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Precisely controlling the size of engineered biomolecules and pharmaceutical compounds is often critical to their function. Standard methods for size characterization, such as dynamic light scattering or size exclusion chromatography, can be sample intensive and may not provide the sensitivity needed for mass- or concentration-limited biological systems. Taylor dispersion analysis (TDA) is a proven analytical method for direct, calibration-free size determination which utilizes only nL-pL sample volumes. In TDA, diffusion coefficients, which are mathematically transformed to hydrodynamic radii, are determined by characterizing band broadening of an analyte under well-controlled laminar flow conditions. Here, we describe the design and development of a 3D printed instrument for TDA, which is the first such instrument to offer dual-point laser-induced fluorescence (LIF) detection. The instrument utilized a fully 3D printed eductor as a vacuum source for precise and stable pressure-driven flow within a capillary, evidenced by a linear response in generated static pressure to applied gas pressure (

Lung infections are one of the leading causes of death worldwide, and this situation has been exacerbated by the emergence of COVID-19. Pre-clinical modelling of viral infections has relied on cell cultures that lack 3D structure and the context of lung extracellular matrices. Here, we propose a bioreactor-based, whole-organ lung model of viral infection. The bioreactor takes advantage of an automated system to achieve efficient decellularization of a whole rat lung, and recellularization of the scaffold using primary human bronchial cells. Automatization allowed for the dynamic culture of airway epithelial cells in a breathing-mimicking setup that led to an even distribution of lung epithelial cells throughout the distal regions. In the sealed bioreactor system, we demonstrate proof-of-concept for viral infection with the engineered lung by infecting primary human airway epithelial cells. Moreover, to assess the possibility of drug screening in this model, we demonstrate the efficacy of the broad-spectrum antiviral Remdesivir. This whole-organ scale lung infection model represents a step towards modelling viral infection of human cells in a 3D context, providing a powerful tool to investigate the mechanisms of the early stages of pathogenic infections and the development of effective treatment strategies for respiratory diseases.

Children infected with SARS-CoV-2 rarely progress to respiratory failure, but the risk of mortality in infected people over 85 years of age remains high, despite vaccination and improving treatment options. Here, we take a comprehensive, multidisciplinary approach to investigate differences in the cellular landscape and function of paediatric (70y) nasal epithelial cells experimentally infected with SARS-CoV-2. Our data reveal that nasal epithelial cell subtypes show different tropism to SARS-CoV-2, correlating with age, ACE2 and TMPRSS2 expression. Ciliated cells are a viral replication centre across all age groups, but a distinct goblet inflammatory subtype emerges in infected paediatric cultures, identifiable by high expression of interferon stimulated genes and truncated viral genomes. In contrast, infected elderly cultures show a proportional increase in ITGB6hiprogenitors, which facilitate viral spread and are associated with dysfunctional epithelial repair pathways.Graphical Abstract

Conventional cell culture systems involve growing cells in stationary cultures in the presence of growth medium containing various types of supplements. At confluency, the cells are divided and further expanded in new culture dishes. This passage from confluent monolayer to sparse cultures does not reflect normal physiological conditions and represents quite a drastic physiological change that may affect the natural cell physiobiology. Hollow-fibre bioreactors were in part developed to overcome these limitations and since their inception, they have widely been used in production of monoclonal antibodies and recombinant proteins. These bioreactors are increasingly used to study antibacterial drug effects via simulation of in vivo pharmacokinetic profiles. The use of the hollow-fibre infection model (HFIM) in viral infection studies is less well developed and in this review we have analysed and summarized the current available literature on the use of these bioreactors, with an emphasis on viruses. Our work has demonstrated that this system can be applied for viral expansion, studies of drug resistance mechanisms, and studies of pharmacokinetic/pharmacodynamic (PK/PD) of antiviral compounds. These platforms could therefore have great applications in large-scale vaccine development, and in studies of mechanisms driving antiviral resistance, since the HFIM could recapitulate the same resistance mechanisms and mutations observed in vivo in clinic. Furthermore, some dosage and spacing regimens evaluated in the HFIM system, as allowing maximal viral suppression, are in line with clinical practice and highlight this ‘in vivo-like’ system as a powerful tool for experimental validation of in vitro-predicted antiviral activities.

Background Advance care planning (ACP) empowers people to think about what is important to them and provides an opportunity to record their wishes for future care (Gold Standards Framework, 2018). NICE recognise that people growing older with a learning disability should have the same opportunities as everyone else to plan for the future (National Institute for Health and Care Excellence, 2018). Working collaboratively allowed the ACP Facilitator and Learning Disabilities Liaison Nurse to provide better opportunities for ACP discussions. Aims To provide people with a learning disability living within a care home in the Wyre Forest with an opportunity to explore ACP; ensuring we capture what is important to people and record a summary of clinical recommendations for care and treatment on a ReSPECT form (Reuscitation Council UK, 2021). Provide support and training to the care home staff to increase their knowledge and understanding of ACP and effective communication skills. Ensuring that we include everyone that should be involved in ACP discussions was vitally important and as shown in the LeDeR review not always happening within practice (LeDer, 2020-21). Methods In collaboration with the care homes, relevant healthcare professionals and advocacy services where appropriate to provide residents and their loved ones opportunities to have open and honest ACP discussions. Reviewing all previously recorded ACP to ensure completed correctly and valid. Results Increased uptake of ReSPECT forms and ACPs for residents within the local learning disability care homes. Development of staff communication skills and understanding of ACP. Good verbal and written feedback following teaching sessions and regarding the support provided. Conclusion ACP is recognised as having a very important role for empowering people to think about what is important to them. These discussions are just as important for people who have a learning disability, and they should be given the same opportunities to have open and honest discussions (PCPLD Network & NHS England, 2017). We must ensure we involve all appropriate people. This collaborative working approach has allowed different skills and knowledge to work together to provide better opportunities for residents.

Voting presents a challenge to military personnel and overseas citizens. Mail absentee ballots are often unreliable, and many citizens are unaware of their full range of voting options. Following implementation of a new internet-based ballot delivery system, we assessed the impact of the different email messages used to introduce it. Our findings show that communications with a concise subject line, source credibility, and that feature a citizen's reference groups encourage system usage. These communications also promote greater usage and turnout among citizens located in nations experiencing conflict, in allied nations, and abroad. Our findings have implications for scholars and political practitioners.

Introduction Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. Methods We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air–liquid interface (ALI). Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. Results Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and βENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. Conclusion Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo.

As seen in Chapter 4, the ability of voting legislation to influence aggregate outcomes is somewhat limited. Although there appears to be improvement in Uniform and Overseas Citizens Absentee Voting Act (UOCAVA) ballot return rates and UOCAVA ballot acceptance rates after the implementation of the Military and Overseas Voter Empowerment (MOVE) Act, military and overseas voter turnout remains low. However, public policy influences not only aggregate macro-level outcomes, but also micro-level individual variables. In contrast to Chapter 4, this chapter looks at the impact of public policy on the individual voter. How have voters responded to changes in the voting process? Are voters more satisfied with the process than in previous years? In this chapter, I use data from the Overseas Vote Foundation (OVF) 2008 and 2010 postelection voter surveys (whose methodology is described in detail in Chapter 2) to examine whether the implementation of electronic transmission methods in the MOVE Act impacted the individual’s perception of the voting process.

Throughout This Analysis, the military and overseas civilian voting populations have been treated equally, with the groups being referred to collectively as “UOCAVA voters,” after the Uniformed and Overseas Citizens Absentee Voting Act. However, as noted in Chapter 1, one of the largest shortcomings of the UOCAVA literature is that it does not identify nor discuss important similarities and differences between these voting blocs. In regard to similarities, for example, the size of the voting populations of the two groups is roughly similar, as seen in Chapter 2. As of September 2012, there were approximately 1.4 million active duty military personnel, of which 173,930 (12.5 percent) were stationed abroad (Department of Defense (DoD) 2013, 3, 30). If civilian personnel, reserve personnel, and military spouses and dependents were included with active duty personnel, there are approximately 6 million military personnel and dependents who are potential UOCAVA voters.

Previous chapters have firmly establishedthat military and overseas voters have traditionally experienced considerable trouble completing the voting process, which is reflected, for example, in the low ballot return rates described in Chapter 4. These difficulties have been primarily attributed to the voting time line; that is, because of their geographical distance from their local election officials (LEOs) as well as a reliance on traditional postal methods, voters were often unable to receive a blank ballot and/or return a voted ballot by the appropriate deadline. In short, it simply took too long to vote. As described in Chapter 3, since 2000, a series of legislative fixes at the state level and the passage of the Military and Overseas Voter Empowerment (MOVE) Act in 2009 at the federal level have been implemented in order to reduce the hurdles individuals face when voting, for example, extending the time line for voting to a mandatory 45 days.

During the summer of 2008, presidential candidate Barack Obama was often criticized for his lack of foreign policy experience. In response to this critique, then Senator Obama made his first major speech on the international stage in Berlin, Germany, on July 24, 2008. Approximately 200,000 people gathered at the Siegessaule (Victory Column) in Berlin to listen to Senator Obama speak about “A World That Stands as One.” Spiegel, a popular German magazine, questioned the true audience of the speech, however, and subtitled it: “People of the World, Look at Me,” which highlighted Senator Obamds desire to raise his international profile: The speech wasn’t truly aimed at the audience assembled at the Siegessaule. While Obama shouted the last few lines of his speech into the crowd, his handlers were already escorting the members of the press that travel with him down from the guest stands. The journalists would be given a few moments to speak with Obama. They were all Americans, all 40 of them. CNN, the New York Times, Newsweek, theChicago Sun-Times. Members of the foreign press were explicitly unwelcome. The target audience was America. Sorry, Berlin.1

As described in Chapter 3, the historical development of military and overseas voting policy is best characterized as a series of legislation created in response to a galvanizing event, such as war, a presidential election, or pressure from outside groups. Very few of these policies were developed in the context of data-driven reform, but were rather supported (or not) based on partisan preferences. The American Political Science Association (APSA) report of 1952 and the Federal Voting Assistance Act of 1955 represent an important exception, and a turning point in the development of public policy. However, it would take another 55 years before enough data were available to enable truly data-driven reform, and in 2009, Congress passed the Military and Overseas Voter Empowerment (MOVE) Act in the wake of the statistics and data accessible after the 2008 presidential election.

Chapter 2 explored and attempted toanswer some of the central issues surrounding military and overseas voters, specifically overseas civilians, including questions about their numbers, location, and demographic makeup. Unfortunately, the available data limit our ability to answer these questions, as they are principally composed of survey data based on convenience samples. However, recent research does provide valuable insight into military and overseas voters, who are, in many cases, distinctly different from domestic voters residing within the continental United States. The development of military and overseas voting rights is also unique, and a rather recent phenomenon, with military and overseas voters taking quite divergent paths toward enfranchisement.

Following their role in the disputed Florida recount, military and overseas voters emerged in importance in 2000. In the summer of 2008, then Senator Obama gave an important speech in Berlin, once again shining a light on this voting bloc. By that time, military and overseas voters had a long-established history of voting rights struggles, but still encountered numerous problems while voting, and remained a largely understudied portion of the voting population. Reflecting upon the amount of change in military and overseas voting between the summer of 2008 and the summer of 2014, the differences are remarkable.

One of modern democracy’s biggest challenges is the overseas voter. Current estimates indicate that there are 4 to 6 million Americans permanently or temporarily living abroad. Congress has passed several key pieces of federal legislation to facilitate overseas voting including the Uniformed and Overseas Citizens Absentee Voting Act (UOCAVA) in 1986 and the Help America Vote Act (HAVA) of 2002. This paper investigates the success of these public policy initiatives in reaching overseas voters. Has overseas voting increased as a result of these legislative initiatives? What barriers still exist? What policy changes can be made to promote voting? This paper is divided into three sections. First, in order to capture the variance in state UOCAVA policy, I create an index operationalizing state legislation on overseas voting issues, such as electronic transmission of voting materials. Second, I adapt the variables used in traditional voter turnout studies to create hypotheses, and use data from the Overseas Vote Foundation (OVF) Post-Election Survey to test the hypotheses and identify variables that hinder successful voting. The results demonstrate that variable deadlines combined with postal problems often hamper voters. Furthermore, the impacts of policy are weak and outreach programs remain vitally important. Finally, I interpret these results in order to provide a better direction for future policy changes.

Iodopentamidine isethionate was heated for 70 min with [I-123]- or [I-125]-sodium iodide and ammonium sulphate in the absence of solvent at 140 degrees C. The product, [I-123]- or [I-125]-iodopentamidine isethionate, was purified by ion exchange chromatography. It was stable at room temperature in aqueous solution over several days, in human blood in vitro for at least 24 h (showing no binding to either cells or proteins), in urine over at least 15 h, and during nebulization in both ultrasonic and jet nebulizers.

Molecular chaperone GroEL and cytochrome c were shown to form a stable complex at low ionic strength which was structurally characterised by means of UV-visible, CD and fluorescence spectroscopy. GroEL-bound cytochrome c was demonstrated to be in a compact, non-native state which could correspond to GroEL-bound forms of two well known cytochrome c folding intermediates IHNC and I*. These were selectively released from GroEL using adenosine 5′-triphosphate and co-chaperone GroES. Drawing from these results and our previous data, a simple passive kinetic partitioning mechanism is proposed for molecular chaperone assisted folding/refolding of substrate proteins in which molecular chaperone GroEL binds to substrate proteins in order to control the steady state concentration of substrate protein folding intermediates below the critical threshold for aggregation, thereby encouraging substrate protein folding intermediates to partition kinetically along routes to correctly folded protein in preference to aggregated states. In this way the yield of correctly folded protein may be maximised, unless the extrinsic folding conditions themselves otherwise prevent this happening by promoting protein misfolding through the formation of incorrect intramolecular interactions.

OBJECTIVES: To determine the effects of a policy of using acupuncture, compared with a policy of avoiding acupuncture, on headache in primary care patients with chronic headache disorders. The effects of acupuncture on medication use, quality of life, resource use and days off sick in this population and the cost-effectiveness of acupuncture were also examined. DESIGN: Randomised, controlled trial. SETTING: General practices in England and Wales. PARTICIPANTS: The study included 401 patients with chronic headache disorder, predominantly migraine. INTERVENTIONS: Patients were randomly allocated to receive up to 12 acupuncture treatments over 3 months or to a control intervention offering usual care. MAIN OUTCOME MEASURES: Outcome measures included headache score; assessment of Short Form 36 (SF-36) health status and use of medication at baseline, 3 months and 12 months; assessment of use of resources every 3 months; and assessment of incremental cost per quality-adjusted life-year (QALY) gained. RESULTS: Headache score at 12 months, the primary end-point, was lower in the acupuncture group than in controls. The adjusted difference between means was 4.6. This result was robust to sensitivity analysis incorporating imputation for missing data. Patients in the acupuncture group experienced the equivalent of 22 fewer days of headache per year. SF-36 data favoured acupuncture, although differences reached significance only for physical role functioning, energy and change in health. Compared with controls, patients randomised to acupuncture used 15% less medication, made 25% fewer visits to GPs and took 15% fewer days off sick. Total costs during the 1-year period of the study were on average higher for the acupuncture group than for controls because of the acupuncture practitioners' costs. The mean health gain from acupuncture during the year of the trial was 0.021 QALYs, leading to a base-case estimate of GBP9180 per QALY gained. This result was robust to sensitivity analysis. Cost per QALY dropped substantially when the analysis incorporated likely QALY differences for the years after the trial. CONCLUSIONS: The study suggests that acupuncture leads to persisting, clinically relevant benefits for primary care patients with chronic headache, particularly migraine. It is relatively cost-effective compared with a number of other interventions provided by the NHS. Further studies could examine the duration of acupuncture effects beyond 1 year and the relative benefit to patients with migraine with compared to tension-type headache. Trials are also warranted examining the effectiveness and cost-effectiveness of acupuncture in patients with headache receiving more aggressive pharmacological management.

Objective To determine the effects of a policy of “use acupuncture” on headache, health status, days off sick, and use of resources in patients with chronic headache compared with a policy of “avoid acupuncture.”Design Randomised, controlled trial.Setting General practices in England and Wales.Participants 401 patients with chronic headache, predominantly migraine.Interventions Patients were randomly allocated to receive up to 12 acupuncture treatments over three months or to a control intervention offering usual care.Main outcome measures Headache score, SF-36 health status, and use of medication were assessed at baseline, three, and 12 months. Use of resources was assessed every three months.Results Headache score at 12 months, the primary end point, was lower in the acupuncture group (16.2, SD 13.7, n = 161, 34% reduction from baseline) than in controls (22.3, SD 17.0, n = 140, 16% reduction from baseline). The adjusted difference between means is 4.6 (95% confidence interval 2.2 to 7.0; P = 0.0002). This result is robust to sensitivity analysis incorporating imputation for missing data. Patients in the acupuncture group experienced the equivalent of 22 fewer days of headache per year (8 to 38). SF-36 data favoured acupuncture, although differences reached significance only for physical role functioning, energy, and change in health. Compared with controls, patients randomised to acupuncture used 15% less medication (P = 0.02), made 25% fewer visits to general practitioners (P = 0.10), and took 15% fewer days off sick (P = 0.2).Conclusions Acupuncture leads to persisting, clinically relevant benefits for primary care patients with chronic headache, particularly migraine. Expansion of NHS acupuncture services should be considered.