Cell-free expression of natively folded hydrophobins.

Hydrophobins are a family of cysteine-rich proteins unique to filamentous fungi. The proteins are produced in a soluble form but self-assemble into organised amphipathic layers at hydrophilic:hydrophobic interfaces. These layers contribute to transitions between wet and dry environments, spore dispersal and attachment to surfaces for growth and infection. Hydrophobins are characterised by four disulphide bonds that are critical to their structure and function. Thus, obtaining correctly folded, soluble and functional hydrophobins directly from bacterial recombinant expression is challenging and in most cases, initial denaturation from inclusion bodies followed by oxidative refolding are required to obtain folded proteins. Here, we report the use of cell-free expression with E. coli cell lysate to directly obtain natively folded hydrophobins. All six of the hydrophobins tested could be expressed after optimisation of redox conditions. For some hydrophobins, the inclusion of the disulfide isomerase DsbC further enhanced expression levels. We are able to achieve a yield of up to 1 mg of natively folded hydrophobin per mL of reaction. This has allowed the confirmation of the correct folding of hydrophobins with the use of 15N-cysteine and 15N-1H nuclear magnetic resonance experiments within 24 h of starting from plasmid stocks.

Pharmacogenetics of irinotecan, doxorubicin and docetaxel transporters in Asian and Caucasian cancer patients: a comparative review.

Drug efflux and influx transporters play critical roles in regulating the cellular drug disposition and modulating the pharmacokinetics and pharmacodynamics of anti-cancer agents, which may potentially alter treatment outcomes. The efficiency of drug transport is often dependent on the expression and activity of these membrane-bound proteins, factors which have been shown to be regulated by genes that are known to be highly polymorphic in different ethnic populations. The role of drug transporters becomes even more critical for anti-cancer agents due to the narrow therapeutic windows that separate treatment response and toxicities for these agents. Moreover, high inter-individual variability in the disposition of anti-cancer agents often results in variable treatment outcomes among patients receiving standard doses of the same drug. Such variability has been attributed at least in part to polymorphisms in genes encoding drug-metabolizing enzymes and transporter. To date, numerous pharmacogenetic studies have investigated the associations between variants in the ABC and SLC transporters genes with drug disposition, treatment outcomes and drug-induced toxicities. However, the strengths of these associations and their clinical relevance in different ethnic populations have not been critically examined. This review aims to summarize and evaluate the implications of pharmacogenetic variants in the ABC and SLC transporters genes on the pharmacokinetics and clinical outcomes of three anti-cancer agents: irinotecan, docetaxel and doxorubicin in Caucasian and Asian patients.

Bistable expression of CsgD in Salmonella enterica serovar Typhimurium connects virulence to persistence.

Pathogenic bacteria often need to survive in the host and the environment, and it is not well understood how cells transition between these equally challenging situations. For the human and animal pathogen Salmonella enterica serovar Typhimurium, biofilm formation is correlated with persistence outside a host, but the connection to virulence is unknown. In this study, we analyzed multicellular-aggregate and planktonic-cell subpopulations that coexist when S. Typhimurium is grown under biofilm-inducing conditions. These cell types arise due to bistable expression of CsgD, the central biofilm regulator. Despite being exposed to the same stresses, the two cell subpopulations had 1,856 genes that were differentially expressed, as determined by transcriptome sequencing (RNA-seq). Aggregated cells displayed the characteristic gene expression of biofilms, whereas planktonic cells had enhanced expression of numerous virulence genes. Increased type three secretion synthesis in planktonic cells correlated with enhanced invasion of a human intestinal cell line and significantly increased virulence in mice compared to the aggregates. However, when the same groups of cells were exposed to desiccation, the aggregates survived better, and the competitive advantage of planktonic cells was lost. We hypothesize that CsgD-based differentiation is a form of bet hedging, with single cells primed for host cell invasion and aggregated cells adapted for persistence in the environment. This allows S. Typhimurium to spread the risks of transmission and ensures a smooth transition between the host and the environment.

Expanded Newborn Screening for Inborn Errors of Metabolism in Hong Kong: Results and Outcome of a 7 Year Journey.

Newborn screening (NBS) is an important public health program that aims to identify pre-symptomatic healthy babies that will develop significant disease if left undiagnosed and untreated. The number of conditions being screened globally is expanding rapidly in parallel with advances in technology, diagnosis, and treatment availability for these conditions. In Hong Kong, NBS for inborn errors of metabolism (NBSIEM) began as a pilot program in October 2015 and was implemented to all birthing hospitals within the public healthcare system in phases, with completion in October 2020. The number of conditions screened for increased from 21 to 24 in April 2016 and then to 26 in October 2019. The overall recruitment rate of the NBS program was 99.5%. In the period between October 2015 and December 2022, 125,688 newborns were screened and 295 were referred back for abnormal results. The recall rate was reduced from 0.26% to 0.12% after the implementation of second-tier testing. An inherited metabolic disorder (IMD) was eventually confirmed in 47 infants, making the prevalence of IMD in Hong Kong 1 in 2674. At the time of the NBS result, 78.7% of the newborns with IMD were asymptomatic. There were two deaths reported: one newborn with methylmalonic acidemia cobalamin B type (MMACblB) died after the initial crisis and another case of carnitine palmitoyltransferase II deficiency (CPTII) died at 18 months of age after metabolic decompensation. The most common IMD noted were disorders of fatty acid oxidation metabolism (40%, 19 cases), closely followed by disorders of amino acid metabolism (38%, 18 cases), with carnitine uptake defect (19.1%, 9 cases) and citrullinemia type II (17%, 8 cases) being the two most common IMD picked up by the NBSIEM in Hong Kong. Out of the all the IMDs identified, 19.1% belonged to diverse ethnic groups. False negative cases were reported for citrullinemia type II and congenital adrenal hyperplasia during this period.

Sex Differences in Association Between Gut Microbiome and Essential Hypertension Based on Ambulatory Blood Pressure Monitoring.

BACKGROUND: Sex differences in the pathogenesis of hypertension exist. While gut microbiota (GM) has been associated with hypertension, it is unclear whether there are sex-linked differences in the association between GM and hypertension. METHODS: We conducted a cross-sectional study to investigate the sex differences in associations between GM characterized by shotgun sequencing, GM-derived short-chain fatty acids, and 24-hour ambulatory blood pressure in 241 Hong Kong Chinese (113 men and 128 women; mean age, 54±6 years). RESULTS: The hypertensive group was associated with GM alterations; however, significant differences in ß-diversity and GM composition in hypertensive versus normotensive groups were only observed in women and not in men under various statistical models adjusting for the following covariates: age, sex, body mass index, sodium intake estimated by spot urine analysis, blood glucose, triglycerides, low- and high-density lipoprotein cholesterol, smoking, menopause, and fatty liver status. Specifically, Ruminococcus gnavus, Clostridium bolteae, and Bacteroides ovatus were significantly more abundant in the hypertensive women, whereas Dorea formicigenerans was more abundant in the normotensive women. No bacterial species were found to be significantly associated with hypertension in men. Furthermore, total plasma short-chain fatty acids and propionic acid were independent predictors of systolic and diastolic blood pressure in women but not men. CONCLUSIONS: GM dysregulation was strongly associated with 24-hour ambulatory blood pressure in women but not men, which may be mediated through propionic acid. Our work suggests that sex differences may be an important consideration while assessing the role of GM in the development and treatment of hypertension.

A GMMA-CPS-Based Vaccine for Non-Typhoidal Salmonella.

Non-typhoidal Salmonella are a major cause of gastroenteritis worldwide, as well as causing bloodstream infections in sub-Saharan Africa with a high fatality rate. No vaccine is currently available for human use. Current vaccine development strategies are focused on capsular polysaccharides (CPS) present on the surface of non-typhoidal Salmonella. This study aimed to boost the amount of CPS purified from S. Typhimurium for immunization trials. Random mutagenesis with Tn10 transposon increased the production of CPS colanic acid, by 10-fold compared to wildtype. Immunization with colanic acid or colanic acid conjugated to truncated glycoprotein D or inactivated diphtheria toxin did not induce a protective immune response in mice. However, immunization with Generalized Modules for Membrane Antigens (GMMAs) isolated from colanic acid overproducing isolates reduced Salmonella colonization in mice. Our results support the development of a GMMA-CPS-based vaccine against non-typhoidal Salmonella.

Propagation of Chikungunya Virus Using Mosquito Cells.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that transmits in between a mosquito host vector to a primate host and then back to the mosquito host vector to complete its life cycle. Hence, CHIKV must be able to replicate in both host cellular systems that are genetically and biochemically distinct. The ability to grow and propagate the virus in high titers in the laboratory is fundamentally crucial in order to understand virus replication in different host cellular systems and many other CHIKV research areas. Here, we describe a method on CHIKV propagation using C6/36, a mosquito cell line derived from Aedes albopictus in both serum-containing and serum-free media.

A potent neutralizing IgM mAb targeting the N218 epitope on E2 protein protects against Chikungunya virus pathogenesis.

Chikungunya virus (CHIKV) is a medically important human viral pathogen that causes Chikungunya fever accompanied with debilitating and persistent joint pain. Host-elicited or passively-transferred monoclonal antibodies (mAb) are essential mediators of CHIKV clearance. Therefore, this study aimed to generate and characterize a panel of mAbs for their neutralization efficacy against CHIKV infection in a cell-based and murine model. To evaluate their antigenicity and neutralization profile, indirect enzyme-linked immunosorbent assay (ELISA), an immunofluorescence assay (IFA) and a plaque reduction neutralization test were performed on mAbs of IgM isotype. CHIKV escape mutants against mAb 3E7b neutralization were generated, and reverse genetics techniques were then used to create an infectious CHIKV clone with a single mutation. 3E7b was also administered to neonate mice prior or after CHIKV infection. The survival rate, CHIKV burden in tissues and histopathology of the limb muscles were evaluated. Both IgM 3E7b and 8A2c bind strongly to native CHIKV surface and potently neutralize CHIKV replication. Further analyses of 3E7b binding and neutralization of CHIKV single-mutant clones revealed that N218 of CHIKV E2 protein is a potent neutralizing epitope. In a pre-binding neutralization assay, 3E7b blocks CHIKV attachment to permissive cells, possibly by binding to the surface-accessible E2-N218 residue. Prophylactic administration of 3E7b to neonate mice markedly reduced viremia and protected against CHIKV pathogenesis in various mice tissues. Given therapeutically at 4 h post-infection, 3E7b conferred 100% survival rate and similarly reduced CHIKV load in most mice tissues except the limb muscles. Collectively, these findings highlight the usefulness of 3E7b for future prophylactic or epitope-based vaccine design.

Antiviral Phosphorodiamidate Morpholino Oligomers are Protective against Chikungunya Virus Infection on Cell-based and Murine Models.

Chikungunya virus (CHIKV) infection in human is associated with debilitating and persistent arthralgia and arthritis. Currently, there is no specific vaccine or effective antiviral available. Anti-CHIKV Phosphorodiamidate Morpholino Oligomer (CPMO) was evaluated for its antiviral efficacy and cytotoxcity in human cells and neonate murine model. Two CPMOs were designed to block translation initiation of a highly conserved sequence in CHIKV non-structural and structural polyprotein, respectively. Pre-treatment of HeLa cells with CPMO1 significantly suppressed CHIKV titre, CHIKV E2 protein expression and prevented CHIKV-induced CPE. CPMO1 activity was also CHIKV-specific as shown by the lack of cross-reactivity against SINV or DENV replication. When administered prophylactically in neonate mice, 15 µg/g CPMO1v conferred 100% survival against CHIKV disease. In parallel, these mice demonstrated significant reduction in viremia and viral load in various tissues. Immunohistological examination of skeletal muscles and liver of CPMO1v-treated mice also showed healthy tissue morphology, in contrast to evident manifestation of CHIKV pathogenesis in PBS- or scrambled sCPMO1v-treated groups. Taken together, our findings highlight for the first time that CPMO1v has strong protective effect against CHIKV infection. This warrants future development of morpholino as an alternative antiviral agent to address CHIKV infection in clinical applications.

Quality of life following lung cancer resection: video-assisted thoracic surgery vs thoracotomy.

STUDY OBJECTIVES: Quality of life (QOL) following video-assisted thoracic surgery (VATS) major lung resection has not been systematically studied. This study was designed to evaluate the intermediate to long-term QOL in patients with lung cancer following resection, comparing VATS with thoracotomy. DESIGN: Cross-sectional study, telephone survey. METHODS: Of 136 disease-free surviving patients with non-small cell lung cancer operated on between 1994 and 2000, 45 patients were excluded because of large tumors (> 5 cm) or locally advanced disease, and another 27 patients were excluded because of adjuvant therapy, coexisting cancer from another source, or psychiatric illness. At the time of the survey, 13 patients were found to be either unsuitable or unwilling to participate. This left a total of 51 patients, with 27 patients in the VATS group and 24 patients in the thoracotomy group (open group), for the final analysis. QOL was assessed using Chinese versions of the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire (QLQ)-C30 and the EORTC QLQ-LC13, supplemented with nine self-developed surgery-related questions. RESULTS: Mean follow-up time was 33.5 months in the VATS group (median, 20.8 months; range, 6.0 to 84.2 months) and 39.4 months in the open group (median, 37.7 months; range, 7.0 to 75.1 months). Both groups had good QOL and high levels of functioning despite a fairly high incidence of symptoms. There was a trend for VATS patients to score higher on the QOL and functioning scales and to report fewer symptoms. However, these differences did not lead to statistical significance. CONCLUSIONS: This study showed that lung cancer patients with resectable disease following surgical treatment without recurrence have good QOL and high levels of functioning on intermediate to long-term follow-up, with no significant differences between the VATS and open groups.

Early results of endoscopic lung volume reduction for emphysema.

BACKGROUND: We determined the feasibility, safety, and short-term efficacy of bronchoscopic placement of a one-way endobronchial valve in selected bronchopulmonary segments as an alternative to surgical lung volume reduction. METHODS: A total of 21 patients with incapacitating emphysema who underwent this procedure were studied. All patients had placement of the endobronchial valves into the most emphysematous lung segments. We recorded any major complications or deaths attributed to the procedure and analyzed (1) improvements in the spirometric and functional parameters and quality of life and (2) the radiologic changes compared with the baseline data at 30 and 90 days. RESULTS: A total of 20 patients had complete follow-up data. There was no mortality in the group studied. The forced expiratory volume at 1 second, forced expiratory volume at 1 second (percentage of predicted), forced vital capacity, and forced vital capacity (percentage of predicted) all improved significantly at 90 days (0.73 +/- 0.26 L vs 0.92 +/- 0.34 L [P =.009]; 33.3% +/- 11.9% vs 42.2% +/- 15.0% [P =.006]; 1.94 +/- 0.62 L vs 2.25 +/- 0.61 L [P =.015]; and 63.3% +/- 17.6% vs 73.9% +/- 17.1% [P =.012], respectively). The 6-minute walking distance improved at 30 and 90 days (251.6 +/- 100.2 m vs 306.3 +/- 112.3 m and 322.3 +/- 129.7 m; P =.012 and P =.003). The results of the 36-Item Short-Form Health Survey and the St George Respiratory Questionnaire showed significant improvements at 90 days. The Medical Research Council dyspnea grade also improved significantly at 30 and at 90 days (P =.006 and P =.003, respectively). CONCLUSIONS: Endobronchial valve placement is a safe procedure, with significant short-term improvements in functional status, quality of life, and relief of dyspnea in selected patients with emphysema. A larger study with long-term follow-up is therefore warranted.

Imagination perspective affects ratings of the likelihood of occurrence of autobiographical memories.

Two experiments tested and confirmed the hypothesis that when the phenomenological characteristics of imagined events are more similar to those of related autobiographical memories, the imagined event is more likely to be considered to have occurred. At Time 1 and 2-weeks later, individuals rated the likelihood of occurrence for 20 life events. In Experiment 1, 1-week after Time 1, individuals imagined 3 childhood events from a first-person or third-person perspective. There was a no-imagination control. An increase in likelihood ratings from Time 1 to Time 2 resulted when imagination was from the third-person but not first-person perspective. In Experiment 2, childhood and recent events were imagined from a third- or first-person perspective. A significant interaction resulted. For childhood events, likelihood change scores were greater for third-person than first-person perspective; for recent adult events, likelihood change scores were greater for first-person than third-person perspective, although this latter trend was not significant.

Going with the flow or swimming against the tide: should children with central venous catheters swim?

Children who require long-term parenteral nutrition (PN) have central venous catheters (CVCs) in place to allow the safe and effective infusion of life-sustaining fluids and nutrition. Many consider recreational swimming to be a common part of childhood, but for some, the risk may outweigh the benefit. Children with CVCs may be at increased risk of exit site, tunnel, and catheter-related bloodstream infections (CRBSIs) if these catheters are immersed in water. The purpose of this review is to evaluate the current literature regarding the risk of infection for patients with CVCs who swim and determine if there is consensus among home PN (HPN) programs on this controversial issue. A total 45 articles were reviewed and 16 pediatric HPN programs were surveyed regarding swimming and CVCs. Due to the limited data available, a firm recommendation cannot be made. Recreational water associated outbreaks are well documented in the general public, as is the presence of human pathogens even in chlorinated swimming pools. As a medical team, practitioners can provide information and education regarding the potential risk, but ultimately the decision lies with the parents. If the parents decide swimming is worth the risk, they are encouraged to use products designed for this use and to change their child's dressing immediately after swimming. Due to our experience with a fatal event immediately after swimming, we continue to strongly discourage patients with CVCs from swimming. Further large and well-designed studies regarding the risk of swimming with a CVC are needed to make a strong, evidence-based recommendation.

Expression of plasmid-based shRNA against the E1 and nsP1 genes effectively silenced Chikungunya virus replication.

BACKGROUND: Chikungunya virus (CHIKV) is a re-emerging alphavirus that causes chikungunya fever and persistent arthralgia in humans. Currently, there is no effective vaccine or antiviral against CHIKV infection. Therefore, this study evaluates whether RNA interference which targets at viral genomic level may be a novel antiviral strategy to inhibit the medically important CHIKV infection. METHODS: Plasmid-based small hairpin RNA (shRNA) was investigated for its efficacy in inhibiting CHIKV replication. Three shRNAs designed against CHIKV Capsid, E1 and nsP1 genes were transfected to establish stable shRNA-expressing cell clones. Following infection of stable shRNA cells clones with CHIKV at M.O.I. 1, viral plaque assay, Western blotting and transmission electron microscopy were performed. The in vivo efficacy of shRNA against CHIKV replication was also evaluated in a suckling murine model of CHIKV infection. RESULTS: Cell clones expressing shRNAs against CHIKV E1 and nsP1 genes displayed significant inhibition of infectious CHIKV production, while shRNA Capsid demonstrated a modest inhibitory effect as compared to scrambled shRNA cell clones and non-transfected cell controls. Western blot analysis of CHIKV E2 protein expression and transmission electron microscopy of shRNA E1 and nsP1 cell clones collectively demonstrated similar inhibitory trends against CHIKV replication. shRNA E1 showed non cell-type specific anti-CHIKV effects and broad-spectrum silencing against different geographical strains of CHIKV. Furthermore, shRNA E1 clones did not exert any inhibition against Dengue virus and Sindbis virus replication, thus indicating the high specificity of shRNA against CHIKV replication. Moreover, no shRNA-resistant CHIKV mutant was generated after 50 passages of CHIKV in the stable cell clones. More importantly, strong and sustained anti-CHIKV protection was conferred in suckling mice pre-treated with shRNA E1. CONCLUSION: Taken together, these data suggest the promising efficacy of anti-CHIKV shRNAs, in particular, plasmid-shRNA E1, as a novel antiviral strategy against CHIKV infection.

What research paradigms have cognitive psychologists used to study "false memory," and what are the implications of these choices?

This research examines the methodologies employed by cognitive psychologists to study "false memory," and assesses if these methodologies are likely to facilitate scientific progress or perhaps constrain the conclusions reached. A PsycINFO search of the empirical publications in cognitive psychology was conducted through January, 2004, using the subject heading, "false memory." The search produced 198 articles. Although there is an apparent false memory research bandwagon in cognitive psychology, with increasing numbers of studies published on this topic over the past decade, few researchers (only 13.1% of the articles) have studied false memory as the term was originally intended--to specifically refer to planting memory for an entirely new event that was never experienced in an individual's lifetime. Cognitive psychologists interested in conducting research relevant to assessing the authenticity of memories for child sexual abuse should consider the generalizability of their research to the planting of entirely new events in memory.

Efficacy of contact lens multipurpose solutions against serratia marcescens.

PURPOSE: To compare the susceptibilities of clinical isolates of Serratia marcescens and the standard ISO ATCC 13880 strain to five contact lens multipurpose disinfection solutions (MPDSs). METHODS: Five commercially available MPDSs, containing either a polymeric biguanide or polyquaternium, were tested using ISO/CD 14729 stand-alone test for contact lens care products against four ocular isolates of S. marcescens and the strain ATCC 13880. An average log reduction in bacterial numbers at the manufacturer's minimum recommended disinfection time was determined and compared with the criteria for stand-alone disinfection products for each MPDS against each bacterial strain. RESULTS: All the MPDSs tested met the stand-alone criteria of 3-log reduction of viable bacteria against the ATCC strain of S. marcescens. However, there was more variability in their ability to meet disinfection criteria when tested against the clinical isolates. Two of the clinical isolates were significantly more resistant to disinfection than was the recommended ISO strain (p < or = 0.034). Two of the polyquaternium-1-based disinfection solutions (solutions D and E, p < or = 0.005) were less effective overall than the other MPDSs against S. marcescens. CONCLUSIONS: The importance of strain selection for the testing of MPDSs is indicated, and the use of a single laboratory strain may be insufficient to provide assurance that the disinfection solution will be effective against clinical isolates. Furthermore, clinical isolates of S. marcescens may show increased resistance to disinfection with polyquaternium.

Is knowing believing? The role of event plausibility and background knowledge in planting false beliefs about the personal past.

False memories are more likely to be planted for plausible than for implausible events, but does just knowing about an implausible event make individuals more likely to think that the event happened to them? Two experiments assessed the independent contributions o f plausibility a nd background knowledge to planting false beliefs. In Experiment 1, subjects rated 20 childhood events as to the likelihood of each event having happened to them. The list included the implausible target event "received an enema," a critical target event of Pezdek, Finger, and Hodge (1997). Two weeks later, subjects were presented with (1) information regarding the high prevalence rate of enemas; (2) background information on how to administer an enema; (3) neither type of information; or (4) both. Immediately or 2 weeks later, they rated the 20 childhood events again. Only plausibility significantly increased occurrence ratings. In Experiment 2, the target event was changed from "barium enema administered in a hospital" to "home enema for constipation"; significant effects of both plausibility and background knowledge resulted. The results suggest that providing background knowledge can increase beliefs about personal events, but that its impact is limited by the extent of the individual's familiarity with the context of the suggested target event.