The glutathione biosynthetic pathway of Plasmodium is essential for mosquito transmission

1Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (γ-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that γ-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs− parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs− parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito

Transition of plasmodium sporozoites into liver stage-like forms is regulated by the RNA binding protein pumilio

Many eukaryotic developmental and cell fate decisions that are effected post-transcriptionally involve RNA binding proteins as regulators of translation of key mRNAs. In malaria parasites (Plasmodium spp.), the development of round, non-motile and replicating exo-erythrocytic liver stage forms from slender, motile and cell-cycle arrested sporozoites is believed to depend on environmental changes experienced during the transmission of the parasite from the mosquito vector to the vertebrate host. Here we identify a Plasmodium member of the RNA binding protein family PUF as a key regulator of this transformation. In the absence of Pumilio-2 (Puf2) sporozoites initiate EEF development inside mosquito salivary glands independently of the normal transmission-associated environmental cues. Puf2- sporozoites exhibit genome-wide transcriptional changes that result in loss of gliding motility, cell traversal ability and reduction in infectivity, and, moreover, trigger metamorphosis typical of early Plasmodium intra-hepatic development. These data demonstrate that Puf2 is a key player in regulating sporozoite developmental control, and imply that transformation of salivary gland-resident sporozoites into liver stage-like parasites is regulated by a post-transcriptional mechanism

Development of the piggyBac transposable system for Plasmodium berghei and its application for random mutagenesis in malaria parasites

Background: The genome of a number of species of malaria parasites ( Plasmodium spp.) has been sequenced in the hope of identifying new drug and vaccine targets. However, almost one-half of predicted Plasmodium genes are annotated as hypothetical and are difficult to analyse in bulk due to the inefficiency of current reverse genetic methodologies for Plasmodium. Recently, it has been shown that the transposase piggyBac integrates at random into the genome of the human malaria parasite P. falciparum offering the possibility to develop forward genetic screens to analyse Plasmodium gene function. This study reports the development and application of the piggyBac transposition system for the rodent malaria parasite P. berghei and the evaluation of its potential as a tool in forward genetic studies. P. berghei is the most frequently used malaria parasite model in gene function analysis since phenotype screens throughout the complete Plasmodium life cycle are possible both in vitro and in vivo. Results: We demonstrate that piggyBac based gene inactivation and promoter-trapping is both easier and more efficient in P. berghei than in the human malaria parasite, P. falciparum. Random piggyBac-mediated insertion into genes was achieved after parasites were transfected with the piggyBac donor plasmid either when transposase was expressed either from a helper plasmid or a stably integrated gene in the genome. Characterization of more than 120 insertion sites demonstrated that more than 70 most likely affect gene expression classifying their protein products as non-essential for asexual blood stage development. The non-essential nature of two of these genes was confirmed by targeted gene deletion one of which encodes P41, an ortholog of a human malaria vaccine candidate. Importantly for future development of whole genome phenotypic screens the remobilization of the piggyBac element in parasites that stably express transposase was demonstrated. Conclusion: These data demonstrate that piggyBac behaved as an efficient and random transposon in P. berghei. Remobilization of piggyBac element shows that with further development the piggyBac system can be an effective tool to generate random genome-wide mutation parasite libraries, for use in large-scale phenotype screens in vitro and in viv

Generation of transgenic rodent malaria parasites by transfection of cell culture-derived merozoites

Malaria research is greatly dependent on and has drastically advanced with the possibility of genetically modifying Plasmodium parasites. The commonly used transfection protocol by Janse and colleagues utilizes blood stage-derived Plasmodium berghei schizonts that have been purified from a blood culture by density gradient centrifugation. Naturally, this transfection protocol depends on the availability of suitably infected mice, constituting a time-based variable. In this study, the potential of transfecting liver stage-derived merozoites was explored. In cell culture, upon merozoite development, infected cells detach from the neighbouring cells and can be easily harvested from the cell culture supernatant. This protocol offers robust experimental timing and temporal flexibility. HeLa cells are infected with P. berghei sporozoites to obtain liver stage-derived merozoites, which are harvested from the cell culture supernatant and are transfected using the Amaxa Nucleofector(®) electroporation technology. Using this protocol, wild type P. berghei ANKA strain and marker-free PbmCherryHsp70-expressing P. berghei parasites were successfully transfected with DNA constructs designed for integration via single- or double-crossover homologous recombination. An alternative protocol for Plasmodium transfection is hereby provided, which uses liver stage-derived P. berghei merozoites for transfection. This protocol has the potential to substantially reduce the number of mice used per transfection, as well as to increase the temporal flexibility and robustness of performing transfections, if mosquitoes are routinely present in the laboratory. Transfection of liver stage-derived P. berghei parasites should enable generation of transgenic parasites within 8-18 days

Endocrine and Growth Abnormalities in 4H Leukodystrophy Caused by Variants in POLR3A, POLR3B, and POLR1C.

CONTEXT: 4H or POLR3-related leukodystrophy is an autosomal recessive disorder typically characterized by hypomyelination, hypodontia, and hypogonadotropic hypogonadism, caused by biallelic pathogenic variants in POLR3A, POLR3B, POLR1C, and POLR3K. The endocrine and growth abnormalities associated with this disorder have not been thoroughly investigated to date. OBJECTIVE: To systematically characterize endocrine abnormalities of patients with 4H leukodystrophy. DESIGN: An international cross-sectional study was performed on 150 patients with genetically confirmed 4H leukodystrophy between 2015 and 2016. Endocrine and growth abnormalities were evaluated, and neurological and other non-neurological features were reviewed. Potential genotype/phenotype associations were also investigated. SETTING: This was a multicenter retrospective study using information collected from 3 predominant centers. PATIENTS: A total of 150 patients with 4H leukodystrophy and pathogenic variants in POLR3A, POLR3B, or POLR1C were included. MAIN OUTCOME MEASURES: Variables used to evaluate endocrine and growth abnormalities included pubertal history, hormone levels (estradiol, testosterone, stimulated LH and FSH, stimulated GH, IGF-I, prolactin, ACTH, cortisol, TSH, and T4), and height and head circumference charts. RESULTS: The most common endocrine abnormalities were delayed puberty (57/74; 77% overall, 64% in males, 89% in females) and short stature (57/93; 61%), when evaluated according to physician assessment. Abnormal thyroid function was reported in 22% (13/59) of patients. CONCLUSIONS: Our results confirm pubertal abnormalities and short stature are the most common endocrine features seen in 4H leukodystrophy. However, we noted that endocrine abnormalities are typically underinvestigated in this patient population. A prospective study is required to formulate evidence-based recommendations for management of the endocrine manifestations of this disorder

Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as risk factors for Alzheimer’s disease

Alzheimer’s disease (AD), the leading cause of dementia, has an estimated heritability of approximately 70%1. The genetic component of AD has been mainly assessed using genome-wide association studies, which do not capture the risk contributed by rare variants2. Here, we compared the gene-based burden of rare damaging variants in exome sequencing data from 32,558 individuals—16,036 AD cases and 16,522 controls. Next to variants in TREM2, SORL1 and ABCA7, we observed a significant association of rare, predicted damaging variants in ATP8B4 and ABCA1 with AD risk, and a suggestive signal in ADAM10. Additionally, the rare-variant burden in RIN3, CLU, ZCWPW1 and ACE highlighted these genes as potential drivers of respective AD-genome-wide association study loci. Variants associated with the strongest effect on AD risk, in particular loss-of-function variants, are enriched in early-onset AD cases. Our results provide additional evidence for a major role for amyloid-β precursor protein processing, amyloid-β aggregation, lipid metabolism and microglial function in AD

Exome sequencing identifies rare damaging variants in ATP8B4 and ABCA1 as novel risk factors for Alzheimers Disease

The genetic component of Alzheimer’s disease (AD) has been mainly assessed using Genome Wide Association Studies (GWAS), which do not capture the risk contributed by rare variants. Here, we compared the gene-based burden of rare damaging variants in exome sequencing data from 32,558 individuals —16,036 AD cases and 16,522 controls— in a two-stage analysis. Next to known genes TREM2, SORL1 and ABCA7, we observed a significant association of rare, predicted damaging variants in ATP8B4 and ABCA1 with AD risk, and a suggestive signal in ADAM10. Next to these genes, the rare variant burden in RIN3, CLU, ZCWPW1 and ACE highlighted these genes as potential driver genes in AD-GWAS loci. Rare damaging variants in these genes, and in particular loss-of-function variants, have a large effect on AD-risk, and they are enriched in early onset AD cases. The newly identified AD-associated genes provide additional evidence for a major role for APP-processing, Aβ-aggregation, lipid metabolism and microglial function in AD

Functional equivalence of structurally distinct ribosomes in the malaria parasite, plasmodium berghei

Contains fulltext : 185650.pdf (Publisher’s version ) (Open Access

Delineating genetic pathways of disease progression in head and neck squamous cell carcinoma

Objective: To identify altered gene targets that characterize disease progression in squamous cell carcinoma (SCC) of the head and neck (HNSCC). Genetic alterations in HNSCC cell lines reflect the tumor in vivo and can serve as valuable tools to study the development and progression of HNSCC. Identification of key molecular events may be-useful for more accurate distinction of prognostic groups for selection and targeting of therapy. Design: Individual gene loci were analyzed for genetic alterations using a novel genomewide strategy. Subjects: Head and neck squamous cell carcinoma primary (A) and recurrent or metastatic (B) cell lines UMSCC-11A/11B, UMSCC-17A/17B (previously karyotyped), and UMSCC-81A/81B are described. Results: At the genome level, loss and gain of genetic loci concurred with tumor karyotypes. Several abnormal gene loci not apparent by cytogenetics were also identified. All except 11B indicated loss of CDKN2A (encodes p14 and p16), with concomitant loss of CDKN2B (encodes p15) in 11A, 17B, and 81A. All 6 cell lines showed gain of PIK3CA (encodes a PI3 kinase) located at 3q26.3. Conclusions: We provide evidence for the role of 3 critical pathways in the development and progression of HNSCC. The CDKN2A/B genes encode various components of the Rb and p53 pathways, and the PIK3CA gene makes a catalytic subunit of the protein phosphatidylinositol 3-OH kinase (POK), which is known to be involved in the PI3K/ATK signaling pathways. Molecular events may ultimately serve to achieve genomic alterations that set off an interplay among key gene loci along discrete genetic pathways used by tumor cells in HNSCC