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Despite some evidence indicating diverse roles of whirlin in neurons, the functional corollary of whirlin gene function and behavior has not been investigated or broadly characterized. A single nucleotide variant was identified from our recessive ENU-mutagenesis screen at a donor-splice site in whirlin, a protein critical for proper sensorineural hearing function. The mutation ( head-bob , hb ) led to partial intron-retention causing a frameshift and introducing a premature termination codon. Mutant mice had a head-bobbing phenotype and significant hyperactivity across several phenotyping tests. Lack of complementation of head-bob with whirler mutant mice confirmed the head-bob mutation as functionally distinct with compound mutants having a mild-moderate hearing defect. Utilizing transgenics, we demonstrate rescue of the hyperactive phenotype and combined with the expression profiling data conclude whirlin plays an essential role in activity-related behaviors. These results highlight a pleiotropic role of whirlin within the brain and implicate alternative, central mediated pathways in its function., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Panoramic radiography imaging modality is widely used by dentists for diagnosing dental and jaw conditions. It can also detect carotid artery calcification (CAC), indicative of calcified atherosclerotic plaques in the carotid arteries. This cross-sectional retrospective study at the University of Tennessee Health Science Center investigated the link between CAC identified on panoramic radiograph (PR) and cerebrovascular accident (CVA), coronary artery disease (CAD), and poor oral health. Data from 314 CAC patients collected from 2014 to 2023 included age at diagnosis, gender, and clinical histories of hypertension, hyperlipidemia, diabetes mellitus, CVA, CAD, and the decay, missing, and filled permanent teeth (DMFT) index. These patients were age- and gender-matched with non-CAC patients for analysis. The findings revealed high prevalences of hypertension (86.2%), hyperlipidemia (57.6%), diabetes mellitus (30.7%), CVA (15.5%), and CAD (28.7%) amongst CAC patients and the average DMFT index was 26.6. A comparative analysis of 276 matched controls demonstrated significant differences in hypertension (85.9% vs. 57.6%), hyperlipidemia (58.3% vs. 33.7%), diabetes (32.6% vs. 22.1%), CVA history (14.9% vs. 5.1%), CAD (26.1% vs. 9.8%), and DMFT scores (26.3 vs. 23.7), all indicating strong associations between CAC and these health conditions. The adjusted analysis showed that hypertension (aOR: 3.20 [95% CI: 2.06-5.07]), hyperlipidemia (aOR: 1.70 [95% CI: 1.14-2.50]), CVA (aOR: 2.20 [95% CI: 1.13-4.30]), and CAD (aOR: 2.10 [95% CI: 1.28-3.60]) were significantly associated with CAC. Notably, only 41.7% of the patients received a medical consultation after CAC detection on PR. It is crucial for dentists to refer patients for further evaluation.

Peroxisomal fatty acid α-oxidation is an essential pathway for the degradation of β-carbon methylated fatty acids such as phytanic acid. One enzyme in this pathway is 2-hydroxyacyl CoA lyase (HACL1), which is responsible for the cleavage of 2-hydroxyphytanoyl-CoA into pristanal and formyl-CoA. Hacl1 deficient mice do not present with a severe phenotype, unlike mice deficient in other α-oxidation enzymes such as phytanoyl-CoA hydroxylase deficiency (Refsum disease) in which neuropathy and ataxia are present. Tissues from wild-type and Hacl1 -/- mice fed a high phytol diet were obtained for proteomic and lipidomic analysis. There was no phenotype observed in these mice. Liver, brain, and kidney tissues underwent trypsin digestion for untargeted proteomic liquid chromatography-mass spectrometry analysis, while liver tissues also underwent fatty acid hydrolysis, extraction, and derivatisation for fatty acid gas chromatography-mass spectrometry analysis. The liver fatty acid profile demonstrated an accumulation of phytanic and 2-hydroxyphytanic acid in the Hacl1 -/- liver and significant decrease in heptadecanoic acid. The liver proteome showed a significant decrease in the abundance of Hacl1 and a significant increase in the abundance of proteins involved in PPAR signalling, peroxisome proliferation, and omega oxidation, particularly Cyp4a10 and Cyp4a14. In addition, the pathway associated with arachidonic acid metabolism was affected; Cyp2c55 was upregulated and Cyp4f14 and Cyp2b9 were downregulated. The kidney proteome revealed fewer significantly upregulated peroxisomal proteins and the brain proteome was not significantly different in Hacl1 -/- mice. This study demonstrates the powerful insight brought by proteomic and metabolomic profiling of Hacl1 -/- mice in better understanding disease mechanism in fatty acid α-oxidation disorders.

Igfn1 is a complex locus that codes for multiple splicing variants of Immunoglobulin- and Fibronectin-like domain containing proteins predominantly expressed in skeletal muscle. To reveal possible roles for Igfn1, we applied non-selective knock-down by shRNAs as well as specific targeting of Igfn1 exon 13 by CRISPR/Cas9 mutagenesis in C2C12 cells. Decreased expression of Igfn1 variants via shRNAs against the common 3'-UTR region caused a total blunting of myoblast fusion, but did not prevent expression of differentiation markers. Targeting of N-terminal domains by elimination of exon 13 via CRISPR/Cas9 mediated homologous recombination, also resulted in fusion defects as well as large multinucleated cells. Expression of IGFN1_v1 partially rescued fusion and myotube morphology in the Igfn1 exon 13 knock-out cell line, indicating a role for this variant in myoblast fusion and differentiation. However, in vivo overexpression of IGFN1_v1 or the Igfn1 Exon 13 CRISPR/Cas9 targeting vector did not result in significant size changes in transfected fibres.

Streptococcus pneumoniae is an important human pathogen responsible for a spectrum of diseases including pneumonia. Immunological and pro-inflammatory processes induced in the lung during pneumococcal infection are well documented, but little is known about the role played by immunoregulatory cells and cytokines in the control of such responses. We demonstrate considerable differences in the immunomodulatory cytokine transforming growth factor (TGF)-β between the pneumococcal pneumonia resistant BALB/c and susceptible CBA/Ca mouse strains. Immunohistochemistry and flow cytometry reveal higher levels of TGF-β protein in BALB/c lungs during pneumococcal pneumonia that correlates with a rapid rise in lung Foxp3(+)Helios(+) T regulatory cells. These cells have protective functions during pneumococcal pneumonia, because blocking their induction with an inhibitor of TGF-β impairs BALB/c resistance to infection and aids bacterial dissemination from lungs. Conversely, adoptive transfer of T regulatory cells to CBA/Ca mice, prior to infection, prolongs survival and decreases bacterial dissemination from lungs to blood. Importantly, strong T regulatory cell responses also correlate with disease-resistance in outbred MF1 mice, confirming the importance of immunoregulatory cells in controlling protective responses to the pneumococcus. This study provides exciting new evidence for the importance of immunomodulation during pulmonary pneumococcal infection and suggests that TGF-β signalling is a potential target for immunotherapy or drug design.

The KY protein underlies a form of muscular dystrophy in the mouse but its role in muscle remains elusive. Immunodetection of endogenous KY protein in C2C12-derived myotubes and expression of a recombinant form in neonatal cardiomyocytes indicated that KY is a Z-band associated protein. Moreover, characterization of a KY interacting protein fragment led to the identification of Igfn1 (Immunoglobulin-like and fibronectin type 3 domain containing 1). Igfn1 is a transcriptionally complex locus encoding many protein variants. A yeast two-hybrid screen identified the Z-band protein filamin C (FLNC) as an interacting partner. Consistent with this, expression of an IGFN1 recombinant fragment showed that the three N-terminal globular domains, common to at least five IGFN1 variants, are sufficient to provide Z-band targeting. Taken together, the yeast two-hybrid, biochemical and immunofluorescence data support the notion that KY, IGFN1 and FLNC are part of a Z-band associated protein complex likely to provide structural support to the skeletal muscle sarcomere.

A comprehensive gene-based map of a genome is a powerful tool for genetic studies and is especially useful for the positional cloning and positional candidate approaches. The availability of gene maps for multiple organisms provides the foundation for detailed conserved-orthology maps showing the correspondence between conserved genomic segments. These maps make it possible to use cross-species information in gene hunts and shed light on the evolutionary forces that shape the genome. Here we report a radiation hybrid map of mouse genes, a combined project of the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research, the Medical Research Council UK Mouse Genome Centre, and the National Center for Biotechnology Information. The map contains 11,109 genes, screened against the T31 RH panel and positioned relative to a reference map containing 2,280 mouse genetic markers. It includes 3,658 genes homologous to the human genome sequence and provides a framework for overlaying the human genome sequence to the mouse and for sequencing the mouse genome.

Higher-order chromatin has been implicated in epigenetic gene control and in the functional organization of chromosomes. We have recently discovered mouse (Suv39h1) and human (SUV39H1) histone H3 lysine 9-selective methyltransferases (Suv39h HMTases) and shown that they modulate chromatin dynamics in somatic cells. We describe here the isolation, chromosomal assignment, and characterization of a second murine gene, Suv39h2. Like Suv39h1, Suv39h2 encodes an H3 HMTase that shares 59% identity with Suv39h1 but which differs by the presence of a highly basic N terminus. Using fluorescent in situ hybridization and haplotype analysis, the Suv39h2 locus was mapped to the subcentromeric region of mouse chromosome 2, whereas the Suv39h1 locus resides at the tip of the mouse X chromosome. Notably, although both Suv39h loci display overlapping expression profiles during mouse embryogenesis, Suv39h2 transcripts remain specifically expressed in adult testes. Immunolocalization of Suv39h2 protein during spermatogenesis indicates enriched distribution at the heterochromatin from the leptotene to the round spermatid stage. Moreover, Suv39h2 specifically accumulates with chromatin of the sex chromosomes (XY body) which undergo transcriptional silencing during the first meiotic prophase. These data are consistent with redundant enzymatic roles for Suv39h1 and Suv39h2 during mouse development and suggest an additional function of the Suv39h2 HMTase in organizing meiotic heterochromatin that may even impart an epigenetic imprint to the male germ line.

A physical map of the mouse genome is an essential tool for both positional cloning and genomic sequencing in this key model system for biomedical research. Indeed, the construction of a mouse physical map with markers spaced at an average interval of 300 kb is one of the stated goals of the Human Genome Project. Here we report the results of a project at the Whitehead Institute/MIT Center for Genome Research to construct such a physical map of the mouse. We built the map by screening sequenced-tagged sites (STSs) against a large-insert yeast artificial chromosome (YAC) library and then integrating the STS-content information with a dense genetic map. The integrated map shows the location of 9,787 loci, providing landmarks with an average spacing of approximately 300 kb and affording YAC coverage of approximately 92% of the mouse genome. We also report the results of a project at the MRC UK Mouse Genome Centre targeted at chromosome X. The project produced a YAC-based map containing 619 loci (with 121 loci in common with the Whitehead map and 498 additional loci), providing especially dense coverage of this sex chromosome. The YAC-based physical map directly facilitates positional cloning of mouse mutations by providing ready access to most of the genome. More generally, use of this map in addition to a newly constructed radiation hybrid (RH) map provides a comprehensive framework for mouse genomic studies.

Macrophage production of PDGF-B is believed to be important in the pathogenesis of diseases where chronic lung inflammation develops into fibrosis. Since tuberculosis is characterized by chronic inflammation and tissue fibrosis, we asked if lymphokines from lymphocytes stimulated by the Mycobacterium tuberculosis antigen PPD, contained factors capable of increasing human alveolar macrophage PDGF-B mRNA. Supernatants from both phytohaemagglutinin (PHA)- and purified protein derivative (PPD)-stimulated lymphocytes, when added to macrophages, induced an increase in the mRNA of PDGF-B, but not transforming growth factor-beta (TGF-beta). When lymphocytes from contacts of patients with tuberculosis, patients with tuberculosis, and normal subjects were compared following PPD stimulation, the lymphocytes from the contacts had the greatest proliferation response, the greatest production of interferon-gamma (IFN-gamma), and their lymphokines induced the greatest increase in PDGF-B mRNA in macrophages. Recombinant human IFN-gamma reproduced this ability of lymphokines to increase macrophage PDGF-B mRNA. Finally, the increase in macrophage PDGF-B mRNA following incubation with supernatants from PPD-stimulated lymphocytes was shown to be due to IFN-gamma, when the increase in macrophage PDGF-B mRNA was prevented by addition of anti-human IFN-gamma antibody to the lymphocyte supernatant. This study indicated that antigen-stimulated lymphocytes released IFN-gamma, which in turn resulted in an increase in PDGF-B mRNA in alveolar macrophages. Such a mechanism provides a link between the DTH response and the first stages of a fibrotic reaction, and may offer an explanation for the progression of chronic inflammation to fibrosis, as occurs in the lungs of patients with untreated pulmonary tuberculosis.

Macrophage production of growth factors for fibroblasts, in particular platelet-derived growth factor B [PDGF(B)] and transforming growth factor-beta (TGF-beta), is thought to be central to the pathogenesis of pulmonary fibrosis. In a search for anti-inflammatory agents that might prevent this process, we asked whether colchicine might modulate the abundance of PDGF(B) and TGF-beta mRNA, as well as the mRNA of early growth response gene 2 (EGR2), in human macrophages. Colchicine caused a dose- and time-dependent increase in PDGF(B), but not TGF-beta or EGR2, mRNA in human macrophages derived from culture of peripheral blood monocytes. Similarly, colchicine caused an increase in PDGF(B) mRNA in human alveolar macrophages obtained from normal volunteers. Colchicine also caused an increase in PDGF(B) protein production by macrophages, as determined by enzyme-linked immunosorbent assay. Interferon-gamma further increased the PDGF(B) mRNA abundance in human alveolar but not monocyte-derived macrophages. The effect of coincubation with dibutyryl-cAMP (dBcAMP) was assessed in an attempt to prevent the colchicine-induced increase in PDGF(B) mRNA. dBcAMP alone resulted in no increase in PDGF(B) mRNA or alteration in TGF-beta mRNA but resulted in a reduction in EGR2 mRNA. When added with colchicine, dBcAMP completely abrogated the colchicine-induced increase in PDGF(B) mRNA but had little effect on TGF-beta mRNA. These data, showing that colchicine increased macrophage PDGF(B) mRNA in human macrophages and that this was prevented by coincubation with dBcAMP, lead us to speculate that colchicine may not be helpful in preventing the contribution of macrophage PDGF(B) gene activation to the pathogenesis of lung fibrosis. However, this effect of colchicine may be prevented by increasing intracellular cAMP in macrophages.

Platelet-derived growth factor (B) (PDGF(B)) from alveolar macrophages is thought to play a central role in orchestrating the fibrotic response. Because corticosteroids are widely used in the treatment of patients with lung fibrosis, we asked whether corticosteroids modulated PDGF(B) gene activation in macrophages. PDGF(B) mRNA in alveolar macrophages obtained from smokers was increased after culture in the presence of dexamethasone (P less than 0.05), interferon-gamma (IFN-gamma) (P less than 0.05), or both in combination (P less than 0.05). Dexamethasone did not alter the abundance of mRNA encoding transforming growth factor-beta (TGF-beta), but did decrease the mRNA of early growth response gene 2 (EGR2). These initial experiments required large numbers of cells and thus were performed on macrophages from smokers. The results were reproduced when PDGF(B) mRNA abundance in macrophages from healthy nonsmoking volunteers was measured by the reverse-transcriptase polymerase chain reaction (RT-PCR). There was an increase in PDGF(B) mRNA in macrophages from nonsmokers after stimulation with dexamethasone alone (P less than 0.05) or in combination with IFN-gamma (P less than 0.05). To provide adequate cell numbers for kinetic and dose-response studies, the in vitro model of phorbol ester (TPA)-induced differentiation of HL60 cells to macrophage-like cells was used. In these cells, dexamethasone caused a 20-fold increase in the abundance of PDGF(B) mRNA, which was concentration and time dependent but not associated with changes in TGF-beta or EGR2 mRNA. This study suggests that in addition to their anti-inflammatory effects, corticosteroids may also increase the abundance of PDGF(B) mRNA.

Linkage between Alzheimer's disease and markers on the long arm of chromosome 21 was investigated in six families affected by disease of early onset. Linkage was confirmed and the disease locus shown to be centromeric to the locus D21S1/S11 on the long arm of the chromosome. It is argued that the data are consistent with the notion that all patients with Alzheimer's disease of genetic aetiology have a predisposing locus on chromosome 21.