Adhesion Characterization and Enhancement between Polyimide-Silica Composite and Nodulated Copper for Applications in Next-Generation Microelectronics.

As the need for high-speed electronics continues to rise rapidly, printed wiring board (PWB) requirements become ever-more demanding. A typical PWB is fabricated by bonding dielectric films such as polyimide to electrically conductive copper foil such as rolled annealed (RA) copper and is expected to become thinner, flexible, durable, and compatible with high-frequency 5G performance. Polyimide films inherently feature a higher coefficient of thermal expansion (CTE) than copper foils; this mismatch causes residual thermal stresses. To attenuate the mismatch, silica nanoparticles may be used to reduce the CTE of PI. A nodulated copper surface can be used to enhance the Cu/PI adhesion by additional bonding mechanisms that could include a type of mechanical bonding, which is a focus of this study. In this investigation, a 90° peel test was used to measure the peel strength in copper/polyimide/copper laminates containing nodulated copper and polyimide reinforced with 0, 20, and 40 wt % silica nanoparticles. The influence of silica nanoparticles on the peel strength was quantitatively evaluated. Laminates incorporating polyimide films lacking silica nanoparticles had a ∼3.75× higher peel strength compared with laminates reinforced with 40% silica. Their failure surfaces were analyzed by using scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDX), and X-ray photoelectron spectroscopy to identify the mode of failure and to understand bonding mechanisms. The key bonding mechanism, mechanical interlocking, was achieved when the polyimide surrounded or engulfed the copper nodules when the laminate was created. Post-testing failure surface analysis revealed the presence of copper on the polyimide side and polyimide on the copper side, indicating mixed mode failure. An analytical model was developed to determine the impact of applied pressure, temperature, and time on the polyimide penetration and mechanical interlocking around the copper nodules. The model was validated by measuring the peel strength on another set of specimens fabricated using increased temperature and pressure that showed a 3× increase in peel strength compared to lower temperature/pressure processing conditions. This enhanced adhesion resulted from the lower polymer material viscosity at higher temperatures, which fosters deeper and more complete penetration around the copper nodules during processing at higher pressures for longer durations. The methodology of combining peel testing, viscosity and CTE measurement, SEM/EDX, surface chemical analysis, and penetration depth calculation developed herein enables the calculation of the desired processing parameters to enhance functionality and improve adhesion.

Breast Hyperplasias, Risk Signature, and Breast Cancer.

We address the dilemma faced by oncologists in administering preventative measures to "at risk" patients diagnosed with atypical and nonatypical hyperplasias due to lack of any molecular means of risk stratification and identifying high-risk subjects. Our study purpose is to investigate a four marker risk signature, MMP-1, CEACAM6, HYAL1, and HEC1, using 440 hyperplastic tissues for identifying high-risk subjects who will benefit from preventative therapies. We assayed the markers by IHC and combined their expression levels to obtain a composite value from 0-10, which we called a "Cancer Risk Score." We demonstrate that the four marker-based risk scores predict subsequent cancer development with an accuracy of 91% and 86% for atypical and nonatypical subjects, respectively. We have established a correlation between risk scores and cancer rates by stratifying the samples into low risk (score ≤ 0.5); intermediate risk (score ≤ 5.4), and high risk (score >5.4) groups using Kaplan-Meier survival analysis. We have evaluated cancer rates at 5, 10, and 15 years. Our results show that the average cancer rates in the first 5 years among low- and intermediate-risk groups were 2% and 15%, respectively. Among high-risk group, the average cancer rates at 5 years were 73% and 34% for atypical and nonatypical subjects, respectively. The molecular risk stratification described here assesses a patient's tumor biology-based risk level as low, intermediate, or high and for making informed treatment decisions. The outcomes of our study in conjunction with the available prophylactic measures could prevent approximately 20%-25% of sporadic breast cancers.

Negative-pressure Wound Therapy for Periocular Necrotizing Fasciitis.

Periocular necrotizing fasciitis is a rare, but potentially blinding, or even fatal disease. The authors report a case of a 44-year-old man who presented with quiescent bilateral periocular and facial necrotizing fasciitis. The patient was treated with antibiotics and surgical debridement, followed by negative-pressure wound therapy (NPWT), until the wound bed was thought to be healthy enough to support bilateral upper eyelid full-thickness skin grafts. NPWT appeared to decrease local edema; speed reperfusion and granulation tissue formation; and served to stabilize the skin grafts against the wound bed, while not causing any ocular complications. NPWT can be a safe and effective adjunct treatment for periocular necrotizing fasciitis.

An Immune-Inflammation Gene Expression Signature in Prostate Tumors of Smokers.

Smokers develop metastatic prostate cancer more frequently than nonsmokers, suggesting that a tobacco-derived factor is driving metastatic progression. To identify smoking-induced alterations in human prostate cancer, we analyzed gene and protein expression patterns in tumors collected from current, past, and never smokers. By this route, we elucidated a distinct pattern of molecular alterations characterized by an immune and inflammation signature in tumors from current smokers that were either attenuated or absent in past and never smokers. Specifically, this signature included elevated immunoglobulin expression by tumor-infiltrating B cells, NF-κB activation, and increased chemokine expression. In an alternate approach to characterize smoking-induced oncogenic alterations, we also explored the effects of nicotine in human prostate cancer cells and prostate cancer-prone TRAMP mice. These investigations showed that nicotine increased glutamine consumption and invasiveness of cancer cells in vitro and accelerated metastatic progression in tumor-bearing TRAMP mice. Overall, our findings suggest that nicotine is sufficient to induce a phenotype resembling the epidemiology of smoking-associated prostate cancer progression, illuminating a novel candidate driver underlying metastatic prostate cancer in current smokers.

Tailoring Interfacial Properties by Controlling Carbon Nanotube Coating Thickness on Glass Fibers Using Electrophoretic Deposition.

The electrophoretic deposition (EPD) method was used to deposit polyethylenimine (PEI) functionalized multiwall carbon nanotube (CNT) films onto the surface of individual S-2 glass fibers. By varying the processing parameters of EPD following Hamaker's equation, the thickness of the CNT film was controlled over a wide range from 200 nm to 2 µm. The films exhibited low electrical resistance, providing evidence of coating uniformity and consolidation. The effect of the CNT coating on fiber matrix interfacial properties was investigated through microdroplet experiments. Changes in interfacial properties due to application of CNT coatings onto the fiber surface with and without a CNT-modified matrix were studied. A glass fiber with a 2 µm thick CNT coating and the unmodified epoxy matrix showed the highest increase (58%) in interfacial shear strength (IFSS) compared to the baseline. The increase in the IFSS was proportional to CNT film thickness. Failure analysis of the microdroplet specimens indicated higher IFSS was related to fracture morphologies with higher levels of surface roughness. EPD enables the thickness of the CNT coating to be adjusted, facilitating control of fiber/matrix interfacial resistivity. The electrical sensitivity provides the opportunity to fabricate a new class of sizing with tailored interfacial properties and the ability to detect damage initiation.

Split hemianterior tibialis turndown muscle flap for coverage of distal leg wounds with preservation of function.

OBJECTIVE: A hemisplit turndown tibialis anterior muscle flap is described for coverage of distal leg wounds with preservation of active extensor function for open wounds of the distal ankle is presented. This is a new flap not previously described and is another local option for coverage of selected distal leg wounds. METHODS: A description of the operative procedure and a clinical successful example is presented. RESULTS: The split hemitibialis anterior turndown muscle flap was successful and preserved function of the muscle and tendon. CONCLUSIONS: This is another option for coverage of difficult wounds of the lower extremity without sacrifice of function of the donor muscle.

Effects of interaction volume on X-ray line-scans across an ultrasonically consolidated aluminum/copper interface.

Diffusion as a bonding mechanism for ultrasonic consolidation of metals is widely debated due to the short weld times and low processing temperatures. To quantify interdiffusion coefficients, X-ray energy dispersive spectroscopy (XEDS) line-scans were performed across an Al-Cu interface using the Scanning Electron Microscope (SEM) with accelerating voltages ranging from 6 to 22 KeV in increments of 2 KeV and a step size of 0.05 microns. Higher accelerating voltages resulted in broader concentration profiles, indicating higher apparent interdiffusion coefficients when scanned at the same location of the same sample. This error due to the interaction volume interference was quantified using Monte Carlo simulations. It was found that an accelerating voltage of 22 KeV and diffusion distance less than 5 microns resulted in at least 50% error. Even at a smaller accelerating voltage of 6 KeV, the percent error in calculation of the interdiffusion coefficient for a diffusion distance of 0.5 microns is expected to be 15-20%. An approximate diffusion distance and apparent interdiffusion coefficient for ultrasonically consolidated Al-Cu were 0.503 microns and 0.013 um(2) /s, respectively. In this study, a methodology is presented that allows one to estimate the error in the calculation of an interdiffusion coefficient from the accelerating voltage used and the diffusion distance measured by the SEM XEDS at that accelerating voltage.

Decrease in CD8+ lymphocyte number and altered cytokine profile in human prostate cancer.

The tumor microenvironment is comprised of multiple cell types arranged in a three-dimensional structure. Interactions amongst the various cell components play an important role in neoplasia, including the inflammatory reaction that occurs as part of the host response. In this study, the regional lymphocyte subpopulations and cytokine profiles associated with prostate cancer were examined using a quantitative imaging approach and expression microarray analysis. Lymphocytes were measured in four different epithelial phenotypes in prostate cancer specimens: carcinoma; prostatic intraepithelial neoplasia (PIN); benign prostate hyperplasia (BPH); and normal epithelium. The data indicate that CD8 positive, cytotoxic T lymphocytes are significantly decreased in regions adjacent to hyperplasia and carcinoma as compared to normal epithelium and PIN. In contrast the relative number of CD4 positive and CD20 positive lymphocytes did not change markedly. Parallel mRNA expression array analysis of the normal and tumor microenvironments identified a distinct cytokine profile in cancer, with 24 dysregulated genes in tumor epithelium and nine altered in tumor-associated stroma. Overall, these data indicate that the spatial distribution of CD8 positive, cytotoxic T lymphocytes is dysregulated in human prostate glands that contain cancer, and cytokine profiles are altered at the mRNA level.

Influence of hypoxia induced by minimally invasive prostatectomy on gene expression: implications for biomarker analysis.

Handling and processing of clinical specimens during and after surgical resection may significantly skew the molecular data obtained from analysis of those samples. Minimally invasive prostatectomy was used as a model to specifically study effects of surgical ischemia on gene expression in human clinical samples. Normal prostatic urethra cup biopsies were procured from 12 patients at three time points during laparoscopic radical prostatectomy. Homogeneous cells (stroma and epithelium) were microdissected. Transcript analysis of 3 oxygen-dependent, 3 oxygen-independent, and 3 control class genes was performed using quantitative RT-PCR. Data were analyzed by relative quantitation and two-sided t-test. Patient demographic and time covariates were fit by a linear mixed model. VEGF, an oxygen-dependent gene, showed significant expression alterations across three time points in epithelium (p=0.008), but not in stroma (p=0.66). Expression levels of VHL, STAT5B, and CYPA showed significant changes at the p<0.05 level in the stroma only. Effects of age, PSA, prostate size, Gleason score, surgery type, total surgery time, total ischemia time, and estimated blood loss on VEGF expression over time were not significant at the p<0.01 level. Therefore, surgical manipulation and tissue processing methods need to be taken into account when assessing prostatic biomarkers; however, resection does not dramatically alter mRNA profiles in prostate specimens.

High throughput screening of normal and neoplastic tissue samples.

The capacity to rapidly and efficiently elucidate a reliable set of disease specific biomarkers is paramount to enable a future of personalized medicine. High throughput screening methods applied to human clinical samples for the discovery of diagnostic, prognostic, and therapeutic targets address this need. Although the ability to analyze either thousands of markers from one sample or one marker from thousands of samples is the current state of high throughput screening, it would be ideal to have the ability to analyze thousands of markers from thousands of samples to expedite the early discovery phase of biomarkers and their validation. This review summarizes the current state of high throughput screening of tissue specimens and discusses its applications. In addition, the rationale, difficulties, strategies, and development of new technologies to address the need for improved high throughput capabilities are discussed.

The chemokine CXCL16 and its receptor, CXCR6, as markers and promoters of inflammation-associated cancers.

Clinical observations and mouse models have suggested that inflammation can be pro-tumorigenic. Since chemokines are critical in leukocyte trafficking, we hypothesized that chemokines play essential roles in inflammation-associated cancers. Screening for 37 chemokines in prostate cancer cell lines and xenografts revealed CXCL16, the ligand for the receptor CXCR6, as the most consistently expressed chemokine. Immunohistochemistry and/or immunofluorescence and confocal imaging of 121 human prostate specimens showed that CXCL16 and CXCR6 were co-expressed, both on prostate cancer cells and adjacent T cells. Expression levels of CXCL16 and CXCR6 on cancer cells correlated with poor prognostic features including high-stage and high-grade, and expression also correlated with post-inflammatory changes in the cancer stroma as revealed by loss of alpha-smooth muscle actin. Moreover, CXCL16 enhanced the growth of CXCR6-expressing cancer and primary CD4 T cells. We studied expression of CXCL16 in an additional 461 specimens covering 12 tumor types, and found that CXCL16 was expressed in multiple human cancers associated with inflammation. Our study is the first to describe the expression of CXCL16/CXCR6 on both cancer cells and adjacent T cells in humans, and to demonstrate correlations between CXCL16 and CXCR6 vs. poor both prognostic features and reactive changes in cancer stoma. Taken together, our data suggest that CXCL16 and CXCR6 may mark cancers arising in an inflammatory milieu and mediate pro-tumorigenic effects of inflammation through direct effects on cancer cell growth and by inducing the migration and proliferation of tumor-associated leukocytes.

Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a valuable tool for measuring gene expression in biological samples. However, unique challenges are encountered when studies are performed on cells microdissected from tissues derived from animal models or the clinic, including specimen-related issues, variability of RNA template quality and quantity, and normalization. qRT-PCR using small amounts of mRNA derived from dissected cell populations requires adaptation of standard methods to allow meaningful comparisons across sample sets. The protocol described here presents the rationale, technical steps, normalization strategy and data analysis necessary to generate reliable gene expression measurements of transcripts from dissected samples. The entire protocol from tissue microdissection through qRT-PCR analysis requires approximately 16 h.

Tissue microdissection.

Procurement of pure populations of cells from heterogeneous histological sections can be accomplished utilizing tissue microdissection. At present, a variety of different manual and laser-based dissection tools are available and each method has particular strengths and weaknesses. The types of biomolecular analyses that can be performed on microdissected cells depend not only on the method of cell procurement, but also on the effects of upstream tissue handling and processing. Tissue preparation protocols include two major approaches; snap-freezing, or, fixation and embedding. Snap-freezing generally provides the best quality tissue for subsequent study, including proteomic analyses such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Tissue fixatives include either precipitating reagents or biomolecular cross-linkers. The fixed samples are then further processed and embedded in a wax medium. In general, the biomolecules recovered from fixed and embedded tissue specimens are lower in both quantity and quality than those from snap-frozen specimens, although they are useful for certain types of analyses. The protocols provided here for tissue handling and processing, preparation of tissue sections, and microdissection are derived from our experience at the Pathogenetics Unit of the National Cancer Institute.

Tumor immunobiological differences in prostate cancer between African-American and European-American men.

The incidence and mortality rates of prostate cancer are significantly higher in African-American men when compared with European-American men. We tested the hypothesis that differences in tumor biology contribute to this survival health disparity. Using microarray technology, we obtained gene expression profiles of primary prostate tumors resected from 33 African-American and 36 European-American patients. These tumors were matched on clinical variables. We also evaluated 18 nontumor prostate tissues from seven African-American and 11 European-American patients. The resulting datasets were analyzed for expression differences on the gene and pathway level comparing African-American with European-American patients. Our analysis revealed a significant number of genes, e.g., 162 transcripts at a false-discovery rate of

Assessment of normalization strategies for quantitative RT-PCR using microdissected tissue samples.

Gene expression measurement techniques such as quantitative reverse transcriptase (qRT)-PCR require a normalization strategy to allow meaningful comparisons across biological samples. Typically, this is accomplished through the use of an endogenous housekeeping gene that is presumed to show stable expression levels in the samples under study. There is concern regarding how precisely specific genes can be measured in limited amounts of mRNA such as those from microdissected (MD) tissues. To address this issue, we evaluated three different approaches for qRT-PCR normalization of dissected samples; cell count during microdissection, total RNA measurement, and endogenous control genes. The data indicate that both cell count and total RNA are useful in calibrating input amounts at the outset of a study, but do not provide enough precision to serve as normalization standards. However, endogenous control genes can accurately determine the relative abundance of a target gene relative to the entire cellular transcriptome. Taken together, these results suggest that precise gene expression measurements can be made from MD samples if the appropriate normalization strategy is employed.

Layered peptide array for multiplex immunohistochemistry.

Layered peptide array is a new methodology for multiplex molecular measurements from two-dimensional life science platforms. The technology can be used in several different configurations depending on the needs of the investigator. Described here is an indirect layered peptide array (iLPA) that is capable of measuring proteins on a solid surface, such as target antigens on a tissue section. A prototype iLPA system was developed and subsequently examined for reproducibility and specificity and then compared with standard immunohistochemistry. Semiquantitative, multiplex proteomic analysis of histological sections was achieved with up to 20 membranes. The experimental variability was 18%. Overall, the data suggest that iLPA technology will be a relatively simple and inexpensive method for molecular measurements from tissue sections.

Continuous silica coatings on glass fibers via bioinspired approaches.

Simple methods for producing continuous inorganic coatings on fibers have application in multiple technologies. The application of bioinspired strategies for the formation of particulate inorganic materials has been widely investigated and provides routes to inorganic materials under environmentally benign conditions. In this work, we describe the formation of stable and continuous inorganic coatings on glass fibers via the polymerization of silica in the presence of biopolymers. The formation of both organic and inorganic coatings was investigated via X-ray photoelectron spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, scanning electron microscopy, and energy-dispersive X-ray analysis. The simple route to silica coatings presented herein could be interesting for the development of functional hybrid fibrous materials suitable for catalytic and sensor applications, given the homogeneous nature of the silica films and the benign conditions employed for film formation.

Layered peptide arrays: a diverse technique for antibody screening of clinical samples.

The layered peptide array (LPA) is a recently developed technique designed to measure antibody levels in a multiplex, high-throughput manner. LPAs can assess antibody presence either in fluid samples or from tissues while maintaining the two-dimensional orientation of the life science platform. In this manuscript, we evaluated and assessed the performance of the LPA platform, focusing on throughput capability, sensitivity, and specificity of the assay in several different systems.

Prostate cancer epigenetics: a review on gene regulation.

Prostate cancer is the most common cancer in men in western countries, and its incidence is increasing steadily worldwide. Molecular changes including both genetic and epigenetic events underlying the development and progression of this disease are still not well understood. Epigenetic events are involved in gene regulation and occur through different mechanisms such as DNA methylation and histone modifications. Both DNA methylation and histone modifications affect gene regulation and play important roles either independently or by interaction in tumor initiation and progression. This review will discuss the genes associated with epigenetic alterations in prostate cancer progression: their regulation and importance as possible markers for the disease.