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Summary: Cardiovascular disease (CVD) remains the largest cause of mortality worldwide. The development of new effective therapeutics is a major unmet need. The current review focuses broadly on the concept of nucleic acid (NA)–based therapies, considering the use of various forms of NAs, including mRNAs, miRNAs, siRNA, and guide RNAs, the latter specifically for the purpose of CRISPR-Cas directed gene editing. We describe the current state-of-the-art of RNA target discovery and development, the status of RNA therapeutics in the context of CVD, and some of the challenges and hurdles to be overcome.

Background: Food allergy (FA) and atopic dermatitis (AD) are common conditions that often present in the first year of life. Identification of underlying mechanisms and environmental determinants of FA and AD is essential to develop and implement effective prevention and treatment strategies. Objectives: We sought to describe the design of the Systems Biology of Early Atopy (SunBEAm) birth cohort. Methods: Funded by the National Institute of Allergy and Infectious Diseases (NIAID) and administered through the Consortium for Food Allergy Research (CoFAR), SunBEAm is a US population-based, multicenter birth cohort that enrolls pregnant mothers, fathers, and their newborns and follows them to 3 years. Questionnaire and biosampling strategies were developed to apply a systems biology approach to identify environmental, immunologic, and multiomic determinants of AD, FA, and other allergic outcomes. Results: Enrollment is currently underway. On the basis of an estimated FA prevalence of 6%, the enrollment goal is 2500 infants. AD is defined on the basis of questionnaire and assessment, and FA is defined by an algorithm combining history and testing. Although any FA will be recorded, we focus on the diagnosis of egg, milk, and peanut at 5 months, adding wheat, soy, cashew, hazelnut, walnut, codfish, shrimp, and sesame starting at 12 months. Sampling includes blood, hair, stool, dust, water, tape strips, skin swabs, nasal secretions, nasal swabs, saliva, urine, functional aspects of the skin, and maternal breast milk and vaginal swabs. Conclusions: The SunBEAm birth cohort will provide a rich repository of data and specimens to interrogate mechanisms and determinants of early allergic outcomes, with an emphasis on FA, AD, and systems biology.

Objective: Intimal hyperplasia (IH) is the expansion of the vascular intimal region after intervention, which can lead to stenosis and eventual failure of vascular grafts or interventional procedures such as angioplasty or stent placement. Our goals were to investigate the development of IH in a rabbit open surgical model and to evaluate the associated pathophysiologic processes involving decorin and the platelet-derived growth factor-BB/platelet-derived growth factor receptor-β/mitogen-activated protein kinase (PDGF-BB/PDGFR-β/MAPK) pathway. Methods: We conducted carotid transection and primary anastomosis on five New Zealand white rabbits to induce IH and examined the associated pathophysiologic changes. Tissue was obtained for histological and protein analysis on postoperative day 21 using the contralateral vessel as a control. Intimal medial thickness (IMT) was calculated to measure IH and compared with the unoperated side. Western blot analysis was performed on tissue lysates to determine the expression of decorin core protein, PDGF-BB, PDGFR-β, and phosphorylated-MAPK (ph-MAPK). Immunofluorescence microscopy was used to assess tissue distribution of matrix metalloproteinase-2 (MMP-2) and ph-PDGFR-β. Results: Bilateral carotid arteries were harvested on postoperative day 21. We compared the IMT in operated with unoperated specimens. IMT was significantly elevated in operated arteries vs unoperated arteries in all five animals (148.6 μm ± 9.09 vs 103.40 μm ± 7.08; 135.2 μm ± 8.30 vs 92.40 μm ± 2.35; 203.1 μm ± 30.23 vs 104.00 μm ± 4.52; 236.2 μm ± 27.22 vs 141.50 μm ± 9.95; 226.9 μm ± 11.12 vs 98.8 μm ± 3.78). Western blot analysis revealed degradation of decorin protein in the operated tissue, including loss of a 50 kDa band and the appearance of a cleaved fragment at 10 kDa. Decorin and MMP-2 were observed, via immunofluorescence microscopy, in the neointima of the operated vessels. Western blot analysis also revealed increased PDGF-BB, PDGFR-β, and ph-MAPK levels in operated tissue. Immunofluorescent staining for ph-PDGFR-β primarily localized to the neointima, indicating increased signaling through PDGF in this region. Conclusions: Carotid transection and primary reanastomosis in rabbits induced IH that was associated with MMP-2 activation, degradation of decorin, and activation of the PDGF/PDGFR-β/MAPK pathway. The findings in this study should lead to further mechanistic evaluation of these pathways to better understand the potential to modify the intimal hyperplastic response to surgery. (JVS–Vascular Science 2021;2:2-12.) Clinical Relevance: Intimal hyperplasia remains a significant challenge to the vascular surgeon in open and interventional procedures. Basic science studies have made headway into understanding the process, but this has not translated into many therapeutic options particularly for primary prevention after a procedure. Decorin is gaining popularity as an important mediator of various pathophysiologic processes involving the extracellular matrix. We sought to determine the possible role of decorin in the development of neointimal hyperplasia in an open surgical model. This study provides a replicable model for the development of intimal hyperplasia and potential therapeutic targets going forward.

Summary: Background: The Coalition for Epidemic Preparedness Innovations was established in 2016, to develop vaccines that can contribute to preparedness for outbreaks of epidemic infectious diseases. Evidence on vaccine development costs for such diseases is scarce. Our goal was to estimate the minimum cost for achieving vaccine research and development preparedness targets in a portfolio of 11 epidemic infectious diseases, accounting for vaccine pipeline constraints and uncertainty in research and development preparedness outcomes. Methods: We assembled a pipeline of 224 vaccine candidates from preclinical through to phase 2 for 11 priority epidemic infectious diseases. We used a linear regression model to identify drivers of development costs from preclinical through to end of phase 2a. Drawing from published estimates of vaccine research and development probabilities of success, we simulated costs for advancing these 224 vaccine candidates through to the end of phase 2a. We combined these findings to determine minimum costs for progressing at least one vaccine through to the end of phase 2a per epidemic infectious disease by means of a stochastic optimisation model. Findings: The cost of developing a single epidemic infectious disease vaccine from preclinical trials through to end of phase 2a is US$31–68 million (US$14–159 million range), assuming no risk of failure. We found that previous licensure experience and indirect costs are upward drivers of research and development costs. Accounting for probability of success, the average cost of successfully advancing at least one epidemic infectious disease vaccine through to the end of phase 2a can vary from US$84–112 million ($23 million–$295 million range) starting from phase 2 to $319–469 million ($137 million–$1·1 billion range) starting from preclinical. This cost includes the cumulative cost of failed vaccine candidates through the research and development process. Assuming these candidates and funding were made available, progressing at least one vaccine through to the end of phase 2a for each of the 11 epidemic infectious diseases would cost a minimum of $2·8–3·7 billion ($1·2 billion–$8·4 billion range). Interpretation: Our analysis provides new evidence on vaccine research and development pipelines and associated costs for 11 epidemic infectious diseases, highlighting both funding needs and research and development gaps for achieving vaccine research and development preparedness targets. Funding: This work was partly supported by the Research Council of Norway through the Global Health and Vaccination Programme GLOBVAC.

The AP-1 adaptor complex is found in all eukaryotes, but it has been implicated in different pathways in different organisms. To look directly at AP-1 function, we generated stably transduced HeLa cells coexpressing tagged AP-1 and various tagged membrane proteins. Live cell imaging showed that AP-1 is recruited onto tubular carriers trafficking from the Golgi apparatus to the plasma membrane, as well as onto transferrin-containing early/recycling endosomes. Analysis of single AP-1 vesicles showed that they are a heterogeneous population, which starts to sequester cargo 30 min after exit from the ER. Vesicle capture showed that AP-1 vesicles contain transmembrane proteins found at the TGN and early/recycling endosomes, as well as lysosomal hydrolases, but very little of the anterograde adaptor GGA2. Together, our results support a model in which AP-1 retrieves proteins from post-Golgi compartments back to the TGN, analogous to COPI's role in the early secretory pathway. We propose that this is the function of AP-1 in all eukaryotes., (© 2024 Robinson et al.)

In this perspective, we discuss how one can initiate, image, and disentangle the ultrafast elementary steps of thermal-energy chemical dynamics, building upon advances in technology and scientific insight. We propose that combinations of ultrashort mid-infrared laser pulses, controlled molecular species in the gas phase, and forefront imaging techniques allow to unravel the elementary steps of general-chemistry reaction processes in real time. We detail, for prototypical first reaction systems, experimental methods enabling these investigations, how to sufficiently prepare and promote gas-phase samples to thermal-energy reactive states with contemporary ultrashort mid-infrared laser systems, and how to image the initiated ultrafast chemical dynamics. The results of such experiments will clearly further our understanding of general-chemistry reaction dynamics.

School nurses frequently interact with school-age children and their parents/guardians regarding vaccinations. As a trusted source of information, the school nurse is in a unique position to share vaccine information with parents/guardians and school-age children that may dispel myths and, consequently, improve vaccination rates. Nevertheless, some parents/guardians are still reluctant to vaccinate their school-age children against Human Papilloma Virus (HPV) for a variety of reasons. Common barriers to HPV vaccination include a lack of understanding of the vaccine's purpose, concerns regarding the vaccine's safety, and insufficient recommendation from healthcare workers. However, school nurses have many duties in addition to ensuring vaccine compliance. School nurses may have difficulty remaining up-to-date on evidence-based answers to parents'/guardians' questions about HPV vaccine. Therefore, the purpose of this article is to provide school nurses with a quick reference question and answer guide to parents'/guardians' common HPV-related vaccination questions.

Background: Since the implementation in 1986, there is little research focused on vaccine information statements (VISs) use for vaccine education and parental perception., Purpose: To explore parental reports of dissemination and use of VISs., Methods: Data for this pilot, cross-sectional, descriptive study were collected through an online survey in both English and Spanish., Results: Responses from 130 parents in one school district were analyzed. Most participants (67.7%) reported getting vaccine information from a pediatric health care provider. A majority (71.5%) said that VISs were included in the vaccination process. Approximately one third of participants (37.7%) reported reading some or all the VIS before their child was vaccinated, and more than half (59.3%) read some or all the VIS after their child was vaccinated., Conclusions: While promising that many parents reported receiving a VIS, more than one quarter of parents reported they did not. Inadequate time to read and understand VIS information before an immunization may lead to limited parental understanding. Although some participants reported struggling to understand VISs, more than half said that VISs were helpful and would read another in the future., Implications: Without appropriate use of vaccine education material, providers miss the opportunity to educate parents on the risks and benefits of vaccinating their children. Providers must be aware of literacy levels and vaccine attitudes and create appropriate opportunities for parents to read and learn about vaccines. VISs are valuable educational tools for patients and parents. Improvements are needed to improve both VIS clarity and dissemination., Competing Interests: Competing interests: The authors report no conflicts of interest., (Copyright © 2023 American Association of Nurse Practitioners.)

Apicomplexan parasites have immense impacts on humanity, but their basic cellular processes are often poorly understood. Where endocytosis occurs in these cells, how conserved this process is with other eukaryotes, and what the functions of endocytosis are across this phylum are major unanswered questions. Using the apicomplexan model Toxoplasma, we identified the molecular composition and behavior of unusual, fixed endocytic structures. Here, stable complexes of endocytic proteins differ markedly from the dynamic assembly/disassembly of these machineries in other eukaryotes. We identify that these endocytic structures correspond to the 'micropore' that has been observed throughout the Apicomplexa. Moreover, conserved molecular adaptation of this structure is seen in apicomplexans including the kelch-domain protein K13 that is central to malarial drug-resistance. We determine that a dominant function of endocytosis in Toxoplasma is plasma membrane homeostasis, rather than parasite nutrition, and that these specialized endocytic structures originated early in infrakingdom Alveolata likely in response to the complex cell pellicle that defines this medically and ecologically important ancient eukaryotic lineage., (© 2023. The Author(s).)

In this work, we investigate the relaxation processes of 2-thiouracil after UV photoexcitation to the S 2 state through the use of ultrafast, single-colour, pump-probe UV/UV spectroscopy. We place focus on investigating the appearance and subsequent decay signals of ionized fragments. We complement this with VUV-induced dissociative photoionisation studies collected at a synchrotron, allowing us to better understand and assign the ionisation channels involved in the appearance of the fragments. We find that all fragments appear when single photons with energy > 11 eV are used in the VUV experiments and hence appear through 3+ photon-order processes when 266 nm light is used. We also observe three major decays for the fragment ions: a sub-autocorrelation decay (i.e., sub-370 fs), a secondary ultrafast decay on the order of 300-400 fs, and a long decay on the order of 220 to 400 ps (all fragment dependent). These decays agree well with the previously established S 2 → S 1 → Triplet → Ground decay process. Results from the VUV study also suggest that some of the fragments may be created by dynamics occurring in the excited cationic state.

Ultrafast high-brightness X-ray pulses have proven invaluable for a broad range of research. Such pulses are typically generated via synchrotron emission from relativistic electron bunches using large-scale facilities. Recently, significantly more compact X-ray sources based on laser-wakefield accelerated (LWFA) electron beams have been demonstrated. In particular, laser-driven sources, where the radiation is generated by transverse oscillations of electrons within the plasma accelerator structure (so-called betatron oscillations) can generate highly-brilliant ultrashort X-ray pulses using a comparably simple setup. Here, we experimentally demonstrate a method to markedly enhance the parameters of LWFA-driven betatron X-ray emission in a proof-of-principle experiment. We show a significant increase in the number of generated photons by specifically manipulating the amplitude of the betatron oscillations by using our novel Transverse Oscillating Bubble Enhanced Betatron Radiation scheme. We realize this through an orchestrated evolution of the temporal laser pulse shape and the accelerating plasma structure. This leads to controlled off-axis injection of electrons that perform large-amplitude collective transverse betatron oscillations, resulting in increased radiation emission. Our concept holds the promise for a method to optimize the X-ray parameters for specific applications, such as time-resolved investigations with spatial and temporal atomic resolution or advanced high-resolution imaging modalities, and the generation of X-ray beams with even higher peak and average brightness., (© 2022. The Author(s).)

Objective: Sjögren syndrome (SS) has a significant impact on health-related quality of life (HRQOL). We sought to evaluate how the Patient Reported Outcome Measurement Information System (PROMIS) domains in SS may supplement the European League Against Rheumatism (EULAR) Sjögren Syndrome Patient Reported Index (ESSPRI)., Methods: A cross-sectional evaluation was performed on consecutive adult patients during visits to an SS clinic between March 2018 and February 2020. Each patient completed PROMIS short forms related to HRQOL and the ESSPRI, and had a clinical assessment. Patients were either classified as SS by 2016 American College of Rheumatology (ACR)/EULAR criteria, or as "sicca not otherwise specified (NOS)" and used as a comparison group. Univariable and multivariable linear regression models were used to evaluate predictors of PROMIS fatigue (-F), pain interference (-PI), and ability to participate in social roles and activities (-APS)., Results: Two hundred twenty-seven patients with SS and 85 with sicca NOS were included and did not differ in ESSPRI domains; 26% of the SS and 20% of the sicca NOS group had concurrent autoimmune disease. In SS, PROMIS-PI, PROMIS-F, and PROMIS physical function were at least one-half SD worse than US population normative values. PROMIS-PI ( r = 0.73) and PROMIS-F ( r = 0.80) were highly correlated with ESSPRI pain and fatigue subdomains. Fatigue and pain interference, but not dryness or mood disturbance, were the strongest predictors of social participation in multivariable analysis., Conclusion: In our SS cohort, PROMIS instruments identified a high disease burden of pain interference, fatigue, and physical function. PROMIS-F strongly predicted PROMIS-APS. PROMIS-PI and PROMIS-F scores correlated highly with their respective ESSPRI domains. PROMIS instruments should be considered to identify relevant HRQOL patterns in SS., (Copyright © 2022 The Journal of Rheumatology.)

The conversion of photon energy into other energetic forms in molecules is accompanied by charge moving on ultrafast timescales. We directly observe the charge motion at a specific site in an electronically excited molecule using time-resolved x-ray photoelectron spectroscopy (TR-XPS). We extend the concept of static chemical shift from conventional XPS by the excited-state chemical shift (ESCS), which is connected to the charge in the framework of a potential model. This allows us to invert TR-XPS spectra to the dynamic charge at a specific atom. We demonstrate the power of TR-XPS by using sulphur 2p-core-electron-emission probing to study the UV-excited dynamics of 2-thiouracil. The method allows us to discover that a major part of the population relaxes to the molecular ground state within 220-250 fs. In addition, a 250-fs oscillation, visible in the kinetic energy of the TR-XPS, reveals a coherent exchange of population among electronic states., (© 2022. The Author(s).)

In this paper, we report X-ray absorption and core-level electron spectra of the nucleobase derivative 2-thiouracil at the sulfur L 1 - and L 2,3 -edges. We used soft X-rays from the free-electron laser FLASH2 for the excitation of isolated molecules and dispersed the outgoing electrons with a magnetic bottle spectrometer. We identified photoelectrons from the 2p core orbital, accompanied by an electron correlation satellite, as well as resonant and non-resonant Coster-Kronig and Auger-Meitner emission at the L 1 - and L 2,3 -edges, respectively. We used the electron yield to construct X-ray absorption spectra at the two edges. The experimental data obtained are put in the context of the literature currently available on sulfur core-level and 2-thiouracil spectroscopy.

We have studied soft X-ray photoabsorption in the doubly deprotonated gas-phase oligonucleotide [dTGGGGT-2H] 2- . The dominating decay mechanism of the X-ray induced inner shell vacancy was found to be Auger decay with detachment of at least three electrons, leading to charge reversal of the anionic precursor and the formation of positively charged photofragment ions. The same process is observed in heavy ion (12 MeV C 4+ ) collisions with [dTGGGGT-2H] 2- where inner shell vacancies are generated as well, but with smaller probability. Auger decay of a single K-vacancy in DNA, followed by detachment of three or more low energy electrons instead of a single high energy electron has profound implications for DNA damage and damage modelling. The production of three low kinetic energy electrons with short mean free path instead of one high kinetic energy electron with long mean free path implies that electron-induced DNA damage will be much more localized around the initial K-shell vacancy. The fragmentation channels, triggered by triple electron detachment Auger decay are predominantly related to protonated guanine base loss and even loss of protonated guanine dimers is tentatively observed. The fragmentation is not a consequence of the initial K-shell vacancy but purely due to multiple detachment of valence electrons, as a very similar positive ion fragmentation pattern is observed in femtosecond laser-induced dissociation experiments., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The ultrafast photo-induced ring opening of the oxirane derivative trans-stilbene oxide has been studied through the use of ultrafast UV/UV pump-probe spectroscopy by using photo-ion detection. Single- and multiphoton probe paths and final states were identified through comparisons between UV power studies and synchrotron-based vacuum ultraviolet (VUV) single-photon ionization studies. Three major time-dependent features of the parent ion (sub-450 fs decay, (1.5±0.2) ps, and >100 ps) were observed. These decays are discussed in conjunction with the primary ring-opening mechanism of stilbene oxide, which occurs through C-C dissociation in the oxirane ring. The appearance of fragments relating to the masses of dehydrogenated diphenylmethane (167 amu) and dehydrogenated methylbenzene (90 amu) were also investigated. The appearance of the 167 amu fragment could suggest an alternative ultrafast ring-opening pathway via the dissociation of one of the C-O bonds within the oxirane ring., (© 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells and occurs by coincidence detection, requiring both phosphatidylinositol 3-phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which, on its own, localizes to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15, while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation., (© 2021 Hirst et al.)

Mastcam-Z is a multispectral, stereoscopic imaging investigation on the Mars 2020 mission's Perseverance rover. Mastcam-Z consists of a pair of focusable, 4:1 zoomable cameras that provide broadband red/green/blue and narrowband 400-1000 nm color imaging with fields of view from 25.6° × 19.2° (26 mm focal length at 283 μrad/pixel) to 6.2° × 4.6° (110 mm focal length at 67.4 μrad/pixel). The cameras can resolve (≥ 5 pixels) ∼0.7 mm features at 2 m and ∼3.3 cm features at 100 m distance. Mastcam-Z shares significant heritage with the Mastcam instruments on the Mars Science Laboratory Curiosity rover. Each Mastcam-Z camera consists of zoom, focus, and filter wheel mechanisms and a 1648 × 1214 pixel charge-coupled device detector and electronics. The two Mastcam-Z cameras are mounted with a 24.4 cm stereo baseline and 2.3° total toe-in on a camera plate ∼2 m above the surface on the rover's Remote Sensing Mast, which provides azimuth and elevation actuation. A separate digital electronics assembly inside the rover provides power, data processing and storage, and the interface to the rover computer. Primary and secondary Mastcam-Z calibration targets mounted on the rover top deck enable tactical reflectance calibration. Mastcam-Z multispectral, stereo, and panoramic images will be used to provide detailed morphology, topography, and geologic context along the rover's traverse; constrain mineralogic, photometric, and physical properties of surface materials; monitor and characterize atmospheric and astronomical phenomena; and document the rover's sample extraction and caching locations. Mastcam-Z images will also provide key engineering information to support sample selection and other rover driving and tool/instrument operations decisions., Competing Interests: Conflicts of interest/Competing interestsThe authors declare that they have no conflict of interest., (© The Author(s) 2020.)

CHoP-In (CRISPR/Cas9-mediated Homology-independent PCR-product integration) is a fast, non-homologous end-joining based, strategy for genomic editing in mammalian cells. There is no requirement for cloning in generation of the integration donor, instead the desired integration donor is produced as a polymerase chain reaction (PCR) product, flanked by the Cas9 recognition sequences of the target locus. When co-transfected with the cognate Cas9 and guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon-homologous end joining. The approach is versatile, allowing N-terminal, C-terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a selection of well characterised membrane trafficking proteins. The lack of donor vectors offers advantages over existing methods in terms of both speed and hands-on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems., (© 2019 The Authors. Traffic published by John Wiley & Sons Ltd.)

Adaptor protein (AP) complexes are heterotetramers that select cargo for inclusion into transport vesicles. Five AP complexes (AP-1 to AP-5) have been described, each with a distinct localisation and function. Furthermore, patients with a range of disorders, particularly involving the nervous system, have now been identified with mutations in each of the AP complexes. In many cases this has been correlated with aberrantly localised membrane proteins. In this Cell Science at a Glance article and the accompanying poster, we summarize what is known about the five AP complexes and discuss how this helps to explain the clinical features of the different genetic disorders., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including 'Dynamic Organellar Maps', to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the trans-Golgi network to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the "ATG9A reservoir" required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

Vesicluar transport of proteins from endosomes to the trans-Golgi network (TGN) is an essential cellular pathway, but much of its machinery is still unknown. A screen for genes involved in endosome-to-TGN trafficking produced two hits, the adaptor protein-1 (AP-1 complex), which facilitates vesicle budding, and WDR11. Here we demonstrate that WDR11 forms a stable complex with two other proteins, which localises to the TGN region and does not appear to be associated with AP-1, suggesting it may act downstream from budding. In a vesicle tethering assay, capture of vesicles by golgin-245 was substantially reduced in WDR11-knockout cells. Moreover, structured illumination microscopy and relocation assays indicate that the WDR11 complex is initially recruited onto vesicles rather than the TGN, where it may in turn recruit the golgin binding partner TBC1D23. We propose that the complex acts together with TBC1D23 to facilitate the golgin-mediated capture of vesicles that were generated using AP-1.

The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR), GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5-associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders.

The dense core vesicles (DCVs) of neuroendocrine cells are a rich source of bioactive molecules such as peptides, hormones, and neurotransmitters, but relatively little is known about how they are formed. Using fractionation profiling, a method that combines subcellular fractionation with mass spectrometry, we identified ∼1200 proteins in PC12 cell vesicle-enriched fractions, with DCV-associated proteins showing distinct profiles from proteins associated with other types of vesicles. To investigate the role of clathrin in DCV biogenesis, we stably transduced PC12 cells with an inducible short hairpin RNA targeting clathrin heavy chain, resulting in ∼85% protein loss. DCVs could still be observed in the cells by electron microscopy, but mature profiles were approximately fourfold less abundant than in mock-treated cells. By quantitative mass spectrometry, DCV-associated proteins were found to be reduced approximately twofold in clathrin-depleted cells as a whole and approximately fivefold in vesicle-enriched fractions. Our combined data sets enabled us to identify new candidate DCV components. Secretion assays revealed that clathrin depletion causes a near-complete block in secretagogue-induced exocytosis. Taken together, our data indicate that clathrin has a function in DCV biogenesis beyond its established role in removing unwanted proteins from the immature vesicle., (© 2017 Sahu et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Acidic clusters act as sorting signals for packaging cargo into clathrin-coated vesicles (CCVs), and also facilitate down-regulation of MHC-I by HIV-1 Nef. To find acidic cluster sorting machinery, we performed a gene-trap screen and identified the medium subunit (µ1) of the clathrin adaptor AP-1 as a top hit. In µ1 knockout cells, intracellular CCVs still form, but acidic cluster proteins are depleted, although several other CCV components were either unaffected or increased, indicating that cells can compensate for long-term loss of AP-1. In vitro experiments showed that the basic patch on µ1 that interacts with the Nef acidic cluster also contributes to the binding of endogenous acidic cluster proteins. Surprisingly, µ1 mutant proteins lacking the basic patch and/or the tyrosine-based motif binding pocket could rescue the µ1 knockout phenotype completely. In contrast, these mutants failed to rescue Nef-induced down-regulation of MHC class I, suggesting a possible mechanism for attacking the virus while sparing the host cell., (© 2017 Navarro Negredo et al.)

Vesicular transport was key to the evolution of eukaryotes, and is essential for eukaryotic life today. All modern eukaryotes have a set of vesicle coat proteins, which couple cargo selection to vesicle budding in the secretory and endocytic pathways. Although these coats share common features (e.g. recruitment via small GTPases, β-propeller-α-solenoid proteins acting as scaffolds), the relationships between them are not always clear. Structural studies on the coats themselves, comparative genomics and cell biology in diverse eukaryotes, and the recent discovery of the Asgard archaea and their 'eukaryotic signature proteins' are helping us to piece together how coats may have evolved during the prokaryote-to-eukaryote transition., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Surfactant miscible-displacement (SMD) column experiments are used to measure air-water interfacial area (A I ) in unsaturated porous media, a property that influences solute transport and phase-partitioning. The conventional SMD experiment results in surface tension gradients that can cause water redistribution and/or net drainage of water from the system ("surfactant-induced flow"), violating theoretical foundations of the method. Nevertheless, the SMD technique is still used, and some suggest that experimental observations of surfactant-induced flow represent an artifact of improper control of boundary conditions. In this work, we used numerical modeling, for which boundary conditions can be perfectly controlled, to evaluate this suggestion. We also examined the magnitude of surfactant-induced flow and its impact on A I measurement during multiple SMD flow scenarios. Simulations of the conventional SMD experiment showed substantial surfactant-induced flow and consequent drainage of water from the column (e.g., from 75% to 55% S W ) and increases in actual A I of up to 43%. Neither horizontal column orientation nor alternative boundary conditions resolved surfactant-induced flow issues. Even for simulated flow scenarios that avoided surfactant-induced drainage of the column, substantial surfactant-induced internal water redistribution occurred and was sufficient to alter surfactant transport, resulting in up to 23% overestimation of A I . Depending on the specific simulated flow scenario and data analysis assumptions used, estimated A I varied by nearly 40% and deviated up to 36% from the system's initial A I . We recommend methods for A I determination that avoid generation of surface-tension gradients and urge caution when relying on absolute A I values measured via SMD., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We present results on ultrafast gas electron diffraction (UGED) experiments with femtosecond resolution using the MeV electron gun at SLAC National Accelerator Laboratory. UGED is a promising method to investigate molecular dynamics in the gas phase because electron pulses can probe the structure with a high spatial resolution. Until recently, however, it was not possible for UGED to reach the relevant timescale for the motion of the nuclei during a molecular reaction. Using MeV electron pulses has allowed us to overcome the main challenges in reaching femtosecond resolution, namely delivering short electron pulses on a gas target, overcoming the effect of velocity mismatch between pump laser pulses and the probe electron pulses, and maintaining a low timing jitter. At electron kinetic energies above 3 MeV, the velocity mismatch between laser and electron pulses becomes negligible. The relativistic electrons are also less susceptible to temporal broadening due to the Coulomb force. One of the challenges of diffraction with relativistic electrons is that the small de Broglie wavelength results in very small diffraction angles. In this paper we describe the new setup and its characterization, including capturing static diffraction patterns of molecules in the gas phase, finding time-zero with sub-picosecond accuracy and first time-resolved diffraction experiments. The new device can achieve a temporal resolution of 100 fs root-mean-square, and sub-angstrom spatial resolution. The collimation of the beam is sufficient to measure the diffraction pattern, and the transverse coherence is on the order of 2 nm. Currently, the temporal resolution is limited both by the pulse duration of the electron pulse on target and by the timing jitter, while the spatial resolution is limited by the average electron beam current and the signal-to-noise ratio of the detection system. We also discuss plans for improving both the temporal resolution and the spatial resolution.

Background: To evaluate the effect of varying levels of power on phacoemulsification efficiency using the CENTURION Vision System., Methods: Formalin-soaked porcine lenses were divided into 2-mm cubes; 0.9-mm, balanced tips were used. Torsional power levels were tested from 10% to 100% at 10% intervals. Vacuum was set to 550 mmHg, aspiration to 50 ml/min, and intraocular pressure at 50 mmHg. Efficiency (time to lens removal) and chatter (number of lens fragment repulsions from the tip) were determined., Results: Increasing torsional power up to 60% increased efficiency. This effect was linear from 30 to 60% power (R 2  = .90; P < 0.05). There were no significant differences in efficiency past 60%. Chatter was highest at 10% power and decreased linearly (R 2  = .87; P = 0.007) as power was increased up to 60% power, and chatter did not improve above this power level., Conclusions: Power improved efficiency only up to a 60% power level, and then was negligible. Chatter correlated well with power up to the 60% level, so that as power was increased, chatter decreased. Because there are no additional benefits in efficiency past 60% power, and because chatter is minimal at 60% power, we recommend torsional ultrasound at 60% as the optimal power setting for using the CENTURION System for phacoemulsification.l., (© 2016 Royal Australian and New Zealand College of Ophthalmologists.)

Observing the motion of the nuclear wave packets during a molecular reaction, in both space and time, is crucial for understanding and controlling the outcome of photoinduced chemical reactions. We have imaged the motion of a vibrational wave packet in isolated iodine molecules using ultrafast electron diffraction with relativistic electrons. The time-varying interatomic distance was measured with a precision 0.07 Å and temporal resolution of 230 fs full width at half maximum. The method is not only sensitive to the position but also the shape of the nuclear wave packet.

Non-adiabatic multiconfigurational molecular dynamics simulations have revealed a molecular "Newton's Cradle" that activates on absorption of light in the mid-UV and assists the S 1 /S 0 internal conversion process in 1,2-dithiane, protecting the disulfide bond from photodamage. This communication challenges contemporary understanding of the S 1 /S 0 internal conversion process in 1,2-dithiane and presents a classically-intuitive reinterpretation of experimental evidence.

Exosomes are extracellular vesicles that are released when endosomes fuse with the plasma membrane. They have been implicated in various functions in both health and disease, including intercellular communication, antigen presentation, prion transmission, and tumour cell metastasis. Here we show that inactivating the vacuolar ATPase in HeLa cells causes a dramatic increase in the production of exosomes, which display endocytosed tracers, cholesterol, and CD63. The exosomes remain clustered on the cell surface, similar to retroviruses, which are attached to the plasma membrane by tetherin. To determine whether tetherin also attaches exosomes, we knocked it out and found a 4-fold reduction in plasma membrane-associated exosomes, with a concomitant increase in exosomes discharged into the medium. This phenotype could be rescued by wild-type tetherin but not tetherin lacking its GPI anchor. We propose that tetherin may play a key role in exosome fate, determining whether they participate in long-range or short-range interactions., Competing Interests: The authors declare that no competing interests exist.

Objective: The authors examined patients' acceptance of the Health Technology Program (HTP), an integrative approach to relapse prevention after hospitalization of adults with schizophrenia or related disorders. The program combines use of digital tools with support from a mental health technology coach (MHTC)., Methods: Patients with schizophrenia spectrum disorders received six months of treatment that began within 60 days of a psychiatric hospitalization and included the development of a personalized relapse prevention plan, three digital tools, and contacts with MHTCs., Results: A total of 200 patients (mean±SD age=34.6±10.6 years) had 28.2±2.0 contacts with the MHTC that lasted 38.3±14.2 minutes. The most discussed topic was case management (52%), and digital tools were discussed in 45% of meetings. Altogether, 87% of patients used at least one of the digital tools, with 96% of patients rating the HTP as satisfying to at least some extent., Conclusions: These data suggest very high acceptance of the HTP, a program that integrates available human support with digital tools.

Imaging changes in molecular geometries on their natural femtosecond timescale with sub-Angström spatial precision is one of the critical challenges in the chemical sciences, as the nuclear geometry changes determine the molecular reactivity. For photoexcited molecules, the nuclear dynamics determine the photoenergy conversion path and efficiency. Here we report a gas-phase electron diffraction experiment using megaelectronvolt (MeV) electrons, where we captured the rotational wavepacket dynamics of nonadiabatically laser-aligned nitrogen molecules. We achieved a combination of 100 fs root-mean-squared temporal resolution and sub-Angstrom (0.76 Å) spatial resolution that makes it possible to resolve the position of the nuclei within the molecule. In addition, the diffraction patterns reveal the angular distribution of the molecules, which changes from prolate (aligned) to oblate (anti-aligned) in 300 fs. Our results demonstrate a significant and promising step towards making atomically resolved movies of molecular reactions.

The adaptor protein 4 (AP4) complex (ϵ/β4/μ4/σ4 subunits) forms a non-clathrin coat on vesicles departing the trans-Golgi network. AP4 biology remains poorly understood, in stark contrast to the wealth of molecular data available for the related clathrin adaptors AP1 and AP2. AP4 is important for human health because mutations in any AP4 subunit cause severe neurological problems, including intellectual disability and progressive spastic para- or tetraplegias. We have used a range of structural, biochemical and biophysical approaches to determine the molecular basis for how the AP4 β4 C-terminal appendage domain interacts with tepsin, the only known AP4 accessory protein. We show that tepsin harbors a hydrophobic sequence, LFxG[M/L]x[L/V], in its unstructured C-terminus, which binds directly and specifically to the C-terminal β4 appendage domain. Using nuclear magnetic resonance chemical shift mapping, we define the binding site on the β4 appendage by identifying residues on the surface whose signals are perturbed upon titration with tepsin. Point mutations in either the tepsin LFxG[M/L]x[L/V] sequence or in its cognate binding site on β4 abolish in vitro binding. In cells, the same point mutations greatly reduce the amount of tepsin that interacts with AP4. However, they do not abolish the binding between tepsin and AP4 completely, suggesting the existence of additional interaction sites between AP4 and tepsin. These data provide one of the first detailed mechanistic glimpses at AP4 coat assembly and should provide an entry point for probing the role of AP4-coated vesicles in cell biology, and especially in neuronal function., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The purification of coated vesicles and the discovery of clathrin by Barbara Pearse in 1975 was a landmark in cell biology. Over the past 40 years, work from many labs has uncovered the molecular details of clathrin and its associated proteins, including how they assemble into a coated vesicle and how they select cargo. Unexpected connections have been found with signalling, development, neuronal transmission, infection, immunity and genetic disorders. But there are still a number of unanswered questions, including how clathrin-mediated trafficking is regulated and how the machinery evolved., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The HIV-1-encoded protein, Nef, plays a key role in the development of AIDS. One of Nef's functions is to keep MHC class I off the surface of infected cells, a process that requires the host proteins clathrin and AP-1. To identify other proteins involved in this pathway, we carried out a genome-wide siRNA library screen on HeLa cells co-expressing HLA-A2 and an inducible form of Nef. Out of 21,121 siRNA pools, 100 were selected for further analysis, based on their ability to either inhibit or enhance downregulation of MHC-I by Nef. When cells were treated with the same siRNA pools as those used in the screen, 79% produced a similar phenotype. However, when the cells were treated with different siRNA reagents targeting the same genes, only 16% produced a similar phenotype. This indicates that most of the hits found in the original screen are likely to have been off-target, an important concern that is often not taken into account in siRNA screening studies. Nevertheless, we identified novel host factors involved in Nef-induced downregulation of MHC-I, including four genes, MIIP, CAMSAP3, SLC6A3, and KCTD19, where multiple reagents produced a strong inhibitory effect on Nef activity. Other hits slightly below our very high stringency cutoff point may also deserve further study. Thus, our dataset is a valuable resource for scientists investigating the pathogenesis of HIV.

The precise functions of most of the proteins that participate in clathrin-mediated intracellular trafficking are unknown. We investigated two such proteins, epsinR and gadkin, using the knocksideways method, which rapidly depletes proteins from the available pool by trapping them onto mitochondria. Although epsinR is known to be an N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-specific adaptor, the epsinR knocksideways blocked the production of the entire population of intracellular clathrin-coated vesicles (CCVs), suggesting a more global function. Using the epsinR knocksideways data, we were able to estimate the copy number of all major intracellular CCV proteins. Both sides of the vesicle are densely covered, indicating that CCVs sort their cargo by molecular crowding. Trapping of gadkin onto mitochondria also blocked the production of intracellular CCVs but by a different mechanism: vesicles became cross-linked to mitochondria and pulled out toward the cell periphery. Both phenotypes provide new insights into the regulation of intracellular CCV formation, which could not have been found using more conventional approaches., (© 2015 Hirst et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)