A CRISPR/Cas9 based engineering tool to activate expression of multiple genes individually or in any specific combination

Engineering of cells by overexpression or knock-down/out of individual genes has demonstrated that in most cases the manipulation of single genes is not sufficient to alter a cellular phenotype. Rather, multiple genes involved in a pathway need to be manipulated. Especially in mammalian cells such as CHO, where clonal variation is large, it has been difficult to unequivocally assess whether the observed change in phenotype is due to such clonal variation or the engineered gene. This can in part be overcome by testing multiple subclones, however, once it comes to engineering multiple genes and combinations thereof, the required workload quickly becomes prohibitive. We here present a simple technology for successive and/or specific activation of multiple genes integrated into a single genomic locus, which presents a potential solution to this problem. The technology consists of a vector containing multiple genes to be engineered or copies of the same gene. The promoters of these genes/gene copies are separated from the translation start site by repressor elements, flanked by individual guide RNA (gRNA) target sites. After integration of the construct into the genome and clone selection, these repressor elements can be removed by transfection with Cas9 and the corresponding pair of gRNAs that target the repressor of the gene(s) to be activated. Efficiency of target gene activation was in the range of 20-30% of the population for individual genes. Using 4 different fluorescent genes, the success of the technology was shown by activation of different combinations of these genes, followed by sorting of cells with the correct combination of required target genes activated. For pathway engineering studies, the selected genes can be expressed linked to these fluorescent genes e.g. via an IRES or a 2A self-cleaving peptide and cells with the desired co-expression pattern sorted, thus obviating the necessity to subclone for subsequent phenotypic characterization of the engineered cells. The technology provides a rapid procedure to assess the effect of gene combination on cellular behavior. Please click Additional Files below to see the full abstract

Investigation into the Hybrid Production of a Superelastic Shape Memory Alloy with Additively Manufactured Structures for Medical Implants

The demographic change in and the higher incidence of degenerative bone disease have resulted in an increase in the number of patients with osteoporotic bone tissue causing. amongst other issues, implant loosening. Revision surgery to treat and correct the loosenings should be avoided, because of the additional patient stress and high treatment costs. Shape memory alloys (SMA) can help to increase the anchorage stability of implants due to their superelastic behavior. The present study investigates the potential of hybridizing NiTi SMA sheets with additively manufactured Ti6Al4V anchoring structures using laser powder bed fusion (LPBF) technology to functionalize a pedicle screw. Different scanning strategies are evaluated, aiming for minimized warpage of the NiTi SMA sheet. For biomechanical tests, functional samples were manufactured. A good connection between the additively manufactured Ti6Al4V anchoring structures and NiTi SMA substrate could be observed though crack formation occurring at the transition area between the two materials. These cracks do not propagate during biomechanical testing, nor do they lead to flaking structures. In summary, the hybrid manufacturing of a NiTi SMA substrate with additively manufactured Ti6Al4V structures is suitable for medical implants

What\u27s in a Phenotype?

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Antibodies Against Glutamic Acid Decarboxylase 65 Are Locally Produced in the CSF and Arise During Affinity Maturation

Background and Objectives Antibodies (Abs) against the cytoplasmic protein glutamic acid decarboxylase 65 (GAD65) are detected in patients with neurologic syndromes together referred to as GAD65-Ab spectrum disorders. The response of some of these patients to plasma exchange or immunoglobulins indicates that GAD65-Abs could contribute to disease pathogenesis at least at some stages of disease. However, the involvement of GAD65-reactive B cells in the CNS is incompletely understood. Methods We studied 7 patients with high levels of GAD65-Abs and generated monoclonal Abs (mAbs) derived from single cells in the CSF. Sequence characteristics, reactivity to GAD65, and the role of somatic hypermutations of the mAbs were analyzed. Results Twelve CSF-derived mAbs were generated originating from 3 patients with short disease duration, and 7/12 of these mAbs (58%) were GAD65 reactive in at least 1 detection assay. Four of 12 (33%) were definitely positive in all 3 detection assays. The intrathecal anti-GAD65 response was polyclonal. GAD65-Abs were mostly of the IgG1 subtype and had undergone affinity maturation. Reversion of 2 GAD65-reactive mAbs to their corresponding germlineencoded unmutated common ancestors abolished GAD65 reactivity. Discussion GAD65-specific B cells are present in the CNS and represent a sizable fraction of CSF B cells early in the disease course. The anti-GAD65 response in the CSF is polyclonal and shows evidence of antigen-driven affinity maturation required for GAD65 recognition. Our data support the hypothesis that the accumulation of GAD65-specific B cells and plasma cells in the CSF is an important feature of early disease stages

Persistence of functional memory B cells recognizing SARS-CoV-2 variants despite loss of specific IgG

Although some COVID-19 patients maintain SARS-CoV-2-specific serum immunoglobulin G (IgG) for more than 6 months postinfection, others eventually lose IgG levels. We assessed the persistence of SARS-CoV-2-specific B cells in 17 patients, 5 of whom had lost specific IgGs after 5–8 months. Differentiation of blood-derived B cells in vitro revealed persistent SARS-CoV-2-specific IgG B cells in all patients, whereas IgA B cells were maintained in 11. Antibodies derived from cultured B cells blocked binding of viral receptor-binding domain (RBD) to the cellular receptor ACE-2, had neutralizing activity to authentic virus, and recognized the RBD of the variant of concern Alpha similarly to the wild type, whereas reactivity to Beta and Gamma were decreased. Thus, differentiation of memory B cells could be more sensitive for detecting previous infection than measuring serum antibodies. Understanding the persistence of SARS-CoV-2-specific B cells even in the absence of specific serum IgG will help to promote long-term immunity

Left Shifting of Language Related Activity Induced by Bihemispheric tDCS in Postacute Aphasia Following Stroke

Both anodal transcranial direct current stimulation (tDCS) of the left IFG and cathodal stimulation of the right IFG were shown to improve rehabilitation of stroke patients with Broca’s aphasia. The study aimed at assessing the impact of a bihemispheric IFG stimulation compared to sham on postacute non-fluent aphasia. Twelve patients with non-fluent aphasia were included at least 4 weeks following cerebral stroke. Ten daily sessions of 2 mA bihemispheric verum or sham tDCS (anode on left IFG and cathode on right IFG) were performed concomitantly with individual language therapy in a double-blinded randomized controlled study with parallel group design. Language functions [i.e., communication (ANELT), picture naming and the Aachen aphasia test (AAT)] were assessed up to 1 month following tDCS. The picture naming task significantly improved (increased number of nouns) at the end of the tDCS procedure in the verum but not sham group. Improvements in the picture naming task and the communication task of the AAT at 4 weeks after tDCS procedure were only seen in the verum group. In patients with postacute cerebral stroke, repeated sessions of tDCS applied on both IFG concomitantly with language therapy were able to induce immediate effects on picture naming presumably due to an early left shift of language-associated function that maintained for 4 weeks. Effects on clinically relevant communicative abilities are likely

Genetic Variants in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis: A Bayesian Approach and Systematic Review.

A number of genome-wide association studies (GWASs) and meta-analyses of genetic variants have been performed in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. We reinterpreted previous studies using false-positive report probability (FPRP) and Bayesian false discovery probability (BFDP). This study searched publications in PubMed and Excerpta Medica Database (EMBASE) up to February 2018. Identification of noteworthy associations were analyzed using FPRP and BFDP, and data (i.e., odds ratio (OR), 95% confidence interval (CI), p-value) related to significant associations were separately extracted. Using filtered gene variants, gene ontology (GO) enrichment analysis and protein⁻protein interaction (PPI) networks were performed. Overall, 241 articles were identified, and 7 were selected for analysis. Single nucleotide polymorphisms (SNPs) discovered by GWASs were shown to be noteworthy, whereas only 27% of significant results from meta-analyses of observational studies were noteworthy. Eighty-five percent of SNPs with borderline p-values (5.0 × 10-8 < p < 0.05) in GWASs were found to be noteworthy. No overlapping SNPs were found between PR3-ANCA and MPO-ANCA vasculitis. GO analysis revealed immune-related GO terms, including "antigen processing and presentation of peptide or polysaccharide antigen via major histocompatibility complex (MHC) class II", "interferon-gamma-mediated (IFN-γ) signaling pathway". By using FPRP and BFDP, network analysis of noteworthy genetic variants discovered genetic risk factors associated with the IFN-γ pathway as novel mechanisms potentially implicated in the complex pathogenesis of ANCA-associated vasculitis