Improving Axial Resolution in Confocal Microscopy with New High Refractive Index Mounting Media

Resolution, high signal intensity and elevated signal to noise ratio (SNR) are key issues for biologists who aim at studying the localisation of biological structures at the cellular and subcellular levels using confocal microscopy. The resolution required to separate sub-cellular biological structures is often near to the resolving power of the microscope. When optimally used, confocal microscopes may reach resolutions of 180 nm laterally and 500 nm axially, however, axial resolution in depth is often impaired by spherical aberration that may occur due to refractive index mismatches. Spherical aberration results in broadening of the point-spread function (PSF), a decrease in peak signal intensity when imaging in depth and a focal shift that leads to the distortion of the image along the z-axis and thus in a scaling error. In this study, we use the novel mounting medium CFM3 (Citifluor Ltd., UK) with a refractive index of 1.518 to minimize the effects of spherical aberration. This mounting medium is compatible with most common fluorochromes and fluorescent proteins. We compare its performance with established mounting media, harbouring refractive indices below 1.500, by estimating lateral and axial resolution with sub-resolution fluorescent beads. We show furthermore that the use of the high refractive index media renders the tissue transparent and improves considerably the axial resolution and imaging depth in immuno-labelled or fluorescent protein labelled fixed mouse brain tissue. We thus propose to use those novel high refractive index mounting media, whenever optimal axial resolution is required.     

Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta)

Enzyme immunoassays (EIA) that measure faecal testosterone metabolites (fTM) are useful tools to monitor gonadal activity. The aim of this study was to validate an “in-house” epiandrosterone EIA to monitor fTM in spotted hyenas. FTM were characterised in a male and a female hyena that each received an injection of ³H-testosterone. High-performance liquid chromatography (HPLC) analyses revealed a cluster of highly polar enzyme-hydrolysable hormone metabolite conjugates. We performed hydrolysis using β-glucuronidase to deconjugate metabolites and improve sensitivity of the assay. Because β-glucuronidase from Helix pomatia has been reported to bias testosterone measurements in some species, we compared the enzymatic activity of the commonly used β-glucuronidase extracted from H. pomatia with the same enzyme from Escherichia coli. Our results showed that β-glucuronidases from both sources produced similar results from spotted hyena faeces. We therefore hydrolysed samples with H. pomatia enzymes. HPLC analyses also demonstrated that following hydrolysis the epiandrosterone EIA measured significant amounts of immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. Additionally, HPLC and GC-MS analyses confirmed the presence of epiandrosterone in faeces of spotted hyenas. The biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM levels in response to a testosterone injection within 16 h, (2) no biological responsiveness to an adrenocorticotropic hormone (ACTH) injection and (3) significant differences in fTM levels between juvenile males and adult immigrant males in a free-ranging wild population. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor gonadal activity in spotted hyenas.     

T1- Thresholds in Black Holes Increase Clinical-Radiological Correlation in Multiple Sclerosis Patients

Background

Magnetic Resonance Imaging (MRI) is an established tool in diagnosing and evaluating disease activity in Multiple Sclerosis (MS). While clinical-radiological correlations are limited in general, hypointense T1 lesions (also known as Black Holes (BH)) have shown some promising results. The definition of BHs is very heterogeneous and depends on subjective visual evaluation.

Objective

We aimed to improve clinical-radiological correlations by defining BHs using T1 relaxation time (T1-RT) thresholds to achieve best possible correlation between BH lesion volume and clinical disability.

Method

40 patients with mainly relapsing-remitting MS underwent MRI including 3-dimensional fluid attenuated inversion recovery (FLAIR), magnetization-prepared rapid gradient echo (MPRAGE) before and after Gadolinium (GD) injection and double inversion-contrast magnetization-prepared rapid gradient echo (MP2RAGE) sequences. BHs (BH_vis) were marked by two raters on native T1-weighted (T1w)-MPRAGE, contrast-enhancing lesions (CE lesions) on T1w-MPRAGE after GD and FLAIR lesions (total-FLAIR lesions) were detected separately. BH_vis and total-FLAIR lesion maps were registered to MP2RAGE images, and the mean T1-RT were calculated for all lesion ROIs. Mean T1 values of the cortex (CTX) were calculated for each patient. Subsequently, Spearman rank correlations between clinical scores (Expanded Disability Status Scale and Multiple Sclerosis Functional Composite) and lesion volume were determined for different T1-RT thresholds.

Results

Significant differences in T1-RT were obtained between all different lesion types with highest T1 values in visually marked BHs (BH_vis: 1453.3±213.4 ms, total-FLAIR lesions: 1394.33±187.38 ms, CTX: 1305.6±35.8 ms; p<0.05). Significant correlations between BH_vis/total-FLAIR lesion volume and clinical disability were obtained for a wide range of T1-RT thresholds. The highest correlation for BH_vis and total-FLAIR lesion masks were found at T1-RT>1500 ms (Expanded Disability Status Scale vs. lesion volume: r_BHvis = 0.442 and r_total-FLAIR = 0.497, p<0.05; Multiple Sclerosis Functional Composite vs. lesion volume: r_BHvis = -0.53 and r_total-FLAIR = -0.627, p<0.05).

Conclusion

Clinical-radiological correlations in MS patients are increased by application of T1-RT thresholds. With the short acquisition time of the MP2RAGE sequences, quantitative T1 maps could be easily established in clinical studies.     

Influence of Granulocyte-Macrophage Colony-Stimulating Factor or Influenza Vaccination on HLA-DR,Infection and Delirium Days in Immunosuppressed Surgical Patients: Double Blind,Randomised Controlled Trial

Purpose

Surgical patients are at high risk for developing infectious complications and postoperative delirium. Prolonged infections and delirium result in worse outcome. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and influenza vaccination are known to increase HLA-DR on monocytes and improve immune reactivity. This study aimed to investigate whether GM-CSF or vaccination reverses monocyte deactivation. Secondary aims were whether it decreases infection and delirium days after esophageal or pancreatic resection over time.

Methods

In this prospective, randomized, placebo-controlled, double-blind, double dummy trial setting on an interdisciplinary ICU of a university hospital 61 patients with immunosuppression (monocytic HLA-DR [mHLA-DR] <10,000 monoclonal antibodies [mAb] per cell) on the first day after esophageal or pancreatic resection were treated with either GM-CSF (250 μg/m²/d), influenza vaccination (Mutagrip 0.5 ml/d) or placebo for a maximum of 3 consecutive days if mHLA-DR remained below 10,000 mAb per cell. HLA-DR on monocytes was measured daily until day 5 after surgery. Infections and delirium were followed up for 9 days after surgery. Primary outcome was HLA-DR on monocytes, and secondary outcomes were duration of infection and delirium.

Results

mHLA-DR was significantly increased compared to placebo (p < 0.001) and influenza vaccination (p < 0.001) on the second postoperative day. Compared with placebo, GM-CSF-treated patients revealed shorter duration of infection (p < 0.001); the duration of delirium was increased after vaccination (p = 0.003).

Conclusion

Treatment with GM-CSF in patients with postoperative immune suppression was safe and effective in restoring monocytic immune competence. Furthermore, therapy with GM-CSF reduced duration of infection in immune compromised patients. However, influenza vaccination increased duration of delirium after major surgery.

Trial Registration

www.controlled-trials.com ISRCTN27114642     

Using Co-authorship Networks to Map and Analyse Global Neglected Tropical Disease Research with an Affiliation to Germany

Background

Research on Neglected Tropical Diseases (NTDs) has increased in recent decades, and significant need-gaps in diagnostic and treatment tools remain. Analysing bibliometric data from published research is a powerful method for revealing research efforts, partnerships and expertise. We aim to identify and map NTD research networks in Germany and their partners abroad to enable an informed and transparent evaluation of German contributions to NTD research.

Methodology/Principal Findings

A SCOPUS database search for articles with German author affiliations that were published between 2002 and 2012 was conducted for kinetoplastid and helminth diseases. Open-access tools were used for data cleaning and scientometrics (OpenRefine), geocoding (OpenStreetMaps) and to create (Table2Net), visualise and analyse co-authorship networks (Gephi). From 26,833 publications from around the world that addressed 11 diseases, we identified 1,187 (4.4%) with at least one German author affiliation, and we processed 972 publications for the five most published-about diseases. Of those, we extracted 4,007 individual authors and 863 research institutions to construct co-author networks. The majority of co-authors outside Germany were from high-income countries and Brazil. Collaborations with partners on the African continent remain scattered. NTD research within Germany was distributed among 220 research institutions. We identified strong performers on an individual level by using classic parameters (number of publications, h-index) and social network analysis parameters (betweenness centrality). The research network characteristics varied strongly between diseases.

Conclusions/Significance

The share of NTD publications with German affiliations is approximately half of its share in other fields of medical research. This finding underlines the need to identify barriers and expand Germany’s otherwise strong research activities towards NTDs. A geospatial analysis of research collaborations with partners abroad can support decisions to strengthen research capacity, particularly in low- and middle-income countries, which were less involved in collaborations than high-income countries. Identifying knowledge hubs within individual researcher networks complements traditional scientometric indicators that are used to identify opportunities for collaboration. Using free tools to analyse research processes and output could facilitate data-driven health policies. Our findings contribute to the prioritisation of efforts in German NTD research at a time of impending local and global policy decisions.     

Sympathetic Neurotransmitters Modulate Osteoclastogenesis and Osteoclast Activity in the Context of Collagen-Induced Arthritis

Excessive synovial osteoclastogenesis is a hallmark of rheumatoid arthritis (RA). Concomitantly, local synovial changes comprise neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze if collagen-induced arthritis (CIA) alters bone marrow-derived macrophage (BMM) osteoclastogenesis and osteoclast activity, and how sympathetic neurotransmitters participate in this process. Therefore, BMMs from Dark Agouti rats at different CIA stages were differentiated into osteoclasts in vitro and osteoclast number, cathepsin K activity, matrix resorption and apoptosis were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA) vasoactive intestinal peptide (VIP) and assay-dependent, adenylyl cyclase activator NKH477. We observed modulation of neurotransmitter receptor mRNA expression in CIA osteoclasts without affecting protein level. CIA stage-dependently altered marker gene expression associated with osteoclast differentiation and activity without affecting osteoclast number or activity. Neurotransmitter stimulation modulated osteoclast differentiation, apoptosis and activity. VIP, NA and adenylyl cyclase activator NKH477 inhibited cathepsin K activity and osteoclastogenesis (NKH477, 10^-6M NA) whereas ACh mostly acted pro-osteoclastogenic. We conclude that CIA alone does not affect metabolism of in vitro generated osteoclasts whereas stimulation with NA, VIP plus specific activation of adenylyl cyclase induced anti-resorptive effects probably mediated via cAMP signaling. Contrary, we suggest pro-osteoclastogenic and pro-resorptive properties of ACh mediated via muscarinic receptors.     

Rewriting the Central European Early Bronze Age Chronology: Evidence from Large-Scale Radiocarbon Dating

The transition from the Neolithic to the Early Bronze Age in Central Europe has often been considered as a supra-regional uniform process, which led to the growing mastery of the new bronze technology. Since the 1920s, archaeologists have divided the Early Bronze Age into two chronological phases (Bronze A1 and A2), which were also seen as stages of technical progress. On the basis of the early radiocarbon dates from the cemetery of Singen, southern Germany, the beginning of the Early Bronze Age in Central Europe was originally dated around 2300/2200 BC and the transition to more complex casting techniques (i.e., Bronze A2) around 2000 BC. On the basis of 140 newly radiocarbon dated human remains from Final Neolithic, Early and Middle Bronze Age cemeteries south of Augsburg (Bavaria) and a re-dating of ten graves from the cemetery of Singen, we propose a significantly different dating range, which forces us to re-think the traditional relative and absolute chronologies as well as the narrative of technical development. We are now able to date the beginning of the Early Bronze Age to around 2150 BC and its end to around 1700 BC. Moreover, there is no transition between Bronze (Bz) A1 and Bronze (Bz) A2, but a complete overlap between the type objects of the two phases from 1900–1700 BC. We thus present a revised chronology of the assumed diagnostic type objects of the Early Bronze Age and recommend a radiocarbon-based view on the development of the material culture. Finally, we propose that the traditional phases Bz A1 and Bz A2 do not represent a chronological sequence, but regionally different social phenomena connected to the willingness of local actors to appropriate the new bronze technology.     

Effects of Inhibiting Dipeptidyl Peptidase-4 (DPP4) in Cows with Subclinical Ketosis

The inhibition of dipeptidyl peptidase-4 (DPP4) via specific inhibitors is known to result in improved glucose tolerance and insulin sensitivity and decreased accumulation of hepatic fat in type II diabetic human patients. The metabolic situation of dairy cows can easily be compared to the status of human diabetes and non-alcoholic fatty liver. For both, insulin sensitivity is reduced, while hepatic fat accumulation increases, characterized by high levels of non-esterified fatty acids (NEFA) and ketone bodies.Therefore, in the present study, a DPP4 inhibitor was employed (BI 14332) for the first time in cows. In a first investigation BI 14332 treatment (intravenous injection at dosages of up to 3 mg/kg body weight) was well tolerated in healthy lactating pluriparous cows (n = 6) with a significant inhibition of DPP4 in plasma and liver. Further testing included primi- and pluriparous lactating cows suffering from subclinical ketosis (β-hydroxybutyrate concentrations in serum > 1.2 mM; n = 12). The intension was to offer effects of DPP4 inhibition during comprehensive lipomobilisation and hepatosteatosis. The cows of subclinical ketosis were evenly allocated to either the treatment group (daily injections, 0.3 mg BI 14332/kg body weight, 7 days) or the control group. Under condition of subclinical ketosis, the impact of DPP4 inhibition via BI 14332 was less, as in particular β-hydroxybutyrate and the hepatic lipid content remained unaffected, but NEFA and triglyceride concentrations were decreased after treatment. Owing to lower NEFA, the revised quantitative insulin sensitivity check index (surrogate marker for insulin sensitivity) increased. Therefore, a positive influence on energy metabolism might be quite possible. Minor impacts on immune-modulating variables were limited to the lymphocyte CD4⁺/CD8⁺ ratio for which a trend to decreased values in treated versus control animals was noted. In sum, the DPP4 inhibition in cows did not affect glycaemic control like it is shown in humans, but was able to impact hyperlipemia, as NEFA and TG decreased.     

A Reevaluation of the Voluntary Medical Male Circumcision Scale-Up Plan in Zimbabwe

Background

The voluntary medical male circumcision (VMMC) program in Zimbabwe aims to circumcise 80% of males aged 13–29 by 2017. We assessed the impact of actual VMMC scale-up to date and evaluated the impact of potential alterations to the program to enhance program efficiency, through prioritization of subpopulations.

Methods and Findings

We implemented a recently developed analytical approach: the age-structured mathematical (ASM) model and accompanying three-level conceptual framework to assess the impact of VMMC as an intervention. By September 2014, 364,185 males were circumcised, an initiative that is estimated to avert 40,301 HIV infections by 2025. Through age-group prioritization, the number of VMMCs needed to avert one infection (effectiveness) ranged between ten (20–24 age-group) and 53 (45–49 age-group). The cost per infection averted ranged between $811 (20–24 age-group) and $5,518 (45–49 age-group). By 2025, the largest reductions in HIV incidence rate (up to 27%) were achieved by prioritizing 10–14, 15–19, or 20–24 year old. The greatest program efficiency was achieved by prioritizing 15–24, 15–29, or 15–34 year old. Prioritizing males 13–29 year old was programmatically efficient, but slightly inferior to the 15–24, 15–29, or 15–34 age groups. Through geographic prioritization, effectiveness varied from 9–12 VMMCs per infection averted across provinces. Through risk-group prioritization, effectiveness ranged from one (highest sexual risk-group) to 60 (lowest sexual risk-group) VMMCs per infection averted.

Conclusion

The current VMMC program plan in Zimbabwe is targeting an efficient and impactful age bracket (13–29 year old), but program efficiency can be improved by prioritizing a subset of males for demand creation and service availability. The greatest program efficiency can be attained by prioritizing young sexually active males and males whose sexual behavior puts them at higher risk for acquiring HIV.     

Electrostatic Charge on Flying Hummingbirds and Its Potential Role in Pollination

Electrostatic phenomena are known to enhance both wind- and insect-mediated pollination, but have not yet been described for nectar-feeding vertebrates. Here we demonstrate that wild Anna''s Hummingbirds (Calypte anna) can carry positive charges up to 800 pC while in flight (mean ± s.d.: 66 ± 129 pC). Triboelectric charging obtained by rubbing an isolated hummingbird wing against various plant structures generated charges up to 700 pC. A metal hummingbird model charged to 400 pC induced bending of floral stamens in four plants (Nicotiana, Hemerocallis, Penstemon, and Aloe spp.), and also attracted falling Lycopodium spores at distances of < 2 mm. Electrostatic forces may therefore influence pollen transfer onto nectar-feeding birds.