Although scenario‐based simulation is an established learning method, some potential benefits remain unexplored. These include its provision of unique opportunities for students to acquire the perspective of the patient and those of interprofessional colleagues. Simulation may also facilitate a detailed understanding of pathophysiology through the writing of scenarios. We sought to extend the potential of simulation by asking students to write, deliver and debrief simulation scenarios.During an established 2‐day, scenario‐based programme using a manikin patient , final‐year medical students were asked to write, deliver and debrief a simulation scenario while working in groups.Students were encouraged to base their scenario on any clinical case they had encountered. They were given a standard template with headings for learning objectives, patient details, required learner actions and simulated patient responses to those actions.Students had 1 month to prepare a patient history, script for the patient, hospital medical record, and relevant blood test and radiology results. Preparation required thorough research of the pathophysiology and clinical evolution of the chosen condition, as well as expected responses to pharmacological interventions.Scenarios underwent expert review at an early stage of development and again immediately prior to delivery. Scenarios were delivered by students (‘instructor’ peers), with one student ‘acting’ as the voice of the patient (audible from the manikin's mouth) and others acting as nurses. Another group of students functioned as scenario participants (‘learner’ peers), assessing and treating the simulated patient. Each scenario was followed by an educational debriefing, facilitated by the ‘instructors’. A total of 79 students participated. Groups took turns in ‘instructing’ and ‘learning’, without prior knowledge of the other groups’ scenarios.The delivery of peer‐written and peer‐run simulation scenarios was feasible, although it represented a demanding exercise for the simulation faculty staff. Unanticipated actions on the part of both the student participants in the scenario and the student instructors delivering the scenario required flexibility and spontaneity.Expert review of scenarios prior to delivery was a key factor in successful delivery as students tended to write scenarios that expected an unrealistically high level of performance from their peers.Students were able to perform high‐standard educational debriefing. Preparing for technical debriefing motivated students to research best‐practice management guidelines for their chosen condition. With minimal teaching students were able to provide their peers with feedback on non‐technical behaviours (teamwork and communication) based solely on exposure to previous instructor‐led debriefings.n = 79) gave the exercise an overall mean rating of 4.6 on a scale of 1–5 .All simulation scenarios are evaluated for quality assurance. For the peer‐led simulation exercises, students were asked whether the components of developing, delivering and debriefing the scenario improved their knowledge; students agreed in 94%, 91% and 96% of responses, respectively. Students (Qualitative feedback from students suggested potential benefits not foreseen by the instructors, which are now the subject of an ongoing research project. These included better understanding of the patient's perspective acquired by acting as the voice of the ‘patient’ and improved interprofessional understanding achieved through acting as a nurse.


Global concerns about anthropogenic climate change, energy security and independence, and environmental consequences of continued fossil fuel exploitation are driving significant public and private sector interest and financing to hasten development and deployment of processes to produce renewable fuels, as well as bio‐based chemicals and materials, towards scales commensurate with current fossil fuel‐based production. Over the past two decades, anaerobic microbial production of ethanol from first‐generation hexose sugars derived primarily from sugarcane and starch has reached significant market share worldwide, with fermentation bioreactor sizes often exceeding the million litre scale. More recently, industrial‐scale lignocellulosic ethanol plants are emerging that produce ethanol from pentose and hexose sugars using genetically engineered microbes and bioreactor scales similar to first‐generation biorefineries.et al., Concomitant with the genesis of a lignocellulosic fuel ethanol industry, publicly funded research by the academic and governmental scientific community has largely shifted its research and development emphasis to producing biofuels that can be drop‐in replacements for fossil‐based diesel, gasoline and jet fuels. This research pivot reflects both limitations in infrastructure compatibility and associated  concerns with ethanol as a fuel blendstock, as well as growing promise of new capabilities in microbial genetic modification emerging through advances in metabolic engineering and synthetic biology. For biofuels produced primarily via microbes, most proposed target drop‐in fuels or fuel precursors are or will be produced via pathways ‘deep’ in carbon metabolism, such as fatty acid, isoprenoid or polyketide synthesis pathways  are thermodynamically ‘uphill’ anabolic metabolic pathways that require energy in the form of ATP and NAD(P)H to achieve high production rates, i.e. as opposed to the thermodynamically ‘downhill’ catabolic metabolic pathways used to produce ethanol (or butanol or short‐chain carboxylic acids) by anaerobic fermentation, which are net ATP‐positive, redox balanced, and can be performed anaerobically.2) to a submerged culture and secondarily due to reduced economies of scale for scaling up aerobic versus anaerobic processes. As an example, the higher costs for aerobic versus anaerobic production are illustrated by comparing two recent NREL design case reports based on largely similar process designs: much higher costs are projected to aerobically produce renewable diesel blendstock using oleaginous yeast  concentration, which are not typically significant at bench scale, will be present in full‐scale bioreactors. Ultimately, new process designs may be required to achieve sufficient mixing in the bioreactor vessel so that under proper operation, the magnitude and duration of such gradients will remain within what the production strain can accommodate. Achieving favourable process economics requires extreme cost minimization, however, and will likely include minimizing energy inputs for mixing and aerating the culture, i.e. power for agitation and air/oxygen compression, and lowering these energy inputs will generally translate into larger gradients within the bioreactor. As such, beyond identifying native or engineered strains that possess desired or more desirable metabolic pathways, strain selection needs to place greater focus on identifying production strains capable of maintaining robust performance over a wider range of temperatures and nutrient concentrations than are typically being studied and reported on in recent literature. Simply put, for extremely large‐scale production, strains will be favoured that can remain productive in the presence of larger concentration and temperature gradients within the bioreactor over the course of the production process.Thinking big about what attributes biofuels production strains will need benefits from envisioning at the outset what large‐scale production will look like and thoroughly considering how large‐scale conditions will differ from those typically encountered in smaller scale laboratory bioreactors Blanch, . First, For robust cultivation on lignocellulosic sugars, there are several general, well‐known strain selection criteria. Foremost are the ability to achieve high cell mass growth and biofuels production rates in biomass‐derived hydrolysates, which often contain a variety of inhibitory components, including aromatic compounds derived from lignin, acetate from hemicellulose, and aldehydes from sugar dehydration reactions. Additionally, the ability to utilize a broad range of pentose and hexose sugars is tremendously beneficial, with the caveat that if needed the ability to utilize additional sugars likely can be engineered into a strain, although this is often a non‐trivial undertaking. Lastly, the ability to tolerate high temperatures or low pH (or lack of pH control) is often a key driver in strain selection. This is because operating a bioreactor at higher temperature generally makes temperature control easier and more economical. Higher temperature also favours higher reaction rates, lower cultivation broth viscosities, and in some cases can also be useful to reduce contamination risk during production. Similarly, the ability to tolerate lower pH can be useful to reduce contamination by many bacteria.Escherichia coli, which is advantageous for being genetically tractable and reasonably well understood, but also has many undesirable traits such as relatively low tolerance to acids and low pH, no well‐established coproduct value for its cell mass, and limited thermotolerance. It is also reflected by a dearth of new public domain literature on larger scale bio‐based aerobic production over the past 20+ years. Notably, the literature on oxygen transfer and aeration for submerged production published over the last several decades is dominated by reports of mammalian and microbial cell‐based pharmaceutical/therapeutic production, with bioreactor of volumes in the range of 20 000 L deemed ‘large scale’; this is orders of magnitude smaller than the 1 000 000 L and larger scales considered relevant for submerged aerobic fuel production.In general, the case can be made that, to date, much of the effort to develop strains for large‐scale biofuels production has been focused on what is known and can be carried out today rather than on what needs to be carried out to achieve the unprecedented levels of process efficiency and cost minimization that will be required to economically produce relatively low priced and low margin biofuels in submerged aerobic bioprocesses. The literature shows recent work being carried out predominantly using well‐characterized microbes and idealized experimental conditions we are most familiar with to demonstrate ‘proof of concept’ of new proposed production routes rather than identifying strains that are sufficiently robust to perform well in envisioned large‐scale processing conditions where higher concentrations and larger concentration and temperate gradients will be present. This is exemplified by the predominance of the use of well‐known microbes like 2 gas‐liquid mass transfer) and heat transfer as well as constrain the size to which the process can be economically scaled. For this reason, research on aerobic production has generally used clarified (solid‐free) sugars as a carbon source, i.e. rather than whole slurry hydrolysates as generated by enzymatic hydrolysis of pretreated biomass. Regardless, it is essential that to maintain a sufficiently low broth viscosity that good mixing and O2 transfer can occur, and it is important to minimize the presence of non‐host microbe solids. Similarly, to maintain effective mixing and aeration, a production strain must not form appreciable levels of any coproducts such as exopolysaccharides (EPS) that will significantly increase the viscosity of the culture broth. In general, feedstock carbon should not be directed to non‐target products in any manner that lowers product yield.In addition to the well‐known titre, rate and yield (TRY) performance criteria, another strain/process attribute that can make or break the viability of a large‐scale production process is broth rheology, which if too high can hinder mass transfer , wherein synthetic biology techniques were used to ‘rewire’ central carbon metabolism in Saccharomyces cerevisiae to increase the efficiency of acetyl‐CoA‐based isoprenoid production, with the modified pathway impressively achieving 25% higher product yield on sugar while requiring 75% less oxygen for aeration.Looking forward, another more complex but powerful strategy with growing potential given the synthetic biology pathway engineering tools now available is to re‐engineer biofuels (or biofuels precursors) production metabolic pathways to increase their energy efficiency and thereby reduce their oxygen demand and related aeration energy requirements. A leading example of successfully applying this approach was recently reported by Amyris  technology is emerging as an intriguing possibility for enhancing bioprocess efficiency by enabling renewable energy in the form of green electrons to supply some of the reducing power needed to drive anabolic product synthesis (Schievano


Differing interpretations about evidence on benefits and harms of screening mammography has led to conflicting recommendations among different jurisdictions that range from intensive screening starting at age 40 to no screening at all. Despite broad attention of scientific and nonscientific media, evidence suggests substantial discrepancy between real and perceived benefits of screening mammography among women. In this commentary, underlying concept of mammographic screening, limitations in primary evidence, results from secondary evidence, and existing misunderstandings are underscored with a critical gaze at available information. Most government programs are initiated based on positive theoretical utility, and usually with good intentions. However, once established, such programs become luxury of certain group that has immense interest to preserve them regardless of their eventual practical utility—a reason why we find very few instances of a government program being revoked in the entire history. Screening mammography is one of the examples. Despite a common scientific evidence, recommendations about screening mammography vary among jurisdictions ‐ American Cancer Society updated recently their guidelines revising recommended age of initiation of screening mammogram from 40 to 45 years; Canadian task force on preventive health care recommends it for women starting at age 50; whereas, the Swiss medical board recommended recently against any new mammographic screening programs Localized breast cancer in itself is never fatal. Therefore, the sole purpose of screening mammography is to detect breast cancer early, thereby preventing progression into an advanced form. An analysis from the Surveillance, Epidemiology, and End Results (SEER) database suggests no increase in the incidence of de‐novo metastatic breast cancer since 1975 Concept of mammography relies on a traditional assumption that breast cancer grows in anatomic linearity—first involving the breast, then progressing to locoregional lymph nodes before involving the distant organs. However, breast cancer is an enormously heterogeneous group of diseases. Whereas some breast cancers are slow‐growing and may disseminate linearly, others will have  involved distant organs before they are detected in the breast—identification of such cancers with screening mammogram is either not possible or is unhelpful.Natural history of some screen‐detected breast cancers is likely different from nonscreen‐detected breast cancers. For example, an astonishing phenomenon was observed when upper age limit of screening mammography was updated in the UK from 64 to 69 years in 2001—there was a steep surge in incidence of new breast cancer, although these women were already being screened for many years while they were still under 64 years—such observation can only be explained if screen‐detected cancers regress with time A meta‐analysis of 10 clinical trials involving over 600,000 women by the Cochrane collaboration suggested only a modest reduction in breast cancer deaths attributable to screening, and that was counterbalanced by deaths from other causes without benefits in overall survival And 90% of the clinical trials that evaluated screening mammography are old (conducted before the eighties) and are methodologically poor by modern standards as evidenced by the fact that the trials that report the largest benefits from screening mammography ironically used only one‐view mammography, screened the control groups only after 3–5 years, and screened at substantially prolonged intervals Survey of American women suggests that a vast majority of women (>70%) believed that screening mammography reduces the risk of death from breast cancer by at least half which is far from what the evidence suggests Timing of uptake of screening mammography around the world varies, but the death after diagnosis of breast cancer started to decrease consistently after introduction of tamoxifen and effective chemotherapies If screening mammography is to be ever beneficial, that is possible only in carefully selected women based on individual assessment of their risks rather than of the population as a whole. Women with strong personal or family history and/or genetic conditions that increase the risk of breast cancer should be screened more vigilantly. Evidence‐based tools to estimate variations in harms and benefits have been published and may help inform women Author has no conflict of interest to disclose.


This data article provides time series on euro area industrial new orders and is related to the research article entitled “Modelling industrial new orders”   Specifications TableValue of the data•The data fill the existing data gap of euro area industrial new order statistics. Monthly updates provide timely information for users.•Industrial new orders are used as leading indicator for economic activity  as well as for analysis and monitoring of economic developments.•The indicators for new orders are combined with the formerly “official” series on new orders from Eurostat so that users have access to long time series, an important feature for modeling.1The data on euro area industrial new orders are available in time series format with a fixed base year (2010) in seasonally and calendar adjusted format. The first two columns of 2For those euro area member states that continue with the data collection , national new order data are used as input. For the remaining euro area countries , estimates are derived, based on various model determinants. The detailed model specifications are available in Data on euro area new orders are subject to revisions in real time, like other economic series, i.e. hard data statistics.


MEDLINE and EMBASE, were systematically searched up to July 2015. The methodological quality of studies included in the review was performed according to Preferred Reporting Items for Systematic Reviews and Meta‐analyses guidelines. Twenty‐five studies were included in the review, identifying 241 SLE candidate proteomic biomarkers related to various aspects of the disease including disease diagnosis and activity or pinpointing specific organ involvement. Furthermore, 13 of the 25 studies validated their results for a selected number of biomarkers in an independent cohort, resulting in the validation of 28 candidate biomarkers. It is noteworthy that 11 candidate biomarkers were identified in more than one study. A significant number of potential proteomic biomarkers that are related to a number of aspects of SLE have been identified using mass spectrometry proteomic approaches. However, further studies are required to assess the utility of these biomarkers in routine clinical practice.Advances in mass spectrometry technologies have created new opportunities for discovering novel protein biomarkers in systemic lupus erythematosus (SLE). We performed a systematic review of published reports on proteomic biomarkers identified in SLE patients using mass spectrometry‐based proteomics and highlight their potential disease association and clinical utility. Two electronic databases,  SLE is a chronic autoimmune inflammatory disease with a broad spectrum of clinical manifestations, affecting the majority of organs and tissues. In most cases, vital organs are involved including brain, heart, joints, skin and kidneys The diagnosis of SLE is challenging due to its heterogeneous nature, variable clinical presentation and unpredictable course with periods of remission and flares e.g. anti‐double‐stranded DNA antibodies), are of limited sensitivity and specificity. In lupus nephritis, renal biopsy remains the ‘gold standard’ not only for assessing disease activity, but also for assessing prognosis and monitoring therapy Besides the pressing need to improve the classification of patients with SLE, assessment of disease activity remains another important aspect in the management of patients with SLE. At present, conventional disease assessment methods, including the use of acute phase markers and autoimmune serologic tests  was developed using the following search terms: [ AND ]. The literature search in EMBASE (1988–July 2015) was conducted in a similar way using the terms: [‘Systemic Lupus Erythematosus’ OR ‘SLE’ OR ‘Lupus nephritis’ OR ‘Lupus’) AND ]. Searching results from the two databases were imported in the bibliographic management software Endnote X5 . Duplicates were automatically removed. The articles were reviewed in a two‐stage procedure. In the first stage of the review process, abstracts of all identified articles were screened. Review articles, editorials, case reports, letters to the editor, conference abstracts, notes and news were excluded. In the second stage of the review process, full texts of the remaining studies were evaluated. A checklist of specified inclusion criteria was used to ensure uniformity in the assessment of the identified manuscripts. The final articles that were selected all fulfilled the following eligibility criteria: written in English, used human samples, compared biological fluids from patients with SLE with a control group , referred to SLE, were informative about the type of biological fluid used, provided details about the mass spectrometry technique used as well as the proteomic biomarkers identified. Articles that did not meet one or more of these inclusion criteria were excluded. This review was performed by two independent reviewers (ON and AK) based on Preferred Reporting Items for Systematic Reviews and Meta‐analyses (PRISMA) guidelines The initial literature search identified 1093 records. After duplicate removal, 775 records remained. These were all screened, resulting in a total of 25 selected full‐text articles, published between 2007 and 2015, which were included in this systematic review. The detailed review process with the eligibility criteria used is shown in Figure S1. The main findings of each article are summarized in Table In the 25 selected articles, a variety of specimen types obtained from patients with SLE was used. This can be attributed to the clinical heterogeneity of the disease, affecting almost all organs and tissues. Serum was the most frequently used biological fluid (9 of the 25 studies) i) separation of proteins and peptides present in a complex biological sample using gel‐based or gel‐free techniques, (ii) MS‐based protein identification The term proteomics describes the large‐scale characterization of the whole protein content of a cell, organ or organism at a given time In the current systematic review, 10 studies used 2‐DE, two studies used sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) A variety of MS approaches was used to obtain proteomic data from different biological samples. These include MALDI‐TOF MS, MALDI‐TOF/TOF MS, surface‐enhanced laser desorption/ionization (SELDI)‐TOF MS and liquid chromatography (LC) combined with tandem MS , 7170, 7661, 7740, 5806 A total of 241 candidate biomarkers were identified in the 25 studies included in this review  SLE biomarkers, (ii) LN biomarkers and (iii) biomarkers associated with neuropsychiatric SLE (NPSLE).This review has revealed 241 potential SLE protein biomarkers that have been identified by MS‐based proteomics, using both targeted and untargeted approaches. Due to the large number of proteins detected, herein we are discussing the most promising biomarkers for SLE by focusing on two particular aspects. The first aspect includes studies in which proteins were validated in an independent cohort, using either MS or immunobased techniques, and the second includes the studies that had identified common biomarkers. In order to present a more targeted discussion, we divided the validated protein biomarkers into three main categories, based on their association with different clinical aspects of the disease. These categories are as follows: , and produce increased interferon‐α The first category includes the most promising biomarkers that are suitable for SLE diagnosis or activity assessment. An increased expression of phosphorylated S100A9 isoforms was detected in the proteome of SLE PBMCs, suggesting abnormal S100A9 signalling, as well as reflecting the increased numbers of circulating low‐density granulocytes in these patients SLEDAI, suggesting an association with a favourable clinical course in patients with SLE and could thus be used as a potential biomarker of clinical SLE activity/severity. The mechanisms of action of STRAP in SLE may be attributed to its interaction, either with transforming growth factor‐β receptor Serine‐threonine kinase receptor‐associated protein is another important biomarker that was found to be under‐expressed in PBMCs of active SLE A significant number of biomarkers were found to be associated with LN diagnosis or clinical activity/severity. Five biomarkers for diagnosing LN in kidney biopsies of class IV LN patients including serotransferrin, cytokeratin 18, cytokeratin 19, albumin and annexin A5 were identified Serotransferrin, an iron‐binding transport protein coregulated by interferon‐α, plays a role in iron metabolism and the innate immune system Cytokeratins (CKs) are a family of intermediate filament proteins, comprising 20 known CKs, which are classified into type I keratins (K9−K20) or type II keratins (K1–K8) Annexins are calcium‐dependent, phospholipid‐binding proteins Serum apolipoprotein CIII (apoCIII) was associated with an increased atherosclerotic risk in LN patients 2D‐isomerase (PGDS) was identified as a candidate biomarker for LN activity 2 (PGH2) to PGD2, which is implicated in physiological processes such as sleep regulation, prevention of platelet aggregation, allergy and inflammation de novo synthesis of PGDS in podocytes, and Bowman's capsule of the glomeruli Urinary prostaglandin HHepcidin is a 25‐amino acid peptide hormone mainly produced by hepatocytes and a key regulator of systemic iron homeostasis SLE affects both the central and the peripheral nervous system. NPSLE is a common manifestation of SLE, with a prevalence of 14–80% in adults and 22–95% in children. It has been suggested that NPSLE pathogenesis may be associated with autoantibody‐mediated neuronal dysfunction and vasculopathy Gdi1 gene that encodes αGDI were reported in families with X‐linked non‐specific mental retardation Gdi1‐deficient mice demonstrated damage of associative memory as well as changes in social behaviour without any anatomic abnormality Anti‐αGDI antibody was identified as a potential diagnostic biomarker of psychosis‐associated NPSLE Ubiquitin is a small regulatory protein, member of a family of structurally conserved proteins, which regulate numerous processes in eukaryotic cells. The most significant function of ubiquitin is targeting proteins for degradation This systematic review has also identified 11 biomarkers that were detected in at least two independent studies Table . These bIn this content, A1AT was found in the urine of active LN and during SLE renal flares via limiting the B‐cell IL‐10 production Ezrin was identified as a candidate antigen for serum autoantibodies in proliferative LN forms Elongation factor 1‐alpha 1 (EF‐1A1) was also a candidate antigen in proliferative LN and was downregulated in PBMCs from patients with SLE, compared with healthy controls Another interesting protein, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), was found to be a candidate antigen for antibodies in patients with proliferative LN In addition, alpha‐enolase was identified as a candidate antigen in proliferative LN forms Transthyretin and haptoglobin (Hp) were upregulated in urine samples from active LN HLA, STAT4 and IRF5. However, risk variants identified so far explain only a small fraction of the overall SLE heritability IRF2, IRF5, UBE2L3, IFIHI, TNIP1, TNFAIP3 and BLK, but also enabled the discovery of additional gene variants, especially rare variants that are not identified by GWAS Finally, advances in genomic technologies during the past decade have enabled the identification of numerous risk genetic factors associated with susceptibility to SLE. To date, more than 60 SLE susceptibility loci have been identified by genome‐wide association studies (GWAS) in different population cohorts, including An ideal SLE biomarker would be biologically and pathophysiologically relevant, reproducible, simple to apply in routine practice  and would have a high degree of sensitivity and specificity Although a significant number of SLE biomarker reports have been published to date, there are still many challenges that need to be overcome in future proteomic studies that aim to identify clinically useful SLE biomarkers. Firstly, differences in proteomic results across different studies can be attributed to differences in the selection criteria of the samples. A recent published study uncovered the molecular heterogeneity of SLE, providing an explanation for the failure of the clinical trials Advances in high‐throughput MS technologies have undoubtedly created new avenues for discovering sensitive and specific SLE biomarkers. MS‐based proteomics have been used to study a plethora of biological specimens from patients with SLE, leading to the identification of biomarkers related to disease diagnosis and activity as well as to specific organ involvement. There are already a number of valuable MS‐based proteomic studies in the literature that fulfil most of the requirements for clinical proteomic biomarker reporting. A significant number of potential biomarkers have been identified to be associated with many clinical aspects of SLE, including diagnosis, disease activity and prognosis. It is noteworthy that almost half of these studies have validated their results in an independent cohort. What is lacking and should be addressed in future biomarker studies, is the use of larger patient cohorts, as well as the validation of already identified biomarkers in independent patient cohorts. Furthermore, protein biomarkers, which have been already identified in multiple studies, particularly those that were also detected in non‐invasive biological samples, hold a great promise. Such biomarkers should form the backbone and be at the forefront of executing larger multicentre studies in future using well‐characterized patient cohorts, in order to prove their clinical utility.The authors declare that they have no competing interests.The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.Figure S1 Flow diagram showing the systematic literature search and review process. All methods were applied in accordance with the PRISMA guidelines.Table S1. Requirements for scientific reporting of proteomic biomarker data reported by Mischak et al. 2010 implemented for the 25 articles obtained in the systematic review.Click here for additional data file.


To test the feasibility and assess the uptake and acceptability of implementing a consumer questions programme, AskShareKnow, to encourage consumers to use the questions ‘1. What are my options; 2. What are the possible benefits and harms of those options; 3. How likely are each of those benefits and harms to happen to me?’ These three questions have previously shown important effects in improving the quality of information provided during consultations and in facilitating patient involvement.This single‐arm intervention study invited participants attending a reproductive and sexual health‐care clinic to view a 4‐min video‐clip in the waiting room. Participants completed three questionnaires: (T1) prior to viewing the intervention; (T2) immediately after their consultation; and (T3) two weeks later.A total of 121 (78%) participants viewed the video‐clip before their consultation. Eighty‐four (69%) participants asked one or more questions, and 35 (29%) participants asked all three questions. For those making a decision, 55 (87%) participants asked one or more questions, while 27 (43%) participants asked all three questions. Eighty‐seven (72%) participants recommended the questions. After two weeks, 47 (49%) of the participants recalled the questions.Enabling patients to view a short video‐clip before an appointment to improve information and involvement in health‐care consultations is feasible and led to a high uptake of question asking in consultations.This AskShareKnow programme is a simple and feasible method of training patients to use a brief consumer‐targeted intervention that has previously shown important effects in improving the quality of information provided during consultations and in facilitating patient involvement and use of evidence‐based questions. Shared decision making is considered key to supporting patients to understand treatment and possible options, express values and preferences to make good treatment decisions and better manage their health.To address these challenges, we previously conducted a crossover controlled trial to test whether three generic questions could facilitate communication about treatment options independently of the clinical context.Box 1What are my options?What are the possible benefits and harms of those options?How likely are each of those benefits and harms to happen to me? Including ‘What will happen if I do nothing?’In this prior study involving trained actors as unannounced standardized patients, we showed that these questions significantly increased facilitation of patient involvement in the consultations, as well increasing information given by physicians about options and their associated benefits and harms.®). The intervention included three components: a 4‐min video‐clip that participants were shown in the waiting room ; a pamphlet which incorporated a consultation summary sheet and website information (www.askshareknow.com.au); and a refrigerator magnet as a reminder for future use.This was a single‐arm intervention study to test the feasibility of the intervention, for which we developed a consumer questions training and support programme, now called AskShareKnow (ASK Patient–Clinician Communication ModelThe study was conducted at a metropolitan family planning clinic (part of a statewide non‐government organization) providing reproductive and sexual health‐care services, primarily to women. Patients >18 years of age, and with English fluency, attending appointments at the clinic were invited to participate in the study. Those with infants were excluded for logistical reasons.Family planning consultations are ideal for testing shared decision‐making interventions because they encompass a very wide range of treatment options  with near equipoise of benefits and harms, and are therefore very sensitive to consumer preference. Furthermore, women – the overwhelming consumers in this clinic – are usually the primary decision‐maker of health matters in society and have influence in over 80% of health‐care decisions.Recruitment took place between July and December 2012. The study was approved by the Human Research Ethics Committees of Family Planning NSW and the University of Sydney. Detailed study information was provided to participants and written consent obtained. The trial was registered with the Australian New Zealand Clinical Trials Registry no. ACTRN12610001055099.After providing consent, participants were provided with the three components of AskShareKnow described above, and asked to complete a questionnaire prior to their consultation (T1). They were then given a media tablet with headphones to watch the 4‐min AskShareKnow video‐clip (but not otherwise invited to ask the three questions featured). To avoid disrupting clinic flow, appointment scheduling took precedence over recruitment, so not all who were recruited were exposed to all the intervention. Those who watched the video‐clip prior to their consultation were invited to complete a second questionnaire immediately after the consultation (T2), and then a third questionnaire at 2 weeks (T3) . Questionnaire items included quantitative and qualitative responses. Quantitative responses were analysed using descriptive statistics. Participants were also invited to take part in a semi‐structured telephone interview at four to six weeks to obtain more detailed information about their experience and views of the AskShareKnow programme. Telephone Interviews were audio‐recorded transcribed verbatim and analysed using thematic analysis.The primary outcome was the use of the three questions in the health‐care consultation based on self‐report by the participants. Other outcomes focussed on key demand, acceptability and implementation aspects of AskShareKnowData were collected at three time points. The baseline questionnaire (T1) included demographic information, decision making and information preferencesA total of 197 participants consented to the study; however, participants who had not completed viewing the video‐clip before being called into their consultation see  were excSix study participants and four clinicians participated in semi‐structured telephone interviews exploring their experience of asking, or being asked, the questions.Thirty‐three women (27%) were born outside Australia, 73 (60%) university educated, 70 (58%) aged under 40 years Table . InformaDemographic information of those who consented but did not have sufficient time prior to their consultation to view the video‐clip was similar to the final sample included in the analysis.n = 58), 29 (50%) asked at least one of the Questions and 8 (14%) asking all three Questions. Of the three Questions, 1 and 2 were asked by 66 and 62 participants, respectively, while Question 3 was asked by 49  reported asking at least one of the questions during their consultation, with 35 29%) asking all three Questions (Table 9% askingThe other components of the ASK Patient–Clinician Communication model were less utilized by participants in the study. Thirty‐eight (31%) had time to view the AskShareKnow website prior to their consultation, and 58 (48%) reported reading the pamphlet prior to their consultation, with 19 (16%) reporting using the consultation summary sheet during the consultation.Several of the participants who were interviewed reported that the questions were similar to those they would normally ask, although commented the questions provided additional structure Box i.Box 2n = 4) interviewed commented that the questions covered information that would generally be covered during a consultation, but all stated it was useful for participants to be provided with the questions in the waiting room, although one did raise query whether they were necessary in all consultation types (Box The clinicians (ypes Box ii.n = 6) who were interviewed stated they would ask the questions again, although not necessarily verbatim , reported they would definitely recommend the questions. Two weeks later, 47 (83%) of those completing the final questionnaire would use the questions again. Each of the participants , or somewhat helpful 19 (31%). This reflected comments made by participants who were interviewed Box iv.Participants were asked whether they recalled the three questions two weeks after their consultation T3). Of the 95 participants who responded, 47 (49%) could; of 62 participants who provided details of the questions, 29 (47%) were able to recall all three questions accurately  programme used the three questions as one of its tools to promote SDM. Evaluation of this component was limited to the specific marketing campaign, and although this was positive (patients and clinicians liked it), the effectiveness of the campaign remains uncertain.Between October 2010 and August 2013, the UK Health Foundation through its MAGIC . While recall of the questions was high at 2 weeks, and participants reported that the questions were useful, and that they intended to recommend them to others, whether or not participants would repeat their use is unknown. Additionally, our exclusion of participants who did not have time to complete watching the 4‐min video‐clip reduced our sample; however, this was caused by the requirement in the research setting to gain consent and complete baseline questionnaires in addition to viewing the video‐clip.®) enabled consumers to ask the listed questions in health‐care consultations. Patients were able to view a short video‐clip to promote question asking immediately before their consultation, and most went on to ask at least one of the questions in that consultation.The brief consumer questions training and support programme, AskShareKnow (ASK Patient–Clinician Communication ModelThis brief intervention of three generic questions relating to health‐care decisions has shown important effects in improving the quality of information provided during clinical consultations and in facilitating patient involvement. It demonstrates the utility of the intervention through the ability of patients to use the questions following a brief waiting room video‐clip demonstration. While further evaluation to determine generalizability of study findings to other settings is needed and could add to the evidence by investigating the longer term impact of a larger campaign, should health systems proceed with implementing the AskShareKnow intervention at an institutional, regional or even national level as a simple way for patients and clinicians to share decisions in practice?HS conceived the study. HS, AB, DB, KC, LT, KM, CDM, PB, RM, VE and EW designed the study and obtained funding. AJ coordinated the running of the study and the data collection. KM carried out the statistical analysis. HS wrote the first draft of the manuscript. All authors contributed to the interpretation of the analysis and the writing of the manuscript. All authors are guarantors.This work was supported by Grant #0175‐1 from the Informed Medical Decisions Foundation. Dr. Shepherd received salary support from National Health and Medical Research Council Public Health Training Fellowship ID 568962. Dr. Shepherd had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.


Plasmodium proteases have been shown to be involved in a variety of pathways that are essential for parasite survival. However, pharmacological rather than target‐based approaches have dominated the field of antimalarial drug development, in part due to the challenge of robustly validating Plasmodium targets at the genetic level. Fortunately, over the last few years there has been significant progress in the development of efficient genetic methods to modify the parasite, including several conditional approaches. This progress is finally allowing us not only to validate essential genes genetically, but also to study their molecular functions. In this review, I present our current understanding of the biological role proteases play in the malaria parasite life cycle. I also discuss how the recent advances in Plasmodium genetics, the improvement of protease‐oriented chemical biology approaches, and the development of malaria‐focused pharmacological assays, can be combined to achieve a robust biological, chemical and therapeutic validation of Plasmodium proteases as viable drug targets.Malaria is a devastating parasitic disease affecting half of the world's population. The rapid emergence of resistance against new antimalarial drugs, including artemisinin‐based therapies, has made the development of drugs with novel mechanisms of action extremely urgent. Proteases are enzymes proven to be well suited for target‐based drug development due to our knowledge of their enzymatic mechanisms and active site structures. More importantly,  ABPactivity‐based probeACTartemisinin‐based combination therapyA‐M17M17‐family leucyl aminopeptidaseA‐M18M18‐family aspartyl aminopeptidaseA‐M1M1‐family alanyl aminopeptidaseAMA1apical membrane antigen 1APPaminopeptidase PATG8autophagy 8BPbergheipainCETSAcellular thermal shift assaycKDconditional knock downcKOconditional knock outClpPcaseinolytic proteaseCOFRADICcombined fractional diagonal chromatographyCoSeSuLcounter selection substrate librariesCRISPRclustered regulatory interspaced short palindromic repeatsDARTdrug affinity response target stabilityDiCredimerizable Cre recombinaseDPAPdipeptidyl aminopeptidaseDUBdeubiquitylating enzymeEMP2erythrocyte membrane protein 2ERendoplasmic reticulumEXPexported protein 1FPfalcipainFtsHfilamentous temperature‐sensitive H peptidaseGBP130glycophorin‐binding protein 130HslVheat‐shock locus VHsp70heat‐shock protein 70HUB1homology to ubiquitin 1HyCoSuLhybrid combinatorial substrate librariesiRBCinfected red blood cellMetAPmethionine aminopeptidaseMIPmitochondrial intermediate peptidaseMPPmitochondrial processing peptidaseMSPmerozoite surface proteinNEDD8Neural Precursor Cell Expressed Developmentally Downregulated 8PARLpresenilins‐associated rhomboid‐like proteinPEXELprotein export elementPMplasmepsinPTEXPlasmodium translocon for exported proteinsPTRAMPthrombospondin‐related apical membrane proteinPVMparasitophorous vacuole membranePVparasitophorous vacuoleRAMArhoptry‐associated membrane antigenRAP1rhoptry‐associated protein 1RBCMred blood cell membraneRBCred blood cellRhopH3high molecular weight rhoptry protein 3ROMrhomboidS2Psite‐2 proteaseSARstructure–activity relationshipSCIDsevere combined immunodeficiencySENPsentrin‐specific peptidaseSERAserine repeat antigenSNPsingle‐nucleotide polymorphismSPPaspartyl signal peptide peptidaseSPserine signal peptidaseSUBsubtilisin‐like proteaseSUMOsmall ubiquitin‐like modifierTAILSterminal amine isotopic labelling of substratesTPPthermal proteome profilingTRAPthrombospondin‐related anonymous proteinUBP1ubiquitin peptidase 1UCHubiquitin C‐terminal hydrolaseURM1ubiquitin‐related modifier 1USP14ubiquitin‐specific peptidase 14WTwild‐typeOver the last decade, the world has seen a significant decrease in malaria incidence, from 1 to 2 million deaths in 2000 to an estimate of half a million this year Anopheles mosquitoes inject highly motile parasites (sporozoites) into the skin . These programs lost some of their momentum once advances in malaria genetics showed a high level of proteolytic redundancy in this pathway in vivo malaria models Plasmodium proteases are likely to be viable antimalarial targets.Target‐based approaches have so far not been very successful in developing antimalarial drugs, in part due to the difficulty of genetically validating targets in Plasmodium biology with a strong emphasis on Plasmodium falciparum, being the most virulent and best studied Plasmodium species. I then present my opinion about the need to validate antimalarial targets at the genetic, biological, chemical and therapeutic levels before embarking on long and expensive drug development campaigns. In particular, I emphasise how the combination of conditional genetic methods, malaria‐specific pharmacological assays, and chemical biology approaches can be used to achieve robust target validation.In this review, I provide an overview of the role of proteases in Plasmodium proteases play both regulatory and effector roles in a variety of essential biological processes. However, it is important to consider which among the ~ 170 predicted Plasmodium proteases are likely to be viable therapeutic targets. A priori, proteases that are not conserved in humans are more likely to have parasite‐specific functions, and their inhibitors might be less prone to inactivate host proteases. That said, the following points should be carefully considered before discarding Plasmodium proteases that have human homologues:Plasmodium and human proteases have been documented Plasmodium‐specific inhibitors.Phylogenetic conservation does not always translate into structural conservation of the active site. Indeed, significant differences in substrate and/or inhibitor specificity between Plasmodium proteasome inhibitors can have good antiparasitic activity with minimal toxicity if they do not inhibit the host β2 subunit; inhibition of the host β5 subunit is well tolerated Off‐target inhibition of host proteases does not always need to lead to adverse effects. For example, potent Targeting host proteases might be beneficial. Most malaria symptoms result from a pronounced dysregulation of the immune and inflammatory responses during the parasite erythrocytic cycle. Given the central role of proteases in these processes, it is important to consider whether off‐target inhibition of certain human proteases might not result in beneficial adjuvant effects. Unfortunately, the role of host proteases in malaria pathology is poorly understood. In addition, there are a few examples showing that erythrocytic enzymes play a role in parasite development. For example, human calpain‐1 is thought to be activated at the time of egress to assist parasites escape the iRBC Plasmodium homologues relatively quickly. This also encourages successful collaborations with industry and provides access to valuable medicinal chemistry, pharmacological and structural biology knowledge. For example, the K11777 inhibitor mentioned above was developed by Khepri Pharmaceuticals as a cathepsin S inhibitor. Also, the work that led to the development of Plasmodium‐specific proteasome inhibitors Repurposing compounds from industry is a cost‐effective strategy to tackle neglected diseases. Because pharma industry efforts are mainly focused on targeting noninfectious human disorders , most protease‐oriented drug development programs target human enzymes. Focused libraries of inhibitors can therefore be repurposed to develop potent inhibitors against Plasmodium spp. have also evolved parasite‐specific proteolytic pathways allowing them to replicate within host cells and evade the immune system efficiently. Parasite‐specific proteolytic functions include mechanisms to get in and out of the RBC, pathways to degrade haemoglobin, and mechanisms to modify the erythrocyte cytosol and membrane to acquire nutrients and evade the immune system. Although proteases involved in these key biological processes are potential drug targets, their activities might only be required for a short period of time during the parasite life cycle. On the other hand, proteases that play core biological functions, such as protein homeostasis or protein traffic, are more likely to be essential at all stages of parasite development.Another important point to consider is whether a protease performing a parasite‐specific function is a better target than one performing a function conserved in eukaryotes. Proteases play important roles in a variety of biological processes such as protein homeostasis, trafficking, cell signalling, catabolism or cell death, and most of these are also conserved in apicomplexan parasites. However, Plasmodium and host proteins that are cleaved during the last 6 h leading to parasite egress After egress from iRBCs, merozoites are vulnerable to detection by the immune system and are only viable for a few minutes during which they need to find and invade a new erythrocyte. Egress and invasion are therefore tightly coordinated and regulated processes with proteolysis playing major regulatory and effector roles. Proteases have long been known to be important in these pathways given that disruption of the parasitophorous vacuole and RBC membranes are required for parasite egress Fig. A, generaP. falciparum comprises nine proteins, each containing a papain‐fold catalytic domain. Only three are predicted to be active (SERA6–8) with the remaining six (SERA1–5 and 9) having a Ser instead of a catalytic Cys. SERA5 and SERA6 are the most abundant SERAs in blood stages and the only ones refractory to genetic KO Plasmodium berghei homologue prevents sporozoite egress from oocysts Prior to egress, an increase in cGMP levels activates the cytosolic cGMP‐dependent protein kinase G, which triggers the secretion of proteins from apical organelles (exonemes and micronemes) into the parasitophorous vacuole (PV) and onto the merozoite surface et al. showed that processing of MSP1 triggers a conformational change that allows it to bind spectrin. Interestingly, parasites that endogenously express MSP1 mutants with inefficiently processed SUB1 cleavage sites egress significantly more slowly, suggesting that MSP1 cleavage might be a rate‐limiting step in parasite egress. This study is an excellent example of how the use of conditional systems can drastically change our understanding of parasite biology. MSP1 is the most abundant merozoite surface protein and provides a scaffold for the formation of the MSP1/6/7 complex, which was previously believed to be essential for RBC binding by released merozoites and invasion. However, conditional truncation of MSP1, to convert it to a soluble PV protein that is no longer GPI‐anchored to the merozoite surface, results in a significant egress defect but only a two‐fold decrease in parasite replication rate, thus showing that merozoites lacking MSP1 can invade RBCs. SUB1 is also expressed in liver stages where it has also been shown to play a role in egress using a cKO approach in P. bergheiBreakdown of the parasitophorous vacuole and RBC membranes (PVM and RBCM) allows the parasites to egress from iRBCs. Current work in the Blackman laboratory using the DiCre cKO system has shown that both SUB1 and SERA6 At the time of egress, human calpain 1 Proteases not only have an active role during invasion but they also ensure proper merozoite maturation before they escape the iRBC Fig. B. The cuPlasmodium rhomboids and subtilisin‐like protease 2 (SUB2) shed the protein coat of the merozoite. Two rhomboids have been studied in detail in P. falciparum: PfROM4 localises at the parasite surface and has been shown to cleave the transmembrane domain of adhesin proteins that are important for parasite attachment to the RBC surface in vitroP. berghei suggest that PbROM1 is not essential during the erythrocytic cycle but that it plays an important role in liver stages, probably during hepatocyte invasion by sporozoites Plasmodium yoelii have shown that PyROM1 is essential for proper PV formation P. bergheiet al. showed that only half of the eight P. berghei rhomboids seem to be essential in blood stages , and PbROM3 is required to produce sporozoites. Nothing is known about the potential functions of ROM6–8, but ROM6 and ROM7 are predicted to localise to the mitochondria and apicoplast, respectively During invasion, 2+‐dependent protease that is secreted from micronemes into the merozoite surface The other important sheddase for RBC invasion is SUB2, a transmembrane CaP. berghei has no effect in blood or insect stages P. berghei their KOs prevent sporozoite formation Note that plasmepsins VII and X (PM‐VII and PM‐X) are also expressed in ookinetes where they have been suggested to play a role in midgut transversal P. falciparum has no apparent effect on parasite development or invasion Finally, although falcipain 1 (FP1) inhibitors seem to block invasion Plasmodium translocon for exported proteins) In order to survive within the RBC, the parasite needs to modify the RBC cytosol and membrane extensively to acquire metabolites, adhere to epithelial cells and evade the immune system. Around 10% of the parasite proteome is exported into the RBC through translocation of PV proteins across the PVM via the PTEX complex , which suggests that the haemoglobin degradation pathway might not be equally important in all Plasmodium spp.It is important to mention that in Most studies on malaria proteases have focused on enzymes that perform parasite‐specific functions that often take place at a specific stage Fig. C. From aP. falciparum, the two serine protease subunits of the signal peptidase complex (SP18 and SP21) have been reported to have proteolytic activity As mentioned above, the majority of proteins destined for the RBC cytosol or membrane are cleaved by PM‐V, which makes it a very promising target as its inhibition would block a variety of biological processes such as protein trafficking to the infected host cell surface, metabolite import, haemoglobin internalisation or Maurer's cleft formation. Similarly, targeting proteases involved in intracellular trafficking will likely disrupt parasite development at any stage. Most soluble proteins in the secretory pathway require co‐translational insertion of a hydrophobic N‐terminal signal peptide into the ER membrane, translocation of the polypeptide chain within the ER, and cleavage of the signal peptide. This is mediated by the signal peptidase complex, which recognises and cleaves the signal peptide in the lumen side of the membrane, and by a transmembrane aspartyl SPP, which cleaves it within the ER membrane. In Plasmodium spp. and is predicted to localise to the apicoplast Plasmodium genomes and predicted to localise to the mitochondria. Proteins destined to the intermembrane mitochondrial space also contain a hydrophobic sorting signal downstream of the MPP cleavage site that is inserted into the inner membrane Plasmodium spp., bioinformatic analysis suggests that SP21 might also localise to the mitochondria. Alternatively, the predicted mitochondrial rhomboid, ROM6, might be able to cleave the sorting peptide within the inner membrane. Finally, the N‐terminus of matrix mitochondrial proteins is often further processed through removal of an octapeptide by the metalloprotease MIP , whose homologue is also found in the Plasmodium genome and predicted to be mitochondrial Proteases involved in the trafficking of apicoplast or mitochondrial proteins might also be potential antimalarial targets. Nuclear‐encoded proteins destined for the apicoplast are directly transferred from the ER to the apicoplast via recognition of a bipartite transit peptide downstream of the N‐terminal signal peptide. In chloroplasts, this transit peptide is cleaved by the stromal processing peptidase Plasmodium species given its complex life cycle and the variety of distinct morphological stages, each requiring a special set of proteins. Indeed, 80% of genes expressed in the erythrocytic cycle are regulated in a cyclic manner The vast majority of proteins within a cell are processed at least twice: first through removal of their N‐terminal methionine by Met‐aminopeptidases (MetAPs), and second, through their degradation by an ATP‐dependent proteolytic system such as the proteasome or in lysosomal organelles. Protein homeostasis is particularly important in Plasmodium spp., and inhibitors of MetAP1b and MetAP2 have been shown to have antiparasitic activity both in vitro and in vivoFive MetAPs have been identified so far in Plasmodium spp. Recent studies on the structure of the P. falciparum proteasome combined with SAR studies have shown that there are significant differences in specificity between the human and malaria proteasomes that can be exploited to design safe P. falciparum proteasome inhibitors The proteasome/ubiquitination/deubiquitination system is not only crucial to regulate protein turnover and degrade misfolded proteins, but also to signal and regulate a variety of biological processes. The proteasome is predicted to be essential in all eukaryotes including Plasmodium, ubiquitination has been linked to a variety of biological process such as the ER‐associated protein degradation (ERAD) pathway, response to oxidative stress, protein trafficking and drug resistance. Bioinformatic analysis predicts close to 30 deubiquitinating enzymes (DUBs) in P. falciparum including proteases predicted to cleave ubiquitin‐like modifiers such as SUMO, NEDD8, HUB1, URM1 or ATG8. Although some of these proteases are likely to be essential, understanding these pathways, which are likely to contain multiple redundant and/or overlapping functionalities, is very challenging, and the main reason why our knowledge of Plasmodium DUB‐like proteases is so sparse: UCH3 Plasmodium homologue of human USP14 was found to associate with the Plasmodium proteasome and has been shown to have DUB activity In Plasmodium spp. A recent study in P. berghei has shown that S2P is expressed throughout the parasite life cycle , localises to the periphery of the nucleus, and its KO results in a significant impairment of parasite development in liver and asexual blood stages. It is not clear whether the second M50 protease plays a redundant or complementary function, nor whether inhibitors with dual specificity might have potent antimalarial activity Finally, site‐2 proteases (S2P), belonging to the M50 family of metalloproteases, are integral membrane proteins that cleave within the transmembrane domain of their substrate. S2Ps generally localise to the Golgi membrane where they have been shown to be involved in the unfolded protein response pathway. Two proteases belonging to the M50 family are present in Plasmodium spp. have two organelles that originated from endosymbiotic events with ancestral prokaryotes: the mitochondrion and the apicoplast. Proteases are required to deliver proteins into these organelles, but also for their biogenesis, protein homeostasis and regulation of protein function. Therefore, mitochondrial and apicoplast proteases might be interesting antimalarial targets, especially if differences in specificity between human and Plasmodium homologues can be identified. Interestingly, a prokaryotic protease that was maintained in Plasmodium spp. but not in mammals is the bacterial proteasome system known as HslUV or ClpQY. This protein complex localises to the mitochondria P. falciparum conditional overexpression of a dominant negative ClpQ mutant results in abnormal mitochondrial morphology, blocks organelle growth and division, and disrupts transcription of mitochondria‐encoded genes Two likely essential rhomboids are predicted to localise to the mitochondria and apicoplast, called PfROM6 and PfROM7, respectively Plasmodium calpain is the only protease that has been shown to localise to the nucleus and have a role in nuclear division Plasmodium spp. On the other hand, metacaspases are present in metazoans, plants, fungi and protozoa and have been shown to have a role in programmed cell death, which has led to the hypothesis that Plasmodium metacaspase‐1 (PfMCA‐1) might have a similar function P. berghei has no effect in parasite development Plasmodium spp. have evolved a programmed cell death pathway.To date, 12 within 72 h and reduce fevers within 24 h. It should also be clinically effective against drug‐resistant strains, prevent relapses due to hypnozoite activation, and block transmission by killing gametocytes, hepatic schizonts and/or the mosquito vector. Although these criteria are unlikely to be fulfilled by blocking any single target, to clinically validate a protease as a target, its inhibitor drug candidate needs to fulfil one or several of these requirements and also be tested in combination with other drugs. However, before investing too much effort in expensive drug development programs, I believe it is essential to validate potential protease targets at the genetic, biological, chemical and therapeutic level.The ubiquitous role of proteases in parasite biology and the proven use of protease‐targeting drugs to treat a variety of diseases make these enzymes very attractive candidates for antimalarial therapy. Medicines for Malaria Venture (MMV) recently published an updated target product and target compound profiles for new antimalarial therapies and drug candidates P. falciparum has been challenging due to the A/T rich nature of its genome, the very low levels of homologous recombination and transfection efficiency, and the scarcity of selection markers. Traditional modification of the haploid asexual blood stages of P. falciparum by single or double crossover homologous recombination usually takes 3–6 months and involved transfection with circular DNA, selection of transfected parasites using a drug resistance marker, lengthy on/off drug cycles to enrich for integrant parasites and to encourage episome loss, and a final limiting dilution cloning step to obtain a population of genetically homogenous modified parasites. Failure to obtain KO lines using this method would only be suggestive of whether a gene is essential, since this inability might be due to inherent input plasmid stability problems or difficulty to target the selected locus. Also, genetic modifications that result in a fitness cost rather than parasite death will allow wild‐type parasites to outcompete the genetically modified ones. A further consideration is that the lengthy selection process might lead to evolutionary adaptation (e.g. by upregulation of genes with complementary functions), thus increasing the risk of mischaracterising the biological relevance of a target. Fortunately, over the last few years several cKO and cKD systems have become available providing much better tools to validate antimalarial targets genetically  and the validation of these cleavage events as being important for parasite development. Global proteomic methods specifically developed to identify protease substrates such as TAILS or COFRADIC in vivo), but also to provide sufficient evidence to show that lead inhibitors are not acting through off‐target or general cytotoxic effects.Chemical validation of a target has to demonstrate that its inhibition by a small molecule at biologically relevant concentrations is sufficient to generate a phenotype. This requires the development of assays that directly measure protease inhibition under living conditions  has allowed to expand the chemical diversity of peptide‐based substrates and provides a very successful approach to identify differences in specificity between proteases in vitro and in vivoActivity‐based probes are excellent tools to measure protease activity under living conditions Fig. A. These Another chemical strategy to determine whether inhibitors act on target is the use of structure–activity relationship (SAR) series. Using one of the assays described above, target inhibition in live parasites can be directly correlated with the expected phenotype in a dose‐dependent manner. If this correlation holds true for different classes of inhibitors, it provides stronger evidence that compounds are acting on target. It is important to emphasise that any inhibitor development program should include the synthesis of negative control compounds in order to confirm that lead inhibitors are acting on target. These should be designed with minimal alteration to the structure of lead compounds: for example, a diastereomer of the inhibitor, a nonreactive version of a covalent inhibitor, or the addition of a sterically clashing methyl group.Finally, a decrease or increase in inhibitor sensitivity upon overexpression or knock down of the target of interest is a good indication that the compound is acting on target. This method was used to demonstrate the on‐target effect of a PM‐V inhibitor by showing that parasites become more or less sensitive to the compound upon cKD or overexpression of PM‐V, respectively Once a phenotype associated with the loss of a particular protease activity has been characterised, an assay can be developed to quantify this phenotype and correlate it with target inhibition in live parasites. If inhibition of a protease is expected to arrest parasite development at a specific stage, this should be confirmed microscopically and/or using FACS‐based assays, which allow high‐throughput quantification of different intraerythrocytic stages. More specific microscopy‐based assays can also be developed if depletion of the protease of interest results in clear morphological changes, such as enlargement of the food vacuole upon falcipain inhibition, or by using fluorescent reporters. For example, episomal expression of a GFP‐tagged truncated and inactive form of ClpP was used to visualise the effect of ClpP inhibition in apicoplast morphology If substrate cleavage mediates a change in its localisation, biochemical fractionation of infected RBCs can be used to monitor this change by western blot or ELISA assays. For example, inhibition of merozoite sheddases should decrease the amount of surface proteins released into the media supernatant after an invasion assay. Expression of fluorescent reporters can also be used to confirm protease inhibition in live parasites. For example, fusion of GFP to a PEXEL‐containing exported protein has been used to confirm PM‐V inhibition by live microscopy, which results in accumulation of GFP signal in the parasitophorous vacuole A common strategy to identify the targets of antimalarial compounds is to culture parasites at increasing compound concentrations to force the emergence of resistance. Full‐genome sequencing allows the identification of point mutations responsible for the resistance phenotype. These SNPs often cluster to genes encoding the targets of the compound A more direct approach to identify the targets of a compound is by using chemical proteomic methods. As mentioned above, activity‐based protein profiling provides a useful tool to determine the specificity of compounds against all members of an enzyme family Fig. B. Howevein vitro understanding of these pharmacological requirements is necessary not only to determine whether a protease will be a viable target, but also to help develop inhibitors with well‐tuned pharmacology.Once a target has been genetically, biologically and chemically validated, the tools used to obtain this information can be repurposed to determine the pharmacological profile a protease inhibitor needs to meet in order to act as an efficient drug. A thorough Proteases are generally expressed as zymogens that need to be activated at a specific time and place to perform their biological function. Protease activity is also tightly regulated through the timely expression and targeted degradation of endogenous inhibitors. Understanding when and where within the parasite life cycle a protease becomes active, and for how long its activity is required, will help define the desired pharmacological profile of inhibitors. The use of ABPs can be instrumental in answering these questions since these chemical tools can differentiate between the active and inactive forms of a protease. Inhibitor treatment at different life stages and for different periods of time can help define for how long a protease needs to be inhibited to block parasite replication, and whether parasites are more sensitive to protease inhibition at a specific stage. For example, food vacuole proteases are constantly being turned over and therefore will require inhibitors able to cross four membranes and sustain target inhibition for several hours in a highly acidic and oxidative environment in vitro, have similar sensitivity to lead protease inhibitors.The development of antimalarial drugs needs always to be evaluated in the context of drug resistance. To avoid its emergence, drug development efforts have been mainly focused on identifying fast‐killing drugs as opposed to compounds that delay or halt parasite development. Specific assays that discriminate between cidal and static effects of compounds 2+‐mediated activation of an internal peroxide that generates toxic carbon‐centred radicals and increases oxidative stress. Because Fe2+ originates from the degradation of haemoglobin, falcipain inhibitors have antagonistic effects with artemisinin An effective strategy to prevent the emergence of resistance is the use of combination therapy. In the context of protease inhibitors, it is important to evaluate what would be a suitable antimalarial drug partner. For example, the mechanism of action of artemisinin involves the Fein vivo models available to evaluate the potential of new antimalarial drug. In addition to the most commonly used P. berghei and P. yoelii murine models, the Plasmodium chabaudi model has been robustly validated as a more accurate immunological model of malaria which can be used to study the acute and chronic phases of infection P. falciparum liver and blood stages within a living host by grafting human hepatocytes and erythrocytes into mice. Although these models do not properly reflect the immune and inflammatory responses to parasite infection, they are valuable tools to assess the efficacy of small molecules in an in vivo system. In theory, cKO/cKD P. falciparum lines could be used in the SCID model to test the essentiality of a target within a living host. Finally, Plasmodium knowlesi is more closely related to Plasmodium vivax, Plasmodium malariae or Plasmodium ovale than P. falciparum, and its recent adaptation to grow in human erythrocytes in vitro model to predict the efficacy of lead inhibitors against these other malaria‐causing species.In recent years, industry and academic groups have invested considerable efforts into developing medium‐ to high‐throughput cell‐based assays to screen compounds that might block transmission. These include assays to monitor gametocyte development and egress, liver stage development, including hypnozoite formation and reactivation, and parasite transmission assays Plasmodium spp genetically. In particular, the development of conditional genetic approaches in P. falciparum is allowing us for the first time to validate essential genes and study their molecular functions. I anticipate that these advances will soon result in a surge of target‐based antimalarial drug development programs.Over the last two decades, the rising interest in tackling neglected infectious diseases has resulted in a substantial increase of funds to study these pathogens, a stronger involvement of the pharma industry, and the creation of nonprofit and public–private partnership institutions to facilitate drug development programs. In the area of malaria, this resulted in a first wave of pharmacology‐based drug development programs including not only large high‐throughput phenotypic screening campaigns, but also development of malaria‐specific pharmacological assays and screening methodology to monitor all parasite stages. In the last few years, there has been a resurgent interest in target‐based approaches In parallel, over the last 30 years there has been a dramatic shift in our understanding of protease function, from being purely degradative enzymes, to the realisation that proteases are, similarly to protein kinases, tightly regulated enzymes playing signalling and effector roles in most biological processes. The ubiquitous role of proteases in human diseases has made them one of the preferred enzyme families for target‐based drug development and has resulted in the implementation of a variety of chemical and proteomic methods to develop specific inhibitors, understand protease regulation and identify their natural substrates.Plasmodium proteases and achieve a robust genetic, biological and chemical validation of these targets. Finally, the variety of malaria‐focused pharmacological assays and in vivo models developed over the last decade will greatly facilitate the therapeutic validation of proteases as viable antimalarial targets.Proteases play crucial roles at all stages of parasite development and are therefore potential antimalarial targets. Importantly, only one‐third of predicted malaria proteases have been studied so far, and only a few of these have been characterised in any detail Table . Thus, t


Research on workforce development for high‐quality dementia care more often focuses on enhancing employee knowledge and skill and less on managing employee stress and coping at work.To review employee stress and coping in response to high job demands in community‐based dementia care organizations in Tasmania, Australia.worker profiles of adjustment.Stress and coping in response to job roles of 25 community‐based dementia care workers were reviewed using self‐report questionnaire data. Data were analysed for descriptive results and at an individual case level. Individual participant scores were reviewed for clinically significant stress and coping factors to create ‘global resilience’ profile, where workers showed positive adjustment and resilience indicating they found their jobs highly rewarding, were very confident in their abilities at work and had a strong match between their personal and organizational values. The second ‘isolated distress’ profile was only found in a minority and included poor opportunities for job advancement, a missmatch in personal and work values or clinically high levels of psychological distress.Two adjustment profiles were found. The Aged care workplaces that advocate employee well‐being and support employees to cope with their work roles may be more likely to retain motivated and committed staff. Future research should consider employee stress and coping at the workforce level, and how this can influence high‐quality care delivery by applying the measures identified for this study. Comparative research across different care settings using meta‐analytic studies may then be possible. Dementia is a debilitating neurodegenerative disease which impacts on every health and social care system in the world.stress and coping as an interactive process. Stress is the psychological and physical response to a situation that is challenging to endure, whereas coping is the strategy or approach an individual applies to manage and endure the stress response or symptoms associated with the situation. Stress and coping factors greatly influence a person's well‐being and psychological adjustment to engage effectively with usual activities in life including work. Research on the well‐being of dementia care workers mostly applies to the residential care setting and not the community‐based arena.The heterogeneous nature of dementia has many implications for the workforce and service providers operating within aged care. In fact, within the older adult group, Pachana and HelmesUnderstanding the stress and coping of the dementia care workforce is of particular concern due to the connection between job stress and turnover.There is uncertainty about the stress aged and dementia care workers experience. A systematic reviewet al.The most difficult aspects of working with people with dementia include coping with, and managing behavioural and psychological symptoms of dementia (including aggression and hostility), particularly when care workers are not trained to manage these situations. The resistive, difficult and unpredictable nature of these behaviours were found to be a challenge for Australian nurses working in residential care.Aged and dementia care workers who stay in their jobs longer experience less stress at work. While early career long‐term  care workers in America experienced stress more than later career workers (employed more than 2 years), they also experienced more hopeful and person‐centred attitudes about people with dementia.Social psychological theory offers a lens for this focus on stress and coping for the aged and dementia care workforce. Self‐efficacy, the belief in one's own ability to successfully accomplish or perform a specific behaviour,The work environment is an essential part of stress and coping for employees. The greater the mismatch between a person's values and the organization's values, the more distress the person experiences.This study aimed to deepen understanding about stress and coping of aged and dementia care workers based in the community. The effects of stress on workers, associated with the dementia care role, were investigated using psychometrically sound measures. Specific stress and coping areas were assessed that have theoretical links to workforce resilience. Results will be discussed in the context of occupational stress literature in dementia care  to review if similarities exist for the community setting.This study formed part of the Work 4 Dementia ProjectThe Work 4 Dementia Project aimed to investigate ways to build capacity and resilience for the dementia care workforce and included several interrelated studies. The study reported here used a cross‐sectional survey design using known constructs of workforce resilience. This method was chosen to further investigate qualitative findings from a previous study within the projectStage 1 of the analysis included descriptive statistics at the group level. Stage 2 of the analysis uniquely applied a case‐based clinical psychology approach, similar to pattern analysis. This method is used frequently in the clinical psychology and psychiatry literature to describe adjustment to a chronic stressor  with an average age of 53 years (SD = 9.6). The majority were women (88%) and employed on a casual basis (64%). Most were employed on average for 6 years (SD = 4.3) and on average worked 25.9 h per week (SD = 10.2). Participants were mostly (n = 11) married (44%) and earned over (n = 9) $65 000 income annually from all sources (35%). Most workers (n = 22) received an orientation to their work on commencement of their jobs (88%). These results are comparable to a recent Australian survey on the aged care workforce which indicated community‐based care workers were mostly aged between 45 and 54 (37.2%) or 55 and 64 (29.7%) years, and worked between 16 and 34 h per week (56.4%). Table Participants were community dementia care workers n = 5 with anOn average most participants were well adapted, but the range in scores shows some participants were not coping  of poor functioning as measured by the questionnaires on areas of psychological well‐being. In addition, workers with global resilient profiles showed clinically significant adjustment on various occupational areas and or psychological well‐being. The isolated distress profile included dysfunction in job demands and or psychological well‐being areas, whereas global resilient profiles showed adaptive function in these areas. While workers in the isolated distress profile showed clinically significant levels of poor adjustment in occupational and or well‐being domains, some workers showed positive adjustment in other areas (e.g. satisfaction with life or positive affect). The term ‘isolated distress’ is therefore used to describe this profile to acknowledge this pattern of scores.We categorized worker's scores on all self‐report measures by Overall, 64 per cent of workers showed resilient profiles of stress and coping as measured by the assessed areas. These employees found their jobs rewarding, including being satisfied with opportunities for promotion . Eighty‐eight per cent of resilient workers were confident in their abilities at work indicated by clinically high levels of occupational self‐efficacy. They also felt their personal values matched those of the organizations  and were committed to working for the organization into the future . Fifty per cent of employees in this profile reported high satisfaction with supervision from a leader in their organization. Twenty‐five per cent had high knowledge of Alzheimer's disease, and 18 per cent showed high satisfaction with pay. Only one worker reported high levels of positive emotions at work demonstrated by scores on positive affect.Workers scoring positively on occupational resources and well‐being were more likely to be in a committed relationship than not, and more likely to have worked for over 5 years, and were currently working above 20 h per week. Most resilient workers had a household income of more than $35 000, Certificate III or above qualifications and had attended training in dementia.likely to have a mild mental disorder range, meaning they were experiencing significant psychological distress. Thirty‐three per cent of workers in the isolated distress profile did not hold the same values as their organization indicated by clinically significantly low scores of congruence with their workplace values. Another 33 per cent of individuals were dissatisfied with life . Two workers reported significantly low levels of opportunities to advance their job, and one worker reported not being committed to the organization in the future indicated by significantly low score on organizational commitment. In the isolated distress profile, 66 per cent of workers had poor knowledge of dementia evidenced by answering less than half of the questions on the assessment of Alzheimer's disease knowledge correctly; however, this was not significantly different from the normative population.Overall, 36 per cent of workers were of interest, as they scored outside the typical resilient profile. Key elements that stood out in this group were high psychological distress, poor match with organizational values, life dissatisfaction and poor opportunities for promotion, as well as low commitment to the organization. Thirty‐three per cent of workers in the isolated distress profile had clinically significantly poor psychological adjustment, as assessed by the K10. In fact, in clinical terms, these workers scored in the Workers scoring poorly on psychological well‐being were not in a committed relationship , employed on a casual basis and had not undertaken training in dementia. These workers were more likely to be women, have Certificate III or below as highest level of education, have household incomes below $35 000 and had employment for <5 years with their current employer. The individual reporting low organizational commitment (i.e. high intention to leave) also met similar criteria, however, had worked for 10 years with their employer and held a degree and a Certificate IV. Overall, workers with poor function on occupational domains and well‐being were more likely to hold Certificate III or above, did not completed training in dementia and were not in a committed relationship.Overall, dementia care workers reported positive function, but some cases showed workers with isolated distress. On average, community dementia care workers showed high occupational self‐efficacy, high job satisfaction in contingent work rewards and promotional opportunities, as well as high congruence with organizational values. Similar results were found for an Australian national survey showing community‐based workers were satisfied with their jobs.Global resilience profiles showed positive adjustment across a range of occupational and intrapersonal domains to suggest that resilient workers were extremely confident in their work ability, believed their personal values matched those of their employer and found their jobs satisfying and rewarding. In comparison, employees with an isolated distress profile faired much worse, as job resources were low , and for some, levels of psychological distress were high . Demographic and occupational differences between profiles showed disparity across workers. Resilient workers were more likely to have longer length of employment (5 years and above), be in a committed relationship, hold higher levels of education and qualifications, earn higher incomes and have dementia‐specific training. These findings are consistent with a World Health Organisationet al.Career status (in terms of length of employment) influenced how workers experienced distress. Later career workers were more likely to identify with a resilient rather than an isolated distress profile, which is supported by findings from Zimmerman occupational communion may have adaptive benefits for workers’ coping and adjustment. In addition, HartlingWhile there was no direct finding of high organizational support, results suggested leadership through supervision, a type of organizational support, may be more important for workers with isolated distress than those with global resilience profiles. Research on residential aged care workers in Australia supports this finding, as supervisor support interacts with job demands to show that when job demands are either too low or too high, well‐being and satisfaction are negatively impacted.Limitations of the study include the generalizability of the findings. While this sample was similar on most national community‐based characteristics including mostly female, born in Australia, worked between 16 and 34 h per week, approximately half attended training in dementia, and approximately one‐fifth with a possible mental illness,The percentage of workers in this study with poor psychological well‐being matched that of the general population, as 20 per cent of Australian adults experience psychological problems such as mental illness in any year.A strength of the study is the use of well validated and reliable psychometric tools to assess work psychological function. Applying the case‐based clinical psychology approach enabled the comparison of dementia care workers function to clinical and normative samples. This approach is distinctive within dementia care research. However, some caution is warranted due to the differences between this sample and the normative comparison samples. Overall, dementia care workers showed good fit with the values and goals of community‐based services and similar findings have been found in other types of effective organizations.Workforce resilience has previously been explored in several occupational groups such as child protection services,This work was supported in part by a grant from the Tasmanian Home and Community Care Program (HACC), a jointly funded State and Australian Government program awarded to Scott et al. (grant number S0017329). This research was also supported in part by a PhD scholarship jointly provided by TIME for Dementia , the Wicking Dementia Research and Education Centre and the University of Tasmania, Australia .