Neural progenitor genes. Germinal zone expression and analysis of genetic overlap in stem cell populations

The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.     

Isolation of macrocyclic and non-macrocyclic trichothecenes (stachybotrys and fusarium toxins) from the Environment of 200 III Sport Horses

Satratoxins H and G, verrucarin J, and roridin E were isolated from the bedding straw of 200 sport horses exhibiting typical symptoms of stachybotryo-toxicosis. At the same time, the oat feed consumed by the horses contained non-macrocyclicFusarium trichothecenes: T-2 toxin and diacetoxyscirpenol.     

CLAME: a new alignment-based binning algorithm allows the genomic description of a novel Xanthomonadaceae from the Colombian Andes

Background

Hot spring bacteria have unique biological adaptations to survive the extreme conditions of these environments; these bacteria produce thermostable enzymes that can be used in biotechnological and industrial applications. However, sequencing these bacteria is complex, since it is not possible to culture them. As an alternative, genome shotgun sequencing of whole microbial communities can be used. The problem is that the classification of sequences within a metagenomic dataset is very challenging particularly when they include unknown microorganisms since they lack genomic reference. We failed to recover a bacterium genome from a hot spring metagenome using the available software tools, so we develop a new tool that allowed us to recover most of this genome.

Results

We present a proteobacteria draft genome reconstructed from a Colombian’s Andes hot spring metagenome. The genome seems to be from a new lineage within the family Rhodanobacteraceae of the class Gammaproteobacteria, closely related to the genus Dokdonella. We were able to generate this genome thanks to CLAME. CLAME, from Spanish “CLAsificador MEtagenomico”, is a tool to group reads in bins. We show that most reads from each bin belong to a single chromosome. CLAME is very effective recovering most of the reads belonging to the predominant species within a metagenome.

Conclusions

We developed a tool that can be used to extract genomes (or parts of them) from a complex metagenome.

     

Secretory phospholipase A2 in SARS-CoV-2 infection and multisystem inflammatory syndrome in children (MIS-C)

Secretory phospholipase 2 (sPLA2) acts as a mediator between proximal and distal events of the inflammatory cascade. Its role in SARS-CoV-2 infection is unknown, but could contribute to COVID-19 inflammasome activation and cellular damage. We present the first report of plasma sPLA2 levels in adults and children with COVID-19 compared with controls. Currently asymptomatic adults with a history of recent COVID-19 infection (≥4 weeks before) identified by SARS-CoV-2 IgG antibodies had sPLA2 levels similar to those who were seronegative (9 ± 6 vs.17 ± 28 ng/mL, P = 0.26). In contrast, children hospitalized with severe COVID-19 had significantly elevated sPLA2 compared with those with mild or asymptomatic SARS-CoV-2 infection (269 ± 137 vs. 2 ± 3 ng/mL, P = 0.01). Among children hospitalized with multisystem inflammatory syndrome in children (MIS-C), all had severe disease requiring pediatric intensive care unit (PICU) admission. sPLA2 levels were significantly higher in those with acute illness <10 days versus convalescent disease ≥10 days (540 ± 510 vs. 2 ± 1, P = 0.04). Thus, sPLA2 levels correlated with COVID-19 severity and acute MIS-C in children, implicating a role in inflammasome activation and disease pathogenesis. sPLA2 may be a useful biomarker to stratify risk and guide patient management for children with acute COVID-19 and MIS-C. Therapeutic compounds targeting sPLA2 and inflammasome activation warrant consideration.     

Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytopathogenicity

The ceIV1 gene encoding a secreted cellulase (CelV1) of Erwinia carotovora subsp. carotovora SCC3193 was cloned and its nucleotide sequence determined. The gene contains an open reading frame of 1511 by and codes for an exported protein of 504 amino acids. The predicted amino acid sequence of Ce1V1 was highly similar to that of CeIV of another E. c. subsp. carotovora strain SCRI193 but completely different from the previously characterized cellulase, CelS, of the strain SCC3193. Gene fusions to the lacZ reporter were employed to characterize the regulation of celV1 and celS. Both genes are coordinately induced in a growth phase-dependent manner and are catabolite repressed. Expression of celV1 but not celS was stimulated by plant extracts. The celS gene was expressed at a much lower level than celV1 under all conditions tested. Inactivation of the celV1 gene in E. c. subsp. carotovora strain SCC3193 by marker exchange showed that celV1 encodes the major cellulase of strain SCC3193, as the resulting mutant strain SCC6001 was devoid of cellulase activity. Ce1Vl mutants exhibited reduced virulence suggesting that CelV1, although not absolutely required for pathogenicity, enhances the ability of strain SCC3193 to macerate plant tissue. Inactivation of the celS gene in the celV1 mutant did not lead to any further decrease in virulence.     

Synthesis of acyloxymethyl ester prodrugs of the transferable protein farnesyl transferase substrate farnesyl methylenediphosphonate

Three isoprenoid diphosphate analogues of farnesyl diphosphate (FPP) where the diphosphate has been replaced by methylene diphosphonate and the negative charges masked by frangible pivaloyloxymethyl (POM) esters were prepared. Farnesyl methylenediphosphonate is a sub-micromolar substrate for protein farnesyl transferase. The tripivaloyloxymethyl esters of isoprenoid methylenediphosphonate have significantly increased lipophilicity and may act as important farnesyl diphosphate prodrugs.     

Heymann antibodies induce complement-dependent injury of rat glomerular visceral epithelial cells

The aim of this study was to investigate the in vitro role of the complement membrane attack complex (MAC) in the injury induced by nephritogenic anti-brush border vesicle (Fx1A) antibodies on rat glomerular visceral epithelial cells (GEC). Both sheep and rabbit anti-rat brush border vesicle IgG-induced complement-dependent lysis of cultured GEC. Fab fragments of sheep anti-rat brush border vesicles and polyclonal or monoclonal gp330 IgG were devoid of lytic activity. Shedding of cell-surface antigens induced by sheep or rabbit anti-rat brush border vesicle IgG protected GEC from subsequent exposure to lytic antibodies and complement, an effect that was not obtained with Fab fragments. When GEC were incubated with sheep or rabbit anti-rat brush border vesicle IgG in capping conditions, the C3 component was co-redistributed with Heymann immune complexes; in contrast, the MAC remained diffusely bound to the cell surface, indicating that it was not associated with the antigen-antibody complexes. The MAC was demonstrated on the surface of GEC by immunofluorescence staining with anti-MAC neoantigen and by electron microscopy of negatively stained membranes showing focal clusters of 110 A MAC lesions. When GEC were treated with sheep IgG or rabbit IgG plus C6-deficient sera, the cells were not lysed and MAC was not demonstrable on the surface; however, lytic activity was restored when C6-deficient sera were reconstituted with purified C6. The results are consistent with the interpretation that injury induced by Heymann antibodies on GEC is MAC-dependent.     

Effect of thiol-functionalised silica on cisplatin adsorption

This study contributes to the investigation related to guest–host interactions between the chemotherapeutic agent cisplatin and a functionalised silica matrix in order to improve and find new materials such as drug carriers. The adsorption of cisplatin and its complexes, cis-[PtCl(NH₃)₂]⁺ and cis-[Pt(NH₃)₂]²⁺, on a SH-functionalised SiO₂(111) surface has been studied by the atom superposition and electron delocalisation method. The adiabatic energy curves for the adsorption of the drug and its products on the delivery system were considered. The electronic structure and bonding analysis were also performed. The molecule and their complex are adsorbed on the functionalised surface resulting in a major absorption of the cis-[Pt(NH₃)₂]²⁺ complex. The molecule–surface interactions are formed via –SH group. The molecule/complexes SH electron-donating effect plays an important role in the catalytic reaction. The more important drug–carrier interactions occur through the Cl–H bond for the adsorption of cis-[PtCl₂(NH₃)₂] and cis-[PtCl(NH₃)₂]⁺, and through the Pt–S and Pt–H interactions for cis-[Pt(NH₃)₂]²⁺ adsorption. When the new interactions are formed, the functionalised carrier maintains their matrix properties while the molecule is the most affected after adsorption. The Pt atomic orbitals present the most important changes during adsorption.     

Selection of phosphate-solubilizing diazotrophic Herbaspirillum and Burkholderia strains and their effect on rice crop yield and nutrient uptake

Background and Aims

Plant growth-promoting bacteria, mainly diazotrophs and phosphate solubilizers, can reduce the use of chemical fertilizers for rice crops. Here, diazotrophic bacteria isolated from rice were screened for their ability to solubilize inorganic P (P_i) in vitro and in association with rice plants cultivated in pots.

Methods

Forty-nine isolates were tested for the ability to solubilize P_i on NBRIP and GL agar plate media and seven selected strains were further evaluated in NBRIP liquid medium. Three of these strains were inoculated in rice plants grown in soil pots containing ¹⁵N-labeled fertilizer and two sources of P: tricalcium phosphate (TCP) or simple superphosphate (SSP). The dry matter, yield, N, P, and the ¹⁵N content accumulated in plant tissues were measured at 135 days after planting.

Results

Seven strains belonging to the genera Herbaspirillum and Burkholderia formed a halo of solubilized P_i on agar plates. The Burkholderia strains showed peak soluble P (around 200 mg P L^?1) on the fifth day when grown in NBRIP liquid medium for 14 days. Inoculation of Herbaspirillum strains (H18, ZA15) and a Burkholderia vietaminensis strain (AR114) increased rice grain yield from 33 to 47 % with TCP and 18 to 44 % with TSS, respectively. The bacterial inoculation led to enhanced N-use efficiency of the ¹⁵N-labeled fertilizer.

Conclusion

These results suggest that the selection and use of P-solubilizing diazotrophic bacteria are a good strategy to promote P solubilization and/or N use efficiency in rice plants.     

Therapeutic use of IL-2 to enhance antiviral T-cell responses in vivo

Interleukin (IL)-2 is currently used to enhance T-cell immunity but can have both positive and negative effects on T cells. To determine whether these opposing results are due to IL-2 acting differently on T cells depending on their stage of differentiation, we examined the effects of IL-2 therapy during the expansion, contraction and memory phases of the T-cell response in lymphocytic choriomeningitis virus (LCMV)-infected mice. IL-2 treatment during the expansion phase was detrimental to the survival of rapidly dividing effector T cells. In contrast, IL-2 therapy was highly beneficial during the death phase, resulting in increased proliferation and survival of virus-specific T cells. IL-2 treatment also increased proliferation of resting memory T cells in mice that controlled the infection. Virus-specific T cells in chronically infected mice also responded to IL-2 resulting in decreased viral burden. Thus, timing of IL-2 administration and differentiation status of the T cell are critical parameters in designing IL-2 therapies.