Cellular feedback to organotelluranes displays modulation of antioxidant proteins gene expression Organotelluranes RT3 and RT4 are thiol reagents that induce mitochondrial transition pore (MTP) opening in a sensitive and insensitive manner to cyclosporin A. Although RT3 and RT4 promote glutathione depletion, paradoxically, they are also an efficient antioxidant for membrane lipids. These compounds antagonistic effects elicited the challenging question of how the gene expression of antioxidant enzymes would respond to treatment with these compounds. The influence of RT3 and RT4 on antioxidant enzyme expression was investigated in cultured aortic smooth muscle cells (ASMC). RT3 and RT4 promoted disruption of ionic calcium homeostasis, mitochondrial transmembrane potential ({Delta}{Psi}), and cell death in a dose-dependent manner. The cell death mechanisms responded qualitatively to the increase of the organotellurane concentration and changed from apoptosis to necrosis. RT3 and RT4 increased the expression of thioredoxin significantly. RT3 also increased the expression of glutaredoxin and glutathione peroxidase, slightly the catalase expression without significant effects on SOD expression. The results are consistent with GSH and protein thiol depletion and discussed based on the cell toxicity mechanism exhibited by these compounds.



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org.highwire.dtl.DTLVardef@52bb82org.highwire.dtl.DTLVardef@19476d9org.highwire.dtl.DTLVardef@6aa99org.highwire.dtl.DTLVardef@1aa605e_HPS_FORMAT_FIGEXP  M_FIG C_FIG
Venomics and antivenomics of Indian spectacled cobra (Naja naja) from the Western Ghats Venom proteome profiling of Naja naja from the Western Ghats region in Kerala was achieved through SDS-PAGE and RP-HPLC followed by Q-TOF LC-MS/MS analysis, incorporating PEAKS and Novor assisted de novo sequencing methodologies. A total of 115 proteins distributed across 17 different enzymatic and non-enzymatic venom protein families were identified through conventional and 39 peptides through homology-driven proteomics approaches. Fourteen peptides derived through de novo complements the Mascot data indicating the importance of homology-driven approaches in improving protein sequence information. Among the protein families identified, glutathione peroxidase and endonuclease were reported for the first time in the Indian cobra venom. Immunological cross-reactivity assessed using Indian polyvalent antivenoms suggested that VINS showed better EC50 (2.48 g/mL) value than that of PSAV (6.04 g/mL) and Virchow (6.03 g/mL) antivenoms. Western blotting experiments indicated that all the antivenoms elicited poor binding specificities, especially towards low molecular mass proteins. Second-generation antivenomics studies revealed that VINS antivenom was less efficient to detect many low molecular mass proteins such as three-finger toxins and Kunitz-type serine protease Inhibitors. Taken together, the present study enabled a large-scale characterization of the venom proteome of Naja naja from the Western Ghats and emphasized the need for developing more efficient antivenoms.

HighlightsO_LIProteomics of cobra venom resulted in the identification of 115 proteins representing 17 snake venom protein families.
C_LIO_LIDe novo approaches exclusively yielded 39 peptides harbouring multiple amino acid mutations.
C_LIO_LIGlutathione peroxidase and endonuclease were identified for the first time in Indian cobra venom.
C_LIO_LIIndian polyvalent antivenoms showed varying cross-reactivity towards cobra venom.
C_LIO_LIVINS antivenom was less efficient to detect many low molecular mass proteins (< 20 kDa).
C_LI
Upregulation of the Cdc42 GTPase limits the replicative lifespan of budding yeast Cdc42, a conserved Rho GTPase, plays a central role in polarity establishment in yeast and animals. Cell polarity is critical for asymmetric cell division, and asymmetric cell division underlies replicative aging of budding yeast. Yet how Cdc42 and other polarity factors impact lifespan is largely unknown. Here, we show by live-cell imaging that the active Cdc42 level is sporadically elevated in wild type during repeated cell divisions but rarely in the long-lived bud8 deletion cells. We find a novel Bud8 localization with cytokinesis remnants, which also recruit Rga1, a Cdc42 GTPase activating protein. Genetic analyses and live-cell imaging suggest that Rga1 and Bud8 oppositely impact lifespan likely by modulating active Cdc42 levels. An rga1 mutant, which has a shorter lifespan, dies at the unbudded state with a defect in polarity establishment. Remarkably, Cdc42 accumulates in old cells, and its mild overexpression accelerates aging with frequent symmetric cell divisions, despite no harmful effects on young cells. Our findings implicate that the interplay among these positive and negative polarity factors limits the lifespan of budding yeast. Mapping the genetic and environmental aetiology of autistic traits in Sweden and the United Kingdom BackgroundAutistic traits are influenced by both genetic and environmental factors, and are known to vary geographically in prevalence. But to what extent does their aetiology also vary from place to place?

MethodsWe applied a novel spatial approach to data on autistic traits from two large twin studies, the Child and Adolescent Twin Study in Sweden (CATSS; N=16,677, including 8,307 twin pairs) and the Twins Early Development Study in the UK (TEDS; N=11,594, including 5,796 twin pairs), to explore how the influence of nature and nurture on autistic traits varies from place to place.

ResultsWe present maps of gene- and environment-by geography interactions in Sweden and the United Kingdom (UK), showing geographical variation in both genetic and environmental influences across the two countries. In Sweden genetic influences appear higher in the far south and in a band running across the centre of the country. Environmental influences appear greatest in the south and north, with reduced environmental influence across the central band. In the UK genetic influences appear greater in the south, particularly in more central southern areas and the southeast, the Midlands and the north of England. Environmental influences appear greatest in the south and east of the UK, with less influence in the north and the west.

ConclusionsWe hope this systematic approach to identifying aetiological interactions will inspire research to examine a wider range of previously unknown environmental influences on the aetiology of autistic traits. By doing so, we will gain greater understanding of how these environments draw out or mask genetic predisposition and interact with other environmental influences in the development of autistic traits. R7 photoreceptor axon targeting depends on the relative levels of lost and found expression in R7 and its synaptic partners As neural circuits form, growing processes select the correct synaptic partners through interactions between cell surface proteins. The presence of such proteins on two neuronal processes may lead to either adhesion or repulsion; however, the consequences of mismatched expression have rarely been explored. Here we show that the Drosophila CUB-LDL protein Lost and found (Loaf) is required in the UV-sensitive R7 photoreceptor for normal axon targeting only when Loaf is also present in its synaptic partners. Although targeting occurs normally in loaf mutant animals, removing loaf from photoreceptors or expressing it in their postsynaptic neurons Tm5a/b or Dm9 in a loaf mutant causes mistargeting of R7 axons. Loaf localizes primarily to intracellular vesicles including endosomes. We propose that Loaf regulates the trafficking or function of one or more cell surface proteins, and an excess of these proteins on the synaptic partners of R7 prevents the formation of stable connections. Sablefish (Anoplopoma fimbria Pallas, 1814) plasma biochemistry and hematology reference intervals including blood cell morphology Plasma biochemistry and hematology reference intervals are integral health assessment tools in all medical fields, including aquatic animal health. As sablefish (Anoplopoma fimbria) are becoming aquaculturally and economically more important, this manuscript provides essential reference intervals (RI) for their plasma biochemistry and hematology along with reference photomicrographs of blood cells in healthy, fasted sablefish. Blood cell morphology can differ between fish species. In addition, blood cell counts and blood chemistry can vary between fish species, demographics, water conditions, seasons, diets, and culture systems, which precludes the use of RIs from other fish species. For this study, blood was collected for plasma biochemistry and hematology analysis between June 20 and July 18, 2019, from healthy, yearling sablefish, hatched and reared in captivity on a commercial diet. Overnight fast of 16-18 hours did not sufficiently reduce lipids in the blood, which led to visible lipemia and frequent rupture of blood cells during analysis. Therefore, sablefish should be fasted for 24 to 36 hours before blood is collected to reduce hematology artifacts or possible reagent interference in plasma biochemistry analysis. Lymphocytes were the most dominant leukocytes (98%), while eosinophils were rare, and basophils were not detected in sablefish. Neutrophils were very large cells with Dohle bodies. In mammals and avian species, Dohle bodies are usually signs of toxic change from inflammation, but no such association was found in these fish. In conclusion, lipemia can interfere with sablefish blood analysis, and available removal methods should be evaluated as fasting for up to 36 h might not always be feasible. Also, more studies are required to establish RI for different developmental stages and rearing conditions. Polycystins are upregulated in fibrotic kidneys and promote renal fibrosis Autosomal polycystic kidney disease (ADPKD) is caused by mutations in PKD1or PKD2 gene, which encodes polycystin-1 or polycystin-2 respectively. Paradoxically, polycystins are up-regulated in human ADPKD kidneys and overexpression of polycystin-1 even induced renal cyst formation in mice, suggesting that up-regulation of polycystin is pathogenic. In this study, we determined the role of polycystin in renal fibrosis and explored its underlying mechanisms. We found that the expression of polycystin-1 or polycystin-2 was positively correlated with the expression of collagen-I in the kidney of three different fibrotic mouse models. Pharmaceutical inhibition or genetic knockout of polycystin-2 reduced the expression of pro-fibrotic markers in fibrotic kidneys of two animal models. Moreover, conditional knockout of Pkd1 gene attenuated renal fibrosis in two mouse models. Enhancer of Zeste homolog 2 (EZH2) is a methyltransferase which plays important role in renal fibrosis. We found that EZH2 was positively correlated with the expression of polycystins in fibrotic kidneys. Conditional knockout of the Ezh2 attenuated the anti-fibrotic responses induced by Pkd1 deletion in fibrotic kidneys.

In conclusion, polycystins are up-regulated in injured kidneys and polycystins promote renal fibrosis through up-regulation of EZH2 in fibrotic kidneys, suggesting that renal fibrosis in polycystic kidneys is not the direct effect of PKD gene mutations.

SignificanceMutations of polycystins cause autosomal polycystic kidney disease (ADPKD) which is associated with increased renal fibrosis in cystic kidneys. However, the role of polycystin in renal fibrosis remains unclear. Here, we showed that the expression of polycystins is positively correlated with the development of renal fibrosis in injured kidneys. Pharmaceutical inhibition or genetic deletion of polycystin-2 attenuated renal fibrosis in fibrotic kidneys. Moreover, genetic deletion of polycystin-1 inhibited renal fibrosis in two different animal models. Finally, we showed that ablation of the methyltransferase EZH2 mitigated the anti-fibrotic effect of polycystin-1 deletion in fibrotic kidneys. We conclude that polycystin promotes through EZH2 and renal fibrosis in cystic kidney is not the direct effect of PKD gene mutations. High school science fair: Experiences of two groups of undergraduate bioscience students Science fairs offer potential opportunities for students to learn first-hand about the practices of science. Over the past six years we have been carrying out voluntary and anonymous surveys with regional and national groups of high school and post high school students to learn about their high school science fair experiences regarding help received, obstacles encountered, and opinions about the value and impact of science fair. Understanding what students think about science fairs will help educators make science fairs more effective learning opportunities. In this paper, we focus on the findings with two national groups of post high school students - undergraduate research fellows (SURF students) who did research at UT Southwestern Medical Center during 2014-2019 and undergraduates biology students attending the 2019 Howard Hughes Medical Institute Science Education Alliance (SEA) summer symposium. About 25% of the students who completed surveys indicated that they had participated in high school science fair, but more than half attended high schools where science fairs were unavailable. Effectively, 6 out of every 10 students participated in science fair if available. Students who could have participated in high school science fair but chose not to do so identified not enough time and coming up with their project idea as major reasons why not. About half the SURF students favored requiring non-competitive science fair regardless whether they themselves had participated in science fair. On the other hand, less than 1 in 5 thought that competitive science fair should be required. Introduction to the scientific process and general learning were mentioned most frequently as the reasons to require non-competitive science fair; these reasons were mentioned rarely in connection with competitive science fair. Unlike the national cohort of high school students we surveyed previously, who mostly did science fair in 9th and 10th grades, SURF students participated in science fair throughout high school and were twice as likely as high school students to have carried out science fair more than once. In conclusion, our findings suggest that participation of the undergraduate bioscience majors in high school science fairs occurs far more frequently than recognized previously. The findings emphasize further the importance of incentivizing rather than requiring science fair participation, especially in 9th and 10th grades, and the potential value of developing non-competitive science fairs. Recommendations for improving statistical inference in population genomics The field of population genomics has grown rapidly in response to the recent advent of affordable, large-scale sequencing technologies. As opposed to the situation during the majority of the 20th century, in which the development of theoretical and statistical population-genetic insights out-paced the generation of data to which they could be applied, genomic data are now being produced at a far greater rate than they can be meaningfully analyzed and interpreted. With this wealth of data has come a tendency to focus on fitting specific (and often rather idiosyncratic) models to data, at the expense of a careful exploration of the range of possible underlying evolutionary processes. For example, the approach of directly investigating models of adaptive evolution in each newly sequenced population or species often neglects the fact that a thorough characterization of ubiquitous non-adaptive processes is a prerequisite for accurate inference. We here describe the perils of these tendencies, present our consensus views on current best practices in population genomic data analysis, and highlight areas of statistical inference and theory that are in need of further attention. Thereby, we argue for the importance of defining a biologically relevant baseline model tuned to the details of each new analysis, of skepticism and scrutiny in interpreting model-fitting results, and of carefully defining addressable hypotheses and underlying uncertainties. Human cortical areas are sparsely connected: Combining histology with diffusion MRI to estimate the absolute number of axons The tracts between cortical areas are conceived as playing a central role in cortical information processing, but their actual numbers have never been determined in humans. Here we estimate the absolute number of axons linking cortical areas from a whole-cortex diffusion-MRI (dMRI) connectome, calibrated using the histologically-measured callosal fiber density. Median connectivity is estimated as ~6200 axons between cortical areas within-hemisphere and ~1300 axons inter-hemispherically, with axons connecting functionally-related areas surprisingly sparse. For example, we estimate that <5% of the axons in the trunk of the arcuate and superior longitudinal fasciculi connect Wernickes and Brocas areas. These results suggest that detailed information is transmitted between cortical areas either via linkage of the dense local connections or via rare, extraordinarily privileged long-range connections. Antibiotic Resistance Genes and Taxa Analysis from Mat and Planktonic Microbiomes of Antarctic Lake Fryxell and Lake Bonney, and a Mat-forming Culture of Rhodoferax antarcticus The Antarctic Taylor Valley Lakes Fryxell and Bonney harbor oligotrophic microbial communities that are separated geographically from other aquatic systems. Their microbiomes include planktonic as well as lift-off mat communities that float to the underside of the perennial ice cover and eventually emerge at the surface. We investigated the antibiotic resistance genes (ARGs) from metagenomes of lift-off mats emerging from ice, from filtered water samples of Lake Fryxell, and from filtered water samples of Lake Bonney. ARG sequence markers were designed by ShortBRED-Identify using the Comprehensive Antibiotic Resistance Database (CARD). The overall proportion of ARG hits in the metagenomes was found to be similar to that found in temperate-zone rural water bodies with moderate human inputs (0.0002-0.0007%). The specific ARGs found showed distinct distributions for the two lakes, and for mat versus planktonic sources. Metagenomic taxa distributions showed that mat phototrophs were mainly cyanobacteria or betaproteobacteria, whereas the water column phototrophs were mainly protists. An enrichment culture of the betaproteobacterium Rhodoferax antarcticus from a Lake Fryxell mat sample showed a mat-forming phenotype not previously reported for this species. Its genome showed no ARGs associated with betaproteobacteria, but had ARGs consistent with a minor Pseudomonas component. The Antarctic lake mats and water showed specific ARGs distinctive to the mat and water sources, but overall ARG levels were similar to those of temperate water bodies with minimal human inputs. Naturally occurring combinations of receptors from single cell transcriptomics in endothelial cells VEGF inhibitor drugs have been successful, especially in ophthalmology, but not all patients respond to them. Combinations of drugs are likely to be needed for more effective therapies of angiogenesis-related diseases. In this paper we describe naturally occurring combinations of receptors in endothelial cells that might help to understand how cells communicate and to identify targets for drug combinations. We also develop and share a new software tool called DECNEO to identify them.

Single-cell gene expression data are used to identify a set of co-expressed endothelial cell receptors, conserved among species (mice and humans) and enriched, within a network, of connections to up-regulated genes. This set includes several receptors previously shown to play a role in angiogenesis. Multiple statistical tests from large datasets, including an independent validation set, support the reproducibility, evolutionary conservation and role in angiogenesis of these naturally occurring combinations of receptors. We also show tissue-specific combinations and, in the case of choroid endothelial cells, consistency with both well-established and recent experimental findings, presented in a separate paper.

The results and methods presented here advance the understanding of signaling to endothelial cells. The methods are generally applicable to the decoding of intercellular combinations of signals. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Chromatin remodelling by INO80 at promoter proximal pause sites promotes premature termination of mRNA synthesis How co-transcriptional RNA quality control is regulated remains poorly understood. Here, we report that in S. cerevisiae premature transcription termination of mRNAs is regulated by the evolutionarily conserved ATP-dependent chromatin remodeling INO80 complex. Loss of INO80 leads to an increase in promoter-proximally paused RNA Polymerase II and defective progression into the gene body. We show that promoter-proximal transcriptional pausing correlates with loading of RNA surveillance and transcription termination factors to mRNA transcripts. Cells lacking INO80 are defective for the Nrd1-Nab3-Sen1 (NNS)-dependent pathway for transcription termination at snRNA genes and promoter-proximally sites of mRNA genes. We demonstrate that INO80 promotes the association of the RNA surveillance and termination factor Nab2 with short promoter-proximal mRNA transcripts. We provide evidence that co-transcriptional recruitment of Nab2 to chromatin is regulated by INO80, which enables the interaction of Nab2 with the histone variant H2A.Z. Our work suggests a chromatin mechanism for premature transcription termination at promoter-proximally pausing sites, linking RNA quality control to the transcriptional process. A combined test for feature selection on sparse metaproteomics data - an alternative to missing value imputation One of the difficulties encountered in the statistical analysis of metaproteomics data is the high proportion of missing values, which are usually treated by imputation. Nevertheless, imputation methods are based on restrictive assumptions regarding missingness mechanisms, namely "at random" or "not at random". To circumvent these limitations in the context of feature selection in a multi-class comparison, we propose a univariate selection method that combines a test of association between missingness and classes, and a test for difference of observed intensities between classes. This approach implicitly handles both missingness mechanisms. We performed a quantitative and qualitative comparison of our procedure with imputation-based feature selection methods on two experimental data sets, as well as simulated data with various scenarios regarding the missingness mechanisms and the nature of the difference of expression (differential intensity or differential missingness). Whereas we observed similar performances in terms of prediction on the experimental data set, the feature ranking and selection from various imputation-based methods were strongly divergent. We showed that the combined test reaches a compromise by correlating reasonably with other methods, and remains efficient in all simulated scenarios unlike imputation-based feature selection methods. Ending animal agriculture would stabilize greenhouse gas levels for 30 years and offset 70 percent of CO2 emissions this century Animal agriculture contributes significantly to global warming through ongoing emissions of the potent greenhouse gases methane and nitrous oxide, and displacement of biomass carbon on the land used to support livestock. However, because estimates of the magnitude of the effect of ending animal agriculture often focus on only one factor, the full potential benefit of a more radical change remains underappreciated. Here we quantify the full "climate opportunity cost" of current global livestock production, by modeling the combined, long-term effects of emission reductions and biomass recovery that would be unlocked by a phaseout of animal agriculture. We show that, even in the absence of any other emission reductions, persistent drops in atmospheric methane and nitrous oxide levels, and slower carbon dioxide accumulation, following a phaseout of livestock production would, through the end of the century, have the same cumulative effect on the warming potential of the atmosphere as a 25 gigaton per year reduction in anthropogenic CO2 emissions, providing half of the net emission reductions necessary to limit warming to 2{degrees}C. The magnitude and rapidity of these potential effects should place the reduction or elimination of animal agriculture at the forefront of strategies for averting disastrous climate change.

Significance StatementThe use of animals to produce food has a negative impact on the climate, but the benefits of a global switch to a plant based diet are underappreciated. We show that the global warming impact, through the rest of this century, of eliminating greenhouse gas emissions from livestock and allowing native ecosystems to regrow on the land currently used to house and feed livestock, would be equivalent to a 68% reduction in carbon dioxide emissions. We hope putting clearer numbers on the "climate opportunity cost" of our continued use of animals as food technology will help policymakers and the public properly prioritize dietary change as a climate defense strategy.

Declaration of Conflict of InterestPatrick Brown is the founder and CEO of Impossible Foods, a company developing alternatives to animals in food-production. Michael Eisen is an advisor to Impossible Foods. Both are shareholders in the company and thus stand to benefit financially from reduction of animal agriculture. Contribution of arm movements to balance recovery after tripping in older adults. Falls are common in daily life, often caused by trips and slips and, particularly in older adults, with serious consequences. Although arm movements play an important role in balance control, there is limited research into the role of arm movements during balance recovery after tripping in older adults. We investigated how older adults use their arms to recover from a trip and the difference in the effects of arm movements between fallers (n=5) and non-fallers (n=11).

Sixteen older males and females (69.7{+/-}2.3 years) walked along a walkway and were occasionally tripped over suddenly appearing obstacles. We analysed the first trip using a biomechanical model based on full-body kinematics and force-plate data to calculate whole body orientation during the trip and recovery phase. With this model, we simulated the effects of arm movements at foot-obstacle impact and during trip recovery on body orientation.

Apart from an increase in sagittal plane forward body rotation at touchdown in fallers, we found no significant differences between fallers and non-fallers in the effects of arm movements on trip recovery. Like earlier studies in young adults, we found that arm movements during the recovery phase had most favourable effects in the transverse plane: by delaying the transfer of angular momentum of the arms to the body, older adults rotated the tripped side more forward thereby allowing for a larger recovery step. Older adults that are prone to falling might improve their balance recovery after tripping by learning to prolong ongoing arm movements. Dynamics of SARS-CoV-2 mutations reveals regional-specificity and similar trends of N501 and High-Frequency mutation N501Y in different levels of control measures Coronavirus disease 2019 (COVID-19) is a contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This disease has spread globally, causing more than 161.5 million cases and 3.3 million deaths to date. Surveillance and monitoring of new mutations in the virus genome are crucial to our understanding of the adaptation of SARS-CoV-2. Moreover, how the temporal dynamics of these mutations is influenced by control measures and non-pharmaceutical interventions (NPIs) is poorly understood. Using 1 058 020 SARS-CoV-2 from sequenced COVID-19 cases from 98 countries (totaling 714 country-month combinations), we perform a normalization by COVID-19 cases to calculate the relative frequency of SARS-CoV-2 mutations and explore their dynamics over time. We found 115 mutations estimated to be present in more than 3 % of global COVID-19 cases and determined three types of mutation dynamics: High-Frequency, Medium-Frequency, and Low-Frequency. Classification of mutations based on temporal dynamics enable us to examine viral adaptation and evaluate the effects of implemented control measures in virus evolution during the pandemic. We showed that Medium-Frequency mutations are characterized by high prevalence in specific regions and/or in constant competition with other mutations in several regions. Finally, taking N501Y mutation as representative of High-Frequency mutations, we showed that level of control measure stringency negatively correlates with the effective reproduction number of SARS-CoV-2 with High-Frequency or not-High-Frequency and both follows similar trends in different levels of stringency. Interfacial tension driven open droplet microfluidics We developed an open channel droplet microfluidic system that autonomously generates droplets at low Ca (~10-4-10-3) by leveraging competing hydrostatic and capillary pressure. With only our open channel polytetrafluoroethylene (PTFE) device, pipettes, and commercially available carrier fluid, we produce hundreds of microliter droplets; tubing, electronics, or pumps are not required, making droplet technology feasible for research labs without external flow generators. Furthermore, we demonstrated conceptual applications that showcase the process of droplet generation, splitting, transport, incubation, mixing, and sorting in our system. Unlike conventional droplet microfluidics, the open nature of the device enables the use of physical tools such as tweezers and styli to directly access the system; with this, we developed a new method of droplet sorting and transfer that capitalizes on the Cheerios effect, the aggregation of buoyant objects along a liquid interface. Our platform offers enhanced usability, direct access to the droplet contents, easy manufacturability, compact footprint, and high customizability. This design is a first step in exploring the space of power-free open droplet microfluidic systems and provide design rules for similar channel designs. ABPP-HT* - Deep meets fast for activity-based profiling of deubiquitylating enzymes using advanced DIA mass spectrometry methods Activity-based protein profiling (ABPP) uses a combination of activity-based chemical probes with mass spectrometry (MS) to selectively characterise a particular enzyme or enzyme class. ABPP has proven invaluable for profiling enzymatic inhibitors in drug discovery. When applied to cell extracts and cells, challenging the ABP-enzyme complex formation with a small molecule can simultaneously inform on potency, selectivity, reversibility/binding affinity, permeability, and stability. ABPP can also be applied to pharmacodynamic studies to inform on cellular target engagement within specific organs when applied to in vivo models. Recently, we established separate high depth and high throughput ABPP (ABPP-HT) protocols for the profiling of deubiquitylating enzymes (DUBs). However, the combination of the two, deep and fast, in one method has been elusive. To further increase the sensitivity of the current ABPP-HT workflow we implemented state-of-the-art data-independent acquisition (DIA) and data-dependent acquisition (DDA) MS analysis tools. Hereby, we describe an improved methodology, ABPP-HT* (enhanced high-throughput-compatible activity-based protein profiling), that in combination with DIA MS methods allowed for the consistent profiling of 35-40 DUBs and provided a reduced number of missing values, whilst maintaining a throughput of 100 samples per day. The AMIGO1 adhesion protein activates Kv2.1 voltage sensors Kv2 voltage-gated potassium channels are modulated by AMIGO neuronal adhesion proteins. Here, we identify steps in the conductance activation pathway of Kv2.1 channels that are modulated by AMIGO1 using voltage clamp recordings and spectroscopy of heterologously expressed Kv2.1 and AMIGO1 in mammalian cell lines. AMIGO1 speeds early voltage sensor movements and shifts the gating charge-voltage relationship to more negative voltages. The gating charge-voltage relationship indicates that AMIGO1 exerts a larger energetic effect on voltage sensor movement than apparent from the midpoint of the conductance-voltage relationship. When voltage sensors are detained at rest by voltage sensor toxins, AMIGO1 has a greater impact on the conductance-voltage relationship. Fluorescence measurements from voltage sensor toxins bound to Kv2.1 indicate that with AMIGO1, the voltage sensors enter their earliest resting conformation, yet this conformation is less stable upon voltage stimulation. We conclude that AMIGO1 modulates the Kv2.1 conductance activation pathway by destabilizing the earliest resting state of the voltage sensors.

Statement of SignificanceKv2 potassium channels activate a potassium conductance that shapes neuronal action potentials. The AMIGO family of adhesion proteins modulate activation of Kv2 conductances, yet, which activation steps are modified is unknown. This study finds that AMIGO1 destabilizes the earliest resting conformation of the Kv2.1 voltage sensors to promote activation of channel conductance. Genomic hallmarks and therapeutic implications of cancer cell quiescence Therapy resistance in cancer is often driven by a subpopulation of cells that are temporarily arrested in a non-proliferative, quiescent state, which is difficult to capture and whose mutational drivers remain largely unknown. We developed methodology to uniquely identify this state from transcriptomic signals and characterised its prevalence and genomic constraints in solid primary tumours. We show quiescence preferentially emerges in the context of more stable, less mutated genomes which maintain TP53 integrity and lack the hallmarks of DNA damage repair deficiency, while presenting increased APOBEC mutagenesis. We employ machine learning to uncover novel genomic dependencies of this process, and validate the role of the centrosomal gene CEP89 as a modulator of proliferation/quiescence capacity. Lastly, we demonstrate that quiescence underlies unfavourable responses to various therapies exploiting cell cycle, kinase signalling and epigenetic mechanisms in single cell data, and propose a signature of quiescence-linked therapeutic resistance to further study and clinically track this state.

STATEMENT OF SIGNIFICANCEWe developed a robust transcriptomic signature of cellular quiescence, and employed it to systematically characterise proliferation/quiescence decisions in solid primary cancers and the genomic events influencing them. We propose CEP89 as a novel target whose suppression increases quiescence. Our expression signature of quiescence could be employed to track resistance to multiple anti-cancer compounds in a drug-tolerant persister cell setting. Ensovibep, a novel trispecific DARPin candidate that protects against SARS-CoV-2 variants SARS-CoV-2 has infected millions of people globally and continues to undergo evolution. Emerging variants can be partially resistant to vaccine induced immunity and therapeutic antibodies, emphasizing the urgent need for accessible, broad-spectrum therapeutics. Here, we report a comprehensive study of ensovibep, the first trispecific clinical DARPin candidate, that can simultaneously engage all three units of the spike protein trimer to potently inhibit ACE2 interaction, as revealed by structural analyses. The cooperative binding of the individual modules enables ensovibep to retain inhibitory potency against all frequent SARS-CoV-2 variants, including Omicron BA.1 and BA.2, as of February 2022. Moreover, viral passaging experiments show that ensovibep, when used as a single agent, can prevent development of escape mutations comparably to a cocktail of monoclonal antibodies (mAb). Finally, we demonstrate that the very high in vitro antiviral potency also translates into significant therapeutic protection and reduction of pathogenesis in Roborovski dwarf hamsters infected with either the SARS-CoV-2 wild-type or the Alpha variant. In this model, ensovibep prevents fatality and provides substantial protection equivalent to the standard of care mAb cocktail. These results support further clinical evaluation and indicate that ensovibep could be a valuable alternative to mAb cocktails and other treatments for COVID-19. A Hierarchy of Biomolecular Proportional-Integral-Derivative Feedback Controllers for Robust Perfect Adaptation and Dynamic Performance Proportional-Integral-Derivative (PID) feedback controllers have been the most widely used controllers in industry for almost a century due to their good performance, simplicity, and ease of tuning. Motivated by their success in various engineering disciplines, PID controllers recently found their way into synthetic biology, where the design of feedback molecular control systems has been identified as an important goal. In this paper, we consider the mathematical realization of PID controllers via biomolecular interactions. We propose an array of topologies that offer a compromise between simplicity and high performance. We first demonstrate that different Proportional-Integral (PI) controllers exhibit different capabilities for enhancing the dynamics and reducing variance (cell-to-cell variability). Next, we introduce several derivative controllers that are realized based on incoherent feedforward loops acting in a feedback configuration. Alternatively, we show that differentiators can be realized by placing molecular integrators in a negative feedback loop--an arrangement that can then be augmented by PI components to yield PID feedback controllers. We demonstrate that the derivative component can be exploited for enhancing system stability, dramatically increasing the molecular control systems dynamic performance, and for reducing the noise effect on the output of interest. The PID controller features are established through various deterministic and stochastic analyses as well as numerical simulations. Finally, we provide an experimental demonstration using a recently developed hybrid setup, the cyberloop, where the controller is implemented in silico to control a biological genetic circuit in vivo. The large array of novel biomolecular PID controllers introduced here forms a basis for the design and construction of advanced high-performance biomolecular control systems that robustly regulate the dynamics of living systems. Robust and Remote Measurement of Heart Rate Based on A Surveillance Camera Arrhythmia is a marked symptom of many cardiovascular diseases (CVDs). The accurate and timely detection of heart rate can greatly reduce the harm of arrhythmia to people. However, it is still a challenge to robustly and remotely measure heart rate in daily life due to the changing environmental conditions during measurement, such as the varying light intensity, the movement of people, and the uncertain distance between the sensor to people. In this study, we propose a method to accurately measure human heart rate within a distance of 4.5 meters under different light intensities by simply using a surveillance camera. After a 20-second color video of a persons hand is captured by the camera, a method based on the Fast Fourier Transform (FFT) algorithm is designed to extract the blood volume pulse wave to calculate the heart rate. According to the comparison between the real heart rate and results measured by electrocardiography (ECG), the proposed method achieves an accuracy of 95.8% when the measurement is performed within a distance of 4.5 meters and 90% when within 5.0 meters. Our experiments show that when the illuminance varies between 100-1000 lux (lighting level indoor), we still get the correct results. Our experiments also demonstrate that the proposed method accurately obtain heart rate even when the light intensity is below 32 lux (300-500 lux in a workplace environment). The methods strong adaptability to changing environmental conditions makes it applicable to many scenarios, such as homes of the elderly, classrooms, and other public spaces. Low photorespiratory capacity is sufficient to block the induction of photosynthetic capacity in high light The induction of high photosynthetic capacity in high light (HL) is a common response among many herbaceous dicot plants, however, the signals that control this response remain largely unknown. Here, multiple independent lines of evidence are presented in support of the conclusion that low photorespiratory capacity acts a negative signal to limit photosynthetic capacity acclimation in HL in Arabidopsis thaliana. Using a panel of natural accessions, primary nitrogen (N) assimilation and photorespiration rates early after a shift to growth in HL, as well as activities for key enzymes in these pathways, were shown to positively correlate with the magnitude of the subsequent induction of photosynthetic capacity, which occurred several days later. Time-resolved metabolomic data during acclimation to HL were collected using a strongly acclimating ecotype and a weakly acclimating ecotype, revealing in greater detail the differences in N assimilation, photorespiration, and triose-phosphate utilization pathways underlying efficient photosynthetic capacity acclimation. When shifted into HL growth conditions under non-photorespiratory conditions, weakly acclimating ecotypes and even photorespiratory mutants gained the ability to strongly induce high photosynthetic capacity in HL. Thus, a negative, photorespiration-dependent signal early in the HL shift appears to block photosynthetic capacity acclimation in accessions with low photorespiratory capacity, whereas accessions with high photorespiratory capacity are licensed to increase photosynthetic capacity.