Low Basal Level Of Expression Research Articles

A tightly controlled gene induction system that contributes to the study of lethal gene function in Streptococcus mutans

ABSTRACT Effective control of gene expression is crucial for understanding gene function in both eukaryotic and prokaryotic cells. While several inducible gene expression systems have been reported in Streptococcus mutans, a conditional pathogen that causes dental caries, the significant non-inducible basal expression in these systems seriously limits their utility, especially when studying lethal gene functions and molecular mechanisms. We introduce a tightly controlled xylose-inducible gene expression system, TC-Xyl, for Streptococcus mutans. Western blot results and fluorescence microscopy analysis indicate that TC-Xyl exhibits an extremely low non-inducible basal expression level and a sufficiently high expression level post-induction. Further, by constructing a mutation in which the only source FtsZ is under the control of TC-Xyl, we preliminarily explored the function of the ftsz gene. We found that FtsZ depletion is lethal to Streptococcus mutans, resulting in abnormal round cell shape and mini cell formation, suggesting FtsZ’s role in maintaining cell shape stability.

Small-Molecule Aptamer for Regulating RNA Functions in Mammalian Cells and Animals.

Synthetic riboswitches that can regulate gene expression by a small molecule recognized by an RNA aptamer in mammalian cells have various potential applications in biotechnology and medicine. However, the variety of small molecules and their cognate aptamers that have been demonstrated to function in mammalian cells is limited. The currently available aptamer-ligand pairs also require high small molecule concentrations to enable gene regulation, making them less desirable for industrial and biomedical applications. We conducted in vitro selection of RNA aptamers against a small molecule ASP7967 whose structure is closely related to ASP2905, a known inhibitor of potassium voltage-gated channel sub-family H member 3 (KCNH3). One of the aptamers selected (AC17-4) was found to be functional in HEK293 cells, and it was used to design aptazyme-based riboswitches that can activate gene expression (>10-fold) in the presence of ASP2905 or ASP7967 at as low as 5 μM in the culture medium. An aptazyme-based riboswitch was successfully used to regulate human erythropoietin expression in mice injected with an adeno-associated virus (AAV8) vector using orally administered ASP7967. Furthermore, by combining aptazyme-based and exon-skipping riboswitch mechanisms, an ON/OFF ratio approaching 300 was achieved with a low basal expression level in cultured cells.

Synthesis of a‐ and α‐series gangliosides during differentiation of leukemic HL‐60 and THP‐1 cells

Synthesis of a‐ and α‐series gangliosides during differentiation of leukemic HL‐60 and THP‐1 cells

SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas.

SUMMARYInherited pathogenic succinate dehydrogenase (SDHx) gene mutations cause the hereditary pheochromocytoma and paraganglioma tumor syndrome. Syndromic tumors exhibit elevated succinate, an oncometabolite that is proposed to drive tumorigenesis via DNA and histone hypermethylation, mitochondrial expansion, and pseudohypoxia-related gene expression. To interrogate this prevailing model, we disrupt mouse adrenal medulla SDHB expression, which recapitulates several key molecular features of human SDHx tumors, including succinate accumulation but not 5hmC loss, HIF accumulation, or tumorigenesis. By contrast, concomitant SDHB and the neurofibromin 1 tumor suppressor disruption yields SDHx-like pheochromocytomas. Unexpectedly, in vivo depletion of the 2-oxoglutarate (2-OG) dioxygenase cofactor ascorbate reduces SDHB-deficient cell survival, indicating that SDHx loss may be better tolerated by tissues with high antioxidant capacity. Contrary to the prevailing oncometabolite model, succinate accumulation and 2-OG-dependent dioxygenase inhibition are insufficient for mouse pheochromocytoma tumorigenesis, which requires additional growth-regulatory pathway activation.

XÁC ĐỊNH VÀ PHÂN TÍCH CÁC GENE HSP90 Ở CÂY ĐU ĐỦ (Carica papaya L.) BẰNG PHƯƠNG PHÁP TIN SINH HỌC

The HSP90 gene family has been shown to play an important role in the tolerance and development of plants. Papaya, which is a fruit crop with high nutritional value, is native to the tropics but now is widely cultivated in many subtropical regions of the world. Therefore, papaya plants have to face many environmental factors during their life. This study aims to identify and analyze the HSP90 gene family in papaya by bioinformatics method. A total of seven HSP90 genes have been identified in the genome of papaya (Carica papaya L.) by using the bioinformatic methods. The full-length genomic sequence of papaya HSP90 genes were ranging from 2650 to 8136 nucleotides, non continuous coding, with number of intro ranging from two to 19. The predicted protein sequences included from 348 to 796 amino acids, according to the molecular weight ranged from 39.92 to 90.61 kDa. Among seven CpHSP90, the two CpHSP90-1 and CpHSP90-4 were considered pseudogenes due to their small size. These proteins were acidic with a pI value ranging from 4.69 to 5.42, except CpHSP90-1 (pI 7.03). Based on the protein structure, subcellular localization and the phylogenic analysis, the papaya HSP90 were divided into two groups, I (cytoplasmic HSP90, four members) and II (organelle HSP90, three members). Analysis of transcriptomes showed that the papaya HSP90s were differentially expressed in different tissues at different development stages. In which, most of the papaya HSP90 is highly expressed in flower buds or in fruits at stage 2 or stage 3. CpHSP90-2 had the highest level of expression, followed by CpHSP90-5. In contrast, CpHSP90-1 was not expressed or very weakly expressed in these studied tissues. All of seven HSP90 genes of papaya were induced by freeze-thaw awakening treatment (in comparison with control treatment), among them, CpHSP90-1 was strongest induced by stress (12.13-folds), however, it was a pseudogene and had a very low level of basal expression. CpHSP90-2 had a high induction level (2.81- folds), and also had a high basal expression level compared to other HSP90 genes of papaya. The results of this work have an important significance and will serve as a base for the further research on gene cloning, functional analysis of HSP90 genes and breeding of papaya in response to environmental abiotic stresses and the development of this fruit crop.

Regulation of Chemosensitivity in Human Medulloblastoma Cells by p53 and the PI3 Kinase Signaling Pathway.

In medulloblastoma, p53 expression has been associated with chemoresistance and radiation resistance and with poor long-term outcomes in the p53-mutated sonic hedgehog, MYC-p53, and p53-positive medulloblastoma subgroups. We previously established a direct role for p53 in supporting drug resistance in medulloblastoma cells with high basal protein expression levels (D556 and DAOY). We now show that p53 genetic suppression in medulloblastoma cells with low basal p53 protein expression levels (D283 and UW228) significantly reduced drug responsiveness, suggesting opposing roles for low p53 protein expression levels. Mechanistically, the enhanced cell death by p53 knockdown in high-p53 cells was associated with an induction of mTOR/PI3K signaling. Both mTOR inhibition and p110α/PIK3CA induction confirmed these findings, which abrogated or accentuated the enhanced chemosensitivity response in D556 cells respectively while converse was seen in D283 cells. Co-treatment with G-actin-sequestering peptide, thymosin β4 (Tβ4), induced p-AKTS473 in both p53-high and p53-low cells, enhancing chemosensitivity in D556 cells while enhancing chemoresistance in D283 and UW228 cells. IMPLICATIONS: Collectively, we identified an unexpected role for the PI3K signaling in enhancing cell death in medulloblastoma cells with high basal p53 expression. These studies indicate that levels of p53 immunopositivity may serve as a diagnostic marker of chemotherapy resistance and for defining therapeutic targeting.

DNMT1 Inhibitor Restores RUNX2 Expression and Mineralization in Periodontal Ligament Cells.

Periodontal ligament cells (PDLCs) have well documented osteogenic potential; however, this commitment can be highly heterogenous, limiting their applications in tissue regeneration. In this study, we use PDLC populations characterized by high and low osteogenic potential (h-PDLCs and l-PDLCs, respectively) to identify possible sources of such heterogeneity and to investigate whether the osteogenic differentiation can be enhanced by epigenetic modulation. In h-PDLCs, low basal expression levels of pluripotency markers (NANOG, OCT4), DNA methyltransferases (DNMT1, DNMT3B), and enzymes involved in active DNA demethylation (TET1, TET3) were prerequisite to high osteogenic potential. Furthermore, these genes were downregulated upon early osteogenesis, possibly allowing for the increase in expression of the key osteogenic transcription factors, Runt-related transcription factor 2 (RUNX2) and SP7, and ultimately, mineral nodule formation. l-PDLCs appeared locked in the multipotent state and this was further enhanced upon early osteogenic stimulation, correlating with low RUNX2 expression and impaired mineralization. Further upregulation of DNMTs was also evident, while pretreatment with RG108, the DNMTs' inhibitor, enhanced the osteogenic program in l-PDLCs through downregulation of DNMTs, increased RUNX2 expression and nuclear localization, accelerated expression of osteogenic markers, and increased mineralization. These findings point toward the role of DNMTs and Ten Eleven Translocations (TETs) in osteogenic commitment and support application of epigenetic approaches to modulate biomineralization in PDLCs.

Quantitative analysis of differential dehydrin regulation in pine and spruce seedlings under water deficit

Dehydrins are well-known components of plant responses to different stresses that cause dehydration, including drought, freezing, salinity, etc. In conifers, the dehydrin gene family is very large, implying that the members of this family have important physiological functions in conifer stress tolerance. However, dehydrin gene expression displays a wide range of responses to stress, from thousand-fold increased expression to decreased expression, and it is generally unknown how regulatory systems are connected at the mRNA and protein levels. Therefore, we studied these aspects of dehydrin regulation in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) H. Karst) seedlings under polyethylene glycol 6000-induced osmotic stress ranging from relatively low (culture medium water potential of −0.15 MPa) to very high (−1.0 MPa) intensities. In pine, the major dehydrin protein was Dhn1 in both the roots and needles, and in spruce, two isoforms of the Dhn4 protein were the major dehydrins; both of these proteins are AESK-type dehydrins. The genes encoding these major proteins were highly expressed even under control conditions; surprisingly, we also observed several highly expressed dehydrin genes that were not abundantly translated. Under osmotic stress, the most prominent expression changes were observed for the dehydrin genes with low basal expression levels, whereas highly expressed genes generally demonstrated rather modest changes in expression. We report proposed constitutive physiological functions of the AESK-type dehydrins in Pinaceae plants.

The therapeutic potential of attenuated diphtheria toxin delivered by an adenovirus vector with survivin promoter on human lung cancer cells

ABSTRACT Adenoviral vectors are superior to plasmid vectors in their gene transport efficiency. The A subunit of the diphtheria toxin (DTA) gene is a popular suicide gene in cancer gene therapy. However, DTA is seldom used in adenoviral therapy due to its great toxicity. The toxicity of DTA is so great that even a single molecule of DTA is enough to kill one cell. To avoid this highly toxic effect on normal cells, DTA should be controlled by tumor-specific promoters. The survivin promoter is a widely used tumor-specific promoter. But genes driven by the survivin promoter show a low level of basal gene expression in non-cancer cells. DTA driven by the survivin promoter in adenoviral vectors may be highly toxic not only to cancer cells but also to normal cells. Therefore, DTA should be attenuated when it is used in adenoviral vectors driven by the survivin promoter. In this study, we compared the three kinds of recombinant adenoviruses that carry DTA or its attenuated forms (DTA176 and DTA197) in the treatment of human lung cancer. The results showed that in comparison with both DTA and DTA176, DTA197 is more suitable for adenoviral cancer therapy controlled by the survivin promoter. In addition, Adsur-DTA197 (DTA197 delivered by an adenoviral vector with the survivin promoter) sensitized human lung cancer cells to cisplatin both in vitro and in vivo. These results indicated that Adsur-DTA197 may be a potential chemosensitizer in cancer therapy.

THE VALUE OF BASAL EXPRESSION LEVEL OF HEMOXYGENASE-1 FOR SENSITIVITY OF HUMAN MELANOMA CELLS TO OXIDATIVE STRESS IN VITRO

Introduction.The molecular basis of radio- and photodynamic therapy (PDT), the mechanism of action of a number of antitumor chemotherapy drugs is oxidative stress (OS). The enzyme hemoxygenase-I (НO-1), a molecular marker of OS, is a key participant in the system of protection and adaptation of tumor cells under stress.Objective.To find out whether the sensitivity of human melanoma tumor cells to OS depends on the basal and modulator-induced levels of НO-1 expressionMaterial and methods.Human melanoma cell lines were used in the study. The expression of mRNA НO-1 in cells was studied by real-time RT-PCR, the reactive oxygen species content in cells – by flow cytometry and the cytotoxicity of drugs – by MTT assay.Results.According to our data, human melanoma cells have different basal levels of HO-1 transcription: high (3.0–3.5 o. u.) in lines MelIL, MelP, medium (1.5 o. u.) in lines MeWo, MelZ, MelIbr and low (0.5 o. e.) – MelMe, A375).There is no direct correlation between the level of basal cell expression of HO-1 and their sensitivity to the OS inducer – Н2О2. The hemin-induced increase in HO-1 expression in cells is accompanied by doubled resistance to Н2О2. It was found that HO-1 repression in the presence of apigenin was registered in melanoma cells with different basal levels, but sensitization to Н2О2 (2–4 times) was observed only for cells with medium (MeWo) and low (A375) levels of basal HO-1 expression. It was found that the decrease in basal expression of HO-1 induced by apigenin is accompanied by an increase in the reactive oxygen species content in cells.Conclusions.The results of our research allow us to recommend natural flavon apigenin, a modulator of HO-1 expression, for inclusion in the chemotherapy and PDT regimens to increase the effectiveness of human melanoma treatment.

Abstract 5107: DDR1 and MT1-MMP expression levels are major determinants of 3D type I collagen-induced apoptosis in basal-like breast carcinoma

Abstract Type I collagen is the major adhesive component in breast interstitial stroma, which represents the first barrier against tumor cell invasion after basement-membrane degradation. Among cellular receptors, type I collagen is able to activate discoidin domain receptors DDR1 and DDR2. We have previously shown that in 3D collagen matrix, DDR1 plays a key role as it promotes cell growth suppression and apoptosis through the upregulation of the pro-apoptotic mediator BIK in noninvasive luminal-like breast carcinoma cells. We have also shown that MT1-MMP is able to rescue these cells and protect them against the effects induced by collagen/DDR1/BIK axis. Our data suggested that the protective effect of MT1-MMP might be mediated through the degradation of type I collagen and/or DDR1 cleavage. Decreased DDR1 expression has been associated with the epithelial to mesenchymal transition process in breast cancer, and its overexpression in aggressive basal-like breast cancer cells reduces their invasiveness in 3D cultures and in vivo. In the present work, we propose to study the role of MT1-MMP in the resistance against collagen-induced apoptosis in basal-like breast carcinoma MDA-MB-231 cells. We aimed to investigate whether MT1-MMP depletion is able to restore apoptosis mediated by collagen/DDR1/BIK axis and to verify if such depletion is able to restore full-length DDR1 expression and phosphorylation. ShRNA strategy against MT1-MMP mRNA was able to partially restore full length DDR1 expression and phosphorylation. This was accompanied by a decrease in cell growth and an upregulation of BIK expression. This suggested that MT1-MMP expression in basal-like breast carcinoma cells, in addition to a low basal level of DDR1 expression, protects these cells against collagen-induced apoptosis via DDR1 cleavage. Since DDR1 was moderately expressed in MDA-MB-231 cells, we then investigated whether overexpression of DDR1 could be able to increase its ability to suppress cell growth and to induce apoptosis. Data showed that overexpression of DDR1 induced a decrease in cell growth and an increase in BIK expression, suggesting that moderate expression level of full length DDR1 in basal-like breast carcinoma provides them with a capacity to resist to collagen-induced cell growth suppression and apoptosis. Finally, the combined overexpression of DDR1 and depletion of MT1-MMP in MDA-MB-231 cells synergistically increased collagen-induced cell growth suppression and apoptosis to a level similar to that observed in luminal breast carcinoma. Taken together, our data suggest that during the acquisition of mesenchymal features, the low level of DDR1 expression should be considered as an important biomarker in the prognosis of basal-like breast carcinoma, conferring them a high rate of cell growth and resistance to BIK-mediated apoptosis induced by the stromal collagen. Citation Format: Charles Saby, Guillaume Collin, Maha Sinane, Emilie Buache, Laurence Van Gulick, Frédéric Saltel, Erik Maquoi, Hamid Morjani. DDR1 and MT1-MMP expression levels are major determinants of 3D type I collagen-induced apoptosis in basal-like breast carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5107.

Role of glucose metabolism in the differential antileukemic effect of melatonin on wild‑type and FLT3‑ITD mutant cells.

The FMS‑like tyrosine kinase 3 internal tandem duplication (FLT3‑ITD) mutation represents the most frequent genetic alteration in acute myeloid leukemia (AML) and is associated with poor prognosis. The mutation promotes cancer cell survival and proliferation, and shifts their glucose metabolism towards aerobic glycolysis, a frequent alteration in cancer. In the present study, the impact of melatonin on the viability of AML cell lines with (MV‑4‑11 and MOLM‑13) or without the FLT3‑ITD mutation (OCI‑AML3 and U‑937) was evaluated. Melatonin induces cell death in AML cells carrying the FLT3‑ITD mutation, but only inhibits the proliferation of AML cells without this mutation. Consistently, melatonin decreases tumor growth and increases animal survival in a xenograft model of FLT3‑ITD AML. Toxicity is related to a decrease in glucose uptake, lactate dehydrogenase activity, lactate production and hypoxia‑inducible factor‑1α activation. Melatonin also regulates the expression of glucose metabolism‑related genes, impairing the balance between anaplerosis and cataplerosis, through the upregulation of the expression of phosphoenolpyruvate carboxykinase 2 (PCK2). Collectively, the present findings highlight the regulation of glucose metabolism, currently considered a possible therapeutic target in cancer, as a key event in melatonin‑induced cytotoxicity, suggesting its potential as a therapeutic tool for the treatment of patients with AML, particularly those carrying the FLT3‑ITD mutation that results in low basal expression levels of PCK2.

A TGF-β- and p63-Responsive Enhancer Regulates IFN-κ Expression in Human Keratinocytes.

Type I IFNs have antiviral and immune-modulating activities. IFN-α/-β have very low basal expression levels but are strongly induced upon activation of pattern recognition receptors. In contrast, IFN-κ is constitutively expressed in uninfected keratinocytes and responds only weakly to pattern recognition receptor activation. IFN-κ expression has been implicated in the pathogenesis of inflammatory skin diseases and in limiting human papillomavirus replication in human keratinocytes. We have identified an enhancer ∼5 kb upstream of the IFNK gene driving its expression in keratinocytes. The enhancer consists of binding sites for the transcription factors jun-B, SMAD3/4, AP-2α/γ, and p63, of which the latter two are key regulators of keratinocyte biology. The jun-B and SMAD3/4 elements confer activation by the TGF-β pathway. Furthermore, inhibition of ERK1/2 kinases activates IFN-κ expression. Our study provides a framework for the cell type-specific, constitutive expression of IFN-κ and its modulation by signal transduction pathways in human keratinocytes.

Design and Application of an Artificial Hybrid PromoterPluxI-lacOin Genetic Circuit to Achieve Lower Basal Expression Level.

Quorum quenching (QQ) enzymes, which degrade signaling molecules so as to disrupt the quorum sensing signaling process, have drawn much attention as alternative antimicrobial agents. However, the screening methods for evolution of such enzymes through constructing genetic circuits remain a challenge for its relatively high false positive rates caused by the higher basal expression level of the naturally acquired promoter. Thus, we presented an improved genetic circuit by introducing an artificial hybrid promoter PluxI-lacO combining PlacO originated from lactose promoter with QS regulatory promoter PluxI to control the expression of reporter gene rfp. Herein, we investigated the effect of various expression strengths of suppressive protein LacI and signaling molecule AHL on the expression of rfp. We found that the effect AHL exerted on the expression of rfp outweighed that from IPTG. The results also demonstrated that our genetic circuit could achieve the lower basal expression level of reporter gene and could respond to the expression of AiiA. The resulting circuits show the potential for screening the evolved AiiA more efficiently by virtue of inherent low basal expression level.

The Phenotype and Secretory Activity of Adipose-Derived Mesenchymal Stem Cells (ASCs) of Patients with Rheumatic Diseases.

Mesenchymal stem/stromal cells (MSCs) have immunosuppressive and regenerative properties. Adipose tissue is an alternative source of MSCs, named adipose-derived mesenchymal stem cells (ASCs). Because the biology of ASCs in rheumatic diseases (RD) is poorly understood, we performed a basic characterization of RD/ASCs. The phenotype and expression of adhesion molecules (intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1) on commercially available healthy donors (HD), ASC lines (n = 5) and on ASCs isolated from patients with systemic lupus erythematosus (SLE, n = 16), systemic sclerosis (SSc, n = 17) and ankylosing spondylitis (AS, n = 16) were analyzed by flow cytometry. The secretion of immunomodulatory factors by untreated and cytokine-treated ASCs was measured by ELISA. RD/ASCs have reduced basal levels of CD90 and ICAM-1 expression, correlated with interleukin (IL)-6 and transforming growth factor (TGF)-β1 release, respectively. Compared with HD/ASCs, untreated and tumour necrosis factor (TNF) + interferon (IFN)-γ (TI)-treated RD/ASCs produced similar amounts of prostaglandin E2 (PGE2), IL-6, leukemia inhibiting factor (LIF), and TGF-β1, more IL-1Ra, soluble human leukocyte antigen G (sHLA-G) and tumor necrosis factor-inducible gene (TSG)-6, but less kynurenines and galectin-3. Basal secretion of galectin-3 was inversely correlated with the patient’s erythrocyte sedimentation rate (ESR) value. IFN-α and IL-23 slightly raised galectin-3 release from SLE/ASCs and AS/ASCs, respectively. TGF-β1 up-regulated PGE2 secretion by SSc/ASCs. In conclusion, RD/ASCs are characterized by low basal levels of CD90 and ICAM-1 expression, upregulated secretion of IL-1Ra, TSG-6 and sHLA-G, but impaired release of kynurenines and galectin-3. These abnormalities may modify biological activities of RD/ASCs.

Identification and functional characterization of the Marshallia (Asteraceae) Clade III Cytokinin Response Factor (CRF)

ABSTRACTCytokinin Response Factor (CRF) genes are a subgroup of AP2/ERF domain-containing transcription factors that are defined by the CRF domain, from which five clades of CRF genes have been identified. Clade III CRFs are strongly induced by cytokinin, as well as other abiotic stress factors, such as oxidative stress. While this appears well studied for the Clade III CRFs in Arabidopsis and tomato, there have been almost no studies done outside of these model systems. This study expands upon that and represents the first CRF research in the Sunflower family, Asteraceae. Fifty Asterid Clade III CRF protein sequences were examined, and novel Clade III CRF C-terminus motifs were identified. Clade III CRF genes of Marshallia mohrii and M. caespitosa were assembled from genome-skimming and transcriptomic data. Expression experiments were conducted on M. caespitosa to test responsiveness to both cytokinin and oxidative stress. Low levels of basal expression for the McCRF1 were found to be strongly induced in both treatment groups. These are the first experiments to show regulation of a nuclear gene in a Marshallia species, and these results suggest there is broad conservation in the sequence, form, and regulation of Clade III CRF genes and proteins.

DDR1 and MT1-MMP Expression Levels Are Determinant for Triggering BIK-Mediated Apoptosis by 3D Type I Collagen Matrix in Invasive Basal-Like Breast Carcinoma Cells.

Type I collagen is the major adhesive component in breast interstitial stroma, which represents the first barrier against tumor cell invasion after basement-membrane degradation. Among cellular receptors, type I collagen is able to activate discoidin domain receptors DDR1 and DDR2. We have previously shown that in 3D collagen matrix, DDR1 plays a key role as it promotes cell growth suppression and apoptosis through the upregulation of the pro-apoptotic mediator BIK in noninvasive luminal-like breast carcinoma cells. We have also shown that MT1-MMP is able to rescue these cells and protect them against the effects induced by collagen/DDR1/BIK axis. Our data suggested that the protective effect of MT1-MMP might be mediated through the degradation of type I collagen and/or DDR1 cleavage. Decreased DDR1 expression has been associated with the epithelial to mesenchymal transition process in breast cancer, and its overexpression in aggressive basal-like breast cancer cells reduces their invasiveness in 3D cultures and in vivo. In the present work, we propose to study the role of MT1-MMP in the resistance against collagen-induced apoptosis in basal-like breast carcinoma MDA-MB-231 cells. We aimed to investigate whether MT1-MMP depletion is able to restore apoptosis mediated by collagen/DDR1/BIK axis and to verify if such depletion is able to restore full-length DDR1 expression and phosphorylation. ShRNA strategy against MT1-MMP mRNA was able to partially restore full length DDR1 expression and phosphorylation. This was accompanied by a decrease in cell growth and an upregulation of BIK expression. This suggested that MT1-MMP expression in basal-like breast carcinoma cells, in addition to a low basal level of DDR1 expression, protects these cells against collagen-induced apoptosis via DDR1 cleavage. Since DDR1 was moderately expressed in MDA-MB-231 cells, we then investigated whether overexpression of DDR1 could be able to increase its ability to suppress cell growth and to induce apoptosis. Data showed that overexpression of DDR1 induced a decrease in cell growth and an increase in BIK expression, suggesting that moderate expression level of full length DDR1 in basal-like breast carcinoma provides them with a capacity to resist to collagen-induced cell growth suppression and apoptosis. Finally, the combined overexpression of DDR1 and depletion of MT1-MMP in MDA-MB-231 cells synergistically increased collagen-induced cell growth suppression and apoptosis to a level similar to that observed in luminal breast carcinoma. Taken together, our data suggest that during the acquisition of mesenchymal features, the low level of DDR1 expression should be considered as an important biomarker in the prognosis of basal-like breast carcinoma, conferring them a high rate of cell growth and resistance to BIK-mediated apoptosis induced by the stromal collagen.

Paralogs of Common Carp Granulocyte Colony-Stimulating Factor (G-CSF) Have Different Functions Regarding Development, Trafficking and Activation of Neutrophils.

Mammalian granulocyte colony-stimulating factor (G-CSF; CSF3) is a primary cytokine that promotes the development, mobilization, and activation of neutrophils and their precursors. Teleosts have been reported to possess two paralogs as a likely result of the teleost-wide whole genome duplication (WGD) event, but functional divergence of G-CSF paralogs remains poorly understood. Common carp are an allotetraploid species owing to an additional WGD event in the carp lineage and here, we report on genomic synteny, sequence similarity, and phylogeny of four common carp G-CSF paralogs (g-csfa1 and g-csfa2; g-csfb1 and g-csfb2). G-csfa1 and g-csfa2 show differential and relatively high gene expression levels, while g-csfb1 and g-csfb2 show low basal gene expression levels in most tissues. All paralogs are expressed higher in macrophages than in other leukocyte sub-types and are highly up-regulated by treatment of macrophages with mitogens. Recombinant G-CSFa1 and G-CSFb1 both promoted the proliferation of kidney hematopoietic cells, while only G-CSFb1 induced the differentiation of kidney cells along the neutrophil-lineage. Colony-forming unit assays revealed that G-CSFb1 alone stimulates the formation of CFU-G colonies from head- and trunk-kidney whereas the combination of G-CSFa1 and G-CSFb1 stimulates the formation of both CFU-G and CFU-GM colonies. Recombinant G-CSFa1 and G-CSFb1 also exhibit chemotactic activity against kidney neutrophils and up-regulation of cxcr1 mRNA expression was highest in neutrophils after G-CSFb1 stimulation. Furthermore, G-CSFb1 more than G-CSFa1 induced priming of kidney neutrophils through up-regulation of a NADPH-oxidase component p47phox. In vivo administration of G-CSF paralogs increased the number of circulating blood neutrophils of carp. Our findings demonstrate that gene duplications in teleosts can lead to functional divergence between paralogs and shed light on the sub-functionalization of G-CSF paralogs in cyprinid fish.

Human Brain Endothelial CXCR2 is Inflammation-Inducible and Mediates CXCL5- and CXCL8-Triggered Paraendothelial Barrier Breakdown.

Chemokines (C-X-C) motif ligand (CXCL) 5 and 8 are overexpressed in patients with multiple sclerosis, where CXCL5 serum levels were shown to correlate with blood–brain barrier dysfunction as evidenced by gadolinium-enhanced magnetic resonance imaging. Here, we studied the potential role of CXCL5/CXCL8 receptor 2 (CXCR2) as a regulator of paraendothelial brain barrier function, using the well-characterized human cerebral microvascular endothelial cell line hCMEC/D3. Low basal CXCR2 mRNA and protein expression levels in hCMEC/D3 were found to strongly increase under inflammatory conditions. Correspondingly, immunohistochemistry of brain biopsies from two patients with active multiple sclerosis revealed upregulation of endothelial CXCR2 compared to healthy control tissue. Recombinant CXCL5 or CXCL8 rapidly and transiently activated Akt/protein kinase B in hCMEC/D3. This was followed by a redistribution of tight junction-associated protein zonula occludens-1 (ZO-1) and by the formation of actin stress fibers. Functionally, these morphological changes corresponded to a decrease of paracellular barrier function, as measured by a real-time electrical impedance-sensing system. Importantly, preincubation with the selective CXCR2 antagonist SB332235 partially prevented chemokine-induced disturbance of both tight junction morphology and function. We conclude that human brain endothelial CXCR2 may contribute to blood–brain barrier disturbance under inflammatory conditions with increased CXCL5 and CXCL8 expression, where CXCR2 may also represent a novel pharmacological target for blood–brain barrier stabilization.

Biolog Phenotype Microarray Is a Tool for the Identification of Multidrug Resistance Efflux Pump Inducers.

Multidrug resistance efflux pumps frequently present low levels of basal expression. However, antibiotic-resistant mutants that overexpress these resistance determinants are selected during infection. In addition, increased expression of efflux pumps can be induced by environmental signals/cues, which can lead to situations of transient antibiotic resistance. In this study, we have applied a novel high-throughput methodology in order to identify inducers able to trigger the expression of the Stenotrophomonas maltophilia SmeVWX and SmeYZ efflux pumps. To that end, bioreporters in which the expression of the yellow fluorescent protein (YFP) is linked to the activity of either smeVWX or smeYZ promoters were developed and used for the screening of potential inducers of the expression of these efflux pumps using Biolog phenotype microarrays. YFP production was also measured by flow cytometry, and the levels of expression of smeV and smeY in the presence of a set of selected compounds were also determined by real-time reverse transcription-PCR (RT-PCR). The expression of smeVWX was induced by iodoacetate, clioquinol, and selenite, while boric acid, erythromycin, chloramphenicol, and lincomycin triggered smeYZ expression. The susceptibility to antibiotics that are known substrates of the efflux pumps decreased in the presence of the inducers. However, the analyzed multidrug efflux systems did not contribute to S. maltophilia resistance to the studied inducers. To sum up, the use of fluorescent bioreporters in combination with Biolog plates is a valuable tool for identifying inducers of the expression of bacterial multidrug resistance efflux pumps, and likely of other bacterial systems whose expression is regulated in response to signals/cues.