BAsE-Seq: a method for obtaining long viral haplotypes from short sequence reads.

We present a method for obtaining long haplotypes, of over 3 kb in length, using a short-read sequencer, Barcode-directed Assembly for Extra-long Sequences (BAsE-Seq). BAsE-Seq relies on transposing a template-specific barcode onto random segments of the template molecule and assembling the barcoded short reads into complete haplotypes. We applied BAsE-Seq on mixed clones of hepatitis B virus and accurately identified haplotypes occurring at frequencies greater than or equal to 0.4%, with >99.9% specificity. Applying BAsE-Seq to a clinical sample, we obtained over 9,000 viral haplotypes, which provided an unprecedented view of hepatitis B virus population structure during chronic infection. BAsE-Seq is readily applicable for monitoring quasispecies evolution in viral diseases

Engineering the First Chimeric Antibody in Targeting Intracellular PRL-3 Oncoprotein for Cancer Therapy in Mice

Antibodies are considered as ‘magic bullets’ because of their high specificity. It is believed that antibodies are too large to routinely enter the cytosol, thus antibody therapeutic approach has been limited to extracellular or secreted proteins expressed by cancer cells. However, many oncogenic proteins are localized within the cell. To explore the possibility of antibody therapies against intracellular targets, we generated a chimeric antibody targeting the intracellular PRL-3 oncoprotein to assess its antitumor activities in mice. Remarkably, we observed that the PRL-3 chimeric antibody could efficiently and specifically reduce the formation of PRL-3 expressing metastatic tumors. We further found that natural killer (NK) cells were important in mediating the therapeutic effect, which was only observed in a nude mouse model (T-cell deficient), but not in a Severe Combined Immunodeficiency’ (scid) mouse model (B- and T-cell deficient), indicating the anticancer effect also depends on host B-cell activity. Our study involving 377 nude and scid mice suggests that antibodies targeting intracellular proteins can be developed to treat cancer

De Novo Design of a Single Chain Diphenylporphyrin Metalloprotein

We describe the computational design of a single-chain four-helix bundle that noncovalently self-assembles with fully synthetic non-natural porphyrin cofactors. With this strategy, both the electronic structure of the cofactor as well as its protein environment may be varied to explore and modulate the functional and photophysical properties of the assembly. Solution characterization (NMR, UV-vis) of the protein showed that it bound with high specificity to the desired cofactors, suggesting that a uniquely structured protein and well-defined site had indeed been created. This provides a genetically expressed single-chain protein scaffold that will allow highly facile, flexible, and asymmetric variations to enable selective incorporation of different cofactors, surface-immobilization, and introduction of spectroscopic probes

Mechanism and kinetics for both thermal and electrochemical reduction of N_2 catalysed by Ru(0001) based on quantum mechanics

The conversion of N_(2(g)) to NH_(3(g)) is an important industrial process that plays a vital role in sustaining the current human population. This chemical transformation relies heavily on the Haber–Bosch process (N2 thermal reduction, N_2TR), which requires enormous quantities of energy (2% of the world supply) and extreme conditions (200 atm and 500 °C). Alternatively, N_(2(g)) can be reduced to NH_(3(g)) through electrochemical means (N_2ER), which may be a less energy intensive and lower-capital approach since the H atoms come from H_2O not H_2. However, N_2ER efficiency is far from satisfactory. In order to provide the basis for developing a new generation of energy efficient processes, we report the detailed atomistic mechanism and kinetics for N_2ER on Ru(0001) along with a comparison to N2TR. We obtained these results using a new electrochemical model for quantum mechanics (QM) calculations to obtain free energy surfaces for all plausible reaction pathways for N_2ER under a constant electrode potential of 0.0 V_(SHE). For both processes, the elementary steps involve several steps of breaking of the NN bonds, hydrogenation of surface N_2H_X or NH_X, and NH_3 release. We find similar energetics for the NN cleavage steps for both systems. However, the hydrogenation steps are very different, leading to much lower free energy barriers for N_2ER compared to N_2TR. Thus, N_2ER favors an associative route where successive hydrogen atoms are added to N_2 prior to breaking the NN bonds rather than the dissociative route preferred by N_2TR, where the NN bonds are broken first followed by the addition of Hs. Our QM results provide the detailed free energy surfaces for N_2ER and N_2TR, suggesting a strategy for improving the efficiency of N_2ER

Mechanism and kinetics for both thermal and electrochemical reduction of N_2 catalysed by Ru(0001) based on quantum mechanics

The conversion of N_(2(g)) to NH_(3(g)) is an important industrial process that plays a vital role in sustaining the current human population. This chemical transformation relies heavily on the Haber–Bosch process (N2 thermal reduction, N_2TR), which requires enormous quantities of energy (2% of the world supply) and extreme conditions (200 atm and 500 °C). Alternatively, N_(2(g)) can be reduced to NH_(3(g)) through electrochemical means (N_2ER), which may be a less energy intensive and lower-capital approach since the H atoms come from H_2O not H_2. However, N_2ER efficiency is far from satisfactory. In order to provide the basis for developing a new generation of energy efficient processes, we report the detailed atomistic mechanism and kinetics for N_2ER on Ru(0001) along with a comparison to N2TR. We obtained these results using a new electrochemical model for quantum mechanics (QM) calculations to obtain free energy surfaces for all plausible reaction pathways for N_2ER under a constant electrode potential of 0.0 V_(SHE). For both processes, the elementary steps involve several steps of breaking of the NN bonds, hydrogenation of surface N_2H_X or NH_X, and NH_3 release. We find similar energetics for the NN cleavage steps for both systems. However, the hydrogenation steps are very different, leading to much lower free energy barriers for N_2ER compared to N_2TR. Thus, N_2ER favors an associative route where successive hydrogen atoms are added to N_2 prior to breaking the NN bonds rather than the dissociative route preferred by N_2TR, where the NN bonds are broken first followed by the addition of Hs. Our QM results provide the detailed free energy surfaces for N_2ER and N_2TR, suggesting a strategy for improving the efficiency of N_2ER

The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding

The validity of the identification and classification of human cancer using antibodies to detect biomarker proteins depends upon antibody specificity. Antibodies that bind to the tumour-suppressor protein p16INK4a are widely used for cancer diagnosis and research. In this study we examined the specificity of four commercially available anti-p16INK4a antibodies in four immunological applications. The antibodies H-156 and JC8 detected the same 16 kDa protein in western blot and immunoprecipitation tests, whereas the antibody F-12 did not detect any protein in western blot analysis or capture a protein that could be recognised by the H-156 antibody. In immunocytochemistry tests, the antibodies JC8 and H-156 detected a predominately cytoplasmic localised antigen, whose signal was depleted in p16INK4a siRNA experiments. F-12, in contrast, detected a predominately nuclear located antigen and there was no noticeable reduction in this signal after siRNA knockdown. Furthermore in immunohistochemistry tests, F-12 generated a different pattern of staining compared to the JC8 and E6H4 antibodies. These results demonstrate that three out of four commercially available p16INK4a antibodies are specific to, and indicate a mainly cytoplasmic localisation for, the p16INK4a protein. The F-12 antibody, which has been widely used in previous studies, gave different results to the other antibodies and did not demonstrate specificity to human p16INK4a. This work emphasizes the importance of the validation of commercial antibodies, aside to the previously reported use, for the full verification of immunoreaction specificity

Depletion of the IKBKAP ortholog in zebrafish leads to hirschsprung disease-like phenotype

© The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved. AIM: To investigate the role of IKBKAP (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase complex-associated protein) in the development of enteric nervous system (ENS) and Hirschsprung disease (HSCR). METHODS: In this study, we injected a morpholino that blocked the translation of ikbkap protein to 1-cell stage zebrafish embryos. The phenotype in the ENS was analysed by antibody staining of the pan-neuronal marker HuC/D followed by enteric neuron counting. The mean numbers of enteric neurons were compared between the morphant and the control. We also studied the expressions of ret and phox2bb, which are involved in ENS development, in the ikbkap morpholino injected embryos by quantitative reverse transcriptase polymerase chain reaction and compared them with the control. RESULTS: We observed aganglionosis (χ2, P < 0.01) and a reduced number of enteric neurons (38.8 ± 9.9 vs 50.2 ± 17.3, P < 0.05) in the zebrafish embryos injected with ikbkap translation-blocking morpholino (morphant) when compared with the control embryos. Specificity of the morpholino was confirmed by similar results obtained using a second non-overlapping morpholino that blocked the translation of ikbkap. We further studied the morphant by analysing the expression levels of genes involved in ENS development such as ret, phox2bb and sox10, and found that phox2bb, the ortholog of human PHOX2B, was significantly down-regulated (0.51 ± 0.15 vs 1.00 ± 0, P < 0.05). Although we also observed a reduction in the expression of ret, the difference was not significant. CONCLUSION: Loss of IKBKAP contributed to HSCR as demonstrated by functional analysis in zebrafish embryos.Link_to_subscribed_fulltex

PRL3-zumab, a first-in-class humanized antibody for cancer therapy

Novel, tumor-specific drugs are urgently needed for a breakthrough in cancer therapy. Herein, we generated a first-in-class humanized antibody (PRL3-zumab) against PRL-3, an intracellular tumor-associated phosphatase upregulated in multiple human cancers, for unconventional cancer immunotherapies. We focused on gastric cancer (GC), wherein elevated PRL-3 mRNA levels significantly correlated with shortened overall survival of GC patients. PRL-3 protein was overexpressed in 85% of fresh-frozen clinical gastric tumor samples examined but not in patient-matched normal gastric tissues. Using human GC cell lines, we demonstrated that PRL3-zumab specifically blocked PRL-3(+), but not PRL-3(–), orthotopic gastric tumors. In this setting, PRL3-zumab had better therapeutic efficacy as a monotherapy, rather than simultaneous combination with 5-fluorouracil or 5-fluorouracil alone. PRL3-zumab could also prevent PRL-3(+) tumor recurrence. Mechanistically, we found that intracellular PRL-3 antigens could be externalized to become “extracellular oncotargets” that serve as bait for PRL3-zumab binding to potentially bridge and recruit immunocytes into tumor microenvironments for killing effects on cancer cells. In summary, our results document a comprehensive cancer therapeutic approach to specific antibody-targeted therapy against the PRL-3 oncotarget as a case study for developing antibodies against other intracellular targets in drug discovery