Association between awareness of vulnerability and disaster preparedness in an infrastructure-resilient city: a population-based study.

OBJECTIVES: Factors associated with an individual's awareness of vulnerability can be modified by the infrastructure of a city. These factors may impact disaster preparedness among local populations in an infrastructure-resilient city, which further influences the health risks of various population subgroups. STUDY DESIGN: This was a population-based study. METHODS: Four population subgroups, which have previously been reported to be related to awareness of vulnerability (i.e. past experiences, sociodemographic deprivation, poor housing conditions and family medical needs), were analysed for their impacts on disaster preparedness. Validated population-based phone interviews (n = 856) were conducted in Hong Kong. Three types of disaster preparedness were studied: (1) physical preparedness; (2) social preparedness; and (3) education preparedness. RESULTS: Previous experience of social hazards, accidental hazards and epidemics increased disaster preparedness among the local population. Specifically, experiences of accidental hazards and social hazards were positively associated with physical preparedness (odds ratios 1.626, 95% confidence interval [95% CI] 1.215, 2.172) and 1.501 [95% CI 1.114, 2.024], respectively). However, experiences of natural hazards did not increase preparedness, even in Hong Kong, which is a city with high 'disaster resilience' because of its well-developed infrastructure. Moreover, individuals with a low educational level or low income had lower education preparedness, unmarried individuals had lower social preparedness, and poor housing conditions of non-private-housing households had negative associations with education preparedness. These findings partially align with local disaster responses to the 2018 Typhoon Mangkhut, the 2019 social unrest and the 2020 COVID-19 pandemic, all of which were observed after the 2018 survey reported in this study. CONCLUSIONS: Social and environmental interventions should be targeted to marginalised subpopulations through location-based community strategies to encourage increased environmental knowledge and participation in disaster preparedness activities.

An update on ABCB1 pharmacogenetics: insights from a 3D model into the location and evolutionary conservation of residues corresponding to SNPs associated with drug pharmacokinetics.

The human ABCB1 protein, (P-glycoprotein or MDR1) is a membrane-bound glycoprotein that harnesses the energy of ATP hydrolysis to drive the unidirectional transport of substrates from the cytoplasm to the extracellular space. As a large range of therapeutic agents are known substrates of ABCB1 protein, its role in the onset of multidrug resistance has been the focus of much research. This role has been of particular interest in the field of pharmacogenomics where genetic variation within the ABCB1 gene, particularly in the form of single nucleotide polymorphisms (SNPs), is believed to contribute to inter-individual variation in ABCB1 function and drug response. In this review we provide an update on the influence of coding region SNPs within the ABCB1 gene on drug pharmacokinetics. By utilizing the crystal structure of the mouse ABCB1 homolog (Abcb1a), which is 87% homologous to the human sequence, we accompany this discussion with a graphical representation of residue location for amino acids corresponding to human ABCB1 coding region SNPs. Also, an assessment of residue conservation, which is calculated following multiple sequence alignment of 11 confirmed sequences of ABCB1 homologs, is presented and discussed. Superimposing a 'heat map' of residue homology to the Abcb1a crystal structure has permitted additional insights into both the conservation of individual residues and the conservation of their immediate surroundings. Such graphical representation of residue location and conservation supplements this update of ABCB1 pharmacogenetics to help clarify the often confounding reports on the influence of ABCB1 polymorphisms on drug pharmacokinetics and response.

Characterization of Plasmodium falciparum co-chaperone p23: its intrinsic chaperone activity and interaction with Hsp90.

It is well known that the co-chaperone p23 regulates Hsp90 chaperone activity in protein folding. In Plasmodium falciparum, a putative p23 (Pfp23) has been identified through genome analysis, but its authenticity has remained unconfirmed since co-immunoprecipitation experiments failed to show its interaction with P. falciparum Hsp90 (PfHsp90). Thus, recombinant Pfp23 and PfHsp90 proteins purified from expressed clones were used in this study. It was clear that Pfp23 exhibited chaperone activity by virtue of its ability to suppress citrate synthase aggregation at 45 degrees C. Pfp23 was also shown to interact with PfHsp90 and to suppress its ATPase activity. Analyses of modeled Pfp23-PfHsp90 protein complex and site-directed mutagenesis further revealed strategically placed amino acid residues, K91, H93, W94 and K96, in Pfp23 to be crucial for binding PfHsp90. Collectively, this study has provided experimental evidence for the inherent chaperone function of Pfp23 and its interaction with PfHsp90, a sequel widely required for client protein activation.

Plasmodium falciparum pyruvate kinase as a novel target for antimalarial drug-screening.

BACKGROUND: Global travellers are increasingly at risk of contracting malaria. The increasing occurrence of drug-resistance in many endemic areas emphasizes the need for novel drug targets for antimalarial-screening. In this study, the use of pyruvate kinase as a drug-target is evaluated. The functional validation of a gene encoding pyruvate kinase (designated PK1) has previously been reported. However, alternative copies of this enzyme encoded by Plasmodium falciparum could also circumvent the role of PK1. A survey of genome data revealed a putative ORF seemingly coding for another pyruvate kinase (designated PK2). METHODS: The expression of PK1 and PK2 in in vitro cultures were investigated by RT-PCR. Biocomputational analysis was carried out to identify structural differences between the P. falciparum pyruvate kinases and the corresponding enzymes from its human host. RESULTS: Both PK1 and PK2 were indeed actively transcribed during the intraerythrocytic stages, suggesting the involvement of both enzymes during infection. A comparison of amino acid residues at the effector binding sites of PK1 and PK2, to those of the human pyruvate kinases revealed some significant differences that could serve as targets for selective inhibitors to be designed against parasitic pyruvate kinases. CONCLUSION: Experimental evidence for the expression of both PK1 and PK2 during the blood stages of malaria infection was provided. Interestingly, phylogenetic analysis revealed that the "PK2" type of enzyme appears to be confined to Apicomplexans, an important observation with respect to the assessment of PK2 as a drug-target.

Evaluation of the first pharmacist-administered vaccinations in Western Australia: a mixed-methods study.

OBJECTIVES: This study evaluated the uptake of Western Australian (WA) pharmacist vaccination services, the profiles of consumers being vaccinated and the facilitators and challenges experienced by pharmacy staff in the preparation, implementation and delivery of services. DESIGN: Mixed-methods methodology with both quantitative and qualitative data through surveys, pharmacy computer records and immuniser pharmacist interviews. SETTING: Community pharmacies in WA that provided pharmacist vaccination services between March and October 2015. PARTICIPANTS: Immuniser pharmacists from 86 pharmacies completed baseline surveys and 78 completed exit surveys; computer records from 57 pharmacies; 25 immuniser pharmacists were interviewed. MAIN OUTCOME MEASURES: Pharmacy and immuniser pharmacist profiles; pharmacist vaccination services provided and consumer profiles who accessed services. RESULTS: 15 621 influenza vaccinations were administered by immuniser pharmacists at 76 WA community pharmacies between March and October 2015. There were no major adverse events, and <1% of consumers experienced minor events which were appropriately managed. Between 12% and 17% of consumers were eligible to receive free influenza vaccinations under the National Immunisation Program but chose to have it at a pharmacy. A high percentage of vaccinations was delivered in rural and regional areas indicating that provision of pharmacist vaccination services facilitated access for rural and remote consumers. Immuniser pharmacists reported feeling confident in providing vaccination services and were of the opinion that services should be expanded to other vaccinations. Pharmacists also reported significant professional satisfaction in providing the service. All participating pharmacies intended to continue providing influenza vaccinations in 2016. CONCLUSIONS: This initial evaluation of WA pharmacist vaccination services showed that vaccine delivery was safe. Convenience and accessibility were important aspects in usage of services. There is scope to expand pharmacist vaccination services to other vaccines and younger children; however, government funding to pharmacists needs to be considered.

ROS1 Kinase Inhibitors for Molecular-Targeted Therapies.

ROS1 is a pivotal transmembrane receptor protein tyrosine kinase which regulates several cellular processes like apoptosis, survival, differentiation, proliferation, cell migration, and transformation. There is increasing evidence supporting that ROS1 plays an important role in different malignancies including glioblastoma, colorectal cancer, gastric adenocarcinoma, inflammatory myofibroblastic tumor, ovarian cancer, angiosarcoma, and non small cell lung cancer; thus, ROS1 has become a potential drug discovery target. ROS1 shares about 49% sequence homology with ALK primary structure; therefore, wide range of ALK kinase inhibitors have shown in vitro inhibitory activity against ROS1 kinase. After Crizotinib approval by FDA for the management of ALK-rearranged lung cancer, ROS1-positive tumors have been focused. Although significant advancements have been achieved in understanding ROS1 function and its signaling pathways plus recent discovery of small molecules modulating ROS1 protein, a vital need of medicinal chemistry efforts is still required to produce selective and potent ROS1 inhibitors as an important therapeutic strategy for different human malignancies. This review focuses on the current knowledge about different scaffolds targeting ROS1 rearrangements, methods to synthesis, and some biological data about the most potent compounds that have delivered various scaffold structures.

Cloning, heterologous expression and purification of an isocitrate lyase from Streptomyces clavuligerus NRRL 3585.

The glyoxylate cycle comprising isocitrate lyase (ICL) and malate synthase (MS) is an anaplerotic pathway essential for growth on acetate as the sole carbon source. The aceB gene, which encodes malate synthase has been previously cloned from Streptomyces clavuligerus NRRL 3585 and characterized. In this study, the aceA gene, encoding ICL from S. clavuligerus NRRL 3585, was obtained via genome walking experiments and PCR. The fully sequenced open reading frame encodes 436 amino acids with a deduced M(r) of 47.5 kDa, consistent with the observed M(r) (49-67.5 kDa) of most ICL enzymes reported so far. The cloned aceA gene was expressed in Escherichia coli BL21(lambdaDE3) cells, from which ICL was purified as a His-tagged product and its functionality demonstrated. Furthermore, the relationship between the carbon sources, growth and ICL activity in S. clavuligerus were investigated. Rapid growth was observed when the cells were cultured on 0.5% (w/v) glycerol, while delayed growth was observed when cells were grown on 0.5% (w/v) acetate. However, in both cases, high levels of ICL activity coincided with a cessation of growth, suggesting a late physiological role played by ICL in the natural host, S. clavuligerus.

Otorhinolaryngeal foreign bodies in children presenting to the emergency department.

INTRODUCTION: Accidents with foreign bodies are common in the paediatric population. It is impossible to mandate that all foreign bodies (FB) in the ear, nose and throat (ENT) of children should be removed by the specialty-trained physicians. This study evaluates the management of ENT FB removal in children achieved by emergency physicians not trained in otolaryngology in an urban tertiary care paediatric emergency department. METHODS: A retrospective study was conducted on consecutive paediatric patients presenting with suspected foreign body in the ear, nose or throat to the children's emergency department (ED) of KK Women's and Children's Hospital over a 10-month period. Removal methods, foreign body types, rates of successful removal and associated complications were evaluated. RESULTS: There were 353 patients, most of whom presented after office hours. An attempt at removal of FB by the emergency physician was made in 76.8 percent of the cases. ENT specialist referral in the ED was made in 1.7 percent of the cases. 50.1 percent of cases were discharged after successful removal of FB in the ED. 4.2 percent of cases were admitted for removal of FB and 44.8 percent of cases were referred to the ENT specialist clinic for further assessment. CONCLUSION: The emergency physician managed most cases in the ED and urgent referral to ENT specialists was not required. Complications and morbidity often occur from repeated attempts at removal of the FB. ENT opinion should be sought whenever there is doubt. The ED physician should be skilled in techniques of FB removal, especially throat FB, which had the lowest rate of success in our study.

Development of a new technique for recovery of cytokines from inflammatory sites in situ.

We attempted to recover cytokines from nasal mucosal surface following allergen challenge. Repeated lavage of nasal mucosa of seven allergic patients was done, but we failed to detect IL-1 beta in the lavage samples even in ten-fold concentrated materials. Therefore, we developed a new technique to recover cytokines using filter strips. Small filter strips were placed on nasal turbinates for 10 min at different time points after allergen challenge. The strips were air-dried, and stored. For recovery of cytokines individual strips were washed with small volumes of Hepes buffer containing 0.3% human serum albumin. Eluates were assayed for the presence of IL-1 beta and GM-CSF using commercially available ELISA. We were able to detect IL-1 beta and GM-CSF in eluates. Both cytokines were consistently detectable in the late phase allergic reaction peaking at 5 h. Nasal challenge with saline failed to detect any cytokine during the 7 h observation period. In standardization experiments known quantities of IL-1 beta and GM-CSF were applied to filter strips and the recovery ranged from 67 to 89%. Thus, we developed a simple technique of recovery of cytokines from inflammatory mucosa in situ.

Hereditary angioedema: its diagnostic and management perspectives.

Although hereditary angioedema accounts for only a small fraction of all cases of angioedema, it is the most common genetically linked clinical disorder caused by the deficiency of a protein associated with complement activation. Attacks may be complicated by incapacitating cutaneous swelling, life-threatening upper airway impediment, and severe gastrointestinal colic. Recent physicochemical and genetic studies have contributed significantly to our understanding of the structure of the inhibitor protein. Measurement of serum C4 titer is an efficacious screening test. Normal levels during symptomatic periods rule out the diagnosis, whereas decreased levels warrant determination of C1 esterase inhibitor titer by immunoassay or functional assay. The functional assay is necessary to ascertain the genetic variant form. The importance of making the correct diagnosis cannot be overemphasized. It can avert potentially fatal consequences, such as upper airway obstruction and unnecessary abdominal surgery. The application of short-term preventive measures can avoid complications associated with trauma. Finally, abatement or elimination of symptoms in patients with incessant and disabling attacks can be attained by long-term therapy with currently available attenuated androgens.

Mutational analysis of tyrosine-191 in the catalysis of Cephalosporium acremonium isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is a key enzyme responsible for the catalytic conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N in the beta-lactam antibiotic biosynthetic pathway. The Aspergillus nidulans IPNS crystal structure implicated amino acid residues tyrosine-189, arginine-279, and serine-281 in the substrate-binding of the valine carboxylate portion of ACV via hydrogen bonds. In previous reports, we provided mutational evidence for the critical involvement of the corresponding arginine-281 and serine-283, which constitute a conserved R-X-S motif, for the catalysis of Cephalosporium acremonium IPNS (cIPNS). In this study, we report the site-directed mutagenesis of the corresponding tyrosine-191 in cIPNS to four amino acids from different amino acid groups, namely, phenylalanine, serine, histidine, and aspartate. The mutants Y191F, Y191H, and Y191R respectively yielded specific activities at levels of 3, 8.6, and 18.8% relative to the wild-type when enzyme bioassays were performed using purified protein fractions. These results were surprising, as previous mutational analyses involving arginine-281 and serine-283 resulted in non-measurable specific activities, thus suggesting that tyrosine-191 is important but not critical for the activity of cIPNS due to its involvement in ACV binding. Hence, it is likely that tyrosine-191 is the least critical of the three residues involved in binding the ACV valine carboxylate moiety.

Site-directed mutagenesis of arginine-89 supports the role of its guanidino side-chain in substrate binding by Cephalosporium acremonium isopenicillin N synthase.

Isopenicillin N synthase (IPNS) catalyses a key step in the penicillin and cephalosporin biosynthetic pathway which involves the oxidative cyclisation of the acyclic peptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N. Based on crystallographic evidence from the Aspergillus nidulans IPNS crystal structure complexed with the substrate ACV (Roach et al. (1997) Nature 387, 827-830), we were able to provide mutational evidence for the critical involvement of the conserved R-X-S motif in ACV binding in IPNS. The crystal structure further implicated arginine-87 in the binding of the aminoadipyl portion of ACV. Thus, in this study, the site-directed mutagenesis of the corresponding arginine-89 in Cephalosporium acremonium IPNS (cIPNS) was performed to ascertain its role in cIPNS. Alteration of arginine-89 to five amino acids from different amino acid groups, namely lysine, serine, alanine, aspartate and leucine, was performed and no activity was detected in all the mutants obtained when enzyme bioassays were performed. Furthermore, the solubility of the mutants was considerably lower than the wild-type cIPNS after expression at 37 degrees C, but could be recovered when the expression temperature was lowered to 25 degrees C. This suggests that arginine-89 could be critical for the activity of cIPNS due to its involvement in ACV binding and the solubility of wild-type enzyme.

Mutational evidence for the role of serine-283 in Cephalosporium acremonium isopenicillin N synthase.

Creation of isopenicillin N from delta-(L-alpha-aminodipyl)-L-cysteinyl-D-valine (ACV) in the penicillin and cephalosporin biosynthetic pathway is catalysed by isopenicillin N synthase (IPNS), a non-heme iron-containing dioxygenase. A tripeptide R-X-S motif which consists of arginine-281 and serine-283 (Cephalosporium acremonium IPNS numbering) was found to be conserved in IPNS and other related proteins. These two amino acids mentioned were proposed to have a role in ACV substrate binding by the recent Aspergillus nidulans IPNS crystal structure. Using site-directed mutagenesis arginine-281 in C. acremonium IPNS (cIPNS) was earlier found to be essential for catalysis by our group. Similarly, serine-283 in cIPNS was also altered by site-directed mutagenesis to determine its role in cIPNS. No measurable activity was detected from the resultant mutant using enzyme bioassays. It is most likely that the eliminatin of the mutant's substrate-binding capability similar to that of arginine-281 lead to the abolishment of the catalytic reaction. This highlights the importance of the R-X-S motif in the functionality of cIPNS.

Glutamine-230 influences enzyme solubility but not catalysis in Streptomyces clavuligerus isopenicillin N synthase.

The conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N is dependent upon the catalytic action of isopenicillin N synthase (IPNS), an important enzyme in the penicillin and cephalosporin biosynthetic pathway. Recent catalytic investigations on the conserved glutamine-230 in the bacterial Streptomyces jumonjinensis IPNS and the corresponding glutamine-234 in the fungal Cephalosporium acremonium IPNS showed contrasting results whereby the former was suggested to be essential for IPNS activity whereas the latter was found not to be so. In order to unravel these conflicting results, we report the site-directed mutagenesis investigation on the corresponding glutamine-230 in a third IPNS isozyme, which is the bacterial Streptomyces clavuligerus IPNS (scIPNS). IPNS enzymatic assays showed that catalytic activity of the mutant Q230L scIPNS was reduced but not eliminated. Moreover, the solubility of the mutant enzyme was also markedly reduced. Hence, we can conclude that glutamine-230 in scIPNS is not essential for catalysis and correspondingly in all IPNS.

Functional analysis of a conserved aspartate D218 in Cephalosporium acremonium isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is instrumental in the catalytic conversion of a tripeptide precursor delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to a bioactive intermediate isopenicillin N in the beta-lactam antibiotic biosynthetic pathway. It has recently been shown that this reaction is dependent on a conserved aspartate, D214, in a bacterial Streptomyces jumonjinensis IPNS. Thus, this study was carried out to provide the experimental evidence for the involvement of a similarly conserved aspartate residue, D218, in a fungal Cephalosporium acremonium IPNS (cIPNS). Initially, alteration of the aspartate residue to generate the mutant D218L cIPNS protein was achieved by site-directed mutagenesis. Subsequent enzyme assays indicated that the catalytic property of the mutant protein was lost, attesting to the need for the corresponding conserved aspartate to maintain IPNS functionality. It is also evident from the observed results that site-directed mutagenesis of this particular aspartate residue in cIPNS can affect its solubility. It is therefore important to take these potential changes into consideration when site-directed mutant proteins are analysed for catalytic function.

Role of leukotriene B4 and interleukin-8 in acute bacterial and viral otitis media.

In order to evaluate the role of polymorphonuclear leukocytes (PMNs) in acute otitis media (AOM), levels of leukotriene B4 (LTB4), a potent inflammatory product of PMNs, and interleukin-8 (IL-8), a PMN chemotactic cytokine, were measured in 271 middle ear fluid (MEF) samples from 106 children with AOM. Forty-two percent of the patients had evidence of respiratory viral infection. At the time of diagnosis, levels of both LTB4 and IL-8 were higher in the MEFs from patients with AOM associated with bacterial or bacterial and viral infection than those MEFs containing no pathogen (p < .05). Antibiotic treatment was not associated with a significant change in levels of LTB4 or IL-8 in the MEFs obtained 2 to 5 days into treatment, compared to those obtained at diagnosis. Bacteriologic failure after 2 to 5 days of treatment was associated with high LTB4 levels in the initial MEFs (p = .05). Recurrence of AOM within 1 month was associated with high IL-8 levels in the initial MEF (p = .04). Our findings suggest that LTB4 and IL-8 are produced during acute infection of the middle ear, and these PMN-related inflammatory substances may play an important role in delaying recovery or in recurrence of AOM. Effective treatment of AOM may require eradication of bacteria by antibiotics, as well as pharmacologic agents that modulate PMN functions.

Survival of Tilletia indica Teliospores in Different Soils.

To determine the potential for Tilletia indica, cause of Karnal bunt of wheat, to survive and become established in new areas, a teliospore longevity study was initiated in Kansas, Maryland, Georgia, and Arizona. Soil from each location was infested with T. indica teliospores and placed in polyester mesh bags. The bags were placed within soil from the same location within polyvinyl chloride pipes. Pipes were buried in the respective plots such that the bags were at 5-, 10-, and 25-cm depths. Each pipe was open at the ends to allow interaction with the outside environment, however fitted with screens preventing possibility of teliospore escape. In the Karnal bunt-quarantine area of Arizona, bags of infested soil also were placed outside the pipes. Teliospore-infested soil from each location was maintained dry in a laboratory. During the first 2 years, viability declined more rapidly in pipes than outside pipes, and more rapidly in fields in Kansas and Maryland than in Georgia or Arizona. After 2 years, viability declined nearly equally. In the laboratory over 3 years, viability decreased significantly more rapidly in dry soil from Kansas or Maryland than in dry soil from Georgia or Arizona, while pure teliospores remained unchanged. We hypothesized that soils, irrespective of weather, affect teliospore longevity.

Site-directed mutagenesis of proline-285 to leucine in Cephalosporium acremonium isopenicillin-N-synthase affects catalysis and increases soluble expression at higher temperatures.

The conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N is dependant on the catalytic action of isopenicillin N-synthase (IPNS), an important enzyme in the penicillin and cephalosporin biosynthetic pathway. One of the amino acid residues suggested by the Aspergillus nidulans IPNS crystal structure for interaction with the valine isopropyl group of ACV is proline-283. Site-directed mutagenesis of the corresponding proline-285 to leucine in Cephalosporium acremonium IPNS resulted in non-measurable activity but an increased soluble expression at higher temperatures in a heterologous E. coli host.

Replacement of tyrosine-197 and the corresponding tyrosine-195 to isoleucine in Cephalosporium acremonium and Streptomyces clavuligerus isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is one of the key enzymes in the penicillin and cephalosporin biosynthetic pathway which catalyses the conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. The IPNS from Penicillium chrysogenum 23X-80-269-37-2, a high penicillin V-producer, was found to possess an isoleucine residue instead of tyrosine at position 195. An attempt to increase the specific activity of IPNS from Cephalosporium acremonium and Streptomyces clavuligerus was undertaken by altering the corresponding tyrosine residue to an isoleucine at the corresponding location. Unfortunately, no apparent increase in specific activity was encountered when the purified mutant enzymes were analysed and thus, this amino acid difference is likely not responsible for high specific activity in IPNS.

A comparison of three site-directed mutagenesis kits.

In this comparative study, three different mutagenesis kits, namely the MutaGene phagemid in vitro mutagenesis kit (Bio-Rad), the Transformerä Site-Directed mutagenesis kit (Clontech) and the Quik-change site-directed mutagenesis kit (Stratagene) were used for the mutagenesis of IPNS genes. However, a large difference in mutation efficiencies among these kits was encountered. Furthermore, these kits employ different strategies with its own individual strengths and weaknesses. Thus, a comparison among these three kits to evaluate their usefulness and improvements on the strategy adopted by the Quik-change site-directed mutagenesis kit, which was the kit of choice for our work, are presented for the benefit of research work.