Miracle-dz/UniFAR-FTUs huggingface dataset:
A forward genetic screen identifies Dolk as a regulator of startle magnitude through the potassium channel subunit Kv1.1

The acoustic startle response is an evolutionary conserved avoidance behavior. Disruptions in startle behavior, in particular startle magnitude, are a hallmark of several human neurological disorders. While the neural circuitry underlying startle behavior has been studied extensively, the repertoire of genes and genetic pathways that regulate this locomotor behavior has not been explored using an unbiased genetic approach. To identify such genes, we took advantage of the stereotypic startle behavior in zebrafish larvae and performed a forward genetic screen coupled with whole genome analysis. This identified mutants in eight genes critical for startle behavior, including two genes encoding proteins associated with human neurological disorders, Dolichol kinase (Dolk), a broadly expressed regulator of the glycoprotein biosynthesis pathway, and the potassium Shaker-like channel subunit Kv1.1. We demonstrate that Kv1.1 acts independently of supraspinal inputs to regulate locomotion, suggesting its site of action is within spinal circuitry. Moreover, we show that Kv1.1 protein is mis-localized in dolk mutants, suggesting they act in a common genetic pathway to regulate movement magnitude. Combined, our results identify a diverse set of eight genes all associated with human disorders that regulate zebrafish startle behavior and reveal a previously unappreciated role for Dolk and Kv1.1 in regulating movement magnitude via a common genetic pathway. Author summary Underlying all animal behaviors are neural circuits, which are controlled by numerous molecular pathways that direct neuron development and activity. To identify and study these molecular pathways that control behavior, we use a simple vertebrate behavior, the acoustic startle response, in the larval zebrafish. In response to an intense noise, larval zebrafish will quickly turn and swim away to escape. From a genetic screen, we have identified a number of mutants that behave in abnormal ways in response to an acoustic stimulus. We cloned these mutants and identified eight genes that regulate startle behavior. All eight genes are associated with human disorders, and here we focus on two genes, dolk and kcna1a, encoding Dolk, a key regulator of protein glycosylation, and the potassium channel Kv1.1, respectively. We demonstrate that loss of dolk or kcna1a causes larval zebrafish to perform exaggerated swim movements and that Dolk is required for Kv1.1 protein localization to axons of neurons throughout the nervous system, providing strong evidence that dolk and kcna1a act in a common molecular pathway. Combined, our studies provide new insights into the genetic regulation of startle behavior.
D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein that mediates SARS-CoV-2 entry into host cells is a major target for vaccines and therapeutics. Thus, insights into its sequence variations are key to understanding the infection and antigenicity of SARS-CoV-2. A dominant mutational variant at position 614 of the S protein (aspartate to glycine, D614G mutation) was observed in the SARS-CoV-2 genome sequence obtained from the Nextstrain database. Using a pseudovirus-based assay, we identified that S-D614 and S-G614 protein pseudotyped viruses share a common receptor, human angiotensin-converting enzyme 2 (ACE2), which could be blocked by recombinant ACE2 with the fused Fc region of human IgG1. However, S-D614 and S-G614 protein demonstrated functional differences. First, S-G614 protein could be cleaved by serine protease elastase-2 more efficiently. Second, S-G614 pseudovirus infected 293T-ACE2 cells significantly more efficiently than did the S-D614 pseudovirus, especially in the presence of elastase-2. Third, an elastase inhibitor approved for clinical use blocked elastase-enhanced S-G614 pseudovirus infection. Moreover, 93% (65/70) convalescent sera from patients with COVID-19 could neutralize both S-D614 and S-G614 pseudoviruses with comparable efficiencies, but about 7% (5/70) convalescent sera showed reduced neutralizing activity against the S-G614 pseudovirus. These findings have important implications for SARS-CoV-2 transmission and immune interventions.
Rapid kinetics of lipid second messengers controlled by a cGMP signalling network coordinates apical complex functions in Toxoplasma tachyzoites

Host cell invasion and subsequent egress by Toxoplasma parasites is regulated by a network of cGMP, cAMP, and calcium signalling proteins. Such eukaryotic signalling networks typically involve lipid second messengers including phosphatidylinositol phosphates (PIPs), diacylglycerol (DAG) and phosphatidic acid (PA). However, the lipid signalling network in Toxoplasma is poorly defined. Here we present lipidomic analysis of a mutant of central flippase/guanylate cyclase TgGC in Toxoplasma, which we show has disrupted turnover of signalling lipids impacting phospholipid metabolism and membrane stability. The turnover of signalling lipids is extremely rapid in extracellular parasites and we track changes in PA and DAG to within 5 seconds, which are variably defective upon disruption of TgGC and other signalling proteins. We then identify the position of each protein in the signal chain relative to the central cGMP signalling protein TgGC and map the lipid signal network coordinating conoid extrusion and microneme secretion for egress and invasion.
Glyphosate used to control invasive Phragmites australis in standing water poses little risk to aquatic biota

When an invasive wetland grass degrades a Ramsar wetland and Important Bird Area, decisive management action is called for. To limit the extent and spread of European Phragmites australis, the Ontario government began the first, large-scale application of glyphosate (Roundup Custom®) over standing water to control an invasive species in Canadian history. Between 2016 and 2018, over 1000 ha of marsh were treated. To assess the risk this herbicide presented to aquatic biota, we measured the concentration of glyphosate, its primary breakdown product aminomethylphosphonic acid (AMPA), and the alcohol ethoxylate-based adjuvant Aquasurf® in water and sediments in areas of the highest exposure risk and up to 150 m into adjacent bays. We never detected glyphosate or AMPA at concentrations exceeding thresholds of toxicological concern. The maximum observed concentration of glyphosate in water was 0.320 ppm, occurring within 24 hr of application. The maximum glyphosate concentration in sediment was 0.250 ppm, occurring within 30 days of application. AMPA was detectable in water and sediment, indicating microbial breakdown of glyphosate in the marsh, but at low concentrations (maxwater = 0.025 ppm, maxsed = 0.012 ppm). The maximum distance from the point of application at which glyphosate was detected in the water was 100 m, vs. 0 m for AMPA. Concentrations in water returned to pre-treatment levels (0.005 ppm) for over one year but less than two years. Concentrations of alcohol ethoxylates were variable in space and time, following a pattern that could not be attributed to Aquasurf® application. The direct, over-water application of Roundup Custom® with Aquasurf® to control invasive P. australis does not pose a toxicological risk to aquatic biota. Highlights Glyphosate-based herbicide was applied directly to >1000 ha of marsh to control invasive P. australis Glyphosate and AMPA did not reach levels of toxicological concern for aquatic biota Aquasurf® exceedances were observed but could not be attributed to P. australis control activity Glyphosate, AMPA, and Aquasurf® dispersed no more than 100 m from the point of application Glyphosate, AMPA and Aquasurf® in water returned to baseline levels within 30 days of application Glyphosate, AMPA and Aquasurf® in sediment returned to baseline levels within 2 years
RECENT CONTRIBUTION OF GENETICS TO THE STUDY OF SPONGE SYSTEMATICS AND BIOLOGY

In this short review we will discuss the recent contributions of genetics to our understanding of the biology and evolution of sponges, particularly on the reappraisal of longstanding beliefs held by sponge taxonomists. The main questions addressed are the following. Are sponges animals? After centuries of controversy, there seems to be a consensus, now, that sponges are metazoans. Phylogenetic studies also indicate that animals are closely related to choanoflagellates. This indicates that choanoflagellate-like structures should not be considered a synapomorphy of the Porifera. Is the phylum Porifera monophyletic? Three main hypotheses are still prevailing: the Porifera are monophyletic; the Porifera are paraphyletic with the Hexactinellida being considered the more basal group of sponges, mostly because of their syncytial nature, or the Demospongiae and the Hexactinellida together, the Calcispongia being a sister-group of the Eumetazoa. Are the currently accepted Classes supported by molecular data? Molecular data confirms the presence of two monophyletic clades within the Calcispongia. On the other hand, the distinction of demosponge classes Tetractinomorpha and Ceractinomorpha, based on an oviparous versus viviparous reproduction, has been rejected by all molecular phylogenies produced so far. Are there true cosmopolitan sponge species? All putative cosmopolitan sponges species have turned out to be, under molecular scrutiny, groups of evolutionary very distinct species. We believe, thus, that the number of true cosmopolitan sponges is likely to be very small. Can sponge populations be homogeneous over large areas? Most sponge species studied to date have shown a rather small capability for long-range dispersal. This indicates that sponge larvae, both from viviparous and oviparous species, do not disperse very much. How important is asexual reproduction in the establishment and maintenance of sponge populations? Molecular markers confirm the presence of extensive asexual reproduction in sponges. The possibility of larval fusion and chimerism has important evolutionary consequences, but has not yet been tested molecularly.
Activation of mitochondria is an acute Akt-dependent response during osteogenic differentiation

Osteogenic differentiation, the process by which bone marrow mesenchymal stem/stromal (a.k.a. skeletal stem) cells and osteoprogenitors form osteoblasts, is a critical event for bone formation during development, fracture repair, and tissue maintenance. Extra- and intracellular signaling pathways triggering osteogenic differentiation are relatively well known; however, the ensuing change in cell energy metabolism is less clearly defined. Here we tested the effect of osteogenic media containing ascorbate and β-glycerol phosphate, or various osteogenic hormones and growth factors on energy metabolism in long bone (ST2)- and calvarial bone (MC3T3-E1)-derived osteoprogenitors. We show that osteogenic media, and differentiation factors, Wnt3a and BMP2, stimulate mitochondrial oxidative phosphorylation (OxPhos) with little effect on glycolysis. The activation of OxPhos occurs acutely, suggesting a metabolic signaling change rather than protein expression change. To this end, we found that the observed mitochondrial activation is Akt-dependent. Akt is activated by osteogenic media, Wnt3a, and BMP2, leading to increased phosphorylation of various mitochondrial Akt targets, a phenomenon known to stimulate OxPhos. In sum, our data provide comprehensive analysis of cellular bioenergetics during osteoinduction in cells of two different origins (mesenchyme vs neural crest) and identify Wnt3a and BMP2 as physiological stimulators of mitochondrial respiration via Akt activation.
Flower initiates a positive feedback loop upon PIP2 enrichment at periactive zones to control bulk endocytosis

Synaptic vesicle (SV) endocytosis is coupled to exocytosis to maintain SV pool size and thus neurotransmitter release. Intense stimulation induces activity-dependent bulk endocytosis (ADBE) to recapture large quantities of SV constituents in large endosomes from which SVs reform. How these consecutive processes are spatiotemporally coordinated remains unknown. Here, we show that the Flower Ca2+ channel-dependent phosphatidylinositol 4,5-bisphosphate (PIP2) compartmentalization governs such control. Strong stimuli trigger PIP2 microdomain formation at periactive zones. Upon exocytosis Flower translocates from SVs to periactive zones, where it increases PIP2 levels via Ca2+ influxes. Remarkably, PIP2 directly enhances Flower channel activity, thereby establishing a positive feedback loop for PIP2 microdomain compartmentalization. The PIP2 microdomains drive ADBE and SV reformation from bulk endosomes. PIP2 further sorts Flower to bulk endosomes, thereby terminating endocytosis. Hence, we propose that the interplay between Flower and PIP2 is the crucial spatiotemporal cue that couples exocytosis to ADBE and subsequent SV reformation.
Beyond Plug and Pray: Context Sensitivity and in silico Design of Artificial Neomycin Riboswitches

Gene regulation in prokaryotes often depends on RNA elements such as riboswitches or RNA thermometers located in the 5’ untranslated region of mRNA. Rearrangements of the RNA structure in response, e. g. , to the binding of small molecules or ions control translational initiation or premature termination of transcription and thus mRNA expression. Such structural responses are amenable to computational modeling, making it possible to rationally design synthetic riboswitches for a given aptamer. Starting from an artificial aptamer, we construct the first synthetic transcriptional riboswitches that respond to the antibiotic neomycin. We show that the switching behavior in vivo critically depends not only on the sequence of the riboswitch itself, but also on its sequence context. We therefore developed in silico methods to predict the impact of the context, making it possible to adapt the design and to rescue non-functional riboswitches. We furthermore analyze the influence of 5’ hairpins with varying stability on neomycin riboswitch activity. Our data highlight the limitations of a simple plug-and-play approach in the design of complex genetic circuits and demonstrate that detailed computational models significantly simplify, improve, and automate the design of transcriptional circuits. Our design software is available under a free license on Github.1
Clonal Diversity of Quaking Aspen (Populus Tremuloides): How Multiple Clones May Add to The resilience and Persistence of this Forest Type

27 Introduction . 28 Methods and Materials . 31 Statistical Analyses . 35 Results . 37 Discussion . 42 References . 46 xi 4. SUMMARY AND CONCLUSSIONS . 52 References . 54 APPENDICES . 55 APPENDIX A . 56 APPENDIX B . 73 APPENDIX C . 76
Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase

Most terpene synthase reactions follow Markovnikov rules for formation of high energy carbenium ion intermediates. However, there are notable exceptions. For example, pentalenene synthase (PS) undergoes an initial anti-Markovnikov cyclization reaction followed by a 1,2-hydride shift to form an intermediate humulyl cation with positive charge on the secondary carbon C9 of the farnesyl diphosphate substrate. The mechanism by which these enzymes stabilize and guide regioselectivity of secondary carbocations has not heretofore been elucidated. In an effort to better understand these reactions, we grew crystals of apo-PS, soaked them with the non-reactive substrate analog 12,13-difluorofarnesyl diphosphate, and solved the x-ray structure of the resulting complex at 2.2 Å resolution. The most striking feature of the active site structure is that C9 is positioned 3.5 Å above the center of the side chain benzene ring of residue F76, perfectly poised for stabilization of the charge through a cation-π interaction. In addition, the main chain carbonyl of I177 and neighboring intramolecular C6,C7-double bond are positioned to stabilize the carbocation by interaction with the face opposite that of F76. Mutagenesis experiments also support a role for residue 76 in cation-π interactions. Most interesting is the F76W mutant which gives a mixture of products that likely result from stabilizing a positive charge on the adjacent secondary carbon C10 in addition to C9 as in the wild-type enzyme. The crystal structure of the F76W mutant clearly shows carbons C9 and C10 centered above the fused benzene and pyrrole rings of the indole side chain, respectively, such that a carbocation at either position could be stabilized in this complex, and two anti-Markovnikov products, pentalenene and humulene, are formed. Finally, we show that there is a rough correlation (although not absolute) of an aromatic side chain (F or Y) at position 76 in related terpene synthases from Streptomyces that catalyze similar anti-Markovnikov addition reactions.
Exposure duration modulates the response of Caribbean corals to global change stressors

Global change is threatening coral reefs, with rising temperatures leading to repeat bleaching events (dysbiosis of coral hosts and their symbiotic algae) and ocean acidification reducing net coral calcification. Although global-scale mass bleaching events are revealing fine-scale patterns of coral resistance and resilience, traits that lead to persistence under environmental stress remain elusive. Here, we conducted a 95-day controlled-laboratory experiment to investigate how duration of exposure to ocean warming (28, 31°C), acidification (pCO2 = 400–2800 μatm), and their interaction influence the physiological responses of two Caribbean reef-building coral species (Siderastrea siderea, Pseudodiploria strigosa) from two reef zones of the Belize Mesoamerican Barrier Reef System. Every 30 days, calcification rate, total host protein and carbohydrate, chlorophyll a pigment concentration, and symbiont cell density were quantified for the same coral colony to characterize acclimatory responses of each genotype. Physiologies of the two species were differentially affected by these stressors, with exposure duration modulating responses. Siderastrea siderea was most affected by extreme pCO2 (~2800 μatm), which resulted in reduced calcification rate, symbiont density, and chlorophyll a concentration. Siderastrea siderea calcification rate initially declined under extreme pCO2 but recovered by the final time point, and overall demonstrated resistance to next-century pCO2 and temperature stress. In contrast, P. strigosa was more negatively impacted by elevated temperature (31°C). Reductions in P. strigosa calcification rate and total carbohydrates were consistently observed over time regardless of pCO2 treatment, with the greatest reductions observed under elevated temperature. However, nearshore colonies of P. strigosa maintained calcification rates under elevated temperature throughout all exposure durations, suggesting individuals from this environment may be locally adapted to the warmer temperatures characterizing their natal reef zone. This experiment highlights how tracking individual coral colony physiology across broad exposure durations can capture acclimatory responses of corals to global change stressors.
Impacts of landscape-scale windthrow and subsequent, variable reforestation on bird communities in Central Europe

With climate change, the area affected by and the intensity of forest disturbances such as windthrow, insect outbreaks and fire will be increasing. Post-disturbance forest management will be varied, and it is difficult to predict how much natural succession will be allowed in comparison to reforestation. Both, disturbance and reforestation will affect forest biodiversity globally, but potential shifts in species distribution, abundance and community composition are poorly understood. We studied the response of breeding bird communities to windthrow and different reforestation strategies in one of Central Europe’s largest contiguous windthrow areas created by storm Kyrill in 2007. A decade after the disturbance, we compared bird species diversity, population densities and community composition on plots in replanted beech, replanted conifers and secondary succession (all salvage-logged after the storm), with undisturbed old Norway spruce Picea abies as a control, in the setting of a natural experiment. Of the stands blown down, 95% were Norway Spruce. Reforestation strategies varied, with Spruce and non-native conifers planted on twice the area that was replanted with European Beech Fagus sylvestris. Large areas were still dominated by successional tree species a decade after the storm, especially birch, mirroring recommendations of sub-national forestry agencies to include secondary succession in future forest development. Birds responded strongly to windthrow, with a pronounced community turnover. Species associated with high conifer stands reached significantly lower densities on sample plots in disturbed areas. Replanted areas were characterized by mostly ubiquitous bird species. Areas dominated by secondary succession, especially birch Betula spp. , were characterized by high densities of long-distance migrants (often species of conservation concern) and shrubland species, among them several indicator species. Our results suggest that an increase of forest disturbance across Central Europe will lead to a pronounced reorganisation of biodiversity. Strategies that allow more secondary succession, and avoid replanting allochthonous tree species are likely to benefit populations of depleted bird species, even at salvage-logged and cleared disturbance sites.