Unraveling micro-architectural modulations in neural tissue upon ischemia by Correlation Tensor MRI Noninvasively detecting and characterizing modulations in cellular scale micro-architecture is a desideratum for contemporary neuroimaging. Diffusion MRI (dMRI) has become the mainstay methodology for probing microstructure, and, in ischemia, its contrasts have revolutionized stroke management. However, the biological underpinnings of the contrasts observed in conventional dMRI in general and in ischemia in particular are still highly debated since the markers only indirectly reporter on microstructure. Here, we present Correlation Tensor MRI (CTI), a method that rather than measuring diffusion, harnesses diffusion correlations as its source of contrast. We show that CTI can resolve the sources of diffusional kurtosis, which in turn, provide dramatically enhanced specificity and sensitivity towards ischemia. In particular, the sensitivity towards ischemia nearly doubles, both in grey matter (GM) and white matter (WM), and unique signatures for neurite beading, cell swelling, and edema are inferred from CTI. The enhanced sensitivity and specificity endowed by CTI bodes well for future applications in biomedicine, basic neuroscience, and in the clinic.
Advanced paternal age diversifies individual trajectories of vocalization patterns in neonatal mice Infant crying is an innate communicative behavior that is frequently impaired in certain neurodevelopmental disorders (NDDs). Since advanced paternal age is a reported risk factor for NDDs in offspring, we evaluated the impact of a fathers age on early vocal development in C57BL/6J mice. We recorded and applied a unique combination of computational analyses to ultrasonic vocalizations (USVs) emitted by mouse pups sired by young and aged fathers. Our data showed that advanced paternal age reduced the number and duration of USVs, and altered the syllable composition in pups. Moreover, pups born to young fathers showed convergent vocal characteristics with a rich repertoire during postnatal development, while those born to aged fathers exhibited more divergent vocal patterns with limited repertoire. Principal component analysis in conjunction with clustering analysis demonstrated that pups from aged fathers deviated from typical trajectories of vocal development, which were considered as atypical individuals. Thus, our study indicates that advanced paternal age has a significant effect on offsprings early vocal development. It is suggested that the trajectories of vocal development could be a useful marker of the NDD-like phenotype associated with the advanced paternal age. In addition, our comprehensive computational analysis described here is an effective approach to characterize the altered individual diversity relevant to neurodevelopmental disorders.

One Sentence SummaryAdvanced paternal age affects vocal development in early postnatal mice, with more pups showing atypical developmental trajectories.
The initial response of females towards congeneric males matches the propensity to hybridise in Ophthalmotilapia. AO_SCPLOWBSTRACTC_SCPLOWCichlid radiations often harbour closely related species with overlapping niches and distribution ranges. Such species sometimes hybridise in nature, which raises the question how can they coexist. This also holds for the Tanganyika mouthbrooders Ophthalmotilapia ventralis and O. nasuta. Earlier studies found indications of asymmetrical hybridisation with females of O. ventralis accepting males of O. nasuta, but not the other way around. We hypothesised that this was due to differences in the capacity for species recognition. Given the higher propensity of O. ventralis females towards hybridisation, we expect a reduced ability for species recognition in O. ventralis females, compared to O. nasuta females. We staged two experiments, one focusing on 22 female O. nasuta and one on 21 female O. ventralis. These fish were placed in one half of a tank and briefly exposed to a conspecific or a heterospecific male, a conspecific female, or nothing (control). Female response was evaluated by scoring six tracking parameters and by noting the occurrence of ten discrete behaviours before and during the encounter. Females always responded to the presence of another fish by approaching it. Remarkably, for both O. nasuta and O. ventralis, we did not find a different response between encounters with conspecific males and females. However, in agreement with our hypothesis, O. nasuta females behaved differently towards conspecific or heterospecific males, whereas O. ventralis females did not. When presented with a heterospecific male, O. nasuta females performed a lower number of  ram behaviours. Additionally, they never displayed the  flee behaviour, a component of the species mating repertoire that was seen in all but one of the presentations with a conspecific male. Our findings show that differences in species recognition at first encounter predict to a large degree the outcome of the mating process, even in the absence of mating behaviour. Antibody Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 The COVID-19 pandemic has ravaged the globe, and its causative agent, SARS-CoV-2, continues to rage. Prospects of ending this pandemic rest on the development of effective interventions. Single and combination monoclonal antibody (mAb) therapeutics have received emergency use authorization1-3, with more in the pipeline4-7. Furthermore, multiple vaccine constructs have shown promise8, including two with ~95% protective efficacy against COVID-199,10. However, these interventions were directed toward the initial SARS-CoV-2 that emerged in 2019. The recent emergence of new SARS-CoV-2 variants B.1.1.7 in the UK11 and B.1.351 in South Africa12 is of concern because of their purported ease of transmission and extensive mutations in the spike protein. We now report that B.1.1.7 is refractory to neutralization by most mAbs to the N-terminal domain (NTD) of spike and relatively resistant to a few mAbs to the receptor-binding domain (RBD). It is not more resistant to convalescent plasma or vaccinee sera. Findings on B.1.351 are more worrisome in that this variant is not only refractory to neutralization by most NTD mAbs but also by multiple individual mAbs to the receptor-binding motif on RBD, largely due to an E484K mutation. Moreover, B.1.351 is markedly more resistant to neutralization by convalescent plasma (9.4 fold) and vaccinee sera (10.3-12.4 fold). B.1.351 and emergent variants13,14 with similar spike mutations present new challenges for mAb therapy and threaten the protective efficacy of current vaccines. Placental uptake and metabolism of 25(OH)Vitamin D determines its activity within the fetoplacental unit Pregnancy 25-hydroxyvitamin D (25(OH)D) concentrations are associated with maternal and fetal health outcomes. Using physiological human placental perfusion and villous explants, we investigate the role of the placenta in regulating the relationships between maternal 25(OH)D and fetal physiology. We demonstrate active placental uptake of 25(OH)D3 by endocytosis, placental metabolism of 25(OH)D3 into 24,25-dihydroxyvitamin D3 and active 1,25-dihydroxyvitamin D [1,25(OH)2D3], with subsequent release of these metabolites into both the maternal and fetal circulations. Active placental transport of 25(OH)D3 and synthesis of 1,25(OH)2D3 demonstrate that fetal supply is dependent on placental function rather than simply the availability of maternal 25(OH)D3. We demonstrate that 25(OH)D3 exposure induces rapid effects on the placental transcriptome and proteome. These map to multiple pathways central to placental function and thereby fetal development, independent of vitamin D transfer. Our data suggest that the underlying epigenetic landscape helps dictate the transcriptional response to vitamin D treatment. This is the first quantitative study demonstrating vitamin D transfer and metabolism by the human placenta, with widespread effects on the placenta itself. These data demonstrate a complex interplay between vitamin D and the placenta and will inform future interventions using vitamin D to support fetal development and maternal adaptations to pregnancy. Absence of cardiolipin from the outer-leaflet of a mitochondrial inner-membrane mimic restricts Opa1-mediated fusion Cardiolipin is a tetra-acylated di-phosphatidylglycerol lipid enriched in the matrix-facing (inner) leaflet of the mitochondrial inner membrane. Cardiolipin plays an important role in regulating mitochondria function and dynamics. Yet, the mechanisms connecting cardiolipin distribution and mitochondrial protein function remain indirect. In our previous work, we established an in vitro system reconstituting mitochondrial inner membrane fusion mediated by Opa1. We found that the long form of Opa1 (l-Opa1) works together with the proteolytically processed short form (s-Opa1) to mediate fast and efficient membrane fusion. Here, we extend our reconstitution system to generate supported lipid bilayers with asymmetric cardiolipin distribution. Using this system, we find the presence of cardiolipin on the inter-membrane space-facing (outer) leaflet is important for membrane tethering and fusion. We discuss how the presence of cardiolipin in this leaflet may influence protein and membrane properties, and future applications for this approach. Natural selection promotes the evolution of recombination 2: during the process of natural selection The ubiquity of sex and recombination in nature has eluded unified explanation since the time of Darwin. Conditions that promote the evolution of recombination, broadly defined as any form of genetic mixing, are fairly well understood: it is favored when genomes tend to contain more selectively mismatched combinations of alleles than can be explained by chance alone. Yet, while a variety of theoretical approaches have been put forth to explain why such conditions would prevail in natural populations, each has turned out to be of limited scope and applicability. Here, we show, simply and surprisingly, that natural selection acting on standing heritable variation always creates conditions favoring the evolution of recombination, in expectation. Specifically, we find that, in expectation: 1) the mean selective advantage of recombinants is non-negative, 2) the mean selective advantage of a recombination-competent modifier is non-negative, and 3) the asymptotic frequency of a recombination-competent modifier is close to one and is independent of the strength of selection. Remarkably, these findings are independent of the distribution of genic fitnesses in the standing heritable variation upon which natural selection acts, implying that the source of this variation is immaterial. Taken together, our findings indicate that: 1) the evolution of recombination should be promoted in expectation wherever natural selection is operating, and 2) sex and recombination may have evolved more as a byproduct than as a catalyst of natural selection. A dysmorphic mouse model reveals developmental interactions of chondrocranium and dermatocranium The cranial endo- and dermal skeletons, which comprise the vertebrate skull, evolved independently over 470 million years ago and form separately during embryogenesis. In mammals, much of the cartilaginous chondrocranium is transient, undergoing endochondral ossification or disappearing, so its role in skull morphogenesis is not well studied and it remains an enigmatic structure. We provide complete three-dimensional (3D) reconstructions of the laboratory mouse chondrocranium from embryonic day 13.5 through 17.5 using a novel methodology of uncertainty-guided segmentation of phosphotungstic enhanced 3D microcomputed tomography images with sparse annotation. We evaluate the embryonic mouse chondrocranium and dermatocranium in 3D and delineate the effects of a Fgfr2 variant on embryonic chondrocranial cartilages and on the association with forming dermal bones using the Fgfr2cC342Y/+ Crouzon syndrome mouse. We show that the dermatocranium develops outside of and in shapes that conform to the chondrocranium. Results reveal direct effects of the Fgfr2 variant on embryonic cartilage, on chondrocranium morphology, and on the association between chondrocranium and dermatocranium development. Histologically we observe a trend of relatively more chondrocytes, larger chondrocytes, and/or more matrix in the Fgfr2cC342Y/+ embryos at all timepoints before the chondrocranium begins to disintegrate at E16.5. The chondrocrania and forming dermatocrania of Fgfr2cC342Y/+ embryos are relatively large, but a contrasting trend begins at E16.5 and continues into early postnatal (P0 and P2) timepoints, with the skulls of older Fgfr2cC342Y/+ mice reduced in most dimensions compared to Fgfr2c+/+ littermates. Our findings have implications for the study and treatment of human craniofacial disease, for understanding the impact of chondrocranial morphology on skull growth, and potentially on the evolution of skull morphology. Centimeter-scale quorum sensing dictates collective survival of differentiating embryonic stem cells Cells can help each other replicate by communicating with diffusible molecules. In cell cultures, molecules may diffuse within a cell colony or between adjacent or distant colonies. Determining which cell helps which cells replication is challenging. We developed a systematic approach, integrating modeling and experiments, for determining the length-scales of cell-cell communication (from microns to centimeters). With this approach, we discovered that differentiating murine ES cells, scattered across centimeters on a dish, communicate over millimeters to form one macroscopic entity that survives if and only if its centimeter-scale population-density is above a threshold value. Single-cell-level measurements, transcriptomics, and modeling revealed that this "macroscopic quorum sensing" arises from differentiating ES cells secreting and sensing survival-promoting FGF4 that diffuses over millimeters and activates YAP1-induced survival mechanisms. Through the same mechanism, a lone macroscopic, but not microscopic, colony survives differentiation. Our work rigorously establishes that in vitro ES-cell differentiation relies on macroscopic cooperation. Fatal COVID-19 and non-COVID-19 Acute Respiratory Distress Syndrome is Associated with Incomplete Alveolar Type 1 Epithelial Cell Differentiation from the Transitional State Without Fibrosis ARDS due to COVID-19 and other etiologies results from injury to the alveolar epithelial cell (AEC) barrier resulting in noncardiogenic pulmonary edema, which causes acute respiratory failure; clinical recovery requires epithelial regeneration. During physiologic regeneration in mice, AEC2s proliferate, exit the cell cycle, and transiently assume a transitional state before differentiating into AEC1s; persistence of the transitional state is associated with pulmonary fibrosis in humans. It is unknown whether transitional cells emerge and differentiate into AEC1s without fibrosis in human ARDS and why transitional cells differentiate into AEC1s during physiologic regeneration but persist in fibrosis. We hypothesized that incomplete but ongoing AEC1 differentiation from transitional cells without fibrosis may underlie persistent barrier permeability and fatal acute respiratory failure in ARDS. Immunostaining of postmortem ARDS lungs revealed abundant transitional cells in organized monolayers on alveolar septa without fibrosis. They were typically cuboidal or partially spread, sometimes flat, and occasionally expressed AEC1 markers. Immunostaining and/or interrogation of scRNAseq datasets revealed that transitional cells in mouse models of physiologic regeneration, ARDS, and fibrosis express markers of cell cycle exit but only in fibrosis express a specific senescence marker. Thus, in severe, fatal early ARDS, AEC1 differentiation from transitional cells is incomplete, underlying persistent barrier permeability and respiratory failure, but ongoing without fibrosis; senescence of transitional cells may be associated with pulmonary fibrosis. The obstacles and potential clues of prime editing applications in tomato, a dicot plant Precision genome editing is highly desired for crop improvement. The recently emerged CRISPR/Cas technology offers great potential applications in precision plant genome engineering. A prime editing (PE) approach combining a reverse transcriptase (RT) with a Cas9 nickase and a "priming" extended guide RNA has shown a high frequency for precise genome modification in mammalian cells and several plant species. Nevertheless, the applications of the PE approach in dicot plants are still limited and inefficient. We designed and tested prime editors for precision editing of a synthetic sequence in a transient assay and for desirable alleles of 10 loci in tomato by stable transformation. Our data obtained by targeted deep sequencing also revealed only low PE efficiencies in both the tobacco and tomato systems. Further assessment of the activities of the PE components uncovered potential reasons for the inefficiency of the PE complexes. Modifying the pegRNA sequences by shortening or introducing mismatches to the primer binding sequences (PBS) in order to reduce their melting temperatures (Tm) did not enhance the PE efficiency at the SlBMP21, SlALC and SlALS1 loci. Our data show challenges of PE approach in tomato, indicating a further improvement of the PE system for the successful applications such as use of improved expression systems. Our work provides an important clue for the successful application of the PE approach in crop improvement. Kinetoplastid-specific X2-family kinesins interact with a kinesin-like pleckstrin homology domain protein that localizes to the trypanosomal microtubule quartet Kinesins are motor proteins found in all eukaryotic lineages that move along microtubules to mediate cellular processes such as mitosis and intracellular transport. In trypanosomatids, the kinesin superfamily has undergone a prominent expansion, resulting in one of the most diverse kinesin repertoires that includes the two kinetoplastid-restricted families X1 and X2. Here, we characterize in Trypanosoma brucei TbKifX2A, an orphaned X2 kinesin. TbKifX2A tightly interacts with TbPH1, a kinesin-like protein with a likely inactive motor domain, a rarely reported occurrence. Both TbKifX2A and TbPH1 localize to the microtubule quartet (MtQ), a characteristic but poorly understood cytoskeletal structure that wraps around the flagellar pocket as it extends to the cell body anterior. The proximal proteome of TbPH1 revealed two other interacting proteins, the flagellar pocket protein FP45 and intriguingly another X2 kinesin, TbKifX2C. Simultaneous ablation of TbKifX2A/TbPH1 results in the depletion of FP45 and TbKifX2C and also an expansion of the flagellar pocket, among other morphological defects. TbKifX2A is the first motor protein to be localized to the MtQ. The observation that TbKifX2C also associates with the MtQ suggests that the X2 kinesin family may have co-evolved with the MtQ, both kinetoplastid-specific traits. Usutu virus escapes langerin-induced restriction to productively infect human Langerhans cells, unlike West Nile virus. Usutu virus (USUV) and West Nile virus (WNV) are phylogenetically close emerging arboviruses transmitted by mosquitoes, and constitute a global public health threat. Since USUV and WNV enter the body through the skin, the first immune cells they encounter are skin-resident dendritic cells, the most peripheral outpost of immune defense. This unique network is composed of Langerhans cells (LCs) and dermal DCs, which reside in the epidermis and the dermis, respectively.

Using human skin explants, we show that while both viruses can replicate in keratinocytes, they can also infect resident DCs with distinct tropism, since WNV preferentially infects DCs in the dermis, whereas USUV has a greater propensity to infect LCs. Using both purified human epidermal LCs (eLCs) and monocyte derived LCs (MoLCs), we confirm that LCs sustain a faster and more efficient replication of USUV compared with WNV and that this correlates with a more intense innate immune response to USUV compared with WNV. Next, we show that ectopic expression of the LC-specific C-type lectin receptor (CLR), langerin, in HEK293T cells allows WNV and USUV to bind and enter, but supports the subsequent replication of USUV only. Conversely, blocking or silencing langerin in MoLCs or eLCs made them resistant to USUV infection, thus demonstrating that USUV uses langerin to enter and replicate in LCs. Altogether, our results demonstrate that LCs constitute privileged target cells for USUV in human skin, because langerin favors its entry and replication. Intriguingly, this suggests that USUV efficiently escapes the antiviral functions of langerin, which normally safeguards LCs from most viral infections. A combined test for feature selection on sparse metaproteomics data - an alternative to missing value imputation One of the difficulties encountered in the statistical analysis of metaproteomics data is the high proportion of missing values, which are usually treated by imputation. Nevertheless, imputation methods are based on restrictive assumptions regarding missingness mechanisms, namely "at random" or "not at random". To circumvent these limitations in the context of feature selection in a multi-class comparison, we propose a univariate selection method that combines a test of association between missingness and classes, and a test for difference of observed intensities between classes. This approach implicitly handles both missingness mechanisms. We performed a quantitative and qualitative comparison of our procedure with imputation-based feature selection methods on two experimental data sets, as well as simulated data with various scenarios regarding the missingness mechanisms and the nature of the difference of expression (differential intensity or differential missingness). Whereas we observed similar performances in terms of prediction on the experimental data set, the feature ranking and selection from various imputation-based methods were strongly divergent. We showed that the combined test reaches a compromise by correlating reasonably with other methods, and remains efficient in all simulated scenarios unlike imputation-based feature selection methods. Computational modeling and quantitative cell physiology reveal central parameters for the brassinosteroid-regulated cell growth of the Arabidopsis root Brassinosteroids (BR) are key hormonal regulators of plant development. However, whereas the individual components of BR perception and signaling are well characterized experimentally, the question of how they can act and whether they are sufficient to carry out the critical function of cellular elongation remains open. Here, we combined computational modeling with quantitative cell physiology to understand the dynamics of the plasma membrane (PM)-localized BR response pathway during the initiation of cell elongation in the epidermis of the Arabidopsis root tip. The model, consisting of ordinary differential equations, comprises the BR induced hyperpolarization of the PM, the acidification of the apoplast and subsequent cell wall swelling. We demonstrated that the competence of the root epidermal cells for the BR response predominantly depends on the amount and activity of H+-ATPases in the PM. The model further predicted that an influx of cations is required to compensate for the shift of positive charges caused by the apoplastic acidification. A potassium channel was subsequently identified and experimentally characterized, fulfilling this function. Thus, we established the landscape of components and parameters capable of triggering and guiding cellular elongation through the fast response to BRs, a central process in plant development. A novel two-generation approach to the population dynamics of gregarious parasitoids Parasitoids, as important natural enemies, help maintain balance in natural ecosystems. Their population dynamics is generally predicted from the number of individuals. Here, using gregarious parasitoids as models, we show that this traditional approach omits one important parameter: the mothers manipulation of offspring fertility due to the clutch size-body size- fertility correlation. As a result of this correlation, when females deliberately adjust the number of offspring laid in a host, they determine not only the number but also the body sizes and reproductive potentials of those offspring. For the first time in the model species Anaphes flavipes, we determined the parasitoids offspring fertility from clutch size. Using this, we experimentally clarified the advantage of specific clutch size combinations and we show that identical fertility in the F1 generation can lead to distinctly different fertility values in the F2 generation. Even with the same number of hosts, lower fertility in the F1 generation can cause higher fertility in the F2 generation. Based on these results, we propose a novel two-generation approach which includes the clutch size-body size-fertility correlation. Our novel approach provides a new perspective for determining the individual fitness levels of gregarious parasitoids with new options for the modelling of parasitoid population dynamics. Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Cancer Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of mitochondrial TCA cycle enzyme, succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect in PC tumors, triggers dysregulation of Ca2+ homeostasis, and aberrantly activates calpain and the protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a cascade of phospho- signaling where GSK3 inhibition inactivates energy sensing by AMP-kinase through dephosphorylation of the AMP-kinase {gamma} subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC tumor formation. A novel Cdk5 inhibitor, MRT3-007, reversed this phospho-cascade, invoking an anti- Warburg effect, cell cycle arrest, and senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important novel mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.

HighlightsO_LILoss of SDHB function in pheochromocytoma causes Ca2+ dysregulation, calpain activation, and aberrant activation of the protein kinase Cdk5.
C_LIO_LIHyperactive Cdk5 deregulates a GSK3/PRKAG2/AMPK signaling cascade.
C_LIO_LIp25 overexpression and consequent aberrant Cdk5 activity in chromaffin cells causes pheochromocytoma.
C_LIO_LIInhibition of Cdk5 activates the PRKAG2/AMPK/p53 signaling to rescue cell senescence and block PC tumor progression.
C_LI

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/475410v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@16f0a48org.highwire.dtl.DTLVardef@10c1c7aorg.highwire.dtl.DTLVardef@21b100org.highwire.dtl.DTLVardef@1eb8674_HPS_FORMAT_FIGEXP  M_FIG C_FIG A rationalized definition of tumor suppressor microRNAsexcludes miR-34a While several microRNAs (miRNAs) have been proposed to act as tumor suppressors, a consensual definition of tumor suppressing miRNAs is still missing. Similarly to coding genes, we propose that tumor suppressor miRNAs must show evidence of genetic or epigenetic inactivation in cancers, and exhibit an anti-tumorigenic (e.g., anti-proliferative) activity under endogenous expression levels. Here we observe that this definition excludes the most extensively studied tumor suppressor candidate miRNA, miR-34a. In analyzable cancer types, miR-34a does not appear to be down-regulated in primary tumors relatively to normal adjacent tissues. Deletion of miR-34a is occasionally found in human cancers, but it does not seem to be driven by an anti-tumorigenic activity of the miRNA, since it is not observed upon smaller, miR-34a-specific alterations. Its anti-proliferative action was observed upon large, supra-physiological transfection of synthetic miR-34a in cultured cells, and our data indicates that endogenous miR-34a levels do not have such an effect. Our results therefore argue against a general tumor suppressive function for miR-34a, providing an explanation to the lack of efficiency of synthetic miR-34a administration against solid tumors. Synthetic Cooling Agents in US-marketed E-cigarette Refill Liquids and Popular Disposable E-cigarettes: Chemical Analysis and Risk Assessment. BackgroundMenthol, through its cooling and pleasant sensory effects, facilitates smoking and tobacco product initiation, resulting in the high popularity of mint/menthol-flavored E-cigarettes. More recently, E-cigarette vendors started marketing synthetic cooling agents as additives that impart a cooling effect but lack a characteristic minty odor. Knowledge about content of synthetic coolants in US-marketed E-cigarette products and associated health risks is limited.

MethodsE-liquid vendor sites were searched with the terms "koolada", "kool/cool", "ice", or WS-3/WS-23, denoting individual cooling agents, and relevant refill E-liquids were purchased. "Ice" flavor varieties of Puffbar, the most popular disposable E-cigarette brand, were compared with non-"Ice" varieties. E-liquids were characterized, and synthetic coolants quantified using GC/MS. Margin of exposure (MOE), a risk assessment parameter, was calculated to assess the risk associated with synthetic coolant exposure from E-cigarette use.

ResultsWS-3 was detected in 24/25 refill E-liquids analyzed. All Puffbar flavor varieties contained either WS-23 (13/14) or WS-3 (5/14), in both "Ice"- and non-"Ice" flavors. Modeling consumption of WS-3 from vaped E-liquids, resulted in MOEs below the safe margin of 100 for most daily use scenarios. MOEs for WS-23 were <100 for 10/13 Puffbar flavors in all use scenarios. Puffbar power specifications are identical to Juul devices.

ConclusionsSynthetic cooling agents (WS-3/WS-23) were present in US-marketed E-cigarettes, at levels that may result in consumer exposures exceeding safety thresholds set by regulatory agencies. Synthetic coolants are not only found in mint-or menthol-flavored products, but also in fruit- and candy-flavored products, including popular disposable E-cigarette products such as Puffbar.

ImplicationsSynthetic cooling agents are widely used in "kool/cool"- and "ice"-flavored E-liquids and in E-liquids without these labels, both as a potential replacement for menthol or to add cooling  notes to non-menthol flavors. These agents may be used to bypass current and future regulatory limits on menthol content in tobacco products, and not just E-cigarettes. Since synthetic cooling agents are odorless, they may not fall under the category of "characterizing flavor", potentially circumventing regulatory measures based on this concept. Regulators need to consider the additional health risks associated with exposure to synthetic cooling agents. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Balancing sensitivity and specificity in preclinical research The success of scientific discovery in preclinical drug development is based on the different roles of exploration and confirmation. Exploration requires sensitivity while confirmation needs to be specific. Based on published empirical data, we simulated two preclinical research trajectories to examine their effectiveness to detect true effects while keeping animal numbers low. We compare a conventional p-value based approach with a method based on an a priori determined smallest effect size of interest (SESOI). Using a SESOI increases transition rates from exploration to confirmation and results in higher detection rates across the trajectory. Specificity in the SESOI trajectory increases if the prior probability of true effects is low. We conclude that employing a SESOI is superior to a p-value based approach. Based on our findings, we propose a reconsideration of planning and conducting preclinical experiments, especially when the prior probability of true hypotheses is low. Microorganisms Characterization in Anaerobic Solventogenic Processing of Volatile Fatty Acids 1This work evaluates four different physicochemical pretreatments (acidic, thermal, acidic-thermal and thermal-acidic) on an anaerobic inoculum used for alcohol production from acetate and butyrate. All experiments were conducted in single batches using acetate and butyrate as substrates at 30 {degrees}C and with a pressurized headspace of pure H2 at 2.15 atm (218.2 MPa). Thermal and acidic-thermal pretreatments lead to higher production of both ethanol and butanol. Mathematical modelling shows that the highest attainable concentrations of ethanol and butanol produced were 122 mg L-1 and 97 mg L-1 for the thermal pretreatment (after 17.5 days) and 87 mg L-1 and 143 mg L-1 for the acidic-thermal pretreatment (after 18.9 days). Acetate was produced in all assays. Thermodynamic data indicated that a high H2 partial pressure favoured solventogenic metabolic pathways. Finally, sequencing data showed that both thermal and acidic-thermal pretreatments selected mainly the bacterial genera Pseudomonas, Brevundimonas and Clostridium. The acidic-thermal pretreatment selected a bacterial community more adapted to the conversion of acetate and butyrate into ethanol and butanol, respectively. Thermal-acidic pretreatment was unstable, showing significant variability between replicates. Acidic pretreatment showed the lowest alcohol production. Cas9-mediated gene disruption in tetraploid Giardia intestinalis CRISPR/Cas9 system is an extremely powerful technique that is extensively used for various genome modifications in different organisms including parasitic protists. Giardia intestinalis, a protist parasite infecting about 280 million people around the world each year, has been eluding the routine use of CRISPR/Cas9 for generating knock-out cell lines due to the presence of four copies of each gene in its two nuclei. Apart from single exception employing rather laborious Cre/loxP system, no full knock-out cell line has been established yet. In this work, we show the ability of in-vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. We further established a cell line stably expressing Cas9 in both G. intestinalis nuclei. Subsequent introduction of a template for homologous recombination containing the transcription units for the resistance marker and gRNA resulted in the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable for the incomplete disruption of an essential gene, as documented by markedly decreased expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a full knock-out cell line in G. intestinalis. Comparative proteome signatures of trace samples by multiplexed Data-Independent Acquisition Single cell transcriptomics has revolutionized our understanding of basic biology and disease. Since transcript levels often do not correlate with protein expression, it is crucial to complement transcriptomics approaches with proteome analyses at single cell resolution. Despite continuous technological improvements in sensitivity, mass spectrometry-based single cell proteomics ultimately faces the challenge of reproducibly comparing the protein expression profiles of thousands of individual cells. Here, we combine two hitherto opposing analytical strategies, DIA and Tandem-Mass-Tag (TMT)-multiplexing, to generate highly reproducible, quantitative proteome signatures from ultra-low input samples. While conventional, data-dependent shotgun proteomics (DDA) of ultra-low input samples critically suffers from the accumulation of missing values with increasing sample-cohort size, data-independent acquisition (DIA) strategies do usually not take full advantage of isotope-encoded sample multiplexing. We developed a novel, identification-independent proteomics data-analysis pipeline that allows to quantitatively compare DIA-TMT proteome signatures across hundreds of samples independent of their biological origin, and to identify cell types and single protein knockouts. We validate our approach using integrative data analysis of different human cell lines and standard database searches for knockouts of defined proteins. These data establish a novel and reproducible approach to markedly expand the numbers of proteins one detects from ultra-low input samples, such as single cells. Including random effects in statistical models in ecology: fewer than five levels? As generalized linear mixed-effects models (GLMMs) have become a widespread tool in ecology, the need to guide the use of such tools is increasingly important. One common guideline is that one needs at least five levels of a random effect. Having such few levels makes the estimation of the variance of random effects terms (such as ecological sites, individuals, or populations) difficult, but it need not muddy ones ability to estimate fixed effects terms - which are often of primary interest in ecology. Here, I simulate ecological datasets and fit simple models and show that having too few random effects terms does not influence the parameter estimates or uncertainty around those estimates for fixed effects terms. Thus, it should be acceptable to use fewer levels of random effects if one is not interested in making inference about the random effects terms (i.e. they are  nuisance parameters used to group non-independent data). I also use simulations to assess the potential for pseudoreplication in (generalized) linear models (LMs), when random effects are explicitly ignored and find that LMs do not show increased type-I errors compared to their mixed-effects model counterparts. Instead, LM uncertainty (and p values) appears to be more conservative in an analysis with a real ecological dataset presented here. These results challenge the view that it is never appropriate to model random effects terms with fewer than five levels - specifically when inference is not being made for the random effects, but suggest that in simple cases LMs might be robust to ignored random effects terms. Given the widespread accessibility of GLMMs in ecology and evolution, future simulation studies and further assessments of these statistical methods are necessary to understand the consequences of both violating and blindly following simple guidelines. High-throughput analysis of DNA replication in single human cells reveals the complex nature of replication timing control DNA replication initiates from replication origins firing throughout S phase. Debate remains about whether origins are a fixed set of loci, or a loose agglomeration of potential sites used stochastically in individual cells, and about how consistent their firing time is. We developed an approach to profile DNA replication from whole-genome sequencing of thousands of single cells, which includes "in silico flow cytometry", a method for discriminating replicating and non-replicating cells. Using two microfluidic platforms, we analyzed up to 2,437 replicating cells from a single sample. The resolution and scale of the data allow focused analysis of replication initiation sites, demonstrating that most occur in confined genomic regions. While initiation order is remarkably similar across cells, we unexpectedly identify several subtypes of initiation regions in late-replicating regions. Taken together, high throughput, high resolution sequencing of individual cells reveals previously underappreciated variability in replication initiation and progression.