SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening ...for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.
Bone metabolism is regulated by the cooperative activity between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the mechanisms mediating the switch between the osteoblastic and ...osteoclastic phases have not been fully elucidated. Here, we identify a specific subset of mature osteoblast-derived extracellular vesicles that inhibit bone formation and enhance osteoclastogenesis. Intravital imaging reveals that mature osteoblasts secrete and capture extracellular vesicles, referred to as small osteoblast vesicles (SOVs). Co-culture experiments demonstrate that SOVs suppress osteoblast differentiation and enhance the expression of receptor activator of NF-κB ligand, thereby inducing osteoclast differentiation. We also elucidate that the SOV-enriched microRNA miR-143 inhibits Runt-related transcription factor 2, a master regulator of osteoblastogenesis, by targeting the mRNA expression of its dimerization partner, core-binding factor β. In summary, we identify SOVs as a mode of cell-to-cell communication, controlling the dynamic transition from bone-forming to bone-resorbing phases in vivo.
The biological significance of micro (mi)RNAs has traditionally been evaluated according to their RNA expression levels based on the assumption that miRNAs recognize and regulate their targets in an ...unvarying fashion. Here we show that a fraction of mature miRNAs including miR-17-5p, -21-5p, and -200c-3p and let-7a-5p harbor methyl marks that potentially alter their stability and target recognition. Importantly, methylation of these miRNAs was significantly increased in cancer tissues as compared to paired normal tissues. Furthermore, miR-17-5p methylation level in serum samples distinguished early pancreatic cancer patients from healthy controls with extremely high sensitivity and specificity. These findings provide a basis for diagnostic strategies for early-stage cancer and add a dimension to our understanding of miRNA biology.
Background
Metagenomic analysis targeting the 16S rRNA gene has made it possible to characterize the vast array of microorganisms contained in the gut.
Aim
The purpose of this study was to evaluate ...how gut microbiota change in intensive care unit (ICU) patients in the acute phase after admission.
Methods
This prospective observational cohort study investigated 12 patients admitted to a single ICU of a large urban tertiary referral hospital. All patients were mechanically ventilated on admission. Fecal samples were collected from patients on days 1–2, 2–4, 5–8, and 7–10 after admission. DNA was extracted from fecal samples, and 16S rRNA deep sequencing was performed to monitor gut changes.
Results
Bacteria belonging to the phyla
Firmicutes
or
Bacteroidetes
were predominant in each sample. We observed serial dynamic changes in the percentages of
Bacteroidetes
and
Firmicutes
that were significantly altered during study period (
p
< 0.05). A ratio of
Bacteroidetes
to
Firmicutes
(B/F ratio) of >10 was seen in four of the six patients who died, whereas a B/F ratio of <0.10 was seen in only one of the six deaths. None of the survivors had a B/F ratio of >10 or <0.10. There was a statistical difference in the B/F ratio between the dead patients and survivors (
p
= 0.022).
Conclusions
Dynamic changes in gut microbiota at the phylum level of ICU patients during the acute phase were identified by high-throughput DNA sequencing. An extreme imbalance in gut microbiota may be associated with prognosis in critically ill patients.
Background
Indigo Naturalis (IN) is used as a traditional herbal medicine for ulcerative colitis (UC). However, the mechanisms of action of IN have not been clarified. We aimed to evaluate the ...efficacy of IN for ameliorating colonic inflammation. We further investigated the mechanisms of action of IN.
Methods
Colitis severity was assessed in dextran sodium sulfate-induced colitis and trinitrobenzene sulfonic acid-induced colitis models with or without the oral administration of IN or indigo, which is a known major component of IN. Colonic lamina propria (LP) mononuclear cells isolated from IN-treated mice were analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry. LP and splenic mononuclear cells cultured in vitro with IN or indigo were also analyzed. The role of the candidate receptor for indigo, the aryl hydrocarbon receptor (AhR), was analyzed using
Ahr
-deficient mice.
Results
Colitis severity was significantly ameliorated in the IN and indigo treatment groups compared with the control group. The mRNA expression levels of interleukin (
Il
)-
10
and
Il-22
in the LP lymphocytes were increased by IN treatment. The treatment of splenocytes with IN or indigo increased the expression of anti-inflammatory cytokines and resulted in the expansion of IL-10-producing CD4
+
T cells and IL-22-producing CD3
−
RORγt
+
cells, but not CD4
+
Foxp3
+
regulatory T cells. The amelioration of colitis by IN or indigo was abrogated in
Ahr
-deficient mice, in association with diminished regulatory cytokine production.
Conclusions
IN and indigo ameliorated murine colitis through AhR signaling activation, suggesting that AhR could be a promising therapeutic target for UC.
Abstract
Anti-dsDNA antibodies are a hallmark of systemic lupus erythematosus and are highly associated with its exacerbation. Cumulative evidence has suggested that somatic hypermutation contributes ...to the high-affinity reactivity of anti-dsDNA antibodies. Our previous study demonstrated that these antibodies are generated from germline precursors with low-affinity ssDNA reactivity through affinity maturation and clonal expansion in patients with acute lupus. This raised the question of whether such precursors could be subjected to immune tolerance. To address this, we generated a site-directed knock-in (KI) mouse line, G9gl, which carries germline-reverted sequences of the VH–DH–JH and Vκ–Jκ regions of patient-derived, high-affinity anti-dsDNA antibodies. G9gl heterozygous mice had a reduced number of peripheral B cells, only 27% of which expressed G9gl B-cell receptor (BCR). The remaining B cells harbored non-KI allele-derived immunoglobulin heavy (IgH) chains or fusion products of upstream mouse VH and the KI gene, suggesting that receptor editing through VH replacement occurred in a large proportion of B cells in the KI mice. G9gl BCR-expressing B cells responded to ssDNA but not dsDNA, and exhibited several anergic phenotypes, including reduced surface BCR and shortened life span. Furthermore, G9gl B cells were excluded from germinal centers (GCs) induced by several conditions. In particular, following immunization with methylated bovine serum albumin-conjugated bacterial DNA, G9gl B cells occurred at a high frequency in memory B cells but not GC B cells or plasmablasts. Collectively, multiple tolerance checkpoints prevented low-affinity precursors of pathogenic anti-dsDNA B cells from undergoing clonal expansion and affinity maturation in GCs.
Regulation of anti-dsDNA antibodies in SLE
Graphical Abstract
Background and Aims
NAFLD is the most common liver disease worldwide. NASH, the progressive form of NAFLD, and advanced fibrosis are associated with poor outcomes. We searched for their noninvasive ...biomarkers.
Approach and Results
Global RNA sequencing of liver tissue from 98 patients with biopsy‐proven NAFLD was performed. Unsupervised hierarchical clustering well distinguished NASH from nonalcoholic fatty liver (NAFL), and patients with NASH exhibited molecular abnormalities reflecting their pathological features. Transcriptomic analysis identified proteins up‐regulated in NASH and/or advanced fibrosis (stage F3‐F4), including matricellular glycoprotein thrombospondin‐2 (TSP‐2), encoded by the thrombospondin 2 (THBS2) gene. The intrahepatic THBS2 expression level showed the highest areas under the receiver operating characteristic curves (AUROCs) of 0.915 and 0.957 for diagnosing NASH and advanced fibrosis, respectively. THBS2 positively correlated with inflammation and ballooning according to NAFLD activity score, serum aspartate aminotransferase and hyaluronic acid (HA) levels, and NAFLD Fibrosis Score (NFS). THBS2 was associated with extracellular matrix and collagen biosynthesis, platelet activation, caspase‐mediated cleavage of cytoskeletal proteins, and immune cell infiltration. Serum TSP‐2 expression was measured in 213 patients with biopsy‐proven NAFLD, was significantly higher in NASH than in NAFL, and increased parallel to fibrosis stage. The AUROCs for predicting NASH and advanced fibrosis were 0.776 and 0.856, respectively, which were comparable to Fibrosis‐4 index, serum HA level, and NFS in advanced fibrosis diagnosis. Serum TSP‐2 level and platelet count were independent predictors of NASH and advanced fibrosis. Serum TSP‐2 levels could stratify patients with NAFLD according to the risk of hepatic complications, including liver cancer and decompensated cirrhotic events.
Conclusions
TSP‐2 may be a useful biomarker for NASH and advanced fibrosis diagnosis in patients with NAFLD.
Excessive intake of animal fat and resultant obesity are major risk factors for prostate cancer. Because the composition of the gut microbiota is known to change with dietary composition and body ...type, we used prostate-specific
knockout mice as a prostate cancer model to investigate whether there is a gut microbiota-mediated connection between animal fat intake and prostate cancer. Oral administration of an antibiotic mixture (Abx) in prostate cancer-bearing mice fed a high-fat diet containing a large proportion of lard drastically altered the composition of the gut microbiota including
and
, inhibited prostate cancer cell proliferation, and reduced prostate
expression and circulating insulin-like growth factor-1 (IGF1) levels. In prostate cancer tissue, MAPK and PI3K activities, both downstream of the IGF1 receptor, were suppressed by Abx administration. IGF1 directly promoted the proliferation of prostate cancer cell lines DU145 and 22Rv1
. Abx administration also reduced fecal levels of short-chain fatty acids (SCFA) produced by intestinal bacteria. Supplementation with SCFAs promoted tumor growth by increasing IGF1 levels. In humans, IGF1 was found to be highly expressed in prostate cancer tissue from obese patients. In conclusion, IGF1 production stimulated by SCFAs from gut microbes influences the growth of prostate cancer via activating local prostate MAPK and PI3K signaling, indicating the existence of a gut microbiota-IGF1-prostate axis. Disrupting this axis by modulating the gut microbiota may aid in prostate cancer prevention and treatment. SIGNIFICANCE: These results suggest that intestinal bacteria, acting through short-chain fatty acids, regulate systemic and local prostate IGF1 in the host, which can promote proliferation of prostate cancer cells.
Hepatocellular carcinoma (HCC) is characterized by high intertumor heterogeneity of genetic drivers. Two multitarget tyrosine kinase inhibitors (TKI), lenvatinib and sorafenib, are used as ...standard-of-care chemotherapeutics in patients with advanced HCC, but a stratification strategy has not been established because of a lack of efficacious biomarkers. Therefore, we sought biomarkers that indicate lenvatinib-susceptible HCC.
We performed genetic screening of HCC driver genes involved in TKI susceptibility using a novel HCC mouse model in which tumor diversity of genetic drivers was recapitulated. A biomarker candidate was evaluated in human HCC cell lines. Secreted proteins from HCC cells were then screened using mass spectrometry. Serum and tumor levels of the biomarker candidates were analyzed for their association and prediction of overall survival in patients with HCC.
We found that lenvatinib selectively eliminated FGF19-expressing tumors, whereas sorafenib eliminated MET- and NRAS-expressing tumors. FGF19 levels and lenvatinib susceptibility were correlated in HCC cell lines, and FGF19 inhibition eliminated lenvatinib susceptibility. Lenvatinib-resistant HCC cell lines, generated by long-term exposure to lenvatinib, showed FGF19 downregulation but were resensitized to lenvatinib by FGF19 reexpression. Thus, FGF19 is a tumor biomarker of lenvatinib-susceptible HCC. Proteome and secretome analyses identified ST6GAL1 as a tumor-derived secreted protein positively regulated by FGF19 in HCC cells. Serum ST6GAL1 levels were positively correlated with tumor FGF19 expression in patients with surgically resected HCC. Among patients with serum ST6GAL1-high HCC who underwent TKI therapy, lenvatinib therapy showed significantly better survival than sorafenib.
Serum ST6GAL may be a novel biomarker that identifies lenvatinib-susceptible FGF19-driven HCC.
The mechanisms underlying metastasis of colorectal cancer (CRC) remain unclear. C14orf159 is a mitochondrial matrix protein converting D-glutamate to 5-oxo-D-proline. Other metabolic functions of ...C14orf159, especially on mitochondrial metabolism, and its contribution to CRC metastasis, are not elucidated.
Metabolome analysis by gas chromatography-mass spectrometry, RNA-sequencing analysis, flow cytometry, migration and invasion assay, sphere-formation assay using C14orf159-knockout and -stable expressing cells, immunohistochemistry of C14orf159 in human CRC specimens, and xenograft experiments using Balb/c nude mice were conducted.
C14orf159 maintained the mitochondrial membrane potential of human CRC cells, and its involvement in amino acid and glutathione metabolism was demonstrated. In human CRC specimens, a decrease in C14orf159 expression at the invasive front of the tumour and in metastasis was determined. C14orf159 was also shown to attenuate the migration, invasion, and spheroid growth of CRC cells in vitro and colorectal tumour growth and metastasis in vivo. Mechanistically, C14orf159 reduced the expression of genes involved in CRC metastasis, including members of the Wnt and MMP family, by maintaining the mitochondrial membrane potential.
Our findings link mitochondrial membrane potential to Wnt/β-catenin signalling and reveal a previously unrecognised function of the mitochondrial matrix protein C14orf159 as a suppressor of CRC metastasis.
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