Miracle-dz/UniFAR-FTUs huggingface dataset:
Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM) for Nanoscale Imaging of Protein-Protein Interactions in Cells

Bimolecular fluorescence complementation (BiFC) has been widely used to visualize protein-protein interactions (PPIs) in cells. Until now, however, the resolution of BiFC has been limited by the diffraction of light to ∼250 nm, much larger than the nanometer scale at which PPIs occur or are regulated. Cellular imaging at the nanometer scale has recently been realized with single molecule superresolution imaging techniques such as photoactivated localization microscopy (PALM). Here we have combined BiFC with PALM to visualize PPIs inside cells with nanometer spatial resolution and single molecule sensitivity. We demonstrated that PAmCherry1, a photoactivatable fluorescent protein commonly used for PALM, can be used as a BiFC probe when split between residues 159 and 160 into two fragments. PAmCherry1 BiFC exhibits high specificity and high efficiency even at 37°C in detecting PPIs with virtually no background from spontaneous reconstitution. Moreover, the reconstituted protein maintains the fast photoconversion, high contrast ratio, and single molecule brightness of the parent PAmCherry1, which enables selective PALM localization of PPIs with ∼18 nm spatial precision. With BiFC-PALM, we studied the interactions between the small GTPase Ras and its downstream effector Raf, and clearly observed nanoscale clustering and diffusion of individual KRas G12D/CRaf RBD (Ras-binding domain) complexes on the cell membrane. These observations provided novel insights into the regulation of Ras/Raf interaction at the molecular scale, which would be difficult with other techniques such as conventional BiFC, fluorescence co-localization or FRET.
Climate-driven shifts in adult sex ratios via sex reversals: the type of sex determination matters

Sex reversals whereby individuals of one genetic sex develop the phenotype of the opposite sex occur in ectothermic vertebrates with genetic sex-determination systems that are sensitive to extreme temperatures during sexual differentiation. Recent rises in global temperatures have led researchers to predict that sex reversals will become more common, resulting in the distortion of many populations' sex ratios. However, it is unclear whether susceptibility to climate-driven sex-ratio shifts depends on the type of sex determination that varies across species. First, we show here using individual-based theoretical models that XX/XY (male-heterogametic) and ZZ/ZW (female-heterogametic) sex-determination systems can respond differentially to temperature-induced sex reversals. Interestingly, the impacts of climate warming on adult sex ratio (ASR) depend on the effects of both genotypic and phenotypic sex on survival and reproduction. Second, we analyse the temporal changes of ASR in natural amphibian populations using data from the literature, and find that ASR shifted towards males in ZZ/ZW species over the past 60 years, but did not change significantly in XX/XY species. Our results highlight the fact that we need a better understanding of the interactions between genetic and environmental sex-determining mechanisms to predict the responses of ectotherms to climate change and the associated extinction risks. This article is part of the themed issue ‘Adult sex ratios and reproductive decisions: a critical re-examination of sex differences in human and animal societies’.
Study of Structure‐active Relationship for Inhibitors of HIV‐1 Integrase LEDGF/p75 Interaction by Machine Learning Methods

HIV‐1 integrase (IN) is a promising target for anti‐AIDS therapy, and LEDGF/p75 is proved to enhance the HIV‐1 integrase strand transfer activity in vitro. Blocking the interaction between IN and LEDGF/p75 is an effective way to inhibit HIV replication infection. In this work, 274 LEDGF/p75‐IN inhibitors were collected as the dataset. Support Vector Machine (SVM), Decision Tree (DT), Function Tree (FT) and Random Forest (RF) were applied to build several computational models for predicting whether a compound is an active or weakly active LEDGF/p75‐IN inhibitor. Each compound is represented by MACCS fingerprints and CORINA Symphony descriptors. The prediction accuracies for the test sets of all the models are over 70 %. The best model Model 3B built by FT obtained a prediction accuracy and a Matthews Correlation Coefficient (MCC) of 81.08 % and 0.62 on test set, respectively. We found that the hydrogen bond and hydrophobic interactions are important for the bioactivity of an inhibitor.
Full-Length HIV-1 Immunogens Induce Greater Magnitude and Comparable Breadth of T Lymphocyte Responses to Conserved HIV-1 Regions Compared with Conserved-Region-Only HIV-1 Immunogens in Rhesus Monkeys

ABSTRACT A global HIV-1 vaccine will likely need to induce immune responses against conserved HIV-1 regions to contend with the profound genetic diversity of HIV-1. Here we evaluated the capacity of immunogens consisting of only highly conserved HIV-1 sequences that are aimed at focusing cellular immune responses on these potentially critical regions. We assessed in rhesus monkeys the breadth and magnitude of T lymphocyte responses elicited by adenovirus vectors expressing either full-length HIV-1 Gag/Pol/Env immunogens or concatenated immunogens consisting of only highly conserved HIV-1 sequences. Surprisingly, we found that the full-length immunogens induced comparable breadth (P = 1.0) and greater magnitude (P = 0.01) of CD8+ T lymphocyte responses against conserved HIV-1 regions compared with the conserved-region-only immunogens. Moreover, the full-length immunogens induced a 5-fold increased total breadth of HIV-1-specific T lymphocyte responses compared with the conserved-region-only immunogens (P = 0.007). These results suggest that full-length HIV-1 immunogens elicit a substantially increased magnitude and breadth of cellular immune responses compared with conserved-region-only HIV-1 immunogens, including greater magnitude and comparable breadth of responses against conserved sequences.
The Uncertainty Reducing Capabilities of Primary Care Physicians’ Video Biographies for Choosing a New Doctor: Is a Video Worth More Than Two Hundred Words?

ABSTRACT Choosing a primary care physician for the first time is an important decision, one that health care systems do not make particularly easy for prospective patients to make solely through the limited information provided on their websites. Without knowledge from others, a new patient is likely to have uncertainty about the physician he or she chooses. Three hundred and twenty participants completed an online experiment and were exposed to two biographies of different doctors with different media and either professional or personal information. Predictions generated by media richness theory revealed greater reductions in uncertainty for video biographies than traditional text biographies. Video biographies, and those containing personal information about the physician, were also related to higher levels of anticipated patient satisfaction and care quality. When asked to choose the physicians they would want to visit, participants overwhelmingly chose the physician with whom they perceived the greatest similarity to themselves, as well as the doctor who provided a video biography. Both theoretical and practical implications of this research are discussed.
Novel de novo variant in EBF3 is likely to impact DNA binding in a patient with a neurodevelopmental disorder and expanded phenotypes: patient report, in silico functional assessment, and review of published cases

Pathogenic variants in EBF3 were recently described in three back-to-back publications in association with a novel neurodevelopmental disorder characterized by intellectual disability, speech delay, ataxia, and facial dysmorphisms. In this report, we describe an additional patient carrying a de novo missense variant in EBF3 (c.487C>T, p.(Arg163Trp)) that falls within a conserved residue in the zinc knuckle motif of the DNA binding domain. Without a solved structure of the DNA binding domain, we generated a homology-based atomic model and performed molecular dynamics simulations for EBF3, which predicted decreased DNA affinity for p.(Arg163Trp) compared with wild-type protein and control variants. These data are in agreement with previous experimental studies of EBF1 showing the paralogous residue is essential for DNA binding. The conservation and experimental evidence existing for EBF1 and in silico modeling and dynamics simulations to validate comparable behavior of multiple variants in EBF3 demonstrates strong support for the pathogenicity of p.(Arg163Trp). We show that our patient presents with phenotypes consistent with previously reported patients harboring EBF3 variants and expands the phenotypic spectrum of this newly identified disorder with the additional feature of a bicornuate uterus.
Antiretroviral Drug Metabolism in Humanized PXR-CAR-CYP3A-NOG Mice

Antiretroviral drug (ARV) metabolism is linked largely to hepatic cytochrome P450 activity. One ARV drug class known to be metabolized by intestinal and hepatic CYP3A are the protease inhibitors (PIs). Plasma drug concentrations are boosted by CYP3A inhibitors such as cobisistat and ritonavir (RTV). Studies of such drug-drug interactions are limited since the enzyme pathways are human specific. While immune-deficient mice reconstituted with human cells are an excellent model to study ARVs during human immunodeficiency virus type 1 (HIV-1) infection, they cannot reflect human drug metabolism. Thus, we created a mouse strain with the human pregnane X receptor, constitutive androstane receptor, and CYP3A4/7 genes on a NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac background (hCYP3A-NOG) and used them to evaluate the impact of human CYP3A metabolism on ARV pharmacokinetics. In proof-of-concept studies we used nanoformulated atazanavir (nanoATV) with or without RTV. NOG and hCYP3A-NOG mice were treated weekly with 50 mg/kg nanoATV alone or boosted with nanoformulated ritonavir (nanoATV/r). Plasma was collected weekly and liver was collected at 28 days post-treatment. Plasma and liver atazanavir (ATV) concentrations in nanoATV/r-treated hCYP3A-NOG mice were 2- to 4-fold higher than in replicate NOG mice. RTV enhanced plasma and liver ATV concentrations 3-fold in hCYP3A-NOG mice and 1.7-fold in NOG mice. The results indicate that human CYP3A-mediated drug metabolism is reduced compared with mouse and that RTV differentially affects human gene activity. These differences can affect responses to PIs in humanized mouse models of HIV-1 infection. Importantly, hCYP3A-NOG mice reconstituted with human immune cells can be used for bench-to-bedside translation.
Identifying the African Wintering Grounds of Hybrid Flycatchers Using a Multi–Isotope (δ 2H, δ 13C, δ 15N) Assignment Approach

Migratory routes and wintering grounds can have important fitness consequences, which can lead to divergent selection on populations or taxa differing in their migratory itinerary. Collared (Ficedula albicollis) and pied (F. hypoleuca) flycatchers breeding in Europe and wintering in different sub-Saharan regions have distinct migratory routes on the eastern and western sides of the Sahara desert, respectively. In an earlier paper, we showed that hybrids of the two species did not incur reduced winter survival, which would be expected if their migration strategy had been a mix of the parent species' strategies potentially resulting in an intermediate route crossing the Sahara desert to different wintering grounds. Previously, we compared isotope ratios and found no significant difference in stable-nitrogen isotope ratios (δ 15N) in winter-grown feathers between the parental species and hybrids, but stable-carbon isotope ratios (δ 13C) in hybrids significantly clustered only with those of pied flycatchers. We followed up on these findings and additionally analyzed the same feathers for stable-hydrogen isotope ratios (δ 2H) and conducted spatially explicit multi-isotope assignment analyses. The assignment results overlapped with presumed wintering ranges of the two species, highlighting the efficacy of the method. In contrast to earlier findings, hybrids clustered with both parental species, though most strongly with pied flycatcher.
Test Driven Development: Advancing Knowledge by Conjecture and Confirmation

Test Driven Development (TDD) is a critical agile software development practice that supports innovation in short development cycles. However, TDD is one of the most challenging agile practices to adopt because it requires changes to work practices and skill sets. It is therefore important to gain an understanding of TDD through the experiences of those who have successfully adopted this practice. We collaborated with an agile team to provide this experience report on their adoption of TDD, using observations and interviews within the product development environment. This article highlights a number of practices that underlie successful development with TDD. To provide a theoretical perspective that can help to explain how TDD supports a positive philosophy of software development, we have revised Northover et al.’s conceptual framework, which is based on a four stage model of agile development, to reinterpret Popper’s theory of conjecture and falsification in the context of agile testing strategies. As a result of our findings, we propose an analytical model for TDD in agile software development which provides a theoretical basis for further investigations into the role of TDD and related practices.
Flotation of Micro-fine Ilmenite Using New Type Collector──ROB

A newly-developed collector--ROB is used for flotation separation of micro-fine ilmenite of Panzhihua mine. In its industrial test, good results such as a concentrate TiO 2 grade of 48% and a recovery of 75% are obtained. Compared with the original collector, the concentrate TiO 2 grade is increased by 0.65%, the recovery is increased by 7.3% and the reagent cost of a ton titanium concentrate is reduced by 40.54 yuan(RMB).
In Vivo Selection of Resistant E. coli after Ingestion of Milk with Added Drug Residues

Antimicrobial resistance represents a major global threat to modern medicine. In vitro studies have shown that very low concentrations of drugs, as frequently identified in the environment, and in foods and water for human and animal consumption, can select for resistant bacteria. However, limited information is currently available on the in vivo impact of ingested drug residues. The objective of our study was to evaluate the effect of feeding preweaned calves milk containing antimicrobial drug residues (below the minimum inhibitory concentration), similar to concentrations detected in milk commonly fed to dairy calves, on selection of resistant fecal E. coli in calves from birth to weaning. At birth, thirty calves were randomly assigned to a controlled feeding trial where: 15 calves were fed raw milk with no drug residues (NR), and 15 calves were fed raw milk with drug residues (DR) by adding ceftiofur, penicillin, ampicillin, and oxytetracycline at final concentrations in the milk of 0.1, 0.005, 0.01, and 0.3 µg/ml, respectively. Fecal samples were rectally collected from each calf once a week starting at birth prior to the first feeding in the trial (pre-treatment) until 6 weeks of age. A significantly greater proportion of E. coli resistant to ampicillin, cefoxitin, ceftiofur, streptomycin and tetracycline was observed in DR calves when compared to NR calves. Additionally, isolates from DR calves had a significant decrease in susceptibility to ceftriaxone and ceftiofur when compared to isolates from NR calves. A greater proportion of E. coli isolates from calves in the DR group were resistant to 3 or more antimicrobial drugs when compared to calves in the ND group. These findings highlight the role that low concentrations of antimicrobial drugs have on the evolution and selection of resistance to multiple antimicrobial drugs in vivo.
Differences in the Clinical Profile and Outcomes of Typical and Atypical Takotsubo Syndrome: Data From the International Takotsubo Registry.

IMPORTANCE Apical ballooning is broadly recognized as the classic form of takotsubo syndrome (TTS). Atypical subtypes of TTS also exist, which constitute about 20% of all cases. To date, clinical profile and course of atypical TTS types have rarely been studied. OBJECTIVE To characterize the clinical profile and outcomes of typical vs atypical types of TTS in a large patient cohort. DESIGN, SETTING, AND PARTICIPANTS Records of 1750 patients from the International Takotsubo Registry, comprising 26 participating cardiovascular centers in 9 different countries, were reviewed and data on clinical profile and outcomes collected from January 1, 2011, to December 31, 2014. MAIN OUTCOMES AND MEASURES Clinical characteristics and in-hospital as well as long-term outcomes were assessed. RESULTS Of 1750 patients diagnosed with TTS between 1998 and 2014, a total of 1430 (81.7%) presented with apical TTS (defined as typical TTS) and 320 (18.3%) with midventricular, basal, or focal TTS (all defined as atypical TTS). Patients with atypical TTS were younger than those with typical TTS (mean [SD], 62.5 [13.3] vs 67.3 [12.9] years; P < .001). Brain natriuretic peptide levels on admission were lower (median factor increase of the upper limit of normal, 4.18 vs 6.59; P = .02) and left ventricular ejection fraction was higher (mean [SD], 43.4% [10.7%] vs 40.6% [12.0%]; P < .001) in patients with atypical than those with typical forms of TTS. ST-segment depression was more prevalent in patients with atypical TTS (31 of 286 [10.8%] vs 90 of 1292 [7.0%]; P = .03), while ST-segment elevation was found more frequently in patients with typical TTS (593 of 1292 [45.9%] vs 97 of 286 [33.9%]; P < .001). Patients with atypical TTS more often had neurologic disorders than those with typical TTS (81 of 274 [29.6%] vs 286 of 1251 [22.9%]; P = .02). While in-hospital mortality was comparable between patients with atypical and typical TTS (10 of 320 [3.1%] vs 62 of 1430 [4.3%]; P = .32), the atypical forms showed a favorable outcome at 1 year (P = .01). However, after adjustment for confounders, only left ventricular ejection fraction less than 45%, atrial fibrillation, and neurologic disease, but not the type of TTS, were independent predictors. After 1 year, patients with both types of TTS showed a similar prognosis at long-term follow-up. CONCLUSIONS AND RELEVANCE Atypical TTS has different characteristics than typical TTS, including younger age of onset, more frequent ST-segment depression, higher prevalence of neurologic diseases, less pronounced reduction in left ventricular ejection fraction, and lower brain natriuretic peptide values on admission. Outcomes are comparable between patients with both types after adjustment for confounders, suggesting that both should be equally monitored.
Preference for Male Traits Differ in Two Female Morphs of the Tree Lizard, Urosaurus ornatus

Non-random female mating preferences may contribute to the maintenance of phenotypic variation in color polymorphic species. However, the effect of female preference depends on the types of male traits used as signals by receptive females. If preference signals derive from discrete male traits (i.e., morph-specific), female preferences may rapidly fix to a morph. However, female preference signals may also include condition-dependent male traits. In this scenario, female preference may differ depending on the social context (i.e., male morph availability). Male tree lizards (Urosaurus ornatus) exhibit a dewlap color polymorphism that covaries with mating behavior. Blue morph males are aggressive and defend territories, yellow males are less aggressive and defend smaller territories, and orange males are typically nomadic. Female U. ornatus are also polymorphic in dewlap color, but the covariation between dewlap color and female behavior is unknown. We performed an experiment to determine how female mate choice depends on the visual and chemical signals produced by males. We also tested whether female morphs differ in their preferences for these signals. Female preferences involved both male dewlap color and size of the ventral color patch. However, the female morphs responded to these signals differently and depended on the choice between the types of male morphs. Our experiment revealed that females may be capable of distinguishing among the male morphs using chemical signals alone. Yellow females exhibit preferences based on both chemical and visual signals, which may be a strategy to avoid ultra-dominant males. In contrast, orange females may prefer dominant males. We conclude that female U. ornatus morphs differ in mating behavior. Our findings also provide evidence for a chemical polymorphism among male lizards in femoral pore secretions.
Resurfaced cell‐penetrating nanobodies: A potentially general scaffold for intracellularly targeted protein discovery

By virtue of their size, functional group diversity, and complex structure, proteins can often recognize and modulate disease‐relevant macromolecules that present a challenge to small‐molecule reagents. Additionally, high‐throughput screening and evolution‐based methods often make the discovery of new protein binders simpler than the analogous small‐molecule discovery process. However, most proteins do not cross the lipid bilayer membrane of mammalian cells. This largely limits the scope of protein therapeutics and basic research tools to those targeting disease‐relevant receptors on the cell surface or extracellular matrix. Previously, researchers have shown that cationic resurfacing of proteins can endow cell penetration. However, in our experience, many proteins are not amenable to such extensive mutagenesis. Here, we report that nanobodies—a small and stable protein that can be evolved to recognize virtually any disease‐relevant receptor—are amenable to cationic resurfacing, which results in cell internalization. Once internalized, these nanobodies access the cytosol. Polycationic resurfacing does not appreciably alter the structure, expression, and function (target recognition) of a previously reported GFP‐binding nanobody, and multiple nanobody scaffolds are amenable to polycationic resurfacing. Given this, we propose that polycationic resurfaced cell‐penetrating nanobodies might represent a general scaffold for intracellularly targeted protein drug discovery.