title: Designing, optimizing, and assessing modular functional near-infrared brain imaging probes using an automated software workflow

abstract:
SignificanceThe exponential growth of research utilizing functional near-infrared spectroscopy (fNIRS) systems has led to the emergence of modular fNIRS systems composed of repeating optical source/detector modules. Compared to conventional fNIRS systems, modular fNIRS systems are more compact and flexible, making wearable and long-time monitoring possible. However, the large number of design parameters makes designing a modular probe a daunting task.

AimWe aim to create a systematic software platform to facilitate the design, characterization, and comparison of modular fNIRS probes.

ApproachOur algorithm automatically tessellates any region-of-interest using user-specified module design parameters and outputs performance metrics such as spatial channel distributions, average brain sensitivity, and sampling rate estimates of the resulting probe. Automated algorithms for spatial coverage, orientation, and routing of repeated modules are also developed.

ResultsWe developed a software platform to help explore a wide range of modular probe features and quantify their performances. We compare full-head probes using three different module shapes and highlight the trade-offs resulting from various module settings. Additionally, we show that one can apply this workflow to improve existing modular probes without needing to re-design or re-manufacture them.

ConclusionOur flexible modular probe design platform shows promise in optimizing existing modular probes and investigating future modular designs.

category: bioengineering


title: Identification of Structures for Ion Channel Kinetic Models

abstract:
Markov models of ion channel dynamics have evolved as experimental advances have improved our understanding of channel function. Past studies have examined various topologies for Markov models of channel dynamics. We present a systematic method for identification of all possible Markov model topologies using experimental data for two types of native voltage-gated ion channel currents: mouse atrial sodium and human left ventricular fast transient outward potassium currents. In addition to optional biophysically inspired restrictions on the number of connections from a state and elimination of long-range connections, this study further suggests successful models have more than minimum number of connections for set number of states. When working with topologies with more than the minimum number of connections, the topologies with three and four connections to the open state tend to serve well as Markov models of ion channel dynamics.

Significance StatementHere, we present a computational routine to thoroughly search for Markov model topologies for simulating whole-cell currents given an experimental dataset. We test this method on two distinct types of voltage-gated ion channels that function in the generation of cardiac action potentials. Particularly successful models have more than one connection between an open state and the rest of the model, and large models may benefit from having even more connections between the open state and the rest of the other states.

category: biophysics


title: Mutations in the B.1.1.7 SARS-CoV-2 spike protein reduce receptor-binding affinity and induce a flexible link to the fusion peptide

abstract:
The B.1.1.7 variant of the SARS-CoV-2 virus shows enhanced infectiousness over the wild type virus, leading to increasing patient numbers in affected areas. A number of single amino acid exchanges and deletions within the trimeric viral spike protein characterize this new SARS-CoV-2 variant. Crucial for viral entry into the host cell is the interaction of the spike protein with the cell surface receptor angiotensin-converting enzyme 2 (ACE2) as well as integration of the viral fusion peptide into the host membrane. Respective amino acid exchanges within the SARS-CoV-2 variant B.1.1.7 affect inter-monomeric contact sites within the spike protein (A570D and D614G) as well as the ACE2-receptor interface region (N501Y), which comprises the receptor-binding domain (RBD) of the viral spike protein. However, the molecular consequences of mutations within B.1.1.7 on spike protein dynamics and stability, the fusion peptide, and ACE2 binding are largely unknown. Here, molecular dynamics simulations comparing SARS-CoV-2 wild type with the B.1.1.7 variant revealed inter-trimeric contact rearrangements, altering the structural flexibility within the spike protein trimer. In addition to reduced flexibility in the N-terminal domain of the spike protein, we found increased flexibility in direct spatial proximity of the fusion peptide. This increase in flexibility is due to salt bridge rearrangements induced by the D614G mutation in B.1.1.7 found in pre- and post-cleavage state at the S2 site. Our results also imply a reduced binding affinity for B.1.1.7 with ACE2, as the N501Y mutation restructures the RBD-ACE2 interface, significantly decreasing the linear interaction energy between the RBD and ACE2.

Our results demonstrate how mutations found within B.1.1.7 enlarge the flexibility around the fusion peptide and change the RBD-ACE2 interface, which, in combination, might explain the higher infectivity of B.1.1.7. We anticipate our findings to be starting points for in depth biochemical and cell biological analyses of B.1.1.7, but also other highly contagious SARS-CoV-2 variants, as many of them likewise exhibit a combination of the D614G and N501Y mutation.


title: Expression of the cancer-associated DNA polymerase ϵ P286R  in fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication

abstract:
Somatic mutations in the proofreading domain of the replicative DNA polymerase {varepsilon} (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, microsatellite stability and a unique mutational signature, with a predominance of C > A transversions in the context TCT. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few frameshift mutations. The mutations are equally distributed across different genomic regions, but they occur within an AT-rich context. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of persistent ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol {kappa}, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype.

AUTHOR SUMMARYCancer is a genetic disease caused by mutations that lead to uncontrolled cell proliferation and other tumour properties. Defects in DNA repair or replication can lead to cancer development by increasing the likelihood that cancer-causing mutations will happen. Here we look at a pathogenic variant of a polymerase involved in genome replication (DNA polymerase POLE-P286R). This variant is associated with highly mutated cancer genomes. By introducing this mutation into the polymerase  gene of a model organism, fission yeast, we show that it causes a large increase in single base substitutions, scattered throughout the genome. The sequence context of mutations is similar in fission yeast and humans, suggesting that the yeast model is useful for understanding how POLE-P286R causes such a high mutation rate. Yeast POLE-P286R cells show slow chromosome replication, suggesting that the polymerase has difficulty in copying certain chromosomal regions. Yeast POLE-P286R cells become inviable when the concentration of dNTP building blocks for DNA synthesis is increased, probably because the mutation rate is pushed to an intolerable level. Interestingly, we find that specialised polymerases that are tolerant of DNA damage contribute to the high mutation rate caused by POLE-P286R. These findings have implications for the therapy of POLE-P286R tumours.

category: cancer biology


title: The molecular interplay of the establishment of an infection - gene expression of Diaphorina citri gut and Candidatus Liberibacter asiaticus

abstract:
Candidatus Liberibacter asiaticus (CLas) is one the causative agents of greening disease in citrus, an unccurable, devastating disease of citrus worldwide. CLas is vectored by Diaphorina citri, and the understanding of the molecular interplay between vector and pathogen will provide additional basis for the development and implementation of successful management strategies. We focused in the molecular interplay occurring in the gut of the vector, a major barrier for CLas invasion and colonization. We investigated the differential expression of vector and CLas genes by analyzing a de novo reference metatranscriptome of the gut of adult psyllids fed of CLas-infected and healthy citrus plants for 1-2, 3-4 and 5-6 days. CLas regulates the immune response of the vector affecting the production of reactive species of oxygen and nitrogen, and the production of antimicrobial peptides. Moreover, CLas overexpressed peroxiredoxin in a protective manner. The major transcript involved in immune expression was related to melanization, a CLIP-domain serine protease we believe participates in the wounding of epithelial cells damaged during infection, which is supported by the down-regulation of pangolin. We also detected that CLas modulates the gut peristalsis of psyllids through the down-regulation of titin, reducing the elimination of CLas with faeces. The up-regulation of the neuromodulator arylalkylamine N-acetyltransferase implies CLas also interferes with the double brain-gut communication circuitry of the vector. CLas colonizes the gut by expressing two Type IVb pilin flp genes and several chaperones that can also function as adhesins. We hypothesized biofil formation occurs by the expression of the cold shock protein of CLas. We also describe the interplay during cell invasion and modification, and propose mechanisms CLas uses to invade the host hemocel. We identified several specific targets for the development of strategies directed to interfere with the successful utilization of the psyllid vector by this pathogen.

Author SummaryHuanglongbing (HLB) or greening is an incurable disease causing severe damage to citrus production, making citrus industrial activity unsustainable in several countries around the world. HLB is caused by three species of Candidatus Liberibacter. Ca. L. asiaticus (CLas), vectored by the psyllid Diaphorina citri, is the prevalent species. Attempts to apply new technologies in the development of strategies for disease and pest management are been made. However, we still miss basic information on this system to efficiently apply the current technologies and envisage the implementation of new approaches for pest control, despite the relevant scientific contribution available. One major gap is regarded to the molecular interplay between CLas and its vector. We focused our attention in the molecular interplay occurring at the first relevant interaction of CLas and D. citri, represented by the gut barrier. We report the transcriptional activity of CLas during the invasion and establishment of the infection in the gut of the vector, as well as the transcriptional activity of the vector in response to the infection. We identified several host genes that are targeted and regulated by CLas as well as several CLas genes that are promising targets for the application of new management strategies.

category: pathology


title: Transcriptome analysis reveals regulatory networks and hub genes in flavonoid metabolism of Rosa roxburghii

abstract: Rosa roxburghii Tratt, the most popular fruit that blooms in the southwest of China, is rich in flavonoids. However, the regulatory network and critical genes involved in the metabolism of flavonoid compounds in R. roxburghii are still unknown. In this study, we revealed that flavonoid, anthocyanin and catechin accumulated at different levels in various tissues of R. roxburghii. We further obtained and analyzed differentially expressed genes (DEGs) involved in flavonoid metabolism from five samples of R. roxburghii by transcriptome sequencing. A total of 1 130 DEGs were identified, including 166 flavonoid pathway biosynthesis genes, 622 transcription factors, 301 transporters, and 221 cytochrome P450 proteins. A weighted gene co-expression network analysis (WGCNA) of the DEGs uncovered different co-expression networks. In terms of biosynthesis enzymes, cytochrome P450 CYP749A22 and CYP72A219 were highlighted in regulation flavonoids content. Anthocyanin 3-O-glucosyltransferase and F3H were the top two critical enzymes for anthocyanin content. In contrast, caffeic acid 3-O-methyltransferase, 4-coumarate-CoA ligase, and shikimate O-hydroxycinnamoyl transferase were essential for catechin accumulation. Additionally, the eigengene network of the "black" module had high correlations with total flavonoid (r= 0.9, p=5e-06). There were 26 eigengenes in the "black" module, including six flavonoid biosynthesis, 14 TFs and six transporters. Among them, besides cytochrome P450 proteins (DN136557_c0_g1, DN135573_c0_g1 and DN145971_c4_g1), isoflavone-hydroxylase (DN143321_c3_g1) was crucial for total flavonoids content based on the high degree of connectivity. The transcription factors RrWRKY45 (DN142829_c1_g5), RrTCP20 (DN146443_c1_g1) and RrERF118 (DN141507_c3_g2) were significantly correlated with flavonoids in R. roxburghii. The present transcriptomic and biochemical data on metabolites should encourage further investigation on functional genomics and breeding of R. roxburghii with strong pharmaceutical potential.

category: plant biology


title: Maize RAMOSA3 accumulates in nuclear condensates enriched in RNA POLYMERASE II isoforms during the establishment of axillary meristem determinacy.

abstract: Brief AbstractMaize meristem determinacy is regulated by Trehalose-6-Phosphate Phosphatase (TPP) metabolic enzymes RAMOSA3 and TPP4. However, this function is independent of their enzymatic activity, suggesting they have an unpredicted, or moonlighting function. Using whole-mount double immunolabeling and imaging processing, we investigated the co-localization of RA3 nuclear speckles with markers for transcription, chromatin state and splicing. We find evidence for RA3 co-localization with RNA POL II, a transcription marker, and not with markers for promoter chromatin remodeling or mRNA processing, suggesting a function of nuclear RA3 in mediating a transcriptional response during meristem determinacy.


title: A Toolbox and Crowdsourcing Platform for Automatic Labeling of Independent Components in Electroencephalography (ALICE)

abstract:
Independent Component Analysis (ICA) is a conventional approach to exclude non-brain signals such as eye-movements and muscle artifacts from electroencephalography (EEG). Due to other possible EEG contaminations, a rejection of independent components (ICs) is usually performed in semiautomatic mode and requires experts involvement. Noteworthy, as also revealed by our study, experts opinion about the nature of a component often disagrees highlighting the need to develop a robust and sustainable automatic system for EEG ICs classification. The current article presents a toolbox and crowdsourcing platform for Automatic Labeling of Independent Components in Electroencephalography (ALICE) available via link http://alice.adase.org/. The ALICE toolbox aims to build a sustainable algorithm not only to remove artifacts but also to find specific patterns in EEG signals using ICA decomposition based on accumulated experts knowledge.

The difference from previous toolboxes is that the ALICE project will accumulate different benchmarks based on crowdsourced visual labeling of ICs collected from publicly available and in-house EEG recordings. The choice of labeling is based on estimation of IC time-series, IC amplitude topography and spectral power distribution. The platform allows supervised ML model training and re-training on available data subsamples for better performance in specific tasks (i.e. movement artifact detection in healthy or autistic children). Also, current research implements the novel strategy for consentient labeling of ICs by several experts. The provided baseline model shows that it can be used not only for detection of noisy IC but also for automatic identifications of components related to the functional brain oscillations such as alpha and mu-rhythm.

The ALICE project implies the creation and constant replenishment of the IC database, which will be used for continuous improvement of ML algorithms for automatic labeling and extraction of non-brain signals from EEG. The toolbox and current dataset are open-source and freely available to the researcher community.

category: neuroscience


title: Visualizing the organization and differentiation of the male-specific nervous system of C. elegans

abstract: Sex differences in the brain are prevalent throughout the animal kingdom and particularly well appreciated in the nematode C. elegans. While 294 neurons are shared between the two sexes, the nervous system of the male contains an additional 93 malespecific neurons, most of which have received very little attention so far. To make these neurons amenable for future study, we describe here how a multicolor, multipromoter reporter transgene, NeuroPAL, is capable of visualizing the distinct identities of all male specific neurons. We used this tool to visualize and characterize a number of features of the male-specific nervous system. We provide several proofs of concept for using NeuroPAL to identify the sites of expression of gfp-tagged reporter genes. We demonstrate the usage of NeuroPAL for cellular fate analysis by analyzing the effect of removal of developmental patterning genes, including a HOX cluster gene (egl-5), a miRNA (lin-4) and a proneural gene (lin-32/Ato), on neuronal identity acquisition within the male-specific nervous system. We use NeuroPAL and its intrinsic cohort of more than 40 distinct differentiation markers to show that, even though male-specific neurons are generated throughout all four larval stages, they execute their terminal differentiation program in a coordinated manner in the fourth larval stage that is concomitant with male tale retraction. This wave of differentiation couples neuronal maturation programs with the appearance of sexual organs. We call this wave "just-in-time" differentiation by its analogy to the mechanism of "just-in-time" transcription of metabolic pathway genes.


title: Descending inhibition of itch by neurokinin 1 receptor (Tacr1) -expressing ON cells in the rostral ventromedial medulla

abstract: The rostral ventromedial medulla (RVM) is important in descending modulation of spinal nociceptive transmission, but it is unclear if the RVM also modulates spinal pruriceptive transmission. RVM ON cells are activated by noxious algesic and pruritic stimuli and are pronociceptive. Many RVM-spinal projection neurons express the neurokinin-1 receptor (Tacr1), and ON-cells are excited by local administration of substance P (SP). We hypothesized that Tacr1-expressing RVM ON cells exert an inhibitory effect on itch opposite to their pronociceptive action. Intramedullary microinjection of SP significantly potentiated RVM ON cells and reduced pruritogen-evoked scratching while producing mild mechanical sensitization. Chemogenetic activation of RVM Tacr1-expressing RVM neurons also reduced acute pruritogen-evoked scratching. Optotagging experiments confirmed RVM Tacr1-expressing neurons to be ON cells. We conclude that Tacr1-expressing ON cells in RVM play a significant role in the modulation of pruriceptive transmission.


title: Inhibition of itch by neurokinin 1 receptor (Tacr1) -expressing ON cells in the rostral ventromedial medulla


title: Antibody response to SARS-CoV-2 mRNA vaccines in pregnant women and their neonates

abstract: Pregnant women were excluded from initial clinical trials for COVID-19 vaccines1-2, thus the immunologic response to vaccination in pregnancy and the transplacental transfer of maternal antibodies are just beginning to be studied4-5.

category: immunology


title: The lysine-rich intracellular loop and cell type-specific co-factors are required for IFITM3 antiviral immunity in hematopoietic stem cells

abstract: The interferon-induced transmembrane protein 3 (IFITM3) inhibits lentiviral gene therapy vector entry into hematopoietic stem cells and can be overcome by Cyclosporine H (CsH), but underlying mechanisms remain unclear. Here, we show that mutating the evolutionarily conserved lysines of the IFITM3 intracellular loop abolishes its antiviral activity without affecting either its localization or its degradation by CsH through non-canonical lysosomal pathways. When confined to the plasma membrane, the lysine-competent IFITM3 lost restriction against VSV-G pseudotyped viral vectors but gained antiviral activity against vectors that fuse directly at the plasma membrane. Interestingly, altering the lysines did not alter IFITM3 homodimerization but impacted higher-order protein complex formation, suggesting loss of interaction with cellular co-factors. In agreement, IFITM3 expression was not sufficient to restrict viral vectors in myeloid K562 cells as opposed to promonocytic THP1 or primary HSC. We exclude the involvement of previously identified factors affecting IFITM3 biology and propose a novel model for IFITM3 restriction that depends on the presence of cellular co-factor(s) that may interact with IFITM3 through the intracellular loop lysine residues. Overall, our work provides significant insight into the mechanisms of action of IFITM3 and CsH that can be exploited for improved gene therapies and broadly acting antiviral strategies.

category: microbiology


title: The strength of the innate immune response drives the within-host competition between highly and low pathogenic avian influenza viruses

abstract: Highly pathogenic avian influenza viruses (HPAIV) emerge from low pathogenic avian influenza viruses (LPAIV) through the introduction of basic amino acids at the hemagglutinin (HA) cleavage site. Following viral evolution, the newly formed HPAIV likely represents a minority variant within the index host, predominantly infected with the LPAIV precursor. Using reverse-genetics engineered H5N8 viruses differing solely at the HA cleavage, we tested the hypothesis that the interaction between the minority HPAIV and the majority LPAIV could modulate the risk of HPAIV emergence and that the nature of the interaction could depend on the host species. In chickens, we observed that the H5N8LP increased H5N8HP replication and pathogenesis. By contrast, the H5N8LP antagonized H5N8HP replication and pathogenesis in ducks. Ducks mounted a more potent antiviral innate immune response than chickens against the H5N8LP, which correlated with H5N8HP inhibition. These data provide experimental evidence that HPAIV may be more likely to emerge in chickens than in ducks and underscore the importance of within-host viral variants interactions in viral evolution.


title: Host factors drive the within-host competition between highly and low pathogenic H5N8 avian influenza viruses


title: Elucidating the crosstalk between inflammation and DNA damage pathways in the pancreatic beta-cell through the diabetes susceptibility gene, TCF19

abstract: TCF19 is a gene that is associated with both type 1 diabetes (T1DM) and type 2 diabetes (T2DM) in genome-wide association studies. Prior studies have demonstrated that TCF19 knockdown impairs {beta}-cell proliferation and increases apoptosis. However, little is known about its role in diabetes pathogenesis or the effects of TCF19 gain-of-function. The aim of this study was to examine the impact of TCF19 overexpression in INS-1 {beta}-cells on proliferation and gene expression. With TCF19 overexpression, there was an increase in nucleotide incorporation without any change in cell cycle gene expression, alluding to an alternate process of nucleotide incorporation. Analysis of RNAseq of TCF19 overexpressing cells revealed increased expression of several DNA damage response (DDR) genes, as well as a tightly linked set of genes involved in cell stress, immune system processes, and inflammation. This connectivity between DNA damage and inflammatory gene expression has not been well studied in the {beta}-cell, and suggests a novel role for TCF19 in regulating these pathways. Future studies determining how TCF19 may modulate these pathways may provide potential targets for {beta}-cell survival.

category: molecular biology


title: Rose Extract Treatment on the CD4+ T lymphocytes

abstract:
Background and AimsTo study the effect of rose extract on CD4+T lymphocytes, and assess the cytokines response after cell treatment. In our previous study on endothelial cells, the rose extract reduced the secretion of inflammatory markers significantly.

MethodsThe red rose extract used in this study was prepared and stored until use at -20{degrees}C. T cells were seeded in 96-well plates at 313500 cells/well in 100l of cell culture medium in duplicate, one half of the wells were used for biomarkers screening in the culture medium, and the other half for cytotoxicity assay. 24h after plating, the cells were treated in duplicate with 100l of red rose extract diluted at 0.5%, 0.1%, 0.05%, 0.01% and 0.005% (v/v) in cell culture medium or with culture medium only as control for 72h. Some other wells were for untreated cells, and cells treated with rose extract at 0.005% for 48h incubation time. After 48h and 72h, the corresponding wells were used for the cytotoxicity assay and from the duplicate wells, the cell culture media were collected and stored at -80{degrees}C until the biomarkers screening assay.

ResultsCytotoxicity assay revealed insignificant changes. IFN-gamma, MCP-1, GRO, RANTES and TIMP, Angiopoietin 1 and MMP-9 were elevated. Except MMP-9 which had fold changes >2 other cytokines were minimally elevated at various concentrations and timing of rose extract treatment. None of the cytokines were less than 0.8-fold.

ConclusionsUnlike in the endothelial cells, there is mild elevation in few inflammatory markers on T lymphocytes treatment by rose extract. Further studies need to be performed to estimate the clinical relevance.

category: cell biology


title: Integrative multi-omics analysis reveals conserved hierarchical mechanisms of FOXO3 pioneer-factor activity

abstract: FOXO transcription factors are critical for cellular homeostasis and have been implicated in longevity in several species. Yet how these factors directly affect aging, particularly in humans, is not well understood. Here, we take an integrated multi-omics approach to identify the chromatin-level mechanisms by which FOXO3 coordinates transcriptional programs. We find that FOXO3 functions as a pioneer factor in human cells, directly altering chromatin accessibility to regulate gene expression. Unexpectedly, FOXO3s pioneer activity at many sites is achieved through a two-step process, in which chromatin accessibility is initially reduced, then transitions to an open conformation. The direct FOXO3 network comprises chromatin remodelers, including the SWI/SNF remodeling complex, which we find is functionally required for FOXO3 activity. We also identify a novel secondary network of activator protein-1 (AP-1) transcription factors deployed by FOXO3, which orchestrate a neuronal-specific subnetwork. Together, this hierarchical FOXO3 pioneer network regulates key cellular processes including metabolism, proteostasis, epigenetics and proliferation, which must be tightly controlled under changing conditions that accompany aging.


title: Anuran Heart Metamorphosis: Anatomical Support for Pulmonary Blood Separation in the Early Aquatic Phase

abstract:
1.BackgroundIn both larval and adult anurans, blood separation and respiratory physiology have remained an enigma. While various blood separation mechanisms have been proposed, the same structure is seen as playing a key role: the conus arteriosus. However, previous findings on its internal structure are contradictory, depending on the specifics of the 2D imaging methods used by different authors. To resolve this problem, we used high-resolution X-ray microtomography of whole Bufo bufo specimens to acquire the first detailed 3D descriptions of this complex structure through metamorphosis.

ResultsIn early tadpoles two small valvular openings develop at the ventricular-conal junction, providing two paths separated by the septum coni and continuing into the aortic arches. Thus, structures to support segregated pulmonary circulation are fully developed well before the lungs appear. The external gills undergo partial resorption and retreat asymmetrically into a gill chamber formed by a hyoidal cover, leaving only a single opening on the left side, the opercular spout.

ConclusionsThe timing of events in Bufo circulatory development does not track the changing modes of respiration used by the developing tadpole. In particular, a system capable of double circulation carries only oxygen-depleted blood for a significant portion of the tadpole stage.

category: developmental biology


title: Colony stimulating factor 1 signaling regulates myeloid fates in zebrafish via distinct action of its receptors and ligands

abstract:
Macrophage colony-stimulating factor receptor (M-CSFR/CSF1R) signaling is crucial for the differentiation, proliferation, and survival of myeloid cells. Therapeutic targeting of the CSF1R pathway is a promising strategy in many human diseases, including neurological disorders or cancer. Zebrafish are commonly used for human disease modeling and preclinical therapeutic screening. Therefore, it is necessary to understand the proper function of cytokine signaling in zebrafish to reliably model human-related diseases. Here, we investigate the roles of zebrafish Csf1rs and their ligands - Csf1a, Csf1b and Il34, in embryonic and adult myelopoiesis. The proliferative effect of exogenous Csf1a on embryonic macrophages is connected to both receptors as it is diminished in both csf1ra{Delta}5bp and csf1rb{Delta}4bp mutants. There is no evident effect of Csf1b in zebrafish embryonic myelopoiesis. Further, we uncover an unknown role of Csf1rb in zebrafish granulopoiesis. Deregulation of Csf1rb signaling leads to failure in myeloid differentiation resulting in neutropenia throughout the whole lifespan. Surprisingly, Il34 signaling through Csf1rb seems to be of high importance as both csf1rb{Delta}4bp and il34{Delta}5bp deficient zebrafish larvae lack granulocytes. Our single-cell RNA sequencing analysis of adult whole kidney marrow (WKM) hematopoietic cells suggests that csf1rb is expressed mainly by blood and myeloid progenitors and that the expression of csf1ra and csf1rb is non-overlapping. We point out differentially expressed genes important in hematopoietic cell differentiation and immune response in selected WKM populations. Our findings could improve the understanding of myeloid cell function and lead to the further study of CSF1R pathway deregulation in disease, mostly in cancerogenesis.

Key pointsO_LIcsf1ra and csf1rb are indispensable for macrophage differentiation and together with csf1a regulate embryonic macrophage fates in zebrafish
C_LIO_LIcsf1rb is important for granulocyte differentiation and migration and together with il34 it regulates embryonic granulocytic fates in zebrafish
C_LI


title: De novo mutations in domestic cat are consistent with an effect of reproductive longevity on both the rate and spectrum of mutations

abstract: The mutation rate is a fundamental evolutionary parameter with direct and appreciable effects on the health and function of individuals. Here, we examine this important parameter in the domestic cat, a beloved companion animal as well as a valuable biomedical model. We estimate a mutation rate of 0.86 x 10-8 per bp per generation for the domestic cat (at an average age of 3.8 years). We find evidence for a strong paternal age effect, with more mutations transmitted by older sires. Our analyses suggest that the cat and the human have accrued similar numbers of mutations in the germline before reaching sexual maturity. The per-generation mutation rate in the cat is slightly lower than what has been observed in humans, but consistent with the shorter generation time in the cat. Using a model of reproductive longevity, which takes into account differences in the reproductive age and time to sexual maturity, we are able to explain much of the difference in per-generation rates between species. We further apply our reproductive longevity model in a novel analysis of mutation spectra and find that the spectrum for the cat resembles the human mutation spectrum at a younger age of reproduction. Together, these results implicate changes in life-history as a driver of mutation rate evolution between species. As the first direct observation of the paternal age effect outside of primates, our results also suggest a phenomenon that may be universal among mammals.

category: evolutionary biology


title: Captive-breeding and catch-and-release's effects on the reproductive success of Atlantic salmon (Salmo salar L.)

abstract: Captive-breeding programs as well as and catch-and-release are among the most commonly adopted conservation practices in recreational fisheries. However, risks and benefits associated with their implementation are rarely evaluated. In the case of Atlantic Salmon, while previous studies revealed that captive-bred fish show reduced fitness compared to their wild counterparts in nature. Yet, few examined the extent and causes of their reduced reproductive success or directly compared their contribution to enhance genetic diversity to that of wild fish, including mature male parr. Furthermore, only one study specifically measured the reproductive success of caught and released Atlantic salmon in natural settings, and no study to date evaluated if released salmon are able to reproduce when released at temperature above 20{degrees}C which is known to increase post-release mortality. Here, we use high-throughput microsatellite sequencing of 38 loci to accurately assign 2500 offspring to a comprehensive set of possible parents from a supplemented Atlantic salmon population in Quebec, Canada. The resolved molecular pedigree provided informative insight on the reproductive pattern of both captive-bred salmon and caught-and-released salmon. Captive-bred salmon had fewer partners than their wild conspecifics which lead to a significant reduction of reproductive success relative to that of their wild counterparts. Supplementation of captive-bred salmon significantly contributed to increase genetic diversity but mature male parr did so to an even greater extent and significantly inflated the number of alleles found among offspring. Moreover, our results showed that that at least 83% of caught-and-released salmon did successfully reproduced although caught-and-released female salmon have a significantly reduced reproductive success, averaging 73% of the reproductive output of non-caught salmon. Reproductive success of released salmon was not influenced by water temperature over 20{degrees}C which suggests either that the studied population is locally adapted to warm waters or that they behaviorally regulated body temperature by accessing nearby thermal refugia. Our results should help refining managers ability to analyze the risks and benefits associated with captive-breeding and catch-and-release, and thus, optimize conservation practices used for the preservation of Atlantic salmon populations.


title: Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity

abstract:
SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA {approx} EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5 position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein.

Author SummaryThe novel coronavirus SARS-CoV-2, the causative agent of the world-wide pandemic of COVID-19, has become one of the greatest threats to human health. While rapid progress has been made in the development of vaccines, drug discovery has lagged, partly due to the lack of atomic-resolution structures of the free and drug-bound forms of the viral proteins. The SARS-CoV-2 envelope (E) protein, with its multiple activities that contribute to viral replication, is widely regarded as a potential target for COVID-19 treatment. As structural information is essential for drug discovery, we established an efficient sample preparation system for biochemical and structural studies of intact full-length SARS-CoV-2 E protein and characterized its structure and dynamics. We also characterized the interactions of amilorides with specific E protein residues and correlated this with their antiviral activity during viral replication. The binding affinity of the amilorides to E protein correlated with their antiviral potency, suggesting that E protein is indeed the likely target of their antiviral activity. We found that residue asparagine15 plays an important role in maintaining the conformation of the amiloride binding site, providing molecular guidance for the design of inhibitors targeting E protein.

category: biochemistry


title: Deep oncopanel sequencing reveals fixation time- and within block position-dependent quality degradation in FFPE processed samples

abstract: Clinical laboratories routinely use formalin-fixed paraffin-embedded (FFPE) tissue or cell block cytology samples in oncology panel sequencing to identify mutations that can predict patient response to targeted therapy. To understand the technical error due to FFPE processing, a robustly characterized normal cell line was used to create FFPE samples with four different pre-tissue processing formalin fixation times. A total of 96 FFPE sections were then distributed to different laboratories for targeted sequencing analysis by four oncopanels, and variants resulting from technical error were identified. Tissue sections that failed more frequently showed low cellularity, lower than recommended library preparation DNA input, or target sequencing depth. Importantly, sections from block surfaces were more likely to show FFPE-specific errors, akin to "edge effects" seen in histology, and the depth of formalin damage was related to fixation time. To assure reliable results, we recommend avoiding the block surface portion and restricting mutation detection to genomic regions of high confidence.


title: Potassium channel-driven bioelectric signaling regulates metastasis in triple-negative breast cancer

abstract: There is a critical need to better understand the mechanisms that drive local cell invasion and metastasis to develop new therapeutics targeting metastatic disease. Bioelectricity is an important mediator of cellular processes and changes in the resting membrane potential (RMP) are associated with increased cancer cell invasion. However, the mechanism is not well understood. Our data demonstrate that altering the RMP of triple-negative breast cancer (TNBC) cells by manipulating potassium channel expression increases in vitro invasion, in vivo tumor growth, and metastasis, and is accompanied by changes in gene expression associated with cell adhesion. We describe a novel mechanism for RMP-mediated cell migration involving cadherin-11 and the MAPK pathway. Importantly, we identify a new strategy to target metastatic TNBC in vivo by repurposing FDA-approved potassium channel blockers. Our results provide an understanding of the mechanisms by which bioelectricity regulates cancer cell invasion and metastasis that could lead to a new class of therapeutics for patients with metastatic disease.


title: The Fatty Acid Methyl Ester (FAME) profile of Phytophthora agathidicida and its potential use as diagnostic tool.

abstract: Phytophthora diseases cause devastation to crops and native ecosystems worldwide. In New Zealand, Phytophthora agathidicida is threatening the survival of kauri, an endemic, culturally and ecologically important tree species. The current method for detecting P. agathidicida is a soil bating assay that is time-consuming and requires high levels of expertise to assess, thus limiting the analytical sample throughput. Here, we characterized the fatty acid methyl ester (FAME) profile of P. agathidicida. We also compared it with the FAME profile of P. cinnamomi and assessed the efficacy of FAME analysis as a diagnostic tool for detecting the pathogen in soil samples. In FAME analysis, the total fatty acid content is isolated from a sample and converted to FAMEs for analysis, a process that takes less than a day. Unique fatty acid acyl chains can serve as biomarkers for specific organisms. We detected 12 fatty acids in P. agathidicida, two of which (20:4{omega}6 and 20:5{omega}3) show promise as potential Phytophthora specific biomarkers. Collectively, these findings advance our fundamental understanding of P. agathidicida biology and provide a promising technique to increase the rate of sample processing and the speed of pathogen detection for P. agathidicida in soil.


title: LncRNA-UCA1 Alleviates Septic Acute Kidney Injury through Regulating Endoplasmic Reticulum Stress in LPS-treated HK-2 Cells

abstract:
ObjectiveSeptic acute kidney injury (AKI) is an important cause of death in patients with sepsis. This study sought to explore the function of the long noncoding RNA, urothelial carcinoma associated 1 (lncRNA-UCA1), in septic AKI and determine the underlying molecular mechanism.

MethodsHK-2 cells were treated with lipopolysaccharide (LPS) to establish an in vitro model of septic AKI. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of lncRNA-UCA1. CCK-8 assay was used to detect the viability of HK-2 cells. Western blotting was utilized to examine protein expression. The contents of SOD, GSH, MDA, and ROS were determined using commercial kits. The apoptosis rate was calculated using TUNEL staining and flow cytometry.

ResultsLncRNA-UCA1 was down-regulated in LPS-treated HK-2 cells. LPS significantly reduced the content of SOD and GSH in HK-2 cells, increased the production of MDA and ROS, and led to an increase in the rate of apoptosis. However, overexpression of lncRNA-UCA1 protected HK-2 cells from oxidative stress and apoptosis. Furthermore, LPS induced endoplasmic reticulum (ER) stress in HK-2 cells, which was inhibited by overexpression of lncRNA-UCA1.

ConclusionOverexpression of lncRNA-UCA1 inhibited LPS-induced oxidative stress and apoptosis of HK-2 cells by suppressing ER stress.


title: Spatial expression programs of the intestinal follicle-associated epithelium

abstract: The intestine is lined with isolated lymphoid follicles (ILFs) that facilitate sampling of luminal antigens to elicit immune responses. Technical challenges related to the scarcity and small sizes of ILFs and their follicle-associated epithelium (FAE) impeded the characterization of their spatial gene expression programs. Here, we combined RNA sequencing of laser capture microdissected tissues with single molecule transcript imaging to obtain a spatial gene expression map of the ILF and its associated FAE in the mouse small intestine. We identified zonated expression programs in both follicles and FAEs, with a decrease in enterocyte anti-microbial and absorption programs and a partial induction of expression programs normally observed at the villus tip. We further identified Lepr+ sub-epithelial telocytes at the FAE top, which are distinct from villus-tip Lgr5+ telocytes. Our analysis exposes the epithelial and mesenchymal cell states associated with ILFs.


title: ALT Neuroblastoma Chemoresistance due to ATM Activation by Telomere Dysfunction is Reversible with the ATM Inhibitor AZD0156

abstract:
Cancers overcome replicative immortality by activating either telomerase or an alternative lengthening of telomeres (ALT) mechanism. ALT occurs in [~] 25% of high-risk neuroblastomas and relapse or progression in ALT neuroblastoma patients during or after front-line therapy is frequent and almost uniformly fatal. Temozolomide + irinotecan is commonly used as salvage therapy for neuroblastoma. Patient-derived cell-lines and xenografts established from relapsed ALT neuroblastoma patients demonstrated de novo resistance to temozolomide + irinotecan (as SN-38 in vitro, P<0.05) and in vivo (mouse event-free survival (EFS) P<0.0001) relative to telomerase-positive neuroblastomas. We observed that ALT neuroblastoma cells manifest constitutive ATM kinase activation due to spontaneous telomere dysfunction while telomerase- positive tumors lacked constitutive ATM activation or spontaneous telomere DNA damage. We demonstrated that induction of telomere dysfunction resulted in ATM activation that in turn conferred resistance to temozolomide + SN-38 (4.2 fold-change in IC50, P<0.001). ATM kinase shRNA knock-down or inhibition using a clinical-stage small molecule inhibitor (AZD0156) reversed resistance to temozolomide + irinotecan in ALT neuroblastoma cell-lines in vitro (P<0.001) and in 4 ALT xenografts in vivo (EFS P<0.0001). AZD0156 showed modest to no enhancement of temozolomide + irinotecan activity in telomerase-positive neuroblastoma cell lines and xenografts. ATR inhibition using AZD6738 did not enhance temozolomide + SN-38 activity in ALT neuroblastoma cell lines. Thus, resistance to chemotherapy in ALT neuroblastoma occurs via ATM kinase activation and was reversed with the ATM inhibitor AZD0156. Combining AZD0156 with temozolomide + irinotecan warrants clinical testing in neuroblastoma.

One Statement SummaryATM activation at telomeres confers resistance to DNA damaging chemotherapy in ALT neuroblastoma that was reversed with ATM knockdown or inhibition.


title: Intron Losses and Gains in Nematodes: Not Eccentric at All

abstract: The evolution of spliceosomal introns has been widely studied among various eukaryotic groups. Researchers nearly reached the consensuses on the pattern and the mechanisms of intron losses and gains across eukaryotes. However, according to previous studies that analyzed a few genes or genomes of nematodes, Nematoda seem to be an eccentric group. Taking advantage of the recent accumulation of sequenced genomes, we carried out an extensive analysis on the intron losses and gains using 104 nematodes genomes across all the five Clades of the phylum. Nematodes have a wide range of intron density, from less than one to more than nine per 1kbp coding sequence. The rates of intron losses and gains exhibit significant heterogeneity both across different nematode lineages and across different evolutionary stages of the same lineage. The frequency of intron losses far exceeds that of intron gains. Five pieces of evidence supporting the model of cDNA-mediated intron loss have been observed in ten Caenorhabditis species, the dominance of the precise intron losses, frequent loss of adjacent introns, and high-level expression of the intron-lost genes, preferential losses of short introns, and the preferential losses of introns close to 3'-ends of genes. Like studies in most eukaryotic groups, we cannot find the source sequences for the limited number of intron gains detected in the Caenorhabditis genomes. All the results indicate that nematodes are a typical eukaryotic group rather than an outlier in intron evolution.

category: genomics


title: Single-cell atlas of human oral mucosa reveals a stromal-neutrophil axis regulating tissue immunity in health and inflammatory disease

abstract: The oral mucosa remains an understudied barrier tissue rich in exposure to antigens, commensals and pathogens. Moreover, it is the tissue where one of the most prevalent human microbe-triggered inflammatory diseases, periodontitis, occurs. To understand this complex environment at the cellular level, we assemble herein a human single-cell transcriptome atlas of oral mucosal tissues in health and periodontitis. Our work reveals transcriptional diversity of stromal and immune cell populations, predicts intercellular communication and uncovers an altered immune responsiveness of stromal cells participating in tissue homeostasis and disease at the gingival mucosa. In health, we define unique populations of CXCL1,2,8-expressing epithelial cells and fibroblasts mediating immune homeostasis primarily through the recruitment of neutrophils. In disease, we further observe stromal, particularly fibroblast hyper-responsiveness linked to recruitment of leukocytes and neutrophil populations. Ultimately, a stromal-neutrophil axis emerges as a key regulator of mucosal immunity. Pursuant to these findings, most Mendelian forms of periodontitis were shown to be linked to genetic mutations in neutrophil and select fibroblast-expressed genes. Moreover, we document previously unappreciated expression of known pattern- and damage-recognition receptors on stromal cell populations in the setting of periodontitis, suggesting avenues for triggering stromal responses. This comprehensive atlas offers an important reference for in-depth understanding of oral mucosal homeostasis and inflammation and reveals unique stromal-immune interactions implicated in tissue immunity.

category: systems biology


title: Charting human subcortical maturation with in vivo 7 T MRI

abstract: The human subcortex comprises hundreds of unique structures. Subcortical functioning is crucial for behavior, and disrupted function is observed in common neurodegenerative diseases. Despite their importance, human subcortical structures continue to be difficult to study in vivo. Here we provide a detailed account of 17 prominent subcortical structures, describing their approximate iron and myelin contents, morphometry, and their age-related changes across the normal adult lifespan. The results provide compelling insights into the heterogeneity and intricate age-related alterations of these structures. They also show that the locations of many structures shift across the lifespan, which is of direct relevance for the use of standard magnetic resonance imaging atlases. The results further our understanding of subcortical morphometry and neuroimaging properties, and of normal aging processes which ultimately can improve our understanding of neurodegeneration.


title: Charting human subcortical maturation across the adult lifespan with in vivo 7 T MRI


title: The MYB33, MYB65, and MYB101 transcription factors affect Arabidopsis and potatoresponses to drought by regulating the ABA signaling pathway

abstract:
AimsDrought is a climate threat limiting crop production. Potato is one of the four most important food crops worldwide and is sensitive to water shortage. The CBP80 gene was shown to affect plant response to drought by regulating the level of microRNA159, and, consequently, the levels of the MYB33 and MYB101 transcription factors. (TF) Our studies aimed to show whether indeed the level of MYB33, MYB65, and MYB101 TFs affects plant response to water shortage.

MethodsArabidopsis transgenic plants exhibiting downregulation and Arabidopsis and potato transgenic plants exhibiting overexpression of selected MYB TFs were obtained. Plants response to drought was mainly measured using relative water content (RWC) and stomata closure upon exogenous ABA.

ResultsThree MYB TFs studied are involved in plant response to drought. When downregulated in Arabidopsis, the MYB33, MYB65 and MYB101 genes cause stomatal hyposensitivity to ABA, leading to reduced tolerance to drought. Transgenic Arabidopsis and potato plants overexpressing a mutated version of these genes with changed miR159 recognition site, show hypersensitivity to ABA and relatively high tolerance to drought conditions.

ConclusionsThe MYB33, MYB65, and MYB101 genes are good be potential targets for innovative breeding to obtain crops with relatively high tolerance to drought.


title: Promoter-sequence determinants and structural basis of primer-dependent transcription initiation in Escherichia coli

abstract: Chemical modifications of RNA 5' ends enable "epitranscriptomic" regulation, influencing multiple aspects of RNA fate. In transcription initiation, a large inventory of substrates compete with nucleoside triphosphates (NTPs) for use as initiating entities, providing an ab initio mechanism for altering the RNA 5' end. In Escherichia coli cells, RNAs with a 5'-end hydroxyl are generated by use of dinucleotide RNAs as primers for transcription initiation, "primer-dependent initiation." Here we use massively systematic transcript end readout ("MASTER") to detect and quantify RNA 5' ends generated by primer-dependent initiation for ~410 (~1,000,000) promoter sequences in E. coli. The results show primer-dependent initiation in E. coli involves any of the 16 possible dinucleotide primers and depends on promoter sequences in, upstream, and downstream of the primer binding site. The results yield a consensus sequence for primer-dependent initiation, YTSS-2NTSS-1NTSSWTSS+1, where TSS is the transcription start site, NTSS-1NTSS is the primer binding site, Y is pyrimidine, and W is A or T. Biochemical and structure-determination studies show that the base pair (nontemplate-strand base:template-strand base) immediately upstream of the primer binding site (Y:RTSS-2, where R is purine) exerts its effect through the base on the DNA template strand (RTSS-2) through inter-chain base stacking with the RNA primer. Results from analysis of a large set of natural, chromosomally-encoded E. coli promoters support the conclusions from MASTER. Our findings provide a mechanistic and structural description of how TSS-region sequence hard-codes not only the TSS position, but also the potential for epitranscriptomic regulation through primer-dependent transcription initiation.


title: The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signalling

abstract: SARS-CoV-2 non-structural protein 3 (Nsp3) contains a macrodomain that is essential for virus replication and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the removal of ADP-ribose modifications catalysed by host poly(ADP-ribose) polymerases (PARPs). Here, we show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this can be used to screen for cell-active macrodomain inhibitors without the requirement for BSL-3 facilities. This IFN-induced ADP-ribosylation is dependent on the PARP9/DTX3L heterodimer, but surprisingly the expression of Nsp3 macrodomain or PARP9/DTX3L deletion do not impair STAT1 phosphorylation or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyse this end product of IFN signalling, and not to suppress the IFN response itself.


title: Septin-microtubule association requires a MAP-like motif unique to Sept9 isoform 1 embedded into septin octamers

abstract: Septins, a family of GTP-binding proteins assembling into higher order structures, interface with the membrane, actin filaments and microtubules, which positions them as important regulators of cytoarchitecture. Septin 9 (Sept9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short MAP-like motif unique to Sept9 isoform 1 (Sept9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring Sept9_i1. Although outside of the MAP-like motif, HNA mutations abrogates this association, identifying a putative regulatory domain. Removal of this domain from Sept9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin associated pathologies.


title: On the Analysis of Transcriptional Noise From RNA-sequencing Data

abstract:
RNA-sequencing (RNA-seq) has revolutionized our understanding of molecular and cellular biology. A central cornerstone in the analysis of RNA-seq is the bioinformatic tools that quantify the data. To evaluate the efficacy of these tools, scientists rely heavily on simulation of RNA-seq. Recently Varabyou et al. took simulation of RNA-seq data to the next level by providing simulated data, that includes simulation of transcriptional noise. While this represents a significant step forward in our ability to perform realistic benchmarks of RNA-seq tools, the data provided by Varabyou et al. need refinement. In the following, I suggest a few improvements with a specific focus on splicing noise.

PrefaceI wrote this paper intending to submit it as a Commentary on the Varabyou et al. 2020 Genome Research paper1, but apparently, Genome Research does not publish correspondence-type articles. That is why it is currently on BioRxiv. If you have suggestions about where this paper could potentially be published do not hesitate to contact me.

category: bioinformatics


title: Explainable Feature Extraction Using a Neural Network with non-Synaptic Memory for Hand-Written Digit Classification

abstract: The human brain recognizes hand-written digits by extracting the features from a few training samples that compose the digit image including horizontal, vertical, and orthogonal lines as well as full or semi-circles. In this study, we present a novel brain-inspired method to extract such features from handwritten digits images in the MNIST database (Modified National Institute of Standards and Technology database). In this study, we developed an explainable feature extraction method for hand written digit classification in which the extracted information are stored inside the neurons as non-synaptic memory manner. For this purpose, a neural network with 10 single neurons was trained to extract features of training images (each neuron represents one digit class). Following that, the trained single neurons are used for the retrieval of information from test images in order to assign them to digit categories. The accuracy of the classification method of test set images is calculated for different number of training samples per digit. The method demonstrates 75 % accuracy using 0.016 % of the training data and maximally shows accuracy 86 % using one epoch of whole training data. The method as an understandable feature extraction method allows users to see how it works and why it does not perform well on some digit classes. To our knowledge, this is the first model that stores information inside single neurons (i.e., non-synaptic memory) instead of storing the information in synapses of connected layers. Due to enabling single neurons to compute individually, it is expected that such class of neural networks show higher performance compared to traditional neural networks used in complicated classification problems.


title: Zebrafish heme oxygenase 1a is necessary for normal development and macrophage migration

abstract: Heme oxygenase function is highly conserved between vertebrates where it plays important roles in normal embryonic development and controls oxidative stress. Expression of the zebrafish heme oxygenase 1 genes are known to be responsive to oxidative stress suggesting a conserved physiological function. Here we generate a knockout allele of zebrafish hmox1a and characterize the effects of hmox1a and hmox1b loss on embryonic development. We find that loss of hmox1a or hmox1b causes developmental defects in only a minority of embryos, in contrast to Hmox1 gene deletions in mice that causes loss of most embryos. Using a tail wound inflammation assay we find a conserved role for hmox1a, but not hmox1b, in normal macrophage migration to the wound site. Together our results indicate zebrafish hmox1a has clearly a partitioned role from hmox1b that is more consistent with conserved functions of mammalian Heme oxygenase 1.


title: Somite morphogenesis is required for axial blood vessel formation

abstract:
Angioblasts that form the major axial blood vessels of the dorsal aorta and cardinal vein migrate towards the embryonic midline from distant lateral positions. Little is known about what controls the precise timing of angioblast migration and their final destination at the midline. Using zebrafish, we found that midline angioblast migration requires neighboring tissue rearrangements generated by somite morphogenesis. The somitic shape changes cause the adjacent notochord to separate from the underlying endoderm, creating a ventral midline cavity that provides a physical space for the angioblasts to migrate into. The anterior to posterior progression of midline angioblast migration is facilitated by retinoic acid induced anterior to posterior somite maturation and the subsequent progressive opening of the ventral midline cavity. Our work demonstrates a critical role for somite morphogenesis in organizing surrounding tissues to facilitate notochord positioning and angioblast migration, which is ultimately responsible for creating a functional cardiovascular system.

Summary statementRetinoic acid induced somite morphogenesis generates a midline cavity that accommodates migrating angioblasts, which form the axial blood vessels.


title: AAV-mediated gene therapy produces fertile offspring in the Lhcgr-deficient mouse model of Leydig cell failure

abstract: Leydig cell failure (LCF) caused by gene mutation results in testosterone deficiency and infertility. Serum testosterone levels can be recovered via testosterone replacement; however, established therapies have shown limited success in restoring fertility. Here, we used a luteinizing hormone/choriogonadotrophin receptor (Lhcgr)-deficient mouse model of genetic LCF to investigate the feasibility of gene therapy for restoring testosterone production and fertility. We screened several adeno-associated virus (AAV) serotypes and identified AAV8 as an efficient vector to drive exogenous Lhcgr expression in progenitor Leydig cells through interstitial injection. We observed considerable testosterone recovery and Leydig cell maturation after AAV8-Lhcgr treatment in pubertal Lhcgr-/- mice. This gene therapy substantially recovered sexual development, partially restored spermatogenesis and effectively produced fertile offspring. Furthermore, these favorable effects could be reproduced in adult Lhcgr-/- mice. Our proof-of-concept experiments in this mouse model demonstrate that AAV-mediated gene therapy may represent a promising therapeutic approach for patients with genetic LCF.


title: Rab10 regulates the sorting of internalised TrkB to retrograde axonal transport

abstract: The extreme, complex morphology of neurons provides an unrivalled model to study the coordination between local signalling and long-range cell responses. A cogent example is provided by the binding of brain-derived neurotrophic factor (BDNF) to its receptor TrkB, which triggers signalling cascades at axon terminals that result in responses at the level of the cell body, including modulation of gene expression. Retrograde propagation of these critical signals relies on the sorting of activated TrkB receptors to retrograde axonal transport organelles termed signalling endosomes. In this work, we show that the small GTPase Rab10 is critical for the sorting of activated TrkB receptors to axonal retrograde carriers and the propagation of neurotrophin signalling from the axon terminal to the soma. Moreover, our data indicate that Rab10 defines a novel class of axonal organelles that are mobilised towards the axon terminal upon BDNF stimulation, thus enabling the axon to dynamically adjust the retrograde signalling flow to changes in BDNF availability at the synapse.


title: Cells of the human intestinal tract mapped across space and time

abstract: The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. To comprehensively map cell lineages in the healthy developing, pediatric and adult human gut from ten distinct anatomical regions, as well as draining lymph nodes, we used singlecell RNA-seq and VDJ analysis of roughly one third of a million cells. This reveals the presence of BEST4+ absorptive cells throughout the human intestinal tract, demonstrating the existence of this cell type beyond the colon for the first time. Furthermore, we implicate IgG sensing as a novel function of intestinal tuft cells, and link these cells to the pathogenesis of inflammatory bowel disease. We define novel glial and neuronal cell populations in the developing enteric nervous system, and predict cell-type specific expression of Hirschsprungs disease-associated genes. Finally, using a systems approach, we identify key cell players across multiple cell lineages driving secondary lymphoid tissue formation in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. These data provide an unprecedented catalogue of intestinal cells, and new insights into cellular programs in development, homeostasis and disease.


title: Towards customized studies of G-quadruplex DNA structures in live cells

abstract: G-quadruplex (G4) DNA structures are implicated in central biological processes and are considered promising therapeutic targets because of their links to human diseases such as cancer. However, functional details of how, when, and why G4 DNA structures form in vivo are largely missing leaving a knowledge gap that requires tailored chemical biology studies in relevant live-cell model systems. Towards this end, we developed a synthetic platform centered around one of the most effective and selective G4 stabilizing compounds, Phen-DC3. We used a structure-based design to equip Phen-DC3 with an amine in a position that does not interfere with G4 interactions. To evaluate the power of the approach, we next used this reactive handle to conjugate a BODIPY fluorophore and a cell-penetrating peptide to Phen-DC3. The BODIPY conjugation generated a fluorescent derivative with retained G4 selectivity, G4 stabilization, and cellular effects that revealed the localization and function of Phen-DC3 in human cells. On the other hand, the cell-penetrating peptide conjugation, while retaining G4 selectivity and stabilization, increased nuclear localization and cellular effects, showcasing the potential of this approach to modulate and direct cellular uptake e.g. as delivery vehicles. The developed platform can thus generate tailored biochemical tools for the studies of G4 biology to uncover molecular details and therapeutic approaches.


title: Evaluation of the Efficacy and Safety of a Clinical Grade Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Patch: A Pre-Clinical Study

abstract:
AimsCardiomyocyte-derived induced pluripotent stem cells (iPSCs) may represent a promising therapeutic strategy for severely damaged myocardium. This study aimed to assess the efficacy and safety of clinical grade human iPSC-derived cardiomyocyte (hiPSC-CM) patches and conduct a pre-clinical proof-of-concept analysis.

Methods and resultsA clinical grade hiPSC line was established from peripheral blood mononuclear cells collected from a healthy volunteer homozygous for human leukocyte antigens and differentiated into cardiomyocytes using cytokines and chemical compounds. hiPSC-CMs were cultured on temperature-responsive culture dishes to fabricate the hiPSC-CM patch. The hiPSC-CMs expressed cardiomyocyte-specific genes and proteins while electrophysiological analyses revealed that hiPSC-CMs were similar to the human myocardium. In vitro safety studies using cell growth, soft agar colony formation, and undifferentiated cell assays indicated that tumourigenic cells were not present. Moreover, no genomic mutations were discovered using whole genome and exome sequencing analysis. Tumour formation was not detected in an in vivo tumourigenicity assay using NOG mice. General toxicity tests also showed no adverse events due to hiPSC-CM patch transplantation. An efficacy study using a porcine model of myocardial infarction demonstrated significantly improved cardiac function with angiogenesis and a reduction in interstitial fibrosis, which was enhanced by cytokine secretion from hiPSC-CM patches after transplantation. No lethal arrhythmias were observed.

ConclusionhiPSC-CM patches show promise for future translational research and clinical trials for ischaemic heart failure.

One-sentence summaryThis pre-clinical study provides a proof-of-concept of the safety and efficacy of hiPSC-CM patches for the treatment of heart failure.

Translational PerspectiveRegenerative therapy using cells and tissues is attractive as a novel approach for treating severe heart failure. We focused on human iPS cell-derived cardiomyocytes (hiPSC-CMs) as a cell source. Using basic research, the characteristics of hiPSC, hiPSC-CMs, and hiPSC-CM patches were determined in vitro and in vivo. We also conducted a pre-clinical study using a porcine model of myocardial infarction that confirmed the safety and efficacy of the hiPSC-CM patch, highlighting its potential for clinical application.


title: delilah, prospero and D-Pax2 constitute a gene regulatory network essential for the development of functional proprioceptors

abstract: Coordinated animal locomotion depends on the development of functional proprioceptors. While early cell-fate determination processes are well characterized, little is known about the terminal differentiation of cells within the proprioceptive lineage and the genetic networks that control them. In this work we describe a gene regulatory network consisting of three transcription factors-Prospero (Pros), D-Pax2 and Delilah (Dei)-that dictates two alternative differentiation programs within the proprioceptive lineage in Drosophila. We show that D-Pax2 and Pros control the differentiation of cap versus scolopale cells in the chordotonal organ lineage by, respectively, activating and repressing the transcription of dei. Normally, D-Pax2 activates the expression of dei in the cap cell but is unable to do so in the scolopale cell where Pros is co-expressed. We further show that D-Pax2 and Pros exert their effects on dei transcription via a 262 bp chordotonal-specific enhancer in which two D-Pax2- and three Pros-binding sites were identified experimentally. When this enhancer was removed from the fly genome, the cap- and ligament-specific expression of dei was lost, resulting in loss of chordotonal organ functionality and defective larval locomotion. Thus, coordinated larval locomotion depends on the activity of a dei enhancer that integrates both activating and repressive inputs for the generation of a functional proprioceptive organ.


title: Myeloid and endothelial cells cooperate to promote hematopoietic stem cells expansion in the fetal niche.

abstract: During embryonic development, very few hematopoietic stem cells (HSCs) are produced from the hemogenic endothelium, that will be expanded in a very specific niche. This fetal HSC niche comprises a complex and dynamic molecular network of interactions between multiple cell types, including endothelial cells (ECs) and mesenchymal stromal cells. It is known that functional changes in the hematopoietic niche, such as aging, vascular cell remodelling or inflammation can directly affect HSCs. Among all these inflammatory regulators, the eicosanoid prostaglandin E (PGE2) has been shown to be very important during embryonic life. However, the precise source of PGE2 in the embryo is still elusive. Here we show that all the genes involved in PGE2 synthesis and transport are expressed by distinct cells of the caudal hematopoietic tissue (CHT) in the zebrafish embryo and in the mouse fetal liver, suggesting that each cell type acts sequentially and collaboratively with the others to produce PGE2 and ultimately expand HSCs. Among these cells, we found myeloid cells (both neutrophils and macrophages) to be absolutely necessary, as they concur to the production of PGH2, the precursor of PGE2. To measure the impact of myeloid cells, we generated a genetic model of myeloid ablation, which caused a loss of HSCs in the CHT, that could be rescued by supplementing zebrafish embryos with PGE2 or PGH2. ECs expressed the slco2b1 transporter to import PGH2, and ptges3, the necessary enzyme to convert this latter into PGE2. Taken altogether, our data show that the triad composed of neutrophils, macrophages and ECs concurs to HSC expansion in the CHT.


title: CD95 expression in triple negative breast cancer blocks induction of an inflammatory state through differential regulation of NF-kB Signaling

abstract: CD95L is expressed by tumor-infiltrating lymphocytes to eliminate CD95-expressing tumor cells and thereby CD95 loss by tumor cells is often considered as a consequence of an immunoediting process. Nonetheless CD95 expression is maintained in most triple negative breast cancers (TNBCs), and we recently reported that CD95 loss in TNBC cells triggers the induction of a pro-inflammatory program promoting the recruitment of cytotoxic NK and CD8+ T-cells and impairing tumor growth. Using a comprehensive proteomic approach, we have identified two yet unknown CD95 interaction partners, Kip1 ubiquitination-promoting complex protein 2 (KPC2) and p65. KPC2 contributes to the partial degradation of p105 (NF{kappa}B1) and the subsequent generation of p50 homodimers, which transcriptionally represses pro-inflammatory NF-{kappa}B-driven gene expression. Mechanistically, KPC2 directly interacts with the C-terminal region of CD95 and links the receptor to RelA (p65) and KPC1, the catalytic subunit of the KPC complex that acts as E3 ubiquitin-protein ligase promoting the partial degradation of p105 into p50. Loss of CD95 in TNBC cells releases KPC2, limiting the formation of the NF-{kappa}B inhibitory homodimer complex (p50/p50), promoting NF-{kappa}B activation and the production of pro-inflammatory cytokines including CSF1, CSF2, CXCL1 and IL1 members, known to promote recruitment and differentiation of certain adaptive and innate immune effector cells.


title: Enabling constrained spherical deconvolution and diffusional variance decomposition with tensor-valued diffusion MRI

abstract: Diffusion tensor imaging (DTI) is widely used to extract valuable tissue measurements and white matter (WM) fiber orientations, even though its lack of specificity is now well-known, especially for WM fiber crossings. Models such as constrained spherical deconvolution (CSD) take advantage of high angular resolution diffusion imaging (HARDI) data to compute fiber orientation distribution functions (fODF) and tackle the orientational part of the DTI limitations. Furthermore, the recent introduction of tensor-valued diffusion MRI allows for diffusional variance decomposition (DIVIDE), opening the door to the computation of measures more specific to microstructure than DTI measures, such as microscopic fractional anisotropy (FA). However, tensor-valued diffusion MRI data is not mathematically compatible with latest versions of CSD and the impacts of such atypical data on fODF reconstruction with CSD are yet to be studied. In this work, we lay down the mathematical and computational foundations of a tensor-valued CSD and use simulated data to explore the effects of various combinations of diffusion encodings on the angular resolution of extracted fOFDs. We also compare the combinations with regards to their performance at producing accurate and precise FA with DIVIDE, and present an optimised protocol for both methods. We show that our proposed protocol enables the reconstruction of both fODFs and FA on in vivo data.


title: Impact of ecotourism on abundance, diversity and activity pattern of medium-large terrestrial mammals at Brownsberg Nature Park, Suriname

abstract:
The impacts of ecotourism on biodiversity are poorly understood and the outcome of research is often contradictory. On the one hand ecotourism could impact the occurrence, survival or behavior of species, on the other hand ecotourism is often mentioned as providing a "human shield" by deterring negative practices like gold mining, logging and hunting.

Brownsberg Nature Park is easily the most visited protected area of Suriname, with a high number of ecotourists visiting from abroad. A four-year study on the impact of ecotourism on medium-large terrestrial mammals was carried out between 2013 and 2016 using 16 camera trapping stations. The area has a clear gradient of tourism pressure, with the pressure decreasing further away from the lodging facilities. Evidently, the impacts of human presence on the mammal communities were more significant in the busiest areas. Most species avoided areas with many hikers or switched to a more nocturnal activity pattern. In these areas the impact was not reflected in species numbers, however it was causing a significant lowering of the diversity of mammals. On the other hand, vehicles had little impact on species avoidance or diversity, but did increase nocturnality even more than hikers. A few species seemed to be "attracted" by hikers and/or traffic. Giant armadillos and spotted pacas used the pools in the road created by traffic. Ocelots, margays and red-rumped agoutis seemed to favor human disturbance probably because of predator release. Some of the most impacted species were the jaguar, puma and lowland tapir, all three species with significant contribution to ecosystem balance. Their avoidance or even disappearance from highly human frequented areas could easily result in ecosystem changes in these areas. Management measures should focus on lowering the number of hikers in popular places and limiting the number of vehicles in recreational or tourist areas.

category: ecology


title: Lignin accumulation in phloem and outer bark is not associated with resistance to mountain pine beetle in high elevation pines

abstract: A key component in understanding plant-insect interactions is the nature of host defenses. Research on defense traits among Pinus species has focused on specialized metabolites and axial resin ducts, but the role of lignin in defense within diverse systems is unclear. We investigated lignin levels in the outer bark and phloem of P. longaeva, P. balfouriana, and P. flexilis; high elevation species in the western United States known to differ in susceptibility to mountain pine beetle (Dendroctonus ponderosae; MPB). Relative to P. flexilis, P. longaeva and P. balfouriana are attacked by MPB less frequently, and MPB brood production in P. longaeva is limited. Because greater lignification of feeding tissues has been shown to provide defense against bark beetles in related genera, such as Picea, we hypothesized that P. longaeva and P. balfouriana would have greater lignin concentrations than P. flexilis. Contrary to expectations, we found that the more MPB-susceptible P. flexilis had greater phloem lignin levels than the less susceptible P. longaeva and P. balfouriana. No differences in outer bark lignin levels among the species were found. We conclude that lignification in Pinus phloem and outer bark is likely not adaptive as a physical defense against MPB.


title: ULTRASTRUCTURAL STUDY OF THE ESOPHAGUS AND STOMACH OF Arapaima gigas (Schinz, 1822), JUVENILE PAICHE, CREATED EXCAVATED TANK

abstract: Paiche (Arapaima gigas) belongs to the Kingdom Animalia, Phylum Chordata, Class Actinopterygii, Order Osteoglossiformes, Family Arapaimidae, Genus Arapaima, and its origin may date to the Jurassic period. The species has natural habitat in the Amazonian rivers, found mainly in marginal lakes, being considered an important fishing resource, with high market value and high demand for meat and leather in both Brazilian and international trade. This study aims to describe the morphology of the esophagus and stomach by light microscopy and scanning electronics microscopy. The esophagus was presented as muscular, short, tubular and fan-shaped in the cranial portion, also presenting deep longitudinal folds, and the entire mucosa is covered by mucus secretory cells with distinct morphological characteristics. Pirarurcus stomach has a J-shape divided into three regions: cardiac with a lighter aspect, fundus portion with few folds in the mucosa, and pyloric with deeper folds, also presenting gastroliths in fundus and pyloric portions. Both microscopy studies highlighted three glandular regions, composed by mucoid columnar epithelial cells, gastric crypts with different shapes and sizes depending on each portion, in which the different shapes of the mucosal folds in each region of the stomach were evident, and digitiform microsaliences were found in the cardiac region, and micro-orifices and desmosome in the fundus region. Also, fundus and pyloric portions produce more mucus than the cardiac. Then morphology found was consistent with the eating habits and management of distinct characteristics of the digestive tract.

category: zoology


title: Consolidation of human skill linked to waking hippocampo-neocortical replay

abstract: The introduction of rest intervals interspersed with practice strengthens wakeful consolidation of skill. The mechanisms by which the brain binds discrete action representations into consolidated, highly temporally-resolved skill sequences during waking rest are not known. To address this question, we recorded magnetoencephalography (MEG) during acquisition and rapid consolidation of a sequential motor skill. We report the presence of highly prominent, fast waking neural replay during the same rest periods in which rapid consolidation occurs. The observed replay was temporally compressed by approximately 20x relative to the acquired skill, occurred in both forward and reverse directions, was selective for the trained sequence and predicted the magnitude of skill consolidation. Replay representations extended beyond the hippocampus and entrorhinal cortex to the contralateral sensorimotor cortex. These results document the presence of robust hippocampo-neocortical replay supporting rapid wakeful consolidation of skill.


title: Intrinsic Timescales Across the Basal Ganglia

abstract: Recent studies have shown that neuronal stability over time can be estimated by the structure of the spike-count autocorrelation of neuronal populations. This estimation, called the intrinsic timescale, has been computed for several cortical areas and can be used to propose a cortical hierarchy reflecting a scale of temporal receptive windows between areas. In this study, we performed an autocorrelation analysis on neuronal populations of three basal ganglia (BG) nuclei, including the striatum and the subthalamic nucleus (STN), the input structures of the BG, and the external globus pallidus (GPe). The analysis was performed during the baseline period of a motivational visuomotor task in which monkeys had to apply different amounts of force to receive a different amount of reward. We found that the striatum and the STN have longer intrinsic timescales than the GPe. Moreover, our results allow for the placement of these subcortical structures within the already-defined scale of cortical temporal receptive windows. Estimates of intrinsic timescales are important in adding further constraints in the development of computational models of the complex dynamics among these nuclei and throughout cortico-BG-thalamo-cortical loops.


title: Dissociation of early and late face-related processes in Autism Spectrum Disorder and Williams syndrome

abstract:
Williams syndrome (WS) and Autism Spectrum Disorders (ASD) are psychiatric conditions associated with atypical but opposite face-to-face interactions patterns: WS patients overly stare at others, ASD individuals escape eye contact. Whether these behaviors result from dissociable visual processes within the occipito-temporal pathways is unknown.

Using high-density electroencephalography, multivariate signal processing algorithms and a protocol designed to identify and extract evoked activities sensitive to facial cues, we investigated how WS (N=14), ASD (N=14) and neurotypical subjects (N=14) decode the information content of a face stimulus.

We found two neural components in neurotypical participants, both strongest when the eye region was projected onto the subjects fovea, simulating a direct eye contact situation, and weakest over more distant regions, reaching a minimum when the focused region was outside the stimulus face. The first component peaks at 170ms, an early signal known to be implicated in low-level face features. The second is identified later, 260ms post-stimulus onset and is implicated in decoding salient face social cues.

Remarkably, both components were found distinctly impaired and preserved in WS and ASD. In WS, we could weakly decode the 170ms signal based on our regressor relative to facial features, probably due to their relatively poor ability to process faces morphology, while the late 260ms component was highly significant. The reverse pattern was observed in ASD participants who showed neurotypical like early 170ms evoked activity but impaired late evoked 260ms signal. Our study reveals a dissociation between WS and ASD patients and point at different neural origins for their social impairments.


title: On the Non-uniqueness Problem in Integrated Information Theory

abstract: Integrated Information Theory is currently the leading mathematical theory of consciousness. The core of the theory relies on the calculation of a scalar mathematical measure of consciousness, {Phi}, which is deduced from the phenomenological axioms of the theory. Here, we show that despite its widespread use, {Phi} is not a well-defined mathematical concept in the sense that the value it specifies is neither unique nor specific. This problem, occasionally referred to as "undetermined qualia", is the result of degeneracies in the optimization routine used to calculate {Phi}, which leads to ambiguities in determining the consciousness of systems under study. As demonstration, we first apply the mathematical definition of {Phi} to a simple AND+OR logic gate system and show 83 non-unique {Phi} values result, spanning a substantial portion of the range of possibilities. We then introduce a Python package called PyPhi-Spectrum which, unlike currently available packages, delivers the entire spectrum of possible {Phi} values for a given system. We apply this to a variety of examples of recently published calculations of {Phi} and show how virtually all {Phi} values from the sampled literature are chosen arbitrarily from a set of non-unique possibilities, the full range of which often includes both conscious and unconscious predictions. Lastly, we review proposed solutions to this degeneracy problem, and find none to provide a satisfactory solution, either because they fail to specify a unique {Phi} value or yield {Phi} = 0 for systems that are clearly integrated. We conclude with a discussion of requirements moving forward for scientifically valid theories of consciousness that avoid these degeneracy issues.


title: Folate bioavailability in reconstructed skin models and effects of folate in a monolayer wound healing assay. Approaches on topic application.

abstract:
In chronic and degenerative diseases impacting the skin folates play an important metabolic role improving wound healing and reducing skin irritations. In contrast to systemic folate administration little is known on skin penetration of folates after topical application. Here the penetration of simple aqueous solutions of reduced folates have been investigated with in-vitro reconstructed skin models mimicking the barrier of native human skin. For up to 24 h, penetration of the epidermis by newly developed folate salts and formulations were investigated. Aqueous and lipophilic solutions of L-formyltetrahydrofolate and L-mefolinate salts were able to penetrate the epidermis. Even more importantly, the skin model revealed the metabolic conversion of L-folinate to L-methyltetrahydrofolate. Exemplarily the effects of these new folate salts have been tested on wound healing in a scratch assay with primary human keratinocytes. All folates applied were able to enhance wound healing compared to the control.

Bioavailability, metabolic conversion and physiologic effectiveness of new folate formulation have been shown successfully in in vitro applications providing evidence for the potent applicability of the new folates in preparations for topic application.


title: Precocious hippocampal structural and functional changes in gestational protein-restricted male elderly offspring: an Alzheimer-simile disease model?

abstract:
BackgroundMaternal undernutrition has been associated with psychiatric and neurological disorders characterized by learning and memory impairment. Considering the lack of evidence for this, we aimed to analyze the effects of gestational protein restriction on learning and memory function later in life. This research associates behavioral findings with hippocampal cell numbers and protein content related to neurodegenerative brain disease.

MethodsExperiments were conducted in animals subjected to a low-protein (LP, 6% casein) or regular-protein (NP, 17% casein) diet throughout their pregnancy. Behavioral tests, isolated hippocampal isotropic fractionator cell studies, immunoblotting, and survival lifetime tests were performed. The results confirmed that the birthweight of LP male pups significantly reduced relative to NP male pups and that hippocampal mass increased in 88-week-old LP compared to age-matched NP offspring. We used the Morris water maze proximity measure, which is the sum of 10 distances each second between rat position and location of a hidden platform target, as a suitable test for assessing age-related learning or memory impairment in aged offspring.

ResultsThe results showed an increased proximity measure in 87-week-old LP rats (52.6 x 104 {+/-} 10.3 x 104 mm) as compared to NP rats (47.0 x 104 {+/-} 10.6 x 103 mm, p = 0.0007). In addition, LP rats exhibited anxiety-like behaviors compared to NP rats at 48 and 86 weeks of life.

Additionally, the estimated neuron number was unaltered in LP rats; however, glial and other cell numbers increased in LP compared to NP rats. Here, we showed unprecedented hippocampal deposition of brain-derived neurotrophic factor, {beta}-amyloid peptide (A{beta}), and tau protein in 88-week-old LP compared to age-matched NP offspring. To date, no predicted studies showed changes in hippocampal neuron and glial cell numbers in maternal protein-restricted elderly offspring. The current data suggest that maternal protein restriction has a high impact on lifespan and brain structure, and function.

Conclusionthe gestational protein restriction may accelerate hippocampal function loss, impacting learning/memory performance, and supposedly developing diseases similar to Alzheimers disease (AD) in elderly offspring. Thus, we propose that maternal protein restriction could be a probable, elegant, and novel method for constructing an AD-like model in adult male offspring.


title: What's the relative humidity in tropical caves?

abstract: Relative humidity (RH) was measured at hourly intervals for approximately one year in two caves at seven stations near Playa del Carmen in Quintana Roo, Mexico. Sistema Muevelo Rico is a 1.1 km long cave with 12 entrances and almost no dark zone. Rio Secreto (Tuch) is a large river cave with more than 40 km of passages, and an extensive dark zone. Given the need for cave specialists to adapt to saturated humidity, presumably by cuticular thinning, the major stress of RH would be its deviation from saturation. RH in Rio Secreto (Tuch) was invariant at three sites and displayed short deviations from 100% RH at the other four sites. These deviations were concentrated at the end of the nortes and beginning of the rainy season. Three of the sites in Sistema Muevelo Rico showed a similar pattern although the timing of the deviations from 100% RH was somewhat displaced. Four sites in Sistema Muevelo Rico were more variable, and were analyzed using a measure of amount of time of deviation from 100% RH for each 24 hour period. Strong seasonality was evident but, remarkably, periods of constant high humidity were not the same at all sites. In most Sistema Muevelo Rico sites, there was a detectable 24 hour cycle in RH, although it was quite weak in about half of them. For Rio Secreto (Tuch) only one site showed any sign of a 24 hour cycle. The troglomorphic fauna was more or less uniformly spread throughout the caves and did not concentrate in any one area or set of RH conditions. Compared to temperature, RH is much more constant, perhaps even more constant than the amount of light.


title: Whole genome sequencing revealed genetic diversity and selection of Guangxi indigenous chickens

abstract: Guangxi chickens play an important role in promoting the high-quality development of the broiler industry in China, but their value and potential are yet to be discovered. To determine the genetic diversity and population structure of Guangxi indigenous chicken, we analyzed the whole genomes of 185 chicken from 8 phenotypically and geographically representative Guangxi chicken breeds, together with 12 RJFt, 12 BRA and 12 WL genomes available from previous studies. Calculation of heterozygosity (Hp), nucleotide diversity ({pi}), and LD level indicated that Guangxi populations were characterized by higher genetic diversity and lower differentiation than RJFt and commercial breeds except HGFC. Population structure analysis also confirmed the introgression from commercial broiler breeds. Each population clustered together while the overall differentiation was small, MA has the richest genetic diversity among all varieties. Selective sweep analysis revealed BCO2, EDN3 and other candidate genes have received strong selection in local breeds, these also provided novel breeding visual and data basis for future breeding.

category: genetics