Does sexuality matter? A cross-sectional study of drug use, social injecting, and access to injection-specific care among men who inject drugs in Melbourne, Australia.

BACKGROUND: Gay, bisexual and other men who have sex with men (GBMSM) are overrepresented in cohorts of people who inject drugs. GBMSM's substance use is usually explored in the context of its contribution to sexual risk. We examined drug use practices, connectedness to other people who inject drugs, peer-to-peer injecting, and access to care among men who inject drugs in Melbourne, Australia. We aim to describe similarities and differences in these parameters for GBMSM and other men. METHODS: Data were drawn from a prospective cohort study of people who inject drugs conducted in Melbourne, Australia, since 2009. This cross-sectional study used data collected between 2016 and 2021. Descriptive statistics were used to assess differences between GBMSM and other men. RESULTS: Of 525 men who injected drugs over the study period, 48 (9%) identified as gay or bisexual, or reported sex with other men in the past 12 months. GBMSM and other men reported similar socio-demographics, drug practices (age of injecting initiation, most injected drug, peer-to-peer injecting, receptive syringe sharing) and access to injecting-specific care (drug treatment, source of needle-syringes). A significantly greater percentage of GBMSM reported past 12-month hepatitis C testing (69% vs. 52%, p = 0.028) and preferring methamphetamine (31% vs. 16%, p = 0.022). A higher percentage of GBMSM reported knowing > 50 other people who inject drugs (46% vs. 37%), but this difference was not statistically significant. Both groups primarily obtained injecting equipment from needle-syringe programs; a minority had accessed injecting-specific primary care. CONCLUSION: Men who injected drugs in this cohort and those who identified as GBMSM reported similar drug and health-seeking practices. The higher prevalence of methamphetamine injecting among GBMSM may warrant different harm reduction support for this group. Health promotion should utilise opportunities to connect men who inject drugs in Melbourne to injecting-specific primary health care.

Assessing individual-level needle and syringe coverage parameters and the measurement of coverage in Melbourne, Australia: methods and impacts.

Background: To assess the structure of individual-level needle and syringe coverage measurement formula, and to estimate the impact of coverage-related behaviours/parameters (instances of syringe acquisition, total syringes acquired, peer-to-peer syringe distribution, injecting frequency) on overall coverage. Methods: Data are drawn from the Melbourne (Australia) injecting drug user cohort study, 2010-16. Data from 518 participants were analysed. We used correlations to explore the relationships between coverage parameters; pooled multiple-linear regression to estimate the effect of each parameter on coverage over time; and exploratory factor analysis to assess the relevance of each parameter within the coverage formula. Results: A 1-unit increase in injecting frequency over time reduced coverage by 10.93 percentage points, almost twice as much as other coverage parameters. Factor analysis results indicated potential improvements to coverage formula structure. Conclusions: Our results suggest that reducing injecting frequency amongst people who inject drugs has the largest improvement in coverage levels, indicating harm reduction services should prioritize it. We also demonstrate that coverage measurement has been inconsistent to date. We sought to refine the method to assist in generating comparable research.

Injecting drug use in low and middle-income countries: Opportunities to improve care and prevent harm.

Inadequate response to injecting drug use (IDU) is a significant problem the world over. Low levels of funding, political inaction, poor levels of health service coverage, high prevalence and incidence of IDU-related blood-borne viruses (BBVs) and ongoing stigmatization/marginalization affect people who inject drugs (PWID) regardless of the income status of the country they reside in. These barriers and system failings are, however, exacerbated in low and middle-income countries (LMICs), meaning that the potential consequences of inaction are more pressing. In this narrative review, we describe the levels of IDU and IDU-specific BBV prevalence in LMICs; levels of harm reduction implementation; the consequences of late or insufficient response, the shortcomings of data collection and dissemination; and the barriers to effective LMIC harm reduction implementation. We also exemplify cases where IDU-related harms and BBV epidemics have been successfully curtailed in LMICs, showing that effective response, despite the barriers, is possible. In conclusion, we suggest four key priorities on the basis of the review: confirming the presence or absence of IDU in LMICs, improving the collection and dissemination of national IDU-specific data, increasing the level of harm reduction programme implementation in LMICs, and increasing both national and international advocacy for PWID and attendant public health interventions.

A Direct Oral Anticoagulant as a Cost Effective Alternative to Warfarin for Treatment of Provoked Venous Thrombosis.

In Ireland, Warfarin is the primary anticoagulant prescribed in the secondary prevention of provoked DVT. We completed a comprehensive cost analysis of a trial group of 24 patients treated with Rivaroxaban (between November 2013 and December 2014), versus a control group treated with Warfarin (between January 2008 and November 2013). The groups were matched for gender (3/7 M/F ratio), DVT type (5 proximal, 19 distal DVTs), provoking factor (20 traumatic, 4 atraumatc), and age. We calculated the cost for each group based on drug administration and clinic costs (labour, sample analysis, and additional costs). Warfarin patients attended clinic 14.58 times; Rivaroxaban patients attended 2.92 times. Overall, the cost per patient on Rivaroxaban is €273.30 versus €260.68 with warfarin. This excludes patient costs which would further increase cost of Warfarin therapy.

Activation of innate anti-viral immune response genes in symptomatic benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is the most common urologic disease in men over age 50. Symptoms include acute urinary retention, urgency to urinate and nocturia. For patients with severe symptoms, surgical treatment is used to remove the affected tissue. Interestingly, the presence of histologic BPH does not always correlate with symptoms. The molecular basis of symptomatic BPH and how it differs from asymptomatic BPH is unknown. Investigation into the molecular players involved in symptomatic BPH will likely give insight into novel therapeutic, and potentially preventative, targets. We determined the expression of genes involved in the innate anti-viral immune response in tissues from patients undergoing surgery to alleviate the symptoms of BPH, and compared the results with prostate tissue with histologic BPH, but from patients with few urinary issues (asymptomatic BPH). We found that expression of complement factor I, apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like protein 3G, oligoadenylate synthetase 2 and interferon-induced tetratricopeptide 1, four genes whose protein products are involved in the innate anti-viral immune response, was significantly transcriptionally upregulated in symptomatic BPH. Additionally, we observe hypomethylation and concomitant expression of ancient retroviral-like sequences, the long interspersed nuclear element 1 retrotransposons, in symptomatic BPH when compared with normal prostate tissue. These findings merit further investigation into the anti-viral immune response in symptomatic BPH.

DNA methylation paradigm shift: 15-lipoxygenase-1 upregulation in prostatic intraepithelial neoplasia and prostate cancer by atypical promoter hypermethylation.

Fifteen (15)-lipoxygenase type 1 (15-LO-1, ALOX15), a highly regulated, tissue- and cell-type-specific lipid-peroxidating enzyme has several functions ranging from physiological membrane remodeling, pathogenesis of atherosclerosis, inflammation and carcinogenesis. Several of our findings support a possible role for 15-LO-1 in prostate cancer (PCa) tumorigenesis. In the present study, we identified a CpG island in the 15-LO-1 promoter and demonstrate that the methylation status of a specific CpG within this island region is associated with transcriptional activation or repression of the 15-LO-1 gene. High levels of 15-LO-1 expression was exclusively correlated with one of the CpG dinucleotides within the 15-LO-1 promoter in all examined PCa cell-lines expressing 15-LO-1 mRNA. We examined the methylation status of this specific CpG in microdissected high grade prostatic intraepithelial neoplasia (HGPIN), PCa, metastatic human prostate tissues, normal prostate cell lines and human donor (normal) prostates. Methylation of this CpG correlated with HGPIN, PCa and metastatic human prostate tissues, while this CpG was unmethylated in all of the normal prostate cell lines and human donor (normal) prostates that either did not display or had minimal basal 15-LO-1 expression. Immunohistochemistry for 15-LO-1 was performed in prostates from PCa patients with Gleason scores 6, 7 [(4+3) and (3+4)], >7 with metastasis, (8-10) and 5 normal (donor) individual males. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect 15-LO-1 in PrEC, RWPE-1, BPH-1, DU-145, LAPC-4, LNCaP, MDAPCa2b and PC-3 cell lines. The specific methylated CpG dinucleotide within the CpG island of the 15-LO-1 promoter was identified by bisulfite sequencing from these cell lines. The methylation status was determined by COBRA analyses of one specific CpG dinucleotide within the 15-LO-1 promoter in these cell lines and in prostates from patients and normal individuals. Fifteen-LO-1, GSTPi and beta-actin mRNA expression in BPH-1, LNCaP and MDAPCa2b cell lines with or without 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin-A (TSA) treatment were investigated by qRT-PCR. Complete or partial methylation of 15-LO-1 promoter was observed in all PCa patients but the normal donor prostates showed significantly less or no methylation. Exposure of LNCAP and MDAPCa2b cell lines to 5-aza-dC and TSA resulted in the downregulation of 15-LO-1 gene expression. Our results demonstrate that 15-LO-1 promoter methylation is frequently present in PCa patients and identify a new role for epigenetic phenomenon in PCa wherein hypermethylation of the 15-LO-1 promoter leads to the upregulation of 15-LO-1 expression and enzyme activity contributes to PCa initiation and progression.

Occipital fixation: effect of inner occipital protuberance alignment on screw position.

Previous authors have demonstrated that the optimum position for screw placement in the occiput is in the inner occipital crest. The position of this structure is usually taken as being in the midline; however, this has not been previously validated. Computerized tomography (CT) of the occipital region was performed prospectively according to a standard protocol. The study included 100 patients (53 female and 47 male, 18-75 years of age). CT images were analyzed to determine the position of the inner occipital crest in relation to the midline. The inner occipital crest is located off the midline (> or = 2 degrees ) in 48% of patients. Preoperative CT evaluation may be helpful prior to occipitocervical fixation on the basis of this study.

Manubriosternal joint dislocation in contact sport.

A 17 year old man developed chest pain and shortness of breath immediately after a scrummage while playing rugby football. A lateral chest radiograph showed a dislocated manubriosternal joint, with no associated injuries. This has not been previously reported in a sporting setting. This injury should be considered in flexion-compression injury of the thorax.

Efficacy and tolerability of a fixed-dose combination of telmisartan plus hydrochlorothiazide in patients uncontrolled with telmisartan monotherapy.

The antihypertensive effects of a telmisartan 80 mg/hydrochlorothiazide (HCTZ) 12.5 mg fixed-dose combination and telmisartan 80 mg monotherapy were compared in patients with a history of mild-to-moderate essential hypertension and inadequate BP control (DBP > or = 90 mm Hg) following 8 weeks of telmisartan monotherapy. At the end of this period, 491 patients (62.9% men; mean age 55.3 years) whose DBP was > or = 90 mm Hg were double-blind randomised to once-daily telmisartan 80 mg/HCTZ 12.5 mg (n = 246) or telmisartan 80 mg (n = 245). Trough (24 h post-dose) clinic BP was measured after 4 and 8 weeks of double-blind therapy. At the end of double-blind treatment, patients receiving telmisartan 80 mg/HCTZ 12.5 mg had significant additional decrements in clinic SBP/DBP over telmisartan 80 mg of -5.7/-3.1 mm Hg (P < 0.01). Most of the additional effect occurred during the first 4 weeks of treatment. The proportion of patients with normalised BP (SBP < 140 mm Hg and DBP < 90 mm Hg) was significantly greater in the telmisartan 80 mg/HCTZ 12.5 mg group than the telmisartan 80 mg group (41.5%vs 26.1%;P < 0.05). Both treatments were well tolerated. The incidence of adverse events was similar except for diarrhoea, which occurred more frequently in the telmisartan 80 mg/HCTZ 12.5 mg group, and oedema, which occurred more frequently in the telmisartan group. Our results indicate that a telmisartan 80 mg/HCTZ 12.5 mg fixed-dose combination confers significant additional BP reductions compared with continuation of telmisartan monotherapy in non-responders.

In vivo suicide gene therapy model using a newly discovered prostate-specific membrane antigen promoter/enhancer: a potential alternative approach to androgen deprivation therapy.

Prostate-specific membrane antigen (PSMA) is a type-2 membrane protein expressed in the prostate, and it is highly expressed in metastatic or poorly differentiated adenocarcinomas. Moreover, PSMA expression is upregulated by androgen deprivation. These advantages make PSMA a useful target for prostate cancer therapy, especially in combination with conventional hormonal treatment. We recently reported that a prostate-specific enhancer is present in the third intron of the PSMA gene. In this study, we have further analyzed the activity of PSMA promoter/enhancer in prostate cancer cells and cells of other tissue origins (breast cancer MCF-7, lung cancer H157, and colorectal cancer HCT8 cells), and we have examined whether this construct could be used for efficient expression of the suicide gene, cytosine deaminase (CD), in vivo. The PSMA promoter/enhancer expressed the luciferase reporter gene in the prostate cancer lines LNCaP and C4-2, with 8- to 20-fold higher expression than the simian virus 40 promoter/enhancer, although it was inactive in the other cell lines. This construct efficiently drove the suicide gene CD, sensitizing C4-2 cells to 5-fluorocytosine (5-FC) with the inhibitory concentration (IC(50)) <300 micromol/L in vitro. Athymic male nude mice bearing the transfected C4-2 cells were treated with intraperitoneal injections of either 5-FC (600 mg/kg) twice a day or saline solution for 3 weeks. C4-2 cell tumors were eliminated by 5-FC when they were expressing our therapeutic construct carrying CD under the regulatory control of the PSMA promoter/enhancer. Our results show the in vivo utility of the PSMA promoter/enhancer in a gene therapy situation targeting prostate cancer.

A tissue-specific enhancer of the prostate-specific membrane antigen gene, FOLH1.

Prostate-specific membrane antigen (PSMA) is an integral membrane protein that is highly expressed on the surface of prostate epithelial cells. It is also expressed on the vascular endothelium of a number of tumor types. We have used an enhancer trap approach with randomly cleaved overlapping DNA fragments from an approximately 55-kb P1 cosmid insert encompassing the 5' half and upstream sequences of the PSMA gene (FOLH1) to isolate an enhancer that strongly activates the FOLH1 core promoter region. The enhancer (PSME) is located in the third intron about 12 kb downstream from the start site of transcription and is characterized by a 72-bp direct repeat within a 331-bp core region. The PSME activates transcription from its own and heterologous promoters in prostate cell lines; enhancement is greatest in the PSMA-expressing cell line LNCaP (>250-fold). The PSME shows essentially no activity in five nonprostate cell lines. PSME-enhanced expression is repressed in the presence of androgen, mimicking the repression of the endogenous FOLH1 gene. The data demonstrate that both cell-type specificity and androgen regulation are intrinsic properties of the enhancer. These properties make the PSME an excellent candidate for regulation of gene expression in gene therapy approaches to prostate cancer.

Drosophila wing development in the absence of dorsal identity.

The developing wing disc of Drosophila is divided into distinct lineage-restricted compartments along both the anterior/posterior (A/P) and dorsal/ventral (D/V) axes. At compartment boundaries, morphogenic signals pattern the disc epithelium and direct appropriate outgrowth and differentiation of adult wing structures. The mechanisms by which affinity boundaries are established and maintained, however, are not completely understood. Compartment-specific adhesive differences and inter-compartment signaling have both been implicated in this process. The selector gene apterous (ap) is expressed in dorsal cells of the wing disc and is essential for D/V compartmentalization, wing margin formation, wing outgrowth and dorsal-specific wing structures. To better understand the mechanisms of Ap function and compartment formation, we have rescued aspects of the ap mutant phenotype with genes known to be downstream of Ap. We show that Fringe (Fng), a secreted protein involved in modulation of Notch signaling, is sufficient to rescue D/V compartmentalization, margin formation and wing outgrowth when appropriately expressed in an ap mutant background. When Fng and alphaPS1, a dorsally expressed integrin subunit, are co-expressed, a nearly normal-looking wing is generated. However, these wings are entirely of ventral identity. Our results demonstrate that a number of wing development features, including D/V compartmentalization and wing vein formation, can occur independently of dorsal identity and that inter-compartmental signaling, refined by Fng, plays the crucial role in maintaining the D/V affinity boundary. In addition, it is clear that key functions of the ap selector gene are mediated by only a small number of downstream effectors.

Overview of evolving strategies incorporating prostate-specific membrane antigen as target for therapy.

Prostate-specific membrane antigen (PSMA) is a potential target in prostate cancer patients because it is very highly expressed and because it has been reported to be upregulated by androgen deprivation. This overview addresses the expression of the PSMA gene in terms of the promoter and enhancer and how that may play a role in gene therapy. We also review PSMA as a target for antibodies for imaging and treatment and the development of a novel hybrid T-cell receptor that combines the specificity of anti-PSMA antibodies with that of T-cell receptor activation when introduced into primary lymphocytes by retroviral-mediated gene transfer. We also discuss our recent findings on the expression of a PSMA-like gene and how that understanding allows specific targeting of PSMA.

A genomics approach to the comprehensive analysis of the glutathione S-transferase gene family in soybean and maize.

By BLAST searching a large expressed sequence tag database for glutathione S-transferase (GST) sequences we have identified 25 soybean (Glycine max) and 42 maize (Zea mays) clones and obtained accurate full-length GST sequences. These clones probably represent the majority of members of the GST multigene family in these species. Plant GSTs are divided according to sequence similarity into three categories: types I, II, and III. Among these GSTs only the active site serine, as well as another serine and arginine in or near the "G-site" are conserved throughout. Type III GSTs have four conserved sequence patches mapping to distinct structural features. Expression analysis reveals the distribution of GSTs in different tissues and treatments: Maize GSTI is overall the most highly expressed in maize, whereas the previously unknown GmGST 8 is most abundant in soybean. Using DNA microarray analysis we observed increased expression among the type III GSTs after inducer treatment of maize shoots, with different genes responding to different treatments. Protein activity for a subset of GSTs varied widely with seven substrates, and any GST exhibiting greater than marginal activity with chloro-2,4 dinitrobenzene activity also exhibited significant activity with all other substrates, suggesting broad individual enzyme substrate specificity.

Prostate-specific suicide gene therapy using the prostate-specific membrane antigen promoter and enhancer.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is abundantly expressed in virtually 100% of prostate cancers and metastases. In addition, unlike prostate-specific antigen (PSA), PSMA is upregulated under conditions of androgen deprivation. Therefore, PSMA is an attractive therapeutic target for advanced prostate cancer. Recently, both the promoter and the enhancer driving prostate-specific expression of the PSMA gene were cloned. We describe here our analysis of the PSMA enhancer for the most active region(s) and present a way of using the enhancer in combination with the E. coli cytosine deaminase gene for suicide-driven gene therapy that converts the nontoxic prodrug 5-fluorocytosine (5-FC) into the cytotoxic drug 5-fluorouracil (5-FU) in prostate cancer cells. METHODS: Deletion constructs of the full-length PSMA enhancer were subcloned into a luciferase reporter vector containing either the PSMA or SV-40 promoter. The most active portion of the enhancer was then determined via luciferase activity in the C4-2 cell line. We then replaced the luciferase gene with the E. coli cytosine deaminase gene in the subclone that showed the most luciferase activity. The specificity of this technique was examined in vitro, using the prostate cancer cell line LNCaP, its androgen-independent derivative C4-2, and a number of nonprostatic cell lines. The toxicity of 5-FC and 5-FU on transiently transfected cell lines was then compared. RESULTS: The enhancer region originally isolated from the PSMA gene was approximately 2 kb. Deletion constructs revealed that at least two distinct regions seem to contribute to expression of the gene in prostate cancer cells, and therefore the best construct for prostate-specific expression was determined to be 1, 648 bp long. The IC(50) of 5-FC was similar in all cell lines tested (>10 mM). However, transfection with the 1648 nt PSMA enhancer and the PSMA promoter to drive the cytosine deaminase gene enhanced toxicity in a dose-dependent manner more than 50-fold, while cells that did not express the PSMA gene were not significantly sensitized by transfection. CONCLUSIONS: Suicide gene therapy using the PSMA enhancer may be of benefit to patients who have undergone androgen ablation therapy and are suffering a relapse of disease.

Fast quantitation of recombinant plasminogen activator inhibitor type 1 in bacterial lysates by micropellicular reversed-phase liquid chromatography.

A rapid reversed-phase HPLC assay is described for quantitating recombinant plasminogen activator inhibitor type 1 (PAI-1) in cultures of Escherichia coli. The assay utilized a short nonporous micropellicular C18 column with an acetonitrile gradient in 0.1% trifluoroacetic acid. The selective resolution of recombinant PAI-1 from major host proteins occurred within 1.3 min. Regeneration of initial chromatography conditions was fast and allowed successive injections every 3 min. The assay's limit of detection for recombinant PAI-1 was 0.5 microg in 5-microl injections of bacterial lysates and the assay was linear to 20 microg. The assay's apparent recovery was between 94 and 108% throughout the quantitative range. In a direct comparison to gel electrophoresis the reversed-phase assay proved superior in monitoring recombinant PAI-1 titers in cultures of E. coli.

Chip is an essential cofactor for apterous in the regulation of axon guidance in Drosophila.

LIM-homeodomain transcription factors are expressed in subsets of neurons and are required for correct axon guidance and neurotransmitter identity. The LIM-homeodomain family member Apterous requires the LIM-binding protein Chip to execute patterned outgrowth of the Drosophila wing. To determine whether Chip is a general cofactor for diverse LIM-homeodomain functions in vivo, we studied its role in the embryonic nervous system. Loss-of-function Chip mutations cause defects in neurotransmitter production that mimic apterous and islet mutants. Chip is also required cell-autonomously by Apterous-expressing neurons for proper axon guidance, and requires both a homodimerization domain and a LIM interaction domain to function appropriately. Using a Chip/Apterous chimeric molecule lacking domains normally required for their interaction, we reconstituted the complex and rescued the axon guidance defects of apterous mutants, of Chip mutants and of embryos doubly mutant for both apterous and Chip. Our results indicate that Chip participates in a range of developmental programs controlled by LIM-homeodomain proteins and that a tetrameric complex comprising two Apterous molecules bridged by a Chip homodimer is the functional unit through which Apterous acts during neuronal differentiation.

Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes.

Isoflavones have drawn much attention because of their benefits to human health. These compounds, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, we used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. We identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the nonleguminous sugarbeet. We expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes.

Axon routing across the midline controlled by the Drosophila Derailed receptor.

In nervous systems with symmetry about the midline, many neurons project axons from one side to the other. Although several of the components controlling midline crossing have been identified, little is known about how axons choose the appropriate pathway when crossing. For example, in the Drosophila embryo axons cross the midline in one of two distinct tracts, the anterior or posterior commissure (AC or PC, respec tively). Here we show that the Derailed (Drl) receptor tyrosine kinase is expressed by neurons that project in the AC, and that in the absence of Drl such neurons often project abnormally into the PC. Conversely, misexpression of Drl in PC neurons forces them to cross in the AC. The behaviour of Drl-misexpressing neurons and the in vivo binding pattern of a soluble Drl receptor probe indicate that Drl acts as a guidance receptor for a repellent ligand present in the PC. Our results show that Drl is a novel component in the control of midline crossing.