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Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3-4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives a rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations., (© 2022. The Author(s).)

To explore the composition of the nirS denitrifying bacterial community during stratification in spring(March to May) in a drinking water reservoir and its relationship with water quality, the water quality and relative abundance and structure of the denitrifying bacterial community were analyzed using in-situ monitoring coupled with Illumina high-throughput sequencing technology in the Lijiahe Reservoir. The results showed that:① through high-throughput sequencing, 4 phyla and 13 genera were identified. The dominant bacterial phylum was Proteobacteria, and its relative abundance was between 52.5% and 70.6%. The overall trend of the relative abundance of Proteobacteria decreased on the time scale ( P <0.05), and its relative abundance in the surface and middle layers was higher than that of the bottom layer on the spatial scale ( P <0.05). There was no difference in the proportion of Proteobacteria between the surface and middle layers ( P >0.05), and the abundance of its bottom layer was relatively stable; eight genera of bacteria with denitrification function were identified, among which the dominant bacterial genera (relative abundance>1%) were Dechloromonas and Pseudomonas . The relative abundance of Dechloromonas showed a trend of first decreasing and then increasing on the time scale, whereas the relative abundance of Pseudomonas showed a trend of increasing first and then decreasing on the time scale. There were no differences on the spatial scale between these two genera ( P >0.05); the changes in bacterial diversity and abundance were basically similar, with a trend of first increasing and then decreasing on the time scale. The highest diversity and abundance of the bacterial community gradually increased with increasing depth on the spatial scale. ② ρ (TN) of the reservoir during stratification was 2.35-2.91 mg·L -1 , and the nitrogen pollution was more serious. In March and April, ρ (TN) on the vertical scale was basically similar and showed a decreasing trend. In May, the content of total nitrogen was higher than that in March and April, and the highest value of total nitrogen content occurred in the surface layer. ③ Redundancy analysis showed that water temperature, dissolved oxygen, nitrate, and ammonia nitrogen were the main driving factors, and ammonia nitrogen showed a significantly negative correlation with Dechlormonas . In summary, the study of nirS -type denitrification communities and related influencing factors will contribute to analyzing the characteristics of denitrifying bacterial community changes in a micro-polluted drinking water reservoir and provide a theoretical research basis for the biological remediation of nitrogen pollution in such reservoirs in the future.

Zinc metalloprotease 1 (Zmp1), a Mycobacterium tuberculosis 75 kDa secreted enzyme, mediates key stages of tuberculosis disease progression. The biological activity of Zmp1 presumably stems from its ability to degrade bacterium- and/or host-derived peptides. The crystal structures of Zmp1 and related M13 metalloproteases, such as neprilysin and endothelin-converting enzyme-1 were determined only in the closed conformation, which cannot capture substrates or release proteolytic products. Thus, the mechanisms of substrate binding and selectivity remain elusive. Here we report two open-state cryo-EM structures of Zmp1, revealed by our SAXS analysis to be the dominant states in solution. Our structural analyses reveal how ligand binding induces a conformational switch in four linker regions to drive the rigid body motion of the D1 and D2 domains, which form the sizable catalytic chamber. Furthermore, they offer insights into the catalytic cycle and mechanism of substrate recognition of M13 metalloproteases for future therapeutic innovations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Although various implants exist for the fixation of isolated greater tuberosity fractures, few implants are specifically designed for such fractures. The purpose of this study was to investigate the clinical and radiologic outcomes of open reduction-internal fixation with a low-profile anatomic locking plate for comminuted greater tuberosity fractures of the proximal humerus., Methods: From November 2012 to February 2018, 24 patients with displaced and comminuted isolated greater tuberosity fractures were treated with the new low-profile anatomic locking plate. To determine clinical outcomes, we evaluated active range of motion; the visual analog scale pain score; the Constant-Murley score; the Disabilities of the Arm, Shoulder and Hand score; radiographs; and complications., Results: In all cases, a mean follow-up period of 29.3 months (range, 18-48 months) was completed. All patients achieved bone union with a mean healing time of 11.3 weeks (range, 8-16 weeks). The mean Constant-Murley score was 91.1 points (range, 69-100 points), with a rate of good to excellent results of 95.8%. The average Disabilities of the Arm, Shoulder and Hand score was 9.9 points (range, 2-25 points), and the mean visual analog scale pain score was 1.1 points (range, 0-4 points). Mean active forward flexion, abduction, external rotation, and internal rotation (level) were 157°, 152°, and 40°, and T11, respectively. Postoperatively, 1 patient had persistent shoulder stiffness, and 1 patient had recurrence of shoulder dislocation because of a falling injury during badminton. No serious complications such as subacromial impingement, malunion, nonunion, loss of reduction, or implant failure occurred., Conclusions: The new low-profile anatomic locking plate was useful for the treatment of comminuted isolated greater tuberosity fractures as it provided reliable stability and satisfactory radiographic and functional results. The described technique is a simple and effective method and provides a new reliable option for the treatment of isolated greater tuberosity fractures., (Copyright © 2020 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

To explore the influence of storm runoff on reservoir organic matter during the flood season, the Lijiahe Reservoir was selected to analyze variations in the content and components of dissolved organic matter (DOM) during four periods (before runoff, flood peak period, 1 week after runoff, and 6 weeks after runoff) using three-dimensional fluorescence spectroscopy parallel factor analysis (EEMs-PARAFAC) and ultraviolet-visible (UV-Vis) spectra. The results showed that:① the turbidity and DOC content of the reservoir increased significantly during the flood peak period ( P <0.01) and gradually decreased thereafter; ② the UV-Vis spectrum characteristics showed that a (254) and a (355) were significantly increased in the flood peak period ( P <0.01) while E2/E3 and E3/E4 were significantly decreased ( P <0.01), indicating that the concentration, relative molecular weight, and degree of DOM humification in the reservoir were increased by storm runoff; ③ four DOM components were identified as terrestrial humus (C1 and C2), microbial humus (C3), and a tryptophan-like component (C4). The fluorescence intensity of the C1-C3 components increased significantly during the flood peak period ( P <0.05), indicating that the increase in the DOM humic-like component was caused by the storm runoff. At the same time, a decrease in the fluorescence intensity of the C1-C4 components was observed after the flood peak period, indicating that DOM continuously settled and degraded after runoff; and ④ Pearson's correlation analyses showed that DOM fluorescence intensity and turbidity were significantly correlated ( r >0.467, P <0.05), indicating that the observed decrease in DOM content was related to the sedimentation of suspended solids. A principal component analysis (PCA) showed that the water quality in the reservoir reflected the observed characteristics during the different runoff periods. Overall, this study reveals the effects of the storm runoff on DOM content and its components over the short and long term, providing scientific support for the management of drinking water quality.

Objective: A passive brain-computer interface recognizes its operator's cognitive state without an explicitly performed control task. This technique is commonly used in conjunction with consumer-grade EEG devices for detecting the conditions of fatigue, attention, emotional arousal, or motion sickness. While it is easy to mount the sensors in the forehead area, which is not covered with hair, the recorded signals become greatly contaminated with eyeblink and movement artifacts, which makes it a challenge to acquire the data of suitable for analysis quality, particularly in few channel systems where a lack of spatial information limits the applicability of sophisticated signal cleaning algorithms. In this article, we demonstrate that by combining the features associated with electrocortical activities and eyeblink recognition analysis, it becomes feasible to design an accurate system for the inattention state prediction using just a single EEG sensor., Approach: Fifteen healthy 22-28 years old participants took part in the experiment that implemented a realistic sustained attention task of nighttime highway driving in a virtual environment. The EEG data were collected using a portable wireless Mindo-4 device, which constitutes an adjustable elastic strip with foam-based sensors, a data-acquisition module, an amplification and digitizing unit, and a Bluetooth[Formula: see text] module., Main Results: The spectral analysis of the EEG samples that immediately preceded the lane departure events revealed alterations in the tonic power spectral density, which accompanied elongations in the drivers' reaction times. The RMSE of the predicted reaction times, which are based on a combination of the brain-related and eyeblink features, is 0.034 ± 0.019 s, and the r 2 is 0.885 ± 0.057 according to a within-session leave-one-trial-out cross-validation., Significance: The drowsiness prediction from a frontal single-channel setup can achieve a comparable performance with using an array of occipital EEG sensors. As a direct result of utilizing a dry sensor placed in the non-covered with hair head area, the proposed approach in this study is low-cost and user-friendly.

Pseudomonas aeruginosa uses a type III secretion system (T3SS) to inject cytotoxic effector proteins into host cells. The promiscuous nucleotidyl cyclase, exoenzyme Y (ExoY), is one of the most common effectors found in clinical P. aeruginosa isolates. Recent studies have revealed that the nucleotidyl cyclase activity of ExoY is stimulated by actin filaments (F-actin) and that ExoY alters actin cytoskeleton dynamics in vitro , via an unknown mechanism. The actin cytoskeleton plays an important role in numerous key biological processes and is targeted by many pathogens to gain competitive advantages. We utilized total internal reflection fluorescence microscopy, bulk actin assays, and EM to investigate how ExoY impacts actin dynamics. We found that ExoY can directly bundle actin filaments with high affinity, comparable with eukaryotic F-actin-bundling proteins, such as fimbrin. Of note, ExoY enzymatic activity was not required for F-actin bundling. Bundling is known to require multiple actin-binding sites, yet small-angle X-ray scattering experiments revealed that ExoY is a monomer in solution, and previous data suggested that ExoY possesses only one actin-binding site. We therefore hypothesized that ExoY oligomerizes in response to F-actin binding and have used the ExoY structure to construct a dimer-based structural model for the ExoY-F-actin complex. Subsequent mutational analyses suggested that the ExoY oligomerization interface plays a crucial role in mediating F-actin bundling. Our results indicate that ExoY represents a new class of actin-binding proteins that modulate the actin cytoskeleton both directly, via F-actin bundling, and indirectly, via actin-activated nucleotidyl cyclase activity., (© 2020 Mancl et al.)

Water is an important pathway for human exposure to heavy metals. Accurate assessment of the health risks that are related to exposure to heavy metals in drinking and household water are required for the preparation of targeted health risk prevention measures. This study explores and identifies the health risks related to exposure to heavy metals via drinking and household water pathways in Xigu District, Lanzhou, northwestern China, using household water samples and survey data obtained during July-September 2015 (wet season) and December 2015-January 2016 (dry season). During each period, drinking water and household water that were available for use by children aged 0-5 and 6-17 years were sampled and a questionnaire on water-related behavior patterns was completed for each household. Cd, Cr, Pb, and As concentrations were analyzed in all water samples, and were used along with water-related exposure factors from the questionnaires to estimate exposure doses and associated health risks using models recommended by the United States Environmental Protection Agency (US EPA). The results showed that the concentrations of Cd, Cr, Pb, and As in both drinking and household water samples did not exceed the relative thresholds defined in China's national water quality standards. The concentrations of heavy metals in household water were more affected by seasonal factors than of those of drinking water. The non-cancer and cancer risks were in the ranges of 2.82×10 -8 -2.43×10 -2 and 7.55×10 -9 -3.62×10 -5 , respectively, which are within acceptable levels, although the non-cancer and cancer risks from drinking water were both higher than of those determined for household water. Furthermore, the non-cancer and cancer risks from household water for children aged 0-5 years were lower than of those for children aged 6-17 years in each period. However, the cancer risk from drinking water for children aged 0-5 years was lower than of that for children aged 6-17 years, whereas the reverse was found for non-cancer risks. This study indicates that Cd, Cr, Pb, and As in drinking and household water did not pose significant detrimental effects to human health, and that the refined exposure assessment used could reduce uncertainties in health risk assessments.

We demonstrate bandwidth broadening in cascaded MgO-doped periodically poled lithium niobate (MgO:PPLN) crystals (Λ=10.3  μm) using the temperature-gradient technique. Up to 2.8 nm bandwidth at 600 nm spectral region is achieved using two 50 mm long cascaded MgO:PPLN crystals via sum frequency generation. This technique combines the merits of high conversion efficiency attributed to cascaded nonlinearity and the reconfigurability of temperature-gradient-induced chirp for broadening of the input wavelength acceptance range.

Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies., Competing Interests: ZZ, WL, LB, YT, HW, AW, MF, VW, LM, DL, WS, RD, BD, DL, AK, SK, AK, SL, BC, CP, WT No competing interests declared, (© 2018, Zhang et al.)

Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the "elbow" regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab-antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This corrects the article DOI: 10.1038/ncomms15375.

In the intestinal epithelium, the aberrant regulation of cell/cell junctions leads to intestinal barrier defects, which may promote the onset and enhance the severity of inflammatory bowel disease (IBD). However, it remains unclear how the coordinated behaviour of cytoskeletal network may contribute to cell junctional dynamics. In this report, we identified ACF7, a crosslinker of microtubules and F-actin, as an essential player in this process. Loss of ACF7 leads to aberrant microtubule organization, tight junction stabilization and impaired wound closure in vitro. With the mouse genetics approach, we show that ablation of ACF7 inhibits intestinal wound healing and greatly increases susceptibility to experimental colitis in mice. ACF7 level is also correlated with development and progression of ulcerative colitis (UC) in human patients. Together, our results reveal an important molecular mechanism whereby coordinated cytoskeletal dynamics contributes to cell adhesion regulation during intestinal wound repair and the development of IBD.

We aim to compare the effects of simvastatin and combination of simvastatin and nylestriol on bone metabolism in ovariectomized (OVX) rats. Fifty healthy Wistar female rats were randomly allocated into 5 groups: sham + saline group (group A), OVX + saline group (group B), OVX + simvastatin (5 mg·kg·d) (group C), OVX + nylestriol (0.01 mg·kg·d) (group D), and OVX + simvastatin (3 mg·kg·d) + nylestriol (0.005 mg·kg·d) (group E). All mice were orally administrated with saline or medicine dissolved in saline for 10 weeks. Body weight of rats before and after the experiment was measured. Twenty-four hours after the experiment, calcium (Ca), creatinine (Cr), and hydroxyproline in urine were detected. Serum levels of osteocalcin (bone Gla-protein, BGP) and alkaline phosphatase (ALP) were measured. Bone mineral density was detected and trabecular bone was observed after the isolation of femur and tibia. Remarkably decreased serum BGP and increased serum ALP levels were detected in group B compared with those in group A. However, notably increased serum BGP and decreased serum ALP levels were found in groups C, D, and E compared with those in group B; femoral and tibial bone mineral density decreased in group B compared with that in group A, but increased in groups C, D, and E compared with that in group B. Simvastatin and combination of simvastatin and nylestriol promote formation of new bone, increase bone density, and improve bone microstructure damage in OVX rats.

Turnover of focal adhesions allows cell retraction, which is essential for cell migration. The mammalian spectraplakin protein, ACF7 (Actin-Crosslinking Factor 7), promotes focal adhesion dynamics by targeting of microtubule plus ends towards focal adhesions. However, it remains unclear how the activity of ACF7 is regulated spatiotemporally to achieve focal adhesion-specific guidance of microtubule. To explore the potential mechanisms, we resolve the crystal structure of ACF7's NT (amino-terminal) domain, which mediates F-actin interactions. Structural analysis leads to identification of a key tyrosine residue at the calponin homology (CH) domain of ACF7, whose phosphorylation by Src/FAK (focal adhesion kinase) complex is essential for F-actin binding of ACF7. Using skin epidermis as a model system, we further demonstrate that the phosphorylation of ACF7 plays an indispensable role in focal adhesion dynamics and epidermal migration in vitro and in vivo. Together, our findings provide critical insights into the molecular mechanisms underlying coordinated cytoskeletal dynamics during cell movement.

CC chemokine ligand 5 (CCL5) and CCL3 are critical for immune surveillance and inflammation. Consequently, they are linked to the pathogenesis of many inflammatory conditions and are therapeutic targets. Oligomerization and glycosaminoglycan (GAG) binding of CCL5 and CCL3 are vital for the functions of these chemokines. Our structural and biophysical analyses of human CCL5 reveal that CCL5 oligomerization is a polymerization process in which CCL5 forms rod-shaped, double-helical oligomers. This CCL5 structure explains mutational data and offers a unified mechanism for CCL3, CCL4, and CCL5 assembly into high-molecular-weight, polydisperse oligomers. A conserved, positively charged BBXB motif is key for the binding of CC chemokines to GAG. However, this motif is partially buried when CCL3, CCL4, and CCL5 are oligomerized; thus, the mechanism by which GAG binds these chemokine oligomers has been elusive. Our structures of GAG-bound CCL5 and CCL3 oligomers reveal that these chemokine oligomers have distinct GAG-binding mechanisms. The CCL5 oligomer uses another positively charged and fully exposed motif, KKWVR, in GAG binding. However, residues from two partially buried BBXB motifs along with other residues combine to form a GAG-binding groove in the CCL3 oligomer. The N termini of CC chemokines are shown to be involved in receptor binding and oligomerization. We also report an alternative CCL3 oligomer structure that reveals how conformational changes in CCL3 N termini profoundly alter its surface properties and dimer-dimer interactions to affect GAG binding and oligomerization. Such complexity in oligomerization and GAG binding enables intricate, physiologically relevant regulation of CC chemokine functions.

We investigated the diagnostic value of the apparent diffusion coefficient (ADC) and fractional anisotropy (FA) of magnetic resonance diffusion tensor imaging (DTI) in patients with spinal cord compression (SCC) using a meta-analysis framework. Multiple scientific literature databases were exhaustively searched to identify articles relevant to this study. Mean values and standardized mean differences (SMDs) were calculated for the ADC and FA in normal and diseased tissues. The STATA version 12.0 software was used for statistical analysis. Of the 41 articles initially retrieved through database searches, 11 case-control studies were eligible for the meta-analysis and contained a combined total of 645 human subjects (394 patients with SCC and 251 healthy controls). All 11 studies reported data on FA, and 9 contained data related to the ADC. The combined SMDs of the ADC and FA showed that the ADC was significantly higher and the FA was lower in patients with SCC than in healthy controls. Subgroup analysis based on the b value showed higher ADCs in patients with SCC than in healthy controls at b values of both ≤500 and >500 s/mm2. In summary, the main findings of this meta-analysis revealed an increased ADC and decreased FA in patients with SCC, indicating that DTI is an important diagnostic imaging tool to assess patients suspected to have SCC.

Spoof surface plasmons derive their properties from structure resonance rather than from electronic resonance, enabling an extremely high degree of freedom for tuning and modulating different resonances. Here, a composite resonator based on multiscale textured metal surface of different grooves is presented, and spoof localized surface plasmons (LSPs) are shown to emerge and interact coherently. Each band of the spoof LSPs resembles those generated by the homogenously textured surface with the corresponding groove. By adjusting the geometry and filling medium of each substructure in the composite system, we find that the multipole resonant modes sustained by one substructure can couple with those in the other, giving rise to multi-band Fano resonances. Such multiple-Fano resonance structures are spatially more compact while spectrally more comprehensive than usual spoof structures. They can be used for unique resonant devices such as microwave antennas and metasurfaces.

Insulin-degrading enzyme (IDE) is a protease that cleaves insulin and other bioactive peptides such as amyloid-β. Knockout and genetic studies have linked IDE to Alzheimer's disease and type-2 diabetes. As the major insulin-degrading protease, IDE is a candidate drug target in diabetes. Here we have used kinetic target-guided synthesis to design the first catalytic site inhibitor of IDE suitable for in vivo studies (BDM44768). Crystallographic and small angle X-ray scattering analyses show that it locks IDE in a closed conformation. Among a panel of metalloproteases, BDM44768 selectively inhibits IDE. Acute treatment of mice with BDM44768 increases insulin signalling and surprisingly impairs glucose tolerance in an IDE-dependent manner. These results confirm that IDE is involved in pathways that modulate short-term glucose homeostasis, but casts doubt on the general usefulness of the inhibition of IDE catalytic activity to treat diabetes.

Objective: To analyze the clinical effect of laparoscopy on patients with endometriosis and a high expression of CA125., Patients and Methods: One hundred cases of endometriosis treated in our hospital from May 2012 to August 2014 were selected as research subjects, after approval from the hospital and the informed consent of patients. The subjects were randomly divided into a control group and a study group with 50 cases in each group. The control group underwent the traditional surgery, while the research group underwent laparoscopy. The clinical curative effect were compared between the two groups of patients., Results: The research group's postoperative 6h, 12h, 24h CA125 levels were compared to those of the control group. There was statistically significant difference between groups (p < 0.05). Surgery and hospitalization time, intraoperative bleeding volume, and and incision length index of the two groups of patients were compared. There were statistically significant differences between the two groups (p < 0.05)., Conclusions: Laparoscopic surgery in endometriosis patients with high expression of CA125 is curative, and the patients'recovery after surgery is promising.

CC chemokine ligands (CCLs) are 8- to 14-kDa signaling proteins involved in diverse immune functions. While CCLs share similar tertiary structures, oligomerization produces highly diverse quaternary structures that protect chemokines from proteolytic degradation and modulate their functions. CCL18 is closely related to CCL3 and CCL4 with respect to both protein sequence and genomic location, yet CCL18 has distinct biochemical and biophysical properties. Here, we report a crystal structure of human CCL18 and its oligomerization states in solution based on crystallographic and small-angle X-ray scattering analyses. Our data show that CCL18 adopts an α-helical conformation at its N-terminus that weakens its dimerization, explaining CCL18's preference for the monomeric state. Multiple contacts between monomers allow CCL18 to reversibly form a unique open-ended oligomer different from those of CCL3, CCL4, and CCL5. Furthermore, these differences hinge on proline 8, which is conserved in CCL3 and CCL4 but is replaced by lysine in human CCL18. Our structural analyses suggest that a mutation of proline 8 to alanine stabilizes a type 1 β-turn at the N-terminus of CCL4 to prevent dimerization but prevents dimers from making key contacts with each other in CCL3. Thus, the P8A mutation induces depolymerization of CCL3 and CCL4 by distinct mechanisms. Finally, we used structural, biochemical, and functional analyses to unravel why insulin-degrading enzyme degrades CCL3 and CCL4 but not CCL18. Our results elucidate the molecular basis for the oligomerization of three closely related CC chemokines and suggest how oligomerization shapes CCL chemokine function., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Insulin degrading enzyme (IDE) is a zinc metalloprotease that degrades small amyloid peptides such as amyloid-â and insulin. So far the dearth of IDE-specific pharmacological inhibitors impacts the understanding of its role in the physiopathology of Alzheimer's disease, amyloid-â clearance, and its validation as a potential therapeutic target. Hit 1 was previously discovered by high-throughput screening. Here we describe the structure-activity study, that required the synthesis of 48 analogues. We found that while the carboxylic acid, the imidazole and the tertiary amine were critical for activity, the methyl ester was successfully optimized to an amide or a 1,2,4-oxadiazole. Along with improving their activity, compounds were optimized for solubility, lipophilicity and stability in plasma and microsomes. The docking or co-crystallization of some compounds at the exosite or the catalytic site of IDE provided the structural basis for IDE inhibition. The pharmacokinetic properties of best compounds 44 and 46 were measured in vivo. As a result, 44 (BDM43079) and its methyl ester precursor 48 (BDM43124) are useful chemical probes for the exploration of IDE's role., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Purpose: To develop a novel injectable strontium-containing calcium phosphate cement with collagen., Methods: A novel calcium phosphate bone cement (CPC) was prepared with the addition of strontium element, collagenl, and modified starch; the injectability, solidification time, microstructure, phase composition, compressive strength, anti-collapsibility and histological properties of material were evaluated., Results: The results showed that the material could be injected with an excellent performance; the modified starch significantly improved the anti-washout property of cement; with the liquid to solid ratio of 0.3, the largest compressive strength of cement was obtained (48.0 MPa ± 2.3 MPa); histological examination of repair tissue showed that the bone was repaired after 16 weeks; the degradation of cement was consistent with the new bone growth., Conclusion: A novel injectable collagen-strontium-containing CPC with excellent compressive strength and suitable setting time was prepared, with addition of modified starch. The CPC showed a good anti-washout property and the degradation time of the cement met with the new bone growing. This material is supposed to be used in orthopedic and maxillofacial surgery for bone defects.

Human presequence protease (hPreP) is an M16 metalloprotease localized in mitochondria. There, hPreP facilitates proteostasis by utilizing an ∼13,300-Å(3) catalytic chamber to degrade a diverse array of potentially toxic peptides, including mitochondrial presequences and β-amyloid (Aβ), the latter of which contributes to Alzheimer disease pathogenesis. Here, we report crystal structures for hPreP alone and in complex with Aβ, which show that hPreP uses size exclusion and charge complementation for substrate recognition. These structures also reveal hPreP-specific features that permit a diverse array of peptides, with distinct distributions of charged and hydrophobic residues, to be specifically captured, cleaved, and have their amyloidogenic features destroyed. SAXS analysis demonstrates that hPreP in solution exists in dynamic equilibrium between closed and open states, with the former being preferred. Furthermore, Aβ binding induces the closed state and hPreP dimerization. Together, these data reveal the molecular basis for flexible yet specific substrate recognition and degradation by hPreP., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: To evaluate the feasibility and safety of a new route of transendocardial stem cell injection., Materials and Methods: Atrial septal puncture was performed in six young male pigs, and then a 6F syringe was passed through the puncture to reach the left atrium. Meanwhile, a guide wire was inserted into the left ventricle through the femoral artery, and echocardiography was used to confirm the relative position of the guide wire with the syringe., Results: After atrial puncture through femoral vein, the syringe could reach the left atrium and finally the left ventricle. Echocardiography confirmed that both the guide wire and the syringe were located in the left ventricle. The diameter of atrial septa puncture and the diameter of the syringe implantation were 4.1 ± 0.5 mm and 8.4 ± 0.7 mm, respectively. But there is no difference in Left ventricle end-systolic dimension (LVES), left ventricle end-diastolic dimension (LVED) and left ventricle ejection fraction (LVEF) before surgery, after atrial septa puncture, after syringe implantation or one month after surgery., Conclusions: It is feasible to perform transendocardial stem cell injection by 6F syringe inserted through femoral vein. The surgery may cause atrial septa tearing but does not jeopardize myocardial function.

Insulin-degrading enzyme (IDE) selectively degrades the monomer of amyloidogenic peptides and contributes to clearance of amyloid β (Aβ). Thus, IDE retards the progression of Alzheimer's disease. IDE possesses an enclosed catalytic chamber that engulfs and degrades its peptide substrates; however, the molecular mechanism of IDE function, including substrate access to the chamber and recognition, remains elusive. Here, we captured a unique IDE conformation by using a synthetic antibody fragment as a crystallization chaperone. An unexpected displacement of a door subdomain creates an ~18-Å opening to the chamber. This swinging-door mechanism permits the entry of short peptides into the catalytic chamber and disrupts the catalytic site within IDE door subdomain. Given the propensity of amyloidogenic peptides to convert into β-strands for their polymerization into amyloid fibrils, they also use such β-strands to stabilize the disrupted catalytic site resided at IDE door subdomain for their degradation by IDE. Thus, action of the swinging door allows IDE to recognize amyloidogenicity by substrate-induced stabilization of the IDE catalytic cleft. Small angle X-ray scattering (SAXS) analysis revealed that IDE exists as a mixture of closed and open states. These open states, which are distinct from the swinging door state, permit entry of larger substrates (e.g., Aβ, insulin) to the chamber and are preferred in solution. Mutational studies confirmed the critical roles of the door subdomain and hinge loop joining the N- and C-terminal halves of IDE for catalysis. Together, our data provide insights into the conformational changes of IDE that govern the selective destruction of amyloidogenic peptides., Competing Interests: The authors declare no conflict of interest.

Connexins (CXs), as a component of gap junction channel, are homologous four transmembrane-domain proteins, with numerous studies confirming their auditory functions. Among a cohort of patients having incurred non-syndromic hearing loss, we identified two novel missense mutations, p.R15G and p.L23H, in the GJC3 gene encoding CX30.2/CX31.3, as causally related to hearing loss in previous study. However, the functional alteration of CX30.2/CX31.3 caused by the mutant GJC3 gene remains unknown. In this study, we compared the intracellular distribution of mutant CX30.2/CX31.3 (p.R15G and p.L23H) with the wild-type (WT) protein in HeLa cells and the effect of the mutant protein had on those cells. Analytical results indicated that p.R15G and p.L23H mutant exhibited continuous staining along apposed cell membranes in the fluorescent localization assay, which is the same with the WT. Moreover, ATP release (hemichannel function) is less in HeLa cells carrying mutant GJC3 genes than those of WT expressing cells. We believe that although p.R15G and p.L23H mutants do not decrease the trafficking of CX proteins, mutations in GJC3 genes result in a loss of hemichannel function of CX30.2/CX31.3 protein, possibly causing hearing loss. Results of this study provide a novel molecular explanation for the role of GJC3 in hearing loss.

Apart from SERPINB2 and SERPINB5, the roles of the remaining 13 members of the human SERPINB family in cancer metastasis are still unknown. In the present study, we demonstrated that most of these genes are differentially expressed in tumor tissues compared to matched normal tissues from lung or breast cancer patients. Overexpression of each SERPINB gene effectively suppressed the invasiveness and motility of malignant cancer cells. Among all of the genes, the SERPINB1, SERPINB5 and SERPINB7 genes were more potent, and the inhibitory effect was further enhanced by co-expression of any two of them. In addition, single treatment of the synthetic peptides corresponding to the P5-P5' sequences of the reactive center loop (RCL) of SERPINB1, SERPINB5 or SERPINB7 markedly suppressed the invasive and migratory properties of the cancer cells in a dose-dependent manner. More significantly, combination treatment of these peptides in cancer cells further improved the suppressive effect by 20-40%. Here, we determined the expression of all SERPINB family members in lung and breast cancer patients, and identified those members with potent inhibitory ability toward invasion and migration, and designed RCL-derived peptides to suppress the malignancy of cancer cells. Forced re-expression of these anti-invasive SERPINB genes or application of the SERPINB RCL-peptides may provide a reasonable strategy against lethal cancer metastasis.

Gap junctional intercellular communication has numerous functions, each of which meets the particular needs of organs, tissues, or groups of cells. Connexins (CXs) are homologous four-transmembrane-domain proteins that are the major components of gap junctions. CX30.2/CX31.3 (GJC3) is a relatively new member of the CX protein family. Until now, however, the functional characteristics of CX30.2/CX31.3 have been unclear. To elucidate the properties of CX30.2/CX31.3 channels, their subcellular localization in HeLa cells, their effectiveness in dye transfer, and function on channels were investigated. In the immunofluorescent assay, cells that express CX30.2/CX31.3-GFP exhibited continuous fluorescence along the apposed cell membranes, rather than punctated fluorescence in contacting membranes between two cells. Surprisingly, dyes that can be capable of being permeated by CX26 GJ, according to a scrape loading dye transfer assay in previous studies, are impermeated by CX30.2/CX31.3 GJ, suggesting a difference between the characteristics of CX30.2/CX31.3 GJ and CX26 GJ. Furthermore, a significant amount of ATP was released from the HeLa cells that stably expressed CX30.2/CX31.3, in a medium with low calcium ion concentration, suggesting a hemichannel-based function for CX30.2/CX31.3. Based on these findings, we suggest that CX30.2/CX31.3 shares functional properties with pannexin (hemi) channels rather than gap junction channels of other CXs.

Aim: To determine the effect of bFGF on extracellular matrix and gene expression on the human de-generated nucleus pulposus cells., Methods: The degenerated intervertebral disc cells were divided into 5 groups. A group: adding 100 pg/L bFGF, B group: adding 200 pg/LbFGF, C group: 500 pg/L bFGF, D group: 1000 pg/L bF-GF, E groups: control group, without interfering factors. The type II collagen and GAG mRNA expression were test, type II collagen content and glycosaminoglycan content of the supernatant were test., Results: bFGF stimulated de-generated nucleus pulposus cells, collagen II, GAG mRNA, collagen type II and glycosaminoglycan expression of the extracellular matrix were increased significantly on the 7 days compared with the control group (P < 0. 05). type II collagen and GAG mRNA were significantly lower than the control group on 14 days, 21 days (P < 0.05). However,collagen type II and glycosaminoglycan expression of the extracellular matrix on 14 days, 21 days were still higher than normal (P <0.05)., Conclusion: bFGF increase GAG and type II collagen mRNA expression, increased extracellular matrix type II collagen and GAG expression in short-term.

Human serine proteinase inhibitor Kazal-type 2 (SPINK2) functions as a trypsin/acrosin inhibitor and is synthesized mainly in the testis and seminal vesicle where its activity is engaged in fertility. The SPINK2 protein contains a typical Kazal domain composed by six cysteine residues forming three disulfide bridges. The expression of SPINK2 is closely related to cancer such as lymphomas, in that a high transcript level of SPINK2 in patients with primary cutaneous follicle center cell lymphomas have better prognosis with lower mortality. To clarify the role of SPINK2 in cancer, we performed quantitative real-time PCR and showed that the expression level of SPINK2 is significantly elevated in most leukemia cell lines except B-lymphoblast TK-6 cells. The molecular function and structural features of SPINK2 were also investigated by employing the recombinant active and mutant inactive SPINK2 proteins to determine its key P2-P2' (Pro(23)-Arg(24)-His(25)-Phe(26)) active site. The inhibition assay results demonstrated that Arg(24) at the P1 site is crucial for the specificity of SPINK2 on target enzyme. Although His(25) at the P1' and Phe(26) at the P2' residues are also involved in trypsin-SPINK2 interaction, Pro(23) at the P2 site may not be directly participated in interacting with trypsin. In addition, we determined the 3D solution structure of SPINK2 and used this structure to predict the SPINK2-proteinase complex structure and binding properties. These studies not only provide critical information about the structural properties and biophysical features of the SPINK2 proteinase inhibitor, but also suggest its important role in tumor progression and response to treatment.

Hemoglobin-based blood substitute is widely studied. As the starting materials, hemoglobin (Hb) is mainly supplied from outdated human blood and animal sources. But there are many disadvantages. As the alternative source, human placenta hemoglobin (PHb) has its own advantages. We take the lead in being occupied in the research of blood substitutes based on the placenta blood. Purified PHb readily undergoes dissociation to form two dimers that are easily filtered by the kidneys and have high 02 affinity (low P50), failing to transport 02 to tissues. As a first step, to enhance PHb stability and P50, we first adopted DBBF to modify deoxyPHb leading to DBBF-alpha PHb. According to detection, we knew that the PHb stability was improved, 02 affinity of the PHb solution was reduced, DBBF-alpha PHb had the appropriate capacity of oxygen-carrying and oxygen-unloading, and DBBF-alpha PHb kept the biological activity well. On the whole DBBF-alpha PHb could be considered as a first step toward the synthesis of a potential blood substitute.

For pilot-scale manufacturing of hemoglobin-based oxygen carrying drugs, we should get highly pure and viral inactivated hemoglobin (Hb) at high recovery. In our method, placenta hemoglobin (PHb) solutions were purified by heating in the presence of reducing agent and deoxygenating conditions so that heat-sensitive proteins were selectively precipitated and virus was inactivated. The optimum preparative condition resulted in highly purified PHb solution (>99% pure) with approximate 90% recovery and less than 2% of MetHb content, maintained oxygen carrying capacity, residual phospholipids less than 1 ppm, free of endotoxin, bacteria, type A&B antigens and virus. Finally, we compared the efficacy of blood exchange on rat with poly-PHb and poly-Hb from adult blood. The results showed no significant difference between two products. Therefore, the placenta Hb obtained from this method could be supplied as materials for oxygen carrying drugs.

Interleukin-22 (IL-22), a member of IL-10 family, plays some important roles in immune response through activation of the STAT 3 signal transduction pathway. Two types of IL-22-binding receptor have been discovered, a membrane-bound receptor and a soluble receptor, both encoded by different genes. IL-22 may be involved in inflammatory processes specifically regulated by soluble receptors. By screening a mouse genomic library for a human IL-22 binding protein homologue, we identified the mouse genomic clone of IL-22 binding protein. Its coding sequence was verified and isolated by RT-PCR. The gene encodes a protein of 230 amino acids that share 67.1% amino-acid sequence identity with human IL-22 binding protein. We designated this receptor 'mouse IL-22 binding protein' (mIL-22BP). mIL-22BP could be upregulated by LPS stimulation in mouse monocytes. mIL-22BP binds to mouse and human IL-22 and neutralizes STAT3 activation induced by both cytokines in human and rat hepatoma cell lines. Treating B cells with mouse IL-22 induces production of reactive oxygen species, which mIL-22BP blocks.

IL-10 is an immunosuppressive cytokine in the immune system. It was in clinical trial as an anti-inflammatory therapy for inflammatory bowel disease and various autoimmune diseases such as psoriasis, rheumatoid arthritis, and multiple sclerosis. IL-19 belongs to the IL-10 family, which includes IL-10, IL-19, IL-20, IL-22, melanoma differentiation-associated gene (MDA-7, IL-24), and AK155 (IL-26). Despite a partial homology in their amino acid sequences, they are dissimilar in their biologic functions. Little is known about the biologic function and gene regulation of IL-19. To understand the gene regulation of human IL-19, we identified a human IL-19 genomic clone and analyzed its promoter region. Five fusion genes containing different regions upstream of exon 1 linked to a luciferase reporter gene were expressed in the canine kidney epithelial-like Madin-Darby canine kidney cells. A fusion gene containing 394 bp showed luciferase activity 7- to 8-fold higher than the negative control of the promoterless fusion gene. We also isolated a full-length mouse cDNA clone. Mouse IL-19 shared 71% amino acid identity with human IL-19. Treatment of monocytes with mouse IL-19 induced the production of IL-6 and TNF-alpha. It also induced mouse monocyte apoptosis and the production of reactive oxygen species. Taken together, our results indicate that mouse IL-19 may play some important roles in inflammatory responses because it up-regulates IL-6 and TNF-alpha and induces apoptosis.

In this paper 12 cases of lumbosacral nerve-root anomalies proved on operations were reported. Two roots with a common conjoined origin entering the common intervertebral foramen separately were seen in two cases; two roots arising respectively from the lateral and ventral sides of the dural sac at the level of upper margin of the pedicle, in which one ran transversely into the foramen and another obliquely ran downward, in one case; two roots with closely adjacent origins, in which one ran transversely into the foramen at the level of upper margin of the pedicle and another obliquely ran downward, in four cases; a single root with abnormally large whereas the adjacent root with abnormally small, in five cases. We proposed a classification method for the sake of appropriate surgical program, which assorted the anomalies into three types: (I) General volume of nerve roots in the spinal canal or nerve-root canal abnormally increased, either the diameter of a single root increases or two roots entered the common. foramen; (II) The origin and course of two roots were anomalous; (III) Combination of I and II.

We improved and made the intramedullary interlocking nail and it's instrument for surgical operation in December 1989 by consulting the reported material of the gamma locking nail. 32 cases of the femoral trochanteric fracture were treated by using this fixation system. The results were satisfactory. The patients could stand up and move about beginning from 6th to 13th day after operation. No mechanical failure of implant, infection and other complications occurred in our series. 29 cases were followed-up for 5 to 19 months and showed all of the fractures were healed. Their hip functions were completely or basically recovered. Coxa vara deformity occurred in two cases and one had a 2.5 cm shortening of the lower limb. We consider that the intramedullary interlocking nail conforms to the principle of human biomechanics and it may be used as an effective surgical treatment with the fracture fixation of superior strength and stability for every type of femoral trochanteric fractures.

The effect of microinjection of 2-amino-5-phosphonovaleric acid (APV), a selective NMDA receptor antagonist, into the rat hippocampal CA3 area on the synaptic efficacy and related conditioned behavior during the acquisition and consolidation of discrimination learning behavior was examined. The results showed: (1) After population spike (PS) amplitude had just increased to the maximum through training i.e. learning-dependent LTP had just formed, APV 1 microliter (2 mmol/L) was injected into CA3 area, then the rats were trained during the time of efficacy of the drug in every experimental block. The result demonstrated that the PS amplitude could not be maintained at the highest level but decreased to the pre-experiment level after 8 blocks. Correct response percentage of rats could not be consolidated with further training but decreased to less than 10%. (2) After the PS amplitude had kept up at the highest level, APV 1 microliter (2 mmol/L) was injected into CA3 area, then the rats were trained during the time of efficacy of the drug in every experimental block, in which case the PS amplitude also could not be maintained at the highest level but decreased to the pre-experiment level after 14 blocks. Correlatively, when the correct response percentage of rats decreased gradually to less than 10%, the conditioned response of the animals extinguished, but its extinction speed was slower than it was in result (1). These results suggest that the NMDA receptor in CA3 area plays an important role in the maintenance of the learning-dependent long-term potentiation.