Production and characterisation of novel recombinant antibody fragments against sialic acid

Sialic acids are a family of acidic monosaccharides that typically reside as terminal moieties on N- and O-linked glycans. These sugars are actively involved in a plethora of biological phenomena, ranging from cell-cell adhesion and recognition, intracellular signalling events, pathogen attack, viral infection and inflammatory disease. In addition, many cancer-related antigens contain terminal sialic acids or altered sialylation patterns. The identification of sialic acid-specific antibodies is important in the fields of basic research and diagnostics. Therefore the primary objective of this thesis was the generation and characterisation of recombinant antisialic acid antibodies.A single-chain antibody fragment (scFv) library was constructed from a chicken that was immunised with a novel synthetic sialic acid protein-conjugate (Neu5Gchumanserum albumin). The scFv library was biopanned using a second novel sialoneoglycoconjugate(Neu5Gc-bovine serum albumin). Anti-sialic scFvs were isolated byphage-display and binding activity, multimeric status, and multivalent properties wereassessed by ELISA, HPLC, FPLC, and SPR. One clone, AE8, displayed the strongest reactivity to a panel of different sialylated structures. In addition, SPR inhibition andkinetic analysis revealed that the AE8 scFv had nanomolar affinity for the Neu5Gc-BSA neoglycoconjugate. The heavy chain gene of the AE8 scFv was assembled with a repertoire of error prone PCR-amplified light chain genes, that originated from the unpanned anti-sialic acid library. The mutant scFv library was biopanned using a depletion protocol and two ovalbumin (OVA) conjugates, Neu5Gc-linker1-OVA and linker1-OVA. ELISA analysis, SPR inhibition and ‘off-rate’ studies identified one mutant clone, E15, which displayed strong affinity for conjugates of sialic acid. A solution-phase SPR-based inhibition study demonstrated that the E15 scFv had an IC50 concentration of 1.6ng/mL for the Neu5Gc-BSA conjugate. When compared to the parental AE8 clone (IC50 of 5.7ng/mL) the mutant E15 scFv exhibited an improvement in sialic acid binding of approximately 3.5-fold. The generation of such highly specific anti-sialic acid antibodies in a recombinant format is exceedingly rare. Most anti-carbohydrate antibodies are not recombinant and display weak carbohydrate-protein interactions. This work shows for the first time, the generation and directed evolution of anti-carbohydrate scFvs that have nanomolar affinity for Neu5Gc-containing structures

Hydrophilic polymeric coatings for enhanced, serial-siphon based flow control on centrifugal lab-on-disc platforms

In this paper, we implement rotational flow control on a polymeric microfluidic “lab-on-a-disc” device by combining serial siphoning and capillary valving for sequential release of on-board stored liquid reagents. The functionality of this integrated, multi-step centrifugal assay platform is tightly linked by the capability to establish reproducible, capillary-driven priming of the innately hydrophobic siphon microchannels. We here demonstrate for the first time that spin-coated hydrophilic polymeric films of poly(vinyl alcohol) and (hydroxylpropyl)methylcellulose provide stable contact angles

The Effects of Psychosis Risk Variants on Brain Connectivity: A Review

In light of observed changes in connectivity in schizophrenia and the highly heritable nature of the disease, neural connectivity may serve as an important intermediate phenotype for schizophrenia. However, how individual variants confer altered connectivity and which measure of brain connectivity is more proximal to the underlying genetic architecture (i.e., functional or structural) has not been well delineated. In this review we consider these issues and the relative sensitivity of imaging methodologies to schizophrenia-related changes in connectivity. We searched PubMed for studies considering schizophrenia risk genes AND functional or structural connectivity. Where data was available, summary statistics were used to determine an estimate of effect size (i.e., Cohen’s d). A random-effects meta-analysis was used to consider (1) the largest effect and (2) all significant effects between functional and structural studies. Schizophrenia risk variants involved in neurotransmission, neurodevelopment and myelin function were found to be associated with altered neural connectivity. On average, schizophrenia risk genes had a large effect on functional (mean d = 0.76) and structural connectivity (mean d = 1.04). The examination of the largest effect size indicated that the outcomes of functional and structural studies were comparable (Q = 2.17, p > 0.05). Conversely, consideration of effect size estimates for all significant effects suggest that reported effect sizes in structural connectivity studies were more variable than in functional connectivity studies, and that there was a significant lack of homogeneity across the modalities (Q = 6.928, p = 0.008). Given the more variable profile of effect sizes associated with structural connectivity, these data may suggest that structural imaging methods are more sensitive to a wider range of effects, as opposed to functional studies which may only be able to determine large effects. These conclusions are limited by methodological considerations, and require further investigation involving larger samples, multiple genes, and novel analysis techniques for confirmation

Requirements engineering in software product line engineering

The final publication is available at Springer via http://dx.doi.org/10.1007/s00766-013-0189-0Many attempts have been made to increase the productivity and quality of software products based on software reuse. Software product line practice is one such approach, one that focuses on developing a family of products which have a majority of features in common. Hence, there are numerous requirements that are common across the family, but others are unique to individual products. Traditional requirements engineering methods were conceived to deal with single product requirements and are usually not flexible enough to address the needs arising from reusing requirements for a family of products. There is also the additional burden of correctly identifying and engineering both product-line-wide requirements and product-specific requirements as well as evolving them. Therefore, in this special issue, we want to highlight the importance and the role of requirements engineering for product line development as well as to provide insights into the state of the art in the field.Insfrán Pelozo, CE.; Chastek, G.; Donohoe, P.; Sampaio Do Prado Leite, JC. (2014). Requirements engineering in software product line engineering. Requirements Engineering. 19(4):331-332. doi:10.1007/s00766-013-0189-0S331332194Clements P, Northrop LM (2001) Software product lines: practices and patterns. Addison-Wesley, BostonDerakhshanmanesh M, Fox J, Ebert J (2012) Adopting feature-centric reuse of requirements assets: an industrial experience report. First international workshop on requirements engineering practices on software product line engineering, Salvador, BrazilKuloor C, Eberlein A (2002) Requirements engineering for software product lines, proceedings of the 15th international conference on software and systems engineering and their applications (ICSSEA’02), Paris, FranceNorthrop LM, Clements P (2013) A framework for software product line practice. Software engineering institute. http://www.sei.cmu.edu/productlines/tools/framework/index.cfm . Accessed 22 July 2013Yu Y, Lapouchnian A, Liaskos S, Mylopoulos J, Leite JCSP (2008) From Goals to High-Variability Software Design. Foundations of Intelligent Systems, 17th International Symposium Proceedings. ISMIS 2008. Springer Lecture Notes in Computer Science, 4994: 1–1

Patient-reported outcome measures (PROMs) after laparoscopic cholecystectomy: systematic review

BACKGROUND: Healthcare requires patient feedback to improve outcomes and experience. This study undertook a systematic review of the depth, variability, and digital suitability of current patient-reported outcome measures (PROMs) in patients undergoing laparoscopic cholecystectomy. METHODS: A PROSPERO-registered (registration number CRD42021261707) systematic review was undertaken for all relevant English language articles using PubMed version of MEDLINE, Scopus, and Web of Science electronic databases in June 2021. The search used Boolean operators and wildcards and included the keywords: laparoscopic cholecystectomy AND patient outcome OR patient-reported outcome OR patient-reported outcome measure OR PRO OR PROM. Medical Subjects Heading terms were used to search PubMed and Scopus. Articles published from 1 January 2011 to 2 June 2021 were included. RESULTS: A total of 4960 individual articles were reviewed in this study, of which 44 were found to evaluate PROMs in patients undergoing laparoscopic cholecystectomy and underwent methodological index for non-randomized studies (MINORS) grading. Twenty-one articles spanning 19 countries and four continents met all inclusion criteria and were included in the qualitative data synthesis. There was significant heterogeneity in PROMs identified with eight different comprehensive PROM tools used in the 21 studies. There was wide variation in the time points at which PROMs were recorded. Fourteen of 21 studies recorded PROMs before and after surgery, and 7 of 21 recorded PROMs only after surgery. Follow-up intervals ranged from 3 days to 2 years after surgery. CONCLUSIONS: This study identified that while post-laparoscopic cholecystectomy PROMs are infrequently measured currently, tools are widely available to achieve this in clinical practice. PROMs may not capture all the outcomes but should be incorporated into future cholecystectomy outcome research. The EQ-5D™ (EuroQoL Group, Rotterdam, the Netherlands) provides a simple platform for the modern digital era

Cognitive predictors of social and occupational functioning in early psychosis : a systematic review and meta-analysis of cross-sectional and longitudinal data

Many individuals with early psychosis experience impairments in social and occupational function. Identification of modifiable predictors of function such as cognitive performance has the potential to inform effective treatments. Our aim was to estimate the strength of the relationship between psychosocial function in early psychosis and different domains of cognitive and social cognitive performance. We conducted a systematic review and meta-analysis of peer-reviewed, cross-sectional, and longitudinal studies examining cognitive predictors of psychosocial function. Literature searches were conducted in PsycINFO, PubMed, and reference lists of relevant articles to identify studies for inclusion. Of the 2565 identified, 46 studies comprising 3767 participants met inclusion criteria. Separate meta-analyses were conducted for 9 cognitive domains. Pearson correlation values between cognitive variables and function were extracted. All cognitive domains were related to psychosocial function both cross-sectionally and longitudinally. Importantly, these associations remained significant even after the effects of symptom severity, duration of untreated psychosis, and length of illness were accounted for. Overall, general cognitive ability and social cognition were most strongly associated with both concurrent and long-term function. Associations demonstrated medium effect sizes. These findings suggest that treatments targeting cognitive deficits, in particular those focusing on social cognition, are likely to be important for improving functional outcomes in early psychosis. [Abstract copyright: © The Author(s) 2021. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center.All rights reserved. For permissions, please email: [email protected].

Mechanics and dynamics of X-chromosome pairing at X inactivation

At the onset of X-chromosome inactivation, the vital process whereby female mammalian cells equalize X products with respect to males, the X chromosomes are colocalized along their Xic (X-inactivation center) regions. The mechanism inducing recognition and pairing of the X’s remains, though, elusive. Starting from recent discoveries on the molecular factors and on the DNA sequences (the so-called "pairing sites") involved, we dissect the mechanical basis of Xic colocalization by using a statistical physics model. We show that soluble DNA-specific binding molecules, such as those experimentally identified, can be indeed sufficient to induce the spontaneous colocalization of the homologous chromosomes but only when their concentration, or chemical affinity, rises above a threshold value as a consequence of a thermodynamic phase transition. We derive the likelihood of pairing and its probability distribution. Chromosome dynamics has two stages: an initial independent Brownian diffusion followed, after a characteristic time scale, by recognition and pairing. Finally, we investigate the effects of DNA deletion/insertions in the region of pairing sites and compare model predictions to available experimental data

Participant perspectives on cognitive remediation and social recovery in early psychosis (CReSt-R): an acceptability study

Aim: Psychosis spectrum disorders continue to rank highly among causes of disability. This has resulted in efforts to expand the range of treatment targets beyond symptom remission to include other recovery markers, including social and occupational function and quality of life. Although the efficacy of psychosocial interventions in early psychosis has been widely reported, the acceptability of these interventions is less well-known. This study explores the participant perspective on a novel, psychosocial intervention combining cognitive remediation and social recovery therapy. Methods: We employed a qualitative research design, based on semi-structured interviews and reflexive thematic analysis. Six participants with early psychosis were recruited from the intervention arm of a randomized pilot study, three women and three men, aged between 22 and 27 years. Results: Four themes were developed through the analytical process, namely, (1) a solid therapeutic foundation, (2) multi-directional flow of knowledge, (3) a tailored toolset, and (4) an individual pathway to recovery. Participants also provided pragmatic feedback about how to improve the delivery of the therapy assessments and intervention. Both the themes and pragmatic feedback are described. Conclusions: People with early psychosis described the intervention as acceptable, engaging, helpful and person-centred, suggesting its potential role in a multicomponent therapy model of early intervention in psychosis services. Participants in this study also highlight the importance of an individualized approach to therapy, the vital role of the therapeutic relationship and the ecological validity and value of adopting an assertive outreach delivery, providing therapy outside a conventional clinic setting

Childhood-Diagnosed ADHD, Symptom Progression, and Reversal Learning in Adulthood

Objective: ADHD persists in up to 60% into adulthood, and the reasons for persistence are not fully understood. The objective of this study was to characterize the neurofunctional basis of decision making in those with a childhood diagnosis of ADHD with either persistent or remitted symptoms in adulthood versus healthy control participants. Method: Thirty-two adults diagnosed with ADHD as children were split into persistent (n = 18) or remitted (n = 14) ADHD groups. Their neural activity and neurofunctional connectivity during a probabilistic reversal learning task were compared with 32 healthy controls. Results: Remitters showed significantly higher neural connectivity in final reversal error and probabilistic error conditions, and persisters depict higher neural connectivity in reversal errors than controls at a family-wise error (FWE) corrected whole-brain corrected threshold. Conclusion: Remitters may have utilized higher neural connectivity than controls to make successful decisions. Also, remitters may have utilized compensatory strategies to override any potential underlying ADHD deficits