Clinicopathological Significance of FOXP3 Expression in Esophageal Squamous Cell Carcinoma

The expression of transcription factor forkhead box protein 3 (FOXP3), a master control gene for regulatory T cells, has been reported to influence patient survival. However, there have been few reports of the relationship between FOXP3 positive cells and esophageal squamous cell carcinoma (ESCC). The aim of this study was to clarify the prognostic value of FOXP3 expression in ESCC. Ninety-five patients who were diagnosed with primary ESCC and underwent subtotal esophagectomy during 2009 and 2010 were retrospectively analyzed. Deepest sections from each tumor were selected for immunohistochemistry and the number of FOXP3 positive cells was counted. The median number was used as a cutoff to divide into FOXP3 positive and FOXP3 negative subgroups. Relationships between FOXP3 expression and clinicopathological features, disease-free survival (DFS) and overall survival (OS) were determined. Statistical values of p < 0.05 were considered significant. FOXP3 positive cells were found in all 95 cases and the number of FOXP3 positive cells was significantly higher in the peri-tumor compartment than in the intra-tumor compartment (p = 0.0006). For this reason, the peri-tumor compartment numbers were used for all of the association studies. Results showed that the FOXP3 positive group had a significantly larger mean tumor size (43.8 ± 4.1mm vs 29.1 ± 4.0mm, p = 0.0055), and the FOXP3 negative group had a significantly higher percentage of deep invasion (T2, T3, T4)(p = 0.0399). There was no significant association for DFS, however, for OS the FOXP3 positive group demonstrated a significantly better prognosis (p = 0.0024). Multivariate analysis showed that peri-tumor FOXP3 expression is an independent prognostic factor for OS (p = 0.0035). Peri-tumoral FOXP3 expression is an independent and favorable prognostic factor for ESCC

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

Tysnd1 Deficiency in Mice Interferes with the Peroxisomal Localization of PTS2 Enzymes, Causing Lipid Metabolic Abnormalities and Male Infertility

<div><p>Peroxisomes are subcellular organelles involved in lipid metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids, among others. Peroxisome matrix proteins are synthesized in the cytoplasm. Targeting signals (PTS or peroxisomal targeting signal) at the C-terminus (PTS1) or N-terminus (PTS2) of peroxisomal matrix proteins mediate their import into the organelle. In the case of PTS2-containing proteins, the PTS2 signal is cleaved from the protein when transported into peroxisomes. The functional mechanism of PTS2 processing, however, is poorly understood. Previously we identified Tysnd1 (Trypsin domain containing 1) and biochemically characterized it as a peroxisomal cysteine endopeptidase that directly processes PTS2-containing prethiolase Acaa1 and PTS1-containing Acox1, Hsd17b4, and ScpX. The latter three enzymes are crucial components of the very-long-chain fatty acids β-oxidation pathway. To clarify the <em>in vivo</em> functions and physiological role of Tysnd1, we analyzed the phenotype of <em>Tysnd1^−/−</em> mice. Male <em>Tysnd1^−/−</em> mice are infertile, and the epididymal sperms lack the acrosomal cap. These phenotypic features are most likely the result of changes in the molecular species composition of choline and ethanolamine plasmalogens. <em>Tysnd1^−/−</em> mice also developed liver dysfunctions when the phytanic acid precursor phytol was orally administered. Phyh and Agps are known PTS2-containing proteins, but were identified as novel Tysnd1 substrates. Loss of <em>Tysnd1</em> interferes with the peroxisomal localization of Acaa1, Phyh, and Agps, which might cause the mild Zellweger syndrome spectrum-resembling phenotypes. Our data established that peroxisomal processing protease Tysnd1 is necessary to mediate the physiological functions of PTS2-containing substrates.</p> </div

Generation of <i>Tysnd1</i>^−/− mice.

<p>A. Map of <i>Tysnd1</i>^−/− targeting constructs. P1, P2 and P3 indicate primers used for genotyping by PCR. B. Identification of genotyping by PCR. Wild type and <i>Tysnd1</i>^−/− genotype were identified by 237 bp and 339 bp PCR products, respectively. C. Relative expression level of <i>Tysnd1</i> mRNA measured by quantitative real-time PCR in liver. D. Tysnd1 protein was absent in the liver of <i>Tysnd1</i>^−/− mice as shown by Western blotting using anti-Tysnd1 antibody. E. The expression of known Tysnd1 substrates in the liver homogenate was detected by Western blotting using anti-Acox1, -ScpX/Scp2, -Hsd17b4 and -Acaa1 antibodies. Processed forms of Tysnd1 substrates were not detected in <i>Tysnd1</i>^−/− mice. Arrows indicate the processed form of each enzyme. F. Peroxisomal β-oxidation activity was measured by [1-C¹⁴]lignoceric acids in 15 weeks old control diet-fed (CE2, Clea Japan) male mice liver homogenate. ***<i>p</i><0.001. Each error bar represents the mean ± SE in <i>n</i> = 3.</p

Male <i>Tysnd1</i>^−/− mice are infertile.

<p>A. Semi-thin (8–10 micron) testes sections of 20 weeks old <i>Tysnd1</i>^+/+ and <i>Tysnd1^−/−</i> mice were stained with hematoxylin-eosin. Abnormal, round sperm heads are visible in the seminiferous tubules of <i>Tysnd1</i>^−/− mice. B. Abnormal morphology of <i>Tysnd1</i>^−/− sperms. Epididymal sperms of 15 weeks-old <i>Tysnd1</i>^+/+ and <i>Tysnd1</i>^−/− mice were stained with mitochondrial stains (MitoFluor Red) and nuclear staining (DAPI). Scale bar = 20 µm. C. Percentage of sperms showing normal morphology in <i>Tysnd1</i>^+/+ (n = 4), <i>Tysnd1</i>^+/− (n = 4) and <i>Tysnd1</i>^−/− (n = 5) mice. Each error bar represents the mean ± SE. D. Anti-MN9 antibody immunostaining and Hoechst nuclear staining of epididymal sperms isolated from a 20 weeks old <i>Tysnd1</i>^−/− mouse (red: acrosome and blue: nucleus). Arrow heads and arrows indicate abnormal round-headed sperms and normal sperms, respectively. Scale bar = 5 µm. E. Acrosomes of a semi-thin testis section from a 10 weeks old <i>Tysnd1</i>^−/− and a heterozygous control mouse were stained with PNA-FITC (green) and Hoechst nuclear stain (blue). F. EM image of a <i>Tysnd1</i>^−/− caudal epididymal sperm. The round-headed sperm lacks the acrosome and shows an abnormal mitochondrial sheath (M) around the nucleus (N). Scale bar = 1 µm. G. EM image showing normal spermatogenesis in <i>Tysnd1</i>^+/− male mice. S7: step 7 round spermatid; S16: step 16 elongated spermatid. Acrosomes (A) are normally formed. Scale bar = 2 µm. H. EM image of <i>Tysnd1</i>^−/− elongated spermatid. S16: step 16 spermatid. In some spermatids the acrosome (*) is detached from the nucleus (N). Scale bar = 2 µm.</p

Tysnd1 processes Agps and Phyh <i>in vitro</i>.

<p>A. COS7 cells were transiently co-transfected with Agps-V5 and Tysnd1 expression plasmids. With increasing amounts of Tysnd1 unprocessed Agps-V5 decreased (arrow). Processed Agps-V5 is indicated by an arrowhead B. Agps processing by Tysnd1 is specific and was affected by MG132 proteasome inhibitor. C. Western blot of testes extract using anti-Agps antibody shows unprocessed (arrow) and processed (arrowhead) forms of Agps. D. COS7 cells were transiently co-transfected with Phyh-V5 and Tysnd1 expression plasmids. With increasing amounts of Tysnd1, unprocessed Phyh-V5 decreased (arrow) and processed Phyh-V5 increased (arrowhead). E. Western blot of liver extract using anti-Phyh antibody shows unprocessed (arrow) and processed (arrowhead) forms of Phyh.</p