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.     

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.     

Correlates of Recent Declines of Rodents in Northern and Southern Australia: Habitat Structure Is Critical

Australia has experienced dramatic declines and extinctions of its native rodent species over the last 200 years, particularly in southern Australia. In the tropical savanna of northern Australia significant declines have occurred only in recent decades. The later onset of these declines suggests that the causes may differ from earlier declines in the south. We examine potential regional effects (northern versus southern Australia) on biological and ecological correlates of range decline in Australian rodents. We demonstrate that rodent declines have been greater in the south than in the tropical north, are strongly influenced by phylogeny, and are consistently greater for species inhabiting relatively open or sparsely vegetated habitat. Unlike in marsupials, where some species have much larger body size than rodents, body mass was not an important predictor of decline in rodents. All Australian rodent species are within the prey-size range of cats (throughout the continent) and red foxes (in the south). Contrary to the hypothesis that mammal declines are related directly to ecosystem productivity (annual rainfall), our results are consistent with the hypothesis that disturbances such as fire and grazing, which occur in non-rainforest habitats and remove cover used by rodents for shelter, nesting and foraging, increase predation risk. We agree with calls to introduce conservation management that limits the size and intensity of fires, increases fire patchiness and reduces grazing impacts at ecological scales appropriate for rodents. Controlling feral predators, even creating predator-free reserves in relatively sparsely-vegetated habitats, is urgently required to ensure the survival of rodent species, particularly in northern Australia where declines are not yet as severe as those in the south.     

Scale Adjustments to Facilitate Two-Dimensional Measurements in OCT Images

Purpose

To address the problem of unequal scales for the measurement of two-dimensional structures in OCT images, and demonstrate the use of intra¬ocular objects of known dimensions in the murine eye for the equal calibration of axes.

Methods

The first part of this work describes the mathematical foundation of major distortion effects introduced by X-Y scaling differences. Illustrations were generated with CorelGraph X3 software. The second part bases on image data obtained with a HRA2 Spectralis (Heidelberg Engineering) in SV129 wild-type mice. Subretinally and intravitreally implanted microbeads, alginate capsules with a diameter of 154±5 μm containing GFP-marked mesenchymal stem cells (CellBeads), were used as intraocular objects for calibration.

Results

The problems encountered with two-dimensional measurements in cases of unequal scales are demonstrated and an estimation of the resulting errors is provided. Commonly, the Y axis is reliably calibrated using outside standards like histology or manufacturer data. We show here that intraocular objects like dimensionally stable spherical alginate capsules allow for a two-dimensional calibration of the acquired OCT raw images by establishing a relation between X and Y axis data. For our setup, a correction factor of about 3.3 was determined using both epiretinally and subretinally positioned beads (3.350 ± 0.104 and 3.324 ± 0.083, respectively).

Conclusions

In this work, we highlight the distortion-related problems in OCT image analysis induced by unequal X and Y scales. As an exemplary case, we provide data for a two-dimensional in vivo OCT image calibration in mice using intraocular alginate capsules. Our results demonstrate the need for a proper two-dimensional calibration of OCT data, and we believe that equal scaling will certainly improve the efficiency of OCT image analysis.     

Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.