O‐12BLAND DYSKARYOSIS: A NEW PITFALL IN THINPREP® LIQUID BASED CYTOLOGY

Liquid based cytology (LBC) has improved cell visualization and preservation in cervical cytology. There has been a reduction in inadequate rate and some data to suggest an increase in sensitivity for dyskaryosis. Training for LBC has focused on differences in distribution of abnormal cells, but in most cases the morphological appearance of the dyskaryotic cells themselves is similar to that seen in conventional cytology. We are describing a new presentation of dyskaryosis which may be a cause of false negative cytology. We have referred to this as ‘Bland dyskaryosis’ because cells appear deceptively bland on low power examination, and can be misinterpreted as metaplastic or endocervical cells. Bland dyskaryosis cells are seen in groups. The architecture of the group is very disorganized, and adjacent cells show variation in size. Cells have a high nuclear/cytoplasmic ratio and smooth nuclear membranes. Chromatin is finely granular and evenly distributed. This is an unusual presentation of high‐grade dyskaryosis and we feel that there is a learning curve in laboratories converting to liquid based cytology. The spectrum of appearances of squamous dyskaryosis needs to be delineated to allow further increases in sensitivity for dyskaryosis.     

Analysis of an intraspecific RIL population uncovers genomic segments harbouring multiple QTL for seed relevant traits in lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> L.)

Improving seed related traits remains key objective in lentil breeding. In recent years, genomic resources have shown great promise to accelerate crop improvement. However, limited genomic resources in lentil greatly restrict the use of genomics assisted breeding. The present investigation aims to build an intraspecific genetic linkage map and identify the QTL associated with important seed relevant traits using 94 recombinant inbreds (WA 8649090 × Precoz). A total of 288 polymorphic DNA markers including simple sequence repeat (SSR), inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) were assayed on mapping population. The resultant genetic linkage map comprised 220 loci spanning 604.2 cM of the lentil genome, with average inter-marker distance of 2.74 cM. QTL mapping in this RIL population uncovered a total of 18 QTL encompassing nine major and nine minor QTL. All major QTL were detected for seed related traits viz., seed diameter (SD), seed thickness (ST), seed weight (SW) and seed plumpness (SP) across two locations. A considerable proportion of the phenotypic variation (PV) was accounted to these QTL. For instance, one major QTL on LG5 controlling SW (QTL 15) explained 50% PV in one location, while the same QTL accounted for 34.18% PV in other location. Importantly, the genomic region containing multiple QTL for different seed traits was mapped to a 17-cM region on LG5. The genomic region harbouring QTL for multiple traits opens up exciting opportunities for genomics assisted improvement of lentil.     

Circulating cortisol‐associated signature of glucocorticoid‐related gene expression in subcutaneous fat of obese subjects

Objective:

Serum cortisol concentrations fluctuate in a circadian fashion, and glucocorticoids exert strong effects on adipose tissue and induce obesity through the glucocorticoid receptor.

Design and Methods:

To examine the impact of physiologic levels of circulating cortisol on subcutaneous adipose tissue, 25 overweight and obese subjects were employed, and their serum levels of morning (AM) and evening (PM) cortisol, AM/PM cortisol ratios, and 24‐h urinary‐free cortisol (UFC) were compared with their clinical parameters, serum cytokine levels, and mRNA expression of 93 receptor action‐regulating and 93 glucocorticoid‐responsive genes in abdominal subcutaneous fat.

Results and Conclusions:

AM cortisol levels did not correlate with mRNA expression of the all genes examined, whereas PM cortisol levels, AM/PM cortisol ratios, and 24‐h UFC were associated with distinct sets of these genes. Body mass index did not significantly correlate with the four cortisol parameters employed. These results suggest that physiologic levels of AM serum cortisol do not solely represent biological effects of circulating cortisol on the expression of glucocorticoid‐related genes in subcutaneous adipose tissue, whereas PM levels, amplitude, and net amounts of the diurnally fluctuating serum cortisol have distinct effects. Through the genes identified in this study, glucocorticoids appear to influence intermediary metabolism, energy balance, inflammation, and local circadian rythmicity in subcutaneous fat. Our results may also explain in part the development of metabolic abnormality and obesity in subjects under stress or patients with melancholic/atypical depression who demonstrate elevated levels of PM serum cortisol.     

The basic framework of VE-cadherin junctions revealed by cryo-EM

Artificial adherens junctions were reconstituted in vitro by assembly of cadherin fragments at the surfaces of liposomes. The architecture of the adherens junctions was revealed by cryo-electron microscopy (cryo-EM). The formation of these artificial adherens junctions was shown to result from the two-dimensional (2D) self-assembly of cadherin fragments at membrane surfaces. The molecular architecture of the junctions was resolved by combining information from several cryo-EM views. This study concludes to the 2D ordered nature of the cadherin assembly and shows that the minimal information required to build up an adherens junction is contained within the extracellular moiety of cadherin molecules.     

Determination of remifentanil in human plasma by liquid chromatography-tandem mass spectrometry utilizing micro extraction in packed syringe (MEPS) as sample preparation

Remifentanil is a synthetic short-acting opioid with a short half-life that is being used during anaesthesia of small children. In this work an LC-MS/MS method for remifentanil quantification in 20 μL volume of human plasma was developed and validated in connection with a clinical study on neonatal children. Sample preparation was performed with micro extraction in packed syringe (MEPS), which is a miniaturization of solid phase extraction. For this method a mixed phase sorbent M1 (C8, cation exchange), and a protocol for basic compound extraction was followed. Remifentanil-(13)C(6) was used as internal standard. For chromatographic separation, a C18 analytical column with gradient elution was used with mobile phase consisting of aqueous 0.1% formic acid and methanol. The total analysis time was 5.0 min and the measuring range was between 0.05 and 50 ng/mL. Precision and accuracy were with acceptance criteria of ±15%. Plasma samples were stable for 5 weeks at -20°C and for 4h at room temperature while 50% was lost after 24h. This method was successfully applied for remifentanil determination in clinical samples and results agreed with a reference method. With this method using MEPS, a low limit of quantification and much reduced sample volume was obtained as compared with previous methods.     

Securinine, a myeloid differentiation agent with therapeutic potential for AML

As the defining feature of Acute Myeloid Leukemia (AML) is a maturation arrest, a highly desirable therapeutic strategy is to induce leukemic cell maturation. This therapeutic strategy has the potential of avoiding the significant side effects that occur with the traditional AML therapeutics. We identified a natural compound securinine, as a leukemia differentiation-inducing agent. Securinine is a plant-derived alkaloid that has previously been used clinically as a therapeutic for primarily neurological related diseases. Securinine induces monocytic differentiation of a wide range of myeloid leukemia cell lines as well as primary leukemic patient samples. Securinine's clinical potential for AML can be seen from its ability to induce significant growth arrest in cell lines and patient samples as well as its activity in significantly impairing the growth of AML tumors in nude mice. In addition, securinine can synergize with currently employed agents such as ATRA and decitabine to induce differentiation. This study has revealed securinine induces differentiation through the activation of DNA damage signaling. Securinine is a promising new monocytic differentiation inducing agent for AML that has seen previous clinical use for non-related disorders.     

Early transcriptional responses of HepG2-A16 liver cells to infection by Plasmodium falciparum sporozoites

Invasion of hepatocytes by Plasmodium sporozoites deposited by Anopheles mosquitoes, and their subsequent transformation into infective merozoites is an obligatory step in the initiation of malaria. Interactions between the sporozoites and hepatocytes lead to a distinct, complex and coordinated cellular and systemic host response. Little is known about host liver cell response to sporozoite invasion, or whether it is primarily adaptive for the parasite, for the host, or for both. Our present study used gene expression profiling of human HepG2-A16 liver cells infected with Plasmodium falciparum sporozoites to understand the host early cellular events and factors influencing parasite infectivity and sporozoite development. Our results show that as early as 30 min following wild-type, non-irradiated sporozoite exposure, the expressions of at least 742 genes was selectively altered. These genes regulate diverse biological functions, such as immune processes, cell adhesion and communications, metabolism pathways, cell cycle regulation, and signal transduction. These functions reflect cellular events consistent with initial host cell defense responses, as well as alterations in host cells to sustain sporozoites growth and survival. Irradiated sporozoites gave very similar gene expression pattern changes, but direct comparative analysis between liver gene expression profiles caused by irradiated and non-irradiated sporozoites identified 29 genes, including glypican-3, that were specifically up-regulated only in irradiated sporozoites. Elucidating the role of this subset of genes may help identify the molecular basis for the irradiated sporozoites inability to develop intrahepatically, and their usefulness as an immunogen for developing protective immunity against pre-erythrocytic stage malaria.     

Engineering the nanoparticle-protein interface: applications and possibilities

Covalent and non-covalent conjugation of proteins to nanoparticles provides access to functional hybrid systems with applications in biotechnology, medicine, and catalysis. The creation of effective conjugates requires the retention of protein structure and function, a challenging task. In this review we discuss successes, challenges and opportunities in the area of protein-nanoparticle bioconjugation.