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This paper presents a model of scheduling of multiunit construction project based on an NP-hard permutation flow shop problem, in which the considered criterion is the sum of the costs of the works’ execution of the project considering the time of the project as a constraint. It is also assumed that each job in the units constituting the project may be realized in up to three different ways with specific time and cost of execution. The optimization task relies on solving the problem with two different decision variables: the order of execution of units (permutation) and a set of ways to carry out the works in units. The task presented in the paper is performed with the use of a created algorithm which searches the space of solutions in which metaheuristic simulated annealing algorithm is used. The paper presents a calculation example showing the applicability of the model in the optimization of sub-contractors’ work in the construction project.

The paper describes the bicriteria discrete optimization problem, that may occur during the scheduling of multiunit construction projects. The multiunit project involves the construction of many civil structures with the same sets of activities needed, but different in size. In the project the deadlines of activities in units are adopted. The missing of them by the contractor causes the payment of the disincentive penalty. The early completion of the activities in units is rewarded extra income for the construction contractor i.e. a incentive bonus. Changing the order of the execution of the units changes the value of the objective functions: the duration of the project and the cost (the sum of the disincentive penalties and incentive bonuses). The proposed model of the project is the bicriteria NP-hard flow shop problem with constraints characteristic for construction projects. The paper presents the method of determining the set of Paretooptimal solutions for small projects. The computational example of the model of the project is also included in the paper.

Introduction: Radial access reduces vascular complications compared to femoral access. Various factors may influence the patient's pain during coronary intervention., Aim: To assess what clinical and periprocedural factors affect discomfort and pain intensity during angiography via the radial artery without use of spasmolytics., Material and Methods: A group of 238 patients (M/F 142/96, mean age: 67 ±10 years) who underwent coronary angiography and interventions through the radial artery approach was studied. Every patient had ultrasound assessment of the radial artery. Pain was assessed according to the numerical pain rating scale, where mild pain is 1-3, moderate pain is 4-7, and severe pain is above 7., Results: We included 238 patients. Most of the participants had a low pain level ( n = 133 (55.88%)), while a smaller number had moderate and severe pain level ( n = 88 (36.97%) and n = 17 (7.14%), respectively). We analyzed 38 characteristics of the patients in terms of the possible influence on the pain level during angiography., Conclusions: In our study we analyzed possible factors which may contribute to the severe pain sensation during percutaneous coronary intervention. We found that masculine gender, greater weight and height as well as diabetes mellitus and myocardial infarction diagnosis on admission correlated with lower pain level. Conversely, greater maximal and minimal diameters of the radial artery correlated with stronger pain level., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2024 Termedia Sp. z o. o.)

Background: In ulcerative colitis, the complexity of mucosal cytokine secretion profiles and how they correlate with endoscopic and clinical scores is still unclear., Methods: In this study, we collected fresh biopsies from UC patients to investigate which cytokines are produced in ex vivo culture conditions, a platform increasingly used for testing of novel drugs. Then, we correlated cytokine production with several scoring indices commonly used to assess the severity of the disease., Results: Increased levels of IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, TNFα and IFNɣ were produced by biopsies of UC patients compared to non-IBD controls. Our results show a better correlation of cytokine levels with Mayo Endoscopic Subscore (MES) and Mayo score, than the more complex Ulcerative Colitis Endoscopic Index of Severity (UCEIS). Out of 10 measured cytokines, eight correlated with MES, six with Mayo score and only three with UCEIS, due to the partial increase in cytokine secretion observed in donors with UCEIS = 7-8. When we analysed individual subscores within the UCEIS, Vascular Network subscore showed a correlation similar to MES (7/10 cytokines), while Bleeding as well as Erosions and Ulcers subscores correlated with only 3/10 cytokines, similarly to the total UCEIS., Conclusions: Our findings suggest that choosing biopsies from donors with MES = 2-3 and UCEIS = 2-6 from areas with no bleeding and no superficial and/or deep ulcers could enable a deeper insight into the cytokine profile of the inflamed tissue and represent a better tool for studying potential therapeutic targets and evaluation of novel therapies., (© 2023 Stichting European Society for Clinical Investigation Journal Foundation.)

Introduction: Telehealth is increasing rapidly as a health care delivery platform, but we lack empirical evidence regarding how telehealth environments can affect patient experiences. The present research determined how physician's telehealth backgrounds affect various patient outcomes. Methods: Participants viewed a 30-s video of a physician with one of six different virtual backgrounds and reported various socioemotional and cognitive responses to the mock telehealth experience. Results: Although the telehealth background manipulation did not impact participants' socioemotional or cognitive responses, participants' subjective perceptions of the telehealth backgrounds were related to important clinical outcomes, such as their ability to remember critical information from the appointment and overall satisfaction with the experience. Discussion: Telehealth environments may result in tradeoffs between patient experience, subjective impressions of clinicians, and information recall. Conclusions: A physician's telehealth background can have measurable impact on patients' telehealth experiences, suggesting a need for careful background selection and design.

Introduction: Due to the reduction in-person visits, the COVID-19 pandemic has led to expansions in the use of telehealth technology to provide patient care, yet clinicians lack evidence-based guidance on how to most effectively use video communication to enhance patient experience and outcomes. Methods: A narrative review was conducted to describe environmental factors derived from research in social psychology and human-computer interaction (HCI) that may guide effective video-based clinician-patient telehealth communication. Results: Factors such as nonverbal cues, spatial proximity, professionalism cues, and ambient features play an important role in patient experience. We present a visual typology of telehealth backgrounds to inform clinical practice and guide future research. Discussion: A growing body of empirical evidence indicates that environmental cues may play an essential role in establishing psychological safety, improving patient experience, and supporting clinical efficacy in these virtual experiences. Conclusion: The expanded use of telehealth visits suggests the need for further research on the relative effects of these environmental factors on patient experience and outcomes.

The physical dimensions of proteins and glycans on cell surfaces can critically affect cell function, for example, by preventing close contact between cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of cell surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call cell surface optical profilometry (CSOP), to quantify the height of key multidomain proteins on a model cell, as well as to capture average protein and glycan heights on native cell membranes. We show that average height of a protein is significantly smaller than its contour length, due to thermally driven bending and rotation on the membrane, and that height strongly depends on local surface and solution conditions. We find that average height increases with cell surface molecular crowding but decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope, based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native cell surface using antibodies and lectins. This versatile method for profiling cell surfaces has the potential to advance understanding of the molecular landscape of cells and the role of the molecular landscape in cell function., Competing Interests: The authors declare no competing interest.

Dynamic organization of microtubule minus ends is vital for the formation and maintenance of acentrosomal microtubule arrays. In vitro, both microtubule ends switch between phases of assembly and disassembly, a behavior called dynamic instability. Although minus ends grow slower, their lifetimes are similar to those of plus ends. The mechanisms underlying these distinct dynamics remain unknown. Here, we use an in vitro reconstitution approach to investigate minus-end dynamics. We find that minus-end lifetimes are not defined by the mean size of the protective GTP-tubulin cap. Rather, we conclude that the distinct tubulin off-rate is the primary determinant of the difference between plus- and minus-end dynamics. Further, our results show that the minus-end-directed kinesin-14 HSET/KIFC1 suppresses tubulin off-rate to specifically suppress minus-end catastrophe. HSET maintains its protective minus-end activity even when challenged by a known microtubule depolymerase, kinesin-13 MCAK. Our results provide novel insight into the mechanisms of minus-end dynamics, essential for our understanding of microtubule minus-end regulation in cells., (© 2019 Strothman et al.)

Macrophages protect the body from damage and disease by targeting antibody-opsonized cells for phagocytosis. Though antibodies can be raised against antigens with diverse structures, shapes, and sizes, it is unclear why some are more effective at triggering immune responses than others. Here, we define an antigen height threshold that regulates phagocytosis of both engineered and cancer-specific antigens by macrophages. Using a reconstituted model of antibody-opsonized target cells, we find that phagocytosis is dramatically impaired for antigens that position antibodies >10 nm from the target surface. Decreasing antigen height drives segregation of antibody-bound Fc receptors from the inhibitory phosphatase CD45 in an integrin-independent manner, triggering Fc receptor phosphorylation and promoting phagocytosis. Our work shows that close contact between macrophage and target is a requirement for efficient phagocytosis, suggesting that therapeutic antibodies should target short antigens in order to trigger Fc receptor activation through size-dependent physical segregation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Quantitative methods and approaches have been playing an increasingly important role in cell biology in recent years. They involve making accurate measurements to test a predefined hypothesis in order to compare experimental data with predictions generated by theoretical models, an approach that has benefited physicists for decades. Building quantitative models in experimental biology not only has led to discoveries of counterintuitive phenomena but has also opened up novel research directions. To make the biological sciences more quantitative, we believe a two-pronged approach needs to be taken. First, graduate training needs to be revamped to ensure biology students are adequately trained in physical and mathematical sciences and vice versa. Second, students of both the biological and the physical sciences need to be provided adequate opportunities for hands-on engagement with the methods and approaches necessary to be able to work at the intersection of the biological and physical sciences. We present the annual Physiology Course organized at the Marine Biological Laboratory (Woods Hole, MA) as a case study for a hands-on training program that gives young scientists the opportunity not only to acquire the tools of quantitative biology but also to develop the necessary thought processes that will enable them to bridge the gap between these disciplines., (© 2014 Shekhar, Zhu, Mazutis, Sgro, Fai, and Podolski. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Stu2 is the budding yeast member of the XMAP215/Dis1 family of microtubule polymerases. It is essential in cell division, allowing proper spindle orientation and metaphase chromosome alignment, as well as spindle elongation during anaphase. Despite Stu2 having a phenotype that suggests it promotes microtubule growth, like the other members of the XMAP215/Dis1 family, previous studies with purified Stu2 indicate only that it antagonizes microtubule growth. One potential explanation for these contradictory findings is that the assays were performed with mammalian brain tubulin, which may not be the right substrate to test the activity of Stu2 given that yeast and brain tubulins are quite divergent in sequence and that the vertebrate tubulins are subject to many post-translational modifications. To test this possibility, we reconstituted the activity of Stu2 with purified budding yeast tubulin. We found that Stu2 accelerated microtubule growth in yeast tubulin by severalfold, similar to the acceleration reported for XMAP215 in porcine brain tubulin. Furthermore, Stu2 accelerated polymerization in yeast tubulin to a much greater extent than in porcine brain tubulin, and the concentration of Stu2 required to reach 50% maximum activity in yeast tubulin was nearly 2 orders of magnitude lower than that in porcine brain tubulin. We conclude that Stu2 is a microtubule polymerase, like its relatives, and that its activity is considerably higher in yeast tubulin compared with mammalian brain tubulin. The biochemical properties of Stu2 reported here account for many of the phenotypes of Stu2 observed in cells., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

In the absence of landmark proteins, hyphae of Aspergillus nidulans lose their direction of growth and show a zigzag growth pattern. Here, we show that the cell-end marker protein TeaA is important for localizing the growth machinery at hyphal tips. The central position of TeaA at the tip correlated with the convergence of the microtubule (MT) ends to a single point. Conversely, in the absence of TeaA, the MTs often failed to converge to a single point at the cortex. Further analysis suggested a functional connection between TeaA and AlpA (an ortholog of the MT polymerase Dis1/CKAP5/XMAP215) for proper regulation of MT growth at hyphal tips. AlpA localized at MT plus-ends, and bimolecular fluorescence complementation assays suggested that it interacted with TeaA after MT plus-ends reached the tip cortex. In vitro MT polymerization assays showed that AlpA promoted MT growth up to sevenfold. Addition of the C-terminal region of TeaA increased the catastrophe frequency of the MTs. Thus, the control of the AlpA activity through TeaA might be a novel principle for MT growth regulation after reaching the cortex. In addition, we present evidence that the curvature of hyphal tips also could be involved in the control of MT growth at hyphal tips.

We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research.

Eryptosis or apoptosis-like death of erythrocytes is characterized by phosphatidylserine exposure and erythrocyte shrinkage, both typical features of nucleated apoptotic cells. Eryptosis is triggered by activation of nonselective Ca(2+)-permeable cation channels with subsequent entry of Ca(2+) and stimulation of Ca(2+)-sensitive scrambling of the cell membrane. The channels are activated and thus eryptosis is triggered by Cl(-) removal, osmotic shock, oxidative stress, or glucose deprivation. The present study has been performed to compare cation channel activity and susceptibility to eryptosis in neonatal and adult erythrocytes. Channel activity was determined by patch-clamp analysis, cytosolic Ca(2+) activity by fluo-3 fluorescence, phosphatidylserine exposure by FITC-labeled annexin V binding, and cell shrinkage by decrease in forward scatter in fluorescence-activated cell sorting analysis. Prostaglandin E(2) (PGE(2)) formation, cation channel activity, Ca(2+) entry, annexin V binding, and decreased forward scatter were triggered by removal of Cl(-) in both adult and neonatal erythrocytes. The effects were, however, significantly blunted in neonatal erythrocytes. Osmotic shock, PGE(2,) and platelet-activating factor similarly increased annexin V binding and decreased forward scatter, effects again significantly reduced in neonatal erythrocytes. On the other hand, spontaneous and oxidative (addition of tert-butylperoxide) stress-induced eryptosis was significantly larger in neonatal erythrocytes. In conclusion, cation channel activity, Ca(2+) leakage, and thus channel-dependent triggering of eryptosis are blunted, whereas spontaneous and oxidative stress-induced eryptosis is more pronounced in neonatal erythrocytes.

Side effects of treatment with chlorpromazine include anaemia which could result from decreased formation or accelerated clearance of circulating erythrocytes. Recently, a novel mechanism leading to erythrocyte clearance has been disclosed. Osmotic shock, oxidative stress and glucose deprivation lead to activation of cation channels, Ca(2+) entry, activation of a Ca(2+)-sensitive erythrocyte scramblase and subsequent exposure of phosphatidylserine at the erythrocyte surface. As macrophages are equipped with phosphatidylserine receptors, they bind, engulf and degrade phosphatidylserine exposing cells. The present experiments have been performed to explore whether chlorpromazine triggers phosphatidylserine exposure of erythrocytes. The phosphatidylserine exposure was estimated from annexin binding as determined in fluorescence activated cell sort (FACS) analysis. A 24 h exposure to glucose-free medium decreased cytosolic ATP levels, decreased cellular levels of reduced glutathione (GSH) and increased annexin binding. The effect on annexin binding and ATP but not on GSH was significantly enhanced in the presence of chlorpromazine (10 microM). Higher concentrations of chlorpromazine (40 microM) increased cytosolic Ca(2+) activity. Osmotic shock and Cl(-) removal similarly increased annexin binding, effects again being enhanced in the presence of chlorpromazine. In conclusion, the present observations point to a novel side effect of chlorpromazine, i.e. increased sensitivity of erythrocytes to glucose deprivation. The effect could well contribute to the known anaemia observed in the treatment with this antipsychotic drug.

Glucose depletion of erythrocytes leads to activation of Ca2+-permeable cation channels, Ca2+ entry, activation of a Ca2+-sensitive erythrocyte scramblase, and subsequent exposure of phosphatidylserine at the erythrocyte surface. Ca2+ entry into erythrocytes was previously shown to be stimulated by phorbol esters and to be inhibited by staurosporine and chelerythrine and is thus thought to be regulated by protein phosphorylation/dephosphorylation, presumably via protein kinase C (PKC) and the corresponding phosphoserine/threonine phosphatases. The present experiments explored whether PKC could contribute to effects of energy depletion on erythrocyte phosphatidylserine exposure and cell volume. Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorter analysis. Removal of extracellular glucose led to depletion of cellular ATP, stimulated PKC activity, led to translocation of PKCalpha, enhanced serine phosphorylation of membrane proteins, decreased cell volume, and increased annexin binding, the latter effect being blunted but not abolished in the presence of 1 microM staurosporine or 50 nM calphostin C. The PKC stimulator phorbol-12-myristate-13-acetate (3 microM) and the phosphatase inhibitor okadaic acid (1-10 microM) mimicked the effect of glucose depletion and similarly led to translocation of PKCalpha and enhanced serine phosphorylation, increased annexin binding, and decreased forward scatter, the latter effects being abrogated by PKC inhibitor staurosporine (1 microM). Fluo-3 fluorescence measurements revealed that okadaic acid also enhanced erythrocyte Ca2+ activity. The present observations suggest that protein phosphorylation and dephosphorylation via PKC and the corresponding protein phosphatases contribute to phosphatidylserine exposure and cell shrinkage after energy depletion.

Osmotic shock, oxidative stress and Cl- removal activate a non-selective Ca2+-permeable cation conductance in human erythrocytes. The entry of Ca2+ leads to activation of a scramblase with subsequent exposure of phosphatidylserine at the cell surface. Phosphatidylserine mediates binding to phosphatidylserine receptors on macrophages which engulf and degrade phosphatidylserine exposing cells. Moreover, phosphatidylserine exposure may lead to adherence of erythrocytes to the vascular wall. In the present study, we explored whether activation of the non-selective cation conductance and subsequent phosphatidylserine exposure might be influenced by catecholamines. Phosphatidylserine exposure has been determined by FITC-annexin V binding while cell volume was estimated from forward scatter in FACS analysis. Removal of Cl- enhanced annexin binding and decreased forward scatter, an effect significantly blunted by the beta agonist isoproterenol (IC50 approx. 1 microM). Fluo-3 fluorescence measurements revealed an increase of cytosolic Ca2+ activity following Cl- removal, an effect again significantly blunted by isoproterenol exposure (10 microM). Whole-cell patch-clamp experiments performed in Cl- free bath solution indeed disclosed a time-dependent inactivation of a non-selective cation conductance following isoproterenol exposure (10 microM). Phenylephrine (IC50<10 microM), dobutamine (IC50 approx. 1 microM) and dopamine (IC50 approx. 3 microM) similarly inhibited the effect of Cl- removal on annexin binding and forward scatter. In conclusion, several catecholamines inhibit the Cl- removal-activated Ca2+ entry into erythrocytes, thus preventing increase of cytosolic Ca2+ activity, subsequent cell shrinkage and activation of erythrocyte scramblase. The catecholamines thus counteract erythrocyte phosphatidylserine exposure and subsequent clearance of erythrocytes from circulating blood.