Labelled dataset for Ultra-Low Temperature Freezer to aid dynamic modelling & fault detection and diagnostics.

Ultra-low temperature (ULT) freezers are used to store perishable biological contents and are among the most energy-intensive equipment in laboratory buildings, biobanks, and similar settings. To ensure reliable and efficient operation, it is essential to implement data-driven fault detection and diagnostic algorithms, along with energy optimization techniques. This study presents labelled and long-term ULT-freezer performance dataset, the first of its kind, derived from 53 ULT freezers featuring two different control strategies. The dataset comprises high-resolution historical operation data spanning up to 10 years. More than 10 attributes are recorded from the freezing chamber and critical locations in the refrigeration systems. The dataset is labelled with regular events, such as door openings, as well as fault events obtained from 46 service reports. A scalable data pipeline, consisting of extraction, transformation, and loading processes, is developed to convert the raw data into a format ready for analysis. The dataset can be utilized to support the development of data-driven models and algorithms that advance the intelligent digital operation of ULT freezers.

An Evaluation of the Supplementation of Rumen-Protected Lysine and Methionine on the Lactation Performance of Fall Parturition Grazing Holstein Cows in Southern Chile.

The aim of this study was to evaluate the effect of the supplementation of rumen-protected (RP) methionine and lysine on milk yield, solids, and body weight over time on fall-calving grazing multiparous Holstein cows from Chile. Four treatment groups were studied and compared for the outcomes over time. The treatments were as follows: (i) CON: control (n = 26); (ii) RP lysine group (LYS; 20 g per cow per day; n = 26); (iii) RP methionine group (MET; 20 g per cow per day; n = 26); and (iv) LYS × MET (RP lysine and RP methionine 20 g of each amino acid per cow per day; n = 26). Data were analyzed with general linear mixed model ANOVAs for repeated measures to primarily test the main effects of each amino acid and their interactions. The supplementation of the amino acids was conducted from 2 to 70 days postpartum. Overall, milk production tended to be higher in the MET and in the LYS × MET group when compared to the control group. Furthermore, CON produced significantly less milk protein (kg) and milk fat (kg) than the LYS, MET, and LYS × MET groups. Milk urea tended to be lower during the entire study in the CON group than the rest of the groups. There was a trend for a reduction in the losses of postpartum body weight in the LYS × MET than the CON. In conclusion, RP methionine and lysine improved milk fat and protein yield in grazing multiparous cows with fall parturitions; consequently, both RP amino acids can be used as a strategy for improving grazing cows' production performance.

Receptor-associated independent cAMP nanodomains mediate spatiotemporal specificity of GPCR signaling.

G protein-coupled receptors (GPCRs) relay extracellular stimuli into specific cellular functions. Cells express many different GPCRs, but all these GPCRs signal to only a few second messengers such as cAMP. It is largely unknown how cells distinguish between signals triggered by different GPCRs to orchestrate their complex functions. Here, we demonstrate that individual GPCRs signal via receptor-associated independent cAMP nanodomains (RAINs) that constitute self-sufficient, independent cell signaling units. Low concentrations of glucagon-like peptide 1 (GLP-1) and isoproterenol exclusively generate highly localized cAMP pools around GLP-1- and ß2-adrenergic receptors, respectively, which are protected from cAMP originating from other receptors and cell compartments. Mapping local cAMP concentrations with engineered GPCR nanorulers reveals gradients over only tens of nanometers that define the size of individual RAINs. The coexistence of many such RAINs allows a single cell to operate thousands of independent cellular signals simultaneously, rather than function as a simple "on/off" switch.

A Versatile Sub-Nanomolar Fluorescent Ligand Enables NanoBRET Binding Studies and Single-Molecule Microscopy at the Histamine H3 Receptor.

The histamine H3 receptor (H3R) is considered an attractive drug target for various neurological diseases. We here report the synthesis of UR-NR266, a novel fluorescent H3R ligand. Broad pharmacological characterization revealed UR-NR266 as a sub-nanomolar compound at the H3R with an exceptional selectivity profile within the histamine receptor family. The presented neutral antagonist showed fast association to its target and complete dissociation in kinetic binding studies. Detailed characterization of standard H3R ligands in NanoBRET competition binding using UR-NR266 highlights its value as a versatile pharmacological tool to analyze future H3R ligands. The low nonspecific binding observed in all experiments could also be verified in TIRF and confocal microscopy. This fluorescent probe allows the highly specific analysis of native H3R in various assays ranging from optical high throughput technologies to biophysical analyses and single-molecule studies in its natural environment. An off-target screening at 14 receptors revealed UR-NR266 as a selective compound.

Determination of G-protein-coupled receptor oligomerization by molecular brightness analyses in single cells.

Oligomerization of membrane proteins has received intense research interest because of their importance in cellular signaling and the large pharmacological and clinical potential this offers. Fluorescence imaging methods are emerging as a valid tool to quantify membrane protein oligomerization at high spatial and temporal resolution. Here, we provide a detailed protocol for an image-based method to determine the number and oligomerization state of fluorescently labeled prototypical G-protein-coupled receptors (GPCRs) on the basis of small out-of-equilibrium fluctuations in fluorescence (i.e., molecular brightness) in single cells. The protocol provides a step-by-step procedure that includes instructions for (i) a flexible labeling strategy for the protein of interest (using fluorescent proteins, small self-labeling tags or bio-orthogonal labeling) and the appropriate controls, (ii) performing temporal and spatial brightness image acquisition on a confocal microscope and (iii) analyzing and interpreting the data, excluding clusters and intensity hot-spots commonly observed in receptor distributions. Although specifically tailored for GPCRs, this protocol can be applied to diverse classes of membrane proteins of interest. The complete protocol can be implemented in 1 month.

Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists.

Although class A G protein-coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer-dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.

Single-molecule analysis reveals agonist-specific dimer formation of µ-opioid receptors.

G-protein-coupled receptors (GPCRs) are key signaling proteins that mostly function as monomers, but for several receptors constitutive dimer formation has been described and in some cases is essential for function. Using single-molecule microscopy combined with super-resolution techniques on intact cells, we describe here a dynamic monomer-dimer equilibrium of µ-opioid receptors (µORs), where dimer formation is driven by specific agonists. The agonist DAMGO, but not morphine, induces dimer formation in a process that correlates both temporally and in its agonist- and phosphorylation-dependence with ß-arrestin2 binding to the receptors. This dimerization is independent from, but may precede, µOR internalization. These data suggest a new level of GPCR regulation that links dimer formation to specific agonists and their downstream signals.

Vibrational spectroscopy as a powerful tool for follow-up immunoadsorption therapy treatment of dilated cardiomyopathy - a case report.

Dilated cardiomyopathy (DCM) is a leading cardiomyopathy condition and is the leading reason for heart transplantation. Due to high etiologic and genetic heterogeneity of the pathologies, different therapeutic treatment strategies are available and have been successful for different treatments. Immunoadsorption (IA) therapy removes the circulating anticardiac antibodies and improves the left ventricular function in substantial proportion of DCM patients. Powerful, non-invasive analytical tools are highly desired to investigate the efficiency and success of IA therapy. In this contribution, we followed the changes of a female DCM patient undergoing IA therapy at different treatment time points in a label-free, non-invasive manner from blood samples (plasma and serum) on the basis of vibrational spectroscopy (Raman scattering and IR absorption). Chemometric methods, including dimension reduction and statistical modeling, were used to interpret spectral data. The impact of different time points of the IA treatment can be identified in both the plasma and serum, using both techniques, with high accuracy. The removal of antibodies of immunoglobulin G (IgG) group during IA therapy and their restoration was reflected in both Raman and FTIR spectra. Relative changes in the spectral bands assigned to IgG agreed well with the immunoturbidimetry measurement of total IgG. Successful clinical treatment was accompanied by spectral differences between vibrational spectra obtained at initial disease state and 11 months after the IA treatment. The long-term follow-up of the patient reveals the stabilization of the health state after therapy. It is noteworthy that the treatment time points were distinguished with a better accuracy using spectra from plasma compared to those from serum samples, which might indicate the involvement of corresponding proteins in the coagulation. Vibrational spectroscopy is a powerful tool for personalized medicine to follow-up the treatment success of IA therapy for the DCM disorder.

Process Improvement in Patient Pathways: A Case Study Applying Accelerated Longitudinal Design With Decomposition Method.

EXECUTIVE SUMMARY: Evaluations of improvements in long chronic-patient pathways must include both short- and long-term effects on patients; that is, effects on the full patient pathway. Otherwise, costs might be cut without considering the long-term effects and, consequently, the overall cost of the pathway could increase. Unfortunately, current methods of evaluation present several issues: (1) they do not provide valid insights regarding the effects of a given improvement effort until several years later, (2) they provide imprecise and biased results, and (3) the aggregated results are not useful for identifying and disseminating the best practices that lead to an improvement. In this article, the accelerated longitudinal design with decomposition of total costs (ALDD) method is applied to evaluate the effects of improvement efforts on inpatient utilization for long cardiac pathways at a Danish hospital. The results show that the ALDD method can deliver valid results much faster than traditional methods and can uncover hidden improvements in the local work processes of clinical teams. Application of the ALDD method at a hospital in Denmark identified a significant reduction (15.4%) in the mean total bed utilization per cardiac pathway and revealed that this reduction was caused by improvements in the work processes.

Towards model predictive control: online predictions of ammonium and nitrate removal by using a stochastic ASM.

Online model predictive control (MPC) of water resource recovery facilities (WRRFs) requires simple and fast models to improve the operation of energy-demanding processes, such as aeration for nitrogen removal. Selected elements of the activated sludge model number 1 modelling framework for ammonium and nitrate removal were included in discretely observed stochastic differential equations in which online data are assimilated to update the model states. This allows us to produce model-based predictions including uncertainty in real time while it also reduces the number of parameters compared to many detailed models. It introduces only a small residual error when used to predict ammonium and nitrate concentrations in a small recirculating WRRF facility. The error when predicting 2 min ahead corresponds to the uncertainty from the sensors. When predicting 24 hours ahead the mean relative residual error increases to ∼10% and ∼20% for ammonium and nitrate concentrations respectively. Consequently this is considered a first step towards stochastic MPC of the aeration process. Ultimately this can reduce electricity demand and cost for water resource recovery, allowing the prioritization of aeration during periods of cheaper electricity.

Molecular details of dimerization kinetics reveal negligible populations of transient µ-opioid receptor homodimers at physiological concentrations.

Various experimental and computational techniques have been employed over the past decade to provide structural and thermodynamic insights into G Protein-Coupled Receptor (GPCR) dimerization. Here, we use multiple microsecond-long, coarse-grained, biased and unbiased molecular dynamics simulations (a total of ~4 milliseconds) combined with multi-ensemble Markov state models to elucidate the kinetics of homodimerization of a prototypic GPCR, the µ-opioid receptor (MOR), embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol lipid bilayer. Analysis of these computations identifies kinetically distinct macrostates comprising several different short-lived dimeric configurations of either inactive or activated MOR. Calculated kinetic rates and fractions of dimers at different MOR concentrations suggest a negligible population of MOR homodimers at physiological concentrations, which is supported by acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments. This study provides a rigorous, quantitative explanation for some conflicting experimental data on GPCR oligomerization.

Structure-Activity Relationships and Computational Investigations into the Development of Potent and Balanced Dual-Acting Butyrylcholinesterase Inhibitors and Human Cannabinoid Receptor 2 Ligands with Pro-Cognitive in Vivo Profiles.

The enzyme butyrylcholinesterase (BChE) and the human cannabinoid receptor 2 (hCB2R) represent promising targets for pharmacotherapy in the later stages of Alzheimer's disease. We merged pharmacophores for both targets into small benzimidazole-based molecules, investigated SARs, and identified several dual-acting ligands with a balanced affinity/inhibitory activity and an excellent selectivity over both hCB1R and hAChE. A homology model for the hCB2R was developed based on the hCB1R crystal structure and used for molecular dynamics studies to investigate binding modes. In vitro studies proved hCB2R agonism. Unwanted µ-opioid receptor affinity could be designed out. One well-balanced dual-acting and selective hBChE inhibitor/hCB2R agonist showed superior in vivo activity over the lead CB2 agonist with regards to cognition improvement. The data shows the possibility to combine a small molecule with selective and balanced GPCR-activity/enzyme inhibition and in vivo activity for the therapy of AD and may help to rationalize the development of other dual-acting ligands.

Cross-Validation of a Glucose-Insulin-Glucagon Pharmacodynamics Model for Simulation Using Data From Patients With Type 1 Diabetes.

BACKGROUND: Currently, no consensus exists on a model describing endogenous glucose production (EGP) as a function of glucagon concentrations. Reliable simulations to determine the glucagon dose preventing or treating hypoglycemia or to tune a dual-hormone artificial pancreas control algorithm need a validated glucoregulatory model including the effect of glucagon. METHODS: Eight type 1 diabetes (T1D) patients each received a subcutaneous (SC) bolus of insulin on four study days to induce mild hypoglycemia followed by a SC bolus of saline or 100, 200, or 300 µg of glucagon. Blood samples were analyzed for concentrations of glucagon, insulin, and glucose. We fitted pharmacokinetic (PK) models to insulin and glucagon data using maximum likelihood and maximum a posteriori estimation methods. Similarly, we fitted a pharmacodynamic (PD) model to glucose data. The PD model included multiplicative effects of insulin and glucagon on EGP. Bias and precision of PD model test fits were assessed by mean predictive error (MPE) and mean absolute predictive error (MAPE). RESULTS: Assuming constant variables in a subject across nonoutlier visits and using thresholds of ±15% MPE and 20% MAPE, we accepted at least one and at most three PD model test fits in each of the seven subjects. Thus, we successfully validated the PD model by leave-one-out cross-validation in seven out of eight T1D patients. CONCLUSIONS: The PD model accurately simulates glucose excursions based on plasma insulin and glucagon concentrations. The reported PK/PD model including equations and fitted parameters allows for in silico experiments that may help improve diabetes treatment involving glucagon for prevention of hypoglycemia.

Collective modes in three-dimensional magnonic vortex crystals.

Collective modes in three-dimensional crystals of stacked permalloy disks with magnetic vortices are investigated by ferromagnetic resonance spectroscopy and scanning transmission X-ray microscopy. The size of the arrangements is increased step by step to identify the different contributions to the interaction between the vortices. These contributions are the key requirement to understand complex dynamics of three dimensional vortex crystals. Both vertical and horizontal coupling determine the collective modes. In-plane dipoles strongly influence the interaction between the disks in the stacks and lead to polarity-dependent resonance frequencies. Weaker contributions discern arrangements with different polarities and circularities that result from the lateral coupling of the stacks and the interaction of the core regions inside a stack. All three contributions are identified in the experiments and are explained in a rigid particle model.

Development of a restricted state space stochastic differential equation model for bacterial growth in rich media.

In the present study, bacterial growth in a rich media is analysed in a Stochastic Differential Equation (SDE) framework. It is demonstrated that the SDE formulation and smoothened state estimates provide a systematic framework for data driven model improvements, using random walk hidden states. Bacterial growth is limited by the available substrate and the inclusion of diffusion must obey this natural restriction. By inclusion of a modified logistic diffusion term it is possible to introduce a diffusion term flexible enough to capture both the growth phase and the stationary phase, while concentration is restricted to the natural state space (substrate and bacteria non-negative). The case considered is the growth of Salmonella and Enterococcus in a rich media. It is found that a hidden state is necessary to capture the lag phase of growth, and that a flexible logistic diffusion term is needed to capture the random behaviour of the growth model. Further, it is concluded that the Monod effect is not needed to capture the dynamics of bacterial growth in the data presented.

A daily intake of approximately 6 microg vitamin B-12 appears to saturate all the vitamin B-12-related variables in Danish postmenopausal women.

BACKGROUND: Recommended daily intakes of vitamin B-12 vary between 2 and 6 microg. OBJECTIVE: The objective was to examine the associations between vitamin B-12 intake and markers of vitamin B-12 deficiency. DESIGN: We studied 98 Danish postmenopausal women aged 41-75 y. Serum cobalamin, transcobalamin (TC) saturated with vitamin B-12 (holo-TC), TC saturation (holo-TC/total TC), methylmalonic acid (MMA), and total homocysteine (tHcy) were measured to assess vitamin B-12 status. Dietary intakes of vitamin B-12 were determined from 7-d weighed food records. Gastric pH measurement and the alkali-challenge test were performed with the use of Heidelberg radiotelemetric capsules. RESULTS: The total intake of vitamin B-12 ranged from 1.2 to 23.9 microg/d. All vitamin B-12-related variables, except gastric pH, correlated significantly with total vitamin B-12 intake. Those taking supplements (54%) had higher circulating concentrations of cobalamin and TC saturation and lower concentrations of MMA and tHcy than did those not taking supplements. All subjects were divided into quintiles according to their total vitamin B-12 intake. For all the variables analyzed, the curves appeared to level off at a daily vitamin B-12 intake of approximately 6 microg; the median (and 25th-75th percentiles) for cobalamin was 380 (270-480) pmol/L, for holo-TC was 119 (92-162) pmol/L, for MMA was 0.12 (0.14-0.17) micromol/L, and for tHcy was 9.75 (8.3-11.40) micromol/L (n = 58). CONCLUSION: A daily vitamin B-12 intake of 6 microg appeared to be sufficient to correct all the vitamin B-12-related variables measured in the postmenopausal Danish women in this study.

Moxifloxacin and azithromycin but not amoxicillin protect human respiratory epithelial cells against streptococcus pneumoniae in vitro when administered up to 6 hours after challenge.

We determined the protective effect of moxifloxacin, azithromycin, and amoxicillin against Streptococcus pneumoniae infection of respiratory cells. Moxifloxacin and azithromycin effectively killed intracellular S. pneumoniae strains and protected respiratory epithelial cells significantly even when given 6 h after S. pneumoniae challenge. Amoxicillin was less effective.

Association of multiple cellular stress pathways with accelerated atherosclerosis in hyperhomocysteinemic apolipoprotein E-deficient mice.

BACKGROUND: A causal relation between hyperhomocysteinemia (HHcy) and accelerated atherosclerosis has been established in apolipoprotein E-deficient (apoE-/-) mice. Although several cellular stress mechanisms have been proposed to explain the atherogenic effects of HHcy, including oxidative stress, endoplasmic reticulum (ER) stress, and inflammation, their association with atherogenesis has not been completely elucidated. METHODS AND RESULTS: ApoE-/- mice were fed a control or a high-methionine (HM) diet for 4 (early lesion group) or 18 (advanced lesion group) weeks to induce HHcy. Total plasma homocysteine levels and atherosclerotic lesion size were significantly increased in early and advanced lesion groups fed the HM diet compared with control groups. Markers of ER stress (GRP78/94, phospho-PERK), oxidative stress (HSP70), and inflammation (phospho-IkappaB-alpha) were assessed by immunohistochemical staining of these atherosclerotic lesions. GRP78/94, HSP70, and phospho-IkappaB-alpha immunostaining were significantly increased in the advanced lesion group fed the HM diet compared with the control group. HSP47, an ER-resident molecular chaperone involved in collagen folding and secretion, was also increased in advanced lesions of mice fed the HM diet. GRP78/94 and HSP47 were predominantly localized to the smooth muscle cell-rich fibrous cap, whereas HSP70 and phospho-IkappaB-alpha were observed in the lipid-rich necrotic core. Increased HSP70 and phospho-IkappaB-alpha immunostaining in advanced lesions of mice fed the HM diet are consistent with enhanced carotid artery dihydroethidium staining. Interestingly, GRP78/94 and phospho-PERK were markedly increased in macrophage foam cells from early lesions of mice fed the control or the HM diet. CONCLUSIONS: Multiple cellular stress pathways, including ER stress, are associated with atherosclerotic lesion development in apoE-/- mice.