Large-scale implementation of pooled RNA extraction and RT-PCR for SARS-CoV-2 detection.

INTRODUCTION: Testing for active SARS-CoV-2 infection is a fundamental tool in the public health measures taken to control the COVID-19 pandemic. Because of the overwhelming use of SARS-CoV-2 reverse transcription (RT)-PCR tests worldwide, the availability of test kits has become a major bottleneck and the need to increase testing throughput is rising. We aim to overcome these challenges by pooling samples together, and performing RNA extraction and RT-PCR in pools. METHODS: We tested the efficiency and sensitivity of pooling strategies for RNA extraction and RT-PCR detection of SARS-CoV-2. We tested 184 samples both individually and in pools to estimate the effects of pooling. We further implemented Dorfman pooling with a pool size of eight samples in large-scale clinical tests. RESULTS: We demonstrated pooling strategies that increase testing throughput while maintaining high sensitivity. A comparison of 184 samples tested individually and in pools of eight samples showed that test results were not significantly affected. Implementing the eight-sample Dorfman pooling to test 26 576 samples from asymptomatic individuals, we identified 31 (0.12%) SARS-CoV-2 positive samples, achieving a 7.3-fold increase in throughput. DISCUSSION: Pooling approaches for SARS-CoV-2 testing allow a drastic increase in throughput while maintaining clinical sensitivity. We report the successful large-scale pooled screening of asymptomatic populations.

Circulating breast-derived DNA allows universal detection and monitoring of localized breast cancer.

BACKGROUND: Tumor-derived circulating cell-free DNA (cfDNA) is present in the plasma of individuals with cancer. Assays aimed at detecting common cancer mutations in cfDNA are being developed for the detection of several cancer types. In breast cancer, however, such assays have failed to detect the disease at a sensitivity relevant for clinical use, in part due to the absence of multiple common mutations that can be co-detected in plasma. Unlike individual mutations that exist only in a subset of tumors, unique DNA methylation patterns are universally present in cells of a common type and therefore may be ideal biomarkers. Here we describe the detection and quantification of breast-derived cfDNA using a breast-specific DNA methylation signature. PATIENTS AND METHODS: We collected plasma from patients with localized breast cancer before and throughout treatment with neoadjuvant chemotherapy and surgery (N = 235 samples). RESULTS: Pretreatment breast cfDNA was detected in patients with localized disease with a sensitivity of 80% at 97% specificity. High breast cfDNA levels were associated with aggressive molecular tumor profiles and metabolic activity of the disease. During neoadjuvant chemotherapy, breast cfDNA levels decreased dramatically. Importantly, the presence of breast cfDNA towards the end of the chemotherapy regimen reflected the existence of residual disease. CONCLUSION: We propose that breast-specific cfDNA is a universal and powerful marker for the detection and monitoring of breast cancer.

Effects of ageing and senescence on pancreatic ß-cell function.

Ageing is generally associated with deterioration of organ function and regenerative potential. In the case of pancreatic ß-cells, an age-related decline in proliferative potential is well documented, and was proposed to contribute to the increased prevalence of type 2 diabetes in the elderly. The effects of ageing on ß-cell function, namely glucose-stimulated insulin secretion (GSIS), have not been studied as extensively. Recent work revealed that, surprisingly, ß-cells of mature mice and humans secrete more insulin than young ß-cells in response to high glucose concentrations, potentially serving to counteract age-related peripheral insulin resistance. This functional change appears to be orchestrated by p16(Ink4A) -driven cellular senescence and downstream remodelling of chromatin structure and DNA methylation, enhancing the expression of genes controlling ß-cell function. We propose that activation of the cellular senescence program drives life-long functional maturation of ß-cells, due to ß-cell hypertrophy, enhanced glucose uptake and more efficient mitochondrial metabolism, in parallel to locking these cells in a non-replicative state. We speculate that the beneficial aspects of this process can be harnessed to enhance GSIS. Other age-related mechanisms, which are currently poorly understood, act to increase basal insulin secretion levels also in low glucose conditions. This leads to an overall reduction in the amplitude of insulin secretion between low and high glucose at old age, which may contribute to a deterioration in metabolic control.

Neurogenin 3+ cells contribute to ß-cell neogenesis and proliferation in injured adult mouse pancreas.

We previously showed that injury by partial duct ligation (PDL) in adult mouse pancreas activates Neurogenin 3 (Ngn3)(+) progenitor cells that can differentiate to ß cells ex vivo. Here we evaluate the role of Ngn3(+) cells in ß cell expansion in situ. PDL not only induced doubling of the ß cell volume but also increased the total number of islets. ß cells proliferated without extended delay (the so-called 'refractory' period), their proliferation potential was highest in small islets, and 86% of the ß cell expansion was attributable to proliferation of pre-existing ß cells. At sufficiently high Ngn3 expression level, upto 14% of all ß cells and 40% of small islet ß cells derived from non-ß cells. Moreover, ß cell proliferation was blunted by a selective ablation of Ngn3(+) cells but not by conditional knockout of Ngn3 in pre-existing ß cells supporting a key role for Ngn3(+) insulin(-) cells in ß cell proliferation and expansion. We conclude that Ngn3(+) cell-dependent proliferation of pre-existing and newly-formed ß cells as well as reprogramming of non-ß cells contribute to in vivo ß cell expansion in the injured pancreas of adult mice.

Glucose metabolism: key endogenous regulator of ß-cell replication and survival.

Recent studies in mice have shown that pancreatic ß-cells have a significant potential for regeneration, suggesting that regenerative therapy for diabetes is feasible. Genetic lineage tracing studies indicate that ß-cell regeneration is based on the replication of fully differentiated, insulin-positive ß-cells. Thus, a major challenge for this field is to identify and enhance the molecular pathways that control ß-cell replication and mass. We review evidence, from human genetics and mouse models, that glucose is a major signal for ß-cell replication. The mitogenic effect of blood glucose is transmitted via glucose metabolism within ß-cells, and through a signalling cascade that resembles the pathway for glucose-stimulated insulin secretion. We introduce the concept that the individual ß-cell workload, defined as the amount of insulin that an individual ß-cell must secrete to maintain euglycaemia, is the primary determinant of replication, survival and mass. We also propose that a cell-autonomous pathway, similar to that regulating replication, appears to be responsible for at least some of the toxic effects of glucose on ß-cells. Understanding and uncoupling the mitogenic and toxic effects of glucose metabolism on ß-cells may allow for the development of effective regenerative therapies for diabetes.

Determinants of pancreatic beta-cell regeneration.

Recent studies have revealed a surprising plasticity of pancreatic beta-cell mass. beta-cell mass is now recognized to increase and decrease in response to physiological demand, for example during pregnancy and in insulin-resistant states. Moreover, we and others have shown that mice recover spontaneously from diabetes induced by killing of 70-80% of beta-cells, by beta-cell regeneration. The major cellular source for new beta-cells following specific ablation, as well as during normal homeostatic maintenance of adult beta-cells, is proliferation of differentiated beta-cells. More recently, it was shown that one form of severe pancreatic injury, ligation of the main pancreatic duct, activates a population of embryonic-type endocrine progenitor cells, which can differentiate into new beta-cells. The molecular triggers for enhanced beta-cell proliferation during recovery from diabetes and for activation of embryonic-type endocrine progenitors remain unknown and represent key challenges for future research. Taken together, recent data suggest that regenerative therapy for diabetes may be a realistic goal.

Alternative pathways of ovarian apoptosis: death for life.

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few, but healthy embryos. Degeneration of the old corpora lutea in each estrus/menstrual cycle by programmed cell death is essential for maintaining the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, play an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows for systematic studies of the mechanisms that control steroidogenesis and apoptosis of granulosa cells. We have discovered that during initial stages of granulosa cell apoptosis progesterone production does not decrease. In contrast, we found that it is elevated for up to 24hr following the onset of the apoptotic stimuli exerted by starvation, cAMP, p53 or tumor necrosis factor alpha stimulation, before total cell collapse. These observations raise the possibility for an alternative unique apoptotic pathway, one that does not involve mitochondrial cytochrome C release associated with the destruction of mitochondrial structure and steroidogenic function. Using mRNA from apoptotic cells and Affymetrix DNA microarray we discovered that Granzyme B, a protease that normally resides in T cytotoxic lymphocytes and natural killer cells of the immune system is expressed and activated in granulosa cells, thereby allowing the apoptotic signals to bypass mitochondrial signals for apoptosis, which can preserve their steroidogenic activity until complete cell destruction. This unique apoptotic pathway assures the cyclicity of estradiol and progesterone release in the estrus/menstrus cycle even during the initial stage of apoptosis.

Hypoxia-inducible factor-2alpha (HIF-2alpha) is involved in the apoptotic response to hypoglycemia but not to hypoxia.

Deprivation of oxygen (hypoxia) and/or glucose (hypoglycemia) represents a serious stress that affects cellular survival. The hypoxia-inducible transcription factor-1alpha (HIF-1alpha), which has been implicated in the cellular response to hypoxia (Semenza, G. L. (1999) Annu. Rev. Cell Dev. Biol. 15, 551-578), mediates apoptosis during hypoxia (Halterman, M. W., Miller, C. C., and Federoff, H. J. (1999) J. Neurosci. 19, 6818-6824 and Carmeliet, P., Dor, Y., Herbert, J. M., Fukumura, D., Brusselmans, K., Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, C. J., Ratcliffe, P., Moons, L., Jain, R. K., Collen, D., and Keshet, E. (1998) Nature 394, 485-490), but the function of its homologue HIF-2alpha remains unknown. Therefore, the role of HIF-2alpha in cellular survival was studied by targeted inactivation of the HIF-2alpha gene (HIF-2alpha(-/-)) in murine embryonic stem (ES) cells. In contrast to HIF-1alpha deficiency, loss of HIF-2alpha did not protect ES cells against apoptosis during hypoxia. Both HIF-1alpha(-/-) and HIF-2alpha(-/-) ES cells were, however, resistant to apoptosis in response to hypoglycemia. When co-cultured with wild type ES cells, HIF-2alpha(-/-) ES cells became rapidly and progressively enriched in hypoglycemia but not in hypoxia. Thus, HIF-1alpha and HIF-2alpha may have distinct roles in responses to environmental stress, and despite its name, HIF-2alpha may be more important in the survival response to environmental variables other than the level of oxygen.

Vascular endothelial growth factor and vascular adjustments to perturbations in oxygen homeostasis.

Development of microvascular networks is set to meet the metabolic requirements of the tissue they perfuse. Accordingly, impairment of oxygen homeostasis, either due to increased oxygen consumption or as a result of blood vessel occlusion, triggers compensatory neovascularization. This feedback reaction is mediated by a hypoxia- and hypoglycemia-induced vascular endothelial growth factor (VEGF). VEGF accumulates under stress as a result of increased hypoxia-inducible factor-1alpha-mediated transcription, stabilization of the mRNA, and the function of a hypoxia-refractory internal ribosome entry site within its 5'-untranslated region. Matching of vascular density to the metabolic needs of the tissue may include a process of hyperoxia-induced vessel regression. Thus newly formed vascular networks may undergo a natural process of vascular pruning that takes place whenever VEGF, acting as a vascular survival factor, is downregulated below the level required to sustain immature vessels. Immature vessels are particularly vulnerable and are selectively obliterated upon withdrawal of VEGF. The plasticity window for vessel regression is determined by a delay in the recruitment of periendothelial cells to the preformed endothelial plexus. Thus fine-tuning of microvascular density takes place mostly in the newly formed plexus, but the mature system is refractory to episodic changes in tissue oxygenation. These regulatory links may malfunction in certain pathological settings.

A novel role for VEGF in endocardial cushion formation and its potential contribution to congenital heart defects.

Normal cardiovascular development is exquisitely dependent on the correct dosage of the angiogenic growth factor and vascular morphogen vascular endothelial growth factor (VEGF). However, cardiac expression of VEGF is also robustly augmented during hypoxic insults, potentially mediating the well-established teratogenic effects of hypoxia on heart development. We report that during normal heart morphogenesis VEGF is specifically upregulated in the atrioventricular (AV) field of the heart tube soon after the onset of endocardial cushion formation (i.e. the endocardium-derived structures that build the heart septa and valves). To model hypoxia-dependent induction of VEGF in vivo, we conditionally induced VEGF expression in the myocardium using a tetracycline-regulated transgenic system. Premature induction of myocardial VEGF in E9.5 embryos to levels comparable with those induced by hypoxia prevented formation of endocardial cushions. When added to explanted embryonic AV tissue, VEGF fully inhibited endocardial-to-mesenchymal transformation. Transformation was also abrogated in AV explants subjected to experimental hypoxia but fully restored in the presence of an inhibitory soluble VEGF receptor 1 chimeric protein. Together, these results suggest a novel developmental role for VEGF as a negative regulator of endocardial-to-mesenchymal transformation that underlies the formation of endocardial cushions. Moreover, ischemia-induced VEGF may be the molecular link between hypoxia and congenital defects in heart septation.

Heat-induced cell cycle arrest of Saccharomyces cerevisiae: involvement of the RAD6/UBC2 and WSC2 genes in its reversal.

The Saccharomyces cerevisiae RAD6 (UBC2 ) gene encodes a ubiquitin-conjugating enzyme that is involved in a wide range of cellular processes including DNA repair, sporulation and N-end rule protein degradation. Under mild heat stress conditions (37-38 degrees C) rad6 null and rad6-149 mutant cells are unable to grow. The molecular basis for this failure to grow is unknown. Here we show that the heat sensitivity of rad6 mutants is not due to cell death but to an inability to progress in the cell cycle. The temperature-induced cell cycle arrest of these mutants is due to a block in a branch of the RAD6 pathway distinct from the DNA repair and the N-end rule protein degradation pathways. Wild-type cells heated to 38 degrees C arrest transiently in the late G1 phase and then resume growth. At 38 degrees C rad6 mutant cells arrest in late G1 but, unlike wild-type cells, are unable to resume cell cycle progression. In both wild-type and in rad6 mutant cells, CLN1 and CLN2 transcript levels fall sharply upon temperature increase. In wild-type cells levels of these transcripts recover rapidly, whereas in the rad6 mutant they recover slowly. As rad6 cells remain arrested even after CLN1 and CLN2 mRNAs regain their preheat stress levels, factors additional to reduced G1 cyclin gene expression must cause the temperature-induced cell cycle block of the mutant. To identify genes involved in the relief of the cell cycle arrest under heat stress, we screened a multicopy yeast genomic library for clones that restore the growth of the rad6-149 mutant. A plasmid was isolated carrying the WSC2 gene, which is closely related to WSC1/SLG1/HCS77, a putative membrane heat sensor. Overexpression of WSC2 reverses the heat-induced cell cycle arrest of rad6-149 but not of rad6 null mutants. Taken together the findings point to the existence of an unidentified heat stress-activated cell cycle checkpoint pathway, which is antagonized by Rad6p by a mechanism also involving Wsc2p.

Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis.

As a result of deprivation of oxygen (hypoxia) and nutrients, the growth and viability of cells is reduced. Hypoxia-inducible factor (HIF)-1alpha helps to restore oxygen homeostasis by inducing glycolysis, erythropoiesis and angiogenesis. Here we show that hypoxia and hypoglycaemia reduce proliferation and increase apoptosis in wild-type (HIF-1alpha+/+) embryonic stem (ES) cells, but not in ES cells with inactivated HIF-1alpha genes (HIF-1alpha-/-); however, a deficiency of HIF-1alpha does not affect apoptosis induced by cytokines. We find that hypoxia/hypoglycaemia-regulated genes involved in controlling the cell cycle are either HIF-1alpha-dependent (those encoding the proteins p53, p21, Bcl-2) or HIF-1alpha-independent (p27, GADD153), suggesting that there are at least two different adaptive responses to being deprived of oxygen and nutrients. Loss of HIF-1alpha reduces hypoxia-induced expression of vascular endothelial growth factor, prevents formation of large vessels in ES-derived tumours, and impairs vascular function, resulting in hypoxic microenvironments within the tumour mass. However, growth of HIF-1alpha tumours was not retarded but was accelerated, owing to decreased hypoxia-induced apoptosis and increased stress-induced proliferation. As hypoxic stress contributes to many (patho)biological disorders, this new role for HIF-1alpha in hypoxic control of cell growth and death may be of general pathophysiological importance.

Ischemia-driven angiogenesis.

New blood vessels usually develop in places where they are most needed. A prime example of neovascularization representing a positive feedback response to insufficient perfusion is the development of collateral blood vessels in the ischemic myocardium and leg. The recent discoveries of hypoxia-inducible transcription and angiogenic factors have provided important mechanistic links between the metabolic consequences of ischemia and compensatory angiogenesis. Vascular endothelial growth factor (VEGF) has emerged as the key mediator of ischemia-driven angiogenesis. Environmental stresses, including hypoxia, hypoglycemia, and hypoferremia, upregulate VEGF expression at both the transcriptional and posttranscriptional levels. VEGF acts in turn on adjacent vascular beds expressing cognate receptors and induces sprouting and capillary growth toward the ischemic tissue. In addition to expanding the vasculature at sites where existing vessels have been occluded or obliterated, VEGF also functions to match the vascular density according to development and physiologic increases in oxygen consumption. Fine adjustment of the vasculature includes a step of oxygen-regulated vascular pruning mediated by VEGF in its capacity as a survival factor for newly formed vessels. Pathologic settings of ischemia-driven angiogenesis include a major component of stress-induced angiogenesis during tumor neovascularization and abnormal vessel growth associated with retinopathies. The latter represents an excessive angiogenic response to conditions of severe retinal ischemia. Further insights into the mechanism of stress-induced angiogenesis are likely to suggest new ways to augment growth of collateral vessels and to restrain unwarranted neovascularization in tumors and retinopathies. (Trends Cardiovasc Med 1997;7:289-294). © 1997, Elsevier Science Inc.

Role of the conserved carboxy-terminal alpha-helix of Rad6p in ubiquitination and DNA repair.

RAD6 in the yeast Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme essential for DNA repair as well as for a number of other biological processes. It is believed that the functions of Rad6p require the ubiquitination of target proteins, but its substrates as well as other interacting proteins are largely unknown. Rad6p homologues of higher eukaryotes have a number of amino acid residues in the C-terminal alpha-helix, which are conserved from yeast to man but are absent from most other yeast ubiquitin-conjugating enzymes (Ubcs). This specific conservation suggests that the C-terminal alpha-helix is important for the unique activities of the Rad6p family of Ubcs. We have investigated the effects of mutating this highly conserved region on the ubiquitination of model substrates in vitro and on error-free DNA repair in vivo. C-terminal point and deletion mutants of Rad6p differentially affected its in vitro activity on various substrates, raising the possibility that Rad6p interacts with its substrates in vivo by similar mechanisms. The distal part of the C-terminal alpha-helix is also essential for error-free DNA repair in vivo. Overexpression of Rad18p, a single-stranded DNA-binding protein that also interacts with Rad6p, alleviates the DNA repair defects of the C-terminal alpha-helix mutants to different degrees. This indicates that the C-terminal alpha-helix of Rad6p mediates its interaction with Rad18p, an essential step in DNA repair. Models of Rad6p action propose that its ubiquitination function is followed by proteolysis of unknown ubiquitinated targets. Mutants affecting several functions of the 26S proteasome retain wild-type capacity for error-free DNA repair. This raises the possibility that ubiquitination by Rad6p in DNA repair does not target proteins for proteasomal degradation.

Chronic GnRH agonist administration down-regulates platelet serotonin transporter in women undergoing assisted reproductive treatment.

The effect of pretreatment with the gonadotropin releasing hormone (GnRH) agonist D-Trp6-LHRH (Decapeptyl) on platelet serotonin transporter in women undergoing assisted reproductive treatment (ART) was investigated and compared with women treated with human menopausal gonadotropin (Pergonal). The study group (n = 10) was exposed for 12 days to 3.2 mg Decapeptyl C.R. while a comparison group (n = 9) was exposed to 11 days of human meno-pausal gonadotropin (Pergonal). All patients were assessed with the Hamilton depression and anxiety scales before and after treatment, and platelet and plasma samples were collected at the same time points. Plasma levels of estradiol, progesterone. FSH and LH were determined by radioimmunoassay (RIA). Platelet serotonin transporter was labeled using high affinity [3H]imipramine binding. The GnRH analogue induced ovarian suppression as reflected by low plasma estradiol levels, while Pergonal administration induced ovarian stimulation. An elevation in the Hamilton depression and anxiety scale scores was observed in the Decapeptyl treated group; this mood alteration was associated with a significant decrease (19%, P < 0.05) in the density (Bmax) of platelet [3H]imipramine binding sites. No significant change was observed in the Bmax of the Pergonal treated group. These results indicate that ovarian suppression (menopausal-like state) in young women is associated with depressed and anxious mood and decreased serotonin transporter density.

Use of 23Na nuclear magnetic resonance spectroscopy to determine the true intracellular concentration of free sodium in a halophilic eubacterium.

We present new data obtained by 23Na nuclear magnetic resonance spectroscopy, which can distinguish free intracellular sodium from cell-bound sodium, showing that the intracellular concentration of Na+ the halophilic eubacterium Vibrio costicola is only 5 to 20% of that in the extracellular medium. Previous methods could not distinguish free intracellular Na+ from that bound to cell structures, and it was believed that in halophilic eubacteria the total monovalent cation concentration inside matched that of the NaCl outside. Information obtained by the newer technology raises fundamental questions about the ways in which these organisms and others which live in hypersaline environments function and cope with osmotic stress.

13C NMR study of the interrelation between synthesis and uptake of compatible solutes in two moderately halophilic eubacteria. Bacterium Ba1 and Vibro costicola.

The synthesis and uptake of intracellular organic osmolytes (compatible solutes) were studied with the aid of natural abundance 13C NMR spectroscopy in two unrelated, moderately halophilic eubacteria: Ba1 and Vibrio costicola. In minimal media containing 1 M NaCl, both microorganisms synthesized the cyclic amino acid, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (trivial name, ectoine) as the predominant compatible solute, provided that no glycine betaine was present in the growth medium. When, however, the minimal medium was supplemented with glycine betaine or the latter was a component of a complex medium, it was transported into the cells and the accumulating glycine betaine replaced the ectoine. In Ba1, grown in a defined medium containing glucose as the single carbon source, ectoine could only be detected if the NaCl concentration in the medium was higher than 0.6 M; the ectoine content increased with the external salt concentration. At NaCl concentrations below 0.6 M, alpha,alpha-trehalose was the major organic osmolyte. The concentration of ectoine reached its peak during the exponential phase and declined subsequently. In contrast, the accumulation of glycine betaine continued during the stationary phase. The results presented here indicate that, at least in the two microorganisms studied, ectoine plays an important role in haloadaptation.

In vitro fertilization and primary embryonic cleavage are possible in 17 alpha-hydroxylase deficiency despite extremely low intrafollicular 17 beta-estradiol.

Congenital adrenal hyperplasia due to 17 alpha-hydroxylase deficiency in genotypic females is characterized by primary amenorrhea and the absence of sexual maturation due to inadequate biosynthesis of ovarian androgens and estrogens. We induced ovarian follicular development in a woman with this syndrome. Ovum pick-up, in vitro fertilization, and primary embryonic development were achieved despite undetectable plasma estradiol and extremely low ovarian androgen concentrations and minute concentrations of these hormones in the ovarian follicular fluid.