Cellular function of the GndA small open reading frame-encoded polypeptide during heat shock.

Over the past 15 years, hundreds of previously undiscovered bacterial small open reading frame (sORF)-encoded polypeptides (SEPs) of fewer than fifty amino acids have been identified, and biological functions have been ascribed to an increasing number of SEPs from intergenic regions and small RNAs. However, despite numbering in the dozens in Escherichia coli, and hundreds to thousands in humans, same-strand nested sORFs that overlap protein coding genes in alternative reading frames remain understudied. In order to provide insight into this enigmatic class of unannotated genes, we characterized GndA, a 36-amino acid, heat shock-regulated SEP encoded within the +2 reading frame of the gnd gene in E. coli K-12 MG1655. We show that GndA pulls down components of respiratory complex I (RCI) and is required for proper localization of a RCI subunit during heat shock. At high temperature GndA deletion (ΔGndA) cells exhibit perturbations in cell growth, NADH+/NAD ratio, and expression of a number of genes including several associated with oxidative stress. These findings suggest that GndA may function in maintenance of homeostasis during heat shock. Characterization of GndA therefore supports the nascent but growing consensus that functional, overlapping genes occur in genomes from viruses to humans.

A transit-amplifying progenitor with biphasic behavior contributes to epidermal renewal.

Transit-amplifying (TA) cells are progenitors that undergo an amplification phase followed by transition into an extinction phase. A long postulated epidermal TA progenitor with biphasic behavior has not yet been experimentally observed in vivo. Here, we identify such a TA population using clonal analysis of Aspm-CreER genetic cell-marking in mice, which uncovers contribution to both homeostasis and injury repair of adult skin. This TA population is more frequently dividing than a Dlx1-CreER-marked long-term self-renewing (e.g. stem cell) population. Newly developed generalized birth-death modeling of long-term lineage tracing data shows that both TA progenitors and stem cells display neutral competition, but only the stem cells display neutral drift. The quantitative evolution of a nascent TA cell and its direct descendants shows that TA progenitors indeed amplify the basal layer before transition and that the homeostatic TA population is mostly in extinction phase. This model will be broadly useful for analyzing progenitors whose behavior changes with their clone age. This work identifies a long-missing class of non-self-renewing biphasic epidermal TA progenitors and has broad implications for understanding tissue renewal mechanisms.

Colloidal Synthesis of Monodisperse High-Entropy Spinel Oxide Nanocrystals.

Synthesis of high-entropy oxide (HEO) nanocrystals has focused on increasing the temperature in the entropy term (T(ΔS)) to overcome the enthalpy term. However, these high temperatures lead to large, polydisperse nanocrystals. In this work, we leverage the low solubility product (Ksp) of metal oxides and optimize the Lewis-acid-catalyzed esterification reaction for equal rate production of the cation monomers to synthesize HEO nanocrystals at low temperatures, producing the smallest (<4 nm) and most monodisperse (<15% size dispersity) HEOs to date. We apply these HEO nanocrystals as electrocatalysts, exhibiting promising activity toward the oxygen evolution reaction in alkaline media, with an overpotential of 345 mV at 10 mA/cm2.

Co-imaging of RelA and c-Rel reveals features of NF-κB signaling for ligand discrimination.

Individual cell sensing of external cues has evolved through the temporal patterns in signaling. Since nuclear factor κB (NF-κB) signaling dynamics have been examined using a single subunit, RelA, it remains unclear whether more information might be transmitted via other subunits. Using NF-κB double-knockin reporter mice, we monitored both canonical NF-κB subunits, RelA and c-Rel, simultaneously in single macrophages by quantitative live-cell imaging. We show that signaling features of RelA and c-Rel convey more information about the stimuli than those of either subunit alone. Machine learning is used to predict the ligand identity accurately based on RelA and c-Rel signaling features without considering the co-activated factors. Ligand discrimination is achieved through selective non-redundancy of RelA and c-Rel signaling dynamics, as well as their temporal coordination. These results suggest a potential role of c-Rel in fine-tuning immune responses and highlight the need for approaches that will elucidate the mechanisms regulating NF-κB subunit specificity.

GABAergic/Glycinergic and Glutamatergic Neurons Mediate Distinct Neurodevelopmental Phenotypes of STXBP1 Encephalopathy.

An increasing number of pathogenic variants in presynaptic proteins involved in the synaptic vesicle cycle are being discovered in neurodevelopmental disorders. The clinical features of these synaptic vesicle cycle disorders are diverse, but the most prevalent phenotypes include intellectual disability, epilepsy, movement disorders, cerebral visual impairment, and psychiatric symptoms ( Verhage and Sørensen, 2020; Bonnycastle et al., 2021; John et al., 2021; Melland et al., 2021). Among this growing list of synaptic vesicle cycle disorders, the most frequent is STXBP1 encephalopathy caused by de novo heterozygous pathogenic variants in syntaxin-binding protein 1 (STXBP1, also known as MUNC18-1; Verhage and Sørensen, 2020; John et al., 2021). STXBP1 is an essential protein for presynaptic neurotransmitter release. Its haploinsufficiency is the main disease mechanism and impairs both excitatory and inhibitory neurotransmitter release. However, the disease pathogenesis and cellular origins of the broad spectrum of neurological phenotypes are poorly understood. Here we generate cell type-specific Stxbp1 haploinsufficient male and female mice and show that Stxbp1 haploinsufficiency in GABAergic/glycinergic neurons causes developmental delay, epilepsy, and motor, cognitive, and psychiatric deficits, recapitulating majority of the phenotypes observed in the constitutive Stxbp1 haploinsufficient mice and STXBP1 encephalopathy. In contrast, Stxbp1 haploinsufficiency in glutamatergic neurons results in a small subset of cognitive and seizure phenotypes distinct from those caused by Stxbp1 haploinsufficiency in GABAergic/glycinergic neurons. Thus, the contrasting roles of excitatory and inhibitory signaling reveal GABAergic/glycinergic dysfunction as a key disease mechanism of STXBP1 encephalopathy and suggest the possibility to selectively modulate disease phenotypes by targeting specific neurotransmitter systems.

UbiNAAT: a multiplexed point-of-care nucleic acid diagnostic platform for rapid at-home pathogen detection.

The COVID-19 pandemic increased demands for respiratory disease testing to facilitate treatment and limit transmission, demonstrating in the process that most existing test options were too complex and expensive to perform in point-of-care or home scenarios. Lab-based molecular techniques can detect viral RNA in respiratory illnesses but are expensive and require trained personnel, while affordable antigen-based home tests lack sensitivity for early detection in newly infected or asymptomatic individuals. The few home RNA detection tests deployed were prohibitively expensive. Here, we demonstrate a point-of-care, paper-based rapid analysis device that simultaneously detects multiple viral RNAs; it is demonstrated on two common respiratory viruses (COVID-19 and influenza A) spiked onto a commercial nasal swab. The automated device requires no sample preparation by the user after insertion of the swab, minimizing user operation steps. We incorporated lyophilized amplification reagents immobilized in a porous matrix, a novel thermally actuated valve for multiplexed fluidic control, a printed circuit board that performs on-device lysis and amplification within a cell-phone-sized disposable device. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) products are visualized via fluorescent dyes using a modified cell phone, resulting in detection of as few as 104 viral copies per swab across both pathogens within 30 minutes. This integrated platform could be commercialized in a form that would be inexpensive, portable, and sensitive; it can readily be multiplexed to detect as many as 8 different RNA or DNA sequences, and adapted to any desired RNA or DNA detection assays.

Scalable Route to Colloidal NixCo3-xS4 Nanoparticles with Low Dispersity Using Amino Acids.

The thiospinel group of nickel cobalt sulfides (NixCo3-xS4) are promising materials for energy applications such as supercapacitors, fuel cells, and solar cells. Solution-processible nanoparticles of NixCo3-xS4 have advantages of low cost and fabrication of high-performance energy devices due to their high surface-to-volume ratio, which increases the electrochemically active surface area and shortens the ionic diffusion path. The current approaches to synthesize NixCo3-xS4 nanoparticles are often based on hydrothermal or solvothermal methods that are difficult to scale up safely and efficiently and that preclude monitoring the reaction through aliquots, making optimization of size and dispersity challenging, typically resulting in aggregated nanoparticles with polydisperse sizes. In this work, we report a scalable "heat-up" method to colloidally synthesize NixCo3-xS4 nanoparticles that are smaller than 15 nm in diameter with less than 15% in size dispersion, using two inexpensive, earth-abundant sulfur sources. Our method provides a reliable synthetic pathway to produce phase-pure, low-dispersity, gram-scale nanoparticles of ternary metal sulfides. This method enhances the current capabilities of NixCo3-xS4 nanoparticles to meet the performance demands to improve renewable energy technologies.

Unannotated microprotein EMBOW regulates the interactome and chromatin and mitotic functions of WDR5.

The conserved WD40-repeat protein WDR5 interacts with multiple proteins both inside and outside the nucleus. However, it is currently unclear whether and how the distribution of WDR5 between complexes is regulated. Here, we show that an unannotated microprotein EMBOW (endogenous microprotein binder of WDR5) dually encoded in the human SCRIB gene interacts with WDR5 and regulates its binding to multiple interaction partners, including KMT2A and KIF2A. EMBOW is cell cycle regulated, with two expression maxima at late G1 phase and G2/M phase. Loss of EMBOW decreases WDR5 interaction with KIF2A, aberrantly shortens mitotic spindle length, prolongs G2/M phase, and delays cell proliferation. In contrast, loss of EMBOW increases WDR5 interaction with KMT2A, leading to WDR5 binding to off-target genes, erroneously increasing H3K4me3 levels, and activating transcription of these genes. Together, these results implicate EMBOW as a regulator of WDR5 that regulates its interactions and prevents its off-target binding in multiple contexts.

General Route to Colloidally Stable, Low-Dispersity Manganese-Based Ternary Spinel Oxide Nanocrystals.

While certain ternary spinel oxides have been well-explored with colloidal nanochemistry, notably the ferrite spinel family, ternary manganese (Mn)-based spinel oxides have not been tamed. A key composition is cobalt (Co)-Mn oxide (CMO) spinel, CoxMn3-xO4, that, despite exemplary performance in multiple electrochemical applications, has few reports in the colloidal literature. Of these reports, most show aggregated and polydisperse products. Here, we describe a synthetic method for small, colloidally stable CMO spinel nanocrystals with tunable composition and low dispersity. By reacting 2+ metal-acetylacetonate (M(acac)2) precursors in an amine solvent under an oxidizing environment, we developed a pathway that avoids the highly reducing conditions of typical colloidal synthesis reactions; these reducing conditions typically push the system toward a monoxide impurity phase. Through surface chemistry studies, we identify organic byproducts and their formation mechanism, enabling us to engineer the surface and obtain colloidally stable nanocrystals with low organic loading. We report a CMO/carbon composite with low organic contents that performs the oxygen reduction reaction (ORR) with a half-wave potential (E1/2) of 0.87 V vs RHE in 1.0 M potassium hydroxide at 1600 rpm, rivaling previous reports for the highest activity of this material in ORR electrocatalysis. We extend the general applicability of this procedure to other Mn-based spinel nanocrystals such as Zn-Mn-O, Fe-Mn-O, Ni-Mn-O, and Cu-Mn-O. Finally, we show the scalability of this method by producing inorganic nanocrystals at the gram scale.

How unexpected events are processed in theory of mind regions: A conceptual replication.

Recent research in social neuroscience has postulated that Theory of Mind (ToM) regions play a role in processing social prediction error (PE: the difference between what was expected and what was observed). Here, we tested whether PE signal depends on the type of prior information people use to make predictions - an agent's prior mental states (e.g., beliefs, desires, preferences) or an agent's prior behavior - as well as the type of information that confirms or violates such predictions. That is, does prior information about mental states (versus behavior) afford stronger predictions about an agent's subsequent mental states or behaviors? Additionally, when information about an agent's prior mental states or behavior is available, is PE signal strongest when information about an agent's subsequent mental state (vs behavior) is revealed? In line with prior research, results suggest that DMPFC, LTPJ, and RTPJ are recruited more for unexpected than expected outcomes. However, PE signal does not seem to discriminate on the basis of prior or outcome information type. These findings suggest that ToM regions may flexibly incorporate any available information to make predictions about, monitor, and perhaps explain, inconsistencies in social agents.

GWAS Explorer: an open-source tool to explore, visualize, and access GWAS summary statistics in the PLCO Atlas.

The Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial is a prospective cohort study of nearly 155,000 U.S. volunteers aged 55-74 at enrollment in 1993-2001. We developed the PLCO Atlas Project, a large resource for multi-trait genome-wide association studies (GWAS), by genotyping participants with available DNA and genomic consent. Genotyping on high-density arrays and imputation was performed, and GWAS were conducted using a custom semi-automated pipeline. Association summary statistics were generated from a total of 110,562 participants of European, African and Asian ancestry. Application programming interfaces (APIs) and open-source software development kits (SKDs) enable exploring, visualizing and open data access through the PLCO Atlas GWAS Explorer website, promoting Findable, Accessible, Interoperable, and Re-usable (FAIR) principles. Currently the GWAS Explorer hosts association data for 90 traits and >78,000,000 genomic markers, focusing on cancer and cancer-related phenotypes. New traits will be posted as association data becomes available. The PLCO Atlas is a FAIR resource of high-quality genetic and phenotypic data with many potential reuse opportunities for cancer research and genetic epidemiology.

Kinetics of mRNA nuclear export regulate innate immune response gene expression.

The abundance and stimulus-responsiveness of mature mRNA is thought to be determined by nuclear synthesis, processing, and cytoplasmic decay. However, the rate and efficiency of moving mRNA to the cytoplasm almost certainly contributes, but has rarely been measured. Here, we investigated mRNA export rates for innate immune genes. We generated high spatio-temporal resolution RNA-seq data from endotoxin-stimulated macrophages and parameterized a mathematical model to infer kinetic parameters with confidence intervals. We find that the effective chromatin-to-cytoplasm export rate is gene-specific, varying 100-fold: for some genes, less than 5% of synthesized transcripts arrive in the cytoplasm as mature mRNAs, while others show high export efficiency. Interestingly, effective export rates do not determine temporal gene responsiveness, but complement the wide range of mRNA decay rates; this ensures similar abundances of short- and long-lived mRNAs, which form successive innate immune response expression waves.

Binary organization of epidermal basal domains highlights robustness to environmental exposure.

Adulte interfollicular epidermis (IFE) renewal is likely orchestrated by physiological demands of its complex tissue architecture comprising spatial and cellular heterogeneity. Mouse tail and back skin display two kinds of basal IFE spatial domains that regenerate at different rates. Here, we elucidate the molecular and cellular states of basal IFE domains by marker expression and single-cell transcriptomics in mouse and human skin. We uncover two paths of basal cell differentiation that in part reflect the IFE spatial domain organization. We unravel previously unrecognized similarities between mouse tail IFE basal domains defined as scales and interscales versus human rete ridges and inter-ridges, respectively. Furthermore, our basal IFE transcriptomics and gene targeting in mice provide evidence supporting a physiological role of IFE domains in adaptation to differential UV exposure. We identify Sox6 as a novel UV-induced and interscale/inter-ridge preferred basal IFE-domain transcription factor, important for IFE proliferation and survival. The spatial, cellular, and molecular organization of IFE basal domains underscores skin adaptation to environmental exposure and its unusual robustness in adult homeostasis.

PLCOjs, a FAIR GWAS web SDK for the NCI Prostate, Lung, Colorectal and Ovarian Cancer Genetic Atlas project.

MOTIVATION: The Division of Cancer Epidemiology and Genetics (DCEG) and the Division of Cancer Prevention (DCP) at the National Cancer Institute (NCI) have recently generated genome-wide association study (GWAS) data for multiple traits in the Prostate, Lung, Colorectal, and Ovarian (PLCO) Genomic Atlas project. The GWAS included 110 000 participants. The dissemination of the genetic association data through a data portal called GWAS Explorer, in a manner that addresses the modern expectations of FAIR reusability by data scientists and engineers, is the main motivation for the development of the open-source JavaScript software development kit (SDK) reported here. RESULTS: The PLCO GWAS Explorer resource relies on a public stateless HTTP application programming interface (API) deployed as the sole backend service for both the landing page's web application and third-party analytical workflows. The core PLCOjs SDK is mapped to each of the API methods, and also to each of the reference graphic visualizations in the GWAS Explorer. A few additional visualization methods extend it. As is the norm with web SDKs, no download or installation is needed and modularization supports targeted code injection for web applications, reactive notebooks (Observable) and node-based web services. AVAILABILITY AND IMPLEMENTATION: code at https://github.com/episphere/plco; project page at https://episphere.github.io/plco.

CK2 alpha prime and alpha-synuclein pathogenic functional interaction mediates synaptic dysregulation in huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the HTT gene for which no therapies are available. HTT mutation causes protein misfolding and aggregation, preferentially affecting medium spiny neurons (MSNs) of the basal ganglia. Transcriptional perturbations in synaptic genes and neuroinflammation are key processes that precede MSN dysfunction and motor symptom onset. Understanding the interplay between these processes is crucial to develop effective therapeutic strategies to treat HD. We investigated the role of protein kinase CK2α', a kinase upregulated in MSNs in HD and previously associated with Parkinson's disease (PD), in the regulation of neuroinflammation and synaptic function in HD. We used the heterozygous knock-in zQ175 HD mouse model and compared that to zQ175 mice lacking one allele of CK2α' (zQ175:CK2α'(±)). CK2α' haploinsufficiency in zQ175 mice resulted in decreased levels of pro-inflammatory cytokines, HTT aggregation, astrogliosis and transcriptional alterations of synaptic genes related to glutamatergic signaling. zQ175:CK2α'(±) mice also presented increased frequency of striatal miniature excitatory postsynaptic currents (mEPSCs), an indicator of synaptic activity, and improved motor coordination compared to zQ175 mice. Neuropathological and phenotypic changes mediated by CK2α' were connected to alpha-synuclein (α-syn) dysregulation and correlated with differences in α-syn serine 129 phosphorylation (pS129-α-syn), a post-translational modification involved in α-synucleinopathy and shown to be regulated by CK2 in PD. pS129-α-syn was increased in the nuclei of MSNs in zQ175 mice and in the striatum of patients with HD, and it decreased in zQ175:CK2α'(±) mice. Collectively, our data established a novel connection between CK2α', neuroinflammation and synaptic gene dysregulation with synucleinopathy in HD and suggested common molecular mechanisms of neurodegeneration between HD and PD. Our results also support CK2α' inhibition as a potential therapeutic strategy to modulate neuronal function and neuroprotection in HD.

Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues.

Macrophages are versatile cells of the innate immune system that perform diverse functions by responding to dynamic changes in their microenvironment. While the effects of soluble cues, including cytokines and chemokines, have been widely studied, the effects of physical cues, including mechanical stimuli, in regulating macrophage form and function are less well understood. In this study, we examined the effects of static and cyclic uniaxial stretch on macrophage inflammatory and healing activation. We found that cyclic stretch altered macrophage morphology and responses to IFNγ/LPS and IL4/IL13. Interestingly, we found that both static and cyclic stretch suppressed IFNγ/LPS induced inflammation. In contrast, IL4/IL13 mediated healing responses were suppressed with cyclic but enhanced with static stretch conditions. Mechanistically, both static and cyclic stretch increased expression of the integrin CD11b (αM integrin), decreased expression of the mechanosensitive ion channel Piezo1, and knock down of either CD11b or Piezo1 through siRNA abrogated stretch-mediated changes in inflammatory responses. Moreover, we found that knock down of CD11b enhanced the expression of Piezo1, and conversely knock down of Piezo1 enhanced CD11b expression, suggesting the potential for crosstalk between integrins and ion channels. Finally, stretch-mediated differences in macrophage activation were also dependent on actin, since pharmacological inhibition of actin polymerization abrogated the changes in activation with stretch. Together, this study demonstrates that the physical environment synergizes with biochemical cues to regulate macrophage morphology and function, and suggests a role for CD11b and Piezo1 crosstalk in mechanotransduction in macrophages.