Surface and solvent influences on protein crystallization

The role of water in protein crystallization was explored by investigating the effects of three factors (salts, point mutations and pressure) on subtilisin crystallization.;Solubility and growth kinetics of three subtilisin mutants in three salt solutions were measured. The decrease of the solubility of Properase RTM and PurafectRTM subtilisin followed the reverse order of the Hofmeister series: SCN- \u3e NO3- \u3e Cl-. The solubility of ProperaseRTM was higher than other two mutants. Crystal morphology changed with the nature of salts and the substitution of surface residues. The required supersaturation (c-s)/s for a given growth rate increased when solubility was decreased. The effect of anion on protein growth was related to the molar Gibbs free energy of hydration of the anion.;Structural and energetic considerations for crystallization of two subtilisin mutants (ProperaseRTM and PurafectRTM) were compared. The average hydrophobicity, solvent accessible surface area (ASA) and the number of hydrogen bonds and salt bridges were calculated to quantify surface properties of proteins in intermolecular contact patches. All three amino acid substitutions are present in the contact patches. Properase RTM lattice involves more atomic contacts and hydrogen bonds and larger accessible surface area, which corresponding to the faster growth of ProperaseRTM crystals. Non-electrostatic interaction energy was calculated for each contact direction and the competition of misoriented molecules with correctly oriented ones was considered to explain the variation of growth kinetics;The increase of solubility with pressure gave a total volume change for crystallization of 37 cm3/mol, whereas the decrease of nucleation rate with pressure gave an activation volume for nucleation of 226 cm 3/mol. 983 water molecules were estimated to attend Properase RTM crystallization.;The second virial coefficients (B2) of Properase RTM and PurafectRTM subtilisin under crystallization conditions were measured by static light scattering as a function of salt type and salt concentration, showing that conditions with slight negative B2 are suitable for protein crystallization. A DLVO-type model was used to fit the effective Hamaker constants for subtilisin and solubility was quantitatively correlated with B2 using a theoretically based correlation

High glucose upregulates connective tissue growth factor expression in human vascular smooth muscle cells

BACKGROUND: Connective tissue growth factor (CTGF) is a potent profibrotic factor, which is implicated in fibroblast proliferation, angiogenesis and extracellular matrix (ECM) synthesis. It is a downstream mediator of some of the effects of transforming growth factor β (TGFβ) and is potentially induced by hyperglycemia in human renal mesangial cells. However, whether high glucose could induce the CTGF expression in vascular smooth muscle cells (VSMCs) remains unknown. Therefore, this study was designed to test whether high glucose could regulate CTGF expression in human VSMC. The effect of modulating CTGF expression on VSMC proliferation and migration was further investigated. RESULTS: Expression of CTGF mRNA was up-regulated as early as 6 hours in cultured human VSMCs after exposed to high glucose condition, followed by ECM components (collagen type I and fibronectin) accumulation. The upregulation of CTGF mRNA appears to be TGFβ-dependent since anti-TGFβ antibody blocks the effect of high glucose on CTGF gene expression. A small interference RNA (siRNA) targeting CTGF mRNA (CTGF-siRNA) effectively suppressed CTGF up-regulation stimulated by high glucose up to 79% inhibition. As a consequence of decreased expression of CTGF gene, the deposition of ECM proteins in the VSMC was also declined. Moreover, CTGF-siRNA expressing vector partially inhibited the high glucose-induced VSMC proliferation and migration. CONCLUSION: Our data suggest that in the development of macrovascular complications in diabetes, CTGF might be an important factor involved in the patho-physiological responses to high glucose in human VSMCs. In addition, the modulatory effects of CTGF-siRNA during this process suggest that specific targeting CTGF by RNA interference could be useful in preventing intimal hyperplasia in diabetic macrovascular complications

Revisiting the TALE repeat

Transcription activator-like (TAL) effectors specifically bind to double stranded (ds) DNA through a central domain of tandem repeats. Each TAL effector (TALE) repeat comprises 33–35 amino acids and recognizes one specific DNA base through a highly variable residue at a fixed position in the repeat. Structural studies have revealed the molecular basis of DNA recognition by TALE repeats. Examination of the overall structure reveals that the basic building block of TALE protein, namely a helical hairpin, is one-helix shifted from the previously defined TALE motif. Here we wish to suggest a structure-based re-demarcation of the TALE repeat which starts with the residues that bind to the DNA backbone phosphate and concludes with the base-recognition hyper-variable residue. This new numbering system is consistent with the α-solenoid superfamily to which TALE belongs, and reflects the structural integrity of TAL effectors. In addition, it confers integral number of TALE repeats that matches the number of bound DNA bases. We then present fifteen crystal structures of engineered dHax3 variants in complex with target DNA molecules, which elucidate the structural basis for the recognition of bases adenine (A) and guanine (G) by reported or uncharacterized TALE codes. Finally, we analyzed the sequence-structure correlation of the amino acid residues within a TALE repeat. The structural analyses reported here may advance the mechanistic understanding of TALE proteins and facilitate the design of TALEN with improved affinity and specificity. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13238-014-0035-2) contains supplementary material, which is available to authorized users

Joint Optimization of DNN Inference Delay and Energy under Accuracy Constraints for AR Applications

The high computational complexity and high energy consumption of artificial intelligence (AI) algorithms hinder their application in augmented reality (AR) systems. This paper considers the scene of completing video-based AI inference tasks in the mobile edge computing (MEC) system. We use multiply-and-accumulate operations (MACs) for problem analysis and optimize delay and energy consumption under accuracy constraints. To solve this problem, we first assume that offloading policy is known and decouple the problem into two subproblems. After solving these two subproblems, we propose an iterative-based scheduling algorithm to obtain the optimal offloading policy. We also experimentally discuss the relationship between delay, energy consumption, and inference accuracy.Comment: 6 pages, 7 figures, accepted by Globecom202

Protective effects of salidroside on chronic heart failure in rats and the underlying mechanisms

The present study aimed to investigate the protective effects of salidroside on chronic heart failure (CHF) in rats and to explore the underlying mechanisms. One hundred SD rats were randomly divided into sham-operated, model, and low-, medium- and high-dose salidroside groups. The CHF model was established in later 4 groups. The later 3 groups were intragastrically administrated with 6, 12 and 24 mg/kg salidroside, respectively, once a day, for continuous 4 weeks. Finally, the serum levels of brain natriuretic peptide (BNP) and interleukin 6 (IL-6), cardiac function indexes, and expression levels of myocardial cysteinyl aspartate-specific proteinase (Caspase)-3, Caspase-9, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) protein were determined. Results showed that, after treatment, compared with model group, in high-dose salidroside group the heart function indexes were significantly improved (P < 0.05), the serum levels of BNP and IL-6 were significantly decreased (P < 0.05), the expression levels of myocardial Caspase-3, Caspase-9 and MMP-1 protein were significantly decreased (P < 0.05), and the expression level of TIMP-1 protein was significantly increased (P < 0.05). In conclusion, salidroside has obvious protective effects on CHF in rats. The mechanisms may be related to its regulation of cardiomyocyte apoptosis and ventricular remodelingregulation related protein expressions

Clinical characteristics of male prolactinoma patients mainly presenting with severe obesity and the metabolic response to dopamine agonist therapy

ObjectiveTo summarize the clinical characteristics of 4 male prolactinoma patients with severe obesity.MethodsThe clinical data of all the patients were retrospectively analyzed.ResultsAll the patients visited our hospital for severe obesity at the age of 16-30 years old with their body mass index (BMI) of 37.9-55.9 kg/m2. All the patients were obese since childhood, even at birth. Hyperprolactinemia (72.3-273.0 ng/ml) was found during the etiological screening of obesity and MRI revealed pituitary adenomas. Additionally, all of them had multiple obesity related complications, such as hyperinsulinemia and dyslipidemia. Treatment of dopamine agonists (DAs) effectively normalized their prolactin level and the pituitary MRI reexamination after 6 months of DAs treatment showed the shrinkage of the pituitary adenomas in 3 patients. Their weight also decreased in different degrees (2.70~19.03% lower than the baseline) with improved metabolic profiles.ConclusionSerum prolactin level should be screened in obese patients, especially those with severe obesity

Specific DNA-RNA Hybrid Recognition by TAL Effectors

SummaryThe transcription activator-like (TAL) effector targets specific host promoter through its central DNA-binding domain, which comprises multiple tandem repeats (TALE repeats). Recent structural analyses revealed that the TALE repeats form a superhelical structure that tracks along the forward strand of the DNA duplex. Here, we demonstrate that TALE repeats specifically recognize a DNA-RNA hybrid where the DNA strand determines the binding specificity. The crystal structure of a designed TALE in complex with the DNA-RNA hybrid was determined at a resolution of 2.5 Å. Although TALE repeats are in direct contact with only the DNA strand, the phosphodiester backbone of the RNA strand is inaccessible by macromolecules such as RNases. Consistent with this observation, sequence-specific recognition of an HIV-derived DNA-RNA hybrid by an engineered TALE efficiently blocked RNase H-mediated degradation of the RNA strand. Our study broadens the utility of TALE repeats and suggests potential applications in processes involving DNA replication and retroviral infections

Distribution of crabs along a habitat gradient on the Yellow Sea coast after Spartina alterniflora invasion

The effects of Spartina alterniflora invasion on macrobenthos have long been of concern; however, there is currently no unified conclusion regarding these effects. Most studies on crabs focus on one species or limited habitat types, and assessments of the community-level effects of S. alterniflora invasion considering multiple species and habitat types have rarely been conducted. In this study, we sampled crabs along a habitat gradient from the shoreline to inland areas on the Yellow Sea coast, including the mudflat, S. alterniflora marsh, Suaeda salsa marsh and Phragmites australis marsh. A total of 10 crab species were found among all habitats, with five species in the mudflat, six species in S. alterniflora marsh, seven species in S. salsa marsh and four species in P. australis marsh. The Shannon index values for the crab communities were similar between S. alterniflora marsh and S. salsa marsh, and these values were significantly higher than those for the mudflat and P. australis marsh. However, the total biomass of crabs was highest in the mudflat, and Metaplax longipes, Philyra pisum and Macrophthalmus dilatatus exclusively preferred the mudflat. The analysis of principal components and similarities showed that the crab community structure in S. alterniflora marsh was most similar to that in S. salsa marsh, while the crab community structure in the mudflat was most different from that in the other habitat types. Our results demonstrate that the distribution of crabs varies across a habitat gradient after S. alterniflora invasion and that the crab community in S. alterniflora marsh is slightly different from that associated with the local vegetation but shows a large difference from that in the mudflat. This study indicates that some crab species may have adapted to habitat containing alien S. alterniflora, while other crab species reject this new marsh type. The effects of the distribution of crabs after S. alterniflora invasion on the regional ecosystem need further study in the future