Glyphosate and its salt formulations are nonselective herbicides that have been extensively used worldwide, both for residential and agricultural purposes. The possible carcinogenicity and ...teratogenicity of glyphosate remain to be elucidated. We developed a sensitive and high-throughput analytical method for urinary glyphosate using liquid chromatography-tandem mass spectrometry with the aim of contributing to glyphosate exposure assessment in epidemiological studies.
After urine dilution (creatinine matching dilution to 0.05 g creatinine/L), glyphosate was extracted using two types of solid phase extraction columns (SCX and NH2) with automated sample preparation instruments. The eluate was dried and dissolved in the mobile phase, followed by liquid chromatography-tandem mass spectrometry analysis. The optimized method was applied to urine samples obtained from 54 Japanese adults and children.
The results from the validation study demonstrated good recoveries (91.0-99.6%), within- and between-run precisions (< 15%), low detection limits (0.1 μg/L), and lower limit of quantification (0.3 μg/L). The detection frequency and median concentration of the urinary glyphosate in Japanese subjects were 59% and 0.25 μg/L (0.34 μg/g creatinine).
Our reliable determination method was successful in measuring urinary glyphosate concentration. Moreover, this is the first biomonitoring report of urinary glyphosate levels in the Japanese general population.
Abstract We found that among four master epithelial-to-mesenchymal transition (EMT)-inducing genes ( ZEB1 , SIP1 , Snail , and Slug ) ZEB1 expression was most significantly correlated with the ...mesenchymal phenotype (high Vimentin and low E-cadherin expression) in non-small cell lung cancer (NSCLC) cell lines and tumors. Furthermore, ZEB1 knockdown with RNA interference in three NSCLC cell lines with high ZEB1 expression suppressed to varying degrees mass culture growth and liquid colony formation but in all cases dramatically suppressed soft agar colony formation. In addition, ZEB1 knockdown induced apoptosis in one of the three lines, indicating that the growth inhibitory effects of ZEB1 knockdown occurs in part through the activation of the apoptosis pathway. These results suggest that inhibiting ZEB1 function may be an attractive target for NSCLC therapeutic development.
BackgroundNear-infrared photoimmunotherapy (NIR-PIT) is a new modality for treating cancer, which uses antibody-photoabsorber (IRDye700DX) conjugates that specifically bind to target tumor cells. ...This conjugate is then photoactivated by NIR light, inducing rapid necrotic cell death. NIR-PIT needs a highly expressed targeting antigen on the cells because of its reliance on antibodies. However, using antibodies limits this useful technology to only those patients whose tumors express high levels of a specific antigen. Thus, to propose an alternative strategy, we modified this phototechnology to augment the anticancer immune system by targeting the almost low-expressed immune checkpoint molecules on tumor cells.MethodsWe used programmed death-ligand 1 (PD-L1), an immune checkpoint molecule, as the target for NIR-PIT. Although the expression of PD-L1 on tumor cells is usually low, PD-L1 is almost expressed on tumor cells. Intratumoral depletion with PD-L1-targeted NIR-PIT was tested in mouse syngeneic tumor models.ResultsAlthough PD-L1-targeted NIR-PIT showed limited effect on tumor cells in vitro, the therapy induced sufficient antitumor effects in vivo, which were thought to be mediated by the ‘photoimmuno’ effect and antitumor immunity augmentation. Moreover, PD-L1-targeted NIR-PIT induced antitumor effect on non-NIR light-irradiated tumors.ConclusionsLocal PD-L1-targeted NIR-PIT enhanced the antitumor immune reaction through a direct photonecrotic effect, thereby providing an alternative approach to targeted cancer immunotherapy and expanding the scope of cancer therapeutics.
Oncogenic KRAS is found in more than 25% of lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC), and is an important target for drug development. To this end, we ...generated four NSCLC lines with stable knockdown selective for oncogenic KRAS. As expected, stable knockdown of oncogenic KRAS led to inhibition of in vitro and in vivo tumor growth in the KRAS-mutant NSCLC cells, but not in NSCLC cells that have wild-type KRAS (but mutant NRAS). Surprisingly, we did not see large-scale induction of cell death and the growth inhibitory effect was not complete. To further understand the ability of NSCLCs to grow despite selective removal of mutant KRAS expression, we conducted microarray expression profiling of NSCLC cell lines with or without mutant KRAS knockdown and isogenic human bronchial epithelial cell lines with and without oncogenic KRAS. We found that although the mitogen-activated protein kinase pathway is significantly downregulated after mutant KRAS knockdown, these NSCLCs showed increased levels of phospho-STAT3 and phospho-epidermal growth factor receptor, and variable changes in phospho-Akt. In addition, mutant KRAS knockdown sensitized the NSCLCs to p38 and EGFR inhibitors. Our findings suggest that targeting oncogenic KRAS by itself will not be sufficient treatment, but may offer possibilities of combining anti-KRAS strategies with other targeted drugs.
Objective Endobronchial ultrasonography with a guide sheath (EBUS-GS) and virtual bronchoscopic navigation (VBN) improves the diagnostic yield in patients with peripheral pulmonary lesions (PPLs). ...Most previous reports on EBUS-GS-guided transbronchial biopsy (TBB) have included patients with benign and malignant diseases. We aimed to determine the factors that predicted a successful diagnosis by EBUS-GS-guided TBB diagnostic in patients with small peripheral lung cancer, with a focus on the high-resolution computed tomography (HRCT) findings before bronchoscopy. Methods We retrospectively reviewed the medical records of 173 consecutive patients with 175 small (≤30 mm) PPLs who were diagnosed with primary lung cancer between June 2010 and October 2013 at Nagoya University Hospital. All patients underwent EBUS-GS-guided TBB with VBN using a ZioStation computer workstation (Ziosoft, Osaka, Japan). We analyzed the patient characteristics, HRCT findings, diagnostic yield, and the diagnostic factors in small peripheral lung carcinoma. Results The EBUS probe position was within the PPL in 83 of the 175 lesions (47%) and 112 (64.0%) cases were successfully diagnosed by EBUS-GS-guided TBB. A univariate analysis revealed that the following factors were associated with a significantly higher diagnostic yield: CT bronchus sign positivity, a lesion of >20 mm in diameter, a solid nodule, and a probe position that was within the lesion. The following factors were not significant: the lesion location, the number of biopsies, and the lung cancer histology. A multivariate analysis revealed that the following factors significantly affected the diagnostic yield: CT bronchus sign positivity odds ratio (OR) =2.479; a probe position that was within the lesion (OR=2.542); and a solid nodule (OR=2.304). Conclusion The significant factors that were significantly associated with a successful diagnosis using EBUS-GS-guided TBB in small peripheral lung carcinoma were as follows: CT bronchus sign positivity, a solid nodule, and a probe position that was within the lesion.
Squamous cell carcinoma (SCC) transformation has been identified as a mechanism of resistance to first-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), gefitinib or ...erlotinib, in EGFR-mutated lung cancer. However, whether second- or third-generation TKIs can overcome resistance due to SCC transformation remains unclear. We herein report an EGFR-mutated lung adenocarcinoma undergoing transformation into SCC that exhibited a durable response to afatinib, which is a second-generation irreversible EGFR-TKI. We suggest that afatinib can be considered as a treatment option for EGFR-mutated tumor undergoing SCC transformation, particularly in the absence of a T790M mutation.
Identification of the causal effects of specific proteins on recurrent and partially reversible hearing loss has been difficult because of the lack of an animal model that provides reversible gene ...knockdown. We have developed the transgenic mouse line Actin-tTS::Nkcc1
for manipulatable expression of the cochlear K
circulation protein, NKCC1. Nkcc1 transcription was blocked by the binding of a tetracycline-dependent transcriptional silencer to the tetracycline operator sequences inserted upstream of the Nkcc1 translation initiation site. Administration of the tetracycline derivative doxycycline reversibly regulated Nkcc1 knockdown. Progeny from pregnant/lactating mothers fed doxycycline-free chow from embryonic day 0 showed strong suppression of Nkcc1 expression (~90% downregulation) and Nkcc1 null phenotypes at postnatal day 35 (P35). P35 transgenic mice from mothers fed doxycycline-free chow starting at P0 (delivery) showed weaker suppression of Nkcc1 expression (~70% downregulation) and less hearing loss with mild cochlear structural changes. Treatment of these mice at P35 with doxycycline for 2 weeks reactivated Nkcc1 transcription to control levels and improved hearing level at high frequency; i.e., these doxycycline-treated mice exhibited partially reversible hearing loss. Thus, development of the Actin-tTS::Nkcc1
transgenic mouse line provides a mouse model for the study of variable hearing loss through reversible knockdown of Nkcc1.
It is suggested that migration of airway smooth muscle (ASM) cells plays an important role in the pathogenesis of airway remodeling in asthma. Increases in intracellular Ca(2+) concentrations ...(Ca(2+)(i)) regulate most ASM cell functions related to asthma, such as contraction and proliferation. Recently, STIM1 was identified as a sarcoplasmic reticulum (SR) Ca(2+) sensor that activates Orai1, the Ca(2+) channel responsible for store-operated Ca(2+) entry (SOCE). We investigated the role of STIM1 in Ca(2+)(i) and cell migration induced by platelet-derived growth factor (PDGF)-BB in human ASM cells. Cell migration was assessed by a chemotaxis chamber assay. Human ASM cells express STIM1, STIM2, and Orai1 mRNAs. SOCE activated by thapsigargin, an inhibitor of SR Ca(2+)-ATPase, was significantly blocked by STIM1 siRNA and Orai1 siRNA but not by STIM2 siRNA. PDGF-BB induced a transient increase in Ca(2+)(i) followed by sustained Ca(2+)(i) elevation. Sustained increases in Ca(2+)(i) due to PDGF-BB were significantly inhibited by a Ca(2+) chelating agent EGTA or by siRNA for STIM1 or Orai1. The numbers of migrating cells were significantly increased by PDGF-BB treatment for 6 h. Knockdown of STIM1 and Orai1 by siRNA transfection inhibited PDGF-induced cell migration. Similarly, EGTA significantly inhibited PDGF-induced cell migration. In contrast, transfection with siRNA for STIM2 did not inhibit the sustained elevation of Ca(2+)(i) or cell migration induced by PDGF-BB. These results demonstrate that STIM1 and Orai1 are essential for PDGF-induced cell migration and Ca(2+) influx in human ASM cells. STIM1 could be an important molecule responsible for airway remodeling.
In mammals, audition is triggered by travelling waves that are evoked by acoustic stimuli in the cochlear partition, a structure containing sensory hair cells and a basilar membrane. When the cochlea ...is stimulated by a pure tone of low frequency, a static offset occurs in the vibration in the apical turn. In the high-frequency region at the cochlear base, multi-tone stimuli induce a quadratic distortion product in the vibrations that suggests the presence of an offset. However, vibrations below 100 Hz, including a static offset, have not been directly measured there. We therefore constructed an interferometer for detecting motion at low frequencies including 0 Hz. We applied the interferometer to record vibrations from the cochlear base of guinea pigs in response to pure tones. When the animals were exposed to sound at an intensity of 70 dB or higher, we recorded a static offset of the sinusoidally vibrating cochlear partition by more than 1 nm towards the scala vestibuli. The offset’s magnitude grew monotonically as the stimuli intensified. When stimulus frequency was varied, the response peaked around the best frequency, the frequency that maximised the vibration amplitude at threshold sound pressure. These characteristics are consistent with those found in the low-frequency region and are therefore likely common across the cochlea. The offset diminished markedly when the somatic motility of mechanosensitive outer hair cells, the force-generating machinery that amplifies the sinusoidal vibrations, was pharmacologically blocked. Therefore, the partition offset appears to be linked to the electromotile contraction of outer hair cells.
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