A novel egg-shell membrane based hybrid nanofibrous scaffold for cutaneous tissue engineering

Abstract Background The main issue in cutaneous regeneration is to develop engineered scaffolds based on natural extracellular matrix to promote dynamics of skin progenitor cells and accelerate differentiation into mature keratinocytes. Methods In this study, nanofibrous scaffolds composed of a blend poly (ɛ-caprolactone) (PCL), silk fibroin (SF), soluble eggshell membrane (SESM), and Aloe vera (AV) gel were developed by electrospinning method and human basal cells were used to examine differentiation capacity toward keratinocyte-like cells. For this propose, cells were allocated to four distinct groups; control, PCL/SF, PCL/SF/SESM, and PCL/SF/SESM/AV. In all groups, cells were incubated with differentiation medium. Morphology, composition, hydrophilicity and mechanical features of PCL/SF, PCL/SF/SESM and PCL/SF/SESM/AV nanofibers were studied by scanning electron microscopy (SEM), Fourier transforms infrared spectroscopy (FT-IR), water contact angle and tensile tests. To examine the orientation of basal cells to mature keratinocytes, we performed immunofluorescence analysis by monitoring cytokeratin-19. The expression of genes such as involucrin, keratin-14 and -5 was monitored by real-time PCR assay. Results PCL/SF, PCL/SF/SESM, and PCL/SF/SESM/AV had suitable physic chemical indices and biological activities to be applied as biomimetic scaffolds for the restoration cutaneous tissue. Compared to control, we found an increased basal cell proliferation at 7 and 14 days after plating on scaffolds and reach maximum levels in group PCL/SF/SESM/AV on day 14 (p < 0.05). Electron microscopy showed cell flattening, morphological adaptation. An integrated cell-to-cell connection was generated after cell seeding on scaffolds in all groups. Immunofluorescence imaging showed the ability of basal cells to synthesize cytokeratin-19 in PCL/SF, PCL/SF/SESM, and positive control cells after exposure to differentiation medium. However, these values were less in PCL/SF/SESM/AV compared to other groups. Real-time PCR analysis showed the potency of all scaffolds to induce the transcription of involucrin, keratin-14 and -5, especially involucrin in PCL/SF/SESM/AV group compared to the negative control. Conclusion Modulation of scaffolds with natural biopolymers could enable us to synthesize structures appropriate for cutaneous regeneration.

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Signal Pathway in Salt-Activated Expression of the Salmonella Pathogenicity Island 1 Type III Secretion System in Salmonella enterica Serovar Typhimurium

Journal of Bacteriology ◽

10.1128/jb.01957-07 ◽

2008 ◽

Vol 190 (13) ◽

pp. 4624-4631 ◽

Cited By ~ 35

Author(s):

Hideaki Mizusaki ◽

Akiko Takaya ◽

Tomoko Yamamoto ◽

Shin-Ichi Aizawa

Keyword(s):

Electron Microscopy ◽

Real Time ◽

Protein Secretion ◽

Real Time Pcr ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Secretion System ◽

Sds Page ◽

Two Component ◽

Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium secretes virulence factors for invasion called Sip proteins or Sips into its hosts through a type III secretion system (T3SS). In the absence of a host, S. enterica induces Sip secretion in response to sucrose or simple salts, such as NaCl. We analyzed induction of host-independent Sip secretion by monitoring protein secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), assembly of needle complexes by electron microscopy, and transcription of virulence regulatory genes by quantitative reverse transcriptase PCR (real-time PCR). SDS-PAGE showed that addition of sucrose or simple salts, such as NaCl, to the growth medium induced Sip secretion without altering flagellar protein secretion, which requires a distinct T3SS. Electron microscopy confirmed that the amount of secreted Sips increased as the number of assembled needle complexes increased. Real-time PCR revealed that added sucrose or NaCl enhanced transcription of hilA, hilC, and hilD, which encode known regulators of Salmonella virulence. However, epistasis analysis implicated HilD and HilA, but not HilC, in the direct pathway from the salt stimulus to the Sip secretion response. Further analyses showed that the BarA/SirA two-component signal transduction pathway, but not the two-component sensor kinase EnvZ, directly activated hilD and hilA transcription and thus Sip secretion in response to either sucrose or NaCl. Finally, real-time PCR showed that salt does not influence transcription of the BarA/SirA-dependent csrB and csrC genes. A model is proposed for the major pathway in which sucrose or salt signals to enhance virulence gene expression.

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Detection of Human Polyomaviruses in Urine from Bone Marrow Transplant Patients: Comparison of Electron Microscopy with PCR

Clinical Chemistry ◽

10.1373/clinchem.2003.024539 ◽

2004 ◽

Vol 50 (2) ◽

pp. 306-312 ◽

Cited By ~ 24

Author(s):

Stefan S Biel ◽

Andreas Nitsche ◽

Andreas Kurth ◽

Wolfgang Siegert ◽

Muhsin Özel ◽

...

Keyword(s):

Electron Microscopy ◽

Bone Marrow ◽

Real Time ◽

Bone Marrow Transplant ◽

Real Time Pcr ◽

Marrow Transplant ◽

Test System ◽

Urine Samples ◽

Clinical Samples ◽

Transplant Patients

Abstract Background: We studied electron microscopy (EM) as an appropriate test system for the detection of polyomavirus in urine samples from bone marrow transplant patients. Methods: We evaluated direct EM, ultracentrifugation (UC) before EM, and solid-phase immuno-EM (SPIEM). The diagnostic accuracy of EM was studied by comparison with a real-time PCR assay on 531 clinical samples. Results: The detection rate of EM was increased by UC and SPIEM. On 531 clinical urine samples, the diagnostic sensitivity of EM was 47% (70 of 149) with a specificity of 100%. We observed a linear relationship between viral genome concentration and the proportion of urine samples positive by EM, with a 50% probability for a positive EM result for urine samples with a polyomavirus concentration of 106 genome-equivalents (GE)/mL; the probability of a positive EM result was 0% for urine samples with <103 GE/mL and 100% for urine samples containing 109 GE/mL. Conclusions: UC/EM is rapid and highly specific for polyomavirus in urine. Unlike real-time PCR, EM has low sensitivity and cannot quantify the viral load.

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Creation of a bovine herpes virus 1 (BoHV-1) quantitative particle standard by transmission electron microscopy and comparison with established standards for use in real-time PCR

Biologicals ◽

10.1016/j.biologicals.2017.03.007 ◽

2017 ◽

Vol 48 ◽

pp. 121-125

Author(s):

Marc Hoferer ◽

Anne Braun ◽

Reinhard Sting

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Real Time ◽

Real Time Pcr ◽

Herpes Virus ◽

Transmission Electron ◽

Bovine Herpes Virus 1 ◽

Herpès Virus

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Life cycle ofRickettsia slovacain L929 cell line studied by quantitative real-time PCR and transmission electron microscopy

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.2009.01510.x ◽

2009 ◽

Vol 293 (1) ◽

pp. 102-106 ◽

Cited By ~ 6

Author(s):

Vojtech BoldiÅ¡ ◽

Jasna Å trus ◽

Elena KocianovÃ¡ ◽

Magda TuÅ¡ek-Å½nidariÄ ◽

KatarÃna Å tefanidesovÃ¡ ◽

...

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Life Cycle ◽

Cell Line ◽

Real Time ◽

Real Time Pcr ◽

L929 Cell ◽

Quantitative Real Time Pcr ◽

Transmission Electron ◽

L929 Cell Line

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Blood cell adhesion to arterial filters analysis by scanning electron microscopy and real-time PCR assay: observational clinical study in cardiac surgery patients

Perfusion ◽

10.1177/0267659120986525 ◽

2021 ◽

pp. 026765912098652

Author(s):

Chiara Scaglioni Tessmer Gatto ◽

Marilde Albuquerque Piccioni ◽

Celia Maria Cassaro Strunz ◽

Idágene Aparecida Cestari ◽

Ligia Cristina Camara Cunha ◽

...

Keyword(s):

Gene Expression ◽

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Cardiac Surgery ◽

Cardiopulmonary Bypass ◽

Real Time ◽

Real Time Pcr ◽

Pcr Assay ◽

Blood Elements ◽

Scanning Electron

Introduction: Arterial filter is the part of the cardiopulmonary bypass circuit where blood cells are exposed to high mechanical stress and where cellular aggregates may fasten in large quantities. The aim of this study was to analyse blood cell adhesiveness in the arterial filter through scanning electron microscopy and real-time PCR assay. Methods: Prospective, clinical and observational study performed on 28 patients undergoing cardiac surgery with cardiopulmonary bypass. Arterial filters were analysed by scanning electron microscopy. Real-time PCR assay was performed in extracted material from the arterial filters for analysis of platelet GPIb and CD45 leucocyte gene expression. Blood coagulation was analysed during cardiopulmonary bypass. Patients were followed until hospital discharge or 28 days after surgery. Results: All studied arterial filters used in the subject patients showed a degree of adhesion from blood elements at scanning electron microscopy. All studied filters were positive for platelets GPIb gene expression and 15% had CD45 leucocyte gene expression. The GPIb platelet gene expression in blood lowered at the end of cardiopulmonary bypass ( p = 0.019). There was negative correlation between blood GPIb platelet gene expression and Clot SR (HEPSCREEN2 ReoRox®) (rho = 0.635; p = 0.027). The filter fields count was correlated to the D-dimer dosage (rho = 0.828; p < 0.001). Conclusion: There was adhesion of blood elements, especially nucleated platelets, on all arterial filters studied. Although the arterial filter worked as a safety device, that possibly prevented arterial embolisation, it may also have caused greater hyperfibrinolysis during cardiopulmonary bypass.

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Habitat visualization, acquisition features and necessity of the gammaproteobacterial symbiont of pistachio stink Bug, Acrosternum heegeri (Hem.: Pentatomidae)

Bulletin of Entomological Research ◽

10.1017/s0007485319000245 ◽

2019 ◽

Vol 110 (1) ◽

pp. 22-33 ◽

Cited By ~ 4

Author(s):

M. Kashkouli ◽

Y. Fathipour ◽

M. Mehrabadi

Keyword(s):

Electron Microscopy ◽

Real Time ◽

Real Time Pcr ◽

Survival Rates ◽

Adult Survival ◽

Bacterial Cells ◽

Stink Bug ◽

Surface Sterilization ◽

Posterior Midgut ◽

Molecular Phylogenetic

AbstractPlant-sucking stinkbugs are especially associated with mutualistic gut bacterial symbionts. Here, we explored the symbiotic relationship of a pistachio stinkbug, Acrosternum heegeri Fieber by histological, fluorescence in situ hybridization (FISH), real-time PCR and molecular phylogenetic techniques. Furthermore, the effects of the symbiont on the resting/wandering behaviors of the newborn nymphs, pre-adult survival rates, and stage compositions were investigated. Transmission electron microscopy and real-time PCR analyses showed that a rod-shaped gammaproteobacterium was persistently located within the posterior midgut crypts. Molecular phylogenetic and FISH techniques strongly suggested that this symbiont should be placed in the genus Pantoea of the Enterobacteriales. Scanning electron microscopy confirmed the presence of the bacterial cells on the egg surface which the surface sterilization of the eggs resulted in the successful removal of the symbiont from the eggs. Symbiotic and aposymbiotic A. heegeri showed no significant differences in the wandering behaviors of the first nymphal stages, while the symbiont-free insects suffered retarded growth and lower survivability. Together, the results highlight the habitat and acquisition features of Pantoea symbiont and its contribution in A. heegeri biology that might help us for better pest management in the future.

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Creating standards for absolute quantification of Coxiella burnetii in real-time PCR – A comparative study based on transmission electron microscopy

Biologicals ◽

10.1016/j.biologicals.2014.10.006 ◽

2015 ◽

Vol 43 (1) ◽

pp. 18-22 ◽

Cited By ~ 1

Author(s):

Reinhard Sting ◽

Kerstin Molz ◽

Marc Hoferer

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Comparative Study ◽

Real Time ◽

Coxiella Burnetii ◽

Real Time Pcr ◽

Absolute Quantification ◽

Transmission Electron

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1,25-Dihydroxyvitamin D3 Attenuates Angiotensin II-Induced Renal Injury by Inhibiting Mitochondrial Dysfunction and Autophagy

Cellular Physiology and Biochemistry ◽

10.1159/000495678 ◽

2018 ◽

Vol 51 (4) ◽

pp. 1751-1762 ◽

Cited By ~ 8

Author(s):

Qiqi Shen ◽

Xiao Bi ◽

Lilu Ling ◽

Wei Ding

Keyword(s):

Electron Microscopy ◽

Western Blot ◽

Mitochondrial Dysfunction ◽

Real Time ◽

Real Time Pcr ◽

Renal Injury ◽

Control Group ◽

Ang Ii ◽

Dihydroxyvitamin D3 ◽

1,25 Dihydroxyvitamin D3

Background/Aims: A recent study has shown that 1,25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D, can ameliorate renal dysfunction. In this study, we aimed to determine the role of 1,25-D3 in angiotensin (Ang II)-induced renal injury and investigate the underlying mechanisms involved. Methods: C57BL/6J mice were treated with Ang II and/or 1,25-D3 (or saline as the control) for 2 weeks. Renal injury was evaluated using transmission electron microscopy and periodic acid-Schiff reagent and Masson’s trichrome staining. The pro-fibrotic and pro-inflammatory factors were assessed using real-time PCR. The renal apoptotic pathway was evaluated with TUNEL staining and western blot. Mitochondrial dysfunction (MtD) was determined using real-time PCR and electron microscopy. The activation of autophagy was detected using western blot. Results: In the Ang II-infused mice, expanded mesangial regions, tubulointerstitial fibrosis, and foot process fusion were observed; the levels of the pro-fibrotic and pro-inflammatory cytokines and MtD were also increased when compared with the control group. However, we found that administration of 1,25-D3 significantly improved renal function and MtD and reduced the pro-fibrotic and pro-inflammatory cytokine levels. Furthermore, 1,25-D3 significantly inhibited Ang II-induced autophagy dysfunction (determined by inhibition of Beclin-1 activation and reduction of the LC3-II/LC3-I ratio). Conclusion: Our findings suggest that 1,25-D3 may attenuate Ang II-induced renal injury by improving MtD and modulating autophagy. 1,25-D3 may be a new therapeutic for the treatment of CKD.

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