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In single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of ZMWs in parallel without sophisticated equipment. Polystyrene 1 μm dia. microbeads self-assemble on a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead interstices. The width of those interstices (and subsequent posts) is adjusted within 100–300 nm by partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology avoiding the availability and expense of sophisticated clean-room environments and equipment. [ABSTRACT FROM AUTHOR]

Proliferating cell nuclear antigen (PCNA) is essential for DNA replication and repair, and cell growth and survival. Previously, we identified a novel class of small molecules that bind directly to PCNA, stabilize PCNA trimer structure, reduce chromatin-associated PCNA, selectively inhibit tumor cell growth, and induce apoptosis. The purpose of this study was to investigate the combinatorial effects of lead compound PCNA-I1S with DNA damaging agents on cell growth, DNA damage, and DNA repair in four lines of human prostate and lung cancer cells. The DNA damage agents used in the study include ionizing radiation source cesium-137 (Cs-137), chemotherapy drug cisplatin (cisPt), ultraviolet-C (UV-C), and oxidative compound H2O2. DNA damage was assessed using immunofluorescent staining of γH2AX and the Comet assay. The homologous recombination repair (HRR) was determined using a plasmid-based HRR reporter assay and the nucleotide excision repair (NER) was indirectly examined by the removal of UV-induced cyclobutane pyrimidine dimers (CPD). We found that PCNA-I1S inhibited cell growth in a dose-dependent manner and significantly enhanced the cell growth inhibition induced by pretreatment with DNA damaging agents Cs-137 irradiation, UV-C, and cisPt. However, the additive growth inhibitory effects were not observed in cells pre-treated with PCNA-I1S, followed by treatment with cisPt. H2O2 enhanced the level of chromatin-bound PCNA in quiescent cells, which was attenuated by PCNA-I1S. DNA damage was induced in cells treated with either PCNA-I1S or cisPt alone and was significantly elevated in cells exposed to the combination of PCNA-I1S and cisPt. Finally, PCNA-I1S attenuated repair of DNA double strand breaks (DSBs) by HRR and the removal of CPD by NER. These data suggest that targeting PCNA with PCNA-I1S may provide a novel approach for enhancing the efficacy of chemotherapy and radiation therapy in treatment of human prostate and lung cancer. [ABSTRACT FROM AUTHOR]

Swim-up is a sperm purification method that is being used daily in andrology labs around the world as a simple step for in vitro sperm selection. This method accumulates the most motile sperm in the upper fraction and leaves sperm with low or no motility in the lower fraction. However, the underlying reasons are not fully understood. In this article, we compare metabolic rate, motility and sperm tail length of bovine sperm cells of the upper and lower fraction. The metabolic assay platform reveals oxygen consumption rates and extracellular acidification rates simultaneously and thereby delivers the metabolic rates in real time. Our study confirms that the upper fraction of bull sperm has not only improved motility compared to the cells in the lower fraction but also shows higher metabolic rates and longer flagella. This pattern was consistent across media of two different levels of viscosity. We conclude that the motility-based separation of the swim-up technique is also reflected in underlying metabolic differences. Metabolic assays could serve as additional or alternative, label-free method to evaluate sperm quality. [ABSTRACT FROM AUTHOR]

Activated platelets release adenosine trisphosphate (ATP) and bioluminescence analysis of ATP release is usually used to monitor activation of platelets induced by various stimulants. However, bioluminescence analysis of ATP possesses poor linearity, the signal is quickly attenuated, and the accuracy of ATP release from platelets is hard to determine accurately enough to be used in a high throughput screening of platelet inhibitors. The present study was designed to optimize bioluminescence analysis of ATP released by platelets and expand its application in high throughput screening of platelet inhibitors. The results showed that accuracy of ATP analysis was significantly improved by adding coenzyme A (CoA) and signal attenuation of ATP analysis was greatly postponed by adding bovine serum albumin (BSA) both in Hank's balanced salt solution (HBSS) and Tyrode's buffer. Furthermore, ATP release of activated platelets and inhibitory effects of Ly294002 and Staurosporine on platelet activation were accurately determined by our optimized bioluminescence analysis of ATP. Thus, we have successfully constructed an optimized bioluminescence analysis of ATP which can be used in high throughput screening of platelet inhibitors. [ABSTRACT FROM AUTHOR]

In nature, protozoa play a major role in controlling bacterial populations. This paper proposes a microfluidic device for the study of protozoa behaviors change due to their chemotactic response in the presence of bacterial cells. A three-channel microfluidic device was designed using a nitrocellulose membrane into which channels were cut using a laser cutter. The membrane was sandwiched between two glass slides; a Euglena suspension was then allowed to flow through the central channel. The two side channels were filled with either, 0.1% peptone as a negative control, or a Listeria suspension respectively. The membrane design prevented direct interaction but allowed Euglena cells to detect Listeria cells as secretions diffused through the nitrocellulose membrane. A significant number of Euglena cells migrated toward the chambers near the bacterial cells, indicating a positive chemotactic response of Euglena toward chemical cues released from Listeria cells. Filtrates collected from Listeria suspension with a series of molecular weight cutoffs (3k, 10k and 100k) were examined in Euglena chemotaxis tests. Euglena cells were attracted to all filtrates collected from the membrane filtration with different molecular weight cutoffs, suggesting small molecules from Listeria might be the chemical cues to attract protozoa. Headspace volatile organic compounds (VOC) released from Listeria were collected, spiked to 0.1% peptone and tested as the chemotactic effectors. It was discovered that the Euglena cells responded quickly to Listeria VOCs including decanal, 3,5- dimethylbenzaldehyde, ethyl acetate, indicating bacterial VOCs were used by Euglena to track the location of bacteria. [ABSTRACT FROM AUTHOR]

Although there is good evidence to indicate a major role of intrinsic impairment of the contractile apparatus in muscle weakness seen in several pathophysiological conditions, the factors responsible for control of myofibrillar function are not fully understood. To investigate the role of mechanical load in myofibrillar function, we compared the skinned fiber force between denervated (DEN) and dexamethasone-treated (DEX) rat skeletal muscles with or without neuromuscular electrical stimulation (ES) training. DEN and DEX were induced by cutting the sciatic nerve and daily injection of dexamethasone (5 mg/kg/day) for 7 days, respectively. For ES training, plantarflexor muscles were electrically stimulated to produce four sets of five isometric contractions each day. In situ maximum torque was markedly depressed in the DEN muscles compared to the DEX muscles (-74% vs. -10%), whereas there was not much difference in the degree of atrophy in gastrocnemius muscles between DEN and DEX groups (-24% vs. -17%). Similar results were obtained in the skinned fiber preparation, with a greater reduction in maximum Ca2+-activated force in the DEN than in the DEX group (-53% vs. -16%). Moreover, there was a parallel decline in myosin heavy chain (MyHC) and actin content per muscle volume in DEN muscles, but not in DEX muscles, which was associated with upregulation of NADPH oxidase (NOX) 2, neuronal nitric oxide synthase (nNOS), and endothelial NOS expression, translocation of nNOS from the membrane to the cytosol, and augmentation of mRNA levels of muscle RING finger protein 1 (MuRF-1) and atrogin-1. Importantly, mechanical load evoked by ES protects against DEN- and DEX-induced myofibrillar dysfunction and these molecular alterations. Our findings provide novel insights regarding the difference in intrinsic contractile properties between DEN and DEX and suggest an important role of mechanical load in preserving myofibrillar function in skeletal muscle. [ABSTRACT FROM AUTHOR]

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments. [ABSTRACT FROM AUTHOR]

Leaves display a remarkable range of forms, from flat sheets with simple outlines to cup-shaped traps. Although much progress has been made in understanding the mechanisms of planar leaf development, it is unclear whether similar or distinctive mechanisms underlie shape transformations during development of more complex curved forms. Here, we use 3D imaging and cellular and clonal analysis, combined with computational modelling, to analyse the development of cup-shaped traps of the carnivorous plant Utricularia gibba. We show that the transformation from a near-spherical form at early developmental stages to an oblate spheroid with a straightened ventral midline in the mature form can be accounted for by spatial variations in rates and orientations of growth. Different hypotheses regarding spatiotemporal control predict distinct patterns of cell shape and size, which were tested experimentally by quantifying cellular and clonal anisotropy. We propose that orientations of growth are specified by a proximodistal polarity field, similar to that hypothesised to account for Arabidopsis leaf development, except that in Utricularia, the field propagates through a highly curved tissue sheet. Independent evidence for the polarity field is provided by the orientation of glandular hairs on the inner surface of the trap. Taken together, our results show that morphogenesis of complex 3D leaf shapes can be accounted for by similar mechanisms to those for planar leaves, suggesting that simple modulations of a common growth framework underlie the shaping of a diverse range of morphologies. [ABSTRACT FROM AUTHOR]

To understand the effect of attention on neuronal dynamics, we propose a multi-module network, with each module consisting of fully interconnected groups of excitatory and inhibitory neurons. This network shows transitive dynamics among quasi-attractors as its typical dynamics. When the release of acetylcholine onto the network is simulated by attention, the transitive dynamics change into stable dynamics in which the system converges to an attractor. We found that this network can reproduce three experimentally observed properties of attention-dependent response modulation, namely an increase in the firing rate, a decrease in the Fano factor of the firing rate, and a decrease in the correlation coefficients between the firing rates of pairs of neurons. Moreover, we also showed theoretically that the release of acetylcholine increases the sensitivity to bottom-up inputs by changing the response function. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are the most effective professional antigen-presenting cell. They ferry antigen from the extremities to T cells and are essential for the initiation of an adaptive immune response. Despite interest in how DCs respond to chemical stimuli, there have been few attempts to model DC migration. In this paper, we simulate the motility of DCs by modeling the generation of forces by filopodia and a force balance on the cell. The direction of fliopodial extension is coupled to differential occupancy of cognate chemokine receptors across the cell. Our model simulates chemokinesis and chemotaxis in a variety of chemical and mechanical environments. Simulated DCs undergoing chemokinesis were measured to have a speed of 5.1 ± 0.07 μm·min-1 and a persistence time of 3.2 ± 0.46 min, consistent with experiment. Cells undergoing chemotaxis exhibited a stronger chemotactic response when exposed to lower average chemokine concentrations, also consistent with experiment. We predicted that when placed in two opposing gradients, cells will cluster in a line, which we call the “line of equistimulation;” this clustering has also been observed. We calculated the effect of varying gradient steepness on the line of equistimulation, with steeper gradients resulting in tighter clustering. Moreover, gradients are found to be most potent when cells are in a gradient of chemokine whose mean concentration is close to the binding of the Kd to the receptor, and least potent when the mean concentration is 0.1Kd. Comparing our simulations to experiment, we can give a quantitative measure of the strength of certain chemokines relative to others. Assigning the signal of CCL19 binding CCR7 a baseline strength of 1, we found CCL21 binding CCR7 had a strength of 0.28, and CXCL12 binding CXCR4 had a strength of 0.30. These differences emerge despite both chemokines having virtually the same Kd, suggesting a mechanism of signal amplification in DCs requiring further study. [ABSTRACT FROM AUTHOR]

Mechanical loading is essential for the maintenance of musculoskeletal homeostasis. Cartilage has been demonstrated to be highly mechanoresponsive, but the mechanisms by which chondrocytes respond to mechanical stimuli are not clearly understood. The goal of the study was to determine how LRP4, LRP5, and LRP6 within canonical Wnt-signaling are regulated in simulated microgravity and cyclic hydrostatic pressure, and to investigate the potential role of LRP 4/5/6 in cartilage degeneration. Rat chondrosacroma cell (RCS) pellets were stimulated using either cyclic hydrostatic pressure (1Hz, 7.5 MPa, 4hr/day) or simulated microgravity in a rotating wall vessel (RWV) bioreactor (11RPM, 24hr/day). LRP4/5/6 mRNA expression was assessed by RT-qPCR and LRP5 protein expression was determined by fluorescent immunostaining. To further evaluate our in vitro findings in vivo, mice were subjected to hindlimb suspension for 14 days and the femoral heads stained for LRP5 expression. We found that, in vitro, LRP4/5/6 mRNA expression is modulated in a time-dependent manner by mechanical stimulation. Additionally, LRP5 protein expression is upregulated in response to both simulated microgravity and cyclic hydrostatic pressure. LRP5 is also upregulated in vivo in the articular cartilage of hindlimb suspended mice. This is the first study to examine how LRP4/5/6, critical receptors within musculoskeletal biology, respond to mechanical stimulation. Further elucidation of this mechanism could provide significant clinical benefit for the identification of pharmaceutical targets for the maintenance of cartilage health. [ABSTRACT FROM AUTHOR]

Taste receptor type 1 (T1r) is responsible for the perception of essential nutrients, such as sugars and amino acids, and evoking sweet and umami (savory) taste sensations. T1r receptors recognize many of the taste substances at their extracellular ligand-binding domains (LBDs). In order to detect a wide array of taste substances in the environment, T1r receptors often possess broad ligand specificities. However, the entire ranges of chemical spaces and their binding characteristics to any T1rLBDs have not been extensively analyzed. In this study, we exploited the differential scanning fluorimetry (DSF) to medaka T1r2a/T1r3LBD, a current sole T1rLBD heterodimer amenable for recombinant preparation, and analyzed their thermal stabilization by adding various amino acids. The assay showed that the agonist amino acids induced thermal stabilization and shifted the melting temperatures (Tm) of the protein. An agreement between the DSF results and the previous biophysical assay was observed, suggesting that DSF can detect ligand binding at the orthosteric-binding site in T1r2a/T1r3LBD. The assay further demonstrated that most of the tested -amino acids, but no -amino acid, induced Tm shifts of T1r2a/T1r3LBD, indicating the broad -amino acid specificities of the proteins probably with several different manners of recognition. The Tm shifts by each amino acid also showed a fair correlation with the responses exhibited by the full-length receptor, verifying the broad amino-acid binding profiles at the orthosteric site in LBD observed by DSF. [ABSTRACT FROM AUTHOR]

The nicotinic acetylcholine receptor (nAChR) is an excitatory pentameric ligand-gated ion channel (pLGIC), homologous to the inhibitory γ-aminobutyric acid (GABA) type A receptor targeted by pharmaceuticals and endogenous sedatives. Activation of the GABAA receptor by the neurosteroid allopregnanolone can be inhibited competitively by thyroid hormone (L-3,3’,5-triiodothyronine, or T3), but modulation of nAChR by T3 or neurosteroids has not been investigated. Here we show that allopregnanolone inhibits the nAChR from Torpedo californica at micromolar concentrations, as do T3 and the anionic neurosteroid pregnenolone sulfate (PS). We test for the role of protein and ligand charge in mediated receptor inhibition by varying pH in a narrow range around physiological pH. We find that both T3 and PS become less potent with increasing pH, with remarkably similar trends in IC50 when T3 is neutral at pH < 7.3. After deprotonation of T3 (but no additional deprotonation of PS) at pH 7.3, T3 loses potency more slowly with increasing pH than PS. We interpret this result as indicating the negative charge is not required for inhibition but does increase activity. Finally, we show that both T3 and PS affect nAChR channel desensitization, which may implicate a binding site homologous to one that was recently indicated for accelerated desensitization of the GABAA receptor by PS. [ABSTRACT FROM AUTHOR]

Predictive processing (e.g., predictive coding) is a predominant paradigm in cognitive neuroscience. This Primer considers the various levels of commitment neuroscientists have to the neuronal process theories that accompany the principles of predictive processing. Specifically, it reviews and contextualises a recent PLOS Biology study of alpha oscillations and travelling waves. We will see that alpha oscillations emerge naturally under the computational architectures implied by predictive coding-and may tell us something profound about recurrent message passing in brain hierarchies. Specifically, the bidirectional nature of forward and backward waves speaks to opportunities to understand attention and how it nuances bottom-up and top-down influences. [ABSTRACT FROM AUTHOR]

Plant trichomes originate from epidermal cell, forming protective structure from abiotic and biotic stresses. Different from the unicellular trichome in Arabidopsis, tomato trichomes are multicellular structure and can be classified into seven different types based on cell number, shape and the presence of glandular cells. Despite the importance of tomato trichomes in insect resistance, our understanding of the tomato trichome morphogenesis remains elusive. In this study, we quantitatively analyzed morphological traits of trichomes in tomato and further performed live imaging of cytoskeletons in stably transformed lines with actin and microtubule markers. At different developmental stages, two types of cytoskeletons exhibited distinct patterns in different trichome cells, ranging from transverse, spiral to longitudinal. This gradual transition of actin filament angle from basal to top cells could correlate with the spatial expansion mode in different cells. Further genetic screen for aberrant trichome morphology led to the discovery of a number of independent mutations in SCAR/WAVE and ARP2/3 complex, which resulted in actin bundling and distorted trichomes. Disruption of microtubules caused isotropic expansion while abolished actin filaments entirely inhibited axial extension of trichomes, indicating that microtubules and actin filaments may control distinct aspects of trichome cell expansion. Our results shed light on the roles of cytoskeletons in the formation of multicellular structure of tomato trichomes. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) were obtained from human bone marrow and amplified in cultures supplemented with human platelet lysate in order to generate myofibroblasts. When MSCs were seeded in solid collagen scaffolds, they differentiated into myofibroblasts that were observed to strongly bind to the substrate, forming a 3D cell scaffold network that developed tension and shortening after KCl stimulation. Moreover, MSC-laden scaffolds recapitulated the Frank-Starling mechanism so that active tension increased in response to increases in the initial length of the contractile system. This constituted a bioengineering tissue that exhibited the contractile properties observed in both striated and smooth muscles. By using the A. F. Huxley formalism, we determined the myosin crossbridge (CB) kinetics of attachment (f1) and detachment (g1 and g2), maximum myosin ATPase activity, molar myosin concentration, unitary CB force and maximum CB efficiency. CB kinetics were dramatically slow, characterizing the non-muscle myosin type IIA (NMMIIA) present in myofibroblasts. When MSCs were seeded in solid collagen scaffolds functionalized with Arg-Gly-Asp (RGD), contractility increased and CB kinetics were modified, whereas the unitary NMMIIA-CB force and maximum CB efficiency did not change. In conclusion, we provided a non-muscle bioengineering tissue whose molecular mechanical characteristics of NMMIIA were very close to those of a non-muscle contractile tissue such as the human placenta. [ABSTRACT FROM AUTHOR]

Many animals are able to sense the earth’s magnetic field, including varieties of arthropods and members of all major vertebrate groups. While the existence of this magnetic sense is widely accepted, the mechanism of action remains unknown. Building from recent work on synthetic magnetoreceptors, we propose a new model for natural magnetosensation based on the rotating magnetocaloric effect (RME), which predicts that heat generated by magnetic nanoparticles may allow animals to detect features of the earth’s magnetic field. Using this model, we identify the conditions for the RME to produce physiological signals in response to the earth’s magnetic field and suggest experiments to distinguish between candidate mechanisms of magnetoreception. [ABSTRACT FROM AUTHOR]

Humans and other animals effortlessly identify natural sounds and categorize them into behaviorally relevant categories. Yet, the acoustic features and neural transformations that enable sound recognition and the formation of perceptual categories are largely unknown. Here, using multichannel neural recordings in the auditory midbrain of unanesthetized female rabbits, we first demonstrate that neural ensemble activity in the auditory midbrain displays highly structured correlations that vary with distinct natural sound stimuli. These stimulus-driven correlations can be used to accurately identify individual sounds using single-response trials, even when the sounds do not differ in their spectral content. Combining neural recordings and an auditory model, we then show how correlations between frequency-organized auditory channels can contribute to discrimination of not just individual sounds but sound categories. For both the model and neural data, spectral and temporal correlations achieved similar categorization performance and appear to contribute equally. Moreover, both the neural and model classifiers achieve their best task performance when they accumulate evidence over a time frame of approximately 1–2 seconds, mirroring human perceptual trends. These results together suggest that time-frequency correlations in sounds may be reflected in the correlations between auditory midbrain ensembles and that these correlations may play an important role in the identification and categorization of natural sounds. [ABSTRACT FROM AUTHOR]

Confined hydration and conformational flexibility are some of the challenges encountered for the rational design of selective antagonists of G-protein coupled receptors. We present a set of C3-substituted (-)-stepholidine derivatives as potent binders of the dopamine D3 receptor. The compounds are characterized biochemically, as well as by computer modeling using a novel molecular dynamics-based alchemical binding free energy approach which incorporates the effect of the displacement of enclosed water molecules from the binding site. The free energy of displacement of specific hydration sites is obtained using the Hydration Site Analysis method with explicit solvation. This work underscores the critical role of confined hydration and conformational reorganization in the molecular recognition mechanism of dopamine receptors and illustrates the potential of binding free energy models to represent these key phenomena. [ABSTRACT FROM AUTHOR]

Achieving global food security for the estimated 9 billion people by 2050 is a major scientific challenge. Crop productivity is fundamentally restricted by the rate of fixation of atmospheric carbon. The dedicated enzyme, RubisCO, has a low turnover and poor specificity for CO2. This limitation of C3 photosynthesis (the basic carbon-assimilation pathway present in all plants) is alleviated in some lineages by use of carbon-concentrating-mechanisms, such as the C4 cycle—a biochemical pump that concentrates CO2 near RubisCO increasing assimilation efficacy. Most crops use only C3 photosynthesis, so one promising research strategy to boost their productivity focuses on introducing a C4 cycle. The simplest proposal is to use the cycle to concentrate CO2 inside individual chloroplasts. The photosynthetic efficiency would then depend on the leakage of CO2 out of a chloroplast. We examine this proposal with a 3D spatial model of carbon and oxygen diffusion and C4 photosynthetic biochemistry inside a typical C3-plant mesophyll cell geometry. We find that the cost-efficiency of C4 photosynthesis depends on the gas permeability of the chloroplast envelope, the C4 pathway having higher quantum efficiency than C3 for permeabilities below 300 μm/s. However, at higher permeabilities the C4 pathway still provides a substantial boost to carbon assimilation with only a moderate decrease in efficiency. The gains would be capped by the ability of chloroplasts to harvest light, but even under realistic light regimes a 100% boost to carbon assimilation is possible. This could be achieved in conjunction with lower investment in chloroplasts if their cell surface coverage is also reduced. Incorporation of this C4 cycle into C3 crops could thus promote higher growth rates and better drought resistance in dry, high-sunlight climates. [ABSTRACT FROM AUTHOR]

Purpose: Diffusion magnetic resonance imaging and tractography has an important role in the visualization of brain white matter and assessment of tissue microstructure. There is a lack of correspondence between diffusion metrics of live tissue, ex vivo tissue, and histological findings. The objective of this study is to elucidate this connection by determining the specific diffusion alterations between live and ex vivo brain tissue. This may have an important role in the incorporation of diffusion imaging in ex vivo studies as a complement to histological sectioning as well as investigations of novel neurosurgical techniques. Methods: This study presents a method of high angular resolution diffusion imaging and tractography of intact and non-fixed ex vivo piglet brains. Most studies involving ex vivo brain specimens have been formalin-fixed or excised from their original biological environment, processes both of which are known to affect diffusion parameters. Thus, non-fixed ex vivo tissue is used. A region-of-interest based analysis of diffusion tensor metrics are compared to in vivo subjects in a selection of major white matter bundles in order to assess the translatability of ex vivo diffusion measurements. Results: Tractography was successfully achieved in both in vivo and ex vivo groups. No significant differences were found in tract connectivity, average streamline length, or apparent fiber density. Significantly decreased diffusivity (mean, axial, and radial; p<0.0005) in the non-fixed ex vivo group and unaltered fractional anisotropy (p>0.059) between groups were observed. Conclusion: This study validates the extrapolation of non-fixed fractional anisotropy measurements to live tissue and the potential use of ex vivo tissue for methodological development. [ABSTRACT FROM AUTHOR]

The therapeutic options for advanced gastric cancer are still limited. Several drugs targeting the epidermal growth factor receptor family have been developed. So far, the HER2 antibody trastuzumab is the only drug targeting the HER-family that is available to gastric cancer patients. The pan-HER inhibitor afatinib is currently investigated in clinical trials and shows promising results in cell culture experiments and patient-derived xenograft (PDX) models. However, some cell lines do not respond to afatinib treatment. The determination of resistance factors in these cell lines can help to find the best treatment option for gastric cancer patients. In this study, we analyzed the role of MET as a resistance factor for afatinib therapy in a gastric cancer cell line. MET expression in afatinib-resistant MET-amplified Hs746T cells was reduced by means of siRNA transfection. The effects of MET knockdown on signal transduction, cell proliferation and motility were examined. In addition to the manual assessment of cell motility, a computational motility analysis involving parameters such as (approximate) average speed, displacement entropy or radial effectiveness was realized. Moreover, the impact of afatinib was compared between MET knockdown cells and control cells. MET knockdown in Hs746T cells resulted in impaired signal transduction and reduced cell proliferation and motility. Moreover, the afatinib resistance of Hs746T cells was reversed after MET knockdown. Therefore, the amplification of MET is confirmed as a resistance factor in gastric cancer cells. Whether MET is a useful resistance marker for afatinib therapy or other HER-targeting drugs in patients should be investigated in clinical trials. [ABSTRACT FROM AUTHOR]

Recent work has shown that weak protein-protein interactions are susceptible to the cellular milieu. One case in point is the binding of heat shock proteins (Hsps) to substrate proteins in cells under stress. Upregulation of the Hsp70 chaperone machinery at elevated temperature was discovered in the 1960s, and more recent studies have shown that ATPase activity in one Hsp70 domain is essential for control of substrate binding by the other Hsp70 domain. Although there are several denaturant-based assays of Hsp70 activity, reports of ATP-dependent binding of Hsp70 to a globular protein substrate under heat shock are scarce. Here we show that binding of heat-inducible Hsp70 to phosphoglycerate kinase (PGK) is remarkably different in vitro compared to in-cell. We use fluorescent-labeled mHsp70 and ePGK, and begin by showing that mHsp70 passes the standard β-galactosidase assay, and that it does not self-aggregate until 50°C in presence of ATP. Yet during denaturant refolding or during in vitro heat shock, mHsp70 shows only ATP-independent non-specific sticking to ePGK, as evidenced by nearly identical results with an ATPase activity-deficient K71M mutant of Hsp70 as a control. Addition of Hsp40 (co-factor) or Ficoll (crowder) does not reduce non-specific sticking, but cell lysate does. Therefore, Hsp70 does not act as an ATP-dependent chaperone on its substrate PGK in vitro. In contrast, we observe only specific ATP-dependent binding of mHsp70 to ePGK in mammalian cells, when compared to the inactive Hsp70 K71M mutant. We hypothesize that enhanced in-cell activity is not due to an unknown co-factor, but simply to a favorable shift in binding equilibrium caused by the combination of crowding and osmolyte/macromolecular interactions present in the cell. One candidate mechanism for such a favorable shift in binding equilibrium is the proven ability of Hsp70 to bind near-native states of substrate proteins in vitro. We show evidence for early onset of binding in-cell. Our results suggest that Hsp70 binds PGK preemptively, prior to its full unfolding transition, thus stabilizing it against further unfolding. We propose a “preemptive holdase” mechanism for Hsp70-substrate binding. Given our result for PGK, more proteins than one might think based on in vitro assays may be chaperoned by Hsp70 in vivo. The cellular environment thus plays an important role in maintaining proper Hsp70 function. [ABSTRACT FROM AUTHOR]

Mutual gliding motility A (MglA), a small Ras-like GTPase; Mutual gliding motility B (MglB), its GTPase activating protein (GAP); and Required for Motility Response Regulator (RomR), a protein that contains a response regulator receiver domain, are major components of a GTPase-dependent biochemical oscillator that drives cell polarity reversals in the bacterium Myxococcus xanthus. We report the crystal structure of a complex of M. xanthus MglA and MglB, which reveals that the C-terminal helix (Ct-helix) from one protomer of the dimeric MglB binds to a pocket distal to the active site of MglA. MglB increases the GTPase activity of MglA by reorientation of key catalytic residues of MglA (a GAP function) combined with allosteric regulation of nucleotide exchange by the Ct-helix (a guanine nucleotide exchange factor [GEF] function). The dual GAP-GEF activities of MglB accelerate the rate of GTP hydrolysis over multiple enzymatic cycles. Consistent with its GAP and GEF activities, MglB interacts with MglA bound to either GTP or GDP. The regulation is essential for cell polarity, because deletion of the Ct-helix causes bipolar localization of MglA, MglB, and RomR, thereby causing reversal defects in M. xanthus. A bioinformatics analysis reveals the presence of Ct-helix in homologues of MglB in other bacterial phyla, suggestive of the prevalence of the allosteric mechanism among other prokaryotic small Ras-like GTPases. [ABSTRACT FROM AUTHOR]

Although information theoretic approaches have been used extensively in the analysis of the neural code, they have yet to be used to describe how information is accumulated in time while sensory systems are categorizing dynamic sensory stimuli such as speech sounds or visual objects. Here, we present a novel method to estimate the cumulative information for stimuli or categories. We further define a time-varying categorical information index that, by comparing the information obtained for stimuli versus categories of these same stimuli, quantifies invariant neural representations. We use these methods to investigate the dynamic properties of avian cortical auditory neurons recorded in zebra finches that were listening to a large set of call stimuli sampled from the complete vocal repertoire of this species. We found that the time-varying rates carry 5 times more information than the mean firing rates even in the first 100 ms. We also found that cumulative information has slow time constants (100–600 ms) relative to the typical integration time of single neurons, reflecting the fact that the behaviorally informative features of auditory objects are time-varying sound patterns. When we correlated firing rates and information values, we found that average information correlates with average firing rate but that higher-rates found at the onset response yielded similar information values as the lower-rates found in the sustained response: the onset and sustained response of avian cortical auditory neurons provide similar levels of independent information about call identity and call-type. Finally, our information measures allowed us to rigorously define categorical neurons; these categorical neurons show a high degree of invariance for vocalizations within a call-type. Peak invariance is found around 150 ms after stimulus onset. Surprisingly, call-type invariant neurons were found in both primary and secondary avian auditory areas. [ABSTRACT FROM AUTHOR]

Mechanical stimulation appears to play a key role in cartilage homeostasis maintenance, but it can also contribute to osteoarthritis (OA) pathogenesis. Accumulating evidence suggests that cartilage loading in the physiological range contributes to tissue integrity maintenance, whereas excessive or reduced loading have catabolic effects. However, how mechanical stimuli can regulate joint homeostasis is still not completely elucidated and few data are available on human cartilage. We aimed at investigating human OA cartilage response to ex vivo loading at physiological intensity. Cartilage explants from ten OA patients were subjected to ex vivo controlled compression, then recovered and used for gene and protein expression analysis of cartilage homeostasis markers. Compressed samples were compared to uncompressed ones in presence or without interleukin 1β (IL-1β) or interleukin 4 (IL-4). Cartilage explants compressed in combination with IL-4 treatment showed the best histological scores. Mechanical stimulation was able to significantly modify the expression of collagen type II (collagen 2), aggrecan, SOX9 transcription factor, cartilage oligomeric matrix protein (COMP), collagen degradation marker C2C and vascular endothelial growth factor (VEGF). Conversely, ADAMTS4 metallopeptidase, interleukin 4 receptor alpha (IL4Rα), chondroitin sulfate 846 epitope (CS846), procollagen type 2 C-propeptide (CPII) and glycosaminoglycans (GAG) appeared not modulated. Our data suggest that physiological compression of OA human cartilage modulates the inflammatory milieu by differently affecting the expression of components and homeostasis regulators of the cartilage extracellular matrix. [ABSTRACT FROM AUTHOR]

Around the world, pansies are one of the most popular garden flowers, but they are generally sensitive to high temperatures, and this limits the practicality of planting them during the warmest days of the year. However, a few pansy germplasms with improved heat tolerance have been discovered or bred, but the mechanisms of their heat resistance are not understood. In this study, we investigated the transcript profiles of a heat-tolerant pansy inbred line, DFM16, in response to high temperatures using RNAseq. Approximately 55.48 Gb of nucleotide data were obtained and assembled into 167,576 unigenes with an average length of 959 bp, of which, 5,708 genes were found to be differentially expressed after heat treatments. Real-time qPCR was performed to validate the expression profiles of the selected genes. Nine metabolic pathways were found to be significantly enriched, in the analysis of the differentially expressed genes. Several potentially interesting genes that encoded putative transcription regulators or key components involving heat shock protein (HSP), heat shock transcription factors (HSF), and antioxidants biosynthesis, were identified. These genes were highlighted to indicate their significance in response to heat stress and will be used as candidate genes to improve pansy heat-tolerance in the future. [ABSTRACT FROM AUTHOR]

The co-evolutionary dynamics of competing populations can be strongly affected by frequency-dependent selection and spatial population structure. As co-evolving populations grow into a spatial domain, their initial spatial arrangement and their growth rate differences are important factors that determine the long-term outcome. We here model producer and free-rider co-evolution in the context of a diffusive public good (PG) that is produced by the producers at a cost but evokes local concentration-dependent growth benefits to all. The benefit of the PG can be non-linearly dependent on public good concentration. We consider the spatial growth dynamics of producers and free-riders in one, two and three dimensions by modeling producer cell, free-rider cell and public good densities in space, driven by the processes of birth, death and diffusion (cell movement and public good distribution). Typically, one population goes extinct, but the time-scale of this process varies with initial conditions and the growth rate functions. We establish that spatial variation is transient regardless of dimensionality, and that structured initial conditions lead to increasing times to get close to an extinction state, called ε-extinction time. Further, we find that uncorrelated initial spatial structures do not influence this ε-extinction time in comparison to a corresponding well-mixed (non-spatial) system. In order to estimate the ε-extinction time of either free-riders or producers we derive a slow manifold solution. For invading populations, i.e. for populations that are initially highly segregated, we observe a traveling wave, whose speed can be calculated. Our results provide quantitative predictions for the transient spatial dynamics of cooperative traits under pressure of extinction. [ABSTRACT FROM AUTHOR]

Dopaminergic neurons (DAs) of the rodent substantia nigra pars compacta (SNc) display varied electrophysiological properties in vitro. Despite this, projection patterns and functional inputs from DAs to other structures are conserved, so in vivo delivery of consistent, well-timed dopamine modulation to downstream circuits must be coordinated. Here we show robust coordination by linear parameter controllers, discovered through powerful mathematical analyses of data and models, and from which consistent control of DA subthreshold oscillations (STOs) and spontaneous firing emerges. These units of control represent coordinated intracellular variables, sufficient to regulate complex cellular properties with radical simplicity. Using an evolutionary algorithm and dimensionality reduction, we discovered metaparameters, which when regressed against STO features, revealed a 2-dimensional control plane for the neuron’s 22-dimensional parameter space that fully maps the natural range of DA subthreshold electrophysiology. This plane provided a basis for spiking currents to reproduce a large range of the naturally occurring spontaneous firing characteristics of SNc DAs. From it we easily produced a unique population of models, derived using unbiased parameter search, that show good generalization to channel blockade and compensatory intracellular mechanisms. From this population of models, we then discovered low-dimensional controllers for regulating spontaneous firing properties, and gain insight into how currents active in different voltage regimes interact to produce the emergent activity of SNc DAs. Our methods therefore reveal simple regulators of neuronal function lurking in the complexity of combined ion channel dynamics. [ABSTRACT FROM AUTHOR]

The identification and separation of cells from heterogeneous populations is critical to the diagnosis of diseases. Label-free methodologies in particular have been developed to manipulate individual cells using properties such as density and morphology. The electrical properties of malignant cells, including the membrane capacitance and cytoplasmic conductivity, have been demonstrated to be altered compared to non-malignant cells of similar origin. Here, we exploit these changes to characterize individual cells in a sequentially-staged in vitro cancer model using electrorotation (EROT)—the rotation of a cell induced by a rotating electric field. Using a microfabricated device, a dielectrophoretic force to suspend cells while measuring their angular velocity resulting from an EROT force applied at frequencies between 3 kHz to 10 MHz. We experimentally determine the EROT response for cells at three stages of malignancy and analyze the resultant spectra by considering models that include the effect of the cell membrane alone (single-shell model) and the combined effect of the cell membrane and nucleus (double-shell model). We find that the cell membrane is largely responsible for a given cell’s EROT response between 3 kHz and 10 MHz. Our results also indicate that membrane capacitance, membrane conductance, and cytoplasmic conductivity increase with an increasingly malignant phenotype. Our results demonstrate the potential of using electrorotation as a means making of non-invasive measurements to characterize the dielectric properties of cancer cells. [ABSTRACT FROM AUTHOR]

The bovine dry period is a non-lactating period between consecutive lactations characterized by mammary gland involution and redevelopment phases to replace senescent mammary epithelial cells with active cells primed for the next lactation. Dairy cows exposed to heat stress during the dry period experience milk yield reductions between 3–7.5 kg/d in the next lactation, partially attributed to processes associated with mammary cell growth and turnover during the dry period. However, the carry-over impact of dry period heat stress on mammary morphology during lactation has yet to be determined. In the current study, we hypothesized that exposure to heat stress during the dry period would alter alveolar microstructure and cellular turnover (i.e. proliferation and apoptosis) during lactation. Cows were either subjected to heat stress (HT, access to shade; n = 12) or cooling (CL, access to shade, fans, and soakers; n = 12) for a 46 d dry period. Upon calving, all cows were treated similarly with access to cooling for their entire lactation. Six cows per treatment were randomly selected for mammary gland biopsies at 14, 42, and 84 days in milk. Tissues were sectioned and stained for histological analysis. During lactation, HT cows produced 4 kg less colostrum and 3.7 kg less milk compared with CL cows. Lactating mammary gland microstructure was impacted after exposure to dry period heat stress; HT cows had fewer alveoli and a higher proportion of connective tissue in the mammary gland relative to CL cows, however alveolar area was similar between treatments. Rates of mammary epithelial cell proliferation and apoptosis were similar between treatment groups. This suggests that heat stress exposure during the dry period leads to reductions in milk yield that could be caused, in part, by a reduction in alveoli number in the lactating mammary gland but not to dynamic alterations in cellular turnover once lactation is established. [ABSTRACT FROM AUTHOR]

Transient receptor potential (TRP) proteins form Ca2+-permeable, nonselective cation channels, but their role in neuronal Ca2+ homeostasis is elusive. In the present paper, we show that TRPC channels potently regulate synaptic plasticity by changing the presynaptic Ca2+-homeostasis of hippocampal neurons. Specifically, loss of TRPC1/C4/C5 channels decreases basal-evoked secretion, decreases the pool size of readily releasable vesicles, and accelerates synaptic depression during high-frequency stimulation (HFS). In contrast, primary TRPC5 channel-expressing neurons, identified by a novel TRPC5–τ-green fluorescent protein (τGFP) knockin mouse line, show strong short-term enhancement (STE) of synaptic signaling during HFS, indicating a key role of TRPC5 in short-term plasticity. Lentiviral expression of either TRPC1 or TRPC5 turns classic synaptic depression of wild-type neurons into STE, demonstrating that TRPCs are instrumental in regulating synaptic plasticity. Presynaptic Ca2+ imaging shows that TRPC activity strongly boosts synaptic Ca2+ dynamics, showing that TRPC channels provide an additional presynaptic Ca2+ entry pathway, which efficiently regulates synaptic strength and plasticity. [ABSTRACT FROM AUTHOR]

In some parts of the Southern Ocean (SO), even though low surface concentrations of iron (Fe) and manganese (Mn) indicate FeMn co-limitation, we still lack an understanding on how Mn and Fe availability influences SO phytoplankton ecophysiology. Therefore, this study investigated the effects of Fe and Mn limitation alone as well as their combination on growth, photophysiology and particulate organic carbon production of the bloom-forming Antarctic diatom Chaetoceros debilis. Our results clearly show that growth, photochemical efficiency and carbon production of C. debilis were co-limited by Fe and Mn as highest values were only reached when both nutrients were provided. Even though Mn-deficient cells had higher photochemical efficiencies than Fe-limited ones, they, however, displayed similar low growth and POC production rates, indicating that Mn limitation alone drastically impeded the cell’s performance. These results demonstrate that similar to low Fe concentrations, low Mn availability inhibits growth and carbon production of C. debilis. As a result from different species-specific trace metal requirements, SO phytoplankton species distribution and productivity may therefore not solely depend on the input of Fe alone, but also critically on Mn acting together as important drivers of SO phytoplankton ecology and biogeochemistry. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a positive-strand RNA virus replicating in a membranous replication organelle composed primarily of double-membrane vesicles (DMVs) having morphological resemblance to autophagosomes. To define the mechanism of DMV formation and the possible link to autophagy, we conducted a yeast two-hybrid screening revealing 32 cellular proteins potentially interacting with HCV proteins. Among these was the Receptor for Activated Protein C Kinase 1 (RACK1), a scaffolding protein involved in many cellular processes, including autophagy. Depletion of RACK1 strongly inhibits HCV RNA replication without affecting HCV internal ribosome entry site (IRES) activity. RACK1 is required for the rewiring of subcellular membranous structures and for the induction of autophagy. RACK1 binds to HCV nonstructural protein 5A (NS5A), which induces DMV formation. NS5A interacts with ATG14L in a RACK1 dependent manner, and with the ATG14L-Beclin1-Vps34-Vps15 complex that is required for autophagosome formation. Both RACK1 and ATG14L are required for HCV DMV formation and viral RNA replication. These results indicate that NS5A participates in the formation of the HCV replication organelle through interactions with RACK1 and ATG14L. [ABSTRACT FROM AUTHOR]

Measuring pressures within complex multi-cellular environments is critical for studying mechanobiology as these forces trigger diverse biological responses, however, these studies are difficult as a deeply embedded yet well-calibrated probe is required. In this manuscript, we use endogenous cell nuclei as pressure sensors by introducing a fluorescent protein localized to the nucleus and confocal microscopy to measure the individual nuclear volumes in 3D multi-cellular aggregates. We calibrate this measurement of nuclear volume to pressure by quantifying the nuclear volume change as a function of osmotic pressure in isolated 2D culture. Using this technique, we find that in multicellular structures, the nuclear compressive mechanical stresses are on the order of MPa, increase with cell number in the cluster, and that the distribution of stresses is homogenous in spherical cell clusters, but highly asymmetric in oblong clusters. This approach may facilitate quantitative mechanical measurements in complex and extended biological structures both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

In the mouse spinal cord, V1 interneurons are a heterogeneous population of inhibitory spinal interneurons that have been implicated in regulating the frequency of the locomotor rhythm and in organizing flexor and extensor alternation. By introducing archaerhodopsin into engrailed-1-positive neurons, we demonstrate that the function of V1 neurons in locomotor-like activity is more complex than previously thought. In the whole cord, V1 hyperpolarization increased the rhythmic synaptic drive to flexor and extensor motoneurons, increased the spiking in each cycle, and slowed the locomotor-like rhythm. In the hemicord, V1 hyperpolarization accelerated the rhythm after an initial period of tonic activity, implying that a subset of V1 neurons are active in the hemicord, which was confirmed by calcium imaging. Hyperpolarizing V1 neurons resulted in an equalization of the duty cycle in flexor and extensors from an asymmetrical pattern in control recordings in which the extensor bursts were longer than the flexor bursts. Our results suggest that V1 interneurons are composed of several subsets with different functional roles. Furthermore, during V1 hyperpolarization, the default state of the locomotor central pattern generator (CPG) is symmetrical, with antagonist motoneurons each firing with an approximately 50% duty cycle. We hypothesize that one function of the V1 population is to set the burst durations of muscles to be appropriate to their biomechanical function and to adapt to the environmental demands, such as changes in locomotor speed. [ABSTRACT FROM AUTHOR]

Congenital nystagmus, involuntary oscillating small eye movements, is commonly thought to originate from aberrant interactions between brainstem nuclei and foveal cortical pathways. Here, we investigated whether nystagmus associated with congenital stationary night blindness (CSNB) results from primary deficits in the retina. We found that CSNB patients as well as an animal model (nob mice), both of which lacked functional nyctalopin protein (NYX, nyx) in ON bipolar cells (BCs) at their synapse with photoreceptors, showed oscillating eye movements at a frequency of 4–7 Hz. nob ON direction-selective ganglion cells (DSGCs), which detect global motion and project to the accessory optic system (AOS), oscillated with the same frequency as their eyes. In the dark, individual ganglion cells (GCs) oscillated asynchronously, but their oscillations became synchronized by light stimulation. Likewise, both patient and nob mice oscillating eye movements were only present in the light when contrast was present. Retinal pharmacological and genetic manipulations that blocked nob GC oscillations also eliminated their oscillating eye movements, and retinal pharmacological manipulations that reduced the oscillation frequency of nob GCs also reduced the oscillation frequency of their eye movements. We conclude that, in nob mice, synchronized oscillations of retinal GCs, most likely the ON-DCGCs, cause nystagmus with properties similar to those associated with CSNB in humans. These results show that the nob mouse is the first animal model for a form of congenital nystagmus, paving the way for development of therapeutic strategies. [ABSTRACT FROM AUTHOR]

Bacterial type IV secretion systems (T4SS) are a highly diversified but evolutionarily related family of macromolecule transporters that can secrete proteins and DNA into the extracellular medium or into target cells. It was recently shown that a subtype of T4SS harboured by the plant pathogen Xanthomonas citri transfers toxins into target cells. Here, we show that a similar T4SS from the multi-drug-resistant opportunistic pathogen Stenotrophomonas maltophilia is proficient in killing competitor bacterial species. T4SS-dependent duelling between S. maltophilia and X. citri was observed by time-lapse fluorescence microscopy. A bioinformatic search of the S. maltophilia K279a genome for proteins containing a C-terminal domain conserved in X. citri T4SS effectors (XVIPCD) identified twelve putative effectors and their cognate immunity proteins. We selected a putative S. maltophilia effector with unknown function (Smlt3024) for further characterization and confirmed that it is indeed secreted in a T4SS-dependent manner. Expression of Smlt3024 in the periplasm of E. coli or its contact-dependent delivery via T4SS into E. coli by X. citri resulted in reduced growth rates, which could be counteracted by expression of its cognate inhibitor Smlt3025 in the target cell. Furthermore, expression of the VirD4 coupling protein of X. citri can restore the function of S. maltophilia ΔvirD4, demonstrating that effectors from one species can be recognized for transfer by T4SSs from another species. Interestingly, Smlt3024 is homologous to the N-terminal domain of large Ca2+-binding RTX proteins and the crystal structure of Smlt3025 revealed a topology similar to the iron-regulated protein FrpD from Neisseria meningitidis which has been shown to interact with the RTX protein FrpC. This work expands our current knowledge about the function of bacteria-killing T4SSs and increases the panel of effectors known to be involved in T4SS-mediated interbacterial competition, which possibly contribute to the establishment of S. maltophilia in clinical and environmental settings. [ABSTRACT FROM AUTHOR]

Circadian rhythms are generated by interlocked transcriptional-translational negative feedback loops (TTFLs), the molecular process implemented within a cell. The contributions, weighting and balancing between the multiple feedback loops remain debated. Dissociated, free-running dynamics in the expression of distinct clock genes has been described in recent experimental studies that applied various perturbations such as slice preparations, light pulses, jet-lag, and culture medium exchange. In this paper, we provide evidence that this “presumably transient” dissociation of circadian gene expression oscillations may occur at the single-cell level. Conceptual and detailed mechanistic mathematical modeling suggests that such dissociation is due to a weak interaction between multiple feedback loops present within a single cell. The dissociable loops provide insights into underlying mechanisms and general design principles of the molecular circadian clock. [ABSTRACT FROM AUTHOR]

At higher latitudes, vertebrates exhibit a seasonal cycle of reproduction in response to changes in day-length, referred to as photoperiodism. Extended day-length induces thyroid-stimulating hormone in the pars tuberalis of the pituitary gland. This hormone triggers the local activation of thyroid hormone in the mediobasal hypothalamus and eventually induces gonadal development. In avian species, light information associated with day-length is detected through photoreceptors located in deep-brain regions. Within these regions, the expressions of multiple photoreceptive molecules, opsins, have been observed. However, even though the Japanese quail is an excellent model for photoperiodism because of its robust and significant seasonal responses in reproduction, a comprehensive understanding of photoreceptors in the quail brain remains undeveloped. In this study, we initially analyzed an action spectrum using photoperiodically induced expression of the beta subunit genes of thyroid-stimulating hormone in quail. Among seven wavelengths examined, we detected maximum sensitivity of the action spectrum at 500 nm. The low value for goodness of fit in the alignment with a template of retinal1-based photopigment, assuming a spectrum associated with a single opsin, proposed the possible involvement of multiple opsins rather than a single opsin. Analysis of gene expression in the septal region and hypothalamus, regions hypothesized to be photosensitive in quail, revealed mRNA expression of a mammal-like melanopsin in the infundibular nucleus within the mediobasal hypothalamus. However, no significant diurnal changes were observed for genes in the infundibular nucleus. Xenopus-like melanopsin, a further isoform of melanopsin in birds, was detected in neither the septal region nor the infundibular nucleus. These results suggest that the mammal-like melanopsin expressed in the infundibular nucleus within the mediobasal hypothalamus could be candidate deep-brain photoreceptive molecule in Japanese quail. Investigation of the functional involvement of mammal-like melanopsin-expressing cells in photoperiodism will be required for further conclusions. [ABSTRACT FROM AUTHOR]

Aims: Glucocorticoid (GC) stimulation has been shown to increase cardiac contractility by elevated intracellular [Ca] but the sources for Ca entry are unclear. This study aims to determine the role of store-operated Ca entry (SOCE) for GC-mediated inotropy. Methods and results: Dexamethasone (Dex) pretreatment significantly increased cardiac contractile force ex vivo in Langendorff-perfused Sprague-Dawley rat hearts (2 mg/kg BW i.p. Dex 24 h prior to experiment). Moreover, Ca transient amplitude as well as fractional shortening were significantly enhanced in Fura-2-loaded isolated rat ventricular myocytes exposed to Dex (1 mg/mL Dex, 24 h). Interestingly, these Dex-dependent effects could be abolished in the presence of SOCE-inhibitors SKF-96356 (SKF, 2 μM) and BTP2 (5 μM). Ca transient kinetics (time to peak, decay time) were not affected by SOCE stimulation. Direct SOCE measurements revealed a negligible magnitude in untreated myocytes but a dramatic increase in SOCE upon Dex-pretreatment. Importantly, the Dex-dependent stimulation of SOCE could be blocked by inhibition of serum and glucocorticoid-regulated kinase 1 (SGK1) using EMD638683 (EMD, 50 μM). Dex preincubation also resulted in increased mRNA expression of proteins involved in SOCE (stromal interaction molecule 2, STIM2, and transient receptor potential cation channels 3/6, TRPC 3/6), which were also prevented in the presence of EMD. Conclusion: Short-term GC-stimulation with Dex improves cardiac contractility by a SOCE-dependent mechanism, which appears to involve increased SGK1-dependent expression of the SOCE-related proteins. Since Ca transient kinetics were unaffected, SOCE appears to influence Ca cycling more by an integrated response across multiple cardiac cycles but not on a beat-to-beat basis. [ABSTRACT FROM AUTHOR]

We compared a published computational model of the action potential of a specific type of human induced pluripotent stem cell -derived cardiomyocytes (hiPSC-CM) with experimental field potential data with regard to their inter-beat interval and the duration of repolarization. In particular, concomitant changes in inter-beat interval and duration of repolarization were calculated after reduction and/or augmentation of specific ion channel conductances as a surrogate for pharmacological manipulation. The observed mismatches between calculations and experimental data indicate that there is information missing about the cellular test system. Based on our results we hypothesize that, among other currents, the actual If (“funny current”) may deviate from the prediction. We show that replacement of the If formulation by alternative equations causes the model predictions to change qualitatively, however, none of the available formulations is actually achieving a satisfactory match with experimental data. We suggest a strategy to clarify whether the mismatch can be completely resolved at all using single cell models and, if yes, how this goal could be reached. [ABSTRACT FROM AUTHOR]

ToxR and TcpP, two winged helix-turn-helix (w-HTH) family transcription factors, co-activate expression of the toxT promoter in Vibrio cholerae. ToxT then directly regulates a number of genes required for virulence. In addition to co-activation of toxT, ToxR can directly activate the ompU promoter and repress the ompT promoter. Based on a previous study suggesting that certain wing residues of ToxR are preferentially involved in toxT co-activation compared to direct ompU activation, we employed alanine-scanning mutagenesis to determine which residues in the wing of ToxR are required for activation of each promoter. All of the ToxR wing residues tested that were critical for transcriptional activation of toxT and/or ompU were also critical for DNA binding. While some ToxR wing mutants had reduced interaction with TcpP, that reduced interaction did not correlate with a specific defect in toxT activation. Rather, such mutants also affected ompU activation and DNA binding. Based on these findings we conclude that the primary role of the wing of ToxR is to bind DNA, along with the DNA recognition helix of ToxR, and this function is required both for direct activation of ompU and co-activation of toxT. [ABSTRACT FROM AUTHOR]

Higher-order genomic architecture varies according to cell type and changes dramatically during differentiation. One of the remarkable examples of spatial genomic reorganization is the rod photoreceptor cell differentiation in nocturnal mammals. The inverted nuclear architecture found in adult mouse rod cells is formed through the reorganization of the conventional architecture during terminal differentiation. However, the mechanisms underlying these changes remain largely unknown. Here, we found that the dynamic deformation of nuclei via actomyosin-mediated contractility contributes to chromocenter clustering and promotes genomic architecture reorganization during differentiation by conducting an in cellulo experiment coupled with phase-field modeling. Similar patterns of dynamic deformation of the nucleus and a concomitant migration of the nuclear content were also observed in rod cells derived from the developing mouse retina. These results indicate that the common phenomenon of dynamic nuclear deformation, which accompanies dynamic cell behavior, can be a universal mechanism for spatiotemporal genomic reorganization. [ABSTRACT FROM AUTHOR]

Physicochemical properties of water molecules as the main compositions of the freezing media can be affected by the electromagnetic fled. The purpose of this study was to apply extremely low repetition rate electromagnetic fields (ELEFs) to change the molecular network of water molecules existing in freezing media used for human sperm cryopreservation. First, different time periods and pulsed electromagnetic fields were used to evaluate the physiochemical properties of water. The lowest rate of cluster size, surface tension, viscosity, and density was observed for water samples exposed to 1000 Hz ELEF for 60 min (P < 0.05) that could be results in small ice crystal formation. Therefore, this treatment was selected for further evaluations in human sperm freezing because there was minimal probability of amorphous ice crystallization in this group. To assess fertilizing potential, human semen samples were subjected to ELEF (1000 Hz) water-made freezing medium and cryopreserved. The highest percentage of total motility, progressive motility, viability, membrane integrity, mitochondrial membrane potential, DNA integrity, and TAC were obtained in frozen ELEF as compared to other groups. The percentage of viable spermatozoa (Annexin V-/PI-) in frozen ELEF was significantly higher than in frozen control. The level of ROS was significantly lower in frozen ELEF when compared to frozen control. It can be concluded that the modification of physicochemical properties of water existing in cryopreservation media by ELEF is a suitable strategy to improve the outcome of cryopreservation. [ABSTRACT FROM AUTHOR]

The incidence of heatstroke has been increasing. Heatstroke has been shown to affect physiological barrier functions. However, there are few studies of the effect of heat stress on the blood-brain barrier (BBB) function. In this study, we investigated the influence of heat stress on brain microvascular endothelial cells in vivo and in vitro. Heatstroke model mice administered Texas Red-dextran showed leakage outside the brain vessel walls. In addition, trans-endothelial electrical resistance (TEER) value was significantly reduced in induced pluripotent stem (iPS) cell-derived brain microvascular endothelial cells under heat stress by reducing claudin-5 expression. In addition, our results showed that the expression level of P-glycoprotein (P-gp) was increased in iPS cell-derived brain microvascular endothelial cells under heat stress. Furthermore, serum from heatstroke model mice could impair the BBB integrity of iPS cell-derived brain microvascular endothelial cells. These results suggest that BBB integrity was affected by heat stress in vivo and in vitro and provide important insights into the development of new therapeutic strategies for heatstroke patients. [ABSTRACT FROM AUTHOR]

Impact of barge movement on phytoplankton abundance and biomass was assessed in the lower stretch of river Ganga, popularly known as Bhagirathi-Hooghly river, during April 2016 to March, 2017. Based on the magnitude of tide, intensity of shipping and boating activities, the stretch from Baranagar to Lalbag (278 km), located at latitude (22°38'33.41"N to 24°10'59.75"N) and longitude (88°21'21.29"E to 88°16'5.65"E) was divided into three zones viz. zone—I (Baranagar to Barrackpore), zone II (Triveni to Balagarh) and zone III (Nabadweep to Lalbag). Water samples were collected randomly from six stations covering 22 barge movements at their passage at three different time intervals viz., 30 minutes before ‘barge movement’, during ‘barge movement’ and 30 minutes after ‘barge movement’. Analysis revealed the presence of 52 phytoplankton taxa belonged to 5 phylum during the study period. The abundance of phytoplankton was highest in zone—I followed by zone III and the zone II. A 44% decrease (1,997 ±1,510 ul-1) in phytoplankton abundance was observed during ‘barge movement’ with respect to normal condition (3,513 ± 2,239 ul-1) which could be due to propeller turbulence in the passage. Cell damage study revealed 21% damage in phytoplankton cell structure in ‘during barge’ followed by ‘after barge’ (10%) condition compared to natural state (6%). Study revealed that phytoplankton biomass (Chlorophyll a) was influenced by ‘barge movement’ in the sampling stretches and the impact was assessed by one way ANOVA. The effect was found significant at Barrackpore (p <0.01), Triveni (p <0.01), Balagarh (p <0.01) and Lalbag (p <0.01) where as it was insignificant at Baranagar and Nabadweep, which may be due to continuous and existing boat trafficking at Baranagar and Nabadweep. Two way ANOVA computed using ‘barge movement’ and sampling stations showed significant (p<0.01) effect on magnitude of Chl a concentrations in the sampling locations. Thus, the ‘barge movement’ influenced phytoplankton abundance and biomass, it had a detrimental effect on phytoplankton cell architecture also. The data set of this work serves as foundation information to understand the ecological implications augmented barge induced environmental disturbances in waterways. This is the first such study which depicts the impact of ‘barge movement’ on aquatic food chain linkages in Bhagirathi- Hooghly river. [ABSTRACT FROM AUTHOR]

Parvalbumin (PV)-positive interneurons modulate cortical activity through highly specialized connectivity patterns onto excitatory pyramidal neurons (PNs) and other inhibitory cells. PV cells are autoconnected through powerful autapses, but the contribution of this form of fast disinhibition to cortical function is unknown. We found that autaptic transmission represents the most powerful inhibitory input of PV cells in neocortical layer V. Autaptic strength was greater than synaptic strength onto PNs as a result of a larger quantal size, whereas autaptic and heterosynaptic PV-PV synapses differed in the number of release sites. Overall, single-axon autaptic transmission contributed to approximately 40% of the global inhibition (mostly perisomatic) that PV interneurons received. The strength of autaptic transmission modulated the coupling of PV-cell firing with optogenetically induced γ-oscillations, preventing high-frequency bursts of spikes. Autaptic self-inhibition represents an exceptionally large and fast disinhibitory mechanism, favoring synchronization of PV-cell firing during cognitive-relevant cortical network activity. [ABSTRACT FROM AUTHOR]

Imaging mass cytometry (IMC) is a technique allowing visualization and quantification of over 40 biological parameters in a single experiment with subcellular spatial resolution, however most IMC experiments are limited to endpoint analysis with antibodies and DNA stains. Small molecules containing tellurium are promising probes for IMC due to their cell permeability, synthetic versatility, and most importantly their application to sequential labelling with isotopologous probes (SLIP) experiments. SLIP experiments with tellurium-containing probes allow quantification of intracellular biology at multiple timepoints with IMC. Despite the promise of tellurium in IMC, there are unique challenges in image processing associated with tellurium IMC data. Here, we address some of these issues by demonstrating the removal of xenon background signal, combining channels to improve signal-to-noise ratio, and calculating isotope transmission efficiency biases. These developments add accuracy to the unique temporal resolution afforded by tellurium IMC probes. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA (mtDNA) genome integrity is essential for proper mitochondrial respiratory chain function to generate cellular energy. Nuclear genes encode several proteins that function at the mtDNA replication fork, including mitochondrial single-stranded DNA-binding protein (SSBP1), which is a tetrameric protein that binds and protects single-stranded mtDNA (ssDNA). Recently, two studies have reported pathogenic variants in SSBP1 associated with hearing loss, optic atrophy, and retinal degeneration. Here, we report a 14-year-old Chinese boy with severe and progressive mitochondrial disease manifestations across the full Pearson, Kearns-Sayre, and Leigh syndromes spectrum, including infantile anemia and bone marrow failure, growth failure, ptosis, ophthalmoplegia, ataxia, severe retinal dystrophy of the rod-cone type, sensorineural hearing loss, chronic kidney disease, multiple endocrine deficiencies, and metabolic strokes. mtDNA genome sequencing identified a single large-scale 5 kilobase mtDNA deletion (m.8629_14068del5440), present at 68% and 16% heteroplasmy in the proband’s fibroblast cell line and blood, respectively, suggestive of a mtDNA maintenance defect. On trio whole exome blood sequencing, the proband was found to harbor a novel de novo heterozygous mutation c.79G>A (p.E27K) in SSBP1. Size exclusion chromatography of p.E27K SSBP1 revealed it remains a stable tetramer. However, differential scanning fluorimetry demonstrated p.E27K SSBP1 relative to wild type had modestly decreased thermostability. Functional assays also revealed p.E27K SSBP1 had altered DNA binding. Molecular modeling of SSBP1 tetramers with varying combinations of mutant subunits predicted general changes in surface accessible charges, strength of inter-subunit interactions, and protein dynamics. Overall, the observed changes in protein dynamics and DNA binding behavior suggest that p.E27K SSBP1 can interfere with DNA replication and precipitate the introduction of large-scale mtDNA deletions. Thus, a single large-scale mtDNA deletion (SLSMD) with manifestations across the clinical spectrum of Pearson, Kearns-Sayre, and Leigh syndromes may result from a nuclear gene disorder disrupting mitochondrial DNA replication. [ABSTRACT FROM AUTHOR]