Social capital of families of children with neurodevelopmental disabilities in South India.

AIM: To investigate the social capital of families with children with neurodevelopmental disabilities in South India receiving a community-based early intervention (Enabling Inclusion®) program and to explore determinants and associations between social capital and program duration, socio-demographic factors, family empowerment, and caregiver burden. METHOD: Using purposive sampling in a cross-sectional study design, 217 families (n = 71 received short Enabling Inclusion [<5 months]; n = 146 received long Enabling Inclusion [>9 months]) were recruited and completed the Short Adapted Social Capital Tool (SASCAT: cognitive, structural), measures of family empowerment, and caregiver strain. Descriptive statistics, regression, and correlations were used for analyses. RESULTS: In 52.1% of participants, low cognitive and structural social capital was observed. Higher odds of low structural social capital were observed for mothers with primary versus secondary education (adjusted odds ratio [OR] = 0.35; 95% confidence interval [CI] 0.13-0.90; p = 0.029); and caregivers of children with cerebral palsy versus autism (OR = 4.66; 95% CI 1.02-21.21; p = 0.046). Significant associations were found between structural social capital, the child's age, and support group membership (χ2 = 6.29; 4.70; degrees of freedom [df] = 2; 1; p = 0.04; p = 0.02 respectively), as well as between cognitive social capital and other disability in the family (χ2 = 4.62, df = 1, p = 0.03). INTERPRETATION: While program duration was not found to mediate social capital, mother's education and child's diagnosis emerged as key influential factors, warranting their consideration in interventions supporting families of children with neurodevelopmental disabilities in low- and-middle-income countries and elsewhere.

Disulfide Bond Formation in the Periplasm of Escherichia coli.

The formation of disulfide bonds is critical to the folding of many extracytoplasmic proteins in all domains of life. With the discovery in the early 1990s that disulfide bond formation is catalyzed by enzymes, the field of oxidative folding of proteins was born. Escherichia coli played a central role as a model organism for the elucidation of the disulfide bond-forming machinery. Since then, many of the enzymatic players and their mechanisms of forming, breaking, and shuffling disulfide bonds have become understood in greater detail. This article summarizes the discoveries of the past 3 decades, focusing on disulfide bond formation in the periplasm of the model prokaryotic host E. coli.

Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes.

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond, including bacterial virulence factors. Thus, proteins involved in disulfide bond formation represent good targets for the development of inhibitors that can act as antibiotics or anti-virulence agents, resulting in the simultaneous inactivation of several types of virulence factors. Here, we present evidence that the disulfide bond forming enzymes, DsbB and VKOR, are required for Pseudomonas aeruginosa pathogenicity and Mycobacterium tuberculosis survival respectively. We also report the results of a HTS of 216,767 compounds tested against P. aeruginosa DsbB1 and M. tuberculosis VKOR using Escherichia coli cells. Since both P. aeruginosa DsbB1 and M. tuberculosis VKOR complement an E. coli dsbB knockout, we screened simultaneously for inhibitors of each complemented E. coli strain expressing a disulfide-bond sensitive ß-galactosidase reported previously. The properties of several inhibitors obtained from these screens suggest they are a starting point for chemical modifications with potential for future antibacterial development.

Identification of the Thioredoxin Partner of Vitamin K Epoxide Reductase in Mycobacterial Disulfide Bond Formation.

Disulfide bonds influence the stability and activity of many proteins. In Escherichia coli, the DsbA and DsbB enzymes promote disulfide bond formation. Other bacteria, including the Actinobacteria, use instead of DsbB the enzyme vitamin K epoxide reductase (VKOR), whose gene is found either fused to or in the same operon as a dsbA-like gene. Mycobacterium tuberculosis and other Gram-positive actinobacteria secrete many proteins with even numbers of cysteines to the cell envelope. These organisms have predicted oxidoreductases and VKOR orthologs. These findings indicate that such bacteria likely form disulfide bonds in the cell envelope. The M. tuberculosisvkor gene complements an E. colidsbB deletion strain, restoring the oxidation of E. coli DsbA. While we have suggested that the dsbA gene linked to the vkor gene may express VKOR's partner in mycobacteria, others have suggested that two other extracytoplasmic oxidoreductases (DsbE or DsbF) may be catalysts of protein disulfide bond formation. However, there is no direct evidence for interactions of VKOR with either DsbA, DsbE, or DsbF. To identify the actual substrate of VKOR, we identified two additional predicted extracytoplasmic DsbA-like proteins using bioinformatics analysis of the M. tuberculosis genome. Using the five potential DsbAs, we attempted to reconstitute disulfide bond pathways in E. coli and in Mycobacterium smegmatis, a close relative of M. tuberculosis Our results show that only M. tuberculosis DsbA is oxidized by VKOR. Comparison of the properties of dsbA- and vkor-null mutants in M. smegmatis shows parallels to the properties of dsb mutations in E. coliIMPORTANCE Disulfide bond formation has a great impact on bacterial pathogenicity. Thus, disulfide-bond-forming proteins represent new targets for the development of antibacterials, since the inhibition of disulfide bond formation would result in the simultaneous loss of the activity of several classes of virulence factors. Here, we identified five candidate proteins encoded by the M. tuberculosis genome as possible substrates of the M. tuberculosis VKOR protein involved in disulfide bond formation. We then reconstituted the mycobacterial disulfide bond formation pathway in E. coli and showed that of the five candidates, only M. tuberculosis DsbA is efficiently oxidized by VKOR in E. coli We also present evidence for the involvement of VKOR in DsbA oxidation in M. smegmatis.

Disulfide bond formation in prokaryotes.

Interest in protein disulfide bond formation has recently increased because of the prominent role of disulfide bonds in bacterial virulence and survival. The first discovered pathway that introduces disulfide bonds into cell envelope proteins consists of Escherichia coli enzymes DsbA and DsbB. Since its discovery, variations on the DsbAB pathway have been found in bacteria and archaea, probably reflecting specific requirements for survival in their ecological niches. One variation found amongst Actinobacteria and Cyanobacteria is the replacement of DsbB by a homologue of human vitamin K epoxide reductase. Many Gram-positive bacteria express enzymes involved in disulfide bond formation that are similar, but non-homologous, to DsbAB. While bacterial pathways promote disulfide bond formation in the bacterial cell envelope, some archaeal extremophiles express proteins with disulfide bonds both in the cytoplasm and in the extra-cytoplasmic space, possibly to stabilize proteins in the face of extreme conditions, such as growth at high temperatures. Here, we summarize the diversity of disulfide-bond-catalysing systems across prokaryotic lineages, discuss examples for understanding the biological basis of such systems, and present perspectives on how such systems are enabling advances in biomedical engineering and drug development.

Aeropyrum pernix membrane topology of protein VKOR promotes protein disulfide bond formation in two subcellular compartments.

Disulfide bonds confer stability and activity to proteins. Bioinformatic approaches allow predictions of which organisms make protein disulfide bonds and in which subcellular compartments disulfide bond formation takes place. Such an analysis, along with biochemical and protein structural data, suggests that many of the extremophile Crenarachaea make protein disulfide bonds in both the cytoplasm and the cell envelope. We have sought to determine the oxidative folding pathways in the sequenced genomes of the Crenarchaea, by seeking homologues of the enzymes known to be involved in disulfide bond formation in bacteria. Some Crenarchaea have two homologues of the cytoplasmic membrane protein VKOR, a protein required in many bacteria for the oxidation of bacterial DsbAs. We show that the two VKORs of Aeropyrum pernix assume opposite orientations in the cytoplasmic membrane, when expressed in E. coli. One has its active cysteines oriented toward the E. coli periplasm (ApVKORo) and the other toward the cytoplasm (ApVKORi). Furthermore, the ApVKORo promotes disulfide bond formation in the E. coli cell envelope, while the ApVKORi promotes disulfide bond formation in the E. coli cytoplasm via a co-expressed archaeal protein ApPDO. Amongst the VKORs from different archaeal species, the pairs of VKORs in each species are much more closely related to each other than to the VKORs of the other species. The results suggest two independent occurrences of the evolution of the two topologically inverted VKORs in archaea. Our results suggest a mechanistic basis for the formation of disulfide bonds in the cytoplasm of Crenarchaea.

Genome Sequences of Five Streptomyces Bacteriophages Forming Cluster BG.

Cluster BG of the actinobacteriophage was formed upon discovery of five novel bacteriophages isolated by enrichment from their host, Streptomyces griseus subsp. griseus strain ATCC 10137. Four members of this cluster (BabyGotBac, Maih, TP1605, and YDN12) share over 89% average nucleotide identity, while the other (Xkcd426) has only 72% similarity to other cluster members.

The Disulfide Bond Formation Pathway Is Essential for Anaerobic Growth of Escherichia coli.

Disulfide bonds are critical to the stability and function of many bacterial proteins. In the periplasm of Escherichia coli, intramolecular disulfide bond formation is catalyzed by the two-component disulfide bond forming (DSB) system. Inactivation of the DSB pathway has been shown to lead to a number of pleotropic effects, although cells remain viable under standard laboratory conditions. However, we show here that dsb strains of E. coli reversibly filament under aerobic conditions and fail to grow anaerobically unless a strong oxidant is provided in the growth medium. These findings demonstrate that the background disulfide bond formation necessary to maintain the viability of dsb strains is oxygen dependent. LptD, a key component of the lipopolysaccharide transport system, fails to fold properly in dsb strains exposed to anaerobic conditions, suggesting that these mutants may have defects in outer membrane assembly. We also show that anaerobic growth of dsb mutants can be restored by suppressor mutations in the disulfide bond isomerization system. Overall, our results underscore the importance of proper disulfide bond formation to pathways critical to E. coli viability under conditions where oxygen is limited.IMPORTANCE While the disulfide bond formation (DSB) system of E. coli has been studied for decades and has been shown to play an important role in the proper folding of many proteins, including some associated with virulence, it was considered dispensable for growth under most laboratory conditions. This work represents the first attempt to study the effects of the DSB system under strictly anaerobic conditions, simulating the environment encountered by pathogenic E. coli strains in the human intestinal tract. By demonstrating that the DSB system is essential for growth under such conditions, this work suggests that compounds inhibiting Dsb enzymes might act not only as antivirulents but also as true antibiotics.

Inhibition of virulence-promoting disulfide bond formation enzyme DsbB is blocked by mutating residues in two distinct regions.

Disulfide bonds contribute to protein stability, activity, and folding in a variety of proteins, including many involved in bacterial virulence such as toxins, adhesins, flagella, and pili, among others. Therefore, inhibitors of disulfide bond formation enzymes could have profound effects on pathogen virulence. In the Escherichia coli disulfide bond formation pathway, the periplasmic protein DsbA introduces disulfide bonds into substrates, and then the cytoplasmic membrane protein DsbB reoxidizes DsbA's cysteines regenerating its activity. Thus, DsbB generates a protein disulfide bond de novo by transferring electrons to the quinone pool. We previously identified an effective pyridazinone-related inhibitor of DsbB enzymes from several Gram-negative bacteria. To map the protein residues that are important for the interaction with this inhibitor, we randomly mutagenized by error-prone PCR the E. coli dsbB gene and selected dsbB mutants that confer resistance to this drug using two approaches. We characterized in vivo and in vitro some of these mutants that map to two areas in the structure of DsbB, one located between the two first transmembrane segments where the quinone ring binds and the other located in the second periplasmic loop of DsbB, which interacts with DsbA. In addition, we show that a mutant version of a protein involved in lipopolysaccharide assembly, lptD4213, is synthetically lethal with the deletion of dsbB as well as with DsbB inhibitors. This finding suggests that drugs decreasing LptD assembly may be synthetically lethal with inhibitors of the Dsb pathway, potentiating the antibiotic effects.

The essential cell division protein FtsN contains a critical disulfide bond in a non-essential domain.

Disulfide bonds are found in many proteins associated with the cell wall of Escherichia coli, and for some of these proteins the disulfide bond is critical to their stability and function. One protein found to contain a disulfide bond is the essential cell division protein FtsN, but the importance of this bond to the protein's structural integrity is unclear. While it evidently plays a role in the proper folding of the SPOR domain of FtsN, this domain is non-essential, suggesting that the disulfide bond might also be dispensable. However, we find that FtsN mutants lacking cysteines give rise to filamentous growth. Furthermore, FtsN protein levels in strains expressing these mutants were significantly lower than in a strain expressing the wild-type allele, as were FtsN levels in strains incapable of making disulfide bonds (dsb- ) exposed to anaerobic conditions. These results strongly suggest that FtsN lacking a disulfide bond is unstable, thereby making this disulfide critical for function. We have previously found that dsb- strains fail to grow anaerobically, and the results presented here suggest that this growth defect may be due in part to misfolded FtsN. Thus, proper cell division in E. coli is dependent upon disulfide bond formation.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase.

Functional overexpression of polytopic membrane proteins, particularly when in a foreign host, is often a challenging task. Factors that negatively affect such processes are poorly understood. Using the mammalian membrane protein vitamin K epoxide reductase (VKORc1) as a reporter, we describe a genetic selection approach allowing the isolation of Escherichia coli mutants capable of functionally expressing this blood-coagulation enzyme. The isolated mutants map to components of membrane protein assembly and quality control proteins YidC and HslV. We show that changes in the VKORc1 sequence and in the YidC hydrophilic groove along with the inactivation of HslV promote VKORc1 activity and dramatically increase its expression level. We hypothesize that such changes correct for mismatches in the membrane topogenic signals between E. coli and eukaryotic cells guiding proper membrane integration. Furthermore, the obtained mutants allow the study of VKORc1 reaction mechanisms, inhibition by warfarin, and the high-throughput screening for potential anticoagulants.

Converting a Sulfenic Acid Reductase into a Disulfide Bond Isomerase.

AIMS: Posttranslational formation of disulfide bonds is essential for the folding of many secreted proteins. Formation of disulfide bonds in a protein with more than two cysteines is inherently fraught with error and can result in incorrect disulfide bond pairing and, consequently, misfolded protein. Protein disulfide bond isomerases, such as DsbC of Escherichia coli, can recognize mis-oxidized proteins and shuffle the disulfide bonds of the substrate protein into their native folded state. RESULTS: We have developed a simple blue/white screen that can detect disulfide bond isomerization in vivo, using a mutant alkaline phosphatase (PhoA*) in E. coli. We utilized this screen to isolate mutants of the sulfenic acid reductase (DsbG) that allowed this protein to act as a disulfide bond isomerase. Characterization of the isolated mutants in vivo and in vitro allowed us to identify key amino acid residues responsible for oxidoreductase properties of thioredoxin-like proteins such as DsbC or DsbG. INNOVATION AND CONCLUSIONS: Using these key residues, we also identified and characterized interesting environmental homologs of DsbG with novel properties, thus demonstrating the capacity of this screen to discover and elucidate mechanistic details of in vivo disulfide bond isomerization.

Compounds targeting disulfide bond forming enzyme DsbB of Gram-negative bacteria.

In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond. These proteins include numerous bacterial virulence factors, and thus bacterial enzymes that promote disulfide bond formation represent targets for compounds inhibiting bacterial virulence. Here, we describe a new target- and cell-based screening methodology for identifying compounds that inhibit the disulfide bond-forming enzymes Escherichia coli DsbB (EcDsbB) or Mycobacterium tuberculosis VKOR (MtbVKOR), which can replace EcDsbB, although the two are not homologs. Initial screening of 51,487 compounds yielded six specifically inhibiting EcDsbB. These compounds share a structural motif and do not inhibit MtbVKOR. A medicinal chemistry approach led us to select related compounds, some of which are much more effective DsbB inhibitors than those found in the screen. These compounds inhibit purified DsbB and prevent anaerobic growth of E. coli. Furthermore, these compounds inhibit all but one of the DsbBs of nine other Gram-negative pathogenic bacteria tested.

A randomized, double-blind, placebo-controlled study of preemptive oral oxycodone with morphine patient-controlled anesthesia for postoperative pain management in patients undergoing uterine artery embolization for symptomatic uterine fibroids.

PURPOSE: To evaluate the analgesic efficacy of oral premedication of oxycodone in a group of patients undergoing elective uterine artery embolization under sedation for fibroid disease. METHODS: Thirty-nine patients (mean age 42.3 years) were prospectively randomized 1:1 to receive 20 mg oxycodone or placebo orally immediately before their procedure. At the commencement of the procedure, patients were provided with a patient-controlled analgesia device for 24 h, programmed to deliver 1 mg boluses of intravenous morphine with a 5 min lockout. Mean visual analog scale pain intensity ratings (0-100 mm) were measured from both groups and evaluated over 0 to 6 h as the primary end point. Other measured parameters included opioid-related side effects and eligibility for discharge (NCT00163930; September 12, 2005). RESULTS: Early pain intensity did not vary significantly between the active and placebo groups [mean (standard deviation): 3.2 (2.5) vs. 3.1 (2.2), p = 0.89]. The oxycodone group, however, experienced significantly more nausea (p = 0.035) and a greater incidence of vomiting (p = 0.044). Overall opioid requirement over 24 h, measured as oral morphine equivalent, was greater in the oxycodone group (median [interquartile range]: 64.5 [45-90] mg vs. 22.5 [15-46.5] mg, p < 0.0001). The number of patients first eligible for discharge at 24 h in the oxycodone group was decreased but not significantly (p = 0.07). CONCLUSION: The addition of preprocedural oral oxycodone to morphine patient-controlled analgesia does not offer any analgesic advantage to patients having uterine artery embolization and may cause a greater incidence of nausea and vomiting.

Folding LacZ in the periplasm of Escherichia coli.

Targeted, translational LacZ fusions provided the initial support for the signal sequence hypothesis in prokaryotes and allowed for selection of the mutations that identified the Sec translocon. Many of these selections relied on the fact that expression of targeted, translational lacZ fusions like malE-lacZ and lamB-lacZ42-1 causes lethal toxicity as folded LacZ jams the translocation pore. However, there is another class of targeted LacZ fusions that do not jam the translocon. These targeted, nonjamming fusions also show toxic phenotypes that may be useful for selecting mutations in genes involved in posttranslocational protein folding and targeting; however, they have not been investigated to the same extent as their jamming counterparts. In fact, it is still unclear whether LacZ can be fully translocated in these fusions. It may be that they simply partition into the inner membrane where they can no longer participate in folding or assembly. In the present study, we systematically characterize the nonjamming fusions and determine their ultimate localization. We report that LacZ can be fully translocated into the periplasm, where it is toxic. We show that this toxicity is likely due to LacZ misfolding and that, in the absence of the periplasmic disulfide bond catalyst DsbA, LacZ folds in the periplasm. Using the novel phenotype of periplasmic ß-galactosidase activity, we show that the periplasmic chaperone FkpA contributes to LacZ folding in this nonnative compartment. We propose that targeted, nonjamming LacZ fusions may be used to further study folding and targeting in the periplasm of Escherichia coli.

Disulfide bond formation in prokaryotes: history, diversity and design.

The formation of structural disulfide bonds is essential for the function and stability of a great number of proteins, particularly those that are secreted. There exists a variety of dedicated cellular catalysts and pathways from archaea to humans that ensure the formation of native disulfide bonds. In this review we describe the initial discoveries of these pathways and report progress in recent years in our understanding of the diversity of these pathways in prokaryotes, including those newly discovered in some archaea. We will also discuss the various successful efforts to achieve laboratory-based evolution and design of synthetic disulfide bond formation machineries in the bacterium Escherichia coli. These latter studies have also led to new more general insights into the redox environment of the cytoplasm and bacterial cell envelope. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.

Identification of YidC residues that define interactions with the Sec Apparatus.

In bacteria, a subset of membrane proteins insert into the membrane via the Sec apparatus with the assistance of the widely conserved essential membrane protein insertase YidC. After threading into the SecYEG translocon, transmembrane segments of nascent proteins are thought to exit the translocon via a lateral gate in SecY, where YidC facilitates their transfer into the lipid bilayer. Interactions between YidC and components of the Sec apparatus are critical to its function. The first periplasmic loop of YidC interacts directly with SecF. We sought to identify the regions or residues of YidC that interact with SecY or with additional components of the Sec apparatus other than SecDF. Using a synthetic lethal screen, we identified residues of YidC that, when mutated, led to dependence on SecDF for viability. Each residue identified is highly conserved among YidC homologs; most lie within transmembrane domains. Overexpression of SecY in the presence of two YidC mutants partially rescued viability in the absence of SecDF, suggesting that the corresponding wild-type YidC residues (G355 and M471) participate in interactions, direct or indirect, with SecY. Staphylococcus aureus YidC complemented depletion of YidC, but not of SecDF, in Escherichia coli. G355 of E. coli YidC is invariant in S. aureus YidC, suggesting that this highly conserved glycine serves a conserved function in interactions with SecY. This study demonstrates that transmembrane residues are critical in YidC interactions with the Sec apparatus and provides guidance on YidC residues of interest for future structure-function analyses.

A cross-sectional exploratory survey of knowledge, attitudes and daily self-reported pain assessment practice among nurses in Mainland China.

AIMS AND OBJECTIVES: To describe the level of knowledge, attitudes, and self-reported quality of practice in pain assessment among nurses of Mainland China and explore links with current hospital pain policy and continuing education. BACKGROUND: Knowledge is necessary for skilled pain assessment among nurses. Little is currently known regarding knowledge, attitude toward, and self reported pain assessment by nurses from Mainland China. METHODS: Quantitative research and cross-sectional convenience sampling assessed nursing knowledge, attitude, and practice among 101 nurses working in high-level hospitals in Mainland China. RESULTS: 81.2% of nurses participating in the survey were from high-level (level three) hospitals in Mainland China. 24.8% of the nurses attended continuing education in pain assessment. No nurses from the 76 hospital staffs surveyed were able to recall any hospital policy regarding pain assessment. Knowledge regarding pain assessment was rated at 1.9 (SD = 1.6) on a (0-7) scale. 27.7% of nurses possessed a positive attitude toward pain assessment. Pain assessment was not routine in most of the hospitals surveyed. Nurses who attended continuing education showed greater knowledge and more positive attitudes regarding pain assessment but did not show improvement in their quality of practice. CONCLUSIONS: This study identified inadequate knowledge and low level of self-reported pain assessment practice among nurses working in high-level hospitals in Mainland China. Current education did not influence nursing self-reported pain assessment practice. Knowledge of pain evaluation should be improved through newer approaches to education. A better policy framework for pain evaluation may also contribute to improvement.

Aggravating genetic interactions allow a solution to redundancy in a bacterial pathogen.

Interactions between hosts and pathogens are complex, so understanding the events that govern these interactions requires the analysis of molecular mechanisms operating in both organisms. Many pathogens use multiple strategies to target a single event in the disease process, confounding the identification of the important determinants of virulence. We developed a genetic screening strategy called insertional mutagenesis and depletion (iMAD) that combines bacterial mutagenesis and RNA interference, to systematically dissect the interplay between a pathogen and its host. We used this technique to resolve the network of proteins secreted by the bacterium Legionella pneumophila to promote intracellular growth, a critical determinant of pathogenicity of this organism. This strategy is broadly applicable, allowing the dissection of any interface between two organisms involving numerous interactions.

Disulfide rearrangement triggered by translocon assembly controls lipopolysaccharide export.

The presence of lipopolysaccharide (LPS) on the cell surface of Gram-negative bacteria is critical for viability. A conserved ß-barrel membrane protein LptD (lipopolysaccharide transport protein D) translocates LPS from the periplasm across the outer membrane (OM). In Escherichia coli, this protein contains two disulfide bonds and forms the OM LPS translocon with the lipoprotein LptE. Here, we identified seven in vivo states on the oxidative-folding pathway of LptD. Proper assembly involved a nonfunctional intermediate containing non-native disulfides. Intermediate formation required the oxidase DsbA, and subsequent maturation to the active form with native disulfides was triggered by LptE. Thus, disulfide bond-dependent protein folding of LptD requires the proper assembly of a two-protein complex to promote disulfide bond rearrangement.