Engineering mammalian cells for solid-state sensor applications.

A fundamental advance in the development and application of cell- and tissue-based biosensors would be the ability to achieve air-dry stabilization of mammalian (especially human) cells with subsequent recovery following rehydration. The would allow for the preparation of sensors with extended shelf lives, only requiring the addition of water for activation. By understanding and subsequently employing the tactics used by desiccation-tolerant extremophiles, it may be possible to design stabilized mammalian cell-based biosensors. The approaches required to realize this goal are discussed and illustrated with several examples.

"New beginnings": a case study in gay men's changing perceptions of quality of life during the course of HIV infection.

This article reports results of an ethnographic study that sought to understand how a cohort of gay men living with HIV infection evaluated and worked to preserve or improve the quality of their lives. Themes of life story narratives are identified, each with an associated stylistic self-orientation to living with HIV infection. Changes in thematic content of a selected participant's life story narratives are discussed, demonstrating how events of his daily life are integrated into the narratives. Resultant concurrent shifting of themes and stylistic orientations is linked to his perception of improved quality of life.

Overexpression of trehalose synthase and accumulation of intracellular trehalose in 293H and 293FTetR:Hyg cells.

A humanized clone containing the trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase (otsA/B) has been constructed. Using the Gateway Cloning System (Invitrogen, Inc.), the otsA/B genes have been placed under the control of the CMV promoter (pEXPcmv-otsA/B) or the CMV promoter and the tet operator (pEXP cmv TetO-otsA/B). The pEXPcmv-otsA/B clone has been introduced into 293H cells using LIPOFECTAMINE 2000 and the intracellular concentration of trehalose has been evaluated. The 293H cells accumulate 4-5 microg trehalose/mg dry weight and this concentration increases to 7-10 microg trehalose/mg dry weight if trehalose is included in the growth medium. The pEXPcmv TetO-otsA/B clone has been transfected into 293FTetR:Hyg cells which contain the tet repressor integrated into the genome. When these transfected cells are grown in the absence of tetracycline, no intracellular trehalose is detected. Inclusion of 0.3 microg/ml tetracycline in the growth medium results in the accumulation of 11-14 microg trehalose/mg dry weight, a value which increases to 19-20 microg trehalose/mg dry weight if trehalose is included in the growth medium. The data for the 293FTetR:Hyg cells indicate that intracellular trehalose accumulates in response to the addition of tetracycline. This system will allow us to manipulate the intracellular concentration of trehalose and to evaluate the desiccation tolerance of these cells as a function of intracellular trehalose concentration.

Dilemmas in conducting qualitative sex research in applied field settings.

Although resources are available to inform researchers of the many technical skills necessary to conduct qualitative research, individuals working in applied field settings often encounter ethical, moral, and sociopolitical dilemmas that cannot be resolved through the application of technical skills. The purpose of this article is to present examples of dilemmas faced by qualitative research methodologists studying sexual behavior in applied field settings. Possible solutions to these dilemmas are discussed within a theoretical and conceptual framework. The examples and discussion are organized around four broad topic areas: informed consent, privacy, confidentiality, and personal relationships.

HIV prevention with young men who have sex with men: what young men themselves say is needed. Medical College of Wisconsin CITY Project Research Team.

Young men who have sex with men (YMSM), and particularly ethnic minority YMSM, experience high incidence HIV infection due to continued patterns of high-risk sexual behaviour. The intent of this research was to systematically solicit input and recommendations from YMSM themselves concerning the kinds of HIV prevention programmes that would best meet their needs and would address risk issues they believed are critical. In-depth qualitative interviews were conducted with a sample of 72 purposively selected YMSM to identify necessary components of HIV prevention targeting YMSM. Respondents noted a need for comprehensive HIV prevention programmes that addressed issues related to dating and intimacy, sexuality and arousal, drugs and alcohol, self-esteem and self-worth, abuse and coercion, and sexual identity. Respondents emphasized the importance of keeping programmes confidential, fun, comfortable, accepting and open to all YMSM regardless of sexual identity. Identified community resource needs included safe havens for youth, more peer educators and older MSM mentors, increased school-based sexuality education, and greater support from the society at large as well as from churches, the gay community and communities of Color. Implications of these findings for HIV prevention are discussed.

Psychosocial Issues in the Era of New AIDS Treatments from the Perspective of Persons Living with HIV.

In the past, HIV disease meant an almost invariably downward health course. New highly active antiretroviral therapy (HAART) regimens have improved the health outlook for many persons living with HIV/AIDS but may create new psychological and coping challenges. In this study, open-ended, in-depth interviews were undertaken with an ethnically diverse sample of 44 purposively selected men and women with HIV disease who were on HAART regimens. The interviews were transcribed and qualitatively coded to identify major themes. While patients responding well to the regimens held optimistic views for their future, some who continued to have detectable viral load exhibited depression and feelings of hopelessness. Many patients reported stress associated with the demands of adhering to complex HAART regimens. Other common themes emerging in the interviews involved concerns about employment, romantic and non-romantic relationship formation, sexual behavior and serostatus disclosure, whether to plan families, and experiences of AIDS-related discrimination. There continue to be critical roles for psychological services in the care of persons living with HIV.

Implications of HIV treatment advances for behavioral research on AIDS: protease inhibitors and new challenges in HIV secondary prevention.

Protease inhibitor combination therapies can reduce HIV viral load, improve immune system functioning, and decrease mortality from AIDS. These medical developments raise a host of critical new issues for behavioral research on HIV/AIDS. This article reviews developments in HIV combination therapy regimens and behavioral factors involved in these regimens and focuses on four key behavioral research areas: (a) the development of interventions to promote treatment adherence, (b) psychological coping with HIV/AIDS in the context of new treatments for the disease, (c) the possible influence of treatment on continued risk behavior, and (d) behavioral research in HIV prevention and care policy areas. Advances in HIV medical care have created important new opportunities for health psychologists to contribute to the well-being of persons with HIV/AIDS.

Cost-effectiveness of post-exposure prophylaxis following sexual exposure to HIV.

OBJECTIVES: To assess the cost-effectiveness, relative to other health-related interventions in the U.S., of post-exposure prophylaxis (PEP) following potential HIV exposure through sexual contact with a partner who may or may not be infected, and to compare the relative cost-effectiveness of dual- and triple-combination PEP. METHODS: Standard techniques of cost-utility analysis were used to assess the cost-effectiveness of PEP with a four-week regimen of zidovudine and lamivudine, or zidovudine, lamivudine, and indinavir. Due to a lack of empirical data on the effectiveness of PEP with combination drug regimens, the analysis assumed that combination PEP was no more effective than PEP with zidovudine alone. The main outcome variable is the cost per quality-adjusted life year (QALY) saved by the program. RESULTS: Providing PEP to a cohort of 10,000 patients who report receptive anal intercourse with a partner of unknown HIV status (who is assumed to be infected with probability equal to 0.18) would prevent about 20 infections, at an average net cost of about US$ 70,000 per infection averted. The cost-utility ratio, US$ 6316 per QALY saved, indicates that PEP is highly cost-effective in this instance. Moreover, triple-combination PEP would need to be about 9% more effective than dual-combination PEP for the addition of indinavir to the regimen to be considered cost-effective. Prophylaxis following receptive vaginal exposure is cost-effective only when it is nearly certain that the partner is infected; PEP for insertive anal and vaginal intercourse does not appear to be cost-effective. CONCLUSIONS: From a purely economic standpoint, PEP should be restricted to partners of infected persons (e.g., serodiscordant couples), to patients reporting unprotected receptive anal intercourse (including condom breakage), and possibly to cases where there is a substantial likelihood that the partner is infected. Providing PEP to all who request it does not appear to be an economically efficient use of limited HIV prevention and treatment resources.

Reaffirming the relevance of culture for nursing.

This article reaffirms the relevance of the concept of culture for nursing and suggests the utility of recent developments in culture theory. Culture has long been considered to play an important role in the practice of nursing. The historical development of the concept of culture is reviewed with emphasis on nurse scholars and anthropologists who have addressed culture as an area of inquiry. The meaning-centered perspective of culture, to include embodiment, is reviewed and recommended as a means of addressing the current emphasis on cultural diversity in nursing. This perspective is seen as having broad implications for both advancing theoretical development in nursing and for nursing practice.

Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants.

Plasmids comprising transgene insertions in four lines of transgenic mice have been retrieved by plasmid rescue into a set of Escherichia coli strains with mutations in different members of the methylation-dependent restriction system (MDRS). Statistical analysis of plasmid rescue frequencies has revealed that the MDRS loci detect differential modifications of the transgene insertions among mouse lines that show distinctive patterns of transgene expression. Plasmids in mice that express hybrid insulin transgenes during development can be readily cloned into E. coli strains carrying mutations in two of the MDRS loci, mcrA and mcrB. In mice in which transgene expression is inappropriately delayed into adulthood, plasmids can only be cloned into E. coli that carry mutations in all known MDRS activities. Differential cloning frequencies in the presence or absence of the various methylation-dependent restriction genes represent a further way to distinguish regions of mammalian chromosomes. These multiply deficient E. coli strains will also facilitate the molecular cloning of modified chromosomal DNA.

Bioassay for specific DNA sequences using a non-radioactive probe.

A novel method for detecting specific DNA sequences is described. The method uses a non-radioactive DNA probe, called a probe-vector, that can transform competent Escherichia coli cells at high efficiency only when it has hybridized to a specific DNA target, thus forming a circular, double-stranded, plasmid-like molecule. The probe-vector carries a plasmid origin of replication and a gene that confers antibiotic resistance on transformed E. coli. The output of the assay--colored bacterial colonies on an agar plate--is quantitative and proportional over a wide range of target concentrations. The utility of the probe-vector method for detecting hepatitis B virus (HBV) DNA in human serum is demonstrated. The assay can detect as little as 0.1 pg HBV DNA. The presence of an internal standard monitors DNA recovery and E. coli transformation efficiency for each sample. The assay has the potential to simultaneously measure the DNA of two or more pathogens within the same clinical sample.

Introduction of bacteriophage lambda into cells of Klebsiella aerogenes.

We have shown that a mutation in the cro gene of phage lambda greatly reduces zygotic induction. This observation has allowed us to move this phage on an episome into cells of Klebsiella aerogenes where it grows as well as in cells of Escherichia coli. This technique should allow the introduction of various derivatives of lambda into any organism which is able to receive deoxyribonucleic acid from E. coli.

Deletion mapping of the polA-metB region of the Escherichia coli chromosome.

A lambdacI857 prophage inserted into one of the genes of the rha locus was used to select deletions unambiguously ordering the markers polA-glnA-rha-pfkA-tpi-metBJF. Transduction with phage P1 indicates at least 70% linkage between glnA and polA. The order of the pfk and tpi markers is reversed from that previously published. Despite the relatively large distance separating the glnA and rha loci, deletions removing this entire region have no obvious phenotype. The isolation of Tn10 transposons integrated at different sites between rha and glnA greatly facilitated this work.

Regulation of glutamine synthetase formation in Escherichia coli: characterization of mutants lacking the uridylyltransferase.

A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of Escherichia coli K-12. Such strains lack uridylyltransferase as a result of an insertion of the TN10 element in the glnD gene. The glnD::Tn10 insertion has been mapped at min 4 on the E. coli chromosome and 98% contransducible by phage P1 with dapD. A lambda transducing phage carrying the glnD gene has been identified. A glnD::Tn10 strain synthesizes highly adenylylated glutamine synthetase under all conditions of growth and fails to accumulate high levels of glutamine synthetase in response to nitrogen limitation. However, this strain, under nitrogen-limiting conditions, allows synthesis of 10 to 20 milliunits of biosynthetically active glutamine synthetase per mg of protein, which is sufficient to allow slow growth in the absence of glutamine. The GlnD phenotype in E. coli can be suppressed by the presence of mutations which increase the quantity of biosynthetically active glutamine synthetase.

Regulation of enzyme synthesis by the glutamine synthetase of Salmonella typhimurium: a factor in addition to glutamine synthetase is required for activation of enzyme formation.

In Klebsiella aerogenes but not in Salmonella typhimurium glutamine synthetase can function during nitrogen-limited growth to increase the rate of synthesis of histidase from the hut genes of S. typhimurium 15-59 (hutS. 15-59). Formation of proline oxidase is also not increased in nitrogen-limited cultures of S. typhimurium. However, in hybrid strains of Escherichia coli or K. aerogenes, the glutamine synthetase of S. typhimurium activates synthesis of histidase from the hutS. 15-59 genes. Apparently, glutamine synthetase is necessary but not sufficient for activation of transcription of the hut genes; another factor must also be present. This factor is active in both K. aerogenes and E. coli but is missing or altered in S. typhimurium.

Regulation of histidase synthesis in intergeneric hybrids of enteric bacteria.

Regulation of the expression of the histidase coded by hutk of Klebsiella aerogenes in Salmonella typhimurium and in Escherichia coli and of the expression of the histidase coded by huts of S. typhimurium in E. coli was investigated. The hutk histidase was found to be sensitive to catabolite repression in K. aerogenes and in E. coli, but insensitive to catabolite repression in S. typhimurium; huts histidase has previously been shown to be catabolite sensitive in all three organisms. The expression of both hutk and huts histidase in E. coli was activated by nitrogen starvation. Apparently, the glutamine synthetase of E. coli may activate the formation of some glutamate- and ammonia-producing enzymes.

Isolation and characterization of catabolite-resistant mutants in the D-serine deaminase system of Escherichia coli K-12.

Two classes of D-serine deaminase (Dsdase)-specific secondary mutants of Escherichia coli K-12 were isolated from a Dsdase low constitutive nonhyperinducible mutant as types which could grow in the presence of both D-serine and glucose. These strains contain cis dominant, nonsuppressible mutations in the dsdO (operator-initiator) region. In the first class of mutants (e.g., FB4010), Dsdase synthesis is completely insensitive to catabolite repression, and synthesis occurs at a high constitutive rate in the absence of cyclic adenosine 5'-monophosphate. In the second class (e.g., FB4005), Dsdase synthesis is partially insensitive to catabolite repression, and catabolite repression is reversed by the addition of cyclic adenosine 5'-monophosphate. Dsdase synthesis in strain FB4005 is partially independent of the cyclic adenosine 5'-monophosphate binding protein, as constitutive synthesis is reduced only 65% (relative to the cap+ strain) in strains unable to synthesize the cyclic adenosine 5'-monophosphate binding protein. Surprisingly, the constitutive rate of Dsdase synthesis is fourfold higher in all mutants of both classes than in the parent, indicating a close interrelationship between the sites of response to induction and catabolite repression.

Isolation and characterization of D-serine deaminase constitutive mutants by utilization of D-serine as sole carbon or nitrogen source.

Mutants constitutive for D-serine deaminase (Dsdase) synthesis were isolated by utilizing D-serine as sole nitrogen or carbon source in the chemostat. This method generated only regulatory constitutive (dsdC) mutants. The altered dsdC gene product in these strains is apparently able to bind D-serine more efficiently than the wild-type dsdC+ gene product--a selective advantage. Constitutive synthesis of Dsdase in all of these dsdC mutants is extremely sensitive to catabolite repression, and catabolite repression is reversed by the addition of D-serine. Of the 15 mutants generated by this method, none are suppressible by supD, supE, or supF. Mutations to a low level of constitutivity (maximal specific activity of 9) occur much more frequently than mutations to a high level (maximal specific activity of 79). High level constitutive synthesis of Dsdase results from the synthesis of an altered dsdC gene product--not from loss of ability to form the dsdC product. Dsdase synthesis is not regulated by the nitrogen supply in the medium, as nitrogen starvation does not result in the derepression of Dsdase synthesis.