Context-specific regulation of extracellular vesicle biogenesis and cargo selection.

To coordinate, adapt and respond to biological signals, cells convey specific messages to other cells. An important aspect of cell-cell communication involves secretion of molecules into the extracellular space. How these molecules are selected for secretion has been a fundamental question in the membrane trafficking field for decades. Recently, extracellular vesicles (EVs) have been recognized as key players in intercellular communication, carrying not only membrane proteins and lipids but also RNAs, cytosolic proteins and other signalling molecules to recipient cells. To communicate the right message, it is essential to sort cargoes into EVs in a regulated and context-specific manner. In recent years, a wealth of lipidomic, proteomic and RNA sequencing studies have revealed that EV cargo composition differs depending upon the donor cell type, metabolic cues and disease states. Analyses of distinct cargo 'fingerprints' have uncovered mechanistic linkages between the activation of specific molecular pathways and cargo sorting. In addition, cell biology studies are beginning to reveal novel biogenesis mechanisms regulated by cellular context. Here, we review context-specific mechanisms of EV biogenesis and cargo sorting, focusing on how cell signalling and cell state influence which cellular components are ultimately targeted to EVs.

Trade-offs between succulent and non-succulent epiphytes underlie variation in drought tolerance and avoidance.

Epiphyte communities comprise important components of many forest ecosystems in terms of biomass and diversity, but little is known regarding trade-offs that underlie diversity and structure in these communities or the impact that microclimate has on epiphyte trait allocation. We measured 22 functional traits in vascular epiphyte communities across six sites that span a microclimatic gradient in a tropical montane cloud forest region in Costa Rica. We quantified traits that relate to carbon and nitrogen allocation, gas exchange, water storage, and drought tolerance. Functional diversity was high in all but the lowest elevation site where drought likely limits the success of certain species with particular trait combinations. For most traits, variation was explained by relationships with other traits, rather than differences in microclimate across sites. Although there were significant differences in microclimate, epiphyte abundance, and diversity, we found substantial overlap in multivariate trait space across five of the sites. We found significant correlations between functional traits, many of which related to water storage (leaf water content, leaf thickness, hydrenchymal thickness), drought tolerance (turgor loss point), and carbon allocation (specific leaf area, leaf dry matter content). This suite of trait correlations suggests that the epiphyte community has evolved functional strategies along with a drought avoidance versus drought tolerance continuum where leaf succulence emerged as a pivotal overall trait.

Generating accurate 3D gaze vectors using synchronized eye tracking and motion capture.

Assessing gaze behavior during real-world tasks is difficult; dynamic bodies moving through dynamic worlds make gaze analysis difficult. Current approaches involve laborious coding of pupil positions. In settings where motion capture and mobile eye tracking are used concurrently in naturalistic tasks, it is critical that data collection be simple, efficient, and systematic. One solution is to combine eye tracking with motion capture to generate 3D gaze vectors. When combined with tracked or known object locations, 3D gaze vector generation can be automated. Here we use combined eye and motion capture and explore how linear regression models generate accurate 3D gaze vectors. We compare spatial accuracy of models derived from four short calibration routines across three pupil data inputs: the efficacy of calibration routines was assessed, a validation task requiring short fixations on task-relevant locations, and a naturalistic object interaction task to bridge the gap between laboratory and "in the wild" studies. Further, we generated and compared models using spherical and Cartesian coordinate systems and monocular (left or right) or binocular data. All calibration routines performed similarly, with the best performance (i.e., sub-centimeter errors) coming from the naturalistic task trials when the participant is looking at an object in front of them. We found that spherical coordinate systems generate the most accurate gaze vectors with no differences in accuracy when using monocular or binocular data. Overall, we recommend 1-min calibration routines using binocular pupil data combined with a spherical world coordinate system to produce the highest-quality gaze vectors.

MAPK- and glycogen synthase kinase 3-mediated phosphorylation regulates the DEAD-box protein modulator Gle1 for control of stress granule dynamics.

Rapid expression of critical stress response factors is a key survival strategy for diseased or stressed cells. During cell stress, translation is inhibited, and a pre-existing pool of cytoplasmic mRNA-protein complexes reversibly assembles into cytoplasmic stress granules (SGs). Gle1 is a conserved modulator of RNA-dependent DEAD-box proteins required for mRNA export, translation, and stress responses. Proper Gle1 function is critical as reflected by some human diseases such as developmental and neurodegenerative disorders and some cancers linked to gle1 mutations. However, the mechanism by which Gle1 controls SG formation is incompletely understood. Here, we show that human Gle1 is regulated by phosphorylation during heat shock stress. In HeLa cells, stress-induced Gle1 hyperphosphorylation was dynamic, primarily in the cytoplasmic pool, and followed changes in translation factors. MS analysis identified 14 phosphorylation sites in the Gle1A isoform, six of which clustered in an intrinsically disordered, low-complexity N-terminal region flanking the coil-coiled self-association domain. Of note, two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), phosphorylated the Gle1A N-terminal domain, priming it for phosphorylation by glycogen synthase kinase 3 (GSK3). A phosphomimetic gle1A6D variant (in which six putative Ser/Thr phosphorylation sites were substituted with Asp) perturbed self-association and inhibited DEAD-box helicase 3 (X-linked) (DDX3) ATPase activity. Expression of alanine-substituted, phosphodeficient GFP-gle1A6A promoted SG assembly, whereas GFP-gle1A6D enhanced SG disassembly. We propose that MAPKs and GSK3 phosphorylate Gle1A and thereby coordinate SG dynamics by altering DDX3 function.

Eye safety implications of high harmonic generation in zinc selenide.

Polycrystalline zinc selenide (ZnSe) has been the subject of many nonlinear optics studies for wavelengths under 4.0 µm including sum/difference frequency generation, harmonic generation, and filamentation. In this report, the conversion efficiency of high harmonic generation (HHG) in ZnSe is quantified for mid-infrared wavelengths ranging from 2.7 µm to 8.0 µm. By increasing the fundamental wavelength, we demonstrate that HHG in thick ZnSe targets is limited by the band gap. The high conversion efficiency of mid-infrared to near-infrared light in ZnSe raises concerns of a nonlinear retinal hazard. We contrast the HHG behavior of ZnSe against the observed harmonic generation of calcium fluoride, BK7, and fused silica over the same wavelengths.

The role of the skin microbiota in acne pathophysiology.

BACKGROUND: The role of skin microbiota in acne remains to be fully elucidated. Initial culture-based investigations were hampered by growth rate and selective media bias. Even with less biased genomic methods, sampling, lysis and methodology, the task of describing acne pathophysiology remains challenging. Acne occurs in sites dominated by Cutibacterium acnes (formerly Propionibacterium acnes) and Malassezia species, both of which can function either as commensal or pathogen. OBJECTIVES: This article aims to review the current state of the art of the microbiome and acne. METHODS: The literature regarding the microbiome and acne was reviewed. RESULTS: It remains unclear whether there is a quantitative difference in microbial community distribution, making it challenging to understand any community shift from commensal to pathogenic nature. It is plausible that acne involves (i) change in the distribution of species/strains, (ii) stable distribution with pathogenic alteration in response to internal (intermicrobe) or external stimuli (host physiology or environmental) or (iii) a combination of these factors. CONCLUSIONS: Understanding physiological changes in bacterial species and strains will be required to define their specific roles, and identify any potential intervention points, in acne pathogenesis and treatment. It will also be necessary to determine whether any fungal species are involved, and establish whether they play a significant role. Further investigation using robust, modern analytic tools in longitudinal studies with a large number of participants, may make it possible to determine whether the microbiota plays a causal role, is primarily involved in exacerbation, or is merely a bystander. It is likely that the final outcome will show that acne is the result of complex microbe-microbe and community-host interplay.

British Neuropathological Society and International Society of Forensic Radiology and Imaging expert consensus statement for post mortem neurological imaging.

AIMS: To develop an expert consensus statement regarding appropriate clinical and forensic post mortem neurological imaging. METHODS: An expert panel of clinicians were recruited from registered members of the British Neuropathological Society (BNS) and the International Society of Forensic Radiology and Imaging (ISFRI) with post mortem expertise. Following a focus group meeting, 16 core statements were incorporated into an online modified Delphi survey and each panellist was asked to score their level of agreement. Following the first iteration, two statements that failed to reach consensus were modified and re-rated. Consensus was predefined as 75% agreement across responders. RESULTS: Seventeen experts joined the panel and 12 (70.6%) attended the focus group meeting; 14 (82%) completed both iterations of the survey. Consensus was reached for need of adequate clinical history, multidisciplinary discussion, establishment of special interest groups to discuss cases, gathering further evidence to inform imaging choices, establishment of methods for quality assessment in reporting standards and adequate funding for imaging services. The panel agreed that pathologists should be responsible for neuroimaging referrals, collating results of ancillary tests, and producing the final post mortem report. Areas requiring further discussion include the impact of double reporting, indications for neuroimaging and utilities of three-dimensional printing. CONCLUSION: The BNS/ISFRI statement represents current views of an expert panel of health professionals engaged in post-mortem neuroimaging. We hope this provides a working guideline for less experienced operators, stimulates discussion and highlights the most pressing clinical and research questions.

Z-scan measurements of water from 1150 to 1400 nm.

Understanding the nonlinear properties of water is essential for laser surgery applications, as well as understanding supercontinuum generation in water. Unfortunately, the nonlinear properties of water for wavelengths longer than 1064 nm are poorly understood. We extend the application of the Z-scan technique in water to determine its nonlinear refractive index (n2) and nonlinear absorption (ß) for wavelengths in the 1150-1400 nm range, where linear absorption is also significant. We observe the wavelength-dependent variation of the nonlinear properties of water around the water absorption band.

Raman spectroscopy compatible PDMS droplet microfluidic culture and analysis platform towards on-chip lipidomics.

Lipids produced by microalgae are viewed as a potential renewable alternative to fossil fuels, however, significant improvements in productivity are required for microalgal biofuels to become economically feasible. Here we present a method that allows for the use of Raman spectroscopy with poly(dimethylsiloxane) (PDMS) droplet microfluidic devices, which not only overcomes the high Raman background of PDMS, but also achieves pairing of the high-throughput single-cell resolution advantages of droplet microfluidics with the direct, chemically specific, label-free, and non-destructive nature of Raman spectroscopy. The platform was successfully utilized for in situ characterization of microalgal lipid production over time within droplets, paving the way towards high-throughput microalgal lipidomics assays.

Reversible morphological changes of assembled supramolecular amphiphiles triggered by pH-modulated host-guest interactions.

Reversible template-directed micellar-size and shape modulation by virtue of host-guest reversible docking of molecular templates at the micellar-solvent interface was achieved in water. By combining a π-electron deficient bipyridinium-based gemini amphiphile which is capable of binding and aligning with a π-electron rich tri(ethylene glycol)-disubstituted 1,5-diaminonaphthalene, a switchable detergent system which operates through the pH-responsive formation of bisammonium dications was realised. The binding of the 1,5-diaminonaphthalene guest to the bipyridinium-based micellar aggregate superstructure can be actuated by the addition of acid and base. Upon the addition of acid, protonation of the guest forming the dication deactivates molecular recognition with the charged head groups of the micellar aggregate by Coulombic repulsion. This process is completely reversible upon the addition of base, whereby the guest reintercalates into the superstructure -again forming donor-acceptor π-π stacks at the micellar-solvent interface amongst contiguous surfactant head groups. Synchrotron small angle X-ray scattering and dynamic laser light scattering confirm that this form of reversible directionally-templated micellisation results in an oblate spheroid-to-lamellar micelle morphological transition with a stabilising net decrease in the free energy of micellisation of 1.4 kcal mol(-1) per hydrophobic tail.

Patient Values and Preferences for Managing Acute Dental Pain Elicited through Online Deliberation.

INTRODUCTION: Patient values and preferences (PVP) are among multiple sources of information panelists synthesize when developing clinical practice guidelines (CPG). Patient and public involvement (PPI) can be critical for learning PVP; however, the methodology for engaging patients in CPG development is lacking. Deliberative engagement is effective for obtaining public views on complex topics that require people to consider ethics, values, and competing perspectives. OBJECTIVE: Elicit comprehensive understanding of PVP concerning oral analgesics for managing acute dental pain consecutive to toothache and simple and surgical dental extractions, with consideration of associated outcomes, both desirable and undesirable. METHODS: Multistage engagement involving 2 electronic surveys and a 90-min online small group deliberative engagement. Adults who have experienced acute dental pain deliberated about 3 hypothetical scenarios stratified according to expected pain intensity, completed a postdeliberation survey, and validated a PVP statement developed by researchers based on review of qualitative data from deliberations and quantitative data from surveys. RESULTS: Participants affirmed the PVP statement reflected their small group deliberations and their individual views. Most indicated that pain relief is critical to deciding which pain relief medicine they would want regardless of expected pain level. Most also identify as critical concerns about substance abuse or misuse, although many believe it unlikely that they will experience these outcomes over the brief prescription timeframe for acute dental pain. Participants identified agency in decision-making, consultation including "better communication" of options, and treatment actions tailored to life circumstances as key values. CONCLUSIONS: Participants preferred nonprescription and nonopioid pain relief options. As expected pain levels increased, more participants expressed willingness to accept opioids, but more also mentioned rescue analgesia as a third outcome critical to decision-making. Online deliberative method provided opportunities for obtaining informed perspectives. Guideline developers and policymakers may find online deliberations useful for eliciting PVP related to health outcomes. KNOWLEDGE TRANSFER STATEMENT: Study results informed the US Food and Drug Administration-funded clinical practice guideline on the management of acute dental pain. Findings may be a resource for clinicians in decision-making conversations with patients regarding expectations for pain relief and positive and negative outcomes of differing pain relief medications. Further research should pursue applicability of online deliberative engagement as a method to elicit patient values and preferences.

Myoclonus-dystonia and Silver-Russell syndrome resulting from maternal uniparental disomy of chromosome 7.

Myoclonus-dystonia (M-D) is a movement disorder that is often associated with mutations in epsilon-sarcoglycan (SGCE), a maternally imprinted gene at 7q21.3. We report a 24-year-old male with short stature (<5th percentile) and a movement disorder clinically consistent with M-D. Single nucleotide polymorphism (SNP) array did not identify significant copy number changes, but revealed three long continuous stretches of homozygosity on chromosome 7 suggestive of uniparental disomy. Parental SNP arrays confirmed that the proband had maternal uniparental disomy of chromosome 7 (mUPD7) with regions of heterodisomy and isodisomy. mUPD7 is the cause of approximately 5-10% of Silver-Russell syndrome (SRS), a disorder characterized by prenatal and postnatal growth retardation. Although SRS was not suspected in our patient, these findings explain his short stature. SGCE methylation testing showed loss of the unmethylated paternal allele. Our findings provide a unifying diagnosis for his short stature and M-D and help to optimize his medication regimen. In conclusion, we show that M-D is a clinical feature that may be associated with SRS due to mUPD7. Individuals with mUPD7 should be monitored for the development of movement disorders. Conversely, individuals with M-D and short stature should be evaluated for SRS.

Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions.

Parkinson disease (PD) is a neurodegenerative disease characterized by tremor, bradykinesia, rigidity and postural instability. Post-mortem examination shows loss of neurons and Lewy bodies, which are cytoplasmic eosinophilic inclusions, in the substantia nigra and other brain regions. A few families have PD caused by mutations (A53T or A30P) in the gene SNCA (encoding alpha-synuclein). Alpha-synuclein is present in Lewy bodies of patients with sporadic PD, suggesting that alpha-synuclein may be involved in the pathogenesis of PD. It is unknown how alpha-synuclein contributes to the cellular and biochemical mechanisms of PD, and its normal functions and biochemical properties are poorly understood. To determine the protein-interaction partners of alpha-synuclein, we performed a yeast two-hybrid screen. We identified a novel interacting protein, which we term synphilin-1 (encoded by the gene SNCAIP). We found that alpha-synuclein interacts in vivo with synphilin-1 in neurons. Co-transfection of both proteins (but not control proteins) in HEK 293 cells yields cytoplasmic eosinophilic inclusions.

Phosphorylation impacts GLE1 nuclear localization and association with DDX1.

Gle1 regulates gene expression at multiple steps from transcription to mRNA export to translation under stressed and non-stressed conditions. To better understand Gle1 function in stressed human cells, specific antibodies were generated that recognized the phosphorylation of threonine residue 102 (T102) in Gle1. A series of in vitro kinase assays indicated that T102 phosphorylation serves as a priming event for further phosphorylation in Gle1's N-terminal low complexity cluster. Indirect immunofluorescence microscopy with the anti-Gle1-pT102 antibodies revealed that basally phosphorylated Gle1 was pre-dominantly nuclear with punctate distribution; however, under sodium arsenite-induced stress, more cytoplasmic localization was detected. Immunoprecipitation with the anti-Gle1-pT102 antibody resulted in co-isolation of Gle1-pT102 with the DEAD-box protein DDX1 in a phosphatase sensitive manner. This suggested Gle1 phosphorylation might be linked to its role in regulating DDX1 during transcription termination. Notably, whereas the total Gle1-DDX1 association was decreased when Gle1 nucleocytoplasmic shuttling was disrupted, co-isolation of Gle1-pT102 and DDX1 increased under the same conditions. Taken together, these studies demonstrated that Gle1 phosphorylation impacts its cellular distribution and potentially drives nuclear Gle1 functions in transcription termination. We propose a model wherein phosphorylation of Gle1 either reduces its nucleocytoplasmic shuttling capacity or increases its binding affinity with nuclear interaction partners.

Analysis of small EV proteomes reveals unique functional protein networks regulated by VAP-A.

Extracellular vesicles (EVs) influence cell phenotypes and functions via protein, nucleic acid and lipid cargoes. EVs are heterogeneous, due to diverse biogenesis mechanisms that remain poorly understood. Our previous study revealed that the endoplasmic reticulum (ER) membrane contact site (MCS) linker protein VAP-A drives biogenesis of a subset of RNA-enriched EVs. Here, we examine the protein content of VAP-A-regulated EVs. Using label-free proteomics, we identified down- and up-regulated proteins in sEVs purified from VAP-A knockdown (KD) colon cancer cells. Gene set enrichment analysis (GSEA) of the data revealed protein classes that are differentially sorted to SEVs dependent on VAP-A. STRING protein-protein interaction network analysis of the RNA-binding protein (RBP) gene set identified several RNA functional machineries that are downregulated in VAP-A KD EVs, including ribosome, spliceosome, mRNA surveillance, and RNA transport proteins. We also observed downregulation of other functionally interacting protein networks, including cadherin-binding, unfolded protein binding, and ATP-dependent proteins.

Systematic Assessment of Small RNA Profiling in Human Extracellular Vesicles.

MOTIVATION: Extracellular vesicles (EVs) are produced and released by most cells and are now recognized to play a role in intercellular communication through the delivery of molecular cargo, including proteins, lipids, and RNA. Small RNA sequencing (small RNA-seq) has been widely used to characterize the small RNA content in EVs. However, there is a lack of a systematic assessment of the quality, technical biases, RNA composition, and RNA biotypes enrichment for small RNA profiling of EVs across cell types, biofluids, and conditions. METHODS: We collected and reanalyzed small RNA-seq datasets for 2756 samples from 83 studies involving 55 with EVs only and 28 with both EVs and matched donor cells. We assessed their quality by the total number of reads after adapter trimming, the overall alignment rate to the host and non-host genomes, and the proportional abundance of total small RNA and specific biotypes, such as miRNA, tRNA, rRNA, and Y RNA. RESULTS: We found that EV extraction methods varied in their reproducibility in isolating small RNAs, with effects on small RNA composition. Comparing proportional abundances of RNA biotypes between EVs and matched donor cells, we discovered that rRNA and tRNA fragments were relatively enriched, but miRNAs and snoRNA were depleted in EVs. Except for the export of eight miRNAs being context-independent, the selective release of most miRNAs into EVs was study-specific. CONCLUSION: This work guides quality control and the selection of EV isolation methods and enhances the interpretation of small RNA contents and preferential loading in EVs.

Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations.

Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination.

Diverse functional responses to drought in a Mediterranean-type shrubland in South Africa.

• Mediterranean-type ecosystems contain 20% of all vascular plant diversity on Earth and have been identified as being particularly threatened by future increases in drought. Of particular concern is the Cape Floral Region of South Africa, a global biodiversity hotspot, yet there are limited experimental data to validate predicted impacts on the flora. In a field rainout experiment, we tested whether rooting depth and degree of isohydry or anisohydry could aid in the functional classification of drought responses across diverse growth forms. • We imposed a 6-month summer drought, for 2 yr, in a mountain fynbos shrubland. We monitored a suite of parameters, from physiological traits to morphological outcomes, in seven species comprising the three dominant growth forms (deep-rooted proteoid shrubs, shallow-rooted ericoid shrubs and graminoid restioids). • There was considerable variation in drought response both between and within the growth forms. The shallow-rooted, anisohydric ericoid shrubs all suffered considerable reductions in growth and flowering and increased mortality. By contrast, the shallow-rooted, isohydric restioids and deep-rooted, isohydric proteoid shrubs were largely unaffected by the drought. • Rooting depth and degree of iso/anisohydry allow a first-order functional classification of drought response pathways in this flora. Consideration of additional traits would further refine this approach.

What women want - quantifying the perception of hair amount: an analysis of hair diameter and density changes with age in caucasian women.

BACKGROUND: It has long been known that women lose satisfaction with their hair with ageing. Our data show that caucasian women perceive a decrease in hair amount in their mid 40s with a further decrease in the mid to late 50s, which leads to this dissatisfaction. Neither loss of density (hairs per cm(2) ) nor shaft diameter alone can fully account for this perception. A new metric, 'hair amount', is proposed as a quantitative metric combining the impact of both density and diameter on the perception of hair loss. OBJECTIVES: Creation of a single parameter combining the contribution of diameter and density to perception of female age-related hair loss. METHODS: In total, 1099 caucasian women (ages 18-66 years) with self-perceived hair loss and 315 caucasian women (ages 17-86 years) with no complaint of hair loss were evaluated. Scalp hair diameter was measured using optical fibre diameter and image analysis. Scalp hair density was measured by phototrichogram with manual or automated counting. RESULTS: Parietal scalp hair diameter increased from ages 20 to 40-45 years, then decreased. Hair density was highest in the youngest group, age 20-30 years, and decreased thereafter with increasing rate. In women self-perceiving hair loss, the rate of decrease in density was significantly faster than for women with no self-perception of hair loss. The combined metric 'hair amount' was relatively constant at younger ages, increasing very slightly to age 35 years, then decreasing significantly. CONCLUSIONS: Increasing hair shaft diameter offsets decreasing hair density through the mid 30s. After that, a lower rate of diameter increase combined with the decrease in density begins to significantly impact the perception of hair amount so that thinning becomes increasingly more noticeable in the mid 40s to the mid to late 50s. Quantitative determination of hair amount is a useful tool to combine the contributions of hair density and diameter to women's perception of age-related hair loss.