The purpose of this study was to investigate the prognostic value of tumour-associated macrophages with a focus on micro-anatomical localisation and determine whether molecular changes of the epidermal growth factor receptor (EGFR) are related to macrophage infiltration in resected non-small cell lung cancer (NSCLC). One hundred and forty-four patients were included in this study. Immunohistochemistry was used to identify CD68+ macrophages in the tumour islet and surrounding stroma. Epidermal growth factor receptor mutations were studied by direct sequencing. The EGFR gene copy number and protein expression were analysed by fluorescence in situ hybridisation and immunohistochemistry. Patients with a high tumour islet macrophage density survived longer than did the patient with a low tumour islet macrophage density (5-year overall survival rate was 63.9 vs 38.9%, P=0.0002). A multivariate Cox proportional hazard analysis revealed that the tumour islet macrophage count was an independent prognostic factor for survival (hazard ratio 0.471, 95% confidence interval 0.300–0.740). However, EGFR mutations, gene copy number, and protein expression were not related to the macrophage infiltration. In conclusion, tumour islet macrophage infiltration was identified as a strong favourable independent prognostic marker for survival but not correlated with the molecular changes of the EGFR in patients with resected NSCLC.
macrophage; non-small cell lung cancer; epidermal growth factor receptor; survival analysis
Cisplatin (cis-diammine-dichloroplatinum; CDDP) is an anticancer drug that induces significant hearing loss and balance dysfunction as side effects. Cilostazol (CS, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl) butoxy]-3, 4-dihydro-2-(1H)-quinolinone) has neuroprotective and antioxidant effects, whereas Ginkgo biloba extract (GbE) has preventive effects on CDDP-induced hearing loss in rats, and GbE enhances the antiatherogenic effect of CS by inhibiting the generation of reactive oxygen species (ROS). The purpose of this study was to investigate the effects of renexin (RXN), which contains GbE and CS, against CDDP-induced cochleo-vestibular dysfunction in rats and to elucidate the mechanism underlying the protective effects of RXN on auditory cells. Rats intraperitoneally injected with CDDP exhibited an increase in hearing threshold and vestibular dysfunction, which agreed with hair cell damage in the Organ of Corti and otoliths. However, these impairments were significantly prevented in a dose-dependent manner by pre- and co-treatment with RXN, and these preventive effects in RXN-treated rats were more prominent than those in GbE-treated rats. In a CDDP pharmacokinetic study, platinum concentration was very similar between CDDP-only treated and RXN+CDDP cotreated rats. RXN markedly attenuated CDDP-induced intracellular ROS and significantly reduced CDDP-activated expression of p-extracellular regulated kinase (ERK), BAX, cytochrome c, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, but increased BCL-XL expression. These results show that RXN may have a synergistic effect by strongly protecting hearing and vestibular dysfunction induced by CDDP by inhibiting ROS production, mitochondrial pathways and the ERK pathway, without interfering with CDDP pharmacokinetics. Therefore, RXN could potentially be used to reduce CDDP-related hearing loss and dizziness.
cisplatin; hearing loss; cilostazol; ginkgo biloba; dizziness
Acinar cell carcinoma (ACC) is a rare pancreatic tumour with a favourable prognosis compared with the more common ductal adenocarcinoma. The radiological findings of this tumour have been described in the literature; however, only limited data are available regarding the metastatic features of ACC of the liver, the most common metastatic site. We report a case of ACC of the pancreas with a hepatic metastasis from a benign-appearing malignant pancreatic lesion.
To determine the prognostic factors and treatment outcomes of patients with early-stage adenocarcinoma (AdCa) of uterine cervix who underwent radical hysterectomy (RH).
Patients with early-stage squamous cell carcinoma (SCCa) of the uterine cervix who underwent RH were compared with patients with AdCa by multivariate analysis.
A total of 1218 patients were eligible, of which 996 (81.8%) had SCCa and 222 (18.2%) had AdCa. In multivariate analysis, parametrial involvement and lymph node metastasis were significant factors for both recurrence-free survival(RFS) and overall survival (OS) of patients with AdCa, whereas age, tumour size, parametrial involvement and lymph node metastasis were significant factors for both RFS and OS of patients with SCCa. After adjusting for significant prognostic factors, patients with AdCa had significantly poorer RFS (odds ratio (OR)=2.07, 95% confidence interval (CI)=1.37–3.12, P=0.001) and OS (OR=2.56, 95% CI=1.65–3.96, P<0.001) than patients with SCCa. Recurrence outside the pelvis was more frequent in AdCa than in those with SCCa (75 vs 57.8%, P=0.084).
Although RH is still acceptable for treatment of patients with AdCa, a more effective systemic adjuvant therapy is required.
early-stage cervical cancer; squamous cell carcinoma; adenocarcinoma; radical hysterectomy; prognosis
Black rice is rich in anthocyanin and is expected to have more healthful dietary potential than white rice. We assessed expression of anthocyanin in black rice cultivars using a newly designed 135 K Oryza sativa microarray. A total of 12,673 genes exhibited greater than 2.0-fold up- or down-regulation in comparisons between three rice cultivars and three seed developmental stages. The 137 transcription factor genes found to be associated with production of anthocyanin pigment were classified into 10 groups. In addition, 17 unknown and hypothetical genes were identified from comparisons between the rice cultivars. Finally, 15 out of the 17 candidate genes were verified by RT-PCR analysis. Among the genes, nine were up-regulated and six exhibited down-regulation. These genes likely play either a regulatory role in anthocyanin biosynthesis or are related to anthocyanin metabolism during flavonoid biosynthesis. While these genes require further validation, the results here underline the potential use of the new microarray and provide valuable insight into anthocyanin pigment production in rice.
Anthocyanin; Black rice; Pigmentation; Rice microarray; Transcription factor
To understand patients' perceptions of clinical trials (CTs) is the principal step in the enrolment of patients to CTs. However, these perceptions in eastern countries are very rare. From 12 February 2007 to 13 April 2007, we consecutively distributed the questionnaire to 842 cancer patients who initiated a first cycle of chemotherapy regardless of each treatment step in the Seoul National University Hospital. Younger age, higher educational degree, higher economic status, and possession of private cancer insurance were related with significantly higher awareness of CTs (P=0.001, P=0.006, P=0.002, and P=0.009, respectively). However, unlike awareness, perceptions on benefits of CTs were not changed according to age, educational degree, and economic status (P=0.709, P=0.920, and P=0.847, respectively). Willingness was also not changed according to age, educational degree, economic status, and private cancer insurance (P=0.381, P=0.775, P=0.887, and P=0.392, respectively). Instead, males and heavily treated patients had more positive perceptions on benefits (P=0.002 and P=0.001, respectively) and more willingness to participate in CTs (OR=1.17, 1.14–2.75: OR=1.59, 1.01–2.51, respectively). In summary, cancer patients' awareness of CTs, perceptions on the benefit in CTs, and willingness to participate are differently influenced by diverse medical and social conditions. This information would be very helpful for investigators to properly conduct CTs in eastern cancer patients.
cancer patient; clinical trial; perception; awareness; willingness
Chronic cough is associated with increased sensitivity to inhaled capsaicin, and both tachykinins and their receptors play important roles in the cough reflex. However, associations between polymorphisms of the tachykinin receptor genes and cough sensitivity in patients with non‐productive chronic cough have not been reported.
Direct sequencing was used to identify single nucleotide polymorphisms (SNPs) in the genes for the neurokinin‐1 and neurokinin‐2 receptors (NK‐1R and NK‐2R, respectively). Informative non‐synonymous SNPs were scored using the single base extension method for 312 patients with chronic cough and for 100 age matched healthy controls. The cough response to capsaicin was recorded for 312 patients with chronic cough, and the potential genetic association between cough sensitivity to capsaicin and the NK‐1R and NK‐2R genotypes was evaluated.
Two informative SNPs were identified in NK‐2R (Gly231Glu and Arg375His), whereas no informative SNP was found in NK‐1R. After adjusting for atopy, sex, age, and smoking, the prevalence of enhanced cough sensitivity to capsaicin was higher in the chronic cough patients with the 231Glu allele (p = 0.004; OR 1.69 (95% CI 1.18 to 2.42)) and the 231Glu_375Arg haplotype (p = 0.003; OR 1.71 (95% CI 1.20 to 2.24)]. Moreover, the lowest capsaicin concentration to cause five consecutive coughs (C5) was significantly lower in patients with 231Glu (mean (SD) 44.1 (53.2) v 60.9 (55.8) μM/l, p = 0.04) and those with 231Glu_375Arg (43.2 (52.7) v 69.6 (52.0) μM/l, p = 0.03).
The results of this study suggest that NK‐2R gene polymorphisms are involved in the enhanced cough sensitivity to capsaicin of patients with chronic cough.
chronic cough; neurokinin receptors; polymorphism; capsaicin sensitivity
Among the factors modulating transplant rejection, chemokines and their respective receptors deserve special attention. Increased expression of monocyte chemoattractant protein-1 (MCP-1) and its corresponding receptor (chemokine receptor-2, CCR2) has been implicated in renal transplant rejection. To determine the impact of the MCP-1-2518G and CCR2-64I genotypes on renal allograft function, 167 Korean patients who underwent transplantation over a 25-year period were evaluated. Genomic DNA was genotyped using polymerase chain reaction followed by restriction fragment length polymorphism analysis. Fifty-five (32.9%) patients were homozygous for the MCP-1-2518G polymorphism. Nine (5.4%) patients were homozygous for the CCR2-64I polymorphism. None of the investigated polymorphism showed a significant shift in long-term allograft survival. However, a significant increase was noted for the risk of late acute rejection in recipients who were homozygous for the MCP-1-2518G polymorphism (OR, 2.600; 95% CI, 1.125–6.012; P = 0.022). There was also an association between the MCP-1-2518G/G genotype and the number of late acute rejection episodes (P = 0.024). Although there was no difference in the incidence of rejection among recipients stratified by the CCR2-V64I genotype, recipients with the CCR2-V64I GG genotype in combination with the MCP-1-2518G/G genotype had a significantly higher risk of acute or late acute rejection among the receptor-ligand combinations (P = 0.006, P = 0.008, respectively). The MCP-1 variant may be a marker for risk of late acute rejection in Korean patients.
Citrus red mite (CRM) is known as the most common sensitizing allergen in subjects with asthma and rhinitis working on citrus farms. The aim of this study is to evaluate the role of specific IgG1 (slgG1) and specific IgG4 (slgG4) to CRM in citrus farmers. Questionnaire survey and skin prick test including CRM antigen was done by 136 workers. Specific IgE (slgE), slgG1 and slgG4 to CRM were detected by enzyme-linked immunosorbent assay (ELISA). CRM-sensitive-asthma was diagnosed upon presence of asthmatic symptoms by questionnaire, airway hyperresponsiveness to methacholine and slgE to CRM. CRM-sensitive rhinitis was diagnosed upon presence of rhinitis symptoms and slgE to CRM. Eleven (8.1%) had CRM-sensitive asthma and 25 (18.4%) had CRM-sensitive rhinitis. Significant association was noted between presence of asthmatic symptoms and slgE or slgG4 (p<0.05, respectively), while no significant association was noted in slgG1 (p>0.05). Significant association was noted in the prevalence between slgG4 and slgE (p<0.05), while no significant association was noted between slgG1 and slgG4 or slgE (p<0.05, respectively). There was a significant correlation between slgE and slgG4 level (r=0.39, p<0.05). These findings suggest that the presence of slgG1 to CRM is response to CRM exposure, and further studies will be needed to evaluate the role of slgG4.
To compare the mediator releasability between atopic and nonatopic asthmatics, we measured basophil histamine releasability (BaHR) using a calcium-ionophore A23187 and anti-IgE in 137 subjects who were treated at Seoul National University Hospital. Subjects were categorized into atopic (group AA, n=77) or nonatopic asthmatics (group NA, n=32), or normal controls (group NC, n=28). Serum total IgE levels were determined and correlation with BaHR was assessed. Anti-IgE-induced maximal BaHR in groups AA, NA, and NC was 41.0+/-3.2, 23.1+/-4.5, and 16.8+/-3.8, respectively (mean+/-SE, %). Anti-IgE-induced BaHR in group AA was significantly higher than that in groups NA and NC (p<0.05). Calcium ionophore A23187-induced maximal BaHR was 43.1+/-2.8, 40.8+/-4.4, and 50.5+/-5.2, respectively (mean+/-SE, %), and there was no significant difference among the groups. Serum total IgE level correlated significantly with anti-IgE-induced maximal BaHR (r=0.281, p<0.01) but not with that induced by calcium ionophore A23187. In conclusion, IgE receptor-related BaHR is higher in atopic asthmatics than in nonatopic asthmatics, and this increased BaHR in atopics is significantly associated with increased serum total IgE level.
CD44 is a member of cell surface glycoproteins which are involved in cell-matrix adhesion and tumor metastasis. Certain types of tumors express complex CD44 isoforms generated by alternative splicing of 2v-10v exons, and their expression appears to promote metastasis of tumor cells. Using a nested RT-PCR, we analyzed expression of CD44 variants in 26 stomach carcinoma, 21 matched normal tissues, and 2 carcinoma cell lines. We observed frequent and complex patterns of CD44 variant expression in tumor tissues. While exons 6v and 7v expression was detected in most normal and tumor tissues, exon 9v was most rarely detected. Exon 5v showed a significantly frequent expression in carcinoma, suggesting that its expression might contribute to the malignant progression. While exon 9v was frequently observed in diffuse-type tumors, the other 8 variant exons including 6v showed more frequent expression in intestinal-type tumors. Exons 9v and 10v were predominantly expressed in advanced tumor tissues and exon 8v was expressed more frequently in tumors of lymph node metastasis. We believe that series with a longer follow-up now need to be tested to clarify the association between CD44 splice variant expression and distant metastasis or long-term prognosis.
Insertion of methyl methacrylate polymer into newly reamed bony cavities has sometimes resulted in profound hypotension, cardiac arrest, or sudden death which are more common in patients with hemodynamic instability or hypovolemia. In paralysis agitans(Parkinson's disease), dramatic worsening of the disease often occurs when another illness or trauma accompanies it. And it is possible that chronic medication with levodopa can cause the loss of ability to support blood pressure. So, it involves some risk to use methyl methacrylate in chronic levodopa-treated paralysis agitans. We present a case of paralysis agitans who demonstrated profound hypotension immediately following insertion of methyl methacrylate polymer in spite of normovolemia and proper anesthetic management.
We report five cases of cytomegalovirus infection in immunocompromised patients which were detected by either cytomegalovirus antigenemia assay or in situ hybridization. Four cases had leukemia and the other had chronic renal failure. All the three BMT recipients suffered from GvHD. Interestingly, there was an unique case of CMV disease without a history of BMT, which reminded us that CMV could attack immunocompromised patients who had not undergone transplantation, too. Four out of five cases died. We think that cytomegalovirus infection or disease should not be regarded as a minor problem in post-transplantation infection in Korea.
A case of malacoplakia of the prostatic gland associated with prostatic nodular hyperplasia from a 69 years old man was presented, and its light and electron microscopic and immunohistochemical features were discussed along with its pathogenesis. This lesion was incidentally found in a transurethral prostatectomy specimen, and consisted of large number of epithelioid cells in which were typical cytoplasmic inclusions known as Michaelis-Gutmann bodies. Ultrastructurally, these inclusions showed a dense, central calcified bodies of various developmental stages. Immunohistochemical study using antilysosomal antibody revealed no lysosomal activity. Based on these findings, we could suspect that main problem for this development of malacoplakia is altered intracellular digestion process of foreign biologic materials.
Zymogram studies of peptide hydrolases from the human intestinal brush border and cytoplasmic fractions produced multiple bands--that is, up to seven--while the brush border membrane produced only a single band of enzyme activity. With all of the substrates tested except L-leucyl-L-leucyl-L-leucine, a band having anodic mobility identical with that produced by the brush border enzymes was produced by the cytoplasmic enzymes. With L-trileucine as a substrate, no overlapping band was produced. This band in the cytoplasmic fraction was heat sensitive, while that in the brush border fraction was not. Thus it would appear that there is a single human intestinal brush border peptide hydrolase capable of hydrolysing a variety of di- and tri-peptides. This peptide hydrolases of the brush border and the cytoplasmic fraction of human intestine are distinct.
Peptide hydrolases, catalyzing the hydrolysis of 13 dipeptides and 5 tripeptides into their respective amino acids, were studied in small intestinal mucosa and other tissues, in man and in the rat.
Studies on the subcellular distribution of these enzymes showed enzyme activities in both the soluble and brush border fractions of the rat small intestinal mucosa, the former constituting 80-90% and the latter 10-15% of the total activity. Zymogram studies of peptide hydrolases, in both fractions, yielded multiple bands indicating multiple zones of enzyme activity. With most substrates a rather broad range of enzyme activities was observed in the soluble fraction differing only slightly from substrate to substrate, the exception being when L-leucyl-L-proline was used: this latter led to a zymogram pattern which was quite distinct. The synthetic substrates, L-leucyl-β-naphthylamide and L-leucinamide appeared to be hydrolyzed by two electrophoretically distinct enzymes, different from those hydrolyzing other leucyl-containing peptide substrates.
Zymogram patterns of the brush border membrane fraction were quite different from those of the soluble fraction of rat small intestine indicating that enzymes from the two sources may be different. No comparable human data were obtained.
Peptide hydrolases in the soluble fractions of various organs from the same species gave similar zymogram patterns, while those from the plasma membrane-bound fractions of different organs in the same species were peculiar to each organ. From these data, it is suggested that peptide hydrolases in the brush border and the soluble fractions of small intestine are distinct enzymes and may play different roles in cellular function.
Recently, we found that Staphylococcus aureus produces extracellular vesicles (EV) that contain pathogenic proteins. Although S. aureus infection has been linked with atopic dermatitis (AD), the identities of the causative agents from S. aureus are controversial. We evaluated whether S. aureus-derived EV are causally related to the pathogenesis of AD.
Extracellular vesicles were isolated by the ultracentrifugation of S. aureus culture media. The EV were applied three times per week to tape-stripped mouse skin. Inflammation and immune dysfunction were evaluated 48 h after the final application in hairless mice. Extracellular vesicles-specific IgE levels were measured by ELISA in AD patients and healthy subjects.
The in vitro application of S. aureus EV increased the production of pro-inflammatory mediators (IL-6, thymic stromal lymphopoietin, macrophage inflammatory protein-1α, and eotaxin) by dermal fibroblasts. The in vivo application of S. aureus EV after tape stripping caused epidermal thickening with infiltration of the dermis by mast cells and eosinophils in mice. These changes were associated with the enhanced cutaneous production of IL-4, IL-5, IFN-γ, and IL-17. Interestingly, the serum levels of S. aureus EV-specific IgE were significantly increased in AD patients relative to healthy subjects.
These results indicate that S. aureus EV induce AD-like inflammation in the skin and that S. aureus-derived EV are a novel diagnostic and therapeutic target for the control of AD.
atopic dermatitis; extracellular vesicles; skin inflammation; Staphylococcus aureus
Previous evidence indicates that inhalation of lipopolysaccharide (LPS)-containing with allergens induced mixed Th1 and Th17 cell responses in the airways. Extracellular vesicles (EVs) are nanometer-sized spherical, lipid-bilayered structures and are recently in the public eye as an intercellular communicator in immune responses.
To evaluate the role of EVs secreted by LPS inhalation in the development of airway immune dysfunction in response to allergens.
Extracellular vesicles in bronchoalveolar lavage fluids of BALB/c mice were isolated and characterized 24 h after applications to the airway of 10 μg of LPS for 3 days. To evaluate the role of LPS-induced EVs on the development of airway immune dysfunction, in vivo and in vitro experiments were performed using the isolated LPS-induced EVs.
The inhalation of LPS enhanced EVs release into the BAL fluid, when compared to the application of PBS. Airway sensitization with allergens and LPS-induced EVs resulted in a mixed Th1 and Th17 cell responses, although that with allergens and PBS-induced EVs induced immune tolerance. In addition, LPS-induced EVs enhanced the production of Th1- and Th17-polarizing cytokines (IL-12p70 and IL-6, respectively) by lung dendritic cells. Moreover, the immune responses induced by the LPS-induced EVs were blocked by denaturation of the EV-bearing proteins.
These data suggest that EVs (especially, the protein components) secreted by LPS inhalation are a key intercellular communicator in the development of airway immune dysfunction to inhaled LPS-containing allergens.
asthma; airway immune dysfunction; extracellular vesicles; lipopolysaccharide
p53 is a key regulator of cell growth and death by controlling cell cycle progression and apoptosis under conditions of stress such as DNA damage or oncogenic stimulation. As these processes are critical for cell function and inhibition of tumor development, p53 regulatory pathways are strictly monitored in cells. Recently, it was recognized that nucleolar proteins, including nucleophosmin/B23, ribosomal protein L11, and alternate reading frame (ARF), form the nucleolus-ARF-murine double minute 2 (MDM2) axis in p53 regulatory pathways, which increases p53 stability by suppressing the activity of MDM2. In this work, we show that nucleolar protein glioma tumor-suppressor candidate region gene 2 (GLTSCR2) translocates to the nucleoplasm under ribosomal stress, where it interacts with and stabilizes p53 and inhibits cell cycle progression without the involvement of the major upstream p53 regulator, ARF. Furthermore, ectopic expression of GLTSCR2 significantly suppressed growth of cancer cells in a xenograft animal model via p53-dependent pathway. Our data identify GLTSCR2 as a new member of the nucleolus–nucleoplasmic axis for p53 regulation. ARF-independent direct regulation of p53 by GLTSCR2 may be a key mechanism and therapeutic target for cell death or growth inhibition when nucleolus-ARF-p53 pathways are inactivated by genetic or epigenetic modifications of ARF, which are the second most common types of genetic change observed in human cancers.
GLTSCR2; p53; tumor suppressor; nucleolus
Salivary duct carcinoma (SDC) is a rare malignancy of high-grade pathological type. We evaluated clinical outcomes and prognostic factors in 35 patients with SDC treated post-operatively with adjuvant radiation.
We retrospectively assessed overall survival, locoregional control and disease-free survival in 35 patients with SDC of the major salivary glands who underwent surgery and were subsequently treated with radiotherapy. The evaluated prognostic factors included gender, age, symptom duration, tumour site, tumour size, TNM classification, and the following pathological features: perineural invasion, lymphovascular invasion, extraparenchymal invasion and resection-margin status.
Of the 35 patients, 30 (85.7%) were male. Median age at initial diagnosis was 62 years (range 38–75 years). The parotid gland was the main site affected in 22 patients (62.9%). 18 patients (51.5%) had pathological T3/T4 tumours, and 26 (74.3%) showed pathological nodal involvement. Actuarial 5-year locoregional control, disease-free survival and overall survival rates were 63.3%, 47.4% and 55.1%, respectively. The cause-specific death rate was 31.4% (n=11). Pathological nodal involvement was correlated with distant metastasis (p=0.011). Lymphovascular invasion was significantly prognostic for distant metastasis-free survival (p=0.049), locoregional control (p=0.012) and overall survival (p=0.003) in a Cox proportional hazard model, whereas perineural invasion was only significantly prognostic for overall survival (p=0.005).
Surgery and post-operative radiotherapy were effective for locoregional control. Lymphovascular invasion and perineural invasion were significant prognostic factors in patients with SDC.
Norovirus genotype II.3 (GII.3) strains are a major cause of sporadic gastroenteritis. Intergenic recombination between the capsid and RNA-dependent RNA polymerase (RdRp) genes is common and results in the acquisition of an alternative RdRp genotype. This study aimed to explore the evolution of the GII.3 capsid gene, focusing on the influence of intergenic recombination. The capsid genes from six GII.3 norovirus strains, collected from Australian children between 2001 and 2010, were sequenced and aligned with 66 GII.3 capsid sequences from GenBank, spanning 1975 to 2010. The GII.3 capsid gene evolved at a rate of 4.16 × 10−3 to 6.97 × 10−3 nucleotide substitutions/site/year from 1975 to 2010 and clustered into five temporally sequential lineages. Clustering of the GII.3 capsid gene sequences was associated with intergenic recombination and switches between RdRp genotypes GII.3, GII.a, GII.b, GII.12, and an undefined ancestral RdRp. Comparison of the substitution rate of the GII.3 and GII.b RdRps suggested that RdRp switching allows a higher evolutionary rate, leading to increased genetic diversity and adaptability. Alignment of GII.3 capsid sequences revealed 36 lineage-specific conserved amino acid substitutions, four of which were under positive selection. Many conserved substitutions were within predicted antibody binding regions and close to host attachment factor binding sites. In conclusion, evolution of GII.3 noroviruses was primarily driven by intergenic recombination. The acquisition of new RdRps may lead to a faster mutation rate and increased genetic diversity, improving overall GII.3 fitness.
Nonlinear dynamics underpin a vast array of physical phenomena ranging from interfacial motion to jamming transitions. In many cases, insight into the nonlinear behavior can be gleaned through exploration of higher order harmonics. Here, a method using band excitation scanning probe microscopy (SPM) to investigate higher order harmonics of the electromechanical response, with nanometer scale spatial resolution is presented. The technique is demonstrated by probing the first three harmonics of strain for a Pb(Zr1-xTix)O3 (PZT) ferroelectric capacitor. It is shown that the second order harmonic response is correlated with the first harmonic response, whereas the third harmonic is not. Additionally, measurements of the second harmonic reveal significant deviations from Rayleigh-type models in the form of a much more complicated field dependence than is observed in the spatially averaged data. These results illustrate the versatility of nth order harmonic SPM detection methods in exploring nonlinear phenomena in nanoscale materials.
Open reading frame 2 (ORF2) of the feline calicivirus (FCV) genome encodes a capsid precursor that is posttranslationally processed to release the mature capsid protein (VP1) and a small protein of 124 amino acids, designated the leader of the capsid (LC). To investigate the role of the LC protein in the virus life cycle, mutations and deletions were introduced into the LC coding region of an infectious FCV cDNA clone. Three cysteine residues that are conserved among all vesivirus LC sequences were found to be critical for the recovery of FCV with a characteristic cytopathic effect in feline kidney cells. A cell-rounding phenotype associated with the transient expression of wild-type and mutagenized forms of the LC correlated with the cytopathic and growth properties of the corresponding engineered viruses. The host cellular protein annexin A2 was identified as a binding partner of the LC protein, consistent with a role for the LC in mediating host cell interactions that alter the integrity of the cell and enable virus spread.