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Dynamic change in subcellular localization of signaling proteins is a general concept that eukaryotic cells evolved for eliciting a coordinated response to stimuli. Mass spectrometry-based proteomics in combination with subcellular fractionation can provide comprehensive maps of spatio-temporal regulation of protein networks in cells, but involves laborious workflows that does not cover the phospho-proteome level. Here we present a high-throughput workflow based on sequential cell fractionation to profile the global proteome and phospho-proteome dynamics across six distinct subcellular fractions. We benchmark the workflow by studying spatio-temporal EGFR phospho-signaling dynamics in vitro in HeLa cells and in vivo in mouse tissues. Finally, we investigate the spatio-temporal stress signaling, revealing cellular relocation of ribosomal proteins in response to hypertonicity and muscle contraction. Proteomics data generated in this study can be explored through https://SpatialProteoDynamics.github.io . Protein activity regulated by phosphorylation can result in subcellular relocation. Here, the authors present a high throughput spatial phosphoproteomics approach to profile six subcellular compartments, providing insights into EGFR and stress signalling dynamics.

Stroke is the fourth leading cause of mortality and is estimated to be one of the major reasons for long-lasting disability worldwide. There are limited studies that describe the application of physical therapy interventions to prevent disabilities in stroke survivors and promote recovery after a stroke. In this review, we have described a wide range of interventions based on impairments, activity limitations, and goals in recovery during different stages of a stroke. This article mainly focuses on stroke rehabilitation tactics, including those for sensory function impairments, motor learning programs, hemianopia and unilateral neglect, flexibility and joint integrity, strength training, hypertonicity, postural control, and gait training. We conclude that, aside from medicine, stroke rehabilitation must address specific functional limitations to allow for group activities and superior use of a hemiparetic extremity. Medical doctors are often surprised by the variety of physiotherapeutic techniques available; they are unfamiliar with the approaches of researchers such as Bobath, Coulter, and Brunnstrom, among others, as well as the scientific reasoning behind these techniques.

Regulation of cell volume is essential for tissue homeostasis and cell viability. In response to hypertonic stress, cells need rapid electrolyte influx to compensate water loss and to prevent cell death in a process known as regulatory volume increase (RVI). However, the molecular component able to trigger such a process was unknown to date. Using a genome-wide CRISPR/Cas9 screen,we identified LRRC8A, which encodes a chloride channel subunit, as the genemost associated with cell survival under hypertonic conditions. Hypertonicity activates the p38 stress-activated protein kinase pathway and its downstream MSK1 kinase, which phosphorylates and activates LRRC8A. LRRC8A-mediated Cl⁻ efflux facilitates activation of the with-no-lysine (WNK) kinase pathway, which in turn, promotes electrolyte influx via Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) and RVI under hypertonic stress. LRRC8A-S217A mutation impairs channel activation by MSK1, resulting in reduced RVI and cell survival. In summary, LRRC8A is key to bidirectional osmotic stress responses and cell survival under hypertonic conditions.

Abstract Serum osmolality is the sum of the osmolalities of every single dissolved particle in the blood such as sodium and associated anions, potassium, glucose, and urea. Under normal conditions, serum sodium concentration is the major determinant of serum osmolality. Effective blood osmolality, so-called blood tonicity, is created by the endogenous (e.g., sodium and glucose) and exogenous (e.g., mannitol) solutes that are capable of creating an osmotic gradient across the membranes. In case of change in effective blood osmolality, water shifts from the compartment with low osmolality into the compartment with high osmolarity in order to restore serum osmolality. The difference between measured osmolality and calculated osmolarity forms the osmolal gap. An increase in serum osmolal gap can stem from the presence of solutes that are not included in the osmolarity calculation, such as hypertonic treatments or toxic alcoholic ingestions. In clinical practice, determination of serum osmolality and osmolal gap is important in the diagnosis of disorders related to sodium, glucose and water balance, kidney diseases, and small molecule poisonings. As blood hypertonicity exerts its main effects on the brain cells, neurologic symptoms varying from mild neurologic signs and symptoms to life-threatening outcomes such as convulsions or even death may occur. Therefore, hypertonic states should be promptly diagnosed and cautiously managed. In this review, the causes and treatment strategies of hyperosmolar conditions including hypernatremia, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, hypertonic treatments, or intoxications are discussed in detail to increase awareness of this important topic with significant clinical consequences.

Osmotic stress represents a major challenge to cells, particularly in the kidney, where tonicity gradients are both physiologically relevant and pathologically altered. The transcription factor nuclear factor of activated T cells 5 (NFAT5) is a key regulator of the osmoadaptive response, yet its downstream metabolic effectors remain incompletely understood. In this study, we identify the glycolytic side-branch enzyme 2,3-bisphosphoglycerate mutase (BPGM) as a transcriptional NFAT5 target that is induced under hypertonic conditions. RNA-seq analysis revealed that Bpgm knockdown significantly alters gene expression under osmotic stress (450 mOsmol/kg), with substantial overlap between BPGM- and NFAT5-responsive transcriptional programs. Bpgm depletion impairs the induction of canonical NFAT5 target genes, suggesting a functional interplay between metabolic and transcriptional adaptation. Promoter enrichment analysis showed that genes regulated by both NFAT5 and BPGM under hypertonic conditions are associated with CpG islands and GC-rich elements, supporting a link to chromatin structure and transcriptional accessibility. Consistently, we show that HIF-1α expression is regulated downstream of NFAT5 and BPGM, indicating a hierarchical organization of osmotic and hypoxic stress responses. We propose that BPGM facilitates NFAT5 function through metabolic-epigenetic coupling, acting as an amplifier of protective gene expression. Notably, this axis is active in BPGM-expressing cells such as those of the distal convoluted tubule. Thus, our findings establish BPGM as a critical node in the osmoadaptive gene regulatory network and highlight how cell type-specific metabolic profiles influence the transcriptional response to hypertonic stress. Graphical abstract

Facial aberrant reinnervation after unilateral facial paralysis is characterized by facial synkinesis and global facial muscle hypertonicity. Therefore, therapy effort is directed on improved facial symmetry by reducing facial synkinesis and the elevated muscle tone. There are no established methods to confirm these aims objectively. Therefore the aim of the present study was to verify if high-resolution surface electromyography (HR-sEMG) mapping of the entire face during standardized facial movements is one such sought-after method. Bilateral HR-sEMG facial mapping was performed in 36 patients (81% women; age range: 24–70 years) with a postparalytic facial nerve syndrome. Participants performed a standard set of standardized facial movement tasks before start (T0) and after nine days of training (T9). A linear mixed-effects model was used to evaluate differences between the facial movement tasks in-between the synkinetic side and the contralateral side at T0 and T9. The overall facial muscle activity was higher on the synkinetic side compared to the contralateral side at T0 ( p  < 0.001) and also at T9, but with reduced difference between sides ( p  ≤ 0.002). The overall muscle activity decreased on the synkinetic side and on the contralateral side (both p  < 0.001). These effects were also verifiable for almost every investigated muscle. HR-sEMG facial mapping proved its suitability as an objective method to confirm facial feedback training effects: A combined visual and EMG-based facial biofeedback training seemed to reduce the facial muscle activity on both facial sides, but markedly more effective on the synkinetic side.

Maintaining internal osmolality constancy is essential for life. Release of arginine vasopressin (AVP) in response to hyperosmolality is critical. Current hypotheses for osmolality sensors in circumventricular organs (CVOs) of the brain focus on mechanosensitive membrane proteins. The present study demonstrated that intracellular protein kinase WNK1 was involved. Focusing on vascular-organ-of-lamina-terminalis (OVLT) nuclei, we showed that WNK1 kinase was activated by water restriction. Neuron-specific conditional KO (cKO) of Wnk1 caused polyuria with decreased urine osmolality that persisted in water restriction and blunted water restriction-induced AVP release. Wnk1 cKO also blunted mannitol-induced AVP release but had no effect on osmotic thirst response. The role of WNK1 in the osmosensory neurons in CVOs was supported by neuronal pathway tracing. Hyperosmolality-induced increases in action potential firing in OVLT neurons was blunted by Wnk1 deletion or pharmacological WNK inhibitors. Knockdown of Kv3.1 channel in OVLT by shRNA reproduced the phenotypes. Thus, WNK1 in osmosensory neurons in CVOs detects extracellular hypertonicity and mediates the increase in AVP release by activating Kv3.1 and increasing action potential firing from osmosensory neurons.

A Maltese dog was presented with a stiff gait, secondary to muscle hypertonicity, affecting the axial and proximal appendicular muscles, which had progressively worsened over the last 4 years, associated with episodes of muscle spasms. Neuroanatomical localization was upper motor neuron (UMN) or generalized neuromuscular system. Cerebrospinal fluid (CSF) analysis was normal. Magnetic resonance imaging (MRI) of the brain and cervical spinal cord was performed and showed hypoplasia of the dorsal part of the left hippocampus, unchanged compared to the MRI performed 4 years earlier, and mild C6–C7 disk extrusion, with no evidence of compression of the spinal cord. Conscious electromyography showed continuous motor unit action potentials (MUAPs) in agonist and antagonist muscles. Indirect immunofluorescence (IFT) detected the presence of antibodies against glutamic acid decarboxylase (GAD). These findings were consistent with a human condition called “Stiff Person Syndrome” (SPS). A condition similar to SPS has only been described once before in a Beagle dog (“Stiff Dog Syndrome”). A therapeutic protocol based on human guidelines for SPS was initiated with a partial improvement. “Stiff Dog Syndrome” (SDS) is a possible cause of muscle hypertonicity and spasms in dogs.

We report the first study assessing human colon manometric features and their correlations with changes in autonomic functioning in patients with refractory chronic constipation prior to consideration of surgical intervention. High-resolution colonic manometry (HRCM) with simultaneous heart rate variability (HRV) was performed in 14 patients, and the resulting features were compared to healthy subjects. Patients were categorized into three groups that had normal, weak, or no high amplitude propagating pressure waves (HAPWs) to any intervention. We found mild vagal pathway impairment presented as lower HAPW amplitude in the proximal colon in response to proximal colon balloon distention. Left colon dysmotility was observed in 71% of patients, with features of (1) less left colon HAPWs, (2) lower left colon HAPW amplitudes (69.8 vs 102.3 mmHg), (3) impaired coloanal coordination, (4) left colon hypertonicity in patients with coccyx injury. Patients showed the following autonomic dysfunction: (1) high sympathetic tone at baseline, (2) high sympathetic reactivity to active standing and meal, (3) correlation of low parasympathetic reactivity to the meal with absence of the coloanal reflex, (4) lower parasympathetic and higher sympathetic activity during occurrence of HAPWs. In conclusion, left colon dysmotility and high sympathetic tone and reactivity, more so than vagal pathway impairment, play important roles in refractory chronic constipation and suggests sacral neuromodulation as a possible treatment.

Drosophila melanogaster exhibits multiple highly sophisticated temperature-sensing systems, enabling its effective response and navigation to temperature changes. Previous research has identified three dorsal organ cool cells (DOCCs) in fly larvae, consisting of two A-type and one B-type cell with distinct calcium dynamics. When subjected to hypertonic conditions, calcium imaging shows that A-type DOCCs maintain their responses to cool temperatures. In contrast, a subset of B-type DOCCs does not exhibit detectable GCaMP baseline signals, and the remaining detectable B-type DOCCs exhibit reduced temperature responses. The activation of both A-type and B-type DOCCs depends on the same members of the ionotropic receptor (IR) family: IR21a, IR93a, and IR25a. A-type DOCCs exhibit a higher somal level of IR93a than B-type DOCCs. Overexpression of Ir93a restores B-type calcium responses to cool temperatures, but not the proportion of B-type cells with a detectable GCaMP baseline, in a hypertonic environment, suggesting a selective role of IR93a in maintaining the temperature responses under hypertonic conditions. Our findings identify a novel function of B-type DOCCs in integrating temperature and tonic stimuli.