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Platinum (Pt) compounds such as oxaliplatin are among the most commonly prescribed anti-cancer drugs. Despite their considerable clinical impact, the molecular basis of platinum cytotoxicity and cancer specificity remain unclear. Here we show that oxaliplatin, a backbone for the treatment of colorectal cancer, causes liquid-liquid demixing of nucleoli at clinically relevant concentrations. Our data suggest that this biophysical defect leads to cell-cycle arrest, shutdown of Pol I-mediated transcription, and ultimately cell death. We propose that instead of targeting a single molecule, oxaliplatin preferentially partitions into nucleoli, where it modifies nucleolar RNA and proteins. This mechanism provides a general approach for drugging the increasing number of cellular processes linked to biomolecular condensates., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

A hallmark pathological feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord 1 . A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing 2-4 . Single nucleotide polymorphisms in UNC13A are among the strongest hits associated with FTD and ALS in human genome-wide association studies 5,6 , but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon-splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines and motor neurons derived from induced pluripotent stem cells resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. The top variants associated with FTD or ALS risk in humans are located in the intron harbouring the cryptic exon, and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD and ALS (UNC13A genetic variants), and loss of TDP-43 function., (© 2022. The Author(s).)

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that leads to the death of upper and lower motor neurons. While most cases of ALS are sporadic, some of the familial forms of the disease are caused by mutations in the gene encoding for the RNA-binding protein FUS. Under physiological conditions, FUS readily phase separates into liquid-like droplets in vivo and in vitro . ALS-associated mutations interfere with this process and often result in solid-like aggregates rather than fluid condensates. Yet, whether cells recognize and triage aberrant condensates remains poorly understood, posing a major barrier to the development of novel ALS treatments. Using a combination of ALS-associated FUS mutations, optogenetic manipulation of FUS condensation, chemically induced stress, and pH-sensitive reporters of organelle acidity, we systematically characterized the cause-effect relationship between the material state of FUS condensates and the sequestering of lysosomes. From our data, we can derive three conclusions. First, regardless of whether we use wild-type or mutant FUS, expression levels (i.e., high concentrations) play a dominant role in determining the fraction of cells having soluble or aggregated FUS. Second, chemically induced FUS aggregates recruit LAMP1-positive structures. Third, mature, acidic lysosomes accumulate only at FUS aggregates but not at liquid-condensates. Together, our data suggest that lysosome-degradation machinery actively distinguishes between fluid and solid condensates. Unraveling these aberrant interactions and testing strategies to manipulate the autophagosome-lysosome axis provides valuable clues for disease intervention., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Trnka, Hoffmann, Wang, Sansevrino, Rankovic, Rost, Schmitz, Schmidt and Milovanovic.)

Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Experimental approaches for dynamically monitoring these intracellular effects are currently lacking. Here, we leverage the environmental sensitivity and structural plasticity of intrinsically disordered protein regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use as environmentally-responsive molecular tools., (© 2021. The Author(s).)

Mutations in RNA-binding proteins (RBPs) such as TDP43 are associated with transcriptome-wide splicing defects and cause severe neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The impact of RBP mutations on splicing function is routinely studied using PCR-based bulk measurements. However, the qualitative and low-throughput nature of this assay make quantitative and systematic analyses, as well as screening approaches, difficult to implement. To overcome this hurdle, we have developed a quantitative, high-throughput flow cytometry assay to investigate TDP43 splicing function on a single-cell level., Competing Interests: Competing interestsThe authors declare no competing interests., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

Liquid-liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix-helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Intrinsically disordered regions (IDRs) are often fast-evolving protein domains of low sequence complexity that can drive phase transitions and are commonly found in many proteins associated with neurodegenerative diseases, including the RNA processing factor TDP43. Yet, how phase separation contributes to the physiological functions of TDP43 in cells remains enigmatic. Here, we combine systematic mutagenesis guided by evolutionary sequence analysis with a live-cell reporter assay of TDP43 phase dynamics to identify regularly-spaced hydrophobic motifs separated by flexible, hydrophilic segments in the IDR as a key determinant of TDP43 phase properties. This heuristic framework allows customization of the material properties of TDP43 condensates to determine effects on splicing function. Remarkably, even a mutant that fails to phase-separate at physiological concentrations can still efficiently mediate the splicing of a quantitative, single-cell splicing reporter and endogenous targets. This suggests that the ability of TDP43 to phase-separate is not essential for its splicing function.

Compared to individuals in lower positions of power, higher-power individuals are theorized to be less motivated to attend to social cues. In support of this theory, previous research has consistently documented negative correlations between social class and emotion perception. Prior studies, however, were limited by the size and diversity of the participant samples as well as the systematicity with which social class and emotion perception were operationalized. Here, we examine the generalizability of prior research across 10,000+ total participants. In an initial modest sample, (n = 179), Study 1 partially replicated past results: emotion identification correlated negativity with subjective social class (β = -0.15, 95% CI = [-0.28,-0.02]) and one of two objective social class measures (participant education β = -0.15, 95% CI = [-0.03,-0.01]). Studies 2-4 followed up on Study 1's mixed results for objective social class in three much larger samples. These results diverged from past literature. In Study 2, complex emotion identification correlated non-significantly with participant education (β = 0.02, p = 0.25; 95% CI = [-0.01, 0.05], n = 2,726), positively with childhood family income (β = 0.03, 95% CI = [0.01,0.06], n = 4,312), and positively with parental education (β = 0.06, 95% CI = [0.04,0.09], n = 4,225). In Study 3, basic emotion identification correlated positively with participant education (β = 0.05, 95% CI = [0.02, 0.09]), n = 2,564). In Study 4, basic emotion discrimination correlated positively with participant education (β = 0.09, 95% CI = [0.05,0.13], n = 2,079), positively with parental education (β = 0.06, 95% CI = [0.02,0.09], n = 3,225), and non-significantly with childhood family income (β = 0.2, 95% CI = [0.01,0.07], n = 3,272). Results remained similar when restricting analyses to U.S.-based participants. Taken together, these findings suggest that previously reported negative correlations between emotion perception and social class may generalize poorly past select samples and/or subjective measures of social class. Data from the three large web-based samples used in Studies 2-4 are available at osf.io/jf7r3 as normative datasets and to support future investigations of these and other research questions., Competing Interests: The authors have declared that no competing interests exist.

TDP-43 is an RNA-binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C-terminal domain which mediates ALS inclusions, TDP-43 has a globular N-terminal domain (NTD). Here, we show that TDP-43 NTD assembles into head-to-tail linear chains and that phosphomimetic substitution at S48 disrupts TDP-43 polymeric assembly, discourages liquid-liquid phase separation (LLPS) in vitro , fluidizes liquid-liquid phase separated nuclear TDP-43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head-to-tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher-order polymerization. These data provide structural detail for the established mechanistic role of the well-folded TDP-43 NTD in splicing and link this function to LLPS. In addition, the fusion-tag solubilized, recombinant form of TDP-43 full-length protein developed here will enable future phase separation and in vitro biochemical assays on TDP-43 function and interactions that have been hampered in the past by TDP-43 aggregation., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Eukaryotic cells contain membrane-less organelles, including nucleoli and stress granules, that behave like liquid droplets. Such endogenous condensates often have internal substructure, but how this is established in the absence of membrane encapsulation remains unclear. We find that the N- and C-terminal domains of TDP43, a heterogeneous nuclear ribonucleoprotein implicated in neurodegenerative diseases, are capable of driving the formation of sub-structured liquid droplets in vivo. These droplets contain dynamic internal "bubbles" of nucleoplasm, reminiscent of membrane-based multi-vesicular endosomes. A conserved sequence embedded within the intrinsically disordered region (IDR) of TDP43 promotes the formation of these multi-phase assemblies. Disease-causing point mutations in the IDR can change the propensity to form bubbles, protein dynamics within the phase, or phase-environment exchange rates. Our results show that a single IDR-containing protein can nucleate the assembly of compartmentalized liquid droplets approximating the morphological complexity of membrane-bound organelles., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Nuclear pore complexes (NPCs) provide a selective passageway for receptor-mediated active transport between nucleus and cytoplasm, while maintaining the distinct molecular compositions of both compartments at large. In this review we discuss how NPCs gain a remarkable sorting selectivity from non-globular FG domains and their phase separation into dense polymer meshworks. The resulting sieve-like FG hydrogels are effective barriers to normal macromolecules but are at the same time highly permeable to shuttling nuclear transport receptors, which bind to FG motifs as well as to their designated cargoes. Phase separation driven by disordered protein domains was recently also recognized as being pivotal to the formation of membraneless organelles, making it an important emerging principle in cell biology., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Nuclear pore complexes (NPCs) conduct massive transport mediated by shuttling nuclear transport receptors (NTRs), while keeping nuclear and cytoplasmic contents separated. The NPC barrier in Xenopus relies primarily on the intrinsically disordered FG domain of Nup98. We now observed that Nup98 FG domains of mammals, lancelets, insects, nematodes, fungi, plants, amoebas, ciliates, and excavates spontaneously and rapidly phase-separate from dilute (submicromolar) aqueous solutions into characteristic 'FG particles'. This required neither sophisticated experimental conditions nor auxiliary eukaryotic factors. Instead, it occurred already during FG domain expression in bacteria. All Nup98 FG phases rejected inert macromolecules and yet allowed far larger NTR cargo complexes to rapidly enter. They even recapitulated the observations that large cargo-domains counteract NPC passage of NTR⋅cargo complexes, while cargo shielding and increased NTR⋅cargo surface-ratios override this inhibition. Their exquisite NPC-typical sorting selectivity and strong intrinsic assembly propensity suggest that Nup98 FG phases can form in authentic NPCs and indeed account for the permeability properties of the pore.

Background: A vast array of parameters has been proposed to predict mortality in chronic heart failure (CHF). Their applicability into clinical practice remains challenging due to economical and availability considerations., Methods and Results: We studied serum uric acid, total cholesterol, and soluble tumour necrosis factor receptor 1 (sTNF-R1) in 114 CHF patients (63.0 ± 1.0 years, NYHA functional class I/II/III/IV: 11/34/54/15) recruited prospectively into a metabolic study program. All patients underwent assessment of left ventricular ejection fraction and measurement of peak oxygen consumption (pVO(2)). Patients were followed for 24 months or until death. A total of 31 patients died; cumulative survival was 78% (95% confidence interval [CI] 70-86%) and 73% (65-81%) at 12 and 24 months, respectively. In single predictor Cox proportional hazard analysis, uric acid, pVO2, sTNFR-1, LVEF (all p<0.0001) and cholesterol (p<0.02) all predicted survival. All parameters remained significant predictors of death after multivariable adjustment (all p<0.02). Receiver-operator characteristic (ROC) curve analyses showed that uric acid and sTNF-R1 are equally strong with regards to their prognostic performance in CHF like pVO(2,) but even better than LVEF. The combination of pVO(2), LVEF, uric acid, and sTNF-R1 in ROC statistics turned out as the best model with the highest prognostic value in CHF (AUC: 0.91, sensitivity: 90.4, specificity: 74.2, p=0.0001)., Conclusion: Including metabolic-immunological parameters into risk assessment might result in a better risk stratification than modeling based on clinical parameters alone, probably due to a better reflection of CHF as multisystem disease. We suggest metabolic-immunological parameters to be tested in larger populations., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The 62 kDa FG repeat domain of the nucleoporin Nsp1p forms a hydrogel-based, sieve-like permeability barrier that excludes inert macromolecules but allows rapid entry of nuclear transport receptors (NTRs). We found that the N-terminal part of this domain, which is characterized by Asn-rich inter-FG spacers, forms a tough hydrogel. The C-terminal part comprises charged inter-FG spacers, shows low gelation propensity on its own, but binds the N-terminal part and passivates the FG hydrogel against nonselective interactions. It was previously shown that a hydrophobic collapse involving Phe residues is required for FG hydrogel formation. Using solid-state NMR spectroscopy, we now identified two additional types of intragel interactions, namely, transient hydrophobic interactions between Phe and methyl side chains as well as intermolecular beta-sheets between the Asn-rich spacer regions. The latter appear to be the kinetically most stable structures within the FG hydrogel. They are also a central feature of neuronal inclusions formed by Asn/Gln-rich amyloid and prion proteins. The cohesive properties of FG repeats and the Asn/Gln-rich domain from the yeast prion Sup35p appear indeed so similar to each other that these two modules interact in trans. Our data, therefore, suggest a fully unexpected cellular function of such interchain beta-structures in maintaining the permeability barrier of nuclear pores. They provide an explanation for how contacts between FG repeats might gain the kinetic stability to suppress passive fluxes through nuclear pores and yet allow rapid NTR passage.

Aims: Acute heart failure syndromes, commonly recognized as de novo heart failure or acute decompensated chronic heart failure (ADHF), are characterized by a rapid onset or change in signs and symptoms of heart failure requiring urgent treatment. Coexisting renal dysfunction is associated with poor prognosis in these patients. We sought to determine whether renal impairment in particular and other admission factors in general predict long-term mortality after hospitalization for ADHF., Methods and Results: We studied 128 patients (age 63 + or - 12 years, 76% male) in NYHA class 2.6 + or - 0.7 with a left ventricular ejection fraction (LVEF) < or = 39%, hospitalized due to ADHF. Mortality rates (per 100 person-years) were 21.9 at 12 months and 12.0 at 60 months. We found that admission serum creatinine level was the best predictor of mortality after 1 (P < 0.001, log-transformed due to skewed distribution) and 5 years (P = 0.001), followed by creatinine clearance, the use of loop diuretics, and digoxin. Moreover, higher NYHA class, decreased body mass index (BMI) and increased levels of urea predicted 1 and 5 years mortality on univariate analysis. In the multivariate analysis, creatinine, NYHA class, and LVEF emerged as independent predictors of mortality after 1 year, whereas BMI and the use of diuretics did not reach significance (joint chi(2) = 29.40, P < 0.001). After 5 years, creatinine and NYHA class independently predicted all-cause mortality (joint chi(2) = 22.71, P < 0.001), but BMI and age did not remain significant., Conclusion: Admission creatinine level strongly predicts medium- and long-term mortality after hospitalization in patients with ADHF, and serves as a cheap and fast clinical marker to identify patients at risk of death.

The disease risk is very high among transition cows that may suffer from poor appetite. The aim of the present study was therefore to investigate the effect of drenching on energy balance, hydration state and selected production parameters in fresh cows. Twenty-one Danish Holstein-Friesian dairy cows in late pregnancy were randomly allocated to either treatment (TG) or control group (CG). TG cows were drenched twice with 20 l of water containing a mixture of calcium propionate, MgSO(4), and KCL specifically developed for prophylactic treatment of fresh cows. The results indicated that cows become dehydrated around calving, losing on average 53 l (TG) and 24.5 l (CG) of extra-cellular fluid, respectively. The drenching volume applied in the present study did not affect the degree of hydration after calving. Based on blood NEFA and BHB values it was shown that drenching caused a reduction in the degree of negative energy balance (NEB). Somatic cell count (SCC) for cows in first lactation was lower in the TG compared to CG. Milk yield was unaffected by treatment. We conclude that prophylactic drenching added little to the health promotion in the transition cows in the present study. Instead, increased focus on management routines would probably be of more value.

Myocardial depression in human sepsis was only unequivocally proven in the 1980s by the group of Parrillo, who used nuclear imaging techniques to measure heart volumes and function in intensive care patients. Heart failure in sepsis is frequently masked by a seemingly normal cardiac output. However, relative to the lowered systemic vascular resistance - resulting in a reduced afterload - cardiac outputs and ventricular ejection fractions are often not adequately enhanced. This septic cardiomyopathy (impairment of the heart within the scope of systemic sepsis) involves both the right and the left ventricles, and is potentially reversible. In response to volume substitution, the heart can be considerably enlarged. The cardiomyopathy is not primarily hypoxic in nature, but may be aggravated by ischemia. Autonomic dysfunction, documented by a reduced heart rate variability and impaired baroreflex and chemoreflex sensitivities, forms part of the disease entity. The severity of myocardial depression correlates with a poor prognosis. Noninfectious systemic inflammatory response syndrome can give rise to an analogous disease entity, namely, systemic inflammatory response syndrome cardiomyopathy.The etiology of septic cardiomyopathy is multifactorial. Several candidates with a potential pathogenetic impact on the heart were identified: bacterial toxins; cytokines and mediators including tumour necrosis factor-alpha, interleukin-1 and nitric oxide; cardiodepressant factors; oxygen reactive species; and catecholamines. Symptomatic treatment consists of volume substitution and catecholamine support; causal therapeutic approaches aiming at an interruption of the proinflammatory mediator cascades are being tested.

Background: The assessment of autonomic function is an important tool for risk stratification in critically ill patients. Peripheral cardiac chemoreflex sensitivity has been considered a marker for increased risk of sudden cardiac death. In normals, the evaluation of peripheral cardiac chemoreflex sensitivity is performed under controlled breathing conditions during inhalation of hypoxic gas. Since this is poorly tolerated by patients, they are commonly studied under hyperoxic conditions, which are not physiological., Methods: We studied 20 healthy volunteers who underwent free and controlled breathing of a hypoxic gas mixture (10% O2 in N2) over 5 min. Values of peripheral cardiac chemoreflex sensitivity, corrected for respiratory influence, were compared with the results obtained experimentally under controlled breathing conditions in the same subjects., Results: We found a substantial difference between values obtained during free and controlled breathing (3.64 +/- 0.81 vs. 1.53 +/- 0.32 ms/mmHg, respectively; P < 0.05). After application of a respiratory correction, described and validated in this article, no significant difference was seen for these values (0.89 +/-0.91 vs. 1.53 +/- 0.32 ms/mmHg, P = 0.46)., Conclusions: This approach allows the evaluation of peripheral cardiac chemoreflex sensitivity in free breathing subjects. This correction could improve the assessment of cardiac chemoreflex sensitivity in patients with cardiorespiratory disorders, who find it difficult to control their breathing according to an experimental protocol.

This study used the "oddball" counting paradigm to examine the possible habituation of the auditory P300 response. Twenty subjects kept a mental record of the number of rarely occurring tone pips presented in a series of more frequently occurring tone pips. Data were collected continuously until responses to 150 rare tone pips were obtained. Findings indicated that the P300 complex decreased in amplitude as a result of repeated stimulation. The decline was logarithmic, not linear, which suggests a stabilization of the amplitude over time. We suggest that the attenuation of amplitude was habituation and not a result of a recovery cycle.

In host range investigations with 152 plant species of 37 families, 96 species from 28 families proved to be experimentally susceptible to asparagus virus 2 (AsV 2). From these, 82 species of 26 families were systematically infectible and 14 species of 10 families only locally. Most frequently were symptomless systemic infections. Of 86 species, their susceptibility for AsV 2 was unknown before. The number of families that contant experimental hosts of the virus increased from 11 to 33. Transmission of AsV 2 was neither possible with Myzus persicae nor with Cuscuta californica or C. campestris. AsV 2-infected mother plants transmitted the virus by about 40 per cent to their seedings. Open-pollinated plants, not infested by AsV 2, showed 3 to 22 per cent of infected seedings. Mechanical back-transmission of AsV 2 from test plants to asparagus succeeded only in one case. The properties of the virus in vitro were as follows: thermal inactivation point between 64 and 66 degrees C, dilution end point between 10(-3) and 10(-4), stability in sap, stored at room temperature, up to 96 hours. Serological investigations demonstrated no relationship to the virus to numerous other viruses. An antiserum with a titer of 1 to 16 was useful for the detection of AvV 2 in asparagus sap. Electron microscopical investigations proved for the virus isometric particles for the first time. In negatively stained preparations they had a diameter of 27 nm.