Concentric lamellae - novel microanatomical structures in the articular calcified cartilage of mice.

The structure, ultrastructure and function of hyaline articular cartilage (HAC) and subchondral bone (SCB), and their involvement in the pathogenesis of osteoarthritis (OA) have been extensively researched. However, much less attention has been focused on the intervening tissue, articular calcified cartilage (ACC) and its role in the initiation and progression of OA. Using both light microscopy (LM) and transmission electron microscopy (TEM), a study of ACC in wild type (WT) mice, and mice with genetic osteoarthropathies (AKU) was undertaken to further understand the role played by ACC in the early stages of OA.Tibio-femoral joints were obtained from BALB/c WT and BALB/c AKU mice aged between 7 and 69 weeks. One joint was processed for routine histological analysis. The tip of the medial femoral condyle (MFC), which contained HAC, ACC, and SCB, was dissected from the contra-lateral joint and processed for TEM.In WT and AKU mice novel microanatomical structures, designated concentric lamellae, were identified surrounding chondrocytes in the ACC. The lamellae appeared to be laid down in association with advancement of the tidemark indicating they may be formed during calcification of cartilage matrix. The lamellae were associated with hypertrophic chondrocytes throughout the ACC.Novel microanatomical structures, termed concentric lamellae, which were present around hypertrophic chondrocytes in the ACC are described for the first time. Their apparent association with mineralisation, advancement of the tidemark, and greater abundance in a model of osteoarthropathy indicate their formation could be important in the pathogenesis of OA and AKU

Impact-based forecasting in South East Asia – what underlies impact perceptions?

The move towards impact-based forecasting presents a challenge for forecasters, who must combine information not just on what the weather might be, but also on what the weather might do. Yet different hazards and impacts are qualitatively distinct, meaning such information cannot be easily or straightforwardly integrated. The present study aimed to provide a way of characterising seemingly disparate impacts. In a collaboration between UK psychologists and partners from three meteorological organisations in Indonesia, Malaysia and the Philippines, the psychometric paradigm was employed to investigate how forecasters and stakeholders perceive weather-related impacts. Participants provided ratings of nine categories of impacts on a total of 10 characteristics, as well as providing an overall impact severity rating. Principal components analysis revealed differing component solutions across countries, which explained around 75% of the variance in perceptions. There were some similarities across all countries, with the characteristics ‘worry’ and ‘destructiveness’ loading positively together, as well as ‘likelihood of harm’ and ‘seriousness of harm’. We did not find strong evidence to indicate that forecasters and stakeholders perceive impacts in different ways. Our results highlight the complex nature of impact perceptions, which are characterised not just by objective factors such as impact scope and duration, but also subjective factors, such as worry and perceived severity

Diversity in Functional Organization of Class I and Class II Biotin Protein Ligase

The cell envelope of Mycobacterium tuberculosis (M.tuberculosis) is composed of a variety of lipids including mycolic acids, sulpholipids, lipoarabinomannans, etc., which impart rigidity crucial for its survival and pathogenesis. Acyl CoA carboxylase (ACC) provides malonyl-CoA and methylmalonyl-CoA, committed precursors for fatty acid and essential for mycolic acid synthesis respectively. Biotin Protein Ligase (BPL/BirA) activates apo-biotin carboxyl carrier protein (BCCP) by biotinylating it to an active holo-BCCP. A minimal peptide (Schatz), an efficient substrate for Escherichia coli BirA, failed to serve as substrate for M. tuberculosis Biotin Protein Ligase (MtBPL). MtBPL specifically biotinylates homologous BCCP domain, MtBCCP87, but not EcBCCP87. This is a unique feature of MtBPL as EcBirA lacks such a stringent substrate specificity. This feature is also reflected in the lack of self/promiscuous biotinylation by MtBPL. The N-terminus/HTH domain of EcBirA has the self-biotinable lysine residue that is inhibited in the presence of Schatz peptide, a peptide designed to act as a universal acceptor for EcBirA. This suggests that when biotin is limiting, EcBirA preferentially catalyzes, biotinylation of BCCP over self-biotinylation. R118G mutant of EcBirA showed enhanced self and promiscuous biotinylation but its homologue, R69A MtBPL did not exhibit these properties. The catalytic domain of MtBPL was characterized further by limited proteolysis. Holo-MtBPL is protected from proteolysis by biotinyl-5′ AMP, an intermediate of MtBPL catalyzed reaction. In contrast, apo-MtBPL is completely digested by trypsin within 20 min of co-incubation. Substrate selectivity and inability to promote self biotinylation are exquisite features of MtBPL and are a consequence of the unique molecular mechanism of an enzyme adapted for the high turnover of fatty acid biosynthesis

Structural Ordering of Disordered Ligand-Binding Loops of Biotin Protein Ligase into Active Conformations as a Consequence of Dehydration

Mycobacterium tuberculosis (Mtb), a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC), an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA). The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants) prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of ∼3.5 Å in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP) domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In-depth analyses of the sequence and the structure also provide answers to the reported lower affinities of Mtb-BirA toward ATP and biotin substrates. This dehydrated crystal structure not only provides key leads to the understanding of the structure/function relationships in the protein in the absence of any ligand-bound structure, but also demonstrates the merit of dehydration of crystals as an inimitable technique to have a glance at proteins in action

Deriving utility scores for co-morbid conditions: a test of the multiplicative model for combining individual condition scores

BACKGROUND: The co-morbidity of health conditions is becoming a significant health issue, particularly as populations age, and presents important methodological challenges for population health research. For example, the calculation of summary measures of population health (SMPH) can be compromised if co-morbidity is not taken into account. One popular co-morbidity adjustment used in SMPH computations relies on a straightforward multiplicative combination of the severity weights for the individual conditions involved. While the convenience and simplicity of the multiplicative model are attractive, its appropriateness has yet to be formally tested. The primary objective of the current study was therefore to examine the empirical evidence in support of this approach. METHODS: The present study drew on information on the prevalence of chronic conditions and a utility-based measure of health-related quality of life (HRQoL), namely the Health Utilities Index Mark 3 (HUI3), available from Cycle 1.1 of the Canadian Community Health Survey (CCHS; 2000–01). Average HUI3 scores were computed for both single and co-morbid conditions, and were also purified by statistically removing the loss of functional health due to health problems other than the chronic conditions reported. The co-morbidity rule was specified as a multiplicative combination of the purified average observed HUI3 utility scores for the individual conditions involved, with the addition of a synergy coefficient s for capturing any interaction between the conditions not explained by the product of their utilities. The fit of the model to the purified average observed utilities for the co-morbid conditions was optimized using ordinary least squares regression to estimate s. Replicability of the results was assessed by applying the method to triple co-morbidities from the CCHS cycle 1.1 database, as well as to double and triple co-morbidities from cycle 2.1 of the CCHS (2003–04). RESULTS: Model fit was optimized at s = .99 (i.e., essentially a straightforward multiplicative model). These results were closely replicated with triple co-morbidities reported on CCHS 2000–01, as well as with double and triple co-morbidities reported on CCHS 2003–04. CONCLUSION: The findings support the simple multiplicative model for computing utilities for co-morbid conditions from the utilities for the individual conditions involved. Future work using a wider variety of conditions and data sources could serve to further evaluate and refine the approach

Sociodemographic variation in the use of chemotherapy and radiotherapy in patients with stage IV lung, oesophageal, stomach and pancreatic cancer: evidence from population-based data in England during 2013-2014.

BACKGROUND: Sociodemographic inequalities in cancer treatment have been generally described, but there is little evidence regarding patients with advanced cancer. Understanding variation in the management of these patients may provide insights into likely mechanisms leading to inequalities in survival. METHODS: We identified 50,232 patients with stage IV lung, oesophageal, pancreatic and stomach cancer from the English national cancer registry. A generalised linear model with a Poisson error structure was used to explore variation in radiotherapy and chemotherapy within 6 months from diagnosis by age, sex, deprivation, ethnicity, cancer site, comorbidity and, additionally, performance status. RESULTS: There was substantial variation by cancer site, large gradients by age, and non-trivial associations with comorbidity and deprivation. After full adjustment, more deprived patients were consistently least likely to be treated with chemotherapy alone or chemotherapy and radiotherapy combined compared with less deprived patients with equally advanced disease stage (treatment rate ratio: 0.82 95% CI (0.78, 0.87) for CT, 0.78 95% CI (0.71, 0.85) for CTRT p < 0.0001). CONCLUSIONS: There was marked variation in the management of patients with stage IV cancer. Routinely collected data could be used for surveillance across all cancers to help reduce treatment variation and optimise outcomes among patients with advanced cancer

An Efficient Vector System to Modify Cells Genetically

The transfer of foreign genes into mammalian cells has been essential for understanding the functions of genes and mechanisms of genetic diseases, for the production of coding proteins and for gene therapy applications. Currently, the identification and selection of cells that have received transferred genetic material can be accomplished by methods, including drug selection, reporter enzyme detection and GFP imaging. These methods may confer antibiotic resistance, or be disruptive, or require special equipment. In this study, we labeled genetically modified cells with a cell surface biotinylation tag by co-transfecting cells with BirA, a biotin ligase. The modified cells can be quickly isolated for downstream applications using a simple streptavidin bead method. This system can also be used to screen cells expressing two sets of genes from separate vectors

Hypertension persisting after pre-eclampsia: a prospective cohort study at Mulago Hospital, Uganda.

BACKGROUND: Pre-eclampsia/eclampsia usually resolves after delivery but sometimes hypertension persists and cardiovascular disease develops later. Our objective was to determine the incidence and maternal socio-demographic and obstetric risk factors for persistence of hypertension in women with pre-eclampsia/eclampsia. METHODS: This was a prospective cohort study conducted from July 2009 to June 2011 at Mulago Hospital labour ward and postnatal clinics. We followed up 188 women admitted with pre-eclampsia/eclampsia until 3 months after delivery. Data was collected using interviewer-administered questionnaires, examination of participants and review of medical records. Stata (version12) software was used for data analysis. Univariable analysis was used to compute the relative risk of persistent hypertension at the 95% confidence level. This was followed by multivariable logistic regression analysis to determine factors independently associated with persistence of hypertension. RESULTS: 64 (34%) out of the 188 women analysed had persistent hypertension three months after delivery. Maternal age, gestational age at delivery and parity were predictors of persistent hypertension. CONCLUSION: The proportion of women with pre-eclampsia/eclampsia at risk of persistent hypertension at three months after delivery was high, with nearly one of three mothers remaining hypertensive. Follow up of mothers who develop pre-eclampsia is important so that early diagnosis and management of chronic hypertension can be made to avoid long term morbidity and mortality