The goal of this work is to determine how GCaMP6m's fluorescence is altered in response to Ca2+-binding. Our detailed spectroscopic study reveals the simplest explanation for how GCaMP6m changes ...fluorescence in response to Ca2+ is with a four-state model, in which a Ca2+-dependent change of the chromophore protonation state, due to a shift in pKa, is the predominant factor. The pKa shift is quantitatively explained by a change in electrostatic potential around the chromophore due to the conformational changes that occur in the protein when calmodulin binds Ca2+ and interacts with the M13 peptide. The absolute pKa values for the Ca2+-free and Ca2+-saturated states of GCaMP6m are critical to its high signal-to-noise ratio. This mechanism has important implications for further improvements to GCaMP6m and potentially for other similarly designed biosensors.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
We present an early version of a Susceptible–Exposed–Infected–Recovered–Deceased (SEIRD) mathematical model based on partial differential equations coupled with a heterogeneous diffusion model. The ...model describes the spatio-temporal spread of the COVID-19 pandemic, and aims to capture dynamics also based on human habits and geographical features. To test the model, we compare the outputs generated by a finite-element solver with measured data over the Italian region of Lombardy, which has been heavily impacted by this crisis between February and April 2020. Our results show a strong qualitative agreement between the simulated forecast of the spatio-temporal COVID-19 spread in Lombardy and epidemiological data collected at the municipality level. Additional simulations exploring alternative scenarios for the relaxation of lockdown restrictions suggest that reopening strategies should account for local population densities and the specific dynamics of the contagion. Thus, we argue that data-driven simulations of our model could ultimately inform health authorities to design effective pandemic-arresting measures and anticipate the geographical allocation of crucial medical resources.
Genetically encoded fluorescent biosensors are powerful tools for studying complex signaling in the nervous system, and now both Ca2+ and voltage sensors are available to study the signaling behavior ...of entire neural circuits. There is a pressing need for improved sensors, but improving them is challenging because testing them involves a low throughput, labor-intensive processes. Our goal was to create synthetic, excitable cells that can be activated with brief pulses of blue light and serve as a medium throughput platform for screening the next generation of sensors. In this live cell system, blue light activates an adenylyl cyclase enzyme (bPAC) that increases intracellular cAMP (Stierl M et al. 2011). In turn, the cAMP opens a cAMP-gated ion channel. This produces slow, whole-cell Ca2+ transients and voltage changes. To increase the speed of these transients, we add the inwardly rectifying potassium channel Kir2.1, the bacterial voltage-gated sodium channel NAVROSD, and Connexin-43. The result is a highly reproducible, medium-throughput, live cell system that can be used to screen voltage and Ca2+ sensors.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The outbreak of COVID-19 in 2020 has led to a surge in interest in the research of the mathematical modeling of epidemics. Many of the introduced models are so-called
compartmental models
, in which ...the total quantities characterizing a certain system may be decomposed into two (or more) species that are distributed into two (or more) homogeneous units called compartments. We propose herein a formulation of compartmental models based on partial differential equations (PDEs) based on concepts familiar to continuum mechanics, interpreting such models in terms of fundamental equations of balance and compatibility, joined by a constitutive relation. We believe that such an interpretation may be useful to aid understanding and interdisciplinary collaboration. We then proceed to focus on a compartmental PDE model of COVID-19 within the newly-introduced framework, beginning with a detailed derivation and explanation. We then analyze the model mathematically, presenting several results concerning its stability and sensitivity to different parameters. We conclude with a series of numerical simulations to support our findings.
Two-photon excitation of fluorescent proteins is an attractive approach for imaging living systems. Today researchers are eager to know which proteins are the brightest and what the best excitation ...wavelengths are. Here we review the two-photon absorption properties of a wide variety of fluorescent proteins, including new far-red variants, to produce a comprehensive guide to choosing the right fluorescent protein and excitation wavelength for two-photon applications.
A dominant histopathological feature in neuromuscular diseases, including amyotrophic lateral sclerosis and inclusion body myopathy, is cytoplasmic aggregation of the RNA-binding protein TDP-43. ...Although rare mutations in TARDBP-the gene that encodes TDP-43-that lead to protein misfolding often cause protein aggregation, most patients do not have any mutations in TARDBP. Therefore, aggregates of wild-type TDP-43 arise in most patients by an unknown mechanism. Here we show that TDP-43 is an essential protein for normal skeletal muscle formation that unexpectedly forms cytoplasmic, amyloid-like oligomeric assemblies, which we call myo-granules, during regeneration of skeletal muscle in mice and humans. Myo-granules bind to mRNAs that encode sarcomeric proteins and are cleared as myofibres mature. Although myo-granules occur during normal skeletal-muscle regeneration, myo-granules can seed TDP-43 amyloid fibrils in vitro and are increased in a mouse model of inclusion body myopathy. Therefore, increased assembly or decreased clearance of functionally normal myo-granules could be the source of cytoplasmic TDP-43 aggregates that commonly occur in neuromuscular disease.
Leaves comprise a number of different cell-types that are patterned in the context of either the epidermal or inner cell layers. In grass leaves, two distinct anatomies develop in the inner leaf ...tissues depending on whether the leaf carries out C3 or C4 photosynthesis. In both cases a series of parallel veins develops that extends from the leaf base to the tip but in ancestral C3 species veins are separated by a greater number of intervening mesophyll cells than in derived C4 species. We have previously demonstrated that the GRAS transcription factor SCARECROW (SCR) regulates the number of photosynthetic mesophyll cells that form between veins in the leaves of the C4 species maize, whereas it regulates the formation of stomata in the epidermal leaf layer in the C3 species rice. Here we show that SCR is required for inner leaf patterning in the C4 species Setaria viridis but in this species the presumed ancestral stomatal patterning role is also retained. Through a comparative mutant analysis between maize, setaria and rice we further demonstrate that loss of NAKED-ENDOSPERM (NKD) INDETERMINATE DOMAIN (IDD) protein function exacerbates loss of function scr phenotypes in the inner leaf tissues of maize and setaria but not rice. Specifically, in both setaria and maize, scr;nkd mutants exhibit an increased proportion of fused veins with no intervening mesophyll cells. Thus, combined action of SCR and NKD may control how many mesophyll cells are specified between veins in the leaves of C4 but not C3 grasses. Together our results provide insight into the evolution of cell patterning in grass leaves and demonstrate a novel patterning role for IDD genes in C4 leaves.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The flexible deployment of developmental regulators is an increasingly appreciated aspect of plant development and evolution. The GRAS transcription factor SCARECROW (SCR) regulates the development ...of the endodermis in Arabidopsis and maize roots, but during leaf development it regulates the development of distinct cell types; bundle-sheath in Arabidopsis and mesophyll in maize. In rice, SCR is implicated in stomatal patterning, but it is unknown whether this function is additional to a role in inner leaf patterning. Here, we demonstrate that two duplicated SCR genes function redundantly in rice. Contrary to previous reports, we show that these genes are necessary for stomatal development, with stomata virtually absent from leaves that are initiated after germination of mutants. The stomatal regulator OsMUTE is downregulated in Osscr1;Osscr2 mutants, indicating that OsSCR acts early in stomatal development. Notably, Osscr1;Osscr2 mutants do not exhibit the inner leaf patterning perturbations seen in Zmscr1;Zmscr1h mutants, and Zmscr1;Zmscr1h mutants do not exhibit major perturbations in stomatal patterning. Taken together, these results indicate that SCR was deployed in different developmental contexts after the divergence of rice and maize around 50 million years ago.
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