A mutation in Tubb2b, a human polymicrogyria gene, leads to lethality and abnormal cortical development in the mouse.

Human cortical malformations, including lissencephaly, polymicrogyria and other diseases of neurodevelopment, have been associated with mutations in microtubule subunits and microtubule-associated proteins. Here we report our cloning of the brain dimple (brdp) mouse mutation, which we recovered from an ENU screen for recessive perinatal phenotypes affecting neurodevelopment. We identify the causal mutation in the tubulin, beta-2b (Tubb2b) gene as a missense mutation at a highly conserved residue (N247S). Brdp/brdp homozygous mutants have significant thinning of the cortical epithelium, which is markedly more severe in the caudo-lateral portion of the telencephalon, and do not survive past birth. The cortical defects are largely due to a major increase in apoptosis and we note abnormal proliferation of the basal progenitors. Adult brdp/+ mice are viable and fertile but exhibit behavioral phenotypes. This allele of Tubb2b represents the most severely affected mouse tubulin phenotype reported to date and this is the first report of a tubulin mutation affecting neuronal proliferation and survival.

Quantitative locus analysis of airway hyperresponsiveness in A/J and C57BL/6J mice.

Airway hyperresponsiveness is a key characteristic of human asthma and a marker for asthma-like conditions in animals. F1 mice derived from A/J and C57BL/6J display a phenotype which resembles the asthma-like phenotype of the A/J mice. Since airway responsiveness failed to segregate as a mendelian trait, we show significant linkage at two loci, Bhr1 (lod = 3.0) and Bhr2 (lod = 3.7) on chromosomes 2 and 15. A third locus, Bhr3 (lod = 2.83), maps to chromosome 17. Each of these loci maps near candidate loci implicated in the pathobiology of asthma. Our study represents the first linkages established through a genome-wide survey of airway hyperresponsiveness in any mammal.

Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene.

Although disorders of iron metabolism are prevalent, iron transport remains poorly understood. To address this problem, we undertook a positional cloning strategy to identify the causative mutation in mice with microcytic anaemia (mk). Homozygous mk/mk mice have microcytic, hypochromic anaemia due to severe defects in intestinal iron absorption and erythroid iron utilization. We report the identification of a strong candidate gene for mk, and suggest that the phenotype is a consequence of a missense mutation in Nramp2 (ref. 5), a previously identified gene of unknown function. Nramp2 is homologous to Nramp1, a gene activa in host defense. If Nramp2 is mk, as the cumulative evidence suggests, our findings have broad implications for the understanding of iron transport and resistance to intracellular pathogens.

Vertebrate genome evolution and the zebrafish gene map.

In chordate phylogeny, changes in the nervous system, jaws, and appendages transformed meek filter feeders into fearsome predators. Gene duplication is thought to promote such innovation. Vertebrate ancestors probably had single copies of genes now found in multiple copies in vertebrates and gene maps suggest that this occurred by polyploidization. It has been suggested that one genome duplication event occurred before, and one after the divergence of ray-finned and lobe-finned fishes. Holland et al., however, have argued that because various vertebrates have several HOX clusters, two rounds of duplication occurred before the origin of jawed fishes. Such gene-number data, however, do not distinguish between tandem duplications and polyploidization events, nor whether independent duplications occurred in different lineages. To investigate these matters, we mapped 144 zebrafish genes and compared the resulting map with mammalian maps. Comparison revealed large conserved chromosome segments. Because duplicated chromosome segments in zebrafish often correspond with specific chromosome segments in mammals, it is likely that two polyploidization events occurred prior to the divergence of fish and mammal lineages. This zebrafish gene map will facilitate molecular identification of mutated zebrafish genes, which can suggest functions for human genes known only by sequence.

Importance and value of adjuvanted influenza vaccine in the care of older adults from a European perspective - A systematic review of recently published literature on real-world data.

BACKGROUND: There is an urgent need for improved influenza vaccines especially for older adults due to the presence of immunosenescence. It is therefore highly relevant to compare enhanced influenza vaccines with traditional influenza vaccines with respect to their effectiveness. OBJECTIVE: To compare vaccine efficacy and effectiveness of adjuvanted influenza vaccines (aTIV/aQIV) vs. non-adjuvanted standard-dose (TIV/QIV) and high-dose (TIV-HD/QIV-HD) influenza vaccines regarding influenza-related outcomes in older adults, complementing findings from the European Centre for Disease Prevention and Control (ECDC)'s systematic review of enhanced seasonal influenza vaccines from February 2020. METHODS: A systematic literature search was conducted in Embase and MEDLINE to identify randomised controlled trials, observational studies and systematic reviews, published since ECDC's systematic review (between 7 February 2020 and 6 September 2021). Included studies were appraised with either the Cochrane Risk of Bias tool, ROBINS-I or AMSTAR 2. RESULTS: Eleven analyses from nine real-world evidence (RWE) studies comprising ∼53 million participants and assessing the relative vaccine effectiveness (rVE) of aTIV vs. TIV, QIV and/or TIV-HD in adults aged ≥65 years over the 2006/07-2008/09 and 2011/12-2019/20 influenza seasons were identified. Nine analyses found that aTIV was significantly more effective than TIV and QIV in reducing influenza-related outcomes by clinical setting and suspected influenza outbreaks (rVE ranging from 7.5% to 25.6% for aTIV vs. TIV and 7.1% to 36.3% for aTIV vs. QIV). Seven analyses found similar effectiveness of aTIV vs. TIV-HD in reducing influenza-related medical encounters, inpatient stays and hospitalisations/emergency room visits. In three analyses, aTIV was significantly more effective than TIV-HD in reducing influenza-related medical encounters and office visits (rVE ranging from 6.6% to 16.6%). Risk of bias of identified studies was moderate to high. CONCLUSIONS: Our study suggests that both adjuvanted and high-dose vaccines are effective alternatives for vaccination programmes in older adults and preferable over conventional standard-dose vaccines.

Identification of a Van der Woude syndrome mutation in the cleft palate 1 mutant mouse.

Mutations in Interferon Regulatory Factor 6 (IRF6) have been identified in two human allelic syndromes with cleft lip and/or palate: Van der Woude (VWS) and Popliteal Pterygium syndromes (PPS). Furthermore, common IRF6 haplotypes and single nucleotide polymorphisms (SNP) alleles are strongly associated with nonsyndromic clefting defects in multiple ethnic populations. Mutations in the mouse often provide good models for the study of human diseases and developmental processes. We identified the cleft palate 1 (clft1) mouse mutant in a forward genetic screen for phenotypes modeling human congenital disease. In the clft1 mutant, we have identified a novel missense point mutation in the mouse Irf6 gene, which confers an amino acid alteration that has been found in a VWS family. Phenotypic comparison of clft1 mutants to previously reported Irf6 mutant alleles demonstrates the Irf6(clft1) allele is a hypomorphic allele. The cleft palate seen in these mutants appears to be due to abnormal adhesion between the palate and tongue. The Irf6(clft1) allele provides the first mouse model for the study of an etiologic IRF6 missense mutation observed in a human VWS family.

Ttc21b is required to restrict sonic hedgehog activity in the developing mouse forebrain.

Organizing centers in the developing brain provide an assortment of instructive patterning cues, including Sonic hedgehog (Shh). Here we characterize the forebrain phenotype caused by loss of Ttc21b, a gene we identified in an ENU mutagenesis screen as a novel ciliary gene required for retrograde intraflagellar transport. The Ttc21b mutant has defects in limb, eye and, most dramatically, brain development. We show that Shh signaling is elevated in the rostral portion of the mutant embryo, including in a domain in or near the zona limitans intrathalamica. We demonstrate here that ciliary defects seen in the Ttc21b mutant extend to the embryonic brain, adding forebrain development to the spectrum of tissues affected by defects in ciliary physiology. We show that development of the Ttc21b brain phenotype is modified by lowering levels of the Shh ligand, supporting our hypothesis that the abnormal patterning is a consequence of elevated Shh signaling. Finally, we evaluate Wnt signaling but do not find evidence that this plays a role in causing the perturbed neurodevelopmental phenotype we describe.

Triggering receptor expressed on myeloid cells-1 (TREM-1) expression on gastric epithelium: implication for a role of TREM-1 in Helicobacter pylori infection.

In Helicobacter pylori gastritis gastric epithelium plays a central role in the innate immunity to H. pylori. However, epithelial receptors interacting with H. pylori have been poorly characterized so far. Recently a new triggering receptor expressed on myeloid cells-1 (TREM-1) has been identified on human neutrophils and monocytes. On these cells TREM-1 triggers innate immunity by stimulating the secretion of interleukin (IL)-8 and tumour necrosis factor (TNF)-alpha and thus amplifies bacterial-induced inflammation. In this study expression and function of TREM-1 in gastric epithelium exposed to H. pylori has been investigated. TREM-1 mRNA and protein were expressed on gastric epithelial cell lines as demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence activated cell sorter analysis. Gastric epithelial TREM-1 expression was up-regulated directly by H. pylori and was independent of epithelial IL-8 induced by H. pylori. Immunohistochemistry and tissue RT-PCR demonstrated significantly stronger TREM-1 expression in H. pylori gastritis compared with the non-inflamed gastric mucosa supporting in vivo that epithelial TREM-1 is up-regulated during H. pylori infection. Stimulation of gastric epithelial TREM-1 receptor resulted in IL-8 up-regulation on mRNA and protein level, as shown by real-time PCR and immunoassay. This is the first study localizing TREM-1 on gastric epithelium. Functional data suggest that TREM-1 expressed on gastric epithelium amplifies inflammation of the underlying gastric mucosa by up-regulation of IL-8.

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

Deletion analysis identifies a region, upstream of the ADH2 gene of Saccharomyces cerevisiae, which is required for ADR1-mediated derepression.

Deletion analysis was used to identify sequences upstream of the ADH2 gene of Saccharomyces cerevisiae that are required for its regulation. 5' and 3' internal deletions of the ADH2 control region were created in vitro, and the fragments were ligated adjacent to the ADH1 promoter and structural gene. Hybrid genes with 3' deletions extending from -119 to -216 (the start site of ADH2 transcription is designated +1) were fully repressed and derepressed to high levels. Hybrid genes with 3' deletions extending from -119 to -257 were repressed but failed to significantly derepress. Hybrid genes lacking the -216 to -257 region also failed to respond to ADR1-5c, a mutant allele of the unlinked regulatory gene ADR1, which confers constitutive expression on ADH2. This implies that the region between these deletion endpoints, which includes a 22-base-pair sequence of dyad symmetry, is required for efficient derepression of an adjacent promoter. Internal deletions extending in the 3' direction from position -1141 confirmed these results. Deletion mutants lacking the region -1141 to -259 were normally regulated, whereas deletions extending from -1141 to -115 were not derepressible. These results support the hypotheses that the ADH2 promoter may normally be in an inactive conformation in the yeast chromosome and that derepression of ADH2 requires positive activation mediated through an upstream activation sequence located between 216 and 257 base pairs 5' to the start site of ADH2 transcription. No evidence for a DNA sequence mediating repression was obtained.

Generation of murine stromal cell lines supporting hematopoietic stem cell proliferation by use of recombinant retrovirus vectors encoding simian virus 40 large T antigen.

The bone marrow is a complex microenvironment made up of multiple cell types which appears to play an important role in the maintenance of hematopoietic stem cell self-renewal and proliferation. We used murine long-term marrow cultures and a defective recombinant retrovirus vector containing the simian virus 40 large T antigen to immortalize marrow stromal cells which can support hematopoiesis in vitro for up to 5 weeks. Such cloned cell lines differentially supported stem cells which, when transplanted, allowed survival of lethally irradiated mice, formed hematopoietic spleen colonies in vivo, and stimulated lymphocyte proliferation in vitro. Molecular and functional analyses of these cell lines did not demonstrate the production of any growth factors known to support the proliferation of primitive hematopoietic stem cells. All cell lines examined produced macrophage colony-stimulating factor. The use of immortalizing retrovirus vectors may allow determination of unique cellular proteins important in hematopoietic stem cell proliferation by the systematic comparison of stromal cells derived from a variety of murine tissues.

Imaging Biomarkers for Adult Medulloblastomas: Genetic Entities May Be Identified by Their MR Imaging Radiophenotype.

BACKGROUND AND PURPOSE: The occurrence of medulloblastomas in adults is rare; nevertheless, these tumors can be subdivided into genetic and histologic entities each having distinct prognoses. This study aimed to identify MR imaging biomarkers to classify these entities and to uncover differences in MR imaging biomarkers identified in pediatric medulloblastomas. MATERIALS AND METHODS: Eligible preoperative MRIs from 28 patients (11 women; 22-53 years of age) of the Multicenter Pilot-study for the Therapy of Medulloblastoma of Adults (NOA-7) cohort were assessed by 3 experienced neuroradiologists. Lesions and perifocal edema were volumetrized and multiparametrically evaluated for classic morphologic characteristics, location, hydrocephalus, and Chang criteria. To identify MR imaging biomarkers, we correlated genetic entities sonic hedgehog (SHH) TP53 wild type, wingless (WNT), and non-WNT/non-SHH medulloblastomas (in adults, Group 4), and histologic entities were correlated with the imaging criteria. These MR imaging biomarkers were compared with corresponding data from a pediatric study. RESULTS: There were 19 SHH TP53 wild type (69%), 4 WNT-activated (14%), and 5 Group 4 (17%) medulloblastomas. Six potential MR imaging biomarkers were identified, 3 of which, hydrocephalus (P = .03), intraventricular macrometastases (P = .02), and hemorrhage (P = .04), when combined, could identify WNT medulloblastoma with 100% sensitivity and 88.3% specificity (95% CI, 39.8%-100.0% and 62.6%-95.3%). WNT-activated nuclear ß-catenin accumulating medulloblastomas were smaller than the other entities (95% CI, 5.2-22.3 cm3 versus 35.1-47.6 cm3; P = .03). Hemorrhage was exclusively present in non-WNT/non-SHH medulloblastomas (P = .04; n = 2/5). MR imaging biomarkers were all discordant from those identified in the pediatric cohort. Desmoplastic/nodular medulloblastomas were more rarely in contact with the fourth ventricle (4/15 versus 7/13; P = .04). CONCLUSIONS: MR imaging biomarkers can help distinguish histologic and genetic medulloblastoma entities in adults and appear to be different from those identified in children.

Impaired homeostasis and phenotypic abnormalities in Prdx6-/-mice lens epithelial cells by reactive oxygen species: increased expression and activation of TGFbeta.

PRDX6, a member of the peroxiredoxins (PRDXs) family, is a key player in the removal of reactive oxygen species (ROS). Using targeted inactivation of the Prdx6 gene, we present evidence that the corresponding protein offsets the deleterious effects of ROS on lens epithelial cells (LECs) and regulates gene expression by limiting its levels. PRDX6-depleted LECs displayed phenotypic alterations and elevated alpha-smooth muscle actin and betaig-h3 expression (markers for cataractogenesis), indistinguishable from transforming growth factor beta (TGFbeta)-induced changes. Biochemical assays disclosed enhanced levels of ROS, as well as high expression and activation of TGFbeta1 in Prdx6-/- LECs. A CAT assay revealed transcriptional repression of lens epithelium-derived growth factor (LEDGF), HSP27, and alphaB-crystallin promoter activities in these cells. A gel mobility shift assay demonstrated the attenuation of LEDGF binding to heat shock or stress response elements present in these genes. A supply of PRDX6 toPrdx6-/- LECs reversed these changes. Based on the above data, we propose a rheostat role for PRDX6 in regulating gene expression by controlling the ROS level to maintain cellular homeostasis.

Choroid plexus carcinoma in a goat.

A 15-year-old female goat suddenly developed right-sided head tilting with anorexia and depression. Post-mortem examination of the brain revealed a large, unilateral, well-demarcated, intraventricular neoplasm which was diagnosed as a choroid plexus carcinoma. The neoplasm, which occupied about 75% of the left lateral ventricle, led to unilateral obstructive hydrocephalus and invaded the white and grey matter of the left piriform lobe, with focal subarachnoid spread and meningeal implantation. Histopathological examination revealed loss of branching papillary architecture, invasive growth, a high mitotic index and marked necrosis in the undifferentiated areas of the tumour. Neoplastic cells expressed vimentin and, multifocally, broad spectrum cytokeratins, but were negative for GFAP, NSE and Sl00 antigen. This is the first report of a choroid plexus carcinoma in a goat.

[Case report: mast cell leukosis in a neonatal calf]. / Fallbericht: Mastzellenleukose bei einem neugeborenen Kalb.

Multicentric mast cell tumours in a newborn Fleckvieh-calf are described. The calf showed clearly pronounced lesions over the whole body. The lesions were multiple raised, cutaneous, greyisch-red and partially ulcerated. It died three hours after birth. Pathohistological examinations resulted in multiple mast cell tumours within the dermis. In addition multifocal to diffuse mast cell aggregations were observed in several internal organs including the lymph nodes and the bone marrow. No evidence for the presence of bovine leukemia virus was found by both investigating a lymph node homogenate of the calf and a blood sample of the mother cow. In this paper the pathomorphology of this rare disease is described, a possible cause is discussed and a short review of the available literature is presented.

Characterization of murine Flt4 ligand/VEGF-C.

Flt4 is a receptor protein tyrosine kinase that is expressed in the adult lymphatic endothelium and high endothelial venules. We have used a BIAcore assay to identify rodent and human cell conditioned media containing the ligand of Flt4 (Flt4-L). Receptor-based affinity chromatography was used to purify this growth factor, followed by amino acid sequencing and molecular cloning of the murine cDNA, the orthologue of human vascular endothelial growth factor-C and vascular endothelial growth factor related protein. The murine flt4-L gene was localized to chromosome 8 and demonstrated to be widely expressed. Flt4-L was found to have a hydrophobic signal sequence and a pro-peptide-like sequence that is removed to generate the mature N-terminus. In addition, the C-terminal region of Flt4-L has four repeats of a cysteine-rich motif that is presumably also proteolytically processed to generate the 21000 Mr polypeptide subunit of the Flt4-L homodimer. Recombinant Flt4-L activated Flt4 as judged by induction of tyrosyl phosphorylation, and induced mitogenesis in vitro of lymphatic endothelial cells.

Cloning, tissue-specific expression, gene structure and chromosomal localization of human phosphatidylcholine transfer protein.

Phosphatidylcholine transfer protein (PC-TP) is a cytosolic protein that catalyzes intermembrane transfer of phosphatidylcholines in vitro. We have cloned a cDNA encoding the human ortholog of PC-TP and have determined its tissue-specific expression as well as genomic organization. Radiation hybrid mapping localized the human gene, PCTP, to chromosome 17q21-22 and PCR-based single strand conformation polymorphism analysis of an interspecific backcross assigned mouse Pctp to the region of syntenic conservation on chromosome 11.

In vivo characterization of the unorthodox BvgS two-component sensor protein of Bordetella pertussis.

Two-component sensor proteins are typically composed of an amino-acid sensory and a carboxy-terminal transmitter domain containing a kinase activity which catalyses the autophosphorylation of a histidine residue. In a second step, the phosphate is transferred to aspartic acid residues located in the receiver domain of the second component, the response regulator. A few sensor proteins such as the BvgS protein of Bordetella pertussis have a more complex structure. BvgS possesses additional C-terminal domains, including receiver and output modules usually found only in the response regulators. The function of these BvgS domains was investigated by mutation and complementation analysis in vivo. BvgS derivatives were constructed lacking the C-terminal domains or containing mutations in conserved amino acids. All mutations caused the inactivation of BvgS as measured by the expression of virulence factors at the transcriptional and translational level after integration of the mutated alleles in the B. pertussis chromosome. However, some of these mutants could be complemented to the wild-type phenotype by the separate expression of various C-terminal BvgS domains in trans indicating a direct interaction of the truncated and complete BvgS proteins. Therefore, the dimerization capacity of the cytoplasmic BvgS domains was analysed using a lambda repressor based dimerization probe system. These results indicated that BvgS has two dimerization regions, one in the transmitter and a second in the C-terminal receiver/output domains. Furthermore, several BvgS hybrid proteins were constructed which contained substitutions of the BvgS receiver and output domains with similar domains of two-component response regulators and of the sensor protein EvgS. It was found that the receiver domain does not carry BvgS-specific functions and can be exchanged by a heterologous receiver domain. However, the BvgS output domain could not be substituted with output domains of the related proteins without inactivation of BvgS.