Impact of Diffusion Barriers to Small Cytotoxic Molecules on the Efficacy of Immunotherapy in Breast Cancer

Molecular-focused cancer therapies, e.g., molecularly targeted therapy and immunotherapy, so far demonstrate only limited efficacy in cancer patients. We hypothesize that underestimating the role of biophysical factors that impact the delivery of drugs or cytotoxic cells to the target sites (for associated preferential cytotoxicity or cell signaling modulation) may be responsible for the poor clinical outcome. Therefore, instead of focusing exclusively on the investigation of molecular mechanisms in cancer cells, convection-diffusion of cytotoxic molecules and migration of cancer-killing cells within tumor tissue should be taken into account to improve therapeutic effectiveness. To test this hypothesis, we have developed a mathematical model of the interstitial diffusion and uptake of small cytotoxic molecules secreted by T-cells, which is capable of predicting breast cancer growth inhibition as measured both in vitro and in vivo. Our analysis shows that diffusion barriers of cytotoxic molecules conspire with γδ T-cell scarcity in tissue to limit the inhibitory effects of γδ T-cells on cancer cells. This may increase the necessary ratios of γδ T-cells to cancer cells within tissue to unrealistic values for having an intended therapeutic effect, and decrease the effectiveness of the immunotherapeutic treatment.     

Bacteriophage Receptor Binding Protein Based Assays for the Simultaneous Detection of Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni and Campylobacter coli are the most common bacterial causes of foodborne gastroenteritis which is occasionally followed by a debilitating neuropathy known as Guillain-Barré syndrome. Rapid and specific detection of these pathogens is very important for effective control and quick treatment of infection. Most of the diagnostics available for these organisms are time consuming and require technical expertise with expensive instruments and reagents to perform. Bacteriophages bind to their host specifically through their receptor binding proteins (RBPs), which can be exploited for pathogen detection. We recently sequenced the genome of C. jejuni phage NCTC12673 and identified its putative host receptor binding protein, Gp047. In the current study, we localized the receptor binding domain to the C-terminal quarter of Gp047. CC-Gp047 could be produced recombinantly and was capable of agglutinating both C. jejuni and C. coli cells unlike the host range of the parent phage which is limited to a subset of C. jejuni isolates. The agglutination procedure could be performed within minutes on a glass slide at room temperature and was not hindered by the presence of buffers or nutrient media. This agglutination assay showed 100% specificity and the sensitivity was 95% for C. jejuni (n = 40) and 90% for C. coli (n = 19). CC-Gp047 was also expressed as a fusion with enhanced green fluorescent protein (EGFP). Chimeric EGFP_CC-Gp047 was able to specifically label C. jejuni and C. coli cells in mixed cultures allowing for the detection of these pathogens by fluorescent microscopy. This study describes a simple and rapid method for the detection of C. jejuni and C. coli using engineered phage RBPs and offers a promising new diagnostics platform for healthcare and surveillance laboratories.     

Rosiglitazone Treatment of Type 2 Diabetic db/db Mice Attenuates Urinary Albumin and Angiotensin Converting Enzyme 2 Excretion

Alterations within the renal renin angiotensin system play a pivotal role in the development and progression of cardiovascular and renal disease. Angiotensin converting enzyme 2 (ACE2) is highly expressed in renal tubules and has been shown to be renoprotective in diabetes. The protease, a disintegrin and metalloprotease (ADAM) 17, is involved in the ectodomain shedding of several transmembrane proteins including ACE2. Renal ACE2 and ADAM17 were significantly increased in db/db mice compared to controls. We investigated the effect of the insulin sensitizer, rosiglitazone, on albuminuria, renal ADAM17 protein expression and ACE2 shedding in db/db diabetic mice. Rosiglitazone treatment of db/db mice normalized hyperglycemia, attenuated renal injury and decreased urinary ACE2 and renal ADAM17 protein expression. Urinary excreted ACE2 is enzymatically active. Western blot analysis of urinary ACE2 demonstrated two prominent immunoreactive bands at approximately 70 & 90 kDa. The predominant immunoreactive band is approximately 20 kDa shorter than the one demonstrated for kidney lysate, indicating possible ectodomain shedding of active renal ACE2 in the urine. Therefore, it is tempting to speculate that renoprotection of rosiglitazone could be partially mediated via downregulation of renal ADAM17 and ACE2 shedding. In addition, there was a positive correlation between blood glucose, urinary albumin, plasma glucagon, and triglyceride levels with urinary ACE2 excretion. In conclusion, urinary ACE2 could be used as a sensitive biomarker of diabetic nephropathy and for monitoring the effectiveness of renoprotective medication.     

Molten Globule of Hemoglobin Proceeds into Aggregates and Advanced Glycated End Products

Conformational alterations of bovine hemoglobin (Hb) upon sequential addition of glyoxal over a range of 0–90% v/v were investigated. At 20% v/v glyoxal, molten globule (MG) state of Hb was observed by altered tryptophan fluorescence, high ANS binding, existence of intact heme, native-like secondary structure as depicted by far-UV circular dichroism (CD) and ATR-FTIR spectra as well as loss in tertiary structure as confirmed by near-UV CD spectra. In addition, size exclusion chromatography analysis depicted that MG state at 20% v/v glyoxal corresponded to expanded pre-dissociated dimers. Aggregates of Hb were detected at 70% v/v glyoxal. These aggregates of Hb had altered tryptophan environment, low ANS binding, exposed heme, increased β-sheet secondary structure, loss in tertiary structure, enhanced thioflavin T (ThT) fluorescence and red shifted Congo Red (CR) absorbance. On incubating Hb with 30% v/v glyoxal for 0–20 days, advanced glycation end products (AGEs) were detected on day 20. These AGEs were characterised by enhanced tryptophan fluorescence at 450 nm, exposure of heme, increase in intermolecular β-sheets, enhanced ThT fluorescence and red shift in CR absorbance. Comet assay revealed aggregates and AGEs to be genotoxic in nature. Scanning electron microscopy confirmed the amorphous structure of aggregates and branched fibrils of AGEs. The transformation of α-helix to β-sheet usually alters the normal protein to amyloidogenic resulting in a variety of protein conformational disorders such as diabetes, prion and Huntington''s.     

Genome-Wide Identification,Characterization and Phylogenetic Analysis of ATP-Binding Cassette (ABC) Transporter Genes in Common Carp (Cyprinus carpio)

The ATP-binding cassette (ABC) gene family is considered to be one of the largest gene families in all forms of prokaryotic and eukaryotic life. Although the ABC transporter genes have been annotated in some species, detailed information about the ABC superfamily and the evolutionary characterization of ABC genes in common carp (Cyprinus carpio) are still unclear. In this research, we identified 61 ABC transporter genes in the common carp genome. Phylogenetic analysis revealed that they could be classified into seven subfamilies, namely 11 ABCAs, six ABCBs, 19 ABCCs, eight ABCDs, two ABCEs, four ABCFs, and 11 ABCGs. Comparative analysis of the ABC genes in seven vertebrate species including common carp, showed that at least 10 common carp genes were retained from the third round of whole genome duplication, while 12 duplicated ABC genes may have come from the fourth round of whole genome duplication. Gene losses were also observed for 14 ABC genes. Expression profiles of the 61 ABC genes in six common carp tissues (brain, heart, spleen, kidney, intestine, and gill) revealed extensive functional divergence among the ABC genes. Different copies of some genes had tissue-specific expression patterns, which may indicate some gene function specialization. This study provides essential genomic resources for future studies in common carp.     

The Sublethal Effects of β-Ecdysterone,a Highly Active Compound from Achyranthes bidentata Blume,on Grape Phylloxera,Daktulosphaira vitifoliae Fitch

Grape phylloxera, Daktulosphaira vitifoliae (Fitch) (Hemiptera, Phylloxeridae), is a very destructive insect pest of grapevines. Intercropping of Achyranthes bidentata Blume (f. Amaranthaceae) and Vitis spp. grapevines can be useful to control this pest. In the present study, the toxicity of 22 compounds, known to be present in A. bidentata, to grape phylloxera was evaluated. All treatments were toxic towards grape phylloxera but the degree of toxicity differed between treatments. Among the 22 tested compounds, several of which proved toxic towards grape phylloxera. However β-ecdysterone had higher toxic effects against grape phylloxera, with LC₅₀ values of 175.73 mg a.i. liter^-1. In addition, we assessed the sublethal effects of LC₁₀, LC₂₀ and LC₄₀ of β-ecdysterone on grape phylloxera. The fourth instar and adult developmental periods and total life span were significantly prolonged by LC₄₀ of β-ecdysterone. Fecundity decreased when grape phylloxera were exposed to LC₂₀ and LC₄₀ of β-ecdysterone. In addition, LC₄₀ of β-ecdysterone decreased the intrinsic rate of increase (r_m) and the finite rate of increase (λ) and prolonged the population doubling time (DT). The net reproductive rate (R₀) was significantly reduced by both the LC₂₀ and LC₄₀ β-ecdysterone treatments. Our results demonstrated that β-ecdysterone had higher toxic effects and significant sublethal effects on grape phylloxera, and showed potential control of grape phylloxera.