Analysis of CLDN14 gene in deaf Moroccan patients with non-syndromic hearing loss

Mutations in the CLDN14 gene, encoding the tight junction claudin 14 protein has been reported to date in an autosomal recessive form of isolated hearing loss DFNB29. In order to identify the contribution of CLDN14 to inherited deafness in Moroccan population, we performed a genetic analysis of this gene in 80 Moroccan familial cases. Our results show the presence of 7 mutations: 6 being conservative and one leading to a missense mutation (C11T) which was found at heterozygous and homozygous states, with a general frequency of 6.87%. The pathogenicity of the resulting T4M substitution is under discussion.     

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC₅₀ < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible.     

Toxin-based in-vitro selection and its potential application to date palm for resistance to the bayoud Fusarium wilt

Date palm (Phoenix dactylifera L.) is qualified as a 'tree' of great ecological and socio-economical importance in desert oases. Unfortunately, it is being decimated, especially in Morocco and Algeria, by a fusariosis wilt called bayoud and caused by Fusarium oxysporum f. sp. albedinis (Fao). Controlling this disease requires the implementation of an integrated management program. Breeding for resistance is one of the most promising component strategies of this program. Few naturally resistant cultivars with a mediocre fruit quality (dates) are known. Conventional and non-conventional methods are under development and have to use the simplest and easiest methods to screen for resistant individuals. The use of pathogen toxins as selective agents at the tissue culture step might be a source of variability that can lead to the selection of individuals with suitable levels of resistance to the toxin and/or to the pathogen among the genetic material available. Foa produces toxins such as fusaric, succinic, 3-phenyl lactic acids and their derivatives, marasmins and peptidic toxins. These toxins can be used bulked or separately as selective agents. The aim of this contribution was to give a brief overview on toxins and their use as a mean to select resistant lines and to initiate a discussion about the potential use of this approach for the date palm-Foa pathosystem. This review does not pretend to be comprehensive or exhaustive and was prepared mainly to highlight the potential use of Foa toxins for selecting date palm individuals with a suitable resistance level to bayoud using toxin-based selective media.     

Association of spermatogenic failure with the b2/b3 partial AZFc deletion

Infertility affects around 1 in 10 men and in most cases the cause is unknown. The Y chromosome plays an important role in spermatogenesis and specific deletions of this chromosome, the AZF deletions, are associated with spermatogenic failure. Recently partial AZF deletions have been described but their association with spermatogenic failure is unclear. Here we screened a total of 339 men with idiopathic spermatogenic failure, and 256 normozoospermic ancestry-matched men for chromosome microdeletions including AZFa, AZFb, AZFc, and the AZFc partial deletions (gr/gr, b1/b3 and b2/b3).AZFa and AZFc deletions were identified in men with severe spermatogenic failure at similar frequencies to those reported elsewhere. Gr/gr deletions were identified in case and control populations at 5.83% and 6.25% respectively suggesting that these deletions are not associated with spermatogenic failure. However, b2/b3 deletions were detected only in men with spermatogenic failure and not in the normospermic individuals. Combined with our previous data this shows an association of the b2/b3 deletion (p = 0.0318) with spermatogenic failure in some populations. We recommend screening for this deletion in men with unexplained spermatogenic failure.     

Using a Genetically Encoded Sensor to Identify Inhibitors of Toxoplasma gondii Ca2+ Signaling

The life cycles of apicomplexan parasites progress in accordance with fluxes in cytosolic Ca²⁺. Such fluxes are necessary for events like motility and egress from host cells. We used genetically encoded Ca²⁺ indicators (GCaMPs) to develop a cell-based phenotypic screen for compounds that modulate Ca²⁺ signaling in the model apicomplexan Toxoplasma gondii. In doing so, we took advantage of the phosphodiesterase inhibitor zaprinast, which we show acts in part through cGMP-dependent protein kinase (protein kinase G; PKG) to raise levels of cytosolic Ca²⁺. We define the pool of Ca²⁺ regulated by PKG to be a neutral store distinct from the endoplasmic reticulum. Screening a library of 823 ATP mimetics, we identify both inhibitors and enhancers of Ca²⁺ signaling. Two such compounds constitute novel PKG inhibitors and prevent zaprinast from increasing cytosolic Ca²⁺. The enhancers identified are capable of releasing intracellular Ca²⁺ stores independently of zaprinast or PKG. One of these enhancers blocks parasite egress and invasion and shows strong antiparasitic activity against T. gondii. The same compound inhibits invasion of the most lethal malaria parasite, Plasmodium falciparum. Inhibition of Ca²⁺-related phenotypes in these two apicomplexan parasites suggests that depletion of intracellular Ca²⁺ stores by the enhancer may be an effective antiparasitic strategy. These results establish a powerful new strategy for identifying compounds that modulate the essential parasite signaling pathways regulated by Ca²⁺, underscoring the importance of these pathways and the therapeutic potential of their inhibition.     

Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase

The Escherichia coli inducible lysine decarboxylase, LdcI/CadA, together with the inner-membrane lysine-cadaverine antiporter, CadB, provide cells with protection against mild acidic conditions (pH～5). To gain a better understanding of the molecular processes underlying the acid stress response, the X-ray crystal structure of LdcI was determined. The structure revealed that the protein is an oligomer of five dimers that associate to form a decamer. Surprisingly, LdcI was found to co-crystallize with the stringent response effector molecule ppGpp, also known as the alarmone, with 10 ppGpp molecules in the decamer. ppGpp is known to mediate the stringent response, which occurs in response to nutrient deprivation. The alarmone strongly inhibited LdcI enzymatic activity. This inhibition is important for modulating the consumption of lysine in cells during acid stress under nutrient limiting conditions. Hence, our data provide direct evidence for a link between the bacterial acid stress and stringent responses.     

The Clp chaperones and proteases of the human malaria parasite Plasmodium falciparum

The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.     

Molecular analysis of the TMPRSS3 gene in Moroccan families with non-syndromic hearing loss

Autosomal recessive non-syndromic hearing impairment (ARNSHI) is the most common type of inherited hearing impairment, accounting for approximately 80% of inherited prelingual hearing impairment. Hearing loss is noted to be both phenotypically and genetically heterogeneous. Mutations in the TMPRSS3 gene, which encodes a transmembrane serine protease, are known to cause autosomal recessive non-syndromic hearing impairment DFNB8/10. In order to elucidate if the TMPRSS3 gene is responsible for ARNSHI in 80 Moroccan families with non-syndromic hearing impairment, the gene was sequenced using DNA samples from these families. Nineteen TMPRSS3 variants were found, nine are located in the exons among which six are missense and three are synonymous. The 10 remaining variations are located in non-coding regions. Missense variants analysis show that they do not have a significant pathogenic effect on protein while pathogenicity of some variant remains under discussion. Thus we show that the TMPRSS3 gene is not a major contributor to non-syndromic deafness in the Moroccan population.