Chimeric Anti-PDPN Antibody ChLpMab-2

Human podoplanin (hPDPN ), a platelet aggregation‐inducing transmembrane glycoprotein, is expressed in different types of tumors, and it binds to C‐type lectin‐like receptor 2 (CLEC ‐2). The overexpression of hPDPN is involved in invasion and metastasis. Anti‐hPDPN monoclonal antibodies (mAbs) such as NZ ‐1 have shown antitumor and antimetastatic activities by binding to the platelet aggregation‐stimulating (PLAG ) domain of hPDPN . Recently, we developed a novel mouse anti‐hPDPN mAb, LpMab‐2, using the cancer‐specific mAb (CasMab) technology. In this study we developed chLpMab‐2, a human–mouse chimeric anti‐hPDPN antibody, derived from LpMab‐2. chLpMab‐2 was produced using fucosyltransferase 8‐knockout (KO ) Chinese hamster ovary (CHO )‐S cell lines. By flow cytometry, chLpMab‐2 reacted with hPDPN ‐expressing cancer cell lines including glioblastomas, mesotheliomas, and lung cancers. However, it showed low reaction with normal cell lines such as lymphatic endothelial and renal epithelial cells. Moreover, chLpMab‐2 exhibited high antibody‐dependent cellular cytotoxicity (ADCC ) against PDPN ‐expressing cells, despite its low complement‐dependent cytotoxicity. Furthermore, treatment with chLpMab‐2 abolished tumor growth in xenograft models of CHO /hPDPN , indicating that chLpMab‐2 suppressed tumor development via ADCC . In conclusion, chLpMab‐2 could be useful as a novel antibody‐based therapy against hPDPN ‐expressing tumors

ATM suppresses c-Myc overexpression in the mammary epithelium in response to estrogen

ATM遺伝子変異による乳癌発症機構を解明 --HBOC症候群の乳腺特異的発癌機構の解明に貢献--. 京都大学プレスリリース. 2023-02-09.ATM gene mutation carriers are predisposed to estrogen-receptor-positive breast cancer (BC). ATM prevents BC oncogenesis by activating p53 in every cell; however, much remains unknown about tissue-specific oncogenesis after ATM loss. Here, we report that ATM controls the early transcriptional response to estrogens. This response depends on topoisomerase II (TOP2), which generates TOP2-DNA double-strand break (DSB) complexes and rejoins the breaks. When TOP2-mediated ligation fails, ATM facilitates DSB repair. After estrogen exposure, TOP2-dependent DSBs arise at the c-MYC enhancer in human BC cells, and their defective repair changes the activation profile of enhancers and induces the overexpression of many genes, including the c-MYC oncogene. CRISPR/Cas9 cleavage at the enhancer also causes c-MYC overexpression, indicating that this DSB causes c-MYC overexpression. Estrogen treatment induced c-Myc protein overexpression in mammary epithelial cells of ATM-deficient mice. In conclusion, ATM suppresses the c-Myc-driven proliferative effects of estrogens, possibly explaining such tissue-specific oncogenesis

Constraints on axion-like polarization oscillations in the cosmic microwave background with POLARBEAR

Very light pseudoscalar fields, often referred to as axions, are compelling dark matter candidates and can potentially be detected through their coupling to the electromagnetic field. Recently a novel detection technique using the cosmic microwave background (CMB) was proposed, which relies on the fact that the axion field oscillates at a frequency equal to its mass in appropriate units, leading to a time-dependent birefringence. For appropriate oscillation periods this allows the axion field at the telescope to be detected via the induced sinusoidal oscillation of the CMB linear polarization. We search for this effect in two years of POLARBEAR data. We do not detect a signal, and place a median $95 \%$ upper limit of $0.65 ^\circ$ on the sinusoid amplitude for oscillation frequencies between $0.02\,\text{days}^{-1}$ and $0.45\,\text{days}^{-1}$, which corresponds to axion masses between $9.6 \times 10^{-22} \, \text{eV}$ and $2.2\times 10^{-20} \,\text{eV}$. Under the assumptions that 1) the axion constitutes all the dark matter and 2) the axion field amplitude is a Rayleigh-distributed stochastic variable, this translates to a limit on the axion-photon coupling $g_{\phi \gamma} < 2.4 \times 10^{-11} \,\text{GeV}^{-1} \times ({m_\phi}/{10^{-21} \, \text{eV}})$.Comment: 17 pages, 5 figures, 2 tables. Published in Physical Review

A pathogenic C terminus-truncated polycystin-2 mutant enhances receptor-activated Ca2+ entry via association with TRPC3 and TRPC7.

Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca2+ release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca2+ influx that may lead to dysregulated cell growth in ADPKD. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.Publisher\u27s version/PDF may be used after 12 months embarg

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

A Novel Intermediate in Initiation Complex Assembly for Fission Yeast DNA Replication

Assembly of initiation factors on individual replication origins at onset of S phase is crucial for regulation of replication timing and repression of initiation by S-phase checkpoint control. We dissected the process of preinitiation complex formation using a point mutation in fission yeast nda4-108/mcm5 that shows tight genetic interactions with sna41(+)/cdc45(+). The mutation does not affect loading of MCM complex onto origins, but impairs Cdc45-loading, presumably because of a defect in interaction of MCM with Cdc45. In the mcm5 mutant, however, Sld3, which is required for Cdc45-loading, proficiently associates with origins. Origin-association of Sld3 without Cdc45 is also observed in the sna41/cdc45 mutant. These results suggest that Sld3-loading is independent of Cdc45-loading, which is different from those observed in budding yeast. Interestingly, returning the arrested mcm5 cells to the permissive temperature results in immediate loading of Cdc45 to the origin and resumption of DNA replication. These results suggest that the complex containing MCM and Sld3 is an intermediate for initiation of DNA replication in fission yeast

Topical Glucose Induces Claudin-1 and Filaggrin Expression in a Mouse Model of Atopic Dermatitis and in Keratinocyte Culture, Exerting Anti-inflammatory Effects by Repairing Skin Barrier Function

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Corticosteroids, which are widely used for AD treatment, have adverse effects, and alternative treatments are urgently needed. This study examined the effect of topical application of high-dose glucose on inflamed skin in a murine model of AD. High-dose glucose treatment on the ear reduced dermatitis scores and ear thicknesses in mite antigen-treated NC/Nga mice. The levels of thymus and activation-regulated chemokine (TARC), Th cytokines (interleukin (IL)-4, IL-5, IL-12, IL-13, and (interferon) IFN-γ), and IgE were decreased in the serum of high-dose glucose-treated mice. Expression of claudin-1 and filaggrin was reduced in the ear epithelium in the NC/Nga mice. However, the reduced expression was restored by topical treatment with high-dose glucose. High-dose glucose also induced the expression of claudin-1 and filaggrin in cultured human skin keratinocytes. Co-stimulation with IL-4, IL-13, and thymic stromal lymphoprotein downregulated the expression of filaggrin in culture. However, high-dose glucose treatment restored the reduced expression of filaggrin. These results suggest that high-dose glucose treatment suppresses inflammation in the skin lesions by improving the skin barrier function