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Metabolic flux investigations of cells and tissue samples are a rapidly advancing tool in diverse research areas. Reliable methods of data normalization are crucial for an adequate interpretation of results and to avoid a misinterpretation of experiments and incorrect conclusions. The most common methods for metabolic flux data normalization are to cell number, DNA and protein. Data normalization may be affected by a variety of factors, such as density, healthy state, adherence efficiency, or proportional seeding of cells. The mussel-derived adhesive Cell Tak is often used to immobilize poorly adherent cells. Here we demonstrate that this coating strongly affects the fluorescent detection of DNA leading to an incorrect and highly variable normalization of metabolic flux data. Protein assays are much less affected and cell counting can virtually completely remove the effect of the coating. Cell-Tak coating also affects cell shape in a cell line-specific manner and may change cellular metabolism. Based on these observations we recommend cell counting as a gold standard normalization method for Seahorse metabolic flux measurements with protein content as a reasonable alternative.

We have previously documented that in liver cells, the multifunctional protein scaffold p62/SQSTM1 is closely associated with IκBα, an inhibitor of the transcriptional activator NF-κB. Such an intimate p62-IκBα association we now document leads to a marked 18-fold proteolytic IκBα-stabilization, enabling its nuclear entry and termination of the NF-κB-activation cycle. In p62-/--cells, such termination is abrogated resulting in the nuclear persistence and prolonged activation of NF-κB following inflammatory stimuli. Utilizing various approaches both classic (structural deletion, site-directed mutagenesis) as well as novel (in cell chemical crosslinking), coupled with proteomic analyses, we have defined the precise structural hotspots of p62-IκBα association. Accordingly, we have identified such IκBα hotspots to reside around N-terminal (K38, K47 and K67) and C-terminal (K238/C239) residues in its 5thankyrin repeat domain. These sites interact with two hotspots in p62: One in its PB-1 subdomain around K13, and the other comprised of a positively charged patch (R183/R186/K187/K189) in the intervening region between its ZZ- and TB-subdomains. APEX proximity analyses upon IκBα co-transfection of cells with and without p62 have enabled the characterization of the p62 influence on IκBα-protein-protein interactions. Interestingly, consistent with p62’s capacity to proteolytically stabilize IκBα, its presence greatly impaired IκBα’s interactions with various 20S/26S proteasomal subunits. Furthermore, consistent with p62-interaction with IκBα on an interface opposite to that of its NF-κB-interacting interface, p62 failed to significantly affect IκBα-NF-κB interactions. These collective findings together with the known dynamic p62 nucleocytoplasmic shuttling, leads us to speculate that it may be involved in “piggy-back” nuclear transport of IκBα following its NF-κB-elicited transcriptional activation andde novosynthesis, required for the termination of the NF-κB-activation cycle. Consequently, mice carrying a liver specific deletion of p62-residues 68-252 harboring its positively charged patch, reveal age-dependent enhanced liver inflammation. Our findings reveal yet another mode of p62-mediated pathophysiologically relevant regulation of NF-κB.Abstract FigureHighlightsp62 binds to and stabilizes IκBα by preventing its proteolytic degradationIn-cell chemical crosslinking/LC-MS/MS identified the inter-crosslinked sitesHotspots of p62-IκBα association are definedAPEX proximity labeling revealed p62 impaired IκBα-interaction with proteasomep62 chaperones newly synthesized IκBα to terminate NF-κB activation.In BriefThe transcriptional activator NF-κB inhibitor, IκBα is proteolytically unstable when uncomplexed. How newly synthesized IκBα escapes degradation to terminate nuclear NF-κB-activation is unknown. Using in-cell chemical crosslinking and proximity labeling MS analyses, we uncovered a novel association of p62 with IκBα via well-defined structural hotspots, which impairs its interaction with the 26S/20S proteasome, extending its life-span and enabling termination of NF-κB-activation. Mice carrying liver-specific genetic deletion of p62-IκBα hotspot exhibit enhanced liver inflammation upon aging, validating this novel p62 role.

The behavior of three copolymers high density polyethylene at high rate loading was investigated. Namely, one unimodal type (HDPE-1) and two bimodal type. Structure of PE samples was characterized by 13C NMR (Carbon-13 nuclear magnetic resonance). The Taylor impact test was used. Specimens in form of a cylinder (14 mm in diameter and 50 mm in length) were fired against a steel bar. Striking velocities achieved up to 116 m/s. No permanent strain of these specimens was detected. Numerical simulation of these tests showed that the material behavior is probably viscoelastic. Results showed that there was no difference in the impact behavior of tested polymers. This conclusion was obtained both for the results in the time domain and the frequency domain.

Background Short-term forecasts of infectious disease burden can contribute to situational awareness and aid capacity planning. Based on best practice in other fields and recent insights in infectious disease epidemiology, one can maximise the predictive performance of such forecasts if multiple models are combined into an ensemble. Here we report on the performance of ensembles in predicting COVID-19 cases and deaths across Europe between 08 March 2021 and 07 March 2022. Methods We used open-source tools to develop a public European COVID-19 Forecast Hub. We invited groups globally to contribute weekly forecasts for COVID-19 cases and deaths reported from a standardised source over the next one to four weeks. Teams submitted forecasts from March 2021 using standardised quantiles of the predictive distribution. Each week we created an ensemble forecast, where each predictive quantile was calculated as the equally-weighted average (initially the mean and then from 26th July the median) of all individual models’ predictive quantiles. We measured the performance of each model using the relative Weighted Interval Score (WIS), comparing models’ forecast accuracy relative to all other models. We retrospectively explored alternative methods for ensemble forecasts, including weighted averages based on models’ past predictive performance. Results Over 52 weeks we collected and combined up to 28 forecast models for 32 countries. We found a weekly ensemble had a consistently strong performance across countries over time. Across all horizons and locations, the ensemble performed better on relative WIS than 84% of participating models’ forecasts of incident cases (with a total N=862), and 92% of participating models’ forecasts of deaths (N=746). Across a one to four week time horizon, ensemble performance declined with longer forecast periods when forecasting cases, but remained stable over four weeks for incident death forecasts. In every forecast across 32 countries, the ensemble outperformed most contributing models when forecasting either cases or deaths, frequently outperforming all of its individual component models. Among several choices of ensemble methods we found that the most influential and best choice was to use a median average of models instead of using the mean, regardless of methods of weighting component forecast models. Conclusions Our results support the use of combining forecasts from individual models into an ensemble in order to improve predictive performance across epidemiological targets and populations during infectious disease epidemics. Our findings further suggest that median ensemble methods yield better predictive performance more than ones based on means. Our findings also highlight that forecast consumers should place more weight on incident death forecasts than incident case forecasts at forecast horizons greater than two weeks. Code and data availability All data and code are publicly available on Github: covid19-forecast-hub-europe/euro-hub-ensemble.

This paper presents 2109 times of minima for 965 objects acquired by 59 members and cooperating observers of the Variable Star and Exoplanet Section of the Czech Astronomical Society (B.R.N.O. Observing project).These observations were submitted to the website of the Variable Star and Exoplanet Section of the Czech Astronomical Society between November 2016 and March 2018.

During cell division, remodelling of the nuclear envelope enables chromosome segregation by the mitotic spindle1. The reformation of sealed nuclei requires ESCRTs (endosomal sorting complexes required for transport) and LEM2, a transmembrane ESCRT adaptor2–4. Here we show how the ability of LEM2 to condense on microtubules governs the activation of ESCRTs and coordinated spindle disassembly. The LEM motif of LEM2 binds BAF, conferring on LEM2 an affinity for chromatin5,6, while an adjacent low-complexity domain (LCD) promotes LEM2 phase separation. A proline–arginine-rich sequence within the LCD binds to microtubules and targets condensation of LEM2 to spindle microtubules that traverse the nascent nuclear envelope. Furthermore, the winged-helix domain of LEM2 activates the ESCRT-II/ESCRT-III hybrid protein CHMP7 to form co-oligomeric rings. Disruption of these events in human cells prevented the recruitment of downstream ESCRTs, compromised spindle disassembly, and led to defects in nuclear integrity and DNA damage. We propose that during nuclear reassembly LEM2 condenses into a liquid-like phase and coassembles with CHMP7 to form a macromolecular O-ring seal at the confluence between membranes, chromatin and the spindle. The properties of LEM2 described here, and the homologous architectures of related inner nuclear membrane proteins7,8, suggest that phase separation may contribute to other critical envelope functions, including interphase repair8–13 and chromatin organization14–17. Following cell division, phase separation of the transmembrane adaptor LEM2 ensures that the ESCRT machinery remodels microtubules and seals the nuclear envelope.

We have previously documented that in liver cells, the multifunctional protein scaffold p62/SQSTM1 is closely associated with IκBα, an inhibitor of the transcriptional activator NF-κB. Such an intimate p62-IκBα association we now document leads to a marked 18-fold proteolytic IκBα-stabilization, enabling its nuclear entry and termination of the NF-κB-activation cycle. In p62-/--cells, such termination is abrogated resulting in the nuclear persistence and prolonged activation of NF-κB following inflammatory stimuli. Utilizing various approaches both classic (structural deletion, site-directed mutagenesis) as well as novel (in-cell chemical crosslinking), coupled with proteomic analyses, we have defined the precise structural hotspots of p62-IκBα association. Accordingly, we have identified such IκBα hotspots to reside around N-terminal (K38, K47, and K67) and C-terminal (K238/C239) residues in its fifth ankyrin repeat domain. These sites interact with two hotspots in p62: One in its PB-1 subdomain around K13, and the other comprised of a positively charged patch (R183/R186/K187/K189) between its ZZ-and TB-subdomains. APEX proximity analyses upon IκBα-cotransfection of cells with and without p62 have enabled the characterization of the p62 influence on IκBα-protein-protein interactions. Interestingly, consistent with p62's capacity to proteolytically stabilize IκBα, its presence greatly impaired IκBα's interactions with various 20S/26S proteasomal subunits. Furthermore, consistent with p62 interaction with IκBα on an interface opposite to that of its NF-κB-interacting interface, p62 failed to significantly affect IκBα-NF-κB interactions. These collective findings together with the known dynamic p62 nucleocytoplasmic shuttling leads us to speculate that it may be involved in "piggy-back" nuclear transport of IκBα following its NF-κB-elicited transcriptional activation and de novo synthesis, required for termination of the NF-κB-activation cycle. Consequently, mice carrying a liver-specific deletion of p62-residues 68 to 252 reveal age-dependent-enhanced liver inflammation. Our findings reveal yet another mode of p62-mediated pathophysiologically relevant regulation of NF-κB.

Unique among metazoan repressive histone methyltransferases, G9a and GLP, which chiefly target histone 3 lysine 9 (H3K9), require dimerization for productive H3K9 mono (me1)- and dimethylation (me2) invivo. Intriguingly, even though each enzyme can independently methylate H3K9, the predominant active form invivo is a heterodimer of G9a and GLP. How dimerization influences the central H3K9 methyl binding ("reading") and deposition ("writing") activity of G9a and GLP and why heterodimerization is essential invivo remains opaque. Here, we examine the H3K9me "reading" and "writing" activities of defined, recombinantly produced homo- and heterodimers of G9a and GLP. We find that both reading and writing are significantly enhanced in the heterodimer. Compared with the homodimers, the heterodimer has higher recognition of H3K9me2, and a striking ∼10-fold increased turnover rate for nucleosomal substrates under multiple turnover conditions, which is not evident on histone tail peptide substrates. Cross-linking Mass Spectrometry suggests that differences between the homodimers and the unique activity of the heterodimer may be encoded in altered ground state conformations, as each dimer displays different domain contacts. Our results indicate that heterodimerization may be required to relieve autoinhibition of H3K9me reading and chromatin methylation evident in G9a and GLP homodimers. Relieving this inhibition may be particularly important in early differentiation when large tracts of H3K9me2 are typically deposited by G9a-GLP, which may require a more active form of the enzyme.

RAS proteins are molecular switches that interact with effector proteins when bound to guanosine triphosphate, stimulating downstream signaling in response to multiple stimuli. Although several canonical downstream effectors have been extensively studied and tested as potential targets for RAS-driven cancers, many of these remain poorly characterized. In this study, we undertook a biochemical and structural approach to further study the role of Sin1 as a RAS effector. Sin1 interacted predominantly with KRAS isoform 4A in cells through an atypical RAS-binding domain that we have characterized by X-ray crystallography. Despite the essential role of Sin1 in the assembly and activity of mTORC2, we find that the interaction with RAS is not required for these functions. Cells and mice expressing a mutant of Sin1 that is unable to bind RAS are proficient for activation and assembly of mTORC2. Our results suggest that Sin1 is a bona fide RAS effector that regulates downstream signaling in an mTORC2-independent manner.

TRIO encodes a cytoskeletal regulatory protein with three catalytic domains-two guanine exchange factor (GEF) domains, GEF1 and GEF2, and a kinase domain-as well as several accessory domains that have not been extensively studied. Function-damaging variants in the TRIO gene are known to be enriched in individuals with neurodevelopmental disorders (NDDs). Disease variants in the GEF1 domain or the nine adjacent spectrin repeats (SRs) are enriched in NDDs, suggesting that dysregulated GEF1 activity is linked to these disorders. We provide evidence here that the Trio SRs interact intramolecularly with the GEF1 domain to inhibit its enzymatic activity. We demonstrate that SRs 6-9 decrease GEF1 catalytic activity both invitro and in cells and show that NDD-associated variants in the SR8 and GEF1 domains relieve this autoinhibitory constraint. Our results from chemical cross-linking and bio-layer interferometry indicate that the SRs primarily contact the pleckstrin homology region of the GEF1 domain, reducing GEF1 binding to the small GTPase Rac1. Together, our findings reveal a key regulatory mechanism that is commonly disrupted in multiple NDDs and may offer a new target for therapeutic intervention for TRIO-associated NDDs.

In a combined experimental and theoretical study, we probe the vibrational excitation of isocyanic acid induced by electron impact in the energy range up to 5 eV. Experimentally, we report differential elastic and vibrationally inelastic cross sections at the scattering angle of ${135}^{\ensuremath{\circ}}$. Theoretically, we characterize the involved resonant states using a regularized analytical continuation method. We also apply a nonlocal resonance model to calculate cross sections for vibrational mode that involves the N-H stretching motion. The model reproduces all the features observed in the experiment: efficient excitation at threshold, sharp cusps in the excitation functions, and the formation of an ${A}^{\ensuremath{'}}$ shape resonance. There is a second $({A}^{\ensuremath{'}\ensuremath{'}})$ resonance visible in the spectra; however, it excites only selected vibrational modes. The origins of this selectivity are discussed.

Background & AimsMallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-κB activation. However, the precise mechanism that links protein aggregation to NFκB-activation and inflammatory response remains unclear.MethodsHerein, we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin, protoporphyrin IX (PPIX), or ZnPPIX) elicited an IκBα-loss with consequent NF-κB activation. We characterized the underlying mechanism in detail using hepatocytes from various knockout mice and MEF cell lines and multiple approaches including immunoblotting, EMSA, RT-PCR, confocal immunofluorescence microscopy, affinity immunoprecipitation, and protein solubility assays. Additionally, we performed rigorous proteomic analyses to identify the proteins aggregating upon PPIX treatment and/or co-aggregating with IκBα.ResultsFour known mechanisms of IκBα-loss were probed and excluded. Immunofluorescence analyses of ZnPPIX-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IκBα-loss was due to its sequestration along with IκBβ into insoluble aggregates. Through proteomic analyses we identified 47 aggregation-prone proteins that co-aggregate with IκBα through direct interaction or proximity. Of these ZnPPIX-aggregation targets, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as likely mediators of IκBα-nuclear import.ConclusionWe discovered a novel mechanism of inflammatory NF-κB activation through IκB-sequestration into insoluble aggregates along with interacting aggregation-prone proteins. This mechanism may account for the protein aggregate-induced inflammation observed in MDB-associated liver diseases, thereby identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is abona fideprotoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.Lay SummaryMallory-Denk-bodies (MDBs) are hepatic protein aggregates commonly featured in many liver diseases. MDB-presence is associated with the induction of inflammatory responses both clinically and in all MDB-inducing models. Similar protein aggregation in neurodegenerative diseases is also known to trigger inflammation and NFκB pathway activation via an as yet to be characterized non-canonical mechanism. Herein using a MDB-inducing cell model, we uncovered a novel mechanism for NFκB activation via cytosolic IκB-sequestration into insoluble aggregates. Furthermore, using a proteomic approach, we identified 47 aggregation-prone proteins that interact and co-aggregate with IκBα. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thereby identifying novel targets for therapeutic intervention.

Histone demethylase KDM5A removes methyl marks from lysine 4 of histone H3 and is often overexpressed in cancer. The in vitro demethylase activity of KDM5A is allosterically enhanced by binding of its product, unmodified H3 peptides, to its PHD1 reader domain. However, the molecular basis of this allosteric enhancement is unclear. Here we show that saturation of the PHD1 domain by the H3 N-terminal tail peptides stabilizes binding of the substrate to the catalytic domain and improves the catalytic efficiency of demethylation. When present in saturating concentrations, differently modified H3 N-terminal tail peptides have a similar effect on demethylation. However, they vary greatly in their affinity towards the PHD1 domain, suggesting that H3 modifications can tune KDM5A activity. Furthermore, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) experiments reveal conformational changes in the allosterically enhanced state. Our findings may enable future development of anti-cancer therapies targeting regions involved in allosteric regulation., The demethylase activity of KDM5A is allosterically enhanced by binding of histone H3 to its PHD1 reader domain, through an unknown mechanism. Here the authors show that the PHD1 domain drives ligand-induced allosteric stimulation by stabilizing the binding of substrate to the catalytic domain.

Purpose: This study was aimed to review the short-term results of endoscopic percutaneous longitudinal tenotomy for noninsertional Achilles tendinopathy using the Centerline Endoscopic Carpal Tunnel Release instrument (Arthrex). This method simplifies the operation technique, allows a good endoscopic visualisation of the Achilles tendon with very promising results. Methods: We performed multiple percutaneous longitudinal tenotomies under local anesthesia in 24 patients (25 tendons) with Achilles tendinopathy or peritendinitis that had failed conservative treatment between January 2013 and September 2016. All ambulatory procedures consisted of paratenon release and longitudinal tenotomies. The results were reviewed in 22 patients (23 tendons) at an average follow-up period of 22.5 months (range 10-36 months). Patients’ satisfaction and functional outcomes were evaluated using the Victorian Institute of Sports Assessment–Achilles (VISA-A) questionnaire, the pain visual analog scale (VAS), and the functional foot index. Results: Initial results are very promising with excellent results in 12 patients, good results in 9 patients, and fair result in 1 patient. One patient developed a postoperative thrombosis of the operated limb. Another patient developed a hypertrophic painful scar of the incision wound. The VAS for pain decreased drastically after the index procedure and averaged to 0.2 (SD 0.447). The VISA-A questionnaire score had improved from 42 ± 7.2 points preoperatively to 96.8 ± 14.3 points postoperatively (P = .004). The functional foot index decreased from 84 (SD 30.517) to 33.4 (SD 6.452) on the follow-up examination. Conclusions: The endoscopic-assisted longitudinal tenotomies procedure of the Achilles tendon is easily feasible and can be performed on an outpatient basis, produces minimal complications and shows excellent results. The described technique is recommended for all surgeons especially for those familiar with endoscopy of the Achilles tendon. Levels of Evidence: Therapeutic, Level IV: Case Series

Aims Hindfoot arthrodesis with retrograde intramedullary nailing has been described as a surgical strategy to reconstruct deformities of the ankle and hindfoot in patients with Charcot arthropathy. This study presents case series of Charcot arthropathy patients treated with two different retrograde intramedullary straight compression nails in order to reconstruct the hindfoot and assess the results over a mid-term follow-up. Patients and Methods We performed a retrospective analysis of 18 consecutive patients and 19 operated feet with Charcot arthropathy who underwent a hindfoot arthrodesis using a retrograde intramedullary compression nail. Patients were ten men and eight women with a mean age of 63.43 years (38.5 to 79.8). We report the rate of limb salvage, complications requiring additional surgery, and fusion rate in both groups. The mean duration of follow-up was 46.36 months (37 to 70). Results The limb salvage rate was 16 of 19 limbs. Three patients had to undergo below-knee amputation due to persistent infection followed by osteomyelitis resistant to parenteral antibiotic therapy and repeated debridement. Complications including infection, hardware removal, nonunion, and persistent ulcers requiring further intervention were also observed. Postoperative functional scores revealed significant improvement compared with preoperative scores on American Orthopaedic Foot and Ankle Society (AOFAS) – Hindfoot scale, Foot Function Index (FFI), visual analogue scale (VAS), and Foot and Ankle Outcome Score (FAOS). Conclusion The use of retrograde intramedullary compression nail results in good rates of limb salvage when used for hindfoot reconstruction in patients with Charcot arthropathy. Cite this article: Bone Joint J 2018;100-B:190–6.

Granulosa cells (GCs) are somatic cells essential for establishing and maintaining bi-directional communication with the oocytes. This connection has a profound importance for the delivery of energy substrates, structural components and ions to the maturing oocyte through gap junctions. Cumulus cells, group of closely associated GCs, surround the oocyte and can diminished the effect of harmful environmental insults. Both GCs and oocytes prefer different energy substrates in their cellular metabolism: GCs are more glycolytic, whereas oocytes rely more on oxidative phosphorylation pathway. The interconnection of these cells is emphasized by the fact that GCs supply oocytes with intermediates produced in glycolysis. The number of GCs surrounding the oocyte and their age affect the energy status of oocytes. This review summarises available studies collaboration of cellular types in the ovarian follicle from the point of view of energy metabolism, signaling and protection of toxic insults. A deeper knowledge of the underlying mechanisms is crucial for better methods to prevent and treat infertility and to improve the technology of in vitro fertilization.

Hyperechogenicity of the substantia nigra (SN) and abnormal dopamine transporter-single-photon emission computed tomography (DAT-SPECT) are biomarkers commonly used in the assessment of prodromal synucleinopathy. Our goals were as follows: (1) to compare echogenicity of SN in idiopathic rapid eye movement (REM) behavior disorder (iRBD), Parkinson's disease (PD) without RBD (PD-noRBD), PD with RBD (PD + RBD), and control subjects; and (2) to examine association between SN degeneration assessed by DAT-SPECT and SN echogenicity.A total of 61 subjects with confirmed iRBD were examined using Movement Disorders Society-unified PD rating scale (MDS-UPDRS), TCS (transcranial sonography) and DAT-SPECT. The results were compared with 44 patients with PD (25% PD + RBD) and with 120 age-matched healthy subjects.The abnormal SN area was found in 75.5% PD, 23% iRBD and 7.3% controls. Median SN echogenicity area in PD (0.27 ± 0.22 cm

Heterochromatin affects genome function at many levels. It enables heritable gene repression, maintains chromosome integrity and provides mechanical rigidity to the nucleus1,2. These diverse functions are proposed to arise in part from compaction of the underlying chromatin2. A major type of heterochromatin contains at its core the complex formed between HP1 proteins and chromatin that is methylated on histone H3, lysine 9 (H3K9me). HP1 is proposed to use oligomerization to compact chromatin into phase-separated condensates3-6. Yet, how HP1-mediated phase separation relates to chromatin compaction remains unclear. Here we show that chromatin compaction by the Schizosaccharomyces pombe HP1 protein Swi6 results in phase-separated liquid condensates. Unexpectedly, we find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a nucleosome. Restraining these dynamics impairs compaction of chromatin into liquid droplets by Swi6. Our results indicate that Swi6 couples its oligomerization to the phase separation of chromatin by a counterintuitive mechanism, namely the dynamic exposure of buried nucleosomal regions. We propose that such reshaping of the octamer core by Swi6 increases opportunities for multivalent interactions between nucleosomes, thereby promoting phase separation. This mechanism may more generally drive chromatin organization beyond heterochromatin.

Congenital hyperinsulinism (CHI) is a neuroendocrine disease with focal or diffuse abnormalities in pancreas. While drug-resistant diffuse forms require near-total pancreatectomy or prolonged pharmacotherapy, focal CHI may be treated by targeted surgical resection. We evaluated the usefulness ofNineteen children (11 boys, 8 girls, aged 2-54 months) with clinical signs of neonatal CHI and positive genetic examinations were enrolled in the study. After i.v. administration ofFocal forms were recorded in 10/19 children and 4 of them underwent surgical resection with complete recovery. Focal uptake was significantly higher than the uptake in the normal pancreatic tissue (p=0.0059). Focal and diffuse forms of CHI did not differ significantly in normal pancreatic tissue uptake. We found no advantage in the measurement of SUV

Mitochondria are highly dynamic organelles involved in many cellular functions. Beyond their central role in metabolism, they also take a part in maintaining calcium homeostasis, cell death, immunity, and ROS production. Changes in these functions have been shown to be crucial for the adaptation and survival of cancer cells. Mitochondria, therefore, constitute a promising target for the development of novel anticancer agents. The triphenylphosphonium (TPP+) moiety has been widely used to target molecules into mitochondria. TPP+ derivatives of a variety of conventional cytostatic drugs, natural substances, metformin, antioxidants or a range of newly synthesized molecules have shown promising results against cancer cells. In this review, we discuss biochemical differences between cancer cells and normal cells with a specific focus on mitochondria, and how mitochondrially targeted molecules can be used to selectively affect mitochondrial function in normal and cancer cells. We summarize the published data on mitochondrially targeted anticancer agents and propose future research avenues.

Highlights • Integrative methods spearhead structural studies of Mediator complex. • 3D architectural models combining data from diverse techniques. • Exhaustive computational sampling produces best macromolecular models. • Complex modularity and transferability encoded in modelling scoring function. • Structure studies of dynamic and disordered transcription complexes tractable., Integrative structural biology combines data from multiple experimental techniques to generate complete structural models for the biological system of interest. Most commonly cross-linking data sets are employed alongside electron microscopy maps, crystallographic structures, and other data by computational methods that integrate all known information and produce structural models at a level of resolution that is appropriate to the input data. The precision of these modelled solutions is limited by the sparseness of cross-links observed, the length of the cross-linking reagent, the ambiguity arisen from the presence of multiple copies of the same protein, and structural and compositional heterogeneity. In recent years integrative structural biology approaches have been successfully applied to a range of RNA polymerase II complexes. Here we will provide a general background to integrative structural biology, a description of how it should be practically implemented and how it has furthered our understanding of the biology of large transcriptional assemblies. Finally, in the context of recent breakthroughs in microscope and direct electron detector technology, where increasingly EM is capable of resolving structural features directly without the aid of other structural techniques, we will discuss the future role of integrative structural techniques.

This study was aimed to study the arthrodesis of the first metatarsophalangeal joint using an oblique interfragmentary lag screw and dorsal plate as an effective option for the treatment of hallux rigidus. Few researchers have studied the outcome of this surgical method over a long follow-up period. We performed a retrospective review of 60 patients status post arthrodesis of the first metatarsophalangeal joint. The mean age was 68.5 years and average follow-up lasted for 47.3 months. Patients’ satisfaction and functional outcomes were evaluated with the American Orthopaedic Foot and Ankle Society Hallux Metatarsophalangeal-Interphalangeal Scale, the foot and ankle questionnaire, the Functional Foot Index, and dynamic pedobarography. Fusion rate of 93.3 % was recorded; 6.7 % of the cases ended up with a painless pseudarthrosis and required no additional surgery. Pedobarographic measurements demonstrated first ray weight bearing function restoration. More physiological foot plantar pressure patterns were observed. Concerning the postoperative outcomes, 71.7 % of the patients were very satisfied and 18.3 % were satisfied. The American Orthopaedic Foot and Ankle Society Hallux Metatarsophalangeal-Interphalangeal Scale increased post-operatively from 40.9 to 79.3. The arthrodesis of the metatarsophalangeal joint using transarticular screw and dorsal nonlocked plate is an effective method for the treatment of severe hallux rigidus with fair patient satisfaction rate and functional outcome.

The heterochromatin protein HP1 is proposed to enable chromatin compaction via liquid droplet formation. Yet, a connection between phase separation and chromatin compaction has not been experimentally demonstrated. More fundamentally, how HP1 action at the level of a single nucleosome drives chromatin compaction remains poorly understood. Here we directly demonstrate that the S.pombeHP1 protein, Swi6, compacts arrays of multiple nucleosomes into phase-separated droplets. Using hydrogen-deuterium exchange, NMR, and mass-spectrometry, we further find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a mononucleosome. Restraining these dynamics via site-specific disulfide bonds impairs the compaction of nucleosome arrays into phase-separated droplets. Our results indicate that chromatin compaction and phase separation can be highly coupled processes. Further, we find that such coupling is promoted by a counter-intuitive function of Swi6, namely disorganization of the octamer core. Phase separation is canonically mediated by weak and dynamic multivalent interactions. We propose that dynamic exposure of buried histone residues increases opportunities for multivalent interactions between nucleosomes, thereby coupling chromatin compaction to phase separation. We anticipate that this new model for chromatin organization may more generally explain the formation of highly compacted chromatin assemblies beyond heterochromatin.

Summary The dicistrovirus, Cricket paralysis virus (CrPV) encodes an RNA interference (RNAi) suppressor, 1A, which modulates viral virulence. Using the Drosophila model, we combined structural, biochemical, and virological approaches to elucidate the strategies by which CrPV-1A restricts RNAi immunity. The atomic resolution structure of CrPV-1A uncovered a flexible loop that interacts with Argonaute 2 (Ago-2), thereby inhibiting Ago-2 endonuclease-dependent immunity. Mutations disrupting Ago-2 binding attenuates viral pathogenesis in wild-type but not Ago-2-deficient flies. CrPV-1A also contains a BC-box motif that enables the virus to hijack a host Cul2-Rbx1-EloBC ubiquitin ligase complex, which promotes Ago-2 degradation and virus replication. Our study uncovers a viral-based dual regulatory program that restricts antiviral immunity by direct interaction with and modulation of host proteins. While the direct inhibition of Ago-2 activity provides an efficient mechanism to establish infection, the recruitment of a ubiquitin ligase complex enables CrPV-1A to amplify Ago-2 inactivation to restrict further antiviral RNAi immunity.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has largely increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats that are generally accepted in the field and that have shown to lead to high-quality results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. From the results obtained, common protocols will be established. Our study serves as basis for establishing best practice guidelines in the field for conducting cross-linking experiments, performing data analysis, and reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.

ISWI family chromatin remodeling motors use sophisticated autoinhibition mechanisms to control nucleosome sliding. Yet how the different autoinhibitory domains are regulated is not well understood. Here we show that an acidic patch formed by histones H2A and H2B of the nucleosome relieves the autoinhibition imposed by the AutoN and the NegC regions of the human ISWI remodeler SNF2h. Further, by single molecule FRET we show that the acidic patch helps control the distance travelled per translocation event. We propose a model in which the acidic patch activates SNF2h by providing a landing pad for the NegC and AutoN auto-inhibitory domains. Interestingly, the INO80 complex is also strongly dependent on the acidic patch for nucleosome sliding, indicating that this substrate feature can regulate remodeling enzymes with substantially different mechanisms. We therefore hypothesize that regulating access to the acidic patch of the nucleosome plays a key role in coordinating the activities of different remodelers in the cell., eLife digest Every human cell contains nearly two meters of DNA, which is carefully packaged to form a dense structure known as chromatin. The building block of chromatin is the nucleosome, a unit composed of a short section of DNA tightly wound up around a spool-like core of proteins called histones. The tight structure of the nucleosome prevents the cell from accessing and ‘reading’ the genes in the packaged DNA, effectively switching off these genes. So the exact placement of nucleosomes helps manage which genes are turned on. Changing the position of the nucleosomes can ‘free’ the DNA and make genes available to the cell. Enzymes called chromatin remodelers move nucleosomes around – for example, they can make the histone core slide on the DNA strand. However, it is still unclear how these enzymes recognize nucleosomes. Previous research indicates that many proteins bind to nucleosomes by using a surface on the histone proteins called the acidic patch. Could chromatin remodelers also work by interacting with this acidic patch? To address this further, Gamarra et al. investigate how a chromatin remodeler enzyme known as SNF2h interacts with a nucleosome. By default, SNF2h is inactive because two of its regions called AutoN and NegC act as brakes. The experiments show that the acidic patch helps to bypass this inactivation and switches on SNF2h. Gamarra et al. propose that, when SNF2h docks on to the nucleosome, the patch provides a landing pad for the AutoN and NegC modules; this interaction activates the enzyme, which can then start remodeling the nucleosome. However, another type of chromatin remodeler also uses the patch to interact with nucleosomes but it does not have the AutoN and NegC regions. This suggests that chromatin remodelers work with the acidic patch in different ways. Overall, the findings deepen our understanding of how DNA is packaged in cells, and how this process may go wrong and cause disease.

Minimally invasive techniques have now become standard for the treatment of many surgical conditions in children. There are a few studies that describe the utility of laparoscopy in BAT in children. In this article, we describe the complete laparoscopic surgical treatment of two patients after a single blunt abdominal trauma, both with bowel perforation. In both cases, the perforation was identified and closed, one laparoscopically with an ongoing suture, the second jejune perforation was closed by laparoscopic-assisted techniques. Both patients had an uneventful postoperative recovery. Therapeutic laparoscopic treatment of patients with upper gastrointestinal perforation is feasible. We hypothesize, that diagnostic laparoscopy provides important information for the treatment of children with abdominal trauma and is accompanied by improved diagnostic accuracy, reduction of nontherapeutic laparotomy rates, and a reduction of morbidity. Minimally invasive surgery in children after BAT is suitable for hemodynamic stable patients, could improve pain scores, cosmetic effect, shorter hospital stays, shorter operative times and shorter return to school/activities. However, at any point in the patients care, in case the unstable hemodynamic is encountered, exploratory laparotomy is the procedure of choice.Key words: miniinvasive surgery blunt abdominal trauma - laparoscopy bowel perforation.

Native electrospray-ionization mass spectrometry (native MS) measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact protein assemblies. However, native spectra derived from these assemblies are often partially obscured by low signal-to-noise as well as broad peak shapes because of residual solvation and adduction after the electrospray process. The wide peak widths together with the fact that sequential charge state series from highly charged ions are closely spaced means that native spectra containing multiple species often suffer from high degrees of peak overlap or else contain highly interleaved charge envelopes. This situation presents a challenge for peak detection, correct charge state and charge envelope assignment, and ultimately extraction of the relevant underlying mass values of the noncovalent assemblages being investigated. In this report, we describe a comprehensive algorithm developed for addressing peak detection, peak overlap, and charge state assignment in native mass spectra, called PeakSeeker. Overlapped peaks are detected by examination of the second derivative of the raw mass spectrum. Charge state distributions of the molecular species are determined by fitting linear combinations of charge envelopes to the overall experimental mass spectrum. This software is capable of deconvoluting heterogeneous, complex, and noisy native mass spectra of large protein assemblies as demonstrated by analysis of (1) synthetic mononucleosomes containing severely overlapping peaks, (2) an RNA polymerase II/α-amanitin complex with many closely interleaved ion signals, and (3) human TriC complex containing high levels of background noise. Graphical Abstract ᅟ.

CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate recruitment, an important step in CYP3A4 proteasomal degradation.

Controllers are a part of larger control systems that influence what and how specific response operations are managed and related command and control work performed. Controllers in emergency and disaster response operations need to be adaptive in multiple dimensions in order to accomplish their goals. The three main dimensions of adaptation are: resource-driven adaptation, control-driven adaptation and adaptation to unexpected disturbances and changes. The chapter describes and discusses the main characteristics of response operations and the related command and control work. First, emergencies and disasters are distinctive phenomena that require specific management of response operations. Secondly, management of this type of response operation is a dynamic control process controlled by controllers. Thirdly, controllers need to be adaptive in multiple dimensions due to the nature of response operation. The adaptation may also concern use of diverse organizational and temporal configurations of command and control work over time.

Gene silencing by heterochromatin is proposed to occur in part as a result of the ability of heterochromatin protein 1 (HP1) proteins to spread across large regions of the genome, compact the underlying chromatin and recruit diverse ligands. Here we identify a new property of the human HP1α protein: the ability to form phase-separated droplets. While unmodified HP1α is soluble, either phosphorylation of its N-terminal extension or DNA binding promotes the formation of phase-separated droplets. Phosphorylation-driven phase separation can be promoted or reversed by specific HP1α ligands. Known components of heterochromatin such as nucleosomes and DNA preferentially partition into the HP1α droplets, but molecules such as the transcription factor TFIIB show no preference. Using a single-molecule DNA curtain assay, we find that both unmodified and phosphorylated HP1α induce rapid compaction of DNA strands into puncta, although with different characteristics. We show by direct protein delivery into mammalian cells that an HP1α mutant incapable of phase separation in vitro forms smaller and fewer nuclear puncta than phosphorylated HP1α. These findings suggest that heterochromatin-mediated gene silencing may occur in part through sequestration of compacted chromatin in phase-separated HP1 droplets, which are dissolved or formed by specific ligands on the basis of nuclear context.

Hintergrund: Diese prospektive Studie untersucht die klinischen und radiologischen Ergebnisse der Chevron-Osteotomie mit Schraubenfixation und distalem Weichteilrelease bis zu einem Intermetatarsalwinkel von 19°. Weiters sollen die Ergebnisse bei Patienten uber dem 70. Lebensjahr prasentiert werden, und ob es in diesen Patientengruppen zu einer erhohten Komplikationsrate kommt. Material und Methode: 86 Fuse von Patienten zwischen 23 und 81 Jahren konnten in die Studie eingeschlossen werden. Neben der Gesamtgruppe wurden die Gruppe mit einem Intermetatarsalwinkel von 16° bis 19° sowie die Patientengruppe uber 70 Jahre evaluiert. Sie wurden sowohl praoperativ als auch beim Follow-up nach durchschnittlich 3,3 Jahren mit dem American Orthopaedic Foot and Ankle Society Score evaluiert. Ergebnisse: Der AOFAS-Score zeigte eine signifikante Verbesserung von 55 Punkten praoperativ auf 90 Punkte bei der Nachuntersuchung. Der praoperative Hallux-valgus-Winkel wurde signifikant von 32° auf 5° und der praoperative Intermetatarsalwinkel signifikant von 14° auf 6° verringert. Die Patientenzufriedenheit der Gesamtgruppe war in 92 % sehr gut und gut. Auch die Patientengruppe von 16–19° und die Patienten uber 70 Jahre zeigten eine signifikante Verbesserung der klinischen und radiologischen Parameter. Die Komplikationsrate war in allen Gruppen sehr gering. Schlussfolgerung: Die Resultate zeigen, dass die Chevron-Osteotomie eine sehr gute und komplikationsarme Operation zur Korrektur bei Spreizfus mit Hallux-valgus-Deformitat ist. Wir konnten zeigen, dass durch die modifizierte Technik mit einem langeren plantaren Schenkel, einer ausgedehnten Weichteiltechnik und durch die Schraubenfixation die Indikation bis zu einem Intermetatarsalwinkel von 19° ausgedehnt werden kann. Weiters zeigte auch die Patientengruppe uber 70 Jahre eine signifikante Verbesserung der klinischen und radiologischen Parameter ohne schwere Komplikationen wie Kopfchenverkippung oder avaskulare Nekrose.

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

AML1-ETO is the most common fusion oncoprotein causing acute myeloid leukemia (AML), a disease with a 5-year survival rate of only 24%. AML1-ETO functions as a rogue transcription factor, altering the expression of genes critical for myeloid cell development and differentiation. Currently, there are no specific therapies for AML1-ETO-positive AML. While known for decades to be the translational product of a chimeric gene created by the stable chromosome translocation t(8;21)(q22;q22), it is not known how AML1-ETO achieves its native and functional conformation or whether this process can be targeted for therapeutic benefit. Here, we show that the biosynthesis and folding of the AML1-ETO protein is facilitated by interaction with the essential eukaryotic chaperonin TRiC (or CCT). We demonstrate that a folding intermediate of AML1-ETO binds to TRiC directly, mainly through its β-strand rich, DNA-binding domain (AML-(1–175)), with the assistance of HSP70. Our results suggest that TRiC contributes to AML1-ETO proteostasis through specific interactions between the oncoprotein's DNA-binding domain, which may be targeted for therapeutic benefit.