Characterization of Curli A Production on Living Bacterial Surfaces by Scanning Probe Microscopy

AbstractCurli are adhesive surface fibers produced by many Enterobacteriaceae, such as Escherichia coli and Salmonella enterica. They are implicated in bacterial attachment and invasion to epithelial cells. In this study, atomic force microscopy was used to determine the effects of curli on topology and mechanical properties of live E. coli cells. Young's moduli of both curli-deficient and curli-overproducing mutants were significantly lower than that of their wild-type (WT) strain, while decay lengths of the former strains were higher than that of the latter strain. Surprisingly, topological images showed that, unlike the WT and curli-overproducing mutant, the curli-deficient mutant produced a large number of flagella-like fibers, which may explain why the strain had a lower Young's modulus than the WT. These results suggest that the mechanical properties of bacterial surfaces are greatly affected by the presence of filamentous structures such as curli and flagella

A potential anti-tumor herbal medicine, Corilagin, inhibits ovarian cancer cell growth through blocking the TGF-β signaling pathways

BACKGROUND: Phyllanthus niruri L. is a well-known hepatoprotective and antiviral medicinal herb. Recently, we identified Corilagin as a major active component with anti-tumor activity in this herbal medicine. Corilagin is a member of the tannin family that has been discovered in many medicinal plants and has been used as an anti-inflammatory agent. However, there have been few reports of the anti-tumor effects of Corilagin, and its anti-tumor mechanism has not been investigated clearly. The aim of the present study is to investigate the anticancer properties of Corilagin in ovarian cancer cells. METHODS: The ovarian cancer cell lines SKOv3ip, Hey and HO-8910PM were treated with Corilagin and analyzed by Sulforhodamine B (SRB) cell proliferation assay, flow cytometry, and reverse phase protein array (RPPA). Corilagin was delivered intraperitoneally to mice bearing SKOv3ip xenografts. RESULTS: Corilagin inhibited the growth of the ovarian cancer cell lines SKOv3ip and Hey, with IC50 values of less than 30 μM, while displaying low toxicity against normal ovarian surface epithelium cells, with IC50 values of approximately 160 μM. Corilagin induced cell cycle arrest at the G2/M stage and enhanced apoptosis in ovarian cancer cells. Immunoblotting assays demonstrated that Cyclin B1, Myt1, Phospho-cdc2 and Phospho-Weel were down-regulated after Corilagin treatment. Xenograft tumor growth was significantly lower in the Corilagin-treated group compared with the untreated control group (P <0.05). More interestingly, Corilagin inhibited TGF-β secretion into the culture supernatant of all tested ovarian cancer cell lines and blocked the TGF-β-induced stabilization of Snail. In contrast, a reduction of TGF-β secretion was not observed in cancer cells treated with the cytotoxic drug Paclitaxel, suggesting that Corilagin specifically targets TGF-β secretion. Corilagin blocked the activation of both the canonical Smad and non-canonical ERK/AKT pathways. CONCLUSIONS: Corilagin extracted from Phyllanthus niruri L. acts as a natural, effective therapeutic agent against the growth of ovarian cancer cells via targeted action against the TGF-β/AKT/ERK/Smad signaling pathways

EGFR/Ras Signaling Controls Drosophila Intestinal Stem Cell Proliferation via Capicua-Regulated Genes

Epithelial renewal in the Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs). Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR) in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. Here we demonstrate that the HMG-box transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic’s nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25), Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt). pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs.This work was supported by the DKFZ, DFG grant SFB 873, and ERC Advanced Grant 268515 to BAE. NH was supported by SFB638, SFB1036. MF and GJ were supported by research grants from the Spanish Government (BFU2011-23611) and Fundació La Marató de TV3 (20131730); GJ is an ICREA investigatorPeer Reviewe

Fez function is required to maintain the size of the animal plate in the sea urchin embryo

Partitioning ectoderm precisely into neurogenic and non-neurogenic regions is an essential step for neurogenesis of almost all bilaterian embryos. Although it is widely accepted that antagonism between BMP and its inhibitors primarily sets up the border between these two types of ectoderm, it is unclear how such extracellular, diffusible molecules create a sharp and precise border at the single-cell level. Here, we show that Fez, a zinc finger protein, functions as an intracellular factor attenuating BMP signaling specifically within the neurogenic region at the anterior end of sea urchin embryos, termed the animal plate. When Fez function is blocked, the size of this neurogenic ectoderm becomes smaller than normal. However, this reduction is rescued in Fez morphants simply by blocking BMP2/4 translation, indicating that Fez maintains the size of the animal plate by attenuating BMP2/4 function. Consistent with this, the gradient of BMP activity along the aboral side of the animal plate, as measured by pSmad1/5/8 levels, drops significantly in cells expressing Fez and this steep decline requires Fez function. Our data reveal that this neurogenic ectoderm produces an intrinsic system that attenuates BMP signaling to ensure the establishment of a stable, well-defined neural territory, the animal plate

Integrative Brain Transcriptome Analysis Reveals Region-Specific and Broad Molecular Changes in Shank3-Overexpressing Mice

Variants of the SH3 and multiple ankyrin repeat domain 3 (SHANK3) gene, encoding excitatory postsynaptic core scaffolding proteins, are causally associated with numerous neurodevelopmental and neuropsychiatric disorders, including autism spectrum disorder (ASD), bipolar disorder, intellectual disability, and schizophrenia (SCZ). Although detailed synaptic changes of various Shank3 mutant mice have been well characterized, broader downstream molecular changes, including direct and indirect changes, remain largely unknown. To address this issue, we performed a transcriptome analysis of the medial prefrontal cortex (mPFC) of adult Shank3-overexpressing transgenic (TG) mice, using an RNA-sequencing approach. We also re-analyzed previously reported RNA-sequencing results of the striatum of adult Shank3 TG mice and of the prefrontal cortex of juvenile Shank3+/ΔC mice with a 50–70% reduction of Shank3 proteins. We found that several myelin-related genes were significantly downregulated specifically in the mPFC, but not in the striatum or hippocampus, of adult Shank3 TG mice by comparing the differentially expressed genes (DEGs) of the analyses side by side. Moreover, we also found nine common DEGs between the mPFC and striatum of Shank3 TG mice, among which we further characterized ASD- and SCZ-associated G protein-coupled receptor 85 (Gpr85), encoding an orphan Gpr interacting with PSD-95. Unlike the mPFC-specific decrease of myelin-related genes, we found that the mRNA levels of Gpr85 increased in multiple brain regions of adult Shank3 TG mice, whereas the mRNA levels of its family members, Gpr27 and Gpr173, decreased in the cortex and striatum. Intriguingly, in cultured neurons, the mRNA levels of Gpr27, Gpr85, and Gpr173 were modulated by the neuronal activity. Furthermore, exogenously expressed GPR85 was co-localized with PSD-95 and Shank3 in cultured neurons and negatively regulated the number of excitatory synapses, suggesting its potential role in homeostatic regulation of excitatory synapses in Shank3 TG neurons. Finally, we performed a gene set enrichment analysis of the RNA-sequencing results, which suggested that Shank3 could affect the directional expression pattern of numerous ribosome-related genes in a dosage-dependent manner. To sum up, these results reveal previously unidentified brain region-specific and broad molecular changes in Shank3-overexpressing mice, further elucidating the complexity of the molecular pathophysiology of SHANK3-associated brain disorders

Depiction of immune heterogeneity of peripheral blood from patients with type II diabetic nephropathy based on mass cytometry

Diabetic nephropathy (DN) is the most prominent cause of chronic kidney disease and end-stage renal failure. However, the pathophysiology of DN, especially the risk factors for early onset remains elusive. Increasing evidence has revealed the role of the innate immune system in developing DN, but relatively little is known about early immunological change that proceeds from overt DN. Herein, this work aims to investigate the immune-driven pathogenesis of DN using mass cytometry (CyTOF). The peripheral blood mononuclear lymphocytes (PBMC) from 6 patients with early-stage nephropathy and 7 type II diabetes patients without nephropathy were employed in the CyTOF test. A panel that contains 38 lineage markers was designed to monitor immune protein levels in PBMC. The unsupervised clustering analysis was performed to profile the proportion of individual cells. t-Distributed Stochastic Neighbor Embedding (t-SNE) was used to visualize the differences in DN patients’ immune phenotypes. Comprehensive immune profiling revealed substantial immune system alterations in the early onset of DN, including the significant decline of B cells and the marked increase of monocytes. The level of CXCR3 was dramatically reduced in the different immune cellular subsets. The CyTOF data classified the fine-grained differential immune cell subsets in the early stage of DN. Innovatively, we identified several significant changed T cells, B cell, and monocyte subgroups in the early-stage DN associated with several potential biomarkers for developing DN, such as CTLA-4, CXCR3, PD-1, CD39, CCR4, and HLA-DR. Correlation analysis further demonstrated the robust relationship between above immune cell biomarkers and clinical parameters in the DN patients. Therefore, we provided a convincible view of understanding the immune-driven early pathogenesis of DN. Our findings exhibited that patients with DN are more susceptible to immune system disorders. The classification of fine-grained immune cell subsets in this present research might provide novel targets for the immunotherapy of DN

Intestinal Stem Cell Pool Regulation in Drosophila

Intestinal epithelial renewal is mediated by intestinal stem cells (ISCs) that exist in a state of neutral drift, wherein individual ISC lineages are regularly lost and born but ISC numbers remain constant. To test whether an active mechanism maintains stem cell pools in the Drosophila midgut, we performed partial ISC depletion. In contrast to the mouse intestine, Drosophila ISCs failed to repopulate the gut after partial depletion. Even when the midgut was challenged to regenerate by infection, ISCs retained normal proportions of asymmetric division and ISC pools did not increase. We discovered, however, that the loss of differentiated midgut enterocytes (ECs) slows when ISC division is suppressed and accelerates when ISC division increases. This plasticity in rates of EC turnover appears to facilitate epithelial homeostasis even after stem cell pools are compromised. Our study identifies unique behaviors of Drosophila midgut cells that maintain epithelial homeostasis

Re-expression of ARHI (DIRAS3) induces autophagy in breast cancer cells and enhances the inhibitory effect of paclitaxel

<p>Abstract</p> <p>Background</p> <p><it>ARHI </it>is a Ras-related imprinted gene that inhibits cancer cell growth and motility. ARHI is downregulated in the majority of breast cancers, and loss of its expression is associated with its progression from ductal carcinoma <it>in situ </it>(DCIS) to invasive disease. In ovarian cancer, re-expression of ARHI induces autophagy and leads to autophagic death in cell culture; however, ARHI re-expression enables ovarian cancer cells to remain dormant when they are grown in mice as xenografts. The purpose of this study is to examine whether ARHI induces autophagy in breast cancer cells and to evaluate the effects of ARHI gene re-expression in combination with paclitaxel.</p> <p>Methods</p> <p>Re-expression of ARHI was achieved by transfection, by treatment with trichostatin A (TSA) or by a combination of TSA and 5-aza-2'-deoxycytidine (DAC) in breast cancer cell cultures and by liposomal delivery of ARHI in breast tumor xenografts.</p> <p>Results</p> <p>ARHI re-expression induces autophagy in breast cancer cells, and ARHI is essential for the induction of autophagy. When ARHI was re-expressed in breast cancer cells treated with paclitaxel, the growth inhibitory effect of paclitaxel was enhanced in both the cell culture and the xenografts. Although paclitaxel alone did not induce autophagy in breast cancer cells, it enhanced ARHI-induced autophagy. Conversely, ARHI re-expression promoted paclitaxel-induced apoptosis and G2/M cell cycle arrest.</p> <p>Conclusions</p> <p>ARHI re-expression induces autophagic cell death in breast cancer cells and enhances the inhibitory effects of paclitaxel by promoting autophagy, apoptosis, and G2/M cell cycle arrest.</p

Altered presynaptic function and number of mitochondria in the medial prefrontal cortex of adult Cyfip2 heterozygous mice

Variants of the cytoplasmic FMR1-interacting protein (CYFIP) gene family, CYFIP1 and CYFIP2, are associated with numerous neurodevelopmental and neuropsychiatric disorders. According to several studies, CYFIP1 regulates the development and function of both pre- and post-synapses in neurons. Furthermore, various studies have evaluated CYFIP2 functions in the postsynaptic compartment, such as regulating dendritic spine morphology; however, no study has evaluated whether and how CYFIP2 affects presynaptic functions. To address this issue, in this study, we have focused on the presynapses of layer 5 neurons of the medial prefrontal cortex (mPFC) in adult Cyfip2 heterozygous (Cyfip2+/−) mice. Electrophysiological analyses revealed an enhancement in the presynaptic short-term plasticity induced by high-frequency stimuli in Cyfip2+/− neurons compared with wild-type neurons. Since presynaptic mitochondria play an important role in buffering presynaptic Ca2+, which is directly associated with the short-term plasticity, we analyzed presynaptic mitochondria using electron microscopic images of the mPFC. Compared with wild-type mice, the number, but not the volume or cristae density, of mitochondria in both presynaptic boutons and axonal processes in the mPFC layer 5 of Cyfip2+/− mice was reduced. Consistent with an identification of mitochondrial proteins in a previously established CYFIP2 interactome, CYFIP2 was detected in a biochemically enriched mitochondrial fraction of the mouse mPFC. Collectively, these results suggest roles for CYFIP2 in regulating presynaptic functions, which may involve presynaptic mitochondrial changes.This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea Government Ministry of Science and ICT (NRF-2018R1C1B6001235, NRF-2018M3C7A1024603, NRF-2017M3C7A1048086, and NRF-2020R1A2C3011464) and the KBRI Basic Research Programs (20-BR01-08 and 20-BR-04-01)

Molecular evolution of the vertebrate TLR1 gene family - a complex history of gene duplication, gene conversion, positive selection and co-evolution

<p>Abstract</p> <p>Background</p> <p>The Toll-like receptors represent a large superfamily of type I transmembrane glycoproteins, some common to a wide range of species and others are more restricted in their distribution. Most members of the Toll-like receptor superfamily have few paralogues; the exception is the TLR1 gene family with four closely related genes in mammals TLR1, TLR2, TLR6 and TLR10, and four in birds TLR1A, TLR1B, TLR2A and TLR2B. These genes were previously thought to have arisen by a series of independent gene duplications. To understand the evolutionary pattern of the TLR1 gene family in vertebrates further, we cloned the sequences of TLR1A, TLR1B, TLR2A and TLR2B in duck and turkey, constructed phylogenetic trees, predicted codons under positive selection and identified co-evolutionary amino acid pairs within the TLR1 gene family using sequences from 4 birds, 28 mammals, an amphibian and a fish.</p> <p>Results</p> <p>This detailed phylogenetic analysis not only clarifies the gene gains and losses within the TLR1 gene family of birds and mammals, but also defines orthologues between these vertebrates. In mammals, we predict amino acid sites under positive selection in TLR1, TLR2 and TLR6 but not TLR10. We detect co-evolution between amino acid residues in TLR2 and the other members of this gene family predicted to maintain their ability to form functional heterodimers. In birds, we predict positive selection in the TLR2A and TLR2B genes at functionally significant amino acid residues. We demonstrate that the TLR1 gene family has mostly been subject to purifying selection but has also responded to directional selection at a few sites, possibly in response to pathogen challenge.</p> <p>Conclusions</p> <p>Our phylogenetic and structural analyses of the vertebrate TLR1 family have clarified their evolutionary origins and predict amino acid residues likely to be important in the host's defense against invading pathogens.</p