Long-term ambient air pollution exposure and cardio-respiratory disease in China: findings from a prospective cohort study.

BACKGROUND: Existing evidence on long-term ambient air pollution (AAP) exposure and risk of cardio-respiratory diseases in China is mainly on mortality, and based on area average concentrations from fixed-site monitors for individual exposures. Substantial uncertainty persists, therefore, about the shape and strength of the relationship when assessed using more personalised individual exposure data. We aimed to examine the relationships between AAP exposure and risk of cardio-respiratory diseases using predicted local levels of AAP. METHODS: A prospective study included 50,407 participants aged 30-79 years from Suzhou, China, with concentrations of nitrogen dioxide (NO2), sulphur dioxide (SO2), fine (PM2.5), and inhalable (PM10) particulate matter, ozone (O3) and carbon monoxide (CO) and incident cases of cardiovascular disease (CVD) (n = 2,563) and respiratory disease (n = 1,764) recorded during 2013-2015. Cox regression models with time-dependent covariates were used to estimate adjusted hazard ratios (HRs) for diseases associated with local-level concentrations of AAP exposure, estimated using Bayesian spatio-temporal modelling. RESULTS: The study period of 2013-2015 included a total of 135,199 person-years of follow-up for CVD. There was a positive association of AAP, particularly SO2 and O3, with risk of major cardiovascular and respiratory diseases. Each 10 µg/m3 increase in SO2 was associated with adjusted hazard ratios (HRs) of 1.07 (95% CI: 1.02, 1.12) for CVD, 1.25 (1.08, 1.44) for COPD and 1.12 (1.02, 1.23) for pneumonia. Similarly, each 10 µg/m3 increase in O3 was associated with adjusted HR of 1.02 (1.01, 1.03) for CVD, 1.03 (1.02, 1.05) for all stroke, and 1.04 (1.02, 1.06) for pneumonia. CONCLUSIONS: Among adults in urban China, long-term exposure to ambient air pollution is associated with a higher risk of cardio-respiratory disease.

Heterogeneity in the diagnosis and prognosis of ischemic stroke subtypes: 9-year follow-up of 22,000 cases in Chinese adults.

BACKGROUND: Reliable classification of ischemic stroke (IS) etiological subtypes is required in research and clinical practice, but the predictive properties of these subtypes in population studies with incomplete investigations are poorly understood. AIMS: To compare the prognosis of etiologically classified IS subtypes and use machine learning (ML) to classify incompletely investigated IS cases. METHODS: In a 9-year follow-up of a prospective study of 512,726 Chinese adults, 22,216 incident IS cases, confirmed by clinical adjudication of medical records, were assigned subtypes using a modified Causative Classification System for Ischemic Stroke (CCS) (large artery atherosclerosis (LAA), small artery occlusion (SAO), cardioaortic embolism (CE), or undetermined etiology) and classified by CCS as "evident," "probable," or "possible" IS cases. For incompletely investigated IS cases where CCS yielded an undetermined etiology, an ML model was developed to predict IS subtypes from baseline risk factors and screening for cardioaortic sources of embolism. The 5-year risks of subsequent stroke and all-cause mortality (measured using cumulative incidence functions and 1 minus Kaplan-Meier estimates, respectively) for the ML-predicted IS subtypes were compared with etiologically classified IS subtypes. RESULTS: Among 7443 IS subtypes with evident or probable etiology, 66% had SAO, 32% had LAA, and 2% had CE, but proportions of SAO-to-LAA cases varied by regions in China. CE had the highest rates of subsequent stroke and mortality (43.5% and 40.7%), followed by LAA (43.2% and 17.4%) and SAO (38.1% and 11.1%), respectively. ML provided classifications for cases with undetermined etiology and incomplete clinical data (24% of all IS cases; n = 5276), with area under the curves (AUC) of 0.99 (0.99-1.00) for CE, 0.67 (0.64-0.70) for LAA, and 0.70 (0.67-0.73) for SAO for unseen cases. ML-predicted IS subtypes yielded comparable subsequent stroke and all-cause mortality rates to the etiologically classified IS subtypes. CONCLUSION: This study highlighted substantial heterogeneity in prognosis of IS subtypes and utility of ML approaches for classification of IS cases with incomplete clinical investigations.

Sec22b is a critical and nonredundant regulator of plasma cell maintenance.

Despite the essential role of plasma cells in health and disease, the cellular mechanisms controlling their survival and secretory capacity are still poorly understood. Here, we identified the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) Sec22b as a unique and critical regulator of plasma cell maintenance and function. In the absence of Sec22b, plasma cells were hardly detectable and serum antibody titers were dramatically reduced. Accordingly, Sec22b-deficient mice fail to mount a protective immune response. At the mechanistic level, we demonstrated that Sec22b contributes to efficient antibody secretion and is a central regulator of plasma cell maintenance through the regulation of their transcriptional identity and of the morphology of the endoplasmic reticulum and mitochondria. Altogether, our results unveil an essential and nonredundant role for Sec22b as a regulator of plasma cell fitness and of the humoral immune response.

Association between pneumonia hospitalisation and long-term risk of cardiovascular disease in Chinese adults: A prospective cohort study.

Background: Lower respiratory tract infections, including pneumonia, have been associated with short-term increased risk of cardiovascular disease (CVD). However, there is only limited evidence about the long-term impact of pneumonia on the cardiovascular system beyond one year. Methods: We conducted a prospective matched cohort study based on data from the China Kadoorie Biobank study of 482,017 adults who were enrolled between June 25, 2004, and July 15, 2008, and were free of CVD at baseline and before pneumonia hospitalization. A total of 24,060 patients hospitalised with pneumonia were identified until December 31, 2018, and were matched on age, sex, urban or rural areas, and decile of the frailty index to 223,875 controls. We used the piecewise Cox proportional hazards model to estimate the hazard ratios (HRs) and their 95% confidence intervals (CIs) for pre-specified incident CVD outcomes, including ischaemic heart disease (IHD), arrhythmia, heart failure (HF), ischaemic stroke (IS), and hemorrhagic stroke (HS), at various time intervals through 10 years after pneumonia hospitalization. Findings: Of the 247,935 pneumonia cases and controls included, the mean age (standard deviation) was 53.5 (10.4), and 40.8% (101,159) were men. During follow-up, 2389 (9.9%) pneumonia cases developed IHD, 489 (2.0%) cases developed arrhythmia, 545 (2.3%) cases developed HF, 1764 (7.3%) cases developed IS, and 348 (1.4%) cases developed HS. After adjustment for sociodemographic characteristics, lifestyle factors, health status and medication, underlying conditions, and family history of CVD, the elevated CVD risk was highest within the first 30 days after pneumonia hospitalisation, with subsequent risk reductions varying by subtypes. The elevated risk remained until the eighth year after pneumonia hospitalisation for IHD, arrhythmia, and HF, with HRs (95% CIs) of 1.48 (1.13-1.93), 2.69 (1.70-4.25), and 4.36 (2.86-6.64), respectively. The risk of stroke associated with pneumonia hospitalisation remained elevated until the seventh year for IS (HR = 1.30; 95% CI: 1.04-1.63), and until the second year for HS (1.39; 1.07-1.80). The above associations were consistently observed across various characteristics of the participants. Interpretation: In middle-aged and older Chinese adults, pneumonia hospitalisation was associated with short- and long-term CVD risk, with the elevated risk of certain CVD outcomes persisting for up to 8 years. Funding: National Natural Science Foundation of China, the National Key R&D Program of China, the Chinese Ministry of Science and Technology, the Kadoorie Charitable Foundation in Hong Kong, the UK Wellcome Trust.

Tobacco smoking and risks of more than 470 diseases in China: a prospective cohort study.

BACKGROUND: Tobacco smoking is estimated to account for more than 1 million annual deaths in China, and the epidemic continues to increase in men. Large nationwide prospective studies linked to different health records can help to periodically assess disease burden attributed to smoking. We aimed to examine associations of smoking with incidence of and mortality from an extensive range of diseases in China. METHODS: We analysed data from the prospective China Kadoorie Biobank, which recruited 512 726 adults aged 30-79 years, of whom 210 201 were men and 302 525 were women. Participants who had no major disabilities were identified through local residential records in 100-150 administrative units, which were randomly selected by use of multistage cluster sampling, from each of the ten diverse study areas of China. They were invited and recruited between June 25, 2004, and July 15, 2008. Upon study entry, trained health workers administered a questionnaire assessing detailed smoking behaviours and other key characteristics (eg, sociodemographics, lifestyle, and medical history). Participants were followed up via electronic record linkages to death and disease registries and health insurance databases, from baseline to Jan 1, 2018. During a median 11-year follow-up (IQR 10-12), 285 542 (55·7%) participants were ever hospitalised, 48 869 (9·5%) died, and 5252 (1·0%) were lost to follow-up during the age-at-risk of 35-84 years. Cox regression yielded hazard ratios (HRs) associating smoking with disease incidence and mortality, adjusting for multiple testing. FINDINGS: At baseline, 74·3% of men and 3·2% of women (overall 32·4%) ever smoked regularly. During follow-up, 1 137 603 International Classification of Diseases, 10th revision (ICD-10)-coded incident events occurred, involving 476 distinct conditions and 85 causes of death, each with at least 100 cases. Compared with never-regular smokers, ever-regular smokers had significantly higher risks for nine of 18 ICD-10 chapters examined at age-at-risk of 35-84 years. For individual conditions, smokers had significantly higher risks of 56 diseases (50 for men and 24 for women) and 22 causes of death (17 for men and nine for women). Among men, ever-regular smokers had an HR of 1·09 (95% CI 1·08-1·11) for any disease incidence when compared with never-regular smokers, and significantly more episodes and longer duration of hospitalisation, particularly those due to cancer and respiratory diseases. For overall mortality, the HRs were greater in men from urban areas than in men from rural areas (1·50 [1·42-1·58] vs 1·25 [1·20-1·30]). Among men from urban areas who began smoking at younger than 18 years, the HRs were 2·06 (1·89-2·24) for overall mortality and 1·32 (1·27-1·37) for any disease incidence. In this population, 19·6% of male (24·3% of men residing in urban settings and 16·2% of men residing in rural settings) and 2·8% of female deaths were attributed to ever-regular smoking. INTERPRETATION: Among Chinese adults, smoking was associated with higher risks of morbidity and mortality from a wide range of diseases. Among men, the future smoking-attributed disease burden will increase further, highlighting a pressing need for reducing consumption through widespread cessation and uptake prevention. FUNDING: British Heart Foundation, Cancer Research UK, Chinese Ministry of Science and Technology, Kadoorie Charitable Foundation, UK Medical Research Council, National Natural Science Foundation of China, Wellcome Trust.

Liver biomarkers, genetic and lifestyle risk factors in relation to risk of cardiovascular disease in Chinese.

Background and aims: Liver biomarkers and metabolic associated fatty liver disease (MAFLD) have been shown to be associated with cardiovascular disease (CVD). However, there is limited evidence on CVD subtypes [myocardial infarction (MI), ischemic stroke (IS), and intracerebral hemorrhage (ICH)], especially in the Chinese population. We examined these associations overall, by genetic predisposition to non-alcoholic fatty liver disease (NAFLD), and by lifestyle risk factors. Approach and results: This is a nested case-control study of CVD (10,298 cases and 5,388 controls) within the China Kadoorie Biobank. Cox regression was used to estimate adjusted hazard ratios (HRs) for CVD associated with liver biomarkers and MAFLD and by stratum of genetic risk and a combined high-risk lifestyle score. For liver enzymes, there were positive associations with MI and IS, but no associations with ICH or carotid plaque. There were positive associations of NAFLD with risks of MI, IS, and ICH (HR 1.43 [95% CI 1.30-1.57], 1.25 [1.16-1.35], and 1.12 [1.02-1.23]) as well as carotid plaque (odds ratio 2.36 [1.12-4.96]). The associations of NAFLD with CVD and carotid plaque were stronger among individuals with a high genetic risk (ICH: p-interaction < 0.05), while the associations with stroke were stronger among those with a favorable lifestyle (p-interaction < 0.05). The results for MAFLD mirrored those for NAFLD. Conclusion: In Chinese adults, liver biomarkers and MAFLD were associated with risk of CVD, with different magnitudes of associations by CVD subtypes. Genetic predisposition to NAFLD and lifestyle factors modified the associations of fatty liver with stroke.

Diagnostic accuracy of major stroke types in Chinese adults: A clinical adjudication study involving 40,000 stroke cases.

Background: Widespread use of brain imaging in suspected stroke cases in Chinese adults has prompted the need for clinical adjudication studies of stroke types. We conducted a clinical adjudication study to assess the reporting and diagnostic accuracy of major stroke types. Methods: The prospective China Kadoorie Biobank recruited >512,000 adults (mean age 52 years, 59% women) from 10 urban and rural areas in China during 2004-2008, and recorded 45,859 first-ever incident stroke cases during an 11-year follow-up. Medical records were retrieved in ∼85%, and clinical details were recorded using a handheld computer for specialist physician adjudicators who applied conventional WHO clinical criteria for diagnosis of stroke. The positive predictive value (PPV) for reported and adjudicated stroke cases was examined for major stroke types (ischaemic stroke [IS], intracerebral haemorrhage [ICH], subarachnoid haemorrhage [SAH]), calendar year, area, and hospital type. Findings: Of 38,823 cases with retrieved medical records, the PPV for reported strokes was 91%. Among 29,952 adjudicated cases, the PPV for adjudicated cases was 81%, with higher PPV for ICH (n = 3391; 98%) and SAH (364; 98%) than for IS (20,473; 79%). Of 5504 cases with a verified IS diagnosis that was refuted on adjudication, 3763 (68%) had silent lacunar infarcts (LACI). The proportion of cases with silent LACI increased from 7.1% in 2004-2008 to 18.2% in 2016-2017. If cases with silent LACI were classified as IS, as advocated by new International Classification of Diseases (ICD-11) diagnostic criteria for stroke involving imaging and clinical rather than clinical criteria alone, the PPV increased to 93%. Interpretation: While the overall reporting and diagnostic accuracy of stroke types in Chinese adults is high, the recent implementation of new diagnostic criteria for IS has important implications for contemporary clinical practice and research on stroke in Chinese populations. Funding: Kadoorie Charitable Foundation, Hong Kong, China, UK Wellcome Trust (212946/Z/18/Z, 202922/Z/16/Z, 104085/Z/14/Z, 088158/Z/09/Z), Chinese National Natural Science Foundation (91843302), and the National Key Research and Development Program of China (2016YFC0900500, 2016YFC0900501, 2016YFC0900504, 2016YFC1303904). British Heart Foundation, and UK Medical Research Council, and Cancer Research UK.

Tobacco smoking and solid fuels for cooking and risk of liver cancer: A prospective cohort study of 0.5 million Chinese adults.

Previous research found tobacco smoking and solid fuel use for cooking to increase the risk of chronic liver disease mortality, but previous cohort studies have not investigated their independent and joint associations with liver cancer incidence in contemporary China. The China Kadoorie Biobank (CKB) study recruited 0.5 million adults aged 30 to 79 years from 10 areas across China during 2004 to 2008. Participants reported detailed smoking and fuel use information at baseline. After an 11.1-year median follow-up via electronic record linkage, we recorded 2997 liver cancer cases. Overall, 29.4% participants were current smokers. Among those who cooked at least once per month, 48.8% always used solid fuels (ie, coal or wood) for cooking. Tobacco smoking and solid fuel use for cooking were independently associated with increased risks of liver cancer, with hazard ratios (95% confidence intervals [CIs]) of 1.28 (1.15-1.42) and 1.25 (1.03-1.52), respectively. The more cigarettes consumed each day, the earlier the age of starting smoking or the longer duration of solid fuels exposure, the higher the risk (Ptrend < .001, =.001, =.018, respectively). Compared with never smokers who had always used clean fuels (ie, gas or electricity), ever-smokers who had always used solid fuels for cooking had a 67% (95% CIs: 1.29-2.17) higher risk. Among Chinese adults, tobacco smoking and solid fuel use for cooking were independently associated with higher risk of liver cancer incidence. Stronger association was observed with higher number of daily cigarette consumption, the earlier age of starting smoking and longer duration of solid fuel use.

Multimorbidity patterns and association with mortality in 0.5 million Chinese adults.

BACKGROUND: Few studies have assessed the relationship between multimorbidity patterns and mortality risk in the Chinese population. We aimed to identify multimorbidity patterns and examined the associations of multimorbidity patterns and the number of chronic diseases with the risk of mortality among Chinese middle-aged and older adults. METHODS: We used data from the China Kadoorie Biobank and included 512,723 participants aged 30 to 79 years. Multimorbidity was defined as the presence of two or more of the 15 chronic diseases collected by self-report or physical examination at baseline. Multimorbidity patterns were identified using hierarchical cluster analysis. Cox regression was used to estimate the associations of multimorbidity patterns and the number of chronic diseases with all-cause and cause-specific mortality. RESULTS: Overall, 15.8% of participants had multimorbidity. The prevalence of multimorbidity increased with age and was higher in urban than rural participants. Four multimorbidity patterns were identified, including cardiometabolic multimorbidity (diabetes, coronary heart disease, stroke, and hypertension), respiratory multimorbidity (tuberculosis, asthma, and chronic obstructive pulmonary disease), gastrointestinal and hepatorenal multimorbidity (gallstone disease, chronic kidney disease, cirrhosis, peptic ulcer, and cancer), and mental and arthritis multimorbidity (neurasthenia, psychiatric disorder, and rheumatoid arthritis). During a median of 10.8 years of follow-up, 49,371 deaths occurred. Compared with participants without multimorbidity, cardiometabolic multimorbidity (hazard ratios [HR] = 2.20, 95% confidence intervals [CI]: 2.14 - 2.26) and respiratory multimorbidity (HR = 2.13, 95% CI:1.97 - 2.31) demonstrated relatively higher risks of mortality, followed by gastrointestinal and hepatorenal multimorbidity (HR = 1.33, 95% CI:1.22 - 1.46). The mortality risk increased by 36% (HR = 1.36, 95% CI: 1.35 - 1.37) with every additional disease. CONCLUSION: Cardiometabolic multimorbidity and respiratory multimorbidity posed the highest threat on mortality risk and deserved particular attention in Chinese adults.

Evaluation of variables influencing success and complication rates in canine total hip replacement: results from the British Veterinary Orthopaedic Association Canine Hip Registry (collation of data: 2010-2012).

The objective of this study was to assess the variables associated with complications of total hip replacement (THR) and report owner-assessed outcomes. Entries into the British Veterinary Orthopaedic Association-Canine Hip Registry (BVOA-CHR) between September 2011 and December 2012 were reviewed separately and in conjunction with previous data (January 2010-August 2011). An outcomes assessment questionnaire was used to collect data from owners. Incidences of surgeon-reported and owner-reported complications were 8.2 per cent and 4.3 per cent, respectively. THR using the BioMedtrix BFX cup/stem prosthesis had a greater incidence of complications compared with THR using the BioMedtrix CFX cup/stem prosthesis (P=0.002); complications were 4.48 times more likely when using the BioMedtrix BFX cup/stem prosthesis versus the BioMedtrix CFX cup/stem prosthesis. THR using the BioMedtrix BFX cup/stem prosthesis had a higher incidence of complications compared with THR using a hybrid prosthesis (BioMedtrix BFX cup/CFX stem, BioMedtrix CFX cup/BFX stem) (P=0.046); complications were 2.85 times more likely when using the BioMedtrix BFX cup/stem prosthesis versus a hybrid prosthesis. In 95 per cent of cases, owner satisfaction with the outcome of THR was 'very good' or 'good'. Complication rates from the BVOA-CHR are similar to previous studies. The data suggest that prosthesis type is associated with complication rate, with BioMedtrix BFX (circa 2012) having a high short-term complication rate.

Rab40b regulates trafficking of MMP2 and MMP9 during invadopodia formation and invasion of breast cancer cells.

Invadopodia-dependent degradation of the basement membrane plays a major role during metastasis of breast cancer cells. Basement membrane degradation is mediated by targeted secretion of various matrix metalloproteinases (MMPs). Specifically, MMP2 and MMP9 (MMP2/9) possess the ability to hydrolyze components of the basement membrane and regulate various aspects of tumor growth and metastasis. However, the membrane transport machinery that mediates targeting of MMP2/9 to the invadopodia during cancer cell invasion remains to be defined. Because Rab GTPases are key regulators of membrane transport, we screened a human Rab siRNA library and identified Rab40b GTPase as a protein required for secretion of MMP2/9. We also have shown that Rab40b functions during at least two distinct steps of MMP2/9 transport. Here, we demonstrate that Rab40b is required for MMP2/9 sorting into VAMP4-containing secretory vesicles. We also show that Rab40b regulates transport of MMP2/9 secretory vesicles during invadopodia formation and is required for invadopodia-dependent extracellular matrix degradation. Finally, we demonstrate that Rab40b is also required for breast cancer cell invasion in vitro. On the basis of these findings, we propose that Rab40b mediates trafficking of MMP2/9 during invadopodia formation and metastasis of breast cancer cells.

Complications and owner assessment of canine total hip replacement: a multicenter internet based survey.

OBJECTIVE: To assess the variables associated with the complications of total hip replacement (THR) and report owner-assessed outcomes, through surgeon-based registration of cases via an online database, informed owner consent, and prospective outcomes assessment using a client-administered clinical metrology instrument. STUDY DESIGN: Prospective case series ANIMALS: Dogs (n = 170) METHODS: Entries into the British Veterinary Orthopaedic Association-Canine Hip Registry (BVOA-CHR) between January 2010 and August 2011 were reviewed. Variables evaluated included dog age, body weight, breed, and indication for THR and prosthesis. Associations between each variable and the incidence of complications were assessed using logistic regression. Additionally, an on-line, owner-administered outcomes assessment questionnaire (modified from the Liverpool Osteoarthritis in Dogs (LOAD) questionnaire) was used to collect data from owners. RESULTS: One hundred and seventy cases met the inclusion criteria. Surgical indications included hip dysplasia and coxofemoral osteoarthritis (n = 150), Legg-Calves-Perthes disease (7), coxofemoral luxation (6), fracture (4), slipped capital physis (2), and femoral head and neck ostectomy revision (1). Surgical implants were from 4 systems. The incidence of surgeon-reported complication was 9.4%. No significant association was identified between weight, age, sex, breed, indication for THR, surgical technique and prosthesis, and the incidence of complications. In 82% of the cases, owners described their satisfaction with the outcome of THR as "very good" and a total of 20% complication rate was reported. There was a statistically significant improvement in owner-assessed questionnaire score before and after THR (P < .001). CONCLUSIONS: The BVOA-CHR offers a novel framework for the prospective studies on THR and on a national/international scale. Initial complication rates from the BVOA-CHR are similar to previous studies.

Structural studies of the allelic wheat glutenin subunits 1Bx7 and 1Bx20 by matrix-assisted laser desorption/ionization mass spectrometry and high-performance liquid chromatography/electrospray ionization mass spectrometry.

Structural studies of the high molecular mass (HMM) glutenin subunits 1Bx7 (from cvs Hereward and Galatea) and 1Bx20 (from cv. Bidi17) of bread wheat were conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). For all three proteins, MALDI-TOFMS analysis showed that the isolated fractions contained a second component with a mass about 650 Da lower than the major component. The testing and correction of the gene-derived amino acid sequences of the three proteins were performed by direct MALDI-TOFMS analysis of their tryptic peptide mixture. Analysis of the digest was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, optimizing the matrix and the acquisition parameters for each mass range. Complementary data were obtained by RP-HPLC/ESI-MS analysis of the tryptic digest. This resulted in coverage of about 98% of the sequences. In contrast to the gene-derived data, the results obtained demonstrate the insertion of the sequence QPGQGQ between Trp716 and Gln717 of subunit 1Bx7 (cv. Galatea) and a possible single amino acid substitution within the T20 peptide of subunit 1Bx20. Moreover, the mass spectrometric data demonstrated that the lower mass components present in all the fractions correspond to the major components but lack about six amino acid residues, which are probably lost from the protein C-terminus. Finally, the results obtained provide evidence for the lack of glycosylation or other post-translational modifications of these subunits.

Identification of the wheat seed protein CM3 as a highly active emulsifier using a novel functional screen.

Lyophilized albumin protein fractions were prepared from flour of four varieties of wheat: Triticum aestivum cvs. Mercia and Riband, Triticum aestivum var. spelta, and Triticum turgidum var. durum (Kamut). The dry powders were redissolved in sodium phosphate buffers at pH 3.0, 6.5, or 8.0 and at ionic strengths of 0.1 or 1.0 M to a concentration of 0.1% (w/v). Emulsions formed by sonication of protein solutions with n-hexadecane were aged at room temperature and separated into aqueous, interstitial, and interfacial phases. The distinct emulsion components were lyophilized and analyzed by RP-HPLC. A protein was observed to be preferentially located in the interfacial component and subsequently purified from a total albumin fraction and identified by N-terminal sequencing as CM3, an alpha-amylase inhibitor subunit. Measurement of the equilibrium surface tension of CM3 as a function of protein concentration demonstrated that it was at least as active as bovine beta-lactoglobulin, an established protein emulsifier. Furthermore, measurement of the surface dilational elastic modulus at an air/water interface demonstrated the formation of a viscoelastic film, while fluorescence and FT-IR spectroscopic measurements on adsorbed and nonadsorbed CM3 suggest that the secondary structure is essentially unchanged upon adsorption to an oil/water interface. It is concluded that functional screening is a valid approach to identify novel protein emulsifiers in complex mixtures.

Structural studies of glutenin subunits 1Dy10 and 1Dy12 by matrix-assisted laser desorption/ionisation mass spectrometry and high-performance liquid chromatography/electrospray ionisation mass spectrometry.

Structural studies of the high molecular weight (HMW) glutenin subunits 1Dy10 and 1Dy12 of bread wheat were conducted using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) and reversed-phase high-performance liquid chromatography/electrospray ionisation mass spectrometry (RP-HPLC/ESI-MS). For both proteins, MALDI-TOFMS analysis showed that the isolated fractions contained a second component with a mass about 500-540 Da lower than the major component. The testing and correction of the gene-derived amino acid sequences of both proteins were performed by direct MALDI-TOFMS analysis of their tryptic peptide mixture and analysis of the digests was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, optimising the matrix and the acquisition parameters for each mass range. Complementary data were obtained by RP-HPLC/ESI-MS analysis of the tryptic digest. This resulted in the coverage of the whole protein sequences except for two short fragments (T1 and T8), which are identical in the two homologous subunits, and for an additional dipeptide (T14) in subunit 1Dy12, which were not detected. It also demonstrated that, in contrast to the gene-derived data, the sequence of subunit 1Dy12 does not include the dipeptide Gly-Gln between residues Gln(454) and Pro(455), and that the lower mass components present in both fractions correspond to the same sequences lacking short peptides that are probably lost from the protein N- or C-termini. Finally, the results obtained provide evidence for the lack of a substantial level of glycosylation or other post-translational modifications of the two subunits, and demonstrate that mass spectrometric mapping is the most useful method presently available for the direct verification of the gene-derived sequences of HMW glutenin subunits and similar proteins.

Investigation and correction of the gene-derived sequence of glutenin subunit 1Dx2 by matrix-assisted laser desorption/ionisation mass spectrometry.

Direct matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) analysis of a mixture of tryptic peptides was used to verify the gene-derived amino acid sequence of the high molecular weight (HMW) subunit 1Dx2 of bread wheat. Analysis of the digest was performed by recording several MALDI mass spectra of the mixture at low, medium and high mass ranges, and optimising the matrix and the acquisition parameters for each mass range. This resulted in coverage of the whole sequence except for a short fragment T3 (3 amino acids), which was not detected. It also allowed the insertion of a Pro residue in position 59 to be identified. The results obtained provide evidence for the lack of a substantial level of glycosylation or other post-translational modifications of subunit 1Dx2, and demonstrate that MALDI-MS is the most useful method presently available for the direct verification of the gene-derived sequences of HMW glutenin subunits and similar proteins.

Study of high molecular weight wheat glutenin subunit 1Dx5 by 13C and 1H solid-state NMR spectroscopy. I. Role of covalent crosslinking.

This work describes a carbon and proton solid-state NMR study of the hydration of a high molecular weight wheat glutenin subunit, 1Dx5. The effect of the presence of disulfide bonds on the hydration behavior of the subunit is investigated by a comparison of the unalkylated and alkylated forms of the protein. Hydration induces partial plasticization of the protein so that some segments become more mobile than others. The 13C cross-polarization and magic-angle spinning (MAS) spectra of the samples in the dry state and at two hydration levels (approximately 40 and approximately 65% D2O) were used to monitor the protein fraction resisting plasticization (trains). Conversely, 13C single pulse excitation and 1H-MAS experiments were used to gain information on the more plasticized segments (loops). The molecular motion of the two protein dynamic populations was further characterized by 13C T1 and 1H T(1rho), T2, and T1 relaxation times. The results suggest that hydration leads to the formation of a network held by a cooperative action of hydrogen bonded glutamines and some hydrophobic interactions. The looser protein segments are suggested to be glycine- and glutamine-rich segments. The primary structure is therefore expected to significantly determine the proportion of trains and loops in the network. The presence of disulfide bonds was observed to promote easier plasticization of the protein and the formation of a more mobile network, probably involving a higher number of loops and/or larger loops.

Study of wheat high molecular weight 1Dx5 subunit by (13)C and (1)H solid-state NMR. II. Roles of nonrepetitive terminal domains and length of repetitive domain.

This work follows a previous article that addressed the role of disulfide bonds in the behavior of the 1Dx5 subunit upon hydration. Here the roles of nonrepetitive terminal domains present and the length of the central repetitive domain in the hydration of 1Dx5 are investigated. This was achieved by comparing the hydration behavior of suitable model samples determined by (13)C- and (1)H-NMR: an alkylated 1Dx5 subunit (alk1Dx5), a recombinant 58-kDa peptide corresponding to the central repetitive domain of 1Dx5 (i.e., lacking the terminal domains), and two synthetic peptides (with 6 and 21 amino acid residues) based on the consensus repeat motifs of the central domain. The (13)C cross-polarization and magic angle spinning (MAS) experiments recorded as a function of hydration gave information about the protein or peptide fractions resisting plasticization. Conversely, (13)C single pulse excitation and (1)H-MAS gave information on the more plasticized segments. The results are consistent with the previous proposal of a hydrated network held by hydrogen-bonded glutamines and possibly hydrophobic interactions. The nonrepetitive terminal domains were found to induce water insolubility and a generally higher network hindrance. Shorter chain lengths were shown to increase plasticization and water solubility. However, at low water contents, the 21-mer peptide was characterized by higher hindrance in the megahertz and kilohertz frequency ranges compared to the longer peptide; and a tendency for a few hydrogen-bonded glutamines and hydrophobic residues to remain relatively hindered was still observed, as for the protein and large peptide. It is suggested that this ability is strongly dependent on the peptide primary structure.