High Cell Turnover Research Articles

Efferocytosis induces a novel SLC program to promote glucose uptake and lactate release.

We turnover billions of apoptotic cells daily, and these are removed by professional and non-professional phagocytes via efferocytosis1. Characterizing the transcriptional program of phagocytes, we discovered a novel solute carrier family (SLC) gene signature (involving 33 SLC members) that is specifically modified during efferocytosis, but not antibody-mediated phagocytosis. Assessing the functional relevance of these SLCs, we noted a robust induction of an aerobic glycolysis program in efferocytic phagocytes, initiated by SLC2A1-mediated glucose uptake, with concurrent suppression of oxidative phosphorylation program. Interestingly, the different steps of phagocytosis2, i.e. smell (‘find-me’ signals/ sensing factors released by apoptotic cells), taste (phagocyte-apoptotic cell contact), and ingestion (corpse internalization), activated different SLCs and other molecules to promote glycolysis. Further, lactate, a natural by-product of aerobic glycolysis3, was released via another SLC (SLC16A1) that was upregulated after corpse uptake. While glycolysis within phagocytes contributed to actin polymerization and the continued uptake of corpses, the lactate released via SLC16A1 influenced the establishment of an anti-inflammatory tissue environment. Collectively, these data reveal a novel SLC program activated during efferocytosis, identify a previously unknown reliance on aerobic glycolysis during apoptotic cell uptake, and that glycolytic byproducts of efferocytosis can also influence other cells in the microenvironment.

Stem Cell Intrinsic Hexosamine Metabolism Regulates Intestinal Adaptation to Nutrient Content

SummaryThe intestine is an organ with an exceptionally high rate of cell turnover, and perturbations in this process can lead to severe diseases such as cancer or intestinal atrophy. Nutrition has a profound impact on intestinal volume and cellular architecture. However, how intestinal homeostasis is maintained in fluctuating dietary conditions remains insufficiently understood. By utilizing the Drosophila midgut model, we reveal a novel stem cell intrinsic mechanism coupling cellular metabolism with stem cell extrinsic growth signal. Our results show that intestinal stem cells (ISCs) employ the hexosamine biosynthesis pathway (HBP) to monitor nutritional status. Elevated activity of HBP promotes Warburg effect-like metabolic reprogramming required for adjusting the ISC division rate according to nutrient content. Furthermore, HBP activity is an essential facilitator for insulin signaling-induced ISC proliferation. In conclusion, ISC intrinsic hexosamine synthesis regulates metabolic pathway activities and defines the stem cell responsiveness to niche-derived growth signals.

More Than Skin Deep: Autophagy Is Vital for Skin Barrier Function.

The skin is a highly organized first line of defense that stretches up to 1.8 m2 and is home to more than a million commensal bacteria. The microenvironment of skin is driven by factors such as pH, temperature, moisture, sebum level, oxidative stress, diet, resident immune cells, and infectious exposure. The skin has a high turnover of cells as it continually bares itself to environmental stresses. Notwithstanding these limitations, it has devised strategies to adapt as a nutrient-scarce site. To perform its protective function efficiently, it relies on mechanisms to continuously remove dead cells without alarming the immune system, actively purging the dying/senescent cells by immunotolerant efferocytosis. Both canonical (starvation-induced, reactive oxygen species, stress, and environmental insults) and non-canonical (selective) autophagy in the skin have evolved to perform astute due-diligence and housekeeping in a quiescent fashion for survival, cellular functioning, homeostasis, and immune tolerance. The autophagic “homeostatic rheostat” works tirelessly to uphold the delicate balance in immunoregulation and tolerance. If this equilibrium is upset, the immune system can wreak havoc and initiate pathogenesis. Out of all the organs, the skin remains under-studied in the context of autophagy. Here, we touch upon some of the salient features of autophagy active in the skin.

Targeting adenosine for cancer immunotherapy

Immune checkpoint antagonists (CTLA-4 and PD-1/PD-L1) and CAR T-cell therapies generate unparalleled durable responses in several cancers and have firmly established immunotherapy as a new pillar of cancer therapy. To extend the impact of immunotherapy to more patients and a broader range of cancers, targeting additional mechanisms of tumor immune evasion will be critical. Adenosine signaling has emerged as a key metabolic pathway that regulates tumor immunity. Adenosine is an immunosuppressive metabolite produced at high levels within the tumor microenvironment. Hypoxia, high cell turnover, and expression of CD39 and CD73 are important factors in adenosine production. Adenosine signaling through the A2a receptor expressed on immune cells potently dampens immune responses in inflamed tissues. In this article, we will describe the role of adenosine signaling in regulating tumor immunity, highlighting potential therapeutic targets in the pathway. We will also review preclinical data for each target and provide an update of current clinical activity within the field. Together, current data suggest that rational combination immunotherapy strategies that incorporate inhibitors of the hypoxia-CD39-CD73-A2aR pathway have great promise for further improving clinical outcomes in cancer patients.

Does Leishmania infantum Use Degenerating Midgut Cells of The Vector as A Nutrient Source?

Ultrastructural evidence from Phlebotomuslangeroni experim-entally infected with Leishmania infantumwas found from day 4 postinfection to support the hypothesis that shed midgutcells act as an important nutrient source for the parasite. Thirteen areas ofdegenerating cells occurred in 12 sections of infected flies compared to nonein complementary controls 8 days post-infection. Parasites penetrateddegenerating cells in 7 of 30 flies sectioned (days 8-11 post-infection), andwere frequently found packed around shed cells in the gut lumen. Nomelanisation was observed, suggesting that although higher cell turnover wasprobably induced by parasites they did not destroy intact cells.

Ingestion of Food Particles Regulates the Mechanosensing Misshapen-Yorkie Pathway in Drosophila Intestinal Growth

The intestinal epithelium has a high cell turnover rate and is an excellent system to study stem cell-mediated adaptive growth. In the Drosophila midgut, the Ste20 kinase Misshapen, which is distally related to Hippo, has a niche function to restrict intestinal stem cell activity. We show here that, under low growth conditions, Misshapen is localized near the cytoplasmic membrane, is phosphorylated at the threonine 194 by the upstream kinase Tao, and is more active toward Warts, which in turn inhibits Yorkie. Ingestion of yeast particles causes a midgut distention and a reduction of Misshapen membrane association and activity. Moreover, Misshapen phosphorylation is regulated by the stiffness of cell culture substrate, changing of actin cytoskeleton, and ingestion of inert particles. These results together suggest that dynamic membrane association and Tao phosphorylation of Misshapen are steps that link the mechanosensing of intestinal stretching after food particle ingestion to control adaptive growth.

Prophylactic strategies to minimize the effect of whole body irradiation on hematopoietic, gastrointestinal and respiratory system leading to morbidity/mortality in animals

Increase in radionuclide application has gone far and wide in the last many decades; though its usage has benefited the society at large, however occasional unplanned exposure to radiation (terrorist/accidental) has also troubled human life. Exposure of humankind to nuclear disaster, accidental and natural background radiation exposure, has created the need to develop complete understanding of the subject and preparedness for having safe countermeasures. In whole-body radiation exposure scenario, all the three organs are responsible for leading the animal to its death; however, hematopoietic (HP) organ is the first to collapse followed by gastrointestinal (GI) and respiratory systems. Radiation-induced basic damage in these organs follows more or less similar pattern. Derangement starts with radiation-induced reactive oxygen species causing damage to DNA, lipids, and proteins and disturbing their regulatory pathways. However, damage in HP and GI is more rapid and severe due to the presence of highly radiosensitive multipotent stem cells essential to meet the need of high cell turnover rate in these organs. To overcome radiation-induced damage to these vital organs, serious efforts are continued globally to find safe remedial measure.

Use of U-STELA for Accurate Measurement of Extremely Short Telomeres.

Telomeres are repetitive genetic materials that protect the chromosomes by capping the ends of chromosomes. Each time a cell divides, telomeres get shorter. Telomere length is mainly maintained by telomerase. This enzyme is present in the embryonic stem cells in high concentrations and declines with age. It is still unclear to what extend there is telomerase in adult stem cells, but considering these are the founder cells to the cells of the all tissues in a body, understanding the telomere dynamics and expression of telomerase in adult stem cells is very important.Telomere length has been implicated as one of the markers for neoplastic transformation in both in vivo and in vitro studies. During cancerogenesis, telomeres shorten due to high cell turnover and repeats are added by active telomerase or alternative lengthening of telomeres (ALT). This gradual shortening is replication driven and does not necessarily explain the presence of ultrashort telomeres. Ultrashort telomeres are observed when there is a sudden shortening in telomeres not related with cell division and may arise from breaks in telomeres due to oxidative damage and replication slippage.Universal STELA is an accurate method for evaluation of ultrashort telomeres in hMSC-telo1 cells. Compared to TRF assay, U-STELA is developed to overcome several problems in detecting abrupt telomere shortening in a single chromosome.

T cell inhibitory mechanisms in a model of aggressive Non-Hodgkin's Lymphoma

ABSTRACTA reduced immune surveillance due to immune deficiency or treatment with immunosuppressive drugs is associated with a higher risk to develop aggressive Non-Hodgkin's lymphoma (NHL). Nevertheless, NHL also develops in immunocompetent patients indicating an escape from the immune system. T cell function in advanced aggressive lymphoma is not well characterized and the molecular mechanisms how malignant B cells influence T cell function are ill-defined. We therefore studied T cell function in Eμ-myc transgenic mice that develop an aggressive B cell lymphoma with some similarities to human Burkitt-lymphoma (BL). In advanced lymphoma, the number of T cells was severely reduced and the remaining CD4+ and CD8+ T cells lost the capacity to produce effector cytokines and expand upon re-stimulation. T cells in lymphoma-bearing mice were characterized by the expression of the immune inhibitory molecules programmed death (PD)-1, 2B4 and lymphocyte activation protein (LAG)-3. The proto-oncogene c-Myc not only drives cell proliferation and disease progression but also induces apoptosis of the malignant cells. We found that apoptotic lymphoma cells release purine metabolites that inhibit T cell function. Taken together, our data document that the characteristic high cell turnover and apoptotic rate in aggressive NHL induce a severe T cell dysfunction mediated by several immune-inhibitory mechanisms including ligation of inhibitory ligands and purine metabolites. Blocking a single mechanism only partially restored T cell function and did not increase survival of lymphoma mice.

A Brain Unfixed: Unlimited Neurogenesis and Regeneration of the Adult Planarian Nervous System.

Powerful genetic tools in classical laboratory models have been fundamental to our understanding of how stem cells give rise to complex neural tissues during embryonic development. In contrast, adult neurogenesis in our model systems, if present, is typically constrained to one or a few zones of the adult brain to produce a limited subset of neurons leading to the dogma that the brain is primarily fixed post-development. The freshwater planarian (flatworm) is an invertebrate model system that challenges this dogma. The planarian possesses a brain containing several thousand neurons with very high rates of cell turnover (homeostasis), which can also be fully regenerated de novo from injury in just 7 days. Both homeostasis and regeneration depend on the activity of a large population of adult stem cells, called neoblasts, throughout the planarian body. Thus, much effort has been put forth to understand how the flatworm can continually give rise to the diversity of cell types found in the adult brain. Here we focus on work using single-cell genomics and functional analyses to unravel the cellular hierarchies from stem cell to neuron. In addition, we will review what is known about how planarians utilize developmental signaling to maintain proper tissue patterning, homeostasis, and cell-type diversity in their brains. Together, planarians are a powerful emerging model system to study the dynamics of adult neurogenesis and regeneration.

Abstract 479: Smc Phenotype Switch Targeting Apoe is a Therapeutic Target in Popliteal Aneurysm

Introduction: Popliteal artery aneurysm (PAA) is an individually highly fatal disease, causing ischemia and eventual major amputation, mainly in 50-70 year old males. It is the most frequent peripheral artery aneurysm and correct treatment is challenging for clinicians, since both open and endovascular repair have only modest success rates, depending on the clinical presentation. In comparison to other aneurysm entities, little is known about its specific pathogenesis. Material and Methods: 30 Human PAA and popliteal artery samples were analyzed by immunohistochemistry, mRNA and miRNA expression analysis and protein assays on the OLink platform in order to identify key features of the disease and crucial pathways involved. Furthermore, a novel murine model of PAA is characterized for future applications in basic research. Results: Vascular smooth muscle cells loose their contractile phenotype along with significant inflammatory and proteolytic changes in the vessel wall architecture. Extensive tissue remodeling with high cell turnover rates is a unique feature in comparison to AAA. This correlates with highly abundant expression of the apolipoproteins E and CI suggestive to have a pro-proliferative effect in peripheral vascular tissue. Additionally, array-based screening on gene and protein level revelaed the pentraxin-related protein 3 as a potential biomarker and soluble inducer of neutrophil homing. Other targets of interest in relation to ApoE were fatty acid binding protein 4 and Insulin growth facto binding protein 2. These findings are emphasized by a specific case of a solely mechanically induced PAA in a young male. Conclusion: Pathogenesis of PAA shows similar histologic features than AAA, yet differs in potential pathways involved. Increased cell turnover in the aneurysmal neck area suggests evaluation of alternative treatment strategies, targeting key processes in its pathogenesis.

Modelling western dietary habits in the mouse: easier said than done.

The recent debate on the relative importance of environmental vs. intrinsic factors in the onset of malignancy has raised major concerns that the general public might conclude that cancer prevention programmes are not worthwhile. In their original report (1), Tomasetti and Vogelstein concluded that a majority of cancers can be explained by the high number of stem cell divisions in tissues with a high cell turn-over, due to generation of random mutations and their accumulation each time DNA replicates. In a subsequent report (2), Wu and colleagues employed a different mathematical model and extended the data set to reach the opposing conclusion that spontaneous mutations occurring during stem cell division rarely reach the level necessary to underlie cancer development. In the majority of cases, exposure to environmental risk factors represents a fundamental requirement for the onset of malignant disease. Of note, in the mathematical approach of the former study (1), the intrinsic rate of stem cell division and the environmental risk factors were regarded as entirely independent variables whereas it is plausible to think that extrinsic factors do affect stem cell homeostasis.

Spontaneous Tumor Lysis Syndrome in Small Cell Lung Cancer

Tumor lysis syndrome (TLS) is a life-threatening oncologic complication caused by the lysis of a vast number of malignant cells resulting in metabolic derangements and organ dysfunction. TLS can occur spontaneously before initiation of any therapies often referred to as spontaneous tumor lysis syndrome (STLS), or shortly after the induction of chemotherapy, radiotherapy, or cytolytic antibody therapy. TLS is vastly seen in patients with hematological malignancies with high rapid cell turnover rates such as Burkitt lymphoma, acute myelogenous leukemia, and acute lymphocytic leukemia, and is rarely observed in solid tumors. However, TLS can occur in solid tumors, and there are multiple reports in the literature on the occurrence of TLS in various solid tumors. In this article, we report a case of STLS in small cell lung cancer followed by a brief review of the occurrence of TLS and STLS in small cell lung cancer.

Significance of Serum Lactate Dehydrogenase in Childhood Acute Lymphoblastic Leukaemia

Introduction: Acute Lymphoblastic Leukaemia (ALL) is the most common childhood malignancy accounting for approximately 30% of childhood malignancies. The incidence rate of leukaemia in various parts of India varies from 0.3-1.2%. Lactate Dehydrogenase (LDH) is a pyridine-linked enzyme which is involved in metabolism of glucose in normal tissue. In leukaemic cells, there is loss of coordination of glycolytic sequence and tricarboxylic acid cycle, which leads to increased utilization of glucose. As there is high cell turnover, this leads to increased cell burden and high levels of serum LDH in ALL and the increase is much more than other haematological malignancies barring Burkitts lymphoma. Aim: To study the level of LDH in ALL cases at the time of diagnosis in comparison with Non ALL cases. Materials and Methods: Fifty five cases of ALL and 23 cases of Non ALL haematological malignancies as control were evaluated based on clinical manifestations, haematological parameters, peripheral blood picture and bone marrow findings. Serum LDH was estimated at the time of presentation by Cobas 6000, a photometrically automated system. Statistical analysis was done using SPSS version 20. Results: In our study, high serum LDH levels were seen in 89.1% (49) cases and normal levels in 10.9% (6) cases. High blast percentage i.e., >20% on peripheral smear (65.5%) was associated with high serum LDH level. On correlation of serum LDH with peripheral smear blast percentage, a p-value of <0.05 was obtained which shows a significant correlation Conclusion: Serum LDH level can be considered as a simple cost-effective tool in the presumptive evaluation of childhood Acute Lymphoblastic Leukaemia. LDH in combination with Uric acid helps in detecting tumourlysis syndrome at an early stage thereby aiding in early management.

Sialic acid as a biomarker of oral potentially malignant disorders and oral cancer.

Carcinogenesis is a multistep process where a healthy cell has initially a precancerous stage and finally an early cancerous stage. The process of carcinogenesis can be divided into three stages of initiation, promotion, and progression. In this process, there is increased turnover, secretion, and/or shedding from malignant cells. Glycoproteins like sialic acid are expressed on the cell surface. In oral potentially malignant disorders (OPMDs) and oral cancer (OC), the sialic acid level is seen to increase due to high cell turnover and shedding of malignant cells which, in turn, results in the release of glycoproteins like sialic acid into circulation. Glycoproteins also form an important constituent of salivary mucins and hence due to the same mechanism, an increase in sialic acid level is also seen in saliva. The aim is to estimate serum and salivary sialic acid levels in healthy controls, patients with OPMDs and patients with OC. In this observational cross-sectional study, serum and salivary sialic acid levels were estimated in thirty healthy controls, thirty patients with OPMDs and thirty patients with OC. Serum and salivary sialic acid levels obtained were subjected to statistical analysis. Post hoc Tukey test was used to compare the serum and salivary sialic acid levels of the two study groups to the control group. ANOVA test was used for the comparison of sialic acid levels between the groups. Pearson's correlation coefficient was used to assess correlation (P < 0.05 was considered statistically significant). The mean serum and salivary sialic acid levels were increased significantly in subjects with OPMDs and OC when compared to healthy controls. This study highlights the high expression of sialic acid on outer cell membranes, due to the significant increase in subjects with OPMDs and OC when compared to healthy controls. A significant increase in sialic acid level is also seen in saliva. Hence, it can be stated that saliva can be used as a reliable, noninvasive tool in diagnosis and management of OPMDs and OC.

Identification and Characterization of the Dermal Panniculus Carnosus Muscle Stem Cells.

SummaryThe dermal Panniculus carnosus (PC) muscle is important for wound contraction in lower mammals and represents an interesting model of muscle regeneration due to its high cell turnover. The resident satellite cells (the bona fide muscle stem cells) remain poorly characterized. Here we analyzed PC satellite cells with regard to developmental origin and purported function. Lineage tracing shows that they originate in Myf5+, Pax3/Pax7+ cell populations. Skin and muscle wounding increased PC myofiber turnover, with the satellite cell progeny being involved in muscle regeneration but with no detectable contribution to the wound-bed myofibroblasts. Since hematopoietic stem cells fuse to PC myofibers in the absence of injury, we also studied the contribution of bone marrow-derived cells to the PC satellite cell compartment, demonstrating that cells of donor origin are capable of repopulating the PC muscle stem cell niche after irradiation and bone marrow transplantation but may not fully acquire the relevant myogenic commitment.

Should tissue structure suppress or amplify selection to minimize cancer risk?

BackgroundIt has been frequently argued that tissues evolved to suppress the accumulation of growth enhancing cancer inducing mutations. A prominent example is the hierarchical structure of tissues with high cell turnover, where a small number of tissue specific stem cells produces a large number of specialized progeny during multiple differentiation steps. Another well known mechanism is the spatial organization of stem cell populations and it is thought that this organization suppresses fitness enhancing mutations. However, in small populations the suppression of advantageous mutations typically also implies an increased accumulation of deleterious mutations. Thus, it becomes an important question whether the suppression of potentially few advantageous mutations outweighs the combined effects of many deleterious mutations.ResultsWe argue that the distribution of mutant fitness effects, e.g. the probability to hit a strong driver compared to many deleterious mutations, is crucial for the optimal organization of a cancer suppressing tissue architecture and should be taken into account in arguments for the evolution of such tissues.ConclusionWe show that for systems that are composed of few cells reflecting the typical organization of a stem cell niche, amplification or suppression of selection can arise from subtle changes in the architecture. Moreover, we discuss special tissue structures that can suppress most types of non-neutral mutations simultaneously.ReviewersThis article was reviewed by Benjamin Allen, Andreas Deutsch and Ignacio Rodriguez-Brenes. For the full reviews, please go to the Reviewers’ comments section.

Acute kidney injury in critically ill cancer patients: an update.

Patients with cancer represent a growing group among actual ICU admissions (up to 20 %). Due to their increased susceptibility to infectious and noninfectious complications related to the underlying cancer itself or its treatment, these patients frequently develop acute kidney injury (AKI). A wide variety of definitions for AKI are still used in the cancer literature, despite existing guidelines on definitions and staging of AKI. Alternative diagnostic investigations such as Cystatin C and urinary biomarkers are discussed briefly. This review summarizes the literature between 2010 and 2015 on epidemiology and prognosis of AKI in this population. Overall, the causes of AKI in the setting of malignancy are similar to those in other clinical settings, including preexisting chronic kidney disease. In addition, nephrotoxicity induced by the anticancer treatments including the more recently introduced targeted therapies is increasingly observed. However, data are sometimes difficult to interpret because they are often presented from the oncological rather than from the nephrological point of view. Because the development of the acute tumor lysis syndrome is one of the major causes of AKI in patients with a high tumor burden or a high cell turnover, the diagnosis, risk factors, and preventive measures of the syndrome will be discussed. Finally, we will briefly discuss renal replacement therapy modalities and the emergence of chronic kidney disease in the growing subgroup of critically ill post-AKI survivors.

In silico study of the impact of cancer stem cell dynamics and radiobiological hypoxia on tumour response to hyperfractionated radiotherapy.

Advanced head and neck carcinomas (HNCs) are aggressive tumours, mainly due to hypoxia and a cancer stem cell (CSC) subpopulation. The aim of this study was to simulate tumour growth and behaviour during radiotherapy of three HNC groups (governed by different growth kinetics, hypoxia levels and CSC division pattern) to determine correlation between resistance factors and responses to hyperfractionated radiotherapy. An in silico HNC model was developed based on biologically realistic input parameters. During radiotherapy simulation, three parameters were studied: growth kinetics, hypoxia and probability of CSC symmetrical division. Both independent and combined effects on tumour response to hyperfractionated radiotherapy were assessed. Oxic and very mildly hypoxic HNCs were revealed to be controlled by hyperfractionated radiotherapy, irrespective of growth kinetics and CSC division pattern. Moderately hypoxic tumours had different responses to radiotherapy: while slowly proliferating HNCs were still controllable, tumours with higher cell turnover were more resistant. In rapidly proliferating tumours, the number of fractions needed for tumour control increased exponentially with the probability of CSC symmetrical division, whereas in moderately growing HNC, this behaviour was linear. Severely hypoxic tumours could not be controlled by radiotherapy alone. Tumours with CSCs in a severely hypoxic niche required adjuvant therapies to be eradicated. Growth kinetics strongly influence tumour responses to treatment. Slowly growing tumours showed linear dependence between dose and hypoxia/CSC, whereas rapidly growing tumours followed exponential behaviour.

A clinical evaluation of the TK 210 ELISA in sera from breast cancer patients demonstrates high sensitivity and specificity in all stages of disease.

Thymidine kinase (TK1) is an enzyme involved in DNA synthesis that leaks into the blood as a result of high cell turnover, particularly in the case of cancer. Serum TK1 activity has been used for prognosis and monitoring of leukemia and lymphoma patients for many years. Here, we describe the first clinical results with the newly developed TK 210 ELISA from AroCell AB. Sera from 124 breast cancer patients with known TNM classification along with sera from 53 healthy females were analyzed by TK 210 ELISA for TK1 protein and TK1 activity levels by the 3[H]-deoxythymidine (dThd) phosphorylation assay. The limit of detection for the TK 210 ELISA was 0.17 ng/ml, and 60 % of the sera from female blood donors were below this value. The median TK1 levels found in sera from breast cancer patients with T1 to T4 stage disease were 0.31, 0.46, 0.47, and 0.55 ng/ml, and these levels significantly differed from healthy controls. The median values of the biomarker CA 15-3 were also increased in patient sera from T1 to T4 patients (16, 34, 36, 40 U/ml, respectively). TK 210 ELISA showed significantly higher sensitivity for the T1 and T2 breast cancer patients compared to the TK activity assay. The combination of the TK1 ELISA and CA 15-3 biomarkers demonstrated a significant increase in sensitivity up to 15 % compared to each marker alone. This evaluation of the TK 210 ELISA strongly suggests that it can provide independent and complementary information for patients with breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-016-5024-z) contains supplementary material, which is available to authorized users.