Exposure of the yeast Saccharomyces cerevisiae to functionalized polystyrene latex nanoparticles: influence of surface charge on toxicity.

Novel nanoparticles with unique physicochemical characteristics are being developed with increasing frequency, leading to higher probability of nanoparticle release and environmental accumulation. Therefore, it is important to assess the potential environmental and biological adverse effects of nanoparticles. In this study, we investigated the toxicity and behavior of surface-functionalized nanoparticles toward yeast (Saccharomyces cerevisiae). The colony count method and confocal microscopy were used to examine the cytotoxicity of manufactured polystyrene latex (PSL) nanoparticles with various functional groups (amine, carboxyl, sulfate, and nonmodified). S. cerevisiae were exposed to PSL nanoparticles (40 mg/L) dispersed in 5-154 mM NaCl solutions for 1 h. Negatively charged nanoparticles had little or no toxic effect. Interestingly, nanoparticles with positively charged amine groups (p-Amine) were not toxic in 154 mM NaCl, but highly toxic in 5 mM NaCl. Confocal microscopy indicated that in 154 mM NaCl, the p-Amine nanoparticles were internalized by endocytosis, whereas in 5 mM NaCl they covered the dead cell surfaces. This demonstrates that nanoparticle-induced cell death might to be related to their adhesion to cells rather than their internalization. Together, these findings identify important factors in determining nanoparticle toxicity that might affect their impact on the environment and human health.

Higher Cardio-Ankle Vascular Index Values in Patients With Vestibular Neuritis May Indicate a Better Prognosis.

BACKGROUND: The presumed etiology of vestibular neuritis (VN), a sudden onset of spontaneous vertigo without auditory or cranial nerve symptoms, includes viral infections and vascular disorders. However, no clinical test for estimating vascular disorders in VN has been reported. Moreover, estimating the etiology of VN is important to predict the prognosis and select appropriate treatment. This study aimed to evaluate the cardio-ankle vascular index (CAVI), which reflects arterial stiffness and elasticity, as an additional indicator for estimating the prognosis and etiology of VN. MATERIALS AND METHODS: Among 207 consecutive patients with suspected VN, 88 patients diagnosed with definite VN were enrolled. Age, initial and final percent canal paresis (CP) in the caloric test, CAVI, presence or absence of vestibular-evoked myogenic potential asymmetry, and medical history were evaluated using univariate and multivariate analyses. RESULTS: Patients with VN with high CAVI had a better prognosis than those with low CAVI. High CAVI was a factor for improvement in percent CP, in addition to younger age and less severe initial percent CP in the Cox proportional hazard model. CONCLUSION: CAVI can be an additional indicator for estimating the prognosis and etiology of VN. This indicator can potentially be applied to other diseases, including vascular disorders with other etiologies.

Lateral medullary infarction presenting as isolated vertigo and unilateral loss of visual suppression.

Isolated vertigo is rare in lateral medullary infarction. We described early diagnostic challenges in such cases by a neuro-otological approach. We report a 56-year-old man who developed a lateral medullary infarction that presented as isolated vertigo. Before the day 4 from disease onset when diffusion-weighted magnetic resonance imaging (MRI) became positive, this patient showed unilateral loss of visual suppression, a central type of vestibular dysfunction. Since MRI abnormalities may not appear in the early few days from disease onset, unilateral loss of visual suppression might become an important diagnostic option for isolated vertigo due to a lateral medullary infarction. This finding is presumably relevant to the inferior olive lesion.

[Clinical evaluation of vertigo in menopausal women].

Vertigo is one of the usual menopausal symptoms. We have often examined some women under the complaint of vertigo related with the menopause. We diagnosed each disease based on neuro-otological examinations and investigated the characteristics of menopausal-associated vertigo. We studied 413 women aged 40-59 years old who complained of vertigo. There were 73 women with menopause symptoms (14 women introduced from the gynecologist in our medical center, 18 women had undergone treatment at another female clinic, and 41 women visited an otorhinolaryngologist first) compared with 340 women without menopause symptoms. In the menopause group, 41 (56.2%) cases were diagnosed as having benign paroxysmal positional vertigo (BPPV), 13 (17.8%) cases had Meniere's disease, sudden deafness with vertigo accounted 2 cases, one was an acoustic tumor, and so on. The percentage of patients with BPPV was almost same ratio between the menopause group (56.2%) and the non-menopause group (52.9%). The percentage of patients with Meniere's disease was higher markedly in the menopausal group (17.8%). than the non-menopause group (9.7%). Menopausal symptoms are caused not only by hot flashes related to a lack of estrogen but also by psychological factors. The onset of Meniere's disease can also be influenced by psychological factors. As for the diagnosis of Meniere's disease, we supposed the reason for the higher percentage in the menopausal group was its relationship with psychological factors. We could diagnose and treat some menopausal women with vertigo. We believe that joint consultation with a gynecologist and otorhinolaryngologist would be necessary to ensure an optimum quality of life for such patients.

Direct measurement of adhesion force between a yeast cell and a lactic acid bacterium cell with atomic force microscopy.

Lactic acid bacteria (LAB) and yeast coexist by providing nutrients as substrates for each other. LAB and yeast cells aggregate via specific interactions between mannose on the yeast surface and the mannose-binding protein (MBP) on the LAB surface. In addition to specific interactions via cell surface proteins, there are also nonspecific interactions related to microbial coaggregation; the extent of their contributions is not clear. Here, microbial coaggregation of yeast and LAB cells was investigated from the view point of particle technology. DLVO theory and thermodynamic approaches predicted that thermodynamically stable coaggregates are not formed because no hydrophobic interaction acts between yeast and LAB. In contrast, optical microscopy revealed that yeast with mannose and LAB with MBP were formed submillimeter-sized coaggregates, whereas deficient strains of yeast and/or LAB were dispersed. Single-cell force spectroscopy revealed that the median adhesion forces were less than 100 pN for all combinations of yeast and LAB. However, some of the adhesion forces between yeast with mannose and LAB with MBP were greater than 400 pN. Furthermore, in the presence of a microbial coaggregation inhibitor, coaggregation of yeast with mannose and LAB with MBP was suppressed, and the adhesion forces were less than 300 pN. These results indicate that the specific interaction rather than the nonspecific interactions acted between mannose on the yeast and the MBP on the LAB formed submillimeter-sized small aggregates. Understanding the contribution of predominant cell-cell interactions may help control microbial behavior in biotechnology.

[Non-specific exercise therapy for benign paroxysmal postural vertigo].

We treated 1145 patients diagnosed as having benign paroxysmal postural vertigo at the Toho University Medical Center Sakura Hospital from August 2007 to July 2009 by the exercise therapy developed by us. The most advantageous characteristic of our method is that patients can perform the exercises themselves at their own pace in their homes, even if the affected side cannot be identified and/or the patients have any orthopedic cervical and/or spinal problems. In 80.7% and 91.7% of the patients in our case series, the vertigo was no longer present at one and three months, respectively. In addition, the vertigo disappeared within two weeks in the patients who were examined within one week of the start of the symptom. The longer the period between the onset of vertigo and the hospital visit, the longer the period needed for control of the symptom.

A novel method of fabrication of latex-stabilized water-core colloidosomes at room temperature.

Colloidosomes have attracted great interest in recent years because of the capability of storage and delivery of useful materials in various fields. In this article, a novel technique for formation of colloidosomes at room temperature suitable for encapsulation of biomaterials was examined. We demonstrate the formation of colloidosomes of 18.0 µm in size at room temperature by adding a small amount of ethanol into the continuous phase of sunflower oil. Poly(methyl methacrylate-co-butyl acrylate) latex particles of 185 nm in size, used in this study, were found to aggregate when ethanol was added to their suspension. We suggest that the shell of the water-core emulsions was locked by the aggregation of latex particles due to the diffusion of ethanol into the aqueous latex suspension.

Synergistic apoptotic effects in cancer cells by the combination of CLK and Bcl-2 family inhibitors.

Emerging evidence indicates that alternative splicing plays a critical role in cancer progression through abnormal expression or mutation of splicing factors. Small-molecule splicing modulators have recently attracted considerable attention as a novel class of cancer therapeutics. CDC-like kinases (CLKs) are central to exon recognition in mRNA splicing and CLK inhibitors exhibit anti-tumour activities. Most importantly, molecular mechanism-based combination strategies for cancer therapy must be considered. However, it remains unclear whether CLK inhibitors modulate expression and splicing of apoptosis-related genes, and whether CLK inhibitors enhance cytotoxicity in combination with apoptosis inducers. Here we report an appropriate mechanism-based drug combination approach. Unexpectedly, we found that the CLK inhibitor T3 rapidly induced apoptosis in A2780 cells and G2/M cell cycle arrest in HCT116 cells. Regardless of the different phenotypes of the two cancer cell types, T3 decreased the levels of anti-apoptotic proteins (cIAP1, cIAP2, XIAP, cFLIP and Mcl-1) for a short period of exposure and altered the splicing of the anti-apoptotic MCL1L and CFLAR isoform in A2780 and HCT116 cells. In contrast, other members of the Bcl-2 family (i.e., Bcl-xL and Bcl-2) were resistant to T3-induced expression and splicing modulation. T3 and a Bcl-xL/Bcl-2 inhibitor synergistically induced apoptosis. Taken together, the use of a CLK inhibitor is a novel therapeutic approach to sensitise cancer cells to Bcl-xL/Bcl-2 inhibitors.

Discovery of a Novel, Highly Potent, and Selective Thieno[3,2-d]pyrimidinone-Based Cdc7 Inhibitor with a Quinuclidine Moiety (TAK-931) as an Orally Active Investigational Antitumor Agent.

In our pursuit of developing a novel, potent, and selective cell division cycle 7 (Cdc7) inhibitor, we optimized the previously reported thieno[3,2-d]pyrimidinone analogue I showing time-dependent Cdc7 kinase inhibition and slow dissociation kinetics. These medicinal chemistry efforts led to the identification of compound 3d, which exhibited potent cellular activity, excellent kinase selectivity, and antitumor efficacy in a COLO205 xenograft mouse model. However, the issue of formaldehyde adduct formation emerged during a detailed study of 3d, which was deemed an obstacle to further development. A structure-based approach to circumvent the adduct formation culminated in the discovery of compound 11b (TAK-931) possessing a quinuclidine moiety as a preclinical candidate. In this paper, the design, synthesis, and biological evaluation of this series of compounds will be presented.

[Examination of dynamic body balance using the body tracking test in cases of vestibular neuronitis].

Body Tracking Test (BTT) is a testing method of the dynamic body balance function wherein movement of the center of gravity in accordance with a moving visual target stimulus is examined to evaluate the tracking function of the body. The objects were the patients who were diagnosed as having vestibular neuronitis at the Department of Otolaryngology in Toho University medical center Sakura hospital, and were undergoing hospital treatment. Lateral BTT was performed in 31 subjects, and antero-posterior (A-P) BTT in 45 subjects. Although gaze nystagmus was noted, inspection was enforced when a standing position posture was possible. In lateral BTT, the average (cm/second) horizontal direction body sway speed was compared, however, no clear lateral difference was noted. In the antero-posterior (A-P) BTT, it inquired using the direction body sway average center displacement (cm) of X. Deviation was seen by the affected side in stabilometry. Deviation was seen by the unaffected side in the antero-posterior (A-P) BTT. This phenomenon differs from the deviation pattern until now and is considered to involve participation of the higher centers.

Benign preparation of aqueous core poly lactic-co-glycolic acid (PLGA) microcapsules.

Poly lactic-co-glycolic acid (PLGA) has attracted considerable attention as a polymer for drug delivery carriers. However, the hydrophobic property of PLGA often leads to the use of harmful organic solvents and poor encapsulation efficiency of hydrophilic materials. To our knowledge, a preparation method of aqueous core PLGA microcapsules without using harmful organic solvents has not been proposed. In this study, we attempted to establish an encapsulation technique of hydrophilic materials in aqueous core biodegradable and biocompatible PLGA microcapsules using vegetable oil as a continuous phase. As a result, the temperature of the oil/water mixture was required to be above the glass transition temperature. In this condition, two different types of morphology were prepared. When the water volume was below the solubility limit, PLGA microcapsules with a smooth shell were formed. In contrast, when the water volume was above the solubility limit, colloidosome-like microcapsules with PLGA nanoparticles assembled at the interface were formed. The obtained microcapsules were then heated at the glass transition temperature. The result is that aqueous core PLGA microcapsules with a smooth shell were prepared using plant oil as a continuous phase. Rhodamine B used as a hydrophilic model encapsulant, was successfully encapsulated in the PLGA microcapsules.

Anti-tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC-dependent vulnerability.

The modulation of pre-mRNA splicing is proposed as an attractive anti-neoplastic strategy, especially for the cancers that exhibit aberrant pre-mRNA splicing. Here, we discovered that T-025 functions as an orally available and potent inhibitor of Cdc2-like kinases (CLKs), evolutionally conserved kinases that facilitate exon recognition in the splicing machinery. Treatment with T-025 reduced CLK-dependent phosphorylation, resulting in the induction of skipped exons, cell death, and growth suppression in vitro and in vivo Further, through growth inhibitory characterization, we identified high CLK2 expression or MYC amplification as a sensitive-associated biomarker of T-025. Mechanistically, the level of CLK2 expression correlated with the magnitude of global skipped exons in response to T-025 treatment. MYC activation, which altered pre-mRNA splicing without the transcriptional regulation of CLKs, rendered cancer cells vulnerable to CLK inhibitors with synergistic cell death. Finally, we demonstrated in vivo anti-tumor efficacy of T-025 in an allograft model of spontaneous, MYC-driven breast cancer, at well-tolerated dosage. Collectively, our results suggest that the novel CLK inhibitor could have therapeutic benefits, especially for MYC-driven cancer patients.

Bioreductive deposition of platinum nanoparticles on the bacterium Shewanella algae.

An environmentally friendly method using the metal ion-reducing bacterium Shewanella algae was proposed to deposit platinum nanoparticles. Resting cells of S. algae were able to reduce aqueous PtCl(6)(2-) ions into elemental platinum at room temperature and neutral pH within 60min when lactate was provided as the electron donor. Biogenic platinum nanoparticles of about 5nm were located in the periplasm--a preferable, cell surface location for easy recovery of biogenic nanoparticles.

Direct determination of oxidation state of gold deposits in metal-reducing bacterium Shewanella algae using X-ray absorption near-edge structure spectroscopy (XANES).

X-ray absorption near-edge structure spectroscopy (XANES) was successfully employed to determine the gold valence in the metal-reducing bacterium Shewanella algae after exposure to a 1 mM aqueous HAuCl4 solution for 10-120 min. XANES spectra revealed the oxidation state of gold in the bacterial cells to be Au(0) without any contribution from Au(III), demonstrating that S. algae cells can reduce AuCl4- ions to elemental gold. Transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) analysis confirmed that gold nanoparticles 5-15 nm in size were deposited in the periplasmic space of the bacterial cells; a preferable, cell surface location for the easy recovery of biogenic nanoparticles.

LSD1 Inhibitor T-3775440 Inhibits SCLC Cell Proliferation by Disrupting LSD1 Interactions with SNAG Domain Proteins INSM1 and GFI1B.

T-3775440 is an irreversible inhibitor of the chromatin demethylase LSD1, which exerts antiproliferative effects by disrupting the interaction between LSD1 and GFI1B, a SNAG domain transcription factor, inducing leukemia cell transdifferentiation. Here, we describe the anticancer effects and mechanism of action of T-3775440 in small-cell lung cancer (SCLC). T-3775440 inhibited proliferation of SCLC cells in vitro and retarded SCLC tumor growth in vivo T-3775440 disrupted the interaction between LSD1 and the transcriptional repressor INSM1, thereby inhibiting expression of neuroendocrine-associated genes, such as ASCL1 INSM1 silencing phenocopied the effects of T-3775440 on gene expression and cell proliferation, consistent with the likelihood T-3775440 mediated its effects in SCLC by inhibiting INSM1. T-3775440 also inhibited proliferation of an SCLC cell line that overexpressed GFI1B, rather than INSM1, by disrupting the interaction between LSD1 and GFI1B. Taken together, our results argue that LSD1 plays an important role in neuroendocrine-associated transcription and cell proliferation of SCLC via interactions with the SNAG domain proteins INSM1 and GFI1B. Targeting these critical interactions with LSD1 inhibitors offers a novel rational strategy to therapeutically manage SCLC. Cancer Res; 77(17); 4652-62. ©2017 AACR.

The Anti-tumor Effect of Cabozantinib on Ovarian Clear Cell Carcinoma In Vitro and In Vivo.

BACKGROUND: Several reports have shown that the overexpression of the MET proto-oncogene, receptor tyrosine kinase (MET), was more frequently observed in clear cell carcinoma (CCC) than in non-CCC. We evaluated the antitumor activity of cabozantinib, that targets MET. MATERIALS AND METHODS: A gene expression analysis of tumors from human ovarian cancers was carried out by transcriptome sequencing. An in vitro 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay (MTT assay) and in vivo experiments were performed to determine the activity of cabozantinib. RESULTS: The MET levels were higher in tumors with CCC than high-grade serous carcinoma (2.2-fold). Cabozantinib inhibited cell viability and phosphorylation of AKT and MAPK under the treatment of hepatocyte growth factor in RMG-I CCC cells. The tumors removed from mice given cabozantinib of 10 mg/kg weighed 70% less than control on day 15, and the immunohistochemical reactivity of phosphorylated MET was reduced compared with control mice. CONCLUSION: Cabozantinib contributes to tumor reduction, and phosphorylated MET represents an attractive target of CCC.

A Novel LSD1 Inhibitor T-3775440 Disrupts GFI1B-Containing Complex Leading to Transdifferentiation and Impaired Growth of AML Cells.

Dysregulation of lysine (K)-specific demethylase 1A (LSD1), also known as KDM1A, has been implicated in the development of various cancers, including leukemia. Here, we describe the antileukemic activity and mechanism of action of T-3775440, a novel irreversible LSD1 inhibitor. Cell growth analysis of leukemia cell lines revealed that acute erythroid leukemia (AEL) and acute megakaryoblastic leukemia cells (AMKL) were highly sensitive to this compound. T-3775440 treatment enforced transdifferentiation of erythroid/megakaryocytic lineages into granulomonocytic-like lineage cells. Mechanistically, T-3775440 disrupted the interaction between LSD1 and growth factor-independent 1B (GFI1B), a transcription factor critical for the differentiation processes of erythroid and megakaryocytic lineage cells. Knockdown of LSD1 and GFI1B recapitulated T-3775440-induced transdifferentiation and cell growth suppression, highlighting the significance of LSD1-GFI1B axis inhibition with regard to the anti-AML effects of T-3775440. Moreover, T-3775440 exhibited significant antitumor efficacy in AEL and AMKL xenograft models. Our findings provide a rationale for evaluating LSD1 inhibitors as potential treatments and indicate a novel mechanism of action against AML, particularly AEL and AMKL. Mol Cancer Ther; 16(2); 273-84. ©2016 AACR.

CLK-dependent exon recognition and conjoined gene formation revealed with a novel small molecule inhibitor.

CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3'-end processing and associated splicing factors.The phosphorylation of serine/arginine-rich proteins by CDC-like kinase is a central regulatory mechanism for RNA splicing reactions. Here, the authors synthesize a novel small molecule CLK inhibitor and map CLK-responsive alternative splicing events and discover an effect on conjoined gene transcription.

Cytotoxicity and colloidal behavior of polystyrene latex nanoparticles toward filamentous fungi in isotonic solutions.

The effects of surface physicochemical properties of functionalized polystyrene latex (PSL) nanoparticles (NPs) and model filamentous fungi Aspergillus oryzae and Aspergillus nidulans cultivated in different environment (aqueous and atmospheric environment) on the colloidal behavior and cytotoxicity were investigated in different isotonic solutions (154 mM NaCl and 292 mM sucrose). When the liquid cultivated fungal cells were exposed to positively charged PSL NPs in 154 mM NaCl solution, the NPs were taken into A. oryzae, but not A. nidulans. Atomic force microscopy revealed that the uptake of NPs was more readily through the cell wall of A. oryzae because of its relatively softer cell wall compared with A. nidulans. In contrast, the positively charged PSL NPs entirely covered the liquid cultivated fungal cell surfaces and induced cell death in 292 mM sucrose solution because of the stronger electrostatic attractive force between the cells and NPs compared with in 154 mM NaCl. When the agar cultivated fungal cells were exposed to the positively charged PSL NPs, both fungal cells did not take the NPs inside the cells. Contact angle measurement revealed that the hydrophobin on the agar cultivated cell surfaces inhibited the uptake of NPs because of its relatively more hydrophobic cell surface compared with the liquid cultivated cells.

Involvement of cathepsins in the invasion, metastasis and proliferation of cancer cells.

Tumor cell invasion and metastasis are associated with the proteolytic activity of various types of proteinases. Among them, cathepsins, which are lysosomal proteinases, have received more attention recently. Since elevated expressions of cathepsins and diminished levels of their inhibitors have been observed in several human cancers, including breast, gastric and prostate cancer, especially in aggressive cancer cells, cathepsins have been suggested to be biological markers of malignant tumors and have proved useful for prognosis of the disease. Furthermore, cathepsins have various roles in cancer progression. Cathepsin D has a mitogenic activity independent of its proteolytic activity and it attenuates the anti-tumor immune response of decaying chemokines to inhibit the function of dendritic cells. Cathepsins B and L have been shown to play an important role in matrix degradation and cell invasion. The administration of their inhibitors prevents the invasion and metastasis of cancer cells. These results indicate that cancer cells orchestrate various cathepsins to progress malignant diseases. Cathepsins may be a potential target for cancer therapy.