Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.     

Simulating transmission and control of Taenia solium infections using a Reed-Frost stochastic model

The transmission dynamics of the human-pig zoonotic cestode Taenia solium are explored with both deterministic and stochastic versions of a modified Reed-Frost model. This model, originally developed for microparasitic infections (i.e. bacteria, viruses and protozoa), assumes that random contacts occur between hosts and that hosts can be either susceptible, infected or 'recovered and presumed immune'. Transmission between humans and pigs is modelled as susceptible roaming pigs scavenging on human faeces infected with T. solium eggs. Transmission from pigs to humans is modelled as susceptible humans eating under-cooked pork meat harbouring T. solium metacestodes. Deterministic models of each scenario were first run, followed by stochastic versions of the models to assess the likelihood of infection elimination in the small population modelled. The effects of three groups of interventions were investigated using the model: (i) interventions affecting the transmission parameters such as use of latrines, meat inspection, and cooking habits; (ii) routine interventions including rapid detection and treatment of human carriers or pig vaccination; and (iii) treatment interventions of either humans or pigs. It is concluded that mass-treatment can result in a short term dramatic reduction in prevalence, whereas interventions targeting interruption of the life cycle lead to long-term reduction in prevalence.     

Molecular Evolution of Peptide Ligands with Custom-Tailored Characteristics for Targeting of Glycostructures

As an advanced approach to identify suitable targeting molecules required for various diagnostic and therapeutic interventions, we developed a procedure to devise peptides with customizable features by an iterative computer-assisted optimization strategy. An evolutionary algorithm was utilized to breed peptides in silico and the “fitness” of peptides was determined in an appropriate laboratory in vitro assay. The influence of different evolutional parameters and mechanisms such as mutation rate, crossover probability, gaussian variation and fitness value scaling on the course of this artificial evolutional process was investigated. As a proof of concept peptidic ligands for a model target molecule, the cell surface glycolipid ganglioside G_M1, were identified. Consensus sequences describing local fitness optima were reached from diverse sets of L- and proteolytically stable D lead peptides. Ten rounds of evolutional optimization encompassing a total of just 4400 peptides lead to an increase in affinity of the peptides towards fluorescently labeled ganglioside G_M1 by a factor of 100 for L- and 400 for D-peptides.     

Comparable low-level mosaicism in affected and non affected tissue of a complex CDH patient

In this paper we present the detailed clinical and cytogenetic analysis of a prenatally detected complex Congenital Diaphragmatic Hernia (CDH) patient with a mosaic unbalanced translocation (5;12). High-resolution whole genome SNP array confirmed a low-level mosaicism (20%) in uncultured cells, underlining the value of array technology for identification studies. Subsequently, targeted Fluorescence In-Situ Hybridization in postmortem collected tissues demonstrated a similar low-level mosaicism, independently of the affected status of the tissue. Thus, a higher incidence of the genetic aberration in affected organs as lung and diaphragm cannot explain the severe phenotype of this complex CDH patient. Comparison with other described chromosome 5p and 12p anomalies indicated that half of the features presented in our patient (including the diaphragm defect) could be attributed to both chromosomal areas. In contrast, a few features such as the palpebral downslant, the broad nasal bridge, the micrognathia, microcephaly, abnormal dermatoglyphics and IUGR better fitted the 5p associated syndromes only. This study underlines the fact that low-level mosaicism can be associated with severe birth defects including CDH. The contribution of mosaicism to human diseases and specifically to congenital anomalies and spontaneous abortions becomes more and more accepted, although its phenotypic consequences are poorly described phenomena leading to counseling issues. Therefore, thorough follow-up of mosaic aberrations such as presented here is indicated in order to provide genetic counselors a more evidence based prediction of fetal prognosis in the future.     

Direct action of endocrine disrupting chemicals on human sperm

Synthetic endocrine disrupting chemicals (EDCs), omnipresent in food, household, and personal care products, have been implicated in adverse trends in human reproduction, including infertility and increasing demand for assisted reproduction. Here, we study the action of 96 ubiquitous EDCs on human sperm. We show that structurally diverse EDCs activate the sperm‐specific CatSper channel and, thereby, evoke an intracellular Ca²⁺ increase, a motility response, and acrosomal exocytosis. Moreover, EDCs desensitize sperm for physiological CatSper ligands and cooperate in low‐dose mixtures to elevate Ca²⁺ levels in sperm. We conclude that EDCs interfere with various sperm functions and, thereby, might impair human fertilization.     

B-Type Natriuretic Peptide-Induced Delayed Modulation of TRPV1 and P2X3 Receptors of Mouse Trigeminal Sensory Neurons

Important pain transducers of noxious stimuli are small- and medium-diameter sensory neurons that express transient receptor vanilloid-1 (TRPV1) channels and/or adenosine triphosphate (ATP)-gated P2X3 receptors whose activity is upregulated by endogenous neuropeptides in acute and chronic pain models. Little is known about the role of endogenous modulators in restraining the expression and function of TRPV1 and P2X3 receptors. In dorsal root ganglia, evidence supports the involvement of the natriuretic peptide system in the modulation of nociceptive transmission especially via the B-type natriuretic peptide (BNP) that activates the natriuretic peptide receptor-A (NPR-A) to downregulate sensory neuron excitability. Since the role of BNP in trigeminal ganglia (TG) is unclear, we investigated the expression of BNP in mouse TG in situ or in primary cultures and its effect on P2X3 and TRPV1 receptors of patch-clamped cultured neurons. Against scant expression of BNP, almost all neurons expressed NPR-A at membrane level. While BNP rapidly increased cGMP production and Akt kinase phosphorylation, there was no early change in passive neuronal properties or responses to capsaicin, α,β-meATP or GABA. Nonetheless, 24 h application of BNP depressed TRPV1 mediated currents (an effect blocked by the NPR-A antagonist anantin) without changing responses to α,β-meATP or GABA. Anantin alone decreased basal cGMP production and enhanced control α,β-meATP-evoked responses, implying constitutive regulation of P2X3 receptors by ambient BNP. These data suggest a slow modulatory action by BNP on TRPV1 and P2X3 receptors outlining the role of this peptide as a negative regulator of trigeminal sensory neuron excitability to nociceptive stimuli.     

First trimester screening for trisomy 21 in gestational week 8-10 by ADAM12-S as a maternal serum marker

Background  

A disintegrin and metalloprotease 12 (ADAM12-S) has previously been reported to be significantly reduced in maternal serum from women with fetal aneuploidy early in the first trimester and to significantly improve the quality of risk assessment for fetal trisomy 21 in prenatal screening. The aim of this study was to determine whether ADAM12-S is a useful serum marker for fetal trisomy 21 using the mixture model.