Intermittent Hypobaric Hypoxia Ameliorates Autistic-Like Phenotypes in Mice.

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by persistent deficits in social communication and stereotyped behaviors. Although major advances in basic research on autism have been achieved in the past decade, and behavioral interventions can mitigate the difficulties that individuals with autism experience, little is known about the many fundamental issues of the interventions, and no specific medication has demonstrated efficiency for the core symptoms of ASD. Intermittent hypobaric hypoxia (IHH) is characterized by repeated exposure to lowered atmospheric pressure and oxygen levels, which triggers multiple physiological adaptations in the body. Here, using two mouse models of ASD, male Shank3B -/- and Fmr1 -/y mice, we found that IHH training at an altitude of 5,000 m for 4 h per day, for 14 consecutive days, ameliorated autistic-like behaviors. Moreover, IHH training enhanced hypoxia inducible factor (HIF) 1α in the dorsal raphe nucleus (DRN) and activated the DRN serotonergic neurons. Infusion of cobalt chloride into the DRN, to mimic IHH in increasing HIF1α expression or genetically knockdown PHD2 to upregulate HIF1α expression in the DRN serotonergic neurons, alleviated autistic-like behaviors in Shank3B -/- mice. In contrast, downregulation of HIF1α in DRN serotonergic neurons induced compulsive behaviors. Furthermore, upregulating HIF1α in DRN serotonergic neurons increased the firing rates of these neurons, whereas downregulation of HIF1α in DRN serotonergic neurons decreased their firing rates. These findings suggest that IHH activated DRN serotonergic neurons via upregulation of HIF1α, and thus ameliorated autistic-like phenotypes, providing a novel therapeutic option for ASD.

Suppression of innate and acquired immunity in severe hand foot and mouth disease caused by EV71 infections in children.

Hand, foot, and mouth disease (HFMD) is a common children infectious disease caused by human enteroviruses. Most of the cases have minimal symptoms, however, some patients may develop serious neurological, cardiac complications, or even death. The pathological mechanism leading to severe HFMD is not clearly understood, and the immunological status of the individual patient may play an important role. Transcriptomes of peripheral blood mononuclear cells from EV71-infected patients (n = 45) and healthy controls (n = 36) were examined. Immune pathways were up-regulated in patients with mild disease symptoms (n = 11, M) compared to the healthy controls (n = 36, H), demonstrating an effective anti-viral response upon EV71 infection. However, in patients with severe symptoms (n = 23, S) as well as severe patients following treatment (n = 11, A), their innate and acquired immune pathways were down-regulated, indicating a global immunity suppression. Such immune suppression characteristics could thus provide an opportunity for early EV-71 infection prognosis prediction. Based on our cohort, an SVM model using RNA-seq expression levels of five genes (MCL1, ZBTB37, PLEKHM1P, IFNAR2 and YEATS2) was developed and achieved a high ROC-AUC (91·3%) in predicting severe HFMD. Meanwhile, qPCR fold-changes method was performed based three genes (MCL1, IFNAR2 and YEATS2) on additional cohort. This qPCR method achieved a ROC-AUC of 78.6% in predicting severe HFMD, which the patients could be distinguished in 2-3 h. Therefore, our models demonstrate the possibility of HFMD severity prediction based on the selected biomarkers that predict severe HFMD effectively.

Synthesis and biological evaluation of water-soluble derivatives of chiral gossypol as HIV fusion inhibitors targeting gp41.

A series of novel or known water-soluble derivatives of chiral gossypol were synthesized and screened in vitro for their anti-HIV-1 activity. (-)-gossypol derivative was more active against HIV-1 than the corresponding (+)-gossypol derivative, respectively. Among these derivatives, d-glucosamine derivative of (-)-gossypol, oligopeptide derivative of (-)-gossypol and taurine derivative of (-)-gossypol, such as compounds 1a, 3a and 14a, showed significant inhibitory activities against HIV-1 replication, HIV-1 mediated cell-cell fusion and HIV gp41 6-helix bundle formation as some amino acid derivatives of (-)-gossypol.

Enantioselective inhibition of reverse transcriptase (RT) of HIV-1 by non-racemic indole-based trifluoropropanoates developed by asymmetric catalysis using recyclable organocatalysts.

Herein, we report the development of efficient inhibitors of reverse transcriptase (RT) of HIV-1 based on indole-alkyl trifluoropyruvate derivatives by a TZM-bl cell assay. The inhibitory activities of the two enantiomers and the corresponding racemic mixture have been compared. TZM-bl cells exhibited strong enantioselective discrimination for the (R)-configuration, among these indole derivatives, the most active compound R-12, with a 5-NO2 substituent, gave the best result when tested in the TZM-bl cells on HIV virus type HIV-1IIIB, with an EC50 value of 0.019 µM, CC50 value of 210.697 µM and SI (selectivity index, CC50/EC50) value of 11,089, respectively. The cell test showed that, in most cases, the R-enantiomer was superior to the Rac-mixture, which was better than the corresponding S-enantiomer. The results indicated that the R-enantiomer is the most favorable configuration as an efficient HIV-1 inhibitor. Molecular modeling studies suggested a structural basis for the enantioselectivity of RT towards this class of molecules.

Correlating electronic transport to atomic structures in self-assembled quantum wires.

Quantum wires, as a smallest electronic conductor, are expected to be a fundamental component in all quantum architectures. The electronic conductance in quantum wires, however, is often dictated by structural instabilities and electron localization at the atomic scale. Here we report on the evolutions of electronic transport as a function of temperature and interwire coupling as the quantum wires of GdSi(2) are self-assembled on Si(100) wire-by-wire. The correlation between structure, electronic properties, and electronic transport are examined by combining nanotransport measurements, scanning tunneling microscopy, and density functional theory calculations. A metal-insulator transition is revealed in isolated nanowires, while a robust metallic state is obtained in wire bundles at low temperature. The atomic defects lead to electron localizations in isolated nanowire, and interwire coupling stabilizes the structure and promotes the metallic states in wire bundles. This illustrates how the conductance nature of a one-dimensional system can be dramatically modified by the environmental change on the atomic scale.

A novel computational method enables RNA editome profiling during human hematopoiesis from scRNA-seq data.

RNA editing is a post-transcriptional modification with a cell-specific manner and important biological implications. Although single-cell RNA-seq (scRNA-seq) is an effective method for studying cellular heterogeneity, it is difficult to detect and study RNA editing events from scRNA-seq data because of the low sequencing coverage. To overcome this, we develop a computational method to systematically identify RNA editing sites of cell types from scRNA-seq data. To demonstrate its effectiveness, we apply it to scRNA-seq data of human hematopoietic stem/progenitor cells (HSPCs) with an annotated lineage differentiation relationship according to previous research and study the impacts of RNA editing on hematopoiesis. The dynamic editing patterns reveal the relevance of RNA editing on different HSPCs. For example, four microRNA (miRNA) target sites on 3' UTR of EIF2AK2 are edited across all HSPC populations, which may abolish the miRNA-mediated inhibition of EIF2AK2. Elevated EIF2AK2 may thus activate the integrated stress response (ISR) pathway to initiate global translational attenuation as a protective mechanism to maintain cellular homeostasis during HSPCs' differentiation. Besides, our findings also indicate that RNA editing plays an essential role in the coordination of lineage commitment and self-renewal of hematopoietic stem cells (HSCs). Taken together, we demonstrate the capacity of scRNA-seq data to exploit RNA editing events of cell types, and find that RNA editing may exert multiple modules of regulation in hematopoietic processes.

Stemness-related genes revealed by single-cell profiling of naïve and stimulated human CD34+ cells from CB and mPB.

BACKGROUND: Hematopoietic stem cells (HSCs) from different sources show varied repopulating capacity, and HSCs lose their stemness after long-time ex vivo culture. A deep understanding of these phenomena may provide helpful insights for HSCs. METHODS: Here, we applied single-cell RNA-seq (scRNA-seq) to analyse the naïve and stimulated human CD34+ cells from cord blood (CB) and mobilised peripheral blood (mPB). RESULTS: We collected over 16 000 high-quality single-cell data to construct a comprehensive inference map and characterised the HSCs under a quiescent state on the hierarchy top. Then, we compared HSCs in CB with those in mPB and HSCs of naïve samples to those of cultured samples, and identified stemness-related genes (SRGs) associated with cell source (CS-SRGs) and culture time (CT-SRGs), respectively. Interestingly, CS-SRGs and CT-SRGs share genes enriched in the signalling pathways such as mRNA catabolic process, translational initiation, ribonucleoprotein complex biogenesis and cotranslational protein targeting to membrane, suggesting dynamic protein translation and processing may be a common requirement for stemness maintenance. Meanwhile, CT-SRGs are enriched in pathways involved in glucocorticoid and corticosteroid response that affect HSCs homing and engraftment. In contrast, CS-SRGs specifically contain genes related to purine and ATP metabolic process, which is crucial for HSC homeostasis in the stress settings. Particularly, when CT-SRGs are used as reference genes for the construction of the development trajectory of CD34+ cells, lymphoid and myeloid lineages are clearly separated after HSCs/MPPs. Finally, we presented an application through a small-scale drug screening using Connectivity Map (CMap) against CT-SRGs. A small molecule, cucurbitacin I, was found to efficiently expand HSCs ex vivo while maintaining its stemness. CONCLUSIONS: Our findings provide new perspectives for understanding HSCs, and the strategy to identify candidate molecules through SRGs may be applicable to study other stem cells.

Synthesis and antiviral activities of novel gossypol derivatives.

In this study, a series of novel gossypol derivatives were synthesized and screened in vitro for their anti-HIV-1 and anti-H(5)N(1) activities, respectively. Replacing the aldehyde groups of gossypol with some amino acids not only reduced the cytotoxicity but also enhanced the activities against HIV-1 and H(5)N(1). Compounds 13-17 showed more potent activities against HIV-1 and H(5)N(1) than the other gossypol derivatives. Meanwhile, these compounds also exhibited more potent activities against H(5)N(1) than 1-adamantylamine. The absence of the COONa group in gossypol derivatives resulted in a loss of anti-HIV-1 activity, suggesting that this group might play an important role in mediating the antiviral activity. Time-of-addition assays indicated that compounds 13-17 had the similar mechanism of anti-HIV-1 action with T20. Molecular modeling analysis demonstrated that compounds 13-17 could fit inside the gp41 hydrophobic pocket through hydrogen bonding network, hydrophobic contacts and strong electrostatic interactions.

Restoration of ß-Globin Expression with Optimally Designed Lentiviral Vector for ß-Thalassemia Treatment in Chinese Patients.

ß-Thalassemia is one of the most prevalent genetic diseases worldwide. The current treatment for ß-thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of matched donors. Gene therapy has been developed as an alternative therapeutic option for transfusion-dependent ß-thalassemia (TDT). However, successful gene therapy for ß-thalassemia patients in China has not been reported. Here, we present the results of preclinical studies of an optimally designed lentiviral vector (LV) named LentiHBBT87Q in hematopoietic stem and progenitor cells (HSPCs) derived from Chinese TDT patients. LentiHBBT87Q was selected from a series of LVs with optimized backbone and de novo cloning strategy. It contains an exogenous T87Q ß-globin gene (HBBT87Q) driven by a specific reconstituted locus control region, and efficiently expresses HBB mRNA and HBB protein in erythroblasts derived from cord blood HSPCs. To facilitate clinical transformation, we manufactured clinical-grade LentiHBBT87Q (cLentiHBBT87Q) and optimized its transduction procedure. Importantly, transduction of cLentiHBBT87Q restored expression of HBB monomer and adult hemoglobin tetramer to relatively normal level in erythroblasts from bone marrow HSPCs of Chinese TDT patients that carry the most common mutation types and cover various genotypes, including ß0/ß0. Furthermore, viral integration sites (VISs) of cLentiHBBT87Q were similar to other LVs safely used in previous clinical trials, and gene-ontology (term) analysis of VIS targeted genes suggests that no tumor-associated pathways were enriched in treated samples. Taken together, we have engineered the cLentiHBBT87Q that can restore ß-globin expression in the HSPCs-derived erythroblasts of Chinese TDT patients with minimal risk of tumorigenesis, providing a favorable starting point for future clinical application.

The route of inoculation determines the tissue tropism of modified vaccinia Tiantan expressing the spike glycoprotein of SARS-CoV in mice.

The live replication-competent modified vaccinia virus Tiantan (MVTT) is an attractive vaccine vector, yet little is known about its tissue tropism and pathology in vivo. Recently, we demonstrated that a recombinant MVTT expressing the spike glycoprotein of SARS-CoV (namely MVTT-S) is superior to the non-replicating modified vaccinia Ankara (MVA-S) for inducing high level of neutralizing antibodies through mucosal vaccination. In this study, we further determined the tissue tropism and safety of MVTT-S after the vaccine was administrated through various routes including: intramuscular (i.m.), intranasal (i.n.), and intravaginal (i.vag.) inoculations, respectively. Using real-time PCR, nested PCR, immunohistochemistry and in situ hybridization assays, we found that MVTT-S was able to produce a transient infection in all cases within 48 hr post-inoculation, yet the major site of viral replication in various tissues or organs was dependent on the route of viral administration. We demonstrated that i.m. injection of MVTT-S primarily targeted draining inguinal lymph nodes, whereas mucosal inoculation had broader range of tissue infections. i.n. inoculation involved infections in lungs, kidneys, spleens and cervix lymph nodes while i.vag. administration targeted uteruses, ovaries, kidneys and spleens. Critically, the infection did not cause severe pathogenic consequences in infected tissues, which was consistent to the attenuated phenotype of MVTT-S. Our findings have implications for the optimization of vaccination route and for studies on the correlation between the magnitude of immune responses and the extent of tissue involvement in vivo.

Transcriptome Analyses of ß-Thalassemia -28(A>G) Mutation Using Isogenic Cell Models Generated by CRISPR/Cas9 and Asymmetric Single-Stranded Oligodeoxynucleotides (assODNs).

ß-thalassemia, caused by mutations in the human hemoglobin ß (HBB) gene, is one of the most common genetic diseases in the world. The HBB -28(A>G) mutation is one of the five most common mutations in Chinese patients with ß-thalassemia. However, few studies have been conducted to understand how this mutation affects the expression of pathogenesis-related genes, including globin genes, due to limited homozygote clinical materials. Therefore, we developed an efficient technique using CRISPR/Cas9 combined with asymmetric single-stranded oligodeoxynucleotides (assODNs) to generate a K562 cell model with HBB -28(A>G) named K562-28(A>G). Then, we systematically analyzed the differences between K562-28(A>G) and K562 at the transcriptome level by high-throughput RNA-seq before and after erythroid differentiation. We found that the HBB -28(A>G) mutation not only disturbed the transcription of HBB, but also decreased the expression of HBG, which may further aggravate the thalassemia phenotype and partially explain the more severe clinical outcome of ß-thalassemia patients with the HBB -28(A>G) mutation. Moreover, we found that the K562-28(A>G) cell line is more sensitive to hypoxia and shows a defective erythrogenic program compared with K562 before differentiation. Importantly, all abovementioned abnormalities in K562-28(A>G) were reversed after correction of this mutation with CRISPR/Cas9 and assODNs, confirming the specificity of these phenotypes. Overall, this is the first time to analyze the effects of the HBB -28(A>G) mutation at the whole-transcriptome level based on isogenic cell lines, providing a landscape for further investigation of the mechanism of ß-thalassemia with the HBB -28(A>G) mutation.

DNB-based on-chip motif finding: A high-throughput method to profile different types of protein-DNA interactions.

Here, we report a sensitive DocMF system that uses next-generation sequencing chips to profile protein-DNA interactions. Using DocMF, we successfully identified a variety of endonuclease recognition sites and the protospacer adjacent motif (PAM) sequences of different CRISPR systems. DocMF can simultaneously screen both 5' and 3' PAMs with high coverage. For SpCas9, we found noncanonical 5'-NAG-3' (~5%) and 5'-NGA-3' (~1.6%), in addition to its common PAMs, 5'-NGG-3' (~89.9%). More relaxed PAM sequences of two uncharacterized Cas endonucleases, VeCas9 and BvCas12a, were extensively characterized using DocMF. Moreover, we observed that dCas9, a DNA binding protein lacking endonuclease activity, preferably bound to the previously reported 5'-NGG-3' sequence. In summary, our studies demonstrate that DocMF is the first tool with the capacity to exhaustively assay both the binding and the cutting properties of different DNA binding proteins.

Selective targeting of the oncogenic KRAS G12S mutant allele by CRISPR/Cas9 induces efficient tumor regression.

Rationale: KRAS is one of the most frequently mutated oncogenes in cancers. The protein's picomolar affinity for GTP/GDP and smooth protein structure resulting in the absence of known allosteric regulatory sites makes its genomic-level activating mutations a difficult but attractive target. Methods: Two CRISPR systems, genome-editing CRISPR/SpCas9 and transcription-regulating dCas9-KRAB, were developed to deplete the KRAS G12S mutant allele or repress its transcription, respectively, with the goal of treating KRAS-driven cancers. Results: SpCas9 and dCas9-KRAB systems with a sgRNA targeting the mutant allele blocked the expression of the mutant KRAS gene, leading to an inhibition of cancer cell proliferation. Local adenoviral injections using SpCas9 and dCas9-KRAB systems suppressed tumor growth in vivo. The gene-depletion system (SpCas9) performed more effectively than the transcription-suppressing system (dCas9-KRAB) on tumor inhibition. Application of both Cas9 systems to wild-type KRAS tumors did not affect cell proliferation. Furthermore, through bioinformatic analysis of 31555 SNP mutations of the top 20 cancer driver genes, the data showed that our mutant-specific editing strategy could be extended to a reference list of oncogenic mutations with high editing potentials. This pipeline could be applied to analyze the distribution of PAM sequences and survey the best alternative targets for gene editing. Conclusion: We successfully developed both gene-depletion and transcription-suppressing systems to specifically target an oncogenic KRAS mutant allele that led to significant tumor regression. These findings show the potential of CRISPR-based strategies for the treatment of tumors with driver gene mutations.

Serum-Free Culture System for Spontaneous Human Mesenchymal Stem Cell Spheroid Formation.

Human mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from a three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from a monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSC spheroids but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stem medium (MiPS) adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR-derived spheroids. Interestingly, genes related to inflammatory response, immune response, and angiogenesis were upregulated in spheroids at day 6 and qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were replated in normal FBS medium, cells formed a typical spindle-shaped morphology and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSC spheroids for clinical research and therapy.

The potent human immunodeficiency virus type 1 (HIV-1) entry inhibitor HR212 blocks formation of the envelope glycoprotein gp41 six-helix bundle.

HR212, a recombinant protein composed of the heptad repeat, is a rationally designed human immunodeficiency virus type 1 (HIV-1) fusion inhibitor. This protein can be easily produced by Escherichia coli at a low cost. Previously, studies indicated that HR212 can efficiently inhibit the entry and replication of both laboratory and clinical HIV-1 strains, and this protein is more stable and less sensitive to proteinases than T20. The procedure of HIV-1 entry into the host cells can be divided into three main steps: gp120-CD4 interactions, coreceptor binding, and gp41 six-helix bundle formation and subsequent membrane fusion. The present study demonstrates that HR212 does not block gp120-CD4 binding or interfere with binding to the coreceptors CXCR4 and CCR5. Instead, HR212 efficiently blocks the six-helix bundle formation between peptides derived from the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) region of gp41. Fluorescence native polyacrylamide gel electrophoresis (FN-PAGE) indicated that HR212 could form a complex with peptide N36 to block gp41 fusogenic core formation. These results suggest that HR212 inhibits HIV-1 entry by targeting the NHR region of gp41. Therefore, HR212 can potentially be developed as a novel, high-efficiency, specific HIV-1 entry inhibitor.

Amino acid derivatives of the (-) enantiomer of gossypol are effective fusion inhibitors of human immunodeficiency virus type 1.

T20 and maraviroc are the only two currently available entry inhibitors that have shown efficacy in treating HIV-1-infected individuals who have failed to respond to first-line antiretroviral drugs. Gossypol is a polyphenolic aldehyde extracted from cotton plants. By modifying the (-) enantiomer of gossypol with a series of small molecules, we have found that neutral amino acids with aliphatic group derivatives of (-) gossypol show the strongest inhibitory activity and the lowest cytotoxicity in vitro among all the derivatives tested. Additionally, the selectivity index of the (-) gossypol-neutral amino acid conjugates is increased 100-fold when compared with (-) gossypol alone. It is widely accepted that gossypol and gossypol derivatives inhibit HIV-1 replication by targeting reverse transcriptase. However, from the results of our time-of-addition assay, HIV-1-mediated cell fusion assay and VSV-G pseudotyped virus assay, we demonstrate that the alanine-(-) gossypol derivative ((-)G-Ala) is an effective HIV-1 entry inhibitor. Further mechanistic analysis revealed that (-)G-Ala neither blocks gp120-CD4 binding nor interacts with the HIV-1 co-receptor CXCR4. Results from sandwich ELISA, native-PAGE and circular dichroism (CD) show that (-)G-Ala inhibits the cell fusion-activated gp41 core domain. Moreover, (-)G-Ala binds to the HIV-5-Helix protein and blocking D-peptide (PIE7) binding to the hydrophobic pocket on the surface of the gp41 internal trimeric coiled-coil domain. The contraceptive properties of (-) gossypol and amino acid derivatives of (-) gossypol are also discussed. Collectively, our results indicate that (-)G-Ala may bind to the gp41 hydrophobic pocket and block the formation of the cell fusion-activated gp41 core to inhibit HIV-1-mediated membrane fusion and subsequent viral entry.

Structural and chemical properties of gold rare earth disilicide core-shell nanowires.

Clear understanding of the relationship between electronic structure and chemical activity will aid in the rational design of nanocatalysts. Core-shell Au-coated dysprosium and yttrium disilicide nanowires provide a model atomic scale system to understand how charges that transfer across interfaces affect other electronic properties and in turn surface activities toward adsorbates. Scanning tunneling microscopy data demonstrate self-organized growth of Au-coated DySi2 nanowires with a nanometer feature size on Si(001), and Kelvin probe force microscopy data measure a reduction of work function that is explained in terms of charge transfer. Density functional theory calculations predict the preferential adsorption site and segregation path of Au adatoms on Si(001) and YSi2. The chemical properties of Au-YSi2 nanowires are then discussed in light of charge density, density of states, and adsorption energy of CO molecules.