Antimicrobial Resistance (AMR) and Multidrug Resistance (MDR): Overview of Current Approaches, Consortia and Intellectual Property Issues

The supply of new diagnostics and treatments is insufficient to keep up with the increase in antimicrobial resistance (AMR) and multidrug resistance (MDR) as older medicines are used more widely and microbes develop resistance to them. At the same time, significant quantities of antibiotics are used on patients and animals that do not need them, while others who do need them lack access. Effective responses to AMR/MDR require effort by both the public and private sectors to develop and disseminate new diagnostics, vaccines and treatments on a global scale, as well as to adapt them to local needs. This calls for good governance to identify priorities, raise awareness and ensure effective stewardship at global, regional and national levels to minimize the development of resistance. Failure to act appropriately in one country will adversely impact all countries as resistance travels fast. Based on a review of recent literature, this WIPO Global Challenges Report includes a broad overview of current approaches and consortia designed to meet the challenge of research and development (R&D) investment for new treatments. It also examines patent applications by both the public and the private sectors as an indicator of innovative activity. This report finds that there is a need to address the unique market challenges and specific uncertainties associated with the development of new diagnostics and treatments, where current approaches are not optimal. An effective global framework that achieves the necessary political support while ensuring effective local implementation is crucial. There is an opportunity to complement this work by formulating mechanisms that drive innovation for results to incentivize success, while feeding expertise and experience into stewardship and access efforts. Intellectual property (IP) could be used in a constructive manner as one element in any reward or prize system for AMR/MDR R&D – both in terms of providing an incentive and governance

Studies on the metabolism and toxicity of hydrazine.

ABSTRACT Hydrazine is extensively utilised in industry and is a m inor metabolite of the clinically used drugs isoniazid and hydralazine. It is toxic, carcinogenic and mutagenic, but the metabolism and biochemical mechanisms of toxicity are not yet fu lly understood. Isolated rat liver microsomes incubated w ith both 2.0 mM and 0.2 m M hydrazine in the presence of NADPH and oxygen at 37°C resulted in the disappearance of hydrazine, which was demonstrated to be due to both enzymatic and chemical oxidation. Boiled microsomes increased the proportion of chemical disappearance whereas incubating the microsomes on ice effectively eliminated it. Further in vitro microsomal studies therefore incorporated samples incubated on ice as controls, allow ing the microsomal enzymatic metabolism of hydrazine to be calculated. Absence of NADPH and oxygen markedly reduced microsomal hydrazine metabolism, as did the presence of each of the cytochrome P450 inhibitors carbon monoxide, piperonyl butoxide and metyrapone, thus indicating that microsomal hydrazine metabolism is catalysed by cytochrome P450. Methimazole, an inhibitor of flavin monooxygenase, also diminished hydrazine metabolism, whereas N ADH in the presence of NADPH, but not alone, increased metabolism. Microsomes prepared from either p-naphthoflavone, acetone or isoniazid pretreated rats did not show significantly increased hydrazine metabolism compared to control microsomes per g protein. However phenobarbitone pretreatment did increase metabolism. Hydrazine metabolism was 20-70% lower in human microsomes prepared from 3 individuals compared to control rats. The dose response for hydrazine hepatotoxicity in vivo, as manifested by triglyceride increase and depletion of ATP and glutathione (GSH), was measured in control rats 6 hr after an i.p. dose. This was then compared to animals which had been pretreated w ith various inhibitors and inducers of cytochrome P450. Pretreatment w ith the inhibitor piperonyl butoxide resulted in an increase in hepatotoxicity, while induction by phenobarbitone (inducer of P450IIB) or p-naphthoflavone (inducer of P450IA) decreased hepatotoxicity. In contrast, acetone or isoniazid (inducers of II PREVIEW x ic /v iixj/ iaiu u ^ u u ii vauovu an 111^1 gaov 111 iic |/a iu iU A iu ijr, i n c 11 i u u u ia u u ii u i hydrazine hepatotoxicity by such pretreatments indicates that different isozymes of cytochrome P450 catalyse the metabolic transformation of hydrazine toxicity by various mechanisms. 6 hr after an acute i.p. hydrazine dose, certain dose related alterations in hepatic microsomal enzyme activity were measured, including a depletion in ethoxyresorufin O-deethylase and p-nitrophenol hydroxylase activity. Repeated adm inistration of 0.78 mM hydrazine in drinking water (2.5 m g.kg^.day'1) had a significant effect on several hepatic biochemical parameters and microsomal enzyme activities after 1,5 and 10 days. This indicated hydrazine to be a probable inducer of cytochrome P450IIE1. Hepatic biochemical parameters and activities of microsomal enzymes were virtually unchanged after repeated administration of 65 pM hydrazine in drinking water (0.25 m g.kg'1.day’1) for 5 or 10 days. In the presence of over 5 pM hydrazine, ATP synthesis in isolated mitochondria was inhibited. Inhibition up to 100 pM hydrazine was found to be dose related and reached a maximum 20-30% inhibition of control. However, above this concentration further inhibition did not occur. Hydrazine was also found to be metabolised by isolated mitochondria, which was not significantly decreased in the presence of either the monoamine oxidase A inhibitor, clorgyline or B inhibitor, pargyline

An open-source solution for advanced imaging flow cytometry data analysis using machine learning

Imaging flow cytometry (IFC) enables the high throughput collection of morphological and spatial information from hundreds of thousands of single cells. This high content, information rich image data can in theory resolve important biological differences among complex, often heterogeneous biological samples. However, data analysis is often performed in a highly manual and subjective manner using very limited image analysis techniques in combination with conventional flow cytometry gating strategies. This approach is not scalable to the hundreds of available image-based features per cell and thus makes use of only a fraction of the spatial and morphometric information. As a result, the quality, reproducibility and rigour of results are limited by the skill, experience and ingenuity of the data analyst. Here, we describe a pipeline using open-source software that leverages the rich information in digital imagery using machine learning algorithms. Compensated and corrected raw image files (.rif) data files from an imaging flow cytometer (the proprietary .cif file format) are imported into the open-source software CellProfiler, where an image processing pipeline identifies cells and subcellular compartments allowing hundreds of morphological features to be measured. This high-dimensional data can then be analysed using cutting-edge machine learning and clustering approaches using “user-friendly” platforms such as CellProfiler Analyst. Researchers can train an automated cell classifier to recognize different cell types, cell cycle phases, drug treatment/control conditions, etc., using supervised machine learning. This workflow should enable the scientific community to leverage the full analytical power of IFC-derived data set. It will help to reveal otherwise unappreciated populations of cells based on features that may be hidden to the human eye that include subtle measured differences in label free detection channels such as bright-field and dark-field imagery

N-Acetyl Cysteine May Support Dopamine Neurons in Parkinson\u27s Disease: Preliminary Clinical and Cell Line Data.

BACKGOUND: The purpose of this study was to assess the biological and clinical effects of n-acetyl-cysteine (NAC) in Parkinson\u27s disease (PD). METHODS: The overarching goal of this pilot study was to generate additional data about potentially protective properties of NAC in PD, using an in vitro and in vivo approach. In preparation for the clinical study we performed a cell tissue culture study with human embryonic stem cell (hESC)-derived midbrain dopamine (mDA) neurons that were treated with rotenone as a model for PD. The primary outcome in the cell tissue cultures was the number of cells that survived the insult with the neurotoxin rotenone. In the clinical study, patients continued their standard of care and were randomized to receive either daily NAC or were a waitlist control. Patients were evaluated before and after 3 months of receiving the NAC with DaTscan to measure dopamine transporter (DAT) binding and the Unified Parkinson\u27s Disease Rating Scale (UPDRS) to measure clinical symptoms. RESULTS: The cell line study showed that NAC exposure resulted in significantly more mDA neurons surviving after exposure to rotenone compared to no NAC, consistent with the protective effects of NAC previously observed. The clinical study showed significantly increased DAT binding in the caudate and putamen (mean increase ranging from 4.4% to 7.8%; p CONCLUSIONS: The results of this preliminary study demonstrate for the first time a potential direct effect of NAC on the dopamine system in PD patients, and this observation may be associated with positive clinical effects. A large-scale clinical trial to test the therapeutic efficacy of NAC in this population and to better elucidate the mechanism of action is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT02445651

Mechanism of subunit interaction at ketosynthase-dehydratase junctions in trans-AT polyketide synthases

Modular polyketide synthases (PKSs) produce numerous structurally complex natural products with diverse applications in medicine and agriculture. They typically consist of several multienzyme subunits that utilize structurally-defined docking domains (DDs) at their N- and C-termini to ensure correct assembly into functional multi-protein complexes. Here we report a fundamentally different mechanism for subunit assembly in trans-AT modular PKSs at the junction between ketosynthase (KS) and dehydratase (DH) domains. This involves direct interaction of a largely unstructured docking domain (DD) at the C-terminus of the KS with the surface of the downstream DH. Acyl transfer assays and mechanism-based cross-linking established that the DD is required for the KS to communicate with the acyl carrier protein appended to the DH. Two distinct regions for binding of the DD to the DH were identified using NMR spectroscopy, carbene foot-printing and mutagenesis, providing a foundation for future elucidation of the molecular basis for interaction specificity

Heme consumption reduces hepatic triglyceride and fatty acid accumulation in a rat model of NAFLD fed westernized diet

Studies have identified that serum-free hemoglobin subunits correlate positively with the severity of nonalcoholic fatty liver disease (NAFLD). However, the role of hemoglobin in the development of NAFLD remains unclear. In the present study, a rat model of NAFLD was developed, using a westernized diet high in saturated fat and refined sugar. Since a westernized diet is also high in red meat, we tested the effect of hemoglobin as a dietary source of heme in our model. Sprague-Dawley rats were fed ad libitum for 4 weeks either control diet (7% fat), westernized diet (WD, 18% fat + 1% cholesterol), hemoglobin diet (7% fat + 2.5% Hb), or westernized and hemoglobin diet (18% fat + 1% cholesterol + 2.5% Hb). Rats fed WD developed features of NAFLD, including insulin resistance and accumulation of liver fatty acids in the form of triglycerides, increased lipid peroxidation (F2-Isoprostanes), and liver fibrotic marker (hydroxyproline). Hemoglobin consumption significantly influenced several biomarkers of NAFLD and hepatic biochemistry, suggesting a possible interaction with diet and/or liver lipid pathways. The complex mechanisms of interaction between WD and hemoglobin in our rat model warrants further studies to examine the role of dietary heme on NAFLD

Pygmy blue whale movement, distribution and important areas in the Eastern Indian Ocean

This study was conducted as part of AIMS’ North West Shoals to Shore Research Program (NWSSRP) and was supported by Santos as part of the company’s commitment to better understand Western Australia’s marine environment. Hydrophone pressure data from Ocean Bottom Seismometers (OBS) were provided by the CANPASS project, jointly funded by the National Natural Science Foundation of China (NSFC grants 91955210, 41625016), and the China Academy of Science (CAS program GJHZ1776). Instruments were provided by the Australian National instrument pool ANSIR (http://ansir.org.au/). ANSIR, OBS data was also made data available from the Geoscience Australia and Shell. Data was sourced from Australia’s Integrated Marine Observing System (IMOS).Pygmy blue whales in the South-east Indian Ocean migrate from the southern coast of Australia to Indonesia, with a significant part of their migration route passing through areas subject to oil and gas production. This study aimed at improving our understanding of the spatial extent of the distribution, migration and foraging areas, to better inform impact assessment of anthropogenic activities in these regions. Using a combination of passive acoustic monitoring of the NW Australian coast (46 instruments from 2006 to 2019) and satellite telemetry data (22 tag deployments from 2009 to 2021) we quantified the pygmy blue whale distribution and important areas during their northern and southern migration. We show extensive use of slope habitat off Western Australia and only minimal use of shelf habitat, compared to southern Australia where use of the continental shelf and shelf break predominates. In addition, movement behaviour estimated by a state-space model on satellite tag data showed that in general pygmy blue whales off Western Australia were mostly engaged in migration, interspersed with mostly relatively short periods (median = 28hours, range = 2 – 1080hours) of low move persistence (slow movement with high turning angles), which is indicative of foraging. Using the spatial overlap of time and number of whales in area analysis of the satellite tracking data (top 50% of grid cells) with foraging movement behaviour, we quantified the spatial extent of pygmy blue whale high use areas for foraging and migration. We compared these areas to the previously described areas of importance to foraging and migrating whales (Biologically Important Areas; BIAs). In some cases these had good agreement with the most important areas we calculated from our data, but others had only low (5%) to moderate (13%) overlap. Month was the most important variable predicting the number of pygmy blue whale units and number of singers (acting as indices of pygmy blue whale density). Whale density was highest in the southern part of the NW Australian coast and whales were present there between April-June, and November-December, a pattern also confirmed by the satellite tracking data. Available data indicated pygmy blue whales spent up to 124 days in Indonesian waters (34% of annual cycle). Since this area may also be the calving ground for this population, inter-jurisdictional management is necessary to ensure their full protection.Publisher PDFPeer reviewe

The Effect of Banisteriopsis caapi (B. caapi) on the Motor Deficits in the MPTP-treated Common Marmoset Model of Parkinson's disease

Banisteriopsis caapi (B. caapi) contains harmine, harmaline, and tetrahydroharmine, has monoamine oxidase inhibitory activity, and has reported antiparkinsonian activity in humans when imbibed as a tea; however, its effects are poorly documented. For this reason, motor function was assessed in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated common marmosets following administration of B. caapi extract (28.4–113.6 mg/kg; po), harmine (0.1 and 0.3 mg/kg; sc), and selegiline (10 mg/kg; sc), alone or with a submaximal dose of L-3,4-dihydroxyphenylalanine (L-DOPA; 4–7 mg/kg). L-DOPA reversed motor disability, increased locomotor activity, and induced moderate dyskinesia. B. caapi did not increase locomotor activity or induce dyskinesia but at 56.8 and 113.6 mg/kg improved motor disability. The L-DOPA response was unaltered by co-administration of B. caapi. Harmine (0.1 and 0.3 mg/kg) produced a mild improvement in motor disability without affecting locomotor activity or dyskinesia but had no effect on the L-DOPA-induced antiparkinsonian response. Selegiline (10 mg/kg) alone improved motor function to the same extent as L-DOPA, but with only mild dyskinesia, and did not alter the response to L-DOPA, although dyskinesia was reduced. The findings suggest that B. caapi alone has a mild antiparkinsonian effect but does not enhance the L-DOPA response or reduce dyskinesia

Using co-inquiry to study co-inquiry: community-university perspectives on research collaboration

In the context of a rapid development of interest in community-university research partnerships, this article argues for a greater focus on collaborative reflexivity to enhance learning from the research process and contribute toward developing sustainable and ethical research collaborations. Incorporating perspectives of community and university participants, the article offers a case study analysis of a UK-based co-inquiry action research group. This group not only studied examples of community-university research collaborations, but also reflected on its own workings as an example of collaborative research in action—scrutinizing relationships of power, responsibility, and boundaries in the group (collaborative reflexivity). This article argues that research projects might be designed with space designated for co-inquiry action research or similar inquiry groups. These co-inquiry groups would serve as replacements or supplements to more traditional steering or advisory groups