A recombinant chimeric spider pyriform-aciniform silk with highly tunable mechanical performance.

Spider silks are natural protein-based biomaterials which are renowned for their mechanical properties and hold great promise for applications ranging from high-performance textiles to regenerative medicine. While some spiders can produce several different types of silks, most spider silk types - including pyriform and aciniform silks - are relatively unstudied. Pyriform and aciniform silks have distinct mechanical behavior and physicochemical properties, with materials produced using combinations of these silks currently unexplored. Here, we introduce an engineered chimeric fusion protein consisting of two repeat units of pyriform (Py) silk followed by two repeat units of aciniform (W) silk named Py2W2. This recombinant ∼86.5 kDa protein is amenable to expression and purification from Escherichia coli and exhibits high α-helicity in a fluorinated acid- and alcohol-based solution used to form a dope for wet-spinning. Wet-spinning enables continuous fiber production and post-spin stretching of the wet-spun fibers in air or following submersion in water or ethanol leads to increases in optical anisotropy, consistent with increased molecular alignment along the fiber axis. Mechanical properties of the fibers vary as a function of post-spin stretching condition, with the highest extensibility and strength observed in air-stretched and ethanol-treated fibers, respectively, with mechanics being superior to fibers spun from either constituent protein alone. Notably, the maximum extensibility obtained (∼157 ± 38 %) is of the same magnitude reported for natural flagelliform silks, the class of spider silk most associated with being stretchable. Interestingly, Py2W2 is also water-compatible, unlike its constituent Py2. Fiber-state secondary structure correlates well with the observed mechanical properties, with depleted α-helicity and increased ß-sheet content in cases of increased strength. Py2W2 fibers thus provide enhanced materials behavior in terms of their mechanics, tunability, and fiber properties, providing new directions for design and development of biomaterials suitable and tunable for disparate applications.

Inhibitory effect and related mechanism of decitabine combined with gemcitabine on proliferation of NK/T cell lymphoma cells.

Background: EBV-associated lymphoma is a neoplasm with a poor prognosis, highly aggressive, and progressive rapidly. There is no standard clinical treatment protocol. Decitabine and gemcitabine are known to have anticancer properties against cells of various cancer, respectively. However, the effect of the combination medication on NK/T cell lymphoma cells and potential mechanisms have not been thoroughly investigated. Methods: Human NK/T cell lymphoma cells NK92MI were treated with decitabine and gemcitabine alone or in combination. Experiments, including the Cell Counting Kit-8 and flow cytometry, were performed to investigate how the combination of decitabine and gemcitabine affects the biological behavior of NK92MI cells in vitro. mRNA sequencing, RT-PCR, and western blotting were used to detect changes in the related signal pathway, mRNA, and protein expressions. Results: Decitabine and gemcitabine significantly inhibited the viability and proliferation of NK92MI cells in a dose-dependent manner. The combination index was less than 1 after treating with two drugs, which was a significant synergistic effect. The decitabine concentration with the best synergistic effect was 4.046 µM, and the gemcitabine concentration was 0.005 µM. Flow cytometry showed that combining two drugs could significantly promote apoptosis and arrest the cell cycle at the S phase. In the combined DAC and GEM group, caspase3 protein levels were higher than in either group alone or the control group. The transcriptome sequence, KEGG, and PPI analysis showed that the differential genes after combined treatment were mainly enriched in signal pathways related to cell proliferation, adhesion, and migration compared with using alone and control groups. Based on the sequencing results, we further investigated the role of DAC and GEM in ferroptosis-related signaling molecules using RT-PCR and Western blot techniques. RT-PCR and western blotting showed that the expression levels of HMOX1 and EBV cleavage gene BRLF1 were higher in the group with combined DAC and GEM than in the group alone and the control group, while the protein and mRNA expression levels of SLC7A11 were lower than the others. In addition, the GPX4 protein expression level in the combination group was lower than in the drug-alone and control groups. In addition, the combination treatment increased the ROS level of NK92MI cells. Conclusion: Our current findings suggested that decitabine had an inhibitory effect on the proliferation of NK92MI cells when co-treated with gemcitabine. This combination may increase the expression of ferroptosis-related signaling molecules, thus inhibiting the proliferation of NK92MI cells. It also promoted apoptosis in NK/T cell lymphoma. For patients with NK/T cell lymphoma, this novel combination may provide clinical benefits.

Photo-functionalized TiO2 film for facile immobilization of EpCAM antibodies and efficient enrichment of circulating tumor cells.

The highly efficient capture of circulating tumor cells (CTCs) in the blood is essential for the screening, treatment, and assessment of the risk of metastasis or recurrence of cancer. Immobilizing specific antibodies, such as EpCAM antibodies, on the material's surface is currently the primary method for efficiently capturing CTCs. However, the strategies for immobilizing antibodies usually have the disadvantages of requiring multiple chemical reagents and a complex pre-treatment process. Herein we developed a simple strategy for the immobilization of EpCAM antibodies without additional chemical reagents. By utilizing the positive charge property of the photo-functionalized titanium dioxide (TiO2), the negatively charged carboxyl terminal of EpCAM antibodies was immobilized by electrostatic interaction, allowing the antibodies to expose the antigen binding site fully. The experimental results showed that the photo-functionalized TiO2 surface had a marked positive charge and super-hydrophilic properties that could immobilize large amounts of EpCAM antibodies and keep excellent activity. CTCs capture experiments in vitro showed that the EpCAM antibodies-modified photo-functionalized TiO2 could efficiently capture CTCs. The results of blood circulation experiments in rabbits showed that the EpCAM antibodies-modified photo-functionalized TiO2 could accurately capture CTCs from the whole body's blood. It was foreseen that the strategy of simple immobilization of EpCAM antibodies based on photo-functionalized TiO2 is expected to serve in the efficient capture of CTCs in the future.

1H, 15N and 13C backbone resonance assignments of the acidic domain of the human MDM2 protein.

The human MDM2 protein regulates the tumor suppressor protein p53 by restricting its transcriptional activity and by promoting p53 degradation. MDM2 is ubiquitously expressed, with its overexpression implicated in many forms of cancer. The inhibitory effects of MDM2 on p53 have been shown to involve its N-terminal p53-binding domain and its C-terminal RING domain. The presence of an intact central acidic domain of MDM2 has also been shown to regulate p53 ubiquitination, with this domain shown to directly interact with the p53 DNA-binding domain to regulate the DNA binding activity of p53. To date, little structural information has been obtained for the MDM2 acidic domain. Thus, to gain insight into the structure and function relationship of this region, we have applied solution-state NMR spectroscopy to characterize the segment of MDM2 spanning residues 215-300. These boundaries for the acidic domain were determined on the basis of consensus observed in multiple sequence alignment. Here, we report the 1H, 15N and 13C backbone and 13Cß chemical shift assignments and steady-state {1H}-15N heteronuclear NOE enhancement factors as a function of residue for the acidic domain of MDM2. We show that this domain exhibits the hallmarks of being a disordered protein, on the basis both of assigned chemical shifts and residue-level backbone dynamics, with localized variation in secondary structure propensity inferred from chemical shift analysis.

Cryptococcus neoformans Prp8 Intein: An In Vivo Target-Based Drug Screening System in Saccharomyces cerevisiae to Identify Protein Splicing Inhibitors and Explore Its Dynamics.

Inteins are genetic mobile elements that are inserted within protein-coding genes, which are usually housekeeping genes. They are transcribed and translated along with the host gene, then catalyze their own splicing out of the host protein, which assumes its functional conformation thereafter. As Prp8 inteins are found in several important fungal pathogens and are absent in mammals, they are considered potential therapeutic targets since inhibiting their splicing would selectively block the maturation of fungal proteins. We developed a target-based drug screening system to evaluate the splicing of Prp8 intein from the yeast pathogen Cryptococcus neoformans (CnePrp8i) using Saccharomyces cerevisiae Ura3 as a non-native host protein. In our heterologous system, intein splicing preserved the full functionality of Ura3. To validate the system for drug screening, we examined cisplatin, which has been described as an intein splicing inhibitor. By using our system, new potential protein splicing inhibitors may be identified and used, in the future, as a new class of drugs for mycosis treatment. Our system also greatly facilitates the visualization of CnePrp8i splicing dynamics in vivo.

The MDMX acidic domain competes with the p53 transactivation domain for MDM2 N-terminal domain binding.

The tumor suppressor protein p53 governs many cellular pathways to control genome integrity, metabolic homeostasis, and cell viability. The critical roles of p53 highlight the importance of proper control over p53 in maintaining normal cellular function, with the negative regulators MDM2 and MDMX playing central roles in regulating p53 activity. The interaction between p53 and either MDM2 or MDMX involves the p53 transactivation domain (p53TD) and the N-terminal domains (NTD) of MDM2 or MDMX. Recently, the acidic domain (AD) of MDMX was found to bind to its own NTD, inhibiting the p53-MDMX interaction. Given the established structural and functional similarity between the MDM2 and MDMX NTDs, we hypothesized that the MDMX AD would also directly bind to MDM2 NTD to inhibit p53-MDM2 interaction. Through solution-state nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we show that the MDMX AD can indeed directly interact with the MDM2 NTD and, as a result, can compete for p53 binding. The MDMX AD is thus able to serve as a regulatory domain to inhibit the MDM2-p53 interaction and may also play a direct role in p53 activation.

1H, 15N and 13C backbone resonance assignments of the acidic domain of the human MDMX protein.

The human MDMX protein, also known as MDM4, plays a pivotal role in regulating the activity of the tumor suppressor protein p53 by restricting p53 transcriptional activity and stimulating the E3 ubiquitin ligase activity of another key regulatory protein, MDM2, to promote p53 degradation. MDMX is ubiquitously expressed in most tissue types and overexpression of MDMX has been implicated in many forms of cancer. MDMX has been shown to require an intact N-terminal p53-binding domain and C-terminal RING domain to exert inhibitory effects on p53. The presence of a tryptophan-rich sequence in the central acidic domain of MDMX has also been implicated in regulating the interaction between MDMX and p53, directly interacting with the p53 DNA-binding domain. To date, little structural information has been obtained for this acidic region of MDMX that encompasses the Trp-rich sequence. In order to gain insight into the structure and function of this region, we have carried out solution-state NMR spectroscopy studies utilizing the segment of MDMX spanning residues 181-300-with bounds specifically chosen through multiple sequence alignment-which encompasses nearly 25% of MDMX. Here, we report the 1H, 15N and 13C backbone chemical shift assignments of the acidic domain of MDMX and show that it exhibits hallmarks of intrinsic disorder and localized variation in inferred secondary structure propensity.

An electrochemical aptasensor for highly sensitive detection of CEA based on exonuclease III and hybrid chain reaction dual signal amplification.

At present, carcinoembryonic antigen (CEA) is considered a broad-spectrum cancer biomarker, and its accurate analysis in clinical samples can assist early cancer diagnosis and treatment. Herein, a novel electrochemical aptasensor has been proposed for CEA detection based on exonuclease III and hybrid chain reaction. The target CEA specifically binds to the aptamer region in hairpin probe 1 (defined as H1) by strong attraction, which leads the rest of the H1 triggering catalytic hairpin assembly to form a high quantity of H1 and hairpin probe 2 (defined as H2) double chain complex (denoted as H1@H2). Subsequently, the exonuclease III digests the complex of H1@H2 and liberates H1 to induce the first signal amplification. Simultaneously, a large number of generated trigger chains initiate a hybrid chain reaction and produce a second signal amplification. This proposed sensor exhibited excellent analytical performance for the detection of CEA, with wide linear range from 10 pg.mL-1 to 100 ng.mL-1 and low limit of detection of 0.84 pg.mL-1. Additionally, the biosensing strategy was successfully verified for direct measurement of CEA in human serum. Therefore, this elaborated sensor provides a new simple method for detecting CEA and exhibits great promise in the early screening of cancer.

Associations of FSHR and LHCGR gene variants with ovarian reserve and clinical pregnancy rates.

RESEARCH QUESTION: Are there any associations between the variants of FSHR c.2039 G>A (p. Ser680Asn, rs6166) and LHCGR c.935A>G (p. Asn312Ser, rs2293275) and ovarian reserve, ovarian response, clinical pregnancy rate and POSEIDON group? DESIGN: A total of 210 infertile women were enrolled in this prospective study. The gene variants were analysed by the Sanger method. The clinical parameters were analysed based on genotypes. RESULTS: The frequency of heterozygous and homozygous G allele for FSHR c.2039 G>A in the low prognosis group was significantly higher than that in other response groups (P = 0.034); there was no significant association between LHCGR c.935 A>G and ovarian response. Moreover, the serum anti-Müllerian hormone (AMH) concentration, antral follicle count (AFC), oocytes retrieved, metaphase II (MII) oocytes and two-pronuclear (2PN) oocytes in patients with AG genotype for FSHR c.2039 G>A were significantly lower than those with AA genotype. The serum LH concentrations and clinical pregnancy rate of fresh embryo transfer in patients with GG genotype for LHCGR c.935 A>G were significantly higher than that of the AG genotype. In POSEIDON analysis, the low prognosis women with AA genotype for FSHR c.2039 G>A were more likely to appear in subgroup 1 (P = 0.038). CONCLUSION: The FSHR c.2039 G>A variant has a significant beneficial influence on ovarian reserve and ovarian response. The LHCGR c.935 A>G variant is associated with increased clinical pregnancy rate of fresh embryo transfer in infertile women. In addition, the low prognosis women with AA genotype for FSHR c.2039 G>A tend to show better ovarian reserve and prognosis.

Site-specific internal protein labeling through trans-splicing.

Atypical S1 and S11 split inteins have been used for N-terminal or C-terminal protein labeling. Here we reported a novel site-specific internal protein labeling method based on two atypical split inteins, Ter DnaE3 S11 and Rma DnaB S1. Protein-peptide trans-splicing activity was first demonstrated in vitro between a short peptide (Flag tag, FLAG) and two recombinant proteins (Maltose binding protein, MBP, and Thioredoxin, Trx) by trans-splicing between MBP-TE3S11N (MBP-N fragment of Ter DnaE3 S11), TE3S11C-FLAG-RBS1N (C fragment of Ter DnaE3 S11-FLAG-N fragment of Rma DnaB S1), and RBS1C-Trx (C fragment of Rma DnaB S1-Trx). To minimize the middle synthetic peptide (TE3S11C-linker-RBS1N), we reduced the number of native extein amino acids, which may play a role in protein trans-splicing. The results showed at least 3 (CKG) native extein amino acids were required for detectable trans-splicing activity. This method was further demonstrated to be effective in facilitating the incorporation of fluorescent probe (FITC) to the internal site of recombinant protein, generating the FITC-labeled protein. Besides the fluorescent group, these two split inteins can also be useful for adding any desirable chemical groups into a protein of interest, which may include biotin, modified and unnatural amino acids, or drug molecules.

A novel missense variant in cathepsin C gene leads to PLS in a Chinese patient: A case report and literature review.

BACKGROUND: Papilon-Lefevre syndrome (PLS; OMIM 245000) is a rare autosomal recessive disease characterized by aggressive periodontitis and palmoplantar keratoderma. The prevalence of PLS in the general population is one to four cases per million. Although the etiology and pathogenic mechanisms underlying PLS remain largely unclear, existing evidence shows loss-of-function mutations of the cathepsin C gene (CTSC; OMIM 602365) could cause PLS. Here we found a novel variant of the CTSC gene in a Chinese PLS family and predicted the effect of the variant on the physic-chemical characters and tertiary structure of the protein. METHODS: The 1-7 coding exons and exon-intron boundaries of CTSC gene of the proband and her family were amplified and sequenced directly, and Chromas was used to read sequencing files. Furthermore, the PolyPhen-2, PROVEAN, and Mutation Taster were utilized to predict the pathogenicity of the variant. Besides, the physic-chemical and structural characters of the protein were analyzed by ProtParam, ProtScale, and SWISS-MODEL. RESULTS: Our study identified a novel homozygous variant c.763T>C (p.Cys255Arg) in exon 6 of the CTSC gene, and it was a likely pathogenic variant as predicted by PolyPhen-2, PROVEAN, and Mutation Taster. Moreover, ProtParam and Protscale revealed the variant increased the isoelectric point and hydrophilicity of the protein, and the SWISS-MODEL analysis suggested the variant was located in a critical domain for protein activity. CONCLUSION: Our study analyzed a Chinese family with PLS and identified a novel missense variant in the CTSC gene. Besides, this study retrospectively summarized 113 variants of CTSC in the world and highlighted the features of 27 CTSC variants in Chinese PLS patients. In addition, this study paid much particular attention to the relationship between CTSC variants and different phenotypes.

Occult Hepatitis B Virus Infection in Maintenance Hemodialysis Patients: Prevalence and Mutations in "a" Determinant.

Background: Occult hepatitis B virus infection (OBI) is defined as undetectable serum hepatitis B surface antigen (HBsAg) with detectable HBV-DNA in the serum or liver. Patients with maintenance hemodialysis (MHD) are at a high risk of OBI. The prevalence of OBI in MHD patients in China is not well evaluated. In this study, we aim to assess the prevalence of OBI in MHD patients in Sichuan Province, Southwest of China and investigate the mutations in the "a" determinant of HBsAg. Methods: A total of 330 patients undergoing MHD at Sichuan Provincial People's Hospital were enrolled. Serum samples were collected for ELISA assay to test the serological markers of HBV infection, real-time PCR assay to identify the presence of HBV-DNA, and nested PCR plus sequencing analysis to investigate the gene mutations. Results: In a total of 330 MHD patients, we found that the prevalence of OBI was 4.2% (7/165) in the test group, 2.1% (7/330) in the overall dialysis cohort. After a follow-up study of 7 MHD patients with OBI for 2 years, 2 (isolated HBcAb+) of them were still detectable for HBV-DNA. By sequencing analysis, we revealed mutations at the "a" determinant of HBsAg, including Q129R, T131N, M133S, F134L and D144E. The Q129R and M133S mutations were first reported. Conclusions: Our study clarifies the prevalence of OBI in MHD patients in Sichuan Province(4.2% in the test group, 2.1% in the overall dialysis cohort), and demonstrate the mutations of Q129R and M133S in the "a" determinant of HBsAg for the first time.

Prevalence and Genotype Distribution of Human Papillomavirus From 9,182 Individuals Participated Health Examination.

BACKGROUND: Human papillomavirus (HPV) is the cause of nearly all cervical cancers and the primary cause of anal cancers. Prevalence of HPV varies largely among countries and regions, and population-based data are largely insufficient. The aim of this study is to determine the prevalence and genotype distribution of HPV infection among the women received a general health check. METHODS: In the years 2015, 2016, and 2017, a total of 553,654 individuals received a general health check in the Sichuan Provincial People's Hospital. Among them, 9,182 unselected and asymptomatic individuals received the HPV screening test. Samples of exfoliated endocervical cells were collected and DNA isolation was performed with a Cell Lysis Kit. Fragments of HPV DNA were amplified by PCR. Twenty-one different HPV genotypes, including HPV 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 68, and CP8304, were detected from PCR products using a GenoArray Diagnostic Hybridization Kit. HPV genotype was read on the colored position on the array. RESULTS: A total of 1,207 individuals were positive for at least one HPV genotype, giving a crude prevalence of 13.2% (95% CI: 12.5 - 13.9%). The prevalence did not differ much among age groups. HPV-positive individuals were 291, 389, and 527 in 2015, 2016, and 2017, respectively. The majority of the HPV-positive participants (960/1,207 = 80%) had one type of virus. Approximately 15% had two genotypes of HPV. One individual had HPV of 6 different genotypes, including 16, 18, 52, 53, 56, and CP8304. The most frequent genotype was 52, followed by CP8304, 58, and 53. The oncogenic types 16 and 18 were found in 112 and 52 participants, corresponding to a prevalence of 0.9% (CI: 0.8 - 1.1%) and 0.4% (CI: 0.3 - 0.6%), respectively, for the 9,182 individuals included in this study. CONCLUSIONS: The prevalence of 13.2% for HPV among unselected and asymptomatic individuals who received a general health check is high in the Sichuan area. Identification of high-risk HPV types is essential for preventing or early detection of cervical cancers and consequently save life.

Long-term treatment with 3, 4-methylenedioxymethamphetamine caused retina damage in C57BL/6 mice / Tratamento a longo prazo com 3, 4-metilenedioximetanfetamina causando dano na retina em camundongos C57BL/6

ABSTRACT Purpose: As a class of psychostimulant drugs, amphetamines are widely abused for their stimulant, euphoric, and hallucinogenic properties. Many of these effects result from acute increases in dopamine and serotonin neurotransmission. Following the onset of these effects, 3,4 methylenedioxymethamphetamine produces persistent damage to dopamine and serotonin nerve terminals, resulting in long-lasting neurotoxicity. The purpose of this investigation was to assess the effects of treatment with low dose of methylenedioxymethamphetamine on retinal function of C57BL/6 mice and its underlying mechanisms. Methods: C57BL/6 mice were divided randomly into two groups (n=10): one group was treated with phosphate buffered saline by intraperitoneal injection daily; the other group was treated with 1 mg/kg methylenedioxymethamphetamine by intraperitoneal injection daily for three months. Electroretinography was used to test retinal function every month. H&E staining and terminal deoxynucleotidyl transferase assay were used to evaluate the retinal morphology and histology. Enzyme-linked immunosorbent assay assays were used to measure markers of oxidative stress and inflammatory factors. Gene and protein expression was detected by real-time PCR and western blot. Results: Three-month treatment with methylenedioxymethamphetamine induced significant retinal dysfunction via photoreceptor cell apoptosis by oxidative stress and inflammatory responses. Conclusions: These results suggest that long-term treatment with methylenedioxymethamphetamine increases inflammatory responses in photoreceptor cells resulting in retinal dysfunction in C57BL/6 mice. Thus, this investigation provides preclinical rationale for the retina damage caused by the methylenedioxymethamphetamine abuse.

RESUMO Objetivos: Como uma classe de drogas psicoesti mulantes, as anfetaminas são amplamente usadas por suas propriedades estimulantes, eufóricas e alucinógenas. Muitos desses efeitos resultam de aumentos agudos na neurotransmissão da dopamina e da serotonina. Após o início desses efeitos, a 3,4-metilenedioximetanfetamina produz danos persistentes nos terminais nervosos de dopamina e serotonina, resultando em neurotoxicidade duradoura. O objetivo desta investigação foi avaliar os efeitos do tratamento baixa dose de metilenedioximetanfetamina na função da retina em camundongos C57BL/6 e seus mecanismos subjacentes. Métodos: Camundongos C57BL/6 foram divididos aleatoriamente em dois grupos (n=10): um grupo foi tratado com solução salina tamponada de fosfato por injeção intraperitoneal diária; o outro grupo foi tratado com 1 mg/kg de metilenedioximetanfetamina por injeção intraperitoneal diária durante 3 meses. Eletroretinografia foi utilizada para testar a função da retina a cada mês. A coloração H&E e análise com deoxinucleotidil terminal transferase foram utilizados para avaliar a morfologia e histologia da retina. Testes de imunoabsorção enzimática foram utilizados para medir marcadores de estresse oxidativo e fatores inflamatórios. A expressão de genes e proteínas foi detectada por PCR em tempo real e western blot. Resultados: O tratamento de três meses com metilenedioximetanfetamina induziu disfunção de retina significativa por apoptose de células fotorreceptoras por estresse oxidativo e resposta inflamatória. Conclusões: Estes resultados sugerem que o tratamento a longo prazo com metilenedioximetanfetamina aumenta as respostas inflamatórias em células fotorreceptoras, resultando em disfunção de retina em camundongos C57BL/6. Assim, a investigação foence uma justificação pré-clínica para os danos na retina causados pelo abuso de metilenedioximetanfetamina.

Structure, amphipathy, and topology of the membrane-proximal helix 8 influence apelin receptor plasma membrane localization.

G-protein coupled receptors (GPCRs) typically have an amphipathic helix ("helix 8") immediately C-terminal to the transmembrane helical bundle. To date, a number of functional roles have been associated with GPCR helix 8 segments, but structure-function analysis for this region remains limited. Here, we examine helix 8 of the apelin receptor (AR or APJ), a class A GPCR with wide physiological and pathophysiological relevance. The 71 residue C-terminal tail of the AR is primarily intrinsically disordered, with a detergent micelle-induced increase in helical character. This helicity was localized to the helix 8 region, in good agreement with the recent AR crystal structure. A series of helix 8 mutants were made to reduce helicity, remove amphipathy, or flip the hydrophobic and hydrophilic faces. Each mutant AR was tested both biophysically, in the isolated C-terminal tail, and functionally in HEK 293 T cells, for full-length AR. In all instances, micelle interactions were maintained, and steady-state AR expression was efficient. However, removal of amphipathy or helical character led to a significant decrease in cell surface localization. Flipping of helix 8 amphipathic topology restored cell surface localization to some degree, but still was significantly reduced relative to wild-type. Structural integrity, amphipathy to drive membrane association, and correct topology of helix 8 membrane association all thus appear important for cell surface localization of the AR. This behavior correlates well to GPCR C-terminal tail sequence motifs, implying that these serve to specify key topological features of helix 8 and its proximity to the transmembrane domain.

Analysis of the Full-Length Pyriform Spidroin Gene Sequence.

Spiders often produce multiple types of silk, each with unique properties suiting them to certain tasks and biological functions. Orb-weaver spiders can generate more than six types of silk fibroins, with pyriform silk used to form attachment discs, adhering silk to other surfaces and substances. The unique higher-order structuring of silk fibroins has been cited as the source of their remarkable biomechanical properties. Even so, only one full-length gene sequence of pyriform silk protein 1 (PySp1) from Argiopeargentata has been reported, and studies on the mechanical properties of natural pyriform silk fibers are also lacking. To better understand the PySp1 family of genes, we used long-distance PCR (LD-PCR) to determine the sequence of PySp1 in the Araneusventricosus species. This full-length PySp1 gene is 11,931 bp in length, encoding for 3976 amino acids residues in non-repetitive N- and C-terminal domains with a central largely repetitive region made up of sixteen remarkably homogeneous units. This was similar to the previously reported A. argentata PySp1 sequence, with PySp1 from A. ventricosus also having a long repetitive N-linker that bridges the N-terminal and repetitive regions. Predictions of secondary structure and hydrophobicity of A. ventricosus PySp1 showed the pyriform silk fiber's functional properties. The amino acid compositions of PySp1 is obviously distinct from other spidroins. Our sequence makes an important contribution to understand pyriform silk protein structure and also provides a new template for recombinant pyriform silk proteins with attractive properties.

A Novel PAX6 Heterozygous Mutation Found in a Chinese Family with Congenital Aniridia.

Purpose: Congenital aniridia is a kind of panocular disorder characterized by the absence of the iris in both eyes. Paired box 6 (PAX6) gene mutations have been identified as the most common cause of congenital aniridia. The aim of this study was to reveal the genetic defect in PAX6 in a Chinese family with congenital aniridia. Methods: Twelve individuals from a three-generation Chinese family were recruited. All the family members underwent comprehensive ophthalmologic examinations. The entire coding region of PAX6 was amplified by polymerase chain reaction, followed by direct Sanger sequencing. Possible structural and functional changes of protein were predicted by bioinformatic analysis using SIFT and Polyohen-2. Results: Among all the 12 members, four were clinically diagnosed with congenital aniridia. A novel heterozygous mutation c.275G>A (p.R92Q) in exon 6 of PAX6 was identified in all the patients, but not in the unaffected individuals or 1186 healthy subjects. This missense mutation is a G-A transition, converting Arginine (R) to Glutamine (Q) at amino acid 92. The substitution of amino acid in the PAX6 protein changed the local charge density and was predicted to damage the normal protein function. Conclusions: Our study identified a novel mutation of PAX6 responsible for congenital aniridia in a Chinese family, which may contribute to understanding the molecular basis and clinical diagnosis of congenital aniridia.

Long-term treatment with 3,4-methylenedioxymethamphetamine caused retina damage in C57BL/6 mice.

PURPOSE: As a class of psychostimulant drugs, amphetamines are widely abused for their stimulant, euphoric, and hallucinogenic properties. Many of these effects result from acute increases in dopamine and serotonin neurotransmission. Following the onset of these effects, 3,4 methylenedioxymethamphetamine produces persistent damage to dopamine and serotonin nerve terminals, resulting in long-lasting neurotoxicity. The purpose of this investigation was to assess the effects of treatment with low dose of methylenedioxymethamphetamine on retinal function of C57BL/6 mice and its underlying mechanisms. METHODS: C57BL/6 mice were divided randomly into two groups (n=10): one group was treated with phosphate buffered saline by intraperitoneal injection daily; the other group was treated with 1 mg/kg methylenedioxymethamphetamine by intraperitoneal injection daily for three months. Electroretinography was used to test retinal function every month. H&E staining and terminal deoxynucleotidyl transferase assay were used to evaluate the retinal morphology and histology. Enzyme-linked immunosorbent assay assays were used to measure markers of oxidative stress and inflammatory factors. Gene and protein expression was detected by real-time PCR and western blot. RESULTS: Three-month treatment with methylenedioxymethamphetamine induced significant retinal dysfunction via photoreceptor cell apoptosis by oxidative stress and inflammatory responses. CONCLUSIONS: These results suggest that long-term treatment with methylenedioxymethamphetamine increases inflammatory responses in photoreceptor cells resulting in retinal dysfunction in C57BL/6 mice. Thus, this investigation provides preclinical rationale for the retina damage caused by the methylenedioxymethamphetamine abuse.

Engineered Ssp DnaX inteins for protein splicing with flanking proline residues.

Inteins are internal protein sequences capable of catalyzing a protein splicing reaction by self-excising from a precursor protein and simultaneously joining the flanking sequences with a peptide bond. Split inteins have separate pieces (N-intein and C-intein) that reassemble non-covalently to catalyze a protein trans-splicing reaction joining two polypeptides. Protein splicing has become increasingly useful tools in many fields of biological research and biotechnology. However, natural and engineered inteins have failed previously to function when being flanked by proline residue at the -1 or +2 positions, which limits general uses of inteins. In this study, different engineered inteins were tested. We found that engineered Ssp DnaX mini-intein and split inteins could carry out protein splicing with proline at the +2 positions or at both -1 and +2 positions. Under in vivo conditions in E. coli cells, the mini-intein, S1 split intein, and S11 split intein spliced efficiently, whereas the S0 split intein did not splice with proline at both -1 and +2 positions. The S1 and S11 split inteins also trans-spliced efficiently in vitro with proline at the +2 positions or at both -1 and +2 positions, but the S0 split intein trans-spliced inefficiently with proline at the +2 position and did not trans-splice with proline at both -1 and +2 positions. These findings contribute significantly to the toolbox of intein-based technologies by allowing the use of inteins in proteins having proline at the splicing point.

Recombinant Silk Fiber Properties Correlate to Prefibrillar Self-Assembly.

Spider silks are desirable materials with mechanical properties superior to most synthetic materials coupled with biodegradability and biocompatibility. In order to replicate natural silk properties using recombinant spider silk proteins (spidroins) and wet-spinning methods, the focus to date has typically been on modifying protein sequence, protein size, and spinning conditions. Here, an alternative approach is demonstrated. Namely, using the same ≈57 kDa recombinant aciniform silk protein with a consistent wet-spinning protocol, fiber mechanical properties are shown to significantly differ as a function of the solvent used to dissolve the protein at high concentration (the "spinning dope" solution). A fluorinated acid/alcohol/water dope leads to drastic improvement in fibrillar extensibility and, correspondingly, toughness compared to fibers produced using a previously developed fluorinated alcohol/water dope. To understand the underlying cause for these mechanical differences, morphology and structure of the two classes of silk fiber are compared, with features tracing back to dope-state protein structuring and preassembly. Specifically, distinct classes of spidroin nanoparticles appear to form in each dope prior to fiber spinning and these preassembled states are, in turn, linked to fiber morphology, structure, and mechanical properties. Tailoring of dope-state spidroin nanoparticle assembly, thus, appears a promising strategy to modulate fibrillar silk properties.