The importance of RT-qPCR primer design for the detection of siRNA-mediated mRNA silencing

<p>Abstract</p> <p>Background</p> <p>The use of RNAi to analyse gene function <it>in vitro </it>is now widely applied in biological research. However, several difficulties are associated with its use <it>in vivo</it>, mainly relating to inefficient delivery and non-specific effects of short RNA duplexes in animal models. The latter can lead to false positive results when real-time RT-qPCR alone is used to measure target mRNA knockdown.</p> <p>Findings</p> <p>We observed that detection of an apparent siRNA-mediated knockdown <it>in vivo </it>was dependent on the primers used for real-time RT-qPCR measurement of the target mRNA. Two siRNAs specific for <it>RRM1 </it>with equivalent activity <it>in vitro </it>were administered to A549 xenografts via intratumoural injection. In each case, apparent knockdown of <it>RRM1 </it>mRNA was observed only when the primer pair used in RT-qPCR flanked the siRNA cleavage site. This false-positive result was found to result from co-purified siRNA interfering with both reverse transcription and qPCR.</p> <p>Conclusions</p> <p>Our data suggest that using primers flanking the siRNA-mediated cleavage site in RT-qPCR-based measurements of mRNA knockdown <it>in vivo </it>can lead to false positive results. This is particularly relevant where high concentrations of siRNA are introduced, particularly via intratumoural injection, as the siRNA may be co-purified with the RNA and interfere with downstream enzymatic steps. Based on these results, using primers flanking the siRNA target site should be avoided when measuring knockdown of target mRNA by real-time RT-qPCR.</p

Glycan Structures Contain Information for the Spatial Arrangement of Glycoproteins in the Plasma Membrane

Glycoconjugates at the cell surface are crucial for cells to communicate with each other and the extracellular microenvironment. While it is generally accepted that glycans are vectorial biopolymers, their information content is unclear. This report provides evidence that distinct N-glycan structures influence the spatial arrangement of two integral membrane glycoproteins, Kv3.1 and E-cadherin, at the adherent membrane which in turn alter cellular properties. Distinct N-glycan structures were generated by heterologous expression of these glycoproteins in parental and glycosylation mutant Chinese hamster ovary cell lines. Unlike the N-linked glycans, the O-linked glycans of the mutant cell lines are similar to those of the parental cell line. Western and lectin blots of total membranes and GFP immunopurified samples, combined with glycosidase digestion reactions, were employed to verify the glycoproteins had predominantly complex, oligomannose, and bisecting type N-glycans from Pro(-)5, Lec1, and Lec10B cell lines, respectively. Based on total internal reflection fluorescence and differential interference contrast microscopy techniques, and cellular assays of live parental and glycosylation mutant CHO cells, we propose that glycoproteins with complex, oligomannose or bisecting type N-glycans relay information for localization of glycoproteins to various regions of the plasma membrane in both a glycan-specific and protein-specific manner, and furthermore cell-cell interactions are required for deciphering much of this information. These distinct spatial arrangements also impact cell adhesion and migration. Our findings provide direct evidence that N-glycan structures of glycoproteins contribute significantly to the information content of cells

The Hubble Constant

I review the current state of determinations of the Hubble constant, which gives the length scale of the Universe by relating the expansion velocity of objects to their distance. There are two broad categories of measurements. The first uses individual astrophysical objects which have some property that allows their intrinsic luminosity or size to be determined, or allows the determination of their distance by geometric means. The second category comprises the use of all-sky cosmic microwave background, or correlations between large samples of galaxies, to determine information about the geometry of the Universe and hence the Hubble constant, typically in a combination with other cosmological parameters. Many, but not all, object-based measurements give $H_0$ values of around 72-74km/s/Mpc , with typical errors of 2-3km/s/Mpc. This is in mild discrepancy with CMB-based measurements, in particular those from the Planck satellite, which give values of 67-68km/s/Mpc and typical errors of 1-2km/s/Mpc. The size of the remaining systematics indicate that accuracy rather than precision is the remaining problem in a good determination of the Hubble constant. Whether a discrepancy exists, and whether new physics is needed to resolve it, depends on details of the systematics of the object-based methods, and also on the assumptions about other cosmological parameters and which datasets are combined in the case of the all-sky methods.Comment: Extensively revised and updated since the 2007 version: accepted by Living Reviews in Relativity as a major (2014) update of LRR 10, 4, 200

Imagining the Lives of Others: Empathy in Public Relations

This paper asks how we might theorise empathy in public relations (PR) in the light of a widespread ‘turn’ towards emotion in the academy, as well as in popular discourse. Two distinct notions of empathy are explored: ‘true’empathy as discussed in intercultural communication, is driven by a human concern for the other in order to understand experiences, feelings and situations that may be different from our own; whereas ‘instrumental’ empathy, reflecting a self orientation, is said to characterise much neoliberal market discourse in which corporations are urged to understand their customers better. Thus, while empathy may seem highly desirable as a means to enter into dialogue with an organisation’s publics, particularly during times of social upheaval and crisis, it is important to pay attention to empathy in public relations discourses including whose goals are served by empathetic engagement; and the type(s) of empathy called upon within a PR context. A literature review identified a socio-cultural definition of empathy as ‘imaginary effort’. A review of the public relations literature, however, found that while empathy is considered an important principle and personal attribute, notions of empathy, with a few exceptions, are under-explored. Nonfunctionalist, socio-cultural research which examines the meanings that practitioners associate with empathy is distinctly lacking; therefore in order to gain further insight into empathy, two sources of data were explored. The analysis of a popular online practitioner blog showed that other-centred empathic skill is discursively framed as instrumental in achieving clients’ business objectives. The analysis of three empathy statements drawn from 12 in-depth interviews with practitioners revealed complex empathic discourse in practitioner-client relationships. While the findings are limited to illustrative analyses only, this paper challenges researchers to develop conceptualisations and perspectives of empathy as imaginary effort in public relations

CD8+ T Cells and IFN-γ Mediate the Time-Dependent Accumulation of Infected Red Blood Cells in Deep Organs during Experimental Cerebral Malaria

Background: Infection with Plasmodium berghei ANKA (PbA) in susceptible mice induces a syndrome called experimental cerebral malaria (ECM) with severe pathologies occurring in various mouse organs. Immune mediators such as T cells or cytokines have been implicated in the pathogenesis of ECM. Red blood cells infected with PbA parasites have been shown to accumulate in the brain and other tissues during infection. This accumulation is thought to be involved in PbA–induced pathologies, which mechanisms are poorly understood. Methods and Findings: Using transgenic PbA parasites expressing the luciferase protein, we have assessed by real-time in vivo imaging the dynamic and temporal contribution of different immune factors in infected red blood cell (IRBC) accumulation and distribution in different organs during PbA infection. Using deficient mice or depleting antibodies, we observed that CD8 + T cells and IFN-c drive the rapid increase in total parasite biomass and accumulation of IRBC in the brain and in different organs 6–12 days post-infection, at a time when mice develop ECM. Other cells types like CD4 + T cells, monocytes or neutrophils or cytokines such as IL-12 and TNF-a did not influence the early increase of total parasite biomass and IRBC accumulation in different organs. Conclusions: CD8 + T cells and IFN-c are the major immune mediators controlling the time-dependent accumulation of P. berghei-infected red blood cells in tissues

The supernatural characters and powers of sacred trees in the Holy Land

This article surveys the beliefs concerning the supernatural characteristics and powers of sacred trees in Israel; it is based on a field study as well as a survey of the literature and includes 118 interviews with Muslims and Druze. Both the Muslims and Druze in this study attribute supernatural dimensions to sacred trees which are directly related to ancient, deep-rooted pagan traditions. The Muslims attribute similar divine powers to sacred trees as they do to the graves of their saints; the graves and the trees are both considered to be the abode of the soul of a saint which is the source of their miraculous powers. Any violation of a sacred tree would be strictly punished while leaving the opportunity for atonement and forgiveness. The Druze, who believe in the transmigration of souls, have similar traditions concerning sacred trees but with a different religious background. In polytheistic religions the sacred grove/forest is a centre of the community's official worship; any violation of the trees is regarded as a threat to the well being of the community. Punishments may thus be collective. In the monotheistic world (including Christianity, Islam and Druze) the pagan worship of trees was converted into the worship/adoration of saints/prophets; it is not a part of the official religion but rather a personal act and the punishments are exerted only on the violating individual

Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1

Myeloproliferative neoplasms (MPNs) originate from genetically transformed hematopoietic stem cells that retain the capacity for multilineage differentiation and effective myelopoiesis. Beginning in early 2005, a number of novel mutations involving Janus kinase 2 (JAK2), Myeloproliferative Leukemia Virus (MPL), TET oncogene family member 2 (TET2), Additional Sex Combs-Like 1 (ASXL1), Casitas B-lineage lymphoma proto-oncogene (CBL), Isocitrate dehydrogenase (IDH) and IKAROS family zinc finger 1 (IKZF1) have been described in BCR-ABL1-negative MPNs. However, none of these mutations were MPN specific, displayed mutual exclusivity or could be traced back to a common ancestral clone. JAK2 and MPL mutations appear to exert a phenotype-modifying effect and are distinctly associated with polycythemia vera, essential thrombocythemia and primary myelofibrosis; the corresponding mutational frequencies are ∼99, 55 and 65% for JAK2 and 0, 3 and 10% for MPL mutations. The incidence of TET2, ASXL1, CBL, IDH or IKZF1 mutations in these disorders ranges from 0 to 17% these latter mutations are more common in chronic (TET2, ASXL1, CBL) or juvenile (CBL) myelomonocytic leukemias, mastocytosis (TET2), myelodysplastic syndromes (TET2, ASXL1) and secondary acute myeloid leukemia, including blast-phase MPN (IDH, ASXL1, IKZF1). The functional consequences of MPN-associated mutations include unregulated JAK-STAT (Janus kinase/signal transducer and activator of transcription) signaling, epigenetic modulation of transcription and abnormal accumulation of oncoproteins. However, it is not clear as to whether and how these abnormalities contribute to disease initiation, clonal evolution or blastic transformation

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

The impact of surgical delay on resectability of colorectal cancer: An international prospective cohort study

AIM: The SARS-CoV-2 pandemic has provided a unique opportunity to explore the impact of surgical delays on cancer resectability. This study aimed to compare resectability for colorectal cancer patients undergoing delayed versus non-delayed surgery. METHODS: This was an international prospective cohort study of consecutive colorectal cancer patients with a decision for curative surgery (January-April 2020). Surgical delay was defined as an operation taking place more than 4 weeks after treatment decision, in a patient who did not receive neoadjuvant therapy. A subgroup analysis explored the effects of delay in elective patients only. The impact of longer delays was explored in a sensitivity analysis. The primary outcome was complete resection, defined as curative resection with an R0 margin. RESULTS: Overall, 5453 patients from 304 hospitals in 47 countries were included, of whom 6.6% (358/5453) did not receive their planned operation. Of the 4304 operated patients without neoadjuvant therapy, 40.5% (1744/4304) were delayed beyond 4 weeks. Delayed patients were more likely to be older, men, more comorbid, have higher body mass index and have rectal cancer and early stage disease. Delayed patients had higher unadjusted rates of complete resection (93.7% vs. 91.9%, P = 0.032) and lower rates of emergency surgery (4.5% vs. 22.5%, P < 0.001). After adjustment, delay was not associated with a lower rate of complete resection (OR 1.18, 95% CI 0.90-1.55, P = 0.224), which was consistent in elective patients only (OR 0.94, 95% CI 0.69-1.27, P = 0.672). Longer delays were not associated with poorer outcomes. CONCLUSION: One in 15 colorectal cancer patients did not receive their planned operation during the first wave of COVID-19. Surgical delay did not appear to compromise resectability, raising the hypothesis that any reduction in long-term survival attributable to delays is likely to be due to micro-metastatic disease

Collection of the thinnest: A unique eggshell assemblage from the Late Cretaceous vertebrate locality of Iharkút (Hungary)

As a result of several years of screen-washing activity, a remarkable assemblage of eggshell fragments has been recovered from the Late Cretaceous vertebrate locality of Iharkút, Hungary. Detailed investigation of the assemblage by multiple visualization techniques (scanning electron microscopy, polarizing light microscopy, X-ray micro-computed tomography), quantitative morphometric analyses, and micro X-ray fluorescence spectrometry revealed a diverse composition of five different eggshell morphotypes (MT I-MT V) and three subcategories within the second morphotype (MT II/a, b, c), with MT I being by far the most abundant (83%) in the assemblage. MT I, MT III, and MT V represent theropod dinosaurian eggshells, whereas MT II and MT IV show characteristics of crocodilian and squamate eggshells, respectively. Hence, despite their fragmentary nature, these eggshells represent the first clear evidence that various sauropsid taxa had nesting sites near the ancient fluvial system of Iharkút. Besides the implied taxonomic diversity, two unique features add to the significance of this eggshell assemblage. First, it contains the thinnest rigid crocodilian (MT II/c) and squamate (MT IV) eggshells ever reported. Moreover, one of the identified theropod morphotypes, MT I, is also among the thinnest fossil dinosaurian eggshells, the thinness of which is only rivalled by the eggshells of the smallest Mesozoic avian eggs known to date. Second, the Iharkút eggshell assemblage consists exclusively of thin eggshells (≤300 μm), a condition unknown from any other fossil eggshell assemblages described to date. Combined with the knowledge acquired from skeletal remains, these peculiarities give additional insights into the paleoecology of the terrestrial sauropsid fauna once inhabiting the ancient island of Iharkút. Finally, the presence of well-preserved eggshells recovered from two different sites representing different depositional environments provides further evidence for previous taphonomic and sedimentological conclusions, and also expands our knowledge of the special conditions that allowed the preservation of these delicate eggshell fragments. © 2017 The Author(s)