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We have generated MLi005-A, a new induced pluripotent stem cell (iPSC) line derived from skin fibroblasts of a male patient with dominant dystrophic epidermolysis bullosa (DDEB). This iPSC line may be used as a model system for studies on skin integrity, the extracellular matrix and skin barrier function. The characterization of the MLi005-A cell line consisted of molecular karyotyping, next-generation sequencing of the COL7A1 alleles, pluripotency and differentiation potentials testing by immunofluorescence of associated markers in vitro. The MLi-005A line has been also tested for ability to differentiate into fibroblasts and keratinocytes and markers associated with these cell types.

We have generated an induced pluripotent stem cell (iPSC) line KCLi003-A (iOP101) from epidermal keratinocytes of a female donor, heterozygous for the loss-of-function mutation p.R501X in the filaggrin gene (FLG), using non-integrating Sendai virus vectors. Derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization and validation of KCLi003-A line included molecular karyotyping, mutation screening using restriction enzyme digestion, next generation sequencing (NGS), while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.

Aim: To determine the effect of nuclear opalescence (NO) on intraoperative parameters during phacoemulsification using the WhiteStar Signature® PRO and to show the impact of preoperative and intraoperative parameters on postoperative corneal edema. Methods: This prospective study included 267 patients selected to undergo phacoemulsification using the WhiteStar Signature® PRO system at the Department of Ophthalmology of the General Hospital “Dr Josip Benčević”, Slavonski Brod, Croatia. NO was graded using the Lens Opacities Classification System III. Preoperative parameters were age, sex, NO and preoperative central corneal thickness. Intraoperative parameters of phacoemulsification included in the study were ultrasound time (UST), phaco time using Ellips FX technology (EFX) and average phaco power (AVG). Patients were followed up on postoperative days 1 and 7 and after two months. The state of the cornea was noted in each follow-up. Results: There was a statistically significant increase of intraoperative parameters with NO. Postoperative corneal edema depended on all measured intraoperative parameters (UST, EFX and AVG, all p < .001), patient’s age (p < .05) and NO (p < .001) on postoperative day 1, while on postoperative day 7, it depended on UST (p = .011) EFX (p = .012) and NO (p < .05). Conclusion: Older patients, higher grade of NO and amount of energy consumed during phacoemulsification using the WhiteStar Signature® PRO are predictive factors for severity of transient corneal edema. We found this information important for better preoperative planning of phacoemulsification, as well as for better postoperative results.

Bulozna epidermoliza (EB) je dedna bolezen krhkosti kože, povezana z mutacijami 18 različnih genov, ki se izražajo v koži. Zaradi raznovrstnosti simptomov se razvršča v 4 osnovne tipe s 30 različnimi podtipi. Osnovni tipi EB so razdeljeni glede na plast kože, ki je z mutacijo prizadeta: EB simpleks, ki je vezana na epidermis, junkcijska EB, ki je vezana na bazalno membrano, ter distrofična EB, ki je vezana na dermis. Poznamo še Kindlerjev sindrom, ki je izredno redka oblika te bolezni. Pri vseh primerih je koža bolnikov zelo krhka, zato so osnovni simptomi mehurji in odprte rane, ki se težko celijo. Bolezen prizadene bolnike telesno in duševno, kronični vnetni procesi pa pogosto povzročijo nastanek agresivnih oblik ploščatoceličnega karcinoma kože. Gre za skupino redkih bolezni z incidenco okoli 1 : 40.000. V Sloveniji imamo 60 bolnikov z EB. V članku predstavljamo trenutno uveljavljen način oskrbe kroničnih ran bolnikov z EB in pregled najnovejših in najsodobnejših genetskih, regenerativnih in farmakoloških pristopov za razvoj načina zdravljenja te še vedno neozdravljive bolezni.

We have generated MLi004-A, a new induced pluripotent stem cell (iPSC) line derived from skin fibroblasts of a female patient with recessive dystrophic epidermolysis bullosa (RDEB). This iPSC line may be used as a model system for studies on skin integrity, the extracellular matrix and skin barrier function. The characterization of the MLi004-A cell line consisted of molecular karyotyping, next-generation sequencing of the COL7A1 alleles, pluripotency and differentiation potentials testing by immunofluorescence of associated markers in vitro. The MLi-004A line has been also tested for ability to differentiate into fibroblasts.

ABSTRACT: We have generated MLi003-A, a new induced pluripotent stem cell (iPSC) line derived from hair follicle keratinocytes of a healthy male characterized with a maximum number of filaggrin tandem repeats, making this iPSC line the best control for studies on skin barrier function. The characterization of the MLi003-A cell line consisted of molecular karyotyping, high-throughput array-based sequencing composed of Fluidigm microfluidics technology and next-generation sequencing of the filaggrin alleles, and pluripotency and differentiation potentials testing by immunofluorescence of associated markers both in vitro and in vivo. The MLi-003A line has been also tested for ability to differentiate into keratinocytes.

Keratin intermediate filaments are the principal structural element of epithelial cells. Their importance in providing bulk cellular stiffness is well recognized, but their role in the mechanics of cell cortex is less understood. In this study, we therefore compared the cortical stiffness of three keratinocyte lines: primary wild type cells (NHEK2), immortalized wild type cells (NEB1) and immortalized mutant cells (KEB7). The cortical stiffness was measured by lateral indentation of cells with AOD-steered optical tweezers without employing any moving mechanical elements. The method was validated on fixed cells and Cytochalasin-D treated cells to ensure that the observed variations in stiffness within a single cell line were not a consequence of low measurement precision. The measurements of the cortical stiffness showed that primary wild type cells were significantly stiffer than immortalized wild type cells, which was also detected in previous studies of bulk elasticity. In addition, a small difference between the mutant and the wild type cells was detected, showing that mutation of keratin impacts also the cell cortex. Thus, our results indicate that the role of keratins in cortical stiffness is not negligible and call for further investigation of the mechanical interactions between keratins and elements of the cell cortex.

We have generated an induced pluripotent stem cell (iPSC) line KCLi003-A (iOP101) from epidermal keratinocytes of a female donor, heterozygous for the loss-of-function mutation p.R501X in the filaggrin gene (FLG), using non-integrating Sendai virus vectors. Derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization and validation of KCLi003-A line included molecular karyotyping, mutation screening using restriction enzyme digestion, next generation sequencing (NGS), while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.

We have generated MLi002-A, a new induced pluripotent stem cell (iPSC) line derived from keratinocytes of a skin punch biopsy of a female patient with the severe epidermolysis bullosa simplex Dowling-Meara phenotype and the keratin K5 E475G mutation. Keratinocytes were reprogrammed using non-integrating Sendai virus vectors, and xeno-free culture conditions were used throughout. The characterization of MLi002-A cell line consisted of molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, and testing of the pluripotency and differentiation potentials by immunofluorescence of associated markers both in vitro and in vivo. This is the first iPSC model of EB Simplex.

Keratin 8 and 18 (K8/K18) mutations have been implicated in the aetiology of certain pathogenic processes of the liver and pancreas. While some K8 mutations (K8 G62C, K8 K464N) are also presumed susceptibility factors for inflammatory bowel disease (IBD), the only K18 mutation (K18 S230T) discovered so far in an IBD patient is thought to be a polymorphism. The aim of our study was to demonstrate that these mutations might also directly affect intestinal cell barrier function. Cell monolayers of genetically engineered human colonocytes expressing these mutations were tested for permeability, growth rate and resistance to heat-stress. We also calculated the change in dissociation constant (Kd, measure of affinity) each of these mutations introduces into the keratin protein, and present the first model of a keratin dimer L12 region with in silico clues to how the K18 S230T mutation may affect keratin function. Physiologically, these mutations cause up to 30% increase in paracellular permeability in vitro. Heat-stress induces little keratin clumping but instead cell monolayers peel off the surface suggesting a problem with cell junctions. K18 S230T has pronounced pathological effects in vitro marked by high Kd, low growth rate and increased permeability. The latter may be due to the altered distribution of tight junction components claudin-4 and ZO-1. This is the first time intestinal cells have been suggested also functionally impaired by K8/K18 mutations. Although an in vitro colonocyte model system does not completely mimic the epithelial lining of the intestine, nevertheless the data suggest that K8/K18 mutations may be also able to produce a phenotype in vivo.

• First fully characterised iPSC line derived from a dominant dystrophic EB patient (DDEB) • Mli005-A carries the most common recurrent autosomal dominant COL7A1 mutation in cases of DDEB. • Mli005-A is a powerful tool for studies on DDEB disease mechanisms, skin barrier function and drug screening and therapy development. We have generated MLi005-A, a new induced pluripotent stem cell (iPSC) line derived from skin fibroblasts of a male patient with dominant dystrophic epidermolysis bullosa (DDEB). This iPSC line may be used as a model system for studies on skin integrity, the extracellular matrix and skin barrier function. The characterization of the MLi005-A cell line consisted of molecular karyotyping, next-generation sequencing of the COL7A1 alleles, pluripotency and differentiation potentials testing by immunofluorescence of associated markers in vitro. The MLi-005A line has been also tested for ability to differentiate into fibroblasts and keratinocytes and markers associated with these cell types. [ABSTRACT FROM AUTHOR]

Abstract Background We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments. Results K14 IBs were electroporated into SW13 cells grown in culture together with a “reporter” plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5. Conclusions Soluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells.

We have generated MLi004-A, a new induced pluripotent stem cell (iPSC) line derived from skin fibroblasts of a female patient with recessive dystrophic epidermolysis bullosa (RDEB). This iPSC line may be used as a model system for studies on skin integrity, the extracellular matrix and skin barrier function. The characterization of the MLi004-A cell line consisted of molecular karyotyping, next-generation sequencing of the COL7A1 alleles, pluripotency and differentiation potentials testing by immunofluorescence of associated markers in vitro. The MLi-004A line has been also tested for ability to differentiate into fibroblasts. [ABSTRACT FROM AUTHOR]

We have generated MLi003-A, a new induced pluripotent stem cell (iPSC) line derived from hair follicle keratinocytes of a healthy male characterized with a maximum number of filaggrin tandem repeats, making this iPSC line the best control for studies on skin barrier function. The characterization of the MLi003-A cell line consisted of molecular karyotyping, high-throughput array-based sequencing composed of Fluidigm microfluidics technology and next-generation sequencing of the filaggrin alleles, and pluripotency and differentiation potentials testing by immunofluorescence of associated markers both in vitro and in vivo. The MLi-003A line has been also tested for ability to differentiate into keratinocytes. [ABSTRACT FROM AUTHOR]

We have generated MLi002-A, a new induced pluripotent stem cell (iPSC) line derived from keratinocytes of a skin punch biopsy of a female patient with the severe epidermolysis bullosa simplex Dowling-Meara phenotype and the keratin K5 E475G mutation. Keratinocytes were reprogrammed using non-integrating Sendai virus vectors, and xeno-free culture conditions were used throughout. The characterization of MLi002-A cell line consisted of molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, and testing of the pluripotency and differentiation potentials by immunofluorescence of associated markers both in vitro and in vivo. This is the first iPSC model of EB Simplex. [ABSTRACT FROM AUTHOR]

An introduction is presented in which the author discusses various reports on topics including breadth and depth of the application of Computational Fluid Dynamics (CFD), multiphase and reactive flow modeling, and industrial problems in the minerals and process industries.

A novel concept for rib fixation is presented that involves the use of a bioresorbable polymer intramedullary telescoping splint. Bone cement is used to anchor each end of the splint inside the medullary canal on each side of the fracture site. In this manner, rib fixation is achieved without fixation device protrusion from the rib, making the splint completely intramedullary. Finite element analysis is used to demonstrate that such a splint/cement composite can preserve rib fixation subjected to cough-intensity force loadings. Computational fluid dynamics and porcine rib experiments were used to study the anchor formation process required to complete the fixation.

Background: Surgical management of fractured ribs with internal fixation is an increasingly accepted therapy. Concurrently, specific rib fixation prostheses are being developed which should improve results and minimise hardware and rib/splint construct failures. The Synthes titanium intramedullary splint lends itself to difficult to access areas such as posterior rib fractures and fractures under the scapula. We analyse a case series of patients in whom this rib fixation prosthesis has been used., Methods: Fifteen patients received 35 intramedullary splints. Follow up at 3 and 6 months was performed with three dimensional computed tomography scanning to assess for bone alignment, callus formation and healing, residual deformity, hardware failure or cut through. Computerized finite element analysis (FEA) was used to model forces acting on a posterior fracture with and without an intramedullary fixation splint in situ., Results: Complete healing (bony union) was noted in only 3 (9 %) of the fractures fixed with splints by 3 months. Partial healing (cartilaginous union) was noted in 28 of the 33 fractures (85 %), and non healing was noted in only 2 (6 %). In both those two patients, failure at the rib / splint interface was noted after both patients reported sneezing. No hardware failures were noted. By 6 months the fractures which had shown partial healing, had all completely healed. There were no late failures (between 3 and 6 months) of either hardware or rib/splint interfaces. FEA modelling identified sites of increased stress in the rib at the rib / splint interface and in a modelled intramedullary splint where it spans the fracture., Conclusions: Further analysis of outcomes with intramedullary splints is warranted as well as further development of intramedullary rib fixation solutions.

Introduction: Recent studies in the literature have highlighted the critical role played by cell signalling in determining haemopoietic stem cell (HSC) fate within ex vivo culture systems. Stimulatory signals can enhance proliferation and promote differentiation, whilst inhibitory signals can significantly limit culture output., Methods: Numerical models of various mitigation strategies are presented and applied to determine effectiveness of these strategies toward mitigation of paracrine inhibitory signalling inherent in these culture systems. The strategies assessed include mixing, media-exchange, fed-batch and perfusion., Results: The models predict that significant spatial concentration gradients exist in typical cell cultures, with important consequences for subsequent cell expansion. Media exchange is shown to be the most effective mitigation strategy, but remains labour intensive and difficult to scale-up for large culture systems. The fed-batch strategy is only effective at very small Peclet number, and its effect is diminished as the cell culture volume grows. Conversely, mixing is effective at high Peclet number, and ineffective at low Peclet number. The models predict that cell expansion in fed-batch cultures becomes independent of increasing dilution rate, consistent with experimental results previously reported in the literature. In contrast, the models predict that increasing the flow rate in perfused cultures will lead to increased cell expansion, indicating the suitability of perfusion for use as an automated, tunable strategy. The effect of initial cell seeding density is also investigated, with the model showing that perfusion outperforms dilution for all densities considered., Conclusions: The models predict that the impact of inhibitory signalling in HSC cultures can be mitigated against using media manipulation strategies, with the optimal strategy dependent upon the protein diffusion time-scale relative to the media manipulation time-scale. The key messages from this study can be applied to any complex cell culture scenario where cell-cell interactions and paracrine signalling networks impact upon cell fate and cell expansion.

The stress on an intramedullary screw rib fixation device holding together a centrally fractured human rib under in vivo force loadings was studied using finite element analysis (FEA). Validation of the FEA modelling using pullout from porcine ribs proved FEA to be suitable for assessing the structural integrity of screw/bone systems such as rib fixated by a screw. In the human rib fixation investigation, it was found that intramedullary bioresorbable Bioretec screws can fixate centrally fractured human ribs under normal breathing conditions. However, under coughing conditions, simulation showed Bioretec fixating screws to bend substantially. High stresses in the screw are mainly the result of flexion induced by the force loading, and are restricted to thin regions on the outside of the screw shaft. Stiffer screws result in less locally intense stress concentrations in bone, indicating that bone failure in the bone/screw contact regions can be averted with improvements in screw stiffness.

Cavitation microstreaming plays a role in the therapeutic action of microbubbles driven by ultrasound, such as the sonoporative and sonothrombolytic phenomena. Microscopic particle-image velocimetry experiments are presented. Results show that many different microstreaming patterns are possible around a microbubble when it is on a surface, albeit for microbubbles much larger than used in clinical practice. Each pattern is associated with a particular oscillation mode of the bubble, and changing between patterns is achieved by changing the sound frequency. Each microstreaming pattern also generates different shear stress and stretch/compression distributions in the vicinity of a bubble on a wall. Analysis of the micro-PIV results also shows that ultrasound-driven microstreaming flows around bubbles are feasible mechanisms for mixing therapeutic agents into the surrounding blood, as well as assisting sonoporative delivery of molecules across cell membranes. Patterns show significant variations around the bubble, suggesting sonoporation may be either enhanced or inhibited in different zones across a cellular surface. Thus, alternating the patterns may result in improved sonoporation and sonothrombolysis. The clear and reproducible delineation of microstreaming patterns based on driving frequency makes frequency-based pattern alternation a feasible alternative to the clinically less desirable practice of increasing sound pressure for equivalent sonoporative or sonothrombolytic effect. Surface divergence is proposed as a measure relevant to sonoporation.