Continuous and Segmented-Flow Microfluidics for Biomolecular Analysis

Segmented flows in microfluidics have attracted much recent attention. Of particular interest is the use of nanolitre droplets as interface tools in microfluidics. A droplet interface connects techniques while leaving them sufficiently independent of each other; a task that difficult to perform within a continuous flow system. The first part of this thesis is dedicated to sample preparation and analysis in continuous flow microfluidic systems. The polymerase chain reaction (PCR) is investigated, using electroosmotic flow to transport the sample across various temperature zones. Additionally, a novel sieving matrix for electrophoretic separation of dsDNA fragments and PCR amplicons on microchip and capillary is demonstrated. The second part of this thesis focuses on interfacing segmented and continuous flows. Two novel interfaces are described and demonstrated. The first interface connects droplet flows and a chip-based electrophoresis device. Using this interface, samples or reactions performed in droplets can be directly transferred to a separation channel without suspending the separation. This allows multiple samples to be analysed in a single separation channel, without cross contamination between droplets. Consequently, PCR reactions and dsDNA calibration ladders can be prepared in droplet format and analysed in high throughput. The second interface links nano-liquid chromatography and MALDI mass spectrometry. Droplet fractionation post nano-LC separation is used to preserve resolution between separated bands. The droplets are subsequently delivered to a MALDI plate for mass spectrometric analysis by removing the continuous oil phase using a hydrophobic oleophilic membrane. The tools developed here reduce manual intervention and provide a link between multiple analytical techniques involved in biomolecule analysis. These innovations will improve reproducibility and reduce cross-contamination between samples

Kajian Populasi Kepiting Kenari Di Pulau Batudaka Kepulauan Togean, Sulawesi Tengah Dan Rekomendasi Manajemen Populasi

This study aimed to quantify the population of Birgus latro in the Batudaka di Togean islands, Central Sulawesi. The research on robber crab was conducted in Batudaka Island, Togean, Tomini Bay, Central Sulawesi. In the study site, 21 plots measuring of 50x50 m2 were created bounded by raffia. Feed in the form of shredded coconut is placed in each plot in the afternoon. At night was performed observations and catchs. In the "base camp" every crab crab carapace caught measured in carapace length and weight. During the study, 277 crabs were caught, consisted of 173 males (62.45%) and 104 (37.55%) females. Based on the formula calculation of Schiller (1992) population figures obtained 821 803 ± 195 030 crabs in Batudaka Island. By regression analysis between carapace length with weight, it was found that the growth of B. latro is negative allometric, i.e., weight gain is faster than the increase length of carapace. The weight gain of female is slightly higher than that of the male. Whether male crab population or female equally composed of 9 age groups. This study showed that 66.7% of male crab and 29.1% of female crab has entered the market size

Reactions from family of origin to the disclosure of lesbian motherhood via donor insemination

Most studies of planned lesbian motherhood via donor insemination (DI) have sampled lesbian mothers in individualistic societies where adults have relatively distant connection to their family of origin. Our study examined the experiences of biological and non-biological lesbian mothers in five families who had children through DI after disclosing their motherhood status to their family networks in Portugal, a familistic society. The first theme identified by thematic analysis, “But why do you want to have a child?” encapsulated the reactions of biological mothers’ family of origin to the announcement of motherhood. Disclosure was mostly met by a shocked response in the extended family, rooted in the belief that lesbian women should not have children. The second theme, “But you weren’t pregnant, how is this your child?” summarized the reactions of non-biological mothers’ family of origin to the disclosure of motherhood status as they considered refusing to recognize their grandchild in the absence of biological connection. Prejudice against lesbian-mother family formation was associated with the specific intersection of lesbianism and motherhood, but relationships between the mothers and their families were largely repaired because of familistic values

Unravelling the metabolic impact of SBS-associated microbial dysbiosis: Insights from the piglet short bowel syndrome model

peer-reviewedLiver disease is a major source of morbidity and mortality in children with short bowel syndrome (SBS). SBS-associated microbial dysbiosis has recently been implicated in the development of SBS-associated liver disease (SBS-ALD), however the pathological implications of this association have not been explored. In this study high-throughput sequencing of colonic content from the well-validated piglet SBS-ALD model was examined to determine alterations in microbial communities, and concurrent metabolic alterations identified in urine samples via targeted mass spectrometry approaches (GC-MS, LC-MS, FIA-MS) further uncovered impacts of microbial disturbance on metabolic outcomes in SBS-ALD. Multi-variate analyses were performed to elucidate contributing SBS-ALD microbe and metabolite panels and to identify microbe-metabolite interactions. A unique SBS-ALD microbe panel was clearest at the genus level, with discriminating bacteria predominantly from the Firmicutes and Bacteroidetes phyla. The SBS-ALD metabolome included important alterations in the microbial metabolism of amino acids and the mitochondrial metabolism of branched chain amino acids. Correlation analysis defined microbe-metabolite clustering patterns unique to SBS-ALD and identified a metabolite panel that correlates with dysbiosis of the gut microbiome in SBS

Transformative spaces in the making: key lessons from nine cases in the Global South

Creating a just and sustainable planet will require not only small changes, but also systemic transformations in how humans relate to the planet and to each other, i.e., social–ecological transformations. We suggest there is a need for collaborative environments where experimentation with new configurations of social–ecological systems can occur, and we refer to these as transformative spaces. In this paper, we seek a better understanding of how to design and enable the creation of transformative spaces in a development context. We analyse nine case studies from a previous special issue on Designing Transformative Spaces that aimed to collect examples of cutting-edge action-oriented research on transformations from the Global South. The analysis showed five design phases as being essential: Problem Definition Phase; Operationalisation Phase; Tactical Phase; Outcome Phase; and Reflection Phase. From this synthesis, we distilled five key messages that should be considered when designing research, including: (a) there are ethical dilemmas associated with creating a transformative space in a system; (b) it is important to assess the readiness of the system for change before engaging in it; (c) there is a need to balance between ‘safe’ and ‘safe-enough’ spaces for transformation; (d) convening a transformative space requires an assemblage of diverse methodological frameworks and tools; and (e) transformative spaces can act as a starting point for institutionalising transformative change. Many researchers are now engaging in transdisciplinary transformations research, and are finding themselves at the knowledge–action interface contributing to transformative space-making. We hope that by analysing experiences from across different geographies we can contribute towards better understanding of how to navigate the processes needed for the urgent global transformations that are being called for to create a more equitable and sustainable planet Earth

Different placement practices for different families? Children's adjustment in LGH adoptive families

Objective: The purpose of this study was to examine the characteristics of children placed with lesbian, gay, and heterosexual adopters, and to examine children’s problem behaviors and positive psychosocial adjustment across the three family types. Background: There is evidence that children with hard-to-place profiles may be more likely to be matched with lesbian and gay parents. In addition, children adopted from care face greater developmental difficulties than children raised by their birth families, although adoptive parents may buffer the negative effects of early adversity on their children’s psychosocial adjustment. Method: A final sample of 149 adoptive families from across the United Kingdom was recruited: 71 heterosexual parented, 39 lesbian parented, and 39 gay parented. Results: The results showed that gay and lesbian parents were more likely than heterosexual parents to be matched with hard-to-place children, partially because they were more open to being matched with children with hard-to-place profiles. However, no differences among the three family types on children’s psychosocial adjustment were found, when controlling for children’s early adversity. Conclusion: Adopted children displayed similar levels of problem behaviors and positive adjustment in lesbian, gay, and heterosexual parented families. Early adversity and having a physical problem/disability accounted for much of the variance in problem behaviors whereas parenting did not. In contrast, it was suggested that parenting processes, namely, parental closeness, may help to explain children’s positive adjustment

Expression and reactivation of HIV in a chemokine induced model of HIV latency in primary resting CD4+ T cells

<p>Abstract</p> <p>Background</p> <p>We recently described that HIV latent infection can be established <it>in vitro </it>following incubation of resting CD4+ T-cells with chemokines that bind to CCR7. The main aim of this study was to fully define the post-integration blocks to virus replication in this model of CCL19-induced HIV latency.</p> <p>Results</p> <p>High levels of integrated HIV DNA but low production of reverse transcriptase (RT) was found in CCL19-treated CD4+ T-cells infected with either wild type (WT) NL4.3 or single round envelope deleted NL4.3 pseudotyped virus (NL4.3- Δenv). Supernatants from CCL19-treated cells infected with either WT NL4.3 or NL4.3- Δenv did not induce luciferase expression in TZM-bl cells, and there was no expression of intracellular p24. Following infection of CCL19-treated CD4+ T-cells with NL4.3 with enhanced green fluorescent protein (EGFP) inserted into the <it>nef </it>open reading frame (NL4.3- Δnef-EGFP), there was no EGFP expression detected. These data are consistent with non-productive latent infection of CCL19-treated infected CD4+ T-cells. Treatment of cells with phytohemagluttinin (PHA)/IL-2 or CCL19, prior to infection with WT NL4.3, resulted in a mean fold change in unspliced (US) RNA at day 4 compared to day 0 of 21.2 and 1.1 respectively (p = 0.01; n = 5), and the mean expression of multiply spliced (MS) RNA was 56,000, and 5,000 copies/million cells respectively (p = 0.01; n = 5). In CCL19-treated infected CD4+ T-cells, MS-RNA was detected in the nucleus and not in the cytoplasm; in contrast to PHA/IL-2 activated infected cells where MS RNA was detected in both. Virus could be recovered from CCL19-treated infected CD4+ T-cells following mitogen stimulation (with PHA and phorbyl myristate acetate (PMA)) as well as TNFα, IL-7, prostratin and vorinostat.</p> <p>Conclusions</p> <p>In this model of CCL19-induced HIV latency, we demonstrate HIV integration without spontaneous production of infectious virus, detection of MS RNA in the nucleus only, and the induction of virus production with multiple activating stimuli. These data are consistent with <it>ex vivo </it>findings from latently infected CD4+ T-cells from patients on combination antiretroviral therapy, and therefore provide further support of this model as an excellent <it>in vitro </it>model of HIV latency.</p

Clinical and cost effectiveness of endoscopic bipolar radiofrequency ablation for the treatment of malignant biliary obstruction: a systematic review

BACKGROUND: Early evidence suggests that using radiofrequency ablation as an adjunct to standard care (i.e. endoscopic retrograde cholangiopancreatography with stenting) may improve outcomes in patients with malignant biliary obstruction. OBJECTIVES: To assess the clinical effectiveness, cost-effectiveness and potential risks of endoscopic bipolar radiofrequency ablation for malignant biliary obstruction, and the value of future research. DATA SOURCES: Seven bibliographic databases, three websites and seven trials registers were searched from 2008 until 21 January 2021. REVIEW METHODS: The study inclusion criteria were as follows: patients with biliary obstruction caused by any form of unresectable malignancy; the intervention was reported as an endoscopic biliary radiofrequency ablation to ablate malignant tissue that obstructs the bile or pancreatic ducts, either to fit a stent (primary radiofrequency ablation) or to clear an obstructed stent (secondary radiofrequency ablation); the primary outcomes were survival, quality of life or procedure-related adverse events; and the study design was a controlled study, an observational study or a case report. Risk of bias was assessed using Cochrane tools. The primary analysis was meta-analysis of the hazard ratio of mortality. Subgroup analyses were planned according to the type of probe, the type of stent (i.e. metal or plastic) and cancer type. A de novo Markov model was developed to model cost and quality-of-life outcomes associated with radiofrequency ablation in patients with primary advanced bile duct cancer. Insufficient data were available for pancreatic cancer and secondary bile duct cancer. An NHS and Personal Social Services perspective was adopted for the analysis. A probabilistic analysis was conducted to estimate the incremental cost-effectiveness ratio for radiofrequency ablation and the probability that radiofrequency ablation was cost-effective at different thresholds. The population expected value of perfect information was estimated in total and for the effectiveness parameters. RESULTS: Sixty-eight studies (1742 patients) were included in the systematic review. Four studies (336 participants) were combined in a meta-analysis, which showed that the pooled hazard ratio for mortality following primary radiofrequency ablation compared with a stent-only control was 0.34 (95% confidence interval 0.21 to 0.55). Little evidence relating to the impact on quality of life was found. There was no evidence to suggest an increased risk of cholangitis or pancreatitis, but radiofrequency ablation may be associated with an increase in cholecystitis. The results of the cost-effectiveness analysis were that the costs of radiofrequency ablation was £2659 and radiofrequency ablation produced 0.18 quality-adjusted life-years, which was more than no radiofrequency ablation on average. With an incremental cost-effectiveness ratio of £14,392 per quality-adjusted life-year, radiofrequency ablation was likely to be cost-effective at a threshold of £20,000 per quality-adjusted life-year across most scenario analyses, with moderate uncertainty. The source of the vast majority of decision uncertainty lay in the effect of radiofrequency ablation on stent patency. LIMITATIONS: Only 6 of 18 comparative studies contributed to the survival meta-analysis, and few data were found concerning secondary radiofrequency ablation. The economic model and cost-effectiveness meta-analysis required simplification because of data limitations. Inconsistencies in standard reporting and study design were noted. CONCLUSIONS: Primary radiofrequency ablation increases survival and is likely to be cost-effective. The evidence for the impact of secondary radiofrequency ablation on survival and of quality of life is limited. There was a lack of robust clinical effectiveness data and, therefore, more information is needed for this indication. FUTURE WORK:  Future work investigating radiofrequency ablation must collect quality-of-life data. Highquality randomised controlled trials in secondary radiofrequency ablation are needed, with appropriate outcomes recorded. STUDY REGISTRATION: This study is registered as PROSPERO CRD42020170233. FUNDING:  This project was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 27, No. 7. See the NIHR Journals Library website for further project information