title: Trade-off between stimulation focality and the number of coils in multi-locus transcranial magnetic stimulation

abstract:
ObjectiveCoils designed for transcranial magnetic stimulation (TMS) must incorporate trade-offs between the required electrical power or energy, focality and depth penetration of the induced electric field (E-field), coil size, and mechanical properties of the coil, as all of them cannot be optimally met at the same time. In multi-locus TMS (mTMS), a transducer consisting of several coils allows electronically targeted stimulation of the cortex without physically moving a coil. In this study, we aimed to investigate the relationship between the number of coils in an mTMS transducer, the focality of the induced E-field, and the extent of the cortical region within which the location and orientation of the maximum of the induced E-field can be controlled.

ApproachWe applied convex optimization to design planar and spherically curved mTMS transducers of different E-field focalities and analyzed their properties. We characterized the trade-off between the focality of the induced E-field and the extent of the cortical region that can be stimulated with an mTMS transducer with a given number of coils.

Main resultsAt the expense of the E-field focality, one can, with the same number of coils, design an mTMS transducer that can control the location and orientation of the peak of the induced E-field within a wider cortical region.

SignificanceWith E-fields of moderate focality, the problem of electronically targeted TMS becomes considerably easier compared with highly focal E-fields; this may speed up the development of mTMS and the emergence of new clinical and research applications.


title: Innate and adaptive immunity to transfused allogenic red blood cells in mice requires MyD88

abstract: Red blood cell (RBC) transfusion therapy is essential for the survival of patients with hematological disorders such as sickle cell anemia. A potentially fatal complication of transfusion is development of non-ABO alloantibodies to polymorphic RBC antigens, yet mechanisms of alloantibody formation remain unclear. Human and mouse RBCs acquire a "storage lesion" prior to transfusion, which in mice contributes to immunogenicity. We previously reported that mouse splenic dendritic cells (DCs) are required for RBC alloimmunization and are activated by sterile and leukoreduced mouse RBCs after storage. Yet how syngeneic RBCs activate innate immune pathways to induce DC activation is unknown. We now show that DC activation to transfused RBCs occurs regardless of alloantigen presence, suggesting that RBC damage induced during storage triggers innate immune receptors. We discovered an unexpected dependence of RBC alloimmunization on the Toll-like receptor (TLR) signaling adaptor molecule MyD88. TLRs are a class of pattern recognition receptors (PRRs) that regulate DC activation and signal through two adaptor molecules, MyD88 and TRIF. We show that the inflammatory cytokine response, DC activation, and the subsequent alloantibody response to transfused syngeneic RBCs require MyD88 but not TRIF, suggesting a restricted set of PRRs are responsible for sensing RBCs and triggering alloimmunization.

category: immunology


title: Directing an mRNA-LNP vaccine toward lymph nodes improves humoral and cellular immunity against SARS-CoV-2

abstract: The exploration and identification of safe and effective vaccines for the SARS-CoV-2 pandemic has captured the worlds attention and remains an ongoing issue in order to protect against emerging variants of concern (VoCs) while generating long lasting immunity. Here, we report the synthesis of a novel messenger ribonucleic acid (mRNA) encoding the spike protein in a lipid nanoparticle formulation (LNP) (STI-7264) that generates robust humoral and cellular immunity following immunization of C57Bl6 mice. In efforts to continually improve immunity, a lymphatic drug delivery device (MuVaxx) was engineered and tested to modulate immune cells at the injection site (epidermis and dermis) and draining lymph node (LN) to elicit adaptive immunity. Using MuVaxx, immune responses were elicited and maintained at a 10-fold dose reduction compared to traditional intramuscular (IM) administration as measured by anti-spike antibodies, cytokine producing CD8 T cells, and neutralizing antibodies against the Washington (Wild Type, WT) and South African (beta) variants. Remarkably, a 4-fold elevated T cell response was observed in MuVaxx administered vaccination as compared to that of IM administered vaccination. Thus, these data support further investigation into STI-7264 and lymphatic mediated delivery using MuVaxx for SARS-CoV-2 and VoCs vaccines.


title: A dynamic rhizosphere interplay between tree roots and soil bacteria under drought

abstract:
O_LIRoot exudates are thought to play an important role in plant-microbial interactions. In return for nutrition, soil bacteria can increase the bioavailability of soil minerals. However, root exudates typically decrease in situations such as drought, calling into question the efficacy of bacteria-dependent mineral uptake in such stress.
C_LIO_LIHere we tested the hypothesis of exudate-driven microbial priming on Cupressus saplings grown in forest soil in custom-made rhizotron boxes. A 1-month imposed drought and concomitant inoculations with Bacillus subtilis and Pseudomonas stutzeri, bacteria species isolated from the forest soil, were applied using factorial design.
C_LIO_LIDirect bacteria counts and visualization by confocal microscopy showed that both bacteria associated with Cupressus roots. Interestingly, root exudation rates increased with bacteria under drought. Forty four metabolites in exudates were significantly different in concentration between irrigated and drought trees, including phenolic acid compounds and quinate, that were shown to be used as carbon and nitrogen sources by both bacterial species. Importantly, soil phosphorous bioavailability was maintained only in inoculated trees, mitigating drought-induced decrease in leaf phosphorus and iron.
C_LIO_LIOur observations of increased root exudation rate when drought and inoculation regimes were combined, support the idea of root recruitment of beneficial bacteria.
C_LI

category: ecology


title: Assembly history modulates vertical root distribution in a grassland experiment

abstract: The order of arrival of plant species during assembly can affect the structure and functioning of grassland communities. These so-called priority effects have been extensively studied aboveground, but we still do not know how they affect the vertical distribution of roots in the soil and the rooting depth of plant communities. To test this hypothesis, we manipulated the order of arrival of three plant functional groups (forbs, grasses and legumes) in a rhizobox experiment. Priority effects were created by sowing one functional group 10 days before the other two. Rhizoboxes in which all functional groups were sown simultaneously were used as controls. During the experiment, the mean rooting depth of plant communities was monitored using image analysis and a new methodological approach using deep learning (RootPainter) for root segmentation. At harvest, we measured aboveground (community and species level) and belowground (community level) biomass, and assessed the vertical distribution of the root biomass in different soil layers. At the community level, all scenarios where one functional group was sown before the other two had similar shoot and root productivity. At the species level, two forbs (Achillea millefolium and Centaurea jacea) benefited from arriving early, and one legume (Trifolium pratense) had a disadvantage when it was sown after the grasses. Priority effect treatments also affected the vertical distribution of roots. When grasses were sown first, plant communities rooted more shallowly than when forbs or legumes were sown first. In addition, roots moved down the soil profile 24% more slowly in grasses-first communities. Our results highlight that plant functional group order of arrival in grassland communities can affect the vertical distribution of roots in the soil and this may have implications for species coexistence.


title: Differential proteomic analysis of laser-microdissected penetration glands of avian schistosome cercariae with a focus on proteins involved in host invasion

abstract:
Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula migrating to final localization. During invasion, cercariae employ histolytic and other bioactive products of specialized holocrine secretory cells - postacetabular (PA) and circumacetabular (CA) penetration glands. Although several studies attempted to characterize protein composition of the in vitro induced gland secretions in Schistosoma mansoni and Schistosoma japonicum, the results were inconsistent and dependent on the method of sample collection and processing. Products of both gland types mixed during their secretion did not allow localization of identified proteins to a particular gland. Here we compared proteomes of separately isolated cercarial gland cells of the avian schistosome Trichobilharzia szidati employing laser-assisted microdissection and shotgun LC-MS/MS, thus obtaining the largest dataset so far concerning the representation and localization of cercarial penetration gland proteins. We optimized the methods of sample processing with cercarial bodies (heads) first. Alizarin-pre-stained, chemically non-fixed samples provided optimal results of MS analyses, and enabled distinguishing PA and CA glands for microdissection. Using 7.5 x 106 m3 sample volume per gland replicate, we identified 3347 peptides assigned to 792 proteins, from which 461 occurred in at least 2 of 3 replicates in either gland type (PA = 455, 40 exclusives; CA = 421, 6 exclusives; 60 proteins differed significantly in their abundance between the glands). Peptidases of 5 catalytic types accounted for ca. 8 % and 6 % of reliably identified proteins in PA and CA glands, respectively. Invadolysin, nardilysin, cathepsins B2 and L3, and elastase 2b orthologs were the major gland endopeptidases. Two cystatins and a serpin were highly abundant peptidase inhibitors in the glands. CA glands were rich in venom allergen-like proteins. The assembled total cercarial body proteome included 1631 identified proteins and revealed additional interesting factors possibly related to tissue invasion.

HighlightsO_LIProteomes of two penetration gland types in schistosome cercariae greatly differ
C_LIO_LIPostacetabular glands possess 40 unique proteins and are abundant in hydrolases
C_LIO_LICircumacetabular glands posses 6 unique proteins and are rich in VAL proteins
C_LIO_LIPeptidases make up 8 % of postacetabular and 6 % of circumacetabular gland proteins
C_LIO_LICercarial elastase is unique to circumacetabular glands of Trichobilharzia szidati
C_LI

Note: Supplementary data associated with this article All supplementary data files can be accessed from the following link: http://www.helminthology.cz/supplementary_files.html

category: bioinformatics


title: High-grade Ovarian Cancer Associated H/ACA snoRNAs Promote Cancer Cell Proliferation and Survival

abstract: Small nucleolar RNAs (snoRNAs) are an omnipresent class of non-coding RNAs involved in the modification and processing of ribosomal RNA (rRNA). As snoRNAs are required for ribosome production, the increase of which is a hallmark of cancer development, their expression would be expected to increase in proliferating cancer cells. However, the nature and extent of snoRNAs contribution to the biology of cancer cells remain largely unexplored. In this study, we examined the abundance patterns of snoRNA in high-grade serous ovarian carcinomas (HGSC) and serous borderline tumours (SBT) and identified a subset of snoRNA associated with increased invasiveness. This subgroup of snoRNA accurately discriminates between SBT and HGSC underlining their potential as biomarkers of tumour aggressiveness. Remarkably, knockdown of HGSC-associated H/ACA snoRNAs, but not their host genes, inhibits cell proliferation and induces apoptosis of model ovarian cancer cell lines. Wound healing and cell migration assays confirmed the requirement of these HGSC-associated snoRNA for cell invasion and increased tumour aggressiveness. Together our data indicate that H/ACA snoRNAs promote tumour aggressiveness through the induction of cell proliferation and resistance to apoptosis.

category: cancer biology


title: Metformin Confers Cardiac and Renal Protection in Sudden Cardiac Arrest via AMPK Activation

abstract:
Sudden cardiac arrest (SCA) affects over 600,000 individuals annually in the United States and is associated with substantial mortality. After resuscitation, multi-system organ damage is common and largely attributable to ischemia-reperfusion injury. The anti-diabetic drug metformin improves cardiac outcomes in models of myocardial ischemia and ischemia-reperfusion. In this study, we evaluated the role of metformin pretreatment in a mouse model of SCA. We found that two weeks of metformin pretreatment protects cardiac ejection fraction and reduces acute kidney injury post-SCA in non-diabetic mice. Further, metformin pretreatment prior to SCA activates AMPK signaling and is associated with altered mitochondrial dynamics and markers of autophagy following arrest. Direct AMPK activation and inhibition studies demonstrate that activation is necessary and sufficient for metformin-mediated protection of cardiac and renal tissues in this model. We were unable to demonstrate cardiac improvement with a single-dose metformin rescue therapy. Importantly, the protective findings translate into patients. We retrospectively evaluated the extent of cardiac and kidney damage in diabetic patients resuscitated from SCA. Metformin-treated patients have less evidence of heart and kidney damage after arrest than diabetics who have not received metformin. Together, these data support AMPK activation as a preventive mechanism in ischemia-reperfusion injury.

Graphical Abstract

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category: physiology


title: Unique spatiotemporal fMRI dynamics in the awake mouse brain

abstract: Human imaging studies have shown that spontaneous brain activity exhibits stereotypic spatiotemporal reorganization in awake, conscious conditions with respect to minimally conscious states. However, whether and how this phenomenon can be generalized to lower mammalian species, remains unclear. Leveraging a robust protocol for resting-state fMRI (rsfMRI) mapping in non-anesthetized, head-fixed mice, we investigated functional network topography and dynamic structure of spontaneous brain activity in wakeful animals. We found that rsfMRI networks in the awake state, while anatomically comparable to those observed under anesthesia, are topologically configured to maximize interregional communication, departing from the underlying community structure of the mouse axonal connectome. We further report that rsfMRI activity in wakeful animals exhibits unique spatiotemporal dynamics characterized by a state-dependent, dominant occurrence of coactivation patterns encompassing a prominent participation of arousal-related forebrain nuclei, and functional anti-coordination between visual-auditory and polymodal cortical areas. We finally show that rsfMRI dynamics in awake mice exhibits a stereotypical temporal structure, in which state-dominant coactivation patterns are configured as network attractors. These findings suggest that spontaneous brain activity in awake mice is critically shaped by state-specific involvement of basal forebrain arousal systems, and document that its dynamic structure recapitulates distinctive, evolutionarily-relevant principles that are predictive of conscious states in higher mammalian species.

category: neuroscience


title: Stochastic Consolidation of Lifelong Memory

abstract: Humans have the remarkable ability to continually store new memories, while maintaining old memories for a lifetime. How the brain avoids catastrophic forgetting of memories due to interference between encoded memories is an open problem in computational neuroscience. Here we present a model for continual learning in a recurrent neural network combining Hebbian learning, synaptic decay and a novel memory consolidation mechanism. Memories undergo stochastic rehearsals with rates proportional to the memorys basin of attraction, causing self-amplified consolidation, giving rise to memory lifetimes that extend much longer than synaptic decay time, and capacity proportional to a power of the number of neurons. Perturbations to the circuit model cause temporally-graded retrograde and anterograde deficits, mimicking observed memory impairments following neurological trauma.


title: Glycation modulates glutamatergic signalling and exacerbates Parkinson's disease-like phenotypes

abstract: Alpha-synuclein (aSyn) is assumed to be a central player in the pathogenesis of synucleinopathies due to its accumulation in typical protein aggregates in the brain. However, it is still unclear how it contributes to neurodegeneration. Type-2 diabetes mellitus is a risk factor for Parkinsons disease and, one common molecular alteration among these disorders is an age-associated increase in protein glycation. Thus, we hypothesized that glycation-induced dysfunction of neuronal pathways might be an underlying molecular cause of synucleinopathies. Here, we evaluated if increased brain glycation modulated motor and/or non-motor phenotypes in a mouse model of synucleinopathies. In addition, we dissected the specific impact of methylglyoxal (MGO, a glycating agent) in mice overexpressing aSyn in the brain, and unveiled the major molecular pathways altered. Age-matched (16 weeks old) male aSyn transgenic (Thy1-aSyn) or WT mice received a single dose of MGO or vehicle via intracerebroventricular (ICV) injection. Behavioural phenotypes were analysed 4 weeks post-treatment, and, at the end of the tests, biochemical and histological studies were conducted on brain tissue. We found that glycation potentiates motor dysfunction, assessed by vertical pole, rotarod and hindlimb clasping tests in Thy1-aSyn mice. In addition, it induces cognitive impairment (Y maze test), olfactory disturbances (block test), and colonic dysfunction. These behavioural changes were accompanied by the accumulation of aSyn in the midbrain, striatum, and prefrontal cortex, and by an overall increase in glycation in the midbrain and cerebellum. Furthermore, MGO induced neuronal and dopaminergic cell loss in the midbrain of Thy1-aSyn mice. Quantitative proteomic analysis revealed that, in Thy1-aSyn mice, MGO mainly impacts on glutamatergic proteins in the midbrain, but not in the prefrontal cortex, where it mainly affects the electron transport chain. Among the altered proteins in the midbrain, we found an upregulation of N-Methyl-D-Aspartate (NMDA) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors levels, glutaminase, vesicle glutamate transporter (VGLUT), and the excitatory amino acid transporter (EAAT1), suggesting potentiation of glutamatergic signalling. Overall, we demonstrated that MGO-induced glycation accelerates Parkinsonian-like sensorimotor and cognitive alterations. The increase in glutamatergic-related proteins in the midbrain may represent a compensatory mechanism to the MGO-induced dopaminergic neurodegeneration. Our study sheds light into the enhanced vulnerability of the midbrain in Parkinsons disease-related synaptic dysfunction that, ultimately leads to cell loss, and provides molecular insight into the observation that glycation suppressors and anti-glutamatergic drugs hold promise as disease-modifying therapies for synucleinopathies.


title: Woodsmoke particulates alter expression of antiviral host response genes in human nasal epithelial cells infected with SARS-CoV-2 in a sex-dependent manner

abstract: We have previously shown that exposure to particulate air pollution, both from natural and anthropogenic sources, alters gene expression in the airways and increases susceptibility to respiratory viral infection. Additionally, we have shown that woodsmoke particulates (WSP) affect responses to influenza in a sex-dependent manner. In the present study, we used human nasal epithelial cells (hNECs) from both sexes to investigate how particulate exposure could modulate gene expression in the context of SARS-CoV-2 infection. We used diesel exhaust particulate (DEP) as well as WSP derived from eucalyptus or red oak wood. HNECs were exposed to particulates at a concentration of 22 g/cm2 for 2 h then immediately infected with SARS-CoV-2 at a MOI (multiplicity of infection) of 0.5. Exposure to particulates had no significant effects on viral load recovered from infected cells. Without particulate exposure, hNECs from both sexes displayed a robust upregulation of antiviral host response genes, though the response was greater in males. However, WSP exposure before infection dampened expression of genes related to the antiviral host response by 72 h post infection. Specifically, red oak WSP downregulated IFIT1, IFITM3, IFNB1, MX1, CCL3, CCL5, CXCL11, CXCL10, and DDX58, among others. After sex stratification of these results, we found that exposure to WSP prior to SARS-CoV-2 infection downregulated anti-viral gene expression in hNECs from females more so than males. These data indicate that WSP, specifically from red oak, alter virus-induced gene expression in a sex-dependent manner and potentially suppress antiviral host defense responses following SARS-CoV-2 infection.

category: pharmacology and toxicology


title: Nucleosome positioning on large tandem DNA repeats of the '601' sequence engineered in Saccharomyces cerevisiae

abstract: The so-called 601 DNA sequence is often used to constrain the position of nucleosomes on a DNA molecule in vitro. Although the ability of the 147 base pair sequence to precisely position a nucleosome in vitro is well documented, in vivo application of this property has been explored only in a few studies and yielded contradictory conclusions. Our goal in the present study was to test the ability of the 601 sequence to dictate nucleosome positioning in Saccharomyces cerevisiae in the context of a long tandem repeat array inserted in a yeast chromosome. We engineered such arrays with three different repeat size, namely 167, 197 and 237 base pairs. Although our arrays are able to position nucleosomes in vitro as expected, analysis of nucleosome occupancy on these arrays in vivo revealed that nucleosomes are not preferentially positioned as expected on the 601-core sequence along the repeats and that the measured nucleosome repeat length does not correspond to the one expected by design. Altogether our results demonstrate that the rules defining nucleosome positions on this DNA sequence in vitro are not valid in vivo, at least in this chromosomal context, questioning the relevance of using the 601 sequence in vivo to achieve precise nucleosome positioning on designer synthetic DNA sequences.

category: synthetic biology


title: The Histone H1-like protein AlgP facilitates even spacing of polyphosphate granules in Pseudomonas aeruginosa

abstract:
Synthesis of polyphosphate (polyP) is an ancient and universal stress and starvation response in bacteria. In many bacteria, polyP chains come together to form granular superstructures within cells. Some species appear to regulate polyP granule subcellular organization. Despite the critical role of polyP in starvation fitness, the composition of these structures, mechanism(s) underpinning their organization, and functional significance of such organization are poorly understood. We previously determined that granules become transiently evenly spaced on the cells long axis during nitrogen starvation in the opportunistic human pathogen Pseudomonas aeruginosa. Here, we developed a granule-enrichment protocol to screen for polyP granule-localizing proteins. We identified AlgP as a protein that associates with polyP granules. We further discovered that AlgP is required for the even spacing of polyP granules. AlgP is a DNA-binding protein with a 154 amino acid C-terminal domain enriched in  KPAA repeats and variants of this repeat, with an overall sequence composition similar to the C-terminal tail of eukaryotic histone H1. Granule size, number, and spacing are significantly perturbed in the absence of AlgP, or when AlgP is truncated to remove the C-terminus. The {Delta}algP and algP{Delta}CTD mutants having fewer, larger granules. We speculate that AlgP may contribute to spacing by tethering polyP granules to the chromosome, thereby inhibiting fusion with neighboring granules. Our discovery that AlgP facilitates granule spacing allows us for the first time to directly uncouple granule biogenesis from even spacing, and will inform future efforts to explore the functional significance of granule organization on fitness during starvation.

IMPORTANCEThe mechanisms underpinning polyPs pleiotropic effects on bacterial starvation physiology remain elusive. This simple polyanions lack of protein binding specificity has impeded validation of bona fide polyP-binding proteins. However, polyP forms granule superstructures with spatial specificity. Our granule enrichment protocol identified a polyP granule-associated protein in Pseudomonas aeruginosa, AlgP. AlgP was originally reported as regulator of alginate, an extracellular polysaccharide important in biofilm formation, including in cystic fibrosis (CF) chronic infections. AlgPs putative role in alginate biosynthesis has recently been called into question. We establish a distinct, previously unknown function for AlgP in modulating the subcellular organization of polyP, another polymer important for pathogenesis. In CF clinical isolates, the C-terminal repeat domain of AlgP is a hotspot for genetic rearrangements. Our finding that the C-terminus of AlgP is required for granule organization lays the groundwork for exploring the functional significance of these mutations in the evolutionary trajectory of chronic infections.

category: microbiology


title: A universal microfluidic approach for quantitative study of bacterial biofilms

abstract: Bacteria usually live in densely packed communities called biofilms, where interactions between the bacteria give rise to complex properties. Quantitative analysis is indispensable in understanding those properties. However, current biofilm culturing approaches impose various limitations to these types of analysis. Here, we developed a microfluidic approach for quantitative study of biofilms, which is universal and can be used to culture biofilms of various bacterial species. To demonstrate the advantages of this approach, we present two examples, both of which revealed new biological insights. In the first example, we explored the response of Escherichia coli biofilms to exogenous hydrogen peroxide; We found the biofilms gained resistance to H2O2, but their growth was slowed down due to the metabolic cost of maintaining the resistance; However, under oxygen limitation, H2O2 can anti-intuitively boost biofilm growth. In the second example, we explored resource retention by Pseudomonas aeruginosa biofilms; We observed a fluorescent substance within the biofilm and identified it as the siderophore pyoverdine; We further showed that the extracellular matrix component Psl acted as a retention barrier for pyoverdine, minimizing its loss into the environment and therefore potentially promoting sharing of pyoverdine within the biofilm.


title: Prochlorococcus exudate stimulates heterotrophic bacterial competition with rival phytoplankton for available nitrogen

abstract:
The marine cyanobacterium Prochlorococcus numerically dominates the phytoplankton community of the nutrient-limited open ocean, establishing itself as the most abundant photosynthetic organism on Earth. This ecological success has been attributed to lower cell quotas for limiting nutrients, superior resource acquisition, and other advantages associated with cell size reduction and genome streamlining. In this study we tested the prediction that Prochlorococcus outcompetes its rivals for scarce nutrients, and that this advantage leads to its numerical success in nutrient-limited waters. Strains of Prochlorococcus and its sister genus Synechococcus grew well in both mono- and co-culture when nutrients were replete. However, in nitrogen-limited medium Prochlorococcus outgrew Synechococcus, but only when heterotrophic bacteria were also present. In the nitrogen-limited medium, the heterotroph Alteromonas macleodii outcompeted Synechococcus for nitrogen, but only if stimulated by exudate released by Prochlorococcus, or if a proxy organic carbon source was provided. Analysis of a nitrate reductase mutant Alteromonas suggested that Alteromonas outcompetes Synechococcus for nitrate, during which co-cultured Prochlorococcus grows on ammonia or other available nitrogen species. We propose that Prochlorococcus can stimulate antagonism between heterotrophic bacteria and potential phytoplankton competitors through a metabolic cross-feeding interaction, and this stimulation could contribute to the numerical success of Prochlorococcus in the nutrient-limited regions of the ocean.

Significance StatementIn nutrient-poor habitats, the competition for limited resources is thought to select for organisms with enhanced ability to scavenge nutrients and utilize them efficiently. Such adaptations characterize the cyanobacterium Prochlorococcus, the most abundant photosynthetic organism in the nutrient-limited open ocean. In this study the competitive superiority of Prochlorococcus over a rival cyanobacterium, Synechococcus, was captured in laboratory culture. Critically, this outcome was achieved only when key aspects of the open ocean were simulated: a limited supply of nitrogen, and the presence of heterotrophic bacteria. Results indicate that Prochlorococcus promotes its numerical dominance over Synechococcus by energizing the heterotrophs ability to outcompete Synechococcus for available nitrogen. This study demonstrates how interactions between trophic groups can influence interactions within trophic groups.


title: Conformation of HIV-1 Envelope governs rhesus CD4 usage and simian-human immunodeficiency virus replication

abstract:
Infection of rhesus macaques with simian-human immunodeficiency viruses (SHIVs) is the preferred model system for vaccine development because SHIVs encode HIV-1 envelope glycoproteins (Env) - a key target of HIV-1 neutralizing antibodies. Since the goal of vaccines is to prevent new infections, SHIVs encoding circulating HIV-1 Env are desired as challenge viruses. Development of such biologically relevant SHIVs has been challenging as they fail to infect rhesus macaques, mainly because most circulating HIV-1 Env do not use rhesus CD4 (rhCD4) receptor for viral entry. Most primary HIV-1 Env exist in a closed conformation and occasionally transit to downstream, open conformation through an obligate intermediate conformation. Here, we provide genetic evidence that open Env conformations can overcome the rhCD4 entry barrier and increase replication of SHIVs in rhesus lymphocytes. Consistent with prior studies, we found that circulating HIV-1 Env do not use rhCD4 efficiently for viral entry. However, using HIV-1 Env with single amino acid substitutions that alter their conformational state, we found that transitions to intermediate and open Env conformation allow usage of physiological levels of rhCD4 for viral entry. We engineered these single amino acid substitutions in the transmitted/founder HIV-1BG505 Env encoded by SHIV-BG505 and found that open Env conformation enhances SHIV replication in rhesus lymphocytes. Lastly, CD4-mediated SHIV pull-down, sensitivity to soluble CD4, and fusogenicity assays indicated that open Env conformation promotes efficient rhCD4 binding and viral-host membrane fusion. These findings identify conformational state of HIV-1 Env as a major determinant for rhCD4 usage, viral fusion, and SHIV replication.

ImportanceRhesus macaques are critical animal model for preclinical testing of HIV-1 vaccine and prevention approaches. However, HIV-1 does not replicate in rhesus macaques, and thus chimeric simian-human immunodeficiency viruses (SHIVs), which encode HIV-1 envelope glycoproteins, are used as surrogate challenge viruses to infect rhesus macaques for modeling HIV-1 infection. Development of SHIVs encoding envelope from clinically relevant, circulating HIV-1 variants has been extremely challenging as such SHIVs replicate poorly, if at all, in rhesus lymphocytes. This is because most circulating HIV-1 envelope do not use rhesus CD4 efficiently for viral entry. In this study, we identify conformational state of HIV-1 envelope as a key determinant for rhesus CD4 usage, viral-host membrane fusion, and SHIV replication in rhesus lymphocytes.


title: Repair of APOBEC3G-mutated retroviral DNA in vivo is facilitated by the host enzyme uracil DNA glycosylase 2

abstract:
Apolipoprotein B mRNA Editing Enzyme Catalytic Subunit 3 (APOBEC3) proteins are critical for the control of infection by retroviruses. These proteins deaminate cytidines in negative strand DNA during reverse transcription, leading to G to A changes in coding strands. Uracil DNA glycosylase (UNG) is a host enzyme that excises uracils in genomic DNA, which the base excision repair machinery then repairs. Whether UNG removes uracils found in retroviral DNA after APOBEC3-mediated mutation is not clear, and whether this occurs in vivo has not been demonstrated. To determine if UNG plays a role in the repair of retroviral DNA, we used APOBEC3G (A3G) transgenic mice which we showed previously had extensive deamination of murine leukemia virus (MLV) proviruses. The A3G transgene was crossed onto an UNG and mouse APOBEC3 knockout background (UNG-/-APO-/-) and the mice were infected with MLV. We found that virus infection levels were decreased in A3G UNG-/-APO-/- compared to A3G APO-/- mice. Deep sequencing of the proviruses showed that there were significantly higher levels of G-to-A mutations in proviral DNA from A3G transgenic UNG-/-APO-/- than A3G transgenic APO-/- mice, suggesting that UNG plays a role in the repair of uracil-containing proviruses. In in vitro studies, we found that cytoplasmic viral DNA deaminated by APOBEC3G was uracilated. In the absence of UNG, the uracil-containing proviruses integrated at higher levels into the genome than did those made in the presence of UNG. Thus, UNG also functions in the nucleus prior to integration by nicking uracil-containing viral DNA, thereby blocking integration. These data show that UNG plays a critical role in the repair of the damage inflicted by APOBEC3 deamination of reverse-transcribed DNA.

ImportanceWhile APOBEC3-mediated mutation of retroviruses is well-established, what role the host base excision repair enzymes play in correcting these mutations is not clear. This question is especially difficult to address in vivo. Here, we use a transgenic mouse developed by our lab that expresses human APOBEC3G and also lacks the endogenous uracil DNA glycosylase (Ung) gene, and show that UNG removes uracils introduced by this cytidine deaminase in MLV reverse transcripts, thereby reducing G-to-A mutations in proviruses. Furthermore, our data suggest that UNG removes uracils at two stages in infection - in unintegrated nuclear viral reverse transcribed DNA, resulting in its degradation and second, in integrated proviruses, resulting in their repair. These data suggest that retroviruses damaged by host cytidine deaminases take advantage of the host DNA repair system to overcome this damage.


title: Bacterial keratitis: similar bacterial and clinical outcomes in female versus male New Zealand White rabbits infected with Serratia marcescens

abstract: Females and males respond differently to a number of systemic viral infections. Differences between females and males with respect to the severity of keratitis caused by Gram-negative bacteria such as Serratia marcescens are less well established. In this study we injected female and male New Zealand White rabbit corneas with a keratitis isolate of S. marcescens and evaluated the eyes after 48 hours for a number of clinical and microbiological parameters. No statistical differences in bacterial burden and corneal scores were recorded between female and male rabbits although there was a non-significant trend toward a higher frequency of female rabbits demonstrating hypopyons. This data suggests that for experimental bacterial keratitis studies involving Gram-negative rods, a single sex or mixed group of rabbit is sufficient for evaluating pathology and bacterial burdens. This will reduce the number of animals used for subsequent studies.


title: Rhomboid protease RHBDL4/RHBDD1 cleaves SREBP-1c at ER monitoring and regulating fatty acids

abstract:
The ER-embedded transcription factors, sterol-regulatory element-binding proteins (SREBPs), master regulators of lipid biosynthesis, are transported to Golgi for proteolytic activation to tune cellular cholesterol levels and regulate lipogenesis. However, mechanisms by which the cell responds to the levels of saturated or unsaturated fatty acids remain underexplored. Here we show that RHBDL4/RHBDD1, a rhomboid family protease, directly cleaves SREBP-1c at ER. The p97/VCP, AAA-ATPase complex then acts as an auxiliary segregase to extract the remaining ER-embedded fragment of SREBP-1c. Importantly, the enzymatic activity of RHBDL4 is enhanced by saturated fatty acids (SFAs), but inhibited by polyunsaturated fatty acids (PUFAs). Genetic deletion of RHBDL4 in mice fed on a Western diet enriched in SFAs and cholesterol prevented SREBP-1c from inducing genes for lipogenesis, particularly for synthesis and incorporation of PUFAs, and secretion of lipoproteins. The RHBDL4-SREBP-1c pathway reveals a regulatory system for monitoring fatty acid composition and maintaining cellular lipid homeostasis.

Graphical Abstract

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category: molecular biology


title: Yeast-expressed Recombinant SARS-CoV-2 Receptor Binding Domain, RBD203-N1 as a COVID-19 Protein Vaccine Candidate

abstract:
BackgroundSARS-CoV-2 protein subunit vaccines are being evaluated by multiple manufacturers to fill the need for low-cost, easy to scale, safe, and effective COVID-19 vaccines for global access. Vaccine candidates relying on the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein have been the focus of our development program. In this paper, we report on the generation of the RBD203-N1 yeast expression construct, which produces a recombinant protein that when formulated with alum and the TLR-9 agonist, CpG1826 elicits a robust immune response and protection in mice.

MethodThe RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity and efficacy were evaluated in mice.

Results, Conclusions, and SignificanceThe RBD203-N1 production process yielded 492.9 {+/-} 3.0 mg/L of protein in the fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 {+/-} 3% (total yield of purified protein: 270.5 {+/-} 13.2 mg/L fermentation supernatant). The protein was characterized as a homogeneous monomer with well-defined secondary structure, thermally stable, antigenic, and when adjuvanted on alum and CpG, it was immunogenic and induced robust levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. These characteristics show that this vaccine candidate is well suited for technology transfer with feasibility of its transition into the clinic to evaluate its immunogenicity and safety in humans.


title: Standard metabolic rate does not associate with age-at-maturity genotype in juvenile Atlantic salmon

abstract:
Atlantic salmon (Salmo salar) is a species with diverse life-history strategies, to which the timing of maturation contributes considerably. Recently, the genome region including the gene vgll3 has gained attention as a locus with a large effect on salmon maturation timing, and recent studies on the vgll3 locus in salmon have indicated that its effect might be mediated through body condition and accumulation of adipose tissue. However, the cellular and physiological pathways leading from vgll3 genotype to phenotype are still unknown. Standard metabolic rate is a potentially important trait for resource acquisition and assimilation and we hypothesized that this trait, being a proxy for the maintenance energy expenditure of an individual, could be an important link in the pathway from vgll3 genotype to maturation-timing phenotype. As a first step to studying links between vgll3 and the metabolic phenotype of Atlantic salmon, we measured the standard metabolic rate of 150 first year Atlantic salmon juveniles of both sexes, originating from 14 different families with either late maturing or early maturing vgll3 genotypes. No significant difference in mass-adjusted standard metabolic rate was detected between individuals with different vgll3 genotypes, indicating that juvenile salmon of different vgll3 genotypes have similar maintenance energy requirements in the experimental conditions used and that the effects of vgll3 on body condition and maturation are not strongly related to maintenance energy expenditure in either sex at this life stage.

Summary statementWe show that vgll3, a gene known to have significant effects on Atlantic salmon (Salmo salar) life-history strategy, does not associate with standard metabolic rate in salmon juveniles.

category: genetics


title: Circulation of third-generation cephalosporin resistant Salmonella Typhi in Mumbai, India

abstract: We report the persistent circulation of third-generation cephalosporin resistant Salmonella Typhi in Mumbai, linked to the acquisition and maintenance of a previously characterized IncX3 plasmid carrying the ESBL gene blaSHV-12 and the fluoroquinolone resistance gene qnrB7 in the genetic context of a triple mutant also associated with fluoroquinolone resistance.

category: genomics


title: CST does not evict elongating telomerase but prevents initiation by ssDNA binding

abstract: The CST complex (CTC1-STN1-TEN1) has been shown to inhibit telomerase extension of the G-strand of telomeres and facilitate the switch to C-strand synthesis by DNA polymerase alpha-primase (pol -primase). Recently the structure of human CST was solved by cryo-EM, allowing the design of mutant proteins defective in telomeric ssDNA binding and prompting the reexamination of CST inhibition of telomerase. The previous proposal that human CST inhibits telomerase by sequestration of the DNA primer was tested with a series of DNA-binding mutants of CST and modeled by a competitive binding simulation. The DNA-binding mutants had substantially reduced ability to inhibit telomerase, as predicted from their reduced affinity for telomeric DNA. These results provide strong support for the previous primer sequestration model. We then tested whether addition of CST to an ongoing processive telomerase reaction would terminate DNA extension. Pulse-chase telomerase reactions with addition of either wild-type CST or DNA-binding mutants showed that CST has no detectable ability to terminate ongoing telomerase extension in vitro. The same lack of inhibition was observed with or without pol -primase bound to CST. These results suggest how the switch from telomerase extension to C-strand synthesis may occur.

category: biochemistry


title: Next generation SARM1 knockout and epitope tagged CRISPR-Cas9-generated isogenic mice reveal that SARM1 does not participate in regulating nuclear transcription, despite confirmation of protein expression in macrophages

abstract: SARM1 is an ancient and highly conserved TIR-domain containing protein, with a diverse range of proposed roles in both innate immunity and neuronal death and degeneration. Murine SARM1 has been reported to regulate the transcription of specific chemokines in both neurons and macrophages, however the extent and mechanism by which SARM1 contributes to transcription regulation remains to be fully understood. Here, using RNA sequencing we identify differential gene expression in bone marrow-derived macrophages (BMDM) from C57BL/6 congenic 129 ES cell-derived Sarm1-/- mice compared to wild type (WT). However, we show that passenger genes which are derived from the 129 donor strain of mice flank the Sarm1 locus, confounding interpretation of results, since many of the identified differentially regulated genes come from the region containing passenger genes. To re-examine the transcriptional role of SARM1 in the absence of such passenger genes, we generated three different Sarm1-/- mice using CRISPR/Cas9 technology. Vincristine treatment of ex vivo cultured post-natal neurons from these mice confirmed SARM1s previously identified key function as an executor of axon degeneration. However, using these mice, we show that the absence of SARM1 has no impact on transcription of genes previously shown to be altered in macrophages or in the brainstem. To gain further insight into SARM1 function, we generated and characterized a mouse expressing epitope-tagged SARM1, as it has been difficult to date to confirm which cells and tissues express SARM1 protein. In these mice we see high SARM1 protein expression in the brain and brainstem, and lower but detectable levels in macrophages. Overall, the generation of these next generation SARM1 knockout and epitope-tagged mice has clarified that SARM1 is expressed in mouse macrophages but has no general role in transcriptional regulation in these cells, and has provided important new animal models to further explore SARM1 function.


title: Enterohaemorrhagic E. coli utilizes an AND-OR logic gate to regulate expression of an outer membrane haem receptor

abstract: To sense the transition from environment to host, bacteria use a range of environmental cues to control expression of virulence genes. Iron is tightly sequestered in host tissues and in the human pathogen enterohaemorrhagic E. coli (EHEC) iron-limitation induces transcription of the outermembrane haem transporter encoded by chuAS. ChuA expression is post-transcriptionally activated at 37{degrees}C by a FourU RNA thermometer ensuring that the haem receptor is only expressed under low iron, high temperature conditions that indicate the host. Here we demonstrate that expression of chuA is also independently regulated by the cAMP-responsive sRNA CyaR and transcriptional terminator Rho. These results indicate that chuAS expression is regulated at the transcription initiation, transcript elongation, and translational level. The natural dependence of these processes creates a hierarchy of regulatory AND and OR logic gates that integrate information about the local environment. We show that the logic of the chuA regulatory circuit is activated under conditions that satisfy low iron AND (low glucose OR high temperature). We speculate that additional sensing of a gluconeogenic environment allows further precision in determining when EHEC is at the gastrointestinal epithelium of the host. With previous studies, it appears that the chuAS transcript is controlled by eight regulatory inputs that control expression through six different transcriptional and post-transcriptional mechanisms. The results highlight the ability of regulatory sRNAs to integrate multiple environmental signals into a conditional hierarchy of signal input.


title: Beehives possess their own distinct microbiomes

abstract: Honey bees use plant material to manufacture their own food. These insect pollinators visit flowers repeatedly to collect nectar and pollen, which are shared with other hive bees to produce honey and beebread. While producing these products, beehives accumulate a tremendous amount of microbes, including bacteria that derive from plants and different parts of the honey bees body. In this study, we conducted 16S rDNA metataxonomic analysis on honey and beebread samples that were collected from 15 beehives in the southeast of England in order to quantify the bacteria associated with beehives. The results highlighted that honeybee products carry a significant variety of bacterial groups that comprise bee commensals, environmental bacteria and pathogens of plants and animals. Remarkably, this bacterial diversity differs amongst the beehives, suggesting a defined fingerprint that is affected, not only by the nectar and pollen gathered from local plants, but also from other environmental sources. In summary, our results show that every hive possesses their own distinct microbiome, and that honeybee products are valuable indicators of the bacteria present in the beehives and their surrounding environment.


title: A mechanistic switch from selective transporter to an ion channel impairs the filamentation signalling capability of ammonium transceptors in yeast

abstract: Ammonium translocation through biological membranes by the ubiquitous Amt-Mep-Rh family of transporters plays a key role in all domains of life. Two highly conserved histidine residues protrude into the lumen of these transporters, forming the familys characteristic Twin-His motif. It has been hypothesized that the motif is essential to confer the selectivity of the transport mechanism. Here, using a combination of in vitro electrophysiology, in vivo yeast functional complementation and in silico molecular dynamics simulations, we demonstrate that variations in the Twin-His motif trigger a mechanistic switch between a specific transporter, depending on ammonium deprotonation, to an unspecific ion channel activity. We therefore propose that there is no selective filter that governs the specificity in Amt-Mep transporters but the inherent mechanism of translocation, dependent on the fragmentation of the substrate, ensures the high specificity of the translocation. We further show that both mechanisms coexist in fungal Mep2 Twin-His variants, disrupting the transceptor function and so inhibiting the filamentation process. These data strongly support a transport mechanism-mediated signalling process in the long-standing debate on the sensory function of Mep2-like transporters.


title: Prediction of standard cell types and functional markers from flow cytometry gating definitions using machine learning

abstract:
BackgroundA key step in clinical flow cytometry data analysis is gating, which involves the identification of cell populations. The process of gating produces a set of reportable results, which are typically described by gating definitions. The non-standardized, non-interpreted nature of gating definitions represents a hurdle for data interpretation and data sharing across and within organizations. Interpreting and standardizing gating definitions for subsequent analysis of gating results requires a curation effort from experts. Machine learning approaches have the potential to help in this process by predicting expert annotations associated with gating definitions.

MethodsWe created a gold-standard dataset by manually annotating thousands of gating definitions with cell type and functional marker annotations. We used this dataset to train and test a machine learning pipeline able to predict standard cell types and functional marker genes associated with gating definitions.

ResultsThe machine learning pipeline predicted annotations with high accuracy for both cell types and functional marker genes. Accuracy was lower for gating definitions from assays belonging to laboratories from which limited or no prior data was available in the training. Manual error review ensured that resulting predicted annotations could be reused subsequently as additional gold-standard training data.

ConclusionsMachine learning methods are able to consistently predict annotations associated with gating definitions from flow cytometry assays. However, a hybrid automatic and manual annotation workflow would be recommended to achieve optimal results.


title: Evolution of host-microbe cell adherence by receptor domain shuffling

abstract: Stable adherence to epithelial surfaces is required for colonization by diverse host-associated microbes. Successful attachment of pathogenic microbes via surface adhesin molecules is also the first step in many devastating infections. Despite the primacy of epithelial adherence in establishing host-microbe associations, the evolutionary processes that shape this crucial interface remain enigmatic. Carcinoembryonic antigen associated cell adhesion molecules (CEACAMs) encompass a multifunctional family of vertebrate cell surface proteins which are recurrent targets of bacterial surface adhesins at epithelial surfaces. Here we show that multiple members of the primate CEACAM family exhibit evidence of repeated natural selection at protein surfaces targeted by bacteria, consistent with pathogen-driven evolution. Inter-species diversity of CEACAM proteins, between even closely-related great apes, determines molecular interactions with a range of bacterial adhesins. Phylogenetic analyses reveal that repeated gene conversion of CEACAM extracellular domains during primate divergence plays a key role in limiting bacterial adhesin tropism. Moreover, we demonstrate that gene conversion has continued to shape CEACAM diversity within human populations, with abundant CEACAM1 variants mediating evasion of adhesins from Neisseria gonorrhoeae, the causative agent of gonorrhea. Together this work reveals a mechanism by which gene conversion shapes first contact between microbes and animal hosts.

category: evolutionary biology


title: Binding of AP endonuclease-1 to G-quadruplex DNA depends on the N-terminal domain, Mg2+ and ionic strength

abstract:
The base excision repair enzyme apurinic/apyrimidinic endonuclease-1 (APE1) is also engaged in transcriptional regulation. APE1 can function in both pathways when the protein binds to a promoter G-quadruplex (G4) bearing an abasic site (modeled with tetrahydrofuran, F) that leads to enzymatic stalling on the non-canonical fold to recruit activating transcription factors. Biochemical and biophysical studies to address APE1s binding and catalytic activity with the vascular endothelial growth factor (VEGF) promoter G4 are lacking, and the present work provides insight on this topic. Herein, the native APE1 was used for cleavage assays, and the catalytically inactive mutant D210A was used for binding assays with double-stranded DNA (dsDNA) versus the native G4 or the G4 with F at various positions, revealing dependencies of the interaction on the cation concentrations K+ and Mg2+ and the N-terminal domain of the protein. Assays in 0, 1, or 10 mM Mg2+ found dsDNA and G4 substrates required the cation for both binding and catalysis, in which G4 binding increased with [Mg2+]. Studies with 50 versus physiological 140 mM K+ ions present showed that F-containing dsDNA was bound and cleaved by APE1; whereas, the G4s with F were poorly cleaved in low salt and not cleaved at all at higher salt while the binding remained robust. Using {Delta}33 or {Delta}61 N-terminal truncated APE1 proteins, the binding and cleavage of dsDNA with F was minimally impacted; in contrast, the G4s required the N-terminus for binding and catalysis. With this knowledge, we found APE1 could remodel the F-containing VEGF promoter dsDNA[->]G4 folds in solution. Lastly, the addition of the G4 ligand pyridostatin inhibited APE1 binding and cleavage of F-containing G4s but not dsDNA. The biological and medicinal chemistry implications of the results are discussed.

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title: Systematic investigation of the link between enzyme catalysis and cold adaptation

abstract: Cold temperature is prevalent across the biosphere and slows the rates of chemical reactions. Increased catalysis has been predicted to be a general adaptive trait of enzymes to reduced temperature, and this expectation has informed physical models for enzyme catalysis and influenced bioprospecting strategies. To broadly test rate as an adaptive trait to cold, we paired kinetic constants of 2223 enzyme reactions with their organisms optimal growth temperature (TGrowth) and analyzed trends of rate as a function of TGrowth. These data do not support a prevalent increase in rate in cold adaptation. In the model enzyme ketosteroid isomerase (KSI), there was prior evidence for temperature adaptation from a change in an active site residue that results in a tradeoff between activity and stability. Here, we found that little of the overall rate variation for 20 KSI variants was accounted for by TGrowth. In contrast, and consistent with prior expectations, we observed a correlation between stability and TGrowth across 433 proteins. These results suggest that temperature exerts a weaker selection pressure on enzyme rate than stability and that evolutionary forces other than temperature are responsible for the majority of enzymatic rate variation.


title: Interactome and structural basis for targeting the human T-cell leukemia virus Tax oncoprotein

abstract: Human T-cell leukemia virus type-1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL). Although ATL is a well-characterized T-cell neoplasm, linked to intermittent expression of the viral Tax-1 protein, there is currently no strategy to target Tax-1 functions using small molecules. Here, we report a comprehensive interaction map between Tax-1 and human PDZ domain-containing proteins (hPDZome). We show that Tax-1 interacts with more than one-third of hPDZome components, including proteins involved in cell cycle, cell-cell junctions, cytoskeleton organization, and membrane complex assembly. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the structural basis of the interaction between the C-terminal PDZ binding motif (PBM) of Tax-1, and the PDZ domains of syntenin-1, an evolutionary conserved hub that controls exosome trafficking. Finally, we have used confocal imaging, molecular modelling, NMR and mammalian cell-based assays to demonstrate that the Tax-1/syntenin-1 interaction is amenable to small-molecule inhibition. Altogether, our study highlights the biological significance of Tax-PDZ interactome and its interplay with exosome formation. It shows a direct link between extracellular vesicles and HTLV-1 transmission, providing a novel framework for the design of targeted therapies for HTLV-1-induced diseases.


title: Structural basis for targeting the human T-cell leukemia virus Tax oncoprotein and syntenin-1 interaction using a small molecule


title: Discovery of novel potent, cell-permeable inhibitors of P. falciparum cGMP-dependent protein kinase.

abstract: The discovery of new targets for treatment of malaria advanced with the demonstration that orally administered inhibitors of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) could clear infection in a murine model. This enthusiasm was tempered by unsatisfactory safety and/or pharmacokinetic issues found with these chemotypes. To address the urgent need for new scaffolds, we recently reported the discovery and optimization of novel, potent isoxazole-based PfPKG inhibitors that lacked any obvious safety warnings. This manuscript presents representative in vitro ADME, hERG characterization and cell-based antiparasitic activity of these PfPKG inhibitors. We also report the discovery and structure-activity relationships of a new series with good potency, low hERG activity and cell-based anti-parasitic activity comparable to a literature standard.


title: Pericyte control of pancreatic islet blood flow impacts glucose homeostasis

abstract: The pancreatic islet depends on blood supply to efficiently sense plasma glucose levels and deliver insulin and glucagon into the circulation. Long thought to be passive conduits of nutrients and hormones, islet capillaries were recently found to be densely covered with contractile pericytes, suggesting local control of blood flow. Here we determined the contribution of islet pericytes to the regulation of islet blood flow, plasma insulin and glucagon levels, and glycemia. Selective optogenetic activation of pericytes in intraocular islet grafts contracted capillaries and diminished blood flow. In awake mice, acute clamping of islet blood flow by optogenetic or pharmacological activation of pericytes disrupted hormonal responses, glycemia, and glucose tolerance. Our findings indicate that pericytes mediate vascular responses in the islet that are required for adequate hormone secretion and glucose homeostasis. Vascular deficiencies commonly seen in the islets of people with type 2 diabetes may impair regulation of islet blood flow and thus precipitate islet dysfunction.


title: Interactions between a mechanosensitive channel and cell wall integrity signaling influence pollen germination in Arabidopsis thaliana

abstract: Cells employ multiple systems to maintain cellular integrity, including mechanosensitive (MS) ion channels and the cell wall integrity (CWI) pathway. Here, we use pollen as a model system to ask how these different mechanisms are interconnected at the cellular level. MscS-Like (MSL)8 is an MS channel required to protect Arabidopsis thaliana pollen from osmotic challenges during in vitro rehydration, germination and tube growth. New CRISPR/Cas9 and artificial microRNA-generated msl8 alleles produced unexpected pollen phenotypes, including the ability to germinate a tube after bursting, dramatic defects in cell wall structure and disorganized callose deposition at the germination site. We document complex genetic interactions between MSL8 and two previously established components of the CWI pathway, MARIS, and ANXUR1/2. Overexpression of MARISR240C-FP suppressed the bursting, germination, and callose deposition phenotypes of msl8 mutant pollen. Null msl8 alleles suppressed the internalized callose structures observed in MARISR240C-FP lines. Similarly, MSL8-YFP overexpression suppressed bursting in the anxur1/2 mutant background, while anxur1/2 alleles reduced the strong rings of callose around ungerminated pollen grains in MSL8-YFP over-expressors. These data show that MS ion channels modulate callose deposition in pollen and provides evidence that cell wall and membrane surveillance systems coordinate in a complex manner to maintain cell integrity.

category: plant biology


title: pH-Triggered Assembly of Endomembrane Multicompartments in Synthetic Cells

abstract: Bottom-up synthetic biology thrives to reconstruct basic cellular processes into a minimalist cellular replica to foster their investigation in greater details with a reduced number of variables. Among these cellular features, the endomembrane system is an important aspect of cells which is at the origin of many of their functions. Still, the reconstruction of these inner compartments within a lipid-based vesicle remains challenging and poorly controlled. Herein, we report the use of pH as external trigger to self-assemble compartmentalized giant unilamellar vesicles (GUVs) by either bulk, or droplet-based microfluidics. By co-encapsulating pH sensitive small unilamellar vesicles (SUVs), negatively charged SUVs and/or proteins, we show that acidification of the droplets efficiently produces GUVs while sequestrating the co-encapsulated material with flexibility and robustness. The method enables the simultaneous reconstruction of more than a single cellular phenotype from the bottom-up, corresponding to an important advancement in the current status quo of bottom-up synthetic biology.


title: A Combinatorial PCR Method for Efficient, Selective Oligo Retrieval from Complex Oligo Pools

abstract: With the rapidly decreasing cost of array-based oligo synthesis, large-scale oligo pools offer significant benefits for advanced applications, including gene synthesis, CRISPR-based gene editing, and DNA data storage. Selectively retrieving specific oligos from these complex pools traditionally uses Polymerase Chain Reaction (PCR), in which any selected oligos are exponentially amplified to quickly outnumber non-selected ones. In this case, the number of orthogonal PCR primers is limited due to interactions between them. This lack of specificity presents a serious challenge, particularly for DNA data storage, where the size of an oligo pool (i.e., a DNA database) is orders of magnitude larger than it is for other applications. Although a nested file address system was recently developed to increase the number of accessible files for DNA storage, it requires a more complicated lab protocol and more expensive reagents to achieve high specificity. Instead, we developed a new combinatorial PCR method that outperforms prior work without compromising the fidelity of retrieved material or complicating wet lab processes. Our method quadratically increases the number of accessible oligos while maintaining high specificity. In experiments, we accessed three arbitrarily chosen files from a DNA prototype database that contained 81 different files. Initially comprising only 1% of the original database, the selected files were enriched to over 99.9% using our combinatorial primer method. Our method thus provides a viable path for scaling up DNA data storage systems and has broader utility whenever scientists need access to a specific target oligo and can design their own primer regions.


title: Physical localization of 45S rDNA in Cymbopogon and the analysis of differential distribution of rDNA in homologous chromosomes of Cymbopogon winterianus.

abstract: Cymbopogon, commonly known as lemon grass, is one of the most important aromatic grasses having therapeutic and medicinal values. FISH signals on somatic chromosome spreads off Cymbopogon species indicated the localization of 45S rDNA on the terminal region of short arms of a chromosome pair. A considerable interspecific variation in the intensity of 45S rDNA hybridization signals was observed in the cultivars of Cymbopogon winterianus and Cymbopogon flexuosus. Furthermore, in all the varieties of Cymbopogon winterianus namely Bio-13, Manjari and Medini, a differential distribution of 45S rDNA was observed in a heterologous pair of chromosome 1. The development of Cymbopogon winterianus var. Manjari through gamma radiation may be responsible for breakage of fragile rDNA site from one of the chromosomes of this heterologous chromosome pair. While, in other two varieties of Cymbopogon winterianus (Bio-13 and Medini), this variability may be because of evolutionary speciation due to natural cross among two species of Cymbopogon which was fixed through clonal propagation. However, in both the situations these changes were fixed by vegetative method of propagation which is general mode of reproduction in the case of Cymbopogon winterianus.


title: Selective localization of Mfn2 near PINK1 enable its preferential ubiquitination by Parkin on mitochondria

abstract: Mutations in Parkin and PINK1 cause an early-onset familial Parkinsons disease. Parkin is a RING-In-Between-RING (RBR) E3 ligase that transfers ubiquitin from an E2 enzyme to a substrate in two steps: 1) thioester intermediate formation on Parkin, and 2) acyl transfer to a substrate lysine. The process is triggered by PINK1, which phosphorylates ubiquitin on damaged mitochondria, which in turn recruits and activates Parkin. This leads to the ubiquitination of outer mitochondrial membrane proteins and clearance of the organelle. While the targets of Parkin on mitochondria are known, the factors determining substrate selectivity remain unclear. To investigate this, we examined how Parkin catalyzes ubiquitin transfer to substrates. We found that His433 in the RING2 domain catalyzes acyl transfer. In cells, mutation of His433 impairs mitophagy. In vitro ubiquitination assays with isolated mitochondria show that Mfn2 is a kinetically preferred substrate. Using proximity-ligation assays, we show that Mfn2 specifically co-localizes with PINK1 and phospho-ubiquitin in U2OS cells upon mitochondrial depolarization. We propose a model whereby ubiquitination of Mfn2 is efficient by virtue of its localization near PINK1, which leads to the recruitment and activation of Parkin via phospho-ubiquitin at these sites.


title: Reduced production of the major allergen Bla g 1 and Bla g 2 in Blattella germanica after antibiotic treatment

abstract:
PurposeAllergens present in the feces or frass of cockroaches can cause allergic sensitization in humans. The use of fecal and frass extracts for immunotherapy has been previously investigated but has not yet been fully standardized. Here, we treated cockroaches with ampicillin to produce extracts with reduced amounts of total bacteria.

MethodsWe performed targeted high-throughput sequencing of 16S rDNA to compare the microbiomes of ampicillin-treated and untreated (control) cockroaches. RNA-seq was performed to identify differentially expressed genes (DEGs) in ampicillin-treated cockroaches.

ResultsAnalysis of the microbiome revealed that alpha diversity was lower in the ampicillin-treated group than in the control group. Beta diversity analysis indicated that ampicillin treatment altered bacterial composition in the microbiome of cockroaches. Quantitative polymerase chain reaction revealed that almost all bacteria were removed from ampicillin-treated cockroaches. RNA-seq analysis revealed 1,236 DEGs in ampicillin-treated cockroaches (compared to untreated cockroaches). Unlike bacterial composition, the DEGs varied between the two groups. Among major allergens, the expression of Bla g 2 decreased significantly in ampicillin-treated cockroaches (compared to untreated group).

ConclusionsIn this study, the reduced level of allergens observed in cockroaches may be related to lower amounts of total bacteria caused by treatment with antibiotics. It is possible to make a protein extract with few bacteria for use in immunotherapy.


title: Histopathological effects and micronucleus assay of glyphosate-based herbicides on cultured african catfish (Clarias Gariepinus, Burchell 1822)

abstract:
Glyphosate, a brand of agricultural herbicides which intensive use has led to widespread contamination of different ecosystems. This study was designed to determine both organotoxicity and genotoxicity of glyphosate on African Catfish (Clarias gariepinus) exposed to different concentrations for 96 hours. Questionnaires were administered (physically and online) to determine the type of glyphosate-based herbicides mostly used by fish farmers. Seventy-five apparently healthy adult Clarias gariepinus (300g) were sourced from a local farmer, transported in a plastic keg to Fish and Wildlife Laboratory at the Department of Veterinary Public Health and Preventive medicine for two (2) weeks of acclimatization. After acclimatization, they were further divided into four (4) groups (T0 (Control), T1 (0.003ml/L), T2 (0.0045ml/L) and T3 (0.006ml/L)) by simple randomization and each group replicated into three (3) treatments. At the expiration of 96 hours of exposure, blood samples and organs (Gills, Kidney, and Liver) were collected for mononuclear assay and histopathological lesions respectively.

Exposed groups showed erratic swimming, splashing, and restlessness. Mortalities rate was dose-dependent (two (2) mortalities at 0.0045ml/L concentration (T2) and five (5) mortalities 0.006ml/L concentration (T3)). Observed histopathological lesions occurred at higher dose treatment (0.0045ml/L (T2) and (0.006ml/L) T3)) concentrations. The gills showed diffuse stunted and eroded secondary lamellae and severe congestion of the blood channel at the core of the primary lamellae. Lesions in the Liver include severe diffuse vacuolation of the hepatocytes, moderate to severe portal congestion and mild diffuse vacuolation of hepatocytes and moderate diffuse vacuolation of hepatocytes, and severe portal congestion. In the kidney, there was mild to moderate congestion of the interstitium and focus of interstitial oedema within the parenchyma. There was presence of micronucleus in the fish nucleated red blood cells at higher dose concentrations.

This study showed that Glyphosate-based herbicides are highly toxic to Clarias gariepinus, therefore their use near the fish farm or in areas close to the aquatic environment should be discouraged. The agricultural community should also be conscious of the potentially adverse effects of pesticides. This is to prevent the water body from the residue of herbicides that would have washed down to the water.


title: Cytogenetic evidence supports Avena insularis being closely related to hexaploid oats.

abstract: Cytogenetic observations, phylogenetic studies and genome analysis using high-density genetic markers have suggested a tetraploid Avena species carrying the C and D genomes (formerly C and A) to be the donor of all hexaploid oats (AACCDD). However, controversy surrounds which of the three extant CCDD tetraploid species - A. insularis, A. maroccana and A. murphyi - is most closely related to hexaploid oats. The present work describes a comparative karyotype analysis of these three CCDD tetraploid species and two hexaploid species, A. sativa and A. byzantina. This involved the use of FISH with six simple sequence repeats (SSRs) with the motifs CT, AAC, AAG, ACG, ATC and ACT, two repeated ribosomal sequences, and C genome-specific repetitive DNA. The hybridization pattern of A. insularis with oligonucleotide (AC)10 was also determined and compared with those previously published for A. sativa and A. byzantina. Significant differences in the 5S sites and SSR hybridization patterns of A. murphyi compared to the other CCDD species rule out its being directly involved in the origin of the hexaploids. In contrast, the repetitive and SSR hybridization patterns shown by the D genome chromosomes, and by most of the C genome chromosomes of A. maroccana and A. insularis, can be equated with the corresponding chromosomes of the hexaploids. Several chromosome hybridization signals seen for A. insularis, but not for A. maroccana, were shared with the hexaploid oats species, especially with A. byzantina. These diagnostic signals add weight to the idea that the extant A. insularis, or a direct ancestor of it, is the most closely related progenitor of hexaploid oats. The similarity of the chromosome hybridization patterns of the hexaploids and CCDD tetraploids was taken as being indicative of homology. A common chromosome nomenclature for CCDD species based on that of the hexaploid species is proposed.


title: Spatially Resolved In Silico Modeling of NKG2D Signaling Kinetics Reveals Key role of NKG2D and Vav1 Co-clustering in Generating Natural Killer cell Activation

abstract: Natural Killer (NK) cells provide key resistance against viral infections and tumors. A diverse set of activating and inhibitory NK cell receptors (NKRs) interact with cognate ligands presented by target host cells, where integration of dueling signals initiated by the ligand-NKR interactions determines NK cell activation or tolerance. Imaging experiments over decades have shown micron and sub-micron scale spatial clustering of activating and inhibitory NKRs. The mechanistic roles of these clusters in affecting downstream signaling and activation are often unclear. To this end, we developed a predictive in silico framework by combining spatially resolved mechanistic agent based modeling, published TIRF imaging data, and parameter estimation to determine mechanisms by which formation and spatial movements of activating NKG2D microclusters affect early time NKG2D signaling kinetics in a human cell line NKL. We show co-clustering of NKG2D and the guanosine nucleotide exchange factor Vav1 in NKG2D microclusters plays a dominant role over ligand (ULBP3) rebinding in increasing production of phospho-Vav1(pVav1), an activation marker of early NKG2D signaling. The in silico model successfully predicts several scenarios of inhibition of NKG2D signaling and time course of NKG2D spatial clustering over a short ([~]3 min) interval. Modeling shows the presence of a spatial positive feedback relating formation and centripetal movements of NKG2D microclusters, and pVav1 production offers flexibility towards suppression of activating signals by inhibitory KIR ligands organized in inhomogeneous spatial patterns (e.g., a ring). Our in silico framework marks a major improvement in developing spatiotemporal signaling models with quantitatively estimated model parameters using imaging data.

category: systems biology


title: Single-cell foundations of live social gaze interaction in the prefrontal cortex and amygdala

abstract: Social gaze interaction powerfully shapes interpersonal communication in humans and other primates. However, little is known about the neural underpinnings of these social behavioral exchanges. Here, we studied neural responses associated with naturalistic, face-to-face, social gaze interactions between pairs of macaques. We examined spiking activity in a large number of neurons spanning four different brain regions involved in social behaviors - the amygdala, orbitofrontal cortex, anterior cingulate cortex, and dorsomedial prefrontal cortex. We observed widespread single-cell representations of social gaze interaction functionalities in these brain regions - social discriminability, social gaze monitoring, and mutual eye contact selectivity. Many of these neurons discriminated looking at social versus non-social stimuli with rich temporal heterogeneity, or parametrically tracked the gaze positions of oneself or the conspecific. Furthermore, many neurons displayed selectivity for mutual eye contact as a function of the initiator or follower of mutual gaze events. Crucially, a significant proportion of neurons coded for more than one of these three signatures of social gaze interaction, supporting the recruitment of partially overlapping neuronal ensembles. Our findings emphasize integrated contributions of the amygdala and prefrontal circuits within the social interaction networks in processing real-life social interactions.


title: Reading Modality Modifies Reading Network: Insights from Neural basis of Braille in Proficient Blind Readers

abstract: The neural basis of reading is highly consistent across many languages and scripts. Are there alternative neural routes to reading? How does the sensory modality of symbols (tactile vs. visual) influence their neural representations? We examined these questions by comparing reading of visual print (sighted group, n=19) and tactile Braille (congenitally blind group, n=19). Blind and sighted readers were presented with written (words, consonant strings, non-letter shapes) and spoken stimuli (words, backward speech) that varied in word-likeness. Consistent with prior work, the ventral occipitotemporal cortex (vOTC) was active during Braille and visual reading. A posterior/anterior vOTC word-form gradient was observed only in sighted readers with more anterior regions preferring larger orthographic units (words). No such gradient was observed in blind readers. Consistent with connectivity predictions, in blind compared to sighted readers, posterior parietal cortices were recruited to a greater degree and contained word-preferring patches. Lateralization of Braille in blind readers was predicted by laterality of spoken language and reading hand. The effect of spoken language increased along a cortical hierarchy, whereas effect of reading-hand waned. These results suggested that the neural basis of reading is influenced by symbol modality and spoken language and support connectivity-based views of cortical function.


title: Reading Modality Shapes Reading Network in Proficient Blind Readers of Braille


title: Effects of Spike Mutations in SARS-CoV-2 Variants of Concern on Human or Animal ACE2-Mediated Virus Entry and Neutralization

abstract:
SARS-CoV-2 is a zoonotic agent capable of infecting humans and a wide range of animal species. Over the duration of the pandemic, mutations in the SARS-CoV-2 Spike protein (S) have arisen in circulating viral populations, culminating in the spread of several variants of concern (VOC) with varying degrees of altered virulence, transmissibility, and neutralizing antibody escape. In this study, we employed lentivirus-based pseudotyped viruses that express specific SARS-CoV-2 S protein substitutions and cell lines that stably express ACE2 from nine different animal species to gain insights into the effects of VOC mutations on viral entry and antibody neutralization capability. All animal ACE2 receptors tested, except mink, support viral cell entry for pseudoviruses expressing the parental (prototype Wuhan-1) S at levels comparable to human ACE2. Most single S substitutions (e.g., 452R, 478K, 501Y) did not significantly change virus entry, although 614G and 484K resulted in a decreased efficiency in viral entry. Conversely, combinatorial VOC substitutions in the S protein were associated with significantly increased entry capacity of pseudotyped viruses compared to that of the parental Wuhan-1 pseudotyped virus. Similarly, infection studies using live ancestral (USA-WA1/2020), Alpha, and Beta SARS-CoV-2 viruses in hamsters revealed a higher replication potential for the Beta variant compared to the ancestral prototype virus. Moreover, neutralizing titers in sera from various animal species, including humans, were significantly reduced by single substitutions of 484K or 452R, double substitutions of 501Y-484K, 452R-484K and 452R-478K and the triple substitution of 501Y-484K-417N, suggesting that 484K and 452R are particularly important for evading neutralizing antibodies in human, cat, and rabbit sera. Cumulatively, this study reveals important insights into the host range of SARS-CoV-2 and the effect of recently emergent S protein substitutions on viral entry, virus replication and antibody-mediated viral neutralization.

Author summaryCells stably expressing ACE2 from various animals and a lentivirus-based SARS-CoV-2 pseudotyped virus assay were established to study SARS-CoV-2 cell entry. The results demonstrated that ACE2 from a wide range of animal species facilitate S-mediated virus entry into cells, which is supported by in silico data as well as natural and experimental infection studies. Pseudotyped viruses containing mutations in the RBD of S representative of the Alpha, Gamma, and especially Beta, variants of concern demonstrated that certain mutations are associated with increased viral entry compared to the parental S. The Beta variant was also observed to have a replicative advantage in vitro and in vivo compared to the prototype virus. Pseudotyped viruses containing combinatorial substitutions of 501Y-484K-417K, 614G-501Y-484K and 614G-501Y-484K-417N increased viral entry via ACE2 across multiple species. The 501Y or 478K single substitution did not significantly affect neutralizing capacity of immune sera compared to the prototype strain, but the addition of 484K or 452R substitutions significantly reduced the neutralizing titers.


title: The B.1.427/1.429 (epsilon) SARS-CoV-2 variants are more virulent than ancestral B.1 (614G) in Syrian hamsters

abstract:
As novel SARS-CoV-2 variants continue to emerge, it is critical that their potential to cause severe disease and evade vaccine-induced immunity is rapidly assessed in humans and studied in animal models. In early January 2021, a novel variant of concern (VOC) designated B.1.429 comprising 2 lineages, B.1.427 and B.1.429, was originally detected in California (CA) and shown to enhance infectivity in vitro and decrease antibody neutralization by plasma from convalescent patients and vaccine recipients. Here we examine the virulence, transmissibility, and susceptibility to pre-existing immunity for B 1.427 and B 1.429 in the Syrian hamster model. We find that both strains exhibit enhanced virulence as measured by increased body weight loss compared to hamsters infected with ancestral B.1 (614G), with B.1.429 causing the most body weight loss among all 3 lineages. Faster dissemination from airways to parenchyma and more severe lung pathology at both early and late stages were also observed with B.1.429 infections relative to B.1. (614G) and B.1.427 infections. In addition, subgenomic viral RNA (sgRNA) levels were highest in oral swabs of hamsters infected with B.1.429, however sgRNA levels in lungs were similar in all three strains. This demonstrates that B.1.429 replicates to higher levels than ancestral B.1 (614G) or B.1.427 in the upper respiratory tract (URT) but not in the lungs. In multi-virus in-vivo competition experiments, we found that epsilon (B.1.427/B.1.429) and gamma (P.1) dramatically outcompete alpha (B.1.1.7), beta (B.1.351) and zeta (P.2) in the lungs. In the URT gamma, and epsilon dominate, but the highly infectious alpha variant also maintains a moderate size niche. We did not observe significant differences in airborne transmission efficiency among the B.1.427, B.1.429 and ancestral B.1 (614G) variants in hamsters. These results demonstrate enhanced virulence and high relative fitness of the epsilon (B.1.427/B.1.429) variant in Syrian hamsters compared to an ancestral B.1 (614G) strain.

Author SummaryIn the last 12 months new variants of SARS-CoV-2 have arisen in the UK, South Africa, Brazil, India, and California. New SARS-CoV-2 variants will continue to emerge for the foreseeable future in the human population and the potential for these new variants to produce severe disease and evade vaccines needs to be understood. In this study, we used the hamster model to determine the epsilon (B.1.427/429) SARS-CoV-2 strains that emerged in California in late 2020 cause more severe disease and infected hamsters have higher viral loads in the upper respiratory tract compared to the prior B.1 (614G) strain. These findings are consistent with human clinical data and help explain the emergence and rapid spread of this strain in early 2021.


title: The secret life (cycle) of temperate bacteriophages

abstract:
Lysogenic induction ends the stable association between a bacteriophage and its host, and the transition to the lytic cycle begins with prophage excision followed by DNA replication and packaging (ERP) - a temporal program that is considered universal for most temperate phages. Here we report that the long-standing ERP program is an artefact of the experimentally favoured Salmonella phage P22 tsc229 heat-inducible mutant, and that wildtype P22 actually follows a replication-packaging-excision (RPE) program. We found that unlike P22 tsc229, P22 delayed excision to just before it was detrimental to phage production. Thus, at minimal expense to itself, P22 has tuned the timing of excision to balance propagation with lateral transduction, powering the evolution of its host through gene transfer in the interest of self-preservation.

One Sentence SummaryGenetic analyses propose a new life cycle for temperate bacteriophages.


title: The omega-3-fatty acid docosahexaenoic acid modulates intracellular myo-inositol and its biosynthetic genes.

abstract: Bipolar disorder is a debilitating mood disorder characterized by recurring episodes of mania and depression. It affects 2.6% of adults and has a lifetime prevalence among adults of 3.9%. Current mood stabilizers are not always effective and/or are not well tolerated by many patients; thus, there is a need to develop or identify more effective and less harmful treatments. Omega-3-fatty acids have been shown to be effective in the treatment of bipolar disorder; however, their mechanism of action is unknown. Myo-inositol depletion has been hypothesized as the mechanism by which mood stabilizers exert their therapeutic effect. Using an enzymatic assay, we determined intracellular myo-inositol levels increased more than 2-fold when cells were grown in the presence of the omega-3 fatty acid docosahexaenoic acid (DHA). RT-qPCR was used to characterize the effects of DHA on genes in the myo-inositol biosynthetic pathway. We show DHA increases relative expression and has a concentration-dependent impact on INO1 and INM1, which encode myo-inositol-1-phosphate synthase and myo-inositol monophosphate 1-phosphatase, respectively. We therefore conclude that the omega-3-fatty acid DHA exerts its therapeutic effect on bipolar disorder by increasing intracellular myo-inositol, which may be accomplished by upregulating its biosynthetic genes.


title: A TET1-PSPC1-Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency

abstract:
TET1 maintains hypomethylation at bivalent promoters through its catalytic activity in embryonic stem cells (ESCs). However, whether and how TET1 exerts catalytic activity-independent functions in regulating bivalent genes is not well understood. Using a proteomics approach, we mapped the TET1 interactome in mouse ESCs and identified PSPC1 as a novel TET1 partner. Genome-wide location analysis reveals that PSPC1 functionally associates with TET1 and Polycomb repressive complex-2 (PRC2) complex. We establish that PSPC1 and TET1 repress, and Neat1, the PSPC1 cognate lncRNA, activates the bivalent gene expression. In ESCs, Neat1 tethers the TET1-PSPC1 pair with PRC2 at bivalent promoters. During the ESC-to-formative epiblast-like stem cell (EpiLC) transition, PSPC1 and TET1 promote PRC2 chromatin occupancy at bivalent gene promoters while restricting Neat1 functions in facilitating PRC2 binding to bivalent gene transcripts. Our study uncovers a novel TET1-PSPC1-Neat1 molecular axis that modulates PRC2 binding affinity to chromatin and bivalent gene transcripts in controlling stem cell bivalency.

In BriefTET1 is a transcriptional repressor for bivalent genes in pluripotent stem cells, but its mechanistic action on stem cell bivalency is unclear. Huang et al. use proteomics and genetic approaches to reveal that catalytic activity-independent functions of TET1, coordinated with the paraspeckle components PSPC1 and its cognate lncRNA Neat1, dynamically regulates stem cell bivalency by modulating PRC2 binding affinity to chromatin and bivalent gene transcripts in pluripotent state transition.

HighlightsO_LIThe TET1 interactome identifies PSPC1 as a novel partner in ESCs
C_LIO_LITET1 and PSPC1 repress bivalent genes by promoting PRC2 chromatin occupancy
C_LIO_LINeat1 facilitates bivalent gene activation by promoting PRC2 binding to their mRNAs
C_LIO_LINeat1 bridges the TET1-PSPC1 and PRC2 complexes in regulating bivalent gene transcription
C_LI

category: developmental biology


title: Strong neutral sweeps occurring during a population contraction

abstract: A strong reduction in diversity around a specific locus is often interpreted as a recent rapid fixation of a positively selected allele, a phenomenon called a selective sweep. Rapid fixation of neutral variants can however lead to similar reduction in local diversity, especially when the population experiences changes in population size, e.g., bottlenecks or range expansions. The fact that demographic processes can lead to signals of nucleotide diversity very similar to signals of selective sweeps is at the core of an ongoing discussion about the roles of demography and natural selection in shaping patterns of neutral variation. Here we quantitatively investigate the shape of such neutral valleys of diversity under a simple model of a single population size change, and we compare it to signals of a selective sweep. We analytically describe the expected shape of such "neutral sweeps" and show that selective sweep valleys of diversity are, for the same fixation time, wider than neutral valleys. On the other hand, it is always possible to parametrize our model to find a neutral valley that has the same width as a given selected valley. We apply our framework to the case of a putative selective sweep signal around the gene Quetzalcoatl in D. melanogaster and show that the valley of diversity in the vicinity of this gene is compatible with a short bottleneck scenario without selection. Our findings provide further insight in how simple demographic models can create valleys of genetic diversity that may falsely be attributed to positive selection.


title: Instruments to assess Evidence-Based Practice among healthcare professionals: a systematic review

abstract: The study makes it possible to select the most appropriate instruments to evaluate the use of Evidence-Based Practice (EBP) among health professionals. The objective of this study was to assess the measurement properties, summarize and describe the instruments that evaluate the use of EBP in health professionals, currently available through the update of the systematic review. The study was conducted and reported according to recommendations of the PRISMA checklist. A systematic search was conducted in the databases: PubMed, Embase, CINAHL and ERIC. In addition, three groups of search terms: EBP terms; evaluation; cross-cultural adaptation and measurement proprieties. They included studies that showed assessment tools of EBP in healthcare workers in general publication of full-text scientific articles, which tested the measurement properties and publication of an article in English. Searches included published studies from 2006 until July 2020. Evaluation of the methodological quality of the studies was conducted according to the COSMIN initiative. 92 studies were included. Forty new instruments have been identified to assess EBP. From these, most were developed for nursing professionals and physiotherapists. More than 48% of studies have American and Australian English as their native language. Only 28% of the studies included students in the samples. Reliability was considered appropriate (sufficient) in 76% of the instruments. The COSMIN checklist classified 7 (seven) instruments as being suitable for use in the target audience. However, Fresno Test remains the most appropriate instrument for assessing the use of EBP in healthcare professionals. 40 new instruments that assess EBP have been identified. Most are consistent and reliable for measuring the use of EBP in healthcare professionals. The Fresno Test, in a list of seven reliable and valid instruments for analysis, remains the most used and the one that most assesses the domains of EBP.

category: scientific communication and education


title: Virtual Touch: Sensing and Feeling with Ultrasound.

abstract: The sense of touch codes the detection and properties of physical objects on the body via mechanoreceptors within the skin. Technological advancements, such as ultrasonic haptic devices, are now able to  touch without touching, claiming this is virtual touch. An initial aim of the study was to investigate subjective intensity and pleasantness ratings of ultrasound stimulation and the influence of top-down factors using the Touch Experience and Attitudes Questionnaire (TEAQ). Self-reported intensity and pleasantness ratings were measured in response to ultrasound stimuli. A second aim was to record from individual low threshold mechanoreceptors using the technique of microneurography in an attempt to determine which mechanoreceptors are activated by ultrasound stimulation of the skin. The major findings here were that microneurography found SAI and SAII units did not respond to ultrasound stimuli; intensity and pleasantness ratings were significantly different between age groups. Ultrasound can produce a variety of sensations with varying intensity and pleasantness ratings. A limitation of the study was the unexpected force difference generated between modulations. These findings have implications for mid-air haptics, somatosensory affective research, and virtual reality. Future research should focus on microneurography investigation of FA fibre responses to ultrasound.


title: Using mass Spectrometry to investigate fluorescent compounds in squirrel fur

abstract: While an array of taxa are capable of producing fluorescent pigments, fluorescence in mammals is a novel and poorly understood phenomenon. We believe that a first step towards understanding the potential adaptive functions of fluorescence in mammals is to develop an understanding of fluorescent compounds, or fluorophores, that are present in fluorescent tissue. Here we use Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) of flying squirrel fur known to fluoresce under ultraviolet (UV) light to identify potentially fluorescent compounds in squirrel fur. All of the potentially fluorescent compounds we identified were either present in non-fluorescent fur or were not present in all species of fluorescent flying squirrel. Therefore, we consider that the compounds responsible for fluorescence in flying squirrels may also be present in non-fluorescent mammal fur. Some factor currently unexplained likely leads to excitation of fluorophores in flying squirrel fur. A recently suggested hypothesis that fluorescence in mammals is widely caused by porphyrins is consistent with our findings.

category: ecology