Effects of Phytolacca decandra on the viability of murine breast adenocarcinoma (4T1 cells) in vitro

Background: Comparative studies in cancer patients using conventional and alternative therapy have demonstrated that Phytolacca decandra in homeopathic potencies increases survival and improves quality of life of patients bearing breast cancer. In vitro studies show the induction of apoptosis pathways in MCF-7, a human breast cancer cells lineage, after treatment with Phytolacca decandra in different homeopathic dilutions (from 30C to 10M). Recently, we observed significant growth reduction of Ehrlich carcinoma in mice treated with Phytolacca decandra 30cH. Aims: To evaluate Phytolacca decandra effect in different homeopathic dilutions on the phenotypic features, apoptosis index, and cell morphology of 4T1 cells (murine carcinoma cell lineage) in vitro. Method: The potencies 6, 12, 30 and 200 CH prepared in sterile pure water were studied. Dynamized sterile pure water was used as control. The cytotoxicity was evaluated after different cell treatments in culture bottles (25ml) with the homeopathic medicines (equal to 10% of total medium volume). Cells were cultured in a cell density of 5 x 105 cells / ml, treated with the respective potency and, after 24 hours, analyzed for the apoptosis index using Annexin V kit and measured using the Countess® System. The morphology of the 4T1 cells was monitored by staining fixed cell smears with hematoxylin-eosin method. Cells were previously adhered to a glass cover slip and fixed with absolute methanol. The samples were evaluated in quadruplicate and the data were analyzed by one-way ANOVA. Results and discussion: The results obtained up to now show that the treatment with Phytolacca decandra 200cH induced increase of apoptosis index in relation to the control. Moreover, morphological changes were observed in the respective cell smears: the presence of multinucleated cells, some of them presenting up to 8 nuclei and the increase of eosinophilic staining pattern of cytoplasm, even in mononucleated cells. Conclusion: The increase in apoptosis index reproduced the results described in the literature with other cell lineages, but the changes in morphology still deserve further evaluation.

A micromechanical cell stretching device compatible with super-resolution microscopy and single protein tracking

Cell mechano-sensing is based on biomolecule deformations and reorganizations, yet the molecular mechanisms are still unclear. Super-resolution microscopy (SRM) and single protein tracking (SPT) techniques reveal the dynamic organization of proteins at the nanoscale. In parallel, stretchable substrates are used to investigate cellular responses to mechanical forces. However, simultaneous combination of SRM/SPT and cell stretching has never been achieved. Here, we present a cell stretching device compatible with SRM and SPT, composed of an ultra-thin Polydimethylsiloxane (PDMS) layer. The PDMS sheet is gliding on a glycerol-lubricated glass cover-slip to ensure flatness during uniaxial stretching, generated with a 3D-printed micromechanical device by a mobile arm connected to a piezoelectric translator. This method enables to obtain super-resolved images of protein reorganization after live stretching, and to monitor single protein deformation and recruitment inside mechanosensitive structures upon stretching. This protocol is related to the publication ‘Cell stretching is amplified by active actin remodeling to deform and recruit proteins in mechanosensitive structures’, in Nature Cell Biology.

Mechanics of Hydra Detachment from Substrates: The Role of Substrate Rigidity and Starvation

Hydra is a fresh water hydrozoan living as a solitary polyp with a sedentary feeder lifestyle attached to a substrate. In times of food shortage they are reported to detach from their substrate and move either by drifting or ‘somer-saulting’. The attachment to the substrate is usually by the basal-body which secretes a mucosal adhesive. The mechanical strength of the adhesion of Hydra has not been quantified so far. Here, we measure the force required to detach Hydra vulgaris and Hydra magnipapillata from a surface and the role of physical and physiological factors. In order to do this, we have developed a flow chamber with a calibrated jet of water. We find H. vulgaris adhering to a hard substrate - a glass cover slip- requires more force to detach it as compared to a soft substrate- polyacrylamide gel. While H. vulgaris after one week of starvation detaches with very similar values of stress, H. magnipapillata detaches more readily when starved. These results suggest that the strength of adhesion is strongly affected by the stiffness of the substrate, while nutritional status dependence of detachment force appears to be species dependent. Given that Hydra detachment is required during locomotion, our measurements on the one hand suggest the magnitude of forces the animal must exert to detach itself. Additionally, our results suggest active detachment of the base might be required for Hydra to achieve movement, and only a small contribution coming from weakening adhesion.

Pyrosequencing on a glass surface

We demonstrated DNA pyrosequencing at the plain hydrophobically coated surface of a microscope glass cover slip using open-surface microfluidics.

Biopolymer-Based Thin Film for Sensor Application

Chitosan is one of the most available biopolymers in nature, which is non-toxic, biocompatible and biodegradable. The crosslinked chitosan solution was synthesized by homogeneous reaction of medium molecular weight chitosan in aqueous acetic acid with glutaraldehyde as crosslinking agent. Then the solution was deposited on glass cover slip by spin coater to form a thin film. The functional group and chemical binding of crosslinked chitosan thin film has been confirmed by X-ray photoelectron spectroscopy (XPS). The chemical interaction between copper ion and the crosslinked chitosan thin film has also been analyzed by XPS. XPS revealed that copper ion adsorbed to the crosslinked chitosan thin film and the functional groups involved in the adsorption mechanisms of copper ion on the thin film were determined. This biopolymer thin film can be incorporated with surface plasmon resonance technique to produce a high potential optical sensor for detection of Cu (II) ion in solution.

High Resolution X-Ray Photoelectron Spectroscopy Study of the Interaction of Copper Ion with Chitosan Thin Film

In this study, high-resolution X-ray photoelectron spectroscopy (XPS) has been used to study the chemical interaction between copper ion and chitosan thin film. The chitosan solution was synthesized by homogeneous reaction of medium molecular weight chitosan in aqueous acetic acid with glutaraldehyde as crosslinking agent. Then the solution was deposited on glass cover slip by spin coater to form a thin film. The functional group and chemical binding of crosslinked chitosan thin film has been confirmed by XPS. XPS revealed that copper ion adsorbed to the crosslinked chitosan thin film and the functional groups involved in the adsorption mechanisms of copper ion on the thin film were determined.

Factors influencing microstructural evolution in nanoparticle sintered Ag die attach

The behaviour of sintered silver die attach at high temperature has been investigated. Assemblies were made by sintering a commercially available paste composed of Ag nanoparticles with zero applied pressure on the die. The morphology of the cross sectioned surface of assemblies remains stable even at temperatures of up to 400 °C. This behaviour remained consistent even inside vacuum or after acid cleaning of the free surface. In contrast, the same sintered Ag material in the interior of a joint or sintered under a glass cover slip showed rapid microstructural changes even at 300 °C. These samples were investigated using an optical microscope to analyse the changes in the microstructure after storage at 200 to 500 °C. The observations showed a 20% increase in silver grain size after only 5 h storage at 300 °C. However, in the case of a free surface, no changes were observed after 60h storage at 400 °C. These observations were combined with DSC experiments in order to suggest the cause of the difference in behaviour. The results suggest ways of stabilizing sintered silver materials so that they can be used in applications up to 400 °C without significant structural changes occurring in the material.

In Vivo Interrogation of a Human Von Willebrand Type A Domain.

Abstract 2176 Proteins containing Von Willebrand A domains play critical roles in hemostasis and arterial thrombosis by promoting cell-cell and cell-ligand interactions. Central to these processes is the formation of an adhesive bond between the A1 domain containing protein Von Willebrand Factor (VWF) and its receptor on platelets known as GPIbα. Although ex-vivo approaches have broadened our understanding of the biophysical properties that govern the interaction between this human receptor–ligand pair, validation of hypotheses based on these intriguing studies will require an appropriate biological model that permits the study of such complex adhesive interactions under appropriate hemodynamic conditions; only then will it be possible to truly ascertain the biological relevance of in vitro observations as they pertain to hemostasis and thrombosis in humans. Moreover, a biological model that permits the study of the function of the human VWF-A1 domain in vivo will be of tremendous value in the development of drugs for use in disorders associated with thrombotic microangiopathy. Given the importance of the interaction between GPIbα and the A1 domain of VWF in hemostasis and thrombus formation in humans, we genetically modified the murine VWF genomic sequence so that it contains the majority of the human A1 domain (VWF HA1) in lieu of its murine counterpart. Mutant mice were viable, born in expected mendelian ratio, and had platelet counts comparable to WT littermates. Importantly, VWF gene transcription, antigen levels, and multimer pattern were also equivalent to WT controls. In contrast, hemostasis was significantly disrupted in homozygous VWF HA1 mice with majority of animals continuing to bleed for >10 minutes after removal of the distal tip of the tail. Evidence that the defect in hemostasis was due to the inability of murine platelets to accumulate at sites of vascular damage is demonstrated by a 4-fold reduction in thrombus size in response to laser-induced arteriolar injury in the microcirculation of the cremaster muscle. To further evaluate the ability of this modified VWF to support interactions with murine platelets, we surfaced immobilized plasma VWF from these animals onto a glass cover slip, which was then incorporated into a parallel plate flow system. WT mouse blood infused over the immobilized substrate was unable to support any significant interactions with plasma VWF HA1 (wall shear rate of 1,600s−1). By contrast, human platelets rapidly accumulated on surface-immobilized VWF HA1 at levels observed for its human plasma counterpart; human platelets administered to VWF HA1 mice were also capable of restoring hemostasis in these animals and formed occlusive thrombi in response to laser-induced arterial injury. Remarkably, the human A1 domain contained within murine VWF was able to support ristocetin-induced human platelet agglutination whereas wild-type murine plasma VWF did not. We are now taking the next important steps in defining the in vivo consequences of altered bond formation between the human VWF-A1 domain and GPIbα as well as evaluating the anti-thrombotic effects of compounds designed to disrupt this interaction in patients with disease-associated thrombotic microangiopathy. Disclosures: No relevant conflicts of interest to declare.

A Novel Low Cost Technique to Perform Concurrent Topside and Backside Analysis of a Bare Die

A novel low cost technique to facilitate concurrent topside and backside imaging of a bare die for optical fault localization purposes is presented. The technique overcomes the restrictions posed by unavailability of a suitable package or limited choices that may be present at a small scale packaging lab. The difficulties imposed on backside preparation of commonly used ceramic packages are overcome by providing an alternative that is relatively less expensive and easier to implement. This is accomplished by mounting the bare die on a glass cover slip using a suitable adhesive and wire bonding the bond pads to a specially designed printed circuit board. This method is being successfully utilized on multiple failure analysis requests received in our lab.

ASSESSMENT OF OPTICAL CLEARING AGENTS USING REFLECTANCE-MODE CONFOCAL SCANNING LASER MICROSCOPY

The mechanism of action of clearing agents to improve optical imaging of mouse skin during reflectance-mode confocal microscopy was tested. The dermal side of excised dorsal mouse skin was exposed for one hour to saline, glycerin, or 80% DMSO, then the clearing agent was removed and the dermis placed against a glass cover slip through which a confocal microscope measured reflectance at 488 nm wavelength. An untreated control was also measured. The axial attenuation of reflectance signal, R(zf) versus increasing depth of focus zf behaved as R = ρ exp (-μzf2G), where ρ is tissue reflectivity and μ is attenuation [cm-1]. The factor 2G accounts for the in/out path of photons, and the numerical aperture of the lens. The ρ, μ data were mapped to values of scattering coefficient (μs [cm-1]) and anisotropy of scattering (g). Images showed that glycerin significantly increased the g of dermis from about 0.7 to about 0.99, with little change in the μs of dermis at about 300 cm-1. DMSO and saline had only slight and inconsistent effects on g and μs.