Use of transmission electron microscopy to identify nanocrystals of challenging protein targets

The current practice for identifying crystal hits for X-ray crystallography relies on optical microscopy techniques that are limited to detecting crystals no smaller than 5 μm. Because of these limitations, nanometer-sized protein crystals cannot be distinguished from common amorphous precipitates, and therefore go unnoticed during screening. These crystals would be ideal candidates for further optimization or for femtosecond X-ray protein nanocrystallography. The latter technique offers the possibility to solve high-resolution structures using submicron crystals. Transmission electron microscopy (TEM) was used to visualize nanocrystals (NCs) found in crystallization drops that would classically not be considered as “hits.” We found that protein NCs were readily detected in all samples tested, including multiprotein complexes and membrane proteins. NC quality was evaluated by TEM visualization of lattices, and diffraction quality was validated by experiments in an X-ray free electron laser.The emergence of X-ray free electron laser (X-FEL)–based serial femtosecond crystallography holds the promise of solving the 3D structure of proteins that can only crystallize as “nanocrystals” (NCs) or are highly sensitive to radiation damage (1–5). NCs appropriate for X-FEL experiments are considered to be 200 nm to 2 μm in size (6). This size is constrained primarily by the requirements of the NC delivery system to the X-FEL beam. In addition to allowing for structure resolution of NCs by X-FEL experiments, they provide the advantage of requiring no crystal cryoprotection because these experiments are performed at room temperature (3, 7). Given the opportunities that X-FELs offer to the field of crystallography, efficient methodologies to detect NCs from single crystallography drops and to optimize these identified conditions yielding NCs will be essential for future developments in structural biology. Current methods to detect the presence of NCs include dynamic light scattering (DLS), bright-field microscopy, birefringence microscopy, and intrinsic tryptophan UV fluorescence imaging, as well as technologies that rely upon second harmonic generation, such as second order nonlinear imaging of chiral crystals (SONICC) (8, 9) and X-ray powder diffraction. However, limitations of these imaging techniques include (i) ineffective detection of crystals smaller than 5 μm (8, 10), (ii) false-positive conditions as a result of interference from precipitate backgrounds (8, 10), and (iii) false-negative conditions resulting from the lack of tryptophan residues in the case of UV fluorescence and from the lack of chiral centers in the case of SONICC (11). Although DLS can accurately measure the size distribution of nanometer-sized protein aggregates, it is unable to distinguish unambiguously between amorphous and crystalline (12). Finally, X-ray powder diffraction, a method that has been applied to evaluate samples for the presence and concentration of NCs, requires more material than is produced in a single crystallization screening drop, and synchrotron radiation is usually required to produce measurable diffraction (13). In this study, we use UV fluorescence microscopy and DLS to detect crystallization drops containing NCs, followed by transmission electron microscopy (TEM) to identify protein NCs accurately and determine NC quality by evaluating the reciprocal lattice reflections in diffraction patterns calculated from images.     

Midterm impact of sleeve gastrectomy, calibrated with a 50-fr bougie, on weight loss, glucose homeostasis, lipid profiles, and comorbidities in morbidly obese patients

Bariatric surgery has been shown to be effective in achieving and maintaining weight change and reducing obesity-related comorbidities. Recent reports have shown that sleeve gastrectomy could have similar resolution rates of the metabolic syndrome than Roux-Y bypass after a short-term follow-up of 1 year. Most surgeons calibrate the sleeve with 32-Fr to 40-Fr bougies. There is little mid- and long-term information available about the evolution of these comorbidities with this procedure and with calibration of the sleeve with a 50-Fr bougie. A retrospective study of all the morbidly obese patients undergoing laparoscopic sleeve gastrectomy, calibrated with a 50-Fr bougie, as bariatric procedure between October 2007 and September 2009 was performed. Mean excessive body mass index loss was 76.9 per cent after 1 year and 79.9 per cent after 2 years. After surgery, 83.3 per cent of patients with Type 2 diabetes mellitus discontinued their hypoglycemic medication at 1 month. All the patients with hypertension discontinued antihypertensive drugs at 6 months. One hundred per cent of patients with hypertriglyceridemia discontinued their hypolipidemic drugs at 3 months. Glucose levels decreased significantly 3 months after surgery (mean reduction of 24.7 mg/dL; 95% confidence interval [CI], 8.8 to 40.7; P = 0.003). Triglyceride levels decreased 3 months after surgery (mean reduction of 54.4 mg/dL; 95% CI, 22.8 to 86.1; P = 0.004). High-density lipoprotein (HDL) cholesterol increased significantly after 12 months (increase of 16.7 mg/dL; 95% CI, 11.7 to 21.7; P < 0.001). The changes observed were maintained 24 months after surgery. Sleeve gastrectomy, calibrated with a 50-Fr bougie, significantly reduced glucose and triglyceride levels and the cardiovascular risk predictor triglyceride/HDL ratio and increased HDL levels after surgery and maintained them under normal ranges for at least 2 years.     

Hb O Padova in a Spanish Family

Haematological and biochemical aspects of a new family with heterozygous Hb O Padova [alpha 30(B11) Glu----Lys] is described. This variant has been found on a screening programme of abnormal haemoglobin in cord blood. It was separated by cellulose acetate, isoelectric focusing, citrate agar, DEAE-cellulose chromatography and by cation exchange high-performance liquid chromatography. The heat stability and the functional properties were found to be normal. This is the third family with Hb O Padova reported, all from the Mediterranean region.     

Vimentin and laminin expression is associated with basal-like phenotype in both sporadic and BRCA1-associated breast carcinomas

AIMS: To determine whether basal-like phenotype and vimentin and/or laminin are related in both sporadic/familial (BRCA1 or BRCA2 mutated) tumours. METHODS: 230 non-familial and 28 hereditary node-negative invasive breast carcinomas were immunohistochemically analysed for oestrogen receptors (ER), progesterone receptors (PR), cytokeratin 5/6 (CK5/6), epidermal growth factor receptors (EGFR), Ki67, p53, vimentin and laminin, using tissue microarrays. Tumours were considered to have basal-like phenotype if they were ER negative and HER2 negative, but positive for CK5/6 and/or EGFR. RESULTS: In sporadic tumours, vimentin expression was found in 77.8% cases with basal-like phenotype and 15.5% of non-basal cases (p<0.001). In familial cases, vimentin was expressed in 83.3% basal-like cancers and 16.7% of non-basal tumours (p<0.001). Vimentin expression was more frequent in BRCA1 than BRCA2 mutation carriers. Vimentin expressing tumours were associated with poor prognosis (p = 0.012) among patients not receiving adjuvant chemotherapy and showed a trend for local recurrence or visceral but not bone metastasis (p = 0.021). Laminin expression was also related to basal-like phenotype in both sporadic/familial cases (p<0.001 and p = 0.007, respectively), but neither with prognosis nor recurrence pattern in sporadic cancers. CONCLUSIONS: Vimentin and laminin expression is associated with basal-like phenotype in breast cancer. Expression of vimentin and laminin is characteristic of BRCA1 associated tumours. Since vimentin and laminin staining is widely used by pathologists for diagnostic purposes, thus demonstrating the robustness of their specific antibodies, the immunohistochemical evaluation of these two molecules could be used in identification of basal-like breast tumours in both sporadic/familial cases.     

Spleen B cells from BALB/c are more prone to activation than spleen B cells from C57BL/6 mice during a secondary immune response to cruzipain

There is an increasing interest in the study of roles that B cells may play in regulating immune responses both in protection and in pathogenesis. However, little is known about additional immune functions of B cells independently of antibody production. In this study, we have assessed how the immunization with T-dependent antigens in different host genetic backgrounds affects several parameters of B cells during secondary immune responses. We have previously reported that BALB/c immunized with cruzipain, induced heart autoimmunity, whereas C57BL/6 mice were resistant. In a comparative study employing the same experimental model, we demonstrated that BALB/c-enriched spleen B cells presented higher ability to proliferate releasing elevated levels of IL-4. Moreover, spleen of immune BALB/c mice presented an increased number of germinal center and plasma cells as well as higher expression of B-cell activation markers (MHC class II, CD40, CD86). These findings demonstrate the influence of genetic background on B-cell activation and emphasize the importance of examining B-cell behavior in the context of the specific immunogens.     

Late-in-life surgery associated with Creutzfeldt-Jakob disease: a methodological outline for evidence-based guidance

Background

There is increasing epidemiological evidence of etiological links between general surgery and sporadic Creutzfeldt-Jakob disease (sCJD) with long incubation periods. The purpose of this study was to identify specific surgical procedures potentially associated with sCJD to be targeted for preventive presurgical-intervention guidance.

Results

We propose a three-step clinical guidance outline where surgical procedures associated with sCJD clinical onset – potentially more contaminant - are taken into account. Data on hospital discharges and surgical procedures were obtained from Danish and Swedish national in-patient hospital registries for 167 sCJD cases, onset 1987–2003, and for 835 matched and 2,224 unmatched population controls. Surgery was allocated to different life-time periods as previously reported, and frequencies were compared using logistic regression analysis. In the year preceding clinical onset, persons with sCJD underwent a statistically significant higher number of minor surgical interventions (OR (95% CI): 17.50 (3.64-84.24)), transluminal endoscopies (OR: 2.73 (1.01–7.37)) and gastrointestinal operations (OR: 3.51 (1.21–10.19)) compared to matched controls. Surgical discharges clustered towards clinical onset. These differences increased during the clinical period, with statistically significant higher frequencies for both endoscopies and minor surgery (OR: 13.91 (5.87-32.95), and for main surgical procedures (OR: 2.10 (1.00-4.39)), particularly gastrointestinal surgery (OR: 6.00 (1.83-19.66)), and surgery contacting skeletal muscle. Comparisons with unmatched controls yielded similar results for neurosurgery in the clinical period (OR: 19.40 (2.22-168.34)).

Conclusions

These results suggest that some types of surgical procedures are associated with sCJD, after clinical onset or particularly just before onset. Selective planning of such surgery to minimize instrument/device contamination or quarantining might be feasible. Conditional to progress in sCJD etiological research, results are relevant for guidance development.

     

Differences in cognitive performance, level of dependency and quality of life (QoL), related to age and cognitive status in a sample of Spanish old adults under and over 80 years of age

The main objective of this study was to analyze the similarities and differences in cognitive performance, level of dependency, cognitive plasticity and QoL in a sample of young-old adults and old-old adults, bearing in mind both the age-group (under or over 80 years) and the cognitive status of the participants. The study population consisted of 220 people living in sheltered accommodation for elderly people in the South of Spain, with an average age of 80.75 years. Participants were evaluated by means of cognitive performance tests, a QoL questionnaire, a depression scale and a dependency assessment scale. The results indicate that the main differences in the variables analyzed are due to the cognitive status of the sample and not to the fact that the participants are under or over 80 years of age. The findings show that major inter-individual differences in this stage of life depend not only on age but also on cognitive status, which is thus an important factor to take into account when working with this sector of the population.