Biphasic Response Curve Research Articles

Hormesis Responses of Photosystem II in Arabidopsis thaliana under Water Deficit Stress.

Since drought stress is one of the key risks for the future of agriculture, exploring the molecular mechanisms of photosynthetic responses to water deficit stress is, therefore, fundamental. By using chlorophyll fluorescence imaging analysis, we evaluated the responses of photosystem II (PSII) photochemistry in young and mature leaves of Arabidopsis thaliana Col-0 (cv Columbia-0) at the onset of water deficit stress (OnWDS) and under mild water deficit stress (MiWDS) and moderate water deficit stress (MoWDS). Moreover, we tried to illuminate the underlying mechanisms in the differential response of PSII in young and mature leaves to water deficit stress in the model plant A. thaliana. Water deficit stress induced a hormetic dose response of PSII function in both leaf types. A U-shaped biphasic response curve of the effective quantum yield of PSII photochemistry (ΦPSII) in A. thaliana young and mature leaves was observed, with an inhibition at MiWDS that was followed by an increase in ΦPSII at MoWDS. Young leaves exhibited lower oxidative stress, evaluated by malondialdehyde (MDA), and higher levels of anthocyanin content compared to mature leaves under both MiWDS (+16%) and MoWDS (+20%). The higher ΦPSII of young leaves resulted in a decreased quantum yield of non-regulated energy loss in PSII (ΦNO), under both MiWDS (-13%) and MoWDS (-19%), compared to mature leaves. Since ΦNO represents singlet-excited oxygen (1O2) generation, this decrease resulted in lower excess excitation energy at PSII, in young leaves under both MiWDS (-10%) and MoWDS (-23%), compared to mature leaves. The hormetic response of PSII function in both young and mature leaves is suggested to be triggered, under MiWDS, by the intensified reactive oxygen species (ROS) generation, which is considered to be beneficial for activating stress defense responses. This stress defense response that was induced at MiWDS triggered an acclimation response in A. thaliana young leaves and provided tolerance to PSII when water deficit stress became more severe (MoWDS). We concluded that the hormesis responses of PSII in A. thaliana under water deficit stress are regulated by the leaf developmental stage that modulates anthocyanin accumulation in a stress-dependent dose.

Bidirectional Responses of Eight Neuroinflammation-Related Transcriptional Factors to 64 Flavonoids in Astrocytes with Transposable Insulated Signaling Pathway Reporters.

Neuroinflammation is implicated in a variety of pathologies and is mechanistically linked to hyperactivation of glial cells in the central nervous system (CNS), predominantly in response to external stimuli. Multiple dietary factors were reported to alter neuroinflammation, but their actions on the relevant transcription factors in glia are not sufficiently understood. Here, an in vitro protocol employing cultured astroglial cells, which carry reporters of multiple signaling pathways associated with inflammation, was developed for screening environmental factors and synthetic drugs. Immortalized rat astrocyte line DI TNC1 was stably transfected with piggyBac transposon vectors containing a series of insulated reporters for the transcriptional activity of NF-κB, AP-1, signal transducer and activator of transcription 1 (STAT1), signal transducer and activator of transcription 3 (STAT3), aromatic hydrocarbon receptor (AhR), Nrf2, peroxisome proliferator-activated receptor γ (PPARγ), and HIF-1α, which is quantified via luciferase assay. Concatenated green fluorescent protein (GFP) expression was employed for simultaneous evaluation of cellular viability. Responses to a set of 64 natural and synthetic monomeric flavonoids representing six main structural classes (flavan-3-ols, flavanones, flavones, flavonols, isoflavones, and anthocyan(id)ins) were obtained at 10 and 50 μM concentrations. Except for HIF-1α, the activity of NF-κB and other transcription factors (TFs) in astrocytes was predominantly inhibited by flavan-3-ols and anthocyan(id)ins, while flavones and isoflavones generally activated these TFs. In addition, we obtained dose-response profiles for 11 flavonoids (apigenin, baicalein, catechin, cyanidin, epigallocatechin gallate, genistein, hesperetin, kaempferol, luteolin, naringenin, and quercetin) within the 1-100 μM range and in the presence of immune-stimulants and immune-suppressors. The flavonoid concentration profiles for TF-activation reveal biphasic response curves from the astrocytes. Apart from epigallocatechin gallate (EGCG), flavonoids failed to inhibit the NF-κB activation by proinflammatory agents [lipopolysaccharide (LPS), cytokines], but most of the tested polyphenols synergized with STAT3 inhibitors (stattic, ruxolitinib) against the activation of this TF in the astrocytes. We conclude that transposable insulated reporters of transcriptional activation represent a convenient neurochemistry tool in screening for activators/inhibitors of signaling pathways.

HDAC Inhibitor Titration of Transcription and Axolotl Tail Regeneration.

New patterns of gene expression are enacted and regulated during tissue regeneration. Histone deacetylases (HDACs) regulate gene expression by removing acetylated lysine residues from histones and proteins that function directly or indirectly in transcriptional regulation. Previously we showed that romidepsin, an FDA-approved HDAC inhibitor, potently blocks axolotl embryo tail regeneration by altering initial transcriptional responses to injury. Here, we report on the concentration-dependent effect of romidepsin on transcription and regeneration outcome, introducing an experimental and conceptual framework for investigating small molecule mechanisms of action. A range of romidepsin concentrations (0–10 μM) were administered from 0 to 6 or 0 to 12 h post amputation (HPA) and distal tail tip tissue was collected for gene expression analysis. Above a threshold concentration, romidepsin potently inhibited regeneration. Sigmoidal and biphasic transcription response curve modeling identified genes with inflection points aligning to the threshold concentration defining regenerative failure verses success. Regeneration inhibitory concentrations of romidepsin increased and decreased the expression of key genes. Genes that associate with oxidative stress, negative regulation of cell signaling, negative regulation of cell cycle progression, and cellular differentiation were increased, while genes that are typically up-regulated during appendage regeneration were decreased, including genes expressed by fibroblast-like progenitor cells. Using single-nuclei RNA-Seq at 6 HPA, we found that key genes were altered by romidepin in the same direction across multiple cell types. Our results implicate HDAC activity as a transcriptional mechanism that operates across cell types to regulate the alternative expression of genes that associate with regenerative success versus failure outcomes.

894-P: Monophasic Glucose Response Curve Is Associated with Increased C-Reactive Protein in Obese Adolescents

Three distinct glucose response curves, biphasic, monophasic and incessant increase, have been demonstrated in adults and adolescents during OGTT. Individuals with incessant increase and monophasic curves have increased risk of type 2 diabetes and decreased insulin sensitivity and secretion and thus are likely at increased risk for other cardiometabolic diseases. We, therefore, examined differences in traditional, and nontraditional cardiometabolic risk factors between obese adolescents with biphasic (n=9; age=12.6±0.9 yr, BMI=31.8±5.9 kg/m2; mean±SD) and monophasic (n=6; age=13.1±0.9; BMI=35.3±6.9) glucose response curves. Traditional risk factors were blood pressure and lipids while non-traditional risk factors included reactive hyperemia (RH), inflammatory markers [c-reactive protein (CRP), interleukin-6 (IL-6)], adiponectin, soluble intracellular adhesion molecule 1 (SICAM1), and insulin sensitivity and secretion (OGTT, Matsuda and insulinogenic indices). Systolic and diastolic blood pressure, triglycerides, HDL and LDL did not differ between groups. RH, Il-6 and Matsuda index also did not differ. Subjects with monophasic curves tended to have increased SICAM1 (260±33 vs. 184±21; mean±SE; p=0.087) and had significantly increased CRP levels (4.40±1.03 vs. 1.66±0.28 ng/ml; p=0.009) compared to subjects with biphasic curves. Adiponection (3570±450 vs. 2350±440 ng/ml; p=0.089) and insulin secretion (4.31±0.52 vs. 2.89±0.53 µU dl/mg ml; p=0.089) tended to be lower in subjects with monophasic curves. The difference in CRP remained significant when BMI was included as a covariate (p=0.02). Obese adolescents with monophasic glucose response curves to OGTT have increased inflammation compared to subjects with biphasic curves. Longitudinal studies are needed to determine whether this early phase of glucose intolerance causes increased inflammation or whether early inflammation reduces insulin secretion and causes early glucose intolerance. Disclosure R. P. Hoffman: Consultant; Self; Provention Bio, Inc., Other Relationship; Self; Medscape, Research Support; Self; Provention Bio, Inc. R. Cazeau: None. L. Rauch: None. J. A. Bauer: None. Funding National Center for Research Resources (UL1RR05755)

Hormetic responses in arbuscular mycorrhizal fungi

The concept of hormesis describes that the application of low concentrations of a toxic compound will stimulate growth and activity of an organism. Since it is unknown whether hormesis occurs in arbuscular mycorrhizal fungi (AMF) the present work was designed to reveal whether two fungicides would generate hormetic response curves for AMF performance. The effect of mancozeb and carbendazim on performance of three AMF in symbioses with pea was investigated. The fungicides were mixed uniformly into irradiated soil at three field dose equivalents, which were 1x, 5x and 25x for mancozeb and 0.01x, 0.1x and 1x for carbendazim. A nil fungicide treatment was included for each fungus and a mesh-enclosed, 33P-labelled soil patch enabled the measurement of AMF P uptake. Both fungicides generated biphasic response curves for AMF root colonization, which was largely enhanced by the two lower doses and suppressed by the highest. Besides, the lowest concentration of both fungicides increased the hyphal length-specific 33P uptake by one of the fungi, while 0.1x carbendazim also increased the 33P uptake by another. In contrast, the length of root-external hyphae was either decreased or unaffected by increasing fungicide doses. The biphasic fungicide responses of root colonization and hyphal P uptake were obtained in irradiated soil without AMF antagonists and therefore probably caused by direct effects on the AMF. Such hormetic response patterns may be common in ecosystems where AMF will usually be exposed to a range of abiotic and biotic stressors.

The Shape of the Glucose Response Curve During an Oral Glucose Tolerance Test: Forerunner of Heightened Glycemic Failure Rates and Accelerated Decline in β-Cell Function in TODAY.

Obese youth without diabetes with monophasic oral glucose tolerance test (OGTT) glucose response curves have lower insulin sensitivity and impaired β-cell function compared with those with biphasic curves. The OGTT glucose response curve has not been studied in youth-onset type 2 diabetes. Here we test the hypothesis that the OGTT glucose response curve at randomization in youth in the TODAY (Treatment Options for Type 2 Diabetes in Adolescents and Youth) study forecasts heightened glycemic failure rates and accelerated decline in β-cell function. OGTTs (n = 662) performed at randomization were categorized as monophasic, biphasic, or incessant increase. Demographics, insulin sensitivity (1/fasting insulin), C-peptide index (△C30/△G30), and β-cell function relative to insulin sensitivity (oral disposition index [oDI]) were compared among the three groups. At randomization, 21.7% had incessant increase, 68.6% monophasic, and 9.7% biphasic glucose response curves. The incessant increase group had similar insulin sensitivity but significantly lower C-peptide index and lower oDI, despite similar diabetes duration, compared with the other two groups. Glycemic failure rates were higher in the incessant increase group (58.3%) versus the monophasic group (42.3%) versus the biphasic group (39.1%) (P < 0.0001). The 6-month decline in C-peptide index (32.8% vs. 18.1% vs. 13.2%) and oDI (32.2% vs. 11.6% vs. 9.1%) was greatest in incessant increase versus monophasic and biphasic with no difference in insulin sensitivity. In the TODAY study cohort, an incessant increase in the OGTT glucose response curve at randomization reflects reduced β-cell function and foretells increased glycemic failure rates with accelerated deterioration in β-cell function independent of diabetes duration and treatment assignment compared with monophasic and biphasic curves. The shape of the OGTT glucose response curve could be a metabolic biomarker prognosticating the response to therapy in youth with type 2 diabetes.

The Shape of the Glucose Response Curve during an Oral Glucose Tolerance Test Was Associated with Muscle Insulin Sensitivity and Visceral Fat Accumulation in Nonobese Healthy Men

Previous studies reported that the shape of glucose response curve during an oral glucose tolerance test (OGTT) could be a marker of insulin sensitivity in obese subjects. The biphasic response curve was defined by a second rise in glucose concentration of &amp;gt;4.5mg/dl after the decline in glucose during OGTT and subjects with biphasic response curve were insulin sensitive and had low risk of type 2 diabetes compared with subjects with monophasic response curve. While type 2 diabetes is often developed in non-obese Asians, it is still unclear whether the subjects with biphasic response curve are also insulin sensitive in non-obese healthy men. To clarify this, we studied 49 non-obese (BMI&amp;lt;25 kg/m2) healthy Japanese men. We evaluated insulin sensitivity in muscle and liver by 2-step hyperinsulinemic euglycemic clamp. We also measured ectopic fat in muscle and liver and subcutaneous and viscera fat areas by 1H-MRS and MRI, respectively. Based on the shape of glucose response curve during OGTT, we divided the subjects into monophasic (n=31) and biphasic group (n=18). The 60- and 90-min glucose levels were significantly lower in biphasic group than monophasic group (60-min:157.5±30.9 vs. 126.4±31.3mg/dl, p&amp;lt;0.01, 90-min:123.4±23.4 vs. 103.8±20.6mg/dl, p&amp;lt;0.01), while glucose levels at other time points and insulinogenic index were comparable between the groups. In addition, biphasic group showed lower visceral fat area (82.7±27.8 vs. 62.6±24.2 cm2, P=0.014) and higher muscle insulin sensitivity (0.22±0.vs. 0.27±0.09 mg/kg FFM·min-1 ·μU-1·ml, P&amp;lt;0.020) compared with monophasic group. Ectopic fat accumulation in muscle and liver, subcutaneous fat area and hepatic insulin sensitivity were comparable between the groups. In conclusion, biphasic glucose response curve is a marker of elevated muscle insulin sensitivity with less visceral fat in non-obese Japanese healthy men. Disclosure H. Kaga: None. Y. Tamura: None. K. Takeno: None. S. Kakehi: None. Y. Someya: None. R. Suzuki: None. S. Kadowaki: None. D. Sugimoto: None. Y. Furukawa: None. T. Funayama: None. R. Kawamori: None. H. Watada: Advisory Panel; Self; AstraZeneca. Consultant; Self; Astellas Pharma US, Inc., AstraZeneca, Boehringer Ingelheim GmbH, Daiichi Sankyo Company, Limited, Sumitomo Dainippon Pharma Co., Ltd., Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceuticals America, Inc., Kyowa Hakko Kirin Co., Ltd., Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk A/S, Novartis AG, Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho Co., Ltd., Takeda Development Center Asia, Pte. Ltd.. Research Support; Self; Abbott, Astellas Pharma US, Inc., AstraZeneca, Bayer AG, Benefit One Health Care Co., Ltd., Boehringer Ingelheim GmbH, Bristol-Myers Squibb Company, Daiichi Sankyo Company, Limited, Sumitomo Dainippon Pharma Co., Ltd., Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceuticals America, Inc., Kyowa Hakko Kirin Co., Ltd., Johnson &amp; Johnson Diabetes Institute, LLC., Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Mochida Pharmaceutical Co., Ltd., Nitto Boseki Co., Ltd., Novartis AG, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Pfizer Inc., Sanofi, Sanwa Kagaku Kenkyusho Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Development Center Asia, Pte. Ltd., Terumo Medical Corporation.

Storage Compartments for Capillary Water Rarely Refill in an Intact Woody Plant.

Water storage is thought to play an integral role in the maintenance of whole-plant water balance. The contribution of both living and dead cells to water storage can be derived from rehydration and water-release curves on excised plant material, but the underlying tissue-specific emptying/refilling dynamics remain unclear. Here, we used x-ray computed microtomography to characterize the refilling of xylem fibers, pith cells, and vessels under both excised and in vivo conditions in Laurus nobilis In excised stems supplied with water, water uptake exhibited a biphasic response curve, and x-ray computed microtomography images showed that high water storage capacitance was associated with fiber and pith refilling as driven by capillary forces: fibers refilled more rapidly than pith cells, while vessel refilling was minimal. In excised stems that were sealed, fiber and pith refilling was associated with vessel emptying, indicating a link between tissue connectivity and water storage. In contrast, refilling of fibers, pith cells, and vessels was negligible in intact saplings over two time scales, 24 h and 3 weeks. However, those compartments did refill slowly when the shoot was covered to prevent transpiration. Collectively, our data (1) provide direct evidence that storage compartments for capillary water refill in excised stems but rarely under in vivo conditions, (2) highlight that estimates of capacitance from excised samples should be interpreted with caution, as certain storage compartments may not be utilized in the intact plant, and (3) question the paradigm that fibers play a substantial role in daily discharge/recharge of stem capacitance in an intact tree.

Male apoE*3-Leiden.CETP mice on high-fat high-cholesterol diet exhibit a biphasic dyslipidemic response, mimicking the changes in plasma lipids observed through life in men.

Physiological adaptations resulting in the development of the metabolic syndrome in man occur over a time span of several decades. This combined with the prohibitive financial cost and ethical concerns to measure key metabolic parameters repeatedly in subjects for the major part of their life span makes that comprehensive longitudinal human data sets are virtually nonexistent. While experimental mice are often used, little is known whether this species is in fact an adequate model to better understand the mechanisms that drive the metabolic syndrome in man. We took up the challenge to study the response of male apoE*3‐Leiden.CETP mice (with a humanized lipid profile) to a high‐fat high‐cholesterol diet for 6 months. Study parameters include body weight, food intake, plasma and liver lipids, hepatic transcriptome, VLDL – triglyceride production and importantly the use of stable isotopes to measure hepatic de novo lipogenesis, gluconeogenesis, and biliary/fecal sterol secretion to assess metabolic fluxes. The key observations include (1) high inter‐individual variation; (2) a largely unaffected hepatic transcriptome at 2, 3, and 6 months; (3) a biphasic response curve of the main metabolic features over time; and (4) maximum insulin resistance preceding dyslipidemia. The biphasic response in plasma triglyceride and total cholesterol appears to mimic that of men in cross‐sectional studies. Combined, these observations suggest that studies such as these can help to delineate the causes of metabolic derangements in patients suffering from metabolic syndrome.

The shape of the glucose concentration curve during an oral glucose tolerance test predicts risk for type 1 diabetes.

We aimed to examine: (1) whether specific glucose-response curve shapes during OGTTs are predictive of type 1 diabetes development; and (2) the extent to which the glucose-response curve is influenced by insulin secretion. Autoantibody-positive relatives of people with type 1 diabetes whose baseline OGTT met the definition of a monophasic or biphasic glucose-response curve were followed for the development of type 1 diabetes (n=2627). A monophasic curve was defined as an increase in OGTT glucose between 30 and 90min followed by a decline of ≥0.25mmol/l between 90 and 120min. A biphasic response curve was defined as a decrease in glucose after an initial increase, followed by a second increase of ≥0.25mmol/l. Associations of type 1 diabetes risk with glucose curve shapes were examined using cumulative incidence curve comparisons and proportional hazards regression. C-peptide responses were compared with and without adjustments for potential confounders. The majority of participants had a monophasic curve at baseline (n=1732 [66%] vs n=895 [34%]). The biphasic group had a lower cumulative incidence of type 1 diabetes (p<0.001), which persisted after adjustments for age, sex, BMI z score and number of autoantibodies (p<0.001). Among the monophasic group, the risk of type 1 diabetes was greater for those with a glucose peak at 90min than for those with a peak at 30min; the difference persisted after adjustments (p<0.001). Compared with the biphasic group, the monophasic group had a lower early C-peptide (30-0min) response, a lower C-peptide index (30-0min C-peptide/30-0min glucose), as well as a greater 2h C-peptide level (p<0.001 for all). Those with biphasic glucose curves have a lower risk of progression to type 1 diabetes than those with monophasic curves, and the risk among the monophasic group is increased when the glucose peak occurs at 90min than at 30min. Differences in glucose curve shapes between the monophasic and biphasic groups appear to be related to C-peptide responses.

Abstract PR04: Mechanisms and pathophysiologic relevance of fluid shear stress resistance in malignant cells

Abstract The mechanobiology of circulating tumor cells, detached from an extracellular matrix, is poorly understood. A longstanding idea in cancer biology is that during metastasis, cancer cells shed into the circulation from epithelial organs are inherently fragile and are mechanically destroyed by hemodynamic forces. To the contrary, we have recently shown that malignant cells, from diverse tissue types, are remarkably resistant to brief (millisecond) pulses of high-level fluid shear stress (FSS) that may be encountered in the microenvironment of the circulation as compared to benign cells [1]. FSS resistance is a phenotype conferred by a variety of oncogenic signaling pathways and involves both an enhanced ability of malignant cells to repair plasma membrane damage as well as to rapidly adapt to prior exposure to FSS to resist subsequent pulses of FSS. However, the mechanisms underlying the FSS resistance phenotype and whether these are relevant to cancer cells in tumor tissue or to circulating tumor cells was unknown. Here we show that cancer cells exposed to FSS exhibit an adaptive response, demonstrating an increased stiffness (Young's modulus) as measured by micropipette aspiration [2]. Increased stiffness of cancer cells is associated with reduced damage to the plasma membrane upon subsequent challenges with FSS. To examine the mechanisms underlying this response, we evaluated the Rho-dependent signaling pathway that controls cell contractility. Knockdown of RhoA, but not RhoC sensitize prostate cancer cells to FSS. Pharmacologic inhibition myosin II-based contractility also reduced prostate cancer cell resistance to FSS. To determine if cancer cells that exist in tumor tissue exhibit resistance to FSS, we isolated cell suspensions from mice bearing prostate-specific PTEN and/or p53 mutations and exposed them to repeated pulses of FSS. We found that, compared to wild-type control mice, prostate cells from tumor-bearing mice exhibited resistance to FSS characteristic of established human prostate cancer cell lines, indicating that our findings extend beyond established cell lines. Moreover, extending our findings to human tumors, we acutely isolated cells from PDX tumors from melanoma patients. 3 independently-derived tumors exhibited a characteristic biphasic response curve when exposed to multiple pulses of FSS. Interestingly, brief exposure to Vemurafenib altered the response profile of two of these isolates. To examine whether FSS impacts circulating tumor cells, we treated prostate cancer cell line acutely with blebbistatin, a myosin II inhibitor, and vehicle control and injected these differentially fluorescently-labeled cells along with 15 micron fluorescent beads. The beads lodge in the microvasculature and act as a reference for cell counts. Within one minute after injection, we euthanized the mice and isolated lungs. Labeled cells and beads were counted in frozen sections and the ratio of cells:beads was counted and compared to pre-injection ratios. ~60% of vehicle treated cancer cells survive intact in the lungs whereas only ~40% of blebbistatin-treated cells survive. This suggests that a single pass through the right mouse heart damages circulating tumor cells, but that a mechanism that depends on myosin II promotes survival of these cells. This is the first evidence that FSS resistance is a contributing factor for the survival of circulating tumor cells. This work is supported by NCI grants CA179981&amp;CA196202.

Cationic uremic toxins affect human renal proximal tubule cell functioning through interaction with the organic cation transporter

Several organic cations, such as guanidino compounds and polyamines, have been found to accumulate in plasma of patients with kidney failure due to inadequate renal clearance. Here, we studied the interaction of cationic uremic toxins with renal organic cation transport in a conditionally immortalized human proximal tubule epithelial cell line (ciPTEC). Transporter activity was measured and validated in cell suspensions by studying uptake of the fluorescent substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium-iodide (ASP(+)). Subsequently, the inhibitory potencies of the cationic uremic toxins, cadaverine, putrescine, spermine and spermidine (polyamines), acrolein (polyamine breakdown product), guanidine, and methylguanidine (guanidino compounds) were determined. Concentration-dependent inhibition of ASP(+) uptake by TPA, cimetidine, quinidine, and metformin confirmed functional endogenous organic cation transporter 2 (OCT2) expression in ciPTEC. All uremic toxins tested inhibited ASP(+) uptake, of which acrolein required the lowest concentration to provoke a half-maximal inhibition (IC50 = 44 ± 2 μM). A Dixon plot was constructed for acrolein using three independent inhibition curves with 10, 20, or 30 μM ASP(+), which demonstrated competitive or mixed type of interaction (K i = 93 ± 16 μM). Exposing the cells to a mixture of cationic uremic toxins resulted in a more potent and biphasic inhibitory response curve, indicating complex interactions between the toxins and ASP(+) uptake. In conclusion, ciPTEC proves a suitable model to study cationic xenobiotic interactions. Inhibition of cellular uptake transport was demonstrated for several uremic toxins, which might indicate a possible role in kidney disease progression during uremia.

When Long-Range Zero-Lag Synchronization is Feasible in Cortical Networks

Many studies have reported long-range synchronization of neuronal activity between brain areas, in particular in the beta and gamma bands with frequencies in the range of 14–30 and 40–80 Hz, respectively. Several studies have reported synchrony with zero phase lag, which is remarkable considering the synaptic and conduction delays inherent in the connections between distant brain areas. This result has led to many speculations about the possible functional role of zero-lag synchrony, such as for neuronal communication, attention, memory, and feature binding. However, recent studies using recordings of single-unit activity and local field potentials report that neuronal synchronization may occur with non-zero phase lags. This raises the questions whether zero-lag synchrony can occur in the brain and, if so, under which conditions. We used analytical methods and computer simulations to investigate which connectivity between neuronal populations allows or prohibits zero-lag synchrony. We did so for a model where two oscillators interact via a relay oscillator. Analytical results and computer simulations were obtained for both type I Mirollo–Strogatz neurons and type II Hodgkin–Huxley neurons. We have investigated the dynamics of the model for various types of synaptic coupling and importantly considered the potential impact of Spike-Timing Dependent Plasticity (STDP) and its learning window. We confirm previous results that zero-lag synchrony can be achieved in this configuration. This is much easier to achieve with Hodgkin–Huxley neurons, which have a biphasic phase response curve, than for type I neurons. STDP facilitates zero-lag synchrony as it adjusts the synaptic strengths such that zero-lag synchrony is feasible for a much larger range of parameters than without STDP.

A comparison of the shock-absorbing properties of cervical disc prosthesis bearing materials

Background Data Cervical arthroplasty offers theoretical advantages over traditional spinal fusion, including elimination of adjacent segment disease and elimination of the risk of pseudoarthrosis formation. Initial studies of cervical arthroplasty have shown promising results, however, the ideal design characteristics for disc replacement constructs have not been determined. The current study seeks to quantify the differences in the shock absorption characteristics of three commonly used materials in cervical disc arthroplasty. Methods Three different nucleus materials, polyurethane (PU), polyethylene (PE) and a titanium-alloy (Ti) were tested in a humidity- and temperature-controlled chamber. Ten of each nucleus type underwent three separate mechanical testing protocols to measure 1) dynamic stiffness, 2) quasi-static stiffness, 3) energy absorption, and 4) energy dissipation. The results were compared using analysis of variance. Results PU had the lowest mean dynamic stiffness (435 ± 13 N/mm, P < .0001) and highest energy absorption (19.4 ± 0.1 N/mm, P < .0001) of all three nucleus materials tested. PU was found to have significantly higher energy dissipation (viscous damping ratio 0.017 ± 0,001, P < .0001) than the PE or TI nuclei. PU had the lowest quasi-static stiffness (598 ± 23 N/mm, P < .0001) of the nucleus materials tested. A biphasic response curve was observed for all of the PU nuclei tests. Conclusions Polyurethane absorbs and dissipates more energy and is less stiff than either polyethylene or titanium. Level of Evidence Basic Science/Biomechanical Study. Clinical Relevance This study characterizes important differences in biomechanical properties of materials that are currently being used for different cervical disc prostheses.

Antibody responses to pertussis toxin display different kinetics after clinical Bordetella pertussis infection than after vaccination with an acellular pertussis vaccine

The measurement of IgG anti-pertussis toxin (IgG anti-PT) antibodies by ELISA is a frequently used method for studying the antibody responses after pertussis vaccination and after Bordetella pertussis infection. Such responses vary according to the different vaccines used as well as to the immunization and infection history of the participants. In the present study, the decay kinetics of the IgG anti-PT antibody response was determined for 71 Danish children and adults with bacteriologically confirmed B. pertussis infection and for 20 Danish adults booster-vaccinated with an acellular pertussis vaccine. For both groups, biphasic decay was seen, but the individual antibody responses varied greatly. No differences related to age were seen. Within each group, individual decay profiles showed parallel log-linear decay for the late part of the response. Antibody half-life was calculated for the late, slower part of the biphasic response curves for both groups (>5 months after diagnosis for individuals with confirmed infection; >3 months for vaccinated individuals). The median half-life for post-infection antibodies was 221 days [interquartile range (IQR) 159-314 days, 36 individuals], and the median half-life for post-vaccination antibodies was 508 days (IQR 428-616 days, 14 individuals). This difference was statistically significant (P<0.0001). Thus, in this setting, we found that the IgG anti-PT antibody decay after an infection with B. pertussis is more than twice as fast as the decay after booster vaccination with an acellular pertussis vaccine. Such knowledge of the IgG anti-PT decay kinetics is crucial for interpretation of serological data that will be used either for diagnosis or for epidemiological studies and surveillance of B. pertussis infections.

Fluorescence optical detection in situ for real‐time monitoring of cytochrome P450 enzymatic activity of liver cells in multiple microfluidic devices

We describe an in situ fluorescence optical detection system to demonstrate real-time and non-invasive detection of reaction products in a microfluidic device while under perfusion within a standard incubator. The detection system is designed to be compact and robust for operation inside a mammalian cell culture incubator for quantitative detection of fluorescent signal from microfluidic devices. When compared to a standard plate reader, both systems showed similar biphasic response curves with two linear regions. Such a detection system allows real-time measurements in microfluidic devices with cells without perturbing the culture environment. In a proof-of-concept experiment, the cytochrome P450 1A1/1A2 activity of a hepatoma cell line (HepG2/C3A) was monitored by measuring the enzymatic conversion of ethoxyresorufin to resorufin. The hepatoma cell line was embedded in Matrigel(TM) construct and cultured in a microfluidic device with medium perfusion. The response of the cells, in terms of P450 1A1/1A2 activity, was significantly different in a plate well system and the microfluidic device. Uninduced cells showed almost no activity in the plate assay, while uninduced cells in Matrigel(TM) with perfusion in a microfluidic device showed high activity. Cells in the plate assay showed a significant response to induction with 3-Methylcholanthrene while cells in the microfluidic device did not respond to the inducer. These results demonstrate that the system is a potentially useful method to measure cell response in a microfluidic system.

Pet stimulates macrophage and epithelial cells MAPKp38 activity within a different time frame

Pet belongs to SPATEs toxin family and it is able to trigger several responses on the host. Such consequences to the host or bacteria itself are under aggressive investigation, as to understand how to support host defense mechanisms. NFκB/MAPK p38 mediated is one of the pathways involved in producing pro‐inflammatory interleukins. The kinase seemingly participates in epithelial survival before challenges. Considering that each cellular type conveys different protection functions for the host, the question arises of whether the pattern response induced by Pet is the same. Here, we established the MAPKp38 activation time course in murine macrophages and in HEp‐2 epithelial cells. A biphasic response curve was obtained for both cell types, although one of the activation peaks was different (30 min/12 hours and 30 min/48 h for macrophages and HEp‐2 cells, respectively). The first activity peak is related to external prompting of MAPKp38, while at least in macrophages, the latter response could be associated either with internalized Pet or a response induced by a product of primed macrophages. Studies of Pet internalization pattern for HEp‐2 cells are being carried out.

A quantitative real-time RT-PCR assay for salmon IGF-I mRNA, and its application in the study of GH regulation of IGF-I gene expression in primary culture of salmon hepatocytes

The hormone insulin-like growth factor-I (IGF-I) regulates vertebrate growth. The liver produces most circulating IGF-I, under the control of pituitary growth hormone (GH) and nutritional status. To study the regulation of liver IGF-I production in salmon, we established a primary hepatocyte culture system and developed a TaqMan quantitative real-time RT-PCR assay for salmon IGF-I gene expression. A portion of the coho salmon acidic ribosomal phosphoprotein P0 (ARP) cDNA was sequenced for use as a reference gene. A systematic bias across the 96 well PCR plate was discovered in an initial IGF-I assay, which was corrected when the assay was redesigned. IGF-I mRNA levels measured with the validated assay correlated well with levels measured with an RNase protection assay, and were highest in liver compared with other tissues. We examined the time course of hepatocyte IGF-I gene expression over 48 h in culture, the response to a range of GH concentrations in hepatocytes from fed and fasted fish, and potential effects of variation in IGF-I in the medium. IGF-I gene expression decreased over time in culture in hepatocytes in plain medium, and in cells treated with 5 nM GH with or without a combination of metabolic hormones (1 μM insulin, 100 nM triiodothyronine, and 0.1 nM dexamethasone). GH stimulated IGF-I gene expression at all time points. In cells treated with GH plus metabolic hormones, IGF-I gene expression was intermediate between the controls and GH alone. Increasing concentrations of GH resulted in biphasic IGF-I gene expression response curves in cells from fed and fasted fish, with the threshold for stimulation from 0.5 to 2.5 nM GH, maximal response from 5 to 50 nM, and a reduced response at 500 nM. Medium IGF-I (5 nM) did not affect basal or GH stimulated IGF-I gene expression. This study shows that primary hepatocyte culture and the TaqMan IGF-I assay can be used to study the regulation of hepatic IGF-I gene expression in salmon, and provides the first evidence of a biphasic response to GH concentration in fish hepatocyte culture.

Functional characterization of alpha(1)-adrenoceptor subtypes in human skeletal muscle resistance arteries.

alpha(1)-adrenoceptor subtypes in human skeletal muscle resistance arteries were characterized using agonists noradrenaline (non-selective) and A61603 (alpha(1A)-selective), the antagonists prazosin (non-selective), 5-methyl-urapidil (alpha(1A)-selective) and BMY7378 (alpha(1D)-selective) and the alkylating agent chloroethylclonidine (preferential for alpha(1B)). Small arteries were obtained from the non-ischaemic skeletal muscle of limbs amputated for critical limb ischaemia and isometric tension recorded using wire myography. Prazosin antagonized responses to noradrenaline with a pA(2) value of 9.18, consistent with the presence of alpha(1)-adrenoceptors, although the Schild slope (1.32) was significantly different from unity. 5-Methyl-urapidil competitively antagonized responses to noradrenaline with a pK(B) value of 8.48 and a Schild slope of 0.99, consistent with the presence of alpha(1A)-adrenoceptors. In the presence of 300 nM 5-methyl-urapidil, noradrenaline exhibited biphasic concentration response curves, indicating the presence of a minor population of a 5-methyl-urapidil-resistant subtype. Contractile responses to noradrenaline were not affected by 1 microM chloroethylclonidine suggesting the absence of alpha(1B)-adrenoceptors. Maximum responses to noradrenaline and A61603 were reduced to a similar extent by 10 microM chloroethylclonidine, suggesting an effect of chloroethylclonidine at alpha(1A)-adrenoceptors at the higher concentration. BMY7378 (10 and 100 nM) had no effect on responses to noradrenaline. BMY7378 (1 microM) poorly shifted the potency of noradrenaline giving a pA(2) of 6.52. These results rule out the presence of the alpha(1D)-subtype. These results show that contractile responses to noradrenaline in human skeletal muscle resistance arteries are predominantly mediated by the alpha(1A)-adrenoceptor subtype with a minor population of an unknown alpha(1)-adrenoceptor subtype.

Cold tolerance in Drosophila: adaptive variations revealed by the analysis of starvation survival reaction norms

Three species of Drosophila were investigated for their capacity to survive without food (starvation tolerance) at seven different temperatures ranging from 0 to 25°C. In all cases biphasic response curves (reaction norms) were observed, corresponding either to special deleterious effects of cold or to a progressive exhaustion of reserves proportional to metabolic rate. The temperature at which survival was longest was called the threshold temperature. The position of the threshold exhibited adaptive changes, either due to acclimation in the same species, or to genetic variations evidenced between species. In D. melanogaster, adults grown at lower temperature (12°C) were more tolerant to cold than adults grown at higher temperatures (21, 25 or 30°C). This acclimation process shifted, in an adaptive way, the position of the threshold temperature from 6.2 to 7.5°C. A comparison of three different species grown at a single developmental temperature (21°C) revealed similar but greater adaptive differences in their threshold temperature: 4.8°C in the temperate D. subobscura, 7°C in the cosmopolitan D. melanogaster and 14.6°C in the tropical D. ananassae.