Summary

Classical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally-based test cases: Tobacco mosaic virus, a rod-shaped virus; and aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multi-component structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures.

We demonstrate a solution to make resonant-waveguide-grating sensing both robust and simpler to optically assess, in the spirit of biochips. Instead of varying wavelength or angle to track the resonant condition, the grating itself has a step-wise variation with typically few tens of neighboring “micropads.” An image capture with incoherent monochromatic light delivers spatial intensity sequences from these micropads. Sensitivity and robustness are discussed using correlation techniques on a realistic model (Fano shapes with noise and local distortion contributions). We confirm through fluid refractive index sensing experiments an improvement over the step-wise maximum position tracking by more than 2 orders of magnitude, demonstrating sensitivity down to 2 × 10−5 RIU, giving high potential development for bioarray imaging.

Botulism, a disease of humans characterized by prolonged paralysis, is caused by botulinum neurotoxins (BoNTs), the most poisonous substances known. There are seven serotypes of BoNT (A–G) which differ from each other by 34–64% at the amino acid level. Each serotype is uniquely recognized by polyclonal antibodies, which originally were used to classify serotypes. To determine if there existed monoclonal antibodies (mAbs) capable of binding two or more serotypes, we evaluated the ability of 35 yeast-displayed single-chain variable fragment antibodies generated from vaccinated humans or mice for their ability to bind multiple BoNT serotypes. Two such clonally related human mAbs (1B18 and 4E17) were identified that bound BoNT serotype A (BoNT/A) and B or BoNT/A, B, E and F, respectively, with high affinity. Using molecular evolution techniques, it proved possible to both increase affinity and maintain cross-serotype reactivity for the 4E17 mAb. Both 1B18 and 4E17 bound to a relatively conserved epitope at the tip of the BoNT translocation domain. Immunoglobulin G constructed from affinity matured variants of 1B18 and 4E17 were evaluated for their ability to neutralize BoNT/B and E, respectively, in vivo. Both antibodies potently neutralized BoNT in vivo demonstrating that this epitope is functionally important in the intoxication pathway. Such cross-serotype binding and neutralizing mAbs should simplify the development of antibody-based BoNT diagnostics and therapeutics.

Aims/hypothesis

FTO harbours the strongest known obesity-susceptibility locus in Europeans. While there is growing evidence for a role for FTO in obesity risk in Asians, its association with type 2 diabetes, independently of BMI, remains inconsistent. To test whether there is an association of the FTO locus with obesity and type 2 diabetes, we conducted a meta-analysis of 32 populations including 96,551 East and South Asians.

Methods

All studies published on the association between FTO-rs9939609 (or proxy [r2 > 0.98]) and BMI, obesity or type 2 diabetes in East or South Asians were invited. Each study group analysed their data according to a standardised analysis plan. Association with type 2 diabetes was also adjusted for BMI. Random-effects meta-analyses were performed to pool all effect sizes.

Results

The FTO-rs9939609 minor allele increased risk of obesity by 1.25-fold/allele (p = 9.0 × 10−19), overweight by 1.13-fold/allele (p = 1.0 × 10−11) and type 2 diabetes by 1.15-fold/allele (p = 5.5 × 10−8). The association with type 2 diabetes was attenuated after adjustment for BMI (OR 1.10-fold/allele, p = 6.6 × 10−5). The FTO-rs9939609 minor allele increased BMI by 0.26 kg/m2 per allele (p = 2.8 × 10−17), WHR by 0.003/allele (p = 1.2 × 10−6), and body fat percentage by 0.31%/allele (p = 0.0005). Associations were similar using dominant models. While the minor allele is less common in East Asians (12–20%) than South Asians (30–33%), the effect of FTO variation on obesity-related traits and type 2 diabetes was similar in the two populations.

Conclusions/interpretation

FTO is associated with increased risk of obesity and type 2 diabetes, with effect sizes similar in East and South Asians and similar to those observed in Europeans. Furthermore, FTO is also associated with type 2 diabetes independently of BMI.

Electronic supplementary material

The online version of this article (doi:10.1007/s00125-011-2370-7) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Pancreas cancer has a grave prognosis and treatment options remain limited despite advancement in anti-cancer chemotherapeutics. This review provides an overview of the emerging therapies for pancreas cancer, focusing on novel signal transduction inhibitors (insulin-like growth factor receptor, hedgehog/Smo, PI3k/Akt/mTOR) and cytotoxics (nab-paclitaxel) that are currently in clinical development. Despite the impact molecularly targeted agents have on other tumor types, their application without cytotoxics in pancreas cancer remains limited. In addition, recent report of the superiority of an intensive cytotoxic regimen using fluorouracil, irinotecan and oxaliplatin (FOLFIRINOX) over gemcitabine reminded us of the importance of cytotoxics in this disease. As such, the future of pancreas cancer therapy may be combination regimens consisting of cytotoxics and molecularly targeted agents.

Botulism is caused by the botulinum neurotoxins (BoNTs), the most poisonous substance known. Because of the high potency of BoNT, development of diagnostic and therapeutic antibodies for botulism requires antibodies of very high affinity. Here we report the use of yeast mating to affinity mature BoNT antibodies by light chain shuffling. A library of immunoglobulin light chains was generated in a yeast vector where the light chain is secreted. The heavy chain variable region and the first domain of the constant region (VH–CH1) from a monoclonal antibody was cloned into a different yeast vector for surface display as a fusion to the Aga2 protein. Through yeast mating of the two haploid yeasts, a library of light chain-shuffled Fab was created. Using this approach, the affinities of one BoNT/A and two BoNT/B scFv antibody fragments were increased from 9- to more than 77-fold. Subcloning the V-genes from the affinity-matured Fab yielded fully human IgG1 with equilibrium binding constants for BoNT/A and BoNT/B of 2.51 × 10−11 M or lower for all three monoclonal antibodies. This technique provides a rapid route to antibody affinity maturation.

Objective: To determine the impact of white matter hyperintensities (WMHs) on physical health and cognitive function in 60–64 year old individuals residing in the community.

Methods: A subsample of 478 persons aged 60–64 from a larger community sample underwent brain magnetic resonance imaging (MRI) scans. WMHs on T2 weighted FLAIR (fluid attenuated inversion recovery) MRI scans were assessed using an automated procedure. Subjects were assessed for global cognitive function, episodic memory, working memory (digit span), information processing speed (Symbol Digit Modalities Test; SDMT), fine motor dexterity (Purdue Pegboard), and grip strength, and completed the Physical Component Summary of the Short Form Health Survey (SF-12). Regression analyses were used to examine the effect of WMHs on physical and cognitive function.

Results: Deep and periventricular WMHs were present in all subjects, with women having slightly more lesions than men. WMHs were significantly associated with poorer reported physical health on the SF-12 scale, after adjusting for depression, cognitive function, and brain atrophy. WMHs were also related to lower scores on the Purdue Pegboard test, grip strength, choice reaction time, and SDMT, but not on tests of episodic memory, working memory, general intellectual function, and global cognitive function. On regression analyses, the Purdue Pegboard test and grip strength were related to physical disability.

Conclusion: WMHs are common, albeit mild, in middle adult life. They are associated with physical disability, possibly through reduced speed, fine motor coordination, and muscular strength. They are also related to slowed information processing speed but not other cognitive functions.

OBJECTIVES—To examine the association between the longest held occupation in a lifetime and risk of disability in activities of daily living (ADL) among elderly people (65 years and older) in northern Taiwan.
METHODS—A case-control design was used nested within two cohorts of a total of 2198 elderly people who had been followed up either between 1993 and 1997 or between 1996 and 1997. Cases were 360 elderly people with ADL disability within the study period. For each case, two sex matched controls were randomly sampled from the pool of elderly people free from ADL disability. Occupational data were collected through interviews conducted in 1997. Performed job contents were classified into occupational categories and occupation based social classes. Unconditional logistic regression techniques were used to estimate relative risk and 95% confidence intervals (95% CIs) of ADL disability.
RESULTS—Compared with people who were former legislators, government administrators, or business executives and managers, workers in agriculture, animal husbandry, forestry, or fishing (odds ratio (OR) 1.9, 95% CI 1.1 to 3.5) and workers in craft and related trades (OR 1.9, 95% CI 1.1 to 3.4) had significantly increased risks of subsequent ADL disability. Differential risks of ADL disability were found across social classes, with a significant dose-response trend in which unskilled blue collar workers had an 1.8 times higher risk of ADL disability than higher social classes of white collar workers.
CONCLUSIONS—After adjustment for education, there was still an inverse relation between risk of ADL disability and social class. Although total control for all the known risk factors for ADL disability among elderly people was impossible, the results tend to suggest a potential for an effect of longest held occupation in a lifetime on risk of ADL disability.


Keywords: activity of daily living; occupation; socioeconomic status

To investigate if decreased exposure to common childhood infections is associated with risk of childhood acute lymphoblastic leukaemia (ALL) we conducted a case–control study of 1842 newly diagnosed and immunophenotypically defined cases of ALL under age 15, and 1986 matched controls in the US. Data regarding day care, sibship size and common childhood infections were obtained through parental interviews. Data were analysed stratified by leukaemia lineage and separately for ‘common’ childhood ALL (age 2–5 years, CD19, CD10-positive). Neither attendance at day care nor time at day care was associated with risk of ALL overall or ‘common’ ALL. Ear infections during infancy were less common among cases, with odds ratios of 0.86, 0.83, 0.71 and 0.69 for 1, 2–4, 5+ episodes, and continuous infections respectively (trend P = 0.026). No effect of sibship size or birth interval was seen. With one exception (ear infections), these data do not support the hypothesis that a decrease in the occurrence of common childhood infection increases risk of ALL. © 2000 Cancer Research Campaign

BACKGROUND: Early discharge after childbirth is widely reported. In this study the authors examined trends in maternal length of hospital stay in Canada from fiscal year 1984-85 through fiscal year 1994-95. They also examined variations in length of stay in 1994-95 in most of the Canadian provinces and the territories. METHODS: Epidemiologic analyses of the temporal and geographic variations in maternal length of hospital stay in Canada from 1984-85 to 1994-95 (even years only), based on hospital discharge data collected by the Canadian Institute for Health Information, with a total of 1,456,800 women for the 6 study years. RESULTS: Mean length of hospital stay decreased during the decade, from 5.3 days in 1984-85 to 3.0 days in 1994-95, with similar trends for both cesarean and vaginal delivery. The decrease resulted from both increasing rates of short stay (less than 2 days) and decreasing rates of long stay (more than 4 days). Substantial temporal and interprovincial variations in several medical and obstetric complications were also observed but did not explain the corresponding variations in length of stay. The reduction in length of hospital stay was not restricted to uncomplicated cases: there was an equivalent decrease in cases with complications. In 1994-95 the average length of hospital stay in Alberta was 2.6 days, 0.3 to 1.7 days shorter than in the other provinces and the territories. INTERPRETATION: Length of hospital stay for childbirth has decreased substantially in Canada in recent years, but there remain important interprovincial variations. These trends and variations are not likely due to changes or differences in patient-specific factors.

OBJECTIVE: To determine, for abdominal aortic aneurysm surgery, whether a previously reported relationship between hospital case volume and mortality rate was observed in Ontario hospitals and to assess the potential impact of age on the mortality rate for elective surgery. DESIGN: Population based observational study using administrative data. SETTING: All Ontario hospitals where repair of abdominal aortic aneurysm as a primary procedure was performed during 1988-92. PATIENTS: These comprised 5492 patients with unruptured abdominal aortic aneurysms and 1203 patients with ruptured abdominal aortic aneurysms admitted to hospital between 1988-92 for repair of abdominal aortic aneurysm as a primary procedure. MAIN OUTCOMES: In-hospital death and length of in-hospital stay. RESULTS: The case fatality rate was 3.8% for unruptured abdominal aortic aneurysms and 40.0% for ruptured abdominal aortic aneurysms. For unruptured cases, after adjustment for patient and hospital covariates, each 10 case per year increase in hospital volume was related to a 6% reduction in relative odds of death (odds ratio (OR) 0.94, 95% confidence intervals 0.88, 0.99) and 0.29 days reduction (95% CI -0.22, -0.35) in postoperative in-hospital stay. Female sex (OR 1.53, 95% CI 1.08, 2.18) and transfer from another acute care hospital (OR 4.37, 95% CI 2.62, 7.29) were associated with increased case fatality rates among patients in the unruptured category. For ruptured cases, neither the case fatality rate nor postoperative in-hospital stay were significantly related to hospital volume. The case fatality rates increased linearly and substantially with advancing age both for unruptured and ruptured aneurysms, and the excess risk of postoperative death in ruptured as compared to unruptured aneurysms was substantially higher in older patients. CONCLUSION: The relationship between hospital volume and mortality or morbidity was very modest and observed only for elective surgery. Case fatality rates in patients with ruptured abdominal aortic aneurysms remained 10 times higher than for patients with unruptured abdominal aortic aneurysms, despite improvements in overall mortality in comparison to previously published data. More effective detection of aneurysms, including elective repair for those once considered "high risk" older patients, might further reduce the toll from ruptured aortic aneurysms.

Continued adverse remodeling of myocardium after infarction may lead to progressive ventricular dilation and heart failure. We tested the hypothesis that exercise training in a healed myocardial infarction-dysfunction rat model can favorably modify the adverse effects of ventricular remodeling including attenuation of abnormal myosin gene expression. Sprague-Dawley rats were subjected to either proximal LAD ligation or sham operation. At 5 wk after the operation, animals were randomly assigned to sedentary conditions or 6 wk of graduated swim training, creating four experimental groups: infarct sedentary (IS), infarct exercise (IE), sham sedentary (SS), and sham exercise (SE). At 11 wk all rats were sacrificed and analyzed. Compared to sedentary infarct controls, exercise training attenuated left ventricular (LV) dilation and allowed more hypertrophy of the non infarct wall. The exercise-trained hearts also showed a reduction in the estimated peak wall tension. Northern blot analysis showed an increase in beta-myosin heavy chain expression in the hearts of the sedentary infarction group soon after infarction when compared to sham controls. However, with exercise training, there was a significant attenuation of the beta-myosin heavy chain expression in the myocardium. Exercise training in a model of left ventricular dysfunction after healed myocardial infarction can improve the adverse remodeling process by attenuating ventricular dilation and reducing wall tension. The abnormal beta-myosin expression was also attenuated in the exercise trained group. This is evidence that abnormal gene expression following severe myocardial infarction dysfunction can be favorably modified by an intervention.

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OBJECTIVE: To test the hypothesis that, with modern diagnostic methods and antibiotics, more conservative use of surgery in cases of suspected appendicitis would not result in increased rates of short-term complications in confirmed cases. DESIGN: Population-based observational study using administrative data. SETTING: All Ontario hospitals in which primary appendectomy was performed from Apr. 1, 1981, to Mar. 31, 1992. PATIENTS: All 126,815 patients admitted to hospital for a primary appendectomy during the study period. OUTCOME MEASURES: Diagnostic accuracy rate (acute appendicitis as the primary diagnosis), perforation rate, in-hospital death rate and length of stay. RESULTS: The diagnostic accuracy rate among the male patients was stable throughout the decade; among the female patients it rose significantly, from 71.7% in 1981 to 75.3% in 1991 (p < 0.01). The perforation rates increased significantly among both the female and male patients (p < 0.01), whereas the mean length of stay decreased (p < 0.05). Despite sex-related differences in the accuracy rates, the male and female patients had similar in-hospital death rates and mean lengths of stay. The institutional diagnostic accuracy rates, as determined from data for 1989-90 to 1991-92, ranged from 50.0% to 96.7%. Multivariate analyses of 27,189 confirmed cases of appendicitis at 175 hospitals revealed that perforation was a strong predictor of in-hospital death (odds ratio [OR] 2.46, 95% confidence interval [CI] 1.24 to 4.88), but comorbidity was the strongest predictor (OR 11.50, 95% CI 5.96 to 22.10). For each 10% increase in the diagnostic accuracy rate, the perforation rate increased 14% (OR 1.14, 95% CI 1.10 to 1.19), but the accuracy rate was not significantly related to the in-hospital death rate or the length of stay. CONCLUSION: A higher diagnostic accuracy rate is associated with more perforated appendixes. Although perforation itself leads to adverse outcomes, a higher accuracy rate does not. This suggests that hospitals with higher accuracy rates incur more perforations, but, with close observation, timely laparotomy and the use of modern antibiotics, these patients have favourable outcomes. This contrasts with adverse effects of perforation among patients at high risk for perforation (especially very young children and elderly people) in centres at all accuracy levels. The variation in hospitals' diagnostic accuracy rates suggests that some proportion of appendectomies could be safely avoided.

Cyclic AMP (cAMP) stimulates the expression of numerous genes through the protein kinase A (PK-A)-mediated phosphorylation of the nuclear factor CREB at Ser-133 (G. A. Gonzalez and M. R. Montminy, Cell 59:675-680, 1989). Like other signal transduction pathways, cAMP induces gene expression with burst-attenuation kinetics; cAMP-dependent transcription and CREB phosphorylation peak within 30 min and decline steadily over the next 4 to 6 h via the protein phosphatase 1-mediated dephosphorylation of CREB (M. Hagiwara, A. Alberts, P. Brindle, J. Meinkoth, J. Feramisco, T. Deng, M. Karin, S. Shenolikar, and M. Montminy, Cell 70:105-113, 1992). Here we characterize a third phase in cAMP-responsive transcription--a refractory period during which hormone-treated cells become transcriptionally unresponsive to subsequent stimulation by cAMP. This refractory period begins 6 to 8 h after stimulation and lasts 3 to 5 days after the removal of hormone. In contrast to the earlier attenuation phase, transcription of cAMP-responsive genes during the refractory period is not restored by inhibitors of protein phosphatase 1 activity. Rather, the establishment and maintenance of this phase rely on a marked reduction in PK-A catalytic subunit expression at the translational level. As overexpression of C-subunit protein can reactive transcription of cAMP-responsive genes during the refractory period, our results suggest that hormone-responsive cells may stimulate, attenuate, and then silence signal-dependent genes through distinct regulatory mechanisms.

Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase.

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The catalytic (C) subunit of cyclic AMP (cAMP) dependent protein kinase (PKA) has previously been shown to enter and exit the nucleus of cells when intracellular cAMP is raised and lowered, respectively. To determine the mechanism of nuclear translocation, fluorescently labeled C subunit was injected into living REF52 fibroblasts either as free C subunit or in the form of holoenzyme (PKA) in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine, respectively. Quantification of nuclear and cytoplasmic fluorescence intensities revealed that free C subunit nuclear accumulation was most similar to that of macromolecules that diffuse into the nucleus. A glutathione S-transferase-C subunit fusion protein did not enter the nucleus following cytoplasmic microinjection. Puncturing the nuclear membrane did not decrease the nuclear concentration of C subunit, and C subunit entry into the nucleus did not appear to be saturable. Cooling or depleting cells of energy failed to block movement of C subunit into the nucleus. Photobleaching experiments showed that even after reaching equilibrium at high [cAMP], individual molecules of C subunit continued to leave the nucleus at approximately the same rate that they had originally entered. These results indicate that diffusion is sufficient to explain most aspects of C subunit subcellular localization.

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Background

Human induced pluripotent stem cells (iPSCs) possess remarkable self-renewal capacity and the potential to differentiate into novel cell types, such as mesenchymal stem cells (MSCs). iPSC-MSCs have been shown to enhance tissue regeneration and attenuate tissue ischaemia; however, their contribution to the immune regulation of Th2-skewed allergic rhinitis (AR) and asthma remains unclear.

Objective

This study compared the immunomodulatory effects of iPSC-MSCs and bone marrow-derived MSCs (BM-MSCs) on lymphocyte proliferation, T-cell phenotypes and cytokine production in peripheral blood mononuclear cells (PBMCs) in patients with AR, and investigated the possible molecular mechanisms underlying the immunomodulatory properties of iPSC-MSCs.

Methods

In co-cultures of PBMCs with iPSC-MSCs or BM-MSCs, lymphocyte proliferation was evaluated using 3H-thymidine (3H-TdR) uptake, carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) assays; the regulatory T-cell (Treg) phenotype was determined by flow cytometry, and cytokine levels were measured using an enzyme-linked immunosorbent assay. The immunomodulatory properties of both MSCs were further evaluated using NS398 and transwell experiments.

Results

Similar to BM-MSCs, we determined that iPSC-MSCs significantly inhibit lymphocyte proliferation and promote Treg response in PBMCs (P < 0.05). Accordingly, the cytokine milieu (IFN-γ, IL-4, IL-5, IL-10 and IL-13) in the supernatants of PBMCs changed significantly (P < 0.05). The immunomodulatory properties of iPSC-MSCs and BM-MSCs were associated with prostaglandin E2 (PGE2) production and cell–cell contact.

Conclusions

These data demonstrate that iPSC-MSCs are capable of modulating T-cell phenotypes towards Th2 suppression through inducing Treg expansion, suggesting that iPSC-MSCs can be used as an alternative candidate to adult MSCs to treat allergic airway diseases.