidized thiols. We demonstrated intrinsic differences between benign and malignant cells.     

Polymerized collagen inhibits fibroblast proliferation via a mechanism involving the formation of a beta1 integrin-protein phosphatase 2A-tuberous sclerosis complex 2 complex that suppresses S6K1 activity

Polymerized type I collagen suppresses fibroblast proliferation. Previous studies have implicated inhibition of fibroblast proliferation with polymerized collagen-mediated suppression of S6K1, but the molecular mechanism of the critical negative feedback loop has not yet been fully elucidated. Here, we demonstrate that polymerized collagen suppresses G(1)/S phase transition and fibroblast proliferation by a novel mechanism involving the formation of a beta1 integrin-protein phosphatase 2A (PP2A)-tuberous sclerosis complex 2 (TSC2) complex that represses S6K1 activity. In response to fibroblast interaction with polymerized collagen, beta1 integrin forms a complex with PP2A that targets TSC2 as a substrate. PP2A represses the level of TSC2 phosphorylation and maintains TSC2 in an activated state. Activated TSC2 negatively regulates the downstream kinase S6K1 and inhibits G(1)/S transit. Knockdown of TSC2 enables fibroblasts to overcome the anti-proliferative properties of polymerized collagen. Furthermore, we show that this reduction in TSC2 and S6K1 phosphorylation occurs largely independent of Akt. Although S6K1 activity was markedly suppressed by polymerized collagen, we found that minimal changes in Akt activity occurred. We demonstrate that up-regulation of Akt by overexpression of constitutively active phosphatidylinositol 3-kinase p110 subunit had minor effects on TSC2 and S6K1 phosphorylation. These findings demonstrate that polymerized collagen represses fibroblast proliferation by a mechanism involving the formation of a beta1 integrin-PP2A-TSC2 complex that negatively regulates S6K1 and inhibits G(1)/S phase transition.     

Effects of PEGylated scFv antibodies against Plasmodium vivax duffy binding protein on the biological activity and stability in vitro

Duffy binding protein (DBP) plays a critical role in Plasmodium vivax invasion of human red blood cells. We previously reported a single-chain antibody fragment (scFv) that was specific to P. vivax DBP (PvDBP). However, the stabilization and the half-life of scFvs have not been studied. Here, we investigated the effect of PEGylated scFvs on their biological activity and stability in vitro. SDS-PAGE analysis showed that three clones (SFDBII-12, -58, and -92) were formed as dimers (about 70 kDa) with PEGylation. Clone SFDBII-58 gave the highest yield of PEGylated scFv. Binding analysis using BIAcore between DBP and scFv showed that both SFDBII-12 and -58 were decreased approximately by two folds at the level of binding affinity to DBP after PEGylation. However, the SFDBII-92 clone still showed a relatively high level of binding affinity (KD=1.02 x 10(-7) M). Binding inhibition assay showed that PEGylated scFv was still able to competitively bind the PvDBP and play a critical role in inhibiting the interactions between PvDBP protein expressed on the surface of Cos-7 cells and Duffy receptor on the surface of erythrocytes. When both scFvs and their PEGylated counterparts were exposed to trypsin, scFv was completely degraded only after 24 h, whereas 35% of PEGylated scFvs remained intact, maintaining their stability against the proteolytic attack of trypsin until 72 h. Taken together, these results suggest that the PEGylated scFvs retain their stability against proteolytic enzymes in vivo, with no significant loss in their binding affinity to target antigen, DBP.     

Synthesis and antifungal activity of 6-arylamino-phthalazine-5,8-diones and 6,7-bis(arylthio)-phthalazine-5,8-diones

6-Arylamino-phthalazine-5,8-diones and 6,7-bis(arylthio)-phthalazine-5,8-diones were synthesized and tested for in vitro antifungal activity against two pathogenic strains of fungi. Among those tested, many compounds showed good antifungal activity. The results suggest that phthalazine-5,8-diones would be potent antifungal agents.     

Complete Genome Sequence of the Bacteriophages ECBP1 and ECBP2 Isolated from Two Different Escherichia coli Strains

Escherichia coli is recognized as one of the most abundant avian bacterial pathogens. In this study, we report the sequencing by the traditional Sanger method of ECBP1 and ECBP2: bacteriophages that infected two different E. coli strains which might be used as therapeutic agents in combination with alternative antibiotics.     

Development and Testing of a Magnetically Actuated Capsule Endoscopy for Obesity Treatment

Intra-gastric balloons (IGB) have become an efficient and less invasive method for obesity treatment. The use of traditional IGBs require complex insertion tools and flexible endoscopes to place and remove the balloon inside the patient’s stomach, which may cause discomfort and complications to the patient. This paper introduces a new ingestible weight-loss capsule with a magnetically remote-controlled inflatable and deflatable balloon. To inflate the balloon, biocompatible effervescent chemicals are used. As the source of the actuation is provided via external magnetic fields, the magnetic capsule size can be significantly reduced compared to current weight-loss capsules in the literature. In addition, there are no limitations on the power supply. To lose weight, the obese subject needs only to swallow the magnetic capsule with a glass of water. Once the magnetic capsule has reached the patient’s stomach, the balloon will be wirelessly inflated to occupy gastric space and give the feeling of satiety. The balloon can be wirelessly deflated at any time to allow the magnetic capsule to travel down the intestine and exit the body via normal peristalsis. The optimal ratio between the acid and base to provide the desired gas volume is experimentally evaluated and presented. A prototype capsule (9.6mm x 27mm) is developed and experimentally validated in ex-vivo experiments. The unique ease of delivery and expulsion of the proposed magnetic capsule is slated to make this development a good treatment option for people seeking to lose excess weight.