The CENP-A nucleosome: where and when it happens during the inner kinetochore's assembly.

CENP-A is an essential histone variant that replaces the canonical H3 at the centromeres and marks these regions epigenetically. The CENP-A nucleosome is the specific building block of centromeric chromatin, and it is recognized by CENP-C and CENP-N, two components of the constitutive centromere-associated network (CCAN), the first protein layer of the kinetochore. Recent proposals of the yeast and human (h)CCAN structures position the assembly on exposed DNA, suggesting an elusive spatiotemporal recognition. We summarize the data on the structural organization of the CENP-A nucleosome and the binding of CENP-C and CENP-N. The latter posits an apparent contradiction in engaging the CENP-A nucleosome versus the CCAN. We propose a reconciliatory model for the assembly of CCAN on centromeric chromatin.

Outcome of Application of Cryopreserved Amniotic Membrane Grafts in the Treatment of Chronic Nonhealing Wounds of Different Origins in Polymorbid Patients: A Prospective Multicenter Study.

To compare the therapeutic efficacy of cryopreserved amniotic membrane (AM) grafts and standard of care (SOC) in treating nonhealing wounds (NHW) through a prospective multicenter clinical trial, 42 patients (76% polymorbid) with 54 nonhealing wounds of various etiologies (mainly venous) and an average baseline size of 20 cm2 were included. All patients were treated for at least 6 weeks in the center before they were involved in the study. In the SOC group, 29 patients (36 wounds) were treated. If the wound healed less than 20% of the baseline size after 6 weeks, the patient was transferred to the AM group (35 patients, 43 wounds). Weekly visits included an assessment of the patient's condition, photo documentation, wound debridement, and dressing. Quality of life and the pain degree were subjectively reported by patients. After SOC, 7 wounds were healed completely, 1 defect partially, and 28 defects remained unhealed. AM application led to the complete closure of 24 wounds, partial healing occurred in 10, and 9 remained unhealed. The degree of pain and the quality of life improved significantly in all patients after AM application. This study demonstrates the effectiveness of cryopreserved AM grafts in the healing of NHW of polymorbid patients and associated pain reduction.

Molecular Mechanism of Nucleosome Recognition by the Pioneer Transcription Factor Sox.

Pioneer transcription factors (PTFs) have the remarkable ability to directly bind to chromatin to stimulate vital cellular processes. In this work, we dissect the universal binding mode of Sox PTF by combining extensive molecular simulations and physiochemistry approaches, along with DNA footprinting techniques. As a result, we show that when Sox consensus DNA is located at the solvent-facing DNA strand, Sox binds to the compact nucleosome without imposing any significant conformational changes. We also reveal that the base-specific Sox:DNA interactions (base reading) and Sox-induced DNA changes (shape reading) are concurrently required for sequence-specific nucleosomal DNA recognition. Among three different nucleosome positions located on the positive DNA arm, a sequence-specific reading mechanism is solely satisfied at the superhelical location 2 (SHL2). While SHL2 acts transparently for solvent-facing Sox binding, among the other two positions, SHL4 permits only shape reading. The final position, SHL0 (dyad), on the other hand, allows no reading mechanism. These findings demonstrate that Sox-based nucleosome recognition is essentially guided by intrinsic nucleosome properties, permitting varying degrees of DNA recognition.

Inter-placental variability is not a major factor affecting the healing efficiency of amniotic membrane when used for treating chronic non-healing wounds.

This study aimed to evaluate the efficacy of cryopreserved amniotic membrane (AM) grafts in chronic wound healing, including the mean percentage of wound closure per one AM application, and to determine whether the healing efficiency differs between AM grafts obtained from different placentas. A retrospective study analyzing inter-placental differences in healing capacity and mean wound closure after the application of 96 AM grafts prepared from nine placentas. Only the placentas from which the AM grafts were applied to patients suffering from long-lasting non-healing wounds successfully healed by AM treatment were included. The data from the rapidly progressing wound-closure phase (p-phase) were analyzed. The mean efficiency for each placenta, expressed as an average of wound area reduction (%) seven days after the AM application (baseline, 100%), was calculated from at least 10 applications. No statistical difference between the nine placentas' efficiency was found in the progressive phase of wound healing. The 7-day average wound reduction in particular placentas varied from 5.70 to 20.99% (median from 1.07 to 17.75) of the baseline. The mean percentage of wound surface reduction of all analyzed defects one week after the application of cryopreserved AM graft was 12.17 ± 20.12% (average ± SD). No significant difference in healing capacity was observed between the nine placentas. The data suggest that if there are intra- and inter-placental differences in AM sheets' healing efficacy, they are overridden by the actual health status of the subject or even the status of its individual wounds.

13 CO2 labelling as a tool for elucidating the mechanism of cuticle development: a case of Clusia rosea.

The plant cuticle is an important plant-atmosphere boundary, the synthesis and maintenance of which represents a significant metabolic cost. Only limited information regarding cuticle dynamics is available. We determined the composition and dynamics of Clusia rosea cuticular waxes and matrix using 13 CO2 labelling, compound-specific and bulk isotope ratio mass spectrometry. Collodion was used for wax collection; gas exchange techniques to test for any collodion effects on living leaves. Cutin matrix (MX) area density did not vary between young and mature leaves and between leaf sides. Only young leaves incorporated new carbon into their MX. Collodion-based sampling discriminated between epicuticular (EW) and intracuticular wax (IW) effectively. Epicuticular differed in composition from IW. The newly synthetised wax was deposited in IW first and later in EW. Both young and mature leaves synthetised IW and EW. The faster dynamics in young leaves were due to lower wax coverage, not a faster synthesis rate. Longer-chain alkanes were deposited preferentially on the abaxial, stomatous leaf side, producing differences between leaf sides in wax composition. We introduce a new, sensitive isotope labelling method and demonstrate that cuticular wax is renewed during leaf ontogeny of C. rosea. We discuss the ecophysiological significance of the new insights.

Broadband Wollaston prism with a large output beam separation based on mercurous halides.

The paper proposes a Wollaston-type crystal polarizer suitable for broadband operation within the visible spectral band up to the far infrared band based on unique optical materials, mercurous halides (Hg2X2). This paper introduces the general characteristics and optical properties of these birefringent tetragonal optical materials, as well as the general description of a Wollaston prism and the process of its parameter optimization. In general, the Wollaston polarizer is constructed from two combined wedge-shaped prisms. The key parameters that affect the properties of the Wollaston polarizer are then the cut angle of these two prisms and the refractive index of the exploited optical cement (immersion) that bonds the prisms together. The optimal prism cut angles and immersion refractive index are investigated to maximize the Wollaston parameters, such as the transmittance of the polarized radiation and the separation angle of the output orthogonally polarized beams. This process is significantly dependent on the characteristics of all selected mercurous halides (Hg2Cl2, Hg2Br2, Hg2I2). The optimal values of the prism cut angle for each material are selected based on the outlined results. In addition, the Wollaston prism behaviour regarding real radiation propagation is modelled in detail via the Zemax optical studio. The presented models aim to aid in the real design and fabrication of a broadband Wollaston polarizer based on mercurous halides.

Dual role of histone variant H3.3B in spermatogenesis: positive regulation of piRNA transcription and implication in X-chromosome inactivation.

The histone variant H3.3 is encoded by two distinct genes, H3f3a and H3f3b, exhibiting identical amino-acid sequence. H3.3 is required for spermatogenesis, but the molecular mechanism of its spermatogenic function remains obscure. Here, we have studied the role of each one of H3.3A and H3.3B proteins in spermatogenesis. We have generated transgenic conditional knock-out/knock-in (cKO/KI) epitope-tagged FLAG-FLAG-HA-H3.3B (H3.3BHA) and FLAG-FLAG-HA-H3.3A (H3.3AHA) mouse lines. We show that H3.3B, but not H3.3A, is required for spermatogenesis and male fertility. Analysis of the molecular mechanism unveils that the absence of H3.3B led to alterations in the meiotic/post-meiotic transition. Genome-wide RNA-seq reveals that the depletion of H3.3B in meiotic cells is associated with increased expression of the whole sex X and Y chromosomes as well as of both RLTR10B and RLTR10B2 retrotransposons. In contrast, the absence of H3.3B resulted in down-regulation of the expression of piRNA clusters. ChIP-seq experiments uncover that RLTR10B and RLTR10B2 retrotransposons, the whole sex chromosomes and the piRNA clusters are markedly enriched of H3.3. Taken together, our data dissect the molecular mechanism of H3.3B functions during spermatogenesis and demonstrate that H3.3B, depending on its chromatin localization, is involved in either up-regulation or down-regulation of expression of defined large chromatin regions.

The healing dynamics of non-healing wounds using cryo-preserved amniotic membrane.

We evaluated the effect of the application of cryo-preserved amniotic membrane on the healing of 26 non-healing wounds (18 patients) with varying aetiologies and baseline sizes (average of 15.4 cm2 ), which had resisted the standard of care treatment for 6 to 456 weeks (average 88.8 weeks). Based on their average general responses to the application of cryo-preserved AM, we could differentiate three wound groups. The first healed group was characterised by complete healing (100% wound closure, maximum treatment period 38 weeks) and represented 62% of treated wounds. The wound area reduction of at least 50% was reached for all wounds in this group within the first 10 weeks of treatment. Exactly 19% of the studied wounds responded partially to the treatment (partially healed group), reaching less than 25% of closure in the first 10 weeks and 90% at maximum for extended treatment period (up to 78 weeks). The remaining 19% of treated wounds did not show any reaction to the AM application (unhealed defects). The three groups have different profiles of wound area reduction, which can be used as a guideline in predicting the healing prognosis of non-healing wounds treated with a cryo-preserved amniotic membrane.

Antifungal and Insecticidal Potential of the Essential Oil from Ocimum sanctum L. against Dangerous Fungal and Insect Species and Its Safety for Non-Target Useful Soil Species Eisenia fetida (Savigny, 1826).

The antifungal and insecticidal effect of the essential oil from Ocimum sanctum L. was evaluated using a model set of harmful organisms hazardous for health and the economy. Toxigenic and plant pathogenic filamentous fungi, including causal agents of human infections, were chosen as exemplary fungal groups-Fusarium verticillioides, Penicillium expansum and Aspergillus flavus. Spodoptera littoralis (African cotton leafworm), Culex quinquefasciatus (Southern house mosquito), the lymphatic filariasis vector and potential Zika virus vector, and the common housefly, Musca domestica were chosen as model insects. Major and minor active substances were detected and quantified using GC/MS analysis. Environmental safety was verified using the non-target useful organism Eisenia fetida. Significant antifungal and insecticidal activity, as well as environmental safety, were confirmed. The essential oil showed the highest efficacy against A. flavus according to MIC50/90, and against S. littoralis larvae according to LD50/90. The monoterpenoid alcohol linalool, t-methyl cinnamate, and estragole as phenylpropanoids were detected as effective major components (85.4%). The essential oil from Ocimum sanctum L. was evaluated as universal and significantly efficient, providing a high potential for use in environmentally safe botanical pesticides.

Quasi-collinear IR AOTF based on mercurous halide single crystals for spatio-spectral hyperspectral imaging.

The paper aims to show the advantages of the infrared-optimised quasi-collinear AOTF (acousto-optic tunable filter) for the spatio-spectral hyperspectral imaging system. The optimisation process is presented based on the selected tetragonal anisotropic materials with exceptional optical and acousto-optical properties in IR (infrared) spectral region. These materials are further compared in terms of their features and suitability for AOTF design. The spectral resolution is considered as the main optimising parameter. Resulting from the analysis, the mercurous chloride (Hg2Cl2) single crystal is selected as a representative of the mercurous halide family for the presentation of the quasi-collinear AOTF model operating in LWIR (long-wave infrared) spectral band. The overall parameters of the AOTF model such as spectral resolution, chromatic field of view, acoustic frequency, and operational power requirements are estimated and discussed in results.

The micronucleus cytome assay - A fast tool for DNA damage screening in human conjunctival epithelial cells.

PURPOSE: To assess whether the micronucleus cytome assay (MCyt) reliably detects DNA damage occurring in control and pathological superficial epithelial cells from human conjunctiva. METHODS: Impression cytology samples from the bulbar conjunctiva of 33 healthy controls, eight patients with conjunctival intraepithelial neoplasia (CIN) and eight with mucous membrane pemphigoid (MMP) were examined using the MCyt modified for the ocular surface. RESULTS: The mean number of micronuclei (MNi) in control samples was 0.94 MNi/1000 epithelial cells, with no significant difference between conjunctival quadrants and independent of sex and age. The MCyt assay applied to CIN-affected eyes showed a significantly higher frequency of MNi (18.63/1000 cells), apoptotic cells, nuclear enlargement, multinucleated cells, and keratolysis compared with the corresponding unaffected paired eyes and with the control value. Although the mean MNi frequency in MMP eyes was also higher (1.73 MNi/1000 cells), it did not prove to be statistically different from the control samples. On the other hand, the MMP-affected eyes revealed significantly elevated percentages of cells with snake-like chromatin, multinucleated cells, apoptotic cells, and nuclear buds compared with controls. CONCLUSIONS: Micronucleus cytome assay was adapted as a rapid screening test for genomic instability on the ocular surface. We have determined reference levels for MNi and other nuclear alterations on healthy conjunctiva and demonstrated that particularly frequencies of MNi are significantly elevated in conjunctiva affected by CIN. We demonstrate that MNi are more specific than other nuclear abnormalities and thus can be used for screening of ocular surface neoplasia.

The NANOTUMOR consortium - Towards the Tumor Cell Atlas.

Cancer is a multi-step disease where an initial tumour progresses through critical steps shaping, in most cases, life-threatening secondary foci called metastases. The oncogenic cascade involves genetic, epigenetic, signalling pathways, intracellular trafficking and/or metabolic alterations within cancer cells. In addition, pre-malignant and malignant cells orchestrate complex and dynamic interactions with non-malignant cells and acellular matricial components or secreted factors within the tumour microenvironment that is instrumental in the progression of the disease. As our aptitude to effectively treat cancer mostly depends on our ability to decipher, properly diagnose and impede cancer progression and metastasis formation, full characterisation of molecular complexes and cellular processes at play along the metastasis cascade is crucial. For many years, the scientific community lacked adapted imaging and molecular technologies to accurately dissect, at the highest resolution possible, tumour and stromal cells behaviour within their natural microenvironment. In that context, the NANOTUMOR consortium is a French national multi-disciplinary workforce which aims at a providing a multi-scale characterisation of the oncogenic cascade, from the atomic level to the dynamic organisation of the cell in response to genetic mutations, environmental changes or epigenetic modifications. Ultimately, this program aims at identifying new therapeutic targets using innovative drug design.

Drug-loading capacity of polylactide-based micro- and nanoparticles - Experimental and molecular modeling study.

Micro- and nanostructures prepared from biodegradable homopolymers and amphiphilic block copolymers (AmBCs) have found application as drug-delivery systems (DDSs). The ability to accumulate a drug is a very important parameter characterizing a given DDS. This work focuses on the impact of DDS size, the packing of polymer chains in the DDS, and drug - polymer matrix compatibility on the hydrophobic drug - loading capacity (DLC) of nano/microcarriers prepared from a biodegradable polymer or its copolymer. Using experimental measurements in combination with atomistic molecular dynamics simulations, an analysis of curcumin encapsulation in microspheres (MSs) from polylactide (PLA) homopolymer and nanoparticles (NPs) from PLA-block-poly(2-methacryloyloxyethylphosphorylcholine) AmBC was performed. The results show that curcumin has good affinity for the PLA matrix due to its hydrophobic nature. However, the DLC value is limited by the fact that curcumin only accumulates in the peripheral part of these structures. Such uneven drug distribution in the PLA matrix results from the non-homogeneous density of MSs (non-uniform packing of the polymer chains in the coil). The results also indicate that the MSs can retain a greater amount of hydrophobic drug compared to the NPs, which is associated with the formation of drug aggregates inside the PLA microparticles.

Nanoscale Dynamic Readout of a Chemical Redox Process Using Radicals Coupled with Nitrogen-Vacancy Centers in Nanodiamonds.

Biocompatible nanoscale probes for sensitive detection of paramagnetic species and molecules associated with their (bio)chemical transformations would provide a desirable tool for a better understanding of cellular redox processes. Here, we describe an analytical tool based on quantum sensing techniques. We magnetically coupled negatively charged nitrogen-vacancy (NV) centers in nanodiamonds (NDs) with nitroxide radicals present in a bioinert polymer coating of the NDs. We demonstrated that the T1 spin relaxation time of the NV centers is very sensitive to the number of nitroxide radicals, with a resolution down to ∼10 spins per ND (detection of approximately 10-23 mol in a localized volume). The detection is based on T1 shortening upon the radical attachment, and we propose a theoretical model describing this phenomenon. We further show that this colloidally stable, water-soluble system can be used dynamically for spatiotemporal readout of a redox chemical process (oxidation of ascorbic acid) occurring near the ND surface in an aqueous environment under ambient conditions.

Cryo-electron microscopy of the chromatin fiber.

The three-dimensional (3D) organization of chromatin plays a crucial role in the regulation of gene expression. Chromatin conformation is strongly affected by the composition, structural features and dynamic properties of the nucleosome, which in turn determine the nature and geometry of interactions that can occur between neighboring nucleosomes. Understanding how chromatin is spatially organized above the nucleosome level is thus essential for understanding how gene regulation is achieved. Towards this end, great effort has been made to understand how an array of nucleosomes folds into a regular chromatin fiber. This review summarizes new insights into the 3D structure of the chromatin fiber that were made possible by recent advances in cryo-electron microscopy.

Phase-plate cryo-EM structure of the Widom 601 CENP-A nucleosome core particle reveals differential flexibility of the DNA ends.

The histone H3 variant CENP-A marks centromeres epigenetically and is essential for mitotic fidelity. Previous crystallographic studies of the CENP-A nucleosome core particle (NCP) reconstituted with a human α-satellite DNA derivative revealed both DNA ends to be highly flexible, a feature important for CENP-A mitotic functions. However, recent cryo-EM studies of CENP-A NCP complexes comprising primarily Widom 601 DNA reported well-ordered DNA ends. Here, we report the cryo-EM structure of the CENP-A 601 NCP determined by Volta phase-plate imaging. The data reveal that one ('left') 601 DNA end is well ordered whereas the other ('right') end is flexible and partly detached from the histone core, suggesting sequence-dependent dynamics of the DNA termini. Indeed, a molecular dynamics simulation of the CENP-A 601 NCP confirmed the distinct dynamics of the two DNA extremities. Reprocessing the image data using two-fold symmetry yielded a cryo-EM map in which both DNA ends appeared well ordered, indicating that such an artefact may inadvertently arise if NCP asymmetry is lost during image processing. These findings enhance our understanding of the dynamic features that discriminate CENP-A from H3 nucleosomes by revealing that DNA end flexibility can be fine-tuned in a sequence-dependent manner.

Generation of Remosomes by the SWI/SNF Chromatin Remodeler Family.

Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of remodeled nucleosomes remain a matter of debates. In this work we compared the type and structure of the products of nucleosome remodeling by SWI/SNF and ACF complexes using high-resolution microscopy combined with novel biochemical approaches. We find that SWI/SNF generates a multitude of nucleosome-like metastable particles termed "remosomes". Restriction enzyme accessibility assay, DNase I footprinting and AFM experiments reveal perturbed histone-DNA interactions within these particles. Electron cryo-microscopy shows that remosomes adopt a variety of different structures with variable irregular DNA path, similar to those described upon RSC remodeling. Remosome DNA accessibility to restriction enzymes is also markedly increased. We suggest that the generation of remosomes is a common feature of the SWI/SNF family remodelers. In contrast, the ACF remodeler, belonging to ISWI family, only produces repositioned nucleosomes and no evidence for particles associated with extra DNA, or perturbed DNA paths was found. The remosome generation by the SWI/SNF type of remodelers may represent a novel mechanism involved in processes where nucleosomal DNA accessibility is required, such as DNA repair or transcription regulation.

Structure of an H1-Bound 6-Nucleosome Array Reveals an Untwisted Two-Start Chromatin Fiber Conformation.

Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.

Antimicrobial efficiency and stability of two decontamination solutions.

Two decontamination solutions, commercially produced BASE•128 and laboratory decontamination solution (LDS), with analogous content of antibiotic and antimycotic agents, were compared in their antimicrobial efficiency and stability (pH and osmolarity). Both solutions were compared immediately after thawing aliquots frozen for 1, 3 or 6 months. Agar well diffusion method was used to test their antimicrobial efficiency against five human pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli and Enterococcus faecalis. The difference in the inhibition of growth between the two decontamination solutions was mostly not statistically significant, with few exceptions. The most pronounced difference between the LDS and BASE•128 was observed in their decontamination efficacy against E. coli and E. faecalis, where the LDS showed to be more efficient than BASE•128. The osmolarity value of LDS decreased with cold-storage, the osmolarity values of the BASE•128 could not be measured as they were below the range of the osmometer. Slight changes were found in pH of the less stable LDS solution, whose pH increased from initial value 7.36 ± 0.07 to 7.72 ± 0.19 after 6 m-storage. We verified that BASE•128 and LDS are similarly efficient in elimination of possible placental bacterial contaminants and may be used for decontamination of various tissues.

Characterization and comparison of human limbal explant cultures grown under defined and xeno-free conditions.

Human limbal epithelial cells (LECs) intended for treatment of limbal stem cell deficiency are commonly cultivated on a 3T3 feeder layer with complex culture medium supplemented with fetal bovine serum (FBS). However, FBS is a xenogeneic component containing poorly characterised constituents and exhibits quantitative and qualitative lot-to-lot variations. Human limbal explants were plated on untreated or fibrin coated plastic plates and cultured in two non-xenogeneic media (supplemented with either human serum or platelet lysate only). Our aim was to find out whether the characteristics of harvested LEC cultures are comparable to those of LEC cultivated in the gold standard - FBS-supplemented complex medium. The growth kinetics, cell proliferation, differentiation, stemness maintenance, apoptosis and contamination by other cell types were evaluated and compared among these conditions. In all of them LECs were successfully cultivated. Stemness was preserved in both xeno-free media. However, cells cultured with human serum on the fibrin-coated plates had the highest growth rate and cell proliferation and very low fibroblast-like cell contamination. These data suggest that xeno-free cell culture conditions can replace the traditional FBS-supplemented medium and thereby provide a safer protocol for ex vivo cultured limbal stem cell transplants.