Genome-wide association study reveals genomic loci of sex differentiation and gonadal development in Plectropomus leopardus.

Introduction: Plectropomus leopardus, a commercially significant marine fish, is primarily found in the Western Pacific regions and along the coast of Southeast Asia. A thorough analysis of the molecular mechanisms involved in sex differentiation is crucial for gaining a comprehensive understanding of gonadal development and improving sex control breeding. However, the relevant fundamental studies of P. leopardus are relatively lacking. Methods: In this study, a genome-wide association study (GWAS) was conducted to investigate the genetic basis mechanism of sex differentiation and gonadal developmental traits in P. leopardus utilizing about 6,850,000 high-quality single-nucleotide polymorphisms (SNPs) derived from 168 individuals (including 126 females and 42 males) by the genome-wide efficient mixed-model association (GEMMA) algorithm. Results: The results of these single-trait GWASs showed that 46 SNP loci (-log10 p > 7) significantly associated with sex differentiation, and gonadal development traits were distributed in multiple different chromosomes, which suggested the analyzed traits were all complex traits under multi-locus control. A total of 1,838 potential candidate genes were obtained by considering a less-stringent threshold (-log10 p > 6) and ±100 kb regions surrounding the significant genomic loci. Moreover, 31 candidate genes were identified through a comprehensive analysis of significant GWAS peaks, gene ontology (GO) annotations, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, including taf7, ddx6, apoeb, sgk1, a2m, usf1, hsd3b7, dll4, xbp1, tet3, esr1, and gli3. These trait-associated genes have been shown to be involved in germline development, male sex differentiation, gonad morphogenesis, hormone receptor binding, oocyte development, male gonad development, steroidogenesis, estrogen-synthetic pathway, etc. Discussion: In the present study, multiple genomic loci of P. leopardus associated with sex differentiation and gonadal development traits were identified for the first time by using GWAS, providing a valuable resource for further research on the molecular genetic mechanism and sex control in P. leopardus. Our results also can contribute to understanding the genetic basis of the sex differentiation mechanism and gonadal development process in grouper fish.

Delineation of the complex microbial nitrogen-transformation network in an anammox-driven full-scale wastewater treatment plant.

Microbial-driven nitrogen removal is a crucial step in modern full-scale wastewater treatment plants (WWTPs), and the complexity of nitrogen transformation is integral to the various wastewater treatment processes. A full understanding of the overall nitrogen cycling networks in WWTPs is therefore a prerequisite for the further enhancement and optimization of wastewater treatment processes. In this study, metagenomics and metatranscriptomics were used to elucidate the microbial nitrogen removal processes in an ammonium-enriched full-scale WWTP, which was configured as an anaerobic-anoxic-anaerobic-oxic system for efficient nitrogen removal (99.63%) on a duck breeding farm. A typical simultaneous nitrification-anammox-denitrification (SNAD) process was established in each tank of this WWTP. Ammonia was oxidized by ammonia-oxidizing bacteria (AOB), archaea (AOA), and nitrite-oxidizing bacteria (NOB), and the produced nitrite and nitrate were further reduced to dinitrogen gas (N2) by anammox and denitrifying bacteria. Visible red anammox biofilms were formed successfully on the sponge carriers submerged in the anoxic tank, and the nitrogen removal rate by anammox reaction was 4.85 times higher than that by denitrification based on 15N isotope labeling and analysis. This supports the significant accumulation of anammox bacteria on the carriers responsible for efficient nitrogen removal. Two distinct anammox bacteria, named "Ca. Brocadia sp. PF01" and "Ca. Jettenia sp. PF02", were identified from the biofilm in this investigation. By recovering their genomic features and their metabolic capabilities, our results indicate that the highly active core anammox process found in PF01, suggests extending its niche within the plant. With the possible contribution of the dissimilatory nitrate reduction to ammonium (DNRA) reaction, enriching PF02 within the biofilm may also be warranted. Collectively, this study highlights the effective design strategies of a full-scale WWTP with enrichment of anammox bacteria on the carrier materials for nitrogen removal and therefore the biochemical reaction mechanisms of the contributing members.

PRAS40 deregulates apoptosis in Ewing sarcoma family tumors by enhancing the insulin receptor/Akt and mTOR signaling pathways.

EWS expression in Ewing sarcoma family tumors (ESFTs) is decreased due to the haploinsufficiency elicited by chromosomal translocation. The abnormal expression levels of EWS and its downstream factors contribute to the manifestation of ESFTs. Previously, we reported that increased Proline-rich Akt substrate of 40 kDa (PRAS40), which is encoded by an EWS mRNA target, promotes the development of ESFTs. However, the mechanism remains elusive. To clarify the role of PRAS40 in ESFTs, we silenced PRAS40 expression in ESFT cells using siRNAs and found increased levels of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. Cleaved caspase 3 levels and cytochrome C release were increased simultaneously. Furthermore, with PRAS40 knockdown, the phosphorylation of Akt and mTOR downstream factors, i.e., S6K and S6, was attenuated notably. Ectopic expression of PRAS40 increased Akt and S6 phosphorylation. Activation of Akt only partially reversed the apoptosis induced by PRAS40 knockdown, and downregulation of S6 phosphorylation by PRAS40 silencing could not be sufficiently restored via Akt activation. Searching the upstream factors in this pathway, the autophosphorylation of insulin receptor (IR) was found to be inhibited significantly by PRAS40 silencing but increased by PRAS40 overexpression. Therefore, PRAS40 may enhance IR phosphorylation to facilitate Akt and mTOR signaling leading to the apoptosis deregulation in ESFTs. Moreover, in vivo results confirmed that PRAS40 deletion suppressed the growth of ESFT xenografts and downregulated IR and S6 phosphorylation. Our findings suggest a novel functioning model for PRAS40, which represents a novel therapeutic target for ESFTs.

Production of high-affinity human monoclonal antibody fab fragments to the 19-kilodalton C-terminal merozoite surface protein 1 of Plasmodium falciparum.

A combinatorial immunoglobulin gene library was constructed from peripheral blood lymphocytes of eight patients infected with Plasmodium falciparum and was screened for the production of human monoclonal antibody Fab fragments to the C-terminal 19-kDa fragment of P. falciparum merozoite surface protein 1 (MSP-1(19)). Three Fab clones recognized recombinant MSP-1(19) under nonreducing conditions. Indirect immunofluorescence microscopy demonstrated that three Fab clones stained the surfaces of late trophozoites/schizonts and merozoites of the FCR3 and 3D7 strains, suggesting the Fabs' reactivities to a conserved epitope. Sequence analysis of the heavy-chain genes revealed that the closest germ line V segments were VH1-8 and VH7-81, with 91% to 98% homology. The closest germ line D segment was D3-10, and the closest germ line J segment was JH4 or JH5, with 90% to 97% homology. In the light-chain genes, the closest germ line V segment was A27 for the Jkappa2, Jkappa4, and Jkappa5 segments. The dissociation constants of these Fab fragments for recombinant MSP-1(19) ranged from 1.09 x 10(-9) to 2.66 x 10(-9) M. The binding of the three Fab fragments to MSP-1(19) was competitively inhibited by the anti-MSP-1(19) mouse monoclonal antibody 12.8, which inhibits erythrocyte invasion by merozoites. However, the human Fab fragment with the highest affinity did not inhibit in vitro growth of P. falciparum. This is the first report of gene analysis and bacterial expression of human monoclonal antibodies to P. falciparum MSP-1(19). The combinatorial immunoglobulin gene library derived from malaria patients provides a potential tool for producing high-affinity human antibodies specific for P. falciparum.

Production of an anti-severe acute respiratory syndrome (SARS) coronavirus human monoclonal antibody Fab fragment by using a combinatorial immunoglobulin gene library derived from patients who recovered from SARS.

A combinatorial human immunoglobulin gene library was constructed from the peripheral lymphocytes of two patients who recovered from severe acute respiratory syndrome (SARS). The library was screened for the production of Fab antibody fragments to a recombinant spike protein of SARS-associated coronavirus (SARS-CoV). One Fab clone, AS3-3, reacted with the spike protein in an enzyme-linked immunosorbent assay. The dissociation constant of AS3-3 was 1.98 x 10(-8) M. Immunofluorescent microscopy revealed that it reacted with SARS-CoV-infected cells. The library seems to be a potent tool for the production of human antibodies to SARS-CoV.