Relationship between shear wave velocity and muscle activation is inconsistent across different muscle types There is an increasing use of shear wave ultrasound elastography to quantify mechanical properties of muscles under various conditions such as changes muscle length and levels of activation in healthy and pathological muscle. However, little is known about the variability in shear wave velocity among muscles as most studies investigate one specific muscle. The purpose of this study was to determine if the relationship between SWV and muscle activation is consistent across muscles with different architectures: biceps brachii, tibialis anterior, and medial gastrocnemius, All measures were made at matching levels of activation and approximately at the optimal length for each muscle to control for length-dependent changes in the relationship between activation and force or stiffness. We also conducted a control experiment to determine how the passive force within a muscle alters the relationship between muscle activation and shear wave velocity. The relationship between shear wave velocity-squared and activation above 10% MVC differed across muscles, with biceps brachii and medial gastrocnemius showing a lower slope than tibialis anterior. Shear wave velocity-squared also differed between muscles at the shortest length (p<0.001) and the increase in shear wave velocity-squared with muscle lengthening also differed between muscle types (p = 0.005) Muscle-specific differences could not be explained by the architectural features such as pennation angle, fiber length, and physiological cross-sectional area. Our results demonstrate that there is not a unique relationship between muscle activation and shear wave velocity, highlighting the importance of understanding the many factors contributing to shear wave propagation in muscle before elastography can be used to make quantitative comparisons across muscle types.
Distributed brain co-processor for tracking electrophysiology and behavior during electrical brain stimulation Early implantable epilepsy therapy devices provided open-loop electrical stimulation without brain sensing, computing, or an interface for synchronized behavioral inputs from patients. Recent epilepsy stimulation devices provide brain sensing but have not yet developed analytics for accurately tracking and quantifying behavior and seizures. Here we describe a distributed brain co-processor providing an intuitive bi-directional interface between patient, implanted neural stimulation and sensing device, and local and distributed computing resources. Automated analysis of continuous streaming electrophysiology is synchronized with patient reports using a hand-held device and integrated with distributed cloud computing resources for quantifying seizures, interictal epileptiform spikes, and patient symptoms during therapeutic electrical brain stimulation. The classification algorithms for interictal epileptiform spikes and seizures were developed and parameterized using long-term ambulatory data from 9 humans and 8 canines with epilepsy, and then implemented prospectively in out-of-sample testing in 2 pet canines and 4 humans with drug resistant epilepsy living in their natural environments.
Critical re-evaluation of the slope factor of the operational model of agonism Background and PurposeAlthough being a relative term, agonist efficacy is a cornerstone in the proper assessment of agonist selectivity and signalling bias. The operational model of agonism (OMA) has become successful in the determination of agonist efficacies and ranking them. In 1985, Black et al. introduced the slope factor to the OMA to make it more flexible and allow for fitting steep as well as flat concentration-response curves.

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

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

Conclusion and ImplicationsThe OMA modified by the Hill coefficient can be used for correct ranking of agonism and subsequent analysis, like quantification of signalling bias. Living with high potassium: a balance between nutrient acquisition and stress signaling during K-induced salt stress O_LIHigh potassium (K) in the growth medium is more toxic to plants than Na at similar concentrations. However, the molecular mechanisms underlying plant responses to K-induced salt stress are virtually unknown.
C_LIO_LIWe examined Arabidopsis thaliana and its extremophyte relative Schrenkiella parvula, using a comparative multi-omics approach to identify cellular processes affected by excess K and understand which deterministic regulatory pathways are active to avoid tissue damage while sustaining growth.
C_LIO_LIA. thaliana showed limited capacity to curb excess K accumulation and prevent nutrient depletion contrasting to S. parvula which could limit excess K accumulation without restricting nutrient uptake. Facilitated by a targeted transcriptomic response, promoting nitrogen uptake along with other key nutrients and uninterrupted N assimilation into primary metabolites during excess K-stress allowed S. parvula to boost its antioxidant and osmolyte pools concurrently leading to sustained growth. Antithetically, A. thaliana showed transcriptional responses indicative of a poor balance between stress signaling, increased ROS levels, and reduced photosynthesis, subsequently leading to inhibited growth.
C_LIO_LIThe ability to regulate independent nutrient uptake and a coordinated transcriptomic response to avoid non-specific stress signaling are two main deterministic steps towards building stress resilience to excess K+-induced salt stress.
C_LI Fast ATP-dependent Subunit Rotation in reconstituted FoF1-ATP synthase trapped in Solution FoF1-ATP synthases are ubiquitous membrane-bound, rotary motor enzymes that can catalyze ATP synthesis and hydrolysis. Their enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, by mechanical inhibitory mechanisms, but also by the electrochemical potential of protons across the membrane. Single-molecule Forster resonance energy transfer (smFRET) has been used to detect subunit rotation within FoF1-ATP synthases embedded in freely diffusing liposomes. We now report that kinetic monitoring of functional rotation can be prolonged from milliseconds to seconds by utilizing an Anti-Brownian electrokinetic trap (ABEL trap). These extended observation times allowed us to observe fluctuating rates of functional rotation for individual FoF1-liposomes in solution. Broad distributions of ATP-dependent catalytic rates were revealed. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores or uncouplers, the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second. This was much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 complexes uncoupled from the membrane-embedded Fo complex. Further application of ABEL trap measurements should help resolve the mechanistic causes of such fluctuating rates of subunit rotation.

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O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/436299v5_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@1bd800org.highwire.dtl.DTLVardef@228aeorg.highwire.dtl.DTLVardef@1bce39forg.highwire.dtl.DTLVardef@3f01a_HPS_FORMAT_FIGEXP  M_FIG O_FLOATNOFigure 1.C_FLOATNO C_FIG Microbial colonization of tannin-rich tropical plants: interplay between degradability, methane production and tannin disappearance in the rumen AO_SCPLOWBSTRACTC_SCPLOWCondensed tannins in plants are found free and attached to protein and fibre but it is not known whether these fractions influence rumen degradation and microbial colonization. This study explored the rumen degradation of tropical tannins-rich plants and the relationship between condensed tannins fractions disappearance and microbial communities colonising plant particles. Leaves from Calliandra calothyrsus, Gliricidia sepium, and Leucaena leucocephala, pods from Acacia nilotica and the leaves of two agricultural by-products: Manihot esculenta and Musa spp. were incubated in situ in the rumen of dairy cows. An in vitro approach was also used to assess the effects of these plants on rumen fermentation parameters. All plants contained more than 100 g/kg of condensed tannins with a large proportion (32 to 61%) bound to proteins. Calliandra calothyrsus had the highest concentration of condensed tannins at 361 g/kg, whereas Acacia nilotica was particularly rich in hydrolysable tannins (350 g/kg). Hydrolysable and free condensed tannins from all plants completely disappeared after 24 h incubation in the rumen. Disappearance of protein-bound condensed tannins was variable with values ranging from 93% for Gliricidia sepium to 21% for Acacia nilotica. In contrast, fibre-bound condensed tannins disappearance averaged ~82% and did not vary between plants. Disappearance of bound fractions of condensed tannins was not associated with degradability of plant fractions. The presence of tannins interfered with the microbial colonisation of plants. Each plant had distinct bacterial and archaeal communities after 3 and 12 h of incubation in the rumen and distinct protozoal communities at 3 h. Adherent communities in tannin-rich plants had a lower relative abundance of fibrolytic microbes, notably Fibrobacter spp. Whereas, archaea diversity was reduced in high tannin-containing Calliandra calothyrsus and Acacia nilotica at 12 h of incubation. Concurrently, in vitro methane production was lower for Calliandra calothyrsus, Acacia nilotica and Leucaena leucocephala although for the latter total volatile fatty acids production was not affected and was similar to control. Here we show that the total amount of hydrolysable and condensed tannins contained in a plant govern the interaction with rumen microbes affecting degradability and fermentation. The effect of protein- and fibre-bound condensed tannins on degradability is less important. The Functional Nanopore Screen: A Versatile High-throughput Assay to Study and Engineer Protein Nanopores in Escherichia coli Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the Functional Nanopore (FuN) screen now provides a quantitative and dynamic read-out of nanopore assembly and function in E. coli. The assay is based on genetically-encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta- and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology and synthetic biology. Frameshift and wild-type proteins are highly similar because the genetic code and genomes were optimized for frameshift tolerance Frameshift protein sequences encoded by alternative reading frames of coding genes have been considered meaningless, and frameshift mutations have been considered of little importance for the molecular evolution of coding genes and proteins. However, functional frameshifts have been found widely existing. It was puzzling how a frameshift protein kept its structure and functionality while its amino-acid sequence was changed substantially. Here we show that frame similarities between frameshifts and wild types are higher than random similarities and are defined at the genetic code, gene, and genome levels. In the standard genetic code, frameshift codon substitutions are more conservative than random substitutions. The frameshift tolerability of the standard genetic code ranks in the top 2.0-3.5% of alternative genetic codes, showing that the genetic code is nearly optimal for frameshift tolerance. Furthermore, frameshift-resistant codons (codon pairs) appear more frequently than expected in many genes and certain genomes, showing that the frameshift optimality is reflected not only in the genetic code but more importantly, in its allowance of further optimizing the frameshift tolerance of a particular gene or genome, which shed light on the role of frameshift mutations in molecular and genomic evolution. Predicting gene regulatory networks from multi-omics to link genetic risk variants and neuroimmunology to Alzheimers disease phenotypes BackgroundGenome-wide association studies have found many genetic risk variants associated with Alzheimers disease (AD). However, how these risk variants affect deeper phenotypes such as disease progression and immune response remains elusive. Also, our understanding of cellular and molecular mechanisms from disease risk variants to various phenotypes is still limited. To address these problems, we performed an integrative multi-omics analysis of genotype, transcriptomics, and epigenomics for revealing gene regulatory mechanisms from disease variants to AD phenotypes.

MethodFirst, given the population gene expression data of a cohort, we construct and cluster its gene co-expression network to identify gene co-expression modules for various AD phenotypes. Next, we predict transcription factors (TFs) regulating co-expressed genes and AD risk SNPs that interrupt TF binding sites on regulatory elements. Finally, we construct a gene regulatory network (GRN) linking SNPs, interrupted TFs, and regulatory elements to target genes and gene modules for each phenotype in the cohort. This network thus provides systematic insights into gene regulatory mechanisms from risk variants to AD phenotypes.

ResultsOur analysis predicted GRNs in three major AD-relevant regions: Hippocampus, Dorsolateral Prefrontal Cortex (DLPFC), Lateral Temporal Lobe (LTL). Comparative analyses revealed cross-region-conserved and region-specific GRNs, in which many immunological genes are present. For instance, SNPs rs13404184 and rs61068452 disrupt SPI1 binding and regulation of AD gene INPP5D in the Hippocampus and LTL. However, SNP rs117863556 interrupts bindings of REST to regulate GAB2 in DLPFC only. Driven by emerging neuroinflammation in AD, we used Covid-19 as a proxy to identify possible regulatory mechanisms for neuroimmunology in AD. To this end, we looked at the GRN subnetworks relating to genes from shared AD-Covid pathways. From those subnetworks, our machine learning analysis prioritized the AD-Covid genes for predicting Covid-19 severity. Decision Curve Analysis also validated our AD-Covid genes outperform known Covid-19 genes for classifying severe Covid-19 patients. This suggests AD-Covid genes along with linked SNPs can be potential novel biomarkers for neuroimmunology in AD. Finally, our results are open-source available as a comprehensive functional genomic map for AD, providing a deeper mechanistic understanding of the interplay among multi-omics, brain regions, gene functions like neuroimmunology, and phenotypes. Cytosolic EZH2-IMPDH2 complexes regulate melanoma progression via GTP The enhancer of zeste homolog 2 (EZH2) oncoprotein is a histone methyltransferase that functions canonically as a catalytic subunit of the polycomb repressive complex 2 (PRC2) to tri-methylate histone H3 at Lys 27 (H3K27me3). Although targeting EZH2 methyltransferase is a promising therapeutic strategy against cancer, methyltransferase-independent oncogenic functions of EZH2 are described. Moreover, pharmacological EZH2 methyltransferase inhibition was only variably effective in pre-clinical and clinical studies, suggesting that targeting EZH2 methyltransferase alone may be insufficient. Here, we demonstrate a non-canonical mechanism of EZH2s oncogenic activity characterized by interactions with inosine monophosphate dehydrogenase 2 (IMPDH2) and downstream promotion of guanosine-5-triphosphate (GTP) production. EZH2-IMPDH2 interactions identified by Liquid Chromatography-Mass Spectrometry (LC-MS) of EZH2 immunoprecipitates from melanoma cells were verified to occur between the N-terminal EED-binding domain of cytosolic EZH2 and the CBS domain of IMPDH2 in a methyltransfersase-independent manner. EZH2 silencing reduced cellular GTP, ribosome biogenesis, RhoA-mediated actomyosin contractility and melanoma cell proliferation and invasion by impeding the activity of IMPDH2. Guanosine, which replenishes GTP, reversed these effects and thereby promoted invasive and clonogenic cell states even in EZH2 silenced cells. IMPDH2 silencing antagonized the proliferative and invasive effects of EZH2, also in a guanosine-reversible manner. In human melanomas, high cytosolic EZH2 and IMPDH2 expression were associated with nucleolar enlargement, a marker of ribosome biogenesis. EZH2-IMPDH2 complexes were also observed in a range of cancers in which Sappanone A (SA), which inhibits EZH2-IMPDH2 interactions, was anti-tumorigenic, although notably non-toxic in normal cells. These findings illuminate a previously unrecognized, non-canonical, methyltransferase-independent, and GTP-dependent mechanism by which EZH2 regulates tumorigenicity in melanoma and other cancers, opening new avenues for development of anti-EZH2 therapeutics.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/467024v2_ufig1.gif" ALT="Figure 1">
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org.highwire.dtl.DTLVardef@153dd35org.highwire.dtl.DTLVardef@dfeb43org.highwire.dtl.DTLVardef@ce8148org.highwire.dtl.DTLVardef@189d8da_HPS_FORMAT_FIGEXP  M_FIG Graphical Abstract C_FIG HighlightsO_LIEZH2 has non-canonical methyltransferase-independent and GTP-dependent tumorigenic and metastatic functions in melanoma.
C_LIO_LIThe N-terminal EED-binding domain of EZH2 interacts with the CBS domain of IMPDH2 in a polycomb repressive complex 2- (PRC2-) and methylation-independent manner.
C_LIO_LIEZH2 accumulates with IMPDH2 in the cytoplasm and increases IMPDH2s tetramerization-mediated activity independently of EZH2 methyltransferase.
C_LIO_LIEZH2 upregulates GTP synthesis by IMPDH2 activation and thereby activates ribosome biogenesis via rRNA synthesis and actomyosin contractility via RhoA GTPase.
C_LIO_LISappanone A (SA) inhibits IMPDH2-EZH2 interactions and is anti-proliferative across a range of cancers including melanoma, but not in normal cells.
C_LI Consequences of alanine-126 mutations in helix-3 on structure and functions of Rad6 E2 ubiquitin-conjugating enzymes Rad6, an E2 ubiquitin-conjugating enzyme conserved from yeast to humans, functions in transcription, genome maintenance and proteostasis. The contributions of many conserved secondary structures of Rad6 and its human homologs UBE2A and UBE2B to their biological functions are not understood. A mutant RAD6 allele with a missense substitution at alanine-126 (A126) of helix-3 that causes defects in telomeric gene silencing, DNA repair and protein degradation was reported over two decades ago. Here, using a combination of genetics, biochemical, biophysical, and computational approaches, we discovered that helix-3 A126 mutations compromise the ability of Rad6 to ubiquitinate target proteins without disrupting interactions with partner E3 ubiquitin-ligases that are required for their various biological functions in vivo. Explaining the defective in vitro or in vivo ubiquitination activities, molecular dynamics simulations and NMR showed that helix-3 A126 mutations cause local disorder of the catalytic pocket of Rad6 in addition to disorganizing the global structure of the protein to decrease its stability in vivo. We also show that helix-3 A126 mutations deform the structures of UBE2A and UBE2B, the human Rad6 homologs, and compromise the in vitro ubiquitination activity and folding of UBE2B. In summary, our studies reveal that the conserved helix-3 is a crucial structural constituent that controls the organization of catalytic pockets, enzymatic activities, and biological functions of the Rad6-family E2 ubiquitin-conjugating enzymes. Nuclear Progestin Receptor Mediated Linkage of Blood Coagulation and Ovulation Ovulation is a dramatic remodeling process that includes rupture of blood capillaries and clotting, but coagulation is not thought to directly regulate this process. Herein, we report remarkable increases of coagulation factors V (f5, [~]3145-fold) and tissue factor (f3a, [~]120-fold) in zebrafish ovarian follicle cells during ovulation. This increase was mediated through the nuclear progestin receptor (Pgr), which is essential for ovulation in zebrafish, and was totally abolished in ovarian follicular cells from pgr-/- mutants. In addition, promoter activities of f5 and f3a were significantly enhanced by progestin (DHP) via Pgr. Similar regulation of human F5 promoter activity was induced via human PGRB, suggesting a conserved mechanism. Site-directed mutagenesis of the zebrafish f5 promoter further demonstrated a direct regulation of coagulation factors via progestin response elements. Moreover, a stark increase of erythrocytes occurred in capillaries meshed in wildtype preovulatory follicles but was absent in pgr-/- mutants. Interestingly, anticoagulants significantly inhibited ovulation both in vitro and in vivo, respectively. Furthermore, reduced fecundity was observed in f5+/- female zebrafish. Taken together, our study provides plausible evidence for steroid regulation of coagulation factors, and a new hypothesis for blood clotting triggered ovulation in vertebrates. Hierarchical Bayesian Integrated Model for Estimating Migratory Bird Harvest in Canada The Canadian Wildlife Service (CWS) requires reliable estimates of the harvest of migratory game birds, including waterfowl, to effectively manage populations of these hunted species. The National Harvest Survey is an annual survey of hunters who purchase Canadas mandatory migratory game bird hunting permit, integrating information from a survey of hunting activity with information from a separate survey of species composition in the harvest. We use these survey data to estimate the number of birds harvested for each species, as well as hunting activity metrics such as the number of active hunters and days spent hunting. The analytical methods used to generate these estimates have not changed since the survey was first designed in the early 1970s. Here we describe a new hierarchical Bayesian integrated model, which replaces the series of ratio estimators that comprised the old model. We are now using this new model to generate estimates for migratory bird harvests as of the 2019-2020 hunting season, and to generate updated estimates for all earlier years. The hierarchical Bayesian model uses over-dispersed Poisson distributions to model mean hunter activity and harvest (zero inflated Poisson and zero truncated Poisson, respectively). It also includes multinomial distributions to model some key components including, variation in total harvest across periods of the hunting season, the species composition of the harvest within each of those periods, and the age and sex composition in the harvests of a given species. We estimated the parameters of the Poisson and the multinomial distributions for each year as random effects using first-difference time-series. This time-series component allows the model to share information across years and reduces the sensitivity of the estimates to annual sampling noise. The new model estimates are generally very similar to those from the old model, particularly for the species that occur most commonly in the harvest, and so the results do not suggest any major changes to harvest management decisions and regulations. However, estimates for all species from the new model are more precise and less susceptible to annual sampling error, particularly for species that occur less commonly in the harvest (e.g., sea ducks and other species of conservation concern). This new model, with its hierarchical Bayesian framework, will also facilitate future improvements and elaborations, allowing the incorporation of prior information from the rich literature and knowledge in game bird management and biology. Outsourced hearing in an orb-weaving spider that uses its web as an auditory sensor Hearing is a fundamental sense of many animals, including all mammals, birds, some reptiles, amphibians, fish, and arthropods. The auditory organs of these animals are extremely diverse in anatomy after hundreds of millions of years of evolution, yet all are made up of cellular tissues and are morphologically part of bodies of animals. Here we show hearing in the orb-weaving spider, Larinioides sclopetarius is not constrained by the organisms body but is extended through outsourcing hearing to its extended phenotype, the proteinaceous, self-manufactured orb-web. We find the wispy, wheel-shaped orb-web acts as a hyperacute acoustic "antenna" to capture the sound-induced air particle movements that approach the maximum physical efficiency, better than the acoustic responsivity of all previously known eardrums. By sensing the motion of web threads, the spider remotely detects and localizes the source of an incoming airborne acoustic wave such as those emitted by approaching prey or predators. By outsourcing its acoustic sensors to its web, the spider is released from body size constraints and permits the araneid spider to increase its sound-sensitive surface area enormously, up to 10,000 times greater than the spider itself. The spider also enables the flexibility to functionally adjust and regularly regenerate its "external eardrum" according to its needs. The "outsourcing" and "supersizing" of auditory function in spiders provides unique features for studying extended and regenerative sensing, and designing novel acoustic flow detectors for precise fluid dynamic measurement and manipulation. SARS-CoV-2 B.1.617.2 Delta variant replication, sensitivity to neutralising antibodies and vaccine breakthrough The SARS-CoV-2 B.1.617.2 (Delta) variant was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha). In vitro, B.1.617.2 is 6-fold less sensitive to serum neutralising antibodies from recovered individuals, and 8-fold less sensitive to vaccine-elicited antibodies as compared to wild type Wuhan-1 bearing D614G. Serum neutralising titres against B.1.617.2 were lower in ChAdOx-1 versus BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies against the receptor binding domain (RBD) and N-terminal domain (NTD), in particular to the clinically approved bamlavinimab and imdevimab monoclonal antibodies. B.1.617.2 demonstrated higher replication efficiency in both airway organoid and human airway epithelial systems as compared to B.1.1.7, associated with B.1.617.2 spike being in a predominantly cleaved state compared to B.1.1.7. Additionally we observed that B.1.617.2 had higher replication and spike mediated entry as compared to B.1.617.1, potentially explaining B.1.617.2 dominance. In an analysis of over 130 SARS-CoV-2 infected healthcare workers across three centres in India during a period of mixed lineage circulation, we observed substantially reduced ChAdOx-1 vaccine efficacy against B.1.617.2 relative to non-B.1.617.2. Compromised vaccine efficacy against the highly fit and immune evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era. Nuclear alpha-synuclein is present in the human brain and is modified in dementia with Lewy bodies Dementia with Lewy bodies is pathologically defined by the cytoplasmic accumulation of alpha-synuclein within neuronal cells in the brain. Alpha-synuclein is predominately pre-synaptic, but has been reported present in various subcellular compartments in cell and animal models. In particular, nuclear alpha-synuclein is evident in-vitro and in disease models and has been associated with altered DNA integrity, gene transcription, nuclear homeostasis. However, owing to various factors, the presence of alpha-synuclein in the nuclei of human brain cells remains controversial, as does its role in synucleinopathies.

Here, we close this gap and provide a unique demonstration confirming the presence of nuclear alpha-synuclein in post-mortem brain tissue obtained from cases of dementia with Lewy bodies as well as from controls via immunohistochemistry, immunoblot, and label-free mass-spectrometry.

Discrete intra-nuclear alpha-synuclein puncta reactive against phosphorylated serine 129-alpha-synuclein and pan-alpha-synuclein antibodies were observed in cortical neurons and non-neuronal cells in fixed brain sections and in isolated nuclear preparations from Dementia with Lewy bodies cases and matched controls. Subsequent biochemical analysis of subcellular fractionated tissue confirmed alpha-synuclein as present in a nuclear fraction at levels ~ 10-fold lower than in the cytoplasm. Critically, however, an increase in monomeric nuclear alpha-synuclein phosphorylated as serine 129 was observed in cases of dementia with Lewy bodies alongside higher molecular weight pan- and phosphorylation reactive alpha-synuclein species, consistent with the formation of intranuclear phosphorylated alpha-synuclein oligomers. Furthermore, the presence of nuclear alpha-synuclein was confirmed via label free mass spectrometry, as 6 unique alpha-synuclein derived peptide sequences were identified in nuclear fractions (71.4% sequence coverage).

Collectively, our data confirm the presence of nuclear alpha-synuclein in human brain tissue and describe nuclear pathology associated with dementia with Lewy bodies. These findings address a major controversy in the synucleinopathy field by confirming the presence of nuclear alpha-synuclein in autoptic human brain tissue and, for the first time, identify that alpha-synuclein is aggregated into novel and potentially pathological assemblies in the nucleus as part of the disease process associated with dementia with Lewy bodies and thus may contribute to the disease phenotype. Omicron booster in ancestral strain vaccinated mice augments protective immunities against both the Delta and Omicron variants A booster vaccination is called for constraining the evolving epidemic of SARS-CoV-2. However, the necessity of a new COVID-19 vaccine is currently unclear. To compare the effect of an Omicron-matched S DNA vaccine and an ancestral S DNA vaccine in boosting cross-reactive immunities, we firstly immunized mice with two-dose of a DNA vaccine encoding the spike protein of the ancestral Wuhan strain. Then the mice were boosted with DNA vaccines encoding spike proteins of either the Wuhan strain or the Omicron variant. Specific antibody and T cell responses were measured at 4 weeks post boost. Our data showed that the Omicron-matched vaccine efficiently boosted RBD binding antibody and neutralizing antibody responses against both the Delta and the Omicron variants. Of note, antibody responses against the Omicron variant elicited by the Omicron-matched vaccine were much stronger than those induced by the ancestral S DNA vaccine. Meanwhile, CD8+ T cell responses against both the ancestral Wuhan strain and the Omicron strain also tended to be higher in mice boosted by the Omicron-matched vaccine than those in mice boosted with the ancestral S DNA vaccine, albeit no significant difference was observed. Our findings suggest that an Omicron-matched vaccine is preferred for boosting cross-reactive immunities. Loss of Resf1 reduces the efficiency of embryonic stem cell self-renewal and germline entry Retroelement silencing factor 1 (RESF1) interacts with the key regulators of mouse embryonic stem cells (ESCs) OCT4 and NANOG, and its absence results in sterility of mice. However, the function of RESF1 in ESCs and germ line specification is poorly understood. In this study, we used Resf1 knockout cell lines to determine the requirements of RESF1 for ESCs self-renewal and for in vitro specification of ESCs into primordial germ cell-like cells (PGCLCs). We found that deletion of Resf1 in ESCs cultured in serum and LIF reduces self-renewal potential whereas episomal expression of RESF1 has a modest positive effect on ESC self-renewal. In addition, RESF1 is not required for the capacity of NANOG and its downstream target ESRRB to drive self-renewal in the absence of LIF. However, Resf1 deletion reduces efficiency of PGCLC differentiation in vitro. These results identify Resf1 as a novel player in the regulation of pluripotent stem cells and germ cell specification. A liquid +TIP-network regulates microtubule dynamics through tubulin condensation Regulation of microtubule dynamics is essential for diverse cellular functions, and proteins that bind to dynamic microtubule ends can regulate network dynamics. Here we show, that two conserved microtubule end-binding proteins, CLIP-170 and EB3, undergo phase separation and form dense liquid-networks. When CLIP-170 and EB3 act together the multivalency of the network increases, which synergistically increases the amount of protein in the dense phase. In vitro and in cells these liquid networks can condense tubulin. In vitro in the presence of microtubules, EB3/CLIP-170 phase separation can co-condense tubulin all along the microtubule. At this condition microtubule growth speed increases up to two-fold and depolymerization events are strongly reduced, compared to conditions with phase separation deficient networks. Our data show that phase separated EB3/CLIP-170 networks impact microtubule growth dynamics beyond direct protein-microtubule interactions. Native proline-rich motifs exploit sequence context to target actin-remodeling Ena/VASP proteins The human proteome is replete with short linear motifs (SLiMs) of 4-6 residues that are critical for protein-protein interactions, yet the importance of the sequence surrounding such motifs is underexplored. We devised a proteomic screen to systematically examine the influence of SLiM sequence context on protein-protein interactions. Focusing on the EVH1 domain of ENAH, an actin regulator that is upregulated in invasive cancers, we screened 36-residue proteome-derived peptides for binding. We discovered a pocket on the ENAH EVH1 domain that diverged from its orthologs to recognize extended SLiMs, and we found that proteins with two EVH1-binding SLiMs can wrap around a single domain. We also found that the ciliary protein PCARE uses an extended 23-residue region to obtain higher affinity than any known ENAH EVH1-binding motif. Our screen provides a way to uncover the effects of broader proteomic context on motif-mediated interactions, revealing diverse mechanisms of contextual control over EVH1 interactions and establishing that SLiMs cant be fully understood outside of their native context. How voluntary and involuntary attention differentially shape spatial resolution Attention alters perception across the visual field. Typically, endogenous (voluntary) and exogenous (involuntary) attention similarly improve performance in many visual tasks, but they have differential effects in some tasks. Extant models of visual attention assume that the effects of these two types of attention are identical and consequently do not explain differences between them. Here, we develop a model of spatial resolution and attention that distinguishes between endogenous and exogenous attention. We focus on texture-based segmentation as a model system because it has revealed a clear dissociation between both attention types. For a texture for which performance peaks at parafoveal locations, endogenous attention improves performance across eccentricity, whereas exogenous attention improves performance where the resolution is low (peripheral locations) but impairs it where the resolution is high (foveal locations) for the scale of the texture. Our model emulates sensory encoding to segment figures from their background and predict behavioral performance. To explain attentional effects, endogenous and exogenous attention require separate operating regimes across visual detail (spatial frequency). Our model reproduces behavioral performance across several experiments and simultaneously resolves three unexplained phenomena: (1) the parafoveal advantage in segmentation, (2) the uniform improvements across eccentricity by endogenous attention and (3) the peripheral improvements and foveal impairments by exogenous attention. Overall, we unveil a computational dissociation between each attention type and provide a generalizable framework for predicting their effects on perception across the visual field. Using Hierarchical Similarity To Examine The Genetics of Behcet's disease Behcets disease (BD) is a multisystem inflammatory disease that affects patients along the historic silk road. Thus far, the pathogenesis of the disease has proved elusive due to the complex genetic interactions and unknown environmental or viral triggering factors of the disease. In this paper, we seek to clarify the genetic factors of the disease while also uncovering other diseases of interest that present with a similar genotype as BD. To do this, we employ a convergent functional genomics approach by leveraging the hierarchical similarity tool available in Geneweaver. Through our analysis, we were able to ascertain 7 BD consensus genes and 16 autoimmune diseases with genetic overlap with BD. The results of our study will inform further research into the pathogenesis of Behcets Disease. High school science fair: Experiences of two groups of undergraduate bioscience students Science fairs offer potential opportunities for students to learn first-hand about the practices of science. Over the past six years we have been carrying out voluntary and anonymous surveys with regional and national groups of high school and post high school students to learn about their high school science fair experiences regarding help received, obstacles encountered, and opinions about the value and impact of science fair. Understanding what students think about science fairs will help educators make science fairs more effective learning opportunities. In this paper, we focus on the findings with two national groups of post high school students - undergraduate research fellows (SURF students) who did research at UT Southwestern Medical Center during 2014-2019 and undergraduates biology students attending the 2019 Howard Hughes Medical Institute Science Education Alliance (SEA) summer symposium. About 25% of the students who completed surveys indicated that they had participated in high school science fair, but more than half attended high schools where science fairs were unavailable. Effectively, 6 out of every 10 students participated in science fair if available. Students who could have participated in high school science fair but chose not to do so identified not enough time and coming up with their project idea as major reasons why not. About half the SURF students favored requiring non-competitive science fair regardless whether they themselves had participated in science fair. On the other hand, less than 1 in 5 thought that competitive science fair should be required. Introduction to the scientific process and general learning were mentioned most frequently as the reasons to require non-competitive science fair; these reasons were mentioned rarely in connection with competitive science fair. Unlike the national cohort of high school students we surveyed previously, who mostly did science fair in 9th and 10th grades, SURF students participated in science fair throughout high school and were twice as likely as high school students to have carried out science fair more than once. In conclusion, our findings suggest that participation of the undergraduate bioscience majors in high school science fairs occurs far more frequently than recognized previously. The findings emphasize further the importance of incentivizing rather than requiring science fair participation, especially in 9th and 10th grades, and the potential value of developing non-competitive science fairs. Heat-induced transposition increases drought tolerance in Arabidopsis Eukaryotic genomes contain a vast diversity of transposable elements (TEs). Formerly often described as selfish and parasitic DNA sequences, TEs are now recognized as a source of genetic diversity and powerful drivers of evolution. Yet, because their mobility is tightly controlled by the host, studies experimentally assessing how fast TEs may mediate the emergence of adaptive traits are scare. Here, we show that the heat-induced transposition of a low-copy TE increases phenotypic diversity and leads to the emergence of drought-tolerant individuals in Arabidopsis thaliana. We exposed high-copy TE lines (hcLines) with up to [~]8 fold increased copy numbers of the heat-responsive ONSEN TE (AtCOPIA78) to drought as a straightforward and ecologically highly relevant selection pressure. We provide evidence for increased drought tolerance in five out of the 23 tested hcLines and further pinpoint one of the causative mutations to an exonic insertion of ONSEN in the ribose-5-phosphate-isomerase 2 gene. The resulting loss-of-function mutation caused a decreased rate of photosynthesis and water consumption. This is one of the rare empirical examples substantiating the adaptive potential of mobilized stress-responsive TEs in eukaryotes. Our work sheds light on the relationship between TEs and their hosts and demonstrates the importance of TE-mediated loss-of-function mutations in stress adaptation, particularly in the face of global warming. Isotopically Nonstationary 13C Metabolic Flux Analysis in Resting and Activated Human Platelets Platelet metabolism is linked to platelet hyper- and hypoactivity in numerous human diseases. Developing a detailed understanding of the link between metabolic shifts and platelet activation state is integral to improving human health. Here, we show the first application of isotopically nonstationary 13C metabolic flux analysis to quantitatively measure carbon fluxes in both resting and thrombin activated platelets. Resting platelets primarily metabolize glucose to lactate via glycolysis, while acetate is oxidized to fuel the tricarboxylic acid cycle. Upon activation with thrombin, a potent platelet agonist, platelets increase their uptake of glucose 3-fold. This results in an absolute increase in flux throughout central metabolism, but when compared to resting platelets they redistribute carbon dramatically. Activated platelets decrease relative flux to the oxidative pentose phosphate pathway and TCA cycle from glucose and increase relative flux to lactate. These results provide the first report of reaction-level carbon fluxes in platelets and allow us to distinguish metabolic fluxes with much higher resolution than previous studies. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly.