[Regulatory effects of bio-intensity electric field on microtubule acetylation in human epidermal cell line HaCaT].

Objective: To investigate the regulatory effects of bio-intensity electric field on directional migration and microtubule acetylation in human epidermal cell line HaCaT, aiming to provide molecular theoretical basis for the clinical treatment of wound repair. Methods: The experimental research methods were used. HaCaT cells were collected and divided into simulated electric field group (n=54) placed in the electric field device without electricity for 3 h and electric field treatment group (n=52) treated with 200 mV/mm electric field for 3 h (the same treatment methods below). The cell movement direction was observed in the living cell workstation and the movement velocity, trajectory velocity, and direction of cosθ of cell movement within 3 h of treatment were calculated. HaCaT cells were divided into simulated electric field group and electric field treatment 1 h group, electric field treatment 2 h group, and electric field treatment 3 h group which were treated with 200 mV/mm electric field for corresponding time. HaCaT cells were divided into simulated electric field group and 100 mV/mm electric field group, 200 mV/mm electric field group, and 300 mV/mm electric field group treated with electric field of corresponding intensities for 3 h. The protein expression of acetylated α-tubulin was detected by Western blotting (n=3). HaCaT cells were divided into simulated electric field group and electric field treatment group, and the protein expression of acetylated α-tubulin was detected and located by immunofluorescence method (n=3). Data were statistically analyzed with Kruskal-Wallis H test,Mann-Whitney U test, Bonferroni correction, one-way analysis of variance, least significant difference test, and independent sample t test. Results: Within 3 h of treatment, compared with that in simulated electric field group, the cells in electric field treatment group had obvious tendency to move directionally, the movement velocity and trajectory velocity were increased significantly (with Z values of -8.53 and -2.05, respectively, P<0.05 or P<0.01), and the directionality was significantly enhanced (Z=-8.65, P<0.01). Compared with (0.80±0.14) in simulated electric field group, the protein expressions of acetylated α-tubulin in electric field treatment 1 h group (1.50±0.08) and electric field treatment 2 h group (1.89±0.06) were not changed obviously (P>0.05), while the protein expression of acetylated α-tubulin of cells in electric field treatment 3 h group (3.37±0.36) was increased significantly (Z=-3.06, P<0.05). After treatment for 3 h, the protein expressions of acetylated α-tubulin of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 300 mV/mm electric field group were 1.63±0.05, 2.24±0.08, and 2.00±0.13, respectively, which were significantly more than 0.95±0.27 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of acetylated α-tubulin in 200 mV/mm electric field group and 300 mV/mm electric field group were increased significantly (P<0.01); the protein expression of acetylated α-tubulin of cells in 300 mV/mm electric field group was significantly lower than that in 200 mV/mm electric field group (P<0.05). After treatment for 3 h, compared with that in simulated electric field group, the acetylated α-tubulin of cells had enhanced directional distribution and higher protein expression (t=5.78, P<0.01). Conclusions: Bio-intensity electric field can induce the directional migration of HaCaT cells and obviously up-regulate the level of α-ubulin acetylation after treatment at 200 mV/mm bio-intensity electric field for 3 h.

Technical note: Survey of extracellular and cell-pellet-associated DNA from 'touch'/trace samples.

The goal of this study was to characterize the reproducibility of extracellular and cell pellet associated DNA yields recovered from handled substrates. Results showed that extracellular DNA yields were extremely variable between contributors-ranging between 0 and >10ng-and tended to dwarf cell pellet yields, which varied between 0 and ∼230pg. DNA yields across multiple samples from the same contributor on different days showed similar levels of variability in both DNA fractions, indicating that extracellular DNA yield is largely influenced by extrinsic and/or environmental factors and is not a contributor-specific attribute. Microscopic surveys of cells from the pellet fraction as well as fingerprints from the same contributor samples were conducted following treatment with fluorescent DNA stain. Nearly all imaged cells exhibited diffuse fluorescence across the cell without discernable evidence of nuclei. This is consistent with the limited nature of DNA recovery from the pellet fraction and the prevalence of extracellular DNA in these samples.

Conical epidermal cells cause velvety colouration and enhanced patterning in Mandevilla flowers.

The majority of angiosperms have flowers with conical epidermal cells, which are assumed to have various functions, such as enhancing the visual signal to pollinators, but detailed optical studies on how conical epidermal cells determine the flower's visual appearance are scarce. Here we report that conical epidermal cells of Mandevilla sanderi flowers effectively reduce surface gloss and create a velvety appearance. Owing to the reduction in surface gloss, the flower further makes more efficient use of floral pigments and light scattering structures inside the flower. The interior backscattering yields a cosine angular dependence of reflected light, meaning that the flowers approximate near-perfect (Lambertian) diffusers, creating a visual signal that is visible across a wide angular space. Together with the large flowers and the tilted corolla tips, this generates a distinct visual pattern, which may enhance the visibility to pollinators.

Cell wall structural changes lead to separation and shedding of biofouled epidermal cell wall layers by the brown alga Ascophyllum nodosum.

Marine plants control the accumulation of biofouling organisms (epibionts) on their surfaces by various chemical and physical means. Ascophyllum nodosum is a perennial multicellular brown alga known to shed patches of epidermal material, thus removing epibionts and exposing unfouled surfaces to another cycle of colonization. While surface shedding is documented in multiple marine macroalgae, the cell and developmental biology of the phenomenon is almost unexplored. A previous investigation of Ascophyllum not only revealed regular cycles of epibiont accumulation and epidermal shedding but also stimulated the development of methods to detect the corresponding changes in epidermal (meristoderm) cells that are reported here. Confocal laser scanning microscopy of cell walls and cytoplasm fluorescently stained with Solophenyl Flavine 7GFE (Direct Yellow 96) and the lipophilic dye Rhodamine B (respectively) was combined with light and electron microscopy of chemically fixed or freeze-substituted tissues. As epibionts accumulated, epidermal cells generated thick, apical cell walls in which differentially stained central layers subsequently developed, marking the site of future cell wall separation. During cell wall separation, the outermost part of the cell wall and its epibionts plus the upper parts of the anticlinal walls between neighboring cells detached in a layer from multiple epidermal cells, exposing the remaining inner part of the cell wall to new colonizing organisms. These findings highlight the dynamic nature of apical cell wall structure and composition in response to colonizing organisms and lay a foundation for further investigations on the periodic removal of biofouling epibionts from the surface of Ascophyllum fronds.

Long non­coding RNA HOTAIR promotes burn wound healing by regulating epidermal stem cells.

Local transplantation of epidermal stem cells (ESCs) exerts a therapeutic effect on burn wounds. However, cell viability can impede their clinical application. HOX antisense intergenic RNA (HOTAIR) is involved in regulating adult tissue stem cells, as well as in developmental patterning and pluripotency. However, little is known about its role in regulating ESCs. The present study was performed to investigate the effects of HOTAIR in the modulation of ESCs and wound repair. Firstly, reverse transcription­quantitative PCR was used to detect the relative expression of HOTAIR during burn wound healing in mice to determine whether HOTAIR is associated with wound healing. Subsequently, ESCs derived from mouse skin were transfected with a lentiviral vector to overexpress or knockdown HOTAIR. The effects of HOTAIR on cell proliferation and differentiation were measured by 5­bromodeoxyuridine and MTT assays, and by assessing NANOG mRNA expression. Lastly, mice with burns were administered a subcutaneous injection of HOTAIR­overexpressing ESCs. Images were captured and histological analyses were performed to evaluate wound healing. The results revealed that the expression of HOTAIR gradually increased and peaked at day 7 post­burn and maintained at relatively high levels until day 14 post­burn during wound healing. Furthermore, overexpression of HOTAIR promoted ESC proliferation and maintained the stem cell state in vitro. By contrast, suppression of HOTAIR inhibited cell proliferation and cell stemness. It was also identified that HOTIR­overexpressing ESCs accelerated re­epithelialization and facilitated burn wound repair. In conclusion, the present findings confirmed an essential role of HOTAIR in the regulation of ESC proliferation and stemness. Therefore, targeting HOTAIR in ESCs may be a potentially promising therapy for burn wound healing.

Stomatal traits as a determinant of superior salinity tolerance in wild barley.

Wild barley Hordeum spontaneum (WB) is the progenitor of a cultivated barley Hordeum vulgare (CB). Understanding efficient mechanisms evolved by WB to cope with abiotic stresses may open prospects of transferring these promising traits to the high yielding CB genotypes. This study aimed to investigate the strategies that WB plants utilise in regard to the control of stomatal operation and ionic homeostasis to deal with salinity stress, one of the major threats to the global food security. Twenty-six genotypes of WB and CB were grown under glasshouse conditions and exposed to 300 mM NaCl salinity treatment for 5 weeks followed by their comprehensive physiological assessment. WB had higher relative biomass than CB when exposed to salinity stress. Under saline conditions, WB plants were able to keep constant stomatal density (SD) while SD significantly decreased in CB. The higher SD in WB also resulted in a higher stomatal conductance (gs) under saline conditions, with gs reduction being 51% and 72% in WB and CB, respectively. Furthermore, WB showed faster stomatal response to light, indicating their better ability to adapt to changing environmental conditions. Experiments with isolated epidermal strips indicated that CB genotypes have the higher stomatal aperture when incubated in 80 mM KCl solution, and its aperture declined when KCl was substituted by NaCl. On the contrary, WB genotype had the highest stomatal aperture being exposed to 80 mM NaCl suggesting that WB plants may use Na+ instead of K+ for stomata movements. Overall, our data suggest that CB employ a stress-escaping strategy by reducing stomata density, to conserve water, when grown under salinity conditions. WB, on a contrary, is capable of maintaining relatively constant stomata density, faster stomatal movement and higher gs under saline conditions.

Evaluation of the DNA damage in lymphocytes, sperm and buccal cells of workers under environmental and occupational boron exposure conditions.

Industrial production and use of boron compounds have increased during the last decades, especially for the manufacture of borosilicate glass, fiberglass, metal alloys and flame retardants. This study was conducted in two districts of Balikesir; Bandirma and Bigadic, which geographically belong to the Marmara Region of Turkey. Bandirma is the production and exportation zone for the produced boric acid and some borates and Bigadic has the largest B deposits in Turkey. 102 male workers who were occupationally exposed to boron from Bandirma and 110 workers who were occupationally and environmentally exposed to boron from Bigadic participated to our study. In this study the DNA damage in the sperm, blood and buccal cells of 212 males was evaluated by comet and micronucleus assays. No significant increase in the DNA damage in blood, sperm and buccal cells was observed in the residents exposed to boron both occupationally and environmentally (p = 0.861) for Comet test in the sperm samples, p = 0.116 for Comet test in the lymphocyte samples, p = 0.042 for micronucleus (MN) test, p = 0.955 for binucleated cells (BN), p = 1.486 for condensed chromatin (CC), p = 0.455 for karyorrhectic cells (KHC), p = 0.541 for karyolitic cells (KLY), p = 1.057 for pyknotic cells (PHC), p = 0.331 for nuclear bud (NBUD)). No correlations were seen between blood boron levels and tail intensity values of the sperm samples, lymphocyte samples, frequencies of MN, BN, KHC, KYL, PHC and NBUD. The results of this study came to the same conclusions of the previous studies that boron does not induce DNA damage even under extreme exposure conditions.

Sensitive and Selective Amino Acid Profiling of Minute Tissue Amounts by HPLC/Electrospray Negative Tandem Mass Spectrometry Using 9-Fluorenylmethoxycarbonyl (Fmoc-Cl) Derivatization.

A method for selective and sensitive quantification of amino acids is described. The combination of established derivatization procedures of secondary and primary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) and subsequent detection of derivatized amino acids by LC-ESI-MS/MS using multiple reaction monitoring provides high selectivity. The attachment of an apolar moiety enables purification of derivatized amino acids from matrix by a single solid-phase extraction step, which increases sensitivity by reduced ion suppression during LC-ESI-MS/MS detection. Additionally, chromatography of all amino acids can be performed on reversed-phase HPLC columns using eluents without additives, which are known to cause significant decreases in signal to noise ratios. The method has been routinely applied for amino acid profiling of low amounts of liquids and tissues of various origins with a sample throughput of about 50-100 samples a day. In addition to a detailed description of the method, some representative examples are presented.

pH in nature, humans and skin.

The pH plays an important physiological role in nature and humans. pH varies from 1 to 8 in human organs with tight regulation in blood and epithelia of barrier organs. The physiological pH of the stratum corneum is 4.1-5.8 and several mechanisms contribute to its formation: filaggrin degradation, fatty acid content, sodium-hydrogen exchanger (NHE1) activation and melanosome release. First, the acidic pH of the stratum corneum was considered to present an antimicrobial barrier preventing colonization (e.g. by Staphylococcus aureus and Malassezia). Later on, it was found that the pH influences skin barrier function, lipid synthesis and aggregation, epidermal differentiation and desquamation. Enzymes of ceramide metabolism (e.g. ß-glucocerebrosidase or acid sphingomyelinase) as well as proteases (e.g. chymotryptic enzyme or cathepsin D linked to epidermal differentiation and desquamation) are regulated by the pH. Experimental disruption of the physical barrier leads to an increase of pH, returning to normal levels only after many hours. Inflammatory skin diseases and diseases with an involvement of the epidermis exhibit a disturbed skin barrier and an increased pH. This is known for atopic dermatitis, irritant contact dermatitis, ichthyosis, rosacea and acne, but also for aged and dry skin. Normalizing the pH by acidification through topical treatment helps to establish a physiological microbiota, to repair skin barrier, to induce epidermal differentiation and to reduce inflammation.

Changes in nano-mechanical properties of human epidermal cornified cells depending on their proximity to the skin surface.

During formation of the stratum corneum (SC) barrier, terminally differentiated keratinocytes continue their maturation process within the dead superficial epidermal layer. Morphological studies of isolated human corneocytes have revealed differences between cornified envelopes purified from the deep and superficial SC. We used atomic force microscopy to measure the mechanical properties of native human corneocytes harvested by tape-stripping from different SC depths. Various conditions of data acquisition have been tested and optimized, in order to obtain exploitable and reproducible results. Probing at 200 nN allowed us to investigate the total stiffness of the cells (at 50 nm indentation) and that of the cornified envelopes (at 10 to15 nm), and lipid envelopes (at 5 to 10 nm). The obtained data indicated statistically significant differences between the superficial (more rigid) and deep (softer) corneocytes, thus confirming the existence of depth and maturation-related morphological changes within the SC. The proposed approach can be potentially used for minimally invasive evaluation of various skin conditions such as aging, skin hydration, and pathologies linked to SC.