Feasibility, acceptability, concerns, and challenges of implementing supervised injection services at a specialty HIV hospital in Toronto, Canada: perspectives of people living with HIV.

BACKGROUND: Substance use significantly impacts health and healthcare of people living with HIV/AIDS (PLHIV), especially their ability to remain in hospital following admission. Supervised injection services (SIS) reduce overdoses and drug-related harms, but are not often provided within hospitals/outpatient programs. Leading us to question, what are PLHIV's perceptions of hospital-based SIS? METHODS: This mixed-methods study explored feasibility and acceptability of implementing SIS at Casey House, a Toronto-based specialty HIV hospital, from the perspective of its in/outpatient clients. We conducted a survey, examining clients' (n = 92) demand for, and acceptability of, hospital-based SIS. Following this, we hosted two focus groups (n = 14) and one-on-one interviews (n = 8) with clients which explored benefits/drawbacks of in-hospital SIS, wherein participants experienced guided tours of a demonstration SIS space and/or presentations of evidence about impacts of SIS. Data were analysed using descriptive statistics and thematic analysis. RESULTS: Among survey participants, 76.1% (n = 70) identified as cis-male and over half (n = 49;54.4%) had been a hospital client for 2 years or less. Nearly half (48.8%) knew about clients injecting in/near Casey House, while 23.6% witnessed it. Survey participants were more supportive of SIS for inpatients (76.1%) than for outpatients (68.5%); most (74.7%) reported SIS implementation would not impact their level of service use at Casey House, while some predicted coming more often (16.1%) and others less often (9.2%). Most focus group/interview participants, believed SIS would enhance safety by reducing health harms (e.g. overdose), increasing transparency between clients and clinicians about substance use, and helping retain clients in care. Debate arose about who (e.g., in/outpatients vs. non-clients) should have access to hospital-based SIS and how implementation may shift organizational priorities/resources away from services not specific to drug use. CONCLUSIONS: Our data showed widespread support of, and need for, hospital-based SIS among client stakeholders; however, attempts to reduce negative impacts on non-drug using clients need to be considered in the balance of implementation plans. Given the increased risks of morbidity and mortality for PLHIV who inject drugs as well as the problems in retaining them in care in a hospital setting, SIS is a key component of improving care for this marginalized group.

STR profiling of epithelial cells identified by X/Y-FISH labelling and laser microdissection using standard and elevated PCR conditions.

During the investigation of allegations of sexual assault, samples are frequently encountered that contain DNA from a female and a male donor. These may represent contributions of DNA from the complainant and potentially, the offender. Many semen stained samples successfully undergo DNA analysis and interpretation using a differential extraction method that separates sperm from the epithelial cells present in the stain. However, for those mixed cell samples that contain only epithelial cells, separation of any male cells from female cells is problematic. This paper describes the application of fluorescent in situ hybridisation (FISH) for the gender identification of epithelial cells and subsequent recovery of target cells using laser microdissection (LMD). The profiling results obtained from samples of known cell numbers using the Identifiler™ multiplex at standard 28-cycle PCR conditions and, when cell numbers are low, the SGM Plus™ multiplex at elevated 34-cycle PCR conditions (also known as Low Copy Number DNA analysis (LCN)) are described.

The use of photographs to record variation in bruising response in humans.

There is considerable value in developing tools capable of accurately and reliably determining when bruises were inflicted in humans. Previous work has focused on the visual changes observed in a bruise as the injury develops and heals. However, due to variables such as how and where on the body the bruise was inflicted, differing tissue compositions at the injured skin site between individuals and inter- and intra-observer variation; a technique sufficiently robust for use in a clinical or medicolegal setting has not yet been identified. In this study we present a series of photographs taken under controlled conditions illustrating standardised bruises induced on participants using a weight dropping mechanism. We show that variation in the appearance of bruises over time across individuals is large and, although photography may be a suitable technique for the recording of injuries, it is not sufficiently reliable for determining the age of a bruise.

A modified method using the SonoPrep ultrasonic skin permeation system for sampling human interstitial fluid is compatible with proteomic techniques.

BACKGROUND: The use of biomarkers in skin is a novel diagnostic tool. Interstitial fluid (ISF) from skin provides a snapshot of proteins secreted at the time of sampling giving insights into the patient's health status. METHODS: A minimally invasive technique for the transdermal collection of human ISF proteins. A low frequency ultrasonic skin permeation device (SonoPrep ultrasonic skin permeation system) was used to produce micropores in the stratum corneum through which ISF was extracted using a portable pulsed vacuum ISF collection device. RESULTS: On average, protein concentrations recovered ranged between 0.064 and 4.792 µg/µL (mean 1.258 µg/µL). Two-dimensional gel electrophoresis revealed that this sample type was amenable to this type of analysis. Gel images indicated that both highly abundant proteins and lower abundance proteins were isolated from the skin. Western blot analysis confirmed the presence of proteins commonly found in plasma and the epidermis. CONCLUSION: A minimally invasive method for the transdermal recovery of ISF proteins has been developed. We have demonstrated that ISF samples obtained using this approach can be analysed with proteomic techniques, such as two-dimensional gel electrophoresis and western blots, providing another tool for the identification of disease specific protein biomarkers.

Efficacy of several candidate protein biomarkers in the differentiation of vaginal from buccal epithelial cells.

Currently, there is no accurate method to differentiate vaginal epithelial cells from buccal epithelial cells in biological samples typically encountered in forensic casework. This study tested the expression of a selection of candidate proteins in buccal and vaginal epithelial cells. We investigated six candidate biomarkers, such as loricrin, vimentin, stratifin, cytokeratin 4, cytokeratin 13, small proline-rich protein 2, and involucrin, using Western blot analysis on whole protein extracts and immunohistochemistry (IHC) on intact cells in an attempt to identify cell-specific markers that would differentiate these cells by microscopy. Involucrin, loricrin, and stratifin showed differential expression during Western blot analysis and were carried through to IHC. Although proteins unique to vaginal epithelial cells and buccal epithelial cells were not identified from among the proteins tested, the increased expression levels of two proteins, loricrin and stratifin in vaginal cells, when compared to buccal cells, do provide encouraging results in the search for epithelial cell-specific markers.

Effects of histological staining on the analysis of human DNA from archived slides.

Archived slides of cell smears treated with histological stains for sperm detection are often the only source of DNA available when cold cases are reopened. There have been conflicting reports as to the negative effects of particular histological stains on DNA recovery and quality from human cells, making stain selection an important consideration for forensic laboratories. This study investigates the effect of several staining systems on DNA recovery from histological slide samples stored from 0 to 10 weeks. DNA profiles obtained after analysis of these samples with AmpFlSTR(®) Identifiler™ and increased cycle AmpFlSTR(®) SGM Plus™ short tandem repeat (STR) profiling systems and the effects that these stains have on DNA quantity and quality over time are described. Results indicate that Christmas Tree and Hematoxylin and Eosin stains do not have significantly different effects on DNA quality after 10-week storage of slides. This research will assist scientists to select staining systems that have minimal deleterious effects on the DNA recovered.

The Expression of Petunia Strigolactone Pathway Genes is Altered as Part of the Endogenous Developmental Program.

Analysis of mutants with increased branching has revealed the strigolactone synthesis/perception pathway which regulates branching in plants. However, whether variation in this well conserved developmental signaling system contributes to the unique plant architectures of different species is yet to be determined. We examined petunia orthologs of the ArabidopsisMAX1 and MAX2 genes to characterize their role in petunia architecture. A single ortholog of MAX1, PhMAX1 which encodes a cytochrome P450, was identified and was able to complement the max1 mutant of Arabidopsis. Petunia has two copies of the MAX2 gene, PhMAX2A and PhMAX2B which encode F-Box proteins. Differences in the transcript levels of these two MAX2-like genes suggest diverging functions. Unlike PhMAX2B, PhMAX2A mRNA levels change in leaves of differing age/position on the plant. Nonetheless, this gene functionally complements the Arabidopsismax2 mutant indicating that the biochemical activity of the PhMAX2A protein is not significantly different from MAX2. The expression of the petunia strigolactone pathway genes (PhCCD7, PhCCD8, PhMAX1, PhMAX2A, and PhMAX2B) was then further investigated throughout the development of wild-type petunia plants. Three of these genes showed changes in mRNA levels over a development series. Alterations to the expression patterns of these genes may influence the branching growth habit of plants by changing strigolactone production and/or sensitivity. These changes could allow both subtle and dramatic changes to branching within and between species.

Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE7 is involved in the production of negative and positive branching signals in petunia.

One of the key factors that defines plant form is the regulation of when and where branches develop. The diversity of form observed in nature results, in part, from variation in the regulation of branching between species. Two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes, CCD7 and CCD8, are required for the production of a branch-suppressing plant hormone. Here, we report that the decreased apical dominance3 (dad3) mutant of petunia (Petunia hybrida) results from the mutation of the PhCCD7 gene and has a less severe branching phenotype than mutation of PhCCD8 (dad1). An analysis of the expression of this gene in wild-type, mutant, and grafted petunia suggests that in petunia, CCD7 and CCD8 are coordinately regulated. In contrast to observations in Arabidopsis (Arabidopsis thaliana), ccd7ccd8 double mutants in petunia show an additive phenotype. An analysis using dad3 or dad1 mutant scions grafted to wild-type rootstocks showed that when these plants produce adventitious mutant roots, branching is increased above that seen in plants where the mutant roots are removed. The results presented here indicate that mutation of either CCD7 or CCD8 in petunia results in both the loss of an inhibitor of branching and an increase in a promoter of branching.

Analysis of the DECREASED APICAL DOMINANCE genes of petunia in the control of axillary branching.

Control of branch development is a major determinant of architecture in plants. Branching in petunia (Petunia hybrida) is controlled by the DECREASED APICAL DOMINANCE (DAD) genes. Gene functions were investigated by plant grafting, morphology studies, double-mutant characterization, and gene expression analysis. Both dad1-1 and dad3 increased branching mutants can be reverted to a near-wild-type phenotype by grafting to a wild-type or a dad2 mutant root stock, indicating that both genes affect the production of a graft-transmissible substance that controls branching. Expression of the DAD1 gene in the stems of grafted plants, detected by quantitative reverse transcription-polymerase chain reaction correlates with the branching phenotype of the plants. The dad2-1 mutant cannot be reverted by grafting, indicating that this gene acts predominantly in the shoot of the plant. Double-mutant analysis indicates that the DAD2 gene acts in the same pathway as the DAD1 and DAD3 genes because the dad1-1dad2-1 and dad2-1dad3 double mutants are indistinguishable from the dad2-1 mutant. However, the dad1-1dad3 double mutant has an additive phenotype, with decreased height of the plants, delayed flowering, and reduced germination rates compared to the single mutants. This result, together with the observation that the dad1-1 and dad3 mutants cannot be reverted by grafting to each other, suggests that the DAD1 and DAD3 genes act in the same pathway, but not in a simple stepwise fashion.

Heat-induced oxidative activity protects suspension-cultured plant cells from low temperature damage.

A short heat pre-treatment (1 h at 38°C) was found to protect both suspension-cultured apple fruit cells and tobacco cells from cold-induced cell death. Tobacco cells were more sensitive to low temperatures than apple cells, with significant cell death after 48 h at 0 or -2°C. Real-time measurements of H2O2 levels during the heat pre-treatment revealed a substantial burst of this reactive oxygen species in both cell types. Real-time and longer-term measurements also showed a large burst of H2O2 production from tobacco cells, but not apple cells, when exposed to low temperatures. Lower temperatures reduced levels of peroxidase activity (both total and intracellular), with the heat pre-treatment preventing some of the cold-induced reduction of this activity in both apple and tobacco cells. The greater sensitivity to low temperature of the tobacco cells may be related to higher H2O2 production, with the heat treatment maintaining higher peroxidase activity. The lesser sensitivity of the apple cells may be due to the lack of a H2O2 burst and maintenance of peroxidase activity by the heat treatment. These results support a role for oxidative metabolism in the beneficial effects of heat in inducing low temperature tolerance.

The Decreased apical dominance1/Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE8 gene affects branch production and plays a role in leaf senescence, root growth, and flower development.

Carotenoids and carotenoid cleavage products play an important and integral role in plant development. The Decreased apical dominance1 (Dad1)/PhCCD8 gene of petunia (Petunia hybrida) encodes a hypothetical carotenoid cleavage dioxygenase (CCD) and ortholog of the MORE AXILLARY GROWTH4 (MAX4)/AtCCD8 gene. The dad1-1 mutant allele was inactivated by insertion of an unusual transposon (Dad-one transposon), and the dad1-3 allele is a revertant allele of dad1-1. Consistent with its role in producing a graft-transmissible compound that can alter branching, the Dad1/PhCCD8 gene is expressed in root and shoot tissue. This expression is upregulated in the stems of the dad1-1, dad2, and dad3 increased branching mutants, indicating feedback regulation of the gene in this tissue. However, this feedback regulation does not affect the root expression of Dad1/PhCCD8. Overexpression of Dad1/PhCCD8 in the dad1-1 mutant complemented the mutant phenotype, and RNA interference in the wild type resulted in an increased branching phenotype. Other differences in phenotype associated with the loss of Dad1/PhCCD8 function included altered timing of axillary meristem development, delayed leaf senescence, smaller flowers, reduced internode length, and reduced root growth. These data indicate that the substrate(s) and/or product(s) of the Dad1/PhCCD8 enzyme are mobile signal molecules with diverse roles in plant development.