Translation, cross-cultural adaptation, and validation of the Duke Activity Status Index (DASI) to Sinhala language.

BACKGROUND: Duke Activity Status Index (DASI) is a widely used tool to assess functional capacity among patients, but there is no Sinhala version validated for patients in Sri Lanka. This study aimed to cross-culturally adapt and test the validity and reliability of the Sinhala version of DASI (DASI-S). METHODS: The translation and cross-cultural adaptation of the DASI questionnaire were conducted following the standard guidelines. It was pre-tested on ten pre-operative patients and further modified. The construct validity and reliability of DASI-S were evaluated by administering the modified final DASI-S, which comprised 12 items, along with the physical functioning sub-scale of the 36-item short-form health survey (SF-36), consisting of 10 items to eighty-one patients who were awaiting non-cardiac surgeries at university surgical wards, National Hospital of Sri Lanka (NHSL), and Colombo North Teaching Hospital (CNTH), Sri Lanka. Reliability was assessed through Cronbach alpha, while the validity was evaluated using factor analysis and Spearman's correlation. The ethical approval was obtained from the Ethics Review Committee, Faculty of Medicine, University of Colombo, Sri Lanka. RESULTS: The mean age of the participants was 46.2 (± 16.6) years and the majority were females (54.3%). The mean height, weight, and body mass index of the sample were 160.5 (± 9.6) cm, 60.3 (± 11.9) kg, and 23.4 (± 4.5) kgm-2 respectively. The Cronbach's alpha coefficient for the internal consistency of DASI-S was 0.861. The concurrent validity of DASI-S was substantiated by positively correlating (p < 0.01, rs = 0.466) with the physical sub-scale of SF-36. There was a significant difference (p < 0.01) in the total score of DASI-S between the two age groups. CONCLUSIONS: Sinhala version of the DASI appears to be a valid, reliable and easy-to-administer tool to assess functional capacity among patients who are awaiting non-cardiac surgeries.

Mucosal IL-4R antagonist HIV vaccination with SOSIP-gp140 booster can induce high-quality cytotoxic CD4+/CD8+ T cells and humoral responses in macaques.

Inducing humoral, cellular and mucosal immunity is likely to improve the effectiveness of HIV-1 vaccine strategies. Here, we tested a vaccine regimen in pigtail macaques using an intranasal (i.n.) recombinant Fowl Pox Virus (FPV)-gag pol env-IL-4R antagonist prime, intramuscular (i.m.) recombinant Modified Vaccinia Ankara Virus (MVA)-gag pol-IL-4R antagonist boost followed by an i.m SOSIP-gp140 boost. The viral vector-expressed IL-4R antagonist transiently inhibited IL-4/IL-13 signalling at the vaccination site. The SOSIP booster not only induced gp140-specific IgG, ADCC (antibody-dependent cellular cytotoxicity) and some neutralisation activity, but also bolstered the HIV-specific cellular and humoral responses. Specifically, superior sustained systemic and mucosal HIV Gag-specific poly-functional/cytotoxic CD4+ and CD8+ T cells were detected with the IL-4R antagonist adjuvanted strategy compared to the unadjuvanted control. In the systemic compartment elevated Granzyme K expression was linked to CD4+ T cells, whilst Granzyme B/TIA-1 to CD8+ T cells. In contrast, the cytotoxic marker expression by mucosal CD4+ and CD8+ T cells differed according to the mucosal compartment. This vector-based mucosal IL-4R antagonist/SOSIP booster strategy, which promotes cytotoxic mucosal CD4+ T cells at the first line of defence, and cytotoxic CD4+ and CD8+ T cells plus functional antibodies in the blood, may prove valuable in combating mucosal infection with HIV-1 and warrants further investigation.

A hierarchical role of IL-25 in ILC development and function at the lung mucosae following viral-vector vaccination.

This study demonstrates that modulation of IL-25 and IL-33 cytokines responsible for innate lymphoid cell 2 (ILC2) activation/function can differentially regulate ILC profiles at the vaccination site, in a vaccine route-dependent manner. Specifically, recombinant fowlpox (rFPV) vector-based vaccine co-expressing an adjuvant that transiently sequestered IL-25 (FPV-HIV-IL-25 binding protein), delivered intramuscularly (i.m.) was able to induce significantly lower IL-25R+ ILC2-deived IL-13 and ILC1/ILC3-derived IFN-γ expression with significantly elevated IL-17A in muscle. In contrast, intranasal (i.n.) delivery was able to induce all three known ILC2 subsets (ST2/IL-33R+, IL-25R+, and TSLPR+ ILC2) to express varying amounts of IL-13 in lung, and also the TSLPR+ ILC2 to express IL-4, unlike the unadjuvanted control, which only showed ST2/IL-33R+ ILC2 to express IL-13. Interestingly, the sequestration of IL-25 in lung also induced a unique lineage- ST2/IL-33R- IL-25R- TSLPR- ILC2 population to express elevated IL-13 and IL-4. Moreover, both i.m. and, i.n. FPV-HIV-IL-25BP vaccination induced significantly elevated ILC1/ILC3-derived IL-17A in lung, indicating that ILC2 could directly impact ILC1/ILC3 activity. To our surprise, transient sequestration of IL-33 at the lung mucosae did not alter the lung ILC2 profiles or activity. These inhibitor studies showed that in the context of i.n. viral vector vaccination, IL-25 plays a predominant role in early ILC development/regulation than IL-33, and likely acts as a master regulator of ILC. Our previous findings have indicated that level of IL-4/IL-13 at the vaccination site impacts the quality/avidity of T cell immunity. Taken together data suggest that IL-25 binding protein could be used as an effective i.m. not an i.n. adjuvant to enhance quality of vaccine-specific T cell immunity. These findings evoke the notion that route-dependent manipulation of ILCs according to the target pathogen could be exploited to design more effective vaccines against chronic pathogens in the future.

Viral vector and route of administration determine the ILC and DC profiles responsible for downstream vaccine-specific immune outcomes.

This study demonstrates that route and viral vector can significantly influence the innate lymphoid cells (ILC) and dendritic cells (DC) recruited to the vaccination site, 24 h post delivery. Intranasal (i.n.) vaccination induced ST2/IL-33R+ ILC2, whilst intramuscular (i.m.) induced IL-25R+ and TSLPR+ (Thymic stromal lymphopoietin protein receptor) ILC2 subsets. However, in muscle a novel ILC subset devoid of the known ILC2 markers (IL-25R- IL-33R- TSLPR-) were found to express IL-13, unlike in lung. Different viral vectors also influenced the ILC-derived cytokines and the DC profiles at the respective vaccination sites. Both i.n. and i.m. recombinant fowlpox virus (rFPV) priming, which has been associated with induction of high avidity T cells and effective antibody differentiation exhibited low ILC2-derived IL-13, high NKp46+ ILC1/ILC3 derived IFN-γ and low IL-17A, together with enhanced CD11b+ CD103- conventional DCs (cDC). In contrast, recombinant Modified Vaccinia Ankara (rMVA) and Influenza A vector priming, which has been linked to low avidity T cells, induced opposing ILC derived-cytokine profiles and enhanced cross-presenting DCs. These observations suggested that the former ILC/DC profiles could be a predictor of a balanced cellular and humoral immune outcome. In addition, following i.n. delivery Rhinovirus (RV) and Adenovius type 5 (Ad5) vectors that induced elevated ILC2-derived IL-13, NKp46+ ILC1/ILC3-derived-IFN-γ and no IL-17A, predominantly recruited CD11b- B220+ plasmacytoid DCs (pDC). Knowing that pDC are involved in antibody differentiation, we postulate that i.n. priming with these vectors may favour induction of effective humoral immunity. Our data also revealed that vector-specific replication status and/or presence or absence of immune evasive genes can significantly alter the ILC and DC activity. Collectively, our findings suggest that understanding the route- and vector-specific ILC and DC profiles at the vaccination site may help tailor/design more efficacious viral vector-based vaccines, according to the pathogen of interest.

Plasmonic p-n Junction for Infrared Light to Chemical Energy Conversion.

Infrared (IR) light represents an untapped energy source accounting for almost half of all solar energy. Thus, there is a need to develop systems to convert IR light to fuel and make full use of this plentiful resource. Herein, we report photocatalytic H2 evolution driven by near- to shortwave-IR light (up to 2500 nm) irradiation, based on novel CdS/Cu7S4 heterostructured nanocrystals. The apparent quantum yield reached 3.8% at 1100 nm, which exceeds the highest efficiencies achieved by IR light energy conversion systems reported to date. Spectroscopic results revealed that plasmon-induced hot-electron injection at p-n heterojunctions realizes exceptionally long-lived charge separation (>273 µs), which results in efficient IR light to hydrogen conversion. These results pave the way for the exploration of undeveloped low-energy light for solar fuel generation.

Study protocol: a randomised controlled trial of supervised resistance training versus aerobic training in Sri Lankan adults with type 2 diabetes mellitus: SL-DART study.

BACKGROUND: The prevalence of type 2 diabetes mellitus (T2DM) and cardiovascular diseases (CVD) is rising globally. T2DM is particularly problematic in South Asia with an estimated 10-15% of Sri Lankans diagnosed with the disease. Exercise is known to improve blood glucose, lipid profiles, blood pressure and adiposity, key goals in the management of T2DM. However, much of the evidence to date has been gained from white Caucasians who have a different body composition and disease profile compared to South Asians. Similarly, the recreational exercise culture is new to Sri Lankans and the effects of exercise on T2DM has not been studied in this population. METHODS: The Sri Lanka Diabetes Aerobic and Resistance Training (SL-DART) Study will be comprised of 2 components. Component 1 is a 12-week randomized controlled trial (RCT) to compare the effects of a supervised progressive resistance exercise program (RT) and aerobic exercise program (AT) with standard treatment/control (CN). Sedentary Sri Lankan adults with T2DM (aged 35-65 years) and with no contraindications to exercise will be randomized into one of 3 groups (AT, RT, CN). Exercise sessions will be conducted 2 days/week for 3 months. Baseline and post-intervention biochemical (glycemic control, lipid and liver profiles, inflammatory markers), anthropometric (height, weight, body circumferences), body composition, physical fitness, food preference (liking and wanting food) and quality of life parameters will be measured and compared between groups. Component 2 will be a qualitative study conducted immediately post-intervention via in-depth interviews to assess the barriers and facilitators for adherence to each exercise program. DISCUSSION: SL-DART Study represents one of the first adequately powered methodologically sound RCTs conducted in South Asia to assess the effects of resistance and aerobic exercise in participants with T2DM. Triangulation of quantitative and qualitative outcomes will enable the design of a culturally appropriate therapeutic physical activity intervention for Sri Lankans with T2DM, and the initiation of a professionally driven and specialized clinical exercise prescription service. TRIAL REGISTRATION: Sri Lanka Clinical Trials Registry; SLCTR/2016/017 . Date registered 17.06.2016. Universal trial number U1111-1181-7561.

Preparation of bone-implants by coating hydroxyapatite nanoparticles on self-formed titanium dioxide thin-layers on titanium metal surfaces.

Preparation of hydroxyapatite coated custom-made metallic bone-implants is very important for the replacement of injured bones of the body. Furthermore, these bone-implants are more stable under the corrosive environment of the body and biocompatible than bone-implants made up of pure metals and metal alloys. Herein, we describe a novel, simple and low-cost technique to prepare biocompatible hydroxyapatite coated titanium metal (TiM) implants through growth of self-formed TiO2 thin-layer (SFTL) on TiM via a heat treatment process. SFTL acts as a surface binder of HA nanoparticles in order to produce HA coated implants. Colloidal HA nanorods prepared by a novel surfactant-assisted synthesis method, have been coated on SFTL via atomized spray pyrolysis (ASP) technique. The corrosion behavior of the bare and surface-modified TiM (SMTiM) in a simulated body fluid (SBF) medium is also studied. The highest corrosion rate is found to be for the bare TiM plate, but the corrosion rate has been reduced with the heat-treatment of TiM due to the formation of SFTL. The lowest corrosion rate is recorded for the implant prepared by heat treatment of TiM at 700 °C. The HA-coating further assists in the passivation of the TiM in the SBF medium. Both SMTiM and HA coated SMTiM are noncytotoxic against osteoblast-like (HOS) cells and are in high-bioactivity. The overall production process of bone-implant described in this paper is in high economic value.

Unique IL-13Rα2-based HIV-1 vaccine strategy to enhance mucosal immunity, CD8(+) T-cell avidity and protective immunity.

We have established that mucosal immunization can generate high-avidity human immunodeficiency virus (HIV)-specific CD8(+) T cells compared with systemic immunization, and interleukin (IL)-13 is detrimental to the functional avidity of these T cells. We have now constructed two unique recombinant HIV-1 vaccines that co-express soluble or membrane-bound forms of the IL-13 receptor α2 (IL-13Rα2), which can "transiently" block IL-13 activity at the vaccination site causing wild-type animals to behave similar to an IL-13 KO animal. Following intranasal/intramuscular prime-boost immunization, these IL-13Rα2-adjuvanted vaccines have shown to induce (i) enhanced HIV-specific CD8(+) T cells with higher functional avidity, with broader cytokine/chemokine profiles and greater protective immunity using a surrogate mucosal HIV-1 challenge, and also (ii) excellent multifunctional mucosal CD8(+) T-cell responses, in the lung, genito-rectal nodes (GN), and Peyer's patch (PP). Data revealed that intranasal delivery of these IL-13Rα2-adjuvanted HIV vaccines recruited large numbers of unique antigen-presenting cell subsets to the lung mucosae, ultimately promoting the induction of high-avidity CD8(+) T cells. We believe our novel IL-13R cytokine trap vaccine strategy offers great promise for not only HIV-1, but also as a platform technology against range of chronic infections that require strong sustained high-avidity mucosal/systemic immunity for protection.

A DNA prime-live vaccine boost strategy in mice can augment IFN-gamma responses to mycobacterial antigens but does not increase the protective efficacy of two attenuated strains of Mycobacterium bovis against bovine tuberculosis.

The Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine has variable efficacy for both human and bovine tuberculosis. There is a need for improved vaccines or vaccine strategies for control of these diseases. A recently developed prime-boost strategy was investigated for vaccination against M. bovis infection in mice. BALB/c and C57BL/6 mice were primed with a DNA vaccine, expressing two mycobacterial antigens, ESAT-6 and antigen 85 A and boosted with attenuated M. bovis strains, BCG or WAg520, a newly attenuated strain, prior to aerosol challenge. Before challenge, the antigen-specific production of interferon-gamma (IFN-gamma) was evaluated by ELISPOT and antibody responses were measured. The prime-boost stimulated an increase in the numbers of IFN-gamma producing cells compared with DNA or live vaccination alone, but this varied according to the attenuated vaccine strain, time of challenge and the strain of mouse used. Animals vaccinated with DNA alone generated the strongest antibody response to mycobacterial antigens, which was predominantly IgG1. BCG and WAg520 alone generally gave a 1-2 log10 reduction in bacterial load in lungs or spleen, compared to non-vaccinated or plasmid DNA only control groups. The prime-boost regimen was not more effective than BCG or WAg520 alone. These observations demonstrate the comparable efficacy of BCG and WAg520 in a mouse model of bovine tuberculosis. However, priming with the DNA vaccine and boosting with an attenuated M. bovis vaccine enhanced IFN-gamma immune responses compared to vaccinating with an attenuated M. bovis vaccine alone, but did not increase protection against a virulent M. bovis infection.

Screening for novel cry genes by hybridization.

AIMS: To develop an efficient and sensitive method to facilitate the search for novel Cry toxins. METHODS AND RESULTS: The method uses a cocktail of cry gene sequences as a hybridization probe to screen Bacillus thuringiensis (Bt) strains and gene libraries prepared from them. Under the hybridization and washing conditions used, cross-hybridization between genes from different cry families was not observed. Probes containing either partial or complete cry gene sequences produced similar patterns when hybridized to genomic DNA of several Bt strains although the pattern produced by the probe composed of entire gene coding regions was somewhat more complex. CONCLUSION: As a tool for gene library screening, hybridization with a mixture of cry gene sequences is an efficient means of detecting clones containing a diverse range of cry genes in a single step. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique greatly improves the ease and efficiency of novel toxin gene discovery compared to previous methods.

Isolation and characterisation of a cytochrome b5 cDNA clone from Helicoverpa armigera (Hubner): possible involvement of cytochrome b5 in cytochrome P450 CYP6B7 activity towards pyrethroids.

A cDNA clone specific for cytochrome b5 was isolated from Helicoverpa armigera. This sequence corresponded to a mRNA of an estimated 544 nucleotides in length excluding the poly A tail. The mRNA contained an open reading frame of 381 nucleotides encoding a protein of 127 amino acid residues with a molecular weight of 14,564 Daltons. The encoded protein sequence showed 51% protein sequence identity with cytochrome b5 from M. domestica and 36-37% identity with mammalian and avian cytochrome b5 sequences. Northern analysis of larval RNA using this cDNA as probe, revealed that cytochrome b5 mRNA expression is tissue specific with the mRNAs being expressed in abundance in the midguts of larvae, at a lower level in fatbody but is not detectable in larval integument. During normal development this mRNA was undetectable in eggs but was present at similar levels from first to fifth instar larvae. The mRNA was expressed at very low levels in pupae and adult moths. The cytochrome b5 mRNA was found to be inducible by treatment with the monoterpene, a-pinene, and to be over-expressed in some individuals of a pyrethroid resistant population of H. armigera. The induction and over-expression patterns were identical to the cytochrome P450, CYP6B7 mRNA. The present data suggests that cytochrome b5 may be involved in CYP6B7 mediated pyrethroid resistance in H. armigera.

Induction of cytochrome P450 CYP6B7 and cytochrome b5 mRNAs from Helicoverpa armigera (Hubner) by pyrethroid insecticides in organ culture.

An organ culture system derived from Helicoverpa armigera has been used to study the expression of cytochrome P450 and cytochrome b5 mRNAs. Northern analysis showed that levels of the mRNAs for cytochrome P450s, CYP6B2, CYP6B6 and CYP6B7, and cytochrome b5 in control tissue were commensurate with those in the tissue of whole larvae. Substantial induction of cytochrome P450, CYP6B7 and cytochrome b5 mRNAs by alpha-pinene, and the pyrethroids, fenvalerate, cypermethrin and permethrin were observed in fat body culture. Neither mRNA was induced, either in midgut or integument organ cultures. In contrast, the relatively water-soluble compound phenobarbital, could induce CYP6B7 mRNA but not cytochrome b5 mRNA in fat body cultures. As for pyrethroids, phenobarbital had no effect on the other tissues in culture. These results confirm a previous conclusion that pyrethroids could induce CYP6B7 mRNA, which was based upon a very slight induction observed in living insects. Because many cytochrome P450 substrates can act as their inducers, these results support a previous conclusion that CYP6B7 could be the enzyme that is involved in pyrethroid resistance in H. armigera.

Isolation and characterization of two cytochrome P450 cDNA clones for CYP6B6 and CYP6B7 from Helicoverpa armigera (Hubner): possible involvement of CYP6B7 in pyrethroid resistance.

Two new cDNA clones specific for members of the CYP6B gene family, CYP6B6 and CYP6B7 have been isolated from Helicoverpa armigera. The sequences correspond to mRNAs of an estimated 1962 and 2411 nucleotides in length respectively excluding the poly A tails. The two mRNAs have open reading frames encoding proteins of 504 amino acid residues with molecular weights of 57,564 and 58,181 Daltons. Both putative proteins contain the conserved cysteine and surrounding regions characteristics of all cytochrome P450s. The encoded protein sequences show 84-88% protein sequence identity between them and with the previously published cytochrome P450 sequence of H. armigera. CYP6B2. The sequences of cDNA clones of CYP6B6 and CYP6B2 show a very high degree of identity within the first 340 nucleotides which may be the result of a gene conversion event. Two major bands are visible after northern analysis of larval RNA using cDNA clones for CYP6B6, CYP6B7, or the previously published CYP6B2 as probes, due to strong cross-hybridization. Analysis with specific oligonucleotide probes and 3' non-coding regions indicated that the cDNAs for CYP6B6 and CYP6B7 correspond to the smaller and large mRNA bands respectively. The previously identified sequence of CYP6B2, contrary to the previous suggestion, corresponds to a rare mRNA of similar size to that for CYP6B6. The mRNA for CYP6B7 was found to be induced by treatment with the monoterpene, alpha-pinene, and to be over-expressed in some individuals of pyrethroid resistant population of H. armigera. We suggest that CYP6B7 is the form responsible for pyrethroid metabolism in H. armigera.

Cycle DNA sequencing with [alpha-35S]dATP demonstrates polymorphism of a surface antigen in malaria parasites from Sri Lankan patients.

Structural diversity in a 45 kDa surface antigen on Plasmodium falciparum merozoites (termed GYMSSA, MSP-2 or MSA-2) and other candidate molecules for developing a malaria vaccine need to be investigated in parasites obtained directly from patients in different malaria endemic countries. A double-stranded DNA sequencing method suitable for this purpose, and also for studying diversity in genes of other haploid cells, is described. A first round polymerase chain reaction (PCR) on DNA isolated from blood was carried out with a primer containing the GCN4 binding site to amplify and subsequently purify the coding region of the MSA-2 gene on GCN4 coated tubes. A second round PCR with more internal primers incorporating M13 forward and reverse primer sequences was then performed. Cycle sequencing was done with unlabelled M13 primers and [alpha-35S]dATP by the dideoxynucleotide procedure. Two different allelic forms of MSA-2 were identified in samples of Plasmodium falciparum from patients in Sri Lanka.

Shortcomings of some currently available DNA probes for malaria detection.

A non-radioactive DNA probe based-method for detecting malaria will greatly aid epidemiological studies. Using putative Plasmodium falciparum and P. vivax-specific 18S ribosomal RNA directed oligonucleotides, different enzymatic and chemiluminescent detection methods were attempted without success. The sensitivity of the corresponding 32P-labelled probes was found to be inadequate. A published procedure based on chemiluminescent detection of repetitive DNA sequences of P. falciparum was found to be adequately sensitive but lacking in specificity.

High seasonal malaria transmission rates in the intermediate rainfall zone of Sri Lanka.

Malaria transmission was studied at Nikawehera, a long-established farming village, located in the intermediate rainfall zone of Sri Lanka. Observations were made over a 12-month period (October-September) that included the main rainy season which occurred during the north-east monsoon in November-January. Anolpheles culicifacies, the recognized vector of malaria in Sri Lanka, was the predominant anopheline mosquito collected by human night baiting at Nikawehera. High entomological inoculation rates with An. culcifacies (0.12/hour for Plasmodium vivax) were observed during the height of the transmission season which occurs during, and immediately after, the north-east monsoon. Anolpheles subpictus was identified as a possible additional vector at Nikawehera. Anopheles annularis, a major vector at Weheragala, a site in a new irrigation development (the Mahaweli Scheme) located 70 km away in the dry zone, was not collected by human baiting at Nikawehera. Clinical, entomological and parasitological data suggest that malaria is hyperendemic at Nikawehera, with high seasonal transmission rates.