Identification and Characterization of the Amphioxus Lck and Its Associated Tyrosine Phosphorylation-Dependent Inhibitory LRR Receptor

Amphioxus (e.g., Branchiostoma belcheri, Bb) has recently emerged as a new model for studying the origin and evolution of vertebrate immunity. Mammalian lymphocyte-specific tyrosine kinase (Lck) plays crucial roles in T cell activation, differentiation and homeostasis, and is reported to phosphorylate both the ITIM and ITSM of PD-1 to induce the recruitment of phosphatases and thus the inhibitory function of PD-1. Here, we identified and cloned the amphioxus homolog of human Lck. By generating and using an antibody against BbLck, we found that BbLck is expressed in the amphioxus gut and gill. Through overexpression of BbLck in Jurkat T cells, we found that upon TCR stimulation, BbLck was subjected to tyrosine phosphorylation and could partially rescue Lck-dependent tyrosine phosphorylation in Lck-knockdown T cells. Mass spectrometric analysis of BbLck immunoprecipitates from immunostimulants-treated amphioxus, revealed a BbLck-associated membrane-bound receptor LRR (BbLcLRR). By overexpressing BbLcLRR in Jurkat T cells, we demonstrated that BbLcLRR was tyrosine phosphorylated upon TCR stimulation, which was inhibited by Lck knockdown and was rescued by overexpression of BbLck. By mutating single tyrosine to phenylalanine (Y-F), we identified three tyrosine residues (Y539, Y655, and Y690) (3Y) of BbLcLRR as the major Lck phosphorylation sites. Reporter gene assays showed that overexpression of BbLcLRR but not the BbLcLRR-3YF mutant inhibited TCR-induced NF-κB activation. In Lck-knockdown T cells, the decline of TCR-induced IL-2 production was reversed by overexpression of BbLck, and this reversion was inhibited by co-expression of BbLcLRR but not the BbLcLRR-3YF mutant. Sequence analysis showed that the three tyrosine-containing sequences were conserved with the tyrosine-based inhibition motifs (ITIMs) or ITIM-like motifs. And TCR stimulation induced the association of BbLcLRR with tyrosine phosphatases SHIP1 and to a lesser extent with SHP1/2. Moreover, overexpression of wild-type BbLcLRR but not its 3YF mutant inhibited TCR-induced tyrosine phosphorylation of multiple signaling proteins probably via recruiting SHIP1. Thus, we identified a novel immunoreceptor BbLcLRR, which is phosphorylated by Lck and then exerts a phosphorylation-dependent inhibitory role in TCR-mediated T-cell activation, implying a mechanism for the maintenance of self-tolerance and homeostasis of amphioxus immune system and the evolutionary conservatism of Lck-regulated inhibitory receptor pathway.

Application of long-read sequencing for robust identification of correct alleles in genome edited animals

Recent developments in CRISPR/Cas9 genome editing tools have facilitated the introduction of more complex alleles, often spanning genetic intervals of several kilobases, directly into the embryo. These techniques often produce mosaic founder animals and the introduction of donor templates, via homologous directed repair, can be erroneous or incomplete. Newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the desired edit(s) together with founder mosaicism. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore long read sequencing of the targeted locus. Taking advantage of sequencing the entire length of the segment in each single read, we were able to determine whether the entire intended mutant sequence was present in both mosaic founders and their offspring.

Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. The 3272–26A>G and 3849+10kbC>T CFTR mutations alter the correct splicing of the CFTR gene, generating new acceptor and donor splice sites respectively. Here we develop a genome editing approach to permanently correct these genetic defects, using a single crRNA and the Acidaminococcus sp. BV3L6, AsCas12a. This genetic repair strategy is highly precise, showing very strong discrimination between the wild-type and mutant sequence and a complete absence of detectable off-targets. The efficacy of this gene correction strategy is verified in intestinal organoids and airway epithelial cells derived from CF patients carrying the 3272–26A>G or 3849+10kbC>T mutations, showing efficient repair and complete functional recovery of the CFTR channel. These results demonstrate that allele-specific genome editing with AsCas12a can correct aberrant CFTR splicing mutations, paving the way for a permanent splicing correction in genetic diseases.

−254C>G SNP in the TRPC6 Gene Promoter Influences Its Expression via Interaction with the NF-κB Subunit RELA in Steroid-Resistant Nephrotic Syndrome Children

This study is aimed at exploring the mechanism by which the −254C>G single nucleotide polymorphism (SNP) on the transient receptor potential cation channel 6 (TRPC6) gene promoter could increase its activation in steroid-resistant nephrotic syndrome children of China. Plasmids containing the TRPC6 promoter region (with the −254C or G allele) were constructed and then transfected into human embryonic kidney (HEK) 293T cells and human podocytes. Luciferase assays were used to test the promoter activity in both cell lines with or without tumor necrosis factor-α (TNF-α) treatment, and chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) analysis was used to verify the transcription factor that could bind to this mutant sequence. Luciferase results indicate that the activity of the mutant promoter was greater than that of the normal promoter of the TRPC6 gene in both cell lines. We further predicted and verified that this variation was mediated by the nuclear factor kappa B (NF-κB) subunit RELA, and TNF-α significantly enhanced the transcription activity of TRPC6 with the −254G allele. In conclusion, the −254C>G SNP is a gain-of-function variation of the TRPC6 gene, and it is also an early and effective factor for predicting steroid-resistant nephrotic syndrome (SRNS) in Chinese children.

A Highly Verified Assay for KRAS Mutation Detection in Tissue and Plasma of Lung, Colorectal, and Pancreatic Cancer

Context.— KRAS Mutation Test v2 is used for the qualitative detection and identification of 28 mutations in exons 2, 3, and 4 of the human KRAS gene. Objective.— To verify the performance of KRAS Mutation Test v2 and to evaluate its accuracy by correlation with a next-generation sequencing method on Illumina MiSeq. Design.— In this study, we used formalin-fixed, paraffin-embedded tissue and plasma specimens from non–small cell lung cancer, colorectal cancer, and pancreatic cancer patients. Results of specificity, precision, analytical sensitivity, and accuracy as compared with a MiSeq method are reported. Results.— The KRAS Mutation Test v2 demonstrated exquisite sensitivity and specificity and broad coverage of KRAS mutations. Precision was 100% (108 of 108) across all samples, operators, and instruments for formalin-fixed, paraffin-embedded tissue and 99.8% (615 of 616) for plasma. Analytical sensitivity was high with detection of 1% mutant sequence in formalin-fixed, paraffin-embedded tissue samples and as low as 25 mutant sequence copies/mL for plasma samples. The test also showed high overall concordance for formalin-fixed, paraffin-embedded tumor tissue as well as for plasma specimens when compared with MiSeq sequencing results. Conclusions.— The KRAS Mutation Test v2 is a highly robust, reproducible, and sensitive test for the qualitative detection of 28 mutations in exons 2, 3, and 4 of the KRAS gene in both solid (tissue) and liquid (plasma) biopsies from colorectal cancer, non–small cell lung cancer, and pancreatic cancer, and is a convenient option for KRAS mutation testing.

Improved Detection of Mutated Human Cytomegalovirus UL97 by Pyrosequencing

ABSTRACT Ganciclovir (GCV) resistance frequently occurs upon prolonged treatment of ongoing active human cytomegalovirus (HCMV) infection in individuals with immature or compromised immune functions (e.g., recipients of solid-organ and hematopoietic stem cell transplants). Using pyrosequencing (PSQ), we established fast and sensitive detection of GCV resistance-associated mutations occurring in the HCMV open reading frame UL97. These mutations have been repeatedly associated with clinical treatment failure. We designed four PSQ assays and evaluated them by analyzing mixtures of plasmids or bacterial artificial chromosome-derived viruses containing UL97 wild-type and mutant sequences. A minimum level of 6% mutant sequence variants could be detected in these mixtures. In order to further evaluate the novel PSQ assays, we tested clinical specimens from patients with active HCMV infections. The results were compared with those obtained by conventional dideoxy chain terminator sequencing. As the PSQ method was more sensitive in detecting minor HCMV mutant fractions in a wild-type population, it is suggested that pyrosequencing is a useful tool for the early detection of emerging GCV-resistant HCMV in GCV-treated patients.

Loss of the N-Linked Glycan at Residue 173 of Human Parainfluenza Virus Type 1 Hemagglutinin-Neuraminidase Exposes a Second Receptor-Binding Site

ABSTRACT BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-1 (rSeV[hPIV-1HN]) (I. V. Alymova, G. Taylor, T. Takimoto, T. H. Lin., P. Chand, Y. S. Babu, C. Li, X. Xiong, and A. Portner, Antimicrob. Agents Chemother. 48:1495-1502, 2004). The ability of BCX 2798 to select drug-resistant variants in vivo was examined. A variant with an Asn-to-Ser mutation at residue 173 (N173S) in HN was recovered from mice after a second passage of rSeV(hPIV-1HN) in the presence of BCX 2798 (10 mg/kg of body weight daily). The N173S mutant remained sensitive to BCX 2798 in neuraminidase inhibition assays but was more than 10,000-fold less sensitive to the compound in hemagglutination inhibition tests than rSeV(hPIV-1HN). Its susceptibility to BCX 2798 in plaque reduction assays was reduced fivefold and did not differ from that of rSeV(hPIV-1HN) in mice. The N173S mutant failed to be efficiently eluted from erythrocytes and released from cells. It demonstrated reduced growth in cell culture and superior growth in mice. The results for gel electrophoresis analysis were consistent with the loss of the N-linked glycan at residue 173 in the mutant. Sequence and structural comparisons revealed that residue 173 on hPIV-1 HN is located close to the region of the second receptor-binding site identified in Newcastle disease virus HN. Our study suggests that the N-linked glycan at residue 173 masks a second receptor-binding site on hPIV-1 HN.

Protein-tyrosine kinase CAKβ/PYK2 is activated by binding Ca2+/calmodulin to FERM F2 α2 helix and thus forming its dimer

CAKβ (cell adhesion kinase β)/PYK2 (proline-rich tyrosine kinase 2) is the second protein-tyrosine kinase of the FAK (focal adhesion kinase) subfamily. It is different from FAK in that it is activated following an increase in cytoplasmic free Ca2+. In the present study we have investigated how Ca2+ activates CAKβ/PYK2. Calmodulin-agarose bound CAKβ/PYK2, but not FAK, in the presence of CaCl2. An α-helix (F2-α2) present in the FERM (band four-point-one, ezrin, radixin, moesin homology) F2 subdomain of CAKβ/PYK2 was the binding site of Ca2+/calmodulin; a mutant of this region, L176A/Q177A (LQ/AA) CAKβ/PYK2, bound to Ca2+/calmodulin much less than the wild-type. CAKβ/PYK2 is known to be prominently tyrosine phosphorylated when overexpressed from cDNA. The enhanced tyrosine phosphorylation was inhibited by W7, an inhibitor of calmodulin, and by a cell-permeable Ca2+ chelator and was almost defective in the LQ/AA-mutant CAKβ/PYK2. CAKβ/PYK2 formed a homodimer on binding of Ca2+/calmodulin, which might then induce a conformational change of the kinase, resulting in transphosphorylation within the dimer. The dimer was formed at a free-Ca2+ concentration of 8–12 μM and was stable at 500 nM Ca2+, but dissociated to a monomer in a Ca2+-free buffer. The dimer formation of CAKβ/PYK2 FERM domain was partially defective in the LQ/AA-mutant FERM domain and was blocked by W7 and by a synthetic peptide with amino acids 168–188 of CAKβ/PYK2, but not by a peptide with its LQ/AA-mutant sequence. It is known that the F2-α2 helix is found immediately adjacent to a hydrophobic pocket in the FERM F2 lobe, which locks, in the autoinhibited FAK, the C-lobe of the kinase domain. Our results indicate that Ca2+/calmodulin binding to the FERM F2-α2 helix of CAKβ/PYK2 releases its kinase domain from autoinhibition by forming a dimer.

New Detection Method of V617F JAK2 Mutation; Its Use in a Clinical Setting.

Background: A single point mutation in the Janus 2 tyrosine kinase gene leading to a V617F substitution has been described in a large group of hematological pathologies such as Polycythemia Vera (PV), Essential Thrombocytaemia (ET), Idiopathic Myelofibrosis (IM) and unclassified Myeloproliferative disorders (MPDs). Mutated JAK2 is an essential biomarker which improves the understanding and classification of MPDs and offers a new target for specific therapeutics. Methods: We adapted the V617F genotyping by amplification mutation system (ARMS) PCR described by AmyV Jones and Nicholas C.P Cross and combined it with a capillary electrophoresis performed on the Agilent Bioanalyseur 2100. DNA quantification was determined by βglobin gene amplification by quantitative PCR.100ng of samples and controls were genotyped by a DNA ARMS assay, using a primer pair that specifically amplifies the mutant sequence. We chose the homozygous HEL cell line as a positive control systematically tested in each run. We investigated the sensitivity of the method by testing serial dilutions of 100% V617F homozygous HEL DNA into a non mutated DNA of PBL. We compared the sensitivity of simplex ARMS PCR method using a primer pair that only amplifies the mutant sequence versus a multiplex ARMS PCR method using a two primer pairs to specifically amplify the normal and mutant sequence plus a positive control band in a single reaction. We genotyped over 6 months, 70 patients who had been screened for MPDs, BCR-ABL fusion negative, PV, ET, and isolated polycythaemia. Furthermore, we intend to collaborate with IPSOGEN SAS to compare our results with their new licensed JAK2 mutation test based on the pioneering work of Dr.Vainchenker. Results: Sensitivity of the mutliplex and simplex techniques respectively ranged from 100% to 1% and 100% to 0.1%. We found evidence that the sensitivity of the technique was imrpoved by using a simplex ARMS PCR followed by a capillary electrophoresis than a multiplex one. Of the 70 samples with a known or suspected diagnosis of MPD, 43(61%) were negative for V617F JAK2 mutation and 27(39%) were positive. The V617F JAK2 mutation was detected in 43% (3/7) PV, 42% (22/53) MPDs, BCR-ABL fusion gene negative, 25% (2/8) ET and none of isolated polycythaemia. Conclusions: The poor frequency of positive V617F V617F JAK2 mutation in patients screened for an evocated PV might be due to the inclusion in PV group of patients who do not meet diagnostic criteria for PV. The V617F JAK2 mutation in MPDs can be detected by various methods. ARMS associated with a capillary electrophoresis seems to be easy to use and reproductible for detection of single base G → T substitution in JAK2 mutation. In addition, the simplex approach improves the sensitivity of the technique leading to V617F JAK2 mutation. This qualitative test leads to the conclusions: presence of the V617F JAK2 mutation “or absence of detection of the V617F JAK2 mutation and is essential in diagnosis and classification of MPDs whereas quantitative approach will improve the management of therapeutic response when targeted therapy against V617F JAK2 mutation will be defined.