Differences in Cellular Clearing Mechanisms of Aggregates of Two Subtypes of HLA-B27

Ankylosing spondylitis (AS) belongs to a group of diseases, called spondyloarthropathies (SpA), that are strongly associated with the genetic marker HLA-B27. AS is characterized by inflammation of joints and primarily affects the spine. Over 160 subtypes of HLA-B27 are known, owing to high polymorphism. Some are strongly associated with disease (e.g., B*2704), whereas others are not (e.g., B*2709). Misfolding of HLA-B27 molecules [as dimers, or as high-molecular-weight (HMW) oligomers] is one of several hypotheses proposed to explain the link between HLA-B27 and AS. Our group has previously established the existence of HMW species of HLA-B27 in AS patients. Still, very little is known about the mechanisms underlying differences in pathogenic outcomes of different HLA-B27 subtypes. We conducted a proteomics-based evaluation of the differential disease association of HLA B*2704 and B*2709, using stable transfectants of genes encoding the two proteins. A clear difference was observed in protein clearance mechanisms: whereas unfolded protein response (UPR), autophagy, and aggresomes were involved in the degradation of B*2704, the endosome–lysosome machinery was primarily involved in B*2709 degradation. These differences offer insights into the differential disease association of B*2704 and B*2709.

Functional analysis of XRCC4 mutations in reported microcephaly and growth defect patients in terms of radiosensitivity

Non-homologous end joining is one of the main pathways for DNA double-strand break (DSB) repair and is also implicated in V(D)J recombination in immune system. Therefore, mutations in non-homologous end-joining (NHEJ) proteins were found to be associated with immunodeficiency in human as well as in model animals. Several human patients with mutations in XRCC4 were reported to exhibit microcephaly and growth defects, but unexpectedly showed normal immune function. Here, to evaluate the functionality of these disease-associated mutations of XRCC4 in terms of radiosensitivity, we generated stable transfectants expressing these mutants in XRCC4-deficient murine M10 cells and measured their radiosensitivity by colony formation assay. V83_S105del, R225X and D254Mfs*68 were expressed at a similar level to wild-type XRCC4, while W43R, R161Q and R275X were expressed at even higher level than wild-type XRCC4. The expression levels of DNA ligase IV in the transfectants with these mutants were comparable to that in the wild-type XRCC4 transfectant. The V83S_S105del transfectant and, to a lesser extent, D254Mfs*68 transfectant, showed substantially increased radiosensitivity compared to the wild-type XRCC4 transfectant. The W43R, R161Q, R225X and R275X transfectants showed a slight but statistically significant increase in radiosensitivity compared to the wild-type XRCC4 transfectant. When expressed as fusion proteins with Green fluorescent protein (GFP), R225X, R275X and D254Mfs*68 localized to the cytoplasm, whereas other mutants localized to the nucleus. These results collectively indicated that the defects of XRCC4 in patients might be mainly due to insufficiency in protein quantity and impaired functionality, underscoring the importance of XRCC4’s DSB repair function in normal development.

Development of a Novel Pipette Tip-Aided Cell Cloning Method for The Effective Isolation of Genome-Edited Porcine Cell

Direct colony cloning of adherent mammalian cells using rings or dilution cloning has been used frequently for obtaining stable transfectants after gene delivery. As an alternative to these methods, successful isolation of the cells in a single colony is possible by placing a trypsin-immersed small paper disk onto the colony and subsequently picking up the paper with the assumption that it carries the trypsinized cells. However, the cloning success using this technique largely relies on the cell type used. In the present study, a novel, simple, and non-invasive technique for the isolation of cells from single colonies using a disposable pipette tip was developed. Using this technique, success was achieved in isolating the clonal populations of genome-edited porcine fibroblastic cells with 100% efficiency after co-transfection with the clustered, regularly interspaced, short palindromic repeats-CRISPR associated protein 9 (CRISPR/Cas9)-based genome editing components [for targeting the porcine GGTA1 that encodes α-1,3-galactosyltransferase (α-GalT)] and the piggyBac-based gene delivery components [to enable efficient chromosomal integration of the transgene carrying the cDNA of enhanced green fluorescent protein (EGFP)]. A toxin-based, drug-free selection system involving saporin (plant toxin)-conjugated BS-I-B4 lectin (IB4SAP) was employed in the present study. Since IB4SAP binds specifically to the cell-surface α-Gal epitope (synthesized by α-GalT), it is supposed that treatment with IB4SAP theoretically eliminates the untransfected or genome-edited porcine cells with a mono-allelic knockout (KO) phenotype, while all the surviving clones have a bi-allelic GGTA1 mutation. A total of 16 clones were isolated in the present study, all of which exhibited loss of the α-Gal epitope (a cell-surface carbohydrate synthesized by α-GalT), suggesting that all the clones had a bi-allelic KO phenotype. Moreover, 75% of these clones expressed EGFP uniformly, while the remainder had mosaic or no EGFP expression. These findings indicate the fidelity of the developed pipette tip-aided cell cloning approach for the efficient isolation of genome-edited porcine fibroblast clones.

CHI3L1 Inhibits Expression of NIS by Activating MEK/ERK1/2 Signal Pathway in Thyroid Cancer

Purpose To study the differentially expressed protein between thyroid cancer and radioactive iodine refractory differentiated thyroid cancer (RR-DTC), which presents highly aggressive and unfavorable prognosis. Meanwhile, to search for a potential radiotherapeutic target for patients suffer from RR-DTC. Methods Totally 6 metastatic lymph nodes of RR-DTC and DTC were collected during lymph node dissection for proteomics studies. Immunohistochemical staining was performed to verify the expression of Chitinase-3-like 1 (CHI3L1) in RR-DTC and DTC tissues. Western blotting was used to detect the expression of sodium-iodine symporter (NIS), MEK, and ERK1/2 in CHI3L1 over-expression stable transfectants, control stable transfectants, and PTC-K1 cells. Results CHI3L1 was demonstrated to be significantly up-regulated in RR-DTC by immunohistochemical staining. Besides, CHI3L1 over expression would inhibit expression of sodium-iodine symporter, a key protein for iodine accumulation, by activating MEK/ERK1/2 signal pathway. Conclusion CHI3L1 might be a potential molecular target for RR-DTC due to its over expression in RR-DTC and membrane location.

Knockdown of RNF2 enhances the radiosensitivity of squamous cell carcinoma in lung

A previous study has reported that knockdown of RING finger protein 2 (RNF2) increases the radiosensitivity of esophageal cancer cells both in vitro and in vivo. However, the effect of RNF2 knockdown on radiosensitivity in squamous cell carcinoma (SqCC) remains unknown. For this, NCI-H226 and SK-MES-1 cells were exposed to X-ray irradiation and then RNF2 levels were determined. RNF2 was knocked-down and stable transfectants were selected. Radiosensitivity, cell proliferation, apoptosis, cell cycle, and γ-H2AX foci formation were evaluated. Interaction among ataxia telangiectasia mutated protein (ATM), mediator of DNA damage checkpoint 1 (MDC1), and H2AX were examined. Xenograft models were used to explore the effect of RNF2 knockdown on radiosensitivity in vivo. The results showed that RNF2 expression was significantly increased by X-ray irradiation. RNF2 knockdown combined with X-ray irradiation markedly inhibited cell proliferation, caused cell cycle arrest at the G1 phase, and induced cell apoptosis. In addition, RNF2 knockdown enhanced the radiosensitivity of SqCC cells, inhibited irradiation-induced γ-H2AX foci formation, and impaired the interactions among ATM, MDC1, and H2AX. Furthermore, combination of RNF2 knockdown and X-ray irradiation suppressed tumor growth and promoted tumor cell apoptosis in vivo. RNF2 may be a new therapeutic target to enhance the radiosensitivity of SqCC cells in lung.

Generation of a Chimeric Antibody against a Synthetic Peptide Derived From IL-23p19 with Potential Therapeutic Application

<p><strong>Aim</strong>: To construct an express a mouse-human chimeric antibody against IL-23p19 using a<br />synthetic peptide as immunogen.</p><p><strong>Methods</strong>: Immunization of mice with a synthetic peptide derived from the IL-23p19 sequence and generation of hybridoma secreting specific antibodies. The chimeric antibody was created using two eukaryotic plasmid constructions; one of them carrying the light mouse-human chain and the other the heavy mouse-human chain. CHO-K1 cells were cotransfected with both plasmids and stable transfectants were grown in selective culture medium.</p><p><strong>Results</strong>: A chimeric version of anti-IL-23p19 was successfully constructed and expressed in eukaryotic cells. The expressed chimeric antibody showed specific recognition not only of the peptide used as immunogen but also the subunit p19 and the complete interleukin Il-23.</p><p><strong>Conclusion</strong>: A Chimeric antibody that was developed against a synthetic peptide, which is able to recognize the parent protein IL23 biologically active, could be developed into a targeted therapy in diseases with chronic inflammation.</p>