The Striatum Organizes 3D Behavior via Moment-to-Moment Action Selection.

Many naturalistic behaviors are built from modular components that are expressed sequentially. Although striatal circuits have been implicated in action selection and implementation, the neural mechanisms that compose behavior in unrestrained animals are not well understood. Here, we record bulk and cellular neural activity in the direct and indirect pathways of dorsolateral striatum (DLS) as mice spontaneously express action sequences. These experiments reveal that DLS neurons systematically encode information about the identity and ordering of sub-second 3D behavioral motifs; this encoding is facilitated by fast-timescale decorrelations between the direct and indirect pathways. Furthermore, lesioning the DLS prevents appropriate sequence assembly during exploratory or odor-evoked behaviors. By characterizing naturalistic behavior at neural timescales, these experiments identify a code for elemental 3D pose dynamics built from complementary pathway dynamics, support a role for DLS in constructing meaningful behavioral sequences, and suggest models for how actions are sculpted over time.

Structure of a Complete Mediator-RNA Polymerase II Pre-Initiation Complex.

A complete, 52-protein, 2.5 million dalton, Mediator-RNA polymerase II pre-initiation complex (Med-PIC) was assembled and analyzed by cryo-electron microscopy and by chemical cross-linking and mass spectrometry. The resulting complete Med-PIC structure reveals two components of functional significance, absent from previous structures, a protein kinase complex and the Mediator-activator interaction region. It thereby shows how the kinase and its target, the C-terminal domain of the polymerase, control Med-PIC interaction and transcription.

Tracking chromatin state changes using nanoscale photo-proximity labelling.

Interactions between biomolecules underlie all cellular processes and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects1,2. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health1. However, in the complex environment of the nucleus, it is challenging to determine protein-protein interactions owing to low abundance, transient or multivalent binding and a lack of technologies that are able to interrogate these interactions without disrupting the protein-binding surface under study3. Here, we describe a method for the traceless incorporation of iridium-photosensitizers into the nuclear micro-environment using engineered split inteins. These Ir-catalysts can activate diazirine warheads through Dexter energy transfer to form reactive carbenes within an approximately 10 nm radius, cross-linking with proteins in the immediate micro-environment (a process termed µMap) for analysis using quantitative chemoproteomics4. We show that this nanoscale proximity-labelling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. µMap improves our fundamental understanding of nuclear protein-protein interactions and, in doing so, is expected to have a significant effect on the field of epigenetic drug discovery in both academia and industry.

Mediator structure and conformation change.

Mediator is a universal adaptor for transcription control. It serves as an interface between gene-specific activator or repressor proteins and the general RNA polymerase II (pol II) transcription machinery. Previous structural studies revealed a relatively small part of Mediator and none of the gene activator-binding regions. We have determined the cryo-EM structure of the Mediator at near-atomic resolution. The structure reveals almost all amino acid residues in ordered regions, including the major targets of activator proteins, the Tail module, and the Med1 subunit of the Middle module. Comparison of Mediator structures with and without pol II reveals conformational changes that propagate across the entire Mediator, from Head to Tail, coupling activator- and pol II-interacting regions.

Bumping up kinase activity with an ATP-derived neo-substrate.

Pharmacologic agents capable of increasing kinase function would be useful for treating diseases associated with reduced kinase activity, such as inherited forms of Parkinson's disease. In this issue, Hertz et al. report an innovative approach for activating the Parkinson's-associated kinase PINK1 in cells with an ATP-derived neo-substrate.

Atezolizumab for Advanced Alveolar Soft Part Sarcoma.

BACKGROUND: Alveolar soft part sarcoma (ASPS) is a rare soft-tissue sarcoma with a poor prognosis and no established therapy. Recently, encouraging responses to immune checkpoint inhibitors have been reported. METHODS: We conducted an investigator-initiated, multicenter, single-group, phase 2 study of the anti-programmed death ligand 1 (PD-L1) agent atezolizumab in adult and pediatric patients with advanced ASPS. Atezolizumab was administered intravenously at a dose of 1200 mg (in patients ≥18 years of age) or 15 mg per kilogram of body weight with a 1200-mg cap (in patients <18 years of age) once every 21 days. Study end points included objective response, duration of response, and progression-free survival according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, as well as pharmacodynamic biomarkers of multistep drug action. RESULTS: A total of 52 patients were evaluated. An objective response was observed in 19 of 52 patients (37%), with 1 complete response and 18 partial responses. The median time to response was 3.6 months (range, 2.1 to 19.1), the median duration of response was 24.7 months (range, 4.1 to 55.8), and the median progression-free survival was 20.8 months. Seven patients took a treatment break after 2 years of treatment, and their responses were maintained through the data-cutoff date. No treatment-related grade 4 or 5 adverse events were recorded. Responses were noted despite variable baseline expression of programmed death 1 and PD-L1. CONCLUSIONS: Atezolizumab was effective at inducing sustained responses in approximately one third of patients with advanced ASPS. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT03141684.).

Profiling dynamic RNA-protein interactions using small-molecule-induced RNA editing.

RNA-binding proteins (RBPs) play an important role in biology, and characterizing dynamic RNA-protein interactions is essential for understanding RBP function. In this study, we developed targets of RBPs identified by editing induced through dimerization (TRIBE-ID), a facile strategy for quantifying state-specific RNA-protein interactions upon rapamycin-mediated chemically induced dimerization and RNA editing. We performed TRIBE-ID with G3BP1 and YBX1 to study RNA-protein interactions during normal conditions and upon oxidative stress-induced biomolecular condensate formation. We quantified editing kinetics to infer interaction persistence and show that stress granule formation strengthens pre-existing RNA-protein interactions and induces new RNA-protein binding events. Furthermore, we demonstrate that G3BP1 stabilizes its targets under normal and oxidative stress conditions independent of stress granule formation. Finally, we apply our method to characterize small-molecule modulators of G3BP1-RNA binding. Taken together, our work provides a general approach to profile dynamic RNA-protein interactions in cellular contexts with temporal control.

Massively parallel, computationally guided design of a proenzyme.

Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design. For a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effectively designed variants shows that they are inhibited by ∼80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro- and catalytic domains based on the design models increases the degree of inhibition to 98% but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing proenzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the prodomain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of proproteins with precise spatial regulation.

Highly Pathogenic Avian Influenza A(H5N1) Viruses from Multispecies Outbreak, Argentina, August 2023.

We report full-genome characterization of highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b virus from an outbreak among sea lions (August 2023) in Argentina and possible spillover to fur seals and terns. Mammalian adaptation mutations in virus isolated from marine mammals and a human in Chile were detected in mammalian and avian hosts.

Efficient Photochemical Vapor Generation from Low Concentration Formic Acid Media.

Herein, we report on surprisingly efficient photochemical vapor generation (PVG) of Ru, Re, and especially Ir, achieved from very dilute HCOOH media employing a thin-film flow-through photoreactor operated in flow injection mode. In the absence of added metal ion sensitizers, efficiencies near 20% for Ir and approximately 0.06% for Ru and Re occur in a narrow range of HCOOH concentrations (around 0.01 M), significantly higher than previously reported from conventionally optimized HCOOH concentrations (1-20 M). A substantial enhancement in efficiency, to around 9 and 1.5%, could be realized for Ru and Re, respectively, when 0.005 M HCOONa served as the PVG medium. The addition of metal ion sensitizers (particularly Cd2+ and Co2+) to 0.01 M HCOOH significantly enhanced PVG efficiencies to 17, 2.2, and 81% for Ru, Re, and Ir, respectively. Possible mechanistic aspects occurring in dilute HCOOH media are discussed, wherein this phenomenon is attributed to the action of 185 nm radiation available in the thin-film flow-through photoreactor. An extended study of PVG of Fe, Co, Ni, As, Se, Mo, Rh, Te, W, and Bi from both dilute HCOOH and CH3COOH was undertaken, and several elements for which a similar phenomenon appears were identified (i.e., Co, As, Se, Te, and Bi). Although use of dilute HCOOH media is attractive for practical analytical applications employing PVG, it is less tolerant toward dissolved gases and interferents in the liquid phase due to the likely lower concentrations of free radicals and aquated electrons required for analyte ion reduction and product synthesis.

Deuterium metabolic imaging for 3D mapping of glucose metabolism in humans with central nervous system lesions at 3T.

PURPOSE: To explore the potential of 3T deuterium metabolic imaging (DMI) using a birdcage 2 H radiofrequency (RF) coil in both healthy volunteers and patients with central nervous system (CNS) lesions. METHODS: A modified gradient filter, home-built 2 H volume RF coil, and spherical k-space sampling were employed in a three-dimensional chemical shift imaging acquisition to obtain high-quality whole-brain metabolic images of 2 H-labeled water and glucose metabolic products. These images were acquired in a healthy volunteer and three subjects with CNS lesions of varying pathologies. Hardware and pulse sequence experiments were also conducted to improve the signal-to-noise ratio of DMI at 3T. RESULTS: The ability to quantify local glucose metabolism in correspondence to anatomical landmarks across patients with varying CNS lesions is demonstrated, and increased lactate is observed in one patient with the most active disease. CONCLUSION: DMI offers the potential to examine metabolic activity in human subjects with CNS lesions with DMI at 3T, promising for the potential of the future clinical translation of this metabolic imaging technique.

Chemoproteomic Approaches to Studying RNA Modification-Associated Proteins.

The function of cellular RNA is modulated by a host of post-transcriptional chemical modifications installed by dedicated RNA-modifying enzymes. RNA modifications are widespread in biology, occurring in all kingdoms of life and in all classes of RNA molecules. They regulate RNA structure, folding, and protein-RNA interactions, and have important roles in fundamental gene expression processes involving mRNA, tRNA, rRNA, and other types of RNA species. Our understanding of RNA modifications has advanced considerably; however, there are still many outstanding questions regarding the distribution of modifications across all RNA transcripts and their biological function. One of the major challenges in the study of RNA modifications is the lack of sequencing methods for the transcriptome-wide mapping of different RNA-modification structures. Furthermore, we lack general strategies to characterize RNA-modifying enzymes and RNA-modification reader proteins. Therefore, there is a need for new approaches to enable integrated studies of RNA-modification chemistry and biology.In this Account, we describe our development and application of chemoproteomic strategies for the study of RNA-modification-associated proteins. We present two orthogonal methods based on nucleoside and oligonucleotide chemical probes: 1) RNA-mediated activity-based protein profiling (RNABPP), a metabolic labeling strategy based on reactive modified nucleoside probes to profile RNA-modifying enzymes in cells and 2) photo-cross-linkable diazirine-containing synthetic oligonucleotide probes for identifying RNA-modification reader proteins.We use RNABPP with C5-modified cytidine and uridine nucleosides to capture diverse RNA-pyrimidine-modifying enzymes including methyltransferases, dihydrouridine synthases, and RNA dioxygenase enzymes. Metabolic labeling facilitates the mechanism-based cross-linking of RNA-modifying enzymes with their native RNA substrates in cells. Covalent RNA-protein complexes are then isolated by denaturing oligo(dT) pulldown, and cross-linked proteins are identified by quantitative proteomics. Once suitable modified nucleosides have been identified as mechanism-based proteomic probes, they can be further deployed in transcriptome-wide sequencing experiments to profile the substrates of RNA-modifying enzymes at nucleotide resolution. Using 5-fluorouridine-mediated RNA-protein cross-linking and sequencing, we analyzed the substrates of human dihydrouridine synthase DUS3L. 5-Ethynylcytidine-mediated cross-linking enabled the investigation of ALKBH1 substrates. We also characterized the functions of these RNA-modifying enzymes in human cells by using genetic knockouts and protein translation reporters.We profiled RNA readers for N6-methyladenosine (m6A) and N1-methyladenosine (m1A) using a comparative proteomic workflow based on diazirine-containing modified oligonucleotide probes. Our approach enables quantitative proteome-wide analysis of the preference of RNA-binding proteins for modified nucleotides across a range of affinities. Interestingly, we found that YTH-domain proteins YTHDF1/2 can bind to both m6A and m1A to mediate transcript destabilization. Furthermore, m6A also inhibits stress granule proteins from binding to RNA.Taken together, we demonstrate the application of chemical probing strategies, together with proteomic and transcriptomic workflows, to reveal new insights into the biological roles of RNA modifications and their associated proteins.

Investigating the origin of the 13 C lactate signal in the anesthetized healthy rat brain in vivo after hyperpolarized [1-13 C]pyruvate injection.

The goal of this study was to investigate the origin of brain lactate (Lac) signal in the healthy anesthetized rat after injection of hyperpolarized (HP) [1-13 C]pyruvate (Pyr). Dynamic two-dimensional spiral chemical shift imaging with flow-sensitizing gradients revealed reduction in both vascular and brain Pyr, while no significant dependence on the level of flow suppression was detected for Lac. These results support the hypothesis that the HP metabolites predominantly reside in different compartments in the brain (i.e., Pyr in the blood and Lac in the parenchyma). Data from high-resolution metabolic imaging of [1-13 C]Pyr further demonstrated that Lac detected in the brain was not from contributions of vascular signal attributable to partial volume effects. Additionally, metabolite distributions and kinetics measured with dynamic imaging after injection of HP [1-13 C]Lac were similar to Pyr data when Pyr was used as the substrate. These data do not support the hypothesis that Lac observed in the brain after Pyr injection was generated in other organs and then transported across the blood-brain barrier (BBB). Together, the presented results provide further evidence that even in healthy anesthetized rats, the transport of HP Pyr across the BBB is sufficiently fast to permit detection of its metabolic conversion to Lac within the brain.

Characterization of arsenic species by liquid sampling-atmospheric pressure glow discharge ionization mass spectrometry.

A liquid sampling-atmospheric pressure glow discharge (LS-APGD) ionization source operating at a nominal power of 30 W and solution flow rate of 30 µL min-1 and supported in a He sheath gas flow rate of 500 mL min-1 was interfaced to an Orbitrap mass spectrometer and assessed for use in rapid identification of inorganic and organic arsenic species, including As(III), As(V), monomethylarsonic acid, dimethylarsinic acid, and arsenobetaine in a 2% (v/v) nitric acid medium. Mass spectral acquisition in low-resolution mode, using only the ion trap analyzer, provided detection of protonated molecular ions for AsBet (m/z 179), DMA (m/z 139), MMA (m/z 141), and As(V) (m/z 143). As(III) is oxidized to As(V), likely due to in-source processes. Typical fragmentation of these compounds resulted in the loss of either water or methyl groups, as appropriate, i.e., introducing DMA also generated ions corresponding to MMA and As(V) as dissociation products. Structure assignments were also confirmed by high-resolution Orbitrap measurements. Spectral fingerprint assignments were based on the introduction of solutions containing 5 µg mL-1 of each arsenic compound.

Chemoproteomic Profiling of 8-Oxoguanosine-Sensitive RNA-Protein Interactions.

Cellular nucleic acids are subject to assault by endogenous and exogenous agents that can perturb the flow of genetic information. Oxidative stress leads to the accumulation of 8-oxoguanine (8OG) in DNA and RNA. 8OG lesions on mRNA negatively impact translation, but their effect on global RNA-protein interactions is largely unknown. Here, we apply an RNA chemical proteomics approach to investigate the effect of 8OG on RNA-protein binding. We find proteins that bind preferentially to 8OG-modified RNA, including IGF2BP1-3 and hnRNPD, and proteins that are repelled by 8OG such as RBM4. We characterize these interactions using biochemical and biophysical assays to quantify the effect of 8OG on binding and show that a single 8OG abolishes the binding of RBM4 to its preferred CGG-containing substrate. Taken together, our work establishes the molecular consequences of 8OG on cellular RNA-protein binding and provides a framework for interrogating the role of RNA oxidation in biological systems.

Global Discovery of Covalent Modulators of Ribonucleoprotein Granules.

Stress granules (SGs) and processing-bodies (PBs, P-bodies) are ubiquitous and widely studied ribonucleoprotein (RNP) granules involved in cellular stress response, viral infection, and the tumor microenvironment. While proteomic and transcriptomic investigations of SGs and PBs have provided insights into molecular composition, chemical tools to probe and modulate RNP granules remain lacking. Herein, we combine an immunofluorescence (IF)-based phenotypic screen with chemoproteomics to identify sulfonyl-triazoles (SuTEx) capable of preventing or inducing SG and PB formation through liganding of tyrosine (Tyr) and lysine (Lys) sites in stressed cells. Liganded sites were enriched for RNA-binding and protein-protein interaction (PPI) domains, including several sites found in RNP granule-forming proteins. Among these, we functionally validate G3BP1 Y40, located in the NTF2 dimerization domain, as a ligandable site that can disrupt arsenite-induced SG formation in cells. In summary, we present a chemical strategy for the systematic discovery of condensate-modulating covalent small molecules.

Highly Efficient Photochemical Vapor Generation for Sensitive Determination of Iridium by Inductively Coupled Plasma Mass Spectrometry.

Herein, we describe the highly efficient photochemical vapor generation (PVG) of a volatile species of Ir (presumably iridium tetracarbonyl hydride) for subsequent detection by inductively coupled plasma mass spectrometry (ICPMS). A thin-film flow-through photoreactor, operated in flow injection mode, provided high efficiency following optimization of identified key PVG parameters, notably, irradiation time, pH of the reaction medium, and the presence of metal sensitizers. For routine use and analytical application, PVG conditions comprising 4 M formic acid as the reaction medium, the presence of 10 mg L-1 Co2+ and 25 mg L-1 Cd2+ as added sensitizers, and an irradiation time of 29 s were chosen. An almost 90% overall PVG efficiency for both Ir3+ and Ir4+ oxidation states was accompanied by excellent repeatability of 1.0% (n = 15) of the peak area response from a 50 ng L-1 Ir standard. Limits of detection ranged from 3 to 6 pg L-1 (1.5-3 fg absolute), dependent on use of the ICPMS reaction/collision cell. Interferences from several transition metals and metalloids as well as inorganic acids and their anions were investigated, and outstanding tolerance toward chloride was found. Accuracy of the developed methodology was verified by analysis of NIST SRM 2556 (Used Auto Catalyst) following peroxide fusion for sample preparation. Practical application was further demonstrated by the direct analysis of spring water, river water, lake water, and two seawater samples with around 100% spike recovery and no sample preparation except the addition of formic acid and the sensitizers.

Activity-based RNA-modifying enzyme probing reveals DUS3L-mediated dihydrouridylation.

Epitranscriptomic RNA modifications can regulate RNA activity; however, there remains a major gap in our understanding of the RNA chemistry present in biological systems. Here we develop RNA-mediated activity-based protein profiling (RNABPP), a chemoproteomic strategy that relies on metabolic RNA labeling, mRNA interactome capture and quantitative proteomics, to investigate RNA-modifying enzymes in human cells. RNABPP with 5-fluoropyrimidines allowed us to profile 5-methylcytidine (m5C) and 5-methyluridine (m5U) methyltransferases. Further, we uncover a new mechanism-based crosslink between 5-fluorouridine (5-FUrd)-modified RNA and the dihydrouridine synthase (DUS) homolog DUS3L. We investigate the mechanism of crosslinking and use quantitative nucleoside liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and 5-FUrd-based crosslinking and immunoprecipitation (CLIP) sequencing to map DUS3L-dependent dihydrouridine (DHU) modifications across the transcriptome. Finally, we show that DUS3L-knockout (KO) cells have compromised protein translation rates and impaired cellular proliferation. Taken together, our work provides a general approach for profiling RNA-modifying enzyme activity in living cells and reveals new pathways for epitranscriptomic RNA regulation.

Haemoproteus jenniae (Haemoproteidae, Haemosporida) infects gulls (Larus spp.) in South Africa, with redescription of Haemoproteus skuae.

Haemoproteus spp. are dipteran-borne protozoa that infect erythrocytes and reticulo-endothelial cells of birds. These parasites are not usually transmitted between birds belonging to different orders. The suborder Lari (order Charadriiformes) comprises ~170 avian species, the majority of which are aquatic, including gulls, terns, auklets, murres and skuas, among others. In spite of the diversity of this avian group, there is limited known diversity of haemosporidian parasites, with only 4 recorded Haemoproteus morphospecies thus far. We examined the blood smears of 21 kelp gulls (Larus dominicanus) captured at a breeding colony in South Africa, as well as Haemoproteus-positive archival blood smears of 15 kelp gulls and 1 Hartlaub's gull (Larus hartlaubii) sampled while under care at seabird rehabilitation facilities in South Africa. Haemoproteus sp. infection was detected in 19% of wild-caught kelp gulls. All parasites from the gulls were morphologically identified as Haemoproteus jenniae, a species previously recorded in Lari birds at the Galapagos Islands (Ecuador), Rocas Atoll (Brazil) and Poland. Gene sequencing uncovered a new cytochrome b lineage, LARDOM01, which was closely related to the previously reported H. jenniae lineage CREFUR01. Additionally, we evaluated a hapantotype blood smear of Haemoproteus skuae, which had been described infecting a brown skua (Catharacta antarctica) in South Africa. We provide a redescription of H. skuae and discuss the morphological characters distinguishing it from H. jenniae. Further research is necessary to improve our knowledge about the host and geographic distribution, health effects and phylogeny of H. jenniae and H. skuae.

Uncoupling Promoter Opening from Start-Site Scanning.

Whereas RNA polymerase II (Pol II) transcription start sites (TSSs) occur about 30-35 bp downstream of the TATA box in metazoans, TSSs are located 40-120 bp downstream in S. cerevisiae. Promoter melting begins about 12 bp downstream in all eukaryotes, so Pol II is presumed to "scan" further downstream before starting transcription in yeast. Here we report that removal of the kinase complex TFIIK from TFIIH shifts the TSS in a yeast system upstream to the location observed in metazoans. Conversely, moving the normal TSS to an upstream location enables a high level of TFIIK-independent transcription in the yeast system. We distinguish two stages of the transcription initiation process: bubble formation by TFIIH, which fills the Pol II active center with single-stranded DNA, and subsequent scanning downstream, also driven by TFIIH, which requires displacement of the initial bubble. Omission of TFIIK uncouples the two stages of the process.