To understand the effects of substitution patterns on photosensitizing the ability of boron dipyrromethene (BODIPY), two structural variations that either investigate the effectiveness of various iodinated derivatives to maximize the "heavy atom effect" or focus on the effect of extended conjugation at the 4-pyrrolic position to red-shift their activation wavelengths were investigated. Compounds with conjugation at the 4-pyrrolic position were less photocytotoxic than the parent unconjugated compound, while those with an iodinated BODIPY core presented better photocytotoxicity than compounds with iodoaryl groups at the meso-positions. The potency of the derivatives generally correlated well with their singlet oxygen generation level. Further studies of compound 5 on HSC-2 cells showed almost exclusive localization to mitochondria, induction of G(2)/M-phase cell cycle block, and onset of apoptosis. Compound 5 also extensively occluded the vasculature of the chick chorioallantoic membrane. Iodinated BODIPY structures such as compound 5 may have potential as new photodynamic therapy agents for cancer.
In our screening for photosensitizers from natural resources, 15(1)-hydroxypurpurin-7-lactone ethyl methyl diester (compound 1) was isolated for the first time from an Araceae plant. To evaluate the efficacy of compound 1 as a photosensitizer for head and neck cancers, compound 1 was studied in reference to a known photosensitizer pheophorbide-a (Pha), in terms of photophysical properties, singlet oxygen generation and in in vitro experiments (intracellular uptake and phototoxicity assays) in two oral (HSC2 and HSC3) and two nasopharyngeal (HK1 and C666-1) cancer cell lines. In this study, compound 1 exhibited higher intracellular uptake over 24 h compared with Pha in both HSC3 and HK1 cells. When activated by ≥4.8 J cm(-2) of light, compound 1 was slightly more potent as a photosensitizer than Pha by consistently having marginally lower IC(50) values across different cell lines. In flow cytometry experiments to study the mechanism of photoactivated cell death in HSC3, compound 1 was observed to induce more pronounced apoptosis compared with Pha, which may have been driven by the transient G(2)/M cell cycle block which was also observed. These promising results on compound 1 warrant its further investigation as a clinically useful photodynamic therapy agent for head and neck cancer.
Adipose tissue-derived stromal cells (ASCs) natively reside in a relatively low-oxygen tension (i.e., hypoxic) microenvironment in human body. Low oxygen tension (i.e., in situ normoxia), has been known to enhance the growth and survival rate of ASCs, which, however, may lead to the risk of tumourigenesis. Here, we investigated the tumourigenic potential of ASCs under their physiological condition to ensure their safe use in regenerative therapy. Human ASCs isolated from subcutaneous fat were cultured in atmospheric O2 concentration (21% O2) or in situ normoxia (2% O2). We found that ASCs retained their surface markers, tri-lineage differentiation potential, and self-renewal properties under in situ normoxia without altering their morphology. In situ normoxia displayed a higher proliferation and viability of ASCs with less DNA damage as compared to atmospheric O2 concentration. Moreover, low oxygen tension significantly up-regulated VEGF and bFGF mRNA expression and protein secretion while reducing the expression level of tumour suppressor genes p16, p21, p53, and pRb. However, there were no significant differences in ASCs telomere length and their relative telomerase activity when cultured at different oxygen concentrations. Collectively, even with high proliferation and survival rate, ASCs have a low tendency of developing tumour under in situ normoxia. These results suggest 2% O2 as an ideal culture condition for expanding ASCs efficiently while maintaining their characteristics.
Adipose-derived stem cells (ASCs) have been found adapted to a specific niche with low oxygen tension (hypoxia) in the body. As an important component of this niche, oxygen tension has been known to play a critical role in the maintenance of stem cell characteristics. However, the effect of O2 tension on their functional properties has not been well determined. In this study, we investigated the effects of O2 tension on ASCs stemness, differentiation and proliferation ability. Human ASCs were cultured under normoxia (21% O2) and hypoxia (2% O2). We found that hypoxia increased ASC stemness marker expression and proliferation rate without altering their morphology and surface markers. Low oxygen tension further enhances the chondrogenic differentiation ability, but reduces both adipogenic and osteogenic differentiation potential. These results might be correlated with the increased expression of HIF-1α under hypoxia. Taken together, we suggest that growing ASCs under 2% O2 tension may be important in expanding ASCs effectively while maintaining their functional properties for clinical therapy, particularly for the treatment of cartilage defects.
Agitation speed was found to influence the tannase production and fungal growth of Aspergillus niger FETL FT3. The optimal agitation speed was at 200 rpm which produced 1.41 U/ml tannase and 3.75 g/l of fungal growth. Lower or higher agitation speeds than 200 rpm produced lower enzyme production and fungal growth. Based on the SEM and TEM micrograph observation, there was a significant correlation between agitation speed and the morphology of the fungal mycelia. The results revealed an increase of the enzyme production with the change of the fungal growth morphology from filamentous to pelleted growth forms. However, the exposure to higher shear stress with an increasing agitation speed of the shaker also resulted in lower biomass yields as well as enzyme production.
The effects of sulfide on the energy metabolism of Boleophthalmus boddaerti in normoxia and hypoxia were examined. The 24-, 48-, and 96-h LC50 values of sulfide for B. boddaerti with body weight ranging from 11.6 to 14.2 g were 0.786, 0.567, and 0.467 mM, respectively. The tolerance of B. boddaerti to sulfide was not due to the presence of a sulfide-insensitive cytochrome c oxidase. There was no accumulation of lactate in the muscle and liver of specimens exposed to sulfide in normoxia. In addition, the levels of ATP, AMP, and energy charge in both the muscle and the liver were unaffected. These results indicate that B. boddaerti was able to sustain the energy supply required for its metabolic needs via mainly aerobic respiration when exposed to sulfide (up to 0.4 mM) in normoxia. Exposure of B. boddaerti simultaneously to hypoxia and 0.2 mM sulfide for 48 h resulted in decreases in the ATP levels in the muscle and liver. However, the energy charge in both tissues remained unchanged, and the level of lactate accumulated in the muscle was too low to have any major contribution to the energy budget of the fish. Our results reveal that B. boddaerti possesses inducible mechanisms to detoxify sulfide in an ample supply or a lack of O2. In normoxia, it detoxified sulfide to sulfate, sulfite, and thiosulfate. There were significant increases in the activities of sulfide oxidase in the muscle and liver of specimens exposed to sulfide, with that in the liver being >13-fold higher than that in the muscle. However, in hypoxia, sulfide oxidase activity in the liver was suppressed in response to environmental sulfide. In such conditions, there were significant increases in the activities of sulfane sulfur-forming enzyme(s) in the muscle and liver that were not observed in specimens exposed to sulfide in normoxia. Correspondingly, there were no changes in the levels of sulfate or sulfite in the muscle or liver. Instead, B. boddaerti detoxified sulfide mainly to sulfane sulfur in hypoxia. In conclusion, B. boddaerti was able to activate different mechanisms to detoxify sulfide, producing different types of detoxification products in normoxia and hypoxia.
Regular endurance training improves muscle oxidative capacity and reduces the risk of age-related disorders. Understanding the molecular networks underlying this phenomenon is crucial. Here, by exploiting the power of computational modeling, we show that endurance training induces profound changes in gene regulatory networks linking signaling and selective control of translation to energy metabolism and tissue remodeling. We discovered that knockdown of the mTOR-independent factor Eif6, which we predicted to be a key regulator of this process, affects mitochondrial respiration efficiency, ROS production, and exercise performance. Our work demonstrates the validity of a data-driven approach to understanding muscle homeostasis.
Developing experimental models to study ischemic heart disease is necessary for understanding of biological mechanisms to improve the therapeutic approaches for restoring cardiomyocytes function following injury. The aim of this study was to develop an in vitro hypoxic/re-oxygenation model of ischemia using primary human cardiomyocytes (HCM) and define subsequent cytotoxic effects. HCM were cultured in serum and glucose free medium in hypoxic condition with 1% O2 ranging from 30 min to 12 h. The optimal hypoxic exposure time was determined using Hypoxia Inducible Factor 1α (HIF-1α) as the hypoxic marker. Subsequently, the cells were moved to normoxic condition for 3, 6 and 9 h to replicate the re-oxygenation phase. Optimal period of hypoxic/re-oxygenation was determined based on 50% mitochondrial injury via 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide assay and cytotoxicity via lactate dehydrogenase (LDH) assay. It was found that the number of cells expressing HIF-1α increased with hypoxic time and 3 h was sufficient to stimulate the expression of this marker in all the cells. Upon re-oxygenation, mitochondrial activity reduced significantly whereas the cytotoxicity increased significantly with time. Six hours of re-oxygenation was optimal to induce reversible cell injury. The injury became irreversible after 9 h as indicated by > 60% LDH leakage compared to the control group cultured in normal condition. Under optimized hypoxic reoxygenation experimental conditions, mesenchymal stem cells formed nanotube with ischemic HCM and facilitated transfer of mitochondria suggesting the feasibility of using this as a model system to study molecular mechanisms of myocardial injury and rescue.
Tissue engineering scaffolds with oxygen generating elements have shown to be able to increase the level of oxygen and cell survivability in specific conditions. In this study, biphasic calcium phosphate (BCP) scaffolds with the composition of 60% hydroxyapatite (HA) and 40% beta-tricalcium phosphate (β-TCP), which have shown a great potential for bone tissue engineering applications, were fabricated by a direct-write assembly (robocasting) technique. Then, the three-dimensional (3D)-printed scaffolds were coated with different ratios of an oxygen releasing agent, calcium peroxide (CPO), which encapsulated within a polycaprolactone (PCL) matrix through dip-coating, and used for in situ production of oxygen in the implanted sites. The structure, composition and morphology of the prepared scaffolds were characterized by different techniques. The oxygen release kinetics and biological investigations of the scaffolds were also studied in vitro. The results showed that oxygen release behaviour was sustained and dependant on the concentration of CPO encapsulated in the PCL coating matrix. It was also demonstrated that the coated scaffolds, having 3% CPO in the coating system, could provide a great potential for promoting bone ingrowth with improving osteoblast cells viability and proliferation under hypoxic conditions. The findings indicated that the prepared scaffolds could play a significant role in engineering of large bone tissue implants with limitations in oxygen diffusion.
The illegal administration of recombinant human erythropoietin (rHuEPO) among athletes is largely preferred over blood doping to enhance stamina. The advent of recombinant DNA technology allowed the expression of EPO-encoding genes in several eukaryotic hosts to produce rHuEPO, and today these performance-enhancing drugs are readily available. As a mimetic of endogenous EPO (eEPO), rHuEPO augments the oxygen carrying capacity of blood. Thus, monitoring the illicit use of rHuEPO among athletes is crucial in ensuring an even playing field and maintaining the welfare of athletes. A number of rHuEPO detection methods currently exist, including measurement of hematologic parameters, gene-based detection methods, glycomics, use of peptide markers, electrophoresis, isoelectric focusing (IEF)-double immunoblotting, aptamer/antibody-based methods, and lateral flow tests. This review gleans these different strategies and highlights the leading molecular recognition elements that have potential roles in rHuEPO doping detection.
Cell-based regenerative therapies, based on in vitro propagation of stem cells, offer tremendous hope to many individuals suffering from degenerative diseases that were previously deemed untreatable. Due to the self-renewal capacity, multilineage potential, and immunosuppressive property, mesenchymal stem cells (MSCs) are considered as an attractive source of stem cells for regenerative therapies. However, poor growth kinetics, early senescence, and genetic instability during in vitro expansion and poor engraftment after transplantation are considered to be among the major disadvantages of MSC-based regenerative therapies. A number of complex inter- and intracellular interactive signaling systems control growth, multiplication, and differentiation of MSCs in their niche. Common laboratory conditions for stem cell culture involve ambient O₂ concentration (20%) in contrast to their niche where they usually reside in 2-9% O₂. Notably, O₂ plays an important role in maintaining stem cell fate in terms of proliferation and differentiation, by regulating hypoxia-inducible factor-1 (HIF-1) mediated expression of different genes. This paper aims to describe and compare the role of normoxia (20% O₂) and hypoxia (2-9% O₂) on the biology of MSCs. Finally it is concluded that a hypoxic environment can greatly improve growth kinetics, genetic stability, and expression of chemokine receptors during in vitro expansion and eventually can increase efficiency of MSC-based regenerative therapies.
Municipal leachate was treated in an experimental unit of constructed wetlands of subsurface flow type. The parameters studied were organics (BOD and COD), solids and heavy metals (Zn, Ni, Cu, Cr and Pb). Using two types of emergent plants of Scirpus globulosus and Eriocaulon sexangulare, more than 80% removal was achieved for all the parameters. E. sexangulare removed organics and heavy metals better than Scirpus globulosus. A higher concentration of heavy metals in the influent did not change the removal efficiency.
Copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] has been the center of attention in the bio-industrial fields, as it possesses superior mechanical properties compared to poly(3-hydroxybutyrate) [P(3HB)]. The usage of oleic acid and 1-pentanol was exploited as the carbon source for the production of P(3HB-co-3HV) copolymer by using a locally isolated strain Cupriavidus sp. USMAA2-4. In this study, the productivity of polyhydroxyalkanoate (PHA) was improved by varying the frequency of feeding in fed-batch culture. The highest productivity (0.48 g/L/h) that represents 200 % increment was obtained by feeding the carbon source and nitrogen source three times and also by considering the oxygen uptake rate (OUR) and oxygen transfer rate (OTR). A significantly higher P(3HB-co-3HV) concentration of 25.7 g/L and PHA content of 66 wt% were obtained. The 3-hydroxyvalerate (3HV) monomer composition obtained was 24 mol% with the growth of 13.3 g/L. The different frequency of feeding carried out has produced a blend copolymer and has broadened the monomer distribution. In addition, increase in number of granules was also observed as the frequency of feeding increases. In general, the most glaring increment in productivity offer advantage for industrial P(3HB-co-3HV) production, and it is crucial in developing cost-effective processes for commercialization.
The fluctuation of domestic wastewater characteristic inhibits the current conventional microbial-based treatment. The bioremediation fungi has received attention and reported to be an effective alternative to treat industrial wastewater. Similar efficient performance is envisaged for domestic wastewater whereby assessed performance of fungi for varying carbon-to-nitrogen ratios in domestic wastewater is crucial. Thus, the performance of pre-grown wild-Serbian Ganoderma lucidum mycelial pellets (GLMPs) was evaluated on four different synthetic domestic wastewaters under different conditions of initial pH (pH 4, 5, and 7) and chemical oxygen demand (COD) to nitrogen (COD/N) ratio of 3.6:1, 7.1:1, 14.2:1, and 17.8:1 (C3.6N1, C7.1N1, C14.2N1, and C17.8N1). The COD/N ratios with a constant concentration of ammonia-nitrogen (NH3-N) were chosen on the basis of the urban domestic wastewater characteristics sampled at the inlet basin of a sewage treatment plant (STP). The parameters of pH, COD, and NH3-N were measured periodically during the experiment. The wild-Serbian GLMPs efficiently removed the pollutants from the synthetic sewage. The COD/N ratio of C17.8N1 wastewater had the best COD and NH3-N removal, as compared to the lower COD/N ratio, and the shortest treatment time was obtained in an acidic environment at pH 4. The highest percentage for COD and NH3-N removal achieved was 96.0% and 93.2%, respectively. The results proved that the mycelium of GLMP has high potential in treating domestic wastewater, particularly at high organic content as a naturally sustainable bioremediation system.
Phthalocyanines are excellent photosensitizers for photodynamic therapy as they have strong absorbance in the near infra-red region which is most relevant for in vivo activation in deeper tissular regions. However, most phthalocyanines present two major challenges, ie, a strong tendency to aggregate and low water-solubility, limiting their effective usage clinically. In the present study, we evaluated the potential enhancement capability of glycerol substitution on the photodynamic properties of zinc (II) phthalocyanines (ZnPc). Three glycerol substituted ZnPc, 1-3, (tetra peripherally, tetra non-peripherally and mono iodinated tri non-peripherally respectively) were evaluated in terms of their spectroscopic properties, rate of singlet oxygen generation, partition coefficient (log P), intracellular uptake, photo-induced cytotoxicity and vascular occlusion efficiency. Tetrasulfonated ZnPc (ZnPcS4) was included as a reference compound. Here, we showed that 1-3 exhibited 10-100 nm red-shifted absorption peaks with higher molar absorptivity, and at least two-fold greater singlet oxygen generation rates compared to ZnPcS4. Meanwhile, phthalocyanines 1 and 2 showed more hydrophilic log P values than 3 consistent with the number of glycerol attachments but 3 was most readily taken up by cells compared to the rest. Both phthalocyanines 2 and 3 exhibited potent phototoxicity against MCF-7, HCT-116 and HSC-2 cancer cell-lines with IC50 ranging 2.8-3.2 µM and 0.04-0.06 µM respectively, while 1 and ZnPcS4 (up to 100 µM) failed to yield determinable IC50 values. In terms of vascular occlusion efficiency, phthalocyanine 3 showed better effects than 2 by causing total occlusion of vessels with diameter <70 µm of the chorioallantoic membrane. Meanwhile, no detectable vascular occlusion was observed for ZnPcS4 with treatment under similar experimental conditions. These findings provide evidence that glycerol substitution, in particular in structures 2 and 3, is able to improve the photodynamic properties of ZnPc.
Volumetric mass transfer coefficient (kLa) is an important parameter in bioreactors handling viscous fermentations such as xanthan gum production, as it affects the reactor performance and productivity. Published literatures showed that adding an organic phase such as hydrocarbons or vegetable oil could increase the kLa. The present study opted for palm oil as the organic phase as it is plentiful in Malaysia. Experiments were carried out to study the effect of viscosity, gas holdup, and kLa on the xanthan solution with different palm oil fractions by varying the agitation rate and aeration rate in a 5 L bench-top bioreactor fitted with twin Rushton turbines. Results showed that 10% (v/v) of palm oil raised the kLa of xanthan solution by 1.5 to 3 folds with the highest kLa value of 84.44 h(-1). It was also found that palm oil increased the gas holdup and viscosity of the xanthan solution. The kLa values obtained as a function of power input, superficial gas velocity, and palm oil fraction were validated by two different empirical equations. Similarly, the gas holdup obtained as a function of power input and superficial gas velocity was validated by another empirical equation. All correlations were found to fit well with higher determination coefficients.
The aeration strategy ranging from intermittent to continuous aeration in the REACT period of moving bed sequencing batch reactor (MBSBR) was evaluated for simultaneous removal of 4-chlorophenol (4-CP) and nitrogen. The results show that the removal rates of 4-CP and ammonium nitrogen (NH(4)(+)-N) increased with the increase of continuous aeration period. In the presence of 4-CP, NH(4)(+)-N removal was mainly by the assimilation process. The removal of NH(4)(+)-N to oxidized nitrogen via oxidation was only observed after 4-CP was completely degraded with sufficient aeration period provided indicating the inhibitory effect of 4-CP on nitrification. As the intermittent aeration strategy would lead to slower 4-CP degradation resulting in the delay of nitrification process, continuous aeration would be the preferred strategy in the simultaneous removal of 4-CP and nitrogen in the MBSBR system.
The potential use of n-dodecane and n-hexadecane as oxygen vectors for enhancing hyaluronic acid (HA) biosynthesis by Streptococcus zooepidemicus ATCC 39920 was investigated using a 2-L stirred-tank bioreactor equipped with helical ribbon or Rushton turbine impellers. The volumetric fraction of the oxygen vector influenced the gas-liquid volumetric oxygen transfer coefficient (K(L)a) positively. Batch HA fermentation with 1% (v/v) n-dodecane or 0.5% (v/v) n-hexadecane addition was carried out at different impeller tip speeds. Even though cell growth was lower in the fermentation with oxygen vector addition, the HA productivity and molecular weight were higher when compared to the fermentation without oxygen vector at low impeller tip speed. The highest HA concentration (4.25 gHA/l) and molecular weight (1.54 × 10(7) Da) were obtained when 0.5% (v/v) n-hexadecane and 0.785 m/s impeller tip speed of helical ribbon were used.
A two-level fractional factorial design (FFD) was used to determine the effects of six factors, i.e. substrate (domestic wastewater sludge - DWS) and co-substrate concentration (wheat flour - WF), temperature, initial pH, inoculum size and agitation rate on the production of cellulase enzyme by Trichoderma harzianum in liquid state bioconversion. On statistical analysis of the results from the experimental studies, optimum process conditions were found to be temperature 32.5 degrees C, substrate concentration (DWS) 0.75% (w/w), co-substrate (WF) concentration 2% (w/w), initial pH 5, inoculum size 2% (v/w) and agitation 175 rpm. Analysis of variance (ANOVA) showed a high coefficient of determination (R2) of 0.975. Cellulase activity reached 10.2 FPU/ml at day 3 during the fermentation process which indicated about 1.5-fold increase in production compared to the cellulase activity obtained from the results of design of experiment (6.9 FPU/ml). Biodegradation of DWS was also evaluated to verify the efficiency of the bioconversion process as a waste management method.