NRL^{-/-} gene edited human embryonic stem cells generate rod-deficient retinal organoids enriched in S-cone-like photoreceptors

Organoid cultures represent a unique tool to investigate the developmental complexity of tissues like the human retina. NRL is a transcription factor required for the specification and homeostasis of mammalian rod photoreceptors. In Nrl‐deficient mice, photoreceptor precursor cells do not differentiate into rods, and instead follow a default photoreceptor specification pathway to generate S‐cone‐like cells. To investigate whether this genetic switch mechanism is conserved in humans, we used CRISPR/Cas9 gene editing to engineer an NRL‐deficient embryonic stem cell (ESC) line (NRL^{−/−}), and differentiated it into retinal organoids. Retinal organoids self‐organize and resemble embryonic optic vesicles (OVs) that recapitulate the natural histogenesis of rods and cone photoreceptors. NRL−/− OVs develop comparably to controls, and exhibit a laminated, organized retinal structure with markers of photoreceptor synaptogenesis. Using immunohistochemistry and quantitative polymerase chain reaction (qPCR), we observed that NRL^{−/−} OVs do not express NRL, or other rod photoreceptor markers directly or indirectly regulated by NRL. On the contrary, they show an abnormal number of photoreceptors positive for S‐OPSIN, which define a primordial subtype of cone, and overexpress other cone genes indicating a conserved molecular switch in mammals. This study represents the first evidence in a human in vitro ESC‐derived organoid system that NRL is required to define rod identity, and that in its absence S‐cone‐like cells develop as the default photoreceptor cell type. It shows how gene edited retinal organoids provide a useful system to investigate human photoreceptor specification, relevant for efforts to generate cells for transplantation in retinal degenerative diseases

Plasmodium berghei Circumvents Immune Responses Induced by Merozoite Surface Protein 1- and Apical Membrane Antigen 1-Based Vaccines

BACKGROUND: Two current leading malaria blood-stage vaccine candidate antigens for Plasmodium falciparum, the C-terminal region of merozoite surface protein 1 (MSP1(19)) and apical membrane antigen 1 (AMA1), have been prioritized because of outstanding protective efficacies achieved in a rodent malaria Plasmodium yoelii model. However, P. falciparum vaccines based on these antigens have had disappointing outcomes in clinical trials. Discrepancies in the vaccine efficacies observed between the P. yoelii model and human clinical trials still remain problematic. METHODOLOGY AND RESULTS: In this study, we assessed the protective efficacies of a series of MSP1(19)- and AMA1-based vaccines using the P. berghei rodent malarial parasite and its transgenic models. Immunization of mice with a baculoviral-based vaccine (BBV) expressing P. falciparum MSP1(19) induced high titers of PfMSP1(19)-specific antibodies that strongly reacted with P. falciparum blood-stage parasites. However, no protection was achieved following lethal challenge with transgenic P. berghei expressing PfMSP1(19) in place of native PbMSP1(19). Similarly, neither P. berghei MSP1(19)- nor AMA1-BBV was effective against P. berghei. In contrast, immunization with P. yoelii MSP1(19)- and AMA1-BBVs provided 100% and 40% protection, respectively, against P. yoelii lethal challenge. Mice that naturally acquired sterile immunity against P. berghei became cross-resistant to P. yoelii, but not vice versa. CONCLUSION: This is the first study to address blood-stage vaccine efficacies using both P. berghei and P. yoelii models at the same time. P. berghei completely circumvents immune responses induced by MSP1(19)- and AMA1-based vaccines, suggesting that P. berghei possesses additional molecules and/or mechanisms that circumvent the host's immune responses to MSP1(19) and AMA1, which are lacking in P. yoelii. Although it is not known whether P. falciparum shares these escape mechanisms with P. berghei, P. berghei and its transgenic models may have potential as useful tools for identifying and evaluating new blood-stage vaccine candidate antigens for P. falciparum

Study of the reaction e^{+}e^{-} -->J/psi\pi^{+}\pi^{-} via initial-state radiation at BaBar

We study the process $e^+e^-\to J/\psi\pi^{+}\pi^{-}$ with initial-state-radiation events produced at the PEP-II asymmetric-energy collider. The data were recorded with the BaBar detector at center-of-mass energies 10.58 and 10.54 GeV, and correspond to an integrated luminosity of 454 $\mathrm{fb^{-1}}$. We investigate the $J/\psi \pi^{+}\pi^{-}$ mass distribution in the region from 3.5 to 5.5 $\mathrm{GeV/c^{2}}$. Below 3.7 $\mathrm{GeV/c^{2}}$ the $\psi(2S)$ signal dominates, and above 4 $\mathrm{GeV/c^{2}}$ there is a significant peak due to the Y(4260). A fit to the data in the range 3.74 -- 5.50 $\mathrm{GeV/c^{2}}$ yields a mass value $4244 \pm 5$ (stat) $\pm 4$ (syst)$\mathrm{MeV/c^{2}}$ and a width value $114 ^{+16}_{-15}$ (stat)$\pm 7$(syst)$\mathrm{MeV}$ for this state. We do not confirm the report from the Belle collaboration of a broad structure at 4.01 $\mathrm{GeV/c^{2}}$. In addition, we investigate the $\pi^{+}\pi^{-}$ system which results from Y(4260) decay

Construction of Transgenic Plasmodium berghei as a Model for Evaluation of Blood-Stage Vaccine Candidate of Plasmodium falciparum Chimeric Protein 2.9

BACKGROUND:The function of the 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1-19) expressed by Plasmodium has been demonstrated to be conserved across distantly related Plasmodium species. The green fluorescent protein (GFP) is a reporter protein that has been widely used because it can be easily detected in living organisms by fluorescence microscopy and flow cytometry. METHODOLOGY AND RESULTS:In this study, we used gene targeting to generate transgenic P. berghei (Pb) parasites (designated as PfMSP1-19Pb) that express the MSP1-19 of P. falciparum (Pf) and the GFP reporter protein simultaneously. The replacement of the PbMSP1-19 locus by PfMSP1-19 was verified by PCR and Southern analysis. The expression of the chimeric PbfMSP-1 and the GFP was verified by Western blot and fluorescence microscopy, respectively. Moreover, GFP-expressing transgenic parasites in blood stages can be readily differentiated from other blood cells using flow cytometry. A comparison of growth rates between wild-type and the PfMSP1-19Pb transgenic parasite indicated that the replacement of the MSP1-19 region and the expression of the GFP protein were not deleterious to the transgenic parasites. We used this transgenic mouse parasite as a murine model to evaluate the protective efficacy in vivo of specific IgG elicited by a PfCP-2.9 malaria vaccine that contains the PfMSP1-19. The BALB/c mice passively transferred with purified rabbit IgG to the PfCP-2.9 survived a lethal challenge of the PfMSP1-19Pb transgenic murine parasites, but not the wild-type P. berghei whereas the control mice passively transferred with purified IgG obtained from adjuvant only-immunized rabbits were vulnerable to both transgenic and wild-type infections. CONCLUSIONS:We generated a transgenic P. berghei line that expresses PfMSP1-19 and the GFP reporter gene simultaneously. The availability of this parasite line provides a murine model to evaluate the protective efficacy in vivo of anti-MSP1-19 antibodies, including, potentially, those elicited by the PfCP-2.9 malaria vaccine in human volunteers

Measuring substance use in the club setting: a feasibility study using biochemical markers

<p>Abstract</p> <p>Background</p> <p>During the last few decades the use of club drugs (e.g., cocaine, amphetamines) has been of increased concern in nightlife settings. Traditionally, surveys have been used to estimate the use of club drugs, however, they mostly rely on self-reports which may not be accurate. Recent advances have allowed for readily accessible drug testing methods such as oral fluid drug testing. Nevertheless, research using oral fluid sampling to measure the frequency of drug use in the club environment is scarce. The objective of this study is to evaluate the feasibility of measuring the frequency of alcohol and drug use among Swedish clubbers using breath alcohol and oral fluid drug testing.</p> <p>Method</p> <p>The setting was a 40 hour electronic music dance event (EMDE) on a cruise ship on the Baltic Sea, departing from Sweden, with 875 passengers. Groups of participants at the EMDE were randomly invited to participate. Data were collected with face-to-face and self-administered questionnaires. Further, oral fluid samples were collected to determine illicit drug use, and blood alcohol concentration (BAC) levels were measured using a breath analyzer.</p> <p>Results</p> <p>A total of 422 passengers were asked to participate in the study whereof 21 declined (5.0% refusal rate). Of the 401 study participants (accounting for 45.8% of all attendees), 5 declined oral fluid drug testing. Results show that there was a discrepancy between self-reported and actual drug use as 10.1% of the participants were positive on illicit drug use (amphetamines, ecstasy/MDMA, cannabis, cocaine), while only 3.7% of the participants reported drug use during the last 48 hours. The average BAC level was 0.10% and 23.7% had BAC levels ≥ 0.15%, while 5.9% had levels below the detection limit. The mean BAC levels for the illicit drug users were significantly higher (<it>p </it>= 0.004) than for non-drug users (0.13% vs. 0.10%). Self-reported AUDIT-C scores (using a threshold of ≥ 5 for men and ≥ 4 for women) revealed that 76.0% of the men and 80.7% of the women had risky alcohol consumption patterns.</p> <p>Conclusion</p> <p>This study indicates that it is feasible to conduct breath alcohol and oral fluid drug testing in a Swedish club setting.</p

MetaPIGA v2.0: maximum likelihood large phylogeny estimation using the metapopulation genetic algorithm and other stochastic heuristics

<p>Abstract</p> <p>Background</p> <p>The development, in the last decade, of stochastic heuristics implemented in robust application softwares has made large phylogeny inference a key step in most comparative studies involving molecular sequences. Still, the choice of a phylogeny inference software is often dictated by a combination of parameters not related to the raw performance of the implemented algorithm(s) but rather by practical issues such as ergonomics and/or the availability of specific functionalities.</p> <p>Results</p> <p>Here, we present MetaPIGA v2.0, a robust implementation of several stochastic heuristics for large phylogeny inference (under maximum likelihood), including a Simulated Annealing algorithm, a classical Genetic Algorithm, and the Metapopulation Genetic Algorithm (metaGA) together with complex substitution models, discrete Gamma rate heterogeneity, and the possibility to partition data. MetaPIGA v2.0 also implements the Likelihood Ratio Test, the Akaike Information Criterion, and the Bayesian Information Criterion for automated selection of substitution models that best fit the data. Heuristics and substitution models are highly customizable through manual batch files and command line processing. However, MetaPIGA v2.0 also offers an extensive graphical user interface for parameters setting, generating and running batch files, following run progress, and manipulating result trees. MetaPIGA v2.0 uses standard formats for data sets and trees, is platform independent, runs in 32 and 64-bits systems, and takes advantage of multiprocessor and multicore computers.</p> <p>Conclusions</p> <p>The metaGA resolves the major problem inherent to classical Genetic Algorithms by maintaining high inter-population variation even under strong intra-population selection. Implementation of the metaGA together with additional stochastic heuristics into a single software will allow rigorous optimization of each heuristic as well as a meaningful comparison of performances among these algorithms. MetaPIGA v2.0 gives access both to high customization for the phylogeneticist, as well as to an ergonomic interface and functionalities assisting the non-specialist for sound inference of large phylogenetic trees using nucleotide sequences. MetaPIGA v2.0 and its extensive user-manual are freely available to academics at <url>http://www.metapiga.org</url>.</p

An EGF-like Protein Forms a Complex with PfRh5 and Is Required for Invasion of Human Erythrocytes by Plasmodium falciparum

Invasion of erythrocytes by Plasmodium falciparum involves a complex cascade of protein-protein interactions between parasite ligands and host receptors. The reticulocyte binding-like homologue (PfRh) protein family is involved in binding to and initiating entry of the invasive merozoite into erythrocytes. An important member of this family is PfRh5. Using ion-exchange chromatography, immunoprecipitation and mass spectroscopy, we have identified a novel cysteine-rich protein we have called P. falciparum Rh5 interacting protein (PfRipr) (PFC1045c), which forms a complex with PfRh5 in merozoites. Mature PfRipr has a molecular weight of 123 kDa with 10 epidermal growth factor-like domains and 87 cysteine residues distributed along the protein. In mature schizont stages this protein is processed into two polypeptides that associate and form a complex with PfRh5. The PfRipr protein localises to the apical end of the merozoites in micronemes whilst PfRh5 is contained within rhoptries and both are released during invasion when they form a complex that is shed into the culture supernatant. Antibodies to PfRipr1 potently inhibit merozoite attachment and invasion into human red blood cells consistent with this complex playing an essential role in this process