Biology subjects

Bianco, M.

Publications and source records attributed to Bianco, M..

4 recordsLinked to original sources

bioRxiv2021

Influence of anatomical features of different brain regions on the spatial localization of fiber photometry signals

Fiber photometry is widely used in neuroscience labs for in vivo detection of functional fluorescence from optical indicators of neuronal activity with a simple optical fiber. The fiber is commonly placed next to the region of interest to both excite and collect the fluorescence signal. However, the path of both excitation and fluorescence photons is altered by the uneven optical properties of the brain, due to local variation of the refractive index, different cellular types, densities and shapes. Nonetheless, the effect of the local anatomy on the actual shape and extent of the volume of tissue that interfaces with the fiber has received little attention so far. To fill this gap, we measured the size and shape of fiber photometry efficiency field in the primary motor and somatosensory cortex, in the hippocampus and in the striatum of the mouse brain, highlighting how their substructures determine the detected signal and the depth at which photons can be mined. Importantly, we show that the information on the spatial expression of the fluorescent probes alone is not sufficient to account for the contribution of local subregions to the overall collected signal, and it must be combined with the optical properties of the tissue adjacent to the fiber tip.

neuroscience↗

bioRxiv2021

A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosyl-Lmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyze SAM- dependent arsenite methylation with formation of monomethylarsenite (MMAs) and dimethylarsenite (DMAs). In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9- based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus {Delta}TtarsM genome was substituted by a gene encoding a stabilized yellow fluorescent protein (sYFP) to create a sensitive genome-based bioreporter system for the detection of arsenic ions. ImportanceWe here describe the discovery of an unknown protein by using a proteomic approach with a functionally related protein as bait. Remarkably, we successfully obtained a novel type of enzyme through the interaction with a transcription regulator, controlling the expression of this enzyme. Employing this strategy, we isolated TtArsM, the first thermophilic prokaryotic arsenite methyltransferase, as a new enzyme of the arsenic resistance mechanism in T. thermophilus HB27. The atypical arsenite binding site of TtArsM categorizes the enzyme as the first member of a new arsenite methyltransferase type, exclusively present in the Thermus genus. The enzyme methylates arsenite producing MMAs and DMAs. Furthermore, we developed an hyperthermophilic Cas9-based genome-editing tool, active up to 65{degrees}C. The tool allowed us to perform highly efficient, marker-free modifications (either gene deletion or insertion) in the T. thermophilus genome. With these modifications, we confirmed the critical role of TtArsM in the arsenite detoxification system and developed a sensitive whole cell bioreporter for arsenic ions. We anticipate that the developed tool can be easily adapted for editing the genomes of other thermophilic bacteria, significantly boosting fundamental and metabolic engineering in hyperthermophilic microorganisms.

biochemistry↗

bioRxiv2021

Games of risk and reward in carnivore communities

Mesopredator release theory suggests that dominant predators suppress subordinate carnivores and ultimately shape community dynamics, but the assumption that subordinate species are only negatively affected ignores the possibility of facilitation through scavenging. We examined the interplay within a carnivore community consisting of cougars, coyotes, black bears, and bobcats using contemporaneous Global Positioning System telemetry data from 51 individuals, diet analysis from 972 DNA-metabarcoded scats, and data from 128 physical investigations of cougar kill sites, 28 of which were monitored with remote cameras. Resource provisioning from competitively-dominant cougars to coyotes through scavenging was so prolific as to be an overwhelming determinant of coyote behavior, space use, and resource acquisition. This was evident via strong attraction of coyotes to cougar kill sites, frequent scavenging of cougar-killed prey, and coyote diets that nearly matched cougars in the magnitude of ungulate consumption. Yet coyotes were often killed by cougars and used space to minimize encounters, complicating the fitness benefits gained from scavenging. We estimated that 23% (95% CI: 8-55%) of the coyote population in our study area was killed by cougars annually suggesting that coyote interactions with cougars are a complex behavioral game of risk and reward. In contrast, we found no indication that bobcat space use or diet was influenced by cougars. Black bears avoided cougars, but there was no evidence of attraction to cougar kill sites, and much lower levels of ungulate consumption and carcass visitation than for coyotes. Interspecific interactions among carnivores are multifaceted encompassing both suppression and facilitation. Significance StatementAn incomplete understanding of the total influence competitively-dominant predators exert on subordinate species hinders our ability to anticipate the effects that changing carnivore populations will have in ecological communities. Here we show that cougars are the architects of a complex behavioral game of risk and reward, as subordinate or co-occurring carnivores are both provisioned and preyed on by the dominant predators. Each co-occurring carnivore species considered here employed a different strategy to approach the risk-reward tradeoff suggesting there are multiple viable solutions to the game. By not considering the multitude of effects dominant predators have on other carnivores, we are missing important linkages in terrestrial food webs.

ecology↗

bioRxiv2020

Ray tracing models for estimating light collection properties of microstructured tapered optical fibers for optical neural interfaces

Tapered optical fibers (TFs) were recently employed for depth-resolved monitoring of functional fluorescence in sub-cortical brain structures, enabling light collection from groups of a few cells through small optical windows located on the taper edge [1]. Here we present a numerical model to estimate light collection properties of microstructured TFs implanted in scattering brain tissue. Ray tracing coupled with Henyey-Greenstein scattering model enables the estimation of both light collection and fluorescence excitation fields in three dimensions, whose combination is employed to retrieve the volume of tissue probed by the device.

bioengineering↗