Time-resolved imaging-based CRISPRi screening.

Our ability to connect genotypic variation to biologically important phenotypes has been seriously limited by the gap between live-cell microscopy and library-scale genomic engineering. Here, we show how in situ genotyping of a library of strains after time-lapse imaging in a microfluidic device overcomes this problem. We determine how 235 different CRISPR interference knockdowns impact the coordination of the replication and division cycles of Escherichia coli by monitoring the location of replication forks throughout on average >500 cell cycles per knockdown. Subsequent in situ genotyping allows us to map each phenotype distribution to a specific genetic perturbation to determine which genes are important for cell cycle control. The single-cell time-resolved assay allows us to determine the distribution of single-cell growth rates, cell division sizes and replication initiation volumes. The technology presented in this study enables genome-scale screens of most live-cell microscopy assays.

In situ genotyping of a pooled strain library after characterizing complex phenotypes.

In this work, we present a proof-of-principle experiment that extends advanced live cell microscopy to the scale of pool-generated strain libraries. We achieve this by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single-molecule fluorescence time-lapse imaging of Escherichia coli strains harboring barcoded plasmids that express a sgRNA which suppresses different genes in the E. coli genome through dCas9 interference. In general, the method solves the problem of characterizing complex dynamic phenotypes for diverse genetic libraries of cell strains. For example, it allows screens of how changes in regulatory or coding sequences impact the temporal expression, location, or function of a gene product, or how the altered expression of a set of genes impacts the intracellular dynamics of a labeled reporter.

Involvement in Bullying During High School: A Survival Analysis Approach.

Knowledge about the risks of bullying involvement during any year of high school is an important element of interventions for changing the likelihood of being bullied. Three cohorts of Australian students (n = 1,382) were tracked from 7th grade to 11th grade. The study showed that some students continue their involvement in bullying, while in addition, new bullies and new victims emerge during each high school year. The findings indicated that the risk of bullying involvement ranged from 16% (as a bully) to 36% (as a victim), increasing to 54.5% and 56.3%, respectively, if a student was a bully or a victim in 7th grade. The risk to students of becoming victims, bullies, or bully-victims in each year of high school suggests that bullying prevention initiatives should be designed to suit students at different stages of adolescent development.

Selecting one of several mating types through gene segment joining and deletion in Tetrahymena thermophila.

The unicellular eukaryote Tetrahymena thermophila has seven mating types. Cells can mate only when they recognize cells of a different mating type as non-self. As a ciliate, Tetrahymena separates its germline and soma into two nuclei. During growth the somatic nucleus is responsible for all gene transcription while the germline nucleus remains silent. During mating, a new somatic nucleus is differentiated from a germline nucleus and mating type is decided by a stochastic process. We report here that the somatic mating type locus contains a pair of genes arranged head-to-head. Each gene encodes a mating type-specific segment and a transmembrane domain that is shared by all mating types. Somatic gene knockouts showed both genes are required for efficient non-self recognition and successful mating, as assessed by pair formation and progeny production. The germline mating type locus consists of a tandem array of incomplete gene pairs representing each potential mating type. During mating, a complete new gene pair is assembled at the somatic mating type locus; the incomplete genes of one gene pair are completed by joining to gene segments at each end of germline array. All other germline gene pairs are deleted in the process. These programmed DNA rearrangements make this a fascinating system of mating type determination.

Spatial stochastic dynamics enable robust cell polarization.

Although cell polarity is an essential feature of living cells, it is far from being well-understood. Using a combination of computational modeling and biological experiments we closely examine an important prototype of cell polarity: the pheromone-induced formation of the yeast polarisome. Focusing on the role of noise and spatial heterogeneity, we develop and investigate two mechanistic spatial models of polarisome formation, one deterministic and the other stochastic, and compare the contrasting predictions of these two models against experimental phenotypes of wild-type and mutant cells. We find that the stochastic model can more robustly reproduce two fundamental characteristics observed in wild-type cells: a highly polarized phenotype via a mechanism that we refer to as spatial stochastic amplification, and the ability of the polarisome to track a moving pheromone input. Moreover, we find that only the stochastic model can simultaneously reproduce these characteristics of the wild-type phenotype and the multi-polarisome phenotype of a deletion mutant of the scaffolding protein Spa2. Significantly, our analysis also demonstrates that higher levels of stochastic noise results in increased robustness of polarization to parameter variation. Furthermore, our work suggests a novel role for a polarisome protein in the stabilization of actin cables. These findings elucidate the intricate role of spatial stochastic effects in cell polarity, giving support to a cellular model where noise and spatial heterogeneity combine to achieve robust biological function.

Prospective markers for early diagnosis and prognosis of sporadic pancreatic ductal adenocarcinoma.

BACKGROUND AND AIM: Sporadic pancreatic ductal adenocarcinoma (PDA) is a highly lethal cancer. No proven screening strategies are available and frequent cross-sectional imaging studies (CT/MRI) are impractical even in patients thought to be at higher risk than the general population. Few PDA biomarkers have been studied prospectively for screening. Here, we prospectively evaluated the Adnab-9 monoclonal antibody in stool, pancreaticobiliary secretions, and tissue for screening and prognostic value in sporadic PDA. We also evaluated the prognostic value of characterized early biomarkers in pancreaticobiliary secretions. METHODS: Adnab-9 diagnostic ability was tested in stool in 249 and 1,132 patients from China and the US, respectively. Immunohistochemistry was performed in 22 tissue samples with Adnab-9 antibody and anti-Defensin 5, a constituent of Paneth cells. Pancreatobiliary secretions were collected from 12 PDA patients and 9 controls. The enriched PCR method was performed to detect K-ras mutations. ELISA was performed with Adnab-9, anti-Her-2/neu, and monoclonal antibody D4 (anti-Reg I). RESULTS: Adnab-9 alone was diagnostic and prognostic when measured in pancreatic secretions, feces, and tissues of PDA patients compared to controls (p < 0.05). Significantly, Adnab-9 fecal binding can precede the clinical diagnosis by 2.3 years, potentially allowing earlier clinical intervention. In pancreatic secretions, a combination of K-ras and Her-2/neu when appropriately standardized can be diagnostic in 75 % of PDA. CONCLUSIONS: Our study suggests that Adnab-9 may be an effective marker for diagnosis and prognosis of PDA. Adnab-9 may be reflective of the presence of Paneth cells confirmed by Defensin-5 staining. These cells may modulate the biological activity of the cancer and confer a better prognosis.

"The Terrors of Kingly Power": The Unusual Dental Pathology of King Pyrrhus of Epirus.

Pyrrhus of Epirus, widely respected and feared by his contemporaries, was a legendary figure in the ancient world. In this paper, we investigate Plutarch's description of the king's unique dental pathology. There are several possibilities to explain the ancient king's presentation, including several different types of developmental dysplasia. However, our conclusion is that it was likely due to a significant dental calculus overgrowth, often seen in the ancient Greek diet of the time. Whatever the underlying cause, Pyrrhus' intimidating visage helped secure the king a legacy that lasts to this day.

A Case of Esophago-Respiratory Fistula due to Inhalation Smoke Injury Diagnosed by Upper Endoscopy.

Esophago-respiratory fistula (ERF) refers to the formation of a pathological connection between the esophagus and respiratory tract. Acquired ERF is a rare but life-threatening diagnosis in adults. We describe a 79-year-old male who was admitted with an inhalation smoke injury. He was diagnosed with ERF by endoscopic visualization and sampling of the hyaline cartilage within the wall of the esophagus. Percutaneous endoscopic gastrostomy placement and conservative measures were effective in the management of ERF.

Implementation quality of whole-school mental health promotion and students' academic performance.

BACKGROUND: This paper argues for giving explicit attention to the quality of implementation of school-wide mental health promotions and examines the impact of implementation quality on academic performance in a major Australian mental health initiative. METHOD: Hierarchical linear modelling was used to investigate change in standardised academic performance across the 2-year implementation of a mental health initiative in 96 Australian primary (or elementary) schools that was focused on improving student social-emotional competencies. RESULTS: After controlling for differences in socioeconomic background, a significant positive relationship existed between quality of implementation and academic performance. The difference between students in high- and low-implementing schools was equivalent to a difference in academic performance of up to 6 months of schooling. KEY PRACTITIONER MESSAGE: Given the known relationship between student academic achievement and mental health, many nations are mounting school-based mental health interventions: however, the quality of program implementation remains a concernThe Australian KidsMatter primary school mental health intervention enabled the development of an Implementation Index allowing schools to be grouped into low- to high- implementing schoolsThe quality of implementation of KidsMatter appears to be positively associated with the level of student academic achievement, equivalent to 6 months more schooling by Year 7, over and above any influence of socioeconomic background.

A positive feedback loop involving the Spa2 SHD domain contributes to focal polarization.

Focal polarization is necessary for finely arranged cell-cell interactions. The yeast mating projection, with its punctate polarisome, is a good model system for this process. We explored the critical role of the polarisome scaffold protein Spa2 during yeast mating with a hypothesis motivated by mathematical modeling and tested by in vivo experiments. Our simulations predicted that two positive feedback loops generate focal polarization, including a novel feedback pathway involving the N-terminal domain of Spa2. We characterized the latter using loss-of-function and gain-of-function mutants. The N-terminal region contains a Spa2 Homology Domain (SHD) which is conserved from yeast to humans, and when mutated largely reproduced the spa2Δ phenotype. Our work together with published data show that the SHD domain recruits Msb3/4 that stimulates Sec4-mediated transport of Bud6 to the polarisome. There, Bud6 activates Bni1-catalyzed actin cable formation, recruiting more Spa2 and completing the positive feedback loop. We demonstrate that disrupting this loop at any point results in morphological defects. Gain-of-function perturbations partially restored focal polarization in a spa2 loss-of-function mutant without restoring localization of upstream components, thus supporting the pathway order. Thus, we have collected data consistent with a novel positive feedback loop that contributes to focal polarization during pheromone-induced polarization in yeast.

Colorectal cancer in the cotton top tamarin (Saguinus oedipus): how do they evade liver metastasis?

BACKGROUND AND AIM: A major cause of cancer-related deaths is the development of liver metastasis. To better understand the metastatic process, we studied the cotton top tamarin as an animal model, which spontaneously develops colorectal cancer but rarely liver metastasis. METHOD: DNA was extracted from primates and Hot-Start PCR was performed. Sequencing was achieved with Big-Dye Terminator™ Sequencing Kit. Tissue expression and glycosylation studies were also performed for carcinoembryonic antigen family proteins. RESULTS: Sixty-three percent of tamarin carcinoembryonic antigen had PELPK changes essential for carcinoembryonic antigen hepatic uptake. Tamarin carcinoembryonic antigen showed minimal glycosylation. Cotton top tamarin livers showed reduced carcinoembryonic antigen-receptor expression and were devoid of CEACAM1 (BGP) as compared to human liver despite positive expression in cotton top tamarin gallbladder mucosa. Peritumoral regions showed more CEACAM1 in human hepatocyte cytoplasm than in biliary canaliculi (P < 0.05). Therefore, tamarins may evade liver metastasis through mechanisms of decreased hepatic uptake by altered PELPK sequences, reduced glycosylation and reduced carcinoembryonic antigen-receptor expression. Furthermore, the absence of cotton top tamarin hepatocyte CEACAM1 may lead to alteration of the liver milieu creating an inhospitable "infertile-field" for metastases. CONCLUSIONS: Four hypotheses explain a complex mechanism for the lack of liver metastasis: (1) carcinoembryonic antigen PELPK-encoding nucleotide sequence changes, (2) minimal carcinoembryonic antigen glycosylation, (3) reduced carcinoembryonic antigen-receptor expression, and (4) reduced CEACAM1 distribution, a putative vascular endothelial growth factor. While these hypotheses are not necessarily causal they are testable and therefore are feasible targets for prevention of hepatic metastasis in man.

Social groups and children's intergroup attitudes: can school norms moderate the effects of social group norms?

The effects of social group norms (inclusion vs. exclusion vs. exclusion-plus-relational aggression) and school norms (inclusion vs. no norm) on 7- and 10-year-old children's intergroup attitudes were examined. Children (n = 383) were randomly assigned to a group with an inclusion or exclusion norm, and to 1 of the school norm conditions. Findings indicated that children's out-group attitudes reflected their group's norm but, with increasing age, they liked their in-group less, and the out-group more, if the group had an exclusion norm. The school inclusion norm instigated more positive attitudes toward out-group members, but it did not moderate or extinguish contrary group norms. The use of school norms to counteract the effects of children's social group norms is discussed.

The diffusive finite state projection algorithm for efficient simulation of the stochastic reaction-diffusion master equation.

We have developed a computational framework for accurate and efficient simulation of stochastic spatially inhomogeneous biochemical systems. The new computational method employs a fractional step hybrid strategy. A novel formulation of the finite state projection (FSP) method, called the diffusive FSP method, is introduced for the efficient and accurate simulation of diffusive transport. Reactions are handled by the stochastic simulation algorithm.

Effects of information about group members on young children's attitudes towards the in-group and out-group.

Research shows that being a member of a group is sufficient to instigate more positive attitudes towards the in-group than an out-group in young children. The present study assessed whether children's intergroup attitudes during the middle childhood years are moderated by additional information about in-group and out-group members, as proposed by Aboud's (1988) socio-cognitive theory (ST). To a minimal group 6-, 8-, and 10-year-old children (N = 159) were assigned, and received information, or no information, about the interests and activities of the in-group and out-group members. Results indicated that the in-group was always rated more positively than the out-group, and that the in-group's ratings were unaffected by either the in-group or out-group information. In contrast, out-group ratings were affected by out-group information, but only when there was no information available about the in-group. The implications of the findings for ST, and for social identity development theory, are discussed.

The development of an instrument to test pre-service teachers' beliefs consistent and inconsistent with self-regulation theory.

BACKGROUND: Students are more effective when they employ appropriate strategies to regulate their learning processes and outcomes. However, many teachers do not provide, or provide very little, explicit instructions to promote this self-regulated learning (SRL). This suggests that teachers' belief systems may include beliefs that may be inconsistent with SRL. AIMS: This paper reported the validation of the 78-item Beliefs about Teaching and Learning (BALT) instrument designed to measure pre-service teachers' beliefs about learning and teaching with particular emphasis on SRL. SAMPLES: Out of the 78 items included in the BALT instrument, 50 of them were administered in BALT 1 and 53 in BALT2 with 25 items administered in both surveys and were common to both instruments. The BALT 1 was administered to 430 pre-service teachers. BALT 2 was administered to 366 pre-service students. METHODS: Confirmatory factor analysis and the Rasch model were used to investigate the unidimensionality of the scales and psychometric properties of the items. In addition, the summated rating scale procedure was used to create composite scores for the examination of the coexistence of beliefs both consistent and inconsistent with SRL. RESULTS: Pre-service teachers expressed high overall agreement with items related to beliefs consistent with SRL and low agreement with items related to beliefs inconsistent with SRL. However, many of them also indicated high agreement with items related to beliefs in Transmissive Teaching. CONCLUSIONS: The study resulted in the development of a 58-item instrument, which was balanced with respect to the inclusion of SRL-consistent and SRL-inconsistent items. This study also provided evidence for the coexistence of pre-service teachers' beliefs in Constructive Teaching and Transmissive Teaching.

A Phase IIa Trial of Metformin for Colorectal Cancer Risk Reduction among Individuals with History of Colorectal Adenomas and Elevated Body Mass Index.

Obesity is associated with risk of colorectal adenoma (CRA) and colorectal cancer. The signaling pathway activated by metformin (LKB1/AMPK/mTOR) is implicated in tumor suppression in ApcMin/+ mice via metformin-induced reduction in polyp burden, increased ratio of pAMPK/AMPK, decreased pmTOR/mTOR ratio, and decreased pS6Ser235/S6Ser235 ratio in polyps. We hypothesized that metformin would affect colorectal tissue S6Ser235 among obese patients with recent history of CRA. A phase IIa clinical biomarker trial was conducted via the U.S. National Cancer Institute-Chemoprevention Consortium. Nondiabetic, obese subjects (BMI ≥30) ages 35 to 80 with recent history of CRA were included. Subjects received 12 weeks of oral metformin 1,000 mg twice every day. Rectal mucosa biopsies were obtained at baseline and end-of-treatment (EOT) endoscopy. Tissue S6Ser235 and Ki-67 immunostaining were analyzed in a blinded fashion using Histo score (Hscore) analysis. Among 32 eligible subjects, the mean baseline BMI was 34.9. Comparing EOT to baseline tissue S6Ser235 by IHC, no significant differences were observed. Mean (SD) Hscore at baseline was 1.1 (0.57) and 1.1 (0.51) at EOT; median Hscore change was 0.034 (P = 0.77). Similarly, Ki-67 levels were unaffected by the intervention. The adverse events were consistent with metformin's known side-effect profile. Among obese patients with CRA, 12 weeks of oral metformin does not reduce rectal mucosa pS6 or Ki-67 levels. Further research is needed to determine what effects metformin has on the target tissue of origin as metformin continues to be pursued as a colorectal cancer chemopreventive agent.

Development and verification of a high-fidelity computational fluid dynamics model of canine nasal airflow.

The canine nasal cavity contains a complex airway labyrinth, dedicated to respiratory air conditioning, filtering of inspired contaminants, and olfaction. The small and contorted anatomical structure of the nasal turbinates has, to date, precluded a proper study of nasal airflow in the dog. This study describes the development of a high-fidelity computational fluid dynamics (CFD) model of the canine nasal airway from a three-dimensional reconstruction of high-resolution magnetic resonance imaging scans of the canine anatomy. Unstructured hexahedral grids are generated, with large grid sizes ((10-100) x 10(6) computational cells) required to capture the details of the nasal airways. High-fidelity CFD solutions of the nasal airflow for steady inspiration and expiration are computed over a range of physiological airflow rates. A rigorous grid refinement study is performed, which also illustrates a methodology for verification of CFD calculations on complex unstructured grids in tortuous airways. In general, the qualitative characteristics of the computed solutions for the different grid resolutions are fairly well preserved. However, quantitative results such as the overall pressure drop and even the regional distribution of airflow in the nasal cavity are moderately grid dependent. These quantities tend to converge monotonically with grid refinement. Lastly, transient computations of canine sniffing were carried out as part of a time-step study, demonstrating that high temporal accuracy is achievable using small time steps consisting of 160 steps per sniff period. Here we demonstrate that acceptable numerical accuracy (between approximately 1% and 15%) is achievable with practical levels of grid resolution (approximately 100 x 10(6) computational cells). Given the popularity of CFD as a tool for studying flow in the upper airways of humans and animals, based on this work we recommend the necessity of a grid dependence study and quantification of numerical error when presenting CFD results in complicated airways.

Stool DNA and occult blood testing for screen detection of colorectal neoplasia.

BACKGROUND: Stool DNA testing is a new approach to colorectal cancer detection. Few data are available from the screening setting. OBJECTIVE: To compare stool DNA and fecal blood testing for detection of screen-relevant neoplasia (curable-stage cancer, high-grade dysplasia, or adenomas >1 cm). DESIGN: Blinded, multicenter, cross-sectional study. SETTING: Communities surrounding 22 participating academic and regional health care systems in the United States. PARTICIPANTS: 4482 average-risk adults. MEASUREMENTS: Fecal blood and DNA markers. Participants collected 3 stools, smeared fecal blood test cards and used same-day shipment to a central facility. Fecal blood cards (Hemoccult and HemoccultSensa, Beckman Coulter, Fullerton, California) were tested on 3 stools and DNA assays on 1 stool per patient. Stool DNA test 1 (SDT-1) was a precommercial 23-marker assay, and a novel test (SDT-2) targeted 3 broadly informative markers. The criterion standard was colonoscopy. RESULTS: Sensitivity for screen-relevant neoplasms was 20% by SDT-1, 11% by Hemoccult (P = 0.020), 21% by HemoccultSensa (P = 0.80); sensitivity for cancer plus high-grade dysplasia did not differ among tests. Specificity was 96% by SDT-1, compared with 98% by Hemoccult (P < 0.001) and 97% by HemoccultSensa (P = 0.20). Stool DNA test 2 detected 46% of screen-relevant neoplasms, compared with 16% by Hemoccult (P < 0.001) and 24% by HemoccultSensa (P < 0.001). Stool DNA test 2 detected 46% of adenomas 1 cm or larger, compared with 10% by Hemoccult (P < 0.001) and 17% by HemoccultSensa (P < 0.001). Among colonoscopically normal patients, the positivity rate was 16% with SDT-2, compared with 4% with Hemoccult (P = 0.010) and 5% with HemoccultSensa (P = 0.030). LIMITATIONS: Stool DNA test 2 was not performed on all subsets of patients without screen-relevant neoplasms. Stools were collected without preservative, which reduced detection of some DNA markers. CONCLUSION: Stool DNA test 1 provides no improvement over HemoccultSensa for detection of screen-relevant neoplasms. Stool DNA test 2 detects significantly more neoplasms than does Hemoccult or HemoccultSensa, but with more positive results in colonoscopically normal patients. Higher sensitivity of SDT-2 was particularly apparent for adenomas.

Modeling the Drosophila melanogaster circadian oscillator via phase optimization.

The circadian clock, which coordinates daily physiological behaviors of most organisms, maintains endogenous (approximately 24 h) cycles and simultaneously synchronizes to the 24-h environment due to its inherent robustness to environmental perturbations coupled with a sensitivity to specific environmental stimuli. In this study, the authors develop a detailed mathematical model that characterizes the Drosophila melanogaster circadian network. This model incorporates the transcriptional regulation of period, timeless, vrille , PAR-domain protein 1, and clock gene and protein counterparts. The interlocked positive and negative feedback loops that arise from these clock components are described primarily through mass-action kinetics (with the exception of regulated gene expression) and without the use of explicit time delays. System parameters are estimated via a genetic algorithm-based optimization of a cost function that relies specifically on circadian phase behavior since amplitude measurements are often noisy and do not account for the unique entrainment features that define circadian oscillations. Resulting simulations of this 29-state ordinary differential equation model comply with fitted wild-type experimental data, demonstrating accurate free-running (23.24-h periodic) and entrained (24-h periodic) circadian dynamics. This model also predicts unfitted mutant phenotype behavior by illustrating short and long periodicity, robust oscillations, and arrhythmicity. This mechanistic model also predicts light-induced circadian phase resetting (as described by the phase-response curve) that are in line with experimental observations.