Fast Benchtop Fabrication of Laminar Flow Chambers for Advanced Microscopy Techniques

Background: Fluid handling technology is acquiring an ever more prominent place in laboratory science whether it is in simple buffer exchange systems, perfusion chambers, or advanced microfluidic devices. Many of these applications remain the providence of laboratories at large institutions with a great deal of expertise and specialized equipment. Even with the expansion of these techniques, limitations remain that frequently prevent the coupling of controlled fluid flow with other technologies, such as coupling microfluidics and high-resolution position and force measurements by optical trapping microscopy. Method: Here we present a method for fabrication of multiple-input laminar flow devices that are optically clear [glass] on each face, chemically inert, reusable, inexpensive, and can be fabricated on the benchtop in approximately one hour. Further these devices are designed to allow flow regulation by a simple gravity method thus requiring no specialized equipment to drive flow. Here we use these devices to perform total internal reflection fluorescence microscopy measurements as well as position sensitive optical trapping experiments. Significance: Flow chamber technology needs to be more accessible to the general scientific community. The method presented here is versatile and robust. These devices use standard slides and coverslips making them compatible with nearly all types and models of light microscopes. These devices meet the needs of groups doing advanced optical trapping experiments, but could also be adapted by nearly any lab that has a function for solution flow coupled with microscopy

Detection of the mosquito-borne flaviviruses, West Nile, Dengue, Saint Louis Encephalitis, Ilheus, Bussuquara, and Yellow Fever in free-ranging black howlers (Alouatta caraya) of Northeastern Argentina

Several medically important mosquito-borne flaviviruses have been detected in Argentina in recent years: Dengue (DENV), St. Louis encephalitis (SLEV), West Nile (WNV) and Yellow Fever (YFV) viruses. Evidence of Bussuquara virus (BSQV) and Ilheus virus (ILHV) activity were found, but they have not been associated with human disease. Non-human primates can act as important hosts in the natural cycle of flaviviruses and serological studies can lead to improved understanding of virus circulation dynamics and host susceptibility. From July–August 2010, we conducted serological and molecular surveys in free–ranging black howlers (Alouatta caraya) captured in northeastern Argentina. We used 90% plaque-reduction neutralization tests (PRNT90) to analyze 108 serum samples for antibodies to WNV, SLEV, YFV, DENV (serotypes 1and 3), ILHV, and BSQV. Virus genome detection was performed using generic reverse transcription (RT)-nested PCR to identify flaviviruses in 51 antibody-negative animals. Seventy animals had antibodies for one or more flaviviruses for a total antibody prevalence of 64.8% (70/108). Monotypic (13/70, 19%) and heterotypic (27/70, 39%) patterns were differentiated. Specific neutralizing antibodies against WNV, SLEV, DENV-1, DENV-3, ILHV, and BSQV were found. Unexpectedly, the highest flavivirus antibody prevalence detected was to WNV with 9 (8.33%) monotypic responses. All samples tested by (RT)-nested PCR were negative for viral genome. This is the first detection of WNV-specific antibodies in black howlers from Argentina and the first report in free-ranging non-human primates from Latin-American countries. Given that no animals had specific neutralizing antibodies to YFV, our results suggest that the study population remains susceptible to YFV. Monitoring of these agents should be strengthened to detect the establishment of sylvatic cycles of flaviviruses in America and evaluate risks to wildlife and human health.Fil: Morales, Maria Alejandra. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbran". Instituto Nacional de Enfermedades Virales Humanas; ArgentinaFil: Fabbri, Cintia M.. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbran". Instituto Nacional de Enfermedades Virales Humanas; ArgentinaFil: Zunino, Gabriel Eduardo. Universidad Nacional de General Sarmiento. Instituto del Conurbano; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kowalewski, Miguel Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia". Estación Biológica de Usos Múltiples (Sede Corrientes); ArgentinaFil: Luppo, Victoria C.. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbran". Instituto Nacional de Enfermedades Virales Humanas; ArgentinaFil: Enría, Delia A.. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbran". Instituto Nacional de Enfermedades Virales Humanas; ArgentinaFil: Levis, Silvana C.. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbran". Instituto Nacional de Enfermedades Virales Humanas; ArgentinaFil: Calderón, Gladys Ethel. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbran". Instituto Nacional de Enfermedades Virales Humanas; Argentin

Integrated methodological framework fos assesing the risk of failure in water supply incorporating drought forecast. Case study: Andean regulated river basin

[EN] Hydroclimatic drought conditions can affect the hydrological services offered by mountain river basins causing severe impacts on the population, becoming a challenge for water resource managers in Andean river basins. This study proposes an integrated methodological framework for assessing the risk of failure in water supply, incorporating probabilistic drought forecasts, which assists in making decisions regarding the satisfaction of consumptive, non-consumptive and environmental requirements under water scarcity conditions. Monte Carlo simulation was used to assess the risk of failure in multiple stochastic scenarios, which incorporate probabilistic forecasts of drought events based on a Markov chains (MC) model using a recently developed drought index (DI). This methodology was tested in the Machángara river basin located in the south of Ecuador. Results were grouped in integrated satisfaction indexes of the system (DSIG). They demonstrated that the incorporation of probabilistic drought forecasts could better target the projections of simulation scenarios, with a view of obtaining realistic situations instead of optimistic projections that would lead to riskier decisions. Moreover, they contribute to more effective results in order to propose multiple alternatives for prevention and/or mitigation under drought conditions.This study was part of the doctoral thesis of Aviles A. at the Technical University of Valencia. This research was funded by the University of Cuenca through its Research Department (DIUC) and the Municipal public enterprise of telecommunications, drinking water, sewage and sanitation of Cuenca (ETAPA) through the projects: BIdentificacion de los procesos hidrometeorologicos que desencadenan inundaciones en la ciudad de Cuenca usando un radar de precipitacion" and "Ciclos meteorologicos y evapotranspiracion a lo largo de una gradiente altitudinal del Parque Nacional Cajas". The authors also thank INAMHI and the CBRM for providing the information for this study. The authors wish to thank the Spanish Ministry of Economy and Competitiveness for its financial support through the ERAS project (CTM2016-77804-P). We thank Angel Vazquez, who helped in the programming of the multiple simulations. Also we thank to the TropiSeca project.Avilés-Añazco, A.; Solera Solera, A.; Paredes Arquiola, J.; Pedro Monzonís, M. (2018). Integrated methodological framework fos assesing the risk of failure in water supply incorporating drought forecast. Case study: Andean regulated river basin. Water Resources Management. 32(4):1209-1223. https://doi.org/10.1007/s11269-017-1863-7S12091223324Andreu J, Capilla J, Sanchís E (1996) AQUATOOL, a generalized decision-support system for water-resources planning and operational management. 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Palisades, NYCancelliere A, Di Mauro G, Bonaccorso B, Rossi G (2007) Drought forecasting using the standardized precipitation index. Water Resour Manag 21(5):801–819. https://doi.org/10.1007/s11269-006-9062-yCancelliere A, Nicolosi V, Rossi G (2009) Assessment of drought risk in water supply systems in coping with drought risk in agriculture and water supply systems. Advances in natural and technological hazards research 26. In: Coping with drought risk in agriculture. Springer, pp 93–109. https://doi.org/10.1007/978-1-4020-9045-5_8Chen YD, Zhang Q, Xiao M, Singh VP, Zhang S (2016) Probabilistic forecasting of seasonal droughts in the Pearl River basin, China. Stoch Environ Res Risk Assess 30(7):2031–2040. https://doi.org/10.1007/s00477-015-1174-6Gong G, Wang L, Condon L, Shearman A, Lall U (2010) A simple framework for incorporating seasonal Streamflow forecasts into existing water resource management practices. 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J Appl Meteorol Climatol 48(7):1289–1301. https://doi.org/10.1175/2009JAMC2071.1Kao S-C, Govindaraju RS (2010) A copula-based joint deficit index for droughts. J Hydrol 380(1-2):121–134. https://doi.org/10.1016/j.jhydrol.2009.10.029Keyantash JA, Dracup JA (2004) An aggregate drought index: assessing drought severity based on fluctuations in the hydrologic cycle and surface water storage. Water Resour Res 40(9):1–13. https://doi.org/10.1029/2003WR002610Khadr M (2016) Forecasting of meteorological drought using hidden Markov model (case study: the upper Blue Nile river basin, Ethiopia). Ain Shams Eng J 7(1):47–56. https://doi.org/10.1016/j.asej.2015.11.005Madadgar S, Moradkhani H (2013) A Bayesian framework for probabilistic seasonal drought forecasting. J Hydrometeorol 14(6):1685–1706. https://doi.org/10.1175/JHM-D-13-010.1Madadgar S, Moradkhani H (2014) Spatio-temporal drought forecasting within Bayesian networks. J Hydrol 512:134–146. https://doi.org/10.1016/j.jhydrol.2014.02.039Mahmoudzadeh H, Mahmoudzadeh H, Afshar M, Yousefi S (2016) Applying first-order Markov chains and SPI drought index to monitor and forecast drought in West Azerbaijan Province of Iran. Int J Geo Sci Environ Plan 1:44–53. 10.22034/ijgsep.2016.40669Mishra AK, Singh VP (2010) Review paper a review of drought concepts. J Hydrol 391(1-2):202–216. https://doi.org/10.1016/j.jhydrol.2010.07.012Nalbantis I, Tsakiris G (2009) Assessment of hydrological drought revisited. Water Resour Manag 23(5):881–897. https://doi.org/10.1007/s11269-008-9305-1Ochola WO, Kerkides P (2003) A Markov chain simulation model for predicting critical wet and dry spells in Kenya: Analysing rainfall events in the kano plains. Irrig Drain 52(4):327–342. https://doi.org/10.1002/ird.094Paulo AA, Pereira LS (2007) Prediction of SPI drought class transitions using Markov chains. Water Resour Manag 21(10):1813–1827. https://doi.org/10.1007/s11269-006-9129-9Phan TD, Smart JCR, Capon SJ, Hadwen WL, Sahin O (2016) Applications of Bayesian belief networks in water resource management: a systematic review. Environ Model Softw 85:98–111. https://doi.org/10.1016/j.envsoft.2016.08.006Pouget L, Roldán T, Gómez M et al (2015) Use of seasonal climate predictions in the water sector—preliminary results from the EUPORIAS project. In: Andreu J, Solera A, Paredes J et al (eds) Drought: research and science-policy interfacing. Taylor & Francis Group, London, UK, p 247Rossi G, Cancelliere A (2013) Managing drought risk in water supply systems in Europe: a review. Int J Water Resour Dev 29(2):272–289. https://doi.org/10.1080/07900627.2012.713848Rossi G, Caporali E, Garrote L (2012) Definition of risk indicators for reservoirs management optimization. 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Dendritic Slow Dynamics Enables Localized Cortical Activity to Switch between Mobile and Immobile Modes with Noisy Background Input

Mounting lines of evidence suggest the significant computational ability of a single neuron empowered by active dendritic dynamics. This motivates us to study what functionality can be acquired by a network of such neurons. The present paper studies how such rich single-neuron dendritic dynamics affects the network dynamics, a question which has scarcely been specifically studied to date. We simulate neurons with active dendrites networked locally like cortical pyramidal neurons, and find that naturally arising localized activity – called a bump – can be in two distinct modes, mobile or immobile. The mode can be switched back and forth by transient input to the cortical network. Interestingly, this functionality arises only if each neuron is equipped with the observed slow dendritic dynamics and with in vivo-like noisy background input. If the bump activity is considered to indicate a point of attention in the sensory areas or to indicate a representation of memory in the storage areas of the cortex, this would imply that the flexible mode switching would be of great potential use for the brain as an information processing device. We derive these conclusions using a natural extension of the conventional field model, which is defined by combining two distinct fields, one representing the somatic population and the other representing the dendritic population. With this tool, we analyze the spatial distribution of the degree of after-spike adaptation and explain how we can understand the presence of the two distinct modes and switching between the modes. We also discuss the possible functional impact of this mode-switching ability

Multiple-Color Optical Activation, Silencing, and Desynchronization of Neural Activity, with Single-Spike Temporal Resolution

The quest to determine how precise neural activity patterns mediate computation, behavior, and pathology would be greatly aided by a set of tools for reliably activating and inactivating genetically targeted neurons, in a temporally precise and rapidly reversible fashion. Having earlier adapted a light-activated cation channel, channelrhodopsin-2 (ChR2), for allowing neurons to be stimulated by blue light, we searched for a complementary tool that would enable optical neuronal inhibition, driven by light of a second color. Here we report that targeting the codon-optimized form of the light-driven chloride pump halorhodopsin from the archaebacterium Natronomas pharaonis (hereafter abbreviated Halo) to genetically-specified neurons enables them to be silenced reliably, and reversibly, by millisecond-timescale pulses of yellow light. We show that trains of yellow and blue light pulses can drive high-fidelity sequences of hyperpolarizations and depolarizations in neurons simultaneously expressing yellow light-driven Halo and blue light-driven ChR2, allowing for the first time manipulations of neural synchrony without perturbation of other parameters such as spiking rates. The Halo/ChR2 system thus constitutes a powerful toolbox for multichannel photoinhibition and photostimulation of virally or transgenically targeted neural circuits without need for exogenous chemicals, enabling systematic analysis and engineering of the brain, and quantitative bioengineering of excitable cells

Variants in the FFAR1 Gene Are Associated with Beta Cell Function

The FFAR1 receptor is expressed mainly in pancreatic beta cells and is activated by medium to long chain free fatty acids (FFAs), as well as by thiazolidinediones, resulting in elevated Ca(2+) concentrations and promotion of insulin secretion. These properties suggest that FFAR1 could be a mediator of lipotoxicity and a potential candidate gene for Type 2 diabetes (T2D). We therefore investigated whether variations at the FFAR1 locus are associated with T2D and beta cell function.We re-sequenced the FFAR1 region in 96 subjects (48 healthy and 48 T2D individuals) and found 13 single nucleotide polymorphisms (SNPs) 8 of which were not previously described. Two SNPs located in the upstream region of the FFAR1 gene (rs1978013 and rs1978014) were chosen and genotyped in 1929 patients with T2D and 1405 healthy control subjects. We observed an association of rs1978013 and rs1978014 with insulinogenic index in males (p = 0.024) and females (p = 0.032), respectively. After Bonferroni corrections, no association with T2D was found in the case-control material, however a haplotype consisting of the T-G alleles conferred protection against T2D (p = 0.0010).Variation in the FFAR1 gene may contribute to impaired beta cell function in T2D

Infection of XC Cells by MLVs and Ebola Virus Is Endosome-Dependent but Acidification-Independent

Inhibitors of endosome acidification or cathepsin proteases attenuated infections mediated by envelope proteins of xenotropic murine leukemia virus-related virus (XMRV) and Ebola virus, as well as ecotropic, amphotropic, polytropic, and xenotropic murine leukemia viruses (MLVs), indicating that infections by these viruses occur through acidic endosomes and require cathepsin proteases in the susceptible cells such as TE671 cells. However, as previously shown, the endosome acidification inhibitors did not inhibit these viral infections in XC cells. It is generally accepted that the ecotropic MLV infection in XC cells occurs at the plasma membrane. Because cathepsin proteases are activated by low pH in acidic endosomes, the acidification inhibitors may inhibit the viral infections by suppressing cathepsin protease activation. The acidification inhibitors attenuated the activities of cathepsin proteases B and L in TE671 cells, but not in XC cells. Processing of cathepsin protease L was suppressed by the acidification inhibitor in NIH3T3 cells, but again not in XC cells. These results indicate that cathepsin proteases are activated without endosome acidification in XC cells. Treatment with an endocytosis inhibitor or knockdown of dynamin 2 expression by siRNAs suppressed MLV infections in all examined cells including XC cells. Furthermore, endosomal cathepsin proteases were required for these viral infections in XC cells as other susceptible cells. These results suggest that infections of XC cells by the MLVs and Ebola virus occur through endosomes and pH-independent cathepsin activation induces pH-independent infection in XC cells

Long-Term Infection and Vertical Transmission of a Gammaretrovirus in a Foreign Host Species

Increasing evidence has indicated natural transspecies transmission of gammaretroviruses; however, viral-host interactions after initial xeno-exposure remain poorly understood. Potential association of xenotropic murine leukemia virus-related virus (XMRV) in patients with prostate cancer and chronic fatigue syndrome has attracted broad interests in this topic. Although recent studies have indicated that XMRV is unlikely a human pathogen, further understanding of XMRV xenoinfection would allow in vivo modeling of the initial steps of gammaretroviral interspecies transmission, evolution and dissemination in a new host population. In this study, we monitored the long-term consequences of XMRV infection and its possible vertical transmission in a permissive foreign host, wild-derived Mus pahari mice. One year post-infection, XMRV-infected mice showed no notable pathological changes, while proviral DNA was detected in three out of eight mice. XMRV-infected mice remained seropositive throughout the study although the levels of gp70 Env- and p30 capsid-specific antibodies gradually decreased. When vertical XMRV transmission was assessed, no viremia, humoral immune responses nor endogenization were observed in nine offspring from infected mothers, yet one offspring was found PCR-positive for XMRV-specific sequences. Amplified viral sequences from the offspring showed several mutations, including one amino acid deletion in the receptor binding domain of Env SU. Our results therefore demonstrate long-term asymptomatic infection, low incidence of vertical transmission and limited evolution of XMRV upon transspecies infection of a permissive new host, Mus pahari

An automated Raman-based platform for the sorting of live cells by functional properties

Stable-isotope probing is widely used to study the function of microbial taxa in their natural environment, but sorting of isotopically labelled microbial cells from complex samples for subsequent genomic analysis or cultivation is still in its early infancy. Here, we introduce an optofluidic platform for automated sorting of stable-isotope-probing-labelled microbial cells, combining microfluidics, optical tweezing and Raman microspectroscopy, which yields live cells suitable for subsequent single-cell genomics, mini-metagenomics or cultivation. We describe the design and optimization of this Raman-activated cell-sorting approach, illustrate its operation with four model bacteria (two intestinal, one soil and one marine) and demonstrate its high sorting accuracy (98.3 ± 1.7%), throughput (200-500 cells h-1; 3.3-8.3 cells min-1) and compatibility with cultivation. Application of this sorting approach for the metagenomic characterization of bacteria involved in mucin degradation in the mouse colon revealed a diverse consortium of bacteria, including several members of the underexplored family Muribaculaceae, highlighting both the complexity of this niche and the potential of Raman-activated cell sorting for identifying key players in targeted processes.</p