Significance Photoactivatable proteins are powerful tools for studying biological processes. Light-induced dimers are especially useful because they can be turned on and off with high spatial and ...temporal resolution in living systems, allowing for control of protein localization and activity. Here, we develop and apply methods for identifying mutations that improve the effectiveness of a light-induced dimer. The engineered switch is modular, can be used in most organisms, has more than 50-fold change in binding affinity upon light stimulation, and can be used to initiate signaling pathways in a specific region of a cell.
The discovery of light-inducible protein–protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa . In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
Light-activatable proteins allow precise spatial and temporal control of biological processes in living cells and animals. Several approaches have been developed for controlling protein localization ...with light, including the conditional inhibition of a nuclear localization signal (NLS) with the Light Oxygen Voltage (AsLOV2) domain of phototropin 1 from Avena sativa. In the dark, the switch adopts a closed conformation that sterically blocks the NLS motif. Upon activation with blue light the C-terminus of the protein unfolds, freeing the NLS to direct the protein to the nucleus. A previous study showed that this approach can be used to control the localization and activity of proteins in mammalian tissue culture cells. Here, we extend this result by characterizing the binding properties of a LOV/NLS switch and demonstrating that it can be used to control gene transcription in yeast. Additionally, we show that the switch, referred to as LANS (light-activated nuclear shuttle), functions in the C. elegans embryo and allows for control of nuclear localization in individual cells. By inserting LANS into the C. elegans lin-1 locus using Cas9-triggered homologous recombination, we demonstrated control of cell fate via light-dependent manipulation of a native transcription factor. We conclude that LANS can be a valuable experimental method for spatial and temporal control of nuclear localization in vivo.
Background: The Automated Self-Administered 24-hour Recall (ASA24), a freely available Web-based tool, was developed to enhance the feasibility of collecting high-quality dietary intake data from ...large samples.Objective: The purpose of this study was to assess the criterion validity of ASA24 through a feeding study in which the true intake for 3 meals was known.Design: True intake and plate waste from 3 meals were ascertained for 81 adults by inconspicuously weighing foods and beverages offered at a buffet before and after each participant served him- or herself. Participants were randomly assigned to complete an ASA24 or an interviewer-administered Automated Multiple-Pass Method (AMPM) recall the following day. With the use of linear and Poisson regression analysis, we examined the associations between recall mode and 1) the proportions of items consumed for which a match was reported and that were excluded, 2) the number of intrusions (items reported but not consumed), and 3) differences between energy, nutrient, food group, and portion size estimates based on true and reported intakes.Results: Respondents completing ASA24 reported 80% of items truly consumed compared with 83% in AMPM (P = 0.07). For both ASA24 and AMPM, additions to or ingredients in multicomponent foods and drinks were more frequently omitted than were main foods or drinks. The number of intrusions was higher in ASA24 (P < 0.01). Little evidence of differences by recall mode was found in the gap between true and reported energy, nutrient, and food group intakes or portion sizes.Conclusions: Although the interviewer-administered AMPM performed somewhat better relative to true intakes for matches, exclusions, and intrusions, ASA24 performed well. Given the substantial cost savings that ASA24 offers, it has the potential to make important contributions to research aimed at describing the diets of populations, assessing the effect of interventions on diet, and elucidating diet and health relations. This trial was registered at clinicaltrials.gov as NCT00978406.
The development of acute kidney injury (AKI) during an intensive care unit (ICU) admission is associated with increased morbidity and mortality.
Our objective was to develop and validate a data ...driven multivariable clinical predictive model for early detection of AKI among a large cohort of adult critical care patients. We utilized data form the Medical Information Mart for Intensive Care III (MIMIC-III) for all patients who had a creatinine measured for 3 days following ICU admission and excluded patients with pre-existing condition of Chronic Kidney Disease and Acute Kidney Injury on admission. Data extracted included patient age, gender, ethnicity, creatinine, other vital signs and lab values during the first day of ICU admission, whether the patient was mechanically ventilated during the first day of ICU admission, and the hourly rate of urine output during the first day of ICU admission.
Utilizing the demographics, the clinical data and the laboratory test measurements from Day 1 of ICU admission, we accurately predicted max serum creatinine level during Day 2 and Day 3 with a root mean square error of 0.224 mg/dL. We demonstrated that using machine learning models (multivariate logistic regression, random forest and artificial neural networks) with demographics and physiologic features can predict AKI onset as defined by the current clinical guideline with a competitive AUC (mean AUC 0.783 by our all-feature, logistic-regression model), while previous models aimed at more specific patient cohorts.
Experimental results suggest that our model has the potential to assist clinicians in identifying patients at greater risk of new onset of AKI in critical care setting. Prospective trials with independent model training and external validation cohorts are needed to further evaluate the clinical utility of this approach and potentially instituting interventions to decrease the likelihood of developing AKI.
Microbial interactions in harmful algal bloom (HAB) communities have been examined in marine systems, but are poorly studied in fresh waters. To investigate HAB-microbe interactions, we isolated ...bacteria with close associations to bloom-forming cyanobacteria, Microcystis spp., during a 2017 bloom in the western basin of Lake Erie. The genomes of five isolates (Exiguobacterium sp. JMULE1, Enterobacter sp. JMULE2, Deinococcus sp. JMULE3, Paenibacillus sp. JMULE4, and Acidovorax sp. JMULE5.) were sequenced on a PacBio Sequel system. These genomes ranged in size from 3.1 Mbp (Exiguobacterium sp. JMULE1) to 5.7 Mbp (Enterobacter sp. JMULE2). The genomes were analyzed for genes relating to critical metabolic functions, including nitrogen reduction and carbon utilization. All five of the sequenced genomes contained genes that could be used in potential signaling and nutrient exchange between the bacteria and cyanobacteria such as Microcystis. Gene expression signatures of algal-derived carbon utilization for two isolates were identified in Microcystis blooms in Lake Erie and Lake Tai (Taihu) at low levels, suggesting these organisms are active and may have a functional role during Microcystis blooms in aggregates, but were largely missing from whole water samples. These findings build on the growing evidence that the bacterial microbiome associated with bloom-forming algae have the functional potential to contribute to nutrient exchange within bloom communities and interact with important bloom formers like Microcystis.
Highlights • Gaze following behaviors, a behavior foundational to social cognition, can be reliably elicited by naturalistic video stimuli using rhesus monkeys in laboratory settings. • Oxytocin ...(OT), previously demonstrated to enhance pro-social behaviors across several species, robustly increases the frequency of gaze following behaviors in rhesus monkeys. • Increased time spent viewing eyes was observed using presentation of static images, but not videos, suggesting gaze following behaviors provide a robust metric for the effects of OT, and thus may be utilized for future experiments exploring the neural mechanisms of OT in primate models.
Unstable distal clavicular fractures treated surgically are associated with high failure rates and hardware-related complications. Newer techniques have shown promising early clinical results with ...fewer hardware complications; however, their biomechanical performance has not been assessed. This study biomechanically compared a distal-third locking plate with 3 newer techniques that incorporate coracoid fixation into the construct.
The study randomized 36 adult fresh frozen cadaveric shoulders to 4 groups: (1) distal-third locking plate (P); (2) distal-third locking plate with a coracoid button augmentation (P + CB); (3) coracoclavicular button (CB); and (4) coracoclavicular button with coracoclavicular ligament reconstruction using semitendinosus allograft (CB + CC). After fixation, each specimen was stressed in the coronal plane. Cyclic displacement, load at 10-mm displacement, and ultimate load to failure were measured.
All 3 experimental groups biomechanically outperformed the locking plate. Mean load to failure was significantly higher in the CB (343 ± 76 N) and CB + CC (349 ± 94 N) groups compared with the P group (193 ± 52 N). There was also significantly less cyclic displacement in the CB (4.3 ± 1.9 mm) and CB + CC (4.4 ± 1.9 mm) groups compared with the P group (8.2 ± 2.9 mm). With respect to load at 10 mm of displacement, which essentially measures a clinical failure, the P + CB (235 ± 112 N), CB (253 ± 111 N), and CB+CC (238 ± 76 N) experimental groups significantly outperformed the P group (96 ± 29 N).
CB and CB + CC techniques demonstrated more than 75% greater strength than the traditional locking plate alone. Coupled with greater overall construct strength and lower-profile hardware, these newer techniques may result in improved clinical outcome and fewer hardware-related complications.
Pancreatic ductal adenocarcinoma is an unsolved health problem with nearly 75% of patients diagnosed with advanced disease and an overall 5-year survival rate near 5%. Despite the strong link between ...mortality and malignancy, the mechanisms behind pancreatic cancer dissemination and metastasis are poorly understood. Correlative pathological and cell culture analyses suggest the chemokine receptor CXCR4 plays a biological role in pancreatic cancer progression. In vivo roles for the CXCR4 ligand CXCL12 in pancreatic cancer malignancy were investigated. CXCR4 and CXCR7 were consistently expressed in normal and cancerous pancreatic ductal epithelium, established cell lines, and patient-derived primary cancer cells. Relative to healthy exocrine ducts, CXCL12 expression was pathologically repressed in pancreatic cancer tissue specimens and patient-derived cell lines. To test the functional consequences of CXCL12 silencing, pancreatic cancer cell lines stably expressingthe chemokine were engineered. Consistent with a role for CXCL12 as a tumor suppressor, cells producing the chemokine wereincreasingly adherent and migration deficient in vitro and poorly metastatic in vivo, compared to control cells. Further, CXCL12 reintroduction significantly reduced tumor growth in vitro, with significantly smaller tumors in vivo, leading to a pronounced survival advantage in a preclinical model. Together, these data demonstrate a functional tumor suppressive role for the normal expression of CXCL12 in pancreatic ducts, regulating both tumor growth andcellulardissemination to metastatic sites.
Underlying the study of animal welfare is the assumption that animals experience emotional states. Although there has been a bias towards studying negative emotions, research into positive emotions ...is necessary for an overall welfare assessment. The aim of the current study was to find behavioural expressions specific for anticipation of different events in domestic fowl,
Gallus gallus domesticus. To this aim, we used a Pavlovian conditioning paradigm by which we induced anticipation of a positive, neutral and negative event. We investigated whether birds were able to discriminate between sound cues signalling these events with different valences and, if so, whether anticipation of different events is reflected in different behavioural responses. The birds showed a response of increased attention to all sound cues. In anticipation of the negative event, the birds showed more head movements and locomotion than in anticipation of both the neutral and positive event, possibly reflecting the aversive nature of the negative event. In anticipation of the positive event, the birds showed more comfort behaviours, such as preening and wing flapping, which have been associated with a state of relaxation. Our study shows that laying hens are able to anticipate differentially a positive, neutral and negative event announced by different sound cues. It is also the first study to identify comfort behaviours as specifically associated with anticipation of a positive event in domestic fowl. Comfort behaviours may therefore be associated with a positive emotional state in domestic fowl.
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