Mn-Anti-CTLA4-CREKA-Sericin Nanotheragnostics for Enhanced Magnetic Resonance Imaging and Tumor Immunotherapy.

The integration of magnetic resonance imaging (MRI), cGAS-STING, and anti-CTLA-4 (aCTLA-4) based immunotherapy offers new opportunities for tumor precision therapy. However, the precise delivery of aCTLA-4 and manganese (Mn), an activator of cGAS, to tumors remains a major challenge for enhanced MRI and active immunotherapy. Herein, a theragnostic nanosphere Mn-CREKA-aCTLA-4-SS (MCCS) is prepared by covalently assembling Mn2+, silk sericin (SS), pentapeptide CREKA, and aCTLA-4. MCCS are stable with an average size of 160 nm and is almost negatively charged or neutral at pH 5.5/7.4. T1-weighted images showed MCCS actively targeted tumors to improve the relaxation rate r1 and contrast time of MRI. This studies demonstrated MCCS raises reactive oxygen species levels, activates the cGAS-STING pathway, stimulates effectors CD8+ and CD80+ T cells, reduces regulatory T cell numbers, and increases IFN-γ and granzyme secretion, thereby inducing tumor cells autophagy and apoptosis in vitro and in vivo. Also, MCCS are biocompatible and biosafe. These studies show the great potential of Mn-/SS-based integrative material MCCS for precision and personalized tumor nanotheragnostics.

Impact of a group-based intervention program on physical activity and health-related outcomes in worksite settings.

BACKGROUND: The benefits of physical activity (PA) have been well documented, and the worksite is a promising setting for PA promotion. The aims of this study were as follows: 1. To evaluate the effect of a group-based worksite intervention on PA and health-related outcomes by using pedometers. 2. To examine the associations between the change in vigorous physical activity (VPA)/moderate physical activity (MPA)/walking and health related outcomes. METHODS: A total of 398 participants (221 in the intervention group (IG) and 177 in the control group (CG)) from 17 worksites were recruited for a prospective self-controlled trial of a worksite physical activity intervention program in China. In the IG, a pedometer was utilized to self-monitor the PA, together with group competition, goal setting, and other incentives. No intervention was applied to the CG. Physical activity, sedentary behavior, and health-related outcomes were measured at baseline and immediately after the 100-day period intervention. RESULTS: A total of 262 participants completed the program (68.3% adherence). Adherence in the intervention group was 67.9% (n = 150/221). Improvements between baseline and follow-up among intervention participants were observed in the following parameters: VPA (+ 109.7 METs/week; p < 0.05), walking (+ 209.2 METs/week; p < 0.01), systolic blood pressure (SBP; - 2.1 mmHg; p < 0.01), waist circumference (WC; - 2.3 cm; p < 0.01), body fat percentage (BF); - 1.0%; p < 0.01), and body mass index (BMI; - 0.5 kg/m2; p < 0.01). VPA was related to changes in body fat percentage (p < 0.05) and body mass index (p < 0.05). CONCLUSION: This integrated group-based intervention program contributed to comprehensive improvement in health-related outcomes. The study was useful for establishing associations between change in VPA/MPA/walking and health-related outcomes in a natural setting. Long-term evaluation is required to examine the potential of such an integrated intervention to promote PA. REGISTRATION: This study was prospectively registered in the Chinese Clinical Trial Registry. TRIAL REGISTRATION NUMBER: ChiCTR-1,800,015,529. Date of registration: April 5, 2018.

Rational Design of Ag/ZnO Hybrid Nanoparticles on Sericin/Agarose Composite Film for Enhanced Antimicrobial Applications.

Silver-based hybrid nanomaterials are receiving increasing attention as potential alternatives for traditional antimicrobial agents. Here, we proposed a simple and eco-friendly strategy to efficiently assemble zinc oxide nanoparticles (ZnO) and silver nanoparticles (AgNPs) on sericin-agarose composite film to impart superior antimicrobial activity. Based on a layer-by-layer self-assembly strategy, AgNPs and ZnO were immobilized on sericin-agarose films using the adhesion property of polydopamine. Scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray powder diffraction spectroscopy were used to show the morphology of AgNPs and ZnO on the surface of the composite film and analyze the composition and structure of AgNPs and ZnO, respectively. Water contact angle, swelling ratio, and mechanical property were determined to characterize the hydrophilicity, water absorption ability, and mechanical properties of the composite films. In addition, the antibacterial activity of the composite film was evaluated against Gram-positive and Gram-negative bacteria. The results showed that the composite film not only has desirable hydrophilicity, high water absorption ability, and favorable mechanical properties but also exhibits excellent antimicrobial activity against both Gram-positive and Gram-negative bacteria. It has shown great potential as a novel antimicrobial biomaterial for wound dressing, artificial skin, and tissue engineering.

Factors Associated With Colonoscopy Compliance Based on Health Belief Model in a Community-Based Colorectal Cancer Screening Program Shanghai, China.

Screening can help early detection of colorectal cancer (CRC) in the general population. However, colonoscopy compliance in screening program is low in China. The study aimed to identify factors associated with colonoscopy compliance based on Health Belief Model (HBM). An investigation was conducted in Huangpu District, Shanghai in 2015. High-risk individuals of CRC received an in-person interview with physicians to fill out a questionnaire. The questionnaires assessing predictors of colonoscopy compliance were collected, and status of colonoscopy participation was determined. Univariate and multiple logistic regression analyses were conducted. Among 2,568 high-risk population (20.68%), 531 subjects underwent colonoscopy. Participants with both risk assessment and fecal immunochemical test positive were most likely to undergo colonoscopy. Based on HBM, colonoscopy compliance was positively associated with higher perceived severity (odds ratio [OR] = 1.05, 95% confidence interval [CI] = [1.00, 1.10]). Higher perceived barriers (OR = 0.97, 95% CI = [0.94, 0.99]), without prior colonoscopy (OR = 0.35, 95% CI = [0.26, 0.47]), not knowing anyone who underwent colonoscopy before (OR = 0.74, 95% CI = [0.58, 0.96]), without health-care provider recommendation on colonoscopy (OR = 0.58, 95% CI = [0.44, 0.77]), and without psychosocial support from someone for colonoscopy (OR = 0.27, 95% CI = [0.21, 0.35]) were shown to be associated with colonoscopy noncompliance. The colonoscopy compliance was low in this CRC screening program in Shanghai, China. The influencing factors were positive results in primary screening, perceived severity, perceived barriers, personal or others' experiences in colonoscopy, health-care provider recommendation, and psychosocial supports. Effective education campaign and facilitated communication between health-care providers and high-risk population were suggested in the future interventions.

A Novel Adenosine Kinase from Bombyx mori: Enzymatic Activity, Structure, and Biological Function.

Adenosine kinase (ADK) is the first enzyme in the adenosine remediation pathway that catalyzes adenosine phosphorylation into adenosine monophosphate, thus regulating adenosine homeostasis in cells. To obtain new insights into ADK from Bombyx mori (BmADK), we obtained recombinant BmADK, and analyzed its activity, structure, and function. Gel-filtration showed BmADK was a monomer with molecular weight of approximately 38 kDa. Circular dichroism spectra indicated BmADK had 36.8% α-helix and 29.9% ß-strand structures, respectively. The structure of BmADK was stable in pH 5.0-11.0, and not affected under 30 °C. The melting temperature and the enthalpy and entropy changes in the thermal transition of BmADK were 46.51 ± 0.50 °C, 253.43 ± 0.20 KJ/mol, and 0.79 ± 0.01 KJ/(mol·K), respectively. Site-directed mutagenesis demonstrated G68, S201, E229, and D303 were key amino acids for BmADK structure and activity. In particular, S201A mutation significantly increased the α-helix content of BmADK and its activity. BmADK was located in the cytoplasm and highly expressed in the silk gland during the pre-pupal stage. RNA interference revealed the downregulation of BmADK decreased ATG-8, Caspase-9, Ec-R, E74A, and Br-C expression, indicating it was likely involved in 20E signaling, apoptosis, and autophagy to regulate silk gland degeneration and silkworm metamorphosis. Our study greatly expanded the knowledge on the activity, structure, and role of ADK.

Rational Design and Fabrication of ZnONPs Functionalized Sericin/PVA Antimicrobial Sponge.

The interests of developing antimicrobial biomaterials based on silk sericin from Bombyx mori cocoon, have been shooting up in the last decades. Sericin is a valuable natural protein owing to its hydrophilicity, biodegradability, and biocompatibility. Here, we fabricated a sponge with antibacterial capacities for potential wound dressing application. By co-blending of sericin, polyvinyl alcohol (PVA) and zinc oxide nanoparticles (ZnONPs), the ZnONPs-sericin/PVA composite sponge (ZnONPs-SP) was successfully prepared after freeze-drying. Scanning electron microscopy showed the porous structure of ZnONPs-SP. Energy dispersive spectroscopy indicated the existence of Zn in the sponge. X-ray diffractometry revealed the hexagonal wurtzite structure of ZnONPs. Fourier transform infrared spectroscopy showed the biologic coupling of ZnONPs and sericin resulted in a decrease of α-helix and random coil contents, and an increase of ß-sheet structure in the sponge. The swelling experiment suggested ZnONPs-SP has high porosity, good hydrophilicity, and water absorption capability. The plate bacterial colony counting coupled with growth curve assays demonstrated that the composite sponge has an efficiently bacteriostatic effect against Staphylococcus aureus and Escherichia coli, respectively. Furthermore, the cell compatibility analysis suggested the composite sponge has excellent cytocompatibility on NIH3T3 cells. In all, ZnONPs-SP composite sponge has significant potentials in biomaterials such as wound dressing and tissue engineering.

Polydopamine-Based Surface Modification of ZnO Nanoparticles on Sericin/Polyvinyl Alcohol Composite Film for Antibacterial Application.

Silk sericin (SS) is a type of natural macromolecular protein with excellent hydrophilicity, biocompatibility and biodegradability, but also has very poor mechanical properties. To develop sericin-based wound dressings, we utilized polyvinyl alcohol (PVA) to reinforce the mechanical property of sericin by blending PVA and sericin, then modified zinc oxide nanoparticles (ZnO NPs) on SS/PVA film with the assistance of polydopamine (PDA) to endow SS/PVA film with antibacterial activity. Scanning electron microscopy, energy dispersive spectroscopy and X-ray powder diffraction demonstrated ZnO NPs were well grafted on PDA-SS/PVA film. Fourier transform infrared spectra suggested PDA coating and ZnONPs modification did not alter the structure of sericin and PVA. Water contact angle and swelling tests indicated the excellent hydrophilicity and swellability of ZnO NPs-PDA-SS/PVA composite film. Mass loss analysis showed ZnO NPs-PDA-SS/PVA film had excellent stability. The mechanical performance test suggested the improved tensile strength and elongation at break could meet the requirement of ZnO NPs-PDA-SS/PVA film in biomaterial applications. The antibacterial assay suggested the prepared ZnO NPs-PDA-SS/PVA composite film had a degree of antimicrobial activity against Escherichia coli and Staphylococcus aureus. The excellent hydrophilicity, swellability, stability, mechanical property and antibacterial activity greatly promote the possibility of ZnO NPs-PDA-SS/PVA composite film in antibacterial biomaterials application.

A Convenient, Rapid, Sensitive, and Reliable Spectrophotometric Assay for Adenylate Kinase Activity.

Enzymatic activity assays are essential and critical for the study of enzyme kinetics. Adenylate kinase (Adk) plays a fundamental role in cellular energy and nucleotide homeostasis. To date, assays based on different principles have been used for the determination of Adk activity. Here, we show a spectrophotometric analysis technique to determine Adk activity with bromothymol blue as a pH indicator. We analyzed the effects of substrates and the pH indicator on the assay using orthogonal design and then established the most optimal assay for Adk activity. Subsequently, we evaluated the thermostability of Adk and the inhibitory effect of KCl on Adk activity with this assay. Our results show that this assay is simple, rapid, and precise. It shows great potential as an alternative to the conventional Adk activity assay. Our results also suggest that orthogonal design is an effective approach, which is very suitable for the optimization of complex enzyme reaction conditions.

A rapid and sensitive colorimetric assay for the determination of adenosine kinase activity.

Adenosine kinase (ADK) plays an important role in the growth and development of organisms. A convenient, quick, reliable, sensitive and low-cost assay for ADK activity is of great significance. Here, we found the reaction system with bromothymol blue as the pH indicator had a maximum absorption peak at 614 nm. The absorbance change in 614 nm was positively correlated with the generated hydrogen ions in the reaction catalyzed by ADK. Then, we demonstrated this assay was feasible for ADK activity. Further, we analyzed the effects of buffer, bromothymol blue concentrations on the sensitivity of the assay, and investigated the sensitivity of ADK contents and adenosine concentration on the assay. Finally, we calculated the Km and Vmax of ADK from Bombyx mori with this assay. Our results suggested this assay was quick, convenient, reliable, sensitive and economic for the activity of ADK. It is an excellent alternative for the conventional ADK assays.

Polydopamine-Assisted Silver Nanoparticle Self-Assembly on Sericin/Agar Film for Potential Wound Dressing Application.

Silver nanoparticles (AgNPs) are extensively applied for their broad-spectrum and excellent antibacterial ability in recent years. Polydopamine (PDA) has great advantages for synthesizing large amounts of AgNPs, as it has multiple sites for silver ion binding and phenolic hydroxyl structure to reduce silver ions to AgNPs. Here, we mixed sericin and agar solution and dried at 65 °C to prepare a sericin (SS)/Agar composite film, and then coated polydopamine (PDA) on the surface of SS/Agar film by soaking SS/Agar film into polydopamine solution, subsequently synthesizing high-density AgNPs with the assistance of PDA to yield antibacterial AgNPs-PDA- SS/Agar film. Scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) spectra indicated the successful synthesis of high-density AgNPs on the surface of PDA-SS/Agar film. PDA coating and AgNPs modification did not affect the structure of sericin and agar. Furthermore, water contact angle, water absorption and mechanical property analysis showed that AgNPs-PDA-SS/Agar film had excellent hydrophilicity and proper mechanical properties. Inhibition zone and growth curve assays suggested the prepared film had excellent and long-lasting antibacterial ability. In addition, it had excellent cytocompatibility on the fibroblast NIH/3T3 cells. The film shows great potential as a novel kind of wound dressing.

A Novel AgNPs/Sericin/Agar Film with Enhanced Mechanical Property and Antibacterial Capability.

Silk sericin is a protein from a silkworm's cocoon. It has good biocompatibility, hydrophilicity, bioactivity, and biodegradability. However, sericin could not be used in biomedical materials directly because of its frangible characteristic. To develop multifunctional sericin-based materials for biomedical purposes, we prepared a sericin/agar (SS/agar) composite film through the blending of sericin and agar and repetitive freeze-thawing. Then, we synthesized silver nanoparticles (AgNPs) in situ on the surface of the composite film to endow it with antibacterial activity. Water contact angle, swelling and losing ratio, and mechanical properties analysis indicated that the composite film had excellent mechanical property, hydrophilicity, hygroscopicity, and stability. Scanning electron microscopy and X-ray photoelectron spectroscopy analysis confirmed the successful modification of AgNPs on the composite film. X-ray powder diffraction showed the face-centered cubic structures of the AgNPs. This AgNPs modified composite film exhibited an excellent antibacterial capability against Escherichia coli and Staphylococcus aureus. Our study develops a novel AgNPs/sericin/agar composite film with enhanced mechanical performance and an antimicrobial property for potential biomedical applications.

One-Step Synthesis of Silver Nanoparticles on Polydopamine-Coated Sericin/Polyvinyl Alcohol Composite Films for Potential Antimicrobial Applications.

Silk sericin has great potential as a biomaterial for biomedical applications due to its good hydrophilicity, reactivity, and biodegradability. To develop multifunctional sericin materials for potential antibacterial application, a one-step synthesis method for preparing silver nanoparticles (AgNPs) modified on polydopamine-coated sericin/polyvinyl alcohol (PVA) composite films was developed. Polydopamine (PDA) acted as both metal ion chelating and reducing agent to synthesize AgNPs in situ on the sericin/PVA composite film. Scanning electron microscopy and energy dispersive spectroscopy analysis revealed that polydopamine could effectively facilitate the high-density growth of AgNPs as a 3-D matrix. X-ray diffractometry studies suggested the synthesized AgNPs formed good face-centered cubic crystalline structures. Contact angle measurement and mechanical test indicated AgNPs modified PDA-sericin/PVA composite film had good hydrophilicity and mechanical property. The bacterial growth curve and inhibition zone assays showed the AgNPs modified PDA-sericin/PVA composite film had long-term antibacterial activities. This work develops a new method for the preparation of AgNPs modified PDA-sericin/PVA film with good hydrophilicity, mechanical performance and antibacterial activities for the potential antimicrobial application in biomedicine.

Decoding silkworm spinning programmed by pH and metal ions.

Silk is one of the toughest fibrous materials known despite spun at ambient temperature and pressure with water as a solvent. It is a great challenge to reproduce high-performance artificial fibers comparable to natural silk by bionic for the incomplete understanding of silkworm spinning in vivo. Here, we found that amphipol and digitonin stabilized the structure of natural silk fibroin (NSF) by a large-scale screening in vitro, and then studied the close-to-native ultrastructure and hierarchical assembly of NSF in the silk gland lumen. Our study showed that NSF formed reversible flexible nanofibrils mainly composed of random coils with a sedimentation coefficient of 5.8 S and a diameter of about 4 nm, rather than a micellar or rod-like structure assembled by the aggregation of globular NSF molecules. Metal ions were required for NSF nanofibril formation. The successive pH decrease from posterior silk gland (PSG) to anterior silk gland (ASG) resulted in a gradual increase in NSF hydrophobicity, thus inducing the sol-gelation transition of NSF nanofibrils. NSF nanofibrils were randomly dispersed from PSG to ASG-1, and self-assembled into anisotropic herringbone patterns at ASG-2 near the spinneret ready for silkworm spinning. Our findings reveal the controlled self-assembly mechanism of the multi-scale hierarchical architecture of NSF from nanofibrils to herringbone patterns programmed by metal ions and pH gradient, which provides novel insights into the spinning mechanism of silk-secreting animals and bioinspired design of high-performance fibers.

POUM2 homeostasis regulates intimal remodeling and cells fate in the anterior silk gland of the silkworm.

Tissue/organ remodeling and cells fate determination play key roles in the life cycle of animals. However, they are still poorly understood in insects, especially in the silkworm. The anterior silk gland (ASG) of the silkworm is essential for the formation and performance of silk fibers, but the regulatory mechanism of ASG remodeling and cells fate determination is less known. Here we found that silencing of POUM2 caused shorter ASG length, intimal structural defects, silkworm spinning failure, and the resultant naked pupae death, but cells number was not affected. Cells staining showed that DNA endoreduplication was not affected in the ASG. Transmission electron microscopy and chitin staining showed cuticle proteins and chitin were greatly reduced in the ASG during the molting period. Transcriptional analysis showed the expression profiles of cuticle proteins and chitin synthase were similar to that of POUM2 during the molting period, and POUM2 down-regulation reduced the expression of cuticle proteins, chitin synthase, autophagy and apoptosis-related genes. While the phenotype resulting from POUM2 over-expression was similar to that of POUM2 down-regulation. Cells staining revealed marked cells apoptosis with cells number reduction and inhibition of DNA endoreduplication in the ASG. Transcriptional analysis showed the expression of autophagy and apoptosis-related genes, and some cuticle proteins and chitin synthase were significantly up-regulated. The results suggest that POUM2 homeostasis regulates ASG intimal remodeling and cells fate, thus affecting ASG development, silkworm spinning and metamorphosis. Our studies not only offer potential molecular targets for genetic improvement of silk performance and molecular breeding of the silkworm, but also provide new insights into POU factor-mediated tissue remodeling and cells fate determination in insects.

POU-M2 promotes juvenile hormone biosynthesis by directly activating the transcription of juvenile hormone synthetic enzyme genes in Bombyx mori.

Juvenile hormone (JH) plays a key role in preventing larval precocious metamorphosis, maintaining larval state, controlling adult sexual development and promoting insect egg maturation. Genetic studies have shown that POU factor ventral veins lacking regulates JH synthesis to control the timing of insect metamorphosis. However, how POU factor regulates JH synthesis is largely unknown. Here, we found POU-M2 was highly expressed in corpora allata (CA) and specifically localized in the nucleus of CA. The overexpression of POU-M2 promoted the expression of JH synthase genes and kr-h1 and enhanced the activity of JH synthase genes promoter. Further, POU-M2 promoted the transcription of JH acid O-methyltransferase (JHAMT) by directly binding to the key cis-regulatory elements -207, -249 and -453 within the proximal regions of JHAMT promoter. Both the POU domain and homeodomain were vital for the activation of POU-M2 on JHAMT transcription. Our study reveals the mechanism by which POU-M2 regulates JHAMT transcription.

Fabrication of antibacterial sericin based hydrogel as an injectable and mouldable wound dressing.

Hydrogel-based wound dressings are ideal materials as they could provide a moist environment to promote wound healing. Silk sericin is a natural macromolecular protein produced by silkworms and has positive effects on fibroblasts and keratinocytes. Sodium alginate is a natural polysaccharide known as a mouldable matrix. To develop novel hydrogel wound dressings, sodium alginate was functionalized with silk sericin to yield an injectable and mouldable semi-interpenetrating network hydrogel (SA/Se) using calcium ions as the crosslinking agents. Meanwhile, silver nanoparticles (AgNPs) were synthesized in situ by the phenolic hydroxyl group of tyrosine in sericin to enhance the antibacterial activity of the hybrid hydrogel. UV-visible spectroscopy, fluorescence spectroscopy, transmission electron microscope, X-ray photoelectron spectroscopy and energy dispersive spectroscopy demonstrated the successful synthesis of AgNPs with average size of 15.7 nm in SA/Se-Ag0.2. Field emission scanning electron microscopy showed the macroporous structure of the hydrogel. The hybrid hydrogel had a swelling ratio of 32 and more than 5% of retention ratio after 50 h. It exhibited effective antibacterial activity through irreversibly disrupting bacterial membrane structure to result in bacterial lysis and death. Cell counting kit-8 assay suggested the hybrid hydrogel was highly cytocompatible. The animal experiment showed the wound contraction ratio in SA/Se-Ag0.2 group was 99% after 12 days, indicating it can effectively promote wound healing. This novel hydrogel is promising for wound healing and tissue engineering.

Effect of Ficoll 70 on thermal stability and structure of creatine kinase.

The effect of Ficoll 70 on the thermal stability and structure of creatine kinase (CK) was studied using far-UV CD spectra and intrinsic fluorescence spectra. The thermal transition curves monitored by CD spectra were fitted to a two-state model using a modified form of the van't Hoff equation to obtain the transition temperature (T(m)) and enthalpy change (DeltaH(u)) of thermally induced denaturation of CK in the absence and presence of Ficoll 70. An increase in T(m) with constant DeltaH(u) was observed with increasing Ficoll 70 concentration, suggesting that Ficoll 70 enhances the thermal stability of CK. Fluorescence spectral measurements confirmed this protective effect of Ficoll 70 on CK structure. In addition, we observed a crowding-induced compaction effect on the structure of both native state and thermally denatured state of CK in the presence of Ficoll 70, which is more obvious on the structure of the denatured ensemble compared to that of the native ensemble. Our observations qualitatively accord with the predictions of previously proposed crowding theory for the effect of intermolecular excluded volume on protein stability and structure. These findings imply that the effects of macromolecular crowding are essential to our understanding of protein folding and unfolding occurring in vivo.

Effects of SDS on the activity and conformation of protein tyrosine phosphatase from thermus thermophilus HB27.

Deciphering the activity-conformation relationship of PTPase is of great interest to understand how PTPase activity is determined by its conformation. Here we studied the activity and conformational transitions of PTPase from thermus thermophilus HB27 in the presence of sodium dodecyl sulfate (SDS). Activity assays showed the inactivation of PTPase induced by SDS was in a concentration-dependent manner. Fluorescence and circular dichroism spectra suggested SDS induced significant conformational transitions of PTPase, which resulted in the inactivation of PTPase, and the changes of α-helical structure and tertiary structure of PTPase. Structural analysis revealed a number of hydrophobic and charged residues around the active sites of PTPase may be involved in the hydrophobic and ionic bonds interactions of PTPase and SDS, which are suggested to be the major driving force to result in PTPase inactivation and conformational transitions induced by SDS. Our results suggested the hydrophobic and charged residues around the active sites were essential for the activity and conformation of PTPase. Our study promotes a better understanding of the activity and conformation of PTPase.

Expression, purification and characterization of recombinant protein tyrosine phosphatase from Thermus thermophilus HB27.

The low-molecular-weight protein tyrosine phosphatases (PTPase) exist ubiquitously in prokaryotes and eukaryotes and play important roles in the regulation of physiological activities. We report here the expression, purification and characterization of an active and soluble PTPase from Thermus thermophilus HB27 in Escherichia coli. This PTPase has an optimum pH range of 2.8-4.8 when using p-nitrophenyl phosphate as the substrate. The thermal inactivation results indicate a high thermal stability of this enzyme, with the optimum temperature of 75 degrees C for activity. It can be activated by Mn(2+), Mg(2+), Ca(2+), Ba(2+), and Ni(2+), but inhibited by Zn(2+), Cu(2+), Cl(1-), and SO(4)(2-). These results suggest that this heat-resistant PTPase may play important roles in vivo in the adaptation of the microorganism to extreme temperatures and specific nutritional conditions.

Cross-talk between juvenile hormone and ecdysone regulates transcription of fibroin modulator binding protein-1 in Bombyx mori.

Juvenile hormone (JH) and 20-hydroxyecdysone (20E) are the most important hormones in silkworm and play vital roles in silkworm development, metamorphosis, and silk protein synthesis. Fibroin modulator binding protein-1 (FMBP-1) is a novel transcription factor regulating fibroin heavy chain (fib-H) transcription in Bombyx mori. The roles of JH and 20E on FMBP-1 transcription are less known. Here, we show FMBP-1 transcription is repressed by juvenile hormone analog (JHA) and activated by 20E. We identify two Krüppel homolog 1 (Kr-h1) binding sites (KBS1 and KBS2) and an E74A binding site (EBS) in the promoter of FMBP-1. We demonstrate Kr-h1 directly binds to KBS1 and KBS2 to repress FMBP-1 transcription, and 20E promotes FMBP-1 transcription through E74A. In the presence of JH and 20E, E74A abolishes the repression of Kr-h1 and activates FMBP-1 transcription through direct binding to EBS between KBS1 and KBS2 in FMBP-1 promoter. Further, JHA and 20E treatment and RNA interference confirm the effects of JH and 20E on FMBP-1 transcription in vivo, thus affecting fib-H transcription. Our results reveal the molecular mechanism of FMBP-1 transcription regulated by the cross-talk between JH and 20E in Bombyx mori, and provide novel insights into FMBP-1 transcriptional regulation and silk protein synthesis.