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Based on the National Educational Panel Study (NEPS), the study provides an overview of the distribution of digital literacy in Germany up to the beginning of the Covid pandemic. Already in childhood and adolescence, there are systematic differences in digital literacy depending on socio-economic background. Children with a migration background and those with unemployed parents show particularly low digital literacy. Gender-specific differences in digital literacy are small in childhood and adolescence, but clearly pronounced among adults. In addition, people with little formal education and people with a migration background have systematically lower digital competences in adulthood. The education sector should therefore promote the digital competences of children and young people at an early stage in order to compensate for the apparently low level of competence development outside the formal education sector. Educational opportunities for digitally less competent adults should also be strengthened to enable older generations to continue to participate in the changing spheres of life, education, and work.

Objective: To examine trends in sex behaviors and STD prevalence over time among heterosexual STD clinic populations from 3 urban STD clinics in the United States. Study Design: Cross-sectional analysis comparing baseline data on risk (self-reported) and STDs (laboratory defined) from 2 randomized controlled trials evaluating counseling efficacy conducted about 5 years apart, Project RESPECT (1993-1995) and RESPECT-2 (19992000). Results: The participants from RESPECT (n = 2457) and RESPECT-2 (n = 3080) were demographically similar. However, the proportion of participants reporting any unprotected anal sex was much higher in RESPECT-2 (women: 7% vs. 18%; men: 7% vs. 17%). Also, substantially more participants reported a new sex partner in RESPECT-2 (women: 43% vs. 61%; men: 54% vs. 72%). In addition, more women reported 2 or more partners (37% vs. 48%) and a partner with another concurrent sex partner (19% vs. 32%). Slightly more women and men in RESPECT-2 reported 2 protective behaviors, having an HIV test and any condom use; however, consistent condom use did not differ. Conversely, the proportion of participants with bacterial STDs (chlamydia, gonorrhea, or syphilis) was much lower in RESPECT-2 (women: 24% vs. 18%; men: 38% vs. 24%). Conclusions: Despite substantial promotion of safer sex behaviors over the past decade, many risk behaviors were stable over time, and some behaviors, such as unprotected anal sex, appeared substantially higher. Even in the absence of widespread behavior change, the prevalence of common bacterial STDs appeared to have decreased appreciably.

Background and Objective: HIV prevention counseling has changed behavior and reduced incident sexually transmitted diseases (STDs) in research participants. Goal: This article assesses whether counselor demographics or counselor-client dyad characteristics influenced prevention counseling in Project RESPECT as measured by intervention completion and incident STD after counseling. Study Design: We analyzed data from Project RESPECT, a randomized, controlled trial of HIV counseling interventions in STD clinics. Results: There was no significant association between client failure to complete the intervention and demographic characteristics of the 32 counselors or dyad characteristics. Clients who did not complete the intervention were significantly more likely to acquire a new STD infection by the 12-month visit than those who completed the intervention (adjusted odds ratio, 1.7; confidence interval, 1.2-2.4). There was no significant association between new STDs and counselor characteristics or dyad characteristics. Conclusions: Counselor or counselor-client dyad characteristics evaluated in this study were not associated with intervention completion or the prevention of new STDs.

Background: For two decades, treatment guidelines for sexually transmitted diseases (STDs) have recommended empirical co-treatment for chlamydia when patients are treated for gonorrhea. Because the epidemiology of and diagnostic testing for STDs have changed over time, co-treatment may no longer be needed as a clinical or public health strategy. Objective: To assess the prevalence of chlamydia among patients at STD clinics who are infected with and treated for Neisseria gonorrhoeae and to determine whether co-treatment recommendations are still justified. Design: Cross-sectional analysis of data from a multisite study. Setting: Five public STD clinics (Baltimore, Maryland; Denver, Colorado; Long Beach, California; Newark, New Jersey; and San Francisco, California). July 1993 through October 1995. Patients: 3885 heterosexual patients (2184 men and 1701 women) who agreed to participate in a trial of counseling interventions and had conclusive results from diagnostic tests for gonorrhea and chlamydia performed routinely as part of the trial. Measurements: Infection with Chlamydia trachomatis as determined by polymerase chain reaction. Results: Chlamydia trachomatis was detected in 20% (95% CI, 16% to 24%) of 411 men and 42% (CI, 35% to 50%) of 151 women with laboratory-confirmed N. gonorrhoeae. Chlamydia trachomatis was detected in 19% (CI, 15% to 22%) of 410 men and 35% (CI, 28% to 43%) of 154 women with treatment indications for gonorrhea who would not otherwise have been treated for chlamydia: chlamydia prevalence among these patients was significantly higher than among patients without treatment indications for either gonorrhea or chlamydia: 7% in men and 9% in women (relative risk, 2.58 [CI, 1.92 to 3.47] and 4.12 [CI, 3.05 to 5.57], respectively). Conclusion: The frequent presence of chlamydia among patients at STD clinics who received treatment for gonorrhea, including sex partners of gonorrhea-infected patients, supports continuing current recommendations for co-treatment.

The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective; (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition; (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. Our instrument achieves a resolution of 450 nm laterally and 2 μm axially over an imaging volume of 3,000 × 800 × 300 μm. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophilaegg chamber development, zebrafish whole-brain activity and zebrafish embryonic development – up to nine embryos at a time.

Background: Growing support for the focus of the 1994 HIV Counseling, Testing, and Referral Guidelines on early recognition of HIV infection and the findings of a multicenter, randomized controlled trial establishing the efficacy of the client-centered model of the Centers for Disease Control and Prevention have placed a new focus on the need for the effective delivery of HIV prevention counseling. Goal: The goal of this study was to compare published national guidelines on HIV counseling, testing, and referral with actual practice. Study Design: The study employed a cross-sectional design and involved 51 interviews. Results: Sixty-one percent of sites routinely completed personalized risk-reduction plans. Thirty-one percent of respondents indicated that HIV-positive clients spoke more than the counselor during posttest counseling, and 23% said the same for HIV-negative clients. Sixty-eight percent of respondents indicated that individual risks were discussed in the typical counseling session, whereas 30% reported discussing a combination of general information and individual risks. Most sites met referral standards (86%), found and notified HIV-positive clients who did not return for their test results (85%), and had at least one counselor observation per year (79%). Conclusions: Several measures indicated areas in which practice did not conform to guidelines, which may compromise the potential benefits of the HIV counseling, testing, and referral services.

Background: Many studies measure sex behavior to determine the efficacy of sexually transmitted disease (STD)/HIV prevention interventions. Goal: To determine how well measured behavior reflects STD incidence. Study Design: Data from a trial (Project RESPECT) were analyzed to compare behavior and incidence of STD (gonorrhea, chlamydia, syphilis, HIV) during two 6-month intervals. Results: A total of 2879 persons had 5062 six-monthly STD exams and interviews; 8.9% had a new STD in 6 months. Incidence was associated with demographic factors but only slightly associated with number of partners and number of unprotected sex acts with occasional partners. Many behaviors had paradoxical associations with STD incidence. After combining behavior variables to compare persons with highest and lowest risk behaviors, the STD incidence ratio was only 1.7. Conclusion: Behavioral interventions have prevented STD. We found people tend to have safe sex with risky partners and risky sex with safe partners. Therefore, it is difficult to extrapolate the disease prevention efficacy of an intervention from a measured effect on behavior alone.

Background: In a previous study, we did follow-up on 418 patients who were exposed to tryptophan in 1989, of whom 47 (1 1%) had definite and 63 (9%) possible eosinophilia-myalgia syndrome (EMS). Methods: We assessed mortality and clinical spectrum of illness since 1989 for 242 (58%) of the 418 tryptophan-exposed patients from the original study. To assess outcomes, we used hospital and death records, interviewer-administered questionnaires, physical examinations, and laboratory tests. Results: During the follow-up interval, mortality from all causes was 19% in those with definite EMS, 7% in possible EMS, and 3% in those who were not ill. The age- and sex-adjusted mortality in those with definite EMS was more than 3 times that of the general population or of tryptophan users in the practice who were not ill. Six deaths (66%) among the definite EMS case patients occurred during the 18 months immediately after symptom onset. Compared with the tryptophan users who were not ill, survivors with definite EMS continued to report excess morbidity for 6 major EMS symptoms (myalgia, arthralgia, weakness, rush, alopecia, and sclerodermiform skin changes), but they also reported that the symptom number and severity diminished with time. None of the tryptophan users who were not ill in 1989 developed a symptom complex suggesting new EMS during the follow-up interval. Conclusions: This study assessing a tryptophan-exposed population found those persons who developed EMS during the 1989 epidemic were at increased risk for death, particularly early after disease onset. Survivors reported improvement or resolution of major symptoms, suggesting that the severity of EMS diminishes with time. We found no evidence of delayed onset of EMS in tryptophan users who were not ill in 1989, regardless of the brand used. (Arch Intern Med. 1996;156:973-979)

Supplemental Digital Content is available in the text.Next-generation T-cell therapies will likely continue to utilize T-cell receptors (TCRs) and chimeric antigen receptors (CARs) because each receptor type has advantages. TCRs often possess exceptional properties even when tested unmodified from patients’ T cells. CARs are generally less sensitive, possibly because their ligand-binding domains are grafted from antibodies selected for binding affinity or avidity and not broadly optimized for a functional response. Because of the disconnect between binding and function among these receptor types, the ultimate potential of CARs optimized for sensitivity and selectivity is not clear. Here, we focus on a thoroughly studied immuno-oncology target, the HLA-A*02/HPV-E629–38complex, and show that CARs can be optimized by a combination of high-throughput binding screens and low-throughput functional assays to have comparable activity to clinical TCRs in acute assays in vitro. These results provide a case study for the challenges and opportunities of optimizing high-performing CARs, especially in the context of targets utilized naturally by TCRs.

In 1989, an unusual illness developed in several people in New Mexico; investigators soon discovered the common feature that linked these cases was the use of L-tryptophan. L-tryptophan is a natural amino acid that serves not only as a building block of proteins, but also as the starting point for the biochemical synthesis of important chemicals in the brain. The substance was used as a dietary supplement among people suffering from insomnia, depression, and other disorders. Within a year, at least 27 people died of what is now referred to as eosinophilia-myalgia syndrome. A study was conducted of the heart tissues of three patients who died of this syndrome to determine if pathological features were present that might account for their sudden deaths. Several such features were identified. Numerous abnormalities were found in the arteries of the heart. Many arteries had a condition called fibromuscular dysplasia, an abnormal tissue buildup, which caused narrowing of the arteries serving the heart muscle. Considerable inflammation of the arteries was also observed, both in the lining and, in some cases, throughout the artery. Inflammation and pathological changes were not limited to the arteries, however. Inflammation was observed in the nerves of the heart, and similar inflammation was seen in the conduction system, which is responsible for distributing signals to maintain regular heart muscle contractions. Abnormalities of arteries were especially common within the heart conduction system. These abnormalities in the electrical conduction system, which controls the heart beat, may have played a key role in the demise of the patients. Similar abnormalities may also be present in living patients who are severely affected by eosinophilia-myalgia syndrome. Therefore, clinicians should be sensitive for signs of electrical instability in patients with eosinophilia-myalgia syndrome. Furthermore, in cases of sudden death, eosinophilia-myalgia syndrome should be considered as a possible cause, and inquiries should be made about the possibility of tryptophan use. (Consumer Summary produced by Reliance Medical Information, Inc.)

Supplemental Digital Content is available in the text.In 2013, an innovative MAGE-A3-directed cancer therapeutic of great potential value was terminated in the clinic because of neurotoxicity. The safety problems were hypothesized to originate from off-target T-cell receptor activity against a closely related MAGE-A12 peptide. A combination of published and new data led us to test this hypothesis with current technology. Our results call into question MAGE-A12 as the source of the neurotoxicity. Rather, the data imply that an alternative related peptide from EPS8L2 may be responsible. Given the qualities of MAGE-A3 as an onco-testis antigen widely expressed in tumors and largely absent from normal adult tissues, these findings suggest that MAGE-A3 may deserve further consideration as a cancer target. As a step in this direction, the authors isolated 2 MAGE-A3 peptide-major histocompatibility complex-directed chimeric antigen receptors, 1 targeting the same peptide as the clinical T-cell receptor. Both chimeric antigen receptors have improved selectivity over the EPS8L2 peptide that represents a significant risk for MAGE-A3-targeted therapeutics, showing that there may be other options for MAGE-A3 cell therapy.

Women are among the fastest-growing groups of persons with the acquired immunodeficiency syndrome (AIDS) in the United States, accounting for 6,571 cases in 1992, or 13.4% of all reported AIDS [...]

Objective: The objective of this study was to evaluate counseling efficacy among high-risk groups. Study: We conducted a subset analysis of data collected from July 1993 through September 1996 during a randomized, controlled trial (Project RESPECT). Participants (n = 4328) from 5 public U.S. sexually transmitted disease (STD) clinics were assigned to enhanced counseling, brief counseling, or educational messages. For 9 subgroups (sex, age, city, education, prior HIV test, STD at enrollment, race/ ethnicity, injection drug use, exchanging sex for money/drugs), we compared STD outcomes for those assigned either type of counseling with STD outcomes for those assigned educational messages. Results: After 12 months, all subgroups assigned counseling (brief or enhanced) had fewer STDs than those assigned educational messages. STD incidence was similar for most subgroups assigned enhanced or brief counseling. All subgroups had an appreciable number of STDs prevented per 100 persons counseled, especially adolescents (9.4 per 100) and persons with STD at enrollment (8.4 per 100). Conclusions: HIV/STD prevention counseling (brief or enhanced counseling) resulted in fewer STDs than educational messages for all subgroups of STD clinic clients, including high-risk groups such as adolescents and persons with STDs at enrollment.

Perinatal transmission is the major mode of hepatitis B virus (HBV) transmission and drives HBV endemicity in the US territory of Guam. We assessed correlates of prenatal hepatitis B surface antigen (HBsAg) screening and HBsAg positivity among pregnant women and evaluated the care of infants of HBsAg-positive women.

Background: Errors in the classification of male circumcision status could bias studies linking infection to lack of circumcision. Goal: To determine the frequency and factors associated with the reproducibility of reporting circumcision status. Study Design: Secondary analysis of data using logistic regression modeling from a multicenter randomized controlled trial was performed. Results: At follow-up assessment, 15.6% of clinician reports on circumcision status disagreed with baseline reports. Disagreement was more common if both clinicians were women than if both were men (odds ratio [OR], 2.8; 95% CI, 1.9-4.1). As compared with whites reported as circumcised (4%, 19/532 visits), the highest disagreement involved uncircumcised Hispanic (OR, 3.3; 95% CI, 1.7-6.3), white (OR, 12.2; 95% CI, 5.8-25.6), or black (OR, 17.1; 95% CI, 10.4-27.9) men. Conclusions: This is one study among a small number of studies examining the reproducibility of clinician-reported circumcision status by comparing multiple clinical examinations of the same patient. The magnitude of the misclassification discovered could bias results and indicates the need for greater accuracy in reporting circumcision status in future studies.

ObjectivesThe WHO recommends pregnant women receive both HIV and syphilis testing at their first antenatal care visit, as untreated maternal infections can lead to severe, adverse pregnancy outcomes. One strategy for increasing testing for both HIV and syphilis is the use of point-of-care (rapid) diagnostic tests that are simple, proven effective and inexpensive. In Malawi, pregnant women routinely receive HIV testing, but only 10% are tested for syphilis at their first antenatal care visit. This evaluation explores stakeholder perceptions of a novel, dual HIV/syphilis rapid diagnostic test and potential barriers to national scale-up of the dual test in Malawi.MethodsDuring June and July 2015, we conducted 15 semistructured interviews with 25 healthcare workers, laboratorians, Ministry of Health leaders and partner agency representatives working in prevention of mother-to-child transmission in Malawi. We asked stakeholders about the importance of a dual rapid diagnostic test, concerns using and procuring the dual test and recommendations for national expansion.ResultsStakeholders viewed the test favourably, citing the importance of a dual rapid test in preventing missed opportunities for syphilis diagnosis and treatment, improving infant outcomes and increasing syphilis testing coverage. Primary technical concerns were about the additional procedural steps needed to perform the test, the possibility that testers may not adhere to required waiting times before interpreting results and difficulty reading and interpreting test results. Stakeholders thought national scale-up would require demonstration of cost-savings, uniform coordination, revisions to testing guidelines and algorithms, training of testers and a reliable supply chain.ConclusionsStakeholders largely support implementation of a dual HIV/syphilis rapid diagnostic test as a feasible alternative to current antenatal testing. Scale-up will require addressing perceived barriers; negotiating changes to existing algorithms and guidelines; and Ministry of Health approval and funding to support training of staff and procurement of supplies.

BackgroundMother-to-child transmission (MTCT) of syphilis and HIV continue to be important yet preventable causes of perinatal and infant morbidity and mortality.ObjectivesTo systematically review, critically appraise and perform a meta-analysis to evaluate the operational characteristics of dual rapid diagnostic tests (RDTs) for HIV/syphilis and evaluate whether they are cost effective, acceptable and easy to use.DesignSystematic review and meta-analysis.Data sourcesWe searched seven electronic bibliographic databases from 2012 to December 2016 with no language restrictions. Search keywords included HIV, syphilis and diagnosis.Review methodsWe included studies that evaluated the operational characteristics of dual HIV/syphilis RDTs. Outcomes included diagnostic test accuracy, cost effectiveness, ease of use and interpretation and acceptability. All studies were assessed against quality criteria and assessed for risk of bias.ResultsOf 1914 identified papers, 18 were included for the meta-analysis of diagnostic accuracy for HIV and syphilis. All diagnostic accuracy evaluation studies showed a very high sensitivity and specificity for HIV and a lower, yet adequate, sensitivity and specificity for syphilis, with some variation among types of test. Dual screening for HIV and syphilis was more cost effective than single rapid tests for HIV and syphilis and prevented more adverse pregnancy outcomes. Qualitative data suggested dual RDTs were highly acceptable to clients, who cited time to result, cost and the requirement of a single finger prick as important characteristics of dual RDTs.ConclusionThe results of this systematic review and meta-analysis can be used by policy-makers and national programme managers who are considering implementing dual RDTs for HIV and syphilis.Trial registration numberPROSPERO 2016:CRD42016049168.

The Human Impacts Database (www.anthroponumbers.org) is a curated, searchable resource housing quantitative data relating to the diverse anthropogenic impacts on our planet, with topics ranging from sea-level rise to livestock populations, greenhouse gas emissions, fertilizer use, and beyond. Each entry in the database reports a quantitative value (or a time series of values) along with clear referencing of the primary source, the method of measurement or estimation, an assessment of uncertainty, and links to the underlying data, as well as a permanent identifier called a Human Impacts ID (HuID). While there are other databases that house some of these values, they are typically focused on a single topic area, like energy usage or greenhouse gas emissions. The Human Impacts Database facilitates access to carefully curated data, acting as a quantitative resource pertaining to the myriad ways in which humans have an impact on the Earth, for practicing scientists, the general public, and those involved in education for sustainable development alike. We outline the structure of the database, describe our curation procedures, and use this database to generate a graphical summary of the current state of human impacts on the Earth, illustrating both their numerical values and their intimate interconnections.

The CEACAM5gene product [carcinoembryonic antigen (CEA)] is an attractive target for colorectal cancer because of its high expression in virtually all colorectal tumors and limited expression in most healthy adult tissues. However, highly active CEA-directed investigational therapeutics have been reported to be toxic, causing severe colitis because CEA is expressed on normal gut epithelial cells. Here, we developed a strategy to address this toxicity problem: the Tmod dual-signal integrator. CEA Tmod cells use two receptors: a chimeric antigen receptor (CAR) activated by CEA and a leukocyte Ig-like receptor 1 (LIR-1)–based inhibitory receptor triggered by human leukocyte antigen (HLA)-A*02. CEA Tmod cells exploit instances of HLA heterozygous gene loss in tumors to protect the patient from on-target, off-tumor toxicity. CEA Tmod cells potently killed CEA-expressing tumor cells in vitro and in vivo. But in contrast to a traditional CEA-specific T cell receptor transgenic T cell, Tmod cells were highly selective for tumor cells even when mixed with HLA-A*02–expressing cells. These data support further development of the CEA Tmod construct as a therapeutic candidate for colorectal cancer.

In April 2007, the Centers for Disease Control and Prevention (CDC) held a two-day consultation with a broad spectrum of stakeholders to obtain input on the potential role of male circumcision (MC) in preventing transmission of human immunodeficiency virus (HIV) in the U.S. Working groups summarized data and discussed issues about the use of MC for prevention of HIV and other sexually transmitted infections among men who have sex with women, men who have sex with men (MSM), and newborn males. Consultants suggested that (1) sufficient evidence exists to propose that heterosexually active males be informed about the significant but partial efficacy of MC in reducing risk for HIV acquisition and be provided with affordable access to voluntary, high-quality surgical and risk-reduction counseling services; (2) information about the potential health benefits and risks of MC should be presented to parents considering infant circumcision, and financial barriers to accessing MC should be removed; and (3) insufficient data exist about the impact (if any) of MC on HIV acquisition by MSM, and additional research is warranted. If MC is recommended as a public health method, information will be required on its acceptability and uptake. Especially critical will be efforts to understand how to develop effective, culturally appropriate public health messages to mitigate increases in sexual risk behavior among men, both those already circumcised and those who may elect MC to reduce their risk of acquiring HIV.

ObjectiveTo assess Ltryptophan LT dose, age, sex, and immunogenetic markers as possible risk or protective factors for the development of LTassociated eosinophilia–myalgia syndrome EMS and related clinical findings.MethodsHLA–DRB1 and DQA1 allele typing and GmKm phenotyping were performed on a cohort of 94 white subjects with documented LT ingestion and standardized evaluations. Multivariate analyses compared LT dose, age, sex, and alleles among groups of subjects who ingested LT and subsequently developed surveillance criteria for EMS, developed EMS or characteristic features of EMS EMS spectrum disorder, or developed no features of EMS unaffected.ResultsConsidering all sources of LT, higher LT dose odds ratio OR 1.4, 95 confidence interval 95 CI 1.1–1.8, age >45 years OR 3.0, 95 CI 1.0–8.8, and HLA–DRB103 OR 3.9, 95 CI 1.2–15.2, DRB104 OR 3.9, 95 CI 1.1–16.4, and DQA10601 OR 13.7, 95 CI 1.3–1.8 were risk factors for the development of EMS, whereas DRB107 OR 0.12, 95 CI 0.02–0.48 and DQA10501 OR 0.23, 95 CI 0.05–0.85 were protective. Similar risk and protective factors were seen for developing EMS following ingestion of implicated LT, except that DRB103 was not a risk factor and DQA10201 was an additional protective factor. EMS spectrum disorder also showed similar findings, but with DRB104 being a risk factor and DRB107 and DQA10201 being protective. There were no differences in sex distribution, GmKm allotypes, or GmKm phenotypes among any groups.ConclusionIn addition to the xenobiotic dose and subject age, polymorphisms in immune response genes may underlie the development of certain xenobioticinduced immunemediated disorders, and these findings may have implications for future related epidemics.

Background:Chimeric Antigen Receptor (CAR) T-cell therapy is a proven, powerful clinical modality. However, it is still limited by the fundamental obstacle of cancer therapy: discriminating cancer from normal cells. Current FDA-approved CAR T-cell therapies eliminate normal B cells, leaving patients with B cell aplasia, hypogammaglobulinemia, and susceptible to infection.

Genomics and proteomics have unveiled a plethora of protein-protein interactions that may control cellular processes involved in disease development. Many of these interactions involve non-traditional candidate targets (i.e., neither enzymes nor cell surface receptors / channels). To date, non-traditional targets have largely been ignored by the pharmaceutical industry or have failed to lead to drugs. This review focuses on the use of transdominant genetically encoded agents and specialized small-molecule drugs to explore this void.

Presents an article on doing an investigative report on the failure of police officers in Washington State to solve cases of missing persons. Factors that prevent the police from solving the cases; Research conducted on unsolved homicide cases in the state; Key findings from the report.

Screens for cytostasis/cytoxicity have considerable value for the discovery of therapeutic agents and the investigation of the biology of apoptosis. For instance, genetic screens for proteins, protein fragments, peptides, RNAs, or chemicals that kill tissue culture cells may aid in identifying new cancer therapeutic targets. A microplate assay for cell death is needed to achieve throughputs sufficient to sift through thousands of agents from expression or chemical libraries. The authors describe a homogeneous assay for cell death in tissue culture cells compatible with 96- or 384-well plates. In combination with a previously described system for retroviral packaging and transduction, nearly 6000 expression library clones could be screened per week in a 96-well plate format. The screening system may also prove useful for chemical screens. (Journal of Biomolecular Screening2003:283-291)

Transdominant genetic selections can yield protein fragment and peptide modulators of specific biochemical pathways. In yeast, such screens have been highly successful in targeting the MAP (mitogen-activated protein) kinase growth-control pathway. We performed a similar type of selection aimed at recovery of modulators of the mammalian MAP kinase cascade. Two pathway activators were identified, fragments of the TrkB and Raf-1 kinases. In a second selection directed at the β-catenin growth-control pathway, three different clones encoding cadherin fragments were recovered. In neither selection were peptide inhibitors observed. We conclude that some transdominant selections in mammalian cells can readily yield high-penetrance protein fragments, but may be less amenable to isolation of peptide inhibitors.

Retroviruses are useful for genetics studies to deliver genes that express proteins, peptides, and RNAs. Several steps, including DNA preparation, transfection, packaging, transduction, and assay, are required to execute screens using retroviral constructs. Unlike screens with purified components, whole-cell assays using retroviral constructs need a large number of steps with microplate manipulations. The nature of these steps, especially the involvement of cultured mammalian cells, limits the throughput of such screens. To improve the efficiency of genetic experiments with retroviral expression vectors, an automated system for retroviral screening in microplates was devised and tested. The system, called Somata, provides high throughputs and robust, reproducible performance.

Fluorescent proteins have become invaluable reporters in many areas of cellular and developmental biology. An enhanced version of the Aequorea victoria green fluorescent protein (AvEGFP) is the most widely used fluorescent protein. For a variety of reasons, it is useful to have alternative fluorescent proteins to AvEGFP. The cDNA sequences for enhanced variants of the Anemonia cyan fluorescent protein (AmCyan1), as well as the Zoanthus green (ZsGreen1) and yellow (ZsYellow1) fluorescent proteins, were cloned downstream of a constitutive cytomegalovirus (CMV) promoter within a retroviral expression vector. NIH3T3, HEK293, SW620, and WM35 cells were transduced with recombinant retroviruses at a low multiplicity of infection (MOI) to bias for single-copy integration. Both unselected and stably selected cells transduced with the retroviral expression constructs were characterized. Expression of each fluorescent protein in cells was detected using flow cytometry and fluorescence microscopy with filter sets typically used for AvEGFP/fluorescein isothiocyanate (FITC) detection and was compared with the expression of AvEGFP. In addition, a fluorescence plate reader with several excitation and emission filter sets was used for detection. Expression of each protein was observable by fluorescence microscopy. Under given conditions of flow cytometry, the ZsGreen1 mean fluorescence was approximately 3-fold, 10-fold, and 50-fold greater than that of AvEGFP, ZsYellow1, and AmCyan1, respectively. AmCyan1, ZsGreen1, and AvEGFP were detected by a fluorescence plate reader. We determined that fluorescent proteins from Anthozoa species are detectable using a standard flow cytometer and fluorescence microscope. All of the mammalian cell lines tested expressed detectable levels of fluorescent proteins from stable integrated provirus. In cell lines where the AvEGFP protein is toxic or poorly expressed, these Anthozoa fluorescent proteins may serve as alternative fluorescent reporters. Cytometry 48:106–112, 2002. © 2002 Wiley-Liss, Inc.

From libraries of peptides and protein fragments, several inhibitors that block pheromone response in Saccharomyces cerevisiae have been isolated previously. In many cases, the inhibitors are displayed as part of a scaffold, such as green fluorescent protein. Each of the inhibitors has a characteristic physiological strength or genetic penetrance. In this report, the roles of expression level and display scaffold on the activities of a subset of pheromone-response pathway inhibitors were examined. Special consideration was given to the relationship between expression levels of specific inhibitors, which may exceed 50 µM in some instances, and penetrance. Copyright © 2002 John Wiley & Sons, Ltd.

From libraries of peptides and protein fragments, several inhibitors that block pheromone response in Saccharomyces cerevisiaehave been isolated previously. In many cases, the inhibitors are displayed as part of a scaffold, such as green fluorescent protein. Each of the inhibitors has a characteristic physiological strength or genetic penetrance. In this report, the roles of expression level and display scaffold on the activities of a subset of pheromone‐response pathway inhibitors were examined. Special consideration was given to the relationship between expression levels of specific inhibitors, which may exceed 50 µMin some instances, and penetrance. Copyright © 2002 John Wiley & Sons, Ltd.

Pseudotyped retroviral vectors combine the advantages of broad host range, high expression, stable chromosomal integration, and ease of preparation. These vectors greatly facilitate delivery into mammalian cells of sequences encoding individual peptide inhibitors—including those with therapeutic utility—and inhibitor libraries. However, retroviral vectors vary in behavior, particularly with respect to expression levels in different cell lines. Expression level is especially important in transdominant experiments because the concentration of an inhibitor (for example, an expressed peptide) is one of the key determinants in the degree of complex formation between the inhibitor and its target. Thus, inhibitor concentration should have an impact on the expressivity and/or penetrance of an induced phenotype. Here, we compare several retroviral vectors and human cell lines for relative expression levels using a green fluorescent protein reporter. We show for a subset of these lines that cellular protein concentrations produced by single-copy vectors range up to about 2 μM. We also examine other variables that contribute to expression level, such as the nature of the expressed protein's carboxy terminus. Finally, we test the effect of increased concentration on phenotype with a nine-amino-acid peptide derived from the human papilloma virus protein E7 which overcomes E7-mediated cell growth.Molecular Therapy (2001) 4, 398–406; doi: 10.1006/mthe.2001.0476

Flow cytometry, in combination with retroviral expression libraries, is a powerful tool for genetic experimentation in mammalian cells. Expression libraries are transduced into cells engineered with a fluorescent reporter. Sorting for either bright or dim cells allows enrichment for specific inhibitors that alter reporter activity. This strategy has been used to isolate peptides and RNAs that either activate or suppress defined biochemical pathways. Several variables contribute to the enrichment process: (1) the background of the fluorescence bioassay; (2) the mean fluorescence ratio between the induced and noninduced reporter cell populations; (3) the genetic penetrance, or strength, of the inhibitor; and (4) the multiplicity of infection (MOI). An experimental and theoretical analysis, including computer modeling, of these issues in the context of a mammalian cell bioassay was undertaken. MOI measurements were shown to be problematic. High MOI had little effect on enrichment early in the cycling process but a significant effect at later stages. Penetrance and background were critical throughout the process. Enrichments within about twofold of the theoretical maximum were observed. Caution should be exercised in MOI determination because of the danger of significant underestimation. High MOI is potentially advantageous early in the selection process but hinders enrichment in the later rounds. Modeling shows that MOI, assay background and clone penetrance are the principal variables that determine the success of transdominant selections by FACS. Cytometry 45:87–95, 2001. © 2001 Wiley-Liss, Inc.

Signalling pathways typically convert a graded, analogue signal into a binary cellular output. In the several eukaryotic systems that have been investigated to date, including MAP kinase cascade activation in Xenopus oocytes, analogue-to-digital conversion occurs at points in the pathway between receptor activation and the effector mechanism. We used flow cytometry combined with an intracellular fluorescent reporter to examine the characteristics of the yeast pheromone-response pathway. Surprisingly, pheromone response in yeast, which relies on the MAP kinase cascade, behaved in a fundamentally graded manner. Expression of certain exogenous dominant inhibitors of the pathway converted the response to graded-or-none behaviour. These results have implications for the dissection of biological response mechanisms in cells and illustrate how signalling pathways, even homologous ones, may have strikingly different signal propagation/amplification characteristics. Copyright © 2001 John Wiley & Sons, Ltd.

PCR is ubiquitous in molecular biology. It is used to amplify single sequences from large genomes, or populations of sequences from complex mixtures such as cDNA libraries in mammalian cells. These cDNA libraries are often employed in subsequent labor-intensive experiments such as genetic screens, the outcome of which depends on library quality. The use of PCR to amplify diverse sequence populations raises important technical issues. One question is whether or not PCR is capable of maintaining population diversity, specifically with respect to template selection in the first rounds of the amplification process (i.e., the possibility that rare sequences in a complex mixture are lost because of amplification failure at the outset of the PCR). Here, we analyze the properties of PCR in the context of template selection in complex mixtures and show that it is an excellent method for preserving diversity.

Background:Male circumcision status has been shown to be associated with sexually transmitted disease (STD) acquisition in some, but not all, studies. Most studies have been cross sectional.Objectives:We examined the association between circumcision status and the prevalence and incidence of gonorrhoea, chlamydia, and syphilis.Methods:We analysed cross sectional and cohort study data from a multicentre controlled trial in the United States. Between July 1993 and September 1996, 2021 men visiting public inner city STD clinics in the United States were examined by a clinician at enrolment and 1456 were examined at follow up visits 6 and 12 months later. At each visit, men had laboratory tests for gonorrhoea, chlamydia, and syphilis and were examined for circumcision status. We used multiple logisitic regression to compare STD risk among circumcised and uncircumcised men adjusted for potentially confounding factors.Results:Uncircumcised men were significantly more likely than circumcised men to have gonorrhoea in the multivariate analyses, adjusted for age, race, and site, in both the cross sectional (odds ratio (OR), 1.3; 95% confidence interval (CI), 0.9 to 1.7) and in the cohort analysis (OR, 1.6; 95% CI, 1.0 to 2.6). There was no association between lack of circumcision and chlamydia in either the cross sectional (OR, 1.0; 95% CI 0.7-1.4) or the cohort analysis (OR, 0.9; 95% CI 0.5-1.5). The magnitude of association between lack of circumcision and syphilis was similar in the cross sectional (OR, 1.4; 95% CI 0.6 to 3.3) and cohort analysis (OR, 1.5; 95% CI 0.4 to 6.1).Conclusion:Uncircumcised men in the United States may be at increased risk for gonorrhoea and syphilis, but chlamydia risk appears similar in circumcised and uncircumcised men. Our results suggest that risk estimates from cross sectional studies would be similar to cohort findings.

Transdominant genetics using expression libraries can identify proteins and peptides that affect cell division. In conjunction with these libraries, oligo-nucleotide-conjugated beads and flow cytometry were used to test a strategy that potentially expands the range of such genetic studies. The experimental approach involved creation of tagged expression libraries, introduction of these libraries into cells, growth of the cultured cells for several generations and recovery on oligonucleotide-conjugated beads of sequences that encode growth-modulatory proteins or peptides. Experiments in Saccharomyces cerevisiae demonstrating the feasibility of the strategy are presented.

We present an approach to the gene identification phase of positional cloning that combines sparse sampling of DNA sequences from large genomic regions with computational analysis. We call the method "software trapping." The goal is to find coding exons while avoiding massive DNA sequence determination and contig assembly. Instead, rapid sequence sampling is combined with exon screening software such as a newly developed package called XPOUND to identify coding sequences. We have tested the approach using a set of model genomic sequences with known intron/exon structures as well as with bona fide P1 genomic clones. The results suggest that the strategy is a useful complement to other methods for finding genes in poorly characterized regions of genomes.

Positional cloning often requires isolation of candidate genes from a large, genetically defined region. Hybrid selection (direct cDNA selection, solution hybrid capture) is a rapid, simple procedure that has been used to identify expressed sequence tags (ESTs) from cloned genomic DNA. We used hybrid selection to screen a 600-kb region that includes the BRCA1 gene. From a set of 931 sequenced clones, we obtained 118 nonoverlapping candidate ESTs from ovary and lymphocyte cDNA. We analyzed the results of our hybrid selection experiments with particular attention to the overall completeness, efficiency, and background noise of the experiment. We introduce simple parameters that serve as measures of important aspects of the hybrid selection process in the context of positional cloning.

The VH1-related human protein (VHR) gene was localized to human chromosome 17q21 in a region thought to contain the BRCA1 locus, a locus that confers susceptibility to breast and ovarian cancer. VHR encodes a phosphatase with dual specificity for tyrosine and serine residues. Thus it is a plausible candidate for a tumor suppressor gene such as BRCA1. To test this possibility, the VHR coding sequence was screened in individuals with familial breast cancer and in sporadic breast tumor and breast cancer cell lines. No mutations were detected, suggesting that the VHR gene is not BRCA1.

The p21 region of human chromosome 9 is thought to contain a gene (MLM) involved in genetic susceptibility to melanoma and a gene or genes that influence progression of certain other tumors. Genomic clones that span a large region in 9p21 surrounding the presumptive tumor suppressor gene(s) have been isolated. A set of sequence-tagged sites in this region has been developed. By using these markers and others previously reported, the 9p21 region has been studied by physical mapping in 84 melanoma cell lines. A putative tumor suppressor gene, perhaps MLM itself, has been localized to a region of less than 40 kb that lies proximal (centromeric) to the alpha-interferon gene cluster.