Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression BackgroundChimeric antigen receptor (CAR) T cells traditionally harbor viral vector-based sequences that encode the CAR transgene in the genome. These T cell products have yet to show consistent anti-tumor activity in patients with solid tumors. Further, viral vector manufacturing is resource intensive, suffers from batch-to-batch variability, and includes several animal components, adding regulatory and supply chain pressures.

MethodsAnti-GD2 CAR T cells were generated using CRISPR/Cas9 within nine days using recombinant Cas9 protein and nucleic acids, without any viral vectors or animal components. The CAR was specifically targeted to the T Cell Receptor Alpha Constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, scRNA-seq, spectral cytometry, and ELISA assays. Functionality was evaluated in vivo in an NSG xenograft neuroblastoma model.

ResultsIn comparison to traditional retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory phenotype, and low tonic signaling prior to infusion arising in part from the the knockout of the TCR. Upon exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibited specific cytotoxicity against GD2+ cells in vitro and induced solid tumor regression in vivo, with robust homing, persistence, and low exhaustion against a human neuroblastoma xenograft model.

ConclusionsThis proof-of-principle study leveraging virus-free genome editing technology could enable flexible manufacturing of clinically relevant, high-quality CAR T cells to treat cancers, including solid tumors.
Use of lignocellulosic residue from second-generation ethanol production to enhance methane production through co-digestion This is a pioneer study evaluating the methane (CH4) production potential from residues of integrated 1st (vinasse and filter cake) and 2nd (deacetylation pretreatment liquor from straw) generation (1G2G) sugarcane biorefinery, providing a fully chemical characterization of them and their relation with the anaerobic digestion (AD) process. Small-scale assays provided fundamentals for basing the co-digestion optimization by assessing the optimal co-substrates synergistic conditions. Biochemical Methane Potential (BMP) tests showed co-digestion enhanced CH4 yield of isolated substrates, reaching up to 605 NmLCH4 gVS-1. The association of vinasse and deacetylation liquor as co-substrates increased the BMP by ~38% mostly by nutritionally benefiting the methanogenic activity. The kinetic analysis confirmed that the deacetylation liquor was the co-substrate responsible for improving the CH4 production in the co-digestion systems due to the highest CH4 conversion rate. The alkaline characteristic of the liquor (pH~12) also prevented alkalizing from being added to the co-digestion, an input that normally makes the process economically unfeasible to implement on an industrial scale due to the large quantities required for buffering the reactor. The filter cake had the lowest BMP (262 NmLCH4 gVS-1) and digestibility ([&le;] 40%), further limited by the required stirring to improve the mass transfer of biochemical reactions. The present study drives towards more sustainable use of vinasse, the most voluminous waste from the sugarcane industry, and lignin-rich residues derived from pre-treatment alkaline methods, aiming at an energy-efficient utilization, by at least 16% when compared to the traditional vinasse AD. The experimental and modeling elements from this work indicated the lignin-rich liquor is the main responsible for putting the co-digestion as a disruptive technological arrangement within the 1G2G sugarcane biorefineries, reinforcing the biogas production as the hub of the bioeconomy in the agroindustrial sector.
Untargeted metabolomic analyses reveal the diversity and plasticity of the specialized metabolome in seeds of different Camelina sativa genotypes Despite the essential role of Specialized Metabolites (SMs) in the interaction of plants with the environment, studying the ability of crop seeds to produce these protective compounds has been neglected. Furthermore, seeds produce a myriad of SMs providing an interesting model to investigate their diversity and plasticity. Camelina sativa gained a lot of interest in the past few years as rustic oil seed crop. A characterization of seed SM landscapes in six camelina genotypes grown in the field and harvested during five growing seasons has been undertaken in this work. This allowed a comprehensive annotation of seed SMs combining analyses that cluster SMs based on their chemical structures and co-accumulation patterns. These data showed broad effects of the environment on the stimulation of the seed-specialized metabolome. Among well annotated compounds, flavonols were identified as the metabolic class characterised by high plasticity, revealing significant variable accumulation according to the year and/or the genotype. Lastly, a deeper characterisation of primary metabolites and lipids in two selected genotypes has been performed. We showed that, in addition to flavonols, alkaloids and glucosinolates displayed a higher phenotypic plasticity with respect to most of the primary metabolites, including some sugars and major storage compounds such as fatty acids, proteins and most lipid classes (e.g. DAG, TAG), but similar plasticity compared to free aminoacids and carboxylic acids. This work highlighted major and unexplored effects of the environment on the seed specialized metabolome demonstrating that seeds exhibit a dynamic and plastic metabolism, with an impact on seed quality.

Significance statementSeeds produce a myriad of Specialized Metabolites (SMs) with an essential role in the interaction of plants with the environment. We characterized SM landscapes, primary metabolites and lipid composition in the seeds of camelina genotypes grown in the open field in five consecutive growing seasons. Our results showed the predominant effect of the environment on the regulation of the seed - specialized metabolome, with a potential impact on seed quality of camelina that may also occur in other oilseed crops. Probing Patterning in Microbial Consortia with picCASO: a Cellular Automaton for Spatial Organisation Microbial consortia exhibit spatial patterning across diverse environments. Since probing the self-organization of natural microbial communities is limited by their inherent complexity, synthetic models have emerged as attractive alternatives. In this study, we develop novel frameworks of bacterial communication and explore the emergent spatiotemporal organization of microbes. Specifically, we build quorum sensing-mediated models of microbial growth that are utilized to characterize the dynamics of communities from arbitrary initial configurations and establish the effectiveness of our communication strategies in coupling the growth rates of microbes. Our simulations indicate that the behavior of quorum sensing-coupled consortia can be most effectively modulated by the rates of secretion of AHLs. Such a mechanism of control enables the construction of desired relative populations of constituent species in spatially organized populations. Our models accurately recapitulate previous experiments that have investigated pattern formation in synthetic multi-cellular systems. Additionally, our software tool enables the easy implementation and analysis of our frameworks for a variety of initial configurations and simplifies the development of sophisticated gene circuits facilitating distributed computing. Overall, we demonstrate the potential of spatial organization as a tunable parameter in synthetic biology by introducing a communication paradigm based on the location and strength of coupling of microbial strains.

Author SummaryInteracting microorganisms that coexist in a given environment tend to have well-defined spatial arrangements. While the emergence of such organization is seen across different microbiomes in nature, it is hitherto not well understood. Decoding the inherent spatial patterning of microbes is constrained by the complexity of their natural habitats. Here, we take advantage of synthetic models of microbial communities to study the dynamics of emergent spatial organization. Our framework of bacterial communication utilizes modular synthetic devices to couple growth rates. In addition to uncovering potential principles of spatial organization, this work enables the construction of complex genetic circuits distributed across communicating strains. Additionally, we have developed a software tool, picCASO, that streamlines the investigation of microbial systems communicating through such frameworks. Col6a1+/CD201+ mesenchymal cells regulate intestinal morphogenesis and homeostasis Intestinal mesenchymal cells encompass multiple subsets, whose origins, functions, and pathophysiological importance are still not clear. Here, we used the Col6a1Cre mouse, which targets telocytes and perivascular cells that can be further distinguished by the combination of the CD201, PDGFR and SMA markers. Developmental studies revealed that the Col6a1Cre mouse also targets mesenchymal aggregates that are crucial for intestinal morphogenesis and patterning, suggesting an ontogenic relationship between them and homeostatic telocytes. Cell depletion experiments in adulthood showed that Col6a1+/CD201+ mesenchymal cells regulate homeostatic enteroendocrine cell differentiation and epithelial proliferation. During acute colitis, they expressed an inflammatory and extracellular matrix remodeling gene signature, but they also retained their properties and topology. Notably, both in homeostasis and tissue regeneration, they were dispensable for normal organ architecture, while CD34+ mesenchymal cells expanded, localised at the top of the crypts and showed increased expression of villous-associated morphogenetic factors, providing thus evidence for the plasticity potential of distinct mesenchymal populations in the intestine. Our results provide a comprehensive analysis of the identities, origin, and functional significance of distinct mesenchymal populations in the intestine. A nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, as an olfactory chemoattractant for the grass puffer Toxic puffers contain the potent neurotoxin, tetrodotoxin (TTX). Although TTX is considered to serve as a defense substance, previous behavioral studies have demonstrated that TTX acts as an attractive pheromone for some toxic puffers. To elucidate the physiological mechanism of putative pheromonal action of TTX, we examined whether grass puffers Takifugu alboplumbeus can detect TTX. Electroolfactogram (EOG) results suggest that the olfactory epithelium (OE) of grass puffers responded to a type of TTX analog (5,6,11-trideoxyTTX), although it did not respond to TTX. We also examined the attractive action of 5,6,11-trideoxyTTX on grass puffers by recording their swimming behavior under dark conditions. Grass puffers preferred to stay on the side of the aquarium where 5,6,11-trideoxyTTX was administered, and their swimming speed decreased. Additionally, odorant-induced labeling of olfactory sensory neurons by immunohistochemistry against neural activity marker (phosphorylated extracellular signal regulated kinase; pERK) revealed that labeled olfactory sensory neurons were localized in the region surrounding "islets" where there was considered as nonsensory epithelium. 5,6,11-trideoxyTTX has been known to accumulate in grass puffers, but its toxicity is much lower (almost nontoxic) than TTX. Our results suggest that toxic puffers may positively use this unknown function TTX analog as an olfactory chemoattractant. Omicron-specific mRNA vaccination alone and as a heterologous booster against SARS-CoV-2 The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has high transmissibility and recently swept the globe. Due to the extensive number of mutations, this variant has high level of immune evasion, which drastically reduced the efficacy of existing antibodies and vaccines. Thus, it is important to test an Omicron-specific vaccine, evaluate its immune response against Omicron and other variants, and compare its immunogenicity as boosters with existing vaccine designed against the reference wildtype virus (WT). Here, we generated an Omicron-specific lipid nanoparticle (LNP) mRNA vaccine candidate, and tested its activity in animals, both alone and as a heterologous booster to existing WT mRNA vaccine. Our Omicron-specific LNP-mRNA vaccine elicited strong and specific antibody response in vaccination-naive mice. Mice that received two-dose WT LNP-mRNA, the one mimicking the commonly used Pfizer/Moderna mRNA vaccine, showed a >40-fold reduction in neutralization potency against Omicron variant than that against WT two weeks post second dose, which further reduced to background level >3 months post second dose. As a booster shot for two-dose WT mRNA vaccinated mice, a single dose of either a homologous booster with WT LNP-mRNA or a heterologous booster with Omicron LNP-mRNA restored the waning antibody response against Omicron, with over 40-fold increase at two weeks post injection as compared to right before booster. Interestingly, the heterologous Omicron LNP-mRNA booster elicited neutralizing titers 10-20 fold higher than the homologous WT booster against the Omicron variant, with comparable titers against the Delta variant. All three types of vaccination, including Omicron mRNA alone, WT mRNA homologous booster, and Omicron heterologous booster, elicited broad binding antibody responses against SARS-CoV-2 WA-1, Beta, and Delta variants, as well as other Betacoronavirus species such as SARS-CoV, but not Middle East respiratory syndrome coronavirus (MERS-CoV). These data provided direct proof-of-concept assessments of an Omicron-specific mRNA vaccination in vivo, both alone and as a heterologous booster to the existing widely-used WT mRNA vaccine form. A tool for assessing alignment of biomedical data repositories with open, FAIR, citation and trustworthy principles Increasing attention is being paid to the operation of biomedical data repositories in light of efforts to improve how scientific data is handled and made available for the long term. Multiple groups have produced recommendations for functions that biomedical repositories should support, with many using requirements of the FAIR data principles as guidelines. However, FAIR is but one set of principles that has arisen out of the open science community. They are joined by principles governing open science, data citation and trustworthiness, all of which are important aspects for biomedical data repositories to support. Together, these define a framework for data repositories that we call OFCT: Open, FAIR, Citable and Trustworthy. Here we developed an instrument using the open source PolicyModels toolkit that attempts to operationalize key aspects of OFCT principles and piloted the instrument by evaluating eight biomedical community repositories listed by the NIDDK Information Network (dkNET.org). Repositories included both specialist repositories that focused on a particular data type or domain, in this case diabetes and metabolomics, and generalist repositories that accept all data types and domains. The goal of this work was both to obtain a sense of how much the design of current biomedical data repositories align with these principles and to augment the dkNET listing with additional information that may be important to investigators trying to choose a repository, e.g., does the repository fully support data citation? The evaluation was performed from March to November 2020 through inspection of documentation and interaction with the sites by the authors. Overall, although there was little explicit acknowledgement of any of the OFCT principles in our sample, the majority of repositories provided at least some support for their tenets. Accurate prediction of functional states of cis-regulatory modules reveals the common epigenetic rules in humans and mice We proposed a two-step approach for predicting active cis-regulatory modules (CRMs) in a cell/tissue type. We first predict a map of CRM loci in the genome using all available transcription factor binding data in the organism, and then predict functional states of all the putative CRMs in any cell/tissue type using few epigenetic marks. We have recently developed a pipeline dePCRM2 for the first step, and now presented machine-learning methods for the second step. Our approach substantially outperforms existing methods. Our results suggest common epigenetic rules for defining functional states of CRMs in various cell/tissue types in humans and mice. Chromosome breaks in breast cancers occur near herpes tumor virus sequences and explain why cancer drugs stop working One explanation for why breast cancer has a relentless tendency to come back after treatment is that breast cancer phenomes are fluid, so it is difficult for therapy to target new phenotypes that arise. In breast cancer, viral interactions cause chromosome breakage and fusion, producing anaphase bridges containing two centromeres. Cell division then leads to more chromosome breaks, more mutations, and new cancer phenotypes. Analyses of public data from about 2100 breast cancers implicate Epstein-Barr virus (EBV or Human Herpes Virus 4) in the cycles that break chromosomes and produce anaphase bridges. EBV generates breakage cycles with highly fragmented chromosomes (chromothripsis) as an inherent feature. These episodes continually produce new cancer driver mutations. DNA at chromothripsis boundaries in breast cancers consistently matches EBV tumor variants. Breakpoints are often near known EBV binding sites, and breast cancer shares an EBV methylation signature with other known EBV cancers. Breast cancer breakpoints on all chromosomes cluster around the same positions as in cancers definitively associated with EBV infection (nasopharyngeal cancer and Burkitts lymphoma). The status of EBV in the microbiome and the presence of anaphase bridges are phenotypic markers of breast cancer that are not explicitly targeted by chemotherapy. The breast cancer phenome continually supplies new cancer driver genes that foster breast cancer recurrence.

SummaryBreaks in breast cancer chromosomes and in fragments of anaphase bridges occur mainly near regions that resemble Epstein-Barr virus (EBV) tumor variants

Extensive breaks are an inherent outcome of anaphase bridges and the reassembly of highly fragmented chromosomes continually generates new cancer driver mutations and new cancer phenotypes.

Treatment does not explicitly target the underlying breakage and reassembly cycles so breast cancer often comes back.

The breast cancer phenome is not fixed and can continuously generate new phenotypes

Graphical Abstract

O_FIG O_LINKSMALLFIG WIDTH=154 HEIGHT=200 SRC="FIGDIR/small/467751v4_ufig1.gif" ALT="Figure 1">
View larger version (38K):
org.highwire.dtl.DTLVardef@6537cforg.highwire.dtl.DTLVardef@132fa50org.highwire.dtl.DTLVardef@1cfcb04org.highwire.dtl.DTLVardef@11979aa_HPS_FORMAT_FIGEXP  M_FIG C_FIG Aldosterone and Dexamethasone Activate African Lungfish Mineralocorticoid Receptor: Increased Activation After Removal of the Amino-Terminal Domain Aldosterone, the main physiological mineralocorticoid in humans and other terrestrial vertebrates, first appears in lungfish, which are lobe-finned fish that are forerunners of terrestrial vertebrates. Aldosterone activation of the MR regulates internal homeostasis of water, sodium and potassium, which was critical in the conquest of land by vertebrates. We studied transcriptional activation of the slender African lungfish MR by aldosterone, other corticosteroids and progesterone and find that aldosterone, 11-deoxycorticosterone, 11-deoxycortisol and progesterone have half-maximal responses (EC50s) below 1 nM and are potential physiological mineralocorticoids. In contrast, EC50s for corticosterone and cortisol were 23 nM and 66 nM, respectively. Unexpectedly, truncated lungfish MR, consisting of the DNA-binding, hinge and steroid-binding domains, had a stronger response to corticosteroids and progesterone than full-length lungfish MR, indicating that the N-terminal domain represses steroid activation of lungfish MR, unlike human MR in which the N-terminal domain contains an activation function. BLAST searches of GenBank did not retrieve a GR ortholog, leading us to test dexamethasone and triamcinolone for activation of lungfish MR. At 10 nM, both synthetic glucocorticoids are about 4-fold stronger than 10 nM aldosterone in activating full-length lungfish MR, leading us to propose that lungfish MR also functions as a GR. An alternative UPF1 isoform drives conditional remodeling of nonsense-mediated mRNA decay The nonsense-mediated mRNA decay (NMD) pathway monitors translation termination to degrade transcripts with premature stop codons and regulate thousands of human genes. Here we show that an alternative mammalian-specific isoform of the core NMD factor UPF1, termed UPF1LL, enables condition-dependent remodeling of NMD specificity. Previous studies indicate that the extension of a conserved regulatory loop in the UPF1LL helicase core confers a decreased propensity to dissociate from RNA upon ATP hydrolysis relative to the major UPF1 isoform, designated UPF1 SL. Using biochemical and transcriptome-wide approaches, we find that UPF1LL overcomes the protective RNA binding proteins PTBP1 and hnRNP L to preferentially bind and down-regulate long 3UTRs normally shielded from NMD. Unexpectedly, UPF1LL supports induction of NMD on new populations of substrate mRNAs in response to activation of the integrated stress response and impaired translation efficiency. Thus, while canonical NMD is abolished by moderate translational repression, UPF1LL activity is enhanced, providing a mechanism to rapidly rewire NMD specificity in response to cellular stress. Effects of hydroelectric dam construction on spatio-temporal changes of land use land cover in Bui National Park, Ghana The construction of hydroelectric dams in forest reserves has become a matter of concern for biodiversity conservationists. Visibly among which is the potential to cause changes in climate and land cover and subsequently affect fauna and flora composition. Spatio-temporal changes of climate and land cover in the Bui National Park was analyzed using indices calculations of the landscape based on land cover maps obtained from Landsat satellite images for pre-and post-dam construction periods. Significant changes in land cover following the dam construction were observed. Notable changes include the built-up areas and water body, which recorded an increase of 315.64 % and 4593.43 % respectively, while the forest area decreased. Significant reduction in rainfall (U = 24,{rho} < 0.05) and increase in temperature (U = 22.5,{rho} < 0.05) were observed between the pre-and post-dam construction periods. Increased human activities such as illegal mining, indiscriminate tree felling, uncontrolled cattle grazing and charcoal production within the reserve results from inadequate monitoring and law enforcement after the dam construction could likely compound the changes in land cover. Hidden dynamics of proteasome autoregulation discovered by cryo-EM data-driven deep learning The cellular functions are executed by macromolecular complexes in nonequilibrium dynamic processes, which exhibit a vast diversity of conformational states. Solving conformational continuum of important complexes at atomic level remains a key challenge in the methodology of computational biophysics. Here we introduce a deep-learning framework, named AlphaCryo4D, which enables atomic-level cryogenic electron microscopy reconstructions of nonequilibrium conformational continuum. AlphaCryo4D integrates 3D deep residual learning with manifold embedding of energy landscapes, which simultaneously improves 3D classification accuracy and reconstruction resolution via an energy-based particle-voting algorithm. Applications of this methodology to six experimental datasets demonstrated its generality in breaking resolution barrier of visualizing dynamic components of functional complexes, in choreographing continuous inter-subunit motions and in exploring their  hidden conformational space. Importantly, AlphaCryo4D identified 64 conformers of the substrate-bound human 26S proteasome and reconstituted single-nucleotide-exchange dynamics of proteasomal AAA-ATPase motor during translocation initiation, which together illuminates a grand hierarchical allostery for proteasome autoregulation. The Hippocampus uses cryptographic principles rather than plasticity to enable animals to secretly cache and retrieve their food For animals, the ability to hide and retrieve valuable information, such as the location of food, can mean the difference between life and death. Here, we propose that to achieve this, their brain uses spatial cells similarly to how we utilize encryption for data security. Some animals are able to cache hundreds of thousands of food items annually by each individual and later retrieve most of what they themselves stashed. Rather than memorizing their cache locations as previously suggested, we propose that they use a single cryptographic-like mechanism during both caching and retrieval. The model we developed is based on hippocampal spatial cells, which respond to an animals positional attention, such as when the animal enters a specific region (place-cells) or gazes at a particular location (spatial-view-cells). We know that the region that activates each spatial cell remains consistent across subsequent visits to the same area but not between areas. This remapping, combined with the uniqueness of cognitive maps, produces a persistent crypto-hash function for both food caching and retrieval. We also show that the model stores temporal information that helps animals in food caching order preference as previously observed. This behavior, which we refer to as crypto-taxis, might also explain consistent differences in decision-making when animals are faced with a large number of alternatives such as in foraging. Only 2 of 12 now-deleted genes of 19 identified and predicted Myb family DNA binding proteins are directly involved in rice Magnaporthe oryzae pathogenicity Proteins with DNA binding Myb domains have been suggested to be involved in regulating development and stress responses in Eukaryotes. Magnaporthe oryzae is considered the most destructive fungal pathogen of rice, and we screened the genome for all genes encoding proteins containing Myb domains since these could be essential during pathogenesis. We found 19 genes Myb1-19. Only a few have previously been investigated, and one has been shown to be involved in pathogenesis. We tried to delete the other 18 genes and succeeded with 12. RT-qPCR showed that all 19 genes are expressed with different expression profiles during pathogenesis. Only deletions of the genes encoding proteins MoMyb2, MoMyb13, and MoMyb15 showed growth, conidiation, or infection phenotypes, indicating that they are singly needed for successful infection. Even more important, the lack of phenotypes for 9 mutants surprised us, and we extended the analysis to expression co-regulation of all 19 Myb proteins and found 5 co-regulated groups of predicted Myb-domain proteins. We point to likely compensatory regulations of other genes compensating and hiding the effect of many deletions. Further studies of the Myb-family genes are thus of interest since revealing their functions with a possible effect on pathogenicity could be targeted for future measures to control M. oryzae in rice. Cell-specific Bioorthogonal Tagging of Glycoproteins Altered glycosylation is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function. Cell Cycle Delay, Pro-metaphase Arrest and C-metaphase Inducing Effects of Petroleum Ether Fraction of Leaf Aqueous Extract of Clerodendrum viscosum Vent. Clerodendrum viscosum is a traditionally used medicinal plant. The present study aimed to analyze cell cycle delay, pro-metaphase arrest, and c-metaphase inducing effects of the petroleum ether fraction (AQPEF) of leaf aqueous extract of C. Viscosum (LAECV). The LAECV was fractionated with petroleum ether and its metaphase arrest, cell cycle delay, and c-metaphase inducing activities were tested on A. cepa root tip cells. The AQPEF induced cell cycle delay, and colchicine like metaphase, c-metaphase, in A. cepa root tip cells. Thus, the present study explores AQPEF as an active fraction of LAECV having metaphase arresting, cell cycle delay, and c-metaphase inducing potentials. Cell-type specific circadian bioluminescence rhythms in Dbp reporter mice Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre-recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues. Robust and efficient online auditory psychophysics Most human auditory psychophysics research has historically been conducted in carefully controlled environments with calibrated audio-equipment, and over potentially hours of repetitive testing with expert listeners. Here, we operationally define such conditions as having high  auditory hygiene. Hereof, conducting auditory psychophysical paradigms online presents a serious challenge, in that results may hinge on absolute sound presentation level, reliably estimated perceptual thresholds, and sustained motivation and attention. We introduce a set of procedures that address these challenges and facilitate auditory hygiene for online auditory psychophysics. First, we establish a simple means of setting sound presentation levels. Across a set of four level-setting conditions conducted in person, we demonstrate the stability and robustness of this amplitude setting procedure in open air and controlled settings. Second, we test participants tone-in-noise thresholds using widely adopted online experiment platforms and demonstrate that reliable threshold estimates can be derived online in approximately one minute of testing. Third, using these amplitude and threshold setting procedures to establish participant-specific stimulus conditions, we show that an online implementation of the classic probe-signal paradigm can be used to demonstrate frequency-selective attention on an individual participant basis, using a third of the trials used in recent in-lab experiments. Finally, we show how threshold and attentional measures relate to well-validated assays of online participants in-task motivation, fatigue, and confidence. This demonstrates the promise of online auditory psychophysics for asking new auditory neuroscience questions quickly, efficiently, and with more diverse samples. Code for the tests is publicly available through Pavlovia and Gorilla. Production of Offspring from Azoospermic Mice with Meiotic Failure: Precise Biparental Meiosis within Halved Oocytes While the large volume of mammalian oocytes is necessary for embryo development, it can lead to error-prone chromosomal segregation during meiosis. Conversely, we hypothesized that smaller oocytes would have a great unidentified potential to stabilize unstable meiosis and improve the development of the resultant embryos. Here, we show that reducing ooplasmic volume can rescue highly error-prone fertilization using primary spermatocytes by preventing segregation errors of chromosomes during biparental meiosis. High-resolution live-imaging analysis revealed that erroneous chromosome segregation occurred in most (90%) spermatocyte-injected oocytes of normal size, but could be ameliorated to 40% in halved oocytes. The birth rate improved remarkably from 1% to 19% (P < 0.0001). Importantly, this technique enabled the production of offspring from azoospermic mice with spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans. Thus, contrary to popular opinion, oocytes inherently have a strong potential for precise meiotic divisions, which can be evoked by reduction of the ooplasmic volume. Their potential might help rescue cases of untreatable human azoospermia with spermatocyte arrest. High-throughput analysis of single human cells reveals the complex nature of DNA 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. Tardigrade CAHS Proteins Act as Molecular Swiss Army Knives to Mediate Desiccation Tolerance Through Multiple Mechanisms Tardigrades, also known as water bears, make up a phylum of small but extremely hardy animals, renowned for their ability to survive extreme stresses, including desiccation. How tardigrades survive desiccation is one of the enduring mysteries of animal physiology. Here we show that CAHS D, an intrinsically disordered protein belonging to a unique family of proteins possessed only by tardigrades, undergoes a liquid-to-gel phase transition in a concentration dependent manner. Unlike other gelling proteins, such as gelatin, our data support a mechanism in which gel formation of CAHS D is driven by intermolecular {beta}-{beta} interactions. We find that gel formation corresponds with strong coordination of water and slowing of water diffusion. The degree of water coordination correlates with the ability of CAHS D to protect lactate dehydrogenase from unfolding when dried. This implies that the mechanism for unfolding protection can be attributed to a combination of hydration and slowed molecular motion. Conversely, rapid diffusion leading to efficient molecular shielding appears to be the predominant mechanism preventing protein aggregation. Our study demonstrates that distinct mechanisms are required for holistic protection during desiccation, and that protectants, such as CAHS D, can act as molecular  Swiss Army Knives capable of providing protection through several different mechanisms simultaneously. Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation ObjectiveUltraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKC-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKC nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.

MethodsWe studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKC nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.

ResultsOur time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKC in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKC nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNF, IFN{gamma}, and IFN in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.

ConclusionsROCK regulated NETosis through modulation of PKC nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB- induced skin inflammation by attenuation of NETosis and NET-associated cytokines. 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. Parent-offspring inference in inbred populations Genealogical relationships are fundamental components of genetic studies. However, it is often challenging to infer correct and complete pedigrees even when genome-wide information is available. For example, inbreeding can obfuscate genetic differences between individuals, making it difficult to even distinguish first-degree relatives such as parent-offspring from full siblings. Similarly, genotyping errors can interfere with the detection of genetic similarity between parents and their offspring. Inbreeding is common in natural, domesticated, and experimental populations and genotyping of these populations often has more errors than in human datasets, so efficient methods for building pedigrees under these conditions are necessary. Here, we present a new method for parent-offspring inference in inbred pedigrees called SPORE (Specific Parent-Offspring Relationship Estimation). SPORE is vastly superior to existing pedigree-inference methods at detecting parent-offspring relationships, in particular when inbreeding is high or in the presence of genotyping errors, or both. SPORE therefore fills an important void in the arsenal of pedigree inference tools.

Author SummaryKnowing the genealogical relationships among individuals is critical for genetic analyses, such as for identifying the mutations that cause diseases or that contribute to valuable agricultural traits such as milk production. Although many tools exist for establishing pedigrees using genetic information, these tools fail when individuals are highly inbred, such as in domesticated animals, or in groups of people in which consanguineous matings are common. Furthermore, existing tools do not work well when genetic information has errors at levels observed in modern datasets. Here, we present a novel approach to solve these problems. Our method is significantly more accurate than existing tools and more tolerant of errors in the genetic data. We expect that our method, which is simple to use and computationally efficient, will be a useful tool in a diversity of settings, from the studies of human and natural populations, to agricultural and experimental settings.