iPS-cell-derived microglia promote brain organoid maturation via cholesterol transfer.

Microglia are specialized brain-resident macrophages that arise from primitive macrophages colonizing the embryonic brain1. Microglia contribute to multiple aspects of brain development, but their precise roles in the early human brain remain poorly understood owing to limited access to relevant tissues2-6. The generation of brain organoids from human induced pluripotent stem cells recapitulates some key features of human embryonic brain development7-10. However, current approaches do not incorporate microglia or address their role in organoid maturation11-21. Here we generated microglia-sufficient brain organoids by coculturing brain organoids with primitive-like macrophages generated from the same human induced pluripotent stem cells (iMac)22. In organoid cocultures, iMac differentiated into cells with microglia-like phenotypes and functions (iMicro) and modulated neuronal progenitor cell (NPC) differentiation, limiting NPC proliferation and promoting axonogenesis. Mechanistically, iMicro contained high levels of PLIN2+ lipid droplets that exported cholesterol and its esters, which were taken up by NPCs in the organoids. We also detected PLIN2+ lipid droplet-loaded microglia in mouse and human embryonic brains. Overall, our approach substantially advances current human brain organoid approaches by incorporating microglial cells, as illustrated by the discovery of a key pathway of lipid-mediated crosstalk between microglia and NPCs that leads to improved neurogenesis.

Induced-Pluripotent-Stem-Cell-Derived Primitive Macrophages Provide a Platform for Modeling Tissue-Resident Macrophage Differentiation and Function.

Tissue macrophages arise during embryogenesis from yolk-sac (YS) progenitors that give rise to primitive YS macrophages. Until recently, it has been impossible to isolate or derive sufficient numbers of YS-derived macrophages for further study, but data now suggest that induced pluripotent stem cells (iPSCs) can be driven to undergo a process reminiscent of YS-hematopoiesis in vitro. We asked whether iPSC-derived primitive macrophages (iMacs) can terminally differentiate into specialized macrophages with the help of growth factors and organ-specific cues. Co-culturing human or murine iMacs with iPSC-derived neurons promoted differentiation into microglia-like cells in vitro. Furthermore, murine iMacs differentiated in vivo into microglia after injection into the brain and into functional alveolar macrophages after engraftment in the lung. Finally, iPSCs from a patient with familial Mediterranean fever differentiated into iMacs with pro-inflammatory characteristics, mimicking the disease phenotype. Altogether, iMacs constitute a source of tissue-resident macrophage precursors that can be used for biological, pathophysiological, and therapeutic studies.

Staphylococcus aureus Conquers Host by Hijacking Mitochondria via PFKFB3 in Epithelial Cells.

Staphylococcus aureus (S. aureus) persists within mammary epithelial cells for an extended duration, exploiting the host metabolic resources to facilitate replication. This study revealed a mechanism by which intracellular S. aureus reprograms host metabolism, with PFKFB3 playing a crucial role in this process. Mechanistically, S. aureus induced mitochondrial damage, leading to increased levels of mitochondrial reactive oxygen species (mROS) and dysfunction in electron transport chain (ETC). Moreover, S. aureus shifted the balance of mitochondrial dynamics from fusion to fission, subsequently activating PINK1-PRKN-dependent mitophagy, causing loss of the sirtuin 3 (SIRT3) to stabilize hypoxic inducible factor 1α (HIF1α), and shifting the host metabolism toward enhanced glycolysis. The inhibition of PFKFB3 reversed the mitochondrial damage and degradation of SIRT3 induced by S. aureus. Overall, our findings elucidate the mechanism by which S. aureus reprograms host metabolism and offer insights into the treatment of S. aureus infection.

Key Roles of Interfacial OH- ion Distribution on Proton Coupled Electron Transfer Kinetics Toward Urea Oxidation Reaction.

Enhancing alkaline urea oxidation reaction (UOR) activity is essential to upgrade renewable electrolysis systems. As a core step of UOR, proton-coupled electron transfer (PCET) determines the overall performance, and accelerating its kinetic remains a challenge. In this work, a newly raised electrocatalyst of NiCoMoCuOx Hy with derived multi-metal co-doping (oxy)hydroxide species during electrochemical oxidation states is reported, which ensures considerable alkaline UOR activity (10/500 mA cm-2 at 1.32/1.52 V vs RHE, respectively). Impressively, comprehensive studies elucidate the correlation between the electrode-electrolyte interfacial microenvironment and the electrocatalytic urea oxidation behavior. Specifically, NiCoMoCuOx Hy featured with dendritic nanostructure creates a strengthened electric field distribution. This structural factor prompts the local OH- enrichment in electrical double layer (EDL), so that the dehydrogenative oxidation of the catalyst is directly reinforced to facilitate the subsequent PCET kinetics of nucleophilic urea, resulting in high UOR performance. In practical utilization, NiCoMoCuOx Hy -driven UOR coupled cathodic hydrogen evolution reaction (HER) and carbon dioxide reduction reaction (CO2 RR), and harvested high value-added products of H2 and C2 H4 , respectively. This work clarifies a novel mechanism to improve electrocatalytic UOR performance through structure-induced interfacial microenvironment modulation.

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in GLS.

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.

A pyrylium salt-based fluorescent probe for the highly sensitive detection of methylamine vapour.

The amine vapour produced by microorganisms is an important indicator of food spoilage. Amines are also ubiquitous in the chemical industry. The development of molecule-based ion sensors has been a pivotal issue that is currently receiving considerable attention. In this work, a new pyrylium salt-based fluorescent probe MTPY has been developed as a rapid, highly sensitive, and selective sensor for methylamine vapour. MTPY exhibits an obvious fluorescence response from yellow to cyan towards CH3NH2 vapour. The calibration curve of titration analysis shows a linear relationship of the fluorescence intensity at 514 nm versus the methylamine concentration in the range of 0.1-2 ppm. In addition to the linearity (R2 = 0.974) and short response time with a low detection limit (2.6 ppt, 8.4 × 10-8 M), the sensing mechanism was traced using mass spectrometry.

A benzophenoxazine-based NIR fluorescent probe for the detection of hydrogen sulfide and imaging in living cells.

Hydrogen sulfide (H2S) plays a vital role in regulating many important physiological functions. However, H2S-containing industrial wastewater is inevitably pumped into the environment which seriously contaminates water supplies and foodstuffs. So it is crucial to develop a single fluorescent probe for H2S detection with high sensitivity and selectivity. Herein, a colorimetric and turn-on near infrared (NIR) fluorescent probe NRDNP based on benzophenoxazine was designed and synthesized by thiolysis of 2,4-dinitrophenyl (DNP) ether as the specific reaction site strategy to achieve highly specific H2S detection in living systems. The studies demonstrated that the probe NRDNP exhibited excellent sensing performance toward H2S with an about 80-fold NIR fluorescence enhancement, a rapid response within 10 min, excellent sensitivity with a detection limit of 19 nM and good selectivity. Furthermore, the NRDNP is an optical sensor which visually changes in terms of the fluorescence colour/intensity upon sensing gaseous H2S molecules. NRDNP has low cytotoxicity and has been successfully applied in the fluorescence imaging of H2S in living cells, suggesting that it would be an effective tool for H2S detection in living systems.

Immune response variations and intestinal flora changes in mastitis induced by three Streptococcus uberis strains.

Streptococcus uberis is a common cause of mastitis. The pathogenicity among different strains of S. uberis and the resultant host immune responses remain to be elucidated. Herein, we document immune responses among three strains of S. uberis, and preliminary explore whether and how intestinal immunity plays a role in host anti-infection processes. Mice have been proved to be effective experimental animals for bovine mastitis, so utilizing a mouse intramammary infection model, we assay immune responses and gut flora changes of three S. uberis strains by histopathologic examination, RT-PCR, Western blot, and 16s ribosomal DNA sequencing. We find that the immune responses among the three sequence-type (ST) S. uberis strains may be linked to the hasA/B and lbp virulence genes, and the beta diversity of the intestine may be independent of the ST of S. uberis. Twenty phyla and 30 genera of intestinal flora were identified, with Verrucomicrobia and Akkermansia being the most prominent phylum and genus, respectively. These bacteria have strong anti-inflammatory and protective effects against S. uberis challenge. These data provide a foundation for further studies to elucidate gut flora function and exploration of therapeutic targets for mastitis.

miR-1205/DNAJB1 reverses docetaxel chemoresistance in human triple negative breast carcinoma cells via regulation of mutp53/TAp63 signaling.

Chemoresistance is the major cause of therapeutic failure in human triple negative breast carcinoma (TNBC). Docetaxel (DOC), a first-line therapeutic drug in TNBC treatment, is limited for long-term use due to the development of chemoresistance. Thus, overcoming chemoresistance of DOC remains an important challenge to improve patient's outcome of TNBC. In this study, we aimed to investigate the molecular mechanism behind DOC chemoresistance and the possible therapeutic effects of miRNAs. Utilizing qRT-PCR analysis, we discovered that miR-1205 is gradually downregulated in human triple negative breast carcinoma MDA-MB-231 and docetaxel-resistant MDA-MB-231 (MDA-MB-231/DOC) cells compared with Hs 578Bst normal human breast fibroblasts. Cell viability, cell cycle and apoptosis assays in MDA-MB-231/DOC cells indicated that miR-1205 overexpression enhances docetaxel sensitivity by reducing cell viability as well as inducing G2/M cell cycle arrest and cell apoptosis. Western blot analysis, dual-luciferase reporter assay, co-immunoprecipitation assay and chromatin immunoprecipitation assay revealed that miR-1205 overexpression disrupts the stable complex formation of DNAJB1, mutp53 and TAp63 by directly reducing DNAJB1 expression, which abates the sequestrating effect of mutp53 on TAp63, thereby leading to the enhanced DOC sensitivity in MDA-MB-231/DOC cells. Our findings demonstrate the role of the miR-1205/DNAJB1 axis in the docetaxel resistance of TNBC, which may offer a promising therapeutic approach to resolve docetaxel resistance in TNBC.

[In vitro screening of natural compounds with antitumor activity by trypsin].

In order to investigate the feasibility of in vitro screening the antitumor activity of natural compounds by trypsin, porcine trypsin was used to for screening test, which is marked by inhibition of enzyme activity. Four compounds, namely daidzin, genistin, matrine and oxymatrine, were selected as test subjects. The natural antitumor drug camptothecin was used as the control. The inhibitory effect was detected by two experimental methods: direct detection of trypsin activity inhibition and hydrolysis of bovine serum albumin by trypsin. The results showed the inhibitory effects of the four natural compounds on trypsin, and the inhibition rates of the four natural compounds were significantly different. The enzyme activity assay showed that the inhibitory effect of matrine was better than that of oxymatrine, indicating that trypsin had a good screening resolution. The inhibitory effect was significantly increased with the increased ratio of sample to trypsin, suggesting the structure-activity correlation and dose-effect correlation of the screening methods. Altogether, the experimental method of screening antitumor activity of natural compounds by trypsin has good application values. Since porcine trypsin is similar to human trypsin in terms of molecular structure and performance, it is more applicable for screening of antitumor efficacy of natural pharmacodynamic compounds.

Distinct Disease Severity Between Children and Older Adults With Coronavirus Disease 2019 (COVID-19): Impacts of ACE2 Expression, Distribution, and Lung Progenitor Cells.

BACKGROUND: Children and older adults with coronavirus disease 2019 (COVID-19) display a distinct spectrum of disease severity yet the risk factors aren't well understood. We sought to examine the expression pattern of angiotensin-converting enzyme 2 (ACE2), the cell-entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the role of lung progenitor cells in children and older patients. METHODS: We retrospectively analyzed clinical features in a cohort of 299 patients with COVID-19. The expression and distribution of ACE2 and lung progenitor cells were systematically examined using a combination of public single-cell RNA-seq data sets, lung biopsies, and ex vivo infection of lung tissues with SARS-CoV-2 pseudovirus in children and older adults. We also followed up patients who had recovered from COVID-19. RESULTS: Compared with children, older patients (>50 years.) were more likely to develop into serious pneumonia with reduced lymphocytes and aberrant inflammatory response (P = .001). The expression level of ACE2 and lung progenitor cell markers were generally decreased in older patients. Notably, ACE2 positive cells were mainly distributed in the alveolar region, including SFTPC positive cells, but rarely in airway regions in the older adults (P < .01). The follow-up of discharged patients revealed a prolonged recovery from pneumonia in the older (P < .025). CONCLUSIONS: Compared to children, ACE2 positive cells are generally decreased in older adults and mainly presented in the lower pulmonary tract. The lung progenitor cells are also decreased. These risk factors may impact disease severity and recovery from pneumonia caused by SARS-Cov-2 infection in older patients.

Role of long polar fimbriae type 1 and 2 in pathogenesis of mammary pathogenic Escherichia coli.

Escherichia coli is a leading cause of bovine mastitis worldwide. The bacteria can rapidly grow in milk and elicit a strong lipopolysaccharide (LPS)/toll-like receptor-4 (TLR4)-dependent inflammatory response. Recently, the long polar fimbriae (LPF) were identified as a promising virulence factor candidate widely distributed in mammary pathogenic E. coli (MPEC) strains. Mammary pathogenic E. coli possess 2 lpf loci encoding LPF1 and LPF2, respectively. By deleting the major fimbrial subunit gene, lpfA, we found that both LPF1 and LPF2 contribute to MPEC adhesion, invasion, and biofilm formation in vitro. The lpf1A and lpf2A mutants showed reduced cytotoxicity in our in vitro cell infection model. Furthermore, we observed that LPF2 induced a mild TLR4-independent proinflammatory response. The median lethal dose (LD50) of both ∆lpf2A and ∆lpf1A∆lpf2A mutants to BALB/c mice increased by 0.38 and 0.15 logs, respectively, whereas that of wild-type strain MPJS13 was 8.69 logs. In contrast, LPF1 deficiency significantly enhanced the LPS/TLR4-mediated inflammatory response in mammary epithelial cells, and the LD50 of the mutant decreased to 8.18 logs. In conclusion, our data suggested that LPF are important in MPEC colonization of mammary cells and may provide a benefit to bacterial intracellular survival that induces persistent bovine mastitis.

A theoretical study of atomically dispersed MN4/C (M = Fe or Mn) as a high-activity catalyst for the oxygen reduction reaction.

Carbon-based, non-noble metal catalysts for the oxygen reduction reaction (ORR) are crucial for the large-scale application of metal-air batteries and fuel cells. Density functional theory calculations were performed to explore the potential of atomically dispersed MN4/C (M = Fe or Mn) as an ORR catalyst in an acidic electrolyte and the ORR mechanism on MN4/C was systematically studied. The results indicated MN4 as the active site of MN4/C and a four-electron OOH transformation pathway as the preferred ORR mechanism on the MN4/C surface. The Gibbs free energy diagram showed that the rate-determining step of the FeN4/C and MnN4/C catalysts is the formation of the second H2O molecule and OOH*, respectively. FeN4/C exhibited higher thermodynamic limiting potential (0.79 V) and, thus, higher ORR activity than MnN4/C (0.52 V) in an acidic environment; its excellent catalytic performance is due to the nice electron structure and adsorption properties of the FeN4 site. Therefore, this work demonstrates that atomically dispersed MN4/C is a promising catalyst for the ORR.

Long polar fimbriae contribute to pathogenic Escherichia coli infection to host cells.

Long polar fimbria (LPF) is one of the few fimbrial adhesins of enterohemorrhagic Escherichia coli (E. coli) O157:H7 associated with colonization on host intestine, and both two types of LPF (including LPF1 and LPF2) play essential roles during the bacterial infection process. Though the fimbriae had been well studied in intestinal pathogenic E. coli strains, new evidences from our research revealed that it might be the key virulence for bovine mastitis pathogenic E. coli (MPEC) as well. This article summarizes the current knowledge on the LPF in E. coli, focusing on its genetic characteristics, prevalence, expression regulation, and adherence mechanism in different pathotypes of E. coli strains.

Development of a novel oil-in-water emulsion and evaluation of its potential adjuvant function in a swine influenza vaccine in mice.

BACKGROUND: Vaccination is the principal strategy for prevention and control of diseases, and adjuvant use is an effective strategy to enhance vaccine efficacy. Traditional mineral oil-based adjuvants have been reported with post-immunization reactions. Developing new adjuvant formulations with improved potency and safety will be of great value. RESULTS: In the study reported herein, a novel oil-in-water (O/W) Emulsion Adjuvant containing Squalane (termed EAS) was developed, characterized and investigated for swine influenza virus immunization. The data show that EAS is a homogeneous nanoemulsion with small particle size (~ 105 nm), low viscosity (2.04 ± 0.24 cP at 20 °C), excellent stability (at least 24 months at 4 °C) and low toxicity. EAS-adjuvanted H3N2 swine influenza vaccine was administrated in mice subcutaneously to assess the adjuvant potency of EAS. The results demonstrated that in mice EAS-adjuvanted vaccine induced significantly higher titers of hemagglutination inhibition (HI) and IgG antibodies than water-in-oil (W/O) vaccines or antigen alone, respectively, at day 42 post vaccination (dpv) (P < 0.05). EAS-adjuvanted vaccine elicited significantly stronger IgG1 and IgG2a antibodies and higher concentrations of Th1 (IFN-γ and IL-2) cytokines compared to the W/O vaccine or antigen alone. Mice immunized with EAS-adjuvanted influenza vaccine conferred potent protection after homologous challenge. CONCLUSION: The O/W emulsion EAS developed in the present work induced potent humoral and cellular immune responses against inactivated swine influenza virus, conferred effective protection after homologous virus challenge and showed low toxicity in mice, indicating that EAS is as good as the commercial adjuvant MF59. The superiority of EAS to the conventional W/O formulation in adjuvant activity, safety and stability will make it a potential veterinary adjuvant.

Tbx3 improves the germ-line competency of induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells can be obtained by the introduction of defined factors into somatic cells. The combination of Oct4 (also known as Pou5f1), Sox2 and Klf4 (which we term OSK) constitutes the minimal requirement for generating iPS cells from mouse embryonic fibroblasts. These cells are thought to resemble embryonic stem cells (ESCs) on the basis of global gene expression analyses; however, few studies have tested the ability and efficiency of iPS cells to contribute to chimaerism, colonization of germ tissues, and most importantly, germ-line transmission and live birth from iPS cells produced by tetraploid complementation. Using genomic analyses of ESC genes that have roles in pluripotency and fusion-mediated somatic cell reprogramming, here we show that the transcription factor Tbx3 significantly improves the quality of iPS cells. iPS cells generated with OSK and Tbx3 (OSKT) are superior in both germ-cell contribution to the gonads and germ-line transmission frequency. However, global gene expression profiling could not distinguish between OSK and OSKT iPS cells. Genome-wide chromatin immunoprecipitation sequencing analysis of Tbx3-binding sites in ESCs suggests that Tbx3 regulates pluripotency-associated and reprogramming factors, in addition to sharing many common downstream regulatory targets with Oct4, Sox2, Nanog and Smad1. This study underscores the intrinsic qualitative differences between iPS cells generated by different methods, and highlights the need to rigorously characterize iPS cells beyond in vitro studies.

Patient-specific induced-pluripotent stem cells-derived cardiomyocytes recapitulate the pathogenic phenotypes of dilated cardiomyopathy due to a novel DES mutation identified by whole exome sequencing.

In this paper, we report a novel heterozygous mutation of A285V codon conversion on exon 4 of the desmin (DES), using whole exome sequencing (WES) in an isolated proband with documented dilated cardiomyopathy (DCM). This mutation is predicted to cause three-dimensional structure changes of DES. Immunohistological and electron microscopy studies demonstrated diffuse abnormal DES aggregations in DCM-induced-pluripotent stem cell (iPSC)-derived cardiomyocytes, and control-iPSC-derived cardiomyocytes transduced with A285V-DES. DCM-iPSC-derived cardiomyocytes also exhibited functional abnormalities in vitro. This is the first demonstration that patient-specific iPSC-derived cardiomyocytes can be used to provide histological and functional confirmation of a suspected genetic basis for DCM identified by WES.

The effects of H3N2 swine influenza virus infection on TLRs and RLRs signaling pathways in porcine alveolar macrophages.

BACKGROUND: Swine influenza is an economically important respiratory disease of swine resulting from infection with influenza A virus. Swine influenza virus (SIV) becomes the focus as pigs have been hypothesized to serve as an intermediate host for the adaptation of avian influenza viruses to humans or as mixing vessels for the generation of genetically reassortant viruses. The ability of the innate immune system to detect and respond to pathogens is important for survival. Therefore, there is a critical need to evaluate the immediate response to viral infection, especially the role of the toll-like receptors (TLRs) and RNA helicase RIG-I-like receptors (RLRs) innate immunity signaling pathways in H3N2 swine influenza virus infection. METHOD: In this study, porcine alveolar macrophages (PAMs) were obtained from porcine lungs and were infected with SIV at a multiplicity of infection (MOI) of 5 in vitro. The changes of the related receptors, signaling proteins and effector molecules of TLRs and RLRs signaling pathways post H3N2 virus infection of PAMs were quantified by Real-time quantitative RT-PCR and western blotting. RESULTS: The results showed that H3N2 SIV infection significantly increased mRNA expression of TLR-3, TLR-7, RIG- I and MDA5 after 4 hpi (P < 0.05). Western blotting showed that the protein levels of TLR-3, TLR-7 and RIG-I also had a significantly increase after PAM exposed to virus. A significant change of MyD88, MAVS, IRF-3 and IRF-7 mRNA expression were present at 8 hpi. More than a 4-fold increase was induced for TNF-α and IL-1ß mRNA expression. And the concentration of TNF-α and IL-1ß peaked at 12 and 24 hpi, respectively. IFN-α, IFN-ß mRNA and protein levels increased after SIV infection and significant differences was observed at 8, 12 and 24 hpi. CONCLUSION: These results indicate that H3N2 swine influenza virus infection significantly influences the expression of the receptors, adapter proteins and downstream effector molecules of RLRs and TLRs signaling pathways. This study enhances our understanding of innate immunity signaling pathways in PAM anti-infection of H3N2 SIV.

Prevalence and fimbrial genotype distribution of poultry Salmonella isolates in China (2006 to 2012).

In this study, a total of 323 Salmonella enterica strains were isolated from 3,566 rectal swab samples of 51 poultry farms in seven regions of 12 provinces of China between 2006 and 2012. The prevalences of Salmonella sp. carriage were 12.4% in geese (66 positive/533 samples), 10.4% in turkeys (32/309), 9.8% in chickens (167/1,706), 6.8% in ducks (41/601), and 4.1% in pigeons (17/417), respectively. These isolates belonged to 20 serovars, in which the most frequent serovars were S. enterica serovar Gallinarum biovar Pullorum (herein, S. Pullorum) (55 isolates, 17.0%), S. enterica serovar Typhimurium (50 isolates, 15.5%), and S. enterica serovar Enteritidis (39 isolates, 12.1%). Overall, S. Typhimurium was the most commonly detected serovar; among the individual species, S. Pullorum was most commonly isolated from chickens, S. Enteritidis was most common in ducks, S. Typhimurium was most common in geese and pigeons, and S. enterica serovar Saintpaul was most common in turkeys. PCR determination of 20 fimbrial genes demonstrated the presence of bcfD, csgA, fimA, stdB, and sthE genes and the absence of staA and stgA genes in these isolates, and other loci were variably distributed, with frequency values ranging from 11.8 to 99.1%. These 323 Salmonella isolates were subdivided into 41 different fimbrial genotypes, and of these isolate, 285 strains (88.2%) had 12 to 14 fimbrial genes. Our findings indicated that the Salmonella isolates from different poultry species were phenotypically and genetically diverse and that some fimbrial genes are more frequently associated with serovars or serogroups.