Solutes unmask differences in clustering versus phase separation of FET proteins.

Phase separation and percolation contribute to phase transitions of multivalent macromolecules. Contributions of percolation are evident through the viscoelasticity of condensates and through the formation of heterogeneous distributions of nano- and mesoscale pre-percolation clusters in sub-saturated solutions. Here, we show that clusters formed in sub-saturated solutions of FET (FUS-EWSR1-TAF15) proteins are affected differently by glutamate versus chloride. These differences on the nanoscale, gleaned using a suite of methods deployed across a wide range of protein concentrations, are prevalent and can be unmasked even though the driving forces for phase separation remain unchanged in glutamate versus chloride. Strikingly, differences in anion-mediated interactions that drive clustering saturate on the micron-scale. Beyond this length scale the system separates into coexisting phases. Overall, we find that sequence-encoded interactions, mediated by solution components, make synergistic and distinct contributions to the formation of pre-percolation clusters in sub-saturated solutions, and to the driving forces for phase separation.

Dorsal Root Ganglion Stimulation for the Management of Phantom Bladder Pain: A Case Report.

Phantom bladder pain, a rare condition following cystectomy, can pose a challenge to pain management providers. We present the case of a 43-year-old male who developed severe phantom bladder pain post-cystectomy. Despite multiple treatments, his symptoms persisted, significantly affecting his quality of life. Dorsal root ganglion stimulation (DRGS) was attempted after conventional therapies failed. The DRGS trial provided significant relief, leading to permanent implantation and a 90% reduction in pain. This case highlights DRGS as a potential treatment for phantom bladder pain, expanding its applications beyond traditional uses. Further research is needed to elucidate its mechanisms and broader applicability.

Doxorubicin Conjugated γ-Globulin Functionalised Gold Nanoparticles: A pH-Responsive Bioinspired Nanoconjugate Approach for Advanced Chemotherapeutics.

Developing successful nanomedicine hinges on regulating nanoparticle surface interactions within biological systems, particularly in intravenous nanotherapeutics. We harnessed the surface interactions of gold nanoparticles (AuNPs) with serum proteins, incorporating a γ-globulin (γG) hard surface corona and chemically conjugating Doxorubicin to create an innovative hybrid anticancer nanobioconjugate, Dox-γG-AuNPs. γG (with an isoelectric point of ~7.2) enhances cellular uptake and exhibits pH-sensitive behaviour, favouring targeted cancer cell drug delivery. In cell line studies, Dox-γG-AuNPs demonstrated a 10-fold higher cytotoxic potency compared to equivalent doxorubicin concentrations, with drug release favoured at pH 5.5 due to the γ-globulin corona's inherent pH sensitivity. This bioinspired approach presents a novel strategy for designing hybrid anticancer therapeutics. Our study also explored the intricacies of the p53-mediated ROS pathway's role in regulating cell fate, including apoptosis and necrosis, in response to these treatments. The pathway's delicate balance of ROS emerged as a critical determinant, warranting further investigation to elucidate its mechanisms and implications. Overall, leveraging the robust γ-globulin protein corona on AuNPs enhances biostability in harsh serum conditions, augments anticancer potential within pH-sensitive environments, and opens promising avenues for bioinspired drug delivery and the design of novel anticancer hybrids with precise targeting capabilities.

Glutamate helps unmask the differences in driving forces for phase separation versus clustering of FET family proteins in sub-saturated solutions.

Multivalent proteins undergo coupled segregative and associative phase transitions. Phase separation, a segregative transition, is driven by macromolecular solubility, and this leads to coexisting phases above system-specific saturation concentrations. Percolation is a continuous transition that is driven by multivalent associations among cohesive motifs. Contributions from percolation are highlighted by the formation of heterogeneous distributions of clusters in sub-saturated solutions, as was recently reported for Fused in sarcoma (FUS) and FET family proteins. Here, we show that clustering and phase separation are defined by a separation of length- and energy-scales. This is unmasked when glutamate is the primary solution anion. Glutamate is preferentially excluded from protein sites, and this enhances molecular associations. Differences between glutamate and chloride are manifest at ultra-low protein concentrations. These differences are amplified as concentrations increase, and they saturate as the micron-scale is approached. Therefore, condensate formation in supersaturated solutions and clustering in sub-saturated are governed by distinct energy and length scales. Glutamate, unlike chloride, is the dominant intracellular anion, and the separation of scales, which is masked in chloride, is unmasked in glutamate. Our work highlights how components of cellular milieus and sequence-encoded interactions contribute to amplifying distinct contributions from associative versus segregative phase transitions.

Prostate Cancer Progression Relies on the Mitotic Kinase Citron Kinase.

Prostate cancer remains the second leading cause of cancer death in men in Western cultures. A deeper understanding of the mechanisms by which prostate cancer cells divide to support tumor growth could help devise strategies to overcome treatment resistance and improve survival. Here, we identified that the mitotic AGC family protein kinase citron kinase (CIT) is a pivotal regulator of prostate cancer growth that mediates prostate cancer cell interphase progression. Increased CIT expression correlated with prostate cancer growth induction and aggressive prostate cancer progression, and CIT was overexpressed in prostate cancer compared with benign prostate tissue. CIT overexpression was controlled by an E2F2-Skp2-p27 signaling axis and conferred resistance to androgen-targeted treatment strategies. The effects of CIT relied entirely on its kinase activity. Conversely, CIT silencing inhibited the growth of cell lines and xenografts representing different stages of prostate cancer progression and treatment resistance but did not affect benign epithelial prostate cells or nonprostatic normal cells, indicating a potential therapeutic window for CIT inhibition. CIT kinase activity was identified as druggable and was potently inhibited by the multikinase inhibitor OTS-167, which decreased the proliferation of treatment-resistant prostate cancer cells and patient-derived organoids. Isolation of the in vivo CIT substrates identified proteins involved in diverse cellular functions ranging from proliferation to alternative splicing events that are enriched in treatment-resistant prostate cancer. These findings provide insights into the regulation of aggressive prostate cancer cell behavior by CIT and identify CIT as a functionally diverse and druggable driver of prostate cancer progression. SIGNIFICANCE: The poorly characterized protein kinase citron kinase is a therapeutic target in prostate cancer that drives tumor growth by regulating diverse substrates, which control several hallmarks of aggressive prostate cancer progression. See related commentary by Mishra et al., p. 4008.

Debunking myths in headache diagnosis for the pain practitioner.

With headache being one of the most common chief complaints, it is essential for pain practitioners to interpret and differentiate a variety of headache characteristics to accurately diagnose and treat specific headache disorders. Certain misconceptions often lead to misdiagnosis. This article presents and discusses six myths about several common headache disorders (migraine, tension-type headache, cluster headache, cervicogenic headache, sinus headache, and occipital neuralgia) often encountered in clinical practice. The discussion is based primarily on the International Classification of Headache Disorders, 3rd edition and the latest studies. Recognizing and understanding the intricacies behind key headache diagnoses will help providers devise appropriate plans to better care for their patients.

Crowder titrations enable the quantification of driving forces for macromolecular phase separation.

Macromolecular solubility is an important contributor to the driving forces for phase separation. Formally, the driving forces in a binary mixture comprising a macromolecule dissolved in a solvent can be quantified in terms of the saturation concentration, which is the threshold macromolecular concentration above which the mixture separates into coexisting dense and dilute phases. In addition, the second virial coefficient, which measures the effective strength of solvent-mediated intermolecular interactions provides direct assessments of solvent quality. The sign and magnitude of second virial coefficients will be governed by a combination of solution conditions and the nature of the macromolecule of interest. Here, we show, using a combination of theory, simulation, and in vitro experiments, that titrations of crowders, providing they are true depletants, can be used to extract the intrinsic driving forces for macromolecular phase separation. This refers to saturation concentrations in the absence of crowders and the second virial coefficients that quantify the magnitude of the incompatibility between macromolecules and the solvent. Our results show how the depletion-mediated attractions afforded by crowders can be leveraged to obtain comparative assessments of macromolecule-specific, intrinsic driving forces for phase separation.

Glutamate helps unmask the differences in driving forces for phase separation versus clustering of FET family proteins in sub-saturated solutions.

Multivalent proteins undergo coupled segregative and associative phase transitions. Phase separation, a segregative transition, is driven by macromolecular solubility, and this leads to coexisting phases above system-specific saturation concentrations. Percolation is a continuous transition that is driven by multivalent associations among cohesive motifs. Contributions from percolation are highlighted by the formation of heterogeneous distributions of clusters in sub-saturated solutions, as was recently reported for Fused in sarcoma (FUS) and FET family proteins. Here, we show that clustering and phase separation are defined by a separation of length- and energy-scales. This is unmasked when glutamate is the primary solution anion. Glutamate is preferentially excluded from protein sites, and this enhances molecular associations. Differences between glutamate and chloride are manifest at ultra-low protein concentrations. These differences are amplified as concentrations increase, and they saturate as the micron-scale is approached. Therefore, condensate formation in supersaturated solutions and clustering in sub-saturated are governed by distinct energy and length scales. Glutamate, unlike chloride, is the dominant intracellular anion, and the separation of scales, which is masked in chloride, is unmasked in glutamate. Our work highlights how components of cellular milieus and sequence-encoded interactions contribute to amplifying distinct contributions from associative versus segregative phase transitions.

Polymeric and biological membranes for organ-on-a-chip devices.

Membranes are fundamental elements within organ-on-a-chip (OOC) platforms, as they provide adherent cells with support, allow nutrients (and other relevant molecules) to permeate/exchange through membrane pores, and enable the delivery of mechanical or chemical stimuli. Through OOC platforms, physiological processes can be studied in vitro, whereas OOC membranes broaden knowledge of how mechanical and chemical cues affect cells and organs. OOCs with membranes are in vitro microfluidic models that are used to replace animal testing for various applications, such as drug discovery and disease modeling. In this review, the relevance of OOCs with membranes is discussed as well as their scaffold and actuation roles, properties (physical and material), and fabrication methods in different organ models. The purpose was to aid readers with membrane selection for the development of OOCs with specific applications in the fields of mechanistic, pathological, and drug testing studies. Mechanical stimulation from liquid flow and cyclic strain, as well as their effects on the cell's increased physiological relevance (IPR), are described in the first section. The review also contains methods to fabricate synthetic and ECM (extracellular matrix) protein membranes, their characteristics (e.g., thickness and porosity, which can be adjusted depending on the application, as shown in the graphical abstract), and the biological materials used for their coatings. The discussion section joins and describes the roles of membranes for different research purposes and their advantages and challenges.

Crowder titrations enable the quantification of driving forces for macromolecular phase separation.

Macromolecular solubility is an important contributor to the driving forces for phase separation. Formally, the driving forces in a binary mixture comprising a macromolecule dissolved in a solvent can be quantified in terms of the saturation concentration, which is the threshold macromolecular concentration above which the mixture separates into coexisting dense and dilute phases. Additionally, the second virial coefficient, which measures the effective strength of solvent-mediated intermolecular interactions provides direct assessments of solvent quality. The sign and magnitude of second virial coefficients will be governed by a combination of solution conditions and the nature of the macromolecule of interest. Here, we show, using a combination of theory, simulation, and in vitro experiments, that titrations of crowders, providing they are true depletants, can be used to extract the intrinsic driving forces for macromolecular phase separation. This refers to saturation concentrations in the absence of crowders and the second virial coefficients that quantify the magnitude of the incompatibility between macromolecules and the solvent. Our results show how the depletion-mediated attractions afforded by crowders can be leveraged to obtain comparative assessments of macromolecule-specific, intrinsic driving forces for phase separation. SIGNIFICANCE: Phase separation has emerged as a process of significant relevance to sorting macromolecules into distinct compartments, thereby enabling spatial and temporal control over cellular matter. Considerable effort is being invested into uncovering the driving forces that enable the separation of macromolecular solutions into coexisting phases. At its heart, this process is governed by the balance of macromolecule-solvent, inter-macromolecule, and solvent-solvent interactions. We show that the driving forces for phase separation, including the coefficients that measure interaction strengths between macromolecules, can be extracted by titrating the concentrations of crowders that enable macromolecules to phase separate at lower concentrations. Our work paves the way to leverage specific categories of measurements for quantitative characterizations of driving forces for phase separation.

Maternal Embryonic Leucine Zipper Kinase is Associated with Metastasis in Triple-negative Breast Cancer.

Triple-negative breast cancer (TNBC) has high relapse and metastasis rates and a high proportion of cancer stem-like cells (CSC), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, is known to promote CSC maintenance and malignant transformation. However, the role of MELK in TNBC metastasis is unknown; we sought to address this in the current study. We found that MELK mRNA levels were higher in TNBC tumors [8.11 (3.79-10.95)] than in HR+HER2- tumors [6.54 (2.90-9.26)]; P < 0.001]. In univariate analysis, patients with breast cancer with high-MELK-expressing tumors had worse overall survival (P < 0.001) and distant metastasis-free survival (P < 0.01) than patients with low-MELK-expressing tumors. In a multicovariate Cox regression model, high MELK expression was associated with shorter overall survival after adjusting for other baseline risk factors. MELK knockdown using siRNA or MELK inhibition using the MELK inhibitor MELK-In-17 significantly reduced invasiveness, reversed epithelial-to-mesenchymal transition, and reduced CSC self-renewal and maintenance in TNBC cells. Nude mice injected with CRISPR MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and improved overall survival compared with mice injected with control cells (P < 0.05). Furthermore, MELK-In-17 suppressed 4T1 tumor growth in syngeneic BALB/c mice (P < 0.001). Our findings indicate that MELK supports metastasis by promoting epithelial-to-mesenchymal transition and the CSC phenotype in TNBC. Significance: These findings indicate that MELK is a driver of aggressiveness and metastasis in TNBC.

Wireless High-Frequency Peripheral Nerve Stimulation for Chronic Refractory Knee Pain Post-total Knee Replacement.

The number of joint replacement surgeries, especially knee replacement surgeries, is rising with the rising geriatric population. Chronic unremitting knee pain post-total knee replacement surgery is a common phenomenon. Usually, the pain responds to conservative measures, including physical therapy and medical management. In some patients, the pain post-knee replacement surgery can be refractory and unremitting. In such scenarios, peripheral nerve stimulation, or neuromodulation, can be an effective option.

Dorsal Root Ganglion Stimulation Therapy for Refractory Idiopathic Pudendal Neuralgia.

Dorsal root ganglion stimulation is a relatively new treatment option for chronic pain conditions such as pudendal neuralgia, which is a chronic pain condition affecting the pudendal nerve in the pelvic region. Pudendal neuralgia is a debilitating condition that can significantly affect the patient's quality of life. In dorsal root ganglion stimulation, a small device is implanted that delivers electrical impulses to the dorsal root ganglion to modulate pain signals coming from the pudendal nerve. The procedure is considered investigational and has been investigated in case series and case reports with promising results. However, more research is needed to fully understand its safety and effectiveness. This case report highlights the potential of dorsal root ganglion stimulation as a treatment option for pudendal neuralgia and the need for further research to establish it as a standard treatment option.

Abernethy Malformation Masquerading as Congenital Heart Disease: A Boy With Cyanosis, Clubbing, and Hypoxia.

Abernethy malformation is an extrahepatic congenital portosystemic shunt characterized by the diversion of the portal blood away from the liver through a shunt that drains directly into the inferior vena cava. We present a case of a male child with Abernethy malformation, which was initially diagnosed as cyanotic heart disease due to pulmonary arteriovenous malformation. However, after proper clinical evaluation and investigations, the diagnosis of Abernethy malformation was established. Thereafter, the patient was successfully treated with endovascular embolization. At one year follow-up, marked relief in exertional dyspnea and improvement in physical growth was achieved with no observable complications.

Injectable Bone Cement Reinforced with Gold Nanodots Decorated rGO-Hydroxyapatite Nanocomposites, Augment Bone Regeneration.

Interest in the development of new generation injectable bone cements having appropriate mechanical properties, biodegradability, and bioactivity has been rekindled with the advent of nanoscience. Injectable bone cements made with calcium sulfate (CS) are of significant interest, owing to its compatibility and optimal self-setting property. Its rapid resorption rate, lack of bioactivity, and poor mechanical strength serve as a deterrent for its wide application. Herein, a significantly improved CS-based injectable bone cement (modified calcium sulfate termed as CSmod ), reinforced with various concentrations (0-15%) of a conductive nanocomposite containing gold nanodots and nanohydroxyapatite decorated reduced graphene oxide (rGO) sheets (AuHp@rGO), and functionalized with vancomycin, is presented. The piezo-responsive cement exhibits favorable injectability and setting times, along with improved mechanical properties. The antimicrobial, osteoinductive, and osteoconductive properties of the CSmod cement are confirmed using appropriate in vitro studies. There is an upregulation of the paracrine signaling mediated crosstalk between mesenchymal stem cells and human umbilical vein endothelial cells seeded on these cements. The ability of CSmod to induce endothelial cell recruitment and augment bone regeneration is evidenced in relevant rat models. The results imply that the multipronged activity exhibited by the novel-CSmod cement would be beneficial for bone repair.

Endovascular embolisation of a complex mandibular AVM: a hybrid transarterial and transvenous approach.

Mandibular arteriovenous malformations (AVMs) are rare lesions which can present with life-threatening haemorrhage. Endovascular embolisation can be life saving for these patients. We discuss a patient of mandibular AVM, who initially presented with reports of massive oral bleeding. The lesion was only partially embolised via transarterial route, as the nidus could not be penetrated completely. In view of rebleeding within 72 hours from the same site, a second embolisation procedure was done via combined, transarterial and transvenous approaches. Coils and onyx were used as embolising agents. Complete embolisation was achieved via combined approach. No further bleeding episodes were seen at 1-year follow-up. Endovascular embolisation of mandibular AVMs can be technically challenging and, hence, a sound knowledge of the anatomy as well as the possible modification of technique is essential to achieve complete obliteration of the lesion and to maximise the benefit of embolisation and to avoid major radical surgery.

Ultrasound-Guided Erector Spinae Block for Refractory Abdominal Pain Due to Acute on Chronic Pancreatitis.

Erector spinae blocks (ESBs) are a type of fascial pain block that has been safely used for various applications, including post-operative and post-trauma pain in several thoracic and abdominal surgeries. Pain related to an acute flare-up of chronic pancreatitis is usually challenging to control and impacts patient comfort and discharge planning. This case report describes an application of ESBs for the effective treatment of refractory abdominal pain associated with acute exacerbation of chronic pancreatitis. Application of ESB in the emergency room setting can potentially decrease hospital admission for this common condition and increase patient satisfaction.

A Microbiological Assay of Common Operating Room (OR) Tapes: Developing a Culture for Patient Safety.

BACKGROUND: There are no existing practices or methods to ensure cleanliness, sterility, or prevent cross-contamination when it comes to common operating room (OR) tape. The authors hypothesized that adhesive tapes used by anesthesia providers in ORs and off-site surgical areas might be colonized by microorganisms and that culturing these tape rolls would reveal significant monomicrobial and polymicrobial contamination.  Material and Methods: The primary objective of this observational cohort study was to report and compare contamination rate including polymicrobial contamination rate between tape specimens collected from storage site and specimen from the ORs, off-sites, and after use on a patient. The outcome measures were the culture reports of the adhesive tapes. The authors then designed an intervention that integrated anesthesia providers' hand hygiene and maintenance of a barrier between the OR tapes and OR surfaces. RESULTS: The authors reported gross contamination and cross-contamination among the OR off-site tapes. The contamination rates reported for tapes from OR, off-site specimens, and patient specimens were 68.2%,63.2%, and 100%, respectively. The authors again cultured adhesive tapes after the intervention and reported improved outcomes. CONCLUSIONS: The current quality improvement (QI) project identified the potential for OR tapes to serve as microbial vectors. The authors advocate environmental decontamination and anesthesia providers' hand hygiene in parallel as a part of routine anesthesia care in their practice and agree that the endotracheal tubes (ETTs) and orogastric or nasogastric tubes should be pre-packaged with single-use tape, which can be used for securing devices.