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.

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.

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...

No abstract

Although bacterial polyketides are of considerable biomedical interest, the molecular biology of polyketide biosynthesis in Bacillus spp., one of the richest bacterial sources of bioactive natural products, remains largely unexplored. Here we assign for the first time complete polyketide synthase (PKS) gene clusters to Bacillus antibiotics. Three giant modular PKS systems of the trans-acyltransferase type were identified in Bacillus amyloliquefaciens FZB 42. One of them, pks1, is an ortholog of the pksX operon with a previously unknown function in the sequenced model strain Bacillus subtilis 168, while the pks2 and pks3 clusters are novel gene clusters. Cassette mutagenesis combined with advanced mass spectrometric techniques such as matrixassisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-electrospray ionization mass spectrometry revealed that the pks1 (bae) and pks3 (dif) gene clusters encode the biosynthesis of the polyene antibiotics bacillaene and difficidin or oxydifficidin, respectively. In addition, B. subtilis OKB105 (pheA sfp 0 ), a transformant of the B. subtilis 168 derivative JH642, was shown to produce bacillaene, demonstrating that the pksX gene cluster directs the synthesis of that polyketide.Environmental Bacillus amyloliquefaciens strain FZB 42 is distinguished from the domesticated model organism Bacillus subtilis 168 (23) by several features important for rhizosphere competence particularly by its abilities to suppress competitive organisms present in the plant rhizosphere (17, 21) and to promote plant growth (16). In a previous contribution (20), we have reported that B. amyloliquefaciens FZB 42 is a producer of three families of lipopeptides, surfactins, bacillomycins D, and fengycins, which are well-known secondary metabolites with mainly antifungal activity. They are also produced by numerous B. subtilis strains (48). Furthermore, three giant gene clusters containing genes with homology to polyketide synthase (PKS) genes of modular organization were identified but not assigned functional roles. Mutants of FZB 42 deficient in the synthesis of cyclic lipopeptides were unable to suppress phytopathogenic fungi but still retained their antibacterial potency.Polyketides belong to a large family of secondary metabolites that include many bioactive compounds with antibacterial, immunosuppressive, antitumor, or other physiologically relevant bioactivities. Their biosynthesis is accomplished by stepwise decarboxylative Claisen condensations between the extender unit and the growing polyketide chain, generating enzyme-bound ␤-ketoacyl intermediates. Before a subsequent round of chain extension, a variable set of modifying enzymes can locally introduce structural variety. Similar to the nonribosomal synthesis of peptides, the PKS multienzyme system uses acyl carrier proteins (ACPs) that are posttranslationally modified with the 4Ј-phosphopantetheine prosthetic group to channel the growing polyketide intermediate during elongation processes (3). Type I PKSs...

We describe the first potent and selective blocker of the class E Ca2+channel. SNX-482, a novel 41 amino acid peptide present in the venom of the African tarantula, Hysterocrates gigas, was identified through its ability to inhibit human class E Ca2+ channels stably expressed in a mammalian cell line. An IC50 of 15-30 nM was obtained for block of the class E Ca2+ channel, using either patch clamp electrophysiology or K+-evoked Ca2+ flux. At low nanomolar concentrations, SNX-482 also blocked a native resistant or R-type Ca2+ current in rat neurohypophyseal nerve terminals, but concentrations of 200-500 nM had no effect on R-type Ca2+ currents in several types of rat central neurons. The peptide has the sequence GVDKAGCRYMFGGCSVNDDCCPRLGCHSLFSYCAWDLTFSD-OH and is homologous to the spider peptides grammatoxin S1A and hanatoxin, both peptides with very different ion channel blocking selectivities. No effect of SNX-482 was observed on the following ion channel activities: Na+ or K+ currents in several cultured cell types (up to 500 nM); K+ current through cloned potassium channels Kv1.1 and Kv1. 4 expressed in Xenopus oocytes (up to 140 nM); Ca2+ flux through L- and T-type Ca2+ channels in an anterior pituitary cell line (GH3, up to 500 nM); and Ba2+ current through class A Ca2+ channels expressed in Xenopus oocytes (up to 280 nM). A weak effect was noted on Ca2+ current through cloned and stably expressed class B Ca2+ channels (IC50 > 500 nM). The unique selectivity of SNX-482 suggests its usefulness in studying the diversity, function, and pharmacology of class E and/or R-type Ca2+ channels.

Zn anodes suffer from poor Coulombic efficiency (CE) and serious dendrite formation due to the unstable anode/electrolyte interface (AEI). The electrical double layer (EDL) structure formed before cycling is of great significance for building stable solid electrolyte interphase (SEI) on Zn surface but barely discussed in previous research about the stabilization of Zn anode. Herein, saccharin (Sac) is introduced as electrolyte additive for regulating the EDL structure on the AEI. It is found that Sac derived anions are preferentially adsorbed on the Zn metal surface instead of water dipole, creating a new H2O‐poor EDL structure. Moreover, the unique SEI is also detected on the Zn surface due to the decomposition of Sac anions. Both are proved to be capable of modulating Zn deposition behavior and preventing side reactions. Encouragingly, Zn|Zn symmetric cells using Sac additive deliver a high cumulative plated capacity of 2.75 Ah cm−2 and a high average CE of 99.6% under harsh test condition (10 mA cm−2, 10 mAh cm−2). The excellent stability is also achieved at a high rate of 40 mA cm−2. The effectiveness of this Sac additive is further demonstrated in the Zn‐MnO2 full cells.