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Laboratory automation effectively increases the throughput in sample analysis, reduces human errors in sample processing, as well as simplifies and accelerates the overall logistics. Automating diagnostic testing workflows in peripheral laboratories and also in near-patient settings -like hospitals, clinics and epidemic control checkpoints- is advantageous for the simultaneous processing of multiple samples to provide rapid results to patients, minimize the possibility of contamination or error during sample handling or transport, and increase efficiency. However, most automation platforms are expensive and are not easily adaptable to new protocols. Here, we address the need for a versatile, easy-to-use, rapid and reliable diagnostic testing workflow by combining open-source modular automation (Opentrons) and automation-compatible molecular biology protocols, easily adaptable to a workflow for infectious diseases diagnosis by detection on paper-based diagnostics. We demonstrated the feasibility of automation of the method with a low-cost Neisseria meningitidis diagnostic test that utilizes magnetic beads for pathogen DNA isolation, isothermal amplification, and detection on a paper-based microarray. In summary, we integrated open-source modular automation with adaptable molecular biology protocols, which was also faster and cheaper to perform in an automated than in a manual way. This enables a versatile diagnostic workflow for infectious diseases and we demonstrated this through a low-cost N. meningitidis test on paper-based microarrays., QC 20240715

QC 20231128

3D bioprinting is an acclaimed technology to develop architecturally significant tissue models, however most bioinks require a secondary support structure to create a clinically relevant sized model. In this work, we develop a 3D sacrificial support structure of polyvinyl alcohol (PVA) with decellularized liver extracellular matrix (dECM) bioink with HepG2 cells. The PVA backbone imparts its 3D structure to dECM and dissolves in cell culture media. We evaluated the PVA dissolution using refractometry and microscopic observation. We further tested the crosslinking of dECM with a PEG-based crosslinker using scanning electron microscopy (SEM). Alamar blue assay and gene expression analysis results demonstrated an increase in cell proliferation within the 3D structure., Part of ISBN 9781733419048QC 20231116

The liver exhibits complex geometrical morphologies of hepatic cells arranged in a hexagonal lobule with an extracellular matrix (ECM) organized in a specific pattern on a multi-scale level. Previous studies have utilized 3D bioprinting and microfluidic perfusion systems with various biomaterials to develop lobule-like constructs. However, they all lack anatomical relevance with weak control over the size and shape of the fabricated structures. Moreover, most biomaterials lack liver-specific ECM components partially or entirely, which might limit their biomimetic mechanical properties and biological functions. Here, we report 3D bioprinting of a sacrificial PVA framework to impart its trilobular hepatic structure to the decellularized liver extracellular matrix (dLM) hydrogel with polyethylene glycol-based crosslinker and tyrosinase to fabricate a robust multi-scale 3D liver construct. The 3D trilobular construct exhibits higher crosslinking, viscosity (182.7 ± 1.6 Pa·s), and storage modulus (2554 ± 82.1 Pa) than non-crosslinked dLM. The co-culture of HepG2 liver cells and NIH 3T3 fibroblast cells exhibited the influence of fibroblasts on liver-specific activity over time (7 days) to show higher viability (90–91.5%), albumin secretion, and increasing activity of four liver-specific genes as compared to the HepG2 monoculture. This technique offers high lumen patency for the perfusion of media to fabricate a densely populated scaled-up liver model, which can also be extended to other tissue types with different biomaterials and multiple cells to support the creation of a large functional complex tissue., QC 20221108

Bioprinting is an acclaimed technique that allows the scaling of 3D architectures in an organized pattern but suffers from a scarcity of appropriate bioinks. Decellularized extracellular matrix (dECM) from xenogeneic species has garnered support as a biomaterial to promote tissue-specific regeneration and repair. The prospect of developing dECM-based 3D artificial tissue is impeded by its inherent low mechanical properties. In recent years, 3D bioprinting of dECM-based bioinks modified with additional scaffolds has advanced the development of load-bearing constructs. However, previous attempts using dECM were limited to low-temperature bioprinting, which is not favorable for a longer print duration with cells. Here, we report the development of a multi-material decellularized liver matrix (dLM) bioink reinforced with gelatin and polyethylene glycol to improve rheology, extrudability, and mechanical stability. This shear-thinning bioink facilitated extrusion-based bioprinting at 37 degrees C with HepG2 cells into a 3D grid structure with a further enhancement for long-term applications by enzymatic crosslinking with mushroom tyrosinase. The heavily crosslinked structure showed a 16-fold increase in viscosity (2.73 Pa s(-1)) and a 32-fold increase in storage modulus from the non-crosslinked dLM while retaining high cell viability (85-93%) and liver-specific functions. Our results show that the cytocompatible crosslinking of dLM bioink at physiological temperatures has promising applications for extended 3D-printing procedures., QC 20220812

Objectives: In many resource-limited health systems, point-of-care tests (POCTs) are the only means for clinical patient sample analyses. However, the speed and simplicity of POCTs also makes their use appealing to clinicians in high-income countries (HICs), despite greater laboratory accessibility. Although also part of the clinical routine in HICs, clinician perceptions of the utility of POCTs are relatively unknown in such settings as compared with others. In a Swedish paediatric emergency department (PED) where POCT use is routine, we aimed to characterise healthcare providers' perspectives on the clinical utility of POCTs and explore their implementation in the local setting; to discuss and compare such perspectives, to those reported in other settings; and finally, to gather requests for ideal novel POCTs. Design: Qualitative focus group discussions study. A data-driven content analysis approach was used for analysis. Setting: The PED of a secondary paediatric hospital in Stockholm, Sweden. Participants: Twenty-four healthcare providers clinically active at the PED were enrolled in six focus groups. Results: A range of POCTs was routinely used. The emerging theme Utility of our POCT use is double-edged illustrated the perceived utility of POCTs. While POCT services were considered to have clinical and social value, the local routine for their use was named to distract clinicians from the care for patients. Requests were made for ideal POCTs and their implementation. Conclusion: Despite their clinical integration, deficient implementation routines limit the benefits of POCT services to this well-resourced paediatric clinic. As such deficiencies are shared with other settings, it is suggested that some characteristics of POCTs and of their utility are less related to resource level and more to policy deficiency. To address this, we propose the appointment of skilled laboratory personnel as ambassadors to hospital clinics offering POCT services, to ensure higher

QC 20211108

(1) Immunization with pneumococcal conjugate vaccines has decreased the burden of community-acquired pneumonia (CAP) in children and likely led to a shift in CAP etiology. (2) The Trial of Respiratory infections in children for ENhanced Diagnostics (TREND) enrolled children 1-59 months with clinical CAP according to the World Health Organization (WHO) criteria at Sachs' Children and Youth Hospital, Stockholm, Sweden. Children with rhonchi and indrawing underwent "bronchodilator challenge". C-reactive protein and nasopharyngeal PCR detecting 20 respiratory pathogens, were collected from all children. Etiology was defined according to an a priori defined algorithm based on microbiological, biochemical, and radiological findings. (3) Of 327 enrolled children, 107 (32%) required hospitalization; 91 (28%) received antibiotic treatment; 77 (24%) had a chest X-ray performed; and 60 (18%) responded to bronchodilator challenge. 243 (74%) episodes were classified as viral, 11 (3%) as mixed viral-bacterial, five (2%) as bacterial, two (0.6%) as atypical bacterial and 66 (20%) as undetermined etiology. After exclusion of children responding to bronchodilator challenge, the proportion of bacterial and mixed viral-bacterial etiology was 1% and 4%, respectively. (4) The novel TREND etiology algorithm classified the majority of clinical CAP episodes as of viral etiology, whereas bacterial etiology was uncommon. Defining CAP in children <5 years is challenging, and the WHO definition of clinical CAP is not suitable for use in children immunized with pneumococcal conjugate vaccines.

Decellularized extracellular matrix is a tissue-specific biomaterial that recapitulates the complexity of the inherent tissue environment to elicit cellular response. Here, a multi-material decellularized liver (dLM)-based bioink with gelatin is developed and polyethylene glycol crosslinking is used to enhance the viscoelasticity of the dLM. We evaluated the necessity of a post-printing secondary cross-linker mushroom tyrosinase to improve robustness and long term stability. We further demonstrate it's biocompatibility using liver specific gene analysis of HepG2 cells., Part of ISBN 9781733419031QC 20230614

While diagnostics are critical for accurate and timely diagnosis, gold-standard diagnostic tests are commonly high- performance laboratory-based tests that require multi-step protocols for complex sample processing and highly trained personnel, both scarce in low-resource settings. Here, we address the need for an easy-to-use, rapid and reliable diagnostic testing pipeline by presenting a solution combining open-source modular automation and automation compatible microfluidics, easily adaptable to a pipeline for infectious diseases diagnosis. We demonstrate an automation compatible microfluidics pipeline for Neisseria meningitidis diagnosis by on-chip nucleic acids isolation and isothermal amplification, as well as pathogen detection on a paper-based microarray., QC 20211002

Cranial radiotherapy in children has detrimental effects on cognition, mood, and social competence in young cancer survivors. Treatments harnessing hippocampal neurogenesis are currently of great relevance in this context. Lithium, a well-known mood stabilizer, has both neuroprotective, pro-neurogenic as well as antitumor effects, and in the current study we introduced lithium treatment 4 weeks after irradiation. Female mice received a single 4 Gy whole-brain radiation dose on postnatal day (PND) 21 and were randomized to 0.24% Li2CO3 chow or normal chow from PND 49 to 77. Hippocampal neurogenesis was assessed on PND 77, 91, and 105. We found that lithium treatment had a pro-proliferative effect on neural progenitors, but neuronal integration occurred only after it was discontinued. Also, the treatment ameliorated deficits in spatial learning and memory retention observed in irradiated mice. Gene expression profiling and DNA methylation analysis identified two novel factors related to the observed effects, Tppp, associated with microtubule stabilization, and GAD2/65, associated with neuronal signaling. Our results show that lithium treatment reverses irradiation-induced loss of hippocampal neurogenesis and cognitive impairment even when introduced long after the injury. We propose that lithium treatment should be intermittent in order to first make neural progenitors proliferate and then, upon discontinuation, allow them to differentiate. Our findings suggest that pharmacological treatment of cognitive so-called late effects in childhood cancer survivors is possible., QC 20200408

The architecture and functional interactions of the cerebral cortex are fascinating in their complexity of billions of neurons and glial cells connected in intricate circuitries and spatial regions. During development, transcription factors and chromatin modifiers work together to coordinate gene expression programs that drive the formation of the cerebral cortex. Notably, neurological aberrations can arise when perturbation occurs in these tightly regulated programs. Although our understanding of corticogenesis has advanced with the identification of master regulators of neural development, we only have rudimentary knowledge of the molecular mechanisms these factors use to execute the developmental programs. The aim of this thesis is to investigate the transcriptional mechanisms of gene regulation by key transcription factors involved in telencephalic development and disease, using primarily human neural progenitors as an in vitro model of development. Study I reports a novel mechanism through an interaction between the co-repressor NCOR and the transcription factor FOXP2. Genome-wide mapping of common binding sites of NCOR/FOXP2 in human iPS-derived neural progenitors included two putative regulatory elements in the proximity of the SLITRK gene cluster. Chromosome conformation capture (3C) confirmed the interaction between the SLITRK cluster gene promoters and the regulatory elements where FOXP2/NCOR binds, which proposes a possible role for this regulatory mechanism in accurate development and possibly evolution of vocal and motor skills. Study II demonstrates that the transcription factor PAX6 can function as a repressor and recruit the histone demethylase KDM5C to repress a subset of genes involved in Notch signaling, which is critical for several neuronal functions, proposing that neurodevelopmental aberrations by PAX6 and/or KDM5C mutations maybe be associated with defects in Notch signaling. Study III explores the gene regulation effects on pluripotency medi

QCR 20221108