Abstract: A bio-sensing method, based on the plasmonic property of gold nano-islands, has been developed for detection of exosomes in a clinical setting. The position of the gold plasmon band in the UV-Visible spectrum depends on the size and shape of gold nanoparticles as well as on the surrounding environment. By adsorbing various chemical entities, or binding them, the gold plasmon band will shift toward longer wavelengths and the shift is proportional to the concentration. Exosomes transport cargoes of molecules and genetic materials to proximal and distal cells. Presently, the standard method for their isolation and quantification from body fluids is by ultracentrifugation, not a practical method to be implemented in a clinical setting. Thus, a versatile and cutting-edge platform is required to selectively detect and isolate exosomes for further analysis at clinical level. The new sensing protocol, instead of antibodies, makes use of a specially synthesized polypeptide (Vn96), to capture and quantify the exosomes from different media, by binding the heat shock proteins from exosomes. The protocol has been established and optimized by using a glass substrate, in order to facilitate the next stage, namely the transfer of the protocol to a microfluidic environment. After each step of the protocol, the UV-Vis spectrum was recorded and the position of gold Localized Surface Plasmon Resonance (LSPR) band was measured. The sensing process was modelled, taking into account the characteristics of the nano-island structure, prepared by thermal convection and annealing. The optimal molar ratios of the most important chemical entities, involved in the detection of exosomes were calculated as well. Indeed, it was found that the results of the sensing process depend on the two major steps: the molar ratios of streptavidin to biotin-PEG-Vn96 and, the final step, the capture of exosomes by the biotin-PEG-Vn96 complex. The microfluidic device designed for sensing of exosomes consists of a glass substrate, sealed by a PDMS layer that contains the channel and a collecting chamber. In the device, the solutions of linker, cross-linker, etc., are pumped over the gold nano-islands and an Ocean Optics spectrometer is used to measure the position of the Au plasmon band at each step of the sensing. The experiments have shown that the shift of the Au LSPR band is proportional to the concentration of exosomes and, thereby, exosomes can be accurately quantified. An important advantage of the method is the ability to discriminate between exosomes having different origins.
Abstract: Adult mesenchymal stem cells (MSCs) have been investigated using preclinical approaches for tissue regeneration. Porcine MSCs (pMSCs) are capable of growing and attaching to plastic with a fibroblast-like morphology and then differentiating into bone, adipose, and cartilage tissues in vitro. This study was conducted to investigate the proliferating abilities, differentiation potentials, and multipotency of miniature pig adipose tissue-derived MSCs (mpAD-MSCs) with or without long-term cryopreservation, considering that cryostorage has the potential for use in clinical applications. After confirming the characteristics of the mpAD-MSCs, we examined the effect of long-term cryopreservation (> 2 years) on expression of cell surface markers (CD34, CD90 and CD105), proliferating abilities (cumulative population doubling level, doubling time, colony-forming unit, and MTT assay) and differentiation potentials into mesodermal cell lineages. As a result, the expression of cell surface markers is similar between thawed and fresh mpAD-MSCs. However, long-term cryopreservation significantly lowered the differentiation potentials (adipogenic, chondrogenic, and osteogenic) of mpAD-MSCs. When compared with fresh mpAD-MSCs, thawed mpAD-MSCs exhibited lower expression of mesodermal cell lineage-related genes such as peroxisome proliferator-activated receptor-g2, lipoprotein lipase, collagen Type II alpha 1, osteonectin, and osteocalcin. Interestingly, long-term cryostoraged mpAD-MSCs exhibited significantly higher cell viability than the fresh mpAD-MSCs. Long-term cryopreservation induced a 30% increase in the cell viability of mpAD-MSCs when compared with the fresh mpAD-MSCs at 5 days after thawing. However, long-term cryopreservation significantly lowered expression of stemness markers such as Oct3/4, Sox2, and Nanog. Furthermore, long-term cryopreservation negatively affected expression of senescence-associated genes such as telomerase reverse transcriptase and heat shock protein 90 of mpAD-MSCs when compared with the fresh mpAD-MSCs. The results from this study might be important for the successful application of MSCs in clinical trials after long-term cryopreservation.
Abstract: The present study was designed to investigate the
cardio protective role of chronic oral administration of alcoholic
extract of Terminalia arjuna in in-vivo ischemic reperfusion injury
and the induction of HSP72. Rabbits, divided into three groups, and
were administered with the alcoholic extract of the bark powder of
Terminalia arjuna (TAAE) by oral gavage [6.75mg/kg: (T1) and
9.75mg/kg: (T2), 6 days /week for 12 weeks]. In open-chest
Ketamine pentobarbitone anaesthetized rabbits, the left anterior
descending coronary artery was occluded for 15 min of ischemia
followed by 60 min of reperfusion. In the vehicle-treated group,
ischemic-reperfusion injury (IRI) was evidenced by depression of
global hemodynamic function (MAP, HR, LVEDP, peak LV (+) & (-
) (dP/dt) along with depletion of HEP compounds. Oxidative stress
in IRI was evidenced by, raised levels of myocardial TBARS and
depletion of endogenous myocardial antioxidants GSH, SOD and
catalase. Western blot analysis showed a single band corresponding
to 72 kDa in homogenates of hearts from rabbits treated with both the
doses. In the alcoholic extract of the bark powder of Terminalia
arjuna treatment groups, both the doses had better recovery of
myocardial hemodynamic function, with significant reduction in
TBARS, and rise in SOD, GSH, catalase were observed. The results
of the present study suggest that the alcoholic extract of the bark
powder of Terminalia arjuna in rabbit induces myocardial HSP 72
and augments myocardial endogenous antioxidants, without causing
any cellular injury and offered better cardioprotection against
oxidative stress associated with myocardial IR injury.
Abstract: Heat-inducible gene expression vectors are useful for hyperthermia-induced cancer gene therapy, because the combination
of hyperthermia and gene therapy can considerably improve the therapeutic effects. In the present study, we developed an enhanced
heat-inducible transgene expression system in which a heat-shock
protein (HSP) promoter and tetracycline-responsive transactivator
were combined. When the transactivator plasmid containing the
tetracycline-responsive transactivator gene was co-transfected with
the reporter gene expression plasmid, a high level of heat-induced gene expression was observed compared with that using the HSP
promoter without the transactivator. In vitro evaluation of the
therapeutic effect using HeLa cells showed that heat-induced therapeutic gene expression caused cell death in a high percentage of
these cells, indicating that this strategy is promising for cancer gene therapy.