A Chemical Reaction Network Drives Complex Population Dynamics in Oscillating Self-Reproducing Vesicles.

We report chemically fueled oscillations of vesicles. The population cycling of vesicles is driven by their self-reproduction and collapse within a biphasic reaction network involving the interplay of molecular and supramolecular events. We studied the oscillations on the molecular and supramolecular scales and tracked vesicle populations in time by interferometric scattering microscopy and dynamic light scattering. Complex supramolecular events were observed during oscillations─including vesicle reproduction, growth, and decomposition─and differences in the number, size, and mass of aggregates can often be observed within and between pulses. This system's dynamic behavior is reminiscent of a reproductive cycle in living cells.

The "Inherent Vices" of Policy Design: Uncertainty, Maliciousness, and Noncompliance.

Policy designs must not only "work" in the sense of accomplishing their goals but must also work in their intended fashion. Most research to date has focused on the former topic and dwells on the technical aspects of how various tools and instruments could be utilized to achieve the aims and goals of policymakers. This branch of research tends to underemphasize the difficulties inherent to policy making including policy contexts that are often highly uncertain, policymakers who fall short of an idealized version of high capacity, well-intentioned decisionmakers grappling with relevant public problems, and policy-takers who fail to comply with government wishes. These "inherent vices" of policy making are factors which contribute to policy volatility or the risk of policy failure. The paper stresses the need for improved risk management and mitigation strategies in policy formulation and policy designs to take these risks into account. It sets out and develops an approach borrowed from product failure management (in manufacturing) and portfolio management (in finance) to help better assess and manage these risks.

The north-south policy divide in transnational healthcare: a comparative review of policy research on medical tourism in source and destination countries.

Medical tourism occupies different spaces within national policy frameworks depending on which side of the transnational paradigm countries belong to, and how they seek to leverage it towards their developmental goals. This article draws attention to this policy divide in transnational healthcare through a comparative bibliometric review of policy research on medical tourism in select source (Canada, United States and United Kingdom) and destination countries (Mexico, India, Thailand, Malaysia and Singapore), using a systematic search of the Web of Science (WoS) database and review of grey literature. We assess cross-national differences in policy and policy research on medical tourism against contextual policy landscapes and challenges, and examine the convergence between research and policy. Our findings indicate major disparities in development agendas and national policy concerns, both between and among source and destination countries. Further, we find that research on medical tourism does not always address prevailing policy challenges, just as the policy discourse oftentimes neglects relevant policy research on the subject. Based on our review, we highlight the limited application of theoretical policy paradigms in current medical tourism research and make the case for a comparative policy research agenda for the field.

Kinetic Treatments for Catalyst Activation and Deactivation Processes based on Variable Time Normalization Analysis.

Progress reaction profiles are affected by both catalyst activation and deactivation processes occurring alongside the main reaction. These processes complicate the kinetic analysis of reactions, often directing researchers toward incorrect conclusions. We report the application of two kinetic treatments, based on variable time normalization analysis, to reactions involving catalyst activation and deactivation processes. The first kinetic treatment allows the removal of induction periods or the effect of rate perturbations associated with catalyst deactivation from kinetic profiles when the quantity of active catalyst can be measured. The second treatment allows the estimation of the activation or deactivation profile of the catalyst when the order of the reactants for the main reaction is known. Both treatments facilitate kinetic analysis of reactions suffering catalyst activation or deactivation processes.

Does Point-of-Care Ultrasonography Improve Clinical Outcomes in Emergency Department Patients With Undifferentiated Hypotension? An International Randomized Controlled Trial From the SHoC-ED Investigators.

STUDY OBJECTIVE: Point-of-care ultrasonography protocols are commonly used in the initial management of patients with undifferentiated hypotension in the emergency department (ED). There is little published evidence for any mortality benefit. We compare the effect of a point-of-care ultrasonography protocol versus standard care without point-of-care ultrasonography for survival and clinical outcomes. METHODS: This international, multicenter, randomized controlled trial recruited from 6 centers in North America and South Africa and included selected hypotensive patients (systolic blood pressure <100 mm Hg or shock index >1) randomized to early point-of-care ultrasonography plus standard care versus standard care without point-of-care ultrasonography. Diagnoses were recorded at 0 and 60 minutes. The primary outcome measure was survival to 30 days or hospital discharge. Secondary outcome measures included initial treatment and investigations, admissions, and length of stay. RESULTS: Follow-up was completed for 270 of 273 patients. The most common diagnosis in more than half the patients was occult sepsis. We found no important differences between groups for the primary outcome of survival (point-of-care ultrasonography group 104 of 136 patients versus standard care 102 of 134 patients; difference 0.35%; 95% binomial confidence interval [CI] -10.2% to 11.0%), survival in North America (point-of-care ultrasonography group 76 of 89 patients versus standard care 72 of 88 patients; difference 3.6%; CI -8.1% to 15.3%), and survival in South Africa (point-of-care ultrasonography group 28 of 47 patients versus standard care 30 of 46 patients; difference 5.6%; CI -15.2% to 26.0%). There were no important differences in rates of computed tomography (CT) scanning, inotrope or intravenous fluid use, and ICU or total length of stay. CONCLUSION: To our knowledge, this is the first randomized controlled trial to compare point-of-care ultrasonography to standard care without point-of-care ultrasonography in undifferentiated hypotensive ED patients. We did not find any benefits for survival, length of stay, rates of CT scanning, inotrope use, or fluid administration. The addition of a point-of-care ultrasonography protocol to standard care may not translate into a survival benefit in this group.

The response of the Irish pharmaceutical manufacturing sector to the COVID-19 pandemic with a focus on Lean and JIT.

Background: Just in Time (JIT) and Lean manufacturing are concepts that originated in the automotive industry and were then adopted by pharmaceutical and biopharmaceutical companies during the 1990s. However, the Covid-19 pandemic and the urgent demand for pharmaceutical treatment challenged JIT and Lean manufacturing processes. Production of Covid-19-related medicines increased, putting pressure on global supply chains and operations. This also hindered the production of medicines using the same or similar materials. Thus, questions are raised concerning JIT and Lean supply chains in the pharmaceutical industry. Objectives: The present study aimed to explore (1) if material and supply constraints occurred due to the Covid-19 pandemic, (2) how companies were impacted and managed and (3) if changes are required to future proof the JIT supply chain approach for future global events. Design: A mixed-method cross-sectional survey design was used and focused on material supply, qualification and validation in Irish pharmaceutical manufacturing sites. Methods: Employees working in the Irish pharmaceutical manufacturing industry were recruited using convenience sampling through online advertisement using the social media platform 'LinkedIn'. Quantitative data was analysed using percentages and qualitative data from free-text responses were used to add context to the quantitative survey questions. Results: A total of 41 participants were recruited. The results suggested that the pandemic had a negative effect on material availability according to 81% of participants. This translated to delays or stoppage of production activity and was mainly handled by sourcing new materials (70%). To cope with future global crises, 60% of participants recommended more flexibility in future validation processes while 78% of participants acknowledged the importance of validating additional suppliers. A hybrid model of manufacturing and supply chain management was also a preferred approach to exclusive Lean and JIT (42%). Conclusions: The production of non-Covid-19 medicines was adversely affected by the Covid-19 pandemic, but the pharmaceutical industry in Ireland demonstrated resilience and collaboration in response to these challenges. This study suggests that the JIT and Lean manufacturing model should be adjusted to ensure medicine supply chains are not disrupted during future global events.

From autocatalysis to survival of the fittest in self-reproducing lipid systems.

Studying autocatalysis - in which molecules catalyse their own formation - might help to explain the emergence of chemical systems that exhibit traits normally associated with biology. When coupled to other processes, autocatalysis can lead to complex systems-level behaviour in apparently simple mixtures. Lipids are an important class of chemicals that appear simple in isolation, but collectively show complex supramolecular and mesoscale dynamics. Here we discuss autocatalytic lipids as a source of extraordinary behaviour such as primitive chemical evolution, chemotaxis, temporally controllable materials and even as supramolecular catalysts for continuous synthesis. We survey the literature since the first examples of lipid autocatalysis and highlight state-of-the-art synthetic systems that emulate life, displaying behaviour such as metabolism and homeostasis, with special consideration for generating structural complexity and out-of-equilibrium models of life. Autocatalytic lipid systems have enormous potential for building complexity from simple components, and connections between physical effects and molecular reactivity are only just beginning to be discovered.

Policy instruments used by States seeking to improve school food environments.

US legislatures and program administrators have sought to control the sale of foods offered outside of federally funded meal programs in schools, but little is known about which policies, if any, will prevent obesity in children. We used a theoretical policy science typology to understand the types of policy instruments used by US state governments from 2001 to 2006. We coded 126 enacted bills and observed several types of instruments prescribed by state legislatures to influence the foods sold in schools and improve the school food environment. Our study helps to better understand the various instruments used by policymakers and sets the stage to examine the effectiveness of the policy instruments used to prevent obesity.

An autonomously oscillating supramolecular self-replicator.

A key goal of chemistry is to develop synthetic systems that mimic biology, such as self-assembling, self-replicating models of minimal life forms. Oscillations are often observed in complex biological networks, but oscillating, self-replicating species are unknown, and how to control autonomous supramolecular-level oscillating systems is also not yet established. Here we show how a population of self-assembling self-replicators can autonomously oscillate, so that simple micellar species repeatedly appear and disappear in time. The interplay of molecular and supramolecular events is key to observing oscillations: the repeated formation and disappearance of compartments is connected to a reaction network where molecular-level species are formed and broken down. The dynamic behaviour of our system across different length scales offers the opportunities for mass transport, as we demonstrate via reversible dye uptake. We believe these findings will inspire new biomimetic systems and may unlock nanotechnology systems such as (supra)molecular pumps, where compartment formation is controlled in time and space.

Emergency department occupancy is useful as a simple real-time measure of crowding.

INTRODUCTION: Emergency department (ED) crowding compromises patient outcomes. Existing crowding measures are complex and difficult to use in real-time. This study evaluated readily available single flow variables as crowding measures. METHODS: Over 2 weeks in a tertiary Canadian ED, we recorded the following potential crowding measures during 168 consecutive two-hour study intervals: total ED patients (census), patients in beds, patients in waiting rooms, patients in treatment areas awaiting MD assessment; number of inpatients boarding, and ED occupancy. We also calculated four complex crowding scores-NEDOCS, EDWIN, ICMED, and a local modification of NEDOCS. We performed ROC analyses to assess the predictive validity of these measures against a reference standard of physician perception of crowding. RESULTS: We gathered data for 144 (63.9%) of 168 study intervals. ED census correlated strongly with crowding (AUC = 0.82, 95% CI 0.76-0.89), as did ED occupancy (AUC = 0.75, 95% CI 0.66-0.83). Their performance was similar to NEDOCS (AUC = 0.80) and to the local modification of NEDOCS (AUC = 0.83). CONCLUSION: ED occupancy as a single measure has similar predictive accuracy to complex crowding scores and is easily generalizable to diverse emergency departments. Real-time tracking of this simple indicator could be used to prompt investigation and implementation of crowding interventions.

RéSUMé: INTRODUCTION: L'encombrement des services d'urgence (SU) compromet les résultats pour les patients. Les mesures d'encombrement existantes sont complexes et difficiles à utiliser en temps réel. Cette étude a évalué des variables de débit unique facilement disponibles comme mesures d'encombrement. LES MéTHODES: Pendant deux semaines dans un service d'urgence tertiaire canadien, nous avons enregistré les mesures d'encombrement potentiel suivantes au cours de 168 intervalles d'étude consécutifs de deux heures : nombre total de patients dans le service d'urgence (recensement), patients dans les lits, patients dans les salles d'attente, patients dans les zones de traitement en attente d'une évaluation médicale ; nombre de patients hospitalisés en internat et occupation du service d'urgence. Nous avons également calculé quatre scores de surpeuplement complexes : NEDOCS, EDWIN, ICMED et une modification locale de NEDOCS. Nous avons effectué des analyses ROC pour évaluer la validité prédictive de ces mesures par rapport à une norme de référence de perception du surpeuplement par les médecins. RéSULTATS: Nous avons recueilli des données pour 144 (63,9 %) des 168 intervalles d'étude. Le recensement des urgences est fortement corrélé avec le surpeuplement (ASC = 0.82, IC 95 % = 0.76­0.89), tout comme l'occupation des urgences (ASC = 0.75, IC 95 % = 0.66­0.83). Leur performance était similaire à celle des NEDOCS (ASC = 0.80) et à la modification locale des NEDOCS (ASC = 0.83). CONCLUSION: L'occupation des urgences en tant que mesure unique a une précision prédictive similaire aux scores complexes de surpeuplement et est facilement généralisable à divers services d'urgence. Le suivi en temps réel de ce simple indicateur pourrait être utilisé pour accélérer l'enquête et la mise en œuvre des interventions en cas de surpeuplement.

Pathway-dependent isotope fractionation during aerobic and anaerobic degradation of monochlorobenzene and 1,2,4-trichlorobenzene.

Stable carbon isotope fractionation is a valuable tool for monitoring natural attenuation and to establish the fate of groundwater contaminants. In this study, we measured carbon isotope fractionation during aerobic and anaerobic degradation of two chlorinated benzenes: monochlorobenzene (MCB) and 1,2,4-trichlorobenzene (1,2,4-TCB). MCB isotope fractionation was measured in anaerobic methanogenic microcosms, while 1,2,4-TCB isotope experiments were carried out in both aerobic and anaerobic microcosms. Large isotope fractionation was observed in both the anaerobic microcosm experiments. Enrichment factors (ε) for anaerobic reductive dechlorination of MCB and 1,2,4-TCB were -5.0‰ ± 0.2‰ and -3.0‰ ± 0.4‰, respectively. In contrast, no significant isotope fractionation was found during aerobic microbial degradation of 1,2,4-TCB. The cleavage of a C-Cl σ bond occurs during anaerobic reductive dechlorination of MCB and 1,2,4-TCB, while no σ bond cleavage is involved during aerobic degradation via dioxygenase. The difference in isotope fractionation for aerobic versus anaerobic biodegradation of MCB and 1,2,4-TCB can be explained by the difference in the initial step of aerobic versus anaerobic biodegradation pathways.

Selection between Competing Self-Reproducing Lipids: Succession and Dynamic Activation.

Models of chemical evolution are central to advancing origins of life research. To design more lifelike systems, we must expand our understanding of molecular selection mechanisms. Here, we show two selection modes that produce evolving populations of self-reproducing species, formed through thiol-disulfide exchange. Competition between thiol precursors can give clear succession patterns based on steric factors, an intrinsic property. A separate, emergent selection mechanism-dynamic activating metathesis-was found when exploring competing disulfide precursors. These experiments reveal that additional species generated in the mixture open up alternative reaction pathways to form self-reproducing products. Thus, increased compositional complexity provides certain species with a unique competitive advantage at the expense of others.

Where is the policy? A bibliometric analysis of the state of policy research on medical tourism.

Background: It is imperative that researchers studying medical tourism connect their work with policy, so that its real-world challenges can be better understood, and more effectively addressed. This article gauges the scope and evolution of policy thinking in medical tourism research through a bibliometric review of published academic literature, to establish the extent to which researchers apply public policy theories and frameworks in their investigation of medical tourism, or consider the policy imperatives of their work. Methods: A Boolean search of the Web of Science (WoS) Core Collection was performed to identify policy-related publications on medical tourism. We analyzed the results using bibliometrics and a data visualization software called VOSviewer to identify patterns in knowledge production and underlying network linkages in policy research on the subject. Results: Our findings suggest that only a small proportion of medical tourism research explicitly addresses policy issues or applies policy paradigms in their study approach. Field-specialized journals serving practitioners publish less research as compared to interdisciplinary social and health policy journals. Moreover, there are significant geographical and disciplinary disparities in the policy-orientation of research, and a predilection towards select policy areas such as reproductive and transplant tourism to the neglect of more holistic governance and health system considerations. Conclusion: This article is a call to action for greater engagement by policy scholars on medical tourism, and for health researchers to more explicitly consider how their research might contribute to the understanding and resolution of contemporary policy challenges of medical tourism. Failure to clearly and consistently make the policy connection is a lost opportunity for researchers to frame the public debate, and influence policy thinking on medical tourism.

Mobilizing Policy (In)Capacity to Fight COVID-19: Understanding Variations in State Responses.

The objective of this collection of essays is to gain insights into the different national-level state responses to COVID-19 around the world and the conditions that shaped them. The pandemic offers a natural experiment wherein the policy problem governments faced was the same but the responses they made were different, creating opportunities for comparison of both the kinds of policy tools being used and the factors that accounted for their choice. Accordingly, after surveying on-line databases of policy tools used in the pandemic and subjecting these to topic modelling to reveal the characteristics of a 'standard' national pandemic response, we discuss the similarities and differences found in specific responses. This is done with reference to the nature and level of policy capacity of respective governments, highlighting the critical roles played by (in)adequate preparation and lesson-drawing from past experiences with similar outbreaks or crises. Taken together the articles show how the national responses to the COVID-19 pandemic were shaped by the opportunity and capacity each government had to learn from previous pandemics and their capacity to operationalize and build political support for the standard portfolio of policy measures deployed to deal with the crisis. However, they also show how other factors such as the nature of national leadership, the organization of government and civil society, and blindspots towards the vulnerabilities of certain population segments also helped to shape policy responses to the pandemic.

Does Point of Care Ultrasound Improve Resuscitation Markers in Undifferentiated Hypotension? An International Randomized Controlled Trial From The Sonography in Hypotension and Cardiac Arrest in the Emergency Department (SHoC-ED) Series.

Introduction Point of Care Ultrasound (PoCUS) protocols are commonly used to guide resuscitation for patients with undifferentiated hypotension, yet there is a paucity of evidence for any outcome benefit. We undertook an international multicenter randomized controlled trial (RCT) to assess the impact of a PoCUS protocol on key clinical outcomes. Here we report on resuscitation markers.  Methods Adult patients presenting to six emergency departments (ED) in Canada and South Africa with undifferentiated hypotension (systolic blood pressure (SBP) <100mmHg or a Shock Index >1.0) were randomized to receive a PoCUS protocol or standard care (control). Reported physiological markers include shock index (SI), and modified early warning score (MEWS), with biochemical markers including venous bicarbonate and lactate, at baseline and four hours.  Results A total of 273 patients were enrolled, with data collected for 270. Baseline characteristics were similar for each group. Improvements in mean values for each marker during initial treatment were similar between groups: Shock Index; mean reduction in Control 0.39, 95% CI 0.34 to 0.44 vs. PoCUS 0.33, 0.29 to 0.38; MEWS, mean reduction in Control 2.56, 2.22 to 2.89 vs. PoCUS 2.91, 2.49 to 3.32; Bicarbonate, mean reduction in Control 2.71 mmol/L, 2.12 to 3.30 mmol/L vs. PoCUS 2.30 mmol/L, 1.75 to 2.84 mmol/L, and venous lactate, mean reduction in Control 1.39 mmol/L, 0.93 to 1.85 mmol/L vs. PoCUS 1.31 mmol/L, 0.88 to 1.74 mmol/L. Conclusion We found no meaningful difference in physiological and biochemical resuscitation markers with or without the use of a PoCUS protocol in the resuscitation of undifferentiated hypotensive ED patients. We are unable to exclude improvements in individual patients or in specific shock types.

Enantioselective reduction of N-alkyl ketimines with frustrated Lewis pair catalysis using chiral borenium ions.

Enantioselective reduction of ketimines was demonstrated using chiral N-heterocyclic carbene (NHC)-stabilised borenium ions in frustrated Lewis pair catalysis. High levels of enantioselectivity were achieved for substrates featuring secondary N-alkyl substituents. Comparative reactivity and mechanistic studies identify key determinants required to achieve useful enantioselectivity and represent a step forward in the further development of enantioselective FLP methodologies.

Introduction of an Extracorporeal Cardiopulmonary Resuscitation Eligibility Protocol for Paramedics in Atlantic Canada: A Pilot Knowledge Translation Project.

Introduction There is currently no protocol for the initiation of extracorporeal cardiopulmonary resuscitation (ECPR) for out of hospital cardiac arrest (OHCA) in Atlantic Canada. Advanced care paramedics (ACPs) perform advanced cardiac life support in the prehospital setting often completing the entire resuscitation on-scene. Implementation of ECPR will present a novel intervention that is only available at the receiving hospital. Our objective is to determine if an educational program can improve identification of ECPR candidates by paramedics. Establishing paramedic competence will ensure rapid transfer of eligible patients for a potentially life-saving intervention. Methods An educational program was delivered to paramedics including a short seminar and pocket card coupled with simulated OHCA cases. A before-and-after study design using a case-based survey was employed. Paramedics were scored on their ability to correctly identify patients suffering OHCA who met the inclusion criteria for our ECPR protocol. A Wilcoxon matched-pairs signed rank test was employed to compare paramedics' scores before and after the education delivery. A six-month follow-up is planned to assess retention. Qualitative data was also collected from paramedics during simulation to help identify practical issues, potential barriers, and to refine inclusion and exclusion criteria prior to the implementation of our protocol in the prehospital setting. Results The median score pre-education was 10 (IQR: 9-10.5) compared to 14 (IQR: 13-15) after education delivery. The median difference between groups was 5. The Wilcoxon matched-pairs test demonstrated a significant improvement in the paramedics' ability to correctly identify ECPR candidates after completing our educational program z = -2.67, p = 0.0039. Conclusion Paramedic training through a didactic session coupled with a pocket card and simulation appeared to be a feasible method of knowledge translation. Six-month follow-up data will help ensure knowledge retention is achieved.

How Feasible is Extracorporeal Cardiopulmonary Resuscitation in a Medium Urban Population Centre?

Background Patients suffering from out-of-hospital cardiac arrest (OHCA) experience poor survival and neurological outcomes, with rates remaining relatively unchanged despite advancements. Extracorporeal membrane oxygenation (ECMO), termed extracorporeal cardiopulmonary resuscitation (ECPR) in arrests, may offer improved outcomes. We developed local screening criteria for ECPR and then estimated the frequency of use by applying those criteria retrospectively to a cardiac arrest database. The purpose was to determine if an ECPR program is feasible in a medium urban population centre in Atlantic Canada. Methods A three-round modified Delphi survey, building upon data from a literature review, was conducted in collaboration with external experts. The resulting selection criteria for potential ECPR candidates were applied to a pre-existing local cardiac arrest database, supplemented by health records review, identifying potential candidates eligible for ECPR. Results Consensus inclusion criteria included witnessed cardiac arrest, age <70, refractory arrest, no-flow time <10min, total downtime <60min, and presumed cardiac or selected non-cardiac etiologies. Consensus exclusion criteria were an unwitnessed arrest, asystole, and select etiologies and comorbidities. Simplified criteria were developed to facilitate emergency medical services transport. Historically, 20.0% (95% CI 16.2-24.3%) of OHCA would be transported to the Emergency Department (ED), with 4.9% (95% CI 3.0% to 7.6%) qualifying for ECPR. Conclusion Despite conservative estimates based upon historically small numbers of select cardiac arrest patients meeting eligibility for transport and initiation of ECPR, a dedicated program may be feasible in our regional hospital setting. Patient care volumes suggest it would not be resource intensive yet would be sufficiently busy to maintain competency.

Does Point-of-care Ultrasonography Change Emergency Department Care Delivered to Hypotensive Patients When Categorized by Shock Type? A Post-Hoc Analysis of an International Randomized Controlled Trial from the SHoC-ED Investigators.

Introduction Our previously reported randomized-controlled-trial of point-of-care ultrasound (PoCUS) for patients with undifferentiated hypotension in the emergency department (ED) showed no survival benefit with PoCUS. Here, we examine the data to see if PoCUS led to changes in the care delivered to patients with cardiogenic and non-cardiogenic shock. Methods A post-hoc analysis was completed on a database of 273 hypotensive ED patients randomized to standard care or PoCUS in six centres in Canada and South Africa. Shock categories recorded one hour after the ED presentation were used to define subcategories of shock. We analyzed initial intravenous fluid volumes, as well as rates of inotrope use and procedures. Results  261 patients could be classified as cardiogenic or non-cardiogenic shock types. Although there were expected differences in the mean fluid volume administered between patients with non-cardiogenic and cardiogenic shock (p-value<0.001), there was no difference between the control and PoCUS groups (mean non-cardiogenic control 1881mL (95% CI 1567-2195mL) vs non-cardiogenic PoCUS 1763mL (1525-2001mL); and cardiogenic control 680mL (28.4-1332mL) vs. cardiogenic PoCUS 744mL (370-1117mL; p= 0.67). Likewise, there were no differences in rates of inotrope administration nor procedures for any of the subcategories of shock between the control group and PoCUS group patients. Conclusion Despite differences in care delivered by subcategory of shock, we did not find any difference in key elements of emergency department care delivered between patients receiving PoCUS and those who did not. This may help explain the previously reported lack of outcome differences between groups.