Role of vinculin in regulating focal adhesion turnover

Although vinculin (-/-) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in vinculin null compared to wild-type cells. Expression of vinculin rescued the spreading defect and resulted in larger and more stable FAs. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a role in vinculin activation by relieving an intramolecular association between the vinculin head (Vh) and tail (Vt) that masks the ligand binding sites in Vh and Vt. To investigate the role of the vinculin/PIP2 interaction in FA dynamics, we used a vinculin mutant lacking the C-terminal arm (residues 1053-1066) and referred to as the deltaC mutation. This mutation reduced PIP2 binding to a Vt deltaC polypeptide by >90% compared to wild type without affecting binding to Vh or F-actin. Interestingly, cells expressing the vinculin deltaC mutant assembled remarkably stable FAs. The results suggest that vinculin inhibits cell migration by stabilising FAs, and that binding of inositol phospholipids to Vt plays an important role in FA turnover.     

Cryopreservation of economically valuable marine micro-algae in the classes Bacillariophyceae, Chlorophyceae, Cyanophyceae, Dinophyceae, Haptophyceae, Prasinophyceae, and Rhodophyceae

The ability to routinely cryopreserve micro-algal species reduces costs associated with maintaining large culture collections and reduces the risks of losing particular strains or species through contamination and genetic drift. Cryopreservation is also a useful adjunct in aquaculture hatcheries for strains of micro-algae where the nutritional status may change as a result of continuous sub-culture. In this study, cryopreservation of isolates from seven micro-algal classes was investigated. Successful candidates included the marine dinoflagellates Amphidinium carterae, Amphidinium trulla, and Gymnodinium simplex, and the haptophytes Chrysochromulina simplex, Prymnesium parvum, Prymnesium parvum f. patelliferum, Isochrysis galbana, and Pavlova lutheri. Also successfully cryopreserved were the planktonic diatoms Chaetoceros calcitrans, Chaetoceros muelleri, Chaetoceros sp., and the benthic Nitzschia ovalis, the chlorophyte Chlamydomonas coccoides, the rhodophyte Porphyridium purpureum, the prasinophytes Tetraselmis chuii, and Tetraselmis suecica, and the cyanophytes Raphidiopsis sp., and Aphanizomenon flos-aquae. All species were successfully cryopreserved using 15% Me2SO.     

Diagnostic accuracy of case-finding questions to identify perinatal depression

Background:

Guidelines for perinatal mental health care recommend the use of two case-finding questions about depressed feelings and loss of interest in activities, despite the absence of validation studies in this context. We examined the diagnostic accuracy of these questions and of a third question about the need for help asked of women receiving perinatal care.

Methods:

We evaluated self-reported responses to two case-finding questions against an interviewer-assessed diagnostic standard (DSM-IV criteria for major depressive disorder) among 152 women receiving antenatal care at 26–28 weeks’ gestation and postnatal care at 5–13 weeks after delivery. Among women who answered “yes” to either question, we assessed the usefulness of asking a third question about the need for help. We calculated sensitivity, specificity and likelihood ratios for the two case-finding questions and for the added question about the need for help.

Results:

Antenatally, the two case-finding questions had a sensitivity of 100% (95% confidence interval [CI] 77%–100%), a specificity of 68% (95% CI 58%–76%), a positive likelihood ratio of 3.03 (95% CI 2.28–4.02) and a negative likelihood ratio of 0.041 (95% CI 0.003–0.63) in identifying perinatal depression. Postnatal results were similar. Among the women who screened positive antenatally, the additional question about the need for help had a sensitivity of 58% (95% CI 38%–76%), a specificity of 91% (95% CI 78%–97%), a positive likelihood ratio of 6.86 (95% CI 2.16–21.7) and a negative likelihood ratio of 0.45 (95% CI 0.25–0.80), with lower sensitivity and higher specificity postnatally.

Interpretation:

Negative responses to both of the case-finding questions showed acceptable accuracy for ruling out perinatal depression. For positive responses, the use of a third question about the need for help improved specificity and the ability to rule in depression.The occurrence of depressive symptoms during the perinatal period is well-recognized. The estimated prevalence is 7.4%–20% antenatally^1,2 and up to 19.2% in the first three postnatal months.³ Antenatal depression is associated with malnutrition, substance and alcohol abuse, poor self-reported health, poor use of antenatal care services and adverse neonatal outcomes.⁴ Postnatal depression has a substantial impact on the mother and her partner, the family, mother–baby interaction and on the longer-term emotional and cognitive development of the baby.⁵Screening strategies to identify perinatal depression have been advocated, and specific questionnaires for use in the perinatal period, such as the Edinburgh Postnatal Depression Scale,⁶ were developed. However, in their current recommendations, the UK National Screening Committee⁷ and the US Committee on Obstetric Practice⁸ state that there is insufficient evidence to support the implementation of universal perinatal screening programs. The initial decision in 2001 by the National Screening Committee to not support universal perinatal screening⁹ attracted particular controversy in the United Kingdom; some service providers subsequently withdrew resources for treatment of postnatal depression, and subsequent pressure by perinatal community practitioners led to modification of the screening guidance in order to clarify the role of screening questionnaires in the assessment of perinatal depression.¹⁰In 2007, the National Institute for Health and Clinical Excellence issued clinical guidelines for perinatal mental health care in the UK, which included guidance on the use of questionnaires to identify antenatal and postnatal depression.¹¹ In this guidance, a case-finding approach to identify perinatal depression was strongly recommended; it involved the use of two case-finding questions (sometimes referred to as the Whooley questions), and an additional question about the need for help asked of women who answered “yes” to either of the initial questions (Box 1).

Box 1:

Case-finding questions recommended for the identification of perinatal depression¹⁰

• “During the past month, have you often been bothered by feeling down, depressed or hopeless?”
• “During the past month, have you often been bothered by having little interest or pleasure in doing things?”
• A third question should be considered if the woman answers “yes” to either of the initial screening questions: “Is this something you feel you need or want help with?”

Useful case-finding questions should be both sensitive and specific so they accurately identify those with and without the condition. The two case-finding questions have been validated in primary care samples^12,13 and examined in other clinical populations^14–16 and are endorsed in recommendations by US and Canadian bodies for screening depression in adults.^17,18 However, at the time the guidance from the National Institute for Health and Clinical Excellence was issued, there were no validation studies conducted in perinatal populations. A recent systematic review¹⁹ identified one study conducted in the United States that validated the two questions against established diagnostic criteria in 506 women attending well-child visits postnatally;²⁰ sensitivity and specificity of the questions were 100% and 44% respectively at four weeks. The review failed to identify studies that validated the two questions and the additional question about the need for help against a gold-standard measure.We conducted a validation study to assess the diagnostic accuracy of this brief case-finding approach against gold-standard psychiatric diagnostic criteria for depression in a population of women receiving perinatal care.     

A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response

Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.     

A rapid, sensitive and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry method for determination of fenretinide (4-HPR) in plasma

A simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method using an atmospheric pressure chemical ionization source (APCI) for the quantification of fenretinide (4-HPR) in mouse plasma was developed and validated. After a simple protein precipitation of plasma sample by acetonitrile, 4-HPR was analyzed by LC-APCI-MS/MS. High-performance liquid chromatography (HPLC) separation was conducted on a Hypurity C18 column (50mmx2.1mm, 5microm) with a flow rate 0.60mL/min using a gradient mobile phase comprised of 0.05% formic acid in water (A) and methanol (B), and a run time of 4.5min. The elimination of a tedious sample preparation process and a shorter run time substantially reduced total analysis time. The method was linear over the range 0.5-100ng/mL, with r>0.998. The intra- and inter-assay precisions were 1.4-9.2% and 5.1-8.2%, respectively, and the intra- and inter-assay accuracies were 93.9-98.6% and 92.7-95.3%, respectively. The absolute recoveries were 90.3% (1.5ng/mL), 97.0% (7.5ng/mL) and 92.1% (75.0ng/mL) for 4-HPR, and 99.1% for the internal standard (150ng/mL). The analytical method had excellent sensitivity using a small sample volume (30microL) with the lower limit of quantification (LLOQ) 0.5ng/mL. This method is robust and has been successfully employed in a pharmacokinetic study of 4-HPR in a mouse xenograft model of neuroblastoma.     

Novel fluorinated acridone derivatives. Part 1: synthesis and evaluation as potential anticancer agents

We report on the synthesis of a novel series of fluorinated acridones from 5-trifluoromethyl-1,3-cyclohexanedione. The cytotoxic activities of the compounds were studied in several cancer cells. Compounds 9a, 9c, 9e, 9f, and 9h exhibited significant anticancer activities in selected cell lines. Compound 9c is the most active showing GI₅₀ that ranged in values from 0.13 to 26 μM, covering a wide range of cancer cell lines.     

Chloroplast development and the synthesis of chlorophyll and protochlorophyllide in Zostera transferred to darkness

Intact plants and isolated leaves of Zostera capricornii Martens ex Aschers were transferred from daylight to darkness. Substantial amounts of chloropyll a and b continued to accumulate in immature and mature tissue in the same ratio as in the light and were incorporated into chlorophyll-protein complexes in the thylakoids. A small amount of protochlorophyllide also accumulated in immature tissue in the dark. Proplastids and immature chloroplasts continued to develop into mature chloroplasts in the dark in the normal manner but prolamellar bodies, which are a conspicuous feature of immature chloroplasts, took longer to disperse than in the light. Protochlorophyllide accumulation and prolamellar-body formation were not correlated. The results indicate that Zostera has a genetic capacity for dark chlorophyll synthesis which is expressed in immature and mature leaf tissue and enables this plant to continue synthesising chlorophyll and assembling chloroplasts at night.Abbreviations Chl chlorophyll - T _o time of transfer to darkness