Loss of TDP-43 in male germ cells causes meiotic failure and impairs fertility in mice

Meiotic arrest is a common cause of human male infertility, but the causes of this arrest are poorly understood. Transactive response DNA-binding protein of 43 kDa (TDP-43) is highly expressed in spermatocytes in the preleptotene and pachytene stages of meiosis. TDP-43 is linked to several human neurodegenerative disorders wherein its nuclear clearance accompanied by cytoplasmic aggregates underlies neurodegeneration. Exploring the functional requirement for TDP-43 for spermatogenesis for the first time, we show here that conditional KO (cKO) of the Tardbp gene (encoding TDP-43) in male germ cells of mice leads to reduced testis size, depletion of germ cells, vacuole formation within the seminiferous epithelium, and reduced sperm production. Fertility trials also indicated severe subfertility. Spermatocytes of cKO mice showed failure to complete prophase I of meiosis with arrest at the midpachytene stage. Staining of synaptonemal complex protein 3 and γH2AX, markers of the meiotic synaptonemal complex and DNA damage, respectively, and super illumination microscopy revealed nonhomologous pairing and synapsis defects. Quantitative RT–PCR showed reduction in the expression of genes critical for prophase I of meiosis, including Spo11 (initiator of meiotic double-stranded breaks), Rec8 (meiotic recombination protein), and Rad21L (RAD21-like, cohesin complex component), as well as those involved in the retinoic acid pathway critical for entry into meiosis. RNA-Seq showed 1036 upregulated and 1638 downregulated genes (false discovery rate <0.05) in the Tardbp cKO testis, impacting meiosis pathways. Our work reveals a crucial role for TDP-43 in male meiosis and suggests that some forms of meiotic arrest seen in infertile men may result from the loss of function of TDP-43.     

Fungemia por Candida lipolytica: a propósito de 2 casos

Candida lipolytica has rarely been reported as a human pathogen. We observed two cases of fungemia caused by C. lipolytica, one of them in a 12-year-old child with cystic pancreatic fibrosis in advanced phase and another in a 86-year-old woman who presented vesical neoformation with peritoneal fibrosis, bilateral hydronephrosis and recurrent urinary tract infections. After antifungal treatment and catheter removal, the fungemia appeared to be finished and blood cultures were negative.     

Isolation and characterization of a strain of Lichtheimia corymbifera (ex Absidia corymbifera) from a case of bovine abortion

Background  

Lichtheimia corymbifera (previously Absidia corymbifera) is a filamentous zygomycetes belonging to the order Mucorales and to the family Lichtheimiaceae. Members of genus Lichtheimia spp. are cosmopolitan and ubiquitous in nature. Lichtheimia corymbifera is a recognized agent of diseases in man and animals. In cattle it causes abortion and mastitis. Three cases of bovine abortion occurred in a herd located in the Po Valley. Serological examinations were performed on fetal and mother's blood. One of the aborted fetus was referred to our laboratory. The paper describes the isolation and characterization of Lichtheimia corymbifera from a bovine aborted fetus.     

Cre-loxP–mediated Inactivation of the α6A Integrin Splice Variant In Vivo: Evidence for a Specific Functional Role of α6A in Lymphocyte Migration but Not in Heart Development

Two splice variants of the α6 integrin subunit, α6A and α6B, with different cytoplasmic domains, have previously been described. While α6B is expressed throughout the development of the mouse, the expression of α6A begins at 8.5 days post coitum and is initially restricted to the myocardium. Later in ontogeny, α6A is found in various epithelia and in certain cells of the immune system. In this study, we have investigated the function of α6A in vivo by generating knockout mice deficient for this splice variant. The Cre- loxP system of the bacteriophage P1 was used to specifically remove the exon encoding the cytoplasmic domain of α6A in embryonic stem cells, and the deletion resulted in the expression of α6B in all tissues that normally express α6A. We show that α6A−/− mice develop normally and are fertile. The substitution of α6A by α6B does not impair the development and function of the heart, hemidesmosome formation in the epidermis, or keratinocyte migration. Furthermore, T cells differentiated normally in α6A−/− mice. However, the substitution of α6A by α6B leads to a decrease in the migration of lymphocytes through laminin-coated Transwell filters and to a reduction of the number of T cells isolated from the peripheral and mesenteric lymph nodes. Lymphocyte homing to the lymph nodes, which involves various types of integrin–ligand interactions, was not affected in the α6A knockout mice, indicating that the reduced number of lymph node cells could not be directly attributed to defects in lymphocyte trafficking. Nevertheless, the expression of α6A might be necessary for optimal lymphocyte migration on laminin in certain pathological conditions.     

The Saharan isolate Saccharothrix algeriensis NRRL B-24137 induces systemic resistance in Arabidopsis thaliana seedlings against Botrytis cinerea

Background and aim

Saccharothrix algeriensis NRRL B-24137, isolated from a Saharan soil, has been described as a potential biocontrol agent against Botrytis cinerea and other phytopathogens. However, the plant protection mechanisms involved still need to be described. The aim of this study was to determine this protection phenomenon as well as parts of the mechanisms involved, using Arabidopsis thaliana seedlings and B. cinerea.

Methods

The bacterial colonization process was evaluated on A. thaliana seedlings using fluorescence in situ hybridization. Protection of A. thaliana seedlings inoculated with NRRL B-24137 against B. cinerea was then evaluated. Parts of the mechanisms involved in the systemic protection against B. cinerea were evaluated using known mutants of genes involved in jasmonate (JA)/ethylene (ET)/salicylic acid (SA) signaling. Other Arabidopsis mutants, AtrhbohD-3, AtrhbohF-3, and ups1-1 were also screened to determine other parts of the mechanisms involved.

Results

The results showed that the strain NRRL B-24137 colonized, epi- and endophytically, the roots of Arabidopsis seedlings but the strain was not a systemic colonizer during the time of the experiment. The strain NRRL B-24137 also reduced B. cinerea symptoms and the protection was linked to known mechanisms of induced systemic resistance (ISR; JA/ET signaling), as well as to functionality of AtrbohF oxidase and of UPS1. Crosstalk between ET/JA and SA signaling could also be involved.

Conclusions

The isolate NRRL B-24137, after colonizing the root systems of A. thaliana, induces an ISR against B. cinerea, which is JA/ET dependent, but could also require SA crosstalk and protection could also require NAPDH oxidases and UPS1 functionalities.     

Grapevine MATE-Type Proteins Act as Vacuolar H+-Dependent Acylated Anthocyanin Transporters

In grapevine (Vitis vinifera), anthocyanins are responsible for most of the red, blue, and purple pigmentation found in the skin of berries. In cells, anthocyanins are synthesized in the cytoplasm and accumulated into the vacuole. However, little is known about the transport of these compounds through the tonoplast. Recently, the sequencing of the grapevine genome allowed us to identify genes encoding proteins with high sequence similarity to the Multidrug And Toxic Extrusion (MATE) family. Among them, we selected two genes as anthocyanin transporter candidates and named them anthoMATE1 (AM1) and AM3. The expression of both genes was mainly fruit specific and concomitant with the accumulation of anthocyanin pigment. Subcellular localization assays in grapevine hairy roots stably transformed with AM1 or AM3green fluorescent protein fusion protein revealed that AM1 and AM3 are primarily localized to the tonoplast. Yeast vesicles expressing anthoMATEs transported acylated anthocyanins in the presence of MgATP. Inhibitor studies demonstrated that AM1 and AM3 proteins act in vitro as vacuolar H⁺-dependent acylated anthocyanin transporters. By contrast, under our experimental conditions, anthoMATEs could not transport malvidin 3-O-glucoside or cyanidin 3-O-glucoside, suggesting that the acyl conjugation was essential for the uptake. Taken together, these results provide evidence that in vitro the two grapevine AM1 and AM3 proteins mediate specifically acylated anthocyanin transport.     

The Genetic Basis of Laboratory Adaptation in Caulobacter crescentus

The dimorphic bacterium Caulobacter crescentus has evolved marked phenotypic changes during its 50-year history of culture in the laboratory environment, providing an excellent system for the study of natural selection and phenotypic microevolution in prokaryotes. Combining whole-genome sequencing with classical molecular genetic tools, we have comprehensively mapped a set of polymorphisms underlying multiple derived phenotypes, several of which arose independently in separate strain lineages. The genetic basis of phenotypic differences in growth rate, mucoidy, adhesion, sedimentation, phage susceptibility, and stationary-phase survival between C. crescentus strain CB15 and its derivative NA1000 is determined by coding, regulatory, and insertion/deletion polymorphisms at five chromosomal loci. This study evidences multiple genetic mechanisms of bacterial evolution as driven by selection for growth and survival in a new selective environment and identifies a common polymorphic locus, zwf, between lab-adapted C. crescentus and clinical isolates of Pseudomonas aeruginosa that have adapted to a human host during chronic infection.Colonization of new environments or changes in resource availability, predatory regime, or climate can drive adaptive evolution. Determining the genetic basis of these changes informs our understanding of the evolution of diversity and the nature of selection. Domestication of crop plants, adaptive radiations, and in-host evolution during chronic microbial infection are characterized by the evolution of a suite of phenotypes that are advantageous in the new environment. Recent work has successfully identified several of the polymorphisms responsible for this type of adaptive evolution in a variety of species (3, 7, 11, 12, 15, 22, 25, 35-37). With comparative genome sequencing emerging as a powerful tool for identifying genetic polymorphism (5, 14, 23), these studies are becoming faster and easier. Still, large genome sizes and countless sequence differences between individuals, isolates, strains, and species have made comprehensive analyses intractable.Upon isolation and introduction into the laboratory, model research organisms experience extreme environmental changes, with associated selection pressures. Indeed, adaptation to life in captivity has been observed in a wide range of domesticated and model research organisms (2) and in zoo populations of endangered species (31). Many phenotypes that evolve in these nonnative environments do so repeatedly and become common features of human-cultured, -raised, or -cultivated organisms (2), providing evidence of positive selection. Likewise, the aquatic bacterium Caulobacter crescentus has evolved marked phenotypic changes during the 50 years it has been cultured in the laboratory environment. At least six phenotypic differences (Fig. ​(Fig.1)1) between two closely related strains (NA1000 and CB15) derived from the same common ancestor have evolved over decades of laboratory cultivation. It is presumed that these phenotypes evolved in response to the dynamic culture conditions and associated selection pressures experienced by bacteria in the laboratory environment. However, the extent of genetic divergence between these strains was uncharacterized, and it was not known whether the phenotypes could be explained by a few single nucleotide polymorphisms (SNPs), insertions/deletions, or genome rearrangements or by the accumulation of many mutations, each with a small contribution to particular phenotypes. In an effort to comprehensively characterize their divergence, we identified the genetic basis of all known phenotypic differences between two laboratory strains (NA1000 and CB15) of C. crescentus.Open in a separate windowFIG. 1.Evolved phenotypic differences between CB15 (Crosson₂) and NA1000 (Crosson₁). (A) Caulobacter cells divide asymmetrically to yield a swarmer and a stalked cell, which are mixed in culture. NA1000 stalked and predivisional cells (light gray) pellet less efficiently than swarmer cells (dark gray), allowing them to be physically separated. Synchrony capacity is quantified by calculating the proportion of cultured cells remaining in suspension. Error bars are ±standard errors of the mean (SEM). (B) When patched and grown on high-sugar media, NA1000 colonies develop a mucoid morphology, while CB15 colonies do not. (C) The transducing phage φCR30 efficiently infects and lyses CB15 cells, resulting in clear plaques, while infection of NA1000 with the same phage lysate results in fewer plaques that are visually turbid. (D) Holdfast-mediated attachment to a surface can be measured using a crystal violet assay. CB15 cells attach, resulting in robust staining, while NA1000 exhibits negligible adherence. (E) Upon continued aeration and incubation of stationary-phase Caulobacter cultures, NA1000 (▪) loses viability more rapidly than CB15 (○). Error bars are ±SEM. (F) In glucose minimal medium, NA1000 generation time is 20% shorter than that of CB15. Error bars are ±SEM.Our study revealed 11 coding, noncoding, and insertion/deletion polymorphisms between these two strains, five of which completely account for the evolved differences between the strains. The results presented herein provide insight into prokaryotic evolution driven by selection for growth and survival in a research laboratory and demonstrate the utility of combining whole-genome sequencing and alignment with molecular genetic tools to reveal the genetic basis of multiple derived phenotypes. Our work demonstrates that rapid adaptation of C. crescentus to the laboratory environment occurred in both strain lineages and is characterized by relatively few genetic changes, including nonsynonymous mutation, noncoding regulatory changes, acquisition of new genes, and inactivation of existing genes, each with a large phenotypic effect.     

Evolution of bacterial phosphoglycerate mutases: non-homologous isofunctional enzymes undergoing gene losses, gains and lateral transfers

Background

The glycolytic phosphoglycerate mutases exist as non-homologous isofunctional enzymes (NISE) having independent evolutionary origins and no similarity in primary sequence, 3D structure, or catalytic mechanism. Cofactor-dependent PGM (dPGM) requires 2,3-bisphosphoglycerate for activity; cofactor-independent PGM (iPGM) does not. The PGM profile of any given bacterium is unpredictable and some organisms such as Escherichia coli encode both forms.

Methods/Principal Findings

To examine the distribution of PGM NISE throughout the Bacteria, and gain insight into the evolutionary processes that shape their phyletic profiles, we searched bacterial genome sequences for the presence of dPGM and iPGM. Both forms exhibited patchy distributions throughout the bacterial domain. Species within the same genus, or even strains of the same species, frequently differ in their PGM repertoire. The distribution is further complicated by the common occurrence of dPGM paralogs, while iPGM paralogs are rare. Larger genomes are more likely to accommodate PGM paralogs or both NISE forms. Lateral gene transfers have shaped the PGM profiles with intradomain and interdomain transfers apparent. Archaeal-type iPGM was identified in many bacteria, often as the sole PGM. To address the function of PGM NISE in an organism encoding both forms, we analyzed recombinant enzymes from E. coli. Both NISE were active mutases, but the specific activity of dPGM greatly exceeded that of iPGM, which showed highest activity in the presence of manganese. We created PGM null mutants in E. coli and discovered the ΔdPGM mutant grew slowly due to a delay in exiting stationary phase. Overexpression of dPGM or iPGM overcame this defect.

Conclusions/Significance

Our biochemical and genetic analyses in E. coli firmly establish dPGM and iPGM as NISE. Metabolic redundancy is indicated since only larger genomes encode both forms. Non-orthologous gene displacement can fully account for the non-uniform PGM distribution we report across the bacterial domain.