Computational chemistry approaches in studies on industrial enzymes / 生物工程学报

We review major computational chemistry techniques applied in industrial enzyme studies, especially approaches intended for guiding enzyme engineering. These include molecular mechanics force field and molecular dynamics simulation, quantum mechanical and combined quantum mechanical/molecular mechanical approaches, electrostatic continuum models, molecular docking, etc. These approaches are essentially introduced from the following two angles for viewing: one is about the methods themselves, including the basic concepts, the primary computational results, and potential advantages and limitations; the other is about obtaining valuable information from the respective calculations to guide the design of mutants and mutant libraries.

Englerin A-sensing charged residues for transient receptor potential canonical 5 channel activation

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or GTPγS. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.

A De Novo RAPGEF2 Variant Identified in a Sporadic Amyotrophic Lateral Sclerosis Patient Impairs Microtubule Stability and Axonal Mitochondria Distribution

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is frequently linked to microtubule abnormalities and mitochondrial trafficking defects. Whole exome sequencing (WES) of patient-parent trios has proven to be an efficient strategy for identifying rare de novo genetic variants responsible for sporadic ALS (sALS). Using a trio-WES approach, we identified a de novo RAPGEF2 variant (c.4069G>A, p.E1357K) in a patient with early-onset sALS. To assess the pathogenic effects of this variant, we have used patient-derived skin fibroblasts and motor neuron-specific overexpression of the RAPGEF2-E1357K mutant protein in Drosophila. Patient fibroblasts display reduced microtubule stability and defective microtubule network morphology. The intracellular distribution, ultrastructure, and function of mitochondria are also impaired in patient cells. Overexpression of the RAPGEF2 mutant in Drosophila motor neurons reduces the stability of axonal microtubules and disrupts the distribution of mitochondria to distal axons and neuromuscular junction (NMJ) synapses. We also show that the recruitment of the pro-apoptotic protein BCL2-associated X (BAX) to mitochondria is significantly increased in patient fibroblasts compared with control cells. Finally, increasing microtubule stability through pharmacological inhibition of histone deacetylase 6 (HDAC6) rescues defects in the intracellular distribution of mitochondria and BAX. Overall, our data suggest that the RAPGEF2 variant identified in this study can drive ALS-related pathogenic effects through microtubule dysregulation.

Functional Identification of Compound Heterozygous Mutations in the CYP17A1 Gene Resulting in Combined 17α-Hydroxylase/17,20-Lyase Deficiency

BACKGROUND: We previously reported a patient with congenital adrenal hyperplasia (CAH) with compound heterozygous mutations in the cytochrome P450 17A1 (CYP17A1) gene. One allele had a p.His373Leu and the other a new p.Glu383fsX36 mutation. The aim of this study was to investigate the functional properties of a new allele present in a compound heterozygote of CYP17A1. METHODS: To understand how p.His373Leu and p.Glu383fsX36 affect P450c17 enzymatic activity, wild type and mutant CYP17A1 cDNAs were cloned into flag-tagged pcDNA3 vector and introduced into human embryonic kidney cells 293T (HEK293T) cells. Protein expression levels of CYP17A1 were then analyzed. And the activities of 17α-hydroxylase and 17,20-lyase of CYP17A1 were evaluated by measuring the conversion of progesterone to 17α-hydroxyprogesterone and of 17α-hydroxypregnenolone to dehydroepiandrosterone, respectively. In addition a computer model was used to create the three-dimensional structure of the mutant CYP17A1 enzymes. RESULTS: Production of the p.His373Leu mutant protein was significantly lower than that of the wild type protein, and the p.Glu383fsX36 protein was hardly produced. Similarly the enzymatic activity derived from the p.His373Leu mutant vector was significantly lower than that obtained from the wild type vector, and little activity was obtained from the p.Glu383fsX36 vector. Three-dimensional modeling of the enzyme showed that p.His373 was located in region important for heme-binding and proper folding. Neither the p.His373Leu nor the p.Glu383fsX36 mutant protein formed a heme-binding structure. CONCLUSION: Enzyme activity measured in both mutants disappeared completely in both 17α-hydroxylase and 17,20-lyase. This result accounts for the clinical manifestations of the patient with the compound heterozygous CYP17A1 mutations.

Utility of BRAF VE1 Immunohistochemistry as a Screening Tool for Colorectal Cancer Harboring BRAF V600E Mutation

BACKGROUND: BRAF mutation has been recognized as an important biomarker of colorectal cancer (CRC) for targeted therapy and prognosis prediction. However, sequencing for every CRC case is not cost-effective. An antibody specific for BRAF V600E mutant protein has been introduced, and we thus examined the utility of BRAF VE1 immunohistochemistry for evaluating BRAF mutations in CRC. METHODS: Fifty-one BRAF-mutated CRCs and 100 age and sexmatched BRAF wild-type CRCs between 2005 and 2015 were selected from the archives of Asan Medical Center. Tissue microarrays were constructed and stained with BRAF VE1 antibody. RESULTS: Forty-nine of the 51 BRAF-mutant CRCs (96.1%) showed more than moderate cytoplasmic staining, except for two weakly stained cases. Six of 100 BRAF wild-type cases also stained positive with BRAF VE1 antibody; four stained weakly and two stained moderately. Normal colonic crypts showed nonspecific weak staining, and a few CRC cases exhibited moderate nuclear reactivity (3 BRAF-mutant and 10 BRAF wild-type cases). BRAF-mutated CRC patients had higher pathologic stages and worse survival than BRAF wild-type patients. CONCLUSIONS: BRAF VE1 immunohistochemistry showed high sensitivity and specificity, but occasional nonspecific staining in tumor cell nuclei and normal colonic crypts may limit their routine clinical use. Thus, BRAF VE1 immunohistochemistry may be a useful screening tool for BRAF V600E mutation in CRCs, provided that additional sequencing studies can be done to confirm the mutation in BRAF VE1 antibody-positive cases.

Evaluation of the VE1 Antibody in Thyroid Cytology Using Ex Vivo Papillary Thyroid Carcinoma Specimens

BACKGROUND: Recently, VE1, a monoclonal antibody against the BRAFV600E mutant protein, has been investigated in terms of its detection of the BRAFV600E mutation. Although VE1 immunostaining and molecular methods used to assess papillary thyroid carcinoma in surgical specimens are in good agreement, evaluation of VE1 in thyroid cytology samples is rarely performed, and its diagnostic value in cytology has not been well established. In present study, we explored VE1 immunoexpression in cytology samples from ex vivo papillary thyroid carcinoma specimens in order to minimize limitations of low cellularity and sampling/targeting errors originated from thyroid fineneedle aspiration and compared our results with those obtained using the corresponding papillary thyroid carcinoma tissues. METHODS: The VE1 antibody was evaluated in 21 cases of thyroid cytology obtained directly from ex vivo thyroid specimens. VE1 immunostaining was performed using liquid-based cytology, and the results were compared with those obtained using the corresponding tissues. RESULTS: Of 21 cases, 19 classic papillary thyroid carcinomas had BRAFV600E mutations, whereas two follicular variants expressed wild-type BRAF. VE1 immunoexpression varied according to specimen type. In detection of the BRAFV600E mutation, VE1 immunostaining of the surgical specimen exhibited 100% sensitivity and 100% specificity, whereas VE1 immunostaining of the cytology specimen exhibited only 94.7% sensitivity and 0% specificity. CONCLUSIONS: Our data suggest that VE1 immunostaining of a cytology specimen is less specific than that of a surgical specimen for detection of the BRAFV600E mutation, and that VE1 immunostaining of a cytology specimen should be further evaluated and optimized for clinical use.

Immunohistochemical evaluation of mutant p53 protein over-expression in non-mucinous adenocarcinoma in-situ and invasive adenocarcinoma, NOS of lung / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the over-expression of mutant p53 protein in non-mucinous adenocarcinoma in-situ (NMAIS) and invasive adenocarcinoma, NOS of lung.</p><p><b>METHODS</b>Immunohistochemical study for p53 protein was performed on 17 cases of NMAIS and 70 cases of invasive adenocarcinoma, NOS of lung. The difference in p53 over-expression between the two tumor subtypes was analyzed.</p><p><b>RESULTS</b>The over-expression of mutant p53 protein was observed in 0 case (0%) of NMAIS and 37 cases (52.9%) of invasive adenocarcinoma, NOS of lung. The difference was of statistical significance (P = 0.000).</p><p><b>CONCLUSION</b>Mutant p53 protein over-expression may play a role in the progression of NMAIS to invasive adenocarcinoma, NOS.</p>

Development of cell models for high-throughput screening system of Charcot-Marie-Tooth disease type 1

PURPOSE: Charcot-Marie-Tooth disease (CMT) is a peripheral neuropathy mainly divided into CMT type 1 (CMT1) and CMT2 according to the phenotype and genotype. Although molecular pathologies for each genetic causative have not been revealed in CMT2, the correlation between cell death and accumulation of misfolded proteins in the endoplasmic reticulum (ER) of Schwann cells is well documented in CMT1. Establishment of in vitro models of ER stress-mediated Schwann cell death might be useful in developing drug-screening systems for the treatment of CMT1. MATERIALS AND METHODS: To develop high-throughput screening (HTS) systems for CMT1, we generated cell models using transient expression of mutant proteins and chemical induction. RESULTS: Overexpression of wild type and mutant peripheral myelin protein 22 (PMP22) induced ER stress. Similar results were obtained from mutant myelin protein zero (MPZ) proteins. Protein localization revealed that expressed mutant PMP22 and MPZ proteins accumulated in the ER of Schwann cells. Overexpression of wild type and L16P mutant PMP22 also reduced cell viability, implying protein accumulation-mediated ER stress causes cell death. To develop more stable screening systems, we mimicked the ER stress-mediated cell death in Schwann cells using ER stress inducing chemicals. Thapsigargin treatment caused cell death via ER stress in a dose dependent manner, which was measured by expression of ER stress markers. CONCLUSION: We have developed genetically and chemically induced ER stress models using Schwann cells. Application of these models to HTS systems might facilitate the elucidation of molecular pathology and development of therapeutic options for CMT1.

Construction of an iss deleted mutant strain from a native avian pathogenic Escherichia coli O78: K80 and in vitro serum resistance evaluation of mutant

Colibacillosis, caused by different serotypes of avian pathogenic Escherichia coli [APEC], is one of the important diseases in poultry industry. The isolate O78 is the most prevalent serotype of APEC in Iran. One of the APEC virulence factors, increased serum survival [iss] gene, is related to serum resistance. The usual form of colibacillosis in avian is extraintestinal, and serum resistance is applied one way by APEC to reach internal organs; hence, it appears that the control of colibacillosis in poultry regarding the deletion of iss and the construction of a serum sensitive APEC strain is beneficial. Additionally, the knowledge about APEC serum resistance could be extended using mutant strains. The present study was an attempt to generate an iss mutant strain from native APEC-O78 strain |c|1378 and to study the level of serum resistance of native APEC-O78 strain c1378 in comparison with its mutant [APEC-O78 strain c1378|D|iss]. The lambda red recombinase system was utilized to delete iss gene in native APEC-O78 strain c1378. This strain was first transformed with the plasmid pkD46 to introduce the lambda red recombinase system and then the PCR product with sequence homology to the iss gene and a kanamycin resistance marker was transformed into the APEC-O78 strain c1378. Serum sensitivity of mutant and wild type strain was investigated by microtiter test. The generation of mutant was successful and the iss was replaced with kanamycin resistance cassette. Also, it was observed that the mutant was sensitive to serum. However, serum sensitivity of iss deleted mutant was not statistically different from its parents. Application of lambda red recombination could be a simple and useful technique for production of a precisely defined gene deletion. Also, there may be some genes that compensate the activity of iss gene

A novel reporter system monitoring sortase A catalyzed protein ligation efficiency / 生物工程学报

Efforts on directed evolution of sortase A to optimize its catalytic properties have been undertaken and shown the promise. To facilitate screening of sortase A mutants with expected catalytic properties, a novel ligation efficiency monitoring system, including reporter substrates GFP-LPETG and GGGYK-Biotin, was developed. GFP-LPETG, wild type sortase A, and a recently reported high activity sortase A mutant were prepared recombinantly from Escherichia coli BL21 (DE3). Taking advantage of the newly designed reporter system, the ligation efficiency catalyzed by wild type and mutant form of sortase A could be sensitively monitored via SDS-PAGE directly. Consistent with previous report, the mutant sortase A displayed much higher catalytic activity compared to wild type enzyme, indicating the new reporter system is easily and fast handled and sensitive. The application of this reporter system into systemic screening will facilitate future directed optimization of sortase A.

Prokaryotic expression and characterization of truncated mutant capsid protein of genotype IV hepatitis E virus / 生物工程学报

A truncated mutant of the Open Reading Frame 2 (ORF2, aa384-606) was amplified from cDNA of genotype IV hepatitis E virus (HEV) by polymerase chain reaction (PCR), subcloned to expression plasmid pTO-T7, and expressed in Escherichia coli. SDS-PAGE and Western blotting were used to detect and identify the recombinant protein, namely rP24. After washing of inclusion bodies, dissolving in denaturing agents, refoldeding by dialysis, ion exchange chromatography and gel chromatography, dynamic light scatter was used to study the hydrated radius of rP24. Western blotting was applied to detect the immunoreactivity of rP24, and mouse immunity test and indirect enzyme linked immunosorbent assay (ELISA) were applied to evaluate the immunogenicity and the detection rate of HEV positive and negative serum. SDS-PAGE and Western blotting show that rP24 was highly expressed in the form of inclusion bodies after induction, and had strong immunoreactivity to monoclonal antibody (McAb) 15B2. After a multi-step purification of rP24, Western blotting indicated that the purified rP24 also had strong immunoreactivity to neutralizing McAb 8C11 and HEV positive serum, suggesting that rP24 simulated the nature structure of HEV capsid protein. Dynamic light scatter demonstrated that the average hydration radius of purified rP24 was 7.48 nm. The mouse immunity test showed that the purified rP24 also had good immunogenicity, and the period of serum antibodies converted from negative to positive was very short, but the antibodies maintained more than 20 weeks. Indirect ELISA tests showed that the detection rate of was the same as anti-HEV-IgG diagnostic kit (Wan Tai corporation). Taken together, the rP24 simulated the neutralizing epitopes of natural HEV, and had strong immunoreactivity and immunogenicity. It provided a basis for the further investigation of the difference of infection mechanism between genotype I and genotype IV HEV.

Correlation of chromosome 1p and 19q status and expression of R132H mutant IDH1 protein in oligodendroglial tumors / 中华病理学杂志

<p><b>OBJECTIVE</b>To correlate the presence of chromosome 1p/19q deletion with the expression of R132H mutant IDH1 status in oligodendroglial tumors, and to explore molecular markers for predicting chemosensitivity of oligodendroglial tumors.</p><p><b>METHODS</b>The study included 75 oligodendroglial tumors (38 oligodendrogliomas and 37 oligoastrocytomas). Immunohistochemistry was used to detect the expression of R132H mutant IDH1 protein, and fluorescence in situ hybridization (FISH) was employed to detect 1p/19q deletion.</p><p><b>RESULTS</b>Deletion of chromosome 1p and/or 19q was detected in 37 cases (37/75, 49.3%), among which co-deletion of 1p and 19q was seen in 34 cases (closely correlated, P < 0.01). Oligodendrogliomas WHOIIhad a slightly higher deletion rate than oligodendrogliomas WHO III, although without statistical significance. Oligodendrogliomas WHO IIand WHO III had a significantly higher deletion rate of chromosome 1p/19q than oligoastrocytomas WHO II and WHO III (P < 0.05). While combined loss of 1p/19q was always detected in oligodendrogliomas when FISH was positive, isolated 1p or 19q deletion was only found in oligoastrocytomas. The expression of R132H mutant IDH1 was detected in 51 of 75 cases (68.0%), in which oligodendrogliomas had a higher positive rate than oligoastrocytomas. Statistical analysis demonstrated a significant correlation between the expression of R132H mutant IDH1 protein and the presence of combined 1p/19q deletion in oligodendrogliomas (P < 0.05).</p><p><b>CONCLUSIONS</b>A significant correlation was observed between the expression of R132H mutant protein and 1p/19q LOH.Expression of 132H mutant IDH1 protein is the potential biomarker for predicating the presence of 1p/19q deletion in oligodendrogliomas.</p>

IDH1 mutation and MGMT expression in astrocytoma and the relationship with prognosis after radiotherapy / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the correlation between IDH1 mutation, MGMT expression, clinicopathologic features and post-radiotherapy prognosis in patients with astrocytoma.</p><p><b>METHODS</b>Detection of IDH1 mutation and MGMT expression was carried out in 48 cases of astrocytoma (WHO grade II to III) by EnVision method with immunohistochemical staining. Follow-up data, including treatment response and overall survival time, were analyzed.</p><p><b>RESULTS</b>The rates of IDH1 mutation and MGMT expression in astrocytomas were 62.7% (30/48) and 47.9% (23/48), respectively. There was a negative correlation between IDH1 mutation and MGMT expression (r = -0.641, P < 0.01). The age of patients with IDH1 mutation was younger at disease onset. The IDH1 mutation rate in patients with WHO grade II astrocytoma was higher than that in patients with WHO grade III tumor (P < 0.05). The age at onset was an independent factor affecting the expression of mutant IDH1. After radiotherapy, patients with IDH1 mutation+/MGMT- tumor carried a longer overall survival time than patients with IDH1 mutation-/MGMT+ tumor (P < 0.05).</p><p><b>CONCLUSIONS</b>There is a correlation between IDH1 mutation and MGMT expression in WHO grade II to III astrocytoma. Age at onset is an independent factor affecting the expression of mutant IDH1. Tumors with IDH1+/MGMT- pattern show better response to radiotherapy than tumors with IDH1-/MGMT+ pattern. Detection of IDH1 mutation and MGMT protein expression can provide some guidance in choice of treatment modalities in patients with astrocytoma.</p>

Increasing of product specificity of gamma-cyclodextrin by mutating the active domain of alpha-cyclodextrin glucanotransferase from Paenibacillus macerans sp. 602-1 / 生物工程学报

We studied the mutation effect of subsites -3(Lys47), -7(146-152), and cyclization center (Tyr195) in active domain on product specificity of alpha-cyclodextrin glucanotransferase (alpha-CGTase) from Paenibacillus macerans sp. 602-1. The Lys47 was replaced by Thr47 and Tyr195 by Ile195, and the amino acids from 146 to 152 were replaced by Ile (named as delta6). All these mutant alpha-CGTases were actively expressed in E. coli BL21. Compared with the wild-type alpha-CGTase, the starch-degrading activities of all the mutant enzymes were declined. For mutant Y195I, the percentage of alpha-CD was decreased from 68% to 30%, and beta-CD was raised from 22.2% to 33.3%. Interestingly, gamma-CD was increased from 8.9% to 36.7% and became the main product, while the actual yield was increased from 0.4 g/L to 1.1 g/L. Mutant K47T and delta6 still produced alpha-CD as main product though the percentage of beta- and gamma-CD increased. Purified Y195I CGTase showed similar optimum temperature with the wild-type alpha-CGTase, but its optimum pH shifted from 5.0 to 6.0 with better pH stability. In summary, mutant Y195I CGTase has the potential to produce gamma-CD as the main product.

Enhanced SEC2 mutants and their superantigen activities / 生物工程学报

As a superantigen protein, Staphylococcal enterotoxin C2 (SEC2) activates the immune system effectively even in extremely low concentrations, and this property could be applied in adjuvant therapy against tumors and infectious diseases. In order to enhance the superantigen activity of SEC2, the residues at position 102-106 of native SEC2 were substituted for WWH, WWT and WWP by over-lap PCR, and three mutants named ST-1, ST-2 and ST-3 were obtained. Stimulating activity to murine lymphocytes proliferation and inhibiting activity to tumor cell growth of the three mutants were significantly improved compared with the native SEC2. Febrile activities of ST-1 and ST-3 were comparable with the native SEC2, but ST-2 showed markedly increased febrile activity than native SEC2. Moreover, the levels of IL-2, IFN-gamma and TNF-alpha secreted by T cells stimulated with the three mutants were significantly improved, which might be the possible reason for enhanced tumor cell growth inhibition activities. Furthermore, mVbeta8.2 gene transcription levels of murine splenocytes stimulated by the three mutants were dramatically increased compared with native SEC2, suggesting their increased affinities to TCR mVbeta8.2 molecular, which might be the main reason for their enhanced superantigen activities.

Key aromatic amino acids of anti-hepatoma activity on Parasporin-2 / 生物工程学报

Nine mutants (P2M1-9) were obtained using PCR with 5-BU based on DNA template (P2Y) encoding the active region of Parasporin-2. Mutant proteins were purified after expressing in E. coli BL21 cells, followed by assayed against hepatoma cells and normal liver cells by MTT. They showed diverse anti-hepatoma activities, in which two mutant proteins, P2M1 and P2M8, exhibited high cytotoxicity against hepatoma cell lines SMMC7721 and Be17402, meanwhile leaving normal liver cells Chang-liver unaffected. Structural comparison among P2Y, P2M1 and P2M8 showed that the length of beta-sheet or beta-fold, and the amount of alpha helix greatly affected the anti-hepatoma activity of Parasporin-2. Results based on amino acid alignment, molecular docking between P2Y, P2M1 or P2M8 and receptor, and mimic mutation demonstrated that amino acid residues at the sites of 52, 56, 58 and 208 on P2Y, especially the aromatic amino acids such as Trp, Phe, and Tyr were involved in the interactions.

Improvement of detection of paternally inherited fetal mutant genes for β-globin in maternal plasma by PNA clamp / 中华血液学杂志

<p><b>OBJECTIVE</b>To study improvement of detection of paternally herited fetal mutant genes for β-globin in maternal plasma by PNA clamp to seek a noninvasive prenatal diagnostic method for β-thalassemia.</p><p><b>METHODS</b>A total of 38 maternal blood samples were collected at 7 to 20 weeks of gestation, samples in which the father carried CD41-42 mutation and mother carried normal gene or the other point mutation for β-thalassemia were examined. The results of fetal DNA in amniotic fluid, cord blood or peripheral blood of newborns were used as the gold standard for comparison. In the study group, the total cell-free DNA was extracted from maternal plasma using QIAamp DNA Blood Mini Kit. After extraction, the total cell-free DNA was separated by agarose gel (1%) electrophoresis, and the cell-free DNA with a size of 100-300 bp was retrieved from the gel slice. Then, the retrieved DNA-free cell underwent PCR amplified with a PNA clamp. The genotype was confirmed by the conventional method (reverse dot blot hybridization), and the results were compared to gold standard. Simultaneously, two control groups with different PCR procedures were set up. The PCR procedure of control group A was amplified with the extracted total cell-free DNA and PNA clamp, and the PCR procedure of control group B was amplified with the retrieved size-fractionated DNA-free cell without PNA clamp.</p><p><b>RESULTS</b>Plasma samples from 38 pregnant women were detected using PCR products for hybridization, the results were compared with the gold standard. Regarding the 21 samples confirmed by gold standard with fetal genotype 41-42M/N, 19, 8, 12 cases were detected as fetal genotype 41-42M in study group, control group A and control group B respectively, the sensitivity was 90.5% (19/21), 38.1% (8/21) and 57.1% (12/21) respectively;Concerning the 17 samples confirmed by gold standard with fetal normal genotype, the amount of false positive cases were 1, 2 and 1 respectively. The respective specificity was 94.1% (16/17), 94.1% (16/17) and 88.2% (15/17) respectively. The respective accuracies were 92.1% (35/38), 63.2% (24/38) and 71.1% (27/38) respectively. The difference in sensitivity and specificity was pairwise compared by means of McNemar's test. There was significant difference between new study group and control group A or control group B (all P﹤0.05).</p><p><b>CONCLUSION</b>The method of detection of paternally inherited fetal mutation genes for β-thalassemia using small size of fetal DNA-free cell in maternal plasma with PNA clamp had several advantages of reliable sensitivity, specificity and accuracy, indicating its potential of clinical practicality.</p>

Sonic hedgehog signaling enhanced the expression of histone demethylase, lysine-specific demethylase 8 in the head and neck squamous cell carcinoma cell line SCC-6 / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To determine whether the sonic hedgehog (Shh) signaling could regulate the expression of histone demethylases in the head and neck squamous cell carcinoma(SCC).</p><p><b>METHODS</b>Human recombinant SHH-N protein or over-expression of the mutant 2 smoothened (M2-SMO) was applied to activate the Shh signaling in tongue squamous cell carcinoma cell line-SCC-6 in this study. Cyclopamine was used to block the Shh signaling in SCC-6. The real-time reverse transcription (RT)-PCR was used to detect the expression of histone demethylases at the mRNA level.</p><p><b>RESULTS</b>The data showed that activation of the Shh signaling up-regulated the expression of histone demethylase, lysine-specific demethylase 8 (KDM-8) at the mRNA level by human recombinant SHH-N protein (1.841 ∼ 3.591 fold compare with untreated group; P < 0.01), over-expression of the M2-SMO also increased the expression of KDM-8 (1.358 ∼ 3.013 fold compared with empty vector group; P < 0.05), and after the Shh signaling was blocked by Cyclopamine, the expression of KDM-8 was down regulated (decreased 25.6% ∼ 66.6% compared with control cells, P < 0.05).</p><p><b>CONCLUSIONS</b>Histone demethylase KDM-8 was downstream target gene of Shh signaling in head and neck squamous cell carcinoma cell line SCC-6, and its expression was positively regulated by the Shh signaling.</p>

Structure and receptor-binding properties of an airborne transmissible avian influenza A virus hemagglutinin H5 (VN1203mut)

Avian influenza A virus continues to pose a global threat with occasional H5N1 human infections, which is emphasized by a recent severe human infection caused by avian-origin H7N9 in China. Luckily these viruses do not transmit efficiently in human populations. With a few amino acid substitutions of the hemagglutinin H5 protein in the laboratory, two H5 mutants have been shown to obtain an air-borne transmission in a mammalian ferret model. Here in this study one of the mutant H5 proteins developed by Kawaoka's group (VN1203mut) was expressed in a baculovirus system and its receptor-binding properties were assessed. We herein show that the VN1203mut had a dramatically reduced binding affinity for the avian α2,3-linkage receptor compared to wild type but showed no detectable increase in affinity for the human α2,6-linkage receptor, using Surface Plasmon Resonance techonology. Further, the crystal structures of the VN1203mut and its complexes with either human or avian receptors demonstrate that the VN1203mut binds the human receptor in the same binding manner (cis conformation) as seen for the HAs of previously reported 1957 and 1968 pandemic influenza viruses. Our receptor binding and crystallographic data shown here further confirm that the ability to bind the avian receptor has to decrease for a higher human receptor binding affinity. As the Q226L substitution is shown important for obtaining human receptor binding, we suspect that the newly emerged H7N9 binds human receptor as H7 has a Q226L substitution.

ULK1 and JNK are involved in mitophagy incurred by LRRK2 G2019S expression

Mutations in LR RK2 (Leucine rich repeat kinase 2) are a major cause of Parkinson's disease (PD). We and others reported recently that expression of the pathogenic gainof-function mutant form of LRRK2, LRRK2 G2019S, induces mitochondrial fission in neurons through DLP1. Here we provide evidence that expression of LRRK2 G2019S stimulates mitochondria loss or mitophagy. We have characterized several LRRK2 interacting proteins and found that LRRK2 interacts with ULK1 which plays an essential role in autophagy. Knockdown of either ULK1 or DLP1 expression with shRNAs suppresses LRRK2 G2019S expression-induced mitochondrial clearance, suggesting that LRRK2 G2019S expression induces mitochondrial fission through DLP1 followed by mitophagy via an ULK1 dependent pathway. In addition to ULK1, we found that LRRK2 interacts with the endogenous MKK4/7, JIP3 and coordinates with them in the activation of JNK signaling. Interestingly, LRRK2 G2019S-induced loss of mitochondria can also be suppressed by 3 different JNK inhibitors, implying the involvement of the JNK pathway in the pathogenic mechanism of mutated LRRK2. Thus our findings may provide an insight into the complicated pathogenesis of PD as well as some clues to the development of novel therapeutic strategies.