The palisade layer of the poxvirus core is composed of flexible A10 trimers.

Due to its asymmetric shape, size and compactness, the structure of the infectious mature virus (MV) of vaccinia virus (VACV), the best-studied poxvirus, remains poorly understood. Instead, subviral particles, in particular membrane-free viral cores, have been studied with cryo-electron microscopy. Here, we compared viral cores obtained by detergent stripping of MVs with cores in the cellular cytoplasm, early in infection. We focused on the prominent palisade layer on the core surface, combining cryo-electron tomography, subtomogram averaging and AlphaFold2 structure prediction. We showed that the palisade is composed of densely packed trimers of the major core protein A10. Trimers display a random order and their classification indicates structural flexibility. A10 on cytoplasmic cores is organized in a similar manner, indicating that the structures obtained in vitro are physiologically relevant. We discuss our results in the context of the VACV replicative cycle, and the assembly and disassembly of the infectious MV.

The Potential Role of RANTES in Post-Stroke Therapy.

One of the key response mechanisms to brain damage, that results in neurological symptoms, is the inflammatory response. It triggers processes that exacerbate neurological damage and create the right environment for the subsequent repair of damaged tissues. RANTES (Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted) chemokine(C-C motif) ligand 5 (CCL5) is one of the chemokines that may have a dual role in stroke progression involving aggravating neuronal damage and playing an important role in angiogenesis and endothelial repair. This study concerned patients with ischemic stroke (AIS), whose CCL5 concentration was measured at various time intervals and was compared with the control group. In addition, the effect of this biomarker on neurological severity and functional prognosis was investigated. Compared to healthy patients, a higher concentration of this chemokine was demonstrated in less than 4.5 h, 24 h and on the seventh day. Differences in CCL5 levels were found to be dependent on the degree of disability and functional status assessed according to neurological scales (modified Rankin Scale, National Institutes of Health Stroke Scale). In addition, differences between various subtypes of stroke were demonstrated, and an increase in CCL5 concentration was proven to be a negative predictor of mortality in patients with AIS. The deleterious effect of CCL5 in the acute phase of stroke and the positive correlation between the tested biomarkers of inflammation were also confirmed.

Co-translational binding of importins to nascent proteins.

Various cellular quality control mechanisms support proteostasis. While, ribosome-associated chaperones prevent the misfolding of nascent chains during translation, importins were shown to prevent the aggregation of specific cargoes in a post-translational mechanism prior the import into the nucleoplasm. Here, we hypothesize that importins may already bind ribosome-associated cargo in a co-translational manner. We systematically measure the nascent chain association of all importins in Saccharomyces cerevisiae by selective ribosome profiling. We identify a subset of importins that bind to a wide range of nascent, often uncharacterized cargoes. This includes ribosomal proteins, chromatin remodelers and RNA binding proteins that are aggregation prone in the cytosol. We show that importins act consecutively with other ribosome-associated chaperones. Thus, the nuclear import system is directly intertwined with nascent chain folding and chaperoning.

Visualizing the disordered nuclear transport machinery in situ.

The approximately 120 MDa mammalian nuclear pore complex (NPC) acts as a gatekeeper for the transport between the nucleus and cytosol1. The central channel of the NPC is filled with hundreds of intrinsically disordered proteins (IDPs) called FG-nucleoporins (FG-NUPs)2,3. Although the structure of the NPC scaffold has been resolved in remarkable detail, the actual transport machinery built up by FG-NUPs-about 50 MDa-is depicted as an approximately 60-nm hole in even highly resolved tomograms and/or structures computed with artificial intelligence4-11. Here we directly probed conformations of the vital FG-NUP98 inside NPCs in live cells and in permeabilized cells with an intact transport machinery by using a synthetic biology-enabled site-specific small-molecule labelling approach paired with highly time-resolved fluorescence microscopy. Single permeabilized cell measurements of the distance distribution of FG-NUP98 segments combined with coarse-grained molecular simulations of the NPC allowed us to map the uncharted molecular environment inside the nanosized transport channel. We determined that the channel provides-in the terminology of the Flory polymer theory12-a 'good solvent' environment. This enables the FG domain to adopt expanded conformations and thus control transport between the nucleus and cytoplasm. With more than 30% of the proteome being formed from IDPs, our study opens a window into resolving disorder-function relationships of IDPs in situ, which are important in various processes, such as cellular signalling, phase separation, ageing and viral entry.

Phosphorylation of the receptor protein Pex5p modulates import of proteins into peroxisomes.

Peroxisomes are organelles with vital functions in metabolism and their dysfunction is associated with human diseases. To fulfill their multiple roles, peroxisomes import nuclear-encoded matrix proteins, most carrying a peroxisomal targeting signal (PTS) 1. The receptor Pex5p recruits PTS1-proteins for import into peroxisomes; whether and how this process is posttranslationally regulated is unknown. Here, we identify 22 phosphorylation sites of Pex5p. Yeast cells expressing phospho-mimicking Pex5p-S507/523D (Pex5p2D) show decreased import of GFP with a PTS1. We show that the binding affinity between a PTS1-protein and Pex5p2D is reduced. An in vivo analysis of the effect of the phospho-mimicking mutant on PTS1-proteins revealed that import of most, but not all, cargos is affected. The physiological effect of the phosphomimetic mutations correlates with the binding affinity of the corresponding extended PTS1-sequences. Thus, we report a novel Pex5p phosphorylation-dependent mechanism for regulating PTS1-protein import into peroxisomes. In a broader view, this suggests that posttranslational modifications can function in fine-tuning the peroxisomal protein composition and, thus, cellular metabolism.

Increased Oxidative Stress Markers in Acute Ischemic Stroke Patients Treated with Thrombolytics.

One of the most common neurological disorders involving oxidative stress is stroke. During a stroke, the balance of redox potential in the cell is disturbed, and, consequently, protein oxidation or other intracellular damage occurs, ultimately leading to apoptosis. The pineal gland hormone, melatonin, is one of the non-enzymatic antioxidants. It not only modulates the perianal rhythm but also has anti-inflammatory properties and protects against stress-induced changes. The focus of this research was to evaluate the concentration of the carbonyl groups and melatonin metabolite in time in patients with acute ischemic stroke that were treated with intravenous thrombolysis. This included a comparison of the functional status of patients assessed according to neurological scales with the control sample comprising healthy people. The studies showed that the serum concentrations of carbonyl groups, which were elevated in patients with ischemic stroke (AIS) in comparison to the control samples, had an impact on the patients' outcome. A urine concentration of the melatonin metabolite, which was lower in patients than controls, was related to functional status after 24 h from cerebral thrombolysis. It shows that determination of carbonyl groups at different time intervals may be an important potential marker of protein damage in patients with AIS treated with cerebral thrombolysis, and that impaired melatonin metabolism induces a low antioxidant protection. Thus, due to the neuroprotective effects of melatonin, attention should also be paid to the design and conduct of clinical trials and hormone supplementation in AIS patients to understand the interactions between exogenous melatonin and its endogenous rhythm, as well as how these relationships may affect patient outcomes.

Selected Mediators of Inflammation in Patients with Acute Ischemic Stroke.

During a stroke, a series of biochemical and metabolic changes occur which eventually lead to the death of cells by necrosis or apoptosis. This is a multi-stage process involving oxidative stress and an inflammatory response from the first signs of occlusion of a blood vessel until the late stages of regeneration and healing of ischemic tissues. The purpose of the research was to assess the concentration of pro-inflammatory cytokines IL-6 and TNF-α in the blood serum of patients with ischemic stroke (AIS) and to investigate their role as new markers in predicting functional prognosis after thrombolytic therapy. The researches have shown that the concentrations of the measured biomarkers were higher compared to the control group. Serum levels of IL-6 and THF-α before the initiation of intravenous thrombolysis were lower in the subgroup of patients with a favourable functional result (mRS: 0−2 pts) compared to the group of patients with an unfavourable functional result (mRS: 3−6 pts). A positive correlation was found between the concentration of IL-6 and TNF-α in patients with AIS during <4.5 h and on one day after the onset of stroke, which means that the concentration of IL-6 increases with the increase in TNF-α concentration. It has also been shown that higher levels of IL-6 in the acute phase of stroke and on the first and seventh days, and TNF-α during onset, were associated with poorer early and late prognosis in patients treated with intravenous thrombolysis. A relationship was found between the level of IL-6 and TNF-α in the subacute AIS and the severity of the neurological deficit. It has been shown that the investigated biomarkers may be a prognostic factor in the treatment of thrombolytic AIS.

AI-based structure prediction empowers integrative structural analysis of human nuclear pores.

INTRODUCTION The eukaryotic nucleus pro-tects the genome and is enclosed by the two membranes of the nuclear envelope. Nuclear pore complexes (NPCs) perforate the nuclear envelope to facilitate nucleocytoplasmic transport. With a molecular weight of ∼120 MDa, the human NPC is one of the larg-est protein complexes. Its ~1000 proteins are taken in multiple copies from a set of about 30 distinct nucleoporins (NUPs). They can be roughly categorized into two classes. Scaf-fold NUPs contain folded domains and form a cylindrical scaffold architecture around a central channel. Intrinsically disordered NUPs line the scaffold and extend into the central channel, where they interact with cargo complexes. The NPC architecture is highly dynamic. It responds to changes in nuclear envelope tension with conforma-tional breathing that manifests in dilation and constriction movements. Elucidating the scaffold architecture, ultimately at atomic resolution, will be important for gaining a more precise understanding of NPC function and dynamics but imposes a substantial chal-lenge for structural biologists. RATIONALE Considerable progress has been made toward this goal by a joint effort in the field. A synergistic combination of complementary approaches has turned out to be critical. In situ structural biology techniques were used to reveal the overall layout of the NPC scaffold that defines the spatial reference for molecular modeling. High-resolution structures of many NUPs were determined in vitro. Proteomic analysis and extensive biochemical work unraveled the interaction network of NUPs. Integra-tive modeling has been used to combine the different types of data, resulting in a rough outline of the NPC scaffold. Previous struc-tural models of the human NPC, however, were patchy and limited in accuracy owing to several challenges: (i) Many of the high-resolution structures of individual NUPs have been solved from distantly related species and, consequently, do not comprehensively cover their human counterparts. (ii) The scaf-fold is interconnected by a set of intrinsically disordered linker NUPs that are not straight-forwardly accessible to common structural biology techniques. (iii) The NPC scaffold intimately embraces the fused inner and outer nuclear membranes in a distinctive topol-ogy and cannot be studied in isolation. (iv) The conformational dynamics of scaffold NUPs limits the resolution achievable in structure determination. RESULTS In this study, we used artificial intelligence (AI)-based prediction to generate an exten-sive repertoire of structural models of human NUPs and their subcomplexes. The resulting models cover various domains and interfaces that so far remained structurally uncharac-terized. Benchmarking against previous and unpublished x-ray and cryo-electron micros-copy structures revealed unprecedented accu-racy. We obtained well-resolved cryo-electron tomographic maps of both the constricted and dilated conformational states of the hu-man NPC. Using integrative modeling, we fit-ted the structural models of individual NUPs into the cryo-electron microscopy maps. We explicitly included several linker NUPs and traced their trajectory through the NPC scaf-fold. We elucidated in great detail how mem-brane-associated and transmembrane NUPs are distributed across the fusion topology of both nuclear membranes. The resulting architectural model increases the structural coverage of the human NPC scaffold by about twofold. We extensively validated our model against both earlier and new experimental data. The completeness of our model has enabled microsecond-long coarse-grained molecular dynamics simulations of the NPC scaffold within an explicit membrane en-vironment and solvent. These simulations reveal that the NPC scaffold prevents the constriction of the otherwise stable double-membrane fusion pore to small diameters in the absence of membrane tension. CONCLUSION Our 70-MDa atomically re-solved model covers >90% of the human NPC scaffold. It captures conforma-tional changes that occur during dilation and constriction. It also reveals the precise anchoring sites for intrinsically disordered NUPs, the identification of which is a prerequisite for a complete and dy-namic model of the NPC. Our study exempli-fies how AI-based structure prediction may accelerate the elucidation of subcellular ar-chitecture at atomic resolution. [Figure: see text].

Does experience of the surgeon affect surgical margins in head and neck basal cell carcinoma?

<b>Aim:</b> The aim of our study was to evaluate the impact of surgical experience in a high volume head and neck surgery department on basal cell carcinoma margin status. </br></br> <b>Material and methods:</b> A retrospective analysis of 546 patients surgically treated for primary basal cell carcinoma of the head and neck region was carried out. Resections were performed by 4 specialists with equal experience in head and neck surgery and 4 ENT residents at the same level of surgical training. A margin of 3-5 mm was chosen, according to guidelines. </br></br> <b>Results:</b> The study consisted of 304 males and 242 females, mean age of 69 (range 26-100). Most of the tumors were loca-ted on the nose (165 pts; 30.2%) and auricle (119; 21.7%). The most common histological subtype was nodular (119; 21.7%). Tumor size was up to 20 mm in 394 cases (72%). Positive surgical margins were found in 112 cases (20.5%). There was no difference in terms of positive surgical margins between residents (19/119 cases; 15.9%) and specialists (93/426; 21.8%; p = 0.161). </br></br> <b>Conclusions:</b> The results of our study have shown that adequate surgical training in a dedicated head and neck surgery de-partment is an efficient factor in obtaining free surgical margins in head and neck basal cell carcinoma.

Nuclear pores dilate and constrict in cellulo.

In eukaryotic cells, nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes and mediate nucleocytoplasmic exchange. They are made of 30 different nucleoporins and form a cylindrical architecture around an aqueous central channel. This architecture is highly dynamic in space and time. Variations in NPC diameter have been reported, but the physiological circumstances and the molecular details remain unknown. Here, we combined cryo­electron tomography with integrative structural modeling to capture a molecular movie of the respective large-scale conformational changes in cellulo. Although NPCs of exponentially growing cells adopted a dilated conformation, they reversibly constricted upon cellular energy depletion or conditions of hypertonic osmotic stress. Our data point to a model where the nuclear envelope membrane tension is linked to the conformation of the NPC.

ColBuilder: A server to build collagen fibril models.

Type I collagen is the main structural component of many tissues in the human body. It provides excellent mechanical properties to connective tissue and acts as a protein interaction hub. There is thus a wide interest in understanding the properties and diverse functions of type I collagen at the molecular level. A precondition is an atomistic collagen I structure as it occurs in native tissue. To this end, we built full-atom models of cross-linked collagen fibrils by integrating the low-resolution structure of collagen fibril available from x-ray fiber diffraction with high-resolution structures of short collagen-like peptides from x-ray crystallography and mass spectrometry data. We created a Web resource of collagen models for 20 different species with a large variety of cross-link types and localization within the fibril to facilitate structure-based analyses and simulations of type I collagen in health and disease. To easily enable simulations, we provide parameters of the modeled cross-links for an Amber force field. The repository of collagen models is available at https://colbuilder.h-its.org.

The m6A RNA Modification Quantity and mRNA Expression Level of RNA Methylation-Related Genes in Head and Neck Squamous Cell Carcinoma Cell Lines and Patients.

RNA methylation at the nitrogen sixth of adenosine (m6A, N6-methyladenosine) is the most abundant RNA modification which plays a crucial role in all RNA metabolic aspects. Recently, m6A modification has been assigned to mediate the biological processes of cancer cells, but their significance in HNSCC development is still poorly described. Thus, the main aim of this study was to globally quantify m6A modification by the mass spectrometry approach and determine the mRNA expression level of selected m6A RNA methyltransferase (METTL3), demethylase (FTO), and m6A readers (YTHDF2, YTHDC2) in 45 HNSCC patients and 4 cell lines (FaDu, Detroit 562, A-253 and SCC-15) using qPCR. In the results, we have not observed differences in the global amount of m6A modification and the mRNA level of the selected genes between the cancerous and paired-matched histopathologically unchanged tissues from 45 HNSCC patients. However, we have found a positive correlation between selected RNA methylation machinery genes expression and m6A abundance on total RNA and characterized the transcript level of those genes in the HNSCC cell lines. Moreover, the lack of global m6A differences between cancerous and histopathologically unchanged tissues suggests that m6A alterations in specific RNA sites may specifically influence HNSCC tumorigenesis.

Mechanoradicals in tensed tendon collagen as a source of oxidative stress.

As established nearly a century ago, mechanoradicals originate from homolytic bond scission in polymers. The existence, nature and biological relevance of mechanoradicals in proteins, instead, are unknown. We here show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species, essential biological signaling molecules. Electron-paramagnetic resonance (EPR) spectroscopy of stretched rat tail tendon, atomistic molecular dynamics simulations and quantum-chemical calculations show that the radicals form by bond scission in the direct vicinity of crosslinks in collagen. Radicals migrate to adjacent clusters of aromatic residues and stabilize on oxidized tyrosyl radicals, giving rise to a distinct EPR spectrum consistent with a stable dihydroxyphenylalanine (DOPA) radical. The protein mechanoradicals, as a yet undiscovered source of oxidative stress, finally convert into hydrogen peroxide. Our study suggests collagen I to have evolved as a radical sponge against mechano-oxidative damage and proposes a mechanism for exercise-induced oxidative stress and redox-mediated pathophysiological processes.

Mechanism of conditional partner selectivity in MITF/TFE family transcription factors with a conserved coiled coil stammer motif.

Interrupted dimeric coiled coil segments are found in a broad range of proteins and generally confer selective functional properties such as binding to specific ligands. However, there is only one documented case of a basic-helix-loop-helix leucine zipper transcription factor-microphthalmia-associated transcription factor (MITF)-in which an insertion of a three-residue stammer serves as a determinant of conditional partner selectivity. To unravel the molecular principles of this selectivity, we have analyzed the high-resolution structures of stammer-containing MITF and an engineered stammer-less MITF variant, which comprises an uninterrupted symmetric coiled coil. Despite this fundamental difference, both MITF structures reveal identical flanking in-phase coiled coil arrangements, gained by helical over-winding and local asymmetry in wild-type MITF across the stammer region. These conserved structural properties allow the maintenance of a proper functional readout in terms of nuclear localization and binding to specific DNA-response motifs regardless of the presence of the stammer. By contrast, MITF heterodimer formation with other bHLH-Zip transcription factors is only permissive when both factors contain either the same type of inserted stammer or no insert. Our data illustrate a unique principle of conditional partner selectivity within the wide arsenal of transcription factors with specific partner-dependent functional readouts.

Hybrid Kinetic Monte Carlo/Molecular Dynamics Simulations of Bond Scissions in Proteins.

Proteins are exposed to various mechanical loads that can lead to covalent bond scissions even before macroscopic failure occurs. Knowledge of these molecular breakages is important to understand mechanical properties of the protein. In regular molecular dynamics (MD) simulations, covalent bonds are predefined, and reactions cannot occur. Furthermore, such events rarely take place on MD time scales. Existing approaches that tackle this limitation either rely on computationally expensive quantum calculations (e.g., QM/MM) or complex bond order formalisms in force fields (e.g., ReaxFF). To circumvent these limitations, we present a new reactive kinetic Monte Carlo/molecular dynamics (KIMMDY) scheme. Here, bond rupture rates are calculated based on the interatomic distances in the MD simulation and then serve as an input for a kinetic Monte Carlo step. This easily scalable hybrid approach drastically increases the accessible time scales. Using this new technique, we investigate bond ruptures in a multimillion atom system of tensed collagen, a structural protein found in skin, bones, and tendons. Our findings show a clear concentration of bond scissions near chemical cross-links in collagen. We also examine subsequent dynamic relaxation steps. Our method exhibits only a minor slowdown compared to classical MD and is straightforwardly applicable to other complex (bio)materials under load and related chemistries.

PepComposer: computational design of peptides binding to a given protein surface.

There is a wide interest in designing peptides able to bind to a specific region of a protein with the aim of interfering with a known interaction or as starting point for the design of inhibitors. Here we describe PepComposer, a new pipeline for the computational design of peptides binding to a given protein surface. PepComposer only requires the target protein structure and an approximate definition of the binding site as input. We first retrieve a set of peptide backbone scaffolds from monomeric proteins that harbor the same backbone arrangement as the binding site of the protein of interest. Next, we design optimal sequences for the identified peptide scaffolds. The method is fully automatic and available as a web server at http://biocomputing.it/pepcomposer/webserver.

Cocoa polyphenols are absorbed in Caco-2 cell model of intestinal epithelium.

Cocoa is an abundant source of polyphenols, mainly flavan-3-ol monomers and polymers. In the literature, there are contradictory data on the absorption limit of procyanidins in humans. In our study, the Caco-2 cell model of intestinal epithelium was used to determine the absorption and secretion of cocoa flavan-3-ols. Three compounds: (+)-catechin, (-)-epicatechin and procyanidin B2 were detected and quantified at the receiver side of Caco-2 monolayer after 2h transport experiment. The obtained results of apparent permeability coefficient suggest paracellular route of transport of investigated compounds. Additionally, the results suggest that compounds of cocoa powder purified extract are able to affect tight junction functioning.

Phenolic compound profiles and antioxidant capacity of Persea americana Mill. peels and seeds of two varieties.

Avocado processing by the food and cosmetic industries yields a considerable amount of phenolic-rich byproduct such as peels and seeds. Utilization of these byproducts would be favorable from an economic point of view. Methanolic (80%) extracts obtained from lyophilized ground peels and seeds of avocado (Persea americana Mill.) of the Hass and Shepard varieties were characterized for their phenolic compound profiles using the HPLC-PAD technique. The structures of the identified compounds were subsequently unambiguously confirmed by ESI-MS. Compositional analysis revealed that the extracts contained four polyphenolic classes: flavanol monomers, proanthocyanidins, hydroxycinnamic acids, and flavonol glycosides. The presence of 3-O-caffeoylquinic acid, 3-O-p-coumaroylquinic acid, and procyanidin A trimers was identified in seeds of both varieties. Intervarietal differences were apparent in the phenolic compound profiles of peels. Peels of the Shepard variety were devoid of (+)-catechin and procyanidin dimers, which were present in the peels of the Hass variety. Peels of both varieties contained 5-O-caffeoylquinic acid and quercetin derivatives. The differences in the phenolic profiles between varietals were also apparent in the different antioxidant activity of the extracts. The peel extracts had a higher total phenolic compound content and antioxidant activity when compared to the seed extracts. The highest TEAC and ORAC values were apparent in peels of the Haas variety in which they amounted to 0.16 and 0.47 mmol Trolox/g DW, respectively. No significant (p > 0.05) differences were apparent between the TEAC values of seeds of the two varieties but the ORAC values differed significantly (p < 0.05). Overall these findings indicate that both the seeds and peel of avocado can be utilized as a functional food ingredient or as an antioxidant additive.

Structure and operation of the DNA-translocating type I DNA restriction enzymes.

Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.