Timer-based proteomic profiling of the ubiquitin-proteasome system reveals a substrate receptor of the GID ubiquitin ligase.

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.

Two intrinsic timing mechanisms set start and end times for dendritic arborization of a nociceptive neuron.

Choreographic dendritic arborization takes place within a defined time frame, but the timing mechanism is currently not known. Here, we report that the precisely timed lin-4-lin-14 regulatory circuit triggers an initial dendritic growth activity, whereas the precisely timed lin-28-let-7-lin-41 regulatory circuit signals a subsequent developmental decline in dendritic growth ability, hence restricting dendritic arborization within a set time frame. Loss-of-function mutations in the lin-4 microRNA gene cause limited dendritic outgrowth, whereas loss-of-function mutations in its direct target, the lin-14 transcription factor gene, cause precocious and excessive outgrowth. In contrast, loss-of-function mutations in the let-7 microRNA gene prevent a developmental decline in dendritic growth ability, whereas loss-of-function mutations in its direct target, the lin-41 tripartite motif protein gene, cause further decline. lin-4 and let-7 regulatory circuits are expressed in the right place at the right time to set start and end times for dendritic arborization. Replacing the lin-4 upstream cis-regulatory sequence at the lin-4 locus with a late-onset let-7 upstream cis-regulatory sequence delays dendrite arborization, whereas replacing the let-7 upstream cis-regulatory sequence at the let-7 locus with an early-onset lin-4 upstream cis-regulatory sequence causes a precocious decline in dendritic growth ability. Our results indicate that the lin-4-lin-14 and the lin-28-let-7-lin-41 regulatory circuits control the timing of dendrite arborization through antagonistic regulation of the DMA-1 receptor level on dendrites. The LIN-14 transcription factor likely directly represses dma-1 gene expression through a transcriptional means, whereas the LIN-41 tripartite motif protein likely indirectly promotes dma-1 gene expression through a posttranscriptional means.

From Time to Timer in Wound Healing Through the Regeneration.

Hard-to-heal wounds are an important public health issue worldwide, with a significant impact on the quality of life of patients. It is estimated that approximately 1-2% of the global population suffers from difficult wounds, which can be caused by a variety of factors such as trauma, infections, chronic diseases like diabetes or obesity, or poor health conditions. Hard-to-heal wounds are often characterized by a slow and complicated healing process, which can lead to serious complications such as infections, pressure ulcers, scar tissue formation, and even amputations. These complications can have a significant impact on the mobility, autonomy, and quality of life of patients, leading to an increase in healthcare and social costs associated with wound care. The preparation of the wound bed is a key concept in the management of hard-to-heal wounds, with the aim of promoting an optimal environment for healing. The TIME (Tissue, Infection/Inflammation, Moisture, Edge) model is a systematic approach used to assess and manage wounds in a targeted and personalized way. The concept of TIMER, expanding the TIME model, further focuses on regenerative processes, paying particular attention to promoting tissue regeneration and wound healing in a more effective and comprehensive way. The new element introduced in the TIMER model is "Regeneration", which highlights the importance of activating and supporting tissue regeneration processes to promote complete and lasting wound healing. Regenerative therapies can include a wide range of approaches, including cellular therapies, growth factors, bioactive biomaterials, stem cell therapies, and growth factor therapies. These therapies aim to promote the formation of new healthy tissues, reduce inflammation, improve vascularization, and stimulate cellular proliferation to accelerate wound closure and prevent complications. Thanks to continuous progress in research and development of regenerative therapies, more and more patients suffering from difficult wounds can benefit from innovative and promising solutions to promote faster and more effective healing, improve quality of life, and reduce the risk of long-term complications.

Is immunology doing well? A look at 100 immune-mediated inflammatory diseases for 100 years of the Journal.

It is now 60 years since Ian Mackay and Macfarlane Burnet published their seminal text "The Autoimmune Diseases" in which they examined the full scope of human inflammatory pathology as a manifestation of the underlying structure and function of the immune system. Here I revisit this approach to ask to what extent has the promise of Mackay and Burnet's work been exploited in clinical medicine as currently practiced. In other words, is immunology doing well? Despite spectacular headline contributions of immunology in clinical medicine, I present evidence suggesting a performance ceiling in our capacity to answer the relatively straightforward questions that patients frequently ask about their own diseases and find that this ceiling exists across almost all of the 100 immune-mediated inflammatory diseases examined. I propose that these questions are difficult, not so much because the immune system is overwhelmingly complex but rather that we have more to learn about the relatively simple agents and rules that may underpin self-organizing complex interacting systems as revealed in studies from other disciplines. The way that the immune system has evolved to exploit the ancient machinery determining three independent cell fate timers as described in this Journal would be a great place to start to decode the self-organizing principles that underpin the emergent pathology that we observe in the clinic.

Deviations from temporal scaling support a stage-specific regulation for C. elegans postembryonic development.

BACKGROUND: After embryonic development, Caenorhabditis elegans progress through for larval stages, each of them finishing with molting. The repetitive nature of C. elegans postembryonic development is considered an oscillatory process, a concept that has gained traction from regulation by a circadian clock gene homologue. Nevertheless, each larval stage has a defined duration and entails specific events. Since the overall duration of development is controlled by numerous factors, we have asked whether different rate-limiting interventions impact all stages equally. RESULTS: We have measured the duration of each stage of development for over 2500 larvae, under varied environmental conditions known to alter overall developmental rate. We applied changes in temperature and in the quantity and quality of nutrition and analysed the effect of genetically reduced insulin signalling. Our results show that the distinct developmental stages respond differently to these perturbations. The changes in the duration of specific larval stages seem to depend on stage-specific events. Furthermore, our high-resolution measurement of the effect of temperature on the stage-specific duration of development has unveiled novel features of temperature dependence in C. elegans postembryonic development. CONCLUSIONS: Altogether, our results show that multiple factors fine tune developmental timing, impacting larval stages independently. Further understanding of the regulation of this process will allow modelling the mechanisms that control developmental timing.

ETAM: Next Generation Event Timer for Picosecond-Precision Time and Amplitude Measurements.

Riga Event Timers have the ability to measure the interval between events with high resolution, on the order of picoseconds. However, they have several drawbacks, such as sensitivity to environmental temperature changes and an inability to capture the amplitude of the events. In this work, we present the ETAM: a next generation Event Timer. Its innovative features include adaptive correction of measurement errors based on an internal temperature sensor, and integrated peak-detector circuit to determine the amplitude of nanosecond-duration pulses. Evaluation shows that the ETAM has high thermal stability with a root mean square error (RMSE) of <3 ps in a temperature range between 0 and +40 °C, and accurate event amplitude measurement capability, with <2.3 mV RMSE in the 100-1000 mV range. These improvements allow the ETAM to be used in satellite laser ranging, optical time-domain reflectometry, and other field applications that require temperature- and amplitude-based time correction in addition to high robustness, performance, and stability.

Blue-to-Red TagFT, mTagFT, mTsFT, and Green-to-FarRed mNeptusFT2 Proteins, Genetically Encoded True and Tandem Fluorescent Timers.

True genetically encoded monomeric fluorescent timers (tFTs) change their fluorescent color as a result of the complete transition of the blue form into the red form over time. Tandem FTs (tdFTs) change their color as a consequence of the fast and slow independent maturation of two forms with different colors. However, tFTs are limited to derivatives of the mCherry and mRuby red fluorescent proteins and have low brightness and photostability. The number of tdFTs is also limited, and there are no blue-to-red or green-to-far-red tdFTs. tFTs and tdFTs have not previously been directly compared. Here, we engineered novel blue-to-red tFTs, called TagFT and mTagFT, which were derived from the TagRFP protein. The main spectral and timing characteristics of the TagFT and mTagFT timers were determined in vitro. The brightnesses and photoconversions of the TagFT and mTagFT tFTs were characterized in live mammalian cells. The engineered split version of the TagFT timer matured in mammalian cells at 37 °C and allowed the detection of interactions between two proteins. The TagFT timer under the control of the minimal arc promoter, successfully visualized immediate-early gene induction in neuronal cultures. We also developed and optimized green-to-far-red and blue-to-red tdFTs, named mNeptusFT and mTsFT, which were based on mNeptune-sfGFP and mTagBFP2-mScarlet fusion proteins, respectively. We developed the FucciFT2 system based on the TagFT-hCdt1-100/mNeptusFT2-hGeminin combination, which could visualize the transitions between the G1 and S/G2/M phases of the cell cycle with better resolution than the conventional Fucci system because of the fluorescent color changes of the timers over time in different phases of the cell cycle. Finally, we determined the X-ray crystal structure of the mTagFT timer and analyzed it using directed mutagenesis.

The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer.

Genetically encoded monomeric blue-to-red fluorescent timers (mFTs) change their fluorescent color over time. mCherry-derived mFTs were used for the tracking of the protein age, visualization of the protein trafficking, and labeling of engram cells. However, the brightness of the blue and red forms of mFTs are 2-3- and 5-7-fold dimmer compared to the brightness of the enhanced green fluorescent protein (EGFP). To address this limitation, we developed a blue-to-red fluorescent timer, named mRubyFT, derived from the bright mRuby2 red fluorescent protein. The blue form of mRubyFT reached its maximum at 5.7 h and completely transformed into the red form that had a maturation half-time of 15 h. Blue and red forms of purified mRubyFT were 4.1-fold brighter and 1.3-fold dimmer than the respective forms of the mCherry-derived Fast-FT timer in vitro. When expressed in mammalian cells, both forms of mRubyFT were 1.3-fold brighter than the respective forms of Fast-FT. The violet light-induced blue-to-red photoconversion was 4.2-fold less efficient in the case of mRubyFT timer compared to the same photoconversion of the Fast-FT timer. The timer behavior of mRubyFT was confirmed in mammalian cells. The monomeric properties of mRubyFT allowed the labeling and confocal imaging of cytoskeleton proteins in live mammalian cells. The X-ray structure of the red form of mRubyFT at 1.5 Å resolution was obtained and analyzed. The role of the residues from the chromophore surrounding was studied using site-directed mutagenesis.

Unique closure of Gustilo IIIA fracture with a hard-to-heal lower extremity wound.

Consistently achieving wound closure requires a broad understanding of wound physiology, anatomy and wound healing phases. The multifaceted principles of wound closure are comprised of: perfusion evaluation; diabetes control; nutritional optimisation; infection control; mechanical stress avoidance; oedema management; wound bed preparation; and community care. Optimisation of each element is crucial to timely and durable resolution of acute and hard-to-heal wounds. This objective is realisable only through an interdisciplinary approach to wound healing. The reconstructive ladder represents the graduation of complex wound management as applied by the specialty of plastic surgery. The approach to reconstruction typically begins with the least invasive option, which is considered reliable. However, there are instances when the most reliable option on the reconstructive ladder is not a viable option and creative solutions for wound closure are required. The following case report demonstrates a unique approach to lower extremity salvage in a subacute compound fracture surgical site infection using a limited reconstructive ladder.

Wound assessment and dressing selection: an overview.

Wound healing consists of four overlapping phases. Holistic assessment of a wound is essential and can confirm whether healing is progressing as anticipated. Frameworks can assist the clinician to conduct the assessment in a systematic way and to plan appropriate care for the patient. Dressings form a relatively small part of the overall care plan, but with such an array available the choice can be overwhelming. This article provides an update on wound assessment using the TIMERS framework and considers the factors influencing dressing choice.

Bicoid gradient formation mechanism and dynamics revealed by protein lifetime analysis.

Embryogenesis relies on instructions provided by spatially organized signaling molecules known as morphogens. Understanding the principles behind morphogen distribution and how cells interpret locally this information remains a major challenge in developmental biology. Here, we introduce morphogen-age measurements as a novel approach to test models of morphogen gradient formation. Using a tandem fluorescent timer as a protein age sensor, we find a gradient of increasing age of Bicoid along the anterior-posterior axis in the early Drosophila embryo. Quantitative analysis of the protein age distribution across the embryo reveals that the synthesis-diffusion-degradation model is the most likely model underlying Bicoid gradient formation, and rules out other hypotheses for gradient formation. Moreover, we show that the timer can detect transitions in the dynamics associated with syncytial cellularization. Our results provide new insight into Bicoid gradient formation and demonstrate how morphogen-age information can complement knowledge about movement, abundance, and distribution, which should be widely applicable to other systems.

TIMERS: undertaking wound assessment in the community.

Community nurses regularly treat patients with chronic wounds (those persisting over 6 weeks); with the complexity of both the patients' health needs and the wound itself, this often becomes a highly time-consuming task for the nurse. Wound assessment tools are designed to support all qualified nurses, regardless of whether the nurse possesses specialist wound care knowledge or not, in delivering safe and appropriate wound care. The wound assessment tool, using the acronym TIME, has been recently amended to now be known as TIMERS (Tissue, Infection/Inflammation, Moisture, Wound edge, Repair/Regeneration, Social). This article will examine what the newly amended wound assessment tool TIMERS represents, in addition to looking at the practical issues around its implementation in community settings.

Monitoring Protein Dynamics in Protein O-Mannosyltransferase Mutants In Vivo by Tandem Fluorescent Protein Timers.

For proteins entering the secretory pathway, a major factor contributing to maturation and homeostasis is glycosylation. One relevant type of protein glycosylation is O-mannosylation, which is essential and evolutionarily-conserved in fungi, animals, and humans. Our recent proteome-wide study in the eukaryotic model organism Saccharomyces cerevisiae revealed that more than 26% of all proteins entering the secretory pathway receive O-mannosyl glycans. In a first attempt to understand the impact of O-mannosylation on these proteins, we took advantage of a tandem fluorescent timer (tFT) reporter to monitor different aspects of protein dynamics. We analyzed tFT-reporter fusions of 137 unique O-mannosylated proteins, mainly of the secretory pathway and the plasma membrane, in mutants lacking the major protein O-mannosyltransferases Pmt1, Pmt2, or Pmt4. In these three pmtΔ mutants, a total of 39 individual proteins were clearly affected, and Pmt-specific substrate proteins could be identified. We observed that O-mannosylation may cause both enhanced and diminished protein abundance and/or stability when compromised, and verified our findings on the examples of Axl2-tFT and Kre6-tFT fusion proteins. The identified target proteins are a valuable resource towards unraveling the multiple functions of O-mannosylation at the molecular level.

A minute makes a difference: Apply countdown timers to cognitive well-being surveys.

This paper implemented the use of countdown timers in online subjective well-being (SWB) surveys via an online experiment. The study involved 600 US residents who were equally divided into two groups: a control group and an experimental group. Both groups were posed with the same question, "All things considered; how do you rate your own life satisfaction?" However, the experimental group was subjected to a 1-minute countdown timer before submitting their responses, while the control group was not. Our findings indicate that incorporating timers into online surveys can effectively prevent participants from mis-responding by distinguishing between their affective and cognitive well-being. Furthermore, the use of timers resulted in more comprehensive responses, as participants were able to engage in deeper reflection on their life and consider a wider range of factors.

Feedback between a retinoid-related nuclear receptor and the let-7 microRNAs controls the pace and number of molting cycles in C. elegans.

Animal development requires coordination among cyclic processes, sequential cell fate specifications, and once-a-lifetime morphogenic events, but the underlying timing mechanisms are not well understood. Caenorhabditis elegans undergoes four molts at regular 8 to 10 hour intervals. The pace of the cycle is governed by PERIOD/lin-42 and other as-yet unknown factors. Cessation of the cycle in young adults is controlled by the let-7 family of microRNAs and downstream transcription factors in the heterochronic pathway. Here, we characterize a negative feedback loop between NHR-23, the worm homolog of mammalian retinoid-related orphan receptors (RORs), and the let-7 family of microRNAs that regulates both the frequency and finite number of molts. The molting cycle is decelerated in nhr-23 knockdowns and accelerated in let-7(-) mutants, but timed similarly in let-7(-) nhr-23(-) double mutants and wild-type animals. NHR-23 binds response elements (ROREs) in the let-7 promoter and activates transcription. In turn, let-7 dampens nhr-23 expression across development via a complementary let-7-binding site (LCS) in the nhr-23 3' UTR. The molecular interactions between NHR-23 and let-7 hold true for other let-7 family microRNAs. Either derepression of nhr-23 transcripts by LCS deletion or high gene dosage of nhr-23 leads to protracted behavioral quiescence and extra molts in adults. NHR-23 and let-7 also coregulate scores of genes required for execution of the molts, including lin-42. In addition, ROREs and LCSs isolated from mammalian ROR and let-7 genes function in C. elegans, suggesting conservation of this feedback mechanism. We propose that this feedback loop unites the molting timer and the heterochronic gene regulatory network, possibly by functioning as a cycle counter.

High-Throughput Analysis of Protein Turnover with Tandem Fluorescent Protein Timers.

Tandem fluorescent protein timers (tFTs) are versatile reporters of protein dynamics. A tFT consists of two fluorescent proteins with different maturation kinetics and provides a ratiometric readout of protein age, which can be exploited to follow intracellular trafficking, inheritance and turnover of tFT-tagged proteins. Here, we detail a protocol for high-throughput analysis of protein turnover with tFTs in yeast using fluorescence measurements of ordered colony arrays. We describe guidelines on optimization of experimental design with regard to the layout of colony arrays, growth conditions, and instrument choice. Combined with semi-automated genetic crossing using synthetic genetic array (SGA) methodology and high-throughput protein tagging with SWAp-Tag (SWAT) libraries, this approach can be used to compare protein turnover across the proteome and to identify regulators of protein turnover genome-wide.

Natural Alpha-Glucosidase Inhibitors Rapid Fishing from Cyperus Rotundus Using Immobilized Enzyme Affinity Screening Combined with UHPLC-QTOF MS.

An efficient and rapid affinity-based screening method for directly fishing out natural alpha-glucosidase inhibitors from Cyperus rotundus extract by using immobilized enzyme technology combined with UHPLC-QTOF MS analysis was established. As a result without time-consuming and laborious isolation workload and false positive interference, five natural alpha-glucosidase inhibitors were successfully recognized and identified from only 400 uL of C. rotundus extracts within only a couple of hours, which suggested that the screening method was rapid, economical, sensitive and feasible. In addition, the captured compounds were isolated and characterized as stilbenoids oligomers, and were proved to be strong alpha-glucosidase inhibitors by inhibitory assay in-vitro. Among them, 3 stilbenoids trimers were reported to be potent α-glucosidase inhibitors for the first time. This method could be modified and have the potential for rapidly screening of active compounds extracts against some new targets by immobilizing some other biomacromolecules.

Display of required crossing speed improves pedestrian judgment of crossing possibility at clearance phase.

At crosswalks with countdown timers, pedestrians arriving at the clearance phase tend to start crossing when the remaining time is too short. It is unclear whether this phenomenon is due to errors in judging the possibility to finish crossing before signal lights turning red. This study evaluated and compared pedestrians' accuracy in judgment of crossing possibility based on two cues: the amount of remaining time, and the minimum required speed to finish crossing within clearance phase (road width / remaining time). The results showed that pedestrians overestimated crossing possibility when they made judgments based on remaining time, especially when the road was narrow. By contrast, the display of required speed resulted in higher overall accuracy and lower false alarm rate, due to higher sensitivity to different crossing possibilities and more conservative set of response criterion. This advantage is consistent across different road widths. These findings suggest that pedestrians' risky decisions based on the countdown timers are partly induced by overestimation of crossing possibilities. The advantages of required-speed display over traditional countdown timers indicate a strong possibility to improve pedestrian judgments by information design.

Stochastically Timed Competition Between Division and Differentiation Fates Regulates the Transition From B Lymphoblast to Plasma Cell.

In response to external stimuli, naïve B cells proliferate and take on a range of fates important for immunity. How their fate is determined is a topic of much recent research, with candidates including asymmetric cell division, lineage priming, stochastic assignment, and microenvironment instruction. Here we manipulate the generation of plasmablasts from B lymphocytes in vitro by varying CD40 stimulation strength to determine its influence on potential sources of fate control. Using long-term live cell imaging, we directly measure times to differentiate, divide, and die of hundreds of pairs of sibling cells. These data reveal that while the allocation of fates is significantly altered by signal strength, the proportion of siblings identified with asymmetric fates is unchanged. In contrast, we find that plasmablast generation is enhanced by slowing times to divide, which is consistent with a hypothesis of competing timed stochastic fate outcomes. We conclude that this mechanistically simple source of alternative fate regulation is important, and that useful quantitative models of signal integration can be developed based on its principles.

Time-series intervention analysis of pedestrian countdown timer effects.

Pedestrians account for 40-50% of traffic fatalities in large cities. Several previous studies based on relatively small samples have concluded that Pedestrian Countdown Timers (PCT) may reduce pedestrian crashes at signalized intersections, but other studies report no reduction. The purposes of the present article are to (1) describe a new methodology to evaluate the effectiveness of introducing PCT signals and (2) to present results of applying this methodology to pedestrian crash data collected in a large study carried out in Detroit, Michigan. The study design incorporated within-unit as well as between-unit components. The main focus was on dynamic effects that occurred within the PCT unit of 362 treated sites during the 120 months of the study. An interrupted time-series analysis was developed to evaluate whether change in crash frequency depended upon of the degree to which the countdown timers penetrated the treatment unit. The between-unit component involved comparisons between the treatment unit and a control unit. The overall conclusion is that the introduction of PCT signals in Detroit reduced pedestrian crashes to approximately one-third of the preintervention level. The evidence for this reductionis strong and the change over time was shown to be a function of the extent to which the timers were introduced during the intervention period. There was no general drop-off in crash frequency throughout the baseline interval of over five years; only when the PCT signals were introduced in large numbers was consistent and convincing crash reduction observed. Correspondingly, there was little evidence of change in the control unit.