High-resolution structural and thermodynamic analysis of extreme stabilization of human procarboxypeptidase by computational protein design

Recent efforts to design de novo or redesign the sequence and structure of proteins using computational techniques have met with significant success. Most, if not all, of these computational methodologies attempt to model atomic-level interactions, and hence high-resolution structural characterization of the designed proteins is critical for evaluating the atomic-level accuracy of the underlying design force-fields. We previously used our computational protein design protocol RosettaDesign to completely redesign the sequence of the activation domain of human procarboxypeptidase A2. With 68% of the wild-type sequence changed, the designed protein, AYEdesign, is over 10 kcal/mol more stable than the wild-type protein. Here, we describe the high-resolution crystal structure and solution NMR structure of AYEdesign, which show that the experimentally determined backbone and side-chains conformations are effectively superimposable with the computational model at atomic resolution. To isolate the origins of the remarkable stabilization, we have designed and characterized a new series of procarboxypeptidase mutants that gain significant thermodynamic stability with a minimal number of mutations; one mutant gains more than 5 kcal/mol of stability over the wild-type protein with only four amino acid changes. We explore the relationship between force-field smoothing and conformational sampling by comparing the experimentally determined free energies of the overall design and these focused subsets of mutations to those predicted using modified force-fields, and both fixed and flexible backbone sampling protocols.     

Effects of Ballota nigra on glucose and insulin in alloxan-diabetic albino rats

The hypoglycemic effect of Ballota nigra extract on albino rats was investigated. Alloxan-induced diabetes mellitus was accompanied by several fold increases in plasma glucose. Administration of aqueous extract of B. nigra extract significantly reduced glucose in both healthy and diabetic rats. These results suggest that B. nigra possess hypoglycemic effects in rats and therefore, can be useful for the treatment of diabetes mellitus.     

Beauveria bassiana for the control of Sunn Pest (Eurygaster integriceps) (Hemiptera: Scutelleridae) and aspects of the insect's daily activity relevant to a mycoinsecticide

A series of investigations was carried out at ICARDA during April-June 2004 and May-June 2005 to investigate the use of the entomopathogenic fungus Beauveria bassiana to control Eurygaster integriceps and to determine aspects of the insect's biology that could be relevant to control with the fungus. Application in 2004 of an oil-based formulation of B. bassiana showed distribution of the spray decreasing from the top of the wheat to the bottom, although the proportion of tillers with coverage at the base was still 80%. Seventy percent of Sunn Pest had received direct hits, with the highest amount on the dorsal surface, followed by the ventral surface, then the legs and antennae. Mortality of field-treated, then laboratory-maintained insects, ranged from 47.6% for insects exposed only to the B. bassiana spray, 74.2% for unsprayed insects exposed to treated wheat and 86.4% for insects receiving direct spray and being exposed to treated wheat. Some field results demonstrated mortality. Mortality assessments following the 2005 treatments were conducted solely in the field, there was no apparent effect upon Sunn Pest numbers. The majority of studies on Sunn Pest biology were carried out on untreated insects outside the trial plots. Different assessment methods resulted in varying estimates of adults and juveniles in the field. The numbers of Sunn Pest found and the proportion of these at the top of the wheat crop, declined after early morning and increased again in the early evening, suggesting sheltering deeper in the crop or in the soil during the middle part of the day. Mobility and location of marked insects was followed in micro-plots and by mark and recapture studies, demonstrating a range of activities, from movement around the crop for a number of metres, including contact with numerous tillers to almost total immobility; Sunn Pest generally avoided movement on the soil. The potential for Sunn Pest control with a mycoinsecticide and how Sunn Pest behaviour is relevant to a mycoinsecticide application are discussed.     

Molecular profiling of adult acute myeloid and lymphoid leukemia in a major referral center in Lebanon: a 10-year experience report and review of the literature

Molecular Biology Reports - Recurrent genetic abnormalities confer distinct morphologic features and play a role in determining the clinical behavior, prognosis and adequate treatment of acute...     

Association of Residual Plasma Viremia and Intima-Media Thickness in Antiretroviral-Treated Patients with Controlled Human Immunodeficiency Virus Infection

Background

While residual plasma viremia is commonly observed in HIV-infected patients undergoing antiretroviral treatment (ART), little is known about its subclinical consequences.

Methods

This cross-sectional study included 47 male, never-smoking, non-diabetic patients with ≥4 years of ART and controlled HIV-replication (HIV-viral load, VL <20 copies/mL for ≥1 year). Residual HIV-VL was measured using an ultrasensitive assay (quantification limit: 1 copy/ml). Patients were categorized as having detectable (D; 1-20 copies/mL, n = 14) or undetectable (UD; <1 copies/mL, n = 33) HIV-VL. Linear regression was used to model the difference in total carotid intima-media thickness [c-IMT, measures averaged across common carotid artery (cca), bifurcation, and internal carotid artery] and cca-IMT alone across detection groups. Multivariable models were constructed for each endpoint in a forward-stepwise approach.

Results

No significant differences were observed between viremia groups with respect to median ART-duration (9.6 years, IQR = 6.8–10.9), nadir CD4+T-cell (208/mm³, IQR = 143–378), and CD4+T-cell count (555/mm³, IQR = 458–707). Median adjusted inflammatory markers tended to be higher in patients with D- than UD-viremia, with differences in IL-10 being significant (p = 0.03). After adjustment on age, systolic blood pressure, and insulin resistance, mean cca-IMT was significantly lower in patients with undetectable (0.668 mm±0.010) versus detectable viremia (0.727 mm±0.015, p = 0.002). Cca-IMT was also independently associated with age and insulin resistance. Mean adjusted total c-IMT was no different between viremia groups (p = 0.2), however there was large variability in bifurcation c-IMT measurements.

Conclusions

Higher cca-IMT was observed in patients with detectable, compared to undetectable, HIV-VL in never-smoking ART-controlled patients, suggesting that residual HIV viremia may be linked to atherosclerosis.     

Inorganic Phosphate Accelerates the Migration of Vascular Smooth Muscle Cells: Evidence for the Involvement of miR-223

Backgound

An elevated serum inorganic phosphate (Pi) level is a major risk factor for kidney disease and downstream vascular complications. We focused on the effect of Pi levels on human aortic vascular smooth muscle cells (VSMCs), with an emphasis on the role of microRNAs (miRNAs).

Methodology/Principal Findings

Exposure of human primary VSMCs in vitro to pathological levels of Pi increased calcification, migration rate and concomitantly reduced cell proliferation and the amount of the actin cytoskeleton. These changes were evidenced by significant downregulation of miRNA-143 (miR-143) and miR-145 and concomitant upregulation of their targets and key markers in synthetic VSMCs, such as Krüppel-like factors−4 and −5 and versican. Interestingly, we also found that miR-223 (a marker of muscle damage and a key factor in osteoclast differentiation) is expressed in VSMCs and is significantly upregulated in Pi-treated cells. Over-expressing miR-223 in VSMCs increased proliferation and markedly enhanced VSMC migration. Additionally, we found that the expression of two of the known miR-223 targets, Mef2c and RhoB, was highly reduced in Pi treated as well as miR-223 over-expressing VSMCs. To complement these in vitro findings, we also observed significant downregulation of miR-143 and miR-145 and upregulation of miR-223 in aorta samples collected from ApoE knock-out mice, which display vascular calcification.

Conclusions/Significance

Our results suggest that (i) high levels of Pi increase VSMC migration and calcification, (ii) altered expression levels of miR-223 could play a part in this process and (iii) miR-223 is a potential new biomarker of VSMC damage.