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Matricryptins are collagen fragments proteolytically released from the extracellular matrix (ECM) with biological activity that can regulate several processes involved in ECM remodeling. Vessel wall matrix reorganization after lesion is important to the recovery of vascular function. This study aimed to analyze the effect of the peptide p1158/59 (Lindsey, 2015) on thrombosis, neointimal formation, and vascular remodeling of C57BL6 mice abdominal aorta. We used a FeCl

Valproic acid/sodium valproate (VPA) constitutes a widely prescribed drug for the treatment of seizure disorders and is a well-known epigenetic agent, inducing the acetylation of histones and affecting the methylation status of DNA and histones, with consequences on gene expression. Because this drug has been recently reported to exert affinity for histone H1, and to a minor degree for DNA, in this work, we investigated a possible interaction of sodium valproate with DNA and histones H1 and H3 using high-performance polarization microscopy and Fourier-transform infrared (FTIR) microspectroscopy. The preparations under examination consisted of hemispheres resulting from drop-casting samples containing VPA-DNA and VPA-histone mixtures. The results indicated that VPA may interact with DNA and histones, inducing changes in the textural superstructure and molecular order of the DNA possibly through van der Waals forces, and in histone H1 and H3 conformations, probably as a result of electrostatic binding between the drug and protein amino acid residues. These results contribute to a better understanding of the pharmacological potential of VPA. The precise sites and mechanisms involved in these interactions would certainly benefit from investigations provided by complementary methodologies.

Background: Valproic acid/sodium valproate (VPA), a well-known anti-epileptic agent, in-hibits histone deacetylases, induces histone hyperacetylation, promotes DNA demethylation, and affects the histone methylation status in some cell models. Histone methylation profiles have been described as potential markers for cervical cancer prognosis. However, histone methylation markers that can be studied in a cervical cancer cell line, like HeLa cells, have not been investigated following treatment with VPA. Method: In this study, the effect of 0.5 mM and 2.0 mM VPA for 24 h on H3K4me2/me3, H3K9me/me2 and H3K27me/me3 signals as well as on KMT2D, EZH2, and KDM3A gene expression was investigated using confocal microscopy, Western blotting, and RT-PCR. Histone methylation changes were also in-vestigated by Fourier-transform infrared spectroscopy (FTIR). Result: We found that VPA induces increased levels of H3K4me2/me3 and H3K9me, which are indic-ative of chromatin activation. Particularly, H3K4me2 markers appeared intensified close to the nuclear periphery, which may suggest their implication in increased transcriptional memory. The abundance of H3K4me2/me3 in the presence of VPA was associated with increased methyltransferase KMT2D gene expression. VPA induced hypomethylation of H3K9me2, which is associated with gene silencing, and concomitant with the demethylase KDM3A, it increased gene expression. Although VPA induces in-creased H3K27me/me3 levels, it is suggested that the role of the methyltransferase EZH2 in this context could be affected by interactions with this drug. Conclusion: Histone FTIR spectra were not affected by VPA under present experimental conditions. Whether our epigenetic results are consistent with VPA affecting the aggressive tumorous state of HeLa cells, further investigation is required.

Optical anisotropy properties and infrared spectra of collagen and DNA as individual entities have been widely described. However, they have not been studied in collagen-DNA complex biofilms. We investigated whether collagen type I and DNA birefringence evaluated using polarization microscopy, and the vibrational patterns of their chemical groups identified in Fourier transform-infrared (FT-IR) spectra are retained in collagen-DNA complexes. Collagen gel rods extruded into a DNA saline solution and films dried from drops using mixtures of a 10-μL (14 μg) collagen solution and a 50-μL (305 μg) DNA solution and of a 50-μL (70 μg) collagen solution and a 10-μL (61 μg) DNA solution were used. Changes in the collagen fibers' stereoarrangement occurred in the gel rods interacting with the DNA. Even when the DNA concentration (305 μg) was significantly higher than the collagen concentration (14 μg) and the DNA played the main role in establishing the overall oriented geometry in the collagen-DNA films (intertwisted fibrillary structure and negative birefringence), some collagen markers were present in the FT-IR spectra of the complex. When collagen and DNA concentrations were similar, DNA denaturation may occur. Present observations suggest formation of collagen-DNA complexes even if highly organized collagen macromolecules are offered to DNA.

The larvae of the two distantly related nonsocial bees Ericrocis lata (Apidae) and Hesperapis (Carinapis) rhodocerata (Melittidae), which develop mostly under arid desert areas of North America, and that differ in that they either spin (E. lata) or do not spin (H. rhodocerata) protective cocoons before entering diapause, produce transparent films that cover the larval integument. To understand the nature of these films, their responses to topochemical tests and their characteristics when examined with fluorescence and high-performance polarization microscopy and microspectroscopy were studied. A positive staining by Sudan black B, birefringence of negative sign, and a Fourier transform-infrared (FT-IR) spectrum typical of lipids were detected for the integument covering of both species. The FT-IR signature, particularly, suggests a wax chemical composition for these lipid coverings, resembling the waxes that are used as construction materials in the honey cells produced by social bees. Considering the arid environmental conditions under which these larvae develop, we hypothesize that their covering films may have evolved as protection against water depletion. This hypothesis seems especially appropriate for H. rhodocerata larvae, which are capable of undergoing a long diapause period in the absence of a protective cocoon.

The control of DNA packaging has been reported to be dependent on an ordered liquid-crystalline state. However, the textural characteristics that are typical of crystals and that resemble mesophases have not been reported for highly polymerized or even shorter types of DNA filaments under in vitro conditions that favor crystallization. Because DNA crystals are expected to exhibit particular textural optical anisotropies, pure and highly polymerized calf thymus DNA and simpler λ phage DNA were crystallized from solution drops and were analyzed using high-performance polarization microscopy with and without differential interference contrast (DIC) optics. Both types of DNA formed crystals that exhibited chiral supramolecular textures resembling the twist-grain boundary (TGB) columnar mesophases described for liquid crystals and exhibited intrinsic negative birefringence. To the best of our knowledge, this is the first observation using polarization/interference optics of pure DNA crystals that have TGB columnar mesophase-like textural characteristics. A comparison of the crystals formed from the highly polymerized calf thymus DNA and those formed from the shorter phage DNA strands revealed textural differences. Compared to the phage DNA crystals, the crystals of highly polymerized thymus DNA exhibited a more intertwisted columnar distribution and a fibrous texture between their columnar structures. In addition, a form birefringence phenomenon was detected only in the thymus DNA crystals. These characteristics are presumed to reflect the higher level of supramolecular order, self-assembly and chirality in highly polymerized calf thymus DNA crystals relative to that of crystals formed from the simpler, shorter, λ phage DNA. The higher-order supramolecular organization revealed here for in vitro DNA preparations raises the possibility that this structure could also occur, possibly to a smaller degree, during DNA self-aggregation under specific in vivo conditions. Whether the DNA crystal properties presently described play a role in the establishment of higher-order levels of hierarchical chromatin structure and consequently in chromatin physiology, should be further investigated.

The Feulgen reaction has been proposed by Robert Feulgen and Heinrich Rossenbeck for the identification of DNA nearly a hundred years ago. Since then, many other applications of this cytochemical/topochemical procedure at qualitative and quantitative level have been proposed in relation to DNA and its role in chromatin in human, animal and plant cells. In this article, we briefly review some fundamental aspects of the Feulgen reaction and current applications of such a method in studies of altered chromatin texture, including its association with or preceding changes in transcriptional activities and effect on epigenetic marks. Further perspectives on the use of the Feulgen reaction will depend of the proposal of innovative biological questions in which its reveals appropriate.

A previous study has not revealed the participation of a mucous component in the cocoon wall of the solitary bee, Lithurgus chrysurus, differing from the cocoon structure reported for many other bee species. However, uncertainty remains, because only the median and rear zones of this cocoon type have thus far been analyzed. Here, we studied the front zone of this cocoon, searching its components and their organization, to fill this knowledge gap. Topochemical assays, polarization microscopy and Fourier transform-infrared (FT-IR) microspectroscopy were used to study cross sections from L. chrysurus cocoon. Three main layers differing in structural organization were found to compose the cocoon wall. Silk fibroins were assumed to constitute the filamentous threads of the inner and outer layers and the laminar structure of the intermediate layer. Deduced from its topochemical properties and FT-IR spectral signature, a foamy material containing mucin glycoproteins and carboxylated acid glycosaminoglycans was found in the intermediate layer. FT-IR analysis using a Savitzky-Golay 2nd-derivative and absence of linear dichroism and birefringence phenomena suggest that a random-coil secondary structure predominates in the foam component. Co-existence of α-helical and β-sheet conformations is also hypothesized for the fibroin component of this cocoon. It is thus concluded that in addition to fibroin elements, a mucous component, likely contributed by a Malpighian tubule secretion, integrates the composition of the front zone of the cocoon wall of L. chrysurus. In addition, the FT-IR analysis of the inner layer silk of this cocoon suggests significant differences in comparison to the silk fibroins of the silkworm, and some minor spectral differences in comparison to published data on the honeybee silk, with respect to protein secondary structure.

Tissue engineering and cell-based therapy combine techniques that create biocompatible materials for cell survival, which can improve tendon repair. This study seeks to use a new fibrin sealant (FS) derived from the venom of Crotalus durissus terrificus, a biodegradable three-dimensional scaffolding produced from animal components only, associated with adipose-derived stem cells (ASC) for application in tendons injuries, considered a common and serious orthopedic problem. Lewis rats had tendons distributed in five groups: normal (N), transected (T), transected and FS (FS) or ASC (ASC) or with FS and ASC (FS + ASC). The in vivo imaging showed higher quantification of transplanted PKH26-labeled ASC in tendons of FS + ASC compared to ASC on the 14th day after transection. A small number of Iba1 labeled macrophages carrying PKH26 signal, probably due to phagocytosis of dead ASC, were observed in tendons of transected groups. ASC up-regulated the Tenomodulin gene expression in the transection region when compared to N, T and FS groups and the expression of TIMP-2 and Scleraxis genes in relation to the N group. FS group presented a greater organization of collagen fibers, followed by FS + ASC and ASC in comparison to N. Tendons from ASC group presented higher hydroxyproline concentration in relation to N and the transected tendons of T, FS and FS + ASC had a higher amount of collagen I and tenomodulin in comparison to N group. Although no marked differences were observed in the other biomechanical parameters, T group had higher value of maximum load compared to the groups ASC and FS + ASC. In conclusion, the FS kept constant the number of transplanted ASC in the transected region until the 14th day after injury. Our data suggest this FS to be a good scaffold for treatment during tendon repair because it was the most effective one regarding tendon organization recovering, followed by the FS treatment associated with ASC and finally by the transplanted ASC on the 21st day. Further investigations in long-term time points of the tendon repair are needed to analyze if the higher tissue organization found with the FS scaffold will improve the biomechanics of the tendons.

Toluidine blue (TB) staining either alone or in association with other methodologies has the potential to answer a variety of biological questions regarding the human, animal and plant tissues or cells. In this brief review, we not only report the primary use of TB to detect the anionic substrates and availability of their binding sites, but also unveil the resulting applications of TB staining in biological research. Among these applications, the uses of TB staining to identify the changes in chromatin DNA-protein complexes, nucleolus location, and extracellular matrix proteoglycan complexes associated with different physiological and pathological events are described. The usefulness of TB staining to monitor the effects elicited by environmental insults on chromatin and intercalation of drugs into the DNA is also included.

Tendon injuries represent a clinical challenge in regenerative medicine because their natural repair process is complex and inefficient. The high incidence of tendon injuries is frequently associated with sports practice, aging, tendinopathies, hypertension, diabetes mellitus, and the use of corticosteroids. The growing interest of scientists in using adipose-derived mesenchymal stem cells (ADMSC) in repair processes seems to be mostly due to their paracrine and immunomodulatory effects in stimulating specific cellular events. ADMSC activity can be influenced by GDF-5, which has been successfully used to drive tenogenic differentiation of ADMSC in vitro. Thus, we hypothesized that the application of ADMSC in isolation or in association with GDF-5 could improve Achilles tendon repair through the regulation of important remodeling genes expression. Lewis rats had tendons distributed in four groups: Transected (T), transected and treated with ADMSC (ASC) or GDF-5 (GDF5), or with both (ASC+GDF5). In the characterization of cells before application, ADMSC expressed the positive surface markers, CD90 (90%) and CD105 (95%), and the negative marker, CD45 (7%). ADMSC were also differentiated in chondrocytes, osteoblast, and adipocytes. On the 14th day after the tendon injury, GFP-ADMSC were observed in the transected region of tendons in the ASC and ASC+GDF5 groups, and exhibited and/or stimulated a similar genes expression profile when compared to the in vitro assay. ADMSC up-regulated Lox, Dcn, and Tgfb1 genes expression in comparison to T and ASC+GDF5 groups, which contributed to a lower proteoglycans arrangement, and to a higher collagen fiber organization and tendon biomechanics in the ASC group. The application of ADMSC in association with GDF-5 down-regulated Dcn, Gdf5, Lox, Tgfb1, Mmp2, and Timp2 genes expression, which contributed to a lower hydroxyproline concentration, lower collagen fiber organization, and to an improvement of the rats&rsquo, gait 24 h after the injury. In conclusion, although the literature describes the benefic effect of GDF-5 for the tendon healing process, our results show that its application, isolated or associated with ADMSC, cannot improve the repair process of partial transected tendons, indicating the higher effectiveness of the application of ADMSC in injured Achilles tendons. Our results show that the application of ADMSC in injured Achilles tendons was more effective in relation to its association with GDF-5.

Entheses are specialized biological structures that functionally anchor tendons to bones. The complexity, mechanical characteristics and properties of the entheses, particularly those related to exercise, mechanical load and pathologies, have been extensively analyzed; however, the macromolecular organization of the enthesis fibers, as assessed by polarization microscopy, has not yet been investigated. Morphological and optical anisotropy characteristics, such as birefringence, linear dichroism (LD) and differential interference contrast (DIC-PLM) properties, are thus analyzed in this study of a healthy adult mouse calcaneal tendon-bone enthesis. The molecular and supramolecular order of collagen and GAGs was determined for the collagen bundles of this enthesis. Based on a birefringence plot pattern as well as on metachromasy and linear dichroism after toluidine blue staining at pH 4.0, a similarity between the calcaneal tendon-bone enthesis and cartilage during ossification may be assumed. This similarity is assumed to favor the adequacy of this enthesis to support a compressive load. Considering that the collagen-proteoglycan complexes and the enthesis fibers themselves have a chiral nature, these structures could be acting via reciprocal signaling with the cellular environment of the enthesis.

Although several treatments for tendon lesions have been proposed, successful tendon repair remains a great challenge for orthopedics, especially considering the high incidence of re-rupture of injured tendons. Our aim was to evaluate the pharmacological potential of Aloe vera on the content and arrangement of glycosaminoglycans (GAGs) during tendon healing, which was based on the effectiveness of A. vera on collagen organization previously observed by our group. In rats, a partial calcaneal tendon transection was performed with subsequent topical A. vera application at the injury site. The tendons were treated with A. vera ointment for 7 days and excised on the 7(th) , 14(th) , or 21(st) day post-surgery. Control rats received ointment without A. vera. A higher content of GAGs and a lower amount of dermatan sulfate were detected in the A. vera-treated group on the 14(th) day compared with the control. Also at 14 days post-surgery, a lower dichroic ratio in toluidine blue stained sections was observed in A. vera-treated tendons compared with the control. No differences were observed in the chondroitin-6-sulfate and TGF-β1 levels between the groups, and higher amount of non-collagenous proteins was detected in the A. vera-treated group on the 21(st) day, compared with the control group. No differences were observed in the number of fibroblasts, inflammatory cells and blood vessels between the groups. The application of A. vera during tendon healing modified the arrangement of GAGs and increased the content of GAGs and non-collagenous proteins.

Polarized Fourier transform-infrared (FT-IR) was used to compare the orientation of vibrational chemical groups of bovine tendon collagen bundles (CBs) to that of nylon 6, a simpler polyamide model, in terms of linear dichroism (LD). Subtraction of spectral profiles identified the most significant differences regarding the amide regions. At 1630cm(-1), the CBs displayed higher peak areas and absorbance when positioned perpendicularly (A⊥) to the plane of polarized light, in comparison with nylon 6. In contrast, at the 1526cm(-1) amide II spectral region the inverse occurred. In the amide III region (1232cm(-1)), the LD was positive and higher for CBs. Dichroic ratios (DR=A||/A⊥) calculated from the average of ten measured spectra for CBs and nylon 6 revealed that the values for CBs were1.0 in the 3298-1655cm(-1) wavenumber range and1.0 in the 1536-1234cm(-1) wavenumber range. From 1535 to 1120cm(-1), nylon 6 displayed DR values higher than those of CBs. The LD band integrated areas were higher in CBs than in nylon 6. The LD differences between CBs and nylon 6 are probably due to a more complex chemical composition and supramolecular oriented architecture in CBs in comparison to nylon 6.

The purpose of this study was to investigate the influence of implanting collagen with a supramolecular organization on peripheral nerve regeneration, using the sciatic nerve tubulization technique. For this purpose, adult female Sprague Dawley rats were divided into five groups: (1) TP – sciatic nerve repaired with empty polyethylene tubular prothesis (n = 10), (2) TPCL – nerve repair with empty polycaprolactone (PCL) tubing (n = 8), (3) TPCLF – repair with PCL tubing filled with an implant of collagen with a supramolecular organization (n = 10), (4) AG – animals that received a peripheral nerve autograft (n = 8), and (5) Normal nerves (n = 8). The results were assessed by quantification of the regenerated fibers, nerve morphometry, and transmission electron microscopy, 60 days after surgery. Immunohistochemistry and polarization microscopy were also used to analyze the regenerated nerve structure and cellular elements. The results showed that the AG group presented a larger number of regenerated axons. However, the TPCL and TPCLF groups presented more compact regenerated fibers with a morphometric profile closer to normal, both at the tube midpoint and 2 mm distal to the prosthesis. These findings were reinforced by polarization microscopy, which indicated a better collagen/axons suprastructural organization in the TPCLF derived samples. In addition, the immunohistochemical results obtained using the antibody anti-p75NTR as a Schwann cell reactivity marker demonstrated that the Schwann cells were more reactive during the regenerative process in the TPCLF group as compared to the TPCL group and the normal sciatic nerve. Altogether, the results of this study indicated that the implant of collagen with a supramolecular organization positively influenced and stimulated the regeneration process through the nerve gap, resulting in the formation of a better morphologically arranged tissue.

Regarding the importance of type I collagen in understanding the mechanical properties of a range of tissues, there is still a gap in our knowledge of how proteins perform such work. There is consensus in literature that the mechanical characteristics of a tissue are primarily determined by the organization of its molecules. The purpose of this study was to characterize the organization of non-irradiated and irradiated type I collagen. Irradiation was performed with a linearly polarized HeNe laser (λ = 632.8 nm) and characterization was undertaken using polarized light microscopy to investigate the birefringence and second harmonic generation to analyze nonlinear susceptibility. Rats received laser irradiation (P = 6.0 mW, I = 21.2 mW/cm(2), E ≈ 0.3 J, ED = 1.0 J/cm(2)) on their healthy Achilles tendons, which after were extracted to prepare the specimens. Our results show that irradiated samples present higher birefringence and greater non-linear susceptibility than non-irradiated samples. Under studied conditions, we propose that a red laser with polarization direction aligned in parallel to the tendon long axis promotes further alignment on the ordered healthy collagen fibrils towards the electric field incident. Thus, prospects for biomedical applications for laser polarized radiation on type I collagen are encouraging since it supports greater tissue organization.

article i nfo Article history: Received 8 June 2012 Accepted 4 September 2012 Aims: The tendon is composed of highly organized collagen fibers that form a complex supramolecular struc- ture. After lesions, the organization and composition of the tendon are not completely restored. Our purpose was to evaluate if the application of Aloe vera improves tendon healing, considering the effectiveness in the stimulus of collagen synthesis. Main methods: The calcaneal tendon of male Wistar rats was partially transected with subsequent topical ap- plication of A. vera ointment at the injury. The animals were separated into groups with tendons treated with the A. vera extract for 7 days and excised on the 7th, 14th and 21st days after surgery; control rats received only ointment base without plant extract. Key findings: Morphological analysis using polarization microscopy showed that the entire tendon undergoes a remodeling process, with disorganized collagen fibers by days 7 and 14 in plant-treated and non-treated groups and with a higher birefringence in tendons of the plant-treated group on the 21st day. A higher con- centration of hydroxyproline was found in plant-treated tendons on days 7 and 14 compared with their con- trols. Western blots showed lower amounts of type I collagen in the plant-treated group on day 14 compared with the control. MMP-9 diminished 14 days after lesion and the active isoform of MMP-2 increased on day 21 in plant-treated groups. Significance: The present study indicates a beneficial effect of A. vera in the tissue reorganization in the transected region of the tendon 21 days after injury and is supported by an increase of active MMP-2.

The aim of this study was to investigate the effect of electroacupuncture (EA) on the composition and organization of the extracellular matrix of the rat Achilles tendon after a partial transection during the proliferative phase of healing. Wistar rats were divided into three groups: rats that were not tenotomized (G1), tenotomized rats (G2), and rats that were tenotomized and submitted to EA (G3). EA was applied 15 days after injury at the ST36 and BL57 acupoints for 20 min, three times per week on alternate days for a total of six sessions. Biochemical analyses were performed using non-collagenous proteins, glycosaminoglycans, and hydroxyproline quantifications. An analysis of metalloproteinase-2 was carried out by zymography. The general organization of the extracellular matrix and the metachromasy of the tendons were analyzed under light microscopy. The organization of the bundles of collagen fibers was analyzed by birefringence analysis. The results showed that EA did not alter the concentration of non-collagenous proteins or glycosaminoglycans or the enzymatic activity of metalloproteinase-2 in the transected tendons. However, the concentration of hydroxyproline was significantly increased when these tendons were treated by EA. The analysis of birefringence showed a higher organization of collagen fibers in the group treated by EA. These results indicate, for the first time, that EA may offer therapeutic benefits for the treatment of tendon injuries by increasing the concentration of collagen and by inducing a better molecular organization of the collagen fibers, which may improve the mechanical strength of the tendon after injury.

Tendons are formed by dense connective tissue composed of an abundant extracellular matrix (ECM) that is constituted mainly of collagen molecules, which are organized into fibrils, fibers, fiber bundles and fascicles helicoidally arranged along the largest axis of the tendon. The biomechanical properties of tendons are directly related to the organization of the collagen molecules that aggregate to become a super-twisted cord. In addition to collagen, the ECM of tendons is composed of non-fibrillar components, such as proteoglycans and non-collagenous glycoproteins. The capacity of tendons to resist mechanical stress is directly related to the structural organization of the ECM. Collagen is a biopolymer and presents optical anisotropies, such as birefringence and linear dichroism, that are important optical properties in the characterization of the supramolecular organization of the fibers. The objective of this study was to present a review of the composition and organization of the ECM of tendons and to highlight the importance of the anisotropic optical properties in the study of alterations in the ECM.

Early pregnancy and multiparity are known to reduce the risk of women to develop breast cancer at menopause. Based on the knowledge that the differentiation of the breast induced by the hormones of pregnancy plays a major role in this protection, this work was performed with the purpose of identifying what differentiation-associated molecular changes persist in the breast until menopause. Core needle biopsies (CNB) obtained from the breast of 42 nulliparous (NP) and 71 parous (P) postmenopausal women were analyzed in morphology, immunocytochemistry and gene expression. Whereas in the NP breast, nuclei of epithelial cells were large and euchromatic, in the P breast they were small and hyperchromatic, showing strong methylation of histone 3 at lysine 9 and 27. Transcriptomic analysis performed using Affymetrix HG_U133 oligonucleotide arrays revealed that in CNB of the P breast, there were 267 upregulated probesets that comprised genes controlling chromatin organization, transcription regulation, splicing machinery, mRNA processing and noncoding elements including XIST. We concluded that the differentiation process induced by pregnancy is centered in chromatin remodeling and in the mRNA processing reactome, both of which emerge as important regulatory pathways. These are indicative of a safeguard step that maintains the fidelity of the transcription process, becoming the ultimate mechanism mediating the protection of the breast conferred by full-term pregnancy.

Lysyl oxidases (LOXs) are enzymes that permit the covalent crosslinking of the component chains of collagen and elastin. These enzymes are present inside the nuclei of certain mammalian cells. Previous studies have proposed LOX binding to histone H1 in vitro, and histone H1 is known to control global chromatin compaction and mitotic chromosome architecture. Therefore, in the present study, we analyzed chromatin supraorganizational changes, mitotic abnormalities, mitotic indices and cell death ratios in COS-7 and NRK-49F cells with high and low lox expression levels, respectively. The objective was to support biochemical data of LOX-H1 interaction, by providing evidence of chromatin remodeling in vivo, under different lox expressions. Chromatin decondensation assessed by image analysis was observed in COS-7 cells with increased lox expression. This decondensation is suggested to be promoted by LOX actions on histone H1, which loosens the DNA-H1 complex. In NRK-49F cells transfected with antisense lox or subjected to treatment with beta-aminopropionitrile (BAPN), chromatin condensation and nuclear phenotypic variability were found, which may be due to reduced LOX-H1 interaction. When lox expression was increased in COS-7 cells, the frequency of irregular chromosome plates was not affected, but cell proliferation decreased and "cell death preceded by multinucleation" increased. In NRK-49F cells there was accelerated proliferation induced by transfection with the antisense lox, and confirmed when cells were treated with BAPN. Apoptosis increased in NRK-49F cells only with BAPN treatment whereas cell death preceded by multinucleation increased only after antisense lox transfection. The data presented herein regarding chromatin remodeling indirectly support the hypothesis that LOX binds to histone H1 in vivo. Cell proliferation in COS-7 and NRK-49F cells and cell death at least in COS-7 cells agree with predicted effects of LOX interference in these processes.

Several studies have demonstrated the relationship between exercise and the extracellular matrix of muscle tendons, and have described alterations in their structural and biochemical properties when subjected to strenuous exercise. However, little is known about what happens to tendons when they are subjected to stretching. We evaluated the changes in the composition and structure of rat calcaneal tendons subjected to a stretching program. The animals had their muscles stretched for 30 s with 30 s of rest, with 10 repetitions, three and five times a week for 21 days. For morphological analysis, the sections were stained with hematoxylin-eosin and toluidine blue. For biochemical analysis, the tendons were treated with 4 M guanidine hydrochloride and analyzed in SDS-PAGE. The contents of total proteins and glycosaminoglycans were also measured. In the sections stained with toluidine blue, we could observe an increase of rounded cells, especially in the enthesis region. In the region next to the enthesis was a metachromatic region, which was more intensely stained in the stretched groups. In the tension regions, the cells appeared more aligned. Cellularity increased in both regions. The SDS-PAGE analysis showed a larger amount of collagen in the stretched groups and a polydispersed component of 65 kDa in all the groups. The amounts of proteins and glycosaminoglycans were also larger in the stretched tendons. The agarose-gel electrophoresis confirmed the presence of dermatan sulfate in the tension and compression regions, and of chondroitin sulfate only in the latter. Our results showed that the stretching stimulus changed the cellularity and the amount of the extracellular matrix compounds, confirming that tendons are dynamic structures with a capacity to detect alterations in their load.

I review here form, or textural, birefringence (Δ(F)) in the context of advances in the field, as well as with regard to findings and applications in the physics of photonic devices, fibers maintaining polarization, photonic crystal fibers, and in biopolymers present in extracellular matrices and the myelin sheath. Some advantages of applying knowledge of Δ(F) to biological fields involving biopolymers, especially collagen fibers, are considered in more detail. Tendon and cartilage collagen fibers have been regarded as a model of dense, highly aggregated biopolymers with preferential orientations. Owing to their supramolecular organization, such materials may be used to study molecular order by using anisotropic optical properties, especially Δ(F). Differences between collagen type I- and collagen type II-rich structures, and similarities between collagen crimp and second harmonic generation images are reported. Based on data reported here, it is possible to deduce that collagen type I supramolecular organization has nonlinear optical properties and that tendon segments can conduct red laser light. With respect to nerve fibers, the detection and measurements of Δ(F) have allowed the myelin sheath to be considered a smectic liquid crystal.

Chromatin supraorganization and extensibility and nuclear glycoprotein content have been reported to change in hepatocytes from mice during development and aging, as well as under starvation and refeeding conditions. In non-obese diabetic (NOD) mice, the expression of insulin-dependent diabetes may be accompanied by metabolic changes in the liver. These changes are likely to be similar to those involved in the aging processes of non-diabetic animals. Therefore, we hypothesized that the chromatin organization, as well as the physical properties and compositions of hepatocyte nuclei would also be affected in NOD mice in the same way as those in aged non-diabetic mice. Nuclear image parameters were evaluated by image analysis of Feulgen-stained preparations. Chromatin extensibility in response to gravity was observed with polarized light after lysis and toluidine blue staining. The Con-A response of nuclear glycoproteins was evaluated with scanning microspectrophotometry. These characteristics were assessed using hepatocyte imprints from female NOD mice after a 28-day period of diabetes expression. Observations and measurements were made in comparison to healthy BALB/c mice. Total RNA amounts were determined for livers of NOD and BALB/c mice. Enhanced polyploidy levels, a decrease in chromatin higher-order packing states, an increased frequency of extended chromatin fiber formation, and deeper Con-A-responsive chromatin areas were observed in the hepatocytes of the NOD mice expressing insulin-dependent diabetes. Reduced amounts of total RNA were also found in the livers of these mice. Our findings for NOD mice expressing insulin-dependent diabetes are consistent with previously reported data for old-aged mice of the inbred strain A/Uni and may reflect changes in transcriptional activities associated with the stressful physiological demands on the liver during the expression of diabetes.

In this study, nonenzymatic glycosylation was assessed in aorta extracellular matrix (ECM) from nonobese diabetic (NOD) mice, using nitroblue tetrazolium (NBT). Molecular and structural changes were investigated in elastic lamellae and collagen fibers of diabetic mice aortas after staining with dansyl chloride and anilinonaphthalene sulfonate (ANS). Alterations in arterial autofluorescence and birefringence of collagen fibers were investigated in unstained aortas. Proliferation of smooth muscle cells (SMC) was also investigated by Feulgen reaction staining assessed by confocal microscopy and image analysis. Assessment of nonenzymatic glycosylation demonstrated glycosylation products in the aorta ECM of NOD mice. Elastic lamellae and collagen fibers from NOD mouse aortas presented less intense fluorescence after staining with dansyl chloride and ANS when compared to aortas of control nondiabetic mice. However, unstained NOD aortas showed more intense autofluorescence when compared to controls. Birefringence analysis suggests alterations in the higher molecular packing of the arterial collagen fibers in NOD aortas. In aortas stained by Feulgen reaction, no evidence of SMC proliferation was observed in diabetic aortas.

The immortalized human breast epithelial MCF-10F cell line, although estrogen receptor alpha negative, develops cell proliferating activities and invasiveness indicative of neoplastic transformation, after treatment with 17-beta-estradiol (E-2). These effects are similar to those produced by benzo[a]pyrene (BP). Since we have previously reported changes in the nuclear parameters accompanying BP-induced tumorigenesis in MCF-10F cells, we have examined whether similar alterations occur in E-2-treated cells. We therefore studied DNA amounts and other nuclear parameters in Feulgen-stained MCF-10F cells after treatment with various concentrations of E-2, BP, the estrogen antagonist ICI 182,780, and E-2 in the presence of ICI 182,780. E-2 caused a certain loss of DNA and changes in the nuclear size and chromatin supraorganization of MCF-10F cells. Many of these changes were similar to those produced by BP and were indicative of neoplastic transformation. More intense chromatin remodelling was seen with 70 nM E-2. Since these changes were not abrogated totally or partially by ICI 182,780, the neoplastic transformation of MCF-10F cells stimulated by E-2 involved a process that was independent of estrogen alpha-receptors. The changes produced by ICI 182,780 alone were attributed to effects other than its well-known anti-estrogenic activity.

Summary Optical anisotropy as dispersion of birefringence (DB) (birefringence studied for light of different wavelengths) and linear dichroism (LD) (selective absorption of polarized light) in stained substrates reflects their macromolecular orientation states. Birefringence interference colors of alcian blue (AB)-stained glycosaminoglycans (GAG) and glycoconjugates observed with polarization microscopy have been found to vary, although their staining characteristics under unpolarized light are practically the same. We investigated the optical anisotropy of GAG-AB and some glycoconjugate-AB complexes used as standards, to provide a basis for interpreting results for AB-stained materials in situ. Filamentous preparations of hyaluronic acid (HA), chondroitinsulfates, proteoglycans, and a mucus sulfoglycoconjugate were studied. Anomalous DB (birefringence sign changing with the wavelength of the incident light) was generally observed, but LD was seen only in the AB–HA complex. LD simultaneous to anomalous DB characteristics on the AB–HA complex were assumed to be caused by a maximally oriented helical conformation of the HA. For the other AB–GAG studied, the optical anisotropic characteristics were suggestive of some degree of folding of their chains into a tertiary structure. The profiles of the anomalous DB curves for the AB-stained sulfoglycoconjugate differed from those of the other materials, probably due to different organization of its dye-binding sites.

Changes in DNA/chromatin structure, ploidy degrees and cell death possibly caused by oxidative stress during the insulin-dependent diabetes have been reported for different cell types. However, all these studies have been carried in streptozotocin-induced diabetic rats or mice and showed contradictory results. In this work, nuclear phenotypes and DNA fragmentation were investigated in fibroblasts from mice spontaneously developing insulin- dependent diabetes (NOD) and compared with healthy (BALB/C) mice. Geometric, densitometric and textural pa- rameters obtained for Feulgen-stained nuclei by image analysis were used to define nuclear phenotypes. Significant differences were observed for nuclear sizes and for densitometric and textural parameters of the tendon nuclei. Op- tical density, Feulgen-DNA values, transmittance variability per nucleus and nuclear entropy values were signifi- cantly higher in the NOD mice. The Feulgen-DNA amounts for the NOD and BALB/C mice were found to be dis- tributed into several doubling Feulgen-DNA classes. The frequency of nuclei with the smallest Feulgen-DNA amounts, which may represent DNA fragmentation and loss, was lower in fibroblasts of the NOD mice (2.3%) in comparison to the BALB/C mice (38%). In contrast, the frequency of polyploid nuclei in NOD mice was higher (24.5%) than that in BALB/C mice (1.9%). Based on optical density, transmittance variability per nucleus, and nu- clear entropy data, a larger contrast between highly and less densely packed states, was demonstrated for the fibrob- lasts of the NOD mice. Maybe the deeper condensation of the highly packed chromatin evident in NOD fibroblasts is related to silencing of some genes involved with changes in tendon supraorganization with the diabetes.

Background: How much DNA remains in mouse hepatocyte nuclei after extended chromatin fiber (ECF) formation or whether this content varies within the nuclear population is not known. This information could be relevant to understanding chromatin extensibility as related to chromatin organization, possibly associated with variable nuclear activities in hepatocytes. Methods: A protocol for ECF formation under the gravity action, image analysis of Feulgen-stained unfixed mouse hepatocyte remnants, and DAPI fluorescence were used. Results: Areas, shape, Feulgen-DNA amounts, and chromatin texture were affected in unfixed, lysed nuclei. The Feulgen-DNA values in nuclear remnants represented ∼37% of the content in fixed, nonlysed nuclei in terms of median values; the coefficient of variation of Feulgen-DNA values in the nuclear remnants was much higher than those in controls. Enhancement in DAPI fluorescence was evident in chromocenters of the fixed nuclei and in remnants and some ECF granules of the unfixed, lysed nuclei. Conclusions: The DNA content of the nuclear remnants was much more variable than that assumed from known variability in hepatocyte ploidy degrees. The variable constraint to chromatin extrusion from hepatocyte nuclei is hypothesized to depend on variable chromatin organization with possible involvement of nuclear matrix association, transcriptional activities, and AT-rich DNA-containing heterochromatin. © 2006 International Society for Analytical Cytology

Purpose: To assess whether the use of collagens with lipografts improves graft survival. Materials and Methods: Thirty isogenic New Zealand rabbits were injected in the auricular area with either autologous fat alone or autologous fat associated with collagen. The animals were evaluated at 7, 30, and 90 days by histologic and morphometric methods together with measurements of the graft area and the number of adipocytes. Results: Addition of collagen to the lipoinjection reduced the local inflammatory reaction and improved the graft's survival; e.g., for stress urinary incontinence. Conclusion: These results suggest that it is possible to increase the survival rate of the lipografts and improve their clinical applicability.

Background A relation between the changes in DNA content and chromatin supra-organization and the expression of gradual steps of tumorigenesis has been assessed by image analysis in human breast epithelial cells (MCF-10F) treated with benzo[a]pyrene (BP) (cell lines BP1, BP1-E, BP1-Tras, and others). Methods Because abnormal chromosomes 11 and 17 have been associated with neoplastic progression in BP-transformed MCF-10F cells, image analysis of Feulgen-stained tumorigenic BP1-E cells with the microcell-mediated chromosome transfer of normal chromosomes 11 and 17 was carried out. Results A tendency of DNA amount distribution and nuclear size restoration to values typical of non-transformed MCF-10F cells was demonstrated, especially after the transfer of chromosome 17. No reversion in chromatin texture was found after the transfer of chromosome 11 or 17. Conclusions Although the presence of a normal chromosome 17 should be considered among the necessary steps for tumorigenic human breast epithelial cells to recover their normality, a more complex genome balance is required for the entire nuclear chromatin of these cells to recover its totally normal supra-organization and expression. Cytometry Part A 52A:70–76, 2003. © 2003 Wiley-Liss, Inc.

Prostatic lesions in Brazilian patients with benign prostatic hyperplasia (BPH, 26 cases) or adenocarcinoma (AC, 25 cases) were compared by qualitative microscopy and morphometric analysis. In 12 cases of BPH, prostate regions with no histological alterations were considered as controls (Ct). Archival material consisted of formalin-fixed, paraffin-embedded specimens obtained from prostatic transurethral resection and radical prostatectomy. Haematoxylin/eosin (HE)- stained sections were used to estimate the nuclear areas, perimeters and form factor values. HE-stained sections from AC specimens were also used for Gleason grading. BPH, AC and Ct could be discriminated by their nuclear areas and nuclear perimeters, but not by the nuclear form factor parameter. No significant differences were found when the AC data were compared using the combined version or the predominant grade version of the Gleason score (p = 0.8380 for nuclear area; p = 0.6076 for nuclear perimeter; p = 0.9202 for nuclear form fa...

Valproic acid (VPA), a well-known histone deacetylase inhibitor, has been reported to affect the DNA methylation status in addition to inducing histone hyperacetylation in several cell types. In HeLa cells, VPA promotes histone acetylation and chromatin remodeling. However, DNA demethylation was not checked in this cell model for standing effects longer than those provided by histone acetylation, which is a rapid and transient phenomenon. Demonstration of VPA-induced DNA demethylation in HeLa cells would contribute to understanding the effect of VPA on an aggressive tumor cell line. In the present work, DNA demethylation in VPA-treated HeLa cells was assessed by image analysis of chromatin texture, the abundance of 5-methylcytosine (5mC) immunofluorescence signals and Fourier transform-infrared (FT-IR) microspectroscopy centered on spectral regions related to the vibration of–CH3 groups. Image analysis indicated that increased chromatin unpacking promoted by a 4-h-treatment with 1.0 mM VPA persisted for 24 h in the absence of the drug, suggesting the occurrence of DNA demethylation that was confirmed by decreased 5mC immunofluorescence signals. FT-IR spectra of DNA samples from 1 mM or 20 mM VPA-treated cells subjected to a peak fitting analysis of the spectral window for–CH3 stretching vibrations showed decreased vibrations and energy of these groups as a function of the decreased abundance of 5mC induced by increased VPA concentrations. Only the 20 mM-VPA treatment caused an increase in the ratio of -CH3 bending vibrations evaluated at 1375 cm-1 in relation to in-plane vibrations of overall cytosines evaluated at 1492 cm-1. CH3 stretching vibrations showed to be more sensitive than–CH3 bending vibrations, as detected with FT-IR microspectroscopy, for studies aiming to associate vibrational spectroscopy and changes in DNA 5mC abundance.

Although several treatments for tendon lesions have been proposed, successful tendon repair remains a great challenge for orthopedics, especially considering the high incidence of re-rupture of injured tendons. Our aim was to evaluate the pharmacological potential of Aloe vera on the content and arrangement of glycosaminoglycans (GAGs) during tendon healing, which was based on the effectiveness of A. vera on collagen organization previously observed by our group. In rats, a partial calcaneal tendon transection was performed with subsequent topical A. vera application at the injury site. The tendons were treated with A. vera ointment for 7 days and excised on the 7(th) , 14(th) , or 21(st) day post-surgery. Control rats received ointment without A. vera. A higher content of GAGs and a lower amount of dermatan sulfate were detected in the A. vera-treated group on the 14(th) day compared with the control. Also at 14 days post-surgery, a lower dichroic ratio in toluidine blue stained sections was observed in A. vera-treated tendons compared with the control. No differences were observed in the chondroitin-6-sulfate and TGF-β1 levels between the groups, and higher amount of non-collagenous proteins was detected in the A. vera-treated group on the 21(st) day, compared with the control group. No differences were observed in the number of fibroblasts, inflammatory cells and blood vessels between the groups. The application of A. vera during tendon healing modified the arrangement of GAGs and increased the content of GAGs and non-collagenous proteins.

In tendons, collagen fibers are structured with a helical supra-molecular organization and interact with structured light depicting intrinsic and form birefringence (FB). FB is an optical anisotropy of the permittivity that is being used in the construction of man- made devices to filling up practical purposes. In the present case the used biomaterial displayed highest levels of FB. Linear polarized monochromatic light, after traveling microscopic sections of collagen fibers, exhibit elliptically polarized front of light, with angular momentum, that after trespassing a second polarizer generated vortices images. Advanced polarized microscopy, structured light, was used in this study to detect and characterize the production of vertices images by collagen fiber as structured biomaterial. Microscopic sections used in the present study fulfilled all of the conditions for generating vortices. Here, it is shown, for the first time that vortex structures are generated by chiral supramolecular collagen fibers in birefringent bundles, and can be observed with a polarizing microscope. Additionally, it is hypothesized that the vortices in biological structures are part of a mechanism of signal transference from extracellular space to cells. Considering that the spatial distribution of collagen fibers and bundles in connective tissues may vary, it is assumed that the complexity of the corresponding vortex morphology may also vary. Finally, it is recommended that the structured characteristics of collagen fibers chirality and their optical anisotropic properties could be used as inspiration for new man-made structured devices.

Background Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile. Methodology/Principal Findings The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software. Conclusions/Significance Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including νas –CH3 stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of –CH3 groups. Other spectral differences were found at 1700–1500 cm−1 and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.

Agradecimentos: The author is indebted to Dr. Maria Luiza S. Mello for dis-cussing and reviewing the manuscript. The support of Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil (grants no. 2003/04597-0, 2007/058251-8 and 2013/11078-0) is gratefully acknowledged Abstract: Histological and histochemical observations support the hypothesis that collagen fibers can link to elastic fibers. However, the resulting organization of elastin and collagen type complexes and differences between these materials in terms of macromolecular orientation and frequencies of their chemical vibrational groups have not yet been solved. This study aimed to investigate the macromolecular organization of pure elastin, collagen type I and elastin–collagen complexes using polarized light DIC-microscopy. Additionally, differences and similarities between pure elastin and collagen bundles (CB) were investigated by Fourier transform-infrared (FT-IR) microspectroscopy. Although elastin exhibited a faint birefringence, the elastin–collagen complex aggregates formed in solution exhibited a deep birefringence and formation of an ordered-supramolecular complex typical of collagen chiral structure. The FT-IR study revealed elastin and CB peptide Nsingle bondH groups involved in different types of H-bonding. More energy is absorbed in the vibrational transitions corresponding to single bondCH, single bondCH2 and CH3 groups (probably associated with the hydrophobicity demonstrated by 8-anilino-1-naphtalene sulfonic acid sodium salt [ANS] fluorescence), and to ?CN, dNH and ?CH2 groups of elastin compared to CB. It is assumed that the a-helix contribution to the pure elastin amide I profile is 46.8%, whereas that of the B-sheet is 20% and that unordered structures contribute to the remaining percentage. An FT-IR profile library reveals that the elastin signature within the 1360–1189 cm-1 spectral range resembles that of Conex–Toray aramid fibers FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP Fechado

Increased amounts of chromatin condensation (i.e., localized areas of high DNA density, or chromatin higher order packing state) have been described in NIH 3T3 cells transformed with the Ha‐rasoncogene. The structural basis for this oncogene‐mediated alteration in nuclear organization is unknown. Since DNA methylation is likely to be involved in regulating the nucleosomal level of DNA packaging, we studied the role of DNA methylation in higher‐order chromatin organization induced by Ha‐ras. CpG‐methylated DNA content was estimated in “condensed” chromatin of Ha‐ras‐transformed NIH 3T3 cell lines which differ in ras expression and ras‐induced metastatic ability but present approximately the same values of “condensed” chromatin areas. The question posed was that if DNA methylation were involved with the chromatin higher‐order organization induced by Ha‐ras in these cell lines, the methylated DNA density in the “condensed” chromatin would also be the same. The DNA evaluation was performed by video image analysis in Feulgen‐stained cells previously subjected to treatment withMspI andHpaII restriction enzymes, which distinguish between methylated and non‐methylated DNA. The amount of methylated CpG sequences not digested byHpaII in “condensed” chromatin regions was found to vary in the studied ras‐transformed cell lines. DNA CpG methylation status is thus suggested not to be involved with the higher order chromatin condensation induced byrastransformation in the mentioned NIH 3T3 cell lines.