Heat Treatment Of Cells Research Articles

Slow Spontaneous Efficiency Enhancement of Single-Crystal Perovskite Solar Cells Due to Trapped Solvent

Perovskite solar cells show spontaneous efficiency enhancement (SEE), i.e., their efficiency improves with time over a few days. Many mechanisms have been proposed for explaining such behavior in polycrystalline perovskite solar cells. However, such studies have not been performed on single-crystal perovskite solar cells. Here, we report our observation of slow (∼20 days) SEE in methylammonium lead iodide single-crystal perovskite solar cells. We found that the SEE is due to the slow escape of solvent trapped by the perovskite single-crystal thin films. We show that this process can be substantially accelerated by heat treatment of cells at 40 °C. This work provides a ground for fast-tracking the progress of single-crystal perovskite solar cells.

Which one of the thermal approaches (heating DNA or cells) enhances the gene expression in mammalian cells?

ObjectivesHeat treatment as a physical method could increase the cellular uptake of nucleic acids. In this study, the effects of heat shock were evaluated to enhance the transfection efficiency of three plasmid DNAs into HeLa and TC-1 cancerous, and HEK-293 T and Vero non-cancerous cell lines using lipofectamine 2000 reagent.MethodsTwo methods of cell- and DNA-based heat treatment were used. Heating DNA solution was performed at 94 °C for 5, 10 and 15 min, and also 72 °C for 30, 60 and 120 min, individually. Moreover, heating the cells was done by incubation at 42 °C for 2 h in different times such as before, during and after DNA transfection.ResultsOur data showed that the conformation of plasmid DNAs was changed at different temperatures with increasing time. The heat-treated plasmid DNAs (94 °C for 10 min or 72 °C for 30 min) indicated higher transfection efficiency than untreated plasmid DNAs (p < 0.05). Furthermore, heat treatment of cells before and during the transfection was higher than untreated cells (p < 0.01). Our results demonstrated that DNA transfection efficiency in cancerous cells was less than non-cancerous cells (p < 0.01).ConclusionGenerally, these findings showed that transfection mediated by thermal stimulation could enhance gene transfection in mammalian cell lines.

Abstract B170: Non-lethal heat treatment of cells results in reduction in cancer stem cell fraction and metastatic potential.

Abstract Metastatic disease and recurrence have been hypothesized to result from residual cancer stem cells (CSCs), also referred to as tumor-initiating cells, which evade initial treatment. CSCs are thought to have the ability to recapitulate the heterogeneity of the parental tumor, to self-renew, and to maintain their self-renewing potential over long periods of time. Therapeutic targeting of the CSC population, in addition to differentiated malignant cells, may be critical for the successful treatment and elimination residual disease in human cancer. Hyperthermia has been used as an adjuvant treatment in which body tissue is exposed to high temperatures (up to 45 °C) in combination with radiation or chemotherapy. In this study, we test the hypothesis that a major effect of hyperthermia is to weaken or reduce the CSC population, resulting in reduced invasive metastatic potential. A pulmonary metastasis model was used, where tumor cells are administered to a mouse by i.v injection. 4 × 105 cells/mouse will yield between 50 and 250 pulmonary nodules visible on the lung surface. The tumor nodules can be easily visualized by bioluminescence imaging (BLI). For effects of hyperthermia on metastatic potential, a mouse melanoma (B16F10-Luc.) model was used. Thermal treatment consisted of immersing the cells in a heated water bath with temperature maintained for 25 min at 37 °C or 43 °C. After thermal treatment, viable control or heat treated (HT) cells were injected into the mice via tail vein and BLI used to determine the metastatic disease in the lungs. MDA-MB 231-Luc. breast cancer cells were administrated after HT. Primary tumor volume and lung metastasis were measured by BLI. For comparison, MM 231 and B16 cells were analyzed via FACS following the same regimen of sham or heat treating using the combination CD44+/CD133+ markers to separate and count the CSC population. In the control, metastatic disease resulted in a faintly observed signal in 3 days, and a clearly observed signal in 6 days from the lungs. In contrast, the HT group had no signal at either time point. HT cells similarly showed an initial loss of BLI signal due to denaturation of luciferase, however, this was completely recovered within 4 days. Overall viability was not significantly altered by HT. FACS analysis of the MM 231 cells showed a 52.2 % reduction in CD44 and 29.6 % reduction in CD133 expression compared to controls, from 55.2 % of the total to 26.4 % in CD44 and from 49.2 % of the total to 34.6 % in CD133. B16 cells showed a 32.1 % reduction in CD44 expression, but they showed similar CD133. Both B16 and MM 231 tumor model showed significant changes in tumor growth with HT. The tumor volume of HT group was smaller than control and metastatic disease was also significantly reduced in the HT. These results suggest that hyperthermia may be capable of selectively injuring CSCs thereby reducing the incidence of metastatic disease formation. However, hyperthermia has only minimal impact on overall cancer cell survival numbers, and, due to its limited application in time and space, has no effect on CSCs already seeded or in circulation unless whole-body treatment is possible. These facts may explain hyperthermia's limited clinical efficacy, but also point towards a strategy for leveraging CSC thermal sensitivity to improve outcomes as an adjuvant therapy. Further studies are needed to establish appropriate methods and timing of treatment. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B170. Citation Format: Yoo-Shin Kim, Tae Hoon Lee, Brian E. O'Neill. Non-lethal heat treatment of cells results in reduction in cancer stem cell fraction and metastatic potential. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B170.

Heat Shock and Caloric Restriction Have a Synergistic Effect on the Heat Shock Response in a sir2.1-dependent Manner in Caenorhabditis elegans

The heat shock response (HSR) is responsible for maintaining cellular and organismal health through the regulation of proteostasis. Recent data demonstrating that the mammalian HSR is regulated by SIRT1 suggest that this response may be under metabolic control. To test this hypothesis, we have determined the effect of caloric restriction in Caenorhabditis elegans on activation of the HSR and have found a synergistic effect on the induction of hsp70 gene expression. The homolog of mammalian SIRT1 in C. elegans is Sir2.1. Using a mutated C. elegans strain with a sir2.1 deletion, we show that heat shock and caloric restriction cooperate to promote increased survivability and fitness in a sir2.1-dependent manner. Finally, we show that caloric restriction increases the ability of heat shock to preserve movement in a polyglutamine toxicity neurodegenerative disease model and that this effect is dependent on sir2.1.

Dedifferentiation of Adult Monkey Sertoli Cells through Activation of Extracellularly Regulated Kinase 1/2 Induced by Heat Treatment

Sertoli cells play a key role in triggering and regulating the process of spermatogenesis. Failure of a Sertoli cell to mature functionally will presumably render it incapable of supporting germ cell survival and development that appeared after puberty. Expression of cytokeratin 18 (ck-18) intermediate filaments indicates a state of undifferentiation usually observed in Sertoli cells of prepubertal testis. In this study we demonstrated that local testicular heat treatment of adult monkey with water at 43 C for 30 min once daily for 2 consecutive days was capable of activating reexpression of ck-18 in Sertoli cells, which was coincident with activation of ERK1/2 and Akt kinases. Using primary Sertoli cell culture isolated from adult monkey testis, we further confirmed that the heat treatment of the cells at 43 C could also induce ck-18 reexpression, which was similar to the in vivo treatment. ERK MAPK was also induced by the heat treatment in a time- and protein kinase A (PKA)-dependent manner. After blocking the ERK MAPK signaling pathway, an inhibition of ck-18 expression in the cultured Sertoli cells was observed, and this inhibitory effect was also detected by blocking the PKA activation. However, ck-18 activation in Sertoli cells remained unaltered when the phosphatidylinositol 3-kinase/Akt pathway was blocked. In conclusion, the heat treatment of adult monkey Sertoli cells are capable of inducing a reversible change in the Sertoli cells from an adult differentiated state to an immature-like dedifferentiated state through PKA-ERK MAPK-dependent pathways but not via the phosphatidylinositol 3-kinase/Akt pathway.

A Bichaperone (Hsp70-Hsp78) System Restores Mitochondrial DNA Synthesis following Thermal Inactivation of Mip1p Polymerase

Mitochondrial DNA synthesis is a thermosensitive process in the yeast Saccharomyces cerevisiae. We found that restoration of mtDNA synthesis following heat treatment of cells is dependent on reactivation of the mtDNA polymerase Mip1p through the action of a mitochondrial bichaperone system consisting of the Hsp70 system and the Hsp78 oligomeric protein. mtDNA synthesis was inefficiently restored after heat shock in yeast lacking either functional component of the bichaperone system. Furthermore, the activity of purified Mip1p was also thermosensitive; however, the purified components of the mitochondrial bichaperone system (Ssc1p, Mdj1p, Mge1p, and Hsp78p) were able to protect its activity under moderate heat shock conditions as well as to reactivate thermally inactivated Mip1p. Interestingly, the reactivation of endogenous Mip1p contributed more significantly to the restoration of mtDNA synthesis than did import of newly synthesized Mip1p from the cytosol. These observations suggest an important link between function of mitochondrial chaperones and the propagation of mitochondrial genomes under ever-changing environmental conditions.

A reverse transcriptase-polymerase chain reaction assay for detection of viable Escherichia coli O157:H7: investigation of specific target genes.

To determine suitable target genes for detection of the pathogen Escherichia coli O157:H7 by reverse transcriptase-polymerase chain reaction (RT-PCR). Potential genes used as indicators for viability included rfbE, fliC, stx1, stx2, mobA, eaeA, hly and 16S rRNA. Under normal growth conditions, rfbE, stx1, hly and 16S rRNA amplicons were detected in association with all growth phases. The products of 16S rRNA, mobA, rfbE and stx1 were readily visualized in RNA isolated from viable but non-culturable cells. The 16S rRNA gene was not amplified following heat treatment of cells at 121 degrees C for 15 min and mRNA targets were not amplified after treatment at 60 degrees C for 20 min. In this instance, genes that are not amplified are good targets for determining viability. The results of RT-PCR amplification indicate that, under the conditions examined, the rfbE gene is the most appropriate target for detection of viable E. coli O157:H7. Prior to detection or identification from an environmental or food sample E. coli O157:H7 may be exposed to many harsh conditions that influence nucleic acid (RNA and DNA) stability. This study gives an insight into the effects of temperature and nutrient deprivation on identification of viable cells using RT-PCR. It also suggests that, if RT-PCR is to be used for detection of live cells in a sample without enrichment, 10(7) cfu of the target organism are required.

Saccharomyces cerevisiae mannoproteins that protect wine from protein haze: evaluation of extraction methods and immunolocalization.

Yeast-derived haze-protective mannoprotein material (HPM) offers protection to white wines from commercially unacceptable turbidities. HPM extraction methods have been evaluated using three winemaking strains of Saccharomyces cerevisiae. Digestion with Zymolyase of cells pretreated with DTE and EDTA gave the greatest yields of active material. Heat treatment of cells with SDS also released active material but the quantities were low. Treatment of the cells in an autoclave or with a French pressure device was less effective. A detailed study was conducted on the strain Maurivin PDM. SDS was not necessary to extract HPM from PDM; boiling the cells for 5 min in Tris buffer was sufficient. HPM could also be extracted with EDTA during the pretreatment of the cells prior to Zymolyase digestion. The data suggest that HPM was noncovalently linked to other cell wall components and loosely associated with the cell wall. An immunological investigation showed that a specific mannoprotein with haze-protective activity, HPF1, was located primarily on the outermost and innermost layers of the cell wall.

The phenomenon of salivary protein adsorption onto Streptococcus mitis ATCC 903 cells.

It is thought that salivary proteins on bacterial cells have some influence on the adhesion of oral bacteria onto the surface of oral tissues and on bacterial aggregation. Initially, this study sought to examine the phenomenon of salivary protein adsorption to the surface of Streptococcus mitis ATCC 903 using 3H-labeled salivary proteins. We investigated the effects of hydrophobic level and lectin-ligand binding on adsorption of salivary proteins, and also the influence of cell surface components on adsorption. The results showed that the adsorption of salivary proteins was clearly reduced by the presence of Tween 20, LiCl, NaSCN, Hexadecane and some sugars. The adsorption was also affected by the pH level, and protease treatment or heat treatment of cells also decreased the volume of adsorbed proteins. Although the adsorption of proteins onto heat-treated cells was dramatically reduced by the presence of LiCl and NaSCN, the presence of sugars had little influence on this adsorption. These findings suggest that the main adsorption of salivary proteins is due to hydrophobic factors, and the heat-sensitive surface components of the cells had some relation to lectin-ligand binding. Therefore, it appears important to study the adsorption of salivary proteins onto cells since the salivary proteins on bacterial cells play an important role in their adherence to the saliva-coated oral tissues and bacterial aggregation.

Heat sensitivity of double-stranded DNA-dependent protein kinase (DNA-PK) activity

Purpose: The heat sensitivity of DNA-PK activity in hybrid cells and the possible restoration of this activity with extracts from scid cells (defective in DNA-PKcs), sxi-3 cells (defective in Ku80) and V79-4 (sxi-3 parental wild-type cells) was analysed. Materials and methods: Heat treatment of cells was performed in a water bath at 44 C. The cell extract from scid cells or sxi-3 cells was added to heat-treated hybrid cell extracts, and the DNA-PK activity was assayed. Results: When hybrid cells were heated at 44 C for 15min, DNA-PK activity was reduced to undetectable levels. The decreased DNA-PK activity could be restored in a concentration-dependent manner with the addition of scid cell extract. The sxi-3 cell extract could not restore heat-inactivated DNA-PK activity. Conclusions: DNA-PK was inactivated by heat treatment at 44 C. Ku70/Ku80, but not Ku70 alone, could restore heat-inactivated DNA-PK.

Physical and chemical methods for enhancing rapid detection of viruses and other agents

Viral replication events can be enhanced by physical, chemical, or heat treatment of cells. The centrifugation of cells can stimulate them to proliferate, reduce their generation times, and activate gene expression. Human endothelial cells can be activated to release cyclo-oxygenase metabolites after rocking for 5 min, and mechanical stress can stimulate endothelial cells to proliferate. Centrifugation of virus-infected cultures can increase cytopathic effects (CPE), enhance the number of infected cells, increase viral yields, and reduce viral detection times and may increase viral isolation rates. The rolling of virus-infected cells also has an effect similar to that of centrifugation. The continuous rolling of virus-infected cultures at < or = 2.0 rpm can enhance enterovirus, rhinovirus, reovirus, rotavirus, paramyxovirus, herpesvirus, and vaccinia virus CPE or yields or both. For some viruses, the continuous rolling of infected cell cultures at 96 rpm (1.9 x g) is superior to rolling at 2.0 rpm for viral replication or CPE production. In addition to centrifugation and rolling, the treatment of cells with chemicals or heat can also enhance viral yields or CPE. For example, the treatment of virus-infected cells with dimethyl sulfoxide can enhance viral transformation, increase plaque numbers and plaque size, increase the number of cells producing antigens, and increase viral yields. The infectivity of fowl plague virus is increased by 80-fold when 4% dimethyl sulfoxide is added to culture medium immediately after infection. The heat shocking of virus-infected cells also has been shown to have a stimulatory effect on the replication events of cytomegalovirus, Epstein-Barr virus, and human immunodeficiency virus. The effects of motion, chemicals, or heat treatments on viral replication are not well understood. These treatments apparently activate cells to make them more permissive to viral infection and viral replication. Perhaps heat shock proteins or stress proteins are a common factor for this enhancement phenomenon. The utility of these treatments alone or in combination with other methods for enhancing viral isolation and replication in a diagnostic setting needs further investigation.

Adhesive Properties of a Symbiotic Bacterium from a Wood-Boring Marine Shipworm

Adhesive properties of a cellulolytic, nitrogen-fixing bacterium isolated from a marine shipworm by Waterbury et al. (J. B. Waterbury, C. B. Calloway, and R. D. Turner, Science 221:1401-1403, 1983) are described. S-labeled cells of the shipworm bacterium bound preferentially to Whatman no. 1 cellulose filter paper, compared with its binding to other cellulose substrata or substrata lacking cellulose. The ability of the bacteria to bind to Whatman no. 1 filter paper was significantly reduced by glutaraldehyde or heat treatment of cells. Pretreatment of cells with azide, valinomycin, gramicidin-D, bis-hexafluoroacetylacetone (1799), or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone inhibited adhesion activity. Cells pretreated with pronase or trypsin also exhibited reduced binding activity, but chymotrypsin and peptidase had no effect on adhesion activity. Cellodextrins and methyl cellulose 15 inhibited the adhesion of shipworm bacteria to filter paper, whereas glucose, cellobiose, and soluble carboxymethyl cellulose had no significant effect. The divalent cation chelators EDTA and EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid] had little or no effect on adhesive properties of shipworm bacteria. Also, preabsorbing the substratum with extracellular endoglucanase isolated from the shipworm bacterium or 1% bovine serum albumin had no apparent effect on bacterial binding. Low concentrations (0.01%) of sodium dodecyl sulfate solubilized a fraction from whole cells, which appeared to be involved in cellular binding activity. After removal of sodium dodecyl sulfate, several proteins in this fraction associated with intact cells. These cells exhibited up to 50% enhanced binding to filter paper in comparison to cells which had not been exposed to the sodium dodecyl sulfate-solubilized fraction.

Decreased expression of heat shock protein 70 mRNA and protein after heat treatment in cells of aged rats.

The effect of aging on the induction of heat shock protein 70 (HSP70)-encoding gene expression by elevated temperatures was studied in cultures of lung- or skin-derived fibroblasts from young (5 mo) and old (24 mo) male Wistar rats. Although the kinetics of the heat shock response were found to be similar in the two age groups, we observed lower levels of induction of HSP70 mRNA and HSP70 protein in confluent primary lung and skin fibroblast cultures derived from aged animals. Additional experiments with freshly excised lung tissue showed a similar age-related decline in the heat-induced expression of HSP70.

Production of a heat stable enterotoxin by Plesiomonas shigelloides

Eight strains of Plesiomonas shigelloides were assayed for enterotoxin production in the rabbit ileal loop assay. Seven strains required serial in vivo passage in the rabbit's intestine before enterotoxin activity was detected in the cells' filtrate. Enterotoxin production was readily lost with subculture of these toxinogenic cells. Heat treatment of the cells' filtrate from three strains that had never been passed in vivo led to detectable enterotoxin activity in three of six separate assays. Using LT, CT, STIa, STIb and STII as probes, no homology to the whole cell DNA of the eight strains was detected on Southern hybridization under low stringency conditions. The enterotoxin of P. shigelloides appears to be novel since production is induced by in vivo passage, activity is seen with heat treatment of cells' filtrate and there is no DNA homology to the cloned enterotoxin genes of Escherichia coli and Vibrio choleraea.

Nuclear and nucleolar localization of the 72,000-dalton heat shock protein in heat-shocked mammalian cells.

The intracellular location of the major induced mammalian heat shock (or stress) protein (Mr = 72,000) has been determined by both biochemical and immunological methods. This protein, shown here to be comprised of at least three structurally related isoforms, is produced at high levels within 30 min to 1 h following heat treatment of cells. Biochemical fractionation of cells grown under heat shock showed that following its synthesis a portion of the 72,000-Da protein (and its isoforms) becomes associated with the nucleus while some remains in the cytoplasm. Indirect immunofluorescence studies using antiserum directed against the major isoforms of the 72,000-Da protein were carried out in normal and heat-shocked cells as well as in cells grown under stress by exposure to either an amino acid analogue or to sodium arsenite. Diffuse cytoplasmic and nuclear staining was observed in cells grown at 37 degrees C. In cells grown under heat shock conditions, both the cytoplasmic staining and the nuclear staining were found to increase with the nuclear staining consisting of both granular and patch-like structures, the latter being coincident with phase-dense nucleoli. In the case of cells exposed to amino acid analogues or to sodium arsenite, only cytoplasmic and to a lesser extent nuclear staining was observed, i.e. no localized nucleolar fluorescence was observed. Following return of heat shock-treated cells to normal growth temperatures, both the synthesis of the 72,000-Dalton stress protein and its nucleolar staining were found to diminish.

Effect of Thermotolerance on Thermal Radiosensitization in Hepatoma Cells

The interaction between hyperthermia and X irradiation was determined in cultured Reuber H35 hepatoma cells with different states of thermosensitivity. Incubation at 41 degrees C followed by 4-Gy X rays resulted after 2 hr in a stabilization of cell survival for heat or plus X rays, with a maximum synergism factor of 1.6. Thermotolerance did not develop during incubation at 41.7 or 42.5 degrees C. When heat treatment of cells was followed by irradiation, the synergism factor for thermal radiosensitization increased with both the amount of thermal cell killing and the amount of X-ray cell killing; the influence of thermal exposure on the synergism factor was greater than that of the X-ray dose. Cells were made thermotolerant either by incubation at 42.5 degrees C for 30 or 60 min followed by an interval at 37 degrees C, or by continuous incubation at 41 degrees C. In both cases thermotolerance was measured by incubation at 42.5 degrees C. No difference was observed between the maximum thermotolerance achieved with both methods. When cells were irradiated in addition to the second heat treatment, thermal radiosensitization was strongly reduced concomitant with the decreased sensitivity to killing by heat.