A randomized controlled trial of the impact of the Teen Council peer education program on youth development.

This paper presents results of an impact evaluation of Teen Council, a program that trains youth as peer educators. Teen Council is designed to help peer educators make healthy sexual and reproductive decisions, increase their confidence and abilities to educate their peers and inspire them to advocate for just sexual policies. The program's impact on these educators was evaluated using a randomized controlled trial. Over 5 years, interested high school students in seven states were randomly assigned to a study condition. An intent-to-treat framework using ordinary least square (OLS) regression was employed to measure program effects. Relative to control, Teen Council youth showed enhanced comfort with their own sexuality, greater comfort with and more frequent communication with parents about sexuality and more positive sexual health behaviors, including accessing reproductive health care and adopting more effective means of contraception. Teen Council youth also reported greater confidence in talking with peers about sexuality and more confidence in their civic engagement skills.

Novel axon projection after stress and degeneration in the Dscam mutant retina.

The Down syndrome cell adhesion molecule gene (Dscam) is required for normal dendrite patterning and promotes developmental cell death in the mouse retina. Loss-of-function studies indicate that Dscam is required for refinement of retinal ganglion cell (RGC) axons in the lateral geniculate nucleus, and in this study we report and describe a requirement for Dscam in the maintenance of RGC axon projections within the retina. Mouse Dscam loss of function phenotypes related to retinal ganglion cell axon outgrowth and targeting have not been previously reported, despite the abundance of axon phenotypes reported in Drosophila Dscam1 loss and gain of function models. Analysis of the Dscam deficient retina was performed by immunohistochemistry and Western blot analysis during postnatal development of the retina. Conditional targeting of Dscam and Jun was performed to identify factors underlying axon-remodeling phenotypes. A subset of RGC axons were observed to project and branch extensively within the Dscam mutant retina after eye opening. Axon remodeling was preceded by histological signs of RGC stress. These included neurofilament accumulation, axon swelling, axon blebbing and activation of JUN, JNK and AKT. Novel and extensive projection of RGC axons within the retina was observed after upregulation of these markers, and novel axon projections were maintained to at least one year of age. Further analysis of retinas in which Dscam was conditionally targeted with Brn3b or Pax6α Cre indicated that axon stress and remodeling could occur in the absence of hydrocephalus, which frequently occurs in Dscam mutant mice. Analysis of mice mutant for the cell death gene Bax, which executes much of Dscam dependent cell death, identified a similar axon misprojection phenotype. Deleting Jun and Dscam resulted in increased axon remodeling compared to Dscam or Bax mutants. Retinal ganglion cells have a very limited capacity to regenerate after damage in the adult retina, compared to the extensive projections made in the embryo. In this study we find that DSCAM and JUN limit ectopic growth of RGC axons, thereby identifying these proteins as targets for promoting axon regeneration and reconnection.

Tumourigenic non-small-cell lung cancer mesenchymal circulating tumour cells: a clinical case study.

BACKGROUND: Over the past decade, numerous reports describe the generation and increasing utility of non-small-cell lung cancer (NSCLC) patient-derived xenografts (PDX) from tissue biopsies. While PDX have proven useful for genetic profiling and preclinical drug testing, the requirement of a tissue biopsy limits the available patient population, particularly those with advanced oligometastatic disease. Conversely, 'liquid biopsies' such as circulating tumour cells (CTCs) are minimally invasive and easier to obtain. Here, we present a clinical case study of a NSCLC patient with advanced metastatic disease, a never smoker whose primary tumour was EGFR and ALK wild-type. We demonstrate for the first time, tumorigenicity of their CTCs to generate a patient CTC-derived eXplant (CDX). PATIENTS AND METHODS: CTCs were enriched at diagnosis and again 2 months later during disease progression from 10 ml blood from a 48-year-old NSCLC patient and implanted into immunocompromised mice. Resultant tumours were morphologically, immunohistochemically, and genetically compared with the donor patient's diagnostic specimen. Mice were treated with cisplatin and pemetrexed to assess preclinical efficacy of the chemotherapy regimen given to the donor patient. RESULTS: The NSCLC CDX expressed lung lineage markers TTF1 and CK7 and was unresponsive to cisplatin and pemetrexed. Examination of blood samples matched to that used for CDX generation revealed absence of CTCs using the CellSearch EpCAM-dependent platform, whereas size-based CTC enrichment revealed abundant heterogeneous CTCs of which ∼80% were mesenchymal marker vimentin positive. Molecular analysis of the CDX, mesenchymal and epithelial CTCs revealed a common somatic mutation confirming tumour origin and showed CDX RNA and protein profiles consistent with the predominantly mesenchymal phenotype. CONCLUSIONS: This study shows that the absence of NSCLC CTCs detected by CellSearch (EpCAM(+)) does not preclude CDX generation, highlighting epithelial to mesenchymal transition and the functional importance of mesenchymal CTCs in dissemination of this disease.

Perceptions of D.M.D. student readiness for basic science courses in the United States: can online review modules help?

There can be a disconnect between the level of content covered in undergraduate coursework and the expectations of professional-level faculty of their incoming students. Some basic science faculty members may assume that students have a good knowledge base in the material and neglect to appropriately review, whilst others may spend too much class time reviewing basic material. It was hypothesised that the replacement of introductory didactic physiology lectures with interactive online modules could improve student preparedness prior to lectures. These modules would also allow faculty members to analyse incoming student abilities and save valuable face-to-face class time for alternative teaching strategies. Results indicated that the performance levels of incoming U.S. students were poor (57% average on a pre-test), and students often under-predicted their abilities (by 13% on average). Faculty expectations varied greatly between the different content areas and did not appear to correlate with the actual student performance. Three review modules were created which produced a statistically significant increase in post-test scores (46% increase, P < 0.0001, n = 114-115). The positive results of this study suggest a need to incorporate online review units in the basic science dental school courses and revise introductory material tailored to students' strengths and needs.

Dental student perception and assessment of their clinical knowledge in educating patients about preventive dentistry.

In today's dental school curricula, an increasing amount of time is dedicated to technological advances, and preventive dentistry topics may not be adequately addressed. Freshman (D1) students participated in a new Introduction to Preventive Dentistry course, which consisted of didactic lectures, active learning breakout sessions and case-based studies. The goal of this study was to determine if D1 dental students completing the course had a better knowledge and comfort level with basic preventive dentistry concepts and caries risk assessment than the upcoming graduating senior dental students. Following the completion of the course, D1 students were administered a survey that assessed their comfort level describing preventive dentistry topics to patients. This was immediately followed by an unannounced examination over the same topics. Senior (D4) students, who had not taken a formal course, reported statistically significant higher comfort levels than D1 students. However, the D4s scored significantly lower in all of the examination areas than the D1 students. Higher scores in D1s may have been due to recent exposure to the course material. However, the basic nature of the content-specific questions should be easily answered by novice practitioners educating their patients on oral disease prevention. As the current data shows lower content-specific scores of basic preventive dentistry knowledge amongst graduating D4 students, this may indicate a need for more guidance and education of students during the patient care. This study showed that implementation of a formalised course for D1 students can successfully ameliorate deficiencies in knowledge of preventive dentistry topics.

Tunneled island pedicle flap reconstruction for upper lateral cutaneous lip defects.

BACKGROUND: Esthetic upper lateral cutaneous lip reconstruction preserves the apical triangle, nasolabial fold symmetry, and free margin position. The tunneled island pedicle flap (IPF) is a novel single-stage reconstruction to achieve these goals. OBJECTIVES: Describe the technique and patient and surgeon-reported outcomes for the tunneled IPF reconstruction of upper lateral cutaneous lip defects. METHODS: Retrospective chart review of consecutive tunneled IPF reconstruction following Mohs micrographic surgery (MMS) at a tertiary care center between 2014 and 2020. Patients rated their scars using the validated Patient Scar Assessment Scale (PSAS), and independent surgeons rated scars using the validated Observer Scar Assessment Scale (OSAS). Descriptive statistics were generated for patient demographics and tumor defect characteristics. RESULTS: Twenty upper lateral cutaneous lip defects were repaired with the tunneled IPF. Surgeons rated scars with a composite OSAS score of 11.83 ± 4.29 (mean, SD) [scale of 5 (normal skin) to 50 (worst scar imaginable)] and an overall scar score of 2.81 ± 1.11 [scale of 1 (normal skin) to 10 (worst scar imaginable)]. Patients rated their scars with a composite PSAS score of 10 ± 5.39 [scale of 6 (best possible score) to 60 (worst)] and with an overall score of 2.2 ± 1.78 [scale of 1 (normal skin) and 10 (very different from normal skin)]. One flap was surgically revised for pincushioning, but none experienced necrosis, hematoma, or infection. CONCLUSIONS: The tunneled IPF is a single-stage reconstruction for upper lateral cutaneous lip defects with favorable scar ratings by patients and observers.

Enhanced stability of microRNA expression facilitates classification of FFPE tumour samples exhibiting near total mRNA degradation.

BACKGROUND: As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits the ability to profile mRNA expression, we explored factors predicting the success of mRNA expression profiling of FFPE material and investigated an approach to overcome the limitation. METHODS: Bladder (n=140, stored 3-8 years) and cervix (n=160, stored 8-23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon 1.0ST arrays. Percentage detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. As miR-205 had been identified as a marker of SCC, precursor mir-205 was measured by Exon array and mature miR-205 by qRT-PCR. Genome-wide microRNA (miRNA) expression (Affymetrix miRNA v2.0 arrays) was compared in eight newer FFPE samples with biological signal and eight older samples without. RESULTS: RNA quality controls (QCs) (e.g., RNA integrity (RIN) number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R=-0.30, P<0.01) and cervix (R=-0.69, P<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT-PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; P=1.10 × 10(-5)). Genome-wide miRNA (R=0.95) and small nucleolar RNA (R=0.97) expression correlated well in the eight newer vs older FFPE samples and better than mRNA expression (R=0.72). CONCLUSION: Sample age is the best predictor of successful mRNA profiling of FFPE material, and miRNA profiling overcomes the limitation of age and copes well with older samples.

Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples.

BACKGROUND: Degradation and chemical modification of RNA in formalin-fixed paraffin-embedded (FFPE) samples hamper their use in expression profiling studies. This study aimed to show that useful information can be obtained by Exon-array profiling archival FFPE tumour samples. METHODS: Nineteen cervical squamous cell carcinoma (SCC) and 9 adenocarcinoma (AC) FFPE samples (10-16-year-old) were profiled using Affymetrix Exon arrays. The gene signature derived was tested on a fresh-frozen non-small cell lung cancer (NSCLC) series. Exploration of biological networks involved gene set enrichment analysis (GSEA). Differential gene expression was confirmed using Quantigene, a multiplex bead-based alternative to qRT-PCR. RESULTS: In all, 1062 genes were higher in SCC vs AC, and 155 genes higher in AC. The 1217-gene signature correctly separated 58 NSCLC into SCC and AC. A gene network centered on hepatic nuclear factor and GATA6 was identified in AC, suggesting a role in glandular cell differentiation of the cervix. Quantigene analysis of the top 26 differentially expressed genes correctly partitioned cervix samples as SCC or AC. CONCLUSION: FFPE samples can be profiled using Exon arrays to derive gene expression signatures that are sufficiently robust to be applied to independent data sets, identify novel biology and design assays for independent platform validation.

Increased inherent intestinal granzyme B expression may be associated with SIV pathogenesis in Asian non-human primates.

BACKGROUND: Unlike Asian non-human primates, chronically SIV-infected African non-human primates (NHP) display a non-pathogenic disease course. The different outcomes may be related to the development of an SIV-mediated breach of the intestinal mucosa in the Asian species that is absent in the African animals. METHODS: To examine possible mechanisms that could lead to the gut breach, we determined whether the colonic lamina propria (LP) of SIV-naïve Asian monkeys contained more granzyme B (GrB) producing CD4 T cells than did that of the African species. GrB is a serine protease that may disrupt mucosal integrity by damaging tight junction proteins. RESULTS: We found that the colonic LP of Asian NHP contain more CD4(+) /GrB(+) cells than African NHP. We also observed reduced CD4 expression on LP T cells in African green monkeys. CONCLUSION: Both phenotypic differences could protect against SIV-mediated damage to the intestinal mucosa and could lead to future therapies in HIV(+) humans.

Progesterone implants enhance SIV vaginal transmission and early virus load.

Simian immunodeficiency virus (SIV) can cross the intact vaginal epithelium to establish a systemic infection in macaques (mac). Using this SIVmac model, we found that subcutaneous progesterone implants, which could mimic hormonally based contraceptives, thinned the vaginal epithelium and enhanced SIV vaginal transmission 7.7-fold over that observed in macaques treated with placebo implants and exposed to SIV in the follicular phase of the menstrual cycle. Progesterone treatment also increased the number of SIV DNA-positive cells in the vaginal lamina propria as detected by in situ polymerase chain reaction analysis. Moreover, plasma viral RNA was elevated for the first three months in macaques with progesterone implants, and three of the progesterone-treated macaques developed relatively rapid disease courses. This study shows that SIV genital infection and disease course are enhanced by subcutaneous implants containing progesterone when compared with the rate of vaginal transmission in the follicular phase.

Range of motion measurements diverge with increasing age for COL5A1 genotypes.

Increased and decreased joint range of motion (ROM) are modifiable risk factors for musculoskeletal soft-tissue injuries. Certain heritable disorders of connective tissue, which have a unifying symptom of joint hypermobility, are caused by mutations within the COL5A1 gene. Furthermore, the COL5A1 BstUI restriction fragment length polymorphism (RFLP) sequence variant is associated with ROM measurements in a mixed injured/uninjured cohort. The association between COL5A1 BstUI RFLP and sit and reach (SR) ROM in an apparently healthy and physically active cohort was investigated. The SR test was performed on 325 white subjects (204 males). Subjects were also genotyped for the BstUI RFLP (C/T) within the 3'-untranslated region of the COL5A1 gene. The COL5A1 BstUI RFLP genotype was associated with SR ROM in older (≥35 years) subjects (TT: 225 ± 96 mm, TC: 245 ± 100 mm, CC: 32 ± 108 mm, N=96, P=0.017). Age and COL5A1 BstUI genotype interacted significantly for SR ROM. Sex and COL5A1 genotype accounted for 22.8% of the variance in SR ROM in the older group. The COL5A1 BstUI RFLP is associated with SR ROM, particularly with increasing age and is an important contributing factor to ROM variation, particularly in older, apparently healthy and physically active individuals.

Large meta-analysis of multiple cancers reveals a common, compact and highly prognostic hypoxia metagene.

BACKGROUND: There is a need to develop robust and clinically applicable gene expression signatures. Hypoxia is a key factor promoting solid tumour progression and resistance to therapy; a hypoxia signature has the potential to be not only prognostic but also to predict benefit from particular interventions. METHODS: An approach for deriving signatures that combine knowledge of gene function and analysis of in vivo co-expression patterns was used to define a common hypoxia signature from three head and neck and five breast cancer studies. Previously validated hypoxia-regulated genes (seeds) were used to generate hypoxia co-expression cancer networks. RESULTS: A common hypoxia signature, or metagene, was derived by selecting genes that were consistently co-expressed with the hypoxia seeds in multiple cancers. This was highly enriched for hypoxia-regulated pathways, and prognostic in multivariate analyses. Genes with the highest connectivity were also the most prognostic, and a reduced metagene consisting of a small number of top-ranked genes, including VEGFA, SLC2A1 and PGAM1, outperformed both a larger signature and reported signatures in independent data sets of head and neck, breast and lung cancers. CONCLUSION: Combined knowledge of multiple genes' function from in vitro experiments together with meta-analysis of multiple cancers can deliver compact and robust signatures suitable for clinical application.

rHVDM: an R package to predict the activity and targets of a transcription factor.

SUMMARY: Highly parallel genomic platforms like microarrays often present researchers with long lists of differentially expressed genes but contain little or no information on how these genes are regulated. rHVDM is a novel R package which uses gene expression time course data to predict the activity and targets of a transcription factor. In the first step, rHVDM uses a small number of known targets to derive the activity profile of a given transcription factor. Then, in a subsequent step, this activity profile is used to predict other putative targets of that transcription factor. A dynamic and mechanistic model of gene expression is at the heart of the technique. Measurement error is taken into account during the process, which allows an objective assessment of the robustness of fit and, therefore, the quality of the predictions. The package relies on efficient algorithms and vectorization to accomplish potentially time consuming tasks including optimization and differential equation integration. We demonstrate the efficiency and accuracy of rHVDM by examining the activity of the tumour-suppressing transcription factor, p53. AVAILABILITY: The version of the package presented here (1.8.1) is freely available from the Bioconductor Web site (http://bioconductor.org/packages/2.3/bioc/html/rHVDM.html).

Antiviral CD8+ T cells in the genital tract control viral replication and delay progression to AIDS after vaginal SIV challenge in rhesus macaques immunized with virulence attenuated SHIV 89.6.

The recently failed clinical efficacy trial of an acquired immunodeficiency syndrome (AIDS) vaccine that elicits antiviral CD8(+) T-cell responses has emphasized the challenge of producing an effective vaccine against human immunodeficiency virus (HIV). In the simian immunodeficiency virus (SIV)/ rhesus monkey model of AIDS, live-attenuated lentivirus 'vaccines' provide the best protection from uncontrolled viral replication and clinical disease after pathogenic SIV challenge. This review summarizes a recent series of studies in which we show that after vaginal SIV challenge of rhesus macaques immunized with an attenuated lentivirus protection from uncontrolled viral replication is primarily mediated by CD8(+) T cells in the vaginal mucosa. Immunization with a chimeric simian/human immunodeficiency virus (SHIV) results in a systemic infection that induces a moderate population of SIV-specific CD8(+) and CD4(+) T cells with cytolytic potential in the vaginal mucosa. Depletion of CD8(+) T cells at the time of SIV challenge completely abrogates the protection mediated by prior infection with attenuated SHIV. Further after vaginal SIV challenge, the only significant expansion of SIV-specific T cells occurs in the vagina in these animals. No significant expansion of T-cell responses was observed in systemic lymphoid tissues. Thus, the presence of SIV-specific CD8(+) T cells in the vagina on the day of vaginal SIV challenge and a modest expansion of local effector T cells is sufficient to stop uncontrolled SIV replication. It seems that T-cell based vaccine strategies that can elicit mucosal effector CD8(+) T-cell populations and avoid inducing systemic T-cell proliferation upon exposure to HIV have the greatest potential for mimicking the success of live-attenuated lentiviral vaccines.

Acoustic oscillations in the early universe and today.

During its first approximately 100,000 years, the universe was a fully ionized plasma with a tight coupling by Thompson scattering between the photons and matter. The trade-off between gravitational collapse and photon pressure causes acoustic oscillations in this primordial fluid. These oscillations will leave predictable imprints in the spectra of the cosmic microwave background and the present-day matter-density distribution. Recently, the BOOMERANG and MAXIMA teams announced the detection of these acoustic oscillations in the cosmic microwave background (observed at redshift approximately 1000). Here, we compare these CMB detections with the corresponding acoustic oscillations in the matter-density power spectrum (observed at redshift approximately 0.1). These consistent results, from two different cosmological epochs, provide further support for our standard Hot Big Bang model of the universe.

Engineering poliovirus as a vaccine vector for the expression of diverse antigens.

As a step toward developing poliovirus as a vaccine vector, poliovirus recombinants were constructed by fusing exogenous peptides (up to 400 amino acids) and an artificial cleavage site for viral protease 3Cpro to the amino terminus of the viral polyprotein. Viral replication proceeded normally. An extended polyprotein was produced in infected cells and proteolytically processed into the complete array of viral proteins plus the foreign peptide, which was excluded from mature virions. The recombinants retained exogenous sequences through successive rounds of replication in culture and in vivo. Infection of animals with recombinants elicited a humoral immune response to the foreign peptides.

A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins.

Analysis of rhesus macaque leukocytes disclosed the presence of an 18-residue macrocyclic, tridisulfide antibiotic peptide in granules of neutrophils and monocytes. The peptide, termed rhesus theta defensin-1 (RTD-1), is microbicidal for bacteria and fungi at low micromolar concentrations. Antibacterial activity of the cyclic peptide was threefold greater than that of an open-chain analog, and the cyclic conformation was required for antimicrobial activity in the presence of 150 millimolar sodium chloride. Biosynthesis of RTD-1 involves the head-to-tail ligation of two alpha-defensin-related nonapeptides, requiring the formation of two new peptide bonds. Thus, host defense cells possess mechanisms for synthesis and granular packaging of macrocyclic antibiotic peptides that are components of the phagocyte antimicrobial armamentarium.

Sexual transmission and propagation of SIV and HIV in resting and activated CD4+ T cells.

In sexual transmission of simian immunodeficiency virus, and early and later stages of human immunodeficiency virus-type 1 (HIV-1) infection, both viruses were found to replicate predominantly in CD4(+) T cells at the portal of entry and in lymphoid tissues. Infection was propagated not only in activated and proliferating T cells but also, surprisingly, in resting T cells. The infected proliferating cells correspond to the short-lived population that produces the bulk of HIV-1. Most of the HIV-1-infected resting T cells persisted after antiretroviral therapy. Latently and chronically infected cells that may be derived from this population pose challenges to eradicating infection and developing an effective vaccine.

Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours.

Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low ( approximately 50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)-PCR was confirmed, especially for abundant transcripts, and RT-PCR validated the regulation pattern for 19 of 24 candidate genes (overall R(2)=0.4662). RT-PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET - whose combined expression carried greater prognostic value than tumour grade - and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.

Micron-pore-sized metallic filter tube membranes for filtration of particulates and water purification.

Robust filtering techniques capable of efficiently removing particulates and biological agents from water or air suffer from plugging, poor rejuvenation, low permeance, and high backpressure. Operational characteristics of pressure-driven separations are in part controlled by the membrane pore size, charge of particulates, transmembrane pressure and the requirement for sufficient water flux to overcome fouling. With long term use filters decline in permeance due to filter-cake plugging of pores, fouling, or filter deterioration. Though metallic filter tube development at ORNL has focused almost exclusively on gas separations, a small study examined the applicability of these membranes for tangential filtering of aqueous suspensions of bacterial-sized particles. A mixture of fluorescent polystyrene microspheres ranging in size from 0.5 to 6 microm in diameter simulated microorganisms in filtration studies. Compared to a commercial filter, the ORNL 0.6 microm filter averaged approximately 10-fold greater filtration efficiency of the small particles, several-fold greater permeance after considerable use and it returned to approximately 85% of the initial flow upon backflushing versus 30% for the commercial filter. After filtering several liters of the particle-containing suspension, the ORNL composite filter still exhibited greater than 50% of its initial permeance while the commercial filter had decreased to less than 20%. When considering a greater filtration efficiency, greater permeance per unit mass, greater percentage of rejuvenation upon backflushing (up to 3-fold), and likely greater performance with extended use, the ORNL 0.6 microm filters can potentially outperform the commercial filter by factors of 100-1,000 fold.