Graphene-Based Photodynamic Therapy and Overcoming Cancer Resistance Mechanisms: A Comprehensive Review.

Photodynamic therapy (PDT) is a non-invasive therapy that has made significant progress in treating different diseases, including cancer, by utilizing new nanotechnology products such as graphene and its derivatives. Graphene-based materials have large surface area and photothermal effects thereby making them suitable candidates for PDT or photo-active drug carriers. The remarkable photophysical properties of graphene derivates facilitate the efficient generation of reactive oxygen species (ROS) upon light irradiation, which destroys cancer cells. Surface functionalization of graphene and its materials can also enhance their biocompatibility and anticancer activity. The paper delves into the distinct roles played by graphene-based materials in PDT such as photosensitizers (PS) and drug carriers while at the same time considers how these materials could be used to circumvent cancer resistance. This will provide readers with an extensive discussion of various pathways contributing to PDT inefficiency. Consequently, this comprehensive review underscores the vital roles that graphene and its derivatives may play in emerging PDT strategies for cancer treatment and other medical purposes. With a better comprehension of the current state of research and the existing challenges, the integration of graphene-based materials in PDT holds great promise for developing targeted, effective, and personalized cancer treatments.

Hyperoside as a UV Photoprotective or Photostimulating Compound-Evaluation of the Effect of UV Radiation with Selected UV-Absorbing Organic Compounds on Skin Cells.

Ultraviolet (UV) radiation is a non-ionizing radiation, which has a cytotoxic potential, and it is therefore necessary to protect against it. Human skin is exposed to the longer-wavelength components of UV radiation (UVA and UVB) from the sun. In the present paper, we focused on the study of eight organic UV-absorbing compounds: astragalin, beta-carotene, 2,4-dihydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone, hyperoside, 3-(4-methylbenzylidene)camphor, pachypodol, and trans-urocanic acid, as possible protectives of skin cells against UVA and UVB radiation. Their protective effects on skin cell viability, ROS production, mitochondrial membrane potential, liposomal permeability, and DNA integrity were investigated. Only some of the compounds studied, such as trans-urocanic acid and hyperoside, had a significant effect on the examined hallmarks of UV-induced cell damage. This was also confirmed by an atomic force microscopy study of morphological changes in HaCaT cells or a study conducted on a 3D skin model. In conclusion, hyperoside was found to be a very effective UV-protective compound, especially against UVA radiation. Commonly used sunscreen compounds such as 2,4-dihydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone, and 3-(4-methylbenzylidene)camphor turned out to be only physical UV filters, and pachypodol with a relatively high absorption in the UVA region was shown to be more phototoxic than photoprotective.

Photodynamic therapy: Innovative approaches for antibacterial and anticancer treatments.

Photodynamic therapy is an alternative treatment mainly for cancer but also for bacterial infections. This treatment dates back to 1900 when a German medical school graduate Oscar Raab found a photodynamic effect while doing research for his doctoral dissertation with Professor Hermann von Tappeiner. Unexpectedly, Raab revealed that the toxicity of acridine on paramecium depends on the intensity of light in his laboratory. Photodynamic therapy is therefore based on the administration of a photosensitizer with subsequent light irradiation within the absorption maxima of this substance followed by reactive oxygen species formation and finally cell death. Although this treatment is not a novelty, there is an endeavor for various modifications to the therapy. For example, selectivity and efficiency of the photosensitizer, as well as irradiation with various types of light sources are still being modified to improve final results of the photodynamic therapy. The main aim of this review is to summarize anticancer and antibacterial modifications, namely various compounds, approaches, and techniques, to enhance the effectiveness of photodynamic therapy.

Biological Evaluation of Photodynamic Effect Mediated by Nanoparticles with Embedded Porphyrin Photosensitizer.

Clinically approved photodynamic therapy (PDT) is a minimally invasive treatment procedure that uses three key components: photosensitization, a light source, and tissue oxygen. However, the photodynamic effect is limited by both the photophysical properties of photosensitizers as well as their low selectivity, leading to damage to adjacent normal tissue and/or inadequate biodistribution. Nanoparticles (NPs) represent a new option for PDT that can overcome most of the limitations of conventional photosensitizers and can also promote photosensitizer accumulation in target cells through enhanced permeation and retention effects. In this in vitro study, the photodynamic effect of TPP photosensitizers embedded in polystyrene nanoparticles was observed on the non-tumor NIH3T3 cell line and HeLa and G361 tumor cell lines. The efficacy was evaluated by viability assay, while reactive oxygen species production, changes in membrane mitochondrial potential, and morphological changes before and after treatment were imaged by atomic force microscopy. The tested nanoparticles with embedded TPP were found to become cytotoxic only after activation by blue light (414 nm) due to the production of reactive oxygen species. The photodynamic effect observed in this evaluation was significantly higher in both tumor lines than the effect observed in the non-tumor line, and the resulting phototoxicity depended on the concentration of photosensitizer and irradiation time.

Lifetime risk of autosomal recessive neurodegeneration with brain iron accumulation (NBIA) disorders calculated from genetic databases.

BACKGROUND: Neurodegeneration with brain iron accumulation (NBIA) are a group of clinically and genetically heterogeneous diseases characterized by iron overload in basal ganglia and progressive neurodegeneration. Little is known about the epidemiology of NBIA disorders. In the absence of large-scale population-based studies, obtaining reliable epidemiological data requires innovative approaches. METHODS: All pathogenic variants were collected from the 13 genes associated with autosomal recessive NBIA (PLA2G6, PANK2, COASY, ATP13A2, CP, AP4M1, FA2H, CRAT, SCP2, C19orf12, DCAF17, GTPBP2, REPS1). The allele frequencies of these disease-causing variants were assessed in exome/genome collections: the Genome Aggregation Database (gnomAD) and our in-house database. Lifetime risks were calculated from the sum of allele frequencies in the respective genes under assumption of Hardy-Weinberg equilibrium. FINDINGS: The combined estimated lifetime risk of all 13 investigated NBIA disorders is 0.88 (95% confidence interval 0.70-1.10) per 100,000 based on the global gnomAD dataset (n = 282,912 alleles), 0.92 (0.65-1.29) per 100,000 in the European gnomAD dataset (n = 129,206), and 0.90 (0.48-1.62) per 100,000 in our in-house database (n = 44,324). Individually, the highest lifetime risks (>0.15 per 100,000) are found for disorders caused by variants in PLA2G6, PANK2 and COASY. INTERPRETATION: This population-genetic estimation on lifetime risks of recessive NBIA disorders reveals frequencies far exceeding previous population-based numbers. Importantly, our approach represents lifetime risks from conception, thus including prenatal deaths. Understanding the true lifetime risk of NBIA disorders is important in estimating disease burden, allocating resources and targeting specific interventions. FUNDING: This work was carried out in the framework of TIRCON ("Treat Iron-Related Childhood-Onset Neurodegeneration").

Optimal Light Source Intensity Setting in Endoscopic Ear Surgery.

HYPOTHESIS: Higher light intensity settings do not yield improved image quality in endoscopic ear surgery. BACKGROUND: Light intensity is a parameter with major impact on the quality of digital images. For ear surgery, light produces heat associated with a thermal risk to ear structures and the light source setting should be accordingly optimized. METHODS: Several series of still images were acquired during live middle ear surgery, using cadaveric and plastic temporal bone models and with three-dimensional printed models. Images obtained under varying light intensities were compared with the image acquired at maximum intensity of a light emitting diode light source. We analyzed digital image brightness and noise using quantitative methods. RESULTS: Our measurements revealed significantly decreased image brightness with light intensities set below 20% with an increase in noise at light intensities lower than 30%. CONCLUSION: The optimal light source setting corresponded to 30% intensity in our experimental set-up. Special attention should be given to those cases where faster image quality degradation is expected (dark or bloody scenes or larger cavities). The results were strongly dependent on the equipment used. The methods described in this study can serve as a general guide for determining the optimal light source setting in any specific set-up.

Synthesis of N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines and their photodynamic properties in the human skin melanoma cell line G361.

A small series of N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines was synthesized from easily accessible 1-phenyl-1H-pyrazol-3-ol via 7-iodo-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine and 7-iodo-4-methyl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine intermediates and their subsequent use in palladium catalyzed Buchwald-Hartwig cross-coupling reaction with various anilines. Majority of the compounds were not significantly cytotoxic to melanoma G361 cells in the dark up to 10 µM concentration, but their activity could be increased by irradiation with visible blue light (414 nm). The most active compound 10 possessed EC50 values of 3.5, 1.6 and 0.9 µM in cells irradiated with 1, 5 and 10 J/cm2, respectively. The treatment caused generation of reactive oxygen species in cells and extensive DNA damage, documented by the comet assay and by detection of phosphorylated histone H2A.X, followed by apoptotic cell death. Our results suggest that N-aryl-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-amines could serve as a potential source of photosensitizing compounds with anticancer activities.

The effect of two porphyrine photosensitizers TMPyP and ZnTPPS4 for application in photodynamic therapy of cancer cells in vitro.

Photodynamic therapy (PDT) is one of the treatments for cancer. This therapy uses a combination of a photosensitizer (PS), light irradiation, and oxygen O2, which is converted to cytotoxic 1O2 and other forms of reactive oxygen species (ROS), causing selective damage to the target tissue. In this work, we studied effect of two porphyrin photosensitizers TMPyP and ZnTPPS4 at three different concentrations (0.25, 0.5, 5µM) after two irradiation doses (5 and 25 J/cm2). Photodynamic efect of TMPyP and ZnTPPS4 were confirmed by a battery of in vitro tests including MTT, reactive oxygen species (ROS) production and mitochondrial membrane potential test (MMP). Morphological changes of the cells before and after treatment were imaged by atomic force microscopy (AFM). The most effective combination of irradiation dose and concentration for both PSs showed a concentration of 5 µM and a irradiation dose of 25 J/cm2 in both cell cultures.

Effects of zinc porphyrin and zinc phthalocyanine derivatives in photodynamic anticancer therapy under different partial pressures of oxygen in vitro.

Photodynamic therapy (PDT) is gradually becoming an alternative method in the treatment of several diseases. Here, we investigated the role of oxygen in photodynamically treated cervical cancer cells (HeLa). The effect of PDT on HeLa cells was assessed by exposing cultured cells to disulphonated zinc phthalocyanine (ZnPcS2) and tetrasulphonated zinc tetraphenylporphyrin (ZnTPPS4). Fluorescence microscopy revealed their different localizations within the cells. ZnTPPS4 seems to be mostly limited to the cytosol and lysosomes, whereas ZnPcS2 is most likely predominantly attached to membrane structures, including plasmalemma and the mitochondrial membrane. Phototoxicity assays of PDT-treated cells carried out under different partial pressures of oxygen showed dose-dependent responses. Interestingly, ZnPcS2 was also photodynamically effective at a minimal level of oxygen, under a nitrogen atmosphere. On the other hand, hyperbaric oxygenation did not lead to a higher PDT efficiency of either photosensitizer. Although both photosensitizers can induce a significant drop in mitochondrial membrane potential, ZnPcS2 has a markedly higher effect on mitochondrial respiration that was completely blocked after two short light cycles. In conclusion, our observations suggest that PDT can be effective even in hypoxic conditions if a suitable sensitizer is chosen, such as ZnPcS2, which can inhibit mitochondrial respiration.

The Phenotypic Spectrum of 47 Czech Patients with Single, Large-Scale Mitochondrial DNA Deletions.

BACKGROUND: In this retrospective study, we analysed clinical, biochemical and molecular genetic data of 47 Czech patients with Single, Large-Scale Mitochondrial DNA Deletions (SLSMD). METHODS: The diagnosis was based on the long-range PCR (LX-PCR) screening of mtDNA isolated from muscle biopsy in 15 patients, and from the buccal swab, urinary epithelial cells and blood in 32 patients. RESULTS: A total of 57% patients manifested before the age of 16. We did not find any significant difference between paediatric and adult manifestation in either the proportion of patients that would develop extraocular symptoms, or the timespan of its progression. The survival rate in patients with Pearson Syndrome reached 60%. Altogether, five patients manifested with atypical phenotype not fulfilling the latest criteria for SLSMD. No correlation was found between the disease severity and all heteroplasmy levels, lengths of the deletion and respiratory chain activities in muscle. CONCLUSIONS: Paediatric manifestation of Progressive External Ophthalmoplegia (PEO) is not associated with a higher risk of multisystemic involvement. Contrary to PEO and Kearns-Sayre Syndrome Spectrum, Pearson Syndrome still contributes to a significant childhood mortality. SLSMD should be considered even in cases with atypical presentation. To successfully identify carriers of SLSMD, a repeated combined analysis of buccal swab and urinary epithelial cells is needed.

Toxicological testing of a photoactive phthalocyanine-based antimicrobial substance.

The aim of the study was toxicological testing of an innovative and efficient antimicrobial agent based on photoactive phthalocyanine (Pc) derivative. A promising Aluminium phthalocyanine (AlPc) with efficient and stable antimicrobial effects was subjected to a battery of toxicological tests to avoid local and systemic toxicity hazard. In compliance with the current European legislation restricting the use of experimental animals, the methods comprised exclusively in vitro procedures based on cellular and tissue models of human origin or mimicking human tissues. The battery of toxicological tests to identify local toxicity included skin corrosion/irritation, eye irritation, and phototoxicity. The basic systemic toxicity tests included acute toxicity, skin sensitization, genotoxicity, and endocrine disruption. The results showed that AlPc induced skin and eye irritation, exhibited borderline sensitization potential and mutagenic potential in one test strain of the Ames test, which was not confirmed in the chromosome aberration test. The AlPc was found to be phototoxic. The results from the cytotoxicity test designed for acute oral toxicity estimation were not conclusive, the acute toxicity potential has to be determined by conventional tests in vivo. Regarding endocrine disruption, no agonistic activity of the AlPc on human estrogen receptor α, nor human androgen receptor was observed. The skin penetration/absorption test revealed that the AlPc has not penetrated into the dermis and receptor fluid, confirming no risk of systemic exposure via the bloodstream.

Multisystem mitochondrial diseases due to mutations in mtDNA-encoded subunits of complex I.

BACKGROUND: Maternally inherited complex I deficiencies due to mutations in MT-ND genes represent a heterogeneous group of multisystem mitochondrial disorders (MD) with a unfavourable prognosis. The aim of the study was to characterize the impact of the mutations in MT-ND genes, including the novel m.13091 T > C variant, on the course of the disease, and to analyse the activities of respiratory chain complexes, the amount of protein subunits, and the mitochondrial energy-generating system (MEGS) in available muscle biopsies and cultivated fibroblasts. METHODS: The respiratory chain complex activities were measured by spectrophotometry, MEGS were analysed using radiolabelled substrates, and protein amount by SDS-PAGE or BN-PAGE in muscle or fibroblasts. RESULTS: In our cohort of 106 unrelated families carrying different mtDNA mutations, we found heteroplasmic mutations in the genes MT-ND1, MT-ND3, and MT-ND5, including the novel variant m.13091 T > C, in 13 patients with MD from 12 families. First symptoms developed between early childhood and adolescence and progressed to multisystem disease with a phenotype of Leigh or MELAS syndromes. MRI revealed bilateral symmetrical involvement of deep grey matter typical of Leigh syndrome in 6 children, cortical/white matter stroke-like lesions suggesting MELAS syndrome in 3 patients, and a combination of cortico-subcortical lesions and grey matter involvement in 4 patients. MEGS indicated mitochondrial disturbances in all available muscle samples, as well as a significantly decreased oxidation of [1-14C] pyruvate in fibroblasts. Spectrophotometric analyses revealed a low activity of complex I and/or complex I + III in all muscle samples except one, but the activities in fibroblasts were mostly normal. No correlation was found between complex I activities and mtDNA mutation load, but higher levels of heteroplasmy were generally found in more severely affected patients. CONCLUSIONS: Maternally inherited complex I deficiencies were found in 11% of families with mitochondrial diseases in our region. Six patients manifested with Leigh, three with MELAS. The remaining four patients presented with an overlap between these two syndromes. MEGS, especially the oxidation of [1-14C] pyruvate in fibroblasts might serve as a sensitive indicator of functional impairment due to MT-ND mutations. Early onset of the disease and higher level of mtDNA heteroplasmy were associated with a worse prognosis.

Toxicity of wastewater from health care facilities assessed by different bioassays.

OBJECTIVES: The purpose of this study was to determine toxicity of wastewater from hospitals in the Czech Republic using traditional and alternative toxicological methods. The pilot study comprised weekly dynamics of sewage ecotoxicity of treated wastewater from one hospital in two different seasons. A detailed investigation of wastewater ecotoxicity, genotoxicity and reprotoxicity followed in five different hospitals. METHODS: The seven following bioassays were used in this study: algal growth inhibition test (ISO 8692), Vibrio fischeri test (ISO 11348-2), Daphnia magna acute toxicity test (ISO 6341), Allium cepa assay, Ames test (OECD TG 471), Comet assay and YES/YAS assay. RESULTS: The wastewater ecotoxicity during one week showed no differences in separate working days, however, higher toxicity values were recorded in May compared to November. In the following study, samples from two of the five hospitals were classified as toxic, the others as non toxic. Genotoxicity has not been confirmed in any sample. In several cases, wastewater samples exhibited agonist activity to the estrogen and androgen receptors. CONCLUSION: The study demonstrated different levels of toxicity of treated hospital wastewater. Variable sensitivity of individual bioassays for tested wastewater samples was recognized. A more extensive study including proposal for improvement of hospital wastewater treatment within the Czech Republic can be recommended with the aim to decrease the discharge of toxic chemicals into the local sewage system and the environment.

Synthesis and Profiling of a Novel Potent Selective Inhibitor of CHK1 Kinase Possessing Unusual N-trifluoromethylpyrazole Pharmacophore Resistant to Metabolic N-dealkylation.

Checkpoint-mediated dependency of tumor cells can be deployed to selectively kill them without substantial toxicity to normal cells. Specifically, loss of CHK1, a serine threonine kinase involved in the surveillance of the G2-M checkpoint in the presence of replication stress inflicted by DNA-damaging drugs, has been reported to dramatically influence the viability of tumor cells. CHK1's pivotal role in maintaining genomic stability offers attractive opportunity for increasing the selectivity, effectivity, and reduced toxicity of chemotherapy. Some recently identified CHK1 inhibitors entered clinical trials in combination with DNA antimetabolites. Herein, we report synthesis and profiling of MU380, a nontrivial analogue of clinically profiled compound SCH900776 possessing the highly unusual N-trifluoromethylpyrazole motif, which was envisioned not to undergo metabolic oxidative dealkylation and thereby provide greater robustness to the compound. MU380 is a selective and potent inhibitor of CHK1 which sensitizes a variety of tumor cell lines to hydroxyurea or gemcitabine up to 10 times. MU380 shows extended inhibitory effects in cells, and unlike SCH900776, does not undergo in vivo N-dealkylation to the significantly less selective metabolite. Compared with SCH900776, MU380 in combination with GEM causes higher accumulation of DNA damage in tumor cells and subsequent enhanced cell death, and is more efficacious in the A2780 xenograft mouse model. Overall, MU380 represents a novel state-of-the-art CHK1 inhibitor with high potency, selectivity, and improved metabolic robustness to oxidative N-dealkylation. Mol Cancer Ther; 16(9); 1831-42. ©2017 AACR.

Effect of Porphyrin Sensitizer MgTPPS4 on Cytoskeletal System of HeLa Cell Line-Microscopic Study.

Metalloporphyrins are an important group of sensitizers with a porphyrin skeleton. Their photophysical properties are significantly affected by the nature of the central ion. In this work, we focus on the mechanical properties of a cervix carcinoma cell line which underwent photodynamic treatment (PDT) with MgTPPS4 photosensitzer. Atomic force microscopy alongside confocal microscopy was used to quantify and qualify the structural characteristics before and after PDT. Cells before PDT showed a fine actin network and higher elasticity with the median of Young modulus 12.2 kPa. After PDT, the median of Young modulus was 13.4 kPa and a large redistribution in the actin network was observed.

Reprint of: Cytotoxicity, cell uptake and microscopic analysis of titanium dioxide and silver nanoparticles in vitro.

Commercially manufactured nanomaterials are used massively for modification of products of everyday use, including products intended for children. Therefore their potential risks have to be ultimately studied. Aside from toxicity of nanomaterials with known specific parameters, the end-consumer is potentially endangered by materials with unknown specification. Commercially available products are not usually accompanied by parameter/specification sheet providing the consumer with sufficient chemico-physical parameters allowing the evaluation of possible toxic effects. The aim of this work was to evaluate the declared parameters of commercially available TiO2 and Ag NPs employing chemico-physical methods and consequently in vitro cytotoxicity and genotoxicity tests performed on non-cancer cell lines. Based on the results of our complex study we can conclude that the data provided by the producers are not in good agreement with the performed measurements. Furthermore, all tested NPs penetrated into the SVK14 cells and all NPs had significant effect on the kinetics of ROS production in all cell lines (note: the ROS production has not been established as the major mechanism of cell damage elicited by Ag NPs). The study revealed greater cytotoxic potential of Ag NPs in comparison with TiO2 NPs and all of the studied NPs caused significant DNA damage.

Phthalocyanine-mediated Photodynamic Treatment of Tumoural and Non-tumoural cell lines.

This study deals with the use of cationic far-red absorbing photosensitizers (λ(max) ~740 nm) from the group of the phthalocyanines, in photodynamic therapy. The photosensitizers differed in their central atom, bearing either hydrogen, zinc or magnesium. These photosensitizers were tested in vitro on the tumour cell line HeLa (cervical cancer) and non-tumour cell line NIH3T3 (mouse fibroblast). The following tests were performed: measurement of reactive oxygen species production, viability testing, Comet assay and cell type detection (apoptotic, necrotic and living cells). The best results were achieved with zinc derivative at relatively low half-maximum inhibitory concentration (0.04 µM) and a total radiation dose of 15 J cm(-2).

Cytotoxicity, cell uptake and microscopic analysis of titanium dioxide and silver nanoparticles in vitro.

Commercially manufactured nanomaterials are used massively for modification of products of everyday use, including products intended for children. Therefore their potential risks have to be ultimately studied. Aside from toxicity of nanomaterials with known specific parameters, the end-consumer is potentially endangered by materials with unknown specification. Commercially available products are not usually accompanied by parameter/specification sheet providing the consumer with sufficient chemico-physical parameters allowing the evaluation of possible toxic effects. The aim of this work was to evaluate the declared parameters of commercially available TiO2 and Ag NPs employing chemico-physical methods and consequently in vitro cytotoxicity and genotoxicity tests performed on non-cancer cell lines. Based on the results of our complex study we can conclude that the data provided by the producers are not in good agreement with the performed measurements. Furthermore, all tested NPs penetrated into the SVK14 cells and all NPs had significant effect on the kinetics of ROS production in all cell lines (note: the ROS production has not been established as the major mechanism of cell damage elicited by Ag NPs). The study revealed greater cytotoxic potential of Ag NPs in comparison with TiO2 NPs and all of the studied NPs caused significant DNA damage.

The time factor in the LDI (Laser Doppler Imaging) diagnosis of burns.

BACKGROUND AND OBJECTIVE: The not quite rare occurrence of inaccurate clinical diagnoses of burns in early post-burn days leads to an inappropriate conservative treatment strategy, or unnecessary surgery. LDI (Laser Doppler Imaging) objectively evaluates skin blood circulation, which correlates with the depth of the burn and the length of healing. The aim of this work was to suggest cutoff values for detecting burns without healing potential within 3 weeks, which should have undergone surgery. METHOD: The burned area's average blood perfusion of 148 burns was measured on 115 patients, using the Laser Doppler Imager PIM III. A total of 268 measurements were performed from the one to the ninth post-burn day (PBD). The perfusion values were compared to the healing time or histology in the case of the surgical treatment. Cutoff values indicating surgery were investigated in various post-burn days; the ROC analysis was used. RESULTS: This work suggest statistically significant increasing cutoff values for indication to surgery (P = 0.05). From the third to the fifth day 148.5 perfusion units (PU), from the sixth to the seventh day 186.0 PU, from the eighth to the ninth PBD 269.5 PU. The cutoff value is not possible to establish until the second day. CONCLUSION: LDI is a useful method for wound healing prediction and an indication of the necessity of surgery. We have demonstrated that the diagnosis of the healing capacity of LDI needs to take into account the factor of time.