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The Arg-specific gingipains of Porphyromonas gingivalis RgpA and RgpB have 97% identical sequences in their catalytic domains yet their propeptides are only 76% identical. RgpA isolates as a proteinase-adhesin complex (HRgpA) which hinders direct kinetic comparison of RgpA cat as a monomer with monomeric RgpB. We tested modifications of rgpA identifying a variant that enabled us to isolate histidine-tagged monomeric RgpA (rRgpAH). Kinetic comparisons between rRgpAH and RgpB used benzoyl-L-Arg-4-nitroanilide with and without cysteine and glycylglycine acceptor molecules. With no glycylglycine, values of K m , V max , k cat and k cat /K m for each enzyme were similar, but with glycylglycine K m decreased, V max increased and k cat increased ~ twofold for RgpB but ~ sixfold for rRgpAH. The k cat /K m for rRgpAH was unchanged whereas that of RgpB more than halved. Recombinant RgpA propeptide inhibited rRgpAH and RgpB with K i 13 nM and 15 nM K i respectively slightly more effectively than RgpB propeptide which inhibited rRgpAH and RgpB with K i 22 nM and 29 nM respectively (p < 0.0001); a result that may be attributable to the divergent propeptide sequences. Overall, the data for rRgpAH reflected observations previously made by others using HRgpA, indicating rRgpAH fidelity and confirming the first production and isolation of functional affinity tagged RgpA., (© 2023. The Author(s).)

Study Objective: To evaluate the competency of trained health workers in detecting and managing hypertensive disorders of pregnancy during routine antenatal check-ups (ANCs) at primary care facilities in Bangladesh., Study Design and Settings: Cross-sectional study; conducted in 26 primary care facilities., Outcome Measures: Accurate diagnosis of the hypertensive disorders of pregnancy., Method: In total 1560 ANC consultations provided by primary health workers, known as Family Welfare Visitors (FWVs), were observed using a structured checklist between October 2017 and February 2018. All consultations were reassessed by study physicians for validation., Result: Of the 'true' cases of gestational hypertension (n=32), pre-eclampsia (n=29) and severe pre-eclampsia (n=16), only 3%, 7% and 25%, respectively, were correctly diagnosed by FWVs. Per cent agreement for the diagnosed cases of any hypertensive disorders of pregnancy was 9% and kappa statistics was 0.50 (p value 0.0125). For identification of any hypertensive disorders by FWVs, sensitivity and positive predictive values were 14% and 50%, respectively. There was a moderate positive correlation between the blood pressure measurements taken by FWVs and study physicians. Only 27% of those who had 'some protein' in urine were correctly identified by FWVs. Women diagnosed with any of the hypertensive disorders of pregnancy by FWVs were more likely to be counselled on at least one danger sign of pre-eclampsia (severe headache, blurring of vision and upper abdominal pain) than those without any such diagnosis (41% vs 19%, p value 0.008). All four cases of severe pre-eclampsia diagnosed by FWVs were given a loading dose of intramuscular magnesium sulphate and three among them were referred to a higher facility., Conclusion: The FWVs should be appropriately trained on risk assessment of pregnant women with particular emphasis on accurately assessing the diagnostic criteria of hypertensive disorders of pregnancy and its management., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The cell-surface cysteine proteinases RgpA, RgpB (Arg-gingipain), and Kgp (Lys-gingipain) are major virulence factors of P. gingivalis , a keystone pathogen in the development of destructive periodontal disease. The gingipains function as proteinases and transpeptidases utilising small peptides such as glycylglycine as acceptor molecules. However, the characteristics of the gingipains from most P. gingivalis strains have not been determined. Methods: We determined the phenotypes of a panel of P. gingivalis laboratory strains and global clinical isolates with respect to growth on blood agar plus whole-cell and vesicle-free culture supernatant (VFSN) Arg- and Lys-specific proteinase activities. Results: The P. gingivalis isolates exhibited different growth characteristics and hydrolysis of haemoglobin in solid media. Whole-cell Arg-gingipain Vmax varied 5.8-fold and the whole cell Lys-gingipain V max varied 2.1-fold across the strains. Furthermore, the P. gingivalis strains showed more than 107-fold variance in soluble Arg-gingipain activity in VFSN and more than 371-fold variance in soluble Lys-gingipain activity in VFSN. Glycylglycine and cysteine stimulated Arg- and Lys-specific cleavage activities of all strains. The stimulation by cysteine was in addition to its redox effect consistent with both glycylglycine and cysteine promoting transpeptidation. Conclusion: The global P. gingivalis clinical isolates exhibit different Arg- and Lys‑gingipain activities with substantial variability in the level of soluble proteinases released into the environment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Background: Pregnancy among adolescent girls in Bangladesh is high, with 66% of women under the age of 18 reporting a first birth; this issue is particularly acute in the northern region of Bangladesh, an area that is especially impoverished and where girls are at heightened risk. Using formative research, CARE USA examined the underlying social, individual and structural factors influencing married girls' early first birth and participation in alternative opportunities (such as education or economic pursuits) in Bangladesh., Methods: In July of 2017, researchers conducted in-depth interviews of community members in two sub-districts of northern Bangladesh (Kurigram Sadar and Rajarhat). Participants (n = 127) included adolescent girls (both married and unmarredi), husbands of adolescent girls, influential adults in the girls' lives, community leaders, and health providers. All interviews were transcribed, coded and organized using Dedoose software., Results: Participants recognize the health benefits of delaying first birth, but stigma around infertility and contraceptive use, pressure from mothers-in-law and health provider bias interfere with a girl's ability to delay childbearing. Girls' social isolation, lack of mobility or autonomy, and inability to envision alternatives to early motherhood compound the issue; provider bias may also prevent access to methods. While participants agree that pursuit of education and economic opportunities are important, better futures for girls do not necessarily supersede their marital obligations of childrearing and domestic chores., Conclusions: Findings indicate the need for a multi-level approach to delaying early birth and stimulating girls' participation in economic and educational pursuits. Interventions must mitigate barriers to reproductive health care; train adolescent girls on viable economic activities; and provide educational opportunities for girls. Effective programs should also address contextual issues by including immediate members of the girls' families, particularly the husband and mother-in-law.

Porphyromonas gingivalis is a keystone periodontal pathogen that has been associated with autoimmune disorders. The cell surface proteases Lys-gingipain (Kgp) and Arg-gingipains (RgpA and RgpB) are major virulence factors, and their proteolytic activity is enhanced by small peptides such as glycylglycine (GlyGly). The reaction kinetics suggested that GlyGly may function as an acceptor molecule for gingipain-catalyzed transpeptidation. Purified gingipains and P. gingivalis whole cells were used to digest selected substrates including human hemoglobin in the presence or absence of peptide acceptors. Mass spectrometric analysis of the substrates digested with gingipains in the presence of GlyGly showed that transpeptidation outcompeted hydrolysis, whereas the trypsin-digested controls exhibited predominantly hydrolysis activity. The transpeptidation levels increased with increasing concentration of GlyGly. Purified gingipains and whole cells exhibited extensive transpeptidation activities on human hemoglobin. All hemoglobin cleavage sites were found to be suitable for GlyGly transpeptidation, and this transpeptidation enhanced hemoglobin digestion. The transpeptidation products were often more abundant than the corresponding hydrolysis products. In the absence of GlyGly, hemoglobin peptides produced during digestion were utilized as acceptors leading to the detection of up to 116 different transpeptidation products in a single reaction. P. gingivalis cells were able to digest hemoglobin faster when acceptor peptides derived from human serum albumin were included in the reaction, suggesting that gingipain-catalyzed transpeptidation may be relevant for substrates encountered in vivo. The transpeptidation of host proteins in vivo may potentially lead to the breakdown of immunological tolerance, culminating in autoimmune reactions.

Porphyromonas gingivalis is a keystone pathogen in chronic periodontitis. Its expression of gingipain proteases (Kgp and RgpA/B) is central to the stimulation of chronic inflammation. In this study, we investigated the inflammatory response of oral epithelial cells to P. gingivalis. The cells responded by upregulating the expression of the orphan chemokine CXCL14. The stimulation of CXCL14 expression was largely triggered by the gingipain proteases and was dependent on the host protease-activated receptor PAR-3. Significantly, CXCL14 expression was transcriptionally repressed in response to epidermal growth factor (EGF)-induced activation of the MEK-ERK1/2 pathway. P. gingivalis overcomes the repression of CXCL14 via the gingipain protease-mediated degradation of EGF. Therefore, P. gingivalis not only directly stimulates CXCL14 expression via PAR-3 but also promotes its expression by antagonising EGF signalling. In addition to chemotactic activity, some chemokines also have antimicrobial activities. CXCL14 was demonstrated to have bactericidal activity, against commensal oral streptococci associated with health. Notably though, P. gingivalis was not susceptible to killing by CXCL14, potentially because the gingipain proteases can degrade CXCL14. This suggests that the stimulation of dysregulated CXCL14 expression by P. gingivalis may help promote dysbiosis and the development of chronic periodontitis., (© 2018 John Wiley & Sons Ltd.)

Background: Cardiovascular diseases (CVDs) are the leading cause of global mortality. Among the CVDs, acute vascular events (AVE) mainly ischemic heart diseases and stroke are the largest contributors. To achieve 25% reduction in preventable deaths from CVDs by 2025, health systems need to be equipped with extended service coverage in order to provide person-centered care. The overall goal of this proposed study is to assess access to health care in-terms of service availability, care seeking patterns and barriers to access care after AVE in rural Bangladesh. We will consider myocardial infarction (MI) and stroke as acute vascular events., Methods/design: We will conduct a mixed methods study in rural Matlab, Bangladesh. This study will comprise of a) health facility survey, b) structured questionnaire interview and c) qualitative study. We will assess service availabilities by creating an inventory of public and private health facilities. Readiness of the facilities to deliver services for AVE will be assessed through a health facility survey using 'service availability and readiness assessment' (SARA) tools of the World Health Organization (WHO). We will interview survivors of AVE and caregivers (present and accompanied the person during the event) of person who died from AVE for exploring patterns of care seeking during an AVE. For exploring barriers to access care for AVE, we will conduct in-depth interview with survivors of AVE and caregivers of the person who died from AVE. We will also conduct key informant interviews with the service providers at primary health care (PHC) facilities and government high level officials at central health administration of Bangladesh., Discussion: This study will provide a comprehensive picture of access to primary health care services during acute cardiovascular events as stroke & MI in rural context of Bangladesh. It will explore available service facilities in rural area for management, utilization of services and barriers to access care during an acute emergency. This study will help to generate hypothesis, develop programs and policies for better access to care for AVE in similar rural settings considering barriers of access and improving utilization.

Glass ionomer cements (GIC) are dental restorative materials that are suitable for modification to help prevent dental plaque (biofilm) formation. The aim of this study was to determine the effects of incorporating casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) into a GIC on the colonisation and establishment of Streptococcus mutans biofilms and the effects of aqueous CPP-ACP on established S mutans biofilms. S. mutans biofilms were either established in flow cells before a single ten min exposure to 1% w/v CPP-ACP treatment or cultured in static wells or flow cells with either GIC or GIC containing 3% w/w CPP-ACP as the substratum. The biofilms were then visualised using confocal laser scanning microscopy after BacLight LIVE/DEAD staining. A significant decrease in biovolume and average thickness of S. mutans biofilms was observed in both static and flow cell assays when 3% CPP-ACP was incorporated into the GIC substratum. A single ten min treatment with aqueous 1% CPP-ACP resulted in a 58% decrease in biofilm biomass and thickness of established S. mutans biofilms grown in a flow cell. The treatment also significantly altered the structure of these biofilms compared with controls. The incorporation of 3% CPP-ACP into GIC significantly reduced S. mutans biofilm development indicating another potential anticariogenic mechanism of this material. Additionally aqueous CPP-ACP disrupted established S. mutans biofilms. The use of CPP-ACP containing GIC combined with regular CPP-ACP treatment may lower S. mutans challenge., Competing Interests: The authors have declared that no competing interests exist.

Tryptic digestion of the calcium-sensitive caseins yields casein phosphopeptides (CPP) that contain clusters of phosphorylated seryl residues. The CPP stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the CPP with calcium phosphate using a variety of nuclear magnetic resonance (NMR) techniques. Translational diffusion measurements indicated that the β-CN(1-25)-ACP nanocomplex has a hydrodynamic radius of 1.526 ± 0.044 nm at pH 6.0, which increases to 1.923 ± 0.082 nm at pH 9.0. (1)H NMR spectra were well resolved, and (3)JH(N)-H(α) measurements ranged from a low of 5.5 Hz to a high of 8.1 Hz. Total correlation spectroscopy and nuclear Overhauser effect spectroscopy spectra were acquired and sequentially assigned. Experiments described in this paper have allowed the development of a structural model of the β-CN(1-25)-amorphous calcium phosphate nanocomplex.

The repair of early dental caries lesions has been demonstrated by the application of the remineralisation technology based on casein phosphopeptide-stabilised amorphous calcium phosphate complexes (CPP-ACP). These complexes consist of an amorphous calcium phosphate mineral phase stabilised and encapsulated by the self-assembly of milk-derived phosphopeptides. During topical application of CPP-ACP complexes in the oral cavity, the CPP encounters the enamel pellicle consisting of salivary proteins and peptides. However the interactions of the CPP with the enamel salivary pellicle are not known. The studies presented here reveal that the predominant peptides of CPP-ACP complexes do interact with specific salivary proteins and peptides of the enamel pellicle, and provide a mechanism by which the CPP-ACP complexes are localised at the tooth surface to promote remineralisation.

Background: The aim of this study was to estimate the prevalence of disability and its associated risk factors among adults aged 18 years and over in Bogra district, Bangladesh., Methods: The Rapid Assessment of Disability (RAD) survey was conducted using probability-proportional-to-size sampling to select 66 clusters each with 50 people aged 18 years and older in 2010. Households within clusters were selected through compact segment sampling. Disability was identified based on the responses to the self-assessment of functioning section of the RAD questionnaire. Descriptive and multivariate logistic regression analyses were performed to model the associations between risk factors and disability status., Results: Of 1855 adults who participated in the study, 195 (10.5 %) had disability. Age and gender adjusted prevalence of disability in Bogra district was 8.9 % (95 % CI: 7.7, 10.3). The highest prevalence of functional limitation was related to psychological distress (4.7 %; 95 % CI: 3.8, 5.7) followed by vision (4.4 %; 95 % CI: 3.6, 5.4), and hearing (2.3 %; 95 % CI: 1.7, 3.0) difficulties. The adjusted odds of disability increased with age with approximately eight-fold increase from 2.9 % (95 % CI: 1.6, 5.1) in 18-24 years to 24.5 % (95 % CI: 20.2, 29.4) in 55 years and above. People with poor socio-economic status (OR 1.90; 95 % CI: 1.1, 3.3) and who were unemployed (OR = 4.6; 95 % CI: 1.8, 11.6) were more like to have disability compared to the higher socio-economic status and those who have an occupation respectively., Conclusions: There is a significant need for promoting programs for health, well-being, and rehabilitation, and policies specifically targeting the older population, women, unemployed and poor people in Bangladesh.

Background: An integrated maternal health intervention in a rural sub district of Bangladesh focused on the training and deployment of a required number of Community Based Skilled Birth Attendants (CSBAs). The aim of the study was to assess the effect of the intervention on use of skilled provider care during pregnancy, delivery and after delivery., Methods: The effect compared the skilled providers care in low performing areas with high and medium performing areas through pre-and post-intervention surveys. The post-intervention survey was conducted two years after the completion of the intervention. Village clusters, with populations of approximately 3000, were randomly assigned to the surveys. Mothers who had delivered within the 6 months prior to the surveys, were recruited for the structured interviews. Logistic regression was conducted to compare the effect., Results: Majority of mothers in the low performing areas belonged to a poor economic quintile. The level of skilled attendance for 4+ Antenatal Care (ANC) and delivery increased sharply from baseline to endline survey in low performing areas (ANC: 1.6% to 15.3%, p < 0.0001; skilled birth attendant at delivery: 12.6% to 38.3%, p < 0.0001). Less than 1% of the women received Post Natal Care (PNC) from a skilled provider prior to the intervention, and this proportion increased to 20% at the end of the intervention. Adjusted odds showed that the intervention had an effect on the individual performing area on use of skilled provider care during ANC, delivery and PNC. The increase of 4+ ANC from skilled providers and skilled birth attendants during delivery in the low performing areas due to the integrated maternal health intervention was significant relative to the increase in the high performing areas [4+ ANC from skilled providers - OR: 3.8 (1.9-7.6); skilled birth attendants - OR: 2.8 (2.1-3.8)]., Conclusion: An integrated maternal health care intervention focused on deployment of a required number of CSBAs increased the opportunity for underprivileged women to benefit from skilled providers care during their pregnancy. This integrated intervention might improve the maternal health in developing countries where home delivery with unskilled attendants is common.

Some amino acids are more energetically costly to synthesize de novo, therefore many microbes have evolved to regulate the metabolic expenditure of the cell and reduce the energy burden of extracellular unrecyclable proteins. Several oral bacterial species take up amino acids and peptides obtained from proteolysis of host proteins and hence do not rely only on de novo synthesis. The aim of this study was to investigate if five oral bacterial species implement cost management strategies to reduce the energy burden of extracellular unrecyclable proteins. Since the relative de novo amino acid synthesis costs are proportional to the masses of the amino acids, the energy costs of producing proteins were assessed by calculating the mean amino acid mass for each protein. For Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Prevotella intermedia and Streptococcus sanguinis, the outer membrane/extracellular proteins are made up of a much larger percentage of lower average mass amino acids whereas cytoplasmic proteins are made up of a larger proportion of higher average mass amino acid residues. These results are consistent with the five oral bacterial species employing energy-saving mechanisms in the production of extracellular unrecyclable proteins. Interestingly, the P. gingivalis and S. sanguinis genomes exhibited significantly lower predicted mean amino acid masses compared with those of the genomes of the other three species, suggesting that this may provide them with an energy advantage with respect to protein biosynthetic cost., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The oral pathogen Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Gingipains, the principle virulence factors of P. gingivalis are multidomain, cell-surface proteins containing a cysteine protease domain. The lysine specific gingipain, Kgp, is a critical virulence factor of P. gingivalis. We have determined the X-ray crystal structure of the lysine-specific protease domain of Kgp to 1.6 Å resolution. The structure provides insights into the mechanism of substrate specificity and catalysis., (© 2014 The Protein Society.)

Background: The Rapid Assessment of Disability (RAD) questionnaire measures the magnitude and impact of disability and aims to inform the design of disability inclusive development programs. This paper reports the psychometric evaluation of the RAD., Methods: The initial version of the RAD comprised five sections: 1) demographics, 2) functioning, 3) rights awareness, 4) well-being, and 5) access to the community. Item functioning and construct validity were assessed in a population-based study in Bangladesh. Data were analysed using descriptive statistics (sections 2 and 5) and Rasch modelling (sections 3 and 4). A subsequent case-control study in Fiji tested the refined questionnaire in a cross-cultural setting and assessed the sensitivity and specificity of the RAD section 2 to identify people with disability., Results: 2,057 adults took part in the study (1,855 in Bangladesh and 202 in Fiji). The prevalence of disability estimated using RAD section 2 in Bangladesh was 10.5% (95% CI 8.8-12.2), with satisfactory sensitivity and specificity (62.4% and 81.2%, respectively). Section 3 exhibited multidimensionality and poor differentiation between levels of rights awareness in both Bangladesh (person separation index [PSI] = 0.71) and Fiji (PSI = 0.0), and was unable to distinguish between people with and without disability (Bangladesh p = 0.786, Fiji p = 0.43). This section was subsequently removed from the questionnaire pending re-development. Section 4 had good ability to differentiate between levels of well-being (PSI = 0.82). In both countries, people with disability had significantly worse well-being scores than people without disability (p < 0.001) and also access to all sectors of community except legal assistance, drinking water and toilets (p < 0.001)., Conclusions: Filed-testing in Bangladesh and Fiji confirmed the psychometric robustness of functioning, well-being, and community access sections of the RAD. Information from the questionnaire can be used to inform and evaluate disability inclusive development programs.

Background: Toll free mobile telephone intervention to support mothers in pregnancy and delivery period was tested in one sub district of Bangladesh. Qualitative research was conducted to measure the changes of mobile phone use in increasing communication for maternal and neonatal complications., Methods: In-depth interviews were conducted among twelve Community Skilled Birth Attendants and fourteen mothers along with their husbands prior to intervention. At intervention end, six Community Skilled Birth Attendants were purposively selected for in-depth interview. Semi structured interviews were conducted among all 27 Community Skilled Birth Attendants engaged in the intervention. One Focus Group Discussion was conducted with 10 recently delivered mothers. Thematic analysis and triangulation of different responses were conducted., Results: Prior to intervention, Community Skilled Birth Attendants reported that mobile communication was not a norm. It was also revealed that poor mothers had poor accessibility to mobile services. Mothers, who communicated through mobile phone with providers noted irritability from Community Skilled Birth Attendants and sometimes found phones switched off. At the end of the project, 85% of mothers who had attended orientation sessions of the intervention communicated with Community Skilled Birth Attendants through mobile phones during maternal health complications. Once a complication is reported or anticipated over phone, Community Skilled Birth Attendants either made a prompt visit to mothers or advised for direct referral. More than 80% Community Skilled Birth Attendants communicated with Solution Linked Group for guidance on maternal health management. Prior to intervention, Solution Linked Group was not used to receive phone call from Community Skilled Birth Attendants. Community Skilled Birth Attendants were valued by the mothers. Mothers viewed that Community Skilled Birth Attendants are becoming confident in managing complication due to communication with Solution Linked Group., Conclusions: The use of mobile technology in this intervention took a leap from simply rendering information to providing more rapid services. Active participation of service providers along with mothers' accessibility motivated both the service providers and mothers to communicate through mobile phone for maternal health issues. These altogether made the shift towards adoption of an innovation.

Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis. The organism's cell-surface cysteine proteinases, the Arg-specific proteinases (RgpA, RgpB) and the Lys-specific proteinase (Kgp), which are known as gingipains have been implicated as major virulence factors. All three gingipain precursors contain a propeptide of around 200 amino acids in length that is removed during maturation. The aim of this study was to characterize the inhibitory potential of the Kgp and RgpB propeptides against the mature cognate enzymes. Mature Kgp was obtained from P. gingivalis mutant ECR368, which produces a recombinant Kgp with an ABM1 motif deleted from the catalytic domain (rKgp) that enables the otherwise membrane bound enzyme to dissociate from adhesins and be released. Mature RgpB was obtained from P. gingivalis HG66. Recombinant propeptides of Kgp and RgpB were produced in Escherichia coli and purified using nickel-affinity chromatography. The Kgp and RgpB propeptides displayed non-competitive inhibition kinetics with K(i) values of 2.04 µM and 12 nM, respectively. Both propeptides exhibited selectivity towards their cognate proteinase. The specificity of both propeptides was demonstrated by their inability to inhibit caspase-3, a closely related cysteine protease, and papain that also has a relatively long propeptide. Both propeptides at 100 mg/L caused a 50% reduction of P. gingivalis growth in a protein-based medium. In summary, this study demonstrates that gingipain propeptides are capable of inhibiting their mature cognate proteinases.

Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. The Arg-specific (RgpA/B) and Lys-specific (Kgp) cysteine proteinases of P. gingivalis are major virulence factors for the bacterium. In this study κ-casein(109-137) was identified in a chymosin digest of casein as an inhibiting peptide of the P. gingivalis proteinases. The peptide was synthesized and shown to inhibit proteolytic activity associated with P. gingivalis whole cells, purified RgpA-Kgp proteinase-adhesin complexes, and purified RgpB proteinase. The peptide κ-casein(109-137) exhibited synergism with Zn(II) against both Arg- and Lys-specific proteinases. The active region for inhibition was identified as κ-casein(117-137) using synthetic peptides. Kinetic studies revealed that κ-casein(109-137) inhibits in an uncompetitive manner. A molecular model based on the uncompetitive action and its synergistic ability with Zn(II) was developed to explain the mechanism of inhibition. Preincubation of P. gingivalis with κ-casein(109-137) significantly reduced lesion development in a murine model of infection.

Cysteine proteases are one of the largest groups of proteases and are involved in many important biological functions in all kingdoms of life. They are virulence factors of a range of eukaryotic, bacterial and viral pathogens and are involved in host invasion, pathogen replication and disruption of the host immune response. Their activity is regulated by a range of protease inhibitors. This review discusses the various families of cysteine protease inhibitors, their different modes of inhibition and their evolutionary relationships. These inhibitors as well as the recent discovery of propeptide and propeptide-like inhibitors provide insights into the structures that are important for particular inhibitory mechanisms, thus forming the foundation for the design of future therapeutics.

Dental caries is a highly prevalent diet-related disease and is a major public health problem. A goal of modern dentistry is to manage non-cavitated caries lesions non-invasively through remineralization in an attempt to prevent disease progression and improve aesthetics, strength, and function. Remineralization is defined as the process whereby calcium and phosphate ions are supplied from a source external to the tooth to promote ion deposition into crystal voids in demineralized enamel, to produce net mineral gain. Recently, a range of novel calcium-phosphate-based remineralization delivery systems has been developed for clinical application. These delivery systems include crystalline, unstabilized amorphous, or stabilized amorphous formulations of calcium phosphate. These systems are reviewed, and the technology with the most scientific evidence to support its clinical use is the remineralizing system utilizing casein phosphopeptides to stabilize and deliver bioavailable calcium, phosphate, and fluoride ions. The recent clinical evidence for this technology is presented and the mechanism of action discussed. Biomimetic approaches to stabilization of bioavailable calcium, phosphate, and fluoride ions and the localization of these ions to non-cavitated caries lesions for controlled remineralization show promise for the non-invasive management of dental caries.

Efforts to prevent HIV from becoming widespread among the youth population 15-24 years in Bangladesh are in the early stages. However, conservative religious and cultural norms may curtail the dissemination of needed information about sexuality and condoms. The community-readiness stages model was adopted as a framework for assessing the level of preparedness of community leaders to facilitate planned HIV prevention efforts. Six focus group discussions with three professional groups (teachers, businessmen, drugshop vendors) in Hobiganj district were conducted in late 2005, and a single multi-professional group made up of teachers, imams, and drugshop vendors was convened in early 2007 to assess changes. The audio recordings in Bangla were coded as were English translations. Everyone had heard of AIDS and regarded it as a potential catastrophe for the health, economy and social fabric of Bangladesh. Remarks concerning Stage 1-Vulnerability indicated that most did not believe their community to be at risk, though Bangladesh was. Remarks at Stage 2-Knowledge of Transmission were mostly vague but accurately identified sex, blood and needles as the main means of spread; however sex with sex workers was also mentioned in each group. Remarks at Stage 3-Prevention showed strong opposition to condoms for unmarried males and a preference for current means of forbidding sex outside of marriage. A few in each group recognized the importance of condoms for wayward youth. Stage 4-Planning discussions centered on raising awareness and fear, and a desire for government and media to take the lead. By 2007 participants articulated more realistic strategies that they themselves could, and had, implemented, but also raised barriers that authorities should help them overcome. The findings provide formative information on the constraints and opportunities of community groups as partners in HIV preventive interventions and strategies to help them move to a higher stage of readiness., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Bangladesh initiated an early response to the HIV epidemic starting in the mid-1980s. Since then, the response has been enhanced considerably, and many HIV-prevention interventions among the most at-risk populations and the general youth are being undertaken. Alongside prevention activities, gathering of data has been a key activity fostered by both the Government and individual development partners. This paper reviews available sources of data, including routine surveillance (HIV and behavioural among most at-risk populations), general population surveys, and various research studies with the aim to understand the dynamics of the HIV epidemic in Bangladesh. Available data show that the HIV epidemic is still at relatively low levels and is concentrated mainly among injecting drug users (IDUs) in Dhaka city. In addition, when the passively-reported cases were analyzed, another population group that appears to be especially vulnerable is migrant workers who leave their families and travel abroad for work. However, all sources of data confirm that risk behaviours that make individuals vulnerable to HIV are high--this is apparent within most at-risk populations and the general population (adult males and youth males and females). Based on the current activities and the sources of data, modelling exercises of the future of the HIV epidemic in Dhaka suggest that, if interventions are not enhanced further, Bangladesh is likely to start with an IDU-driven epidemic, similar to other neighbouring countries, which will then move to other population groups, including sex workers, males who have sex with males, clients of sex workers, and ultimately their families. This review reiterates the often repeated message that if Bangladesh wants to be an example of how to avert an HIV epidemic, it needs to act now using evidence-based programming.

The casein phosphopeptides (CPP) are derived from the milk protein casein by tryptic digestion. The CPP, containing the sequence -Pse-Pse-Pse-Glu-Glu- where Pse is a phosphoseryl residue, stabilize calcium and phosphate ions in aqueous solution and make these essential nutrients bioavailable. Under alkaline conditions the calcium phosphate is present as an alkaline amorphous phase complexed by the CPP, referred to as casein phosphopeptide-amorphous calcium phosphate (CPP-ACP). The CPP-ACP complexes readily incorporate fluoride ions forming casein phosphopeptide-amorphous calcium fluoride phosphate (CPP-ACFP). A mechanism is discussed which provides a rationale for the ability of the CPP-ACP to remineralize carious lesions in dental enamel. Clinical applications of the CPP-ACP as agents in the treatment of dental caries and other hypomineralized conditions are reviewed. It is concluded that the CPP are a safe and novel carrier for calcium, phosphate and hydroxide (fluoride) ions to promote enamel remineralization with application in oral care products, dental professional products and foodstuffs.

Neonatal deaths account for about half of all deaths among children under 5 years of age in Bangladesh, making prevention a major priority. This paper reports on a study of neonatal deaths in 12 areas of Bangladesh served by a large NGO programme, which had high coverage of reproductive health outreach services and relatively low neonatal mortality in recent years. The study aimed to identify the main factors associated with neonatal mortality in these areas, with a view to developing appropriate strategies for prevention. A case-control design was adopted for collection of data from mothers whose children, born alive in 2003, died within 28 days postpartum (142 cases), or did not (617 controls). Crude and adjusted odds ratios (AOR) were calculated as estimates of relative risk for neonatal death, using 'neighbourhood' controls (241) and 'non-neighbourhood' controls (376). A similar proportion of case and control mothers had received NGO health education and maternal health services. The main risk factors for neonatal death among 122 singleton babies, based on the two sets of controls, were: complications during delivery [AOR, 2.6 (95% CI: 1.5-4.5) and 3.1 (95% CI: 1.8-5.3)], prematurity [AOR, 7.2 (95% CI: 3.6-14.4) and 8.3 (95% CI: 4.2-16.5)], care for a sick neonate from an unlicensed 'traditional healer' [AOR, 2.9 (95% CI 0.9-9.5 and 5.9 (95% CI: 1.3-26.3)], or care not sought at all [AOR, 23.3 (95% CI: 3.9-137.4)]. The strongest predictor of neonatal death was having a previous sibling not vaccinated against measles [AOR, 5.9 (95% CI: 2.2-15.5) and 12.0 (95% CI: 4.5-31.7)]. The findings of this study indicate the need for identification of babies at high risk and early postpartum interventions (40.2% of the deaths occurred within 24 hours of delivery). Relevant strategies include special counselling during pregnancy for mothers with risk characteristics, training birth attendants in resuscitation, immediate postnatal check-up in the home for high-risk babies identified at delivery, advice for mothers on appropriate care-seeking for sick babies, improving the capacity of sub-district hospitals for emergency obstetric and newborn care, and promotion of institutional deliveries.

Although the neonatal mortality rate (NMR) in Bangladesh remained steady between 1995-99 and 1999-2003 (41-42 deaths per 1,000 live births), evidence from the management information system (MIS) of a large nongovernmental organization (NGO) program indicates that the NMR declined by about 50 percent between 1996 and 2002 in the area served. This study aims to validate the recording of neonatal deaths among the cohort of children registered as born in 2003 and to assess the evidence of a decline in the NMR. It also measures the coverage of reproductive health outreach services, focusing on 12 of the 27 NGOs that have provided services in the same areas since 1996. Field-workers' registers, verbal autopsy reports, and immunization records were checked to confirm infants' survival. Interviews were conducted with 142 mothers of children who died within 28 days postpartum and with a random sample of 109 women with registered stillbirths. Out of 11,253 registered live births in 2003, 210 neonatal deaths were found, compared with 194 deaths that were reported in the MIS for 2003. The corrected NMR was 19 deaths per 1,000 live births, and it was in the range of 15-29 deaths per 1,000 live births in 11 of the NGO areas. Because underreporting of neonatal deaths was probably higher in 1996 when the MIS-reported NMR was 39 deaths per 1,000 live births, the decline in the NMR is likely to have been genuine.

Bovine dentine phosphophoryn (BDP), a protein rich in aspartyl (Asp) and O-phosphoseryl (Ser(P)) residues, is synthesized by odontoblasts and believed to be involved in matrix-mediated biomineralization of dentine. Phosphophoryn was purified from bovine dentine using EDTA extraction, Ca(2+) precipitation, anion exchange and size exclusion chromatography. The purified protein migrated on SDS-PAGGE as a single band. The protein was dephosphorylated using a chelex alkaline dialysis procedure, repurified using anion exchange and size exclusion chromatography and then subjected to cleavage with trypsin. The digest was subjected to reversed-phase HPLC and analysed by Q-TOF mass spectrometry. The only non-trypsin peptides that could be identified were two collagen Type I alpha2 peptides whose sequence was determined by fragmentation analysis. The association of collagen fragments with highly purified phosphophoryn suggests that the EDTA extraction method yields BDP that is strongly bound to collagen fragments. This association now helps explain discrepancies in molecular weight and amino acid composition data for various phosphophoryn preparations compared with the same data calculated from the C-terminal extension of mouse, rat and human dentine sialophosphoprotein (DSPP) gene products. Analysis of the mutation pattern of the clinical disorder Osteogenesis Imperfecta within the region enclosed by the identified collagen fragments reveals that phosphophoryn associates with a segment of collagen that is crucial for structure and/or function.

Context: In rural Bangladesh, family planning services--previously provided through household visits and satellite clinics--were transferred to static community clinics under the government's sectoral program for 1998-2003, but the next sectoral program reversed the change without a formal evaluation. It is important to assess changes in utilization and coverage to inform further development of the service delivery system., Methods: Longitudinal data on use of family planning services and contraceptive methods were collected quarterly in 1998-2002 from married women in about 11,000 households in two rural surveillance areas--Abhoynagar and Mirsarai. Cross-sectional surveys were conducted among women and service providers in 2003 to gather detailed information about the transition to static clinics and women's response to the changes. Quarterly time series graphs of selected indicators were plotted for areas served by community clinics., Results: In a time of considerable change in service delivery and sources of contraceptive supply, contraceptive prevalence remained constant in Abhoynagar and increased in Mirsarai. Community clinics quickly became the source of supplies for one-third of contraceptive users in Abhoynagar and one-fifth in Mirsarai. In wards where community clinics became operational (mostly in 2001-2002), three-quarters of women had used one at some time., Conclusions: Despite cultural constraints on mobility, women do not appear to have become dependent on home delivery of contraceptives.

Bovine dentin phosphophoryn (BDP), a protein rich in aspartyl (Asp) and o-phosphoseryl [Ser(P)] residues, is synthesized by odontoblasts and believed to be involved in matrix-mediated biomineralization of dentin. The elucidation of the structure-function relationship of phosphophoryn has been a challenge because of its high-molecular weight, high negative charge, repetitive sequence, and lability. We have used the dynamic behavior of the (1)H NMR signal at 600 MHz to provide insight into the molecular dynamics of phosphophoryn. Our results indicate that phosphophoryn is a molecule of uniformly high mobility, thus belonging to a recently identified class of intrinsically disordered proteins that are characterized by sequences of low complexity and rich in polar and charged residues. The significance of our results is that phosphophoryn, because of its uniform nature has the potential to be replaced by biomimetic synthetic peptide analogs that together with amorphous calcium phosphate may lead to the development of novel, nontoxic, apatite-based dental restorative materials.

Several proteins associated with mineralised tissue (teeth and bone) or involved in calcium phosphate stabilisation in the body fluids, milk and saliva have been mapped to the q arm of human chromosome 4. These include the dentine/bone proteins dentine sialophosphoprotein (DSPP), dentine matrix protein 1 (DMP1), bone sialoprotein (BSP), matrix extracellular phosphoglycoprotein, osteopontin (OPN), enamelin, ameloblastin, milk caseins, salivary statherin, and proline-rich proteins. The proposed function of those that are multiphosphorylated is: (i) the stabilisation of calcium phosphate in solution (e.g. casein, statherin) preventing spontaneous precipitation and seeded-crystal growth or (ii) promoting biomineralisation (e.g. the phosphophoryn domain of DSPP), where the protein described as a template macromolecule, is proposed to act as a nucleator/promoter of crystal growth. The genes of these proteins have been subjected to conserved chromosomal synteny during mammalian evolution. The multiphosphorylated proteins statherin, caseins, phosphophoryn, BSP and OPN have been characterised as intrinsically disordered. The codon usage patterns for the amino acid serine reveal a bias for AGC and AGT codons within the human genes dspp, dmp1 and bsp, mouse dspp and dmp1 but not significantly for statherin or caseins. This pattern was also observed in the gene encoding hen phosvitin that also contains stretches of multiphosphorylated serines and in the dmp1 gene sequences of mammalian, reptilian and avian classes. In conclusion, these intrinsically disordered multiphosphorylated proteins are the translation products of genes displaying examples of codon usage bias, internal repeats and conserved chromosomal synteny within the mammalian class.

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.

The repair of early tooth enamel lesions has been recently demonstrated by tryptic phosphopeptides derived from milk caseins that associate with amorphous calcium phosphate (ACP) forming stable complexes. These casein phosphopeptides (CPP), containing the cluster sequence-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-, form calcium phosphate delivery vehicles that retard enamel demineralization and promote remineralization. Recently, we have shown that these peptides also stabilize calcium fluoride phosphate as soluble complexes. These complexes designated CPP-ACFP, have the potential to provide superior clinical efficacy in preventing dental caries and treating and repairing early stages of disease. In an approach to determine the ultrastructure of the casein phosphopeptide-amorphous calcium fluoride phosphate complexes, we have studied the structure of the predominant peptide alpha(S1)-CN(59-79) bound to ACFP using nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. The alpha(S1)-CN(59-79) peptide stabilized calcium fluoride phosphate as amorphous nanocomplexes with a hydrodynamic radius of 2.12+/-0.26 nm. The nanocomplexes exhibited stoichiometry of one peptide to 15 calcium, nine phosphate and three fluoride ions. Sequence-specific resonance assignments were determined for the peptide alpha(S1)-CN(59-79) complexed to the ACFP. The secondary structure of the peptide alpha(S1)-CN(59-79) was characterized by sequential (i, i+1), medium-range (i, i+2) nOes and H alpha chemical shifts. The spectral data were compared with that of the peptide alpha(S1)-CN(59-79) bound to calcium ions, revealing that the structurally significant secondary NH and alpha-chemical shifts were similar.

Sequence-specific nuclear magnetic resonance (NMR) assignments have been determined for the peptide alphaS2-CN(2-20) containing the multiphosphorylated motif-8Ser(P)-Ser(P)-Ser(P)-Glu-Glu12- in the presence of molar excess Ca2+. The secondary structure of the peptide was characterized by sequential (i,i + 1), medium-range (i,i + 2/3/4) nOes and H alpha chemical shifts. Molecular modelling of the peptide based on these constraints suggests a nascent helix for residues Ser(P)9 to Glu12. The spectral data for alphaS2-CN(2-20) were compared with those of other casein phosphopeptides beta-CN(1-25) and alphaS1-CN(59-79) that also contain the multiphosphorylated motif. This comparison revealed a similar pattern of secondary amide chemical shifts in the multiphosphorylated motif. However, the patterns of medium-range nOe connectivities in the three peptides suggests they have distinctly different conformations in the presence of Ca2+ despite having a high degree of sequential similarity.

Complete sequence-specific, proton-resonance assignments have been determined for the calcium phosphate-stabilizing tryptic peptide beta-casein-(1-25) containing the phosphorylated sequence motif Ser(P)(17)-Ser(P)-Ser(P)-Glu-Glu(21). Spectra of the peptide have been recorded, in separate experiments, in the presence of excess ammonium ions, sodium ions and calcium ions, and of the dephosphorylated peptide in the presence of excess sodium ions. We observed significant changes to chemical shifts for backbone and side-chain resonances that were dependent upon the nature of the cation present. Medium-range nuclear Overhauser effect (nOe) enhancements, characteristic of small structured regions in the peptide, were observed and also found to be cation dependent. The secondary structure of the peptide was characterized by sequential and medium-range (i, i+2/3/4, which denotes an interaction between residue i and residue i+2, i+3 or i+4 in the peptide) nOe connectivities, and Halpha chemical shifts. Four structured regions were identified in the calcium-bound peptide: residues Arg(1) to Glu(4) were involved in a loop-type structure, and residues Val(8) to Glu(11), Ser(P)(17) to Glu(20) and Glu(21) to Thr(24) were implicated in beta-turn conformations. Comparison of the patterns of medium-range nOe connectivities in beta-casein-(1-25) with those in alpha(S1)-casein-(59-79) suggest that the two peptides have distinctly different conformations in the presence of calcium ions, despite having a high degree of sequential and functional similarity.

Bovine dentin phosphophoryn (BDP), a protein rich in aspartyl (Asp) and O-phosphoseryl (Ser[P]) residues, is synthesized by odontoblasts and is believed to be involved in matrix-mediated biomineralization of dentin. We have purified BDP, using selective precipitation and ion exchange chromatography, from an EDTA soluble dentin extract and converted the Ser(P) residues to S-propylcysteinyl residues that are stable to Edman degradation, facilitating the determination of the amino acid sequence of the N-terminal 38 residues. After the initial Asp-Ser(P)-Pro-Asn-Ser(P)-Ser(P)-Asp-Glu-Ser(P)-Asn-Gly-, the sequence contained the repeated motifs Asp-Ser(P) and Asp-Ser(P)-Ser(P). Purified BDP migrated as a single band on gradient SDS-PAGE with an apparent molecular weight of 156 kDa. This value was consistent with the molecular weight of the dephosphorylated protein of 105 kDa determined by means of MALDI mass spectrometry.