Genomic origin and EGFR-TKI treatments of pulmonary adenosquamous carcinoma.

BACKGROUND: Adenosquamous carcinoma (ASC) of the lung is a heterogeneous disease that is composed of both adenocarcinoma components (ACC) and squamous cell carcinoma components (SCCC). Their genomic profile, genetic origin, and clinical management remain controversial. PATIENTS AND METHODS: Resected ASC and metastatic tumor in regional lymph nodes (LNs) were collected. The ACC and SCCC were separated by microdissection of primary tumor. The 1021 cancer-related genes were evaluated by next-generation sequencing independently in ACC and SCCC and LNs. Shared and private alterations in the two components were investigated. In addition, genomic profiles of independent cohorts of adenocarcinomas and squamous cell carcinomas were examined for comparison. We have also carried out a retrospective study of ASCs with known EGFR mutation status from 11 hospitals in China for their clinical outcomes. RESULTS: The most frequent alterations in 28 surgically resected ASCs include EGFR (79%), TP53 (68%), MAP3K1 (14%) mutations, EGFR amplifications (32%), and MDM2 amplifications (18%). Twenty-seven patients (96%) had shared variations between ACC and SCCC, and pure SCCC metastases were not found in metastatic LNs among these patients. Only one patient with geographically separated ACC and SCCC had no shared mutations. Inter-component heterogeneity was a common genetic event of ACC and SCCC. The genomic profile of ASC was similar to that of 170 adenocarcinomas, but different from that of 62 squamous cell carcinomas. The incidence of EGFR mutations in the retrospective analysis of 517 ASCs was 51.8%. Among the 129 EGFR-positive patients who received EGFR-TKIs, the objective response rate was 56.6% and the median progression-free survival was 10.1 months (95% confidence interval: 9.0-11.2). CONCLUSIONS: The ACC and SCCC share a monoclonal origin, a majority with genetically inter-component heterogeneity. ASC may represent a subtype of adenocarcinoma with EGFR mutation being the most common genomic anomaly and sharing similar efficacy to EGFR TKI.

Reactive atmospheric pressure plasma for highly efficient removal of structure-directing agents from zeolite thin films.

We present a novel approach to remove the structure-directing agent (SDA) from as-synthesized zeolites using an atmospheric pressure plasma jet (APPJ). This reduces the time required to less than 60 seconds as compared to the existing thermal calcination, whose durations range from hours to days. The highly reactive plasma also results in a pronounced Q(3)-to-Q(4) transformation in the pure-silica zeolite MFI.

Müllerian adenosarcoma: a review of cases and literature.

OBJECTIVE: Mullerian adenosarcoma usually originates in the endometrium and grows as a polypoid mass in post-menopausal women presenting as abnormal vaginal bleeding. This report reviewed Miillerian adenosarcoma cases to clarify the clinical and pathologic characteristics. MATERIALS AND METHODS: Fifteen cases ofMiillerian adenosarcoma in two medical centers covering a 15-year period were reviewed. Their clinical characteristics, pathologic findings, treatment, and outcomes were compared. RESULTS: Of the 15 cases, three originated from the endometrium, six arose from uterine adenomyosis, three from the adnexa, and three from the cervix. There was only one post-menopausal case. One case was of breast cancer with tamoxifen (TMX) therapy. There were four Miillerian adenosarcoma with sarcomatous overgrowth (MASO) cases, three of which died within one year after surgery. Only the focal MASO case survived. CONCLUSION: The rare variant of MASO is very aggressive and associated with poor prognosis.

Immune focusing and enhanced neutralization induced by HIV-1 gp140 chemical cross-linking.

Experimental vaccine antigens based upon the HIV-1 envelope glycoproteins (Env) have failed to induce neutralizing antibodies (NAbs) against the majority of circulating viral strains as a result of antibody evasion mechanisms, including amino acid variability and conformational instability. A potential vaccine design strategy is to stabilize Env, thereby focusing antibody responses on constitutively exposed, conserved surfaces, such as the CD4 binding site (CD4bs). Here, we show that a largely trimeric form of soluble Env can be stably cross-linked with glutaraldehyde (GLA) without global modification of antigenicity. Cross-linking largely conserved binding of all potent broadly neutralizing antibodies (bNAbs) tested, including CD4bs-specific VRC01 and HJ16, but reduced binding of several non- or weakly neutralizing antibodies and soluble CD4 (sCD4). Adjuvanted administration of cross-linked or unmodified gp140 to rabbits generated indistinguishable total gp140-specific serum IgG binding titers. However, sera from animals receiving cross-linked gp140 showed significantly increased CD4bs-specific antibody binding compared to animals receiving unmodified gp140. Moreover, peptide mapping of sera from animals receiving cross-linked gp140 revealed increased binding to gp120 C1 and V1V2 regions. Finally, neutralization titers were significantly elevated in sera from animals receiving cross-linked gp140 rather than unmodified gp140. We conclude that cross-linking favors antigen stability, imparts antigenic modifications that selectively refocus antibody specificity and improves induction of NAbs, and might be a useful strategy for future vaccine design.

Regulated expression of neuronal SIRT1 and related genes by aging and neuronal ß2-containing nicotinic cholinergic receptors.

Longevity genes attenuate the aging process, but their expression in the brain during aging remains unknown. Loss of the majority of heteromeric brain nicotinic acetylcholine receptors (nAChRs) results in premature brain aging, and altered regulation of longevity genes could be involved. Using in situ hybridization, the expression of SIRT1, Ku70, Nampt, p53, forkhead Box O3 (FoxO3), and mitochondria uncoupling protein 5 (UCP5) was determined in neocortex and hippocampus of young adult 3-month and middle-aged 18-month-old wild-type (WT), and age-matched mice lacking ß2* heteromeric nAChRs (ß2-/-). Age-related structural changes were detected in WT mice. In particular, cortical thickness was decreased but neuronal density increased, and hippocampal volume increased with age. In contrast, young ß2-/- mice exhibited increased cortical neuronal density, and with age, cortical thickness decreased more dramatically, and hippocampal volume did not increase. Thus, young ß2-/- mice exhibited cortical signs of aging, and aging was accelerated at 18 months. The longevity genes probed exhibited similar expression patterns in frontal brain structures, with strong expression in hippocampus, medial habenula (MHb), and cortex. In WT mice, age significantly decreased expression of all genes except SIRT1 in cortical structures, and a similar pattern was detected in the MHb. Genotype had no effect on expression in young adults in either cortex or MHb, but increased mRNA expression of SIRT1, Nampt, and Ku70 was detected in cortex, hippocampus, and MHb of aged ß2-/- mice compared with WT mice. This is the first study to determine age-related expression of survival genes in forebrain areas. Although, structural changes indicative of accelerated aging are evident in young ß2-/- mice, the data suggest that nAChRs do not directly regulate expression of survival genes. However, loss of ß2* nAChRs could result in augmented cellular stress, which indirectly increases expression of SIRT1, Nampt, and Ku70 as an adaptive response to provide protection against neurodegeneration.

Cryogen spray cooling in conjunction with pulse dye laser treatment of port wine stains of the head and neck.

BACKGROUND: When a cryogen spurt is applied to the skin surface for an appropriately short period of time, the spatial distribution of cooling remains localized in the normal overlying epidermis, while leaving the temperature of deeper port wine stain (PWS) blood vessels unchanged. Furthermore, cooling continues after pulsed laser exposure as cryogen remaining on the surface evaporates and removes heat deposited by light absorption in epidermal melanin. The objective of this study was to evaluate the efficacy and advantages of cryogen spray cooling plus flashlamp-pumping in conjunction with dye laser treatment (CSC-LT) of PWS. METHODS: From 1996 to 2000, a retrospective study was conducted on 63 patients, consisting of 43 women and 20 men, between the ages of 8 and 62 years treated with pulsed dye laser (lambda = 585 nm, tau p = 450 microseconds) over a 4-year period. The duration of cryogen spurts and the delay period between cryogen delivery and laser illumination were controlled. An infrared focal plane array thermodetector measured changes of lesion surface temperature which were recorded. The subject was asked to score discomfort during treatment using a pain scale. The primary efficacy measure was the quantitative assessment of a blanching response score. RESULTS: The ambient skin surface temperature of PWS was 33.31 +/- 1.55 degrees C. The mean pain score for uncooled sites was 39.85 +/- 0.23 compared to 20.18 +/- 0.15 for cooled sites. There was a statistically significant difference in pain elimination between cooled and uncooled sites (p = 0.001). The mean blanching response score of CSC-LT was 3.70. A significant blanching response of PWS when receiving CSC-LT was noted. CONCLUSION: Our clinical studies demonstrate the feasibility of selective epidermal cooling while achieving photothermolysis of blood vessels during pulsed dye laser treatment of PWS.

Drug-targeting strategies in cancer therapy.

Genetic changes in cell-cycle, apoptotic, and survival pathways cause tumorigenesis, leading to significant phenotypic changes in transformed cells. These changes in the tumor environment - elevated expression of surface proteases, increased angiogenesis and glucuronidase activity - can be taken advantage of to improve the therapeutic index of existing cancer therapies. Targeting cytotoxics to tumor cells by enzymatic activation is a promising strategy for improving chemotherapeutics.

Total internal reflection for precision small-angle measurement.

A method for precision small-angle measurement is proposed. This method is based on the total-internal-reflection effect of a light beam at a pair of glass prisms. Angular displacement of the light beam is measured when the intensity change of the reflected beam is detected as a result of the relative phase shift between the s- and the p-polarized beams. An initial phase shift between the s- and the p-polarized components is introduced to increase measurement sensitivity. For increased measurement linearity and reduced effect of laser power fluctuation on the output, a differential method is used in which the light beam is split equally into two beams, each reflected at a prism and detected by a photodiode. The output is obtained as the difference of the two detected intensities divided by their sum. A prototype device was built, which demonstrated a nonlinearity error of 1.3% in a measurement range of ?0.6 degrees or 0.4% in ?0.3 degrees . The peak-to-peak noise level was found to be at approximately 0.5 arc sec. This noise level can be reduced further and resolution increased by a reduction of the measurement range.

Automatic gait recognition via statistical approaches for extended template features.

A gait recognition system using extended template features is presented. A proposed statistical approach is applied for feature extraction from spatial and temporal templates. This method can be used to reduce data dimensionality and to optimize the class separability of different gait sequences simultaneously. Dimensionality reduction is achieved by template extraction followed by principal component analysis. Gait recognition is achieved in the canonical space using a measure of accumulated distance as the metric. By incorporating spatial and temporal information into an extended feature, gait recognition becomes more robust and accurate than using spatial or temporal features alone.

Development of a binding assay for p53/HDM2 by using homogeneous time-resolved fluorescence.

The p53 tumor suppressor protein is activated and stabilized in response to DNA damage, resulting in cell cycle arrest or apoptosis. HMD2 is a negative regulator of p53. Binding of p53 by HDM2 traffics p53 from the nucleus to the cytoplasm where it is recognized and targeted for ubiquitin-mediated degradation (D. A. Freedman, L. Wu, and A. J. Levine, 1999, Cell. Mol. Life Sci. 55, 96-107). Several reports have suggested that disruption of this complex in normal cells results in p53 signaling (V. Böttger, A. Böttger, A. Sparks, W.-L. Liu, S. F. Howard, and D. P. Lane, 1997, Curr. Biol. 7, 860-869; C. Wasylyk, R. Salvi, M. Argentini, C. Dureuil, I. Delumeau, J. Abecassis, L. Debussche, and B. Wasylyk, 1999, Oncogene 18, 1921-1934). A homogeneous time-resolved fluorescence (HTRF) assay has been developed to monitor p53/HDM2 binding. This assay employs a site-specific biotinylated p53 protein, a GST-fused HDM2 protein, and two fluorophore-conjugated detection reagents, streptavidin-XL665 and europium cryptate-labeled anti-GST antibody ¿Eu(K)-anti-GST. Binding of p53 to HDM2 brings the fluorophores into close proximity, allowing fluorescence resonance energy transfer to occur. Development of this assay and comparison to a traditional ELISA are described in this report. The HTRF assay was then utilized to assess the effect of serine phosphorylation within the p53 N-terminus on HDM2 binding, and to determine the relative affinity of a p73 peptide for HDM2.

Blood alcohol concentration among injured drivers in Hualien County.

Vehicle-related injuries are the major cause of death and injuries in Hualien County. Driving under the influence of alcohol plays a major role in such crashes. From December 1997 to May 1998, we determined the blood alcohol concentrations of 750 injured drivers from vehicle crashes, visiting the two emergency rooms of teaching hospitals in Hualien. The objectives of this study were to investigate the incidence of alcohol used in vehicle crashes, to identify the prevalence groups for prevention and to discuss alcohol testing at emergency services. Sixty-four percent were male; 27.5% were aborigines. The mean age was 36.5 +/- 16.5 years. About 54.1% tested positive for blood alcohol concentration (BAC), which ranged from 13 to 611 mg/dL; 38.6% had BAC levels exceeding 50 mg/dL and 21.1% exceeding 200 mg/dL. The mean BAC was 85.9 mg/dL (+/- 118.5). Middle-aged males and aborigines were more likely to drive under the influence of alcohol. We recommend blood alcohol testing to be mandatory at the emergency service and to be used as evidence for prosecution in a court of law. Preventing drunk driving through community programs is imperative, especially in the aboriginal communities.

Use of thin films for high-sensitivity angle measurement.

We propose to use thin films to provide a drastic improvement of measurement sensitivity in the recently developed small-angle measurement method, namely, angle measurement based on the internal-reflection effect. By designing the thin films (single layer or multiple layers) so that they provide an antireflection effect in the vicinity of the critical angle, we show that the sensitivity of angle measurement can be increased exponentially with the increase of the number of thin-film layers. This method provides a new means of designing angle sensors with increased sensitivities without having to increase the number of reflections and therefore the physical size and the required fabrication accuracy of the reflection prisms. We describe the design of the thin films for this particular application and the analysis of measurement sensitivity and range as determined by the material and the number of layers of the thin films. Selection of the optimal initial angle for high linearity performance is also discussed.

Small-angle measurement by use of a single prism.

A new method of angle measurement based on the internal reflection effect is proposed that uses a single right-angle prism. We measure the angular displacement between a laser beam and the prism by detecting the changes in reflectance as a function of the angle of incidence. We achieve high linearity of measurement by taking the inverse of reflectance as the output. The inverse of reflectance is obtained from the intensities of the reflected and the transmitted beams measured by two photodiodes. Experiments with a prototype device have demonstrated that angle measurement with a range of ?500 arc sec, a nonlinearity error of ?0.1%, and a resolution of 0.1 arc sec can be readily achieved. The measurement range can be further increased with some sacrifice of linearity.

Oncogene products as therapeutic targets for cancer.

Enormous progress has been made in the last several years in delineating signal transduction pathways associated with cell proliferation and apoptosis. The components of these pathways, which include both oncogenes and tumor suppressors, may provide viable targets for therapeutic intervention for the treatment of cancer and other diseases. This review highlights some of these recent biologic and pharmacologic advances, focusing on the ras pathway and on p53-dependent apoptosis.

Angle measurement based on the internal-reflection effect using elongated critical-angle prisms.

Elongated critical-angle prisms that provide multiple reflections have been used to increase measurement sensitivity while retaining excellent linearity in the recently developed angle-measurement method, angle measurement based on the internal-reflection effect.

An SH3 domain is required for the mitogenic activity of microinjected phospholipase C-gamma 1.

Phospholipase activity is elevated in dividing cells. In response to growth factor stimulation, phospholipase C-gamma (PLC-gamma) binds to activated tyrosine kinase receptors via SH2 binding domains, resulting in phosphorylation of PLC-gamma and activation of its enzyme activity. These observations suggest that PLC-gamma participates in the signal transduction pathway employed by growth factors to promote mitogenesis. Consistent with this hypothesis, microinjection of purified bovine PLC-gamma into quiescent fibroblasts has been previously reported to initiate a mitogenic response [Smith et al. (1989) Proc. Natl. Acad. Sci. 86, 3659]. We have reproduced this result using recombinant rat PLC-gamma protein. Surprisingly, however, a catalytically inactive mutant of PLC-gamma, H335Q, also elicited a full mitogenic response. The capacity to induce mitogenesis by microinjection of PLC-gamma was mapped to the 'Z' domain of the protein, which contains PLC-gamma's SH2 and SH3 motifs. Inactivation of the phosphorylated tyrosine binding properties of both SH2 domains had no effect on the mitogenic activity of the Z-domain peptide. However, deletion of the SH3 domain resulted in a complete loss of activity. These results suggest that PLC-gamma's mitogenic properties do not require the enzyme's phospholipase activity, but are instead mediated by a novel pathway for mitogenic stimulation which is dependent upon an intact SH3 domain.

Angle measurement based on the internal-reflection effect and the use of right-angle prisms.

A new development in angle measurement based on the internal-reflection effect (AMIRE) is described in which a pair of right-angle prisms is used to replace the previously used elongated critical-angle prisms, resulting in lower costs and a more compact size. Excellent linearity is achieved through careful alignment of the right-angle prisms. The measurement sensitivity and range can be selected through the use of light sources with different polarization states. Experiments with a prototype sensor demonstrated a measurement range of 1.6°, a resolution of 0.04 arcsec, and a nonlinearity error of ±0.1%. Both analytical and experimental results are presented.

Retinoblastoma protein reverses DNA bending by transcription factor E2F.

E2F is a mammalian transcription factor involved in cell cycle regulation. The retinoblastoma gene product, pRB, binds to E2F in a cell cycle-dependent manner and appears to turn E2F from a transcriptional activator into a repressor. We show here that in vitro binding of pRB has three major effects on the DNA binding properties of E2F affinity-purified from HeLa cells; pRB binding increases the half-life of E2F.DNA complexes 10-15-fold, it reduces E2F specific DNA binding in the presence of nonspecific DNA by sequestering E2F, and it partially reverses the DNA bending induced by E2F. Upon specific DNA binding, E2F induces a DNA bend with a flexure angle of 125 degrees. Both full-length pRB105 and the N-terminally truncated pRB60 bind to the E2F.DNA complex with a Kd,app of 150 pM and reduce the apparent DNA bending to less than 80 degrees. DNA footprinting analysis indicates that the nonspecific DNA binding activity of pRB is not involved in this effect. Our biochemical data suggest that transcriptional activation by E2F may involve DNA bending and that the reversal of bending upon binding of pRB may turn E2F into a repressor.

Transcription factor E2F binds DNA as a heterodimer.

E2F is a mammalian transcription factor that appears to play an important role in cell cycle control. DNA affinity column-purified E2F from HeLa cells reproducibly exhibits multiple protein bands when analyzed by SDS/PAGE. After electrophoretic purification, electroelution, and refolding of the individual protein components, the E2F DNA binding activity of the individual proteins was poor. However, upon mixing the individual components together, a dramatic (100- to 1000-fold) increase in specific DNA binding activity was observed. The five protein bands isolated can be separated into two groups based on apparent molecular mass. Optimal reconstitution of activity requires one of the two proteins found in the group of larger molecular mass (approximately 60 kDa) and one of the three proteins in the smaller-sized group (approximately 50 kDa). The reconstituted heterodimer is identical to authentic affinity-purified E2F by three criteria: DNA-binding specificity, DNA pattern, and binding to the retinoblastoma gene product. A recently cloned protein with E2F-like activity, RBP3/E2F-1, is related to the protein components of the group of larger molecular mass, as determined by Western blot analysis and reconstitution experiments. These data suggest that E2F, like many other transcription factors, binds DNA as an oligomeric complex composed of at least two distinct proteins.

Protein domains governing interactions between E2F, the retinoblastoma gene product, and human papillomavirus type 16 E7 protein.

Human papillomaviruses (HPVs) are the etiological agents for genital warts and contribute to the development of cervical cancer in humans. The HPV E7 gene product is expressed in these diseases, and the E7 genes from HPV types 16 and 18 contribute to transformation in mammalian cells. Mutation and deletion analysis of this gene suggests that the transforming activity of the protein product resides in the same domain as that which is directly involved in complex formation with the retinoblastoma gene product (pRB). This domain is one of two conserved regions (designated CRI and CRII) shared by E7 and other viral oncoproteins which bind pRB, including adenovirus E1A protein. Binding of HPV type 16 E7 protein to pRB has previously been shown to affect pRB's ability to bind DNA and to form complexes with other cellular proteins. In the current study, we map the functional interaction between E7 protein and pRB by monitoring the association between a 60-kDa version of the pRB, pRB60, and the cellular transcription factor E2F. We observe that CRII of E7 (amino acids 20 to 29), which completely blocks binding of full-length E7 protein, is necessary but not sufficient to inhibit E2F/pRB60 complex formation. While CRI of E1A (amino acids 37 to 55) appears to be sufficient to compete with E2F for binding to pRB60, the equivalent region of E7 is neither necessary nor sufficient. Only E7 fragments that contained both CRII and at least a portion of the zinc-binding domain (amino acids 60 to 98) inhibited E2F/pRB60 complex formation. These results suggest that pRB60 associates with E7 and E2F through overlapping but distinct domains.