Litopenaeus vannamei oxygen consumption and HSP gene expression at cyclic conditions of hyperthermia and hypoxia

Although Litopenaeus vannamei is a widely studied species, the information on how the organisms respond to natural daily variations of environmental conditions such as temperature and dissolved oxygen, and how such conditions alter the physiological responses, is scarce. In the present work, the strategies used by shrimps to cope with temperature and dissolved oxygen fluctuations during 24 days were investigated through the evaluation of oxygen consumption and heat shock proteins (HSP) gene expression. During daily fluctuations, no change in oxygen consumption in the short-term, but a significant increase in the long-term during hyperthermia conditions was registered, whereas a significant decrease during hypoxia was observed during all the bioassay. On the other hand, HSP70 and HSP90 gene expression increased in gills under thermal stress but was down-regulated under hypoxia, in both the short- and the long-term. This study highlights that to counteract environmental variations of temperature and dissolved oxygen, the shrimps use molecular compensatory mechanisms (HSP gene expression) that are different to those used under constant hypoxic conditions, suggesting that hypoxia can compromise physiological cytoprotection.     

Temperature measurements in a capacitive system of deep loco-regional hyperthermia

Hyperthermia has been shown to be a medically useful procedure applicable for different indications. For the connection between clinical effects and heat, it is important to understand the actual temperatures achieved in the tissue. There are limited temperature data available when using capacitive hyperthermia devices even though this is worldwide the most widespread method for loco-regional heating. Hence, this study examines temperature measurements using capacitive heating. Bioequivalent phantoms were used for the measurements, which, however, do not consider perfusion in live tissue. In general, the required temperature impact for an effective cancer therapy should need an increase of 0.2°C/min, which has been achieved. In the described tests on the non-perfused dummy, on average, the temperature increases by approximately 2°C in the first 12 min. The temperature difference relative to the starting temperature was 10–12°C within a therapy time of 60 min (rising from the initial room temperature between 20–24°C and 32–34°C). The average deviation with three individual measurements each on different days in a specified localization was 2°C. The minimum temperature difference was 4.2°C, and the maximum value was reached in the liver with 10.5°C. These values were achieved with a moderate energy input of 60–150 watts, with much higher performance outputs still available.

These results show that the tested capacitive device is capable of achieving quick temperature increase with a sufficient impact into the depth of a body.     

Inactivation of Pseudomonas aeruginosa PA01 biofilms by hyperthermia using superparamagnetic nanoparticles

The primary goal of this study was to develop a new strategy to inactivate bacterial biofilms using the thermal stress derived from superparamagnetic iron oxide nanoparticles (SPIONs) in an alternating current (AC) magnetic field. A large number of studies have examined the inactivation of bacterial biofilms using antimicrobial agents; however, there have been no attempts to inactivate biofilms by hyperthermia using SPIONs. In this study, a SPION solution was added to Pseudomonas aeruginosa (P. aeruginosa) PA01 biofilm, and heat was generated by placing the nanoparticle-containing biofilm in an AC magnetic field. The heating temperature was dependent on the concentration of the added SPION solution. More than 4 log inactivation of the PA01 biofilm was obtained using a 60 mg mL⁻¹ SPION solution in 8 min, and this resulted in a dramatic disintegration of the bacterial cell membrane in the biofilm. This inactivation was largely due to the thermal effect. Local heating of a specific area is also possible using this method, and the heating temperature can be easily adjusted by controlling the concentration of the SPION solution. Therefore, hyperthermia using magnetic nanoparticles holds promise as an effective tool for inactivating the bacterial biofilm.     

3D in silico study of magnetic fluid hyperthermia of breast tumor using Fe3O4 magnetic nanoparticles

Modeling and simulation of the temperature distribution, the mass concentration, and the heat transfer in the breast tissue are hot issues in magnetic fluid hyperthermia treatment of cancer. The breast tissue can be visualized as a porous matrix with saturated blood. In this paper, 3D in silico study of breast cancer hyperthermia using magnetic nanoparticles (MNPs) is conducted. The 3D FEM models are incorporated to investigate the infusion and backflow of nanofluid in the breast tumor, the diffusion of nanofluid, temperature distribution during the treatment, and prediction of the fraction of tumor necrosis while dealing with the thermal therapy. All the hyperthermia procedures are simulated and analyzed on COMSOL Multiphysics. The sensitivity of frequency and amplitude of the applied magnetic field (AMF) is investigated on the heating effect of the tumor. The mesh dependent solution of Penne's bioheat model is also analyzed. The simulated results demonstrate successful breast cancer treatment using MNPs with minimum side effects. Validation of current simulations results with experimental studies existing in literature advocates the success of our therapy. The increase in the amplitude and frequency of the AMF increases of the temperature in the tumor. The variation of mesh from coarser to finer increased the temperature through small fractions. We have also simulated the magnetic induction problem where the magnetic field is generated by current-carrying coil conductors induce heat in nearby breast tumors due to excitation of MNPs by magnetic flux. This research will aid treatment protocols and real-time clinical breast cancer treatments.     

Antitumor effects of combined therapy of recombinant heat shock protein 70 and hyperthermia using magnetic nanoparticles in an experimental subcutaneous murine melanoma

Heat shock proteins (HSPs) are recognized as significant participants in cancer immunity. We previously reported that HSP70 expression following hyperthermia using magnetic nanoparticles induces antitumor immunity. In the present study, we examine whether the antitumor immunity induced by hyperthermia is enhanced by administration of recombinant HSP70 protein into the tumor in situ. Hyperthermia was conducted using our original magnetite cationic liposomes (MCLs), which have a positive surface charge and generate heat in an alternating magnetic field (AMF) due to hysteresis loss. MCLs and recombinant mouse HSP70 (rmHSP70) were injected into melanoma nodules in C57BL/6 mice, which were subjected to AMF for 30 min. Temperature within the tumor reached 43°C and was maintained by controlling the magnetic field intensity. The combined treatment strongly inhibited tumor growth over a 30-day period and complete regression of tumors was observed in 20% (2/10) of mice. It was also found that systemic antitumor immunity was induced in the cured mice. This study suggests that novel combined therapy using exogenous HSP70 and hyperthermia has great potential in cancer treatment.     

Effect of heat dissipation of superparamagnetic nanoparticles in alternating magnetic field on three human cancer cell lines in magnetic fluid hyperthermia

ABSTRACT

Purpose: The purpose of this study was to propose a method for constructing the software setup required for investigating thermal effect of superparamagnetic nanoparticles on three human cell lines. This article aimed to examine the required nanoparticle dose, frequency, field intensity and the exposure time. Materials and methods: In the present study, first some general details were given about design and construction of the setup required for generating a safe magnetic field in order to examine the thermal effect of superparamagnetic nanoparticles on three human cancer cell lines, cultured under laboratory conditions. Next, a series of experimental tests were conducted to study the effect of magnetic field, on the cells. Finally, by applying three types of iron-based nanoparticles with mean diameters of 8, 15 and 20 nm, for 30 min, the temperature rise and specific absorption rate (SAR) were calculated. Results: By conducting experimental tests, the maximum temperature rise at the resonance frequency of the coil was reported to be 80 kHz, and it was observed that all the cells died when temperature of the cells reached 42°C/30 min. Based on the experiments, it was observed that magnetic field with intensity of 8 kA/m within the frequency range of 80–180 kHz did not have any effect on the cells. Conclusions: Based on the results, it can be concluded that the nanoparticle dose of 80 µg/ml with diameter of 8 nm at the resonance frequency of coil for 30 min was sufficient to destroy all the cancerous cells in the flask.     

Evaluation of a static granular bed reactor using a chemical oxygen demand balance and mathematical modeling

In order to evaluate the static granular bed reactor (SGBR), a chemical oxygen demand (COD) balance was used along with a mathematical model. The SGBR was operated with an organic loading rate (OLR) ranging from 0.8 to 5.5 kg/m³ day at 24 °C. The average COD removal efficiency was 87.4%, and the removal efficiencies of COD, carbohydrates, and proteins increased with an OLR, while the lipids removal efficiency was not a function of an OLR. From the results of the COD balance, the yield of biomass increased with an OLR. The SGBR was modeled using the general transport equation considering advection, diffusion, and degradation by microorganisms, and the first-order reaction rate constant was 0.0166/day. The simulation results were in excellent agreement with experimental data. In addition, the SGBR model provided mechanistic insight into why the COD removal efficiency in the SGBR is proportional to an OLR.     

Invariance of demographic parameters using total or viable eggs

Recently Mou et al. (J. Appl. Entomol., 139, 2015 and 00) recommended that in population studies in which hatch rates vary with maternal age, demographic parameters should be calculated excluding unhatched eggs. A mathematical proof was provided to support this. In this note, we expose a flaw in their proof and show that the demographic parameters do not differ by considering either all eggs or only viable ones, as long as the beginning and end of a generation are defined accordingly.     

Evaluation of the roles of passive and active control of balance using a balance control model

At present there is a lack of consensus regarding the relative roles of passive and active control of quiet upright stance. In the current work, this issue was investigated using two simulation models based on contemporary theories. Specifically, the two models, both of which assumed active control torques to be generated from an optimal neural controller, differed with respect to whether or not passive control torques (stiffness and damping) were included. Model parameters were specified using experimental center-of-pressure (COP) time series obtained during upright stance, and comparisons then made between simulated and actual COP-based measures. Including both active and passive joint torques in the control model did not appear to lead to any improvement in the ability to simulate COP compared with only including active joint torque. Further, simulated passive control torques were typically less than 10% of the active control torques, though some exceptions were found. These results, along with existing empirical evidence, suggest that active control torque is dominant in maintaining balance during upright stance.     

Outdoor continuous culture of Porphyridium cruentum in a tubular photobioreactor: quantitative analysis of the daily cyclic variation of culture parameters

The present work reports on the daily cyclic variation of oxygen generation rates, carbon consumption rates, photosynthetic activities, growth rates and biochemical composition of the biomass in a pilot plant continuous outdoor culture of the microalgae Porphyridium cruentum. A linear relationship between the external irradiance and the average irradiance inside the culture was found. In addition, the oxygen generation and carbon consumption rates were found to be a function of the average irradiance inside the culture. A reduction in photosynthetic activity of the cells at noon and recovery in the afternoon was also observed. Therefore, the cells showed a short-term response of parameters such as oxygen generation rate as well as carbon consumption rate with external and average irradiance; a model of photosynthesis rate considering photoinhibition is proposed. This model is a useful tool for the operation and scaleup of tubular photobioreactors, and can be used for determining CO₂ requirements of the system. The higher the photosynthesis rates, the lower the carbon losses, ranging from 25% at noon to 100% during the night. The growth rate showed a linear relationship with the daily mean average irradiance inside the culture with a long-term response. Likewise, a linear relationship among the oxygen generation rate and the growth rate was obtained. With respect to the biochemical composition of the biomass, the cells showed a long-term response of metabolic routes to mean daily culture conditions. During the illuminated period, energy was stored as carbohydrates and synthesis of proteins was low. During the night, the stored carbohydrates were consumed. The fatty acid dry weight (DW) content decreased during the daylight period, whereas the fatty acid profile, as total fatty acids, was a function of growth rate. A short-term variation of exopolysaccharides synthesis with solar irradiance was also observed, i.e. the higher the external irradiance the higher the excretion of exopolysaccharides as a protection against adverse culture conditions.     

Calcite microcrystals in the pineal gland of the human brain: first physical and chemical studies

A new form of biomineralization has been studied in the pineal gland of the human brain. It consists of small crystals that are less than 20 microm in length and that are completely distinct from the often observed mulberry-type hydroxyapatite concretions. A special procedure was developed for isolation of the crystals from the organic matter in the pineal gland. Cubic, hexagonal, and cylindrical morphologies have been identified using scanning electron microscopy. The crystal edges were sharp whereas their surfaces were very rough. Energy dispersive spectroscopy showed that the crystals contained only the elements calcium, carbon, and oxygen. Selected area electron diffraction and near infrared Raman spectroscopy established that the crystals were calcite. With the exception of the otoconia structure of the inner ear, this is the only known nonpathological occurrence of calcite in the human body. The calcite microcrystals are probably responsible for the previously observed second harmonic generation in pineal tissue sections. The complex texture structure of the microcrystals may lead to crystallographic symmetry breaking and possible piezoelectricity, as is the case with otoconia. It is believed that the presence of two different crystalline compounds in the pineal gland is biologically significant, suggesting two entirely different mechanisms of formation and biological functions. Studies directed toward the elucidation of the formation and functions, and possible nonthermal interaction with external electromagnetic fields are currently in progress.     

Purification of two enolases from human brain using a chromatofocusing column

When a purified preparation of rat αγ enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) was applied to a chromatofocusing column, the enolase was almost completely dissociated and recombined to form αα and γγ enolases, which were eluted at different fractions from the column. Using these phenomena, two homo-dimer forms (αα and γγ) of human brain enolase were purified from a crude preparation of the hybrid αγ enolase by the chromatofocusing, and subsequent chromatography on a QAE-Sephadex column (αα) or a DEAE-Sephadex column (γγ). Each purified preparation showed a single band on SDS-gel electrophoresis with a relative mobility corresponding to a molecular weight of about 50 000. Amino acid analysis, peptide mapping analysis with a limited proteolysis and immunochemical studies of the purified αα and γγ enolases revealed that the two subunits, α and γ, are distinct proteins. The antisera to human αα or γγ enolase cross-reacted with the respective form of rat enolase.     

Method for therapeutic drug monitoring of azole antifungal drugs in human serum using LC/MS/MS

Fungal infections occur in immunocompromised patients. Azole antifungal agents are used for the prophylaxis and treatment of these infections. The interest in therapeutic drug monitoring azole agents has increased over the last few years. Inter- and intra-patient variability of pharmacokinetics, drug–drug interactions, serum concentration related toxicity and success of therapy has stressed the need of frequently therapeutic drug monitoring of the drugs, belonging to the group of azoles. Therefore a simple, rapid and flexible method of analysis is required. This method is based on the precipitation of proteins in human serum with LC/MS/MS detection. Validation was performed according to the guidelines for bioanalytical method validation of the food and drug administration agency. The four most used azole drugs can be detected in human serum within the clinical relevant serum levels with good accuracy and reproducibility at the limit of quantification. Intra- and inter-day validation demonstrated good accuracy and reproducibility. A rapid, sensitive and flexible LC/MS/MS method has been developed and validated to measure voriconazole (VRZ), fluconazole (FLZ), itraconazole (ITZ) and posaconazole (PSZ) in human serum. This new method is suitable for clinical pharmacokinetic studies and routine monitoring in daily practice.     

Modeling human mortality using mixtures of bathtub shaped failure distributions

Aging and mortality is usually modeled by the Gompertz-Makeham distribution, where the mortality rate accelerates with age in adult humans. The resulting parameters are interpreted as the frailty and decrease in vitality with age. This fits well to life data from 'westernized' societies, where the data are accurate, of high resolution, and show the effects of high quality post-natal care. We show, however, that when the data are of lower resolution, and contain considerable structure in the infant mortality, the fit can be poor. Moreover, the Gompertz-Makeham distribution is consistent with neither the force of natural selection, nor the recently identified 'late life mortality deceleration'. Although actuarial models such as the Heligman-Pollard distribution can, in theory, achieve an improved fit, the lack of a closed form for the survival function makes fitting extremely arduous, and the biological interpretation can be lacking. We show, that a mixture, assigning mortality to exogenous or endogenous causes, using the reduced additive and flexible Weibull distributions, models well human mortality over the entire life span. The components of the mixture are asymptotically consistent with the reliability and biological theories of aging. The relative simplicity of the mixture distribution makes feasible a technique where the curvature functions of the corresponding survival and hazard rate functions are used to identify the beginning and the end of various life phases, such as infant mortality, the end of the force of natural selection, and late life mortality deceleration. We illustrate our results with a comparative analysis of Canadian and Indonesian mortality data.     

Evaluation of heritable determinants of blood and brain serotonin homeostasis using recombinant inbred mice

The biogenic amine serotonin (5‐HT, 5‐hydroxytryptamine) exerts powerful, modulatory control over multiple physiological functions in the brain and periphery, ranging from mood and appetite to vasoconstriction and gastrointestinal motility. In order to gain insight into shared and distinct molecular and phenotypic networks linked to variations in 5‐HT homeostasis, we capitalized on the stable genetic variation present in recombinant inbred mouse strains. This family of strains, all derived from crosses between C57BL/6J and DBA/2J (BXD) parents, represents a unique, community resource with approximately 40 years of assembled phenotype data that can be exploited to explore and test causal relationships in silico. We determined levels of 5‐HT and 5‐hydroxyindoleacetic acid from whole blood, midbrain and thalamus/hypothalamus (diencephalon) of 38 BXD lines and both sexes. All 5‐HT measures proved highly heritable in each region, although both gender and region significantly impacted between‐strain correlations. Our studies identified both expected and novel biochemical, anatomical and behavioral phenotypes linked to 5‐HT traits, as well as distinct quantitative trait loci. Analyses of these loci nominate a group of genes likely to contribute to gender‐ and region‐specific capacities for 5‐HT signaling. Analysis of midbrain mRNA variations across strains revealed overlapping gene expression networks linked to 5‐HT synthesis and metabolism. Altogether, our studies provide a rich profile of genomic, molecular and phenotypic networks that can be queried for novel relationships contributing risk for disorders linked to perturbed 5‐HT signaling .     

Targeted enrichment and high‐resolution digital profiling of mitochondrial DNA deletions in human brain

Due largely to the inability to accurately quantify and characterize de novo deletion events, the mechanisms underpinning the pathogenic expansion of mtDNA deletions in aging and neuromuscular disorders remain poorly understood. Here, we outline and validate a new tool termed ‘Digital Deletion Detection’ (3D) that allows for high-resolution analysis of rare deletions occurring at frequencies as low as 1 × 10⁻⁸. 3D is a three-step process that includes targeted enrichment for deletion-bearing molecules, single-molecule partitioning of genomes into thousands of droplets for direct quantification via droplet digital PCR, and breakpoint characterization using massively parallel sequencing. Using 3D, we interrogated over 8 billion mitochondrial genomes to analyze the age-related dynamics of mtDNA deletions in human brain tissue. We demonstrate that the total deletion load increases with age, while the total number and diversity of unique deletions remain constant. Our data provide support for the hypothesis that expansion of pre-existing mutations is the primary factor contributing to age-related accumulation of mtDNA deletions.     

Evaluation of blood-brain barrier thiamine efflux using the in situ rat brain perfusion method

Thiamine is an essential, positively charged (under physiologic conditions), water-soluble vitamin requiring transport into brain. Brain thiamine deficiency has been linked to neurodegenerative disease by subsequent impairment of thiamine-dependent enzymes used in brain glucose/energy metabolism. In this report, we evaluate brain uptake and efflux of [3H]thiamine using the in situ rat brain perfusion technique. To confirm brain distribution was not related to blood-brain barrier endothelial cell uptake, we compared parenchymal and cell distribution of [3H]thiamine using capillary depletion. Our work supports previous literature findings suggesting blood-brain barrier thiamine uptake is via a carrier-mediated transport mechanism, yet extends the literature by redefining the kinetics with more sensitive methodology. Significantly, [3H]thiamine brain accumulation was influenced by a considerable efflux rate. Evaluation of the efflux mechanism demonstrated increased stimulation by the presence of increased vascular thiamine. The influx transport mechanism and efflux rate were each comparable throughout brain regions despite documented differences in glucose and thiamine metabolism. The observation that [3H]thiamine blood-brain barrier influx and efflux is regionally homogenous may have significant relevance to neurodegenerative disease linked to thiamine deficiency.     

Assessment of trace elements in human brain using inductively coupled plasma mass spectrometry

Recent brain research reveals a major role of trace elements in various diseases such as multiple sclerosis, Alzheimer's and Wilson's disease. The majority of published tissue concentrations dates back decades, and was assessed with various methods. Little is known about hemispherical differences, the correlation of trace elements or age-dependent changes in the human brain. Thus, the aim of this study was to examine trace element concentrations in different human brain regions after whole brain formalin fixation.549 samples of 13 brain regions were investigated in 11 deceased subjects without known history of brain pathology. Regional wet-to-dry mass ratios and concentrations of iron, copper, magnesium, manganese, calcium and zinc were determined using inductively coupled plasma mass spectrometry.Cortical gray matter revealed higher water content (wet-to-dry mass ratios 5.84–6.40) than white matter regions (wet-to-dry mass ratios 2.95–3.05). Element concentrations displayed specific regional differences. Good linear correlation of concentrations between elements was found for iron/copper as well as for manganese/magnesium (Spearman's rank correlation coefficient 0.74 and 0.65, respectively). Significant inter-hemispherical differences were found for copper in occipital white matter, for magnesium and calcium in putamen and for iron and copper in temporal white matter. An age dependent increase was seen in cortical gray matter for calcium, for magnesium in all regions except in cortical gray matter, for copper in substantia nigra and for zinc in occipital cortex.The presented trace element concentrations can serve as a fundamental basis for further brain research. Wet-to-dry mass ratios allow a comparison with reference data from other studies.     

Imaging incorporation of circulating docosahexaenoic acid into the human brain using positron emission tomography

Docosahexaenoic acid (DHA; 22:6n-3) is a critical constituent of the brain, but its metabolism has not been measured in the human brain in vivo. In monkeys, using positron emission tomography (PET), we first showed that intravenously injected [1-¹¹C]DHA mostly entered nonbrain organs, with ∼0.5% entering the brain. Then, using PET and intravenous [1-¹¹C]DHA in 14 healthy adult humans, we quantitatively imaged regional rates of incorporation (K*) of DHA. We also imaged regional cerebral blood flow (rCBF) using PET and intravenous [¹⁵O]water. Values of K* for DHA were higher in gray than white matter regions and correlated significantly with values of rCBF in 12 of 14 subjects despite evidence that rCBF does not directly influence K*. For the entire human brain, the net DHA incorporation rate J_in, the product of K*, and the unesterified plasma DHA concentration equaled 3.8 ± 1.7 mg/day. This net rate is equivalent to the net rate of DHA consumption by brain and, considering the reported amount of DHA in brain, indicates that the half-life of DHA in the human brain approximates 2.5 years. Thus, PET with [1-¹¹C]DHA can be used to quantify regional and global human brain DHA metabolism in relation to health and disease.