Intragenic Agrobacterium-mediated gene transfer mimics micro-translocations without foreign DNA.

MAIN CONCLUSION: Agrobacterium-mediated transformation of Nicotiana tabacum, using an intragenic T-DNA region derived entirely from the N. tabacum genome, results in the equivalence of micro-translocations within genomes. Intragenic Agrobacterium-mediated gene transfer was achieved in Nicotiana tabacum using a T-DNA composed entirely of N. tabacum DNA, including T-DNA borders and the acetohydroxyacid synthase gene conferring resistance to sulfonylurea herbicides. Genomic analysis of a resulting plant, with single locus inheritance of herbicide resistance, identified a single insertion of the intragenic T-DNA on chromosome 5. The insertion event was composed of three N. tabacum DNA fragments from other chromosomes, as assembled on the T-DNA vector. This validates that intragenic transformation of plants can mimic micro-translocations within genomes, with the absence of foreign DNA.

A snapshot of injecting drug consumption from the analysis of used syringes within the Medically Supervised Injecting Centre in Sydney, Australia.

INTRODUCTION: The administration of illicit drugs by injection is associated with considerable harm, including an increased risk of overdose. The chemical analysis of used syringes can enhance knowledge on injecting drug consumption beyond traditional data sources (self-report surveys). This additional information may be useful during significant global events like the COVID-19 pandemic. This study aimed to examine a snapshot of the drugs injected at the Medically Supervised Injecting Centre (MSIC) in Sydney, Australia, in 2019-2020. METHODS: Used syringes were collected from MSIC across three periods throughout 2019 and 2020 (February 2019, March-April 2020 and June-September 2020). Drug residues were extracted from used syringes using methanol before detection by gas chromatography-mass spectrometry and ultra-performance liquid chromatography-tandem mass spectrometry. The chemical analysis results were compared to self-report data obtained from MSIC clients. RESULTS: Heroin (46-53%), methamphetamine (24-34%) and pharmaceutical opioids (15-27%) were the most common drug residues detected. The chemically detected drugs had declining coherence with the drugs self-reported by MSIC clients across the time periods examined. DISCUSSION AND CONCLUSIONS: There was no significant change in the drugs injected (heroin, methamphetamine and pharmaceutical opioids) across the three periods collected throughout varying COVID-19 lockdown restrictions. Changes in the frequency of other drugs injected and discrepancies between chemical analysis and self-report were potentially related to regulatory changes, degradation or misinformed sales. Routine chemical analysis of used syringes has provided an alternative information source to promote awareness of current drug trends and aid harm reduction.

Monitoring for fentanyl within Australian supervised injecting facilities: Findings from feasibility testing of novel methods and collaborative workshops.

BACKGROUND: Australia is yet to see widespread fentanyl-contaminated heroin, despite the established presence of fentanyl in other countries. International mortality trends alongside a local cluster of fentanyl-related deaths prompted interest in developing methods to monitor for fentanyl and other potentially harmful novel psychoactive substances (NPS) in Australia. METHODS: We tested novel methods to monitor for fentanyl and other NPS. From 2017-2021, clients from supervised injecting facilities (SIFs) in Melbourne and Sydney, Australia, contributed urine screens (UDS) with BTNX Rapid Response™ fentanyl test strips (FTS) paired with surveys, and injecting equipment associated with opioid overdoses for laboratory analysis. A single site piloted drug checking using FTS with laboratory confirmation. Two workshops were conducted with SIF staff, content experts and people with lived experience to determine how results can inform practices within SIFs. RESULTS: Of the 911 UDS with FTS conducted, less than 1% (n=8) yielded positive results that were not explained by self-reported pharmaceutical fentanyl use, with two laboratory confirmed fentanyl positive results. Injecting equipment from 59 overdoses was tested and neither fentanyl nor other NPS were identified. Drug checking with FTS (n=34) indicated the presence of fentanyl on three tests. Two specimens were subsequently sent for laboratory testing and classified as false positives as the presence of fentanyl was not confirmed. Workshop participants (n=21) felt routine monitoring with FTS currently had limited value. A process for using pre-defined signals to trigger surveillance was developed. CONCLUSION: The high false positive rates with FTS, relative to the small number of positive results and potential for them to undermine confidence in FTS emphasised the need for confirmatory testing. The role of routine surveillance was unclear within the current low-fentanyl context, however, a process was developed to upscale testing should signals of increased fentanyl prevalence in the Australian heroin market emerge.

Infrequent detection of unintentional fentanyl use via urinalysis among people who regularly inject opioids in Sydney and Melbourne, Australia.

BACKGROUND AND AIM: The current phase of the North American 'opioid crisis' is characterised by illicit fentanyl use; however, the presence of illicit fentanyl in Australia is unknown. This study aimed to monitor unintentional fentanyl consumption in Australia. DESIGN: Rapid urine drug screens (UDS) paired with surveys conducted within supervised injecting facilities (SIFs) and confirmatory laboratory testing. SETTING: Sydney and Melbourne, Australia. PARTICIPANTS: Clients who used heroin within the past 2 days (n = 911 tests, 2017-2021). Participants were demographically similar to the overall client base (median age 43, 72% male). MEASUREMENTS: UDS were conducted using BTNX Rapid Response fentanyl urine strip tests with cross-reactivity to numerous fentanyl analogues. Positive urine samples were analysed using liquid chromatography coupled with tandem mass spectrometry. Surveys covered past 3 day drug use and lifetime report of fentanyl in heroin. FINDINGS: Two percent of participants reported intentional use of fentanyl, mostly through fentanyl patches. Of the 911 rapid UDS conducted, 17 (1.9%) yielded positive results. Eight of these (all from Melbourne) were not explained by survey-reported fentanyl use in the past 3 days. Of these 8 unexplained positives, confirmatory laboratory analysis was conducted on 6, with 4 deemed to be false positives, and 2 confirmed for the presence of fentanyl. This represents the first confirmation of unintended use of fentanyl type substances in this population. CONCLUSION: There is limited evidence of unintentional fentanyl use among people in Sydney and Melbourne, Australia who regularly inject heroin, suggesting that, currently, there is very little illicit fentanyl in Australian drug markets accessed by supervised injecting facilities attendees. This study demonstrates the feasibility of quick onsite testing to cost-effectively screen large samples for fentanyl; however, the high false positive rate emphasises the need for confirmation of positive tests through advanced analytical techniques.

Adapting harm reduction services during COVID-19: lessons from the supervised injecting facilities in Australia.

The COVID-19 crisis has had profound impacts on health service provision, particularly those providing client facing services. Supervised injecting facilities and drug consumption rooms across the world have been particularly challenged during the pandemic, as have their client group-people who consume drugs. Several services across Europe and North America closed due to difficulties complying with physical distancing requirements. In contrast, the two supervised injecting facilities in Australia (the Uniting Medically Supervised Injecting Centre-MSIC-in Sydney and the North Richmond Community Health Medically Supervised Injecting Room-MSIR-in Melbourne) remained open (as at the time of writing-December 2020). Both services have implemented a comprehensive range of strategies to continue providing safer injecting spaces as well as communicating crucial health information and facilitating access to ancillary services (such as accommodation) and drug treatment for their clients. This paper documents these strategies and the challenges both services are facing during the pandemic. Remaining open poses potential risks relating to COVID-19 transmission for both staff and clients. However, given the harms associated with closing these services, which include the potential loss of life from injecting in unsafe/unsupervised environments, the public and individual health benefits of remaining open are greater. Both services are deemed 'essential health services', and their continued operation has important benefits for people who inject drugs in Sydney and Melbourne.

Substances injected at the Sydney supervised injecting facility: A chemical analysis of used injecting equipment and comparison with self-reported drug type.

Providing information about substances injected can reduce the negative impact of illicit drug consumption and support people who inject drugs to make informed decisions. In Australia, information about drugs injected relies largely on periodic self-report surveys. For the first time, the analysis of the residual content of used injecting equipment was conducted in a supervised injecting facility (SIF) located in Sydney, Australia. The aim was to gain a better understanding of the substances injected by clients through: (1) chemical analyses of the content of used syringes; (2) comparison of these results with clients' self-reported drug use; and (3) assessing the usefulness of analysing other injecting equipment to detect substances used. During one week in February 2019, syringes and other injecting equipment were collected at the Sydney SIF. Their residual content was analysed by gas-chromatography/mass-spectrometry. Heroin was the most commonly detected substance (present in 51% of syringes), followed by methamphetamine (22%) and oxycodone (10%). In addition to the main psychoactive substance, cutting agents reported in the literature were also detected in used syringes. The main psychoactive substance identified by laboratory analysis reliably corresponded with users' self-reported drug type. Analytical confirmation of substances injected allows for the provision of better targeted harm reduction messaging based on timely and objective data. The approach used is amenable to clients and feasible in the Australian SIF context. Upscaling and wider implementation could be done through Needle and Syringe Programs, and would support the early detection of harmful substances entering drug markets and better inform harm reduction strategies.

Effect of Intranasal vs Intramuscular Naloxone on Opioid Overdose: A Randomized Clinical Trial.

Importance: Previous unblinded clinical trials suggested that the intranasal route of naloxone hydrochloride was inferior to the widely used intramuscular route for the reversal of opioid overdose. Objective: To test whether a dose of naloxone administered intranasally is as effective as the same dose of intramuscularly administered naloxone in reversing opioid overdose. Design, Setting, and Participants: A double-blind, double-dummy randomized clinical trial was conducted at the Uniting Medically Supervised Injecting Centre in Sydney, Australia. Clients of the center were recruited to participate from February 1, 2012, to January 3, 2017. Eligible clients were aged 18 years or older with a history of injecting drug use (n = 197). Intention-to-treat analysis was performed for all participants who received both intranasal and intramuscular modes of treatment (active or placebo). Interventions: Clients were randomized to receive 1 of 2 treatments: (1) intranasal administration of naloxone hydrochloride 800 µg per 1 mL and intramuscular administration of placebo 1 mL or (2) intramuscular administration of naloxone hydrochloride 800 µg per 1 mL and intranasal administration of placebo 1 mL. Main Outcomes and Measures: The primary outcome measure was the need for a rescue dose of intramuscular naloxone hydrochloride (800 µg) 10 minutes after the initial treatment. Secondary outcome measures included time to adequate respiratory rate greater than or equal to 10 breaths per minute and time to Glasgow Coma Scale score greater than or equal to 13. Results: A total of 197 clients (173 [87.8%] male; mean [SD] age, 34.0 [7.82] years) completed the trial, of whom 93 (47.2%) were randomized to intramuscular naloxone dose and 104 (52.8%) to intranasal naloxone dose. Clients randomized to intramuscular naloxone administration were less likely to require a rescue dose of naloxone compared with clients randomized to intranasal naloxone administration (8 [8.6%] vs 24 [23.1%]; odds ratio, 0.35; 95% CI, 0.15-0.66; P = .002). A 65% increase in hazard (hazard ratio, 1.65; 95% CI, 1.21-2.25; P = .002) for time to respiratory rate of at least 10 and an 81% increase in hazard (hazard ratio, 1.81; 95% CI, 1.28-2.56; P = .001) for time to Glasgow Coma Scale score of at least 13 were observed for the group receiving intranasal naloxone compared with the group receiving intramuscular naloxone. No major adverse events were reported for either group. Conclusions and Relevance: This trial showed that intranasally administered naloxone in a supervised injecting facility can reverse opioid overdose but not as efficiently as intramuscularly administered naloxone can, findings that largely replicate those of previous unblinded clinical trials. These results suggest that determining the optimal dose and concentration of intranasal naloxone to respond to opioid overdose in real-world conditions is an international priority. Trial Registration: anzctr.org.au Identifier: ACTRN12611000852954.

Overdose Risk and Client Characteristics Associated With the Injection of Buprenorphine at a Medically Supervised Injecting Center in Sydney, Australia.

Background: Buprenorphine and buprenorphine/naloxone (BNX) were developed to improve the safety profile of opioid substitution treatment (OST) and reduce diversion and injection, yet continue to be injected, despite the risk of harm. Previous studies examining injection of these substances have relied on self-reported injection and overdose. Using data from the Uniting Medically Supervised Injecting Center (MSIC) in Sydney, this study aimed to assess the overdose risk associated with the use of buprenorphine and BNX and identify factors associated with injecting. Methods: Client data routinely collected from MSIC, a drug consumption room where clients can legally inject drugs under supervision, was used. Odds ratios (OR) to assess the risk of overdose and their associated 95% confidence intervals (95%CI) were calculated and compared to other substances. Univariate analysis using χ-square and multivariate logistic regressions were used to determine characteristics associated with buprenorphine and BNX injection. Results: Data from 1,020,782 injections by 15,832 individuals were analyzed. Risk of overdose was low for buprenorphine compared to other substances (OR 0.16; 95%CI: 0.07-0.19) and no overdoses occurred when BNX was injected. Injection of both buprenorphine and BNX was associated with male gender, homelessness, no income/reliance upon government payments, and prior imprisonment. Conclusions: Buprenorphine and BNX continue to be injected, albeit in small numbers. This is the first study to report on injection and overdose risk using direct observation, and has confirmed the lower overdose risk. MSIC clients who inject buprenorphine and BNX tend to be marginalized and may benefit from targeted harm reduction measures.

Urine drug screening for early detection of unwitting use of fentanyl and its analogues among people who inject heroin in Sydney, Australia.

INTRODUCTION AND AIMS: North America has witnessed a dramatic rise in fatal opioid overdoses due to the unwitting consumption of non-pharmaceutical fentanyl and its analogues. While some of the drivers of this crisis-including profitability and access to high-potency opioids through internet sources-also apply in Australia, to our knowledge, there have been no ongoing surveillance studies of local populations. Therefore, this pilot study aimed to detect unintentional fentanyl consumption among people who inject heroin through instant urine screening, and determine the feasibility and acceptability of voluntary urinalysis of clients at the Medically Supervised Injecting Centre, Kings Cross, Sydney. DESIGN AND METHODS: Brief surveys and urine drug screens were conducted with 67 participants in Wave 1 (October 2017) and 51 participants in Wave 2 (March 2018). Urine samples were tested with BTNX Rapid Response™ fentanyl urine strip test at a detection level of 20 ng/mL norfentanyl. These strips also cross-react to numerous fentanyl analogues. RESULTS: There were no cases where positive urine tests suggested unwitting fentanyl use detected in this study. DISCUSSION AND CONCLUSIONS: These negative findings contrast sharply with similar Canadian studies. While no cases of fentanyl-laced heroin use have been detected so far, we have demonstrated that this surveillance design is low-cost, feasible and scalable approach to monitoring the considerable public-health threat of undetected fentanyl and its analogues in Australia. Further validation of cross-reactivity of test strips would strengthen this method.

Somatic cell selection for chlorsulfuron-resistant mutants in potato: identification of point mutations in the acetohydroxyacid synthase gene.

BACKGROUND: Somatic cell selection in plants allows the recovery of spontaneous mutants from cell cultures. When coupled with the regeneration of plants it allows an effective approach for the recovery of novel traits in plants. This study undertook somatic cell selection in the potato (Solanum tuberosum L.) cultivar 'Iwa' using the sulfonylurea herbicide, chlorsulfuron, as a positive selection agent. RESULTS: Following 5 days' exposure of potato cell suspension cultures to 20 µg/l chlorsulfuron, rescue selection recovered rare potato cell colonies at a frequency of approximately one event in 2.7 × 105 of plated cells. Plants that were regenerated from these cell colonies retained resistance to chlorsulfuron and two variants were confirmed to have different independent point mutations in the acetohydroxyacid synthase (AHAS) gene. One point mutation involved a transition of cytosine for thymine, which substituted the equivalent of Pro-197 to Ser-197 in the AHAS enzyme. The second point mutation involved a transversion of thymine to adenine, changing the equivalent of Trp-574 to Arg-574. The two independent point mutations recovered were assembled into a chimeric gene and binary vector for Agrobacterium-mediated transformation of wild-type 'Iwa' potato. This confirmed that the mutations in the AHAS gene conferred chlorsulfuron resistance in the resulting transgenic plants. CONCLUSIONS: Somatic cell selection in potato using the sulfonylurea herbicide, chlorsulfuron, recovered resistant variants attributed to mutational events in the AHAS gene. The mutant AHAS genes recovered are therefore good candidates as selectable marker genes for intragenic transformation of potato.

Expression and purification of the antimicrobial peptide GSL1 in bacteria for raising antibodies.

BACKGROUND: The Gibberellin Stimulated-Like (GSL) or Snakin peptides from higher plants are cysteine-rich, with broad spectrum activity against a range of bacterial and fungal pathogens. To detect GSL peptides in applications such as western blot analysis and enzyme-linked immunosorbent assays (ELISA), specific antibodies that recognise GSL peptides are required. However, the intrinsic antimicrobial activity of these peptides is likely to prevent their expression alone in bacterial or yeast expression systems for subsequent antibody production in animal hosts. RESULTS: To overcome this issue we developed an Escherichia coli expression strategy based on the expression of the GSL1 peptide as a His-tagged thioredoxin fusion protein. The DNA sequence for the mature GSL1 peptide from potato (Solanum tuberosum L.) was cloned into the pET-32a expression vector to produce a construct encoding N-terminally tagged his6-thioredoxin-GSL1. The fusion protein was overexpressed in E. coli to produce soluble non-toxic protein. The GSL1 fusion protein could be easily purified by using affinity chromatography to yield ~1.3 mg of his6-thioredoxin-GSL1 per L of culture. The fusion protein was then injected into rabbits for antibody production. Western blot analysis showed that the antibodies obtained from rabbit sera specifically recognised the GSL1 peptide that had been expressed in a wheat germ cell-free expression system. CONCLUSION: We present here the first report of a GSL1 peptide expressed as a fusion protein with thioredoxin that has resulted in milligram quantities of soluble protein to be produced. We have also demonstrated that a wheat germ system can be used to successfully express small quantities of GSL1 peptide useful as positive control in western blot analysis. To our knowledge this is the first report of antibodies being produced against GSL1 peptide. The antibodies will be useful for analysis of GSL1peptides in western blot, localization by immunohistochemistry (IHC) and quantitation by ELISA.

Structure and expression of GSL1 and GSL2 genes encoding gibberellin stimulated-like proteins in diploid and highly heterozygous tetraploid potato reveals their highly conserved and essential status.

BACKGROUND: GSL1 and GSL2, Gibberellin Stimulated-Like proteins (also known as Snakin-1 and Snakin-2), are cysteine-rich peptides from potato (Solanum tuberosum L.) with antimicrobial properties. Similar peptides in other species have been implicated in diverse biological processes and are hypothesised to play a role in several aspects of plant development, plant responses to biotic or abiotic stress through their participation in hormone crosstalk, and redox homeostasis. To help resolve the biological roles of GSL1 and GSL2 peptides we have undertaken an in depth analysis of the structure and expression of these genes in potato. RESULTS: We have characterised the full length genes for both GSL1 (chromosome 4) and GSL2 (chromosome 1) from diploid and tetraploid potato using the reference genome sequence of potato, coupled with further next generation sequencing of four highly heterozygous tetraploid cultivars. The frequency of SNPs in GSL1 and GSL2 were very low with only one SNP every 67 and 53 nucleotides in exon regions of GSL1 and GSL2, respectively. Analysis of comprehensive RNA-seq data substantiated the role of specific promoter motifs in transcriptional control of gene expression. Expression analysis based on the frequency of next generation sequence reads established that GSL2 was expressed at a higher level than GSL1 in 30 out of 32 tissue and treatment libraries. Furthermore, both the GSL1 and GSL2 genes exhibited constitutive expression that was not up regulated in response to biotic or abiotic stresses, hormone treatments or wounding. Potato transformation with antisense knock-down expression cassettes failed to recover viable plants. CONCLUSIONS: The potato GSL1 and GSL2 genes are very highly conserved suggesting they contribute to an important biological function. The known antimicrobial activity of the GSL proteins, coupled with the FPKM analysis from RNA-seq data, implies that both genes contribute to the constitutive defence barriers in potatoes. The lethality of antisense knock-down expression of GSL1 and GSL2, coupled with the rare incidence of SNPs in these genes, suggests an essential role for this gene family. These features are consistent with the GSL protein family playing a role in several aspects of plant development in addition to plant defence against biotic stresses.

GSL2 over-expression confers resistance to Pectobacterium atrosepticum in potato.

Over-expression of the potato Gibberellin Stimulated-Like 2 ( GSL2 ) gene in transgenic potato confers resistance to blackleg disease incited by Pectobacterium atrosepticum and confirms a role for GSL2 in plant defence. The Gibberellin Stimulated-Like 2 (GSL2) gene (also known as Snakin 2) encodes a cysteine-rich, low-molecular weight antimicrobial peptide produced in potato plants. This protein is thought to play important roles in the innate defence against invading microbes. Over-expression of the GSL2 gene in potato (cultivar Iwa) was achieved using Agrobacterium-mediated gene transfer of a plant expression vector with the potato GSL2 gene under the regulatory control elements of the potato light-inducible Lhca3 gene. The resulting plants were confirmed as being transgenic by PCR, and subsequently analysed for transcriptional expression of the Lhca3-GSL2-Lhca3 chimeric potato gene. Quantitative RT-PCR analysis demonstrated that the majority of the transgenic potato lines over-expressed the GSL2 gene at the mRNA level. Based on qRT-PCR results and evaluation of phenotypic appearance, eight lines were selected for further characterisation and evaluated in bioassays for resistance to Pectobacterium atrosepticum (formerly Erwinia carotovora subsp. atroseptica), the causal agent of blackleg in potato. Three independent pathogenicity bioassays showed that transgenic lines with significantly increased transcriptional expression of the GSL2 gene exhibit resistance to blackleg disease. This establishes a functional role for GSL2 in plant defence against pathogens in potato.

Allele diversity for the apoplastic invertase inhibitor gene from potato.

In planta the enzymatic activity of apoplastic and vacuolar invertases is controlled by inhibitory proteins. Although these invertase inhibitors (apoplastic and vacuolar forms) have been implicated as contributing to resistance to cold-induced sweetening (CIS) in tubers of potato (Solanum tuberosum L.), there is a lack of information on the structure and allelic diversity of the apoplastic invertase inhibitor genes. We have PCR-isolated and sequenced the alleles of the apoplastic invertase inhibitor gene (Stinh1) from three tetraploid potato genotypes: 1021/1 (a genotype with very high tolerance to CIS), 'Karaka' and 'Summer Delight' (two cultivars that are highly susceptible to CIS). In total, five alleles were identified in these genotypes, of which four (Stinh1-c, Stinh1-d, Stinh1-e, Stinh1-f) were novel. An analysis of allele diversity was conducted by incorporating previously published sequences of apoplastic invertase inhibitors from potato. Eight alleles were assessed for sequence polymorphism in the two exons and the single hypervariable intron. Contrary to the hypervariable intron, only 65 single nucleotide polymorphisms were observed in the exons, of which 42 confer amino acid substitutions. Phylogenetic analysis of amino acid sequences indicates that the alleles of the invertase inhibitor are highly conserved amongst members of the Solanaceae family.