Friends or Foes? Emerging Impacts of Biological Toxins.

Toxins are substances produced from biological sources (e.g., animal, plants, microorganisms) that have deleterious effects on a living organism. Despite the obvious health concerns of being exposed to toxins, they are having substantial positive impacts in a number of industrial sectors. Several toxin-derived products are approved for clinical, veterinary, or agrochemical uses. This review sets out the case for toxins as 'friends' that are providing the basis of novel medicines, insecticides, and even nucleic acid sequencing technologies. We also discuss emerging toxins ('foes') that are becoming increasingly prevalent in a range of contexts through climate change and the globalisation of food supply chains and that ultimately pose a risk to health.

Development of Bifunctional, Raman Active Diyne-Girder Stapled α-Helical Peptides.

Stapled peptides are a unique class of cyclic α-helical peptides that are conformationally constrained via their amino acid side-chains. They have been transformative to the field of chemical biology and peptide drug discovery through addressing many of the physicochemical limitations of linear peptides. However, there are several issues with current chemical strategies to produce stapled peptides. For example, two distinct unnatural amino acids are required to synthesize i, i+7 alkene stapled peptides, leading to high production costs. Furthermore, low purified yields are obtained due to cis/trans isomers produced during ring-closing metathesis macrocyclisation. Here we report the development of a new i, i+7 diyne-girder stapling strategy that addresses these issues. The asymmetric synthesis of nine unnatural Fmoc-protected alkyne-amino acids facilitated a systematic study to determine the optimal (S,S)-stereochemistry and 14-carbon diyne-girder bridge length. Diyne-girder stapled T-STAR peptide 29 was demonstrated to have excellent helicity, cell permeability and stability to protease degradation. Finally, we demonstrate that the diyne-girder constraint is a Raman chromophore with potential use in Raman cell microscopy. Development of this highly effective, bifunctional diyne-girder stapling strategy leads us to believe that it can be used to produce other stapled peptide probes and therapeutics.

A Crowdsourced Evaluation of Facial Averageness and Attractiveness.

BACKGROUND: Evolutionary psychologists have demonstrated that humans are attracted to individuals who possess average anatomy for the population. OBJECTIVES: The aim of this study was to prove that a composite of average facial features would be more attractive to raters than the cohort utilized to create the composite. METHODS: The male and female cohorts each consisted of 41 standardized frontal-view monochrome photographs, with 1 composite image derived from the other 40 real images. Amazon Mechanical Turk, a widely used crowdsourcing platform, was utilized to obtain ratings of images ranging from 1 to 7, with 1 and 7 being least and most attractive, respectively. The strength of the preference for the composite over the real images was assessed by the difference between the mean rating of the composite and real images. RESULTS: In total, 870 and 876 respondents were recruited to rate the male and female cohorts, respectively. For the male and female cohorts, the composite image was rated significantly higher than the rest of the cohort overall and across all ages, genders, and countries of residence (all P < 0.0001). For both cohorts, the strength of the preference was significantly higher for European respondents and lower for South American and nonbinary respondents (all P < 0.05). CONCLUSIONS: This study reveals that average facial anatomy is perceived as most attractive across all demographics, a finding that is hoped to serve as a stepping stone for further studies leading to objective cosmetic quantifications and integrating evidence-based medicine into aesthetic surgery.

Robust and automated detection of subcellular morphological motifs in 3D microscopy images.

Rapid developments in live-cell three-dimensional (3D) microscopy enable imaging of cell morphology and signaling with unprecedented detail. However, tools to systematically measure and visualize the intricate relationships between intracellular signaling, cytoskeletal organization and downstream cell morphological outputs do not exist. Here, we introduce u-shape3D, a computer graphics and machine-learning pipeline to probe molecular mechanisms underlying 3D cell morphogenesis and to test the intriguing possibility that morphogenesis itself affects intracellular signaling. We demonstrate a generic morphological motif detector that automatically finds lamellipodia, filopodia, blebs and other motifs. Combining motif detection with molecular localization, we measure the differential association of PIP2 and KrasV12 with blebs. Both signals associate with bleb edges, as expected for membrane-localized proteins, but only PIP2 is enhanced on blebs. This indicates that subcellular signaling processes are differentially modulated by local morphological motifs. Overall, our computational workflow enables the objective, 3D analysis of the coupling of cell shape and signaling.

Heterozygous Mutation of Vegfr3 Reduces Renal Lymphatics without Renal Dysfunction.

BACKGROUND: Lymphatic abnormalities are observed in several types of kidney disease, but the relationship between the renal lymphatic system and renal function is unclear. The discovery of lymphatic-specific proteins, advances in microscopy, and available genetic mouse models provide the tools to help elucidate the role of renal lymphatics in physiology and disease. METHODS: We utilized a mouse model containing a missense mutation in Vegfr3 (dubbed Chy ) that abrogates its kinase ability. Vegfr3 Chy/+ mice were examined for developmental abnormalities and kidney-specific outcomes. Control and Vegfr3 Chy/+ mice were subjected to cisplatin-mediated injury. We characterized renal lymphatics using tissue-clearing, light-sheet microscopy, and computational analyses. RESULTS: In the kidney, VEGFR3 is expressed not only in lymphatic vessels but also, in various blood capillaries. Vegfr3 Chy/+ mice had severely reduced renal lymphatics with 100% penetrance, but we found no abnormalities in BP, serum creatinine, BUN, albuminuria, and histology. There was no difference in the degree of renal injury after low-dose cisplatin (5 mg/kg), although Vegfr3 Chy/+ mice developed perivascular inflammation. Cisplatin-treated controls had no difference in total cortical lymphatic volume and length but showed increased lymphatic density due to decreased cortical volume. CONCLUSIONS: We demonstrate that VEGFR3 is required for development of renal lymphatics. Our studies reveal that reduced lymphatic density does not impair renal function at baseline and induces only modest histologic changes after mild injury. We introduce a novel quantification method to evaluate renal lymphatics in 3D and demonstrate that accurate measurement of lymphatic density in CKD requires assessment of changes to cortical volume.

A Silver Lining? Fewer Non-Severe Acute Respiratory Syndrome Coronavirus 2 Respiratory Viruses During the Coronavirus Disease 2019 Pandemic.

Nonpharmaceutical interventions (NPIs) have "flattened the curve" of the coronavirus disease 2019 pandemic; however the effect of these interventions on other respiratory viruses is unknown. We used aggregate level case count data for 8 respiratory viruses and compared the institutional and statewide case counts before and during the period that NPIs were active. We observed a 61% decrease (incidence rate ratio, 0.39; 95% confidence interval, .37-.41; P < .001) in non-severe acute respiratory syndrome coronavirus 2 respiratory viral infections when NPIs were implemented. This finding, if further verified, should guide future public health initiatives to mitigate viral epidemics.

Effectiveness of Telehealth in Rural and Remote Emergency Departments: Systematic Review.

BACKGROUND: Emergency telehealth has been used to improve access of patients residing in rural and remote areas to specialist care in the hope of mitigating the significant health disparities that they experience. Patient disposition decisions in rural and remote emergency departments (EDs) can be complex and largely dependent on the expertise and experience available at local (receiving-end) hospitals. Although there has been some synthesis of evidence of the effectiveness of emergency telehealth in clinical practice in rural and remote EDs for nonacute presentations, there has been limited evaluation of the influence of contextual factors such as clinical area and acuity of presentation on these findings. OBJECTIVE: The aims of this systematic review are to examine the outcome measures used in studying the effectiveness of telehealth in rural and remote EDs and to analyze the clinical context in which these outcome measures were used and interpreted. METHODS: The search strategy used Medical Subject Headings and equivalent lists of subject descriptors to find articles covering 4 key domains: telehealth or telemedicine, EDs, effectiveness, and rural and remote. Studies were selected using the Population, Intervention, Comparator, Outcomes of Interest, and Study Design framework. This search strategy was applied to MEDLINE (Ovid), Cochrane Library, Scopus, CINAHL, ProQuest, and EconLit, as well as the Centre for Reviews and Dissemination databases (eg, National Health Service Economic Evaluation Database) for the search period from January 1, 1990, to May 23, 2020. Qualitative synthesis was performed on the outcome measures used in the included studies, in particular the clinical contexts within which they were interpreted. RESULTS: A total of 21 full-text articles were included for qualitative analysis. Telehealth use in rural and remote EDs demonstrated effectiveness in achieving improved or equivalent clinical effectiveness, appropriate care processes, and-depending on the context-improvement in speed of care, as well as favorable service use patterns. The definition of effectiveness varied across the clinical areas and contexts of the studies, and different measures have been used to affirm the safety and clinical effectiveness of telehealth in rural and remote EDs. The acuity of patient presentation emerged as a dominant consideration in the interpretation of interlinking time-sensitive clinical effectiveness and patient disposition measures such as transfer and discharge rates, local hospital admission, length of stay, and ED length of stay. These, together with clinical area and acuity of presentation, are the outcome determination criteria that emerged from this review. CONCLUSIONS: Emergency telehealth studies typically use multiple outcome measures to determine the effectiveness of the services. The outcome determination criteria that emerged from this analysis are useful when defining the favorable direction for each outcome measure of interest. The findings of this review have implications for emergency telehealth service design and policies. TRIAL REGISTRATION: PROSPERO CRD42019145903; https://tinyurl.com/ndmkr8ry.

Synthesis and Fluorescent Properties of ß-Pyridyl α-Amino Acids.

The preparation of a new class of ß-pyridyl α-amino acid is described using a highly regioselective, ytterbium-catalyzed hetero-Diels-Alder reaction of enones with vinyl ethers followed by a modified Knoevenagel-Stobbe reaction as the key heterocycle forming steps. Investigation of the properties and applications of these amino acids showed that they could be utilized in solid phase peptide synthesis for the preparation of a biologically relevant hexapeptide, while pyridines bearing electron-rich substituents exhibited strongly fluorescent properties with high quantum yields and MegaStokes shifts. A solvatochromic study with the most fluorogenic amino acid, a p-methoxyphenyl analogue, revealed that this charge-transfer based chromophore is highly sensitive to solvent polarity with a bathochromic shift of 115 nm on changing from THF to phosphate-buffered saline.

A heme-binding domain controls regulation of ATP-dependent potassium channels.

Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels.

Urotensin-II peptidomimetic incorporating a non-reducible 1,5-triazole disulfide bond reveals a pseudo-irreversible covalent binding mechanism to the urotensin G-protein coupled receptor.

The urotensin-II receptor (UTR) is a class A GPCR that predominantly binds to the pleiotropic cyclic peptide urotensin-II (U-II). U-II is constrained by a disulfide bridge that induces a ß-turn structure and binds pseudo-irreversibly to UTR and is believed to result in a structural rearrangement of the receptor. However, it is not well understood how U-II binds pseudo-irreversibly and the nature of the reorganization of the receptor that results in G-protein activation. Here we describe a series of U-II peptidomimetics incorporating a non-reducible disulfide bond structural surrogate to investigate the feasibility that native U-II binds to the G protein-coupled receptor through disulfide bond shuffling as a mechanism of covalent interaction. Disubstituted 1,2,3-triazoles were designed with the aid of computational modeling as a non-reducible mimic of the disulfide bridge (Cys5-Cys10) in U-II. Solid phase synthesis using CuAAC or RuAAC as the key macrocyclisation step provided four analogues of U-II(4-11) incorporating either a 1,5-triazole bridge (5, 6) or 1,4-triazole bridge (9, 10). Biological evaluation of compounds 5, 6, 9 and 10 was achieved using in vitro [125I]UII binding and [Ca2+]i assays at recombinant human UTR. Compounds 5 and 6 demonstrated high affinity (KD ∼ 10 nM) for the UTR and were also shown to bind reversibly as predicted and activate the UTR to increase [Ca2+]i. Importantly, our results provide new insight into the mechanism of covalent binding of U-II with the UTR.

Insights into the Recruitment of Class IIa Histone Deacetylases (HDACs) to the SMRT/NCoR Transcriptional Repression Complex.

Class IIa histone deacetylases repress transcription of target genes. However, their mechanism of action is poorly understood because they exhibit very low levels of deacetylase activity. The class IIa HDACs are associated with the SMRT/NCoR repression complexes and this may, at least in part, account for their repressive activity. However, the molecular mechanism of recruitment to co-repressor proteins has yet to be established. Here we show that a repeated peptide motif present in both SMRT and NCoR is sufficient to mediate specific interaction, with micromolar affinity, with all the class IIa HDACs (HDACs 4, 5, 7, and 9). Mutations in the consensus motif abrogate binding. Mutational analysis of HDAC4 suggests that the peptide interacts in the vicinity of the active site of the enzyme and requires the "closed" conformation of the zinc-binding loop on the surface of the enzyme. Together these findings represent the first insights into the molecular mechanism of recruitment of class IIa HDACs to the SMRT/NCoR repression complexes.

Genomic ancestry estimation quantifies use of wild species in grape breeding.

BACKGROUND: Grapes are one of the world's most valuable crops and most are made into wine. Grapes belong to the genus Vitis, which includes over 60 inter-fertile species. The most common grape cultivars derive their entire ancestry from the species Vitis vinifera, but wild relatives have also been exploited to create hybrid cultivars, often with increased disease resistance. RESULTS: We evaluate the genetic ancestry of some of the most widely grown commercial hybrids from North America and Europe. Using genotyping-by-sequencing (GBS), we generated 2482 SNPs and 56 indels from 7 wild Vitis, 7 V. vinifera, and 64 hybrid cultivars. We used a principal component analysis (PCA) based ancestry estimation procedure and verified its accuracy with both empirical and simulated data. V. vinifera ancestry ranged from 11 % to 76 % across hybrids studied. Approximately one third (22/64) of the hybrids have ancestry estimates consistent with F1 hybridization: they derive half of their ancestry from wild Vitis and half from V. vinifera. CONCLUSIONS: Our results suggest that hybrid grape breeding is in its infancy. The distribution of V. vinifera ancestry across hybrids also suggests that backcrosses to wild Vitis species have been more frequent than backcrosses to V. vinifera during hybrid grape breeding. This pattern is unusual in crop breeding, as it is most common to repeatedly backcross to elite, or domesticated, germplasm. We anticipate our method can be extended to facilitate marker-assisted selection in order to introgress beneficial wild Vitis traits, while allowing for offspring with the highest V. vinifera content to be selected at the seedling stage.

FaRXf1: a locus conferring resistance to angular leaf spot caused by Xanthomonas fragariae in octoploid strawberry.

KEY MESSAGE: Angular leaf spot is a devastating bacterial disease of strawberry. Resistance from two wild accessions is highly heritable and controlled by a major locus on linkage group 6D. Angular leaf spot caused by Xanthomonas fragariae is the only major bacterial disease of cultivated strawberry (Fragaria ×ananassa). While this disease may cause reductions of up to 8 % of marketable yield in Florida winter annual production, no resistant cultivars have been commercialized. Wild accessions US4808 and US4809 were previously identified as resistant to the four genetic clades of X. fragariae, and introgression of the trait into commercial quality perennial-type germplasm was initiated. Previous reports indicated high heritability for the trait but proposed both single-locus and multi-locus inheritance models. The objective of this study was to determine the mode of inheritance of resistance, to identify causal loci, and to begin introgression of resistance into Florida-adapted germplasm. Resistance was observed in two years of field trials with inoculated plants that assayed four full-sib families descended from US4808 to US4809. Resistance segregated 1:1 in all families indicating control by a dominant allele at a single locus. Using a selective genotyping approach with the IStraw90 Axiom(®) SNP array and pedigree-based QTL detection, a single major-effect QTL was identified in two full-sib families, one descended from each resistant accession. High-resolution melt curve analysis validated the presence of the QTL in separate populations. The QTL was delimited to the 33.1-33.6 Mbp (F. vesca vesca v1.1 reference) and 34.8-35.3 Mbp (F. vesca bracteata v2.0 reference) regions of linkage group 6D for both resistance sources and was designated FaRXf1. Characterization of this locus will facilitate marker-assisted selection toward the development of resistant cultivars.

Robust asymmetric synthesis of unnatural alkenyl amino acids for conformationally constrained α-helix peptides.

The efficient asymmetric synthesis of unnatural alkenyl amino acids required for peptide 'stapling' has been achieved using alkylation of a fluorine-modified Ni(II) Schiff base complex as the key step.

A general algorithm for consensus 3D cell segmentation from 2D segmented stacks.

Cell segmentation is the fundamental task. Only by segmenting, can we define the quantitative spatial unit for collecting measurements to draw biological conclusions. Deep learning has revolutionized 2D cell segmentation, enabling generalized solutions across cell types and imaging modalities. This has been driven by the ease of scaling up image acquisition, annotation and computation. However 3D cell segmentation, which requires dense annotation of 2D slices still poses significant challenges. Labelling every cell in every 2D slice is prohibitive. Moreover it is ambiguous, necessitating cross-referencing with other orthoviews. Lastly, there is limited ability to unambiguously record and visualize 1000's of annotated cells. Here we develop a theory and toolbox, u-Segment3D for 2D-to-3D segmentation, compatible with any 2D segmentation method. Given optimal 2D segmentations, u-Segment3D generates the optimal 3D segmentation without data training, as demonstrated on 11 real life datasets, >70,000 cells, spanning single cells, cell aggregates and tissue.

Rotamer-Controlled Dual Emissive α-Amino Acids.

The synthesis and photoluminescent properties of novel α-amino acids are described in which the biaryl benzotriazinone-containing chromophores were found to display dual emission fluorescence via locally excited (LE) and twisted intramolecular charge transfer (TICT) states. The intensity of each emission band could be controlled by the electronics and position of the substituents, and this led to the design of a 2-methoxyphenyl analogue that, due to twisting, displayed bright TICT fluorescence, solvatochromism, and pH sensitivity.

Facial Nerve High-Resolution Microanatomy Analysis of the Distal Intratemporal to Extracranial Pes Segment: Reconstructive Implications.

BACKGROUND: Current knowledge of facial nerve topography between the stylomastoid foramen to the pes anserinus is very limited. Elucidating this segment's intraneural microanatomy may be advantageous in certain clinical settings: the planning of nerve grafts for gaps extending from the proximal facial nerve trunk to distal branches or in determining coaptation sites for hypoglossal jump grafts to provide selective upper and lower facial tone. This study is the first to provide high-definition intraneural topography of the aforementioned segment to optimize reconstructive outcomes. METHODS: Sixteen facial nerves extending from the second genu to the pes anserinus were harvested from eight cadavers en bloc to preserve orientation. Specimens were imaged by micro-computed tomography using a serial 6-µm protocol and digitally reconstructed three-dimensionally to be analyzed using bioinformatic tools. RESULTS: No clinically significant fascicular separation was noted between 14.4 mm proximal to the stylomastoid foramen until 4.4 mm distal to the foramen. Fascicles remained separate throughout the remainder of the specimen and were found to undergo a mean rotation of 45.5 degrees ( P = 0.0002) between 8.9 and 13.7 mm distal to the stylomastoid foramen. This reliable clockwise rotation in left nerves and counterclockwise rotation in right nerves resulted in superficially oriented fascicles entering the upper division of the pes anserinus, whereas deep-oriented fascicles entered the lower division. CONCLUSION: Intraneural facial nerve topography and rotation are consistent from 4 to 14 mm distal to the stylomastoid foramen, enabling surgeons to accurately place grafts targeted to either the upper or lower face, thus optimizing functional accuracy and minimizing synkinesis.

A rapid and bidirectional reporter of neural activity reveals neural correlates of social behaviors in Drosophila.

Neural activity is modulated over different timescales encompassing subseconds to hours, reflecting changes in external environment, internal state and behavior. Using Drosophila as a model, we developed a rapid and bidirectional reporter that provides a cellular readout of recent neural activity. This reporter uses nuclear versus cytoplasmic distribution of CREB-regulated transcriptional co-activator (CRTC). Subcellular distribution of GFP-tagged CRTC (CRTC::GFP) bidirectionally changes on the order of minutes and reflects both increases and decreases in neural activity. We established an automated machine-learning-based routine for efficient quantification of reporter signal. Using this reporter, we demonstrate mating-evoked activation and inactivation of modulatory neurons. We further investigated the functional role of the master courtship regulator gene fruitless (fru) and show that fru is necessary to ensure activation of male arousal neurons by female cues. Together, our results establish CRTC::GFP as a bidirectional reporter of recent neural activity suitable for examining neural correlates in behavioral contexts.