Author Correction: In situ 3D-patterning of electrospun fibers using two-layer composite materials.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In situ 3D-patterning of electrospun fibers using two-layer composite materials.

Polymeric electrospun nanofibers have extensive applications in filtration, sensing, drug delivery, and tissue engineering that often require the fibers to be patterned or integrated with a larger device. Here, we describe a highly versatile in situ strategy for three-dimensional electrospun fiber patterning using collectors with an insulative surface layer and conductive recessed patterns. We show that two-layer collectors with pattern dimensions down to 100-micrometers are easily fabricated using available laboratory equipment. We use finite element method simulation and experimental validation to demonstrate that the fiber patterning strategy is effective for a variety of pattern dimensions and fiber materials. Finally, the potential for this strategy to enable new applications of electrospun fibers is demonstrated by incorporating electrospun fibers into dissolving microneedles for the first time. These studies provide a framework for the adaptation of this fiber patterning strategy to many different applications of electrospun fibers.

Effect of neck flexor muscle activation on impact velocity of the head during backward falls in young adults.

Falls are a common cause of traumatic brain injuries (TBI) across the lifespan. A proposed but untested hypothesis is that neck muscle activation influences impact severity and risk for TBI during a fall. We conducted backward falling experiments to test whether activation of the neck flexor muscles facilitates the avoidance of head impact, and reduces impact velocity if the head contacts the ground. Young adults (n=8) fell from standing onto a 30cm thick gymnastics mat while wearing a helmet. Participants were instructed to fall backward and (a) prevent their head from impacting the mat ("no head impact" trials); (b) allow their head to impact the mat, but with minimal impact severity ("soft impact" trials); and (c) allow their head to impact the mat, while inhibiting efforts to reduce impact severity ("hard impact" trials). Trial type associated with peak magnitude of electromyographic activity of the sternocleidomastoid (SCM) muscles (p<0.017), and with the vertical and horizontal velocity of the head at impact (p<0.001). Peak SCM activations, expressed as percent maximal voluntary isometric contraction (%MVIC), averaged 75.3, 67.5, and 44.5%MVIC in "no head impact", "soft impact", and "hard impact" trials, respectively. When compared to "soft impact" trials, vertical impact velocities in "hard impact" trials averaged 87% greater (3.23 versus 1.73m/s) and horizontal velocities averaged 83% greater (2.74 versus 1.50m/s). For every 10% increase in SCM %MVIC, vertical impact velocity decreased 0.24m/s and horizontal velocity decreased 0.22m/s. We conclude that SCM activation contributes to the prevention and modulation of head impact severity during backward falls.

A High-Resolution Tile-Based Approach for Classifying Biological Regions in Whole-Slide Histopathological Images.

Computational analysis of histopathological whole slide images (WSIs) has emerged as a potential means for improving cancer diagnosis and prognosis. However, an open issue relating to the automated processing of WSIs is the identification of biological regions such as tumor, stroma, and necrotic tissue on the slide. We develop a method for classifying WSI portions (512x512-pixel tiles) into biological regions by (1) extracting a set of 461 image features from each WSI tile, (2) optimizing tile-level prediction models using nested cross-validation on a small (600 tile) manually annotated tile-level training set, and (3) validating the models against a much larger (1.7x106 tile) data set for which ground truth was available on the whole-slide level. We calculated the predicted prevalence of each tissue region and compared this prevalence to the ground truth prevalence for each image in an independent validation set. Results show significant correlation between the predicted (using automated system) and reported biological region prevalences with p < 0.001 for eight of nine cases considered.

Exploration of Genomic, Proteomic, and Histopathological Image Data Integration Methods for Clinical Prediction.

The emergence of large multi-platform and multi-scale data repositories in biomedicine has enabled the exploration of data integration for holistic decision making. In this research, we investigate multi-modal genomic, proteomic, and histopathological image data integration for prediction of ovarian cancer clinical endpoints in The Cancer Genome Atlas (TCGA). Specifically, we study two data integration techniques, simple data concatenation and ensemble classification, to determine whether they can improve prediction of ovarian cancer grade or patient survival. Results indicate that integration via ensemble classification is more effective than simple data concatenation. We also highlight several key factors impacting data integration outcome such as predictability of endpoint, class prevalence, and unbalanced representation of features from different data modalities.

k-Nearest neighbor models for microarray gene expression analysis and clinical outcome prediction.

In the clinical application of genomic data analysis and modeling, a number of factors contribute to the performance of disease classification and clinical outcome prediction. This study focuses on the k-nearest neighbor (KNN) modeling strategy and its clinical use. Although KNN is simple and clinically appealing, large performance variations were found among experienced data analysis teams in the MicroArray Quality Control Phase II (MAQC-II) project. For clinical end points and controls from breast cancer, neuroblastoma and multiple myeloma, we systematically generated 463,320 KNN models by varying feature ranking method, number of features, distance metric, number of neighbors, vote weighting and decision threshold. We identified factors that contribute to the MAQC-II project performance variation, and validated a KNN data analysis protocol using a newly generated clinical data set with 478 neuroblastoma patients. We interpreted the biological and practical significance of the derived KNN models, and compared their performance with existing clinical factors.

Advances in molecular classification of renal neoplasms.

Kidney neoplasms are classified by light microscopy using the World Health Organization (WHO) system. The WHO system defines histopathologic tumor subtypes with distinct clinical behavior and underlying genetic mutations. In adults, the common malignant subtypes are variants of renal cell carcinoma (RCC). Histopathologic classification is critical for clinical management of RCC, but is becoming more complex with recognition of novel tumor subtypes, development of procedures yielding small diagnostic biopsies, and emergence of molecular therapies directed at tumor gene activity. Therefore, classification systems based on gene expression are likely to become essential for diagnosis, prognosis and treatment of kidney tumors. Recent DNA microarray studies have shown that clinically relevant renal tumor subtypes are characterized by distinct gene expression profiles, which are useful for discovery of novel diagnostic and prognostic biomarkers. In this review, we summarize the WHO classification system for renal tumors, general applications of microarray technology in cancer research, and specific microarray studies that have advanced knowledge of renal tumor diagnosis, prognosis, therapy and pathobiology.

An integrated cancer biomarker information system.

Rapid progress in functional genomics is yielding unparalleled opportunities for cancer biomarker discovery. Several procedures including data collection, analysis, verification, and visualization are necessary to identify cancer biomarkers from continuously updated microarray data, which are derived from multiple tissue sources using various methods. BioMarker is designed to integrate these procedures in a user-friendly manner, for practical application by cancer researcher and clinicians. From the feedback of clinical and research application, BioMarker is the first effort to provide a system that facilitates all aspects of biomarker discovery from microarray datasets.

[Tobacco etch virus proteinase: crystal structure of the active enzyme and its inactive mutant]. / Proteinaza virusa gravirovki tabaka: kristallicheskaia struktura aktivnogo fermenta i ego neaktivnogo mutanta.

Tobacco Etch Virus Protease (TEV protease) is widely used as a tool for separation of recombinant target proteins from their fusion partners. The crystal structures of two mutants of TEV protease, active autolysis-resistant mutant TEV-S219D in complex with the proteolysis product, and inactive mutant TEV-C151A in complex with a substrate, have been determined at 1.8 and 2.2 A resolution, respectively. The active sites of both mutants, including their oxyanion holes, have identical structures. The C-terminal residues 217-221 of the enzyme are involved in formation of the binding pockets S3-S6. This indicates that the autolysis of the peptide bond Met218-Ser219 exerts a strong effect on the fine-tuning of the substrate in the enzyme active site, which results in considerable decrease in the enzymatic activity.

Human thymidylate synthase is in the closed conformation when complexed with dUMP and raltitrexed, an antifolate drug.

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms while raltitrexed (Tomudex, ZD1694) is an antifolate inhibitor of TS approved for clinical use in several European countries. The crystal structure of the complex between recombinant human TS, dUMP, and raltitrexed has been determined at 1.9 A resolution. In contrast to the situation observed in the analogous complex of the rat TS, the enzyme is in the closed conformation and a covalent bond between the catalytic Cys 195 and dUMP is present in both subunits. This mode of ligand binding is similar to that of the analogous complex of the Escherichia coli enzyme. The only major differences observed are a direct hydrogen bond between His 196 and the O4 atom of dUMP and repositioning of the side chain of Tyr 94 by about 2 A. The thiophene ring of the drug is disordered between two parallel positions.

Structure of human thymidylate synthase suggests advantages of chemotherapy with noncompetitive inhibitors.

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms. The emergence of resistance to the treatment is often related to the increased levels of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding, a feedback regulatory mechanism, and/or to the increased stability to intracellular degradation of TS.drug complexes (versus unliganded TS). The active site loop of human TS (hTS) has a unique conformation resulted from a rotation by 180 degrees relative to its orientation in bacterial TSs. In this conformation, the enzyme must be inactive, because the catalytic cysteine is no longer positioned in the ligand-binding pocket. The ordered solvent structure obtained from high resolution crystallographic data (2.0 A) suggests that the inactive loop conformation promotes mRNA binding and intracellular degradation of the enzyme. This hypothesis is supported by fluorescence studies, which indicate that in solution both active and inactive forms of hTS are present. The binding of phosphate ion shifts the equilibrium toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active form. Thus, TS inhibition via stabilization of the inactive conformation should lead to less resistance than is observed with presently used drugs, which are analogs of its substrates, dUMP and CH(2)H(4)folate, and bind in the active site, promoting the active conformation. The presence of an extension at the N terminus of native hTS has no significant effect on kinetic properties or crystal structure.

Lipodystrophy in the fld mouse results from mutation of a new gene encoding a nuclear protein, lipin.

Mice carrying mutations in the fatty liver dystrophy (fld) gene have features of human lipodystrophy, a genetically heterogeneous group of disorders characterized by loss of body fat, fatty liver, hypertriglyceridemia and insulin resistance. Through positional cloning, we have isolated the gene responsible and characterized two independent mutant alleles, fld and fld(2J). The gene (Lpin1) encodes a novel nuclear protein which we have named lipin. Consistent with the observed reduction of adipose tissue mass in fld and fld(2J)mice, wild-type Lpin1 mRNA is expressed at high levels in adipose tissue and is induced during differentiation of 3T3-L1 pre-adipocytes. Our results indicate that lipin is required for normal adipose tissue development, and provide a candidate gene for human lipodystrophy. Lipin defines a novel family of nuclear proteins containing at least three members in mammalian species, and homologs in distantly related organisms from human to yeast.

Catalytic cysteine of thymidylate synthase is activated upon substrate binding.

The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in catalysis has been characterized by kinetic and crystallographic studies. Position 167 variants including S167A, S167N, S167D, S167C, S167G, S167L, S167T, and S167V were generated by site-directed mutagenesis. Only S167A, S167G, S167T, and S167C complemented the growth of thymidine auxotrophs of E. coli in medium lacking thymidine. Steady-state kinetic analysis revealed that mutant enzymes exhibited k(cat) values 1.1-95-fold lower than that of the wild-type enzyme. Relative to wild-type TS, K(m) values of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were 1.5-16-fold higher. The rate of dehalogenation of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by TS that does not require CH(2)H(4)folate as cosubstrate, by mutant TSs was analyzed and showed that only S167A and S167G catalyzed the dehalogenation reaction and values of k(cat)/K(m) for the mutant enzymes were decreased by 10- and 3000-fold, respectively. Analysis of pre-steady-state kinetics of ternary complex formation revealed that the productive binding of CH(2)H(4)folate is weaker to mutant TSs than to the wild-type enzyme. Chemical transformation constants (k(chem)) for the mutant enzymes were lower by 1.1-6.0-fold relative to the wild-type enzyme. S167A, S167T, and S167C crystallized in the I2(1)3 space group and scattered X-rays to either 1.7 A (S167A and S167T) or 2.6 A (S167C). The high-resolution data sets were refined to a R(crys) of 19.9%. In the crystals some cysteine residues were derivatized with 2-mercaptoethanol to form S,S-(2-hydroxyethyl)thiocysteine. The pattern of derivatization indicates that in the absence of bound substrate the catalytic cysteine is not more reactive than other cysteines. It is proposed that the catalytic cysteine is activated by substrate binding by a proton-transfer mechanism in which the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the transfer of a proton from the catalytic cysteine to a His 207-Asp 205 diad via a system of ordered water molecules.

A locus conferring resistance to diet-induced hypercholesterolemia and atherosclerosis on mouse chromosome 2.

Dietary cholesterol is known to raise total and low density lipoprotein cholesterol concentrations in humans and experimental animals, but the response among individuals varies greatly. Here we describe a mouse strain, C57BL/6ByJ (B6By), that is resistant to diet-induced hypercholesterolemia, in contrast to the phenotype seen in other common strains of mice including the closely related C57BL/6J (B6J) strain. Compared to B6J, B6By mice exhibit somewhat lower basal cholesterol levels on a chow diet, and show a relatively modest increase in absolute levels of total and LDL/VLDL cholesterol in response to an atherogenic diet containing 15% fat, 1.25% cholesterol, and 0.5% cholate. Correspondingly, B6By mice are also resistant to diet-induced aortic lesions, with less than 15% as many lesions as B6J. Food intake and cholesterol absorption are similar between B6By and B6J mice. To investigate the gene(s) underlying the resistant B6By phenotype, we performed genetic crosses with the unrelated mouse strain, A/J. A genome-wide scan revealed a locus, designated Diet1, on chromosome 2 near marker D2Mit117 showing highly significant linkage (lod = 9.6) between B6By alleles and hypo-response to diet. Examination of known genes in this region suggested that this locus represents a novel gene affecting plasma lipids and atherogenesis in response to diet.

Drug-resistant variants of Escherichia coli thymidylate synthase: effects of substitutions at Pro-254.

Drug-resistant variants of thymidylate synthase (TS) can potentially be used in gene therapy applications to decrease the myelosuppressive side effects of TS-directed anticancer agents or to select genetically modified cells in vivo. Mutations of proline 303 of human TS confer resistance to TS-directed fluoropyrimidines and antifolates (). We generated the corresponding variants in Escherichia coli TS (ecTS), position 254, to better understand the mechanism by which mutations at this residue confer resistance. In addition, because ecTS is intrinsically resistant to several antifolates when compared with human TS, we suspected that greater resistance could be achieved with the bacterial enzyme. The P254L enzyme conferred >100-fold resistance to both raltitrexed and 5-fluoro-2'-deoxyuridine (FdUrd) compared with wild-type ecTS. Four additional mutants (P254F, P254S, P254G, and P254D), each of which complemented growth of a TS-deficient cell line, were generated, isolated, and characterized. Steady-state values of K(m) for dUMP and k(cat) were not substantially different among the variants and were comparable with the wild-type values, but K(m) for methylenetetrahydrofolate (CH(2)H(4)PteGlu) was >10-fold higher for P254D. Values of k(on) and k(off) for nucleotide binding, which were obtained by stopped-flow spectroscopy, were virtually unchanged among the mutants. Drastic differences were observed for CH(2)H(4)PteGlu binding, with K(d) values >15-fold higher than observed with the wild-type enzyme; surprisingly, the proposed isomerization reaction that is very evident for the wild-type enzyme is not observed with P254S. The decrease in affinity for CH(2)H(4)PteGlu correlates well with K(i) values obtained for three TS-directed inhibitors. These results show that mutations at Pro-254 specifically affect the initial binding interactions between enzyme and cofactor and also alter the ability of the mutant enzymes to undergo conformational changes that occur on ternary complex formation. The crystal structure of P254S was determined at 1.5 A resolution and is the most precise structure of TS available. When compared with wild-type TS, the structure shows local conformational changes affecting mostly Asp-253; its carbonyl is rotated approximately 40 degrees, and the side chain forms an ion pair with Arg-225.

Clinical and laboratory features of patients of Vietnamese descent with systemic lupus erythematosus.

The prevalence rate and disease manifestations of systemic lupus erythematosus (SLE) have been noted to vary among different ethnic groups. There has been no description in the English literature of SLE in the Vietnamese population. This is the first report, which details the clinical and laboratory features as well as an estimation of the prevalence of SLE in patients with a Vietnamese ancestry living in the United States. We performed a retrospective chart review of clinical and laboratory features of patients of Vietnamese descent with SLE. The case finding was performed by a review of the rheumatology clinic records at two large teaching hospitals in Santa Clara County searching for patients with SLE with a Vietnamese surname. In addition, we recruited patients by contacting all of the rheumatologists practicing in the county. Twenty-three patients of Vietnamese descent were identified with SLE in Santa Clara County. The estimated prevalence of SLE in the patients of Vietnamese descent was 42 cases per 100 000 persons. Eighty-seven per cent of the cases were born in Vietnam. The clinical and laboratory features of SLE were similar to prior published reviews except for a relatively high prevalence of anti-RNP antibody (54%). The patients with anti-RNP antibody exhibited features of overlap syndrome. There was a high rate of exposure to tuberculosis (TB). Fifty-eight per cent of patients had a positive purified protein derivative (PPD) skin test and 27% of patients had a history of clinical TB. Forty-four per cent of patients had evidence of hepatitis B exposure. The prevalence of SLE in the Vietnamese population in Santa Clara County is similar to that of other Asian populations. There was a relatively high prevalence of anti-RNP antibody in our patient group which was associated with overlap features. As expected in an immigrant population from Southeast Asia, there was a high rate of prior exposure to tuberculosis and hepatitis B. Clinicians should diligently screen for these infections and appropriately prophylaxe and treat patients.

Utility of contrast echocardiography in the diagnosis of aortic dissection.

Determination of ascending aortic involvement in aortic dissection has significant implications in prognosis, immediate surgical referral, and surgical approach. We report 2 cases of the use of an echo contrast agent in enhancing the diagnostic capability of transesophageal echocardiography in suspected ascending aortic dissection. Contrast echocardiography eliminated the linear artifacts often confused with true dissection flaps and allowed easy identification of the presence of true and false lumina.

Genetic, physical, and transcript map of the fld region on mouse chromosome 12.

The fatty liver dystrophy (fld) mutation is manifested in abnormalities of lipid and glucose metabolism and peripheral neuropathy. To identify the gene affected by this mutation, we generated a genetic map of the fld region on chromosome 12 by the analysis of F2 offspring from an intersubspecific cross between strains BALB/cByJ-fld and CAST/EiJ. The results localize fld to the 0.42-cM interval between the microsatellite markers D12Mit170 and D12Mit184. A contig of YACs and BACs covering the nonrecombinant genomic region has been constructed and used for the identification of genes. Expressed sequence tag mapping and exon trapping identified three transcripts within the critical interval: Ctla2b, which encodes a cysteine protease inhibitor, and mouse homologs of KIAA0188 and KIAA0575, two long human transcripts of unknown function. Expression analysis revealed that Kiaa0188 is expressed in wildtype but not in fld liver, implicating this gene as a candidate for harboring the fld mutation.

Outcome in elderly patients undergoing definitive surgery and radiation therapy for supratentorial glioblastoma multiforme at a tertiary care institution.

PURPOSE: To determine the efficacy of definitive surgery and radiation in patients aged 70 years and older with supratentorial glioblastoma multiforme. METHODS AND MATERIALS: We selected elderly patients (> or = 70 years) who had primary treatment for glioblastoma multiforme at our tertiary care institution from 1977 through 1996. The study group (n = 102) included 58 patients treated with definitive radiation, 19 treated with palliative radiation, and 25 who received no radiation. To compare our results with published findings, we grouped our patients according to the applicable prognostic categories developed by the Radiation Therapy Oncology Group (RTOG): RTOG group IV (n = 6), V (n = 70), and VI (n = 26). Patients were retrospectively assigned to prognostic group IV, V, or VI based on age, performance status, extent of surgery, mental status, neurologic function, and radiation dose. Treatment included surgical resection and radiation (n = 49), biopsy alone (n = 25), and biopsy followed by radiation (n = 28). Patients were also stratified according to whether they were optimally treated (gross total or subtotal resection with postoperative definitive radiation) or suboptimally treated (biopsy, biopsy + radiation, surgery alone, or surgery + palliative radiation). Patients were considered to have a favorable prognosis (n = 39) if they were optimally treated and had a Karnofsky Performance Status (KPS) score of at least 70. RESULTS: The median survival for patients according to RTOG groups IV, V, and VI was 9.2, 6.6, and 3.1 months, respectively (log-rank, p < 0.0004). The median overall survival was 5.3 months. The definitive radiation group (n = 58) had a median survival of 7.3 months compared to 4.5 months in the palliative radiation group (n = 19) and 1.2 months in the biopsy-alone group (p < 0.0001). Optimally treated patients had a median survival of 7.4 months compared to 2.4 months in those suboptimally treated (p < 0.0001). The favorable prognosis group had an 8.4-month median survival compared to 2.4 months in the unfavorable group (p < 0.0001). On multivariate analysis, the KPS, RTOG group, favorable/unfavorable prognosis, and optimal treatment/suboptimal treatment were significant predictors of survival. CONCLUSION: Elderly patients with good performance status (> or = 70 KPS) when treated aggressively with maximal resection and definitive radiation had longer survival than those treated with palliative radiation and biopsy. Aggressive treatment in such patients should be considered.