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Multicolor single-molecule tracking (SMT) provides a powerful tool to mechanistically probe molecular interactions in living cells. However, because of the limitations in the optical and chemical properties of currently available fluorophores and the multiprotein labeling strategies, intracellular multicolor SMT remains challenging for general research studies. Here, we introduce a practical method employing a nanopore-electroporation (NanoEP) technique to deliver multiple organic dye-labeled proteins into living cells for imaging. It can be easily expanded to three channels in commercial microscopes or be combined with other in situ labeling methods. Utilizing NanoEP, we demonstrate three-color SMT for both cytosolic and membrane proteins. Specifically, we simultaneously monitored single-molecule events downstream of EGFR signaling pathways in living cells. The results provide detailed resolution of the spatial localization and dynamics of Grb2 and SOS recruitment to activated EGFR along with the resultant Ras activation.

Most resource-constrained project scheduling problems consider resources that are independent of the job processing sequence. However, solving project scheduling problems in some industrial manufacturing is challenging because the required resources depend on the job processing sequence in existing scheduling approaches. To address this issue, this paper proposes a dependent resource-constrained project scheduling problem formulated with two types of models: a conceptual model and a binary integer programming model. The performance of four proposed algorithms – binary integer programming, reduced subproblem of the binary integer programming, job sequence assignment, and job as early as possible – is tested on 68 combinations of numerical simulation experiments. The computational results show that the job as early as possible algorithm can find a better approximate solution for solving real-world problems. This achievement aids project managers in generating activity schedules and finding the approximate minimum makespan for a project with dependent resource constraints. [ABSTRACT FROM AUTHOR]

Many fluorescent proteins are currently available for biological spectroscopy and imaging measurements, allowing a wide range of biochemical and biophysical processes and interactions to be studied at various length scales. However, in applications where a small fluorescence reporter is required or desirable, the choice of fluorophores is rather limited. As such, continued effort has been devoted to the development of amino acid-based fluorophores that do not require a specific environment and additional time to mature and have a large fluorescence quantum yield, long fluorescence lifetime, good photostability, and an emission spectrum in the visible region. Herein, we show that a tryptophan analog, 4-cyanotryptophan, which differs from tryptophan by only two atoms, is the smallest fluorescent amino acid that meets these requirements and has great potential to enable in vitro and in vivo spectroscopic and microscopic measurements of proteins.

Because of its importance in viral replication, the M2 proton channel of the influenza A virus has been the focus of many studies. Although we now know a great deal about the structural architecture underlying its proton conduction function, we know little about its conformational dynamics, especially those controlling the rate of this action. Herein, we employ a single-molecule fluorescence method to assess the dynamics of the inter-helical channel motion of both full-length M2 and the transmembrane domain of M2. The rate of this motion depends not only on the identity of the channel and membrane composition but also on the pH in a sigmoidal manner. For the full-length M2 channel, the rate is increased from approximately 190 μs-1 at high pH to approximately 80 μs-1 at low pH, with a transition midpoint at pH 6.1. Because the latter value is within the range reported for the conducting pKa value of the His37 tetrad, we believe that this inter-helical motion accompanies proton conduction.

Background and Aims: Dehydration is a prevalent and costly healthcare concern, linked to heightened risks of acute kidney injury and in-hospital mortality. Despite its significance, limited evidence exists regarding its prevalence and correlation with renal function decline in apparently healthy individuals. This retrospective cohort study aimed to investigate the prevalence and association of dehydration with renal function decline and the development or progression of chronic kidney disease (CKD) in the general population. Methods: The medical records of subjects undergoing annual health check-ups from 2016 to 2019 at a single center in Taiwan were analyzed, and those with CKD stage V, insufficient data, or an increased estimated glomerular filtration rate (eGFR) were excluded. Serum osmolality, eGFR, and relevant parameters were measured. Logistic regression and Kaplan–Meier analyses were used to assess associations between osmolality and CKD-related outcomes. Results: Among the 4449 eligible subjects, those in the higher osmolality quartiles had an elevated risk of CKD or CKD progression. Multivariate analyses identified age, systolic blood pressure, serum osmolality, uric acid, proteinuria, and a history of diabetes as independent risk factors, with high-density lipoprotein being protective. Cumulative incidence curves demonstrated a significant increase in the risk of CKD with increasing osmolality levels. Restricted cubic spline analyses confirmed a nonlinear relationship between osmolality and CKD risk. Conclusions: Elevated serum osmolality independently predicted renal function decline and CKD development in apparently healthy individuals, and this effect persisted after adjusting for established risk factors. Our findings underscore the importance of addressing dehydration as a modifiable risk factor for CKD. [ABSTRACT FROM AUTHOR]

Excavation of a superlarge diameter tunnel by tunnel boring machine (TBM) is different from that of a shield tunnel with normal dimension, in which the control system of the superlarge TBM is very complicated and difficult to operate. Hence, it is very important to focus on the control and management of significant parameters to ensure excavation stability under uncertainty. In this paper, we (i) utilize a BIM-based big data platform (BIM-BDP) to manage the essential construction data of tunnel project in digital format; (ii) adopt the global sensitivity analysis (SA) to recognize significant parameters for shield excavation based on polynomial chaos expansion (PCE)–extended Fourier amplitude sensitivity test (eFAST) model; and (iii) employ the uncertainty analysis (UA) to discover the correlation between significant parameters from the data of the BIM-BDP. This research contributes to (i) the body of knowledge of proposing a more appropriate research methodology that can cope with aleatory and epistemic uncertainty and support uncertainty and sensitivity analysis (UA/SA) processes based on data from BIM-BDP and (ii) the state of practice by providing a data-driven surrogate model to simulate system behaviors of shield excavation with high reliability and to reduce dependency on domain experts. Here, we pay close attention to the most influential parameters that require priority parameter control, which can help administrators optimize the management of shield parameters during tunnel excavation.