A split horseradish peroxidase for detection of intercellular protein-protein interactions and sensitive visualization of synapses

Intercellular protein-protein interactions (PPIs) enable communication between cells in diverse biological processes, including cell proliferation, immune responses, infection and synaptic transmission, but they are challenging to visualize because existing techniques1,2,3 have insufficient sensitivity and/or specificity. Here we report split horseradish peroxidase (sHRP) as a sensitive and specific tool for detection of intercellular PPIs. The two sHRP fragments, engineered through screening of 17 cut sites in HRP followed by directed evolution, reconstitute into an active form when driven together by an intercellular PPI, producing bright fluorescence or contrast for electron microscopy. Fusing the sHRP fragments to the proteins neurexin (NRX) and neuroligin (NLG), which bind each other across the synaptic cleft4, enabled sensitive visualization of synapses between specific sets of neurons, including two classes of synapses in the mouse visual system. sHRP should be widely applicable for studying mechanisms of communication between a variety of cell types

Production of mannosylglycerate in Saccharomyces cerevisiae by metabolic engineering and bioprocess optimization

Mannosylglycerate (MG) is one of the most widespread compatible solutes among marine microorganisms adapted to hot environments. This ionic solute holds excellent ability to protect proteins against thermal denaturation, hence a large number of biotechnological and clinical applications have been put forward. However, the current prohibitive production costs impose severe constraints towards large-scale applications. All known microbial producers synthesize MG from GDP-mannose and 3-phosphoglycerate via a two-step pathway in which mannosyl-3-phosphoglycerate is the intermediate metabolite. In an early work, this pathway was expressed in Saccharomyces cerevisiae with the goal to confirm gene function (Empadinhas et al. in J Bacteriol 186:4075--4084, 2004), but the level of MG accumulation was low. Therefore, in view of the potential biotechnological value of this compound, we decided to invest further effort to convert S. cerevisiae into an efficient cell factory for MG production.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and also by project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI). Cristiana Faria was supported by a Ph.D. Grant from FCT (Ref. SFRH/ BD/79552/2011).info:eu-repo/semantics/publishedVersio

Object connectedness affects quantity perception in dogs.

Research in humans has shown that connecting a collection of dots with task-irrelevant lines leads to a robust numerical underestimation. To date, this paradigm has not been explored in other animal species. We assessed if connectedness results in an underestimation of quantity in dogs. Dogs (N=30) underwent an initial task, where they were presented with two plates containing different numbers of food items, and were trained to choose the stimulus containing the larger amount of food. Once they reached the learning criterion, dogs were presented with two arrays, each of which contained 30 food pieces, arranged in identical locations. On one array the food pieces were connected by thin inedible lines, forming three clusters of ten elements each. On the other array, identical lines were arranged randomly amongst food elements without being in contact with them. Each dog was presented with the test condition four times, and were expected to maximize food intake, choosing the stimulus where food items appeared more numerous. Thirteen dogs completed the test, the rest of the dogs did not reach the learning criterion in the training phase. An intercept-only GLMM model on dogs’ choice in test trials (connected or unconnected stimulus) revealed that dogs chose the unconnected stimulus more often than expected by chance (35/52, p=0.015). This suggests that connectedness affects quantity judgements by dogs in a similar way to humans. The result is of particular relevance in view of the fact that previous research generally failed to find effects of non-numerical cues on quantity estimation by dogs. Furthermore, dogs are the first non-human species in which the illusion has been assessed. In the light of the scarce susceptibility to quantity illusions by many animal species, it would be interesting to know if susceptibility to this illusion applies also to other species

Attractive serial dependence in numerosity perception in dogs (Canis familiaris).

Recent literature in humans has described a perceptual phenomenon called serial dependence, by which the current stimulus appears similar to the previous one. It has been interpreted as an active stabilization process, integrating stimulus features over time for a stable and seamless conscious experience. Despite the growing number of studies investigating serial dependence across several visual domains in humans, it is not known if the phenomenon extends also to other species. In the current study we aim to fill this gap of knowledge by exploring the behavioral signature of serial dependence in numerosity perception in dogs. We enrolled 5 dogs, who were trained on quantity discrimination prior to being presented with a set of test trials. Simultaneously presented test stimuli included a variable probe (4-16 dots) and a reference (8 dots), which was preceded by a task-irrelevant inducer stimulus (4 or 16) in the identical location. We hypothesized that if dogs are susceptible to the serial dependence, the reference would be perceived either smaller or larger depending on the inducer numerosity. For each dog we fitted a psychometric curve based on the probability of choosing the reference and defined the point of subjective equality (PSE) for the reference preceded by either of the inducer, (PSE(4)=7.10; PSE(16)=10.07). To compare the perceptual effect of the two inducers, we performed an ANOVA, which revealed a significant effect of the inducer (F(2)=23.87; p<0.001), with both of theconditions being significantly different from the control condition (p(4)=0.35; p(16)=0.02, sequential Bonferroni corrections applied). This result suggests an attractive bias in consecutively presented stimuli in dogs, providing the first evidence that the phenomenon of serial dependence extends also to non-human animals. Moreover, the finding expands the similarities between humans and dogs in processing visual stimuli