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To establish and preliminarily validate an individualized reference of carotid stiffness quantified by ultrafast pulse wave velocity (ufPWV), our study included 225 healthy individuals in the modeling cohort and 628 individuals in the validation cohort. All participants underwent assessment of carotid intima-media thickness (cIMT), pulse wave velocity-beginning of systole and pulse wave velocity-end of systole (PWV-ES). A threshold equation of estimated PWV-ES was obtained by multiple linear regression analysis in the modeling cohort as follows: estimated PWV-ES (m/s) = 0.080 × age (y) + 0.767 × low-density lipoprotein (mmol/L) + 0.040 × systolic blood pressure (mm Hg) + 0.372 × sex (male = 1, female = 0) - 2.803. With this equation, the validation cohort was divided into the low PWV-ES (actual PWV-ES ≤ estimated PWV-ES) and high PWV-ES (actual PWV-ESestimated PWV-ES) groups. A clear boundary was found to be present between the low PWV-ES and high PWV-ES groups in the validation cohort. Participants with increasing PWV-ES increased with age gradually. We further subdivided participants into cIMT subgroups using a cutoff thickness of 0.050 cm. Diagnostic performance analysis revealed that the sensitivity and specificity of the threshold equation were 78.9% and 73.9%, respectively. We established and validated a novel individualized reference equation for estimated PWV-ES, which can likely expand the application of prospective ufPWV assessment.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that delays color change during fruit ripening. Whether H2S affects anthocyanin biosynthesis in red-skinned pears (Pyrus L.) remains unclear. Here, we found that H2S substantially inhibits anthocyanin accumulation in red-skinned pears and the expression of several genes encoding transcription factors is affected in response to H2S signaling. For example, PyMYB10 and PyMYB73 were down-regulated, whereas PyMYB114 and PyMYB6 were up-regulated. Bioinformatics analysis showed that PyMYB73 and PyMYB6, each containing an EAR motif, may negatively regulate anthocyanin accumulation. Transient expression analysis showed that PyMYB73 substantially promotes anthocyanin biosynthesis by co-transforming with PyMYB10/PyMYB114 + PybHLH3; however, PyMYB6 inhibited anthocyanin biosynthesis in strawberry (Fragaria vesca) receptacles and pear fruits, and PyMYB73 interacted with PyMYB10 and PyMYB6 but not PyMYB114 or PybHLH3. Further investigation showed that Cys194 and Cys218 of PyMYB10 were modified by persulfidation and that PyMYB10Cys218Ala substantially increased anthocyanin accumulation by a transient transformation system. Co-transformation of PyMYB10Cys218Ala + PyMYB73/PyMYB6 also promoted anthocyanin accumulation in pear fruits. Yeast two-hybrid assays showed that the mutation of PyMYB10 did not affect the interaction between PyMYB10 and PyMYB73, but it inhibited interaction with PyMYB6. Moreover, H2S weakened the interaction between PyMYB10 and PyMYB73 but enhanced the interaction with PyMYB6. Thus, we provided a model in which PyMYB10 undergoes persulfidation at Cys218, enhancing the interaction with PyMYB6 and reducing the interaction with PyMYB73. These subsequently results in lower expression of the anthocyanin biosynthesis-related genes Pyrus dihydroflavonol 4-reductase (PyDFR), Pyrus anthocyanidin synthase (PyANS), Pyrus UDP-glucose: flavonoid 3-glucosyl transferase (PyUFGT) and Pyrus glutathione S-transferase (PyGST), thereby inhibiting anthocyanin accumulation in red-skinned pears. Our findings provided a molecular mechanism for H2S-mediated anthocyanin biosynthesis in red-skinned pears.

Red-skinned pears (Pyrus L.) are preferred to consumers for their attractive color and abundant anthocyanins. Pyrus ETHYLENE RESPONSE FACTOR 3 (PyERF3) positively regulates anthocyanin biosynthesis through interacting with Pyrus myeloblastosis family 114 (PyMYB114) and Pyrus basic helix-loop-helix 3 (PybHLH3) in red-skinned pears. However, the role of APETALA2/ethylene response factors (AP2/ERFs), which negatively regulate anthocyanin biosynthesis, remains unclear in red-skinned pears. Here, we validated that 2 AP2/ERFs, PyERF4.1 and PyERF4.2, screened from the transcriptome data of ‘Starkrimson’ pear (Pyrus communis L.) and its green mutant, inhibit anthocyanin biosynthesis in transgenic pear calli, as well as in overexpression and gene-edited tomato (Solanum lycopersicum) fruits. Meanwhile, the co-transformation of PyERF4.1/PyERF4.2 with PyERF3–PyMYB114–PybHLH3 inhibited anthocyanin biosynthesis in pear fruits and strawberry (Fragaria vesca) receptacles. Further assays showed that PyMYB114 activated the transcription of PyERF4.1/PyERF4.2; PyERF4.1/PyERF4.2 then interacted with PyERF3 to affect the stability of the PyERF3–PyMYB114–PybHLH3 complex, thereby inhibiting the transcription of the anthocyanin biosynthesis gene Pyrus anthocyanidin synthase (PyANS). Furthermore, deletion of the ERF-associated-amphiphilic repression (EAR) motif eliminated the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis, and a mutation of the PyERF4.2-EAR motif (LxLxM to LxLxL) strengthened the inhibitory effect, demonstrating that the EAR motif is indispensable for the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis in pears. Our study has shed light on a feedback regulatory loop mechanism that balances the excessive accumulation of anthocyanins in red-skinned pears, providing insights into the regulatory mechanism of anthocyanin biosynthesis and the regulatory network of coloration in red-skinned pears.

This work explores the relationship between the microstructure and mechanical properties of TC4 titanium alloy using molecular dynamics simulations. The stress-strain curves are calculated from tensile and shear tests. For the TC4 model with initial HCP structure, tensile strength along the [0001] crystal direction is larger than along the [01-10] and [2-1-10] directions. The tensile strength also increases with larger strain rate. During tensile deformation, phase transformation from HCP to BCC structure is observed, which is caused by dislocations along the close-packed direction [-1-120]. A band of amorphous structure is formed in the middle of shearing, which causes the abrupt drop in shear stress. By setting a temperature control, the heating, holding and cooling stages of heat treatment are simulated to mimic the impact of annealing, solid solution and aging. The results confirm a general trend of improved tensile and shear strength with annealing and additional solid solution and aging. With higher solid solution temperature, the amount of α phase also increases, giving rise to higher tensile and shear strength simultaneously. At the same 811 K for aging, shear strength also increases with solid solution temperature. But at the same 1227 K for the solid solution, shear strength decreases with aging temperature, in agreement with experimental results.

PurposeThis study aims to investigate the extent to which overeducation imposes wage effects on university graduates, taking into account the individual heterogeneity due to skills and innate ability.Design/methodology/approachUsing Graduates Occupation and Mobility Survey (GOMS) 2019 and Korea Dictionary of Occupations (KDOT) 2019, the overeducated and adequately educated graduates are differentiated by the job analysis (JA) measure. To unveil the masked results, the unconditional quantile regression (UQR) accompanying skills and field of study mismatches is adopted to explore the wage effects of overeducation across the overall wage distribution.FindingsEmpirical evidence shows that the incidence of overeducation is high; however, overeducated graduates only suffer a 6.5% wage loss relative to their adequately matched peers. The findings indicate that regardless of being derived from either overskilled or field of study mismatch, genuine overeducation impose a higher wage penalty at all percentiles relative to the apparent overeducation. Meanwhile, high-ability men suffer lower-wage penalties than their low-ability peers, whereas the inverted “U” pattern is exhibited for women. The theoretical hypotheses differ depending on the estimated results by gender.Research limitations/implicationsEach measure of educational mismatch has been criticized for its insurmountable shortcoming. The recent graduates are likely to overstate the job requires of skills.Originality/valueThis paper contributes to the insufficient evidence on the multiple aspects of wage effects of overeducation by providing new and rigorous examinations and by focusing on the country experiencing rapid economic growth, industrial upgrading and educational expansion.

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.

Atherosclerosis is a progressive vascular disease triggered by interplay between abnormal shear stress and endothelial lipid retention. A combination of these and, potentially, other factors leads to a chronic inflammatory response in the vessel wall, which is thought to be responsible for disease progression characterized by a buildup of atherosclerotic plaques. Yet molecular events responsible for maintenance of plaque inflammation and plaque growth have not been fully defined. Here we show that endothelial transforming growh factor β (TGF-β) signalling is one of the primary drivers of atherosclerosis-associated vascular inflammation. Inhibition of endothelial TGF-β signalling in hyperlipidemic mice reduces vessel wall inflammation and vascular permeability and leads to arrest of disease progression and regression of established lesions. These proinflammatory effects of endothelial TGF-β signalling are in stark contrast with its effects in other cell types and identify it as an important driver of atherosclerotic plaque growth and show the potential of cell-type-specific therapeutic intervention aimed at control of this disease.

As the properties of wire + arc additively manufactured Al-6.3Cu alloy cannot meet the applying requirements, a double-wire + arc additive manufacturing system was built to add magnesium into Al-Cu deposits for higher mechanical properties. Two commercial binary wires aluminum-copper ER2319 and aluminum-magnesium ER5087 were chosen as the filler metal to build Al-Cu-Mg components with different compositions by adjusting the wire feed speed. The microstructure and morphology of thin wall samples were characterized by optical micrographs (OM), X-ray diffraction (XRD) and scanning electron microscopy (SEM). The Vickers hardness and tensile properties were investigated. The microstructure of Al-Cu-Mg deposits was mainly composed of coarse columnar grains and fine equiaxed grains with non-uniformly distributing characteristics. With higher Cu but lower Mg content, the strengthen phase turned to Al2Cu + Al2CuMg from Al2CuMg, and the micro hardness presented an increasing trend. The isotropic characteristics of ultimate tensile strength (UTS), yield strength (YS) and elongation were revealed in these samples. The UTS was about 280 ± 5 MPa both in horizontal and vertical directions for all samples. The YS showed an increasing trend from 156 MPa to 187 MPa with the same content trend, while elongation decreased from 8.2% to 6%. The fractographs exhibited typical brittle fracture characteristics.

The balance between low carbon development and rapid economic growth is a ‘Gordian knot’ for most countries. This study aims to explore the practicable route of lowering carbon intensity with the expansion of the Information and Communication Technologies (ICT) industry in China. Using the provincial panel data from 2000 to 2017, the paper employs five indicators to empirically investigate the determinant mechanism of carbon intensity based on the refined Stochastic Impact by Regression on Population, Affluence, and Technology (STIRPAT) and spatial econometric models. The findings demonstrate that the ICT industry is the main force in adjusting carbon intensity. Despite constraint by the rebound effect, the net effect of the ICT industry shows a significant positive impact on lowering carbon intensity under the consideration of spatial dependence. Furthermore, compared with developed regions, the contribution of ICT on regional inequality of carbon intensity shows a higher share in developing regions. By applying the Oaxaca-Ransom counterfactual decomposition, the results reveal that there are substantial regional gaps between multi-comparable groups. The composite effect of informatization, industrialization, and urbanization accounts for the major share of the regional gap. Thus, policymakers should pay more attention to enhancing the ICT infrastructure in developing regions, boosting the ICT-related technology investment in specific sectors, coordinating regional cooperation, and balancing the migration of skilled workers.

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are of importance to these processes1. While much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism2,3, little is understood about the role of fibroblast growth factors (FGFs) in this context4. Here we identify FGF receptor (FGFR) signaling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signaling inputs results in decreased glycolysis leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/r3 double mutant mice while HK2 overexpression partially rescues the defects caused by suppression of FGF signaling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development.

Innovative enabling technologies are key drivers of human augmentation. In this paper, we explore a new, conductive, and configurable material made from Polydimethylsiloxane (PDMS) that is capillary doped with silver particles (Ag) using an immiscible secondary fluid to build ultra-stretchable, soft electronics. Bonding silver particles directly with PDMS enables inherently stretchable Ag-PDMS circuits. Compared to previous work, the reduced silver consumption creates significant advantages, e.g., better stretchability and lower costs. The secondary fluid ensures self-assembling conductivity networks. Sensors are 3D-printed ultra-thin (200%. Therefore, printed circuits can attach tightly onto the body. Due to biocompatibility, devices can be implanted (e.g., open wounds treatment). We present a proof of concept on-skin interface that uses the new material to provide six distinct input gestures. Our quantitative evaluation with ten participants shows that we can successfully classify the gestures with a low spatial-resolution circuit. With few training data and a gradient boosting classifier, we yield 83% overall accuracy. Our qualitative material study with twelve participants shows that usability and comfort are well perceived; however, the smooth but easy to adapt surface does not feel tissue-equivalent. For future work, the new material will likely serve to build robust and skin-like electronics.

Materials with advanced conductive properties are in high demand to fulfill the requirements of energy transport in many fields. Here, we present a simple, efficient yet low cost method to produce highly conductive polymer based composites at low filler concentration of 10 vol%. A particle-wetting fluid as a secondary fluid is introduced to bridge plate-shape silver particles within an epoxy base. The electrical and thermal conductivity dependence on network structure in ternary silver adhesive presenting strong capillary attraction was first reported. Both electrical and thermal conductivities are greatly enhanced by introducing the secondary fluid at a low particle volume fraction of 10%. A non-monotonic dependence of the conductivities on secondary fluid content was observed. The maximum electrical and thermal conductivities correspond to the formation of full pendular network and funicular network, respectively. Gradually increasing the secondary fluid content leads to a microstructure evolution from highly dispersed particles, weak gel network, full pendular network, funicular network and ultimately to compact capillary aggregates network. Such structural transition appears to result from a pendular bridge formation and bridges coalescence. Face-face configuration of plate-shaped silver particles was observed in the pendular state. Taking account of the highly non-spherical shape of silver flake, a bridging gelation model is proposed to explain the underlying mechanism of microstructure transition.

Stretchable conductors are required for next-generation soft electronics. Achieving both high electrical conductivity and high stretchability in conductors composed of elastomers and conductive fillers, however, is challenging. Here, a generic, versatile strategy is reported for producing ultrastretchable conductors exhibiting both superior electrical conductivity (>103 S/cm) and stretchability (>1600%). This is achieved by adding small amounts of immiscible secondary fluid into silver (Ag)-filled inks. Capillary forces in these ternary systems induce the self-assembly of conductive particle networks at a low percolation threshold (6-7 vol %), cutting silver consumption by more than 2/3 compared to conventional conductive elastomers. Ag-filled polydimethylsiloxane exhibits superior cyclic durability sustaining 100% tensile strain for 1000 cycles with only a minor loss of conductivity. Ag-filled thermoplastic polyurethane displays unprecedented reversibility with nonretarded switching from conductive to nonconductive states during repeated stretching up to 200% strain. Patterned strain sensors and conductive wirings were 3D-printed to demonstrate the technical feasibility.

Signet-ring cell carcinoma (SRCC) has specific epidemiology and oncogenesis in gastric cancer, however, with no systematical investigation for prognostic genomic features. Here we report a systematic investigation conducted in 1868 Chinese gastric cancer patients indicating that signet-ring cells content was related to multiple clinical characteristics and treatment outcomes. We thus perform whole-genome sequencing on 32 pairs of SRC samples, and identify frequent CLDN18-ARHGAP26/6 fusion (25%). With 797 additional patients for validation, prevalence of CLDN18-ARHGAP26/6 fusion is noticed to be associated with signet-ring cell content, age at diagnosis, female/male ratio, and TNM stage. Importantly, patients with CLDN18-ARHGAP26/6 fusion have worse survival outcomes, and get no benefit from oxaliplatin/fluoropyrimidines-based chemotherapy, which is consistent with the fact of chemo-drug resistance acquired in CLDN18-ARHGAP26 introduced cell lines. Overall, this study provides insights into the clinical and genomic features of SRCC, and highlights the importance of frequent CLDN18-ARHGAP26/6 fusions in chemotherapy response for SRCC., Signet-ring cell carcinoma (SRCC) is a unique type of gastric cancer with no prognostic features. Here, the authors report a CLDN18-ARHGAP26/6 gene fusion in patients with a high signet-ring cell content, poor survival outcomes, and who experience no benefit from platinum/fluoropyrimidines-based chemotherapy.

The widespread application of next generation sequencing (NGS) in clinical settings has enabled testing, diagnosis, treatment and prevention of genetic diseases. However, many issues have arisen in the meanwhile. One of the most pressing issues is the lack of standards for reporting genetic test results across different service providers. The First Forum on Standards and Specifications for Clinical Genetic Testing was held to address the issue in Shenzhen, China, on October 28, 2017. Participants, including geneticists, clinicians, and representatives of genetic testing service providers, discussed problems of clinical genetic testing services across in China and shared opinions on principles, challenges, and standards for reporting clinical genetic test results. Here we summarize expert opinions presented at the seminar and report the consensus, which will serve as a basis for the development of standards and guidelines for reporting of clinical genetic testing results, in order to promote the standardization and regulation of genetic testing services in China.

Hepatocellular carcinoma (HCC) is the fifth most common type of cancers worldwide. However, current therapeutic approaches for this epidemic disease are limited, and its 5-year survival rate hasn't been improved in the past decades. Patient-derived xenograft (PDX) tumor models have become an excellent in vivo system for understanding of disease biology and drug discovery. In order to identify new therapeutic targets for HCC, whole-exome sequencing (WES) was performed on more than 60 HCC PDX models. Among them, four models exhibited protein-altering mutations in JAK1 (Janus Kinase 1) gene. To explore the transforming capability, these mutations were then introduced into HEK293FT and Ba/F3 cells. The results demonstrated that JAK1S703I mutation was able to activate JAK-STAT (Signal Transducer and Activator of Transcription) signaling pathway and drive cell proliferation in the absence of cytokine stimulation in vitro. Furthermore, the sensitivity to the treatment of a JAK1/2 inhibitor, ruxolitinib, was observed in JAK1S703I mutant PDX model, but not in other non-activating mutant or wild type models. Pharmacodynamic analysis showed that phosphorylation of STAT3 in the Ruxolitinib-treated tumor tissues was significantly suppressed. Collectively, our results suggested that JAK1S703I is an activating mutation for JAK-STAT signaling pathway in vitro and in vivo, and JAK-STAT pathway might represent a new therapeutic approach for HCC treatment. Monotherapy using a more potent and specific JAK1 inhibitor and combinatory therapy should be further explored in JAK1 mutant PDX models.

Acute lymphoblastic leukemia (ALL) is a leading cause of cancer-associated death in children. To study the mechanisms of drug resistance in ALL, we performed whole-genome sequencing of diagnosis-relapse-germline trios from 103 Chinese patients and ultra-deep sequencing of 208 serial bone marrow samples from 17 of them. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2), which were predominantly involved in response to thiopurines, glucocorticoids, methotrexate, and other drugs. Four lines of evidence indicate that these resistance mutations frequently developed during treatment, rather than pre-existing at diagnosis. First, two novel, relapse-specific mutational signatures (novel signatures 1 and 2), most likely caused by chemotherapeutic regimens, were detected in 15% and 14% of relapsed cases, respectively. Drug resistance mutations frequently appeared at novel signature-associated trinucleotide contexts, indicating that chemotherapy may directly cause drug resistance mutations in ALL. The signatures were validated in NCI TARGET relapsed ALL samples, 2% and 23% of which harbored novel signatures 1 and 2, respectively. The varying signature prevalence between cohorts may reflect treatment differences. The novel signatures were not detected in >2,000 adult cancers from the PCAWG study. Novel signature 1 induced C>G transversions, particularly at GCC and TCT trinucleotides, and showed transcription-strand bias indicating guanine adducts. Novel signature 2 favored C>T and C>G mutations at CCG, and correlated with relapse-specific dinucleotide variants and structural variants, indicating an agent causing multiple mutation types. The drugs inducing these novel signatures are being explored in vitro. Second, mathematical modeling using growth curves of drug-resistant ALL indicated that drug resistance mutations occur, in some cases, long after diagnosis, during active treatment. Third, some patients acquired multiple drug resistance mutations sequentially through successive relapses, a finding inconsistent with their pre-existence at diagnosis. Indeed, 20% of relapses had multiple drug resistance mutations targeting different drug classes. Fourth, most relapsed ALLs derived from a subclone detected at diagnosis, which then evolved additional mutations, including drug resistance mutations, not detectable at diagnosis using 2000X targeted sequencing. Drug resistance mutations were often subclonal at relapse, suggesting later appearance. Together these data indicate that fully drug-resistant clones may not necessarily pre-exist at diagnosis in ALL, but may be acquired later during treatment. Thus, early intensive or targeted treatment strategies in slow responders may forestall the subsequent development of drug resistance mutations. Citation Format: Benshang Li, Samuel W. Brady, Xiaotu Ma, Shuhong Shen, Yingchi Zhang, Yongjin Li, Yu Liu, Ningling Wang, Diane Flasch, Matthew Myers, Heather Mulder, Lixia Ding, Yanling Lu, Liqing Tian, Kohei Hagiwara, Ke Xu, Edgar Sioson, Tianyi Wang, Liu Yang, Jie Zhao, Hui Zhang, Ying Shao, Hongye Sun, Lele Sun, Jiaoyang Cai, Ting-Nien Lin, Lijuan Du, Fan Yang, Michael Rusch, Michael Edmonson, John Easton, Xiaofan Zhu, Jingliao Zhang, Cheng Cheng, Benjamin Raphael, Jingyan Tang, James Downing, Bin-Bing Zhou, Ching-Hon Pui, Jun Yang, Jinghui Zhang. Acquisition of drug resistance mutations during chemotherapy treatment in pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2872.

Next-generation sequencing analysis of formalin-fixed, paraffin-embedded (FFPE) samples has the potential to lead to major advances in cancer treatment and prevention. However, whole-genome sequencing (WGS) of these samples has remained challenging due to the difficult process of isolating high-quality DNA and distinguishing true variant calls from artifacts. As evidence of these challenges, only four WGS studies of FFPE samples have been published to date. Previous work has attempted to overcome these challenges by using whole-exome sequencing or smaller exon panels at high coverage depths with publications reporting success from less than half to most samples. Thus, there has not been widespread adoption of using FFPE samples for next-generation sequencing. We developed a novel DNA extraction and library prep protocol for whole-genome sequencing of FFPE tissue samples. This method produces larger DNA fragments that can be successfully used for WGS. We previously presented results of a large esophageal cancer cohort where we sequenced over 1,000 samples in tumor-normal pairs with data equivalent to that generated from fresh-frozen tissues. We recently expanded the tumor types, ages, and sources and achieved similar results. We have assayed FFPE tissues derived from breast cancer, colorectal cancer, non-small cell lung cancer, prostate cancer, and melanoma. These samples were sourced from several different labs, which have used variations on the standard fixation protocol. Some of these FFPE samples were over 15 years old. Variant calling provides excellent results without excessive C>T mutations. The coverage uniformity was very good, and CNV calls were made with results comparable to published TCGA data. These samples have been analyzed for MSI and TMB, and the results are comparable to those using fresh tissues. Our method makes new types of clinical studies possible utilizing FFPE samples that have been generated as part of standard hospital procedures. This method also facilitates performing studies comparing primary and recurrent cancers easier to establish and run as recurrent cancer cases can be enrolled and stored FFPE primary samples used to establish a cancer baseline. Citation Format: Shannon T. Bailey, James Lund, Muhammad Ekram, Wanfeng Yu, Richard T. Williams, Hongye Sun, Jeffrey R. Gulcher. High-quality whole-genome sequencing and analysis of FFPE samples from multiplecancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1706.

e13016 Background: Whole-genome sequencing (WGS) of formalin-fixed, paraffin-embedded (FFPE) samples could enable novel insights from archival sample collections, yet robust FFPE WGS is challenged by fragmented DNA, uneven genomic coverage & sequencing artifacts attributed to FFPE fixation. We report our proprietary extraction & library preparation methodology (SeqPlus) with high quality, uniform WGS sequencing performance comparable to that from fresh-frozen samples. Methods: We analyzed 20 paired esophageal carcinoma (EC) samples i.e., primary tumors & matched germline samples to assess SeqPlus performance on 10-15-year-old FFPE tissues, measure variant concordance between WGS and a high-depth sequencing panel (269 genes, 400x coverage) & identify novel genomic features. Results: At a targeted 70x WGS tumor sequencing depth, 93% of the genome was covered by ³ 20 reads, 99% of bases had 10x coverage & average duplicate reads were 31%. We noted similar transition/transversion ratios & mutational spectra as from fresh-frozen EC specimens, suggesting that extraction & library preparation contributes to prior FFPE artifacts. Concordance of tumor-specific SNVs & indels derived from WGS & targeted panel was high at 86%. All 76 targeted panel-detected variants above the WGS limit of detection (mutant allele frequency [MAF] > 10%) were detected by WGS, 2 variants (2 tumors) were detected only by WGS, and 12 variants at MAF ≤ 6% (9 tumors) were only detected by the targeted panel. Tumor WGS yielded SNV, indels & CNV findings beyond variants detected by targeted sequencing. WGS enabled detection of 10.4 putative cancer variants per tumor compared to 12 variants per patient from frozen specimens and a median of 7 (up to 16) cancer-associated variants in genes outside the targeted panel. WGS copy number analysis revealed CCND1, EGFR, TP63, and SOX2amplification, CDKN2A/B deletion and additional unrecognized genomic aberrations. Conclusions: Our study reinforces the utility of high-quality, uniform WGS sequencing of archival FFPE cancer samples with SeqPlus and unlocks the potential for massive-scale retrospective genomic analysis of archived pathology samples with associated clinical & outcomes data.

We have uncovered some unusual thermal interface properties of a three-dimensional, flexible and interconnected graphene foam (GF). The thermal interfacial resistance of GF at Si–Al interface is as low as 0.04 cm2K W−1, which is one order of magnitude lower than conventional thermal grease and thermal paste-based thermal interfacial material (TIM). The thermal contact resistance was found to dominate the overall interfacial resistance of GF-based TIM, in as much as the bulk thermal conductivity of GF is rather high. The contact pressure-dependent thermal interfacial resistance of GF exhibits an asymptotic behavior, which converges into a plateau value at an ultralow contact pressure (∼0.1 MPa). Significantly, the GF-based TIM has shown a superior performance to vertically aligned carbon nanotubes currently held as the gold standard (at least ∼75% improvement in thermal interfacial resistance at Si–Al interface), thus providing a strong candidate for the next generation of high-performance carbon-based TIM.

An ionic liquid type imidazolium catalyst enhanced the conductivity of epoxy-anhydride resin based electrical conductive adhesives (ECAs) by more than two orders of magnitude compared with common imidazole compounds. Percolation growth dynamics study combining with epoxy curing kinetics results indicates that the enhancement of conductivity was attributed to the higher percolation efficiency within ionic liquid catalyzed ECAs. An “interphase layer” model was proposed to explain the percolation mechanism in the thermoset based conductive composite. The present work highlights the importance of cross-linking the polymer matrix with filler interconnections, and provides practical guidance on the control over percolation dynamics in conductive composites in order to achieve enhanced percolation conductivity.

Introduction Relapsed childhood acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death in children and has poor prognosis due to acquired drug resistance. However, the clonal evolution leading to drug resistance at ALL relapse is incompletely understood. Methods We performed whole-genome sequencing (WGS) of samples at diagnosis and relapse from 103 Chinese patients, most of whom were enrolled on the Shanghai Children's Medical Center (SCMC) ALL2005 frontline treatment protocol. We also performed ultra-deep sequencing at 5,000-50,000X coverage of 211 serial bone-marrow samples from 17 of these patients (3-23 per case) collected during ALL therapy. Fifteen ALL subtypes were identified based on copy number variation, structural re-arrangement and gene fusion by WGS and RNA-Seq analysis. Cases were selected based on sample availability with no bias on age, gender, cytogenetics, or immunophenotype compared to the characteristics of all relapsed cases at SCMC; however only one CRLF2-rearranged case was observed suggesting that CRLF2-rearrangement could be a rare event in Asian patients. Functional impact of novel mutations was assessed by ectopic expression in ALL cell lines. Results Relapse-specific somatic alterations were significantly enriched in 12 genes (NT5C2, NR3C1/2, PRPS1/2, TP53, CREBBP, MSH2/6, PMS2, WHSC1, and FPGS) predominantly involved in drug metabolism and response pathways. These somatic alterations were present in over 50% (56/103) of relapsed ALLs and were enriched in patients with early relapse (9-36 months from diagnosis) compared to very early (36 months) patients. NR3C1 mutations resulted in loss of glucocorticoid receptor transcriptional activity and severely impaired glucocorticoid response in vitro, while FPGS mutations led to reduced enzymatic activation of methotrexate. Genome-wide analysis identified 9 mutational signatures, including two novel ones exclusive to relapse. Novel signature 1 was characterized by C>G mutations and was present in 15% of the relapsed cases with an enrichment for hyperdiploid ALL, while novel signature 2, present in 14% of the relapsed cases, was characterized by C mutations (C>T in particular) followed by a G. These novel signatures gave rise to relapse-specific drug resistance mutations, including PRPS1, TP53, NT5C2, and KRAS mutations. The majority (59%) of the cases underwent a selective sweep as the relapse clone arose from a subclone detectable at diagnosis; however, very early relapse cases were less likely to experience a clonal sweep and had multiple lineages present at relapse. In patients tracked serially through multiple relapses, the clonal composition of drug resistance variants evolved substantially in response to chemotherapy. For example, in one patient, two independent NT5C2-mutant clones appeared at relapse, manifesting convergent evolution (Fig. 1a). One of the NT5C2-mutant clones expanded after subsequent treatment while the other diminished in prevalence. Sequential acquisition of multiple drug resistance mutations was also evident, with one patient sequentially acquiring KRAS, FBXW7, NR3C1 and FPGS mutations over a period of 5 years through multiple relapses (Fig. 1b). Indeed, 17% (18/103) of patients acquired multiple drug resistance mutations at relapse. The median time from detecting resistant clones to relapse was 41 days, suggesting that ultra-deep sequencing offers a means for early detection of genetic lesions that could serve as an indicator of relapse to enable earlier therapeutic intervention. Conclusions Very early relapses likely have different resistance mechanisms than early or late relapses, perhaps due to increased intrinsic resistance or the presence of multiple drug-resistant clones. At least a subset of relapse-specific drug resistance mutations may be acquired de novo, rather than pre-existing, as evidenced by resistance mutations bearing the novel relapse-specific mutational signatures which are likely to be therapy-induced. Relapsed ALL can acquire multiple drug resistance mutations targeting different drug classes. Frequent monitoring of disease, such as via cell-free DNA sequencing, may enable earlier detection of relapse and facilitate timely treatment to avoid selection for drug-resistant clones. Disclosures No relevant conflicts of interest to declare.

Next-generation sequencing analysis of formalin-fixed, paraffin-embedded (FFPE) samples has the potential to lead to major advances in cancer treatment and prevention. However, whole-genome sequencing of these samples has remained challenging due to the difficult process of isolating high-quality DNA and distinguishing true variant calls from artifacts. Previous studies have attempted to overcome these challenges by using whole-exome sequencing or smaller exon panels at high coverage depths to analyze FFPE samples. While these approaches have produced useful results, they have had varying degrees of success, tend have coverage biases due to probe capture issues, and are limited to only a fraction of the genome. Thus, there has not been widespread adoption of using FFPE samples for next-generation sequencing. In an attempt to improve on the process of next-generation sequencing of FFPE samples, we first developed an efficient DNA extraction method that produces high quantities of high-quality DNA. Whole-genome sequencing of DNA generated by this method demonstrated uniform high coverage that was comparable to DNA prepared from fresh frozen samples at the same sequencing depth. This result is in contrast with data generated from DNA isolated by a traditional FFPE extraction method, which had lesser sequencing coverage even with twice the sequencing depth. Further analysis of the data generated using our method demonstrated similar numbers of mutations called and mutational signatures without artifact contamination. We extended this analysis to a large cancer cohort consisting of over 1,000 FFPE samples in tumor normal pairs that were up to 6 years old. Whole-genome sequencing of these samples demonstrate that there was an average of 98% of the genome covered at 20X or greater using our method with no drop off for the older samples. Mutational analyses of these samples demonstrate similar findings as that previously published using TCGA datasets. In summary, our study of greater than 1,000 samples is the largest report of whole-genome sequencing of FFPE samples to date. Our results demonstrate the feasibility of using our method to assay tissues stored in archival tissue biobanks, analyze patient samples for clinical trial recruitment, or examining sections as part of normal clinical workflows, all of which hold the potential for greatly benefiting human health. Citation Format: Shannon T. Bailey, Jim Lund, Hao Wang, Weiren Cui, Chen Hao, Hongye Sun, Jeffrey R. Gulcher. High-quality whole-genome sequencing of FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 427.

e13500Background: Next-generation sequencing analysis of formalin-fixed, paraffin-embedded (FFPE) samples has the potential to lead to major advances in cancer treatment and prevention. However, wh...

// Qingyang Gu 1, * , Bin Zhang 1, * , Hongye Sun 2 , Qiang Xu 2 , Yexiong Tan 3 , Guan Wang 2 , Qin Luo 2 , Weiguo Xu 1 , Shuqun Yang 1 , Jian Li 1 , Jing Fu 3 , Lei Chen 3 , Shengxian Yuan 3 , Guibai Liang 1 , Qunsheng Ji 1 , Shu-Hui Chen 1 , Chi-Chung Chan 1 , Weiping Zhou 3 , Xiaowei Xu 4 , Hongyang Wang 3 , Douglas D. Fang 1, 5 1 Discovery Services, WuXi AppTec Co., Ltd., Waigaoqiao Free Trade Zone, Shanghai, 200131 China 2 Genome Center, WuXi AppTec Co., Ltd., Waigaoqiao Free Trade Zone, Shanghai, 200131 China 3 Eastern Hepatobiliary Surgery Hospital/Institute of Shanghai, Shanghai, 200131 China 4 Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA 19104, USA 5 Current address: Cancer Translational Research and In Vivo Pharmacology, China Novartis Institute for Biomedical Research, Zhangjiang Hi-Tech Park, Pudong, Shanghai, 200131 China * These authors have contributed equally to this work Correspondence to: Douglas D. Fang, e-mail: douglasfang@yahoo.com Hongyang Wang, e-mail: hywangk@vip.sina.com Keywords: hepatocellular carcinoma (HCC), patient-derived xenograft (PDX) tumor model, copy number alterations (CNA), single nucleotide polymorphism (SNP), fibroblast growth factor receptor (FGFR) Received: January 25, 2015 Accepted: May 09, 2015 Published: May 21, 2015 ABSTRACT Lack of clinically relevant tumor models dramatically hampers development of effective therapies for hepatocellular carcinoma (HCC). Establishment of patient-derived xenograft (PDX) models that faithfully recapitulate the genetic and phenotypic features of HCC becomes important. In this study, we first established a cohort of 65 stable PDX models of HCC from corresponding Chinese patients. Then we showed that the histology and gene expression patterns of PDX models were highly consistent between xenografts and case-matched original tumors. Genetic alterations, including mutations and DNA copy number alterations (CNAs), of the xenografts correlated well with the published data of HCC patient specimens. Furthermore, differential responses to sorafenib, the standard-of-care agent, in randomly chosen xenografts were unveiled. Finally, in the models expressing high levels of FGFR1 gene according to the genomic data, FGFR1 inhibitor lenvatinib showed greater efficacy than sorafenib. Taken together, our data indicate that PDX models resemble histopathological and genomic characteristics of clinical HCC tumors, as well as recapitulate the differential responses of HCC patients to the standard-of-care treatment. Overall, this large collection of PDX models becomes a clinically relevant platform for drug screening, biomarker discovery and translational research in preclinical setting.

We have used circular dichroism and frequency-domain fluorescence spectroscopy to determine how the site-specific substitution of Tyr138 with either Phe138 or Gln138 affects the structural coupling between the opposing domains of calmodulin (CaM). A double mutant was constructed involving conservative substitution of Tyr99 --> Trp99 and Leu69 --> Cys69 to assess the structural coupling between the opposing domains, as previously described [Sun, H., Yin, D., and Squier, T. C. (1999) Biochemistry 38, 12266-12279]. Trp99 acts as a fluorescence resonance energy transfer (FRET) donor in distance measurements to probe the conformation of the central helix. Cys69 provides a reactive group for the covalent attachment of 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), which functions as a FRET acceptor and permits the measurement of the rotational dynamics of the amino-terminal domain. These CaM mutants demonstrate normal calcium-dependent gel-mobility shifts and changes in their near-UV CD spectra, have similar secondary structures to wild-type CaM following calcium activation, and retain the ability to fully activate the plasma membrane Ca-ATPase. The global folds, therefore, of both the carboxyl- and amino-terminal domains in these CaM mutants are similar to that of wild-type CaM. However, in comparison to wild-type CaM, the substitution of Tyr138 with either Phe138 or Gln138 results in (i) alterations in the average spatial separation and increases in the conformational heterogeneity between the opposing globular domains and (ii) the independent rotational dynamics of the amino-terminal domain. These results indicate that alterations in either the hydrogen bond between Tyr138 and Glu82 or contact interactions between aromatic amino acid side chains have the potential to initiate the structural collapse of CaM normally associated with target protein binding and activation.

We have used fluorescence and spin-label EPR spectroscopy to investigate how the phosphorylation of phospholamban (PLB) by cAMP-dependent protein kinase (PKA) modifies structural interactions between PLB and the Ca2+- and Mg2+-dependent ATPase (Ca-ATPase) that result in enzyme activation. Following covalent modification of N-terminal residues of PLB with dansyl chloride or the spin label 4-isothiocyanato-2,2,6,6-tetramethylpiperidine-N-oxyl (‘ITC-TEMPO’), we have co-reconstituted PLB with affinity-purified Ca-ATPase isolated from skeletal sarcoplasmic reticulum (SR) with full retention of catalytic function. The Ca2+-dependence of the ATPase activity of this reconstituted preparation is virtually identical with that observed using native cardiac SR before and after PLB phosphorylation, indicating that co-reconstituted sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1 (SERCA1) and PLB provide an equivalent experimental model for SERCA2a–PLB interactions. Phosphorylation of PLB in the absence of the Ca-ATPase results in a greater amplitude of rotational mobility, suggesting that the structural linkage between the transmembrane region and the N-terminus is destabilized. However, whereas co-reconstitution with the Ca-ATPase restricts the amplitude of rotational motion of PLB, subsequent phosphorylation of PLB does not significantly alter its rotational dynamics. Thus structural interactions between PLB and the Ca-ATPase that restrict the rotational mobility of the N-terminus of PLB are retained following the phosphorylation of PLB by PKA. On the other hand, the fluorescence intensity decay of bound dansyl is sensitive to the phosphorylation state of PLB, indicating that there are changes in the tertiary structure of PLB coincident with enzyme activation. These results suggest that PLB phosphorylation alters its structural interactions with the Ca-ATPase by inducing structural rearrangements between PLB and the Ca-ATPase within a defined complex that modulates Ca2+-transport function.

To investigate the structural linkage between the opposing globular domains in vertebrate calmodulin (CaM), we have constructed a CaM mutant (CaMX(145)) deficient in the last four amino acids between Met(145) and Lys(148) at the carboxyl terminal. Circular dichroism and fluorescence spectroscopic measurements were used to detect changes in the average secondary and tertiary structure of CaMX(145) in comparison to full-length CaM. Complementary measurements of the maximal calcium-binding stoichiometry and ability to activate the plasma membrane (PM) Ca-ATPase permit an assessment of the functional significance of observed structural changes. In comparison with native CaM, we find that CaMX(145) exhibits (i) a large reduction in alpha-helical content, (ii) a dramatic decrease in the average spatial separation between the opposing globular domains, (iii) the loss of one high-affinity calcium-binding site, and (iv) a diminished binding affinity for the PM-Ca-ATPase. Thus, the sequence near the carboxyl terminus functions to stabilize high-affinity calcium binding at one site and facilitates important intramolecular interactions that maintain CaM in an extended conformation. However, despite the large conformational changes resulting from deletion of the last four amino acids at the carboxyl terminal, CaMX(145) can fully activate the PM-Ca-ATPase. These results indicate that target protein binding can restore the nativelike structure critical to function, emphasizing that the structure of the central helix is not critical to CaM function under equilibrium conditions. Rather, the central helix functions to maintain the spatial separation between the opposing domains in CaM that may be critical to high-affinity binding and the rapid activation of the PM-Ca-ATPase, which are necessary for optimal calcium signaling. Thus, following initial association between CaM and target proteins, structural changes involving the carboxyl-terminal sequence have the potential to play an important role in triggering the structural collapse of CaM that facilitates the rapid and cooperative binding of the opposing globular domains with target proteins, which is important to high-affinity binding and rapid enzyme activation.

We have investigated the mechanisms of activation of the plasma membrane (PM) Ca-ATPase by calmodulin (CaM), which result in enhanced calcium transport rates and the maintenance of low intracellular calcium levels. We have isolated the amino- or carboxyl-terminal domains of CaM (i.e. CaMN or CaMC), permitting an identification of their relative specificity for binding to sites on either the PM Ca-ATPase or a peptide (C28W) corresponding to the CaM-binding sequence. We find that either CaMN or CaMC alone is capable of productive interactions with the PM Ca-ATPase that induces enzyme activation. There are, however, large differences in the affinity and specificity of binding between CaMN and CaMC and either C28W or the PM Ca-ATPase. The initial binding interaction between CaMC and the PM Ca-ATPase is highly specific, having approximately 10,000-fold greater affinity in comparison with CaMN. However, following the initial association of either CaMC or CaMN, there is a 300-fold enhancement in the affinity of CaMN for the secondary binding site. Thus, while CaMC binds with a high affinity to the two CaM-binding sites within the PM Ca-ATPase in a sequential manner, CaMN binds cooperatively with a lower affinity to both binding sites. These large differences in the binding affinities and specificities of the amino- and carboxyl-terminal domains ensure that CaM binding to the PM Ca-ATPase normally involves the formation of a specific complex in which the initial high affinity association of the carboxyl-terminal domain promotes the association of the amino-terminal domain necessary for enzyme activation.

We have investigated the ability of methionine sulfoxide reductase (MsrA) to maintain optimal calmodulin (CaM) function through the repair of oxidized methionines, which have been shown to accumulate within CaM in senescent brain [Gao, J., Yin, D. H., Yao, Y., Williams, T. D., and Squier, T. C. (1998) Biochemistry 37, 9536-9548]. Oxidatively modified calmodulin (CaMox) isolated from senescent brain or obtained by in vitro oxidation was incubated with MsrA. This treatment restores the functional ability of CaMox to activate the plasma membrane (PM) Ca-ATPase, confirming that (i) the decreased ability of CaM isolated from senescent animals to activate the PM Ca-ATPase results solely from methionine sulfoxide formation and (ii) MsrA can repair methionine sulfoxides within cytosolic proteins. We have used electrospray ionization mass spectrometry to investigate the extent and rates of methionine sulfoxide repair within CaMox. Upon exhaustive repair by MsrA, there remains a distribution of methionine sulfoxides within functionally reactivated CaMox, which varies from three to eight methionine sulfoxides. The rates of repair of methionine sulfoxides within individual tryptic fragments of CaMox vary by a factor of 2, where methionine sulfoxides located within hydrophobic sequences are repaired in preference to methionines that are more solvent accessible within the native structure. However, no single methionine sulfoxide is completely repaired in all CaM oxiforms. Decreases in the alpha-helical content and a disruption of the tertiary structure of CaM have previously been shown to result from methionine oxidation. Repair of selected methionine sulfoxides in CaMox by MsrA results in a partial refolding of the secondary structure, suggesting that MsrA repairs methionine sulfoxides within unfolded sequences until native-like structure and function are re-attained. The ability of CaMox isolated from senescent brain to fully activate the PM Ca-ATPase following repair by MsrA suggests the specific activity of MsrA is insufficient to maintain CaM function in aging brain. These results are discussed in terms of the possible regulatory role MsrA may play in the modulation of CaM function and calcium homeostasis under conditions of oxidative stress.

We have used electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), and fluorescence spectroscopy to investigate the secondary and tertiary structural consequences that result from oxidative modification of methionine residues in wheat germ calmodulin (CaM), and prevent activation of the plasma membrane Ca-ATPase. Using ESI-MS, we have measured rates of modification and molecular mass distributions of oxidatively modified CaM species (CaMox) resulting from exposure to H2O2. From these rates, we find that oxidative modification of methionine to the corresponding methionine sulfoxide does not predispose CaM to further oxidative modification. These results indicate that methionine oxidation results in no large-scale alterations in the tertiary structure of CaMox, because the rates of oxidative modification of individual methionines are directly related to their solvent exposure. Likewise, CD measurements indicate that methionine oxidation results in little change in the apparent alpha-helical content at 28 degrees C, and only a small (0.3 +/- 0.1 kcal mol(-1)) decrease in thermal stability, suggesting the disruption of a limited number of specific noncovalent interactions. Fluorescence lifetime, anisotropy, and quenching measurements of N-(1-pyrenyl)-maleimide (PMal) covalently bound to Cys26 indicate local structural changes around PMal in the amino-terminal domain in response to oxidative modification of methionine residues in the carboxyl-terminal domain. Because the opposing globular domains remain spatially distant in both native and oxidatively modified CaM, the oxidative modification of methionines in the carboxyl-terminal domain are suggested to modify the conformation of the amino-terminal domain through alterations in the structural features involving the interdomain central helix. The structural basis for the linkage between oxidative modification and these global conformational changes is discussed in terms of possible alterations in specific noncovalent interactions that have previously been suggested to stabilize the central helix in CaM.

Die attach material (DA) is important to heat dissipation and light output of solid state lighting (SSL) packages. Even though high thermal conductivity benefits to reduce the bulk thermal resistance of die attach, high viscosity will increase the contact thermal resistance in the packages. To face such a dilemma, it is of desirable to develop new technique to increase the filler loading without sacrificing the rheological properties. In this paper, we propose surface treatment of fillers with PDMS to keep the viscosity at a relatively low level while achieve high thermal performance. It has been demonstrated that surface-grafting can significantly reduce the viscosity of the die attach and increase the filler loading. The thermal concutvity of the die attach increased with the increase of the filler loading. However, there wasn't any percolation-like transition of thermal conductivity observed at high loading up to 50%, which might limit achieving better thermal performance in LED packages through surface-grafting technique. It is expected that the decease of percolation threshold of die attach materials and increase of percolative thermal conductivity are the complementing approach to fully exploit the benefit from surface grafting techniques to enhance the thermal performance.

The mechanism of thermal conductivity enhancement of silver epoxy based die attach materials (DA) by iodine treatment is studied. Iodine treatment is found to be able to uniform the polymer network and accelerate the curing of die attach at relatively lower temperature with shorter curing duration. In addition, several metal halide salts are proved to have the same effect as the iodine treatment and be able to improve the conductivity of DA. The developed DA is applied in LED packages. The thermal performance of LED packages is evaluated to demonstrate the effect of iodine treatment of DA.