Automated STED nanoscopy for high-throughput imaging of cellular structures

STimulated Emission Depletion (STED) nanoscopy uniquely combines a high spatial resolution (20-50nm in cells) with relatively fast imaging (frame rate of ∼1-30Hz), straightforward sample preparation and direct image output (no postprocessing required). Although these characteristics in principle make STED very suitable for high-throughput imaging, only few steps towards automation have been made. Here, we have developed fully automated STED imaging, eliminating all manual steps including the selection and characterisation of the relevant (cellular) regions, sample focusing and positioning, and microscope adjustments. This automatic STED image acquisition increases the data output by roughly two orders of magnitude, resulting in a more efficient use of the high-end microscope, and the ability to detect and characterise objects that are only present in a small subset of the sample

Covalent binding studies on the 14C-labeled antitumour compound 2,5-bis(1-aziridinyl)-1,4-benzoquinone. Involvement of semiquinone radical in binding to DNA, and binding to proteins and bacterial macromolecules in situ

2,5-Bis(1-aziridinyl)-1,4-benzoquinone (BABQ) is a compound from which several antitumour drugs are derived, such as Trenimone, Carboquone and Diaziquone (AZQ). The mechanism of DNA binding of BABQ was studied using 14C-labeled BABQ and is in agreement with reduction of the quinone moiety and protonation of the aziridine ring, followed by ring opening and alkylation. The one-electron reduced (semiquinone) form of BABQ alkylates DNA more efficiently than two-electron reduced or non reduced BABQ. Covalent binding to polynucleotides did not unambiguously reveal preference for binding to specific DNA bases. Attempts to elucidate further the molecular structure of DNA adducts by isolation of modified nucleosides from enzymatic digests of reacted DNA failed because of instability of the DNA adducts. The mechanism of covalent binding to protein (bovine serum albumin, BSA) appeared to be completely different from that of covalent binding to DNA. Binding of BABQ to BSA was not enhanced by reduction of the compound and was pH dependent in a way that is opposite to that of DNA alkylation. Glutathione inhibits binding of BABQ to BSA and forms adducts with BABQ in a similar pH dependence as the protein binding. The aziridine group therefore does not seem to be involved in the alkylation of BSA. Incubation of intact E. coli cells, which endogenously reduce BABQ, resulted in binding to both DNA and RNA, but also appreciable protein binding was observed

Cost-effectiveness of ovarian stimulation agents for IUI in couples with unexplained subfertility

Acknowledgements The authors wish to thank M.P. Diamond, R.S. Legro, K. Peeraer, M. Erdem, T. Dankert, and R. Ecochard, on behalf of the IUI IPDMA collaboration, for providing data from their RCTs for the IPDMA of which the results are used in a sensitivity analysis. Funding This work was supported by ZonMw Doelmatigheidsonderzoek, grant 80-85200-98-91072. The funder had no role in the design, conduct or reporting of this work. B.W.M. is supported by a NHMRC Practitioner Fellowship (GNT1082548).Peer reviewedPublisher PD

Mitosene-DNA adducts. Characterization of two major DNA monoadducts formed by 1,10-bis(acetoxy)-7-methoxymitosene upon reductive activation

Reductive activation of racemic 1,10-bis(acetoxy)-7-methoxymitosene WV15 in the presence of DNA, followed by enzymatic digestion and HPLC analysis, revealed the formation of various DNA adducts. Reduction is a necessary event for adduct formation to occur. This reductive activation was performed under hypoxic conditions in various ways:  (1) chemically, using a 2-fold excess of sodium dithionite (Na2S2O4), (2) enzymatically using NADH-cytochrome c reductase, (3) electrochemically on a mercury pool working electrode, and (4) catalytically, using a H2/PtO2 system. Five different mitosene−DNA adducts were detected. These adducts were also present when poly(dG-dC) was used instead of DNA, but were absent with poly(dA-dT). All were shown to be adducts of guanine. Reduction of 1,10-dihydroxymitosene WV14 in the presence of DNA did not result in detectable adduct formation, demonstrating the importance of good leaving groups for efficient adduct formation by these mitosenes. Finally, two of the adducts were isolated and their structures elucidated, using mass spectrometry, 1H NMR and circular dichroism (CD). The structures were assigned as the diastereoisomers N2-(1‘ ‘-n-hydroxymitosen-10‘ ‘-yl), 2‘-deoxyguanosine (n = α or β). These type of adducts, in which the mitosene C-10 is covalently bonded to the N-2 of a guanosylic group, are different from the well-known mitomycin C 2‘-deoxyguanosine monoadducts, that is linked via the mitomycin C C-1 position, demonstrating that the order of reactivity of the C-1 and C-10 in these mitosenes is reversed, as compared to mitomycin C. The 7-methoxy substituent of WV15 is a likely factor causing this switch. Evidence is presented that the 7-substituent of mitosenes also influences their DNA alkylation site. Adducts 4 and 5 represent the first isolated and structurally characterized covalent adducts of DNA and a synthetic mitosene

Late Quaternary loss of genetic diversity in muskox (Ovibos)

BACKGROUND: The modern wildherd of the tundra muskox (Ovibos moschatus) is native only to the New World (northern North America and Greenland), and its genetic diversity is notably low. However, like several other megafaunal mammals, muskoxen enjoyed a holarctic distribution during the late Pleistocene. To investigate whether collapse in range and loss of diversity might be correlated, we collected mitochondrial sequence data (hypervariable region and cytochrome b) from muskox fossil material recovered from localities in northeastern Asia and the Arctic Archipelago of northern North America, dating from late Pleistocene to late Holocene, and compared our results to existing databases for modern muskoxen. RESULTS: Two classes of haplotypes were detected in the fossil material. "Surviving haplotypes" (SHs), closely similar or identical to haplotypes found in modern muskoxen and ranging in age from ~22,000 to ~160 yrbp, were found in all New World samples as well as some samples from northeastern Asia. "Extinct haplotypes" (EHs), dating between ~44,000 and ~18,000 yrbp, were found only in material from the Taimyr Peninsula and New Siberian Islands in northeastern Asia. EHs were not found in the Holocene muskoxen specimens available for this study, nor have they been found in other studies of extant muskox populations. CONCLUSION: We provisionally interpret this evidence as showing that genetic variability was reduced in muskoxen after the Last Glacial Maximum but before the mid-Holocene, or roughly within the interval 18,000-4,000 yrbp. Narrowing this gap further will require the recovery of more fossils and additional genetic information from this interval

Clinical Profile of Dengue in Adult patients aged more than 13 years

Dengue Fever, known commonly as Break bone fever is the most common Arboviral mosquito borne disease in the world. This dengue virus is spread by the bite of infected Aedes mosquito, most commonly Ae. aegypti. Many countries especially the countries of the Indian subcontinent have suffered at the hands of this disease Dengue has a varied and wide spectrum of clinical presentations, often with unpredictable clinical evolution and outcome. This study is an attempt to derive the clinical profile of Dengue infection from the current epidemic that stormed Tirunelveli District. To identify some peculiar features that may help in early recognition and appropriate case management. The present study is an observational study where we studied the clinical profile of 251 serologically proven dengue patients admitted in the Department of Medicine, Tirunelveli Medical College from April 2012 - March 2013. Out of 251 cases, 149 patients (59.40%) belonged to DCF, DHF in 76 patients (30.30%), where as 26 patients (10.40%) belonged to more severe variety of DSS. Majority of cases 40.20% occurred in young adult 60 years of age. DCF (44.30%) and DSS (42.30%) was more common in the younger age group 60 years in this study. Distribution of dengue in females was slightly higher 51.40% when compared to males 48.60%. In this study all patients had fever 100%. Followed by headache 61%, Myalgia 54.60%, chills 46.20%, abdominal pain 43.40%, vomiting 42.20%.The characteristic feature of dengue like bone pain and retro-orbital pain was present in only 3.20% and 32.70% respectively. Atypical clinical feature like seizure was present in 1.20%. Most common sign observed in this study was conjunctival congestion 23.90%, followed by Hepatomegaly 12%, Ascites 10%, Rashes 10%, Pleural effusion 9.60%, Puffiness of the face and Splenomegaly 8.40%. Most common bleeding manifestation encountered was malena 27.50% followed by petechiae 8.40% and gum bleeding in 4.80% less frequent was bleeding manifestation hemoptysis 0.40%. BP is the most important clinical monitor in a case of dengue for identifying onset of complications like shock. Pulse pressure is more important than BP in identifying early stage shock. Narrowed pulse pressure (< 20 mm of Hg) is the most sensitive sign. 87 cases showed enzyme abnormalities in 100% cases with LFT abnormality which was similar to our study. This could be due to direct injury to liver cells by the virus or due to immunological response. Ischemic hepatitis in patients especially in shock could be another possible etiology USG was more sensitive in picking up Pleural effusion. Out of 251 patients mortality rate was 0.8% Recovery rate was 99.20%. The cause of death was intra cerebral bleed/ encephalopathy/seizures/DSS/DIC/Refractory shock in one case. The other case succumbed due to massive malena/ encephalopathy/DSS/DIC/refractory shock

The impact of body mass index on labour management and mode of delivery : A retrospective matched cohort study

FUNDINGBWM is supported by an NHMRC Investigator grant (GNT1176437). BWM reports consultancy, research funding and travel support from Merck.ACKNOWLEDGEMENTSOpen access publishing facilitated by Monash University, as part of the Wiley - Monash University agreement via the Council of Australian University Librarians.Peer reviewedPublisher PD

Healthcare choice: Discourses, perceptions, experiences and practices

Policy discourse shaped by neoliberal ideology, with its emphasis on marketisation and competition, has highlighted the importance of choice in the context of healthcare and health systems globally. Yet, evidence about how so-called consumers perceive and experience healthcare choice is in short supply and limited to specific healthcare systems, primarily in the Global North. This special issue aims to explore how choice is perceived and utilised in the context of different systems of healthcare throughout the world, where choice, at least in policy and organisational terms, has been embedded for some time. The articles are divided into those emphasising: embodiment and the meaning of choice; social processes associated with choice; the uncertainties, risks and trust involved in making choices; and issues of access and inequality associated with enacting choice. These sociological studies reveal complexities not always captured in policy discourse and suggest that the commodification of healthcare is particularly problematic