Design of anti-SARS-CoV-2 molecules from hydroxychloroquine (HCQ)for better binding SARS-CoV-2 N protein Great efforts have led to successfully developing the spike-based vaccines but challenges still exist to completely terminate the SARS-CoV-2 pandemic. SARS-CoV-2 nucleocapsid (N) protein plays the essential roles in almost all key steps of the viral life cycle, thus representing a top drug target. Almost all key functions of N protein including liquid-liquid phase separation (LLPS) depend on its capacity in interacting with nucleic acids. Therefore, only the variants with their N proteins functional in binding nucleic acids might survive and spread in evolution and indeed, the residues critical for binding nucleic acids are highly conserved. Very recently, hydroxychloroquine (HCQ) was shown to prevent the transmission in a large-scale clinical study in Singapore but so far, no specific SARS-CoV-2 protein was experimentally identified to be targeted by HCQ. Here by NMR, we unambiguously decode that HCQ specifically binds NTD and CTD of SARS-CoV-2 N protein with Kd of 112.1 and 57.1 M respectively to inhibit their interaction with nucleic acid, as well as to disrupt LLPS essential for the viral life cycle. Most importantly, HCQ-binding residues are identical in SARS-CoV-2 variants and therefore HCQ is likely effective to them all. The results not only provide a structural basis for the anti-SARS-CoV-2 activity of HCQ, but also renders HCQ to be the first known drug capable of targeting LLPS. Furthermore, the unique structure of the HCQ-CTD complex decodes a promising strategy for further design of better anti-SARS-CoV-2 drugs from HCQ. Therefore, HCQ is a promising candidate to help terminate the pandemic.
Sequence Determines the Switch in the Fibril Forming Regions in the Low-Complexity FUS Protein and Its Variants Residues spanning distinct regions of the low-complexity domain of the RNA-binding protein, Fused in Sarcoma (FUS-LC), form fibril structures with different core morphologies. NMR experiments show that the 214 residue FUS-LC forms a fibril with an S-bend (core-1, residues 39-95), while the rest of the protein is disordered. In contrast, the fibrils of the C-terminal variant (FUS-LC-C; residues 111-214) has a U-bend topology (core-2, residues 112-150). Absence of the U-bend in FUS-LC implies that the two fibril cores do not coexist. Computer simulations show that these perplexing findings could be understood in terms of the population of sparsely-populated fibril-like excited states in the monomer. The propensity to form core-1 is higher compared to core-2. We predict that core-2 forms only in truncated variants that do not contain the core-1 sequence. At the monomer level, sequence-dependent enthalpic effects determine the relative stabilities of the core-1 and core-2 topologies.

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Endogenous but not sensory-driven activity controls migration, morphogenesis and survival of adult-born neurons in the mouse olfactory bulb The development and survival of adult-born neurons is believed to be driven by sensory signaling. By genetically manipulating excitability of adult-born cells (via cell-specific overexpression of either Kv1.2 or Kir2.1 K+ channels), longitudinal in vivo monitoring of their Ca2+ signaling and transcriptome analyses, we show that endogenous but not sensory-driven activity governs migration, morphogenesis, survival, and functional integration of adult-born juxtaglomerular neurons in the mouse olfactory bulb. The proper development of these cells required fluctuations of cytosolic Ca2+ levels, phosphorylation of CREB, and pCREB-mediated gene expression. Attenuating Ca2+ fluctuations via K+ channel overexpression strongly downregulated genes involved in neuronal migration, differentiation, and morphogenesis and upregulated apoptosis-related genes, thus locking adult-born cells in the vulnerable and immature state. Together, the data reveal signaling pathways connecting the endogenous intermittent neuronal activity/Ca2+ fluctuations as well as proper Kv1.2/Kir2.1 K+ channel function to migration, maturation, and survival of adult-born neurons. A cell cycle-dependent GARP-like transcriptional repressor regulates the initiation of differentiation in Giardia lamblia Transcriptional regulation of differentiation is critical for parasitic pathogens to adapt to environmental changes and regulate transmission. In response to encystation stimuli, Giardia lamblia cells shift from G1+S phase of the cell cycle to G2+M. By 2-4 hours, cyst wall proteins are upregulated, indicating that key regulatory steps occur within the first four hours of encystation. However, all characterized transcription factors (TFs) in Giardia have only been investigated at later time points of encystation. How TFs initiate encystation and link it to the cell cycle remains enigmatic. Here, we systematically screened six putative early upregulated TFs for nuclear localization, established their dynamic expression profiles, and determined their functional role in regulating encystation. We found a critical repressor, GLP4 that increases rapidly after 30 min of encystation stimuli and downregulates encystation specific markers, including Cyst Wall Proteins (CWPs) and enzymes in the cyst N-acetyl galactosamine (GalNAc) pathway. Depletion of GLP4 increases cyst production. Importantly, we observe that G2+M cells exhibit higher levels of CWP1 resulting from the activation of MYB2, a TF previously linked to encystation in Giardia. GLP4 upregulation occurs in G1+S cells, suggesting a role in repressing MYB2 and encystation specific genes in the G1+S phase of the cell cycle. Furthermore, we demonstrate that depletion of GLP4 upregulates MYB2 and promotes encystation while overexpression of GLP4 downregulates MYB2 and represses encystation. Together, these results suggest that Giardia employs a dose-dependent transcriptional response that involves the cell cycle regulated repressor GLP4 to orchestrate MYB2 and entry into the encystation pathway.

ImportanceTransition between life cycle stages is a common feature among parasitic pathogens and its regulation must be optimized to balance persistence of infection with transmission. The early transcription factors (TFs) regulating commitment to differentiate are totally unknown in Giardia. In this work, we identified GLP4, a previously uncharacterized GARP-like TF, as an early-acting transcriptional repressor that inhibits G1+S cells from entering the encystation pathway. GLP4 is therefore a key regulator controlling the balance between proliferative growth and terminal differentiation into infective cysts. Citation needed?Wikipedia bibliometrics during the first waveof the COVID-19 pandemic. BackgroundWith the COVID-19 pandemics outbreak, millions flocked to Wikipedia for updated information. Amid growing concerns regarding an "infodemic", ensuring the quality of information is a crucial vector of public health. Investigating if and how Wikipedia remained up to date and in line with science is key to formulating strategies to counter misinformation. Using citation analyses, we asked: which sources informed Wikipedias COVID-19-related articles before and during the pandemics first wave (January-May 2020).

ResultsWe found that coronavirus-related articles referenced trusted media sources and high-quality academic research. Moreover, despite a surge in COVID-19 preprints, Wikipedia had a clear preference for open-access studies published in respected journals and made little use of preprints. Building a timeline of English COVID-19 articles from 2001-2020 revealed a nuanced trade-off between quality and timeliness. It further showed how preexisting articles on key topics related to the virus created a framework for integrating new knowledge. Supported by a rigid sourcing policy, this "scientific infrastructure" facilitated contextualization and regulated the influx of new information. Lastly, we constructed a network of DOI-Wikipedia articles, which showed the shifting landscape of pandemic-related knowledge on Wikipedia and how academic citations create a web of shared knowledge supporting topics like COVID-19 vaccine development.

ConclusionsUnderstanding how scientific research interacts with the digital knowledge-sphere during the pandemic provides insight into how Wikipedia can facilitate access to science. It also reveals how, aided by what we term its "citizen encyclopedists", it successfully fended off COVID-19 disinformation and how this unique model may be deployed in other contexts. 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. Naturally occurring combinations of receptors from single cell transcriptomics in endothelial cells VEGF inhibitor drugs have been successful, especially in ophthalmology, but not all patients respond to them. Combinations of drugs are likely to be needed for more effective therapies of angiogenesis-related diseases. In this paper we describe naturally occurring combinations of receptors in endothelial cells that might help to understand how cells communicate and to identify targets for drug combinations. We also develop and share a new software tool called DECNEO to identify them.

Single-cell gene expression data are used to identify a set of co-expressed endothelial cell receptors, conserved among species (mice and humans) and enriched, within a network, of connections to up-regulated genes. This set includes several receptors previously shown to play a role in angiogenesis. Multiple statistical tests from large datasets, including an independent validation set, support the reproducibility, evolutionary conservation and role in angiogenesis of these naturally occurring combinations of receptors. We also show tissue-specific combinations and, in the case of choroid endothelial cells, consistency with both well-established and recent experimental findings, presented in a separate paper.

The results and methods presented here advance the understanding of signaling to endothelial cells. The methods are generally applicable to the decoding of intercellular combinations of signals. RNA polymerase II is recruited to DNA double-strand breaks for dilncRNA transcription in Drosophila DNA double-strand breaks are among the most toxic lesions that can occur in a genome and their faithful repair is thus of great importance. Recent findings have uncovered local transcription that initiates at the break and forms a non-coding transcript, called damage-induced long non-coding RNA (dilncRNA), which helps to coordinate the DNA transactions necessary for repair. We provide nascent RNA sequencing-based evidence that RNA polymerase II transcribes the dilncRNA in Drosophila and that this is more efficient for DNA breaks in an intron-containing gene, consistent with the higher damage-induced siRNA levels downstream of an intron. The spliceosome thus stimulates recruitment of RNA polymerase II to the break, rather than merely promoting the annealing of sense and antisense RNA to form the siRNA precursor. In contrast, RNA polymerase III nascent RNA libraries did not contain reads corresponding to the cleaved loci and selective inhibition of RNA polymerase III did not reduce the yield of damage-induced siRNAs. Finally, the damage-induced siRNA density was unchanged downstream of a T8 sequence, which terminates RNA polymerase III transcription. We thus found no evidence for a participation of RNA polymerase III in dilncRNA transcription in cultured Drosophila cells. Heterogeneous recruitment abilities to RNA polymerases generate nonlinear scaling of gene expression level with cell volume While most genes expression levels are proportional to cell volumes, some genes exhibit nonlinear scaling between their expression levels and cell volume. Therefore, their mRNA and protein concentrations change as the cell volume increases, which often have crucial biological functions such as cell-cycle regulation. However, the biophysical mechanism underlying the nonlinear scaling between gene expression and cell volume is still unclear. In this work, we show that the nonlinear scaling is a direct consequence of the heterogeneous recruitment abilities of promoters to RNA polymerases based on a gene expression model at the whole-cell level. Those genes with weaker (stronger) recruitment abilities than the average ability spontaneously exhibit superlinear (sublinear) scaling with cell volume. Analysis of the promoter sequences and the nonlinear scaling of Saccharomyces cerevisiaes mRNA levels shows that motifs associated with transcription regulation are indeed enriched in genes exhibiting nonlinear scaling, in concert with our model. Robust retrieval of data stored in DNA by de Bruijn graph-based de novo strand assembly DNA data storage is a rapidly developing technology with great potential due to its high density, long-term durability, and low maintenance cost. The major technical challenges include various errors, such as the strand breaks, rearrangements, and indels that frequently arise during DNA synthesis, amplification, sequencing, and preservation. Through a de novo assembly strategy, we developed an algorithm based on the de Bruijn graph and greedy path search (DBGPS) to address these issues. DBGPS shows distinct advantages in handling DNA breaks, rearrangements, and indels. The robustness of DBGPS is demonstrated by accelerated aging, multiple independent data retrievals, deep error-prone PCR, and large data scale simulations. Remarkably, 6.8 MB of data can be retrieved accurately from a seriously corrupted sample that has been treated at 70 {degrees}C for 70 days. With DBGPS, we were able to achieve a logical density of 1.30 bits/cycle and a physical density of 295 PB/g.

One-Sentence SummaryA de Bruijn graph-based de novo assembly algorithm for DNA data storage enables fast and robust data readouts even with DNA samples that have been severely corrupted. The DC1 domain protein BINUCLEATE POLLEN is required for pollen development in Arabidopsis thaliana Development of the male gametophyte is a tightly regulated process that requires precise control of cell division and gene expression. A relevant aspect to understand the events underlying pollen development regulation constitutes the identification and characterization of the genes required for this process. In this work we showed that the DC1 domain protein BINUCLEATE POLLEN (BNP) is essential for pollen development and germination. Pollen grains carrying the defective BNP allele failed to complete mitosis II and are impaired in pollen germination. By yeast two hybrid screening and bimolecular fluorescence complementation assays, we identified a set of BNP-interacting proteins. Among confirmed interactors we found NAC family transcriptional regulators Vascular plant One-Zinc finger 1 (VOZ1) and VOZ2. VOZ1 localization changes during pollen development, moving to the vegetative nucleus at the tricellular stage. We observed that this relocalization requires BNP, as in their absence in pollen from bnp/BNP plants, VOZ1 nuclear localization resulted impaired. As voz1voz2 double mutants showed the same developmental defect observed in bnp pollen grains, we propose that BNP requirement to complete microgametogenesis could be linked to its interaction with VOZ1/2 proteins. BNP could have a role of scaffold protein, recruiting VOZ1/2 to the endosomal system into assemblies that are required for their further translocation to the nucleus, where they act as transcriptional regulators. Critical re-evaluation of the slope factor of the operational model of agonism Background and PurposeAlthough being a relative term, agonist efficacy is a cornerstone in the proper assessment of agonist selectivity and signalling bias. The operational model of agonism (OMA) has become successful in the determination of agonist efficacies and ranking them. In 1985, Black et al. introduced the slope factor to the OMA to make it more flexible and allow for fitting steep as well as flat concentration-response curves.

Experimental ApproachFunctional analysis of OMA demonstrates that the slope factor implemented by Black et al. affects relationships among parameters of the OMA, leading to wrong estimates of operational efficacy and affinity of an agonist. In contrast, functional analysis of OMA with Hill coefficient as a slope factor has not this flaw.

Key ResultsFitting of the OMA with Black et al. slope factor to concentration-response curves of several models (homotropic allosteric modulation, substrate inhibition and non-competitive auto-inhibition) gives wrong estimates of operational efficacy and affinity. In contrast, fitting of the OMA modified by the Hill coefficient to concentration-response curves of these several models gives correct estimates of operational efficacy and affinity.

Conclusion and ImplicationsThe OMA modified by the Hill coefficient can be used for correct ranking of agonism and subsequent analysis, like quantification of signalling bias. Migrators Within Migrators: Exploring Transposable Element Dynamics in the Monarch Butterfly, Danaus plexippus BackgroundLepidoptera (butterflies and moths) are an important model system in ecology and evolution. A high-quality chromosomal genome assembly is available for the monarch butterfly (Danaus plexippus), but it lacks an in-depth transposable element (TE) annotation, presenting an opportunity to explore monarch TE dynamics and the impact of TEs on shaping the monarch genome.

ResultsWe find 6.21% of the monarch genome is comprised of TEs, a reduction of 6.85% compared to the original TE annotation performed on the draft genome assembly. Monarch TE content is low compared to two closely related species with available genomes, Danaus chrysippus (33.97% TE) and Danaus melanippus (11.87% TE). The biggest TE contributions to genome size in the monarch are LINEs and Penelope-like elements, and three newly identified families, r2-hero_dPle (LINE), penelope-1_dPle (Penelope-like), and hase2-1_dPle (SINE), collectively contribute 34.92% of total TE content. We find evidence of recent TE activity, with two novel Tc1 families rapidly expanding over recent timescales (tc1-1_dPle, tc1- 2_dPle). LINE fragments show signatures of genomic deletions indicating a high rate of TE turnover. We investigate associations between TEs and wing colouration and immune genes and identify a three-fold increase in TE content around immune genes compared to other host genes.

ConclusionsWe provide a detailed TE annotation and analysis for the monarch genome, revealing a considerably smaller TE contribution to genome content compared to two closely related Danaus species with available genome assemblies. We identify highly successful novel DNA TE families rapidly expanding over recent timescales, and ongoing signatures of both TE expansion and removal highlight the dynamic nature of repeat content in the monarch genome. Our findings also suggest that insect immune genes are promising candidates for future interrogation of TE-mediated host adaptation. Predicting the zoonotic capacity of mammals to transmit SARS-CoV-2 Back and forth transmission of SARS-CoV-2 between humans and animals may lead to wild reservoirs of virus that can endanger efforts toward long-term control of COVID-19 in people, and protecting vulnerable animal populations that are particularly susceptible to lethal disease. Predicting high risk host species is key to targeting field surveillance and lab experiments that validate host zoonotic potential. A major bottleneck to predicting animal hosts is the small number of species with available molecular information about the structure of ACE2, a key cellular receptor required for viral cell entry. We overcome this bottleneck by combining species ecological and biological traits with 3D modeling of virus and host cell protein interactions using machine learning methods. This approach enables predictions about the zoonotic capacity of SARS-CoV-2 for over 5,000 mammals -- an order of magnitude more species than previously possible. The high accuracy predictions achieved by this approach are strongly corroborated by in vivo empirical studies. We identify numerous common mammal species whose predicted zoonotic capacity and close proximity to humans may further enhance the risk of spillover and spillback transmission of SARS-CoV-2. Our results reveal high priority areas of geographic overlap between global COVID-19 hotspots and potential new mammal hosts of SARS-CoV-2. With molecular sequence data available for only a small fraction of potential host species, predictive modeling integrating data across multiple biological scales offers a conceptual advance that may expand our predictive capacity for zoonotic viruses with similarly unknown and potentially broad host ranges. 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. A secondary metabolite drives intraspecies antagonism in a gut symbiont that is inhibited by cell wall acetylation The mammalian microbiome encodes numerous secondary metabolite biosynthetic gene clusters, yet their role in microbe-microbe interactions is unclear. Here, we characterized two polyketide synthase gene clusters (fun and pks) in the gut symbiont Limosilactobacillus reuteri. The pks, but not the fun cluster, encodes antimicrobial activity. Forty-one out of 51 L. reuteri strains tested are sensitive to Pks products, which was independent of strains host origin. The sensitivity to Pks was also established in intraspecies competition experiments in gnotobiotic mice. Comparative genome analyses between Pks-resistant and sensitive strains identified an acyltransferase gene (act) that is unique to Pks-resistant strains. Subsequent cell wall analysis of the wild-type and the act mutant strains showed that Act acetylates cell wall components. The pks mutants lost their competitive advantage and act mutants lost their Pks resistance in vivo. Thus, our findings provide insight into how closely related gut symbionts can compete and co- exist in the gastrointestinal tract. A Novel Proposal for an Index for Regional Cerebral Perfusion Pressure - A Theoretical Approach Using Fluid Dynamics Cerebral blood flow (CBF) / cerebral blood volume (CBV) ratio derived by [15O] H2O/ CO2 and CO positron emission tomography (PET) examination has been used empirically as an index for cerebral perfusion pressure (CPP). However, this equation lacks a theoretical basis and furthermore, has not been confirmed to be proportionate to CPP, as direct measurement of local CPP is not practical.

We have developed a new index for CPP using the Poiseuille equation based on a simple model. Our model suggests that CBF/CBV2 is proportionate to CPP and that it is mathematically a more accurate index than CBF/CBV. High Spatial Resolution MALDI Imaging Mass Spectrometry ofFresh-Frozen Bone Bone and bone marrow are vital to mammalian structure, movement, and immunity. These tissues are also commonly subjected to molecular alterations giving rise to debilitating diseases like rheumatoid arthritis, osteoporosis, osteomyelitis, and cancer. Technologies such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) enable the discovery of spatially resolved chemical information in biological tissue samples to help elucidate the complex molecular processes underlying pathology. Traditionally, preparation of native osseous tissue for MALDI IMS has been difficult due to the mineralized composition and heterogenous morphology of the tissue, and compensation for these challenges with decalcification and fixation protocols can remove or delocalize molecular species. Here, sample preparation methods were advanced to enable multimodal MALDI IMS of undecalcified, fresh-frozen murine femurs allowing the distribution of endogenous lipids to be linked to specific tissue structures and cell types. Adhesive-bound bone sections were mounted onto conductive glass slides with a microscopy-compatible glue and freeze-dried to minimize artificial bone marrow damage. Subliming matrix does not induce further bone marrow cracking, and recrystallizing the deposited matrix improves lipid signal. High spatial resolution (10 m) MALDI IMS was employed to characterize lipid distributions in fresh-frozen bone, and use of complementary microscopy modalities aided tissue and cell assignments. For example, various phosphatidylcholines localize to bone marrow, adipose tissue, marrow adipose tissue, and muscle. Further, sphingomyelin(42:1) was abundant in megakaryocytes, whereas sphingomyelin(42:2) was diminished in this cell type. These data reflect the vast molecular and cellular heterogeneity indicative of the bone marrow and the soft tissue surrounding the femur. Multimodal MALDI IMS has the potential to advance bone-related biomedical research by offering deep molecular coverage with spatial relevance in a preserved native bone microenvironment. De-noising Spatial Transcriptomics Data Based on Position and Image Information Spatial transcriptomics (ST) technologies provide gene expression close to or even superior to single-cell resolution while retaining the physical locations of sequencing and often also providing matched pathology images. However, the expression data captured by ST technologies suffer from high noise levels, as a result of the shallow coverage in each sequencing unit. The extra experimental steps for preserving the spatial locations of sequencing could result in even more severe noises, compared to regular single-cell RNA-sequencing (scRNA-seq). Fortunately, such noises could be largely removed by leveraging information from the physical locations of sequencing, and the tissue and cellular organization reflected by corresponding pathology images. In this work, we demonstrated the extensive levels of noise in ST data. We developed a mathematical model, named Sprod, to impute accurate ST gene expression based on latent space and graph learning of matched location and imaging data. We comprehensively validated Sprod and demonstrated its advantages over prior methods for removing drop-outs in scRNA-seq data. We further showed that, after adequate imputation by Sprod, differential expression analyses, pseudotime analyses, and cell-to-cell interaction inferences yield significantly more informative results. Overall, we envision denoising by Sprod to become a key first step to empower ST technologies for biomedical discoveries and innovations. An ALS-associated mutant KIF5A forms oligomers and aggregates and induces neuronal toxicity KIF5A is a kinesin superfamily motor protein that transports various cargos in neurons. Mutations in Kif5a cause familial amyotrophic lateral sclerosis (ALS). These ALS mutations are in the intron of Kif5a and induce mis-splicing of KIF5A mRNA, leading to splicing out of exon 27, which in human KIF5A encodes the cargo-binding tail domain of KIF5A. Therefore, it has been suggested that ALS is caused by loss of function of KIF5A. However, the precise mechanisms regarding how mutations in KIF5A cause ALS remain unclear. Here, we show that an ALS-associated mutant of KIF5A, KIF5A({Delta}exon27), is predisposed to form oligomers and aggregates in cultured mouse cell lines. Interestingly, purified KIF5A({Delta}exon27) oligomers showed more active movement on microtubules than wild type KIF5A in vitro. Purified KIF5A({Delta}exon27) was prone to form aggregates in vitro. Moreover, KIF5A({Delta}exon27)-expressing Caenorhabditis elegans neurons showed morphological defects. These data collectively suggest that ALS-associated mutations of KIF5A are toxic gain-of-function mutations rather than simple loss-of-function mutations. Non-Newtonian Blood Rheology Impacts Left Atrial Stasis in Patient-Specific Simulations The lack of mechanically effective contraction of the left atrium (LA) during atrial fibrillation (AF) disturbs blood flow, increasing the risk of thrombosis and ischemic stroke. Thrombosis is most likely in the left atrial appendage (LAA), a small narrow sac where blood is prone to stagnate. Slow flow promotes the formation of erythrocyte aggregates in the LAA, also known as rouleaux, causing viscosity gradients that are usually disregarded in patient-specific simulations. To evaluate these non-Newtonian effects, we built atrial models derived from 4D computed tomography scans of patients and carried out computational fluid dynamics simulations using the Carreau-Yasuda constitutive relation. We examined six patients, three of whom had AF and LAA thrombosis or a history of transient ischemic attacks (TIAs). We modeled the effects of hematocrit and rouleaux formation kinetics by varying the parameterization of the Carreau-Yasuda relation and modulating non-Newtonian viscosity changes based on residence time. Comparing non-Newtonian and Newtonian simulations indicates that slow flow in the LAA increases blood viscosity, altering secondary swirling flows and intensifying blood stasis. While some of these effects can be subtle when examined using instantaneous metrics like shear rate or kinetic energy, they are manifested in the blood residence time, which accumulates over multiple heartbeats. Our data also reveal that LAA blood stasis worsens when hematocrit increases, offering a potential new mechanism for the clinically reported correlation between hematocrit and stroke incidence. In summary, we submit that hematocrit-dependent non-Newtonian blood rheology should be considered in calculating patient-specific blood stasis indices by computational fluid dynamics. Immediate and sustained effects of cobalt and zinc-containing pigments on macrophages Pigments are among the oldest nanoparticulate products known to mankind, and their use in tattoos is also very old. Nowadays, 25% American people aged 18 to 50 are tattooed, which poses the question of the delayed effects of tattoos. In this article, we investigated three cobalt (Pigment Violet 14 (purple color)) or cobalt alloy pigments (Pigment Blue 28 (blue color), Pigment Green 14 (green color)), and one zinc pigment (Pigment White 4 (white color)) which constitute a wide range of colors found in tattoos. These pigments contain microparticles and a significant proportion of nanoparticles (in either aggregate or free form). Because of the key role of macrophages in the scavenging of particulate materials, we tested the effects of cobalt- and zinc-based pigments on the J774A.1 macrophage cell line. In order to detect delayed effects, we compared two exposure schemes: acute exposure for 24 hours and an exposure for 24 hours followed by a 3-day post-exposure recovery period. The conjunction of these two schemes allowed for the investigation of the delayed or sustained effects of pigments. All pigments induced functional effects on macrophages, most of which were pigment-dependent. For example, Pigment Green 19, Pigment Blue 28, and Pigment White 4 showed a delayed alteration of the phagocytic capacity of cells. Moreover, all the pigments tested induced a slight but significant increase in TNF secretion. This effect, however, was transitory. Conversely, only Pigment Blue 28 induced a short and sustained increase in interleukin 6. Results showed that in response to bacterial stimuli (LPS), the secretion of tumor necrosis factor and interleukin 6 declined after recovery exposure with pigments. The pigments also induced persisting changes in some important macrophage membrane markers such as CD11b, an integrin contributing to cell adhesion and immunological tolerance. In conclusion, the pigments induced functional dis-orders in macrophages, which, in some cases, persist long after exposure, even at non-toxic doses. Sexual selection for males with beneficial mutations Sexual selection is the process by which traits providing a mating advantage are favoured. Theoretical treatments of the evolution of sex by sexual selection propose that it operates by reducing the load of deleterious mutations. Here, we postulate instead that sexual selection primarily acts through females preferentially mating with males carrying beneficial mutations. We used simulation and analytical modelling to investigate the evolutionary dynamics of beneficial mutations in the presence of sexual selection. We found that female choice for males with beneficial mutations had a much greater impact on genetic quality than choice for males with low mutational load. We also relaxed the typical assumption of a fixed mutation rate. For deleterious mutations, mutation rate should always be minimized, but when rare beneficial mutations can occur, female choice for males with those rare beneficial mutations could overcome a decline in average fitness and allow an increase in mutation rate. We propose that sexual selection for beneficial mutations could overcome the  two-fold cost of sex much more readily than choice for males with low mutational load and may therefore be a more powerful explanation for the prevalence of sexual reproduction than the existing theory. If sexual selection results in higher fitness at higher mutation rates and if the variability produced by mutation itself promotes sexual selection, then a feedback loop between these two factors could have had a decisive role in driving adaptation. A MALDI-TOF assay identifies nilotinib as an inhibitor of inflammation in acute myeloid leukaemia Inflammatory responses are important in cancer, particularly in the context of monocyte-rich aggressive myeloid neoplasm. We developed a label-free cellular phenotypic drug discovery assay to identify anti-inflammatory drugs in human monocytes derived from acute myeloid leukaemia (AML), by tracking several biological features ionizing from only 2,500 cells using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. A proof-of-concept screen showed that the BCR-ABL inhibitor nilotinib, but not the structurally similar imatinib, blocks inflammatory responses. In order to identify the cellular (off-)targets of nilotinib, we performed thermal proteome profiling (TPP). Unlike imatinib, nilotinib and other later generation BCR-ABL inhibitors inhibit the p38-MK2/3 signalling axis which suppressed the expression of inflammatory cytokines, cell adhesion and innate immunity markers in activated human monocytes derived from AML. Thus, our study provides a tool for the discovery of new anti-inflammatory drugs, which could contribute to the treatment of inflammation in myeloid neoplasms and other diseases.

Key PointsLabel-free cell-based assay identifies new anti-inflammatory drugs using MALDI-TOF MS. Nilotinib reduces inflammation by inhibition of MAPK14-MK2/3 signalling axis in AML. 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. Conserved organ-specific microbial assemblages in different populations of a terrestrial crab Brachyuran crabs originated in the oceans and evolved specific morphological and physiological adaptations to live in freshwater, intertidal and even terrestrial habitats but the role of a selection mechanism involving symbiotic microorganisms long these colonization processes are not known. In this work we investigated the associated microbiota of three populations of a terrestrial brachyuran crab, Chiromantes haematocheir, to find evidence of a conserved crab-specific microbiome unrelated to the population of origin and dissimilar from environmental microbial assemblages. Bacterial 16S rRNA gene and fungal ITS sequences were obtained from selected crab organs and environmental matrices to profile microbial communities. In spite of the presence of truly marine larval stages and no gregarious behaviour, favouring microbiota exchanges, we found common, organ-specific microbiota, associated to the gut and the gills of the crabs (with more than 15% of the genera detected specifically enriched only in one organ). Our results suggest an early establishment of a new common, stable microbiota in the transition from water to land.