title: Genetic influence on resting state networks in young male and female adults

abstract:
Determining genetic versus environmental influences on the human brain is of crucial importance to understand the healthy brain as well as in a variety of disease and disorder states. Here we propose a unique, minimal assumption, approach to investigate genetic influence on the functional connectivity of the brain using 260 subjects" (65 monozygotic (MZ) and 65 dizygotic (DZ) healthy young adult twin pairs) resting state fMRI (rsfMRI) data from the Human Connectome Project (HCP). For any given resting state connection between twin pairs, the connection strengths across pairs were subtracted from each other in both directions. By applying the F-Test for equality of variances per connection, we found that there were a number of significant connections that demonstrated greater variance among dizygotic pairs in comparison to monozygotic pairs, implying these connections were under significant genetic influence. These population (DZ-MZ) results remained true irrespective of gender, with the caveat that certain connections were significant on a gender-specific basis. This is the first study to our knowledge to assess the heritability across young healthy adults both in general and specific to gender.

Population Results & DiscussionAt the population level, there appears to be a posterior to anterior gradient of more to less genetic influence on brain connections and networks with visual > temporal, parietal > frontal. There was a high density of genetically-influenced functional connections predominantly involving posterior regions or networks of the brain: Visual Networks (VNs - primary visual, early visual, dorsal stream and ventral stream visual cortices, MT+ complex). These posterior regions of the brain with greater genetic influence are implicated for example in visual, perceptual, dorsal ("where") and ventral ("what") visuospatial processing streams (VNs).

There was a low-density or paucity of genetically-influenced functional connections predominantly involving anterior regions or networks of the brain comprising Task Positive Networks (TPNs): FrontoParietal Networks (FPNs - dorsolateral prefrontal, orbital and polar frontal, midcingulate, insular and frontal opercular, superior and inferior parietal cortices); FrontoTemporal Networks (FTNs - inferior frontal, posterior opercular, early auditory, auditory association cortices); Sensorimotor Networks (SMNs - premotor, somatosensory, paralobular, and motor cortices); These anterior regions of the brain with lesser genetic influence are implicated in various TPN processes; for example in high-level cognitive and affective processes such as working memory, executive function, reasoning, attentional and impulse control, emotional judgement and decision making (FPNs); language and auditory processes (FTNs); action-planning and movement processes (SMN).

There was a mix of high (posterior) and low (anterior) density of genetically influenced functional connections involving the extended Default Mode Network (eDMN). Specifically, there was a high density of genetically-influenced functional connections involving predominantly posterior-medial regions of eDMN - hippocampus and precuneus/posterior cingulate cortices; There was a low density of genetically influenced connections involving anterior regions (anterior cingulate and medial prefrontal) and lateral (inferior parietal, temporoparietooccipital) regions of the eDMN. The eDMN is involved in low-level cognitive and affective processes such as those involved in episodic memory retrieval, mental imagery, introspection, rumination, evaluation of self and others.

These differences in genetic influence on posterior (more) vs. anterior (less) brain regions may have implications in terms of the environmental influence (e.g., education, school and work environment, family and home environment, social interaction with friends and peers, medications, nutrition, sports and physical exercise) on posterior (less) vs. anterior (more) portions of the brain during development and later in life.

Gender-Specific Results & DiscussionAs noted at the population level, both males and females were under extensive genetic influence in terms of network interactions involving visual cortices. In addition, males were more genetically influenced in terms of network interactions involving auditory-language related cortices compared to females. This finding suggests that males may be more functionally "hard-wired" and females may be more environmentally influenced and shaped in terms of auditory-language systems than males.

As noted at the population level, both males and females were under extensive genetic influence in terms of interactions involving the eDMN which is considered a central hub of the brain for various processes such as internal monitoring, rumination and evaluation of self and others, as noted previously. In addition, males also were more genetically influenced compared to females in terms of intranetwork and internetwork interactions of eDMN and other brain regions (occipital, temporal, parietal, and frontal regions) involved in various task-oriented processes and attending to and interacting with the environment which comprise part of the Task Positive Networks (TPNs). There were also nearly five times more genetically influenced functional connections in males (310) than females (64) suggesting that male brains are more genetically influenced, i.e. functionally "hard-wired", than females. This result suggests differences in genetic predisposition in males (more) vs. females (less) in terms of interplay of attending to task-oriented interactions with the environment (TPNs) vs. internal and external interactions with self and others (eDMN). This finding may also have implications in terms of brain plasticity differences in males (less) versus females (more) in terms of ability to react or adapt/maladapt to environmental influences (e.g. task completion demands, psychosocial stressors, positive and negative feedback, meditation, cognitive behavioral therapy, pharmacotherapy) and their overall malleability.

These results reveal the similarities and differences of genetics and environmental influences on different connections, areas, and networks of the resting state functional brain in young healthy males and females with implications in development and later in life. This unique method can be applied in healthy as well as in patient populations to reveal the genetic and environmental influences on the brain.

SignificanceThere were high vs. low genetic influences on posterior vs. anterior brain regions involved in low-level visuospatial processes vs. high-level cognitive processes such as reasoning and language respectively. This finding may have implications in terms of the brain to be environmentally influenced (e.g., school, work and home environment) during development and later in life.

There were nearly five times more genetically influenced functional connections in males than females in brain regions involved in task-oriented interactions with environment vs. interactions with self and others. This finding may have implications in terms of brain plasticity differences in males (less) versus females (more) in terms of ability to adapt/maladapt to environmental influences (e.g. task completion demands, psychosocial stressors, various therapies) and their overall malleability. This is the first study to our knowledge to assess the heritability across young healthy adults both in general and specific to gender.


title: Fractal dimension of EEG signal senses complexity of fractal animations

abstract: We are surrounded with many fractal and self-similar patterns which has been area of many researches in the recent years. We can perceive self-similarities in various spatial and temporal scales; however, the underlying neural mechanism needs to be well understood. In this study, we hypothesized that complexity of visual stimuli directly influence complexity of information processing in the brain. Therefore, changes in fractal pattern of EEG signal must follow change in fractal dimension of animation. To investigate this hypothesis, we recorded EEG signal of fifteen healthy participants while they were exposed to several 2D fractal animations. Fractal dimension of each frame of the animation was estimated by box counting method. Subsequently, fractal dimensions of 32 EEG channels were estimated in a frequency specific manner. Then, association between pattern of fractal dimensions of the animations and pattern of fractal dimensions of EEG signals were calculated using the Pearsons correlation algorithm. The results indicated that fractal animation complexity is mainly sensed by changes in fractal dimension of EEG signals at the centro-parietal and parietal regions. It may indicate that when the complexity of visual stimuli increases the mechanism of information processing in the brain also enhances its complexity to better attend and comprehend the stimuli.

category: neuroscience


title: Enduring motor memory of walking on a split-belt treadmill in young children and adults

abstract: Adults and children modify how they move to accommodate persistent changes in their surroundings, called motor adaptation. Walking-related motor adaption can be seen when one walks on a treadmill with two belts running at different speeds, a split-belt treadmill, where a new walking pattern is slowly adopted with practice. This has been suggested as a way to improve left-right symmetry in walking after a stroke. Central to using split-belt walking for rehabilitation is whether the adapted motor pattern is retained over days and weeks, and whether this motor memory is a function of the persons age. When first exposed to split-belt walking, the walking is asymmetric (initial error), resembling a limp. With subsequent exposure, the error is smaller than the initial error, indicating the adaptation was remembered. Here, we explored the persistence of this memory over 24 hours, one week, and two weeks, in young children (3-6 yr old), younger adults (20-30 yr old) and older adults (50-70 yr old). We found that the motor memory declines with the interval duration, but was still evident up to 2 weeks after initial exposure. Retention of the motor memory in children and younger adults was better than older adults. Further, forgetting between trials was seen on the first day of split-belt walking especially in children and older adults, but this forgetting diminished with repeated trials. The results indicate that long-term memory of motor adaptation on the split-belt treadmill is affected by age, but it may be possible to enhance the memory by more frequent and repeated exposure. This suggests that when using split-belt adaptation in rehabilitation, the sessions should ideally be less than one week apart.


title: Memory of split-belt walking endures weeks in young children and younger adults but less so in older adults


title: Tracing Human Amygdala across School Age

abstract: The developmental pattern of the amygdala throughout childhood and adolescence has been inconsistently reported in previous neuroimaging studies. Given the relatively small size of the amygdala on full brain MRI scans, discrepancies may be partly due to methodological differences in amygdalar segmentation. To investigate the impact of volume extraction methods on amygdala volume, we compared FreeSurfer, FSL and volBrain segmentation measurements with those obtained by manual tracing. The manual tracing method, which we used as the  gold standard, exhibited almost perfect intra- and inter-rater reliability. We observed systematic differences in amygdala volumes between automatic (FreeSurfer and volBrain) and manual methods. Specifically, compared with the manual tracing, FreeSurfer estimated larger amygdalae, and volBrain produced smaller amygdalae while FSL demonstrated a mixed pattern. The tracing bias was not uniform, but higher for smaller amygdalae. We further modeled amygdalar growth curves using accelerated longitudinal cohort data from the Chinese Color Nest Project. Trajectory modeling and statistical assessments of the manually traced amygdalae revealed linearly increasing and parallel developmental patterns for both girls and boys, although the amygdalae of boys were larger than those of girls. Compared to these trajectories, the shapes of developmental curves were similar when using the volBrain derived volumes. FreeSurfer derived trajectories had more nonlinearities and appeared flatter. FSL derived trajectories demonstrated an inverted U shape and were significantly different from those derived from manual tracing method. The use of amygdala volumes adjusted for total gray-matter volumes, but not intracranial volumes, resolved the shape discrepancies and led to reproducible growth curves curves between manual tracing and the automatic methods (except FSL). Our findings revealed steady growth of the human amygdala, mirroring its functional development across the school age. Methodological improvements are warranted for current automatic tools to achieve more accurate tracing of the amygdala at school age, calling for next generation tools.


title: Charting the human amygdala development across childhood and adolescence: Manual and automatic segmentation


title: During natural viewing, neural processing of visual targets continues throughout saccades

abstract: Relatively little is known about visual processing during free-viewing visual search in realistic dynamic environments. Free-viewing is characterized by frequent saccades. During saccades, visual processing is thought to be inhibited, yet we know that the pre-saccadic visual content can modulate post-saccadic processing. To better understand these processes in a realistic setting, we study here saccades and neural responses elicited by the appearance of visual targets in a realistic virtual environment. While subjects were being driven through a 3D virtual town they were asked to discriminate between targets that appear on the road. We found that the presence of a target enhances early occipital as well as late frontocentral saccade-related responses. The earlier potential, shortly after 125ms post-saccade onset, was enhanced for targets that appeared in peripheral vision as compared to central vision, suggesting that fast peripheral processing initiated before saccade onset. The later potential, at 195ms post-saccade onset, was strongly modulated by the visibility of the target with a spatial distribution reminiscent of the classic P300 response. Together these results suggest that, during natural viewing, neural processing of the pre-saccadic visual stimulus continues throughout the saccade, apparently unencumbered by saccadic inhibition.


title: Neural indicators of human gut feelings

abstract: Understanding the neural processes governing the human gut-brain connection has been challenging due to the inaccessibility of the bodys interior. Here, we investigated neural responses to gastrointestinal sensation using a minimally invasive mechanosensory probe by quantifying brain, stomach, and perceptual responses following the ingestion of a vibrating capsule. Participants successfully perceived capsule stimulation under two vibration conditions (normal and enhanced), as evidenced by above chance accuracy scores. Perceptual accuracy improved significantly during the enhanced relative to normal stimulation, which was associated with a more rapid detection of the stimulation and reduced reaction time variability. Stomach stimulation induced early and late neural responses in parieto-occipital leads near the midline. Moreover, these  gastric evoked potentials showed intensity-dependent increases in amplitude and were significantly correlated with perceptual accuracy. These findings highlight a unique form of enterically-focused sensory monitoring within the human brain, with implications for understanding gut-brain interactions in healthy and clinical populations.


title: SIPA1L1/SPAR1 is a non-PSD protein involved in GPCR signaling

abstract: SIPA1L1 (also known as SPAR1) has been proposed to regulate synaptic functions that are important in maintaining normal neuronal activities, such as regulating spine growth and synaptic scaling, as a component of the postsynaptic density (PSD)-95/N-methyl-D-aspartate receptor (NMDA-R)-complex. However, contrary to this view, our super-resolution and immunoelectron microscopic analyses demonstrate that SIPA1L1 is mainly localized to general submembranous and cytoplasmic regions in neurons, but scarcely to PSD. Our screening for native interactors of SIPA1L1 identified spinophilin and neurabin-1, regulators of G protein-coupled receptor (GPCR) signaling, but rejected PSD-95/NMDA-R-complex components. Furthermore, Sipa1l1-/- mice showed normal spine size distribution and NMDA-R-dependent synaptic plasticity. Nevertheless, Sipa1l1-/- mice showed aberrant responses to 2-adrenergic receptor (a spinophilin target) or adenosine A1 receptor (a neurabin-1 target) agonist stimulation and striking behavioral anomalies, such as hyperactivity, enhanced anxiety, learning impairments, social interaction deficits, and enhanced epileptic seizure susceptibility. Our findings revealed unexpected properties of SIPA1L1, suggesting a possible association of SIPA1L1 deficiency with neuropsychiatric disorders related to dysregulated GPCR signaling, such as epilepsy, attention deficit hyperactivity disorder (ADHD), autism, or fragile X syndrome.


title: Innate Immune sensing of Influenza A viral RNA through IFI16 promotes pyroptotic cell death

abstract: Programmed cell death pathways are triggered by various stresses or stimuli, including viral infections. The mechanism underlying the regulation of these pathways upon Influenza A virus IAV infection is not well characterized. We report that a cytosolic DNA sensor IFI16 is essential for the activation of programmed cell death pathways in IAV infected cells. We have identified that IFI16 functions as an RNA sensor for influenza A virus by binding to genomic RNA. The activation of IFI16 triggers the production of type I, III interferons, and also other pro-inflammatory cytokines via the STING-TBK1 and Pro-caspase-1 signaling axis, thereby promoting cell death (apoptosis and pyroptosis in IAV infected cells). Whereas, IFI16 knockdown cells showed reduced inflammatory responses and also prevented cell mortality during IAV infection. These results demonstrate the pivotal role of IFI16-mediated IAV sensing and its essential role in activating programmed cell death pathways.

category: immunology


title: Meta-analysis of scRNA seq data to define the landscape of human liver immune homeostasis.

abstract: The liver is unique in both its ability to maintain immune homeostasis and in its potential for immune tolerance following solid organ transplantation. Single-cell RNA sequencing (scRNA seq) is a powerful approach to generate highly dimensional transcriptome data to understand cellular phenotypes. However, when scRNA data is produced by different groups, with different data models, different standards, and samples processed in different ways, it can be challenging to draw meaningful conclusions from the aggregated data. The goal of this study was to establish a method to combine  human liver scRNA seq datasets by 1) characterizing the heterogeneity between studies and 2) using the meta-atlas to define the dominant phenotypes across immune cell subpopulations in healthy human liver. Publicly available scRNA seq data generated from liver samples obtained from a combined total of 17 patients and [~]32,000 cells were analyzed. Liver-specific immune cells (CD45+) were extracted from each dataset, and immune cell subpopulations (myeloid cells, NK and T cells, plasma cells, and B cells) were examined using dimensionality reduction (UMAP), differential gene expression, and ingenuity pathway analysis. All datasets co-clustered, but cell proportions differed between studies. Gene expression correlation demonstrated similarity across all studies, and canonical pathways that differed between datasets were related to cell stress and oxidative phosphorylation rather than immune-related function. Next, a meta-atlas was generated via data integration and compared against PBMC data to define gene signatures for each hepatic immune subpopulation. This analysis defined key features of hepatic immune homeostasis, with decreased expression across immunologic pathways and enhancement of pathways involved with cell death. This method for meta-analysis of scRNA seq data provides a novel approach to broadly define the features of human liver immune homeostasis. Specific pathways and cellular phenotypes described in this human liver immune meta-atlas provide a critical reference point for further study of immune mediated disease processes within the liver.


title: Creation of a single cell RNA seq meta-atlas to define human liver immune homeostasis


title: Fast, efficient and virus-free generation of TRAC-replaced CAR T cells

abstract: Chimeric Antigen Receptor (CAR) redirected T cells are a potent treatment option for certain hematological malignancies. Recently, site-specific insertion of CARs into the T cell receptor (TCR) alpha constant (TRAC) locus using gene editing and adeno-associated viruses was shown to generate CAR T cells with improved functionality over their retrovirally transduced counterparts. However, the development of viruses for gene transfer is complex and associated with extensive costs at early clinical stages. Here, we provide an economical and virus-free method for efficient CAR insertion into the TRAC locus of primary human T cells via CRISPR-Cas mediated homology-directed repair (HDR). While the toxicity induced by transfected double-stranded template (donor) DNA was not fully prevented by pharmacological means, the combination of DNA-sensor inhibitors and HDR enhancers resulted in highly efficient gene editing with TCR-to-CAR replacement rates reaching up to 68%. The resulting TCR-deficient CAR T cells show antigen-specific cytotoxicity and cytokine production in vitro. Our GMP-compatible non-viral platform technology lays the foundation for clinical trials and fast-track generation of novel CAR T cells applicable for autologous or allogeneic off-the-shelf use.


title: In vivo generation of BK and JC polyomavirus defective viral genomes in human urine samples associated with higher viral loads

abstract:
Defective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens. Here, using direct deep sequencing with multiple library preparation strategies and confirmatory ddPCR of urine samples taken from immunosuppressed individuals, we show clinical BKPyV and JCPyV strains contain widespread genomic rearrangements across multiple loci that likely interfere with viral replication. BKPyV DVGs were universally derived from type I subtype BKPyV. The presence of DVGs was associated with specimens containing higher viral loads but never reached clonality, consistent with a model of parasitized replication. These DVGs persisted during clinical infection as evidenced in two separate pairs of samples containing BK virus collected from the same individual up to 302 days apart. In a separate individual, we observed the generation of DVGs after a 57.5-fold increase in viral load. In summary, by extending the presence of DVGs in clinical specimens to DNA viruses, we demonstrate the ubiquity of DVGs in clinical virology.

IMPORTANCEDefective viral genomes (DVGs) can have a significant impact on the production of infectious virus particles. DVGs have only been identified in cultured viruses passaged at high multiplicities of infection and RNA viruses collected from clinical specimens -- no DNA virus in the wild has been shown to contain DVGs. Here, we identified BK and JC polyomavirus DVGs in clinical urine specimens and demonstrated that these DVGs are more frequently identified in samples with higher viral loads. The strains containing DVGs had rearrangements throughout their genomes with the majority affecting genes required for viral replication. Longitudinal analysis showed these DVGs can persist during an infection, but do not reach clonality within the chronically infected host. Our identification of polyomavirus DVGs suggests these parasitic sequences exist across the many classes of viruses capable of causing human disease.

category: microbiology


title: Chemical ecology of a tripartite symbiosis

abstract:
Microbial symbiotic interactions, mediated by small molecule signaling, drive physiological processes of higher order systems. Metabolic analytic technologies advancements provide new avenues to examine how chemical ecology, or conversion of existing biomass to new forms, changes over a symbiotic lifecycle. We examine such processes using the tripartite relationship between nematode host Steinernema carpocapsae, its obligate mutualist bacterium, Xenorhabdus nematophila, and the insects they infect together. We integrate trophic, metabolomics, and gene regulation analyses to understand insect biomass conversion to nematode or bacterium biomass. Trophic analysis established bacteria as the primary insect consumers, with nematodes at trophic position 4.37, indicating consumption of bacteria and likely other nematodes. Significant, discrete metabolic phases were distinguishable from each other, indicating the insect chemical environment changes reproducibly during bioconversion. Tricarboxylic acid cycle components and amino acids were significantly affected throughout infection. These findings contribute to an ongoing understanding of how symbiont associations shape chemical environments.

TeaserEntomopathogenic nematodes act as an apex predator in some ecosystems through altering chemical environments of their prey.


title: Chemical ecology of an apex predator life cycle


title: Genomes of gut bacteria from Nasonia wasps shed light on phylosymbiosis and microbe-assisted hybrid breakdown

abstract:
Phylosymbiosis is a cross-system trend whereby microbial community relationships recapitulate the host phylogeny. In Nasonia parasitoid wasps, phylosymbiosis occurs throughout development, is distinguishable between sexes, and benefits host development and survival. Moreover, the microbiome shifts in hybrids as a rare Proteus bacteria in the microbiome becomes dominant. The larval hybrids then catastrophically succumb to bacterial-assisted lethality and reproductive isolation between the species. Two important questions for understanding phylosymbiosis and bacterial-assisted lethality in hybrids are: (i) Do the Nasonia bacterial genomes differ from other animal isolates and (ii) Are the hybrid bacterial genomes the same as those in the parental species? Here we report the cultivation, whole genome sequencing, and comparative analyses of the most abundant gut bacteria in Nasonia larvae, Providencia rettgeri and Proteus mirabilis. Characterization of new isolates shows Proteus mirabilis forms a more robust biofilm than Providencia rettgeri and when grown in co-culture, Proteus mirabilis significantly outcompetes Providencia rettgeri. Providencia rettgeri genomes from Nasonia are similar to each other and more divergent to pathogenic, human-associates strains. Proteus mirabilis from N. vitripennis, N. giraulti, and their hybrid offspring are nearly identical and relatively distinct from human isolates. These results indicate that members of the larval gut microbiome within Nasonia are most similar to each other, and the strain of the dominant Proteus mirabilis in hybrids is resident in parental species. Holobiont interactions between shared, resident members of the wasp microbiome and the host underpin phylosymbiosis and hybrid breakdown.

IMPORTANCEAnimal and plant hosts often establish intimate relationships with their microbiomes. In varied environments, closely-related host species share more similar microbiomes, a pattern termed phylosymbiosis. When phylosymbiosis is functionally significant and beneficial, microbial transplants between host species or host hybridization can have detrimental consequences on host biology. In the Nasonia parasitoid wasp genus that contains a phylosymbiotic gut community, both effects occur and provide evidence for selective pressures on the holobiont. Here, we show that bacterial genomes in Nasonia differ from other environments and harbor genes with unique functions that may regulate phylosymbiotic relationships. Furthermore, the bacteria in hybrids are identical to parental species, thus supporting a hologenomic tenet that the same members of the microbiome and the host genome impact phylosymbiosis, hybrid breakdown, and speciation.


title: Suppression of Plasmodium MIF-CD74 Signaling Protects Against Severe Malaria

abstract: Malaria begins when mosquito-borne Plasmodium sporozoites invade hepatocytes and usurp host pathways to support the differentiation and multiplication of erythrocyte-infective merozoite progeny. The deadliest complication of infection, cerebral malaria, accounts for the majority of malarial fatalities. Although our understanding of the cellular and molecular mechanisms underlying the pathology remains incomplete, recent studies support the contribution of systemic and neuroinflammation as the cause of cerebral edema and blood-brain barrier (BBB) dysfunction. All Plasmodium species encode an orthologue of the innate cytokine, Macrophage Migration Inhibitory Factor (MIF), which functions in mammalian biology to regulate innate responses. Plasmodium MIF (PMIF) similarly signals through the host MIF receptor CD74, leading to an enhanced inflammatory response. We investigated the PMIF-CD74 interaction in the onset of experimental cerebral malaria (ECM) using CD74 deficient (Cd74-/-) mice, which were found to be protected from ECM. The protection was associated with the inability of brain microvessels from Cd74-/- hosts to present parasite antigen to sequestered Plasmodium-specific CD8+ T cells. Infection of mice with PMIF-deficient sporozoites (PbAmif-) also protected mice from ECM, highlighting the pivotal role of PMIF in the pre-erythrocytic stage of the infection. A novel pharmacologic PMIF-selective antagonist reduced PMIF/CD74 signaling and fully protected mice from ECM. These findings reveal a conserved mechanism for Plasmodium usurpation of host CD74 signaling and suggest a tractable approach for new pharmacologic intervention.


title: A nadA mutation confers nicotinic acid auxotrophy in pro-carcinogenic intestinal Escherichia coli NC101

abstract:
Inflammatory bowel diseases and inflammation-associated colorectal cancer are linked to blooms of adherent-invasive Escherichia coli (AIEC) in the intestinal microbiota. AIEC are functionally defined by their ability to adhere/invade epithelial cells and survive/replicate within macrophages. Changes in micronutrient availability can alter AIEC physiology and interactions with host cells. Thus, culturing AIEC for mechanistic investigations often involves precise nutrient formulation. We observed that the pro-inflammatory and pro-carcinogenic AIEC strain NC101 failed to grow in minimal media (MM). We hypothesized that NC101 was unable to synthesize a vital micronutrient normally found in the host gut. Through nutrient supplementation studies, we identified that NC101 is a nicotinic acid (NA) auxotroph. NA auxotrophy was not observed in the other non-toxigenic E. coli or AIEC strains we tested. Sequencing revealed NC101 has a missense mutation in nadA, a gene encoding quinolinate synthase A that is important for de novo NAD biosynthesis. Correcting the identified nadA point mutation restored NC101 prototrophy without impacting AIEC function, including motility and AIEC-defining survival in macrophages. Our findings, along with the generation of a prototrophic NC101 strain, will greatly enhance the ability to perform in vitro functional studies that are needed for mechanistic investigations on the role of intestinal E. coli in digestive disease.

ImportanceInflammatory bowel diseases (IBD) and colorectal cancer (CRC) are significant global health concerns that are influenced by gut resident microbes, like adherent-invasive Escherichia coli (AIEC). Nutrient availability influences specialized metabolite production, AIEC-defining functional attributes, and AIEC:host interactions. NC101 is a pro-inflammatory and pro-carcinogenic AIEC strain commonly used for studies on IBD and CRC. We identified that NC101 growth in vitro requires a micronutrient found in the host gut. By correcting an identified mutation, we generated an NC101 strain that no longer has micronutrient restrictions. Our findings will facilitate future research that necessitates precise nutrient manipulation, enhancing AIEC functional studies and investigations on other auxotrophic intestinal microbiota members. Broadly, this will improve the study of bacterial:host interactions impacting health and disease.


title: Easy multiple sequential CRISPR/Cas9 knockouts in cell lines using a Cre/LoxP re-cyclable vector

abstract: To easily generate cell lines lacking multiple proteins, we inserted Cre/Lox sites flanking the guide RNA, Cas9, and mCherry fluorescent protein coding regions of a CRISPR/Cas9 lentiviral vector. Cells bearing an inducible Cre recombinase construct can be transfected with the CRISPR/Cas9 lentiviral vector, and mCherry positive cells sorted by flow cytometry. Induction of Cre causes deletion of the guide RNA, Cas9, and mCherry genes, so that mCherry negative cells can be isolated. After confirming successful targeting of the gene, the cells can be re-infected with the same vector bearing a different guide RNA, and mCherry-positive cells sorted once more. In this way, multiple genes can be mutated sequentially using the same vector and selection marker, without persistent expression of the guide RNA or Cas9. We used this system to sequentially mutate two candidate genes, Bak1 and Bcl2, and generated lines that lacked expression of both proteins.

category: molecular biology


title: CRISPR/Cas9 targeting of MCPyV T antigen in Merkel tumors

abstract: Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCPyV) T antigen gene expression, post integration ([~]80% cases), or by UV mediated DNA damage. Viral-positive Merkel tumors are not only caused by but also oncogenically addicted to tumor antigen expression. In this study we used CRISPR-Cas9 based gene-editing to develop a potential therapeutic tool for MCPyV positive MCC. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system is a genome editing technology whereby a guide RNA (gRNA) molecule, targets a Cas endonuclease to a specific genomic site, using sequence homology, and induces a double strand break. To target MCPyV T antigens, we designed a strategy using 2 gRNAs targeting the T antigen genomic region that would cut off a substantial portion of the gene thereby rendering it dysfunctional. We validated the MCPYV T antigen targeting efficiency of our gRNAs, both individually and together by in vitro cleavage assays. Finally, to translate this finding, we delivered this CRISPR system in patient-derived MCC cell lines and show reduction in T antigen gene expression. Our proof-of-concept study shows that 2 MCPyV targeting CRISPR/Cas gRNAs in combination can knock out MCPyV T antigen, thus, being of therapeutic importance. We hope that this CRISPR system can be potentially delivered in vivo for advancing MCPyV positive MCC treatment in the future.


title: Newly developed ad hoc molecular assays shows how eDNA can witness and anticipate the monk seal recolonization of central Mediterranean

abstract: The monk seal Monachus monachus is the most endangered pinniped worldwide and the only one found in the Mediterranean, where its distribution and abundance have suffered a drastic decline in the last few decades. Data on its status are scattered due to both its rarity and evasiveness, and records are biased towards occasional, mostly coastal, encounters. Nowadays molecular techniques allow us to detect and quantify minute amounts of DNA traces released in the environment (eDNA) by any organism. We present three qPCR-assays targeting the monk seal mitogenome. The assays were soundly tested on an extensive and diversified sample set (n=73), including positive controls from Madeira breeding population collected during the peak of abundance, and two opportunistic Mediterranean eDNA-sample collections (offshore/coastal) from on-going projects. Monk seal DNA was detected in 47.2% and 66.7% of the samples collected in the Tyrrhenian from a ferry platform (2018-2019) and in the Pelagie archipelago -Strait of Sicily- (2020) respectively, anticipating (up to 2 year) visual observations occurred subsequently in proximity of the sampled areas. In the Tyrrhenian, detection occurrence increased between 2018 and 2019. Monk seal DNA recoveries were commoner in night-time ferry-samples, suggesting nocturnal predatory activity in pelagic waters. The proposed technique provides a non-invasive and yet highly-sensitive tool for defining the monk seal actual distribution and home range, its recovery rate and pinpoint coastal/offshore localities where prioritizing conservation, research, citizen science and education initiatives.


title: Newly developed ad hoc molecular assays show how eDNA can witness and anticipate the monk seal recolonization of central Mediterranean


title: Reaction of ependymal cells to spinal cord injury: a potential role for oncostatin pathway and microglial cells

abstract: Ependymal cells with stem cell properties reside in the adult spinal cord around the central canal. They rapidly activate and proliferate after spinal cord injury, constituting a source of new cells. They produce neurons and glial cells in lower vertebrates but they mainly generate glial cells in mammals. The mechanisms underlying their activation and their glial-biased differentiation in mammals remain ill-defined. This represents an obstacle to control these cells. We addressed this issue using RNA profiling of ependymal cells before and after injury. We found that these cells activate STAT3 and ERK/MAPK signaling during injury and downregulate cilia-associated genes and FOXJ1, a central transcription factor in ciliogenesis. Conversely, they upregulate 510 genes, six of them more than 20 fold, namely Crym, Ecm1, Ifi202b, Nupr1, Rbp1, Thbs2 and Osmr. OSMR is the receptor for the inflammatory cytokine oncostatin (OSM) and we studied its regulation and role using neurospheres derived from ependymal cells. We found that OSM induces strong OSMR and p-STAT3 expression together with proliferation reduction and astrocytic differentiation. Conversely, production of oligodendrocyte-lineage OLIG1+ cells was reduced. OSM is specifically expressed by microglial cells and was strongly upregulated after injury. We observed microglial cells apposed to ependymal cells in vivo and co-cultures experiments showed that these cells upregulate OSMR in neurosphere cells. Collectively, these results support the notion that microglial cells and OSMR/OSM pathway regulate ependymal cells in injury. In addition, the generated high throughput data provides a unique molecular resource to study how ependymal cell react to spinal cord lesion.

category: cell biology


title: Programmed ER fragmentation drives selective ER inheritance and degradation in budding yeast meiosis

abstract: The endoplasmic reticulum (ER) is a membrane-bound organelle with diverse, essential functions that rely on the maintenance of membrane shape and distribution within cells. ER structure and function are remodeled in response to changes in cellular demand, such as the presence of external stressors or the onset of cell differentiation, but mechanisms controlling ER remodeling during cell differentiation are not well understood. Here, we describe a series of developmentally regulated changes in ER morphology and composition during budding yeast meiosis, a conserved differentiation program that gives rise to gametes. During meiosis, the cortical ER undergoes fragmentation before collapsing away from the plasma membrane at anaphase II. This programmed collapse depends on the meiotic transcription factor Ndt80, conserved ER membrane structuring proteins Lnp1 and reticulons, and the actin cytoskeleton. A subset of ER is retained at the mother cell plasma membrane and excluded from gamete cells via the action of ER-plasma membrane tethering proteins. ER remodeling is coupled to ER degradation by selective autophagy, which is regulated by the developmentally timed expression of the autophagy receptor Atg40. Autophagy relies on ER collapse, as artificially targeting ER proteins to the cortically retained ER pool prevents their degradation. Thus, developmentally programmed changes in ER morphology determine the selective degradation or inheritance of ER subdomains by gametes.


title: A phosphorylation of RIPK3 kinase initiates an intracellular apoptotic pathway that promotes corpus luteum regression

abstract: Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) normally signals to necroptosis by phosphorylating MLKL. We report here that when the cellular RIPK3 chaperone Hsp90/CDC37 level is low, RIPK3 also signals to apoptosis. The apoptotic function of RIPK3 requires phosphorylation of the serine 165/threonine 166 sites on its kinase activation loop, resulting in inactivation of RIPK3 kinase activity while gaining the ability to recruit RIPK1, FADD, and caspase-8 to form a cytosolic caspase-activating complex, thereby triggering apoptosis. We found that PGF2 induces RIPK3 expression in luteal granulosa cells in the ovary to cause luteal regression through this RIPK3-mediated apoptosis pathway. Mice carrying homozygous phosphorylation-resistant RIPK3 S165A/T166A knockin mutations failed to respond to PGF2 but retained pro-necroptotic function, whereas mice with phospho-mimicking S165D/T166E homozygous knockin mutation underwent spontaneous apoptosis in multiple RIPK3-expressing tissues and died shortly after birth. Thus, RIPK3 signals to either necroptosis or apoptosis depending on its serine 165/threonine 166 phosphorylation status.


title: Dynamin-2 controls complement receptor 3- mediated phagocytosis completion and closure

abstract: Phagocytosis is the mechanism of the internalization of large particles, microorganisms and cellular debris. The complement pathway represents one of the first mechanisms of defense against infection and the complement receptor 3 (CR3), which is highly expressed on macrophages, is a major receptor for many pathogens and debris. Key to dissecting the mechanisms by which CR3-mediated phagocytosis occurs, is understanding how the complex actin binding protein machinery and associated regulators interact with actin during phagocytosis, from triggering of receptor, through to phagosome formation and closure. However, how CR3-mediated phagosome completion and closure are orchestrated is not known. Here, we reveal that dynamin-2 is recruited concomitantly with polymerised actin at the site of the nascent phagosomes and accumulates until membrane scission. Inhibition of dynamin activity leads to stalled phagocytic cups and a decrease in the amount of F-actin at the site of phagocytosis. Acute inhibition of dynamin activity in living phagocytosing cells established that dynamin-2 plays a critical role in the effective scission of the CR3-phagosome from the plasma membrane. Thus, dynamin-2 has two distinct roles in CR3-mediated phagocytosis, in the assembly of the F-actin phagocytic cup and during phagosome scission.


title: Ectosome uptake by glia sculpts Caenorhabditis elegans cilia

abstract: Cilia are sensory organelles protruding from cell surface. A tight regulation of membrane receptors traffic in and out of cilia is achieved by the action of Intraflagellar Transport (IFT). Here, we show that ectosomes bud from a subset of C. elegans sensory cilia. Packing and disposal of ciliary receptors in ectosomes complement their retrieval by IFT. Mutations in ciliary retrieval genes increase export of the salt sensor GCY-22 from ASER neurons by ectosomes, preventing its accumulation in ASER cilia. Ectosomes are produced from two ciliary locations: cilia tip and/or cilia base. Ectosomes budding from cilia tip are released in the environment. Ectosomes produced from the cilia base are concomitantly phagocytosed by the associated glial cells. Although ectocytosis does not require glia to occur, ectosome phagocytosis by the contacting glia contributes to maintain cilia shape and sensory function. We suggest this coordinated neuron-glia interaction is required for proper cilia function.


title: Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription

abstract: Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates1-3. The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle4-7, together with variations in substrate docking to sites on associated cyclin and Cks subunits3, 6, 8-10. Many substrates are modified at multiple sites to provide more complex regulation9, 11-14. Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of genes required for mitotic progression15, 16. As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback17-20. Consistent with these findings, we show that low Cdk1 activity promotes CLB2 expression at mitotic entry. We also find, however, that CLB2 expression is inhibited by high levels of Cdk1 activity in a mitotic arrest. Inhibition is accompanied by Ndd1 degradation, and we present evidence that high mitotic Cdk1-Clb2 activity generates phosphodegrons on Ndd1, leading to its degradation. Complete Ndd1 phosphorylation by the Clb2-Cdk1-Cks1 complex requires the phosphothreonine-binding site of Cks1, as well as a novel phosphate-binding pocket on the cyclin Clb221. We therefore propose that initial phosphorylation by Cdk1 primes the protein for secondary phosphorylation at phosphodegrons, resulting in degradation only at high Cdk1 activity. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression.


title: Ferulic acid combined with skeletal stem cells to repair irradiated bone defect

abstract:
The reconstruction of irradiated bone defects after settlement of skeletal tumors remains a significant challenge in clinical applications. In this study, we explored radiation-induced skeletal stem cell (SSC) stemness impairments and rescuing effects of ferulic acid (FA) on SSCs in vitro and in vivo. The immunophenotype, cell renewal, cell proliferation, and differentiation of SSCs in vitro after irradiation were investigated. Mechanistically, the changes in tissue regeneration-associated gene expression and MAPK pathway activation in irradiated SSCs were evaluated. The regenerative capacity of SSCs in the presence of FA in an irradiated bone defect mouse model was also investigated. We found that irradiation reduced CD140a- and CD105-positive cells in skeletal tissues and mouse-derived SSCs. Additionally, irradiation suppressed cell proliferation, colony formation, and osteogenic differentiation of SSCs. The RNA-Seq results showed that tissue regeneration-associated gene expression decreased, and the western blotting results demonstrated the suppression of phosphorylated p38/MAPK and ERK/MAPK in irradiated SSCs. Notably, FA significantly rescued the radiation-induced impairment of SSCs by activating the p38/MAPK and ERK/MAPK pathways. Moreover, the results of imaging and pathological analyses demonstrated that FA enhanced the bone repair effects of SSCs in an irradiated bone defect mouse model substantially. Importantly, inhibition of the p38/MAPK and ERK/MAPK pathways in SSCs by specific chemical inhibitors partially abolished the promotive effect of FA on SSC-mediated bone regeneration. In summary, our findings reveal a novel function of FA in repairing irradiated bone defects by maintaining SSC stemness and suggest that the p38/MAPK and ERK/MAPK pathways contribute to SSC-mediated tissue regeneration post-radiation.

Significance StatementRadiotherapy combined with surgery for the settlement of skeletal tumors usually leads to large bone defects and hampers wound healing. Skeletal stem cells (SSCs) have been defined as tissue-specific stem cells in skeletons and are highlighted in bone development and regeneration. Ferulic acid is a phytochemical with a wide range of therapeutic effects, including alleviation of radiation-induced oxidative stress and promotion of tissue regeneration. In the current study, promising data based on an in vitro cell model and an in vivo animal model demonstrates that ferulic acid alleviates radiation-induced impairment of SSCs and promotes SSC-mediated bone regeneration post-radiation.


title: GRK2/3/5/6 knockout: The impact of individual GRKs on arrestin-binding and GPCR regulation

abstract: G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors and represent major drug targets. Upon ligand stimulation, GPCRs activate G proteins and undergo a complex regulation by interaction with GPCR kinases (GRKs) and formation of receptor-arrestin complexes. For many GPCRs, this mechanism triggers receptor desensitisation, internalisation, and possibly a second intracellular signalling wave. Here we created eleven different HEK293 knockout cell clones for GRK2, 3, 5, and 6 individually and in combination. These include four single, two double, four triple, and the quadruple GRK knockout. The statistical evaluation of {beta}-arrestin1/2 interactions for twelve different receptors grouped the tested GPCRs into two main subsets: those for which {beta}-arrestin interaction was mediated by either GRK2, 3, 5, or 6 and those that are mediated by GRK2 or 3 only. Interestingly, the overexpression of specific GRKs was found to induce a robust, ligand-independent {beta}-arrestin interaction with the V2R and AT1R. Finally, using GRK knockout cells, PKC inhibitors, and {beta}-arrestin mutants, we present evidence for differential AT1R-{beta}-arrestin2 complex configurations mediated by selective engagement of PKC, GRK2, or GRK6. We anticipate our novel GRK-knockout platform to facilitate the elucidation of previously unappreciated details of GRK-specific GPCR regulation and {beta}-arrestin complex formation.


title: An exon junction complex-independent function of Barentsz in neuromuscular synapse growth

abstract: The exon junction complex controls the translation, degradation and localization of spliced mRNAs, and three of its four core subunits also play a role in splicing. Here we show that the fourth subunit, Barentsz, has distinct biological functions within and separate from the exon junction complex in neuromuscular development. Barentsz controls the distribution of mitochondria in larval muscles, a function that also depends on other subunits of the exon junction complex and that is not rescued by a transgene in which residues required for binding to the core subunit eIF4AIII are mutated. In contrast, interactions with the exon junction complex are not required for Barentsz to promote the growth of neuromuscular synapses. We found that the Activin ligand Dawdle shows reduced expression in barentsz mutants and acts downstream of Barentsz to control synapse growth. Both barentsz and dawdle are required in motor neurons, muscles and glia for normal synapse growth, and exogenous Dawdle can rescue synapse growth in the absence of barentsz. These results identify a biological function for Barentsz that is independent of the exon junction complex.

category: developmental biology


title: p53 pro-apoptotic activity is regulated by the G2/M promoting factor Cdk1 in response to DNA damage

abstract: Exposure to genotoxic stress promotes cell-cycle arrest and DNA repair or apoptosis. These "life" or "death" cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, how p53 activity is precisely regulated is essential to maintain tissue homeostasis and to prevent cancer development. Here we demonstrate that Drosophila p53 pro-apoptotic activity is regulated by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation induced apoptosis. We show that the p53 protein is not able to bind to and to activate the expression of the pro-apoptotic genes in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 pro-apoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 pro-apoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.


title: Paths and Pathways that Generate Cell-Type Heterogeneity and Developmental Progression in Hematopoiesis

abstract: Mechanistic studies of Drosophila lymph gland hematopoiesis are limited by the availability of cell-type specific markers. Using a combination of bulk RNA-Seq of FACS-sorted cells, single cell RNA-Seq and genetic dissection, we identify new blood cell subpopulations along a developmental trajectory with multiple paths to mature cell types. This provides functional insights into key developmental processes and signaling pathways. We highlight metabolism as a driver of development, show that graded Pointed expression allows distinct roles in successive developmental steps, and that mature crystal cells specifically express an alternate isoform of Hypoxia-inducible factor (Hif/Sima). Mechanistically, the Musashi-regulated protein Numb facilitates Sima-dependent non-canonical, while inhibiting canonical, Notch signaling. Broadly, we find that prior to making a fate choice, a progenitor selects between alternative, biologically relevant, transitory states allowing smooth transitions reflective of combinatorial expressions rather than stepwise binary decisions. Increasingly, this view is gaining support in mammalian hematopoiesis.


title: Nitrate enhances anaerobic oxidation of methane in boreal lake sediments

abstract: The identity of electron acceptors in promoting anaerobic oxidation of methane (AOM) in the sediments of boreal lakes is currently unknown. Here, we studied the AOM rate of sediment slurries collected from three profundal stations of a nitrate-rich, oligo-mesotrophic, boreal lake (Lake Paajarvi, Finland), under varying nitrate concentrations using 13C-labelling. Furthermore, vertical profiles of the sediment and porewater geochemistry, and the microbial communities (16S rRNA gene and shotgun metagenomic sequencing) were analyzed. Despite geochemical data indicating that simultaneous consumption of nitrate and methane took place at the sediment layers chosen for incubations, AOM rate was not enhanced by nitrate amendments at either of the stations. AOM rate was much higher at the shallow Station 1 (0.9-6.8 nmol C cm-3 d-1) with high contents of labile phytoplankton-derived organic matter, than at the deeper stations, Station 2 (0-0.3 nmol C cm-3 d-1) and 3 (0-0.2 nmol C cm-3 d-1). Accordingly, a higher relative abundance of methanotrophic archaea (Candidatus Methanoperedens) and bacteria (Methylococcales) were observed in the layers chosen for incubations at Station 1 than at the other stations. Besides nitrate, the geochemical profiles indicated that AOM was potentially coupled with iron or sulfate reduction at all stations. Furthermore, putative nitrite-reducing methanotrophs (Ca. Methylomirabilis) were the most abundant methanotrophs above the incubation layer at Station 2 and 3, which suggests that nitrite reduction also plays a role in driving AOM in the study lake. We conclude that AOM is not uniquely coupled to nitrate reduction in sediments of nitrate-rich, oligo-mesotrophic, boreal lakes.

category: ecology


title: Anaerobic oxidation of methane in sediments of a nitrate-rich, oligo-mesotrophic boreal lake


title: Animal soundscapes reveal key markers of Amazon forest degradation from fire and logging

abstract:
Safeguarding tropical forest biodiversity requires solutions for monitoring ecosystem composition over time. In the Amazon, logging and fire reduce forest carbon stocks and alter tree species diversity, but the long-term consequences for wildlife remain unclear, especially for lesser-known taxa. Here, we combined data from multi-day acoustic surveys, airborne lidar, and satellite timeseries covering logged and burned forests (n=39) in the southern Brazilian Amazon to identify acoustic markers of degradation. Our findings contradict theoretical expectations from the Acoustic Niche Hypothesis that animal communities in more degraded habitats occupy fewer  acoustic niches. Instead, we found that habitat structure (e.g., aboveground biomass) was not a consistent proxy for biodiversity based on divergent patterns of acoustic space occupancy (ASO) in logged and burned forests. Full 24-hr soundscapes highlighted a stark and sustained reorganization in community structure after multiple fires; animal communication networks were quieter, more homogenous, and less acoustically integrated in forests burned multiple times than in logged or once-burned forests. These findings demonstrate strong biodiversity co-benefits from protecting Amazon forests from recurrent fire activity. By contrast, soundscape changes after logging were subtle and more consistent with community recovery than reassembly. In both logged and burned forests, insects were the dominant acoustic markers of degradation, particularly during midday and nighttime hours that are not typically sampled by traditional field surveys of biodiversity. The acoustic fingerprints of degradation history were conserved across replicate recording locations at each site, indicating that soundscapes offer a robust, taxonomically inclusive solution for tracking changes in community composition over time.

Significance StatementFire and logging reduce the carbon stored in Amazon forests, but little is known about how human degradation alters animal communities. We recorded thousands of hours of ecosystem sounds to investigate animal community assembly and the associations between biodiversity and biomass following Amazon forest degradation over time. 24-hr patterns of acoustic activity differed between logged and burned forests, and we observed large and sustained breakpoints in community structure after multiple burns. Soundscape differences among degraded forests were clearest during insect-dominated hours rarely sampled in field studies of biodiversity. These findings demonstrate that acoustic monitoring holds promise for routine biodiversity accounting, even by non-experts, to capture a holistic measure of animal communities in degraded tropical forests and benchmark change over time.


title: A macroecological description of alternative stable states reproduces intra- and inter-host variability of gut microbiome

abstract: The most fundamental questions in microbial ecology concern the diversity and variability of communities. Their composition varies widely across space and time, as it is determined by a non-trivial combination of stochastic and deterministic processes. The interplay between non-linear community dynamics and environmental fluctuations determines the rich statistical structure of community variability, with both rapid temporal dynamics fluctuations and non-trivial correlations across habitats. Here we analyze long time-series of gut microbiome and compare intra- and inter-community dissimilarity. Under a macroecological framework we characterize their statistical properties. We show that most taxa have large but stationary fluctuations over time, while a minority is characterized by quick changes of average abundance which cluster in time, suggesting the presence of alternative stable states. We disentangle inter-individual variability in a major stochastic component and a deterministic one, the latter recapitulated by differences in the carrying capacities of taxa. Finally, we develop a model which includes environmental fluctuations and alternative stable states. This model quantitatively predicts the statistical properties of both intra- and inter-individual community variability, therefore summarizing variation in a unique macroecological framework.


title: Influence of warming temperatures on coregonine embryogenesis within and among species

abstract: The greatest known global response of lakes to climate change has been an increase in water temperatures. The responses of many lake fishes to warming water temperatures are projected to be inadequate to counter the speed and magnitude of climate change. We experimentally evaluated the responses of embryos from a group of cold, stenothermic fishes (Salmonidae Coregoninae) to increased incubation temperatures. Study groups included cisco (Coregonus artedi) from lakes Superior and Ontario (USA), and vendace (C. albula) and European whitefish (C. lavaretus) from Lake Southern Konnevesi (Finland). Embryos from artificial crossings were incubated at water temperatures of 2.0, 4.5, 7.0, and 9.0{degrees}C, and their responses were quantified for developmental and morphological traits. Embryo survival, incubation period, and length-at- hatch were inversely related to incubation temperature whereas yolk-sac volume increased with incubation temperature within study groups. However, varying magnitudes of responses among study groups suggested differential levels of developmental plasticity to climate change. Differential levels of parental effects indicate genetic diversity may enable all study groups to adapt to cope with some degree of changing environmental conditions. Our results suggest that the coregonines sampled within and among systems may have a wide range of embryo responses to warming incubation conditions.


title: Tree phylogenetic diversity supports nature's contributions to people, but is at risk from human population growth

abstract: There is growing evidence for a link between biodiversity and ecosystem function, and for a correlation between human population and the species diversity of plants and animals in a region. Here, we suggest these relationships might not be independent. Using a comprehensive phylogeny of southern African trees and structural equation modelling, we show that human population density correlates with tree phylogenetic diversity and show that this relationship is stronger than the correlation with species richness alone. Further, we demonstrate that areas high in phylogenetic diversity support a greater diversity of ecosystem goods and services, indicating that the evolutionary processes responsible for generating variation among living organisms are also key to the provisioning of natures contributions to people. Our results raise the intriguing possibility that the history of human settlement in southern Africa may have been shaped, in part, by the evolutionary history of its tree flora. However, the correlation between human population and tree diversity generates a conflict between people and nature. Our study suggests that future human population growth may threaten the contributions to people provided by intact and phylogenetically diverse ecosystems.


title: Forest loss in Indonesian New Guinea: trends, drivers, and outlook

abstract: The rich forests of Indonesian New Guinea are threatened. We used satellite data to examine annual forest loss, road development and plantation expansion from 2001 to 2019, then developed a model to predict future deforestation in this understudied region. In 2019, 34.29 million hectares (Mha), or 83% of Indonesian New Guinea, supported old-growth forest. Over nineteen years, two percent (0.75 Mha) were cleared: 45% (0.34 Mha) converted to industrial plantations, roads, mine tailings, or other uses near cities; 55% (0.41 Mha) cleared by transient processes including selective natural timber extraction, inland water bodies-related processes, fires, and shifting agriculture. Industrial plantations expanded by 0.23 Mha, with the majority (0.21 Mha; 28% of forest loss) replacing forests and reaching 0.28 Mha in 2019 (97% oil palm; 3% pulpwood). The Trans-Papua Highway, a ~4,000 km national investment project, increased by 1,554 km. Positive correlations between highway and plantations expansion indicate these are linked processes. Plantations and roads grew rapidly after 2011, peaked in 2015/16, and declined thereafter. Indonesian government allocated 2.62 Mha of land for the development of industrial plantations (90% oil palm 10% pulpwood) of which 74% (1.95 Mha) remained forest in 2019. A spatial model predicts that an additional 4.5 Mha of forest could be cleared by 2036 if Indonesian New Guinea follows similar relationships to Indonesian Borneo. We highlight the opportunities for policy reform and the importance of working with indigenous communities, local leaders, and provincial government to protect the biological and cultural richness still embodied in this remarkable region.


title: On the evolution of host specificity: a case study of helminths

abstract: The significant variation in host specificity exhibited by parasites has been separately linked to evolutionary history and ecological factors in specific host-parasite associations. Yet, whether there are any general patterns in the factors that shape host specificity across parasites more broadly is unknown. Here we constructed a molecular phylogeny for 249 helminth species infecting free-range mammals and find that the influence of ecological factors and evolutionary history varies across different measures of host specificity. Whereas the phylogenetic range of hosts a parasite can infect shows a strong signal of evolutionary constraint, the number of hosts a parasite infects does not. Our results shed new light on the evolution of host specificity in parasites, suggesting that phylogenetic breadth may capture the evolutionary potential of a parasite to jump between hosts, whereas the number of hosts may reflect ecological opportunity. Finally, we show parasite phylogenies can also provide an alternative perspective on zoonosis by identifying which hosts are infected by a broad phylogenetic range of parasites.

category: evolutionary biology


title: Phylogenomic subsampling and the search for phylogenetically reliable loci

abstract: Phylogenomic subsampling is a procedure by which small sets of loci are selected from large genome-scale datasets and used for phylogenetic inference. This step is often motivated by either computational limitations associated with the use of complex inference methods, or as a means of testing the robustness of phylogenetic results by discarding loci that are deemed potentially misleading. Although many alternative methods of phylogenomic subsampling have been proposed, little effort has gone into comparing their behavior across different datasets. Here, I calculate multiple gene properties for a range of phylogenomic datasets spanning animal, fungal and plant clades, uncovering a remarkable predictability in their patterns of covariance. I also show how these patterns provide a means for ordering loci by both their rate of evolution and their relative phylogenetic usefulness. This method of retrieving phylogenetically useful loci is found to be among the top performing when compared to alternative subsampling protocols. Relatively common approaches such as minimizing potential sources of systematic bias or increasing the clock-likeness of the data are found to fare worse than selecting loci at random. Likewise, the general utility of rate-based subsampling is found to be limited: loci evolving at both low and high rates are among the least effective, and even those evolving at optimal rates can still widely differ in usefulness. This study shows that many common subsampling approaches introduce unintended effects in off-target gene properties, and proposes an alternative multivariate method that simultaneously optimizes phylogenetic signal while controlling for known sources of bias.


title: How plants minimize somatic evolution

abstract: A remarkable property of plants is their ability to accumulate mutations at a very slow pace despite their potentially long lifespans, during which they continually form buds, each with the potential to become a new branch. Because replication errors in cell division represent an unavoidable source of mutations, minimizing mutation accumulation requires the minimization of cell divisions. Here we show that there exists a well defined theoretical minimum for the branching cost, defined as the number of cell divisions necessary for the creation of each branch. Most importantly, we also show that this theoretical minimum can be closely approached by a simple pattern of cell divisions in the meristematic tissue of apical buds during the generation of novel buds. Both the optimal pattern of cell divisions and the associated branching cost are consistent with recent experimental data, suggesting that plant evolution has led to the discovery of this mechanism.


title: Rapid evolution of complete dosage compensation in Poecilia

abstract:
Dosage compensation balances gene expression between the sexes in systems with diverged heterogametic sex chromosomes. Theory predicts that dosage compensation should rapidly evolve in parallel with the divergence of sex chromosomes to prevent the deleterious effects of dosage imbalances that occur as a result of sex chromosome divergence. Examples of complete dosage compensation, where gene expression of the entire sex chromosome is compensated, are rare and have only been found in relatively ancient sex chromosome systems. Consequently, very little is known about the evolutionary dynamics of complete dosage compensation systems. We recently found the first example of complete dosage compensation in a fish, Poecilia picta. We also found that the Y chromosome degraded substantially in the common ancestor of P. picta and their close relative Poecilia parae. In this study we find that P. parae also have complete dosage compensation, thus complete dosage compensation likely evolved in the short ([~]3.7 my) interval after the split of the ancestor of these two species from P. reticulata, but before they diverged from each other. These data suggest that novel dosage compensation mechanisms can evolve rapidly, thus supporting the longstanding theoretical prediction that such mechanisms arise in parallel with rapidly diverging sex chromosomes.

SIGNIFICANCE STATEMENTIn species with XY sex chromosomes, females (XX) have as many copies of X-linked genes compared to males (XY), leading to unbalanced expression between the sexes. Theory predicts that dosage compensation mechanisms should evolve rapidly as X and Y chromosomes diverge, but examples of complete dosage compensation in recently diverged sex chromosomes are scarce, making this theory difficult to test. Across Poeciliid species the X and Y chromosomes have recently diversified. Here we find complete dosage compensation evolved rapidly as the X and Y diverged in the common ancestor of Poecilia parae and P. picta, supporting that novel dosage compensation mechanisms can evolve rapidly in tandem with diverging sex chromosomes. These data confirm longstanding theoretical predictions of sex chromosome evolution.


title: Delineating the molecular and phenotypic spectrum of the SETD1B-related syndrome

abstract: Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay and seizures. To date, clinical features have been described for eleven patients with (likely) pathogenic SETD1B sequence variants. We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. Selected variants were functionally tested using in vitro and genome-wide methylation assays. Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Interestingly, males are significantly overrepresented and more severely affected, and we speculate that sex-linked traits could affect susceptibility to penetrance and the clinical spectrum of SETD1B variants. Finally, despite the possibility of non-redundant contributions of SETD1B and its paralogue SETD1A to epigenetic control, the clinical phenotypes of the related disorders share many similarities, indicating that elucidating shared and divergent downstream targets of both genes will help to understand the mechanism leading to the neurobehavioral phenotypes. Insights from this extensive cohort will facilitate the counseling regarding the molecular and phenotypic landscape of newly diagnosed patients with the SETD1B-related syndrome.

category: genetics


title: Evaluating Sleep Disturbances in Children with Rare Genetic Neurodevelopmental Syndromes

abstract:
BackgroundAdequate sleep is important for proper neurodevelopment and positive health outcomes. Sleep disturbances are more prevalent in children with genetically determined neurodevelopmental syndromes compared to typically developing counterparts. We characterize sleep behavior in Rett (RTT), Angelman (AS) and Prader-Willi (PWS) syndromes in order to identify effective approaches for treating sleep problems in these populations. We compared sleep-related symptoms across individuals with these different syndromes to each other, and to typically developing controls.

MethodsChildren were recruited from the Rare Diseases Clinical Research Network (RDCRN) consortium registries; unaffected siblings were enrolled as related controls. For each participant, a parent completed multiple sleep questionnaires including: Pediatric Sleep Questionnaire (Sleep-Disordered Breathing [SDB]); Childrens Sleep Habits Questionnaire; Pediatric Daytime Sleepiness Scale.

ResultsSleep data were analyzed from 714 participants, ages 2-18 years. Young children with AS had more reported sleep problems than children with RTT or PWS. Older children with RTT had more reported daytime sleepiness than those with AS or PWS. Finally, all individuals with RTT had more evidence of sleep-disordered breathing when compared to individuals with PWS. Notably, typically developing siblings were also reported to have sleep problems, except for sleep-related breathing disturbances which were associated with each of the genetic syndromes.

ConclusionsIndividuals with RTT, AS and PWS frequently experience sleep problems, including sleep-disordered breathing. Screening for sleep problems in individuals with these and other neurogenetic disorders should be included in clinical assessment and managements. These data may also be useful in developing treatment strategies and in clinical trials.


title: Antisense oligonucleotide-based drug development for Cystic Fibrosis patients carrying the 3849+10kb C-to-T splicing mutation

abstract: Antisense oligonucleotide (ASO)-based drugs for splicing modulation were recently been approved for various genetic diseases with unmet need. Here we aimed to develop an ASO-based splicing modulation therapy for Cystic Fibrosis (CF) patients carrying the 3849+10kb C-to-T splicing mutation in the CFTR gene. We have screened, in FRT cells expressing this mutation, ~30 ASOs chemically modified with 2'-O-Methyl on a phosphrothioate backbone, targeted to prevent the recognition and inclusion of a cryptic exon generated due to the mutation. The screening identified five ASO candidates able to promote CFTR correct splicing and rescue channel activity. Further analyses in well differentiated primary human nasal and bronchial epithelial cells (HNEs, HBEs), derived from patients carrying at least one 3849+10kb C-to-T allele, led to the identification of a highly potent lead ASO. The ASO was efficiently delivered by free uptake into patients HNEs and HBEs and completely restored CFTR function to wild type levels in cells from a homozygous patient and led to 43{+/-}8% of wild type levels in cells from various heterozygous patients. Optimized efficiency was further obtained with 2-Methoxy Ethyl chemical modification. The results demonstrate the therapeutic potential and clinical benefit of ASO-based splicing modulation for genetic diseases caused by splicing mutations.


title: Shrinkage Parameter Estimation in Penalized Logistic RegressionAnalysis of Case-Control Data

abstract:
IntroductionIncreasingly, logistic regression methods for genetic association studies of binary phenotypes must be able to accommodate data sparsity, which arises from unbalanced case-control ratios and/or rare genetic variants. Sparseness leads to maximum likelihood estimators (MLEs) of log-OR parameters that are biased away from their null value of zero and tests with inflated type 1 errors. Different penalized-likelihood methods have been developed to mitigate sparse-data bias. We study penalized logistic regression using a class of log-F priors indexed by a shrinkage parameter m to shrink the biased MLE towards zero.

MethodsWe propose a two-step approach to the analysis of a genetic association study: first, a set of variants that show evidence of association with the trait is used to estimate m; and second, the estimated m is used for log-F -penalized logistic regression analyses of all variants using data augmentation with standard software. Our estimate of m is the maximizer of a marginal likelihood obtained by integrating the latent log-ORs out of the joint distribution of the parameters and observed data. We consider two approximate approaches to maximizing the marginal likelihood: (i) a Monte Carlo EM algorithm (MCEM) and (ii) a Laplace approximation (LA) to each integral, followed by derivative-free optimization of the approximation.

ResultsWe evaluate the statistical properties of our proposed two-step method and compared its performance to other shrinkage methods by a simulation study. Our simulation studies suggest that the proposed log-F -penalized approach has lower bias and mean squared error than other methods considered. We also illustrate the approach on data from a study of genetic associations with "super senior" cases and middle aged controls.

Discussion/ConclusionWe have proposed a method for single rare variant analysis with binary phenotypes by logistic regression penalized by log-F priors. Our method has the advantage of being easily extended to correct for confounding due to population structure and genetic relatedness through a data augmentation approach.


title: Penalized Logistic Regression Analysis for Genetic Association Studies of Binary Phenotypes


title: Epigenetic modulation in the pathogenesis and treatment of inherited aortic aneurysm conditions

abstract: Shprintzen-Goldberg syndrome (SGS) is a rare systemic connective tissue disorder characterized by craniofacial, skeletal, neurodevelopmental, cutaneous, and cardiovascular manifestations, including aortic root aneurysm. It has significant phenotypic overlap with both Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS). We previously reported that SGS is caused by heterozygous mutations in the Sloan-Kettering Institute proto-oncogene (SKI), which encodes a potent suppressor of transforming growth factor beta (TGF{beta}) target gene expression. Herein, we show that mouse lines harboring orthologous amino acid substitutions in Ski recapitulate multiple human SGS phenotypic manifestations, including skin collagen deposition, skeletal kyphosis, behavioral hypoactivity, and aortic root aneurysm. Furthermore, aortic root aneurysm in SGS mice is associated with both increased acetylation of histone H3 at lysine-27 (H3K27) and TGF{beta} target gene expression, all of which can be ameliorated by pharmacological CBP/P300 inhibition in vivo; similar findings were seen in cultured dermal fibroblast from SGS patients. Aortic root growth is also abrogated in a mouse model of MFS by selective CBP/P300 inhibition in association with blunted expression of TGF{beta} target genes. These data document excessive H3K27 acetylation and hence TGF{beta} target gene expression in the pathogenesis of inherited presentations of aortic root aneurysm and the therapeutic potential of pharmacological epigenetic modulation.


title: Constructing Local Cell Sepcific Networks from Single Cell Data

abstract: Single-cell RNA sequencing facilitates investigations of gene co-expression networks at the cellular level potentially yielding unique biological insights, but the estimation problem is challenging. We develop a non-parametric approach to estimate cell-specific networks for each cell and cell type and propose novel downstream analyses. The individual networks preserve the cellular heterogeneity and facilitate testing for differences between cell groups. To further our understanding of autism spectrum disorder, we examine the evolution of gene networks in fetal brain cells and compare the gene networks of cells sampled from case and control subjects to reveal patterns in gene co-expression.

category: genomics


title: Genomic evidence that governmentally produced Cannabis sativa poorly represents genetic variation available in state markets

abstract: The National Institute on Drug Abuse (NIDA) is the sole producer of Cannabis for research purposes in the United States, including medical investigation. Previous research established that cannabinoid profiles in the NIDA varieties lacked diversity and potency relative to the Cannabis produced commercially. Additionally, microsatellite marker analyses have established that the NIDA varieties are genetically divergent form varieties produced in the private legal market. Here, we analyzed the genome of multiple Cannabis varieties from diverse lineages including two produced by NIDA, and we provide further support that NIDAs varieties differ from widely available medical, recreational, or industrial Cannabis. Furthermore, our results suggest that NIDAs varieties lack diversity in the single copy portion of the genome, the maternally inherited genomes, the cannabinoid genes, and in the repetitive content of the genome. Therefore, results based on NIDAs varieties are not generalizable regarding the effects of Cannabis after consumption. For medical research to be relevant, material that is more widely used would have to be studied. Clearly, having research to date dominated by a single, non-representative source of Cannabis has hindered scientific investigation.


title: Female and male Sirex noctilio use age and size to select a mate

abstract: While male mate choice in insects is a widely accepted concept, there is still limited evidence showing that lek formation is compatible with the evolution of male mate choice. In the woodwasp Sirex noctilio, males form leks that are used by females to select a mate. However, males have been observed to ignore certain females, suggesting the presence of male mate choice despite the presence of a lek mating system. In this study we demonstrate that males only attempt to mate with certain females. To understand the criteria used by males and females to select a mate, we also tested the effect of age, size, and male to female size ratio on the number of mating attempts made by males and on female receptivity. We demonstrate that size and age play a role in both male and female mate choice. Our results suggest that males must reach sexual maturity after emergence and are neither receptive nor attractive to females during the first few days of their lives. We also show that older females become less attractive to males, suggesting that female S. noctilio switch to a strict host location phase sometime after emergence. Our results show that male and female size, and the ratio between them, play a role in mate choice. While larger males are more motivated to mate, their large size can physically prevent them from mating with small females. Small females are also more attractive and more receptive to males, consistent with the presence of convenience polyandry in S. noctilio.

category: animal behavior and cognition


title: Cancer blues? A validated judgment bias task suggests pessimism in nude mice with tumors

abstract: In humans, affective states can bias responses to ambiguous information: a phenomenon termed judgment bias (JB). Judgment biases have great potential for assessing affective states in animals, in both animal welfare and biomedical research. New animal JB tasks require construct validation, but for laboratory mice (Mus musculus), the most common research vertebrate, a valid JB task has proved elusive. Here (Experiment 1), we demonstrate construct validity for a novel mouse JB test: an olfactory Go/Go task in which subjects dig for high- or low-value food rewards. In C57BL/6 and Balb/c mice faced with ambiguous cues, latencies to dig were sensitive to high/low welfare housing, environmentally-enriched animals responding with relative  optimism through shorter latencies. Illustrating the versatility of a validated JB task across fields of research, it further allowed us to test hypotheses about the mood-altering effects of cancer (Experiment 2). Male nude mice bearing subcutaneous lung adenocarcinomas responded more pessimistically than healthy controls to ambiguous cues. Similar effects were not seen in females, however. To our knowledge, this is the first validation of a mouse JB task and the first demonstration of pessimism in tumor-bearing animals. This task, especially if refined to improve its sensitivity, thus has great potential for investigating mouse welfare, the links between affective state and disease, depression-like states in animals, and hypotheses regarding the neurobiological mechanisms that underlie affect-mediated biases in judgment.


title: Structure of an infectious mammalian prion

abstract: Classical mammalian prions are assemblies of prion protein molecules that are extraordinarily transmissible, with a microgram of protein containing up to 108 lethal doses of infectivity1,2. Unlike most other pathologic and amyloidogenic proteins, prions typically contain glycolipid anchors 3 and abundant asparagine-linked glycans4-6. The infectious nature, complexity, and biophysical properties of prions have complicated structural analyses and stymied any prior elucidation of 3D conformation at the polypeptide backbone level7. Here we have determined the structure of the core of a fully infectious, brain-derived prion by cryo-electron microscopy with [~]3.1 [A] resolution. The purified prions are amyloid fibrils comprised of monomers assembled with parallel in-register intermolecular beta sheets and connecting chains. Residues [~]95-227 of each monomer provide one rung of the ordered fibril core, with the glycans and glycolipid anchor projecting from the lateral surfaces of the fibril. The fibril ends, where prion growth occurs, are formed by single monomers in an extended serpentine combination of {beta}- arches, a Greek key, and loops that presumably template the refolding of incoming monomers. Our results describe an atomic model to underpin detailed molecular hypotheses of how pathologic prion proteins can propagate as infectious agents, and how such propagation and associated pathogenesis might be impeded.

category: biochemistry


title: Structure and mechanistic analyses of the gating mechanism of elongating ketosynthases

abstract: Ketosynthases (KSs) catalyze carbon-carbon bond forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester tethered substrates to the active sites of KSs. Therefore, protein-protein interactions (PPIs) and KS-mediated substrate recognition events are required for catalysis. Recently, crystal structures of Escherichia coli elongating type II FAS KSs, FabF and FabB, in complex with E. coli ACP, AcpP, revealed distinct conformational states of two active site KS loops. These loops were proposed to operate via a gating mechanism to coordinate substrate recognition and delivery followed by catalysis. Here we interrogate this proposed gating mechanism by solving two additional high-resolution structures of substrate engaged AcpP-FabF complexes, one of which provides the missing AcpP-FabF gate-closed conformation. Clearly defined interactions of one of these active site loops with AcpP are present in both the open and closed conformations, suggesting AcpP binding triggers or stabilizes gating transitions, further implicating PPIs in carrier protein-dependent catalysis. We functionally demonstrate the importance of gating in the overall KS condensation reaction and provide experimental evidence for its role in the transacylation reaction. Furthermore, we evaluate the catalytic importance of these loops using alanine scanning mutagenesis and also investigate chimeric FabF constructs carrying elements found in type I PKS KS domains. These findings broaden our understanding of the KS mechanism which advances future engineering efforts in both FASs and evolutionarily related PKSs.


title: Thiophosphopeptides Instantly Targeting Golgi Apparatus and Selectively Killing Cancer Cells

abstract: Golgi apparatus is emerging as a key signaling hub of cells, but there are few approaches for targeting Golgi and selectively killing cancer cells. Here we show an unexpected result that changing an oxygen atom of the phosphoester bond in phospho-peptides by a sulfur atom enables instantly targeting Golgi apparatus (GA) and selectively killing cancer cells by enzymatic self-assembly. Specifically, conjugating cysteamine S-phosphate to the C-terminal of a self-assembling peptide generates a thiophospho-peptide. Being a substrate of alkaline phosphatase (ALP), the thiophosphopeptide undergoes rapid ALP-catalyzed dephosphorylation to form a thiopeptide that self-assembles. The thiophosphopeptide enters cells via caveolin-mediated endocytosis and macropinocytosis and instantly accumulates in GA because of dephosphorylation and formation of disulfide bonds in Golgi. Moreover, the thiophosphopeptide, targeting Golgi, potently and selectively inhibits cancer cells (e.g., HeLa) with the IC50 (about 3 M), which is an order of magnitude more potent than that of the parent phosphopeptide. This work, as the first report of thiophospho-peptide for targeting Golgi, illustrates a new molecular platform for designing enzyme responsive molecules that target subcellular compartment for functions.


title: SUMOylation of the chromodomain factor MRG-1 in C. elegans affects chromatin-regulatory dynamics

abstract: Epigenetic mechanisms to control chromatin accessibility and structure is important for gene expression in eukaryotic cells. Chromatin regulation ensures proper development and cell fate specification but is also essential later in life. Modifications of histone proteins as an integral component of chromatin can promote either gene expression or repression, respectively. Proteins containing specific domains such as the chromodomain recognize mono-, di- or tri-methylated lysine residues on histone H3. The chromodomain protein MRG-1 in Caenorhabditis elegans is the ortholog of mammalian MRG15, which belongs to the MORF4 Related Gene (MRG) family in humans. In C. elegans MRG-1 predominantly binds methylated histone H3 lysine residues at position 36 (H3K36me3). MRG-1 is important during germline maturation and for safeguarding the germ cell identity. However, it lacks enzymatic activity and depends on protein-protein interaction to cooperate with other factors to regulate chromatin. To elucidate the variety of MRG-1 interaction partners we performed in-depth protein-protein interaction analysis using immunoprecipitations coupled with mass-spectrometry. Besides previously described and novel interactions with other proteins, we also detected a strong association with the Small Ubiquitin-like Modifier (SUMO). Since SUMO is known to be attached to proteins in order to modulate the target proteins activity we assessed whether MRG-1 is post-translationally modified by SUMOylation. Notably, we provide evidence that MRG-1 is indeed SUMOylated and that this post-translational modification influences the chromatin-binding profile of MRG-1 in the C. elegans genome. Our presented study hints towards an important role of SUMOylation in the context of epigenetic regulation via the chromodomain protein MRG-1, which may be a conserved phenomenon also in mammalian species.


title: Nano-hydroxyapatite coating synthesized on quasi-fibrillar superstructures of collagen hydrolysate leads to superior osteoblast proliferation when compared to nano-hydroxyapatite synthesized on collagen fibrils

abstract:
This study had a two-fold objective: To utilize collagen hydrolysate for synthesizing a nanoscale Hydroxyapatite (HA) coating that would act as a superior osteoblast adhesion/proliferation agent compared to collagen-derived HA (C/HA) and to comprehend the significant role played by structural constraints on HA nucleation. Collagen was extracted from pacu skin with a high yield of 65.3% (w/w of tissue). It was digested by collagenase and the hydrolysate (CH) was purified with a high yield of 0.68g/g of collagen. The CH peptides had a mass of 6kDa, a predominant PP-II conformation and formed self-assembling hierarchical structures at physiological pH with dimensions of 842.2{+/-}229nm. The HA synthesized on CH (CH/HA) displayed higher yield when compared to C/HA. Structural analysis of CH/HA revealed that the PP-II peptides coiled to form mimic-helical moieties with reduced intermolecular packing distance of 0.9nm. The mimic helices cross-linked to form a vast quasi-fibrillar network that was comparatively smaller than collagen fibrils but exhibited enhanced stability and greater dynamicity. CH/HA displayed intense calcium-carboxyl interactions, sharper diffraction planes, smaller size of 48{+/-}6.2nm and a Ca/P ratio closer to 1.69 when compared to C/HA along with displaying serrated edge blooming crystals. Because of the small size, the CH/HA nanocrystals displayed significantly better osteoblast adhesion than C/HA and reduced the doubling time of cells. Overall, the results indicated that CH based nanocomposites displayed suitable morphological characteristics and cellular response for potential application as implant and bone graft coating material.

Graphical abstract

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/431097v1_ufig1.gif" ALT="Figure 1">
View larger version (57K):
org.highwire.dtl.DTLVardef@1b020c2org.highwire.dtl.DTLVardef@36a899org.highwire.dtl.DTLVardef@77106aorg.highwire.dtl.DTLVardef@d6b801_HPS_FORMAT_FIGEXP  M_FIG C_FIG


title: Metabolic and Process Engineering for Microbial Production of Protocatechuate from Xylose with Corynebacterium glutamicum

abstract: 3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of interest in the chemical industry as a building block for novel polymers and has wide potential for pharmaceutical applications due to its antioxidant, anti-inflammatory, and antiviral properties. In the present study, we designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of O_SCPLOWDC_SCPLOW-xylose for microbial production of PCA. The engineered strain showed a maximum PCA titer of 62.1 {+/-} 12.1 mM (9.6 {+/-} 1.9 g L-1) from O_SCPLOWDC_SCPLOW-xylose as the primary carbon and energy source. The corresponding yield was [Formula], which corresponds to 38 % of the maximum theoretical yield and is 14-fold higher compared to the parental producer strain on O_SCPLOWDC_SCPLOW-glucose. By establishing a one-pot bioreactor cultivation process followed by subsequent process optimization, the same maximum titer and a total amount of 16.5 {+/-} 1.1 g was reached. Downstream processing of PCA from this fermentation broth was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. Since PCA turned out to be electrochemically unstable in combination with several anode materials, a threechamber electrolysis setup was established to crystallize PCA and to avoid direct anode contact. This resulted in a maximum final purity of 95.4 %. In summary, the established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks.

category: bioengineering