PCR-based gene synthesis to produce recombinant proteins for crystallization

<p>Abstract</p> <p>Background</p> <p>Gene synthesis technologies are an important tool for structural biology projects, allowing increased protein expression through codon optimization and facilitating sequence alterations. Existing methods, however, can be complex and not always reproducible, prompting researchers to use commercial suppliers rather than synthesize genes themselves.</p> <p>Results</p> <p>A PCR-based gene synthesis method, referred to as SeqTBIO, is described to efficiently assemble the coding regions of two novel hyperthermophilic proteins, PAZ (Piwi/Argonaute/Zwille) domain, a siRNA-binding domain of an Argonaute protein homologue and a deletion mutant of a family A DNA polymerase (PolA). The gene synthesis procedure is based on sequential assembly such that homogeneous DNA products can be obtained after each synthesis step without extensive manipulation or purification requirements. Coupling the gene synthesis procedure to <it>in vivo </it>homologous recombination techniques allows efficient subcloning and site-directed mutagenesis for error correction. The recombinant proteins of PAZ and PolA were subsequently overexpressed in <it>E. coli </it>and used for protein crystallization. Crystals of both proteins were obtained and they were suitable for X-ray analysis.</p> <p>Conclusion</p> <p>We demonstrate, by using PAZ and PolA as examples, the feasibility of integrating the gene synthesis, error correction and subcloning techniques into a non-automated gene to crystal pipeline such that genes can be designed, synthesized and implemented for recombinant expression and protein crystallization.</p

Spiropyran modified micro-fluidic chip channels as photonically controlled self-indicating system for metal ion accumulation and release

In this paper, we show how through integrating the beneficial characteristics of micro-fluidic devices and spiropyrans dyes, a simple and very innovative chip configured as an on-line photonically controlled self-indicating system for metal ion accumulation and release can be realised. The micro-fluidic device consists of five independent 94 μm depth, 150 μm width channels fabricated in polydimethylsiloxane. The spiropyran 1’-(3-carboxypropyl)-3,3’-dimethyl-6-nitrospiro-1-benzopyran-2,2’-indoline is immobilised by physical adsorption into a polydimethylsiloxane matrix and covalently on the ozone plasma activated polydimehylsiloxane micro-channel walls. When the colourless, inactive, spiropyran coating absorbs UV light it switches to the highly coloured merocyanine form, which also has an active binding site for certain metal ions. Therefore metal ion uptake can be triggered using UV light and subsequently reversed on demand by shining white light on the coloured complex, which regenerates the inactive spiropyran form, and releases the metal ion. When stock solutions of several metal ions (Ca2+, Zn2+, Hg2+, Cu2+, Co2+) are pumped independently through the five channels, different optical responses were observed for each metal, and the platform can therefore be regarded as a micro-structured device for online self-indicating metal ion complexation, accumulation and release

Refractive change following pseudophakic vitrectomy: a retrospective review

Background To assess the occurrence and magnitude of refractive change in pseudophakic eyes undergoing 20 gauge pars plana vitrectomy without scleral buckling and to investigate possible aetiological factors. Methods Retrospective case note review of 87 pseudophakic eyes undergoing 20 gauge pars plana vitrectomy for a variety of vitreo-retinal conditions over a three-year period. Anterior chamber depth (ACD) was measured before and after vitrectomy surgery in 32 eyes. Forty-three pseudophakic fellow eyes were used as controls. Results Eighty-seven eyes (84 patients) were included in the study. Mean spherical equivalent refraction prior to vitrectomy was -0.20 dioptres, which changed to a mean of -0.65 dioptres postoperatively (standard deviation of refractive change 0.59, range-2.13 to 0.75 dioptres) (p < 0.001). Sixty-one of the 87(70%) eyes experienced a myopic shift and 45(52%) eyes had a myopic shift of -0.5 dioptres or more. Mean fellow eye refraction was -0.19 dioptres preoperatively and -0.17 dioptres postoperatively (p = 0.14)(n = 37) Mean ACD preoperatively was 3.29 mm and postoperatively 3.27 mm (p = 0.53) (n = 32) and there was no significant change in ACD with tamponade use. Regression analysis revealed no statistically significant association between changes in anterior chamber depth, as well as a wide variety of other pre-, intra and postoperative factors examined, and the refractive change observed. Conclusion Significant refractive changes occur in some pseudophakic patients undergoing 20 g pars plana vitrectomy. The mean change observed was a small myopic shift but the range was large. The aetiology of the refractive change is uncertain

Qualitative research design and approaches in radiography

Introduction Radiography, as with other health care professions, aims towards establishing full professional recognition. A recognized research background is considered an essential component of this. Usually, emphasis is placed on quantitative research which focuses on the deductive component of the scientific 'confirmatory' method. However, this situation changed after the emergence of a post-positivist philosophy of medical research, which suggests the need for both quantitative and qualitative research to grasp a better understanding of the reality. The nature of qualitative research, unlike quantitative, is inductive and exploratory, providing insight into certain topics of which little is known and hence complementing quantitative research. Purpose: This article identifies the main qualitative research traditions that can be applied to radiography, providing a review in terms of their applications, areas of inquiry, and strengths and weaknesses according to the nature of the study. Previous radiography studies using qualitative approaches are critically reviewed to illustrate these issues. Their applications are also discussed based on the proposed radiography research framework by Adams and Smith, who identified broad radiography research areas aiming to further the research capacity of radiographers and the profession, especially by means of qualitative research.Methods: A comprehensive literature search was conducted using the CINAHL and ScienceDirect databases and the journal Radiography, and by using the keywords qualitative research to identify articles focusing on qualitative research. Only articles that were related to health care disciplines were then selected for the review. Conclusion: Three approaches, that are grounded theory, phenomenology and ethnography, can all be applied to the proposed radiography research framework to study intra- and inter-professional issues and clinical practice, and patient and health delivery issues. Hence, qualitative research methods can be used to rigorously study these important areas so as to produce high quality outcomes and promote the use of qualitative approaches in the radiography research culture

Assessing social recovery of vulnerable youth in global mental health settings: a pilot study of clinical research tools in Malaysia

Background A social recovery approach to youth mental health focuses on increasing the time spent in valuable and meaningful structured activities, with a view to preventing enduring mental health problems and social disability. In Malaysia, access to mental health care is particularly limited and little research has focused on identifying young people at risk of serious socially disabling mental health problems such as psychosis. We provide preliminary evidence for the feasibility and acceptability of core social recovery assessment tools in a Malaysian context, comparing the experiential process of engaging young Malaysian participants in social recovery assessments with prior accounts from a UK sample. Methods Nine vulnerable young people from low-income backgrounds were recruited from a non-government social enterprise and partner organisations in Peninsular Malaysia. Participants completed a battery of social recovery assessment tools (including time use, unusual experiences, self-schematic beliefs and values). Time for completion and completion rates were used as indices of feasibility. Acceptability was examined using qualitative interviews in which participants were asked to reflect on the experience of completing the assessment tools. Following a deductive approach, the themes were examined for fit with previous UK qualitative accounts of social recovery assessments. Results Feasibility was indicated by relatively efficient completion time and high completion rates. Qualitative interviews highlighted the perceived benefits of social recovery assessments, such as providing psychoeducation, aiding in self-reflection and stimulating goal setting, in line with findings from UK youth samples. Conclusions We provide preliminary evidence for the feasibility and acceptability of social recovery assessment tools in a low-resource context, comparing the experiential process of engaging young Malaysian participants in social recovery assessments with prior accounts from a UK sample. We also suggest that respondents may derive some personal and psychoeducational benefits from participating in assessments (e.g. of their time use and mental health) within a social recovery framework

Views of young people in Malaysia on mental health, help-seeking and unusual psychological experiences

Aim Mental health problems are prevalent among young people in Malaysia yet access to specialist mental health care is extremely limited. More context‐specific research is needed to understand the factors affecting help‐seeking in youth, when mental health problems typically have first onset. We aimed to explore the attitudes of vulnerable young Malaysians regarding mental health problems including unusual psychological experiences, help‐seeking and mental health treatment. Methods In the present study, nine young people (aged 16–23 years) from low‐income backgrounds participated in a semi‐structured interview about their perspectives on mental health problems, unusual psychological experiences and help‐seeking. Results Four themes were developed using thematic analysis. “Is it that they [have] family problems?” reflected participants' explanatory models of mental health problems. “Maybe in Malaysia” was concerned with perceptions of Malaysian culture as both encouraging of open sharing of problems and experiences, but also potentially stigmatizing. “You have to ask for help” emphasized the importance of mental health help‐seeking despite potential stigma. “It depends on the person” addressed the challenges of engaging with psychological therapy. Conclusions We conclude that young people in Malaysia may hold compassionate, non‐stigmatizing views towards people experiencing mental health problems and a desire to increase their knowledge and understandings. Yet societal stigma is a perceived reputational risk that may affect mental health problem disclosure and help‐seeking. We suggest that efforts to improve mental health literacy would be valued by young Malaysians and could support reduced stigma and earlier help‐seeking

An ovine model of hyperdynamic endotoxemia and vital organ metabolism

BACKGROUND: Animal models of endotoxemia are frequently used to understand the pathophysiology of sepsis and test new therapies. However, important differences exist between commonly used experimental models of endotoxemia and clinical sepsis. Animal models of endotoxemia frequently produce hypodynamic shock in contrast to clinical hyperdynamic shock. This difference may exaggerate the importance of hypoperfusion as a causative factor in organ dysfunction. This study sought to develop an ovine model of hyperdynamic endotoxemia and assess if there is evidence of impaired oxidative metabolism in the vital organs. METHODS: Eight sheep had microdialysis catheters implanted into the brain, heart, liver, kidney and arterial circulation. Shock was induced with a 4hr escalating dose infusion of endotoxin. After 3hrs vasopressor support was initiated with noradrenaline and vasopressin. Animals were monitored for 12hrs after endotoxemia. Blood samples were recovered for haemoglobin, white blood cell count, creatinine and proinflammatory cytokines (IL-1Beta, IL-6 & IL-8). RESULTS: The endotoxin infusion was successful in producing distributive shock with the mean arterial pressure decreasing from 84.5 ± 12.8 mmHg to 49 ± 8.03 mmHg (p < 0.001). Cardiac index remained within the normal range decreasing from 3.33 ± 0.56 l/min/m to 2.89l ± 0.36 l/min/m (p = 0.0845). Lactate/pyruvate ratios were not significantly abnormal in the heart, brain, kidney or arterial circulation. Liver microdialysis samples demonstrated persistently high lactate/pyruvate ratios (mean 37.9 ± 3.3). CONCLUSIONS: An escalating dose endotoxin infusion was successful in producing hyperdynamic shock. There was evidence of impaired oxidative metabolism in the liver suggesting impaired splanchnic perfusion. This may be a modifiable factor in the progression to multiple organ dysfunction and death

Bacterial dissolution of fluorapatite as a possible source of elevated dissolved phosphate in the environment

In order to understand the contribution of geogenic phosphorus to lake eutrophication, we have investigated the rate and extent of fluorapatite dissolution in the presence of two common soil bacteria (Pantoea agglomerans and Bacillus megaterium) at T = 25 °C for 26 days. The release of calcium (Ca), phosphorus (P), and rare earth elements (REE) under biotic and abiotic conditions was compared to investigate the effect of microorganism on apatite dissolution. The release of Ca and P was enhanced under the influence of bacteria. Apatite dissolution rates obtained from solution Ca concentration in the biotic reactors increased above error compared with abiotic controls. Chemical analysis of biomass showed that bacteria scavenged Ca, P, and REE during their growth, which lowered their fluid concentrations, leading to apparent lower release rates. The temporal evolution of pH in the reactors reflected the balance of apatite weathering, solution reactions, bacterial metabolism, and potentially secondary precipitation, which was implied in the variety of REE patterns in the biotic and abiotic reactors. Light rare earth elements (LREE) were preferentially adsorbed to cell surfaces, whereas heavy rare earth elements (HREE) were retained in the fluid phase. Decoupling of LREE and HREE could possibly be due to preferential release of HREE from apatite or selective secondary precipitation of LREE enriched phosphates, especially in the presence of bacteria. When corrected for intracellular concentrations, both biotic reactors showed high P and REE release compared with the abiotic control. We speculate that lack of this correction explains the conflicting findings about the role of bacteria in mineral weathering rates. The observation that bacteria enhance the release rate of P and REE from apatite could account for some of the phosphorus burden and metal pollution in aquatic environments

L-theanine in the adjunctive treatment of generalized anxiety disorder: a double-blind, randomised, placebo-controlled trial

Partial or non-response to antidepressants in Generalized Anxiety Disorder (GAD) is common in clinical settings, and adjunctive biological interventions may be required. Adjunctive herbal and nutraceutical treatments are a novel and promising treatment option. L-theanine is a non-protein amino acid derived most-commonly from tea (Camellia sinensis) leaves, which may be beneficial in the treatment of anxiety and sleep disturbance as suggested by preliminary evidence. We conducted a 10-week study (consisting of an 8-week double-blind placebo-controlled period, and 1-week pre-study and 2-week post-study single-blinded observational periods) involving 46 participants with a DSM-5 diagnosis of GAD. Participants received adjunctive L-theanine (450–900 mg) or matching placebo with their current stable antidepressant treatment, and were assessed on anxiety, sleep quality, and cognition outcomes. Results revealed that adjunctive L-theanine did not outperform placebo for anxiety reduction on the HAMA (p = 0.73) nor insomnia severity on the Insomnia Severity Index (ISI; p = 0.35). However, LT treated participants reported greater self-reported sleep satisfaction than placebo (ISI item 4; p = 0.015). Further, a separation in favour of L-theanine was noted on the ISI in those with non-clinical levels of insomnia symptoms (ISI ≤ 14; p = 0.007). No significant cognitive effects (trail making time and the modified emotional Stroop) were revealed. While this preliminary study did not support the efficacy of L-theanine in the treatment of anxiety symptoms in GAD, further studies to explore the application of L-theanine in sleep disturbance are warranted