In vitro wound healing: Inhibition activity of insect‐derived mAb CO17‐1A in human colorectal cancer cell migration

The monoclonal antibody (mAb) CO17‐1A specifically binds to the tumor‐associated cell surface glycoprotein GA733 in colorectal cancer cells. Thus, mAb CO17‐1A has the potential to act as an immune therapeutic protein against colorectal cancer. Recently, it was shown that the baculovirus insect cell expression system produces anti‐colorectal cancer mAb CO17‐1A. In this study, the colorectal cancer antibody mAb CO17‐1A fused to the endoplasmic reticulum (ER) retention signal sequence (KDEL), and the (mAb CO17‐1AK) was expressed in Spodoptera frugiperda Sf9 insect cells. The yield, cell cytotoxicity, and in vitro anti‐tumor activity of mAb CO17‐1AK were verified. Western blotting was performed to confirm that both heavy and light chains of mAb CO17‐1A were expressed in Sf9 insect cells. The insect‐derived mAb (mAb^I) CO17‐1A was purified using a protein G affinity column. An in vitro wound healing assay was conducted to determine the inhibition activity of mAb CO17‐1A during tumor cell migration, showing that mAb^I CO17‐1AK was effective as mammalian‐derived mAb CO17‐1A (mAb^M CO17‐1A). These results suggest that the insect cell expression system can produce and properly assemble mAbs that inhibit tumor cell migration.     

Fuscibacter oryzae gen. nov., sp. nov., a phosphate-solubilizing bacterium isolated from the rhizosphere of rice plant

An ovoid to rod shaped, white to brown pigmented, facultative anaerobic, mesophilic, non-phototrophic, Gram-staining-negative, non-motile, multiply by binary fission designated strain KVB23^T, which was isolated from root of rice plant, near Ilsan, South Korea, was investigated for its taxonomic position by polyphasic approach. Optimal growth was found to occur at 30?C, at pH 6.5 and in the absence of NaCl on R2A. Phylogenetic analysis based on the 16S rRNA gene sequence of strain KVB23^T revealed that it formed a distinct lineage, as a separate deep branch within the family Rhodobacteriaceae, with?<?96.5% sequence similarity to representatives of the genera Rhodobacter, Xinfangfangia, Tabrizicola, Falsirhodobacter, Haematobacter, Paenirhodobacter, Pseudorhodobacter and Pararhodobacter. Based in 16S rRNA sequences strain KVB23^T was most closely related to Tabrizicola fusiformis KCTC 62105 ^T (96.5%) and Rhodobacter thermarum KCTC 52712 ^T (96.2%). The draft genome of strain KVB23^T was 3.80 bp long with a DNA G?+?C content of 63.1%. Genome of strain KVB23^T harboured gene clusters for tryptophan and cobalamin biosynthesis. The strain contained Q-10 as the sole respiratory quinone. The predominant fatty acids were found to consist of C_16:0, C_18:0 and summed feature 8 (comprising C_18:1 ω7c and / or C_18:1 ω6). The polar lipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, seven unidentified phosphoglycolipids, two unidentified aminophosphoglycolipid, one unidentified glycolipid and four unidentified lipids. Phosphate-solubilizing bacteria have the ability to dissolve insoluble phosphates and enhance the soil fertility. Strain KVB23^T can solubilize calcium phosphate tribasic. Phosphate solubilizing and tryptophan biosynthesis property of strain KVB23^T could be a possible factor for the increase in growth of rice plant. Differential phenotypic, chemotaxonomic and genotypic properties, together with the phylogenetic distinctiveness, demonstrated that strain KVB23^T was found to represent a novel genus in the Rhodobacteriaceae family, for which the name Fuscibacter oryzae gen. nov., sp. nov. is proposed, with the type strain KVB23^T(=?KACC 21711 ^T?=?NBRC 114716 ^T).

     

Linoleic acid inhibits Pseudomonas aeruginosa biofilm formation by activating diffusible signal factor‐mediated quorum sensing

Bacterial biofilm formation causes serious problems in various fields of medical, clinical, and industrial settings. Antibiotics and biocide treatments are typical methods used to remove bacterial biofilms, but biofilms are difficult to remove effectively from surfaces due to their increased resistance. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In the present study, we found that linoleic acid (LA), a plant unsaturated fatty acid, inhibits biofilm formation under static and continuous conditions without inhibiting the growth of Pseudomonas aeruginosa. LA also influenced the bacterial motility, extracellular polymeric substance production, and biofilm dispersion by decreasing the intracellular cyclic diguanylate concentration through increased phosphodiesterase activity. Furthermore, quantitative gene expression analysis demonstrated that LA induced the expression of genes associated with diffusible signaling factor‐mediated quorum sensing that can inhibit or induce the dispersion of P. aeruginosa biofilms. These results suggest that LA is functionally and structurally similar to a P. aeruginosa diffusible signaling factor (cis‐2‐decenoic acid) and, in turn, act as an agonist molecule in biofilm dispersion.     

G protein–coupled receptor kinase 2 modifies the ability of Caenorhabditis elegans to survive oxidative stress

Survival and adaptation to oxidative stress is important for many organisms, and these occur through the activation of many different signaling pathways. In this report, we showed that Caenorhabditis (C.) elegans G protein–coupled receptor kinases modified the ability of the organism to resist oxidative stress. In acute oxidative stress studies using juglone, loss-of-function grk-2 mutants were more resistant to oxidative stress compared with loss-of-function grk-1 mutants and the wild-type N2 animals. This effect was Ce-AKT-1 dependent, suggesting that Ce-GRK2 adjusted C. elegans oxidative stress resistance through the IGF/insulin-like signaling (IIS) pathway. Treating C. elegans with a GRK2 inhibitor, the selective serotonin reuptake inhibitor paroxetine, resulted in increased acute oxidative stress resistance compared with another selective serotonin reuptake inhibitor, fluoxetine. In chronic oxidative stress studies with paraquat, both grk-1 and grk-2 mutants had longer lifespan compared with the wild-type N2 animals in stress. In summary, this research showed the importance of both GRKs, especially GRK2, in modifying oxidative stress resistance.     

A Novel Application of Furazolidone: Anti-Leukemic Activity in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common malignant myeloid disorder of progenitor cells in myeloid hematopoiesis and exemplifies a genetically heterogeneous disease. The patients with AML also show a heterogeneous response to therapy. Although all-trans retinoic acid (ATRA) has been successfully introduced to treat acute promyelocytic leukemia (APL), it is rather ineffective in non-APL AML. In our present study, 1200 off-patent marketed drugs and natural compounds that have been approved by the Food and Drug Administration (FDA) were screened for anti-leukemia activity using the retrovirus transduction/transformation assay (RTTA). Furazolidone (FZD) was shown to inhibit bone marrow transformation mediated by several leukemia fusion proteins, including AML1-ETO. Furazolidone has been used in the treatment of certain bacterial and protozoan infections in human and animals for more than sixty years. We investigated the anti-leukemic activity of FZD in a series of AML cells. FZD displayed potent antiproliferative properties at submicromolar concentrations and induced apoptosis in AML cell lines. Importantly, FZD treatment of certain AML cells induced myeloid cell differentiation by morphology and flow cytometry for CD11b expression. Furthermore, FZD treatment resulted in increased stability of tumor suppressor p53 protein in AML cells. Our in vitro results suggest furazolidone as a novel therapeutic strategy in AML patients.     

Suicidal Ideation and Suicide Attempts in Anxious or Depressed Family Caregivers of Patients with Cancer: A Nationwide Survey in Korea

Purpose

To describe the prevalence of suicidal ideation and suicide attempts in family caregivers (FCs) of patients with cancer and to identify the factors associated with suicidal ideation and suicide attempts in FCs with anxiety or depression.

Methods

A national, multicenter survey administered to 897 FCs asked questions concerning suicidal ideation and suicide attempts during the previous year and assessed anxiety, depression, socio–demographic factors, caregiving burden, patient factors, and quality of life (QOL).

Results

A total of 17.7% FCs reported suicidal ideation, and 2.8% had attempted suicide during the previous year. Among FCs with anxiety, 31.9% had suicidal ideation and 4.7% attempted suicide; the corresponding values for FCs with depression were 20.4% and 3.3%, respectively. Compared with FCs without anxiety and depression, FCs with anxiety or depression showed a higher adjusted odds ratios (aOR) for suicidal ideation (aOR  = 4.07 and 1.93, respectively) and attempts (OR  = 3.00 and 2.43, respectively). Among FCs with anxiety or depression, being female, unmarried, unemployed during caregiving, and having a low QOL were associated with increased odds of suicidal ideation. FCs with anxiety who became unemployed during caregiving constituted a high-risk group for suicide. Being unmarried and having a low QOL with respect to financial matters were associated with increased suicide attempts among FCs with depression.

Conclusion

FCs with anxiety or depression were at high risk of suicide. Interventions to enhance social support and to improve perceived QOL may help prevent suicide and manage suicidal ideation in FCs with anxiety or depression.     

Control of Cellular Bcl-xL Levels by Deamidation-Regulated Degradation

The cellular concentration of Bcl-x_L is among the most important determinants of treatment response and overall prognosis in a broad range of tumors as well as an important determinant of the cellular response to several forms of tissue injury. We and others have previously shown that human Bcl-x_L undergoes deamidation at two asparaginyl residues and that DNA-damaging antineoplastic agents as well as other stimuli can increase the rate of deamidation. Deamidation results in the replacement of asparginyl residues with aspartyl or isoaspartyl residues. Thus deamidation, like phosphorylation, introduces a negative charge into proteins. Here we show that the level of human Bcl-x_L is constantly modulated by deamidation because deamidation, like phosphorylation in other proteins, activates a conditional PEST sequence to target Bcl-x_L for degradation. Additionally, we show that degradation of deamidated Bcl-x_L is mediated at least in part by calpain. Notably, we present sequence and biochemical data that suggest that deamidation has been conserved from the simplest extant metazoans through the human form of Bcl-x_L, underscoring its importance in Bcl-x_L regulation. Our findings strongly suggest that deamidation-regulated Bcl-x_L degradation is an important component of the cellular rheostat that determines susceptibility to DNA-damaging agents and other death stimuli.     

How to regulate neutrophils in gout

Most research in gout has concentrated on the proinflammatory mechanisms to explain the inflammation that is generated when leucocytes are in contact with monosodium urate crystals. However, the episodic nature of gout and the absence of inflammation even when crystals are present suggest that there are natural counter-regulatory mechanisms to limit the inflammatory response. Gagné and colleagues showed that myeloid inhibitory C-type lectin, a C-type lectin inhibitory receptor expressed on neutrophils, modulates monosodium urate-induced neutrophil responses in vitro.Neutrophil recruitment and activation play a key role in the acute inflammatory response to monosodium urate (MSU) crystals. In acute gout, our current treatments such as nonsteroidal anti-inflammatory drugs, colchicine or corticosteroids all act on different steps of neutrophil activation. These drugs form part of the first treatment objective in gout – to relieve the painful symptoms of the acute attack – but do not address the second objective, which is to treat the underlying metabolic disorder hyperuricemia. Can neutrophil activation be manipulated or regulated? Are there signals that can be modulated and can this be of clinical relevance?The article by Gagné and colleagues provides evidence for an inhibitory pathway of neutrophil activation that acts through a recently described C-type lectin receptor called the myeloid inhibitory C-type lectin (MICL) [1]. This membrane receptor, also known as CLEC12A, inhibits neutrophil activation when it is engaged. C-type lectin receptors form a large family of proteins that have a common type of carbohydrate-binding domain that mediate cell adhesion and ligand binding in a calcium-dependent manner. Members of the C-type lectin receptors are known to participate in immune regulation, with well-known examples including Dectin-1 (CLE7A), DC-sign (CD209 or CLEC4L) and natural killer cell receptors (Ly49 or KLRA1). The MICL protein is encoded on chromosome 12p13, closely linked to the natural killer gene complex. MICL contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif and is expressed mainly on neutrophils and monocytes. Previous work has shown that the receptor could inhibit cellular activation [2]. The ligands that lead to MICL activation are currently unknown, as there is only a small body of data to show that the receptor interacts with ligands expressed in the bone marrow, thymus and kidney [3].In their studies, Gagné and colleagues showed that MSU crystals as well as a MICL-specific antibody downmodulated MICL expression on neutrophils. Reducing the expression of MICL by transfecting small interfering RNA or by antibody modulation of the receptor led to enhanced production of IL-8 when MSU was added to neutrophils, but no changes in IL-1β secretion were observed. The mechanisms of MICL signaling probably involve tyrosine phosphorylation as well as calcium flux, differing from previous results that showed MICL associated with the phosphatases SHP-1 and SHP-2 [2]. Finally, the addition of colchicine to neutrophils abrogated the negative effect of MSU on MICL expression.These results showed that reduced MICL expression is associated with augmented inflammatory responses from neutrophils, and a higher level of neutrophil MICL expression is associated with a reduced IL-8 production in vitro. As IL-8 is a major neutrophil chemoattractant, this can have important effects on neutrophil recruitment to an inflammatory site in gout. By extrapolation, if MICL expression or signaling could be enhanced or maintained during inflammation, the inhibitory signal may be reinforced and thereby downregulate inflammation. The effect of colchicine in this system is to elevate the expression of MICL, thereby increasing the inhibitory signaling mechanisms that counteract the inflammatory process.A number of caveats need to be mentioned in the interpretation of these results. The data presented were based on in vitro models of inflammation using MSU, and we need to see how this works in vivo before coming to any conclusions, as we have had examples where the in vivo results did not recapitulate the in vitro findings. They convincingly showed that reducing MICL expression on the surface of neutrophils enhanced the proinflammatory signature, but they did not show the converse – that enhanced MICL signaling can further downmodulate inflammation. Furthermore, the ligands that bind and activate MICL are unknown, so we have no idea what is the signal or how to reinforce or manipulate this signaling system. The results presented show that MSU had dual effects on neutrophils – the first is to downregulate MICL expression, and the second is to activate IL-8 production. How are these two mechanisms linked? If MSU acts mainly on the cell membrane internalization of MICL, what is the trigger for the IL-8 secretion? Notwithstanding these uncertainties, the finding that MICL modulates neutrophil activation in gout suggests that there are a number of counter-regulatory mechanisms in operation during an inflammatory process. Identifying these mechanisms may help us to understand the nature of gout as well as open up new therapeutic perspectives.