Analytical validation of a multi-cancer early detection test with cancer signal origin using a cell-free DNA-based targeted methylation assay.

The analytical validation is reported for a targeted methylation-based cell-free DNA multi-cancer early detection test designed to detect cancer and predict the cancer signal origin (tissue of origin). A machine-learning classifier was used to analyze the methylation patterns of >105 genomic targets covering >1 million methylation sites. Analytical sensitivity (limit of detection [95% probability]) was characterized with respect to tumor content by expected variant allele frequency and was determined to be 0.07%-0.17% across five tumor cases and 0.51% for the lymphoid neoplasm case. Test specificity was 99.3% (95% confidence interval, 98.6-99.7%). In the reproducibility and repeatability study, results were consistent in 31/34 (91.2%) pairs with cancer and 17/17 (100%) pairs without cancer; between runs, results were concordant for 129/133 (97.0%) cancer and 37/37 (100%) non-cancer sample pairs. Across 3- to 100-ng input levels of cell-free DNA, cancer was detected in 157/182 (86.3%) cancer samples but not in any of the 62 non-cancer samples. In input titration tests, cancer signal origin was correctly predicted in all tumor samples detected as cancer. No cross-contamination events were observed. No potential interferent (hemoglobin, bilirubin, triglycerides, genomic DNA) affected performance. The results of this analytical validation study support continued clinical development of a targeted methylation cell-free DNA multi-cancer early detection test.

Absence of keratin 8 confers a paradoxical microflora-dependent resistance to apoptosis in the colon.

Keratin 8 (K8) is a major intermediate filament protein present in enterocytes and serves an antiapoptotic function in hepatocytes. K8-null mice develop colonic hyperplasia and colitis that are reversed after antibiotic treatment. To investigate the pathways that underlie the mechanism of colonocyte hyperplasia and the normalization of the colonic phenotype in response to antibiotics, we performed genome-wide microarray analysis. Functional annotation of genes that are differentially regulated in K8(-/-) and K8(+/+) isolated colon crypts (colonocytes) identified apoptosis as a major altered pathway. Exposure of K8(-/-) colonocytes or colon organ ("organoid") cultures, but not K8(-/-) small intestine organoid cultures, to apoptotic stimuli showed, surprisingly, that they are resistant to apoptosis compared with their wild-type counterparts. This resistance is not related to inflammation per se because T-cell receptor α-null (TCR-α(-/-)) and wild-type colon cultures respond similarly upon induction of apoptosis. Following antibiotic treatment, K8(-/-) colonocytes and organ cultures become less resistant to apoptosis and respond similarly to the wild-type colonocytes. Antibiotics also normalize most differentially up-regulated genes, including survivin and ß4-integrin. Treatment of K8(-/-) mice with anti-ß4-integrin antibody up-regulated survivin, and induced phosphorylation of focal adhesion kinase with decreased activation of caspases. Therefore, unlike the proapoptotic effect of K8 mutation or absence in hepatocytes, lack of K8 confers resistance to colonocyte apoptosis in a microflora-dependent manner.

Zebrafish embryology and cartilage staining protocols for high school students.

The Life Sciences-Howard Hughes Medical Institute Outreach Program at Harvard University supports high school science education by offering an on-campus program for students and their teachers to participate in investigative, hands-on laboratory sessions. The outreach program has recently designed and launched a successful zebrafish embryology protocol that we present here. The main objectives of this protocol are to introduce students to zebrafish as a model research organism and to provide students with direct experience with current techniques used in embryological research. The content of the lab is designed to generate discussions on embryology, genetics, fertilization, natural selection, and animal adaptation. The protocol produces reliable results in a time-efficient manner using a minimum of reagents. The protocol presented here consists of three sections: observations of live zebrafish larvae at different developmental stages, cartilage staining of zebrafish larvae, and a mutant hunt involving identification of two zebrafish mutants (nacre and chokh). Here, we describe the protocol, show the results obtained for each section, and suggest possible alternatives for different lab settings.

Patient-provider race-concordance: does it matter in improving minority patients' health outcomes?

OBJECTIVE: To understand if patient-provider race-concordance is associated with improved health outcomes for minorities. DESIGN: A comprehensive review of published research literature (1980-2008) using MEDLINE, HealthSTAR, and CINAHL databases were conducted. Studies were included if they had at least one research question examining the effect of patient-provider race-concordance on minority patients' health outcomes and pertained to minorities in the USA. The database search and data analysis were each independently conducted by two authors. The review was limited to data analysis in tabular and text format. A meta-analysis was not possible due to the discrepancy in methods and outcomes across studies. RESULTS: Twenty-seven studies met the inclusion criteria. Combined, the studies were based on data from 56,276 patients and only 1,756 providers. Whites/Caucasians (37.6%) and Blacks/African Americans (31.5%), followed by Hispanics/Latinos (13.3%), and Asians/Pacific Islanders (4.3%) comprised the majority of the patient sample. The median sample of providers was only 16 for African Americans, 10 for Asians and two for Hispanics. The review presented mixed results. Of the 27 studies, patient-provider race-concordance was associated with positive health outcomes for minorities in only nine studies (33%), while eight studies (30%) found no association of race-concordance with the outcomes studied and 10 (37%) presented mixed findings. Analysis suggested that having a provider of same race did not improve 'receipt of services' for minorities. No clear pattern of findings emerged in the domains of healthcare utilization, patient-provider communication, preference, satisfaction, or perception of respect. CONCLUSIONS: There is inconclusive evidence to support that patient-provider race-concordance is associated with positive health outcomes for minorities. Studies were limited to four racial/ethnic groups and generally employed small samples of minorities. Further research is needed to understand what health outcomes may be more sensitive to cultural proximity between patients and providers, and what patient, provider and setting-level variables may moderate or mediate these outcomes.

The major yolk protein of sea urchins is endocytosed by a dynamin-dependent mechanism.

Sea urchin oocytes grow to 10 times their original size during oogenesis by both synthesizing and importing a specific repertoire of proteins to drive fertilization and early embryogenesis. During the vitellogenic growth period, the major yolk protein (MYP), a transferrin-like protein, is synthesized in the gut, transported into the ovary, and actively endocytosed by the oocytes. Here, we begin to dissect this mechanism by first testing the hypothesis that MYP endocytosis is dynamin-dependent. We have identified a sea urchin dynamin cDNA that is highly similar in amino acid sequence, structure, and size to mammalian dynamin I: it contains an N-terminal GTPase domain, a pleckstrin-homology domain, and a C-terminal proline-rich domain. Sea urchin dynamin is enriched at the cortex of oocytes and colocalizes to MYP endocytic vesicles at the oocyte periphery. To test for a functional relationship between MYP endocytosis and dynamin, we used a dominant-negative human dynamin I mutant protein containing an alteration within the GTPase domain (hDyn(K44A)) to specifically compete for dynamin function. Using a fluorescent MYP construct to follow its endocytosis solely, as well as a general endocytosis marker, we demonstrate that the disruption of dynamin function significantly reduces MYP uptake but does not affect fluid-phase endocytosis. Using this specific biochemical approach, we are able to separate distinct pathways of endocytosis during oogenesis and learn that dynamin-mediated endocytosis is responsible for MYP endocytosis but not fluid-phase uptake.

Selective transport and packaging of the major yolk protein in the sea urchin.

The major yolk protein of sea urchins is an iron-binding, transferrin-like molecule that is made in the adult gut. Its final destination though is the developing oocytes that are embedded in somatic accessory cells and encompassed by two epithelial layers of the ovary. In this study, we address the dynamics of yolk transport, endocytosis, and packaging during the vitellogenic phase of oogenesis in the sea urchin by use of fluorescently labeled major yolk protein (MYP). Incorporation of MYP into the accessory cells of the ovary and its packaging into yolk platelets of developing oocytes is visualized in isolated oocytes, ovary explants, and in whole animals. When MYP is introduced into the coelom of adult females, it is first accumulated by the somatic cells of the ovarian capsule and is then transported to the oocytes and packaged into yolk platelets. This phenomenon is specific for MYP and accurately reflects the endogenous MYP packaging. We find that oocytes cultured in isolation are endocytically active and capable of selectively packaging MYP into yolk platelets. Furthermore, oocytes that packaged exogenous MYP are capable of in vitro maturation, fertilization, and early development, enabling an in vivo documentation of MYP utilization and yolk platelet dynamics. These results demonstrate that the endocytic uptake of yolk proteins in sea urchins does not require a signal from their surrounding epithelial cells and can occur autonomous of the ovary. In addition, these results demonstrate that the entire population of yolk platelets is competent to receive new yolk protein input, suggesting that they are all made simultaneously during oogenesis.

The major yolk protein in sea urchins is a transferrin-like, iron binding protein.

The major yolk protein (MYP) in sea urchins has historically been classified as a vitellogenin based on its abundance in the yolk platelets. Curiously, it is found in both sexes of sea urchins where it is presumed to play a physiological role in gametogenesis, embryogenesis, or both. Here we present the primary structure of MYP as predicted from cDNAs of two sea urchins species, Strongylocentrotus purpuratus and Lytechinus variegatus. The sequence from these two species share identity to one another, but bear no resemblance to other known vitellogenins. Instead the sequence shares identity to members of the transferrin superfamily of proteins. In vitro iron binding assays, including both (59)Fe overlay assays of MYP enriched coelomic fluid and immunoprecipitation of native iron-bound MYP from coelomic fluid, support this classification. We suggest that one of MYP's transferrin-like properties is to shuttle iron to developing germ cells.