In-Silico Analyses of Nonsynonymous Variants in the BRCA1 Gene

BReast CAncer gene 1 (BRCA1)—a tumor suppressor gene plays an important role in the DNA repair mechanism. Several BRCA1 variants perturb its structure and function, including synonymous and nonsynonymous single nucleotide polymorphisms (SNPs). In the present study, we performed in-silico analyses of nonsynonymous SNPs (nsSNPs) of the BRCA1 gene. In total, 122 nsSNPs were retrieved from the NCBI SNP database and in-silico analyses were performed using computational prediction tools: SIFT, PROVEAN, Mutation Taster, PolyPhen-2, MutPred, and ConSurf. Of these tools, SIFT, PROVEAN, and Mutation Taster predicted 61 out of 122 nsSNPs as “damaging”, based on structural homology analysis. PolyPhen-2 classified 22 nsSNPs as “probably damaging”. These nsSNPs were further analyzed by MutPred to predict basic molecular mechanisms of amino acid alteration. ConSurf analysis predicted eleven conserved amino acid residues with structural and functional consequences. We identified five amino acid residues in the RING finger domain (L22, C39, H41, C44, and C47) and two in the BRCT domain (P1771 and I1707) with the potential to deter the BRCA1 protein function. This study provides insights into the effect of nsSNPs and amino acid substitutions in BRCA1.

     

Apolipoprotein E promotes astrocyte colocalization and degradation of deposited amyloid-beta peptides

We have previously shown that apolipoprotein E (Apoe) promotes the formation of amyloid in brain and that astrocyte-specific expression of APOE markedly affects the deposition of amyloid-beta peptides (Abeta) in a mouse model of Alzheimer disease. Given the capacity of astrocytes to degrade Abeta, we investigated the potential role of Apoe in this astrocyte-mediated degradation. In contrast to cultured adult wild-type mouse astrocytes, adult Apoe(-/-) astrocytes do not degrade Abeta present in Abeta plaque-bearing brain sections in vitro. Coincubation with antibodies to either Apoe or Abeta, or with RAP, an antagonist of the low-density lipoprotein receptor family, effectively blocks Abeta degradation by astrocytes. Phase-contrast and confocal microscopy show that Apoe(-/-) astrocytes do not respond to or internalize Abeta deposits to the same extent as do wild-type astrocytes. Thus, Apoe seems to be important in the degradation and clearance of deposited Abeta species by astrocytes, a process that may be impaired in Alzheimer disease.     

Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation

Background

The use of spectrally distinct variants of green fluorescent protein (GFP) such as cyan or yellow mutants (CFP and YFP, respectively) is very common in all different fields of life sciences, e.g. for marking specific proteins or cells or to determine protein interactions. In the latter case, the quantum physical phenomenon of fluorescence resonance energy transfer (FRET) is exploited by specific microscopy techniques to visualize proximity of proteins.

Methodology/Principal Findings

When we applied a commonly used FRET microscopy technique - the increase in donor (CFP)-fluorescence after bleaching of acceptor fluorophores (YFP), we obtained good signals in live cells, but very weak signals for the same samples after fixation and mounting in commercial microscopy mounting fluids. This observation could be traced back to much faster bleaching of CFP in these mounting media. Strikingly, the opposite effect of the mounting fluid was observed for YFP and also for other proteins such as Cerulean, TFP or Venus. The changes in photostability of CFP and YFP were not caused by the fixation but directly dependent on the mounting fluid. Furthermore we made the interesting observation that the CFP-fluorescence intensity increases by about 10 - 15% after illumination at the YFP-excitation wavelength – a phenomenon, which was also observed for Cerulean. This photoactivation of cyan fluorescent proteins at the YFP-excitation can cause false-positive signals in the FRET-microscopy technique that is based on bleaching of a yellow FRET acceptor.

Conclusions/Significance

Our results show that photostability of fluorescent proteins differs significantly for various media and that CFP bleaches significantly faster in commercial mounting fluids, while the opposite is observed for YFP and some other proteins. Moreover, we show that the FRET microscopy technique that is based on bleaching of the YFP is prone to artifacts due to photoactivation of cyan fluorescent proteins under these conditions.     

Sequence context- and temperature-dependent nucleotide excision repair of a benzo[a]pyrene diol epoxide-guanine DNA adduct catalyzed by thermophilic UvrABC proteins

The influence of DNA base sequence context on the removal of a bulky benzo[a]pyrene diol epoxide-guanine adduct, (+)-trans-B[a]P-N2-dG (G*), by UvrABC nuclease from the thermophilic organism Bacillus caldotenax was investigated. The lesion was flanked by either T or C in otherwise identical complementary 43-mer duplexes (TG*T or CG*C, respectively). It was reported earlier that in the CG*C context, a dominant minor groove adduct structure was observed by NMR methods with all Watson-Crick base pairs intact, and the duplex exhibited a rigid bend. In contrast, in the TG*T context, a highly flexible bend was observed, base pairing at G*, and two 5'-base pairs flanking the adduct were impaired, and multiple solvent-accessible adduct conformations were observed. The TG*T-43-mer duplexes are incised with consistently greater efficiency by UvrABC proteins from B. caldotenax by a factor of 2.3 +/- 0.3. The rates of incisions increase with increasing temperature and are characterized by linear Arrhenius plots with activation energies of 27.0 +/- 1.5 and 23.4 +/- 1.0 kcal/mol for CG*C and TG*T duplexes, respectively. These values reflect the thermophilic characteristics of the UVrABC nuclease complex and the contributions of the different DNA substrates to the overall activation energies. These effects are consistent with base sequence context-dependent differences in structural disorder engendered by a loss of local base stacking interactions and Watson-Crick base pairing in the immediate vicinity of the lesions in the TG*T duplexes. The local weakening of base pairing interactions constitutes a recognition element of the UvrABC nucleotide excision repair apparatus.     

Control of Adipose Tissue Expandability in Response to High Fat Diet by the Insulin-like Growth Factor-binding Protein-4

Adipose tissue expansion requires growth and proliferation of adipocytes and the concomitant expansion of their stromovascular network. We have used an ex vivo angiogenesis assay to study the mechanisms involved in adipose tissue expansion. In this assay, adipose tissue fragments placed under pro-angiogenic conditions form sprouts composed of endothelial, perivascular, and other proliferative cells. We find that sprouting was directly stimulated by insulin and was enhanced by prior treatment of mice with the insulin sensitizer rosiglitazone. Moreover, basal and insulin-stimulated sprouting increased progressively over 30 weeks of high fat diet feeding, correlating with tissue expansion during this period. cDNA microarrays analyzed to identify genes correlating with insulin-stimulated sprouting surprisingly revealed only four positively correlating (Fads3, Tmsb10, Depdc6, and Rasl12) and four negatively correlating (Asph, IGFbp4, Ppm1b, and Adcyap1r1) genes. Among the proteins encoded by these genes, IGFbp4, which suppresses IGF-1 signaling, has been previously implicated in angiogenesis, suggesting a role for IGF-1 in adipose tissue expandability. Indeed, IGF-1 potently stimulated sprouting, and the presence of activated IGF-1 receptors in the vasculature was revealed by immunostaining. Recombinant IGFbp4 blocked the effects of insulin and IGF-1 on mouse adipose tissue sprouting and also suppressed sprouting from human subcutaneous adipose tissue. These results reveal an important role of IGF-1/IGFbp4 signaling in post-developmental adipose tissue expansion.     

The use of the long head of triceps interposition muscle flap for treatment of massive rotator cuff tears

Massive rotator cuff tears present a difficult problem for orthopedic surgeons. To address this problem, a long head of triceps muscle interposition flap was proposed. Ten patients underwent repair of massive rotator cuff tears using the triceps muscle flap. The patients' strength and range of motion were tested preoperatively, and a University of California, Los Angeles, shoulder score was assigned. Similar testing was performed 1 year later. Postoperatively, the patients showed significant improvement in the overall shoulder score and in the pain and function components of the score. There was no significant improvement in shoulder range of motion, except for shoulder flexion. An important finding was that there was no loss of strength in elbow extension following the loss of the long head of triceps muscle. It was concluded that the long head of triceps interposition flap is useful in the reconstruction of the massive rotator cuff tear.     

Impacted morselized cancellous bone: mechanical effects of defatting and augmentation with fine hydroxyapatite particles

Geotechnical engineering testing techniques were used to study the mechanical properties of morselized cancellous bone (MCB) and the effects of defatting and augmentation with fine hydroxyapatite (HA) particles. Bovine and human cancellous bone was morselized, rinsed, and manually squeezed to remove excess fluid, producing a standard surgical MCB sample that was also used as a control. Some of the MCB was defatted with heat and detergent and mixed with HA particles in ratios ranging from 0% to 100% HA. Compaction tests were used to determine the effects of moisture content and the amount of MCB that can be packed into a confined space. One-dimensional consolidation tests were used to determine the uniaxial strain behavior, confined modulus, and steady-state creep rate. The compaction tests demonstrated that defatting and adding HA particles significantly increased density. The one-dimensional consolidation tests showed that strain was decreased, modulus was increased and the creep rate was decreased by defatting and adding HA.     

Mechanistic Insights into PTS2-mediated Peroxisomal Protein Import: THE CO-RECEPTOR PEX5L DRASTICALLY INCREASES THE INTERACTION STRENGTH BETWEEN THE CARGO PROTEIN AND THE RECEPTOR PEX7*

The destination of peroxisomal matrix proteins is encoded by short peptide sequences, which have been characterized as peroxisomal targeting signals (PTS) residing either at the C terminus (PTS1) or close to the N terminus (PTS2). PTS2-carrying proteins interact with their cognate receptor protein PEX7 that mediates their transport to peroxisomes by a concerted action with a co-receptor protein, which in mammals is the PTS1 receptor PEX5L. Using a modified version of the mammalian two-hybrid assay, we demonstrate that the interaction strength between cargo and PEX7 is drastically increased in the presence of the co-receptor PEX5L. In addition, cargo binding is a prerequisite for the interaction between PEX7 and PEX5L and ectopic overexpression of PTS2-carrying cargo protein drastically increases the formation of PEX7-PEX5L complexes in this assay. Consistently, we find that the peroxisomal transfer of PEX7 depends on cargo binding and that ectopic overexpression of cargo protein stimulates this process. Thus, the sequential formation of a highly stable trimeric complex involving cargo protein, PEX7 and PEX5L stabilizes cargo binding and is a prerequisite for PTS2-mediated peroxisomal import.     

Prehabilitation before total knee arthroplasty increases strength and function in older adults with severe osteoarthritis

Preparing for the stress of total knee arthroplasty (TKA) surgery by exercise training (prehabilitation) may improve strength and function before surgery and, if effective, has the potential to contribute to postoperative recovery. Subjects with severe osteoarthritis (OA), pain intractable to medicine and scheduled for TKA were randomized into a usual care (UC) group (n = 36) or usual care and exercise (UC + EX) group (n = 35). The UC group maintained normal daily activities before their TKA. The UC + EX group performed a comprehensive prehabilitation program that included resistance training using bands, flexibility, and step training at least 3 times per week for 4-8 weeks before their TKA in addition to UC. Leg strength (isokinetic peak torque for knee extension and flexion) and ability to perform functional tasks (6-minute walk, 30 second sit-to-stand repetitions, and the time to ascend and descend 2 flights of stairs) were assessed before randomization at baseline (T1) and 1 week before the subject's TKA (T2). Repeated-measures analysis of variance indicated a significant group by time interaction (p < 0.05) for the 30-second sit-to-stand repetitions, time to ascend the first flight of stairs, and peak torque for knee extension in the surgical knee. Prehabilitation increased leg strength and the ability to perform functional tasks for UC + EX when compared to UC before TKA. Short term (4-8 weeks) of prehabilitation was effective for increasing strength and function for individuals with severe OA. The program studied is easily transferred to a home environment, and clinicians working with this population should consider prehabilitation before TKA.