Ethical considerations constrain the in vivo study of human hemopoietic stem cells (HSC). To overcome this limitation, small animal models of human HSC engraftment have been used. We report the development and characterization of a new genetic stock of IL-2R common γ-chain deficient NOD/LtSz-scid (NOD-scid IL2Rγnull) mice and document their ability to support human mobilized blood HSC engraftment and multilineage differentiation. NOD-scid IL2Rγnull mice are deficient in mature lymphocytes and NK cells, survive beyond 16 mo of age, and even after sublethal irradiation resist lymphoma development. Engraftment of NOD-scid IL2Rγnull mice with human HSC generate 6-fold higher percentages of human CD45+ cells in host bone marrow than with similarly treated NOD-scid mice. These human cells include B cells, NK cells, myeloid cells, plasmacytoid dendritic cells, and HSC. Spleens from engrafted NOD-scid IL2Rγnull mice contain human Ig+ B cells and lower numbers of human CD3+ T cells. Coadministration of human Fc-IL7 fusion protein results in high percentages of human CD4+CD8+ thymocytes as well human CD4+CD8− and CD4−CD8+ peripheral blood and splenic T cells. De novo human T cell development in NOD-scid IL2Rγnull mice was validated by 1) high levels of TCR excision circles, 2) complex TCRβ repertoire diversity, and 3) proliferative responses to PHA and streptococcal superantigen, streptococcal pyrogenic exotoxin. Thus, NOD-scid IL2Rγnull mice engrafted with human mobilized blood stem cells provide a new in vivo long-lived model of robust multilineage human HSC engraftment.

The environmental strain Bacillus amyloliquefaciens FZB42 promotes plant growth and suppresses plant pathogenic organisms present in the rhizosphere. We sampled sequenced the genome of FZB42 and identified 2,947 genes with >50% identity on the amino acid level to the corresponding genes of Bacillus subtilis 168. Six large gene clusters encoding nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) occupied 7.5% of the whole genome. Two of the PKS and one of the NRPS encoding gene clusters were unique insertions in the FZB42 genome and are not present in B. subtilis 168. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis revealed expression of the antibiotic lipopeptide products surfactin, fengycin, and bacillomycin D. The fengycin (fen) and the surfactin (srf) operons were organized and located as in B. subtilis 168. A large 37.2-kb antibiotic DNA island containing the bmy gene cluster was attributed to the biosynthesis of bacillomycin D. The bmy island was found inserted close to the fen operon. The responsibility of the bmy, fen, and srf gene clusters for the production of the corresponding secondary metabolites was demonstrated by cassette mutagenesis, which led to the loss of the ability to produce these peptides. Although these single mutants still largely retained their ability to control fungal spread, a double mutant lacking both bacillomycin D and fengycin was heavily impaired in its ability to inhibit growth of phytopathogenic fungi, suggesting that both lipopeptides act in a synergistic manner.The rhizosphere colonizing Bacillus amyloliquefaciens strain FZB42 is distinguished from the related model organism Bacillus subtilis 168 by its ability to stimulate plant growth and to suppress plant pathogenic organisms (12,14). However, the basis for successful mutualistic colonization of plant rhizosphere by some Bacillus strains is still unknown. We assume that rhizosphere competence and biocontrol function in bacilli are partly caused by nonribosomally produced cyclic lipopeptides acting against phytopathogenic viruses, bacteria, fungi, and nematodes. These lipopeptides are synthesized at modular multienzymatic templates (33) and consist of a ␤-amino or ␤-hydroxy fatty acid component that is integrated into a peptide moiety.Some of these lipopeptides have been studied in greater detail, including surfactin, fengycins, and several iturins. Surfactin is a heptapeptide with an LLDLLDL chiral sequence linked, via a lactone bond, to a ␤-hydroxy fatty acid with 13 to 15 carbon atoms. Surfactin exerts its antimicrobial and antiviral effect by altering membrane integrity (30). Fengycin and the closely related plipastatin are cyclic lipodecapeptides containing a ␤-hydroxy fatty acid with a side chain length of 16 to 19 carbon atoms. Four D-amino acids and ornithine (a nonproteinogenic residue) have been identified in the peptide portion of fengycin. It is specifically active against filamentous fungi and inhibits phospholipase A 2 (26). Members of the iturin famil...

It has long been assumed that the C-terminal motif, NPXY, is the internalization signal for ␤-amyloid precursor protein (APP) and that the NPXY tyrosine (Tyr 743 by APP751 numbering, Tyr 682 in APP695) is required for APP endocytosis. To evaluate this tenet and to identify the specific amino acids subserving APP endocytosis, we mutated all tyrosines in the APP cytoplasmic domain and amino acids within the sequence GYENPTY (amino acids 737-743). Stable cell lines expressing these mutations were assessed for APP endocytosis, secretion, and turnover. Normal APP endocytosis was observed for cells expressing Y709A, G737A, and Y743A mutations. However, Y738A, N740A, and P741A or the double mutation of Y738A/P741A significantly impaired APP internalization to a level similar to that observed for cells lacking nearly the entire APP cytoplasmic domain (⌬C), arguing that the dominant signal for APP endocytosis is the tetrapeptide YENP. Although not an APP internalization signal, Tyr 743 regulates rapid APP turnover because half-life increased by 50% with the Y743A mutation alone. Secretion of the APP-derived proteolytic fragment, A␤, was tightly correlated with APP internalization, such that A␤ secretion was unchanged for cells having normal APP endocytosis but significantly decreased for endocytosis-deficient cell lines. Remarkably, secretion of the A␤42 isoform was also reduced in parallel with endocytosis from internalization-deficient cell lines, suggesting an important role for APP endocytosis in the secretion of this highly pathogenic A␤ species. APP1 is a transmembrane protein with homology to glycosylated cell surface receptors (1), can reside at the cell surface (2-4) and is reinternalized via clathrin-coated pits (5, 6) to the endosomal-lysosmal pathway (7,8). Some internalized APP remains intact to be recycled to the cell surface plasma membrane (9, 10). However, internalized APP can also be proteolytically processed into several distinct secreted fragments, which include the large secreted N-terminal APP ectodomain (APP s ), and A␤, the major protein component of senile plaques in Alzheimer's disease (AD; reviewed in Ref. 11).Because A␤ deposition may be central to AD pathogenesis, the mechanism by which A␤ is generated from the precursor is an important focus of AD research. At least two species of A␤, differing by two amino acids at the C terminus (A␤40 and A␤42), are released from cells during normal cellular metabolism (12-14). A␤42, which readily aggregates in vitro (reviewed in Ref. 15) appears to be more pathogenic and may serve as a seed for plaque formation in individuals with AD (16), hereditary cerebral hemorrhage with amyloidosis Dutch type (17), and Down's syndrome (18). The source of A␤ deposited in brain tissues is still uncertain. However, cell lines expressing wild type APP can produce and release A␤ primarily after internalization of APP from the cell surface (19,20). Although familial mutations in APP can enhance A␤ secretion (e.g. the Swedish KM 3 NL mutation; Refs. 20 -23), almost all huma...

Background:Although the SARS-CoV-2 viral load detection of respiratory specimen has been widely used for novel coronavirus disease diagnosis, it is undeniable that serum SARS-CoV-2 nucleic acid (RNAaemia) could be detected in a fraction of the COVID-19 patients. However, it is not clear that if the incidence of RNAaemia could be correlated with the occurrence of cytokine storm or with the specific class of patients. Methods:This study enrolled 48 patients with COVID-19 admitted to the General Hospital of Central Theater Command, PLA, a designated hospital in Wuhan, China. The patients were divided into three groups according to the Dia gnosis and Treatment of New Coronavirus Pneumonia (version 6) published by the National Health Commission of China. The clinical and laboratory data were collected. The serum viral load detection and serum IL-6 levels were determined. Except for routine statistical analysis, Generalized Linear Models (GLMs) analysis was used to establish a patient status prediction model based on real-time RT-PCR Ct value. Findings:The Result showed that cases with RNAaemia were exclusively confirmed in critically ill patients group and appeared to reflect the illness severity. Further more, the inflammatory cytokine IL-6 levels were significantly elevated in critically ill patients, which is almost 10-folds higher than those in other patients. More importantly, the extremely high IL-6 level was closely correlated with the incidence of RNAaemia (R=0.902) and the vital signs of COVID-19 patients (R= -0.682). Interpretation:Serum SARS-CoV-2 viral load (RNAaemia) is strongly associated with cytokine storm and can be used to predict the poor prognosis of COVID-19 patients.Moreover, our results strongly suggest that cytokine IL-6 should be considered as a therapeutic target in critically ill patients with excessive inflammatory response. All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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