A Zebrafish Drug-Repurposing Screen Reveals sGC-Dependent and sGC-Independent Pro-Inflammatory Activities of Nitric Oxide

Tissue injury and infection trigger innate immune responses. However, dysregulation may result in chronic inflammation and is commonly treated with corticosteroids and non-steroidal anti-inflammatory drugs. Unfortunately, long-term administration of both therapeutic classes can cause unwanted side effects. To identify alternative immune-modulatory compounds we have previously established a novel screening method using zebrafish larvae. Using this method we here present results of an in vivo high-content drug-repurposing screen, identifying 63 potent anti-inflammatory drugs that are in clinical use for other indications. Our approach reveals a novel pro-inflammatory role of nitric oxide. Nitric oxide affects leukocyte recruitment upon peripheral sensory nervous system or epithelial injury in zebrafish larvae both via soluble guanylate cyclase and in a soluble guanylate cyclase -independent manner through protein S-nitrosylation. Together, we show that our screening method can help to identify novel immune-modulatory activities and provide new mechanistic insights into the regulation of inflammatory processes.     

Oseltamivir Prophylaxis Reduces Inflammation and Facilitates Establishment of Cross-Strain Protective T Cell Memory to Influenza Viruses

CD8⁺ T cells directed against conserved viral regions elicit broad immunity against distinct influenza viruses, promote rapid virus elimination and enhanced host recovery. The influenza neuraminidase inhibitor, oseltamivir, is prescribed for therapy and prophylaxis, although it remains unclear how the drug impacts disease severity and establishment of effector and memory CD8⁺ T cell immunity. We dissected the effects of oseltamivir on viral replication, inflammation, acute CD8⁺ T cell responses and the establishment of immunological CD8⁺ T cell memory. In mice, ferrets and humans, the effect of osteltamivir on viral titre was relatively modest. However, prophylactic oseltamivir treatment in mice markedly reduced morbidity, innate responses, inflammation and, ultimately, the magnitude of effector CD8⁺ T cell responses. Importantly, functional memory CD8⁺ T cells established during the drug-reduced effector phase were capable of mounting robust recall responses. Moreover, influenza-specific memory CD4⁺ T cells could be also recalled after the secondary challenge, while the antibody levels were unaffected. This provides evidence that long-term memory T cells can be generated during an oseltamivir-interrupted infection. The anti-inflammatory effect of oseltamivir was verified in H1N1-infected patients. Thus, in the case of an unpredicted influenza pandemic, while prophylactic oseltamivir treatment can reduce disease severity, the capacity to generate memory CD8⁺ T cells specific for the newly emerged virus is uncompromised. This could prove especially important for any new influenza pandemic which often occurs in separate waves.     

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.     

What Do Pneumocystis Organisms Tell Us about the Phylogeography of Their Hosts? The Case of the Woodmouse Apodemus sylvaticus in Continental Europe and Western Mediterranean Islands

Pneumocystis fungi represent a highly diversified biological group with numerous species, which display a strong host-specificity suggesting a long co-speciation process. In the present study, the presence and genetic diversity of Pneumocystis organisms was investigated in 203 lung samples from woodmice (Apodemus sylvaticus) collected on western continental Europe and Mediterranean islands. The presence of Pneumocystis DNA was assessed by nested PCR at both large and small mitochondrial subunit (mtLSU and mtSSU) rRNA loci. Direct sequencing of nested PCR products demonstrated a very high variability among woodmouse-derived Pneumocystis organisms with a total number of 30 distinct combined mtLSU and mtSSU sequence types. However, the genetic divergence among these sequence types was very low (up to 3.87%) and the presence of several Pneumocystis species within Apodemus sylvaticus was considered unlikely. The analysis of the genetic structure of woodmouse-derived Pneumocystis revealed two distinct groups. The first one comprised Pneumocystis from woodmice collected in continental Spain, France and Balearic islands. The second one included Pneumocystis from woodmice collected in continental Italy, Corsica and Sicily. These two genetic groups were in accordance with the two lineages currently described within the host species Apodemus sylvaticus. Pneumocystis organisms are emerging as powerful tools for phylogeographic studies in mammals.     

Inequalities of visceral leishmaniasis case-fatality in Brazil: A multilevel modeling considering space,time, individual and contextual factors

BackgroundIn Brazil, case-fatality from visceral leishmaniasis (VL) is high and characterized by wide differences between the various political-economic units, the federated units (FUs). This study was designed to investigate the association between factors at the both FU and individual levels with the risk of dying from VL, after analysing the temporal trend and the spatial dependency for VL case-fatality.MethodologyThe analysis was based on individual and aggregated data of the Reportable Disease Information System-SINAN (Brazilian Ministry of Health). The temporal and spatial distributions of the VL case-fatality between 2007 and 2017 (27 FUs as unit of analysis) were considered together with the individual characteristics and many other variables at the FU level (socioeconomic, demographic, access to health and epidemiological indicators) in a mixed effects models or multilevel modeling, assuming a binomial outcome distribution (death from VL).FindingsA linear increasing temporal tendency (4%/year) for VL case-fatality was observed between 2007 and 2017. There was no similarity between the case-fatality rates of neighboring FUs (non-significant spatial term), although these rates were heterogeneous in this spatial scale of analysis. In addition to the known individual risk factors age, female gender, disease’s severity, bacterial co-infection and disease duration, low level schooling and unavailability of emergency beds and health professionals (the last two only in univariate analysis) were identified as possibly related to VL death risk. Lower VL incidence was also associated to VL case-fatality, suggesting that unfamiliarity with the disease may delay appropriate medical management: VL patients with fatal outcome were notified and had VL treatment started 6 and 3 days later, respectively, in relation to VL cured patients. Access to garbage collection, marker of social and economic development, seems to be protective against the risk of dying from VL. Part of the observed VL case-fatality variability in Brazil could not be explained by the studied variables, suggesting that factors linked to the intra FU environment may be involved.ConclusionsThis study aimed to identify epidemiological conditions and others related to access to the health system possibly linked to VL case-fatality, pointing out new prognostic determinants subject to intervention.     

Mutations in CSPP1 Cause Primary Cilia Abnormalities and Joubert Syndrome with or without Jeune Asphyxiating Thoracic Dystrophy

Joubert syndrome (JBTS) is a recessive ciliopathy in which a subset of affected individuals also have the skeletal dysplasia Jeune asphyxiating thoracic dystrophy (JATD). Here, we have identified biallelic truncating CSPP1 (centrosome and spindle pole associated protein 1) mutations in 19 JBTS-affected individuals, four of whom also have features of JATD. CSPP1 mutations explain ∼5% of JBTS in our cohort, and despite truncating mutations in all affected individuals, the range of phenotypic severity is broad. Morpholino knockdown of cspp1 in zebrafish caused phenotypes reported in other zebrafish models of JBTS (curved body shape, pronephric cysts, and cerebellar abnormalities) and reduced ciliary localization of Arl13b, further supporting loss of CSPP1 function as a cause of JBTS. Fibroblasts from affected individuals with CSPP1 mutations showed reduced numbers of primary cilia and/or short primary cilia, as well as reduced axonemal localization of ciliary proteins ARL13B and adenylyl cyclase III. In summary, CSPP1 mutations are a major cause of the Joubert-Jeune phenotype in humans; however, the mechanism by which these mutations lead to both JBTS and JATD remains unknown.     

Functional implications of squamosal suture size in paranthropus boisei

It has been hypothesized that the extensively overlapping temporal and parietal bones of the squamosal sutures in Paranthropus boisei are adaptations for withstanding loads associated with feeding. Finite element analysis (FEA) was used to investigate the biomechanical effects of suture size (i.e., the area of overlap between the temporal and parietal bones) on stress, strain energy, and strain ratio in the squamosal sutures of Pan troglodytes and P. boisei (specimen OH 5) during biting. Finite element models (FEMs) of OH 5 and a P. troglodytes cranium were constructed from CT scans. These models contain sutures that approximate the actual suture sizes preserved in both crania. The FEM of Pan was then modified to create two additional FEMs with squamosal sutures that are 50% smaller and 25% larger than those in the original model. Comparisons among the models test the effect of suture size on the structural integrity of the squamosal suture as the temporal squama and parietal bone move relative to each other during simulated premolar biting. Results indicate that with increasing suture size there is a decreased risk of suture failure, and that maximum stress values in the OH 5 suture were favorable compared to values in the Pan model with the normal suture size. Strain ratios suggest that shear is an important strain regime in the squamosal suture. This study is consistent with the hypothesis that larger sutures help reduce the likelihood of suture failure under high biting loads. Am J Phys Anthropol 153:260–268, 2014. © 2013 Wiley Periodicals, Inc.     

Intraskeletal isotopic compositions (δ13C, δ15N) of bone collagen: Nonpathological and pathological variation

Paleodiet research traditionally interprets differences in collagen isotopic compositions (δ¹³C, δ¹⁵N) as indicators of dietary distinction even though physiological processes likely play some role in creating variation. This research investigates the degree to which bone collagen δ¹³C and δ¹⁵N values normally vary within the skeleton and examines the influence of several diseases common to ancient populations on these isotopic compositions. The samples derive from two medieval German cemeteries and one Swiss reference collection and include examples of metabolic disease (rickets/osteomalacia), degenerative joint disease (osteoarthritis), trauma (fracture), infection (osteomyelitis), and inflammation (periostitis). A separate subset of visibly nonpathological skeletal elements from the German collections established normal intraindividual variation. For each disease type, tests compared bone lesion samples to those near and distant to the lesions sites. Results show that normal (nonpathological) skeletons exhibit limited intraskeletal variation in carbon‐ and nitrogen‐isotope ratios, suggesting that sampling of distinct elements is appropriate for paleodiet studies. In contrast, individuals with osteomyelitis, healed fractures, and osteoarthritis exhibit significant intraskeletal differences in isotope values, depending on whether one is comparing lesions to near or to distant sites. Skeletons with periostitis result in significant intraskeletal differences in nitrogen isotope values only, while those with rickets/osteomalacia do not exhibit significant intraskeletal differences. Based on these results, we suggest that paleodiet researchers avoid sampling collagen at or close to lesion sites because the isotope values may be reflecting both altered metabolic processes and differences in diet relative to others in the population. Am J Phys Anthropol 153:598–604, 2014. © 2013 Wiley Periodicals, Inc.     

Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer

A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP–HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β₄ can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI–MS (electrospray ionization–mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β₄ did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β₄ as well as of other proteins and peptides.