timsTOF HT Improves Protein Identification and Quantitative Reproducibility for Deep Unbiased Plasma Protein Biomarker Discovery.

Mass spectrometry (MS) is a valuable tool for plasma proteome profiling and disease biomarker discovery. However, wide-ranging plasma protein concentrations, along with technical and biological variabilities, present significant challenges for deep and reproducible protein quantitation. Here, we evaluated the qualitative and quantitative performance of timsTOF HT and timsTOF Pro 2 mass spectrometers for analysis of neat plasma samples (unfractionated) and plasma samples processed using the Proteograph Product Suite (Proteograph) that enables robust deep proteomics sampling prior to mass spectrometry. Samples were evaluated across a wide range of peptide loading masses and liquid chromatography (LC) gradients. We observed up to a 76% increase in total plasma peptide precursors identified and a >2-fold boost in quantifiable plasma peptide precursors (CV < 20%) with timsTOF HT compared to Pro 2. Additionally, approximately 4.5 fold more plasma peptide precursors were detected by both timsTOF HT and timsTOF Pro 2 in the Proteograph analyzed plasma vs neat plasma. In an exploratory analysis of 20 late-stage lung cancer and 20 control plasma samples with the Proteograph, which were expected to exhibit distinct proteomes, an approximate 50% increase in total and statistically significant plasma peptide precursors (q < 0.05) was observed with timsTOF HT compared to Pro 2. Our data demonstrate the superior performance of timsTOF HT for identifying and quantifying differences between biologically diverse samples, allowing for improved disease biomarker discovery in large cohort studies. Moreover, researchers can leverage data sets from this study to optimize their liquid chromatography-mass spectrometry (LC-MS) workflows for plasma protein profiling and biomarker discovery. (ProteomeXchange identifier: PXD047854 and PXD047839).

Selective augmentation of corticospinal motor drive with trans-spinal direct current stimulation in the cat.

BACKGROUND: A key outcome for spinal cord stimulation for neurorehabilitation after injury is to strengthen corticospinal system control of the arm and hand. Non-invasive, compared with invasive, spinal stimulation minimizes risk but depends on muscle-specific actions for restorative functions. OBJECTIVE: We developed a large-animal (cat) model, combining computational and experimental techniques, to characterize neuromodulation with transcutaneous spinal direct current stimulation (tsDCS) for facilitation of corticospinal motor drive to specific forelimb muscles. METHODS: Acute modulation of corticospinal function by tsDCS was measured using motor cortex-evoked muscle potentials (MEPs). The effects of current intensity, polarity (cathodal, anodal), and electrode position on specific forelimb muscle (biceps vs extensor carpi radialis, ECR) MEP modulation were examined. Locations of a key target, the motoneuron pools, were determined using neuronal tracing. A high-resolution anatomical (MRI and CT) model was developed for computational simulation of spinal current flow during tsDCS. RESULTS: Effects of tsDCS on corticospinal excitability were robust and immediate, therefore supporting MEPs as a sensitive marker of tsDCS targeting. Varying cathodal/anodal current intensity modulated MEP enhancement/suppression, with higher cathodal sensitivity. Muscle-specificity depended on cathode position; the rostral position preferentially augmented biceps responses and the caudal position, ECR responses. Precise anatomical current-flow modeling, supplemented with target motor pool distributions, can explain tsDCS focality on muscle groups. CONCLUSION: Anatomical current-flow modeling with physiological validation based on MEPs provides a framework to optimize muscle-specific tsDCS interventions. tsDCS targeting of representative motor pools enables muscle- and response-specific neuromodulation of corticospinal motor drive.

A Quantitative Proteomic Approach for the Identification of DNA Guanine Quadruplex-Binding Proteins.

DNA sequences of high guanine (G) content have the potential to form G quadruplex (G4) structures. A more complete understanding about the biological functions of G4 DNA requires the investigation about how these structures are recognized by proteins. Here, we conducted exhaustive quantitative proteomic experiments to profile the interaction proteomes of G4 structures by employing different sequences of G4 DNA derived from the human telomere and the promoters of c-MYC and c-KIT genes. Our results led to the identification of a number of candidate G4-interacting proteins, many of which were discovered here for the first time. These included three proteins that can bind to all three DNA G4 structures and 78 other proteins that can bind selectively to one or two of the three DNA G4 structure(s). We also validated that GRSF1 can bind directly and selectively toward G4 structure derived from the c-MYC promoter. Our quantitative proteomic screening also led to the identification of a number of candidate "antireader" proteins of G4 DNA. Together, we uncovered a number of cellular proteins that exhibit general and selective recognitions of G4 folding patterns, which underscore the complexity of G4 DNA in biology and the importance of understanding fully the G4-interaction proteome.

The fine line of defensive medicine.

Defensive medicine is a practice that has been utilized by clinicians in efforts of preventing patient dissatisfaction and malpractice claims and may be done through either omission or commission. As much as 57% of physicians have disclosed that they practice defensive medicine. However, this practice does not necessarily prevent malpractice claims and more importantly, neither does it equate to good medical practice, with some leading to poor outcomes. Unfortunately, there is a high percentage of malpractice claims lodged against clinicians in both primary care and hospital settings. Specialists such as surgeons, obstetricians, and gynecologists face the highest claims. In particular, during the SARS CoV-2 pandemic, with new challenges and limited treatment algorithms, there is an even greater concern for possible bourgeoning claims. Counteracting defensive medicine can be accomplished through decriminalizing malpractice claims, leaving physician oversight up to state medical boards and hospital claims management committees. Additional tort reform measures must also be taken such as caps on noneconomic damages to ensure emphasis on beneficence and nonmaleficence. Once these are in place, it may well serve to increase clinician-patient trust and improve patient independence in the shared decision-making process of their treatment, allowing clinicians to practice their full scope of practice without feeling wary of potential malpractice claims.

YY1 interacts with guanine quadruplexes to regulate DNA looping and gene expression.

The DNA guanine quadruplexes (G4) play important roles in multiple cellular processes, including DNA replication, transcription and maintenance of genome stability. Here, we showed that Yin and Yang 1 (YY1) can bind directly to G4 structures. ChIP-seq results revealed that YY1-binding sites overlap extensively with G4 structure loci in chromatin. We also observed that the dimerization of YY1 and its binding with G4 structures contribute to YY1-mediated long-range DNA looping. Displacement of YY1 from G4 structure sites disrupts substantially the YY1-mediated DNA looping. Moreover, treatment with G4-stabilizing ligands modulates the expression of not only those genes with G4 structures in their promoters, but also those associated with distal G4 structures that are brought to close proximity via YY1-mediated DNA looping. Together, we identified YY1 as a DNA G4-binding protein, and revealed that YY1-mediated long-range DNA looping requires its dimerization and occurs, in part, through its recognition of G4 structure.

VEZF1-guanine quadruplex DNA interaction regulates alternative polyadenylation and detyrosinase activity of VASH1.

Vascular endothelial zinc finger 1 (VEZF1) plays important roles in endothelial lineage definition and angiogenesis. Vasohibins 1 and 2 (VASH1 and VASH2) can form heterodimers with small vasohibin-binding protein (SVBP) and were recently shown to regulate angiogenesis by acting as tubulin detyrosinases. Here, we showed that VEZF1 binds directly with DNA guanine quadruplex (G quadruplex, G4) structures in vitro and in cells, which modulates the levels of the two isoforms of VASH1 mRNA. Disruption of this interaction, through genetic depletion of VEZF1 or treatment of cells with G4-stabilizing small molecules, led to increased production of the long over short isoform of VASH1 (i.e. VASH1A and VASH1B, respectively) mRNA and elevated tubulin detyrosinase activity in cells. Moreover, disruption of VEZF1-G4 interactions in human umbilical vein endothelial cells resulted in diminished angiogenesis. These results suggest that the interaction between VEZF1 and G4 structures assumes a crucial role in angiogenesis, which occurs through regulating the relative levels of the two isoforms of VASH1 mRNA and the detyrosinase activity of the VASH1-SVBP complex. Together, our work revealed VEZF1 as a G4-binding protein, identified a novel regulatory mechanism for tubulin detyrosinase, and illustrated that the VEZF1- and VASH1-mediated angiogenesis pathways are functionally connected.

Refractory dermatomyositis-systemic lupus erythematosus overlap syndrome and response to tofacitinib.

Dermatomyositis is an autoimmune condition that commonly presents in the form of an overlap syndrome with other rheumatic diseases. The overlap between syndromes with highly variable symptomology makes treatment difficult. We present a case of a 39-year-old woman who presented with a facial rash, arthralgias, and lower-extremity edema and steadily progressed to develop severe proximal muscle weakness and hair loss over the course of a 2.5-month hospitalization. After diagnostic testing, she was found to have a dermatomyositis-systemic lupus erythematosus overlap syndrome. Her symptoms were refractory to initial medical management but finally resolved once she was switched to tofacitinib.

The Quasi-uniform assumption for Spinal Cord Stimulation translational research.

BACKGROUND: Quasi-uniform assumption is a general theory that postulates local electric field predicts neuronal activation. Computational current flow model of spinal cord stimulation (SCS) of humans and animal models inform how the quasi-uniform assumption can support scaling neuromodulation dose between humans and translational animal. NEW METHOD: Here we developed finite element models of cat and rat SCS, and brain slice, alongside SCS models. Boundary conditions related to species specific electrode dimensions applied, and electric fields per unit current (mA) predicted. RESULTS: Clinically and across animal, electric fields change abruptly over small distance compared to the neuronal morphology, such that each neuron is exposed to multiple electric fields. Per unit current, electric fields generally decrease with body mass, but not necessarily and proportionally across tissues. Peak electric field in dorsal column rat and cat were ∼17x and ∼1x of clinical values, for scaled electrodes and equal current. Within the spinal cord, the electric field for rat, cat, and human decreased to 50% of peak value caudo-rostrally (C5-C6) at 0.48 mm, 3.2 mm, and 8 mm, and mediolaterally at 0.14 mm, 2.3 mm, and 3.1 mm. Because these space constants are different, electric field across species cannot be matched without selecting a region of interest (ROI). COMPARISON WITH EXISTING METHOD: This is the first computational model to support scaling neuromodulation dose between humans and translational animal. CONCLUSIONS: Inter-species reproduction of the electric field profile across the entire surface of neuron populations is intractable. Approximating quasi-uniform electric field in a ROI is a rational step to translational scaling.

SLIRP Interacts with Helicases to Facilitate 2'-O-Methylation of rRNA and to Promote Translation.

SRA stem-loop-interacting RNA-binding protein (SLIRP) is a versatile protein that can interact with the stem-loop structure in RNA and with G quadruplex DNA. By using a quantitative proteomic experiment, we found that SLIRP interacts with the majority of the human helicase proteome. We also found that these interactions facilitate 2'-O-methylation of a number of nucleosides in rRNA and promote protein translation. Hence, we uncovered a novel function of SLIRP protein and offered novel mechanistic insights into its function as a RNA chaperone and into the regulation of 2'-O-methylation of rRNA.

Integrated Genomic and Proteomic Analyses Reveal Novel Mechanisms of the Methyltransferase SETD2 in Renal Cell Carcinoma Development.

Clear cell renal cell carcinoma (ccRCC) is the most common type of RCC in humans. SET domain-containing 2 (SETD2), a lysine methyltransferase for histone and other proteins, has been found to be frequently lost in ccRCC. However, the mechanisms through which deficiency in SETD2 contributes to ccRCC development remain largely unknown. Here, we used a human embryonic kidney epithelial cell line with the SETD2 gene being knocked out using CRISPR/Cas9 technology. Using ChIP-seq analysis, we showed that SETD2 loss leads to diminished occupancy of histone H3K36me3 and H4K16ac on actively transcribed genes. Transcriptome sequencing of the knockout cells revealed diminished expression of genes involved in metabolic pathways and elevated expression of genes involved in regulation of RNA polymerase II-mediated transcription. Quantitative proteomic analysis of chromatin-associated proteins showed that genetic ablation of SETD2 leads to elevated chromatin occupancy of proteins involved in chromatin remodeling and RNA polymerase II transcription regulation, and diminished chromatin binding of proteins involved in translation elongation and RNA splicing. Interestingly, we found that SETD2 depletion attenuates cell proliferation, and this can be rescued by knockdown of CDK1. Taken together, we illustrate multiple SETD2-regulated cellular pathways that suppress cancer development and uncover mechanisms underlying aberrant cell cycle regulation in SETD2-depleted cells.

Motor system plasticity after unilateral injury in the developing brain.

In maturity, motor skills depend on the corticospinal tract (CST) and brainstem pathways that together synapse on interneurons and motoneurons in the spinal cord. Descending signals to spinal neurons that mediate voluntary control can be distinguished from peripheral sensory signals, primarily for feedback control. These motor system circuits depend initially on developmental genetic mechanisms to establish their connections and neural activity- and use-dependent synaptic refinement during the early postnatal period to enable motor skills to develop. In this review we consider four key activity-dependent developmental mechanisms that provide insights into how the motor systems establish the proper connections for skilled movement control and how the same mechanisms also inform the mechanisms of motor impairments and developmental plasticity after corticospinal system injury: (1) synaptic competition between the CSTs from each hemisphere; (2) interactions between the CST and spinal cord neurons; (3) synaptic competition between the CST and proprioceptive sensory fibres; and (4) interactions between the developing corticospinal motor system and the rubrospinal tract. Our findings suggest that the corticospinal motor system effectively 'oversees' development of its subcortical targets through synaptic competition and trophic-like interactions and this has important implications for motor impairments after perinatal cortical stroke. WHAT THIS PAPER ADDS: Neural activity-dependent processes inform the brain and spinal cord response to injury. The corticospinal motor system may 'oversee' development of its downstream subcortical targets through activity, trophic-like interactions, and synaptic competition.

Transcription-translation coupling: direct interactions of RNA polymerase with ribosomes and ribosomal subunits.

In prokaryotes, RNA polymerase and ribosomes can bind concurrently to the same RNA transcript, leading to the functional coupling of transcription and translation. The interactions between RNA polymerase and ribosomes are crucial for the coordination of transcription with translation. Here, we report that RNA polymerase directly binds ribosomes and isolated large and small ribosomal subunits. RNA polymerase and ribosomes form a one-to-one complex with a micromolar dissociation constant. The formation of the complex is modulated by the conformational and functional states of RNA polymerase and the ribosome. The binding interface on the large ribosomal subunit is buried by the small subunit during protein synthesis, whereas that on the small subunit remains solvent-accessible. The RNA polymerase binding site on the ribosome includes that of the isolated small ribosomal subunit. This direct interaction between RNA polymerase and ribosomes may contribute to the coupling of transcription to translation.

Identification of SLIRP as a G Quadruplex-Binding Protein.

The guanine quadruplex (G4) structure in DNA is a secondary structure motif that plays important roles in DNA replication, transcriptional regulation, and maintenance of genomic stability. Here, we employed a quantitative mass spectrometry-based approach to profile the interaction proteomes of three well-defined G4 structures derived from the human telomere and the promoters of cMYC and cKIT genes. We identified SLIRP as a novel G4-interacting protein. We also demonstrated that the protein could bind directly with G4 DNA with Kd values in the low nanomolar range and revealed that the robust binding of the protein toward G4 DNA requires its RRM domain. We further assessed, by using CRISPR-Cas9-introduced affinity tag and ChIP-Seq analysis, the genome-wide occupancy of SLIRP, and showed that the protein binds preferentially to G-rich DNA sequences that can fold into G4 structures. Together, our results uncovered a novel cellular protein that can interact directly with G4 DNA, which underscored the complex regulatory networks involved in G4 biology.

1H NMR Metabolic Profiling of Earthworm (Eisenia fetida) Coelomic Fluid, Coelomocytes, and Tissue: Identification of a New Metabolite-Malylglutamate.

Earthworm metabolism is recognized as a useful tool for monitoring environmental insults and measuring ecotoxicity, yet extensive earthworm metabolic profiling using 1H nuclear magnetic resonance (NMR) spectroscopy has been limited in scope. This study aims to expand the embedded metabolic material in earthworm coelomic fluid, coelomocytes, and tissue to aid systems toxicology research. Fifty-nine metabolites within Eisenia fetida were identified, with 47 detected in coelomic fluid, 41 in coelomocytes, and 54 in whole-worm samples and tissue extracts. The newly detected but known metabolites 2-aminobutyrate, nicotinurate, Nδ,Nδ,Nδ-trimethylornithine, and trigonelline are reported along with a novel compound, malylglutamate, elucidated using 2D NMR and high-resolution MS/MS. We postulate that malylglutamate acts as a glutamate/malate store, chelator, and anionic osmolyte and helps to provide electrolyte balance.

Cell and Protein Recognition at a Supported Bilayer Interface via In Situ Cavitand-Mediated Functional Polymer Growth.

Water-soluble deep cavitands embedded in a supported lipid bilayer are capable of anchoring ATRP initiator molecules for the in situ synthesis of primary amine-containing polymethacrylate patches at the water:membrane interface. These polymers can be derivatized in situ to incorporate fluorescent reporters, allow selective protein recognition, and can be applied to the immobilization of nonadherent cells at the bilayer interface.

Motor Cortex Activity Organizes the Developing Rubrospinal System.

The corticospinal and rubrospinal systems function in skilled movement control. A key question is how do these systems develop the capacity to coordinate their motor functions and, in turn, if the red nucleus/rubrospinal tract (RN/RST) compensates for developmental corticospinal injury? We used the cat to investigate whether the developing rubrospinal system is shaped by activity-dependent interactions with the developing corticospinal system. We unilaterally inactivated M1 by muscimol microinfusion between postnatal weeks 5 and 7 to examine activity-dependent interactions and whether the RN/RST compensates for corticospinal tract (CST) developmental motor impairments and CST misprojections after M1 inactivation. We examined the RN motor map and RST cervical projections at 7 weeks of age, while the corticospinal system was inactivated, and at 14 weeks, after activity returned. During M1 inactivation, the RN on the same side showed normal RST projections and reduced motor thresholds, suggestive of precocious development. By contrast, the RN on the untreated/active M1 side showed sparse RST projections and an immature motor map. After M1 activity returned later in adolescent cat development, RN on the active M1/CST side continued to show a substantial loss of spinal terminations and an impaired motor map. RN/RST on the inactivated side regressed to a smaller map and fewer axons. Our findings suggest that the developing rubrospinal system is under activity-dependent regulation by the corticospinal system for establishing mature RST connections and RN motor map. The lack of RS compensation on the non-inactivated side can be explained by development of ipsilateral misprojections from the active M1 that outcompete the RST. Significance statement: Skilled movements reflect the activity of multiple descending motor systems and their interactions with spinal motor circuits. Currently, there is little insight into whether motor systems interact during development to coordinate their emerging functions and, if so, the mechanisms underlying this process. This study examined activity-dependent interactions between the developing corticospinal and rubrospinal systems, two key systems for skilled limb movements. We show that the developing rubrospinal system competes with the corticospinal system in establishing the red nucleus motor map and rubrospinal tract connections. This is the first demonstration of one motor system steering development, and ultimately function, of another. Knowledge of activity-dependent competition between these two systems helps predict the response of the rubrospinal system following corticospinal system developmental injury.

Activity-Based Therapies for Repair of the Corticospinal System Injured during Development.

This review presents the mechanistic underpinnings of corticospinal tract (CST) development, derived from animal models, and applies what has been learned to inform neural activity-based strategies for CST repair. We first discuss that, in normal development, early bilateral CST projections are later refined into a dense crossed CST projection, with maintenance of sparse ipsilateral projections. Using a novel mouse genetic model, we show that promoting the ipsilateral CST projection produces mirror movements, common in hemiplegic cerebral palsy (CP), suggesting that ipsilateral CST projections become maladaptive when they become abnormally dense and strong. We next discuss how animal studies support a developmental "competition rule" whereby more active/used connections are more competitive and overtake less active/used connections. Based on this rule, after unilateral injury the damaged CST is less able to compete for spinal synaptic connections than the uninjured CST. This can lead to a progressive loss of the injured hemisphere's contralateral projection and a reactive gain of the undamaged hemisphere's ipsilateral CST. Knowledge of the pathophysiology of the developing CST after injury informs interventional strategies. In an animal model of hemiplegic CP, promoting injured system activity or decreasing the uninjured system's activity immediately after the period of a developmental injury both increase the synaptic competitiveness of the damaged system, contributing to significant CST repair and motor recovery. However, delayed intervention, despite significant CST repair, fails to restore skilled movements, stressing the need to consider repair strategies for other neural systems, including the rubrospinal and spinal interneuronal systems. Our interventional approaches harness neural activity-dependent processes and are highly effective in restoring function. These approaches are minimally invasive and are poised for translation to the human.

Postnatal maturation of the red nucleus motor map depends on rubrospinal connections with forelimb motor pools.

The red nucleus (RN) and rubrospinal tract (RST) are important for forelimb motor control. Although the RST is present postnatally in cats, nothing is known about when rubrospinal projections could support motor functions or the relation between the development of the motor functions of the rubrospinal system and the corticospinal system, the other major system for limb control. Our hypothesis is that the RN motor map is present earlier in development than the motor cortex (M1) map, to support early forelimb control. We investigated RN motor map maturation with microstimulation and RST cervical enlargement projections using anterograde tracers between postnatal week 3 (PW3) and PW16. Microstimulation and tracer injection sites were verified histologically to be located within the RN. Microstimulation at PW4 evoked contralateral wrist, elbow, and shoulder movements. The number of sites producing limb movement increased and response thresholds decreased progressively through PW16. From the outset, all forelimb joints were represented. At PW3, RST projections were present within the cervical intermediate zone, with a mature density of putative synapses. In contrast, beginning at PW5 there was delayed and age-dependent development of forelimb motor pool projections and putative rubromotoneuronal synapses. The RN has a more complete forelimb map early in development than previous studies showed for M1, supporting our hypothesis of preferential rubrospinal rather than corticospinal control for early movements. Remarkably, development of the motor pool, not intermediate zone, RST projections paralleled RN motor map development. The RST may be critical for establishing the rudiments of motor skills that subsequently become refined with further CST development.

Rapid and persistent impairments of the forelimb motor representations following cervical deafferentation in rats.

Skilled motor control is regulated by the convergence of somatic sensory and motor signals in brain and spinal motor circuits. Cervical deafferentation is known to diminish forelimb somatic sensory representations rapidly and to impair forelimb movements. Our focus was to determine what effect deafferentation has on the motor representations in motor cortex, knowledge of which could provide new insights into the locus of impairment following somatic sensory loss, such as after spinal cord injury or stroke. We hypothesized that somatic sensory information is important for cortical motor map topography. To investigate this we unilaterally transected the dorsal rootlets in adult rats from C4 to C8 and mapped the forelimb motor representations using intracortical microstimulation, immediately after rhizotomy and following a 2-week recovery period. Immediately after deafferentation we found that the size of the distal representation was reduced. However, despite this loss of input there were no changes in motor threshold. Two weeks after deafferentation, animals showed a further distal representation reduction, an expansion of the elbow representation, and a small elevation in distal movement threshold. These changes were specific to the forelimb map in the hemisphere contralateral to deafferentation; there were no changes in the hindlimb or intact-side forelimb representations. Degradation of the contralateral distal forelimb representation probably contributes to the motor control deficits after deafferentation. We propose that somatic sensory inputs are essential for the maintenance of the forelimb motor map in motor cortex and should be considered when rehabilitating patients with peripheral or spinal cord injuries or after stroke.