Convection and extracellular matrix binding control interstitial transport of extracellular vesicles.

Extracellular vesicles (EVs) influence a host of normal and pathophysiological processes in vivo. Compared to soluble mediators, EVs can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins, and their large size (∼30-150 nm) limits diffusion. We isolated EVs from the MCF10 series-a model human cell line of breast cancer progression-and demonstrated increasing presence of laminin-binding integrins α3ß1 and α6ß1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs within a microfluidic device under controlled physiological interstitial flow (0.15-0.75 µm/s) demonstrated that convection was the dominant mechanism of transport. Binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was mitigated by blocking integrins α3ß1 and α6ß1. Our studies demonstrate that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be leveraged in nanotherapeutic design.

Protein profiling of forehead epidermal corneocytes distinguishes frontal fibrosing from androgenetic alopecia.

Protein profiling offers an effective approach to characterizing how far epidermis departs from normal in disease states. The present pilot investigation tested the hypothesis that protein expression in epidermal corneocytes is perturbed in the forehead of subjects exhibiting frontal fibrosing alopecia. To this end, samples were collected by tape stripping from subjects diagnosed with this condition and compared to those from asymptomatic control subjects and from those exhibiting androgenetic alopecia. Unlike the latter, which exhibited only 3 proteins significantly different from controls in expression level, forehead samples from frontal fibrosing alopecia subjects displayed 72 proteins significantly different from controls, nearly two-thirds having lower expression. The results demonstrate frontal fibrosing alopecia exhibits altered corneocyte protein expression in epidermis beyond the scalp, indicative of a systemic condition. They also provide a basis for quantitative measures of departure from normal by assaying forehead epidermis, useful in monitoring response to treatment while avoiding invasive biopsy.

Elucidation of familial relationships using hair shaft proteomics.

This study examines the potential of hair shaft proteomic analysis to delineate genetic relatedness. Proteomic profiling and amino acid sequence analysis provide information for quantitative and statistically-based analysis of individualization and sample similarity. Protein expression levels are a function of cell-specific transcriptional and translational programs. These programs are greatly influenced by an individual's genetic background, and are therefore influenced by familial relatedness as well as ancestry and genetic disease. Proteomic profiles should therefore be more similar among related individuals than unrelated individuals. Likewise, profiles of genetically variant peptides that contain single amino acid polymorphisms, the result of non-synonymous SNP alleles, should behave similarly. The proteomically-inferred SNP alleles should also provide a basis for calculation of combined paternity and sibship indices. We test these hypotheses using matching proteomic and genetic datasets from a family of two adults and four siblings, one of which has a genetic condition that perturbs hair structure and properties. We demonstrate that related individuals, compared to those who are unrelated, have more similar proteomic profiles, profiles of genetically variant peptides and higher combined paternity indices and combined sibship indices. This study builds on previous analyses of hair shaft protein profiling and genetically variant peptide profiles in different real-world scenarios including different human hair shaft body locations and pigmentation status. It also validates the inclusion of proteomic information with other biomolecular substrates in forensic hair shaft analysis, including mitochondrial and nuclear DNA.

Age-Related Changes in Hair Shaft Protein Profiling and Genetically Variant Peptides.

Recent reports highlight possible improvements in individual identification using proteomic information from human hair evidence. These reports have stimulated investigation of parameters that affect the utility of proteomic information. In addition to variables already studied relating to processing technique and anatomic origin of hair shafts, an important variable is hair ageing. Present work focuses on the effect of age on protein profiling and analysis of genetically variant peptides (GVPs). Hair protein profiles may be affected by developmental and physiological changes with age of the donor, exposure to different environmental conditions and intrinsic processes, including during storage. First, to explore whether general trends were evident in the population at different ages, hair samples were analyzed from groups of different subjects in their 20's, 40's and 60's. No significant differences were seen as a function of age, but consistent differences were evident between European American and African American hair profiles. Second, samples collected from single individuals at different ages were analyzed. Mostly, these showed few protein expression level differences over periods of 10 years or less, but samples from subjects at 44 and 65 year intervals were distinctly different in profile. The results indicate that use of protein profiling for personal identification, if practical, would be limited to decadal time intervals. Moreover, batch effects were clearly evident in samples processed by different staff. To investigate the contribution of storage (at room temperature) in affecting the outcomes, the same proteomic digests were analyzed for GVPs. In samples stored over 10 years, GVPs were reduced in number in parallel with the yield of identified proteins and unique peptides. However, a very different picture emerged with respect to personal identification. Numbers of GVPs sufficed to distinguish individuals despite the age differences of the samples. As a practical matter, three hair samples per person provided nearly the maximal number obtained from 5 or 6 samples. The random match probability (where the log increased in proportion to the number of GVPs) reached as high as 1 in 108. The data indicate that GVP results are dependent on the single nucleotide polymorphism profile of the donor genome, where environmental/processing factors affect only the yield, and thus are consistent despite the ages of the donors and samples and batchwise effects in processing. This conclusion is critical for application to casework where the samples may be in storage for long periods and used to match samples recently collected.

Collagen Turnover in Relation to Risk Factors and Hemodynamics in Human Intracranial Aneurysms.

Background and Purpose- Determinants for molecular and structural instability, that is, impending growth or rupture, of intracranial aneurysms (IAs) remain uncertain. To elucidate this, we endeavored to estimate the actual turnover rates of the main molecular constituent in human IA (collagen) on the basis of radiocarbon (14C) birth dating in relation to IA hemodynamics. Methods- Collagen turnover rates in excised human IA samples were calculated using mathematical modeling of 14C birth dating data of collagen in relation to risk factors and histological markers for collagen maturity/turnover in selected IA. Hemodynamics were simulated using image-based computational fluid dynamics. Correlation, logistic regression, and receiver operating characteristic analyses were performed. Results- Collagen turnover rates were estimated in 46 IA (43 patients); computational fluid dynamics could be performed in 20 IA (20 patients). The mean collagen turnover rate (γ) constituted 126% (±1% error) per year. For patients with arterial hypertension, γ was greater than 2600% annually, whereas γ was distinctly lower with 32% (±1% error) per year for patients without risk factors, such as smoking and hypertension. There was a distinct association between histological presence of rather immature collagen in human IA and the presence of modifiable risk factors. Spatial-temporal averaged wall shear stress predicted rapid collagen turnover (odds ratio, 1.6 [95% CI, 1.0-2.7]). Receiver operating characteristic analysis demonstrated a good test accuracy (area under the curve, 0.798 [95% CI, 0.598-0.998]) for average wall shear stress with a threshold ≥4.9 Pa for rapid collagen turnover. Conclusions- Our data indicate that turnover rates and stability of collagen in human IA are strongly associated with the presence of modifiable risk factors and aneurysmal hemodynamics. These findings underline the importance of strict risk factor modification in patients with unruptured IA. Future should include more detailed risk factor data to establish a more causal understanding of hemodynamics and the rupture risk of individual IA.

Deducing signaling pathways from parallel actions of arsenite and antimonite in human epidermal keratinocytes.

Inorganic arsenic oxides have been identified as carcinogens in several human tissues, including epidermis. Due to the chemical similarity between trivalent inorganic arsenic (arsenite) and antimony (antimonite), we hypothesized that common intracellular targets lead to similarities in cellular responses. Indeed, transcriptional and proteomic profiling revealed remarkable similarities in differentially expressed genes and proteins resulting from exposure of cultured human epidermal keratinocytes to arsenite and antimonite in contrast to comparisons of arsenite with other metal compounds. These data were analyzed to predict upstream regulators and affected signaling pathways following arsenite and antimonite treatments. A majority of the top findings in each category were identical after treatment with either compound. Inspection of the predicted upstream regulators led to previously unsuspected roles for oncostatin M, corticosteroids and ephrins in mediating cellular response. The influence of these predicted mediators was then experimentally verified. Together with predictions of transcription factor effects more generally, the analysis has led to model signaling networks largely accounting for arsenite and antimonite action. The striking parallels between responses to arsenite and antimonite indicate the skin carcinogenic risk of exposure to antimonite merits close scrutiny.

Speeding Up Percolator.

The processing of peptide tandem mass spectrometry data involves matching observed spectra against a sequence database. The ranking and calibration of these peptide-spectrum matches can be improved substantially using a machine learning postprocessor. Here, we describe our efforts to speed up one widely used postprocessor, Percolator. The improved software is dramatically faster than the previous version of Percolator, even when using relatively few processors. We tested the new version of Percolator on a data set containing over 215 million spectra and recorded an overall reduction to 23% of the running time as compared to the unoptimized code. We also show that the memory footprint required by these speedups is modest relative to that of the original version of Percolator.

The Beta 2 Adrenergic Receptor Antagonist Timolol Improves Healing of Combined Burn and Radiation Wounds.

In a scenario involving a nuclear detonation during war or a terrorist attack, acute radiation exposure combined with thermal and blast effects results in severe skin injury. Although the cutaneous injury in such a scenario may not be lethal, it may lead to inflammation, delayed wound healing and loss of the skin barrier, resulting in an increased risk of infection. In this study, we tested the potential use of timolol, a beta-adrenergic receptor antagonist, to improve epidermal wound closure after combined burn and radiation injury using an ex vivo human skin culture model. Daily application of 10 µ M timolol after combined injury (burn and 10 Gy ex vivo irradiation) increased wound epithelialization by 5-20%. In addition, exposure to 10 Gy significantly suppressed epidermal keratinocyte proliferation by 46% at 48 h postirradiation. Similar to what has been observed in a thermal burn injury, the enzyme phenylethanolamine N-methyltransferase (PNMT), which generates epinephrine, was elevated in the combined thermal burn and radiation wounds. This likely resulted in elevated tissue levels of this catecholamine, which has been shown to delay healing. Thus, with the addition of timolol to the wound to block the binding of locally generated epinephrine to the beta-adrenergic receptor, healing is improved. This work suggests that by antagonizing local epinephrine action within the wound, a beta-adrenergic receptor antagonist such as timolol may be a useful adjunctive treatment to improve healing in the combined burn and radiation injury.

Proteomic analysis of hair shafts from monozygotic twins: Expression profiles and genetically variant peptides.

Forensic association of hair shaft evidence with individuals is currently assessed by comparing mitochondrial DNA haplotypes of reference and casework samples, primarily for exclusionary purposes. Present work tests and validates more recent proteomic approaches to extract quantitative transcriptional and genetic information from hair samples of monozygotic twin pairs, which would be predicted to partition away from unrelated individuals if the datasets contain identifying information. Protein expression profiles and polymorphic, genetically variant hair peptides were generated from ten pairs of monozygotic twins. Profiling using the protein tryptic digests revealed that samples from identical twins had typically an order of magnitude fewer protein expression differences than unrelated individuals. The data did not indicate that the degree of difference within twin pairs increased with age. In parallel, data from the digests were used to detect genetically variant peptides that result from common nonsynonymous single nucleotide polymorphisms in genes expressed in the hair follicle. Compilation of the variants permitted sorting of the samples by hierarchical clustering, permitting accurate matching of twin pairs. The results demonstrate that genetic differences are detectable by proteomic methods and provide a framework for developing quantitative statistical estimates of personal identification that increase the value of hair shaft evidence.

Myasthenia gravis exacerbation after discontinuing mycophenolate: A single-center cohort study.

OBJECTIVE: To determine whether discontinuation or marked reduction of mycophenolate mofetil (MMF) in patients with myasthenia gravis (MG) causes MG exacerbations. METHODS: We identified 88 patients with MG who took MMF during the 5-year period 2007-2011 at our MG clinic. We then performed detailed chart reviews and recorded all MG exacerbations and their relationship to MMF and other treatment changes. We also recorded demographic data and disease characteristics (including antibody status and Myasthenia Gravis Foundation of America status). RESULTS: There were 14 patients who had an MG exacerbation during the study period. Of these, 13 had discontinued MMF therapy, with a median time until exacerbation of 16 weeks after discontinuation (9 patients) or marked dose reduction (4 patients) of MMF therapy (exacerbation in the absence of change in any other component of the immunosuppressive regimen). Using the cluster option in a Cox regression analysis, the MMF coefficient was -5.32, with a standard error of 1.05 and a p value of 0.0002, corresponding to an estimated hazard ratio of 204. CONCLUSIONS: This retrospective cohort study suggests that discontinuation/marked reduction of MMF therapy may increase the risk of MG exacerbation many fold, supporting the hypothesis that MMF plays a role in the maintenance of MG remission/minimal manifestation status. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that in patients with MG taking MMF, discontinuation or marked reduction of MMF causes MG exacerbation.

The Serum Immunoglobulin G Glycosylation Signature of Gastric Cancer.

Biomarkers may facilitate detection of gastric cancer at an earlier stage and reduce mortality. Here we sought to determine if the glycosylation profile of serum immunoglobulin G (IgG) could distinguish patients with non-atrophic gastritis (NAG), duodenal ulcer (DU) and gastric cancer (GC). Serum IgG was released and analyzed using nano-LC-TOF mass spectrometry. Statistically significant false discovery rate (FDR)-adjusted p-values were observed for 18 glycans, eight that differed significantly between NAG and GC, three that distinguished NAG from DU, and eight that differed between DU and GC. The IgG glycosylation signature may be useful as a predictive marker for gastric cancer.

Targeted therapy with MXD3 siRNA, anti-CD22 antibody and nanoparticles for precursor B-cell acute lymphoblastic leukaemia.

Conventional chemotherapy for precursor B-cell (preB) acute lymphoblastic leukaemia (ALL) has limitations that could be overcome by targeted therapy. Previously, we discovered a potential therapeutic molecular target, MDX3 (MAX dimerization protein 3), in preB ALL. In this study, we hypothesize that an effective siRNA therapy for preB ALL can be developed using antiCD22 antibody (αCD22 Ab) and nanoparticles. We composed nanocomplexes with super paramagnetic iron oxide nanoparticles (SPIO NPs), αCD22 Abs and MXD3 siRNA molecules based on physical interactions between the molecules. We demonstrated that the MXD3 siRNA-αCD22 Ab-SPIO NP complexes entered leukaemia cells and knocked down MXD3, leading the cells to undergo apoptosis and resulting in decreased live cell counts in the cell line Reh and in primary preB ALL samples in vitro. Furthermore, the cytotoxic effects of the MXD3 siRNA-αCD22 Ab-SPIO NP complexes were significantly enhanced by addition of the chemotherapy drugs vincristine or doxorubicin. We also ruled out potential cytotoxic effects of the MXD3 siRNA-αCD22 Ab-SPIO NP complexes on normal primary haematopoietic cells. Normal B cells were affected while CD34-positive haematopoietic stem cells and non-B cells were not. These data suggest that MXD3 siRNA-αCD22 Ab-SPIO NP complexes have the potential to be a new targeted therapy for preB ALL.

Differential network analyses of Alzheimer's disease identify early events in Alzheimer's disease pathology.

In late-onset Alzheimer's disease (AD), multiple brain regions are not affected simultaneously. Comparing the gene expression of the affected regions to identify the differences in the biological processes perturbed can lead to greater insight into AD pathogenesis and early characteristics. We identified differentially expressed (DE) genes from single cell microarray data of four AD affected brain regions: entorhinal cortex (EC), hippocampus (HIP), posterior cingulate cortex (PCC), and middle temporal gyrus (MTG). We organized the DE genes in the four brain regions into region-specific gene coexpression networks. Differential neighborhood analyses in the coexpression networks were performed to identify genes with low topological overlap (TO) of their direct neighbors. The low TO genes were used to characterize the biological differences between two regions. Our analyses show that increased oxidative stress, along with alterations in lipid metabolism in neurons, may be some of the very early events occurring in AD pathology. Cellular defense mechanisms try to intervene but fail, finally resulting in AD pathology as the disease progresses. Furthermore, disease annotation of the low TO genes in two independent protein interaction networks has resulted in association between cancer, diabetes, renal diseases, and cardiovascular diseases.

Genomic characterization of a three-dimensional skin model following exposure to ionizing radiation.

This study aimed at characterizing the genomic response to low versus moderate doses of ionizing radiation (LDIR versus MDIR) in a three-dimensional (3D) skin model, which exhibits a closer tissue complexity to human skin than monolayer cell cultures. EpiDermFT skin plugs were exposed to 0, 0.1 and 1 Gy doses of X-rays and harvested at 5 min, 3, 8 and 24 h post-irradiation (post-IR). RNA was interrogated for global gene expression alteration. Our results show that MDIR modulated a larger number of genes over the course of 24 h compared to LDIR. However, immediately and throughout the first 3h post-IR, LDIR modulated a larger number of genes than MDIR, mostly associated with cell-cell signaling and survival promotion. Significant modulation of pathways was detected only at 3 h post-IR in MDIR with induction of genes promoting apoptosis. Collectively, the data show different dynamics in the response to LDIR versus MDIR, especially in cell-cycle distribution. LDIR-exposed tissues showed signs of attempted cell-cycle re-entry as early as 3 h post-IR, but were arrested beyond 8 h at the G1/S checkpoint. At 24 h, cells appeared to accumulate at the G2/M checkpoint. MDIR-exposed tissues did not exhibit a prolonged G1/S arrest but rather a prolonged G2/M arrest, which was sustained at least up to 24 h. By 24 h cells exhibited signs of recovery in both LDIR- and MDIR-exposed tissues. In summary, the most pronounced difference in the initial cellular response to LDIR versus MDIR is the promotion of protection and survival in LDIR versus the promotion of apoptosis in MDIR.

Comparison of low and high dose ionising radiation using topological analysis of gene coexpression networks.

BACKGROUND: The growing use of imaging procedures in medicine has raised concerns about exposure to low-dose ionising radiation (LDIR). While the disastrous effects of high dose ionising radiation (HDIR) is well documented, the detrimental effects of LDIR is not well understood and has been a topic of much debate. Since little is known about the effects of LDIR, various kinds of wet-lab and computational analyses are required to advance knowledge in this domain. In this paper we carry out an "upside-down pyramid" form of systems biology analysis of microarray data. We characterised the global genomic response following 10 cGy (low dose) and 100 cGy (high dose) doses of X-ray ionising radiation at four time points by analysing the topology of gene coexpression networks. This study includes a rich experimental design and state-of-the-art computational systems biology methods of analysis to study the differences in the transcriptional response of skin cells exposed to low and high doses of radiation. RESULTS: Using this method we found important genes that have been linked to immune response, cell survival and apoptosis. Furthermore, we also were able to identify genes such as BRCA1, ABCA1, TNFRSF1B, MLLT11 that have been associated with various types of cancers. We were also able to detect many genes known to be associated with various medical conditions. CONCLUSIONS: Our method of applying network topological differences can aid in identifying the differences among similar (eg: radiation effect) yet very different biological conditions (eg: different dose and time) to generate testable hypotheses. This is the first study where a network level analysis was performed across two different radiation doses at various time points, thereby illustrating changes in the cellular response over time.

Transcriptional response of ex vivo human skin to ionizing radiation: comparison between low- and high-dose effects.

Although human exposure to low-dose ionizing radiation can occur through a variety of sources, including natural, medical, occupational and accidental, the true risks of low-dose ionizing radiation are still poorly understood in humans. Here, the global transcriptional responses of human skin after ex vivo exposure to low (0.05 Gy) and high (5 Gy) doses of X rays and of time in culture (0 Gy) at 0, 2, 8 and 30 h postirradiation were analyzed and compared. Responses to low and high doses differed quantitatively and qualitatively. Differentially expressed genes fell into three groups: (1) unique genes defined as responsive to either 0.05 or 5 Gy but not both and also responsive to time in culture, (2) specific genes defined as responsive to either 0.05 or 5 Gy but not both and not responsive to time in culture, and (3) dose-independent responsive genes. Major differences observed in ex vivo irradiated skin between transcriptional responses to low or high doses were twofold. First, gene expression modulated by 0.05 Gy was transient, while in response to 5 Gy persistence of modified gene expression was observed for a limited number of genes. Second, neither TP53 nor TGFß target genes were modulated after exposure to an acute low dose, suggesting that the TP53-dependent DNA damage response either was not triggered or was triggered only briefly.

Identification of tissue cyst wall components by transcriptome analysis of in vivo and in vitro Toxoplasma gondii bradyzoites.

The Toxoplasma gondii bradyzoite is essential to establish persistent infection, yet little is known about what factors this developmental form secretes to establish the cyst or interact with its host cell. To identify candidate bradyzoite-secreted effectors, the transcriptomes of in vitro tachyzoites 2 days postinfection, in vitro bradyzoites 4 days postinfection, and in vivo bradyzoites 21 days postinfection were interrogated by microarray, and the program SignalP was used to identify signal peptides indicating secretion. One hundred two putative bradyzoite-secreted effectors were identified by this approach. Two candidates, bradyzoite pseudokinase 1 and microneme adhesive repeat domain-containing protein 4, were chosen for further investigation and confirmed to be induced and secreted by bradyzoites in vitro and in vivo. Thus, we report the first analysis of the transcriptomes of in vitro and in vivo bradyzoites and identify two new protein components of the Toxoplasma tissue cyst wall.

Impact of public reporting of coronary artery bypass graft surgery performance data on market share, mortality, and patient selection.

BACKGROUND: The impact of publicly reporting risk-adjusted outcomes for hospitals and surgeons remains controversial, with particular concern about unintended consequences. OBJECTIVES: We evaluated the impact of 3 reports from the voluntary California CABG Mortality Reporting Program (CCMRP) on hospital market share, hospital mortality, and patient selection for coronary artery bypass graft (CABG) surgery. RESEARCH DESIGN AND PARTICIPANTS: We analyzed data from January 2000 to December 2005 for all patients receiving isolated CABG surgery in California. We compared hospital groups based on their quality classification, including low-mortality outliers ("better"), high-mortality outliers ("worse"), and nonoutliers, as well as participation in the CCMRP. MEASURES: We compared changes in market share, risk-adjusted mortality, and hospital caseload of high-risk patients for isolated CABG surgeries before and after the public release of 3 CCMRP reports (July 2001, August 2003, and February 2005). RESULTS: Low-mortality outlier hospitals experienced significantly increased market share for isolated CABG surgery in the first 6 months after the public release of the CCMRP reports (relative change in adjusted mean market share=8.9%, P=0.002). We found no evidence to suggest reduced risk adjusted mortality after the release of the CCMRP reports, but high-mortality outlier hospitals, on average, operated on less sick patients (relative change in mean expected mortality=25%, P=0.02). CONCLUSIONS: The release of public CABG hospital performance reports in California was associated with increased volume at low-mortality hospitals, and may have reduced referrals of high-risk patients to high-mortality hospitals (or risk avoidance).

Expression of CD44s and CD44v6 in lung cancer and their correlation with prognostic factors.

BACKGROUND: CD44, a transmembrane glycoprotein receptor, plays a major role in tumor progression and metastasis. OBJECTIVE: To evaluate the expression of CD44 standard (CD44s) and its variant 6 (CD44v6) in normal and neoplastic lung tissue and correlate it with prognostic factors in lung cancer. METHODS: The study included 52 non-small cell lung carcinomas (NSCLC) (21 squamous cell carcinomas and 31 adenocarcinomas), 15 small cell lung carcinomas (SCLC) and 8 carcinoid tumors. Expression of CD44s and CD44v6 was evaluated by immunohistochemistry and correlated with lung cancer prognostic factors. RESULTS: All squamous cell carcinomas expressed both CD44s and CD44v6. Adenocarcinomas expressed CD44s in 39% of cases and CD44v6 in 45%. Carcinoid tumors expressed only CD44s in 88% of cases. All SCLCs were negative for both CD44s and CD44v6. A restricted panel consisting of CD44s and CD44v6 will discriminate NSCLC from SCLC with a sensitivity of 67% and a specificity of 100%. In adenocarcinoma CD44s expression was significantly correlated with lymph node metastases (p=0.007) while CD44v6 expression was more significantly associated with tumor size (p=0.0032). CONCLUSIONS: CD44s and CD44v6 are expressed in certain types of lung cancer. In adenocarcinoma CD44s and CD44v6 expression is significantly correlated with lymph node metastases and tumor size.