title: A comprehensive computational investigation into the conserved virulent proteins of Shigella sp unveils potential siRNA candidates as a new therapeutic strategy against shigellosis

abstract: Shigella sp account for the second-leading cause of deaths due to diarrheal diseases among children of less than 5 years of age. Emergence of multi-drug resistant Shigella isolates and the lack of availability of Shigella vaccines have made the efforts in the development of new therapeutic strategies against shigellosis very pertinent. In our study we have analyzed a total of 241 conserved sequences from a 15 different conserved virulence genes of Shigella sp and through extensive rational validation using a plethora of computational algorithms; we primarily obtained fifty eight small-interfering RNA (siRNA) candidates. Further extensive computational validation showed only three siRNA candidates that were found to exhibit substantial functional efficacy, be non-immunogenic and have a thermodynamically stable and sterically feasible and thereby acceptable tertiary structure. These siRNA candidates are intended to suppress the expression of the virulence genes, namely: IpgD (siRNA 9) and OspB (siRNA 15 and siRNA 17) and thus act as a prospective tool in the RNA interference (RNAi) pathway. However, the findings of our study require further wet lab validation and optimization for regular therapeutic use in the treatment of shigellosis.


title: A linear response theory based method for prediction of large scale protein conformational changes upon ligand binding

abstract: Prediction of ligand-induced protein conformational transitions is a challenging task due to a large and rugged conformational space, and limited knowledge of probable direction(s) of structure change. These transitions can involve a large scale, global (at the level of entire protein molecule) structural change and occur on a timescale of milliseconds to seconds, rendering application of conventional molecular dynamics simulations prohibitive even for small proteins. We have developed a computational protocol to efficiently and accurately predict these ligand-induced structure transitions solely from the knowledge of protein apo structure and ligand binding site. Our method involves a series of small scale conformational change steps, where at each step linear response theory is used to predict the direction of small scale global response to ligand binding in the protein conformational space (dLRT) followed by construction of a linear combination of slow (low frequency) normal modes (calculated for the structure from the previous step) that best overlaps with dLRT. Protein structure is evolved along this direction using molecular dynamics with excited normal modes (MDeNM) wherein excitation energy along each normal mode is determined by excitation temperature, mode frequency, and its overlap with dLRT. We show that excitation temperature ({Delta}T) is a very important parameter that allows limiting the extent of structural change in any one step and develop a protocol for automated determination of its optimal value at each step. We have tested our protocol for three protein-ligand systems, namely, adenylate Kinase - di(adenosine-5)pentaphosphate, ribose binding protein - {beta}-D-ribopyranose, and DNA {beta}-glucosyltransferase - uridine-5-diphosphate, that incorporate important differences in type and range of structural changes upon ligand binding. We obtain very accurate prediction for not only the structure of final protein-ligand complex (holo-structure) having a large scale conformational change, but also for biologically relevant intermediates between the apo and the holo structures. Moreover, most relevant set of normal modes for conformational change at each step are an output from our method, which can be used as collective variables for determination of free energy barriers and transition timescales along the identified pathway.

category: biophysics


title: Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting novel and conserved epitopes

abstract: There is an urgent need to develop effective interventions resistant to the evolving variants of SARS-CoV-2. Nanobodies (Nbs) are stable and cost-effective agents that can be delivered by novel aerosolization route to treat SARS-CoV-2 infections efficiently. However, it remains unknown if they possess broadly neutralizing activities against the prevalent circulating strains. We found that potent neutralizing Nbs are highly resistant to the convergent variants of concern that evade a large panel of neutralizing antibodies (Abs) and significantly reduce the activities of convalescent or vaccine-elicited sera. Subsequent determination of 9 high-resolution structures involving 6 potent neutralizing Nbs by cryoelectron microscopy reveals conserved and novel epitopes on virus spike inaccessible to Abs. Systematic structural comparison of neutralizing Abs and Nbs provides critical insights into how Nbs uniquely target the spike to achieve high-affinity and broadly neutralizing activity against the evolving virus. Our study will inform the rational design of novel pan-coronavirus vaccines and therapeutics.


title: Bi-directional flow of the funny current (If) during the pacemaking cycle in murine sinoatrial node myocytes

abstract:
Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current, If, is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. We used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of -30 mV. Despite operating at only 2-5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that {beta}-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously-expressed HCN4 channels and by mathematical models of If. Modelling further suggested that the slow activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions.

Significance StatementCardiac pacemaker cells trigger each heartbeat by virtue of spontaneous oscillations in their membrane voltage. Although the funny current (If) is critical for these oscillations and for setting heart rate, its precise role remains an enigma because it activates mostly outside of the physiological voltage range and quite slowly relative to the pacemaker cycle. Here we show that If is persistently active in pacemaker cells; once opened, the small fraction of ion channels that conduct If do not re-close. Consequently, If flows both inward and outward to help propel the voltage oscillations and it paradoxically conducts a large fraction of the net charge movement. These results establish a new conceptual framework for the role of If in driving cardiac pacemaking.

category: physiology


title: OsbZIP62/OsFD7, a functional ortholog of Flowering Locus D (FD), regulates floral transition and panicle development in rice

abstract:
We have characterized a rice bZIP protein coding gene OsbZIP62/OsFD7 that expresses preferentially in SAM and during early panicle developmental stages in comparison to other OsFDs characterised till date. Surprisingly, unlike OsFD1, OsFD7 interacts directly and more efficiently with OsFTLs; the interaction is strongest with OsFTL1 followed by Hd3a and RFT1, as confirmed by FLIM-FRET analysis. Also, OsFD7 is phosphorylated at its C-terminal end by OsCDPK41 and OsCDPK49 in vitro and this phosphorylated moiety is recognized by OsGF14 proteins. OsFD7 RNAi transgenics were late flowering; the transcript levels of some floral meristem identity genes (e.g. OsMADS14, OsMADS15 and OsMADS18) were also down-regulated. It was quite interesting to note that these RNAi lines exhibited dense panicle morphology with increase in the number of primary and secondary branches resulting in longer panicles and more seeds probably due to downregulation of Sepallata (SEP) family genes. In comparison to other FD-like proteins characterized thus far from rice, it appears that OsFD7 may have undergone diversification during evolution resulting in the acquisition of newer functions and thus playing dual role in floral transition and panicle development in rice.

HighlightOsbZIP62/OsFD7 interacts with major flowering regulators participating in the processes of floral transition as well as panicle and floral organ development.

category: plant biology


title: Mapping of Whole-Brain Resting-State Networks with Half-Millimetre Resolution

abstract: Resting-state fMRI has been used in numerous studies to map networks in the brain that employ spatially disparate regions. However, attempts to map networks with high spatial resolution have been hampered by conflicting technical demands and associated problems. Results from recent fMRI studies have shown that spatial resolution remains around 0.7 x 0.7 x 0.7 mm3, with only partial brain coverage. This work presents a novel fMRI method, TR-external EPI with keyhole (TR-external EPIK), which can provide a nominal spatial resolution of 0.51 x 0.51 x 1.00 mm3 (0.26 mm3 voxel) with whole-brain coverage. TR-external EPIK enabled the identification of various resting-state networks distributed throughout the brain from a single fMRI session, with mapping fidelity onto the grey matter at 7T. The high-resolution functional image further revealed mesoscale anatomical structures, such as small cerebral vessels and the internal granular layer of the cortex within the postcentral gyrus.

category: neuroscience


title: A precise and adaptive neural mechanism for predictive temporal processing in the frontal cortex

abstract: The theory of predictive processing posits that the nervous system uses expectations to process information predictively. Direct empirical evidence in support of this theory however has been scarce and largely limited to sensory areas. Here, we report a precise and adaptive neural mechanism in the frontal cortex of non-human primates consistent with predictive processing of temporal events. We found that the speed at which neural states evolve over time is inversely proportional to the statistical mean of the temporal distribution of an expected stimulus. This lawful relationship was evident across multiple experiments and held true during learning: when temporal statistics underwent covert changes, neural responses underwent predictable changes that reflected the new mean. Together, these results highlight a precise mathematical relationship between temporal statistics in the environment and neural activity in the frontal cortex that could serve as a mechanistic foundation for predictive temporal processing.


title: Structural connectome quantifies tumor invasion and predicts survival in glioblastoma patients

abstract: Glioblastoma widely affects brain structure and function, and remodels neural connectivity. Characterizing the neural connectivity in glioblastoma may provide a tool to understand tumor invasion. Here, using a structural connectome approach based on diffusion MRI, we quantify the global and regional connectome disruptions in individual glioblastoma patients and investigate the prognostic value of connectome disruptions and topological properties. We show that the disruptions in the normal-appearing brain beyond the lesion could mediate the topological alteration of the connectome (P <0.001), associated with worse patient performance (P <0.001), cognitive function (P <0.001), and survival (overall survival: HR: 1.46, P = 0.049; progression-free survival: HR: 1.49, P = 0.019). Further, the preserved connectome in the normal-appearing brain demonstrates evidence of remodeling, where increased connectivity is associated with better overall survival (log-rank P = 0.005). Our approach reveals the glioblastoma invasion invisible on conventional MRI, promising to benefit patient stratification and precise treatment.


title: MEYE: Web-app for translational and real-time pupillometry

abstract: Pupil dynamics alterations have been found in patients affected by a variety of neuropsychiatric conditions, including autism. Studies in mouse models have used pupillometry for phenotypic assessment and as a proxy for arousal. Both in mice and humans, pupillometry is non-invasive and allows for longitudinal experiments supporting temporal specificity, however its measure requires dedicated setups. Here, we introduce a Convolutional Neural Network that performs on-line pupillometry in both mice and humans in a web app format. This solution dramatically simplifies the usage of the tool for non-specialist and non-technical operators. Because a modern web browser is the only software requirement, this choice is of great interest given its easy deployment and set-up time reduction. The tested model performances indicate that the tool is sensitive enough to detect both spontaneous and evoked pupillary changes, and its output is comparable with state-of-the-art commercial devices.


title: Sex, age, tissue, and disease patterns of matrisome expression in GTEx transcriptome data

abstract: The extracellular matrix (ECM) has historically been explored through proteomic methods. Whether or not global transcriptomics can yield meaningful information on the human matrisome is unknown. Gene expression data from 17,382 samples across 52 tissues, were obtained from the Genotype-Tissue Expression (GTEx) project. Additional datasets were obtained from The Cancer Genome Atlas (TCGA) program and the Gene Expression Omnibus for comparisons. Gene expression levels generally recapitulated proteome-derived matrisome expression patterns. Further, matrisome gene expression properly clustered tissue types, with some matrisome genes including SERPIN family members having tissue-restricted expression patterns. Deeper analyses revealed 388 genes varied by age and 222 varied by sex in at least one tissue, with expression correlating with digitally imaged histologic tissue features. A comparison of TCGA tumor, TCGA adjacent normal and GTEx normal tissues demonstrated robustness of the GTEx samples as a generalized control, while also determining a common primary tumor matrisome. Additionally, GTEx tissues served as a useful non-diseased control in a separate study of idiopathic pulmonary fibrosis matrix changes. Altogether, these findings indicate that the transcriptome, in general, and GTEx in particular, has value in understanding the state of organ ECM.

category: molecular biology


title: An objective-based prioritization approach to improve trophic complexity through ecological restoration

abstract:
O_LIReassembling ecological communities and rebuilding habitats through active restoration treatments requires curating the selection of plant species to use in seeding and planting mixes. Ideally, these mixes should be assembled based on attributes that support ecosystem function and services, promote plant and animal species interactions and ecological networks in restoration while balancing project constraints. Despite these critical considerations, it is common for species mixes to be selected opportunistically. Reframing the selection of seed mixes for restoration around ecological objectives is essential for success but accessible methods and tools are needed to support this effort.
C_LIO_LIWe developed a framework to optimize species seed mixes based on prioritizing plant species attributes to best support different objectives for ecosystem functions, services, and trophic relationships such as pollination, seed dispersal, and herbivory. We compared results to approaches where plant species are selected to represent plant taxonomic richness, dominant species, and at random. We tested our framework for 176 plant species found in European alpine grasslands and identified 163 associated attributes affiliated to trophic relationships, ecosystem functions, and services.
C_LIO_LIIn all cases, trophic relationships, ecosystem functions, and services can be captured more efficiently through objective-based prioritization using the functional identity of plant species. Solutions (plant species lists) can be compared quantitatively, in terms of costs, species, or objectives. We confirm that a random draw of plant species from the regional plant species pool cannot be assumed to support other trophic groups and ecosystem functions and services.
C_LIO_LISynthesis and Applications. Our framework is presented as a proof of concept to help restoration practitioners better apply quantitative decision-support to plant species selection in order to meet ecological restoration outcomes. Our approach may be tailored to any restoration initiative and habitat where seeding or planting mixes will be applied in active treatments. As global priority and resources are increasingly placed into restoration, this approach could be advanced to help make efficient decisions for many stages of the restoration process.
C_LI

category: ecology


title: The Intervening Domain Is Required For DNA-binding and Functional Identity of Plant MADS Transcription Factors

abstract: The MADS transcription factors (TF) are an ancient protein family with a high degree of sequence identity that bind almost identical DNA sequences across all eukaryotic kingdoms of life, yet fulfill dramatically different physiological roles. In plants, the family is divided into two main lineages, type I and II, based on sequence conservation of the DNA-binding MADS-box domain (M domain) with yeast and animal M domains. Here, we demonstrate that DNA binding in both lineages absolutely requires a short amino acid sequence C-terminal to the M domain called the Intervening domain (I domain) in type II MADS. Structural elucidation of the MI domains from the floral regulator, SEPALLATA3 (SEP3), shows a highly conserved MADS-box fold with the I domain forming an alpha helix and acting to stabilize the M domain. Based on secondary structure prediction, sequences fulfilling the same function as the SEP3 I domain can be found in both lineages of plant MADS TFs, suggesting the I domain is a conserved and required part of the DNA-binding domain. Using the floral organ identity MADS TFs, SEP3, APETALA1 (AP1) and AGAMOUS (AG), domain swapping demonstrate that the I domain alters DNA-binding specificity based on seq-DAP-seq experiments. Yeast 2-hybrid experiments further revealed the role of the I domain in dimerization specificity. Surprisingly, introducing AG carrying the I domain of AP1 in the Arabidopsis ap1 mutant, resulted in a high degree of complementation and restoration of first and second whorl organs. Taken together, these data demonstrate that the I domain acts both as an integral part of the DNA-binding domain and strongly contributes to the functional identity of the MADS TF.


title: SSR1 is a vital regulator in plant mitochondrial iron-sulfur biosynthesis

abstract: The Arabidopsis SHORT AND SWOLLEN ROOT1 (SSR1) gene encodes a mitochondrial TPR domain-containing protein and was previously reported to function in maintaining mitochondria function. In a screen for suppressors of the short-root phenotype of the loss-of-function mutant ssr1-2, two mutations, sus1 and sus2 (suppressor of ssr1-2), were isolated. sus1 and sus2 result from G87D and T55M single amino acid substitution in HSCA2 (At5g09590) and ISU1 (At4g22220), both of which are core components in iron-sulfur cluster biosynthesis pathway in mitochondria (ISC). We here demonstrated that SSR1 displayed a strong chaperone-like activity and was able to enhance the binding of HSCA2 to ISU1, an essential step for the normal operation of ISC machinery. Accordingly, the enzymatic activities of several iron-sulfur proteins, the mitochondrial membrane potential and ATP content are reduced in ssr1-2. Interestingly, SSR1 appears to exist only in plant lineages, possibly conferring adaptive advantages on plant ISC machinery to environment.


title: Direct sulphate-TOR signalling controls transcriptional reprogramming for shoot apex activation in Arabidopsis

abstract: Photosynthetic plants play a primary role for the global sulphur cycle in the earth ecosystems by reduction of inorganic sulphate from the soil to organic sulphur-containing compounds. How plants sense and transduce the sulphate availability in soil to mediate their growth remains largely unclear. The target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator of nutrient sensing and metabolic signalling to control cell proliferation and growth in all eukaryotes. Here, we discovered that inorganic sulphate exhibits higher potency than organic cysteine and glutathione for activation of TOR and cell proliferation in the leaf primordium to promote true leaf development in Arabidopsis. Chemical genetic analyses further revealed that this sulphate activation of TOR is independent of the sulphate-assimilation process and glucose-energy signalling. Significantly, tissue specific transcriptome analyses uncovered previously unknown sulphate-orchestrating genes involved in DNA replication, cell proliferation, autophagy and various secondary metabolism pathways, which are completely depending on TOR signalling. Systematic comparison between the sulphate- and glucose-TOR controlled transcriptome further revealed that, as the central growth integrator, TOR kinase can sense different upstream nutrient signals to control both shared and unique transcriptome networks, therefore, precisely modulate plant proliferation, growth and stress responses.


title: Identification and characterization of two transmembrane proteins required for virulence of Ustilago maydis

abstract: Smut fungi comprise a large group of biotrophic phytopathogens infecting important crops such as wheat and corn. Through the secretion of effector proteins, the fungus actively suppresses plant immune reactions and modulates its hosts metabolism. Consequently, how soluble effector proteins contribute to virulence is already characterized in a range of phytopathogens. However, membrane-associated virulence factors have been much less studied to date. Here, we investigated six transmembrane (TM) proteins that show elevated gene expression during biotrophic development of the maize pathogen Ustilago maydis. We show that two of the six proteins, named Vmp1 and Vmp2 (virulence-associated membrane protein), are essential for the full virulence of U. maydis. The deletion of the corresponding genes lead to a substantial attenuation in the virulence of U. maydis. Furthermore, both are conserved in various related smuts and contain no domains of known function. Our biochemical analysis clearly shows that Vmp1 and Vmp2 are membrane-associated proteins, potentially localizing to the U. maydis plasma membrane. Mass photometry and light scattering suggest that Vmp1 mainly occurs as a monomer, while Vmp2 is dimeric. Notably, the large and partially unstructured C-terminal domain of Vmp2 is crucial for virulence while not contributing to dimerization. Taken together, we here provide an initial characterization of two membrane proteins as virulence factors of U. maydis.


title: Standing genetic variation of the AvrPm17 avirulence gene in powdery mildew limits the effectiveness of an introgressed rye resistance gene in wheat

abstract:
Introgressions of chromosomal segments from related species into wheat are important sources of resistance against fungal diseases. The durability and effectiveness of introgressed resistance genes upon agricultural deployment is highly variable - a phenomenon that remains poorly understood as the corresponding fungal avirulence genes are largely unknown. Until its breakdown, the Pm17 resistance gene introgressed from rye to wheat provided broad resistance against powdery mildew (Blumeria graminis). Here, we used QTL mapping to identify the corresponding wheat mildew avirulence effector AvrPm17. It is encoded by two paralogous genes that exhibit signatures of re-occurring gene conversion events and are members of a mildew sub-lineage specific effector cluster. Extensive haplovariant mining in wheat mildew and related sub-lineages identified several ancient virulent AvrPm17 variants that were present as standing genetic variation in wheat powdery mildew prior to the Pm17 introgression, thereby paving the way for the rapid breakdown of the Pm17 resistance. QTL mapping in mildew identified a second genetic component likely corresponding to an additional resistance gene present on the 1AL.1RS translocation carrying Pm17. This gene remained previously undetected due to suppressed recombination within the introgressed rye chromosomal segment. We conclude that the initial effectiveness of 1AL.1RS was based on simultaneous introgression of two genetically linked resistance genes. Our results demonstrate the relevance of pathogen-based genetic approaches to disentangle complex resistance loci in wheat. We propose that identification and monitoring of avirulence gene diversity in pathogen populations becomes an integral part of introgression breeding to ensure effective and durable resistance in wheat.

Significance StatementDomesticated and wild wheat relatives provide an important source of new immune receptors for wheat resistance breeding against fungal pathogens. The durability of these resistance genes is variable and difficult to predict, yet it is crucial for effective resistance breeding. We identified a fungal effector protein recognised by an immune receptor introgressed from rye to wheat. We found that variants of the effector allowing the fungus to overcome the resistance are ancient. They were already present in the wheat powdery mildew gene pool before the introgression of the immune receptor and are therefore responsible for the rapid resistance breakdown. Our study demonstrates that the effort to identify new resistance genes should be accompanied by studies of avirulence genes on the pathogen side.


title: A New Protocol for Targeted insertion using CRISPR-Cas9, Oligo Single-Stranded DNA and Protoplast Regeneration

abstract: Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50.0% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60-nt donor containing 27 nt in each homologous arm, 6 of 22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6-bp HindIII site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants.


title: Targeted Gene Editing in Plants using CRISPR-Cas9, Single-Stranded DNA Oligonucleotides, and Protoplast Regeneration

category: genetics


title: Efficient and Economical Targeted Insertion in Plant Genomes via Protoplast Regeneration


title: Discovery of Dynamical Network Models for Genetic Circuits from Time-Series Data with Incomplete Measurements

abstract: Synthetic gene networks are frequently conceptualized and visualized as static graphs. This view of biological programming stands in stark contrast to the transient nature of biomolecular interaction, which is frequently enacted by labile molecules that are often unmeasured. Thus, the network topology and dynamics of synthetic gene networks can be difficult to verify in vivo or in vitro, due to the presence of unmeasured biological states. Here we introduce the dynamical structure function as a new mesoscopic, data-driven class of models to describe gene networks with incomplete measurements. We introduce a network reconstruction algorithm and a code base for reconstructing the dynamical structure function from data, to enable discovery and visualization of graphical relationships in a genetic circuit diagram as time-dependent functions rather than static, unknown weights. We prove a theorem, showing that dynamical structure functions can provide a data-driven estimate of the size of crosstalk fluctuations from an idealized model. We illustrate this idea with numerical examples. Finally, we show how data-driven estimation of dynamical structure functions can explain failure modes in two experimentally implemented genetic circuits, a historical genetic circuit and a new E. coli based transcriptional event detector.

category: synthetic biology


title: Data-Driven Network Models for Genetic Circuits From Time-Series Data with Incomplete Measurements


title: DeepSea: An efficient deep learning model for automated cell segmentation and tracking

abstract: Time-lapse microscopy can directly capture the dynamics and heterogeneity of cellular processes at the single-cell level. Successful application of single-cell live microscopy requires automated segmentation and tracking of hundreds of individual cells over several time points. Recently, deep learning models have ushered in a new era in quantitative analysis of microscopy images. This work presents a versatile and trainable deep-learning-based software, termed DeepSea, that allows for both segmentation and tracking of single cells and their nuclei in sequences of phase-contrast live microscopy images. We show that DeepSea can quantify several cell biological features of mouse embryonic stem cells, such as cell division cycle, mitosis, cell morphology, and cell size, with high precision using phase-contrast images. Using DeepSea, we were able to show that despite their ultrafast cell division cycle, mouse embryonic stem cells exhibit cell size control in the G1 phase of the cell cycle.

category: systems biology


title: Polypide anatomy of hornerid bryozoans (Stenolaemata: Cyclostomatida)

abstract:
Bryozoans are small colonial coelomates whose colonies are made of individual modules (zooids). Like most coelomate animals, bryozoans have a characteristic body wall composition, including epidermis, extracellular matrix (ECM) and coelothelium, all pressed together. The order Cyclostomatida, however, presents the most striking deviation, in which the ECM and the corresponding coelothelium underlying major parts of the skeletal wall epidermis are ";peeled off" to form an independent membranous sac. The polypide anatomy and ultrastructure of this group is best known from one family, the Crisiidae (Articulata). Here we examined four species from the phylogenetically and ecologically contrasting family Horneridae (Cancellata) from New Zealand. Here we provide the first detailed ultrastructural examination of the hornerid polypide, including tentacles, mouth region, digestive system and the funiculus. We were able to trace continuity and transitions of cell and ECM layers throughout the whole polypide. In addition we identified that the funiculus is a lumen-free ECM cord with two associated muscles, disconnected from interzooidal pores. While agreeing with the general cyclostomate body plan, hornerids have some unique traits that make them worthy of additional study.

HighlightsHornerids share a general cyclostomate body plan. The frontal tentacle ECM transitions into oral sphincter ECM, the abfrontal lophophore ECM becomes a septum between coelomic compartments, and the funuculus is a solid ECM cord supplied with muscles.

category: zoology


title: Effect of Voluntary Adolescent Alcohol Consumption on Encoding of Decision-Related Variables in Prefrontal Cortex

abstract: The medial and orbitofrontal regions of prefrontal cortex (PFC) have been implicated in guiding optimal behavior and updating the economic value of rewards that result from choice behaviors. Both regions mature through adolescence into early adulthood and are thus vulnerable to exposure to neurotoxins, such as alcohol, during this critical developmental window. We sought to examine how voluntary alcohol consumption during adolescence would alter long-term PFC function and subsequent decision-making behavior in adulthood. Male and female rats were given adolescent intermittent ethanol (AIE) exposure to provide voluntary access to alcohol during the period of PFC maturation. In adulthood, we assessed the long-term effects on decision-making behavior using a risk task in adulthood, while concurrently recording neural activity in orbitofrontal cortex (OFC) and medial prefrontal cortex (mPFC). While control animals preferences for risky rewards increased with the likelihood of their delivery, AIE animals showed an overall reduction in their preferences for the risky option with higher levels of alcohol consumption, suggesting reduced discriminability of uncertain rewards and a shift away from the potential for reward omission. During task performance, neurons in mPFC and OFC responded to events (lever press, reward delivery). In mPFC, neurons with phasic increases at the time of lever press showed a reversal from larger elevations for risky presses in control animals to larger elevations for certain presses as prior alcohol consumption increased. Neurons in mPFC generally showed less discrimination of reward outcome with increased alcohol consumption as well. In OFC, responses to lever press were largely unaffected by AIE exposure. However, encoding of reward size in OFC showed differential effects in males and females. With higher alcohol consumption in males, OFC neurons showed largest excursions from baseline activity in response to largest reward, and smallest excursions for reward omission. This discrimination was reduced as prior alcohol consumption increased. In females, neurons with increased reward-activity, showed an overall higher level of activity due to stronger responses to certain rewards that were selected more frequently. Collectively the results show diminished capacity of PFC to encode decision-related elements to guide adaptive behavior and further clarify the lasting impact of adolescent alcohol use on neural function and behavior, even in the absence of continued use.


title: Variable autoinhibition among deafness-associated variants of Diaphanous 1 (DIAPH1)

abstract: One of the earliest mapped human deafness genes, DIAPH1, encodes the formin DIAPH1. To date, at least three distinct mutations in the C-terminal domains and two additional mutations in the N-terminal region are associated with autosomal dominant hearing loss. The underlying molecular mechanisms are not known, and the role of formins in the inner ear is not well understood. In this study we use biochemical assays to test the hypotheses that autoinhibition and/or actin assembly activities are disrupted by DFNA1 mutations. Our results indicate that C-terminal mutant forms of DIAPH1 are functional in vitro and promote actin filament assembly. Similarly, N-terminal mutants are well-folded and have quaternary structures and thermal stabilities similar to the WT protein. The strength of the autoinhibitory interactions varies widely among mutants, with the ttaa, A265S and I530S mutations having an affinity similar to WT and the 1213x and {Delta}ag mutations completely abolishing autoinhibition. These data indicate that, in some cases, hearing loss may be linked to reduced inhibition of actin assembly.

category: biochemistry


title: Post-transcriptional regulation of glutamate metabolism of Pichia pastoris and development of a glutamate-inducible yeast expression system

abstract: Pichia pastoris harbours a unique glutamate utilization pathway in which glutamate dehydrogenase 2 (GDH2), aspartate aminotransferase 2 (AAT2) and phosphoenolpyruvate carboxykinase (PEPCK) catalyze the conversion of glutamate to -ketoglutarate, oxaloacetate, and phosphoenolpyruvate respectively in the cytosol. GDH2 and PEPCK are glutamate-inducible enzymes and their synthesis is regulated post-transcriptionally by Rtg1p, a cytosolic basic helix-loop-helix protein via Rtg1p response elements located downstream of TATA box of GDH2 and PEPCK promoters. Glutamate-inducible synthesis of PEPCK is abrogated in{Delta} gdh2 and{Delta} aat2. -ketoglutarate induces PEPCK synthesis in{Delta} gdh2 but not{Delta} aat2. We propose that oxaloacetate derived from glutamate is the inducer of PEPCK synthesis. Enzymes of glutamate utilization pathway are synthesized during carbon starvation and they enable P. pastoris to overcome nutritional stress. Finally, green fluorescent protein can be synthesized efficiently from GDH2 and PEPCK promoters using food-grade monosodium glutamate as inducer indicating that the post-transcriptional regulatory circuit described here can be exploited for the development of glutamate-inducible P. pastoris expression system.


title: A redox-dependent mechanism for AMPK dysregulation interrupts metabolic adaptation of cancer under glucose deprivation

abstract:
Accelerated aerobic glycolysis is a distinctive metabolic property of cancer cells that confers dependency on glucose for survival. However, the selective therapeutic targeting of this vulnerability has yielded mixed results owing to the different sensitivities of each cancer type to glucose removal and lack of insight into the underlying mechanisms of glucose deprivation-induced cell death.

Here, we screened multiple cell lines to determine their sensitivities to glucose deprivation and found that the cell lines most sensitive to glucose deprivation failed to activate AMPK, a major regulator of metabolic adaptation, resulting in metabolic catastrophe.

Notably, glucose deprivation-induced AMPK dysregulation and rapid cell death were observed only in cancer cell lines with high expression of cystine/glutamate antiporter xCT. While this phenomenon was prevented by pharmacological or genetic inhibition of xCT, overexpression of xCT sensitized resistant cancer cells to glucose deprivation. We found that cystine uptake and glutamate export through xCT under glucose deprivation contributes to rapid NADPH depletion, leading to the collapse of the redox system, which subsequently inactivates AMPK by inhibitory oxidation and phosphorylation. This AMPK dysregulation caused a failure of metabolic switch to fatty acid oxidation upon glucose deprivation, resulting in mitochondrial dysfunction and cell death.

Taken together, these findings suggest a novel cross-talk between the metabolic and signal transduction and reveal a metabolic vulnerability in xCT-high expressing cancer cells to glucose deprivation and provide a rationale for targeting glucose metabolism in these cancer cells.

category: cancer biology


title: CRISPR enriches for cells with mutations in a p53-related interactome, and this can be inhibited.

abstract: CRISPR/Cas9 can be used to inactivate or modify genes by inducing double-stranded DNA breaks1-3. As a protective cellular response, DNA breaks result in p53-mediated cell cycle arrest and activation of cell death programs4,5. Inactivating p53 mutations are the most commonly found genetic alterations in cancer, highlighting the important role of the gene6-8. Here, we show that cells deficient in p53, as well as in genes of a core CRISPR-p53 tumor suppressor interactome, are enriched in a cell population when CRISPR is applied. Such enrichment could pose a challenge for clinical CRISPR use. Importantly, we identify that transient p53 inhibition suppresses the enrichment of cells with these mutations. Furthermore, in a data set of >800 human cancer cell lines, we identify parameters influencing the enrichment of p53 mutated cells, including strong baseline CDKN1A expression as a predictor for an active CRISPR-p53 axis. Taken together, our data identify strategies enabling safe CRISPR use.


title: Shank3 influences mammalian sleep development

abstract:
Sleep problems are prevalent in Autism Spectrum Disorder (ASD), can be observed before diagnosis, and are associated with increased restricted and repetitive behaviors. Therefore, sleep abnormalities may be a core feature of the disorder, but the developmental trajectory remains unknown. Animal models provide a unique opportunity to understand sleep ontogenesis in ASD. Previously we showed that adult mice with a truncation in the high-confidence ASD gene Shank3 (Shank3{Delta}C) recapitulate the clinical sleep phenotype. In this study we used longitudinal electro-encephalographic (EEG) recordings to define, for the first time, changes in sleep from weaning to young adulthood in an ASD mouse model. We show that Shank3{Delta}C mice sleep less overall throughout their lifespan, have increased rapid eye movement (REM) sleep early in life despite significantly reduced non-rapid eye movement (NREM) sleep, and have abnormal responses to increased sleep pressure that emerge during a specific developmental period. We demonstrate that the ability to fall asleep quickly in response to sleep loss develops normally between 24 and 30 days in mice. However, mutants are unable to reduce sleep latency after periods of prolonged waking and maintain the same response to sleep loss regardless of age. This phenomenon seems independent of homeostatic NREM sleep slow-wave dynamics. Overall, our study recapitulates both preclinical models and clinical studies showing that reduced sleep is consistently associated with ASD and suggests that problems falling asleep may reflect abnormal development of sleep and arousal mechanisms.

SIGNIFICANCE STATEMENTIn this first longitudinal sleep study in an Autism mouse model, we demonstrate that sleeping less seems a core feature of the disorder while problems falling asleep emerge during development.


title: Development of Equine Immunoglobulin Fragment F(ab')2 with High Neutralizing Capability against SARS-CoV-2

abstract: The ongoing pandemic, COVID-19, caused by SARS-CoV-2 has taken the world, and especially the scientific community by storm. While vaccines are being introduced into the market, there is also a pressing need to find potential drugs and therapeutic modules. Remdesivir is one of the antivirals currently being used with a limited window of action. As more drugs are being vetted, passive immunotherapy in the form of neutralizing antibodies can provide immediate action to combat the increasing numbers of COVID-positive cases. Herein, we demonstrate that equines hyper-immunized with chemically inactivated SARS-CoV-2 generate high titers of antibody with a strong virus neutralizing potential. ELISA performed with pooled antisera displayed highest immunoglobulin titer on 42 days post-immunization, at 1:51,200 dilutions. F(ab)2 immunoglobulin fragments generated from the pools also showed very high, antigen-specific affinity at 1:102,400 dilutions. Finally, in vitro virus neutralization assays confirmed that different pools of F(ab)2 fragments could successfully neutralize SARS-CoV-2 with titers well above 25,000, indicating the potential of this strategy in treating severe COVID-19 cases with high titers. The F(ab)2 was able to cross neutralize another SARS-CoV-2 strain, demonstrating its efficacy against the emerging viral variants and the importance of this approach in our efforts of eradication of COVID-19. In conclusion, this study demonstrates that virus-neutralizing antibodies raised in equines can potentially be used as a treatment regimen in the form of effective passive immunotherapy to combat COVID-19.

category: immunology


title: Bruton's Tyrosine Kinase Supports Gut Mucosal Immunity and Commensal Microbiome Recognition in Autoimmune Arthritis

abstract: Brutons tyrosine kinase (Btk) deficiency preferentially eliminates autoreactive B cells while sparing normal humoral responses, but has not been studied in mucosal immunity. Commensal microbes are essential for arthritis in K/BxN mice, used here to examine how BTK-mediated signaling interfaces with the microbiome. Btk-deficient K/BxN mice were found to have small Peyers Patches with reduced germinal center and IgA+ B cells. Although lamina propria IgA+ plasma cells were numerically normal, intestinal IgA was low and IgA coating of commensal bacteria was reduced. IgA-seq showed a shift in microbes that are normally IgA-coated into the uncoated fraction in Btk-deficient mice. In this altered microbial milieau, the proportion of Parabacteroides distasonis was reduced in Btk-deficient K/BxN mice. To determine whether P. distasonis contributes to arthritis, it was reintroduced into antibiotic-protected K/BxN mice, where it restored disease. This suggests that P. distasonis inability to thrive in Btk-deficient mice may be a factor in disease protection. Thus, BTK supports normal intestinal IgA development, with downstream effects on the microbiome that may contribute to autoimmunity.


title: The dual function monoclonal antibodies VIR-7831 and VIR-7832 demonstrate potent in vitro and in vivo activity against SARS-CoV-2

abstract: Sotrovimab (VIR-7831) and VIR-7832 are dual action monoclonal antibodies (mAbs) targeting the spike glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sotrovimab and VIR-7832 were derived from a parent antibody (S309) isolated from memory B cells of a 2003 severe acute respiratory syndrome coronavirus (SARS-CoV) survivor. Both mAbs contain an "LS" mutation in the Fc region to prolong serum half-life. In addition, VIR-7832 encodes an Fc GAALIE mutation that has been shown previously to evoke CD8+ T-cells in the context of an in vivo viral respiratory infection. Sotrovimab and VIR-7832 neutralize wild-type and variant pseudotyped viruses and authentic virus in vitro. In addition, they retain activity against monoclonal antibody resistance mutations conferring reduced susceptibility to previously authorized mAbs. The sotrovimab/VIR-7832 epitope continues to be highly conserved among circulating sequences consistent with the high barrier to resistance observed in vitro. Furthermore, both mAbs can recruit effector mechanisms in vitro that may contribute to clinical efficacy via elimination of infected host cells. In vitro studies with these mAbs demonstrated no enhancement of infection. In a Syrian Golden hamster proof-of concept wildtype SARS-CoV-2 infection model, animals treated with sotrovimab had less weight loss, and significantly decreased total viral load and infectious virus levels in the lung compared to a control mAb. Taken together, these data indicate that sotrovimab and VIR-7832 are key agents in the fight against COVID-19.


title: Droplet-based single cell RNA sequencing of bacteria identifies known and previously unseen cellular states

abstract: Clonal bacterial populations rely on transcriptional variation to differentiate into specialized cell states that increase the communitys fitness. Such heterogeneous gene expression is implicated in many fundamental microbial processes including sporulation, cell communication, detoxification, substrate utilization, competence, biofilm formation, motility, pathogenicity, and antibiotic resistance1. To identify these specialized cell states and determine the processes by which they develop, we need to study isogenic bacterial populations at the single cell level2,3. Here, we develop a method that uses DNA probes and leverages an existing commercial microfluidic platform (10X Chromium) to conduct bacterial single cell RNA sequencing. We sequenced the transcriptome of over 15,000 individual bacterial cells, detecting on average 365 transcripts mapping to 265 genes per cell in B. subtilis and 329 transcripts mapping to 149 genes per cell in E. coli. Our findings correctly identify known cell states and uncover previously unreported cell states. Interestingly, we find that some metabolic pathways segregate into distinct subpopulations across different bacteria and growth conditions, suggesting that some cellular processes may be more prone to differentiation than others. Our high throughput, highly resolved single cell transcriptomic platform can be broadly used for understanding heterogeneity in microbial populations.

category: microbiology


title: A genome-annotated bacterial collection of the barley rhizosphere microbiota

abstract: We generated a bacterial collection from the rhizosphere of cultivated barley (Hordeum vulgare L. ssp. vulgare) to assess taxonomic distribution of culturable members of the barley microbiota and their plant growth-promoting potential. From this we retrieved strains belonging to the dominant phyla of the plant microbiota-- Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria--and gathered evidence they code for functional genes implicated in nitrogen fixation, hydrogen cyanide channels and phosphate solubilisation. Here we present an initial comparative genomic analysis of the collection revealing that plant growth-promoting potential of the culturable barley bacterial microbiota appears to have a relatively broad phylogenetic base while retaining some strain-specificity.


title: Metapopulation Structure of Diatom-associated Marine Bacteria

abstract: Marine bacteria-phytoplankton interaction ultimately shapes ecosystem productivity. The biochemical mechanisms underlying their interactions become increasingly known, yet how these ubiquitous interactions drive bacterial evolution has not been illustrated. Here, we sequenced genomes of 294 bacterial isolates associated with 19 coexisting diatom cells. These bacteria constitute eight genetically monomorphic populations of the globally abundant Roseobacter group. Six of these populations are members of Sulfitobacter, arguably the most prevalent bacteria associated with marine diatoms. A key finding is that populations varying at the intra-specific level have been differentiated and each are either associated with a single diatom host or with multiple hosts not overlapping with those of other populations. These closely related populations further show functional differentiation; they differ in motility phenotype and they harbor distinct types of secretion systems with implication for mediating organismal interactions. This interesting host-dependent population structure is even evident for demes within a genetically monomorphic population but each associated with a distinct diatom cell, as shown by a greater similarity in genome content between isolates from the same host compared to those from different hosts. Importantly, the intra- and inter-population differentiation pattern remains when the analyses are restricted to isolates from intra-specific diatom hosts, ruling out distinct selective pressures and instead suggesting coexisting microalgal cells as physical barriers of bacterial gene flow. Taken together, microalgae-associated bacteria display a unique microscale metapopulation structure, which consists of numerous small populations whose evolution is driven by random genetic drift.


title: Cannabidiol Inhibits SARS-CoV-2 Replication and Promotes the Host Innate Immune Response

abstract: The rapid spread of COVID-19 underscores the need for new treatments. Here we report that cannabidiol (CBD), a compound produced by the cannabis plant, inhibits SARS-CoV-2 infection. CBD and its metabolite, 7-OH-CBD, but not congeneric cannabinoids, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after cellular infection, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD induces interferon expression and up-regulates its antiviral signaling pathway. A cohort of human patients previously taking CBD had significantly lower SARS-CoV-2 infection incidence of up to an order of magnitude relative to matched pairs or the general population. This study highlights CBD, and its active metabolite, 7-OH-CBD, as potential preventative agents and therapeutic treatments for SARS-CoV-2 at early stages of infection.


title: Myogenetic oligodeoxynucleotide (myoDN) recovers the differentiation of skeletal muscle myoblasts deteriorated by diabetes mellitus

abstract: Sarcopenic obesity is a complication of decreased muscle mass and strength associated with obesity, and sarcopenia associated with diabetes mellitus (DM) is a serious risk factor that may result in mortality. Deteriorated differentiation of muscle precursor cells, called myoblasts, in DM patients is considered to be one of the causes of muscle atrophy. We recently developed myogenetic oligodeoxynucleotides (myoDNs), which are 18-base single-strand DNAs that promote myoblast differentiation by targeting nucleolin. Herein, we report the applicability of a myoDN, iSN04, to myoblasts isolated from patients with type 1 and type 2 DM. Myogenesis of DM myoblasts was exacerbated concordantly with a delayed shift of myogenic transcription and induction of interleukins. Analogous phenotypes were reproduced in healthy myoblasts cultured with excessive glucose or palmitic acid, mimicking hyperglycemia or hyperlipidemia. iSN04 treatment recovered the deteriorated differentiation of plural DM myoblasts by downregulating myostatin and interleukin-8. iSN04 also ameliorated the impaired myogenic differentiation induced by glucose or palmitic acid. These results demonstrate that myoDNs can directly facilitate myoblast differentiation in DM patients, making them novel candidates for nucleic acid drugs to treat sarcopenic obesity.

category: cell biology


title: Courtship ritual of male and female nuclei during fertilisation in Neurospora crassa

abstract: Sexual reproduction is a key process influencing the evolution and adaptation of animals, plants and many eukaryotic microorganisms, such as fungi. Mycologists have described the different fungal fruiting bodies, while geneticists have partly unravelled the regulation of sexual development. However, the sequential fungal cell biology of fertilisation and the associated nuclear dynamics after plasmogamy are poorly understood. Using histone-fluorescent parental isolates, we tracked male and female nuclei during fertilisation in the model ascomycetes Neurospora crassa using live-cell-imaging. This study unravels the behaviour of trichogyne resident female nuclei and the extraordinary manner that male nuclei migrate up the trichogyne to the protoperithecium. Our observations raise new fundamental questions about the modus operandi of nuclei movements during sexual reproduction, male and female nuclear identity, guidance of nuclei within the trichogyne and, unexpectedly, the avoidance of "polyspermy" in fungi. The spatio-temporal dynamics of male nuclei within the trichogyne following plasmogamy are also described, where the speed and the deformation of male nuclei are of the most dramatic observed to date in a living organism.


title: SNHG16 promotes cell proliferation and inhibits cell apoptosis via regulation of the miR-1303p/STARD9 axis in renal cell carcinoma

abstract:
BackgroundRenal cell carcinoma (RCC) is a fatal malignant tumor with high morbidity. Numerous medical studies have suggested that long noncoding RNAs (lncRNAs) exert their biological function on various cancerous progresses. Herein, functions of lncRNA SNHG16 in RCC cells and the mechanism medicated by SNHG16 were investigated.

MethodsThe expression levels of SNHG16 and its downstream genes in RCC cells and tissues were examined utilizing reverse transcription quantitative polymerase chain reaction analyses. Cell counting kit-8 and 5-Ethynyl-2-deoxyuridine assays were carried out to evaluate the proliferation of RCC cells, and flow cytometry analyses were employed to determine the apoptosis of RCC cells. Western blot analysis was applied to examine protein levels associated with cell proliferation and apoptosis. The combination between SNHG16 and miRNA as well as miRNA and its target gene were explored by luciferase reporter, RNA pull down, and RNA immunoprecipitation assays.

ResultsThe significant upregulation of SNHG16 was observed in RCC tissues and cells. SNHG16 downregulation inhibited the proliferation and promoted the apoptosis of RCC cells. In addition, SNHG16 served as a competing endogenous RNA for miR-1301-3p, and STARD9 was a target gene of miR-1301-3p in RCC cells. SNHG16 upregulated STARD9 expression by binding with miR-1301-3p in RCC cells. Rescue assays validated that SNHG16 promoted RCC cell promotion and induced RCC cell apoptosis by upregulating STARD9 expression.

ConclusionsSNHG16 promotes RCC cell proliferation and suppresses RCC cell apoptosis via interaction with miR-1301-3p to upregulate STARD9 expression in RCC cells.


title: Cell-type-specific chromatin occupancy by the pioneer factor Zelda drives key developmental transitions in Drosophila

abstract: During Drosophila embryogenesis, the essential pioneer factor Zelda defines hundreds of cis-regulatory regions and in doing so reprograms the zygotic transcriptome. While Zelda is essential later in development, it is unclear how the ability of Zelda to define cis-regulatory regions is shaped by cell-type-specific chromatin architecture. Asymmetric division of neural stem cells (neuroblasts) in the fly brain provide an excellent paradigm for investigating the cell-type-specific functions of this pioneer factor. We show that Zelda synergistically functions with Notch to maintain neuroblasts in an undifferentiated state. Zelda misexpression reprograms progenitor cells to neuroblasts, but this capacity is limited by transcriptional repressors critical for progenitor commitment. Zelda genomic occupancy in neuroblasts is reorganized as compared to the embryo, and this reorganization is driven by differences in chromatin accessibility and cofactor availability. We propose that Zelda regulates essential transitions in the neuroblasts and embryo through a shared gene-regulatory network by defining cell-type-specific enhancers.

category: developmental biology


title: Lamb1a regulates atrial growth by limiting excessive, contractility-dependent second heart field addition during zebrafish heart development

abstract: During early vertebrate heart development, the heart transitions from a linear tube to a complex asymmetric structure. This process includes looping of the tube and ballooning of the emerging cardiac chambers, which occur simultaneously with growth of the heart. A key driver of cardiac growth is deployment of cells from the Second Heart Field (SHF) into both poles of the heart, with cardiac morphogenesis and growth intimately linked in heart development. Laminin is a core component of extracellular matrix (ECM) basement membranes, and although mutations in specific laminin subunits are linked with a variety of cardiac abnormalities, including congenital heart disease and dilated cardiomyopathy, no role for laminin has been identified in early vertebrate heart morphogenesis. We identified dynamic, tissue-specific expression of laminin subunit genes in the developing zebrafish heart, supporting a role for laminins in heart morphogenesis. lamb1a mutants exhibit cardiomegaly from 2dpf onwards, with subsequent progressive defects in cardiac morphogenesis characterised by a failure of the chambers to compact around the developing atrioventricular canal. We show that loss of lamb1a results in excess addition of SHF cells to the atrium, revealing that Lamb1a functions to limit heart size during cardiac development by restricting SHF addition to the venous pole. lamb1a mutants exhibit hallmarks of altered haemodynamics, and specifically blocking cardiac contractility in lamb1a mutants rescues heart size and atrial SHF addition. Furthermore, we identify that FGF and RA signalling, two conserved pathways promoting SHF addition, are regulated by heart contractility and are dysregulated in lamb1a mutants, suggesting that laminin mediates interactions between SHF deployment, heart biomechanics, and biochemical signalling during heart development. Together, this describes the first requirement for laminins in early vertebrate heart morphogenesis, reinforcing the importance of specialised ECM composition in cardiac development.


title: Cell-specific alterations in Pitx1 regulatory landscape activation caused by the loss of a single enhancer

abstract: Most developmental genes rely on multiple transcriptional enhancers for their accurate expression during embryogenesis. Because enhancers may have partially redundant activities, the loss of one of them often leads to a partial loss of gene expression and concurrent moderate phenotypic outcome, if any. While such a phenomenon has been observed in many instances, the nature of the underlying mechanisms remains elusive. We used the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion in vivo of one of its enhancers (Pen), which normally accounts for 30 percent of Pitx1 expression in hindlimb buds. By combining single cell transcriptomics and a novel in embryo cell tracing approach, we observed that this global decrease in Pitx1 expression results from both an increase in the number of non- or low-expressing cells, and a decrease in the number of high-expressing cells. We found that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. The resulting increase in Pitx1 non/low-expressing cells eventually affects the proximal limb more severely than the distal limb, leading to a clubfoot phenotype likely produced through a localized heterochrony and concurrent loss of irregular connective tissue. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes.


title: Factors Shaping Young and Mature Bacterial Biofilm Communities in Two Drinking Water Distribution Networks

abstract:
The presence of biofilms in drinking water distribution systems (DWDS) can affect both water quality and system integrity; yet these systems remain poorly studied due to lack of accessibility. We established two independent full-scale DWDS Testbeds (A and B) on two different campuses situated in a tropical urban environment and equipped them with online sensors. Testbed B experienced higher levels of monochloramine and lower water age than Testbed A within the campus. Based on long amplicon-sequencing of bacterial 16S rRNA genes extracted from the mature biofilms (MPB) growing on pipes and young biofilms (YSB) growing on the sensors, a core community was identified in the two testbeds. The relative abundances of operational taxonomic units at the family level, including Mycobacteriaceae, Methylobacteriaceae, Rhodospirillaceae, Nitrosomonadaceae, and Moraxellaceae, were consistent for MPB and YSB on each campus. The MPB community was found to be influenced by conductivity, sample age, and pipe diameter as determined by both canonical correlation analysis and fuzzy set ordination. MPB displayed higher -diversity based on Hill numbers than YSB; in general, second order Hill numbers correlated positively with conductivity and sample age, but negatively with ORP and nitrite. Pseudomonas spp. together with Bacillus spp. likely initiated biofilm formation of YSB on Testbed A under conditions of reduced monochloramine and high water age. Significant levels of orthophosphate were detected in YSB samples at two stations and associated with higher levels of stagnation based on long-term differential turbidity measurement (DTM). Orthophosphate and DTM may act as indicators of the biofilm growth potential within DWDS.

Highlights- Established two testbeds to study biofilms in full-scale distribution system
- Biofilms on pipes and sensors had core community
- Temporal effect and higher -diversity for biofilms on pipes
- Water chemistry was related to biofilm community differences

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/434709v1_ufig1.gif" ALT="Figure 1">
View larger version (26K):
org.highwire.dtl.DTLVardef@c392aforg.highwire.dtl.DTLVardef@1d355eborg.highwire.dtl.DTLVardef@1addf37org.highwire.dtl.DTLVardef@19cc22_HPS_FORMAT_FIGEXP  M_FIG Graphical Abstract

C_FIG


title: Drivers of Rift Valley fever virus persistence and the impact of control measures in a spatially heterogeneous landscape: the case of the Comoros archipelago, 2004-2015

abstract: Rift Valley fever (RVF) is one of the many zoonotic arboviral haemorrhagic fevers present in Africa. The ability of the pathogen to persist in multiple geographically distinct regions has raised concerns about its potential for spread to and persistence within currently disease-free areas. However, the mechanisms for which RVF virus persistence occurs at both local and broader geographical scales have yet to be fully understood and rigorously quantified. Here, we developed a mathematical metapopulation model describing RVF virus transmission in livestock across the four islands of the Comoros archipelago and fitted this model in a Bayesian framework to surveillance data conducted in livestock across those islands between 2004 and 2015. In doing so, we estimated the importance of island-specific environmental factors and animal movements between those islands on the persistence of RVF virus in the archipelago, and we further tested the impact of different control scenarios on reducing disease burden. We demonstrated that the archipelago network was able to sustain viral transmission over 10 years after assuming only one introduction event during early 2007. Movement restrictions were only useful to control the disease in Anjouan and Mayotte, as Grande Comore and Moheli were able to self-sustain RVF viral persistence, probably due to local environmental conditions that are more favourable for vectors. We also evidenced that repeated outbreaks during 2004-2020 may have gone under-detected by local surveillance in Grande Comore and Moheli. Strengthened longterm and coordinated surveillance would enable the detection of viral re-emergence and evaluation of different relevant vaccination programmes.


title: Developmental innovations promote species diversification in mushroom-forming fungi

abstract: Fungi evolved complex fruiting body ( mushroom) morphologies as adaptations to efficient spore dispersal in terrestrial habitats. Mushroom-forming fungi (Agaricomycetes) display a graded series of developmental innovations related to fruiting body morphology, however, how these evolved is largely unknown, leaving the functional biology and evolutionary principles of complex multicellularity in the third largest multicellular kingdom poorly known. Here, we show that developmental innovations of mushroom-forming fungi that enclose the spore-producing surface (hymenophore) in a protected environment display significant asymmetry in their evolution and are associated with increased diversification rates.  Enclosed development and related tissues (partial and universal veils) evolved convergently and became a widespread developmental type in clades in which it emerged. This probably mirrors increased fitness for protected fruiting body initials in terrestrial habitats, by better coping with environmental factors such as desiccation or predators, among others. We observed similar patterns in the evolution of complex hymenophore architectures, such as gills, pores or teeth, which optimize biomass-to-propagule number ratios and were found to spur diversification in mushrooms. Taken together, our results highlight new morphological traits associated with the adaptive radiation of mushroom-forming fungi and present formal phylogenetic testing of hypotheses on the reproductive ecology of a poorly known but hyperdiverse clade.

category: evolutionary biology


title: Mitochondrial genome sequencing of marine leukemias reveals cancer contagion between clam species in the Seas of Southern Europe

abstract: Clonally transmissible cancers are tumour lineages that are transmitted between individuals via the transfer of living cancer cells. In marine bivalves, leukemia-like transmissible cancers, called hemic neoplasia, have demonstrated the ability to infect individuals from different species. We performed whole-genome sequencing in eight warty venus clams that were diagnosed with hemic neoplasia, from two sampling points located more than 1,000 nautical miles away in the Atlantic Ocean and the Mediterranean Sea Coasts of Spain. Mitochondrial genome sequencing analysis in tumour tissues from neoplastic animals revealed the coexistence of haplotypes from two different clam species. Phylogenies estimated from mitochondrial and nuclear markers confirmed this leukemia originated in striped venus clams and later transmitted to clams of the species warty venus, in which it survives as a contagious cancer. The analysis of mitochondrial and nuclear gene sequences supports all studied tumours belong to a single neoplastic lineage that spreads in the Seas of Southern Europe.

category: genomics


title: Butyryl/Caproyl-CoA:Acetate CoA-Transferase: Cloning, Expression and Characterization of the Key Enzyme Involved in Medium-Chain Fatty Acid Biosynthesis

abstract: Coenzyme A transferases (CoATs) are important enzymes involved in carbon chain elongation contributing to medium-chain fatty acid (MCFA) biosynthesis. For example, butyryl-CoA:acetate CoA transferase (BCoAT) is responsible for the final step of butyrate synthesis from butyryl-CoA. However, little is known about caproyl-CoA:acetate CoA-transferase (CCoAT), which is responsible for the final step of caproate synthesis from caproyl-CoA. In this study, two CoAT genes from Ruminococcaceae bacterium CPB6 and Clostridium tyrobutyricum BEY8 were identified by gene cloning and expression analysis. The enzyme assays and kinetic studies were carried out using butyryl-CoA or caproyl-CoA as the substrate. CPB6-CoAT can catalyze the conversion of both butyryl-CoA to butyrate and caproyl-CoA to caproate, but its catalytic efficiency with caproyl-CoA as the substrate was 3.8 times higher than that with butyryl-CoA. In contrast, BEY8-CoAT had only BCoAT activity, not CCoAT activity. This demonstrated the existence of a specific CCoAT involved in chain elongation via the reverse {beta}-oxidation pathway. Comparative bioinformatics analysis showed the presence of a highly conserved motif (GGQXDFXXGAXX) in CoATs, which is predicted to be the active center of CoATs. Single point mutations in the conserved motif of CPB6-CoAT (Asp346 and Ala351) led to marked decreases in the activity for butyryl-CoA and caproyl-CoA, indicating that the conserved motif is the active center of CPB6-CoAT, and sites Asp346 and Ala351 were critical residues that affect enzymatic activity. This work provides insight into the function of CCoAT in caproic acid biosynthesis and improves the understanding of the chain elongation pathway for MCFA production.


title: The bacterial amyloids phenol soluble modulins from Staphylococcus aureus catalyze alpha-synuclein aggregation

abstract: Aggregated -synuclein (-syn) is the main constituent of Lewy bodies, the main pathological hallmark of Parkinsons disease (PD). Environmental factors are thought to be potential triggers capable of initiating the aggregation of the otherwise monomeric -syn. Braaks seminal work redirected attention to the intestine and recent reports of dysbiosis have highlighted the potential causative role that the microbiome might play in the pathology of PD. Staphylococcus aureus is a bacterium carried by 30-70% of the general population. It has been shown to produce functional amyloids, called Phenol Soluble Modulins (PSMs). Here, we studied the kinetics of -syn aggregation under quiescent conditions in the presence or absence of four different PSM peptides and observed a remarkable shortening of the lag phase in their presence. Whereas pure -syn monomer did not aggregate up to 450 h after initiation of the experiment in neither neutral nor mildly acidic buffer, the addition of different PSM peptides resulted in an almost immediate increase in the Thioflavin T (ThT) fluorescence. Despite similar peptide sequences, the different PSM peptides displayed distinct effects on the kinetics of -syn aggregation. Kinetic analyses of the data suggest that while all four peptides catalyze -syn aggregation, the underlying mechanisms might differ with a model of nucleation and elongation fitting the -syn aggregation induced by PSM2 but not PSM1. The results of immunogold TEM imply that the aggregates are fibrillar and composed of -syn. Addition of the co-aggregated materials to HEK cells expressing the A53T -syn variant fused to GFP was found to catalyze -syn aggregation and phosphorylation in the cells. Our results provide evidence of a potential trigger of synucleinopathies and could have implications for the prevention of the diseases.


title: Tight junction ZO proteins maintain tissue fluidity, ensuring efficient collective cell migration

abstract: Tight junctions are essential components of epithelial tissues connecting neighboring cells to provide protective barriers. Albeit their general function to seal compartments is well understood, their role in collective cell migration is largely unexplored. Here, the importance of the tight junction proteins ZO1 and ZO2 for epithelial migration is investigated employing video microscopy in conjunction with velocimetry, segmentation, cell tracking, and atomic force microscopy/spectroscopy. The results indicate that ZO proteins are necessary for fast and coherent migration. In particular, ZO1 and 2 loss (dKD) induces actomyosin remodeling away from the central cortex towards the periphery of individual cells, resulting in altered viscoelastic properties. A tug-of-war emerges between two subpopulations of cells with distinct morphological and mechanical properties: 1) smaller and highly contractile cells with an outward-bulged apical membrane, and 2) larger, flattened cells, which, due to tensile stress, display a higher proliferation rate. In response, the cell density increases, leading to crowding-induced jamming and more small cells over time. Co-cultures comprising wildtype and dKD cells display phase separation based on differences in contractility rather than differential adhesion. This study shows that ZO proteins are necessary for efficient collective cell migration by maintaining tissue fluidity and controlling proliferation.

category: biophysics


title: A photoswitchable helical peptide with light-controllable interface / transmembrane topology in lipidic membranes

abstract: According to the three-step model, the spontaneous insertion and folding of helical transmembrane (TM) polypeptides into lipid bilayers is driven by three sequential equilibria: solution-to-membrane interface (MI) partition, unstructured-to-helical folding, and MI-to-TM helix insertion. However, understanding these three steps with molecular detail has been challenged by the lack of suitable experimental approaches to rapidly and reversibly perturb membrane-bound hydrophobic polypeptides out of equilibrium. Here, we report on a 24-residues-long hydrophobic -helical polypeptide, covalently coupled to an azobenzene photoswitch (KCALP-azo), which displays a light-controllable TM/MI equilibrium in hydrated lipid bilayers. FTIR spectroscopy shows that dark-adapted KCALP-azo (trans azobenzene) folds as a TM -helix, with its central TM region displaying an average tilt of 36 {+/-} 4{degrees} with the membrane normal (TM topology). After trans-to-cis photoisomerization of the azobenzene moiety with UV light (reversed with blue light), spectral changes by FTIR spectroscopy indicate that the helical structure of KCALP-azo is maintained but the peptide experiences a more polar environment. Interestingly, pH changes induced similar spectral alterations in the helical peptide LAH4, with a well-characterized pH-dependent TM/MI equilibrium. Polarized experiments confirmed that the membrane topology of KCALP-azo is altered by light, with its helix tilt changing reversibly from 32 {+/-} 5{degrees} (TM topology, blue light) to 79 {+/-} 8{degrees} (MI topology, UV light). Further analysis indicates that, while the trans isomer of KCALP-azo is ~100% TM, the cis isomer exists in a ~90% TM and ~10% MI mixture. Strategies to further increase the perturbation of the TM/MI equilibrium with the light are briefly discussed.


title: Integrated Systems-Analysis of the Human and Murine Pancreatic Cancer Glycomes Reveal a Tumor Promoting Role for ST6GAL1

abstract:
Pancreatic ductal adenocarcinoma (PDA) is the 3rd leading cause of cancer-death in the U.S.. Glycans, such as CA-19-9, are biomarkers of PDA and are emerging as important modulators of cancer phenotypes. Herein, we utilized a systems-based approach integrating glycomic analysis of human PDA and the well-established KC mouse model, with transcriptomic data to identify and probe the functional significance of aberrant glycosylation in pancreatic cancer. We observed both common and distinct patterns of glycosylation in pancreatic cancer across species. Common alterations included increased levels of -2,3- and -2,6-sialic acids, bisecting GlcNAc and poly-LacNAc. However, core fucose, which was increased in human PDAC, was not seen in the mouse, indicating that not all human glycomic changes can be modeled in the KC mouse. In silico a nalysis of bulk and single cell sequencing data identified ST6GAL1, which underlies -2,6-sialic acid, as overexpressed in human PDA, concordant with histological data. Enzymes levels correlated with the stage of clinical disease. To test whether ST6GAL1 promotes pancreatic cancer we created a novel mouse in which a pancreas-specific genetic deletion of this enzyme overlays the KC mouse model. Analysis of our new model showed delayed cancer formation and a significant reduction in fibrosis. Our results highlight the importance of a strategic systems-approach to identifying glycans whose functions can be modeled in mouse, a crucial step in the development of therapeutics targeting glycosylation in pancreatic cancer.

SIGNIFICANCEPancreatic ductal adenocarcinoma (PDA) is the 3rd leading cause of cancer-death in the U.S.. Glycosylation is emerging as an important modulator of cancer phenotype. Herein we use a systems-approach integrating glycomics of human PDA and a well-established PDA mouse model with transcriptomic data to identify ST6GAL1, the enzyme underlying -2,6-sialic acid, as a potential cancer promoter. A pancreatic specific ST6GAL1 knockout in the KC mouse showed delayed cancer formation and a reduction in fibrosis. Our results highlight the importance of a strategic systems-approach to identifying glycans whose functions can be modeled in mouse, a crucial step in the development of therapeutics targeting glycosylation in pancreatic cancer.


title: Multiple introductions of multidrug-resistant typhoid associated with acute infection and asymptomatic carriage, Kenya

abstract: Understanding the dynamics of infection and carriage of typhoid in endemic settings is critical to finding solutions to prevention and control. In a 3 year case-control study, we investigated typhoid among children aged <16 years (4,670 febrile cases and 8,549 age matched controls) living in an informal settlement, Nairobi, Kenya. 148 S. Typhi isolates from cases and 95 from controls (stool culture) were identified; a carriage frequency of 1%. Whole-genome sequencing showed 97% of cases and 88% of controls were genotype 4.3.1 (Haplotype58), with the majority of each (76% and 88%) being multidrug-resistant strains in 3 sublineages of H58 genotype (East Africa 1 (EA1), EA2, and EA3), with sequences from cases and carriers intermingled. The high rate of multidrug-resistant H58 S. Typhi, and the close phylogenetic relationships between carriers and controls, provides evidence for the role of carriers as a reservoir for the community spread of typhoid in this setting.

category: physiology


title: Memory B cell repertoire for recognition of evolving SARS-CoV-2 spike

abstract: Memory B cell reserves can generate protective antibodies against repeated SARS-CoV-2 infections, but with an unknown reach from original infection to antigenically drifted variants. We charted memory B cell receptor-encoded monoclonal antibodies (mAbs) from 19 COVID-19 convalescent subjects against SARS-CoV-2 spike (S) and found 7 major mAb competition groups against epitopes recurrently targeted across individuals. Inclusion of published and newly determined structures of mAb-S complexes identified corresponding epitopic regions. Group assignment correlated with cross-CoV-reactivity breadth, neutralization potency, and convergent antibody signatures. mAbs that competed for binding the original S isolate bound differentially to S variants, suggesting the protective importance of otherwise-redundant recognition. The results furnish a global atlas of the S-specific memory B cell repertoire and illustrate properties conferring robustness against emerging SARS-CoV-2 variants.


title: CASPAM: a triple modality biosensor for multiplexed imaging of caspase network activity

abstract: Understanding signal propagation across biological networks requires to simultaneously monitor the dynamics of several nodes to uncover correlations masked by inherent intercellular variability. To monitor the enzymatic activity of more than two components over short time scales has proven challenging. Exploiting the narrow spectral width of homoFRET-based biosensors, up to three activities can be imaged through fluorescence polarization anisotropy microscopy. We introduce CASPAM (Caspase Activity Sensor by Polarization Anisotropy Multiplexing) a single-plasmid triple-modality-reporter of key nodes of the apoptotic network. Apoptosis provides an ideal molecular framework to study interactions between its three composing pathways (intrinsic, extrinsic and effector). We characterized the biosensor performance and demonstrated the advantages that equimolar expression has both in simplifying experimental procedure and reducing observable variation, thus enabling robust data-driven modelling. Tools like CASPAM become essential to analyze molecular pathways where multiple nodes need to be simultaneously monitored.


title: TCR signaling promotes the assembly of RanBP2/RanGAP1-SUMO1/Ubc9 NPC subcomplex via phosphorylation of RanGAP1 by PKC-θ

abstract: The nuclear pore complex (NPC) is the sole and selective gateway for nuclear transport and its dysfunction has been associated with many diseases. The NPC subcomplex RanBP2, which consists of RanBP2 (Nup358), RanGAP1-SUMO1 and Ubc9, regulates the assembly and function of the NPC. The roles of immune signaling in NPC assembly remain poorly understood. Here, we show that, following TCR stimulation, protein kinase C-{theta} (PKC-{theta}) directly phosphorylates RanGAP1 to facilitate RanBP2 subcomplex assembly and nuclear import and, thus, the nuclear translocation of AP-1 transcription factor. Mechanistically, TCR stimulation induces the translocation of activated PKC-{theta} to the NPC, where it interacts with and phosphorylates RanGAP1 on Ser504 and Ser506. RanGAP1 phosphorylation increases its binding affinity for Ubc9, thereby promoting sumoylation of RanGAP1 and, finally, assembly of the RanBP2 subcomplex. Our findings reveal an unexpected role of PKC-{theta} as a direct regulator of nuclear import and uncover a phosphorylation-dependent sumoylation of RanGAP1, delineating a novel link between TCR signaling and assembly of the RanBP2 NPC subcomplex.


title: The NOTCH3 Downstream Target HEYL Regulates Human Airway Epithelial Club Cell Differentiation

abstract: Basal cells (BC) are the resident stem/progenitor cells of the adult pseudostratified airway epithelium, whose differentiation program is orchestrated by the NOTCH signaling pathway. NOTCH3 receptor mediated signaling regulates BC to club cell differentiation; however, the downstream responses that regulate this process are largely unknown. In the present study we used an in vitro air-liquid interface model of the human pseudostratified airway epithelium to identify the NOTCH3-dependent downstream genes/pathways that regulate human BC to club cell differentiation. Activation of NOTCH3 signaling in BC via lentivirus-mediated over-expression of the active NOTCH3 intracellular domain (NICD3) promoted club cell differentiation. Bulk RNA-seq analysis of control vs NICD3-transduced cells, identified 692 NICD3 responsive genes enriched for pathways linked to airway epithelial biology and differentiation including Wnt/{beta}-catenin Signaling. Expression of the classical NOTCH target HEYL increased in response to NOTCH3 activation and positively correlated with expression of the club cell marker SCGB1A1. Further, using single-cell RNA-seq, we report that HEYL+ cells primarily clustered with SCGB1A1+ and NOTCH3+ cells. Moreover, HEYL protein co-localized with SCGB1A1 in ALI cultures in vitro and in the human and mouse airway epithelium in vivo. siRNA-mediated knockdown of HEYL in BC led to changes in epithelial structure including altered morphology and significant reductions in transepithelial electrical resistance and expression of tight junction related genes. Finally, HEYL knockdown significantly reduced the number of SCGB1A1+ club cells, along with a corresponding increase in KRT8+ BC-intermediate cells. Overall, our data identifies NOTCH3-HEYL signaling as a key regulator of BC to club cell differentiation.


title: The CATP-8/P5A-type ATPase functions in multiple pathways during neuronal patterning

abstract:
The assembly of neuronal circuits involves the migrations of neurons from their place of birth to their final location in the nervous system, as well as the coordinated growth and patterning of axons and dendrites. In screens for genes required for patterning of the nervous system, we identified the catp-8/P5A-ATPase as an important regulator of neural patterning. P5A-ATPases are part of the P-type ATPases, a family of proteins known to serve a conserved function as transporters of ions, lipids and polyamines in unicellular eukaryotes, plants, and humans. While the function of many P-type ATPases is relatively well understood, the function of P5A-ATPases in metazoans remained elusive. We show here, that the Caenorhabditis elegans ortholog catp-8/P5A-ATPase is required for specific aspects of nervous system development. Specifically, the catp-8/P5A-ATPase serves functions in shaping the elaborately sculpted dendritic trees of somatosensory PVD neurons. Moreover, catp-8/P5A-ATPase is required for axonal guidance and repulsion at the midline, as well as embryonic and postembryonic neuronal migrations. Interestingly, not all axons at the midline require catp-8/P5A-ATPase, although the axons run in the same fascicles and navigate the same space. Similarly, not all neuronal migrations require catp-8/P5A-ATPase. A CATP-8/P5A-ATPase reporter is localized to the ER in most if not all tissues and catp-8/P5A-ATPase can function both cell-autonomously and non-autonomously to regulate neuronal development. Genetic analyses establish that catp-8/P5A-ATPase can function in multiple pathways, including the Menorin pathway, previously shown to control dendritic patterning in PVD, and Wnt signaling, which functions to control neuronal migrations. Lastly, we show that catp-8/P5A-ATPase is required for localizing select transmembrane proteins necessary for dendrite morphogenesis. Collectively, our studies suggest that catp-8/P5A-ATPase serves diverse, yet specific roles in different genetic pathways, and may be involved in the regulation or localization of transmembrane proteins to specific subcellular compartments.

AUTHOR SUMMARYP-type ATPases are a large family of transporters that are conserved from unicellular eukaryotes and plants to metazoans. Structurally and functionally, they fall into five subfamilies, P1 to P5, of which the latter is further divided into P5A and P5B-type ATPases. Unlike for other P-type ATPases, no mutant phenotypes for the P5A-type ATPases have been described in metazoans. Here, we show that the catp-8/P5A-ATPase in the nematode Caenorhabditis elegans is involved in multiple aspects of neuronal patterning, including neuronal migrations as well as axon guidance and dendrite patterning. A functional fluorescent reporter fusion shows the CATP-8/P5A-ATPase is expressed in most, if not all, tissues in the endoplasmic reticulum and catp-8 can function both in neurons and surrounding tissues from where it orchestrates neuronal development. Genetically, catp-8 acts in multiple pathways during these processes, including the Wnt signaling and the Menorin pathway. Imaging studies suggest that the catp-8/P5A-ATPase is necessary for proper localization of cell-surface transmembrane molecules to dendrites of sensory neurons, but likely not for their trafficking. In summary, we propose that CATP-8/P5A-ATPase serves a function in the ER during development of select neurons, by localizing certain transmembrane, and possibly, secreted proteins


title: Activation of innate immunity by neutral polysaccharides from broccoli buds

abstract: Edible substances that stimulate the innate immune system are good candidates for functional foods to improve human health. We have previously reported that acidic polysaccharides from broccoli extract exhibit immunostimulatory effects, but neutral polysaccharides have been overlooked. In the present study, we found that neutral polysaccharides have significantly stronger (higher specific activity) immunostimulatory activity than acidic polysaccharides. The hot water extract of broccoli showed the immunostimulatory activity in the silkworm muscle contraction assay, suggesting that it stimulates innate immunity via paralytic peptide pathway. The activity was concentrated in the buds, but not in the stems and stalk. The active substance was recovered in the flow-through fraction of diethylaminoethyl-cellulose column chromatography with neutral polysaccharides. The specific activity of the fraction was significantly higher than that of the acidic polysaccharides from broccoli reported previously. These results suggest that the neutral polysaccharide present in broccoli buds stimulates innate immunity and can be semi-purified by one-step chromatography.


title: Growth differentiation factor 15 increases in both cerebrospinal fluid and serum during pregnancy

abstract:
AimGrowth differentiation factor 15 (GDF15) increases in serum during pregnancy to levels not seen in any other physiological state and is suggested to be involved in pregnancy-induced nausea, weight regulation and glucose metabolism. The main action of GDF15 is regulated through a receptor of the brainstem, i.e., through exposure of GDF15 in both blood and cerebrospinal fluid (CSF). The aim of the current study was to measure GDF15 in both CSF and serum during pregnancy, and to compare it longitudinally to non-pregnant levels.

MethodsWomen were sampled at elective caesarean section (n=45, BMI=28.1{+/-}5.0) and were followed up 5 years after pregnancy (n=25). GDF15, insulin and leptin were measured in CSF and serum. In addition, glucose, adiponectin and Hs-CRP were measured in blood.

ResultsGDF15 levels were higher during pregnancy compared with follow-up in both CSF (385{+/-}128 vs. 115{+/-}32 ng/l, p<0.001) and serum (73789{+/-}29198 vs. 404{+/-}102 ng/l, p<0.001). CSF levels correlated with serum levels during pregnancy (p<0.001), but not in the non-pregnant state (p=0.98). Both CSF and serum GDF15 were highest in women carrying a female fetus (p<0.001), previously linked to pregnancy-induced nausea. Serum GDF15 correlated with the homeostatic model assessment for beta-cell function and placental weight, and CSF GDF15 correlated inversely with CSF insulin levels.

ConclusionThis, the first study to measure CSF GDF15 during pregnancy, demonstrated increased GDF15 levels in both serum and CSF during pregnancy. The results suggest that effects of GDF15 during pregnancy can be mediated by increases in both CSF and serum levels.


title: RNA-silencing induces target gene relocalization toward a specialized nuage domain

abstract:
The authors wish to withdraw this manuscript and apologize for errors in the initial submission. All the original experiments were performed by YY. Unfortunately, JP and members of the Mello lab have not been able to replicate some aspects of the study. JP has failed to independently reproduce the specific results showing RNAi-triggered relocalization of target RNA, and P granule specific accumulation of (the P granule component GLH-1) as reported. The conditions/strains analyzed by JP were as follows: (1) oma-1 FISH on WT worms [control, 6 hr and 12 hr oma-1(RNAi)]. (2) oma-1 FISH on OMA-1:GFP worms [control, 6 hr oma-1(RNAi), or 6 hr gfp(RNAi)]. (3) oma-1 FISH on WT worms [control, 4hr, 6hr, 8 hr, and 10 hr oma-1(RNAi)]. 10-23 gonads were analyzed per experiment. Fixation conditions were essentially as described, with the only known difference being that gonads were not exposed to detergent prior to fixation. Using YYs reagents and protocol the Mello lab has not observed an obvious relocalization of target RNA to P granules (marked by GFP::GLH-1) after 6 hrs oma-1(RNAi); n=92 gonads. CM, JP and DG consider that the published images accurately represent the image stacks provided by YY as representative, raw data, but JP and CM note configurations of FISH signals in germ nuclei and gonad anatomy that they consider unusual. CM, JP and DG have not detected any evidence of image manipulation.

YY states that none of the raw image data were manipulated beyond standard adjustments for brightness and contrast prior to processing images for publication as described. However, YY reports that the images were not representative of the majority of sample gonads, and instead were pre-selected under low magnification for rare examples with asymmetrical, expanded P granules.

Efforts to identify conditions that explain the rare gonads imaged by YY continue in the Mello lab, as do efforts to reproduce independently each of the other reported results; we plan to provide an update in the near future.


title: Effects of hyperinsulinemia on pancreatic cancer development and the immune microenvironment revealed through single-cell transcriptomics

abstract: Hyperinsulinemia is independently associated with increased risk and mortality of pancreatic cancer. We recently reported that a [~]50% reduction in pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in mice could be achieved with reduced insulin production. However, only female mice remained normoglycemic and only the gene dosage of rodent-specific Ins1 alleles was tested in our previous model. Moreover, we did not delve into the molecular and cellular mechanisms associated with modulating hyperinsulinemia. Here, we studied PanIN lesion development in both male and female Ptf1aCreER;KrasLSL-G12D mice lacking the rodent specific Ins1 gene, and possessing one or two alleles of the wild-type Ins2 gene to modulate insulin production. High-fat diet induced hyperinsulinemia was transiently and modestly reduced, without affecting glucose tolerance, in male and female mice with only one allele of Ins2. Genetic reduction of insulin production resulted in mice with a tendency for less PanIN and acinar-to-ductal metaplasia (ADM) lesions. Using single-cell transcriptomics, we found hyperinsulinemia affected multiple cell types in the pancreas, with the most statistically significant effects on local immune cell populations, which were highly represented in our analysis. Specifically, hyperinsulinemia modulated pathways associated with protein translation, MAPK-ERK signaling, and PI3K-AKT signaling, which were changed in epithelial cells and subsets of immune cells. These data suggest a role for the immune microenvironment in hyperinsulinemia-driven PanIN development. Together with our previous work, we propose that mild suppression of insulin levels may be useful in preventing pancreatic cancer by acting on multiple cell types.


title: The cAMP effector PKA mediates Moody GPCR signaling in Drosophila blood-brain barrier formation and maturation

abstract: The blood-brain barrier (BBB) of Drosophila is comprised of a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previously, we identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. Here, we identify cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. We show that PKA is enriched at the basal side of the SPG cell and that this polarized Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity, by precisely regulating the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKAs molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments and not their total length is crucial for generating a proper paracellular seal. Based on these findings, we propose a model that polarized Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity during the continuous morphogenesis of the SPG secondary epithelium, which is critical for maintain tissue size and brain homeostasis during organogenesis.