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Acetaminophen (APAP) overdose can cause severe liver injury and APAP-induced liver injury (AILI) is one of the leading causes of acute liver failure (ALF). Bruton's tyrosine kinase (BTK) is a key tyrosine kinase in immune responses, which plays an important role in many inflammatory diseases. However, its effect on AILI is still not clear. Here, we aimed to assess the effect of BTK on AILI and explore its underlying mechanism.In our study, western blot and immunohistochemistry were used to detect the expression of BTK in AILI. The C57BL/6 mice were used to check the protective effect of BTK inhibition on AILI and the activation of BTK was confirmed in mice macrophages treated with APAP. Immunofluorescence, immunohistochemistry, oxygen consumption rate (OCR) detection, polymerase chain reaction (PCR), flow cytometry and western blot were used to determine the role of BTK in mitochondrial dynamics and function of macrophages and the underlying mechanisms in AILI.Our results showed that BTK upregulated in AILI. BTK inhibition protected mice from AILI and BTK was activated in mice macrophages in response to APAP. Mechanically, BTK inhibition promoted mitochondrial fusion and restored mitochondrial function through phospholipase C gamma 2 (PLCγ2)-reactive oxygen species (ROS)-Optic Atrophy 1(OPA1) pathway in macrophages and finally suppressed the release of proinflammatory cytokines.In conclusion, we found that BTK inhibition protected mice from AILI by restoring the mitochondrial function of macrophages through the improvement of the mitochondrial dynamic imbalance via PLCγ2-ROS-OPA1 signaling pathway, which indicated that BTK might be a potential therapeutic target of AILI.

Genetic screening based on the clustered regularly interspaced palindromic repeat (CRISPR) system has been indicated to be a powerful tool for identifying regulatory genes or cis-elements. However, when applying CRISPR screens to pinpoint functional elements at particular loci, a large number of guide RNA (gRNA) spacers may be required to achieve saturated coverage. Here, we present a controlled template-dependent elongation (CTDE) method relying on reversible terminators to synthesize gRNA libraries with genomic regions of interest. By applying this approach to H3K4me3 chromatin immunoprecipitation (ChIP)-derived DNA of mammalian cells, mega-sized gRNA libraries were synthesized in a tissue-specific manner, with which we conducted screening experiments to annotate essential sites for cell proliferation. Additionally, we confirmed that an essential site within the intron of LINC00339 regulates its own mRNA and that LINC00339 is a novel regulator of the cell cycle that maintains HepG2 proliferation. The CTDE method has the potential to be automated with high efficiency at low cost, and will be widely used to identify functional elements in mammalian genomes.

Nanobodies that neutralize Monoclonal antibodies that bind to the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) show therapeutic promise but must be produced in mammalian cells and need to be delivered intravenously. By contrast, single-domain antibodies called nanobodies can be produced in bacteria or yeast, and their stability may enable aerosol delivery. Two papers now report nanobodies that bind tightly to spike and efficiently neutralize SARS-CoV-2 in cells. Schoof et al. screened a yeast surface display of synthetic nanobodies and Xiang et al. screened anti-spike nanobodies produced by a llama. Both groups identified highly potent nanobodies that lock the spike protein in an inactive conformation. Multivalent constructs of selected nanobodies achieved even more potent neutralization. Science , this issue p. 1473 , p. 1479

Summary Therapeutic and diagnostic efficacies of small biomolecules and chemical compounds are hampered by suboptimal pharmacokinetics. Here, we developed a repertoire of robust and high-affinity antihuman serum albumin nanobodies (NbHSA) that can be readily fused to small biologics for half-life extension. We characterized the thermostability, binding kinetics, and cross-species reactivity of NbHSAs, mapped their epitopes, and structurally resolved a tetrameric HSA-Nb complex. We parallelly determined the half-lives of a cohort of selected NbHSAs in an HSA mouse model by quantitative proteomics. Compared to short-lived control nanobodies, the half-lives of NbHSAs were drastically prolonged by 771-fold. NbHSAs have distinct and diverse pharmacokinetics, positively correlating with their albumin binding affinities at the endosomal pH. We then generated stable and highly bioactive NbHSA-cytokine fusion constructs “Duraleukin” and demonstrated Duraleukin's high preclinical efficacy for cancer treatment in a melanoma model. This high-quality and versatile Nb toolkit will help tailor drug half-life to specific medical needs., Graphical abstract, Highlights • We provide a resource of high-affinity and versatile albumin nanobodies for drug delivery • We systematically map albumin nanobody epitopes by hybrid structural approaches • We parallelly measure the pharmacokinetics of nanobodies in a humanized mouse model • We develop nanobody-cytokine conjugates “Duraleukin” for cancer immunotherapy, Drug delivery system; Biochemical engineering; Biotechnology; Structural biology; Proteomics

Therapeutic and diagnostic efficacies of numerous small biomolecules and chemical compounds are hampered by poor pharmacokinetics. Here we developed a repertoire of distinct and high-affinity albumin-nanobodies (NbHSA) to facilitate drug delivery. Using biophysics and hybrid structural methods, we have systematically characterized the Nb repertoire, mapped the epitopes, and resolved the architecture of a tetrameric Nb-albumin complex. We employed quantitative proteomics for accurate and multiplex pharmacokinetic analysis of dozens of diverse and high-quality NbHSA and confirmed the most stable construct has a 771-fold T1/2 improvement compared to non-albumin binding Nbs. Interestingly, the pharmacokinetics of NbHSA is related to their biophysical and structural properties. To demonstrate the utility of NbHSA, we developed stable NbHSA-cytokine conjugates “Duraleukins” and confirmed the high anticancer efficacy of a Duraluekin in vivo. This high-quality Nb resource may help advance research into novel biotherapeutics.

Epigenome is the set of DNA-associated proteins or chemical modifications to DNA, which regulates gene expression in processes of development and disease. While current advances have allowed researchers to routinely profile epigenomes from given samples, our understandings of the functions of epigenetic hallmarks are nonspecific at best. Applying CRISPR-screening to genome-widely interrogate the function of individual epigenetic hallmarks demands massive sgRNA libraries which are unaffordable via commercial syntheses. Our development consists of a high throughput and cost-effective controlled template-dependent elongation (CTDE) approach which converts source DNA to sgRNA templates. Affiliated screenings encompass 3.8M sgRNAs generated by CTDE targeting all major H3K4me3 and CTCF hallmarks in mESCs and HepG2 and identified 20K essential epigenetic hallmarks, which render the first batch of functional epigenome annotation of H3K4me3 and CTCF hallmarks in mammals. As an application example, we show that a H3K4me3 hallmark orchestrates CDC42 level and cell-cycle progression through promoting LINC00339 expression in HepG2.

Therapeutic and diagnostic efficacies of numerous small biomolecules and chemical compounds are hampered by the short half-lives. Here we report the development of a repertoire of diverse, high-affinity albumin-nanobodies (NbHSA) to facilitate drug delivery. By integrating biophysics, and hybrid structural biology, we have systematically characterized the NbHSAfor albumin binding, mapped the epitopes, and resolved the architecture of a tetrameric Nb-albumin complex. We employed quantitative proteomics for accurate, multiplex Nb pharmacokinetic analysis. Using a humanized albumin mouse model, we found that the NbHSAhas outstanding pharmacokinetics; the most stable NbHSAhas a 771-fold T1/2improvement compared with a control Nb. Interestingly, the pharmacokinetics of NbHSAis related to their biophysical and structural properties. To demonstrate the utility of NbHSA, we developed a highly stable NbHSA-hIL-2 cytokine conjugate “Duraleukin” and confirmed its improved anticancer properties than hIL-2 alone. We envision that this high-quality Nb resource will advance research into novel biotherapeutics.

Current single-cell RNA-seq approaches are hindered by preamplification bias, loss of strand of origin information, and the inability to observe small-RNA and mRNA dual transcriptomes. Here, we introduce a single-cell holo-transcriptome sequencing (Holo-Seq) that overcomes all three hurdles. Holo-Seq has the same quantitative accuracy and uniform coverage with a complete strand of origin information as bulk RNA-seq. Most importantly, Holo-Seq can simultaneously observe small RNAs and mRNAs in a single cell. Furthermore, we acquire small RNA and mRNA dual transcriptomes of 32 human hepatocellular carcinoma single cells, which display the genome-wide super-enhancer activity and hepatic neoplasm kinetics of these cells. Electronic supplementary material The online version of this article (10.1186/s13059-018-1553-7) contains supplementary material, which is available to authorized users.

Aim: The aim was to highlight the clinical complications and to evaluate risk factors of mortality in Chinese HIV/TB patients. Methods: The etiology of clinical deterioration of Chinese HIV/TB patients were evaluated in 180 HIV-infected patients admitted in the Beijing Ditan Hospital between 1 January 2012 and 30 April 2014. Results & conclusion: AIDS-defining illnesses (20.0%) were the most common complication, followed by TB-associated immune reconstitution inflammatory syndrome (16.6%), drug-induced liver injury (11.1%), drug rash (11.1%), non-AIDS-defining illness (5.6%), as well as highly active antiretroviral therapy resistance (3.3%). The risk factors for mortality were tuberculous meningitis associated immune reconstitution inflammatory syndrome (OR: 152.614; CI: 18.324–1263.615; p < 0.001) and non-AIDS-defining illnesses (OR: 114.133; CI: 12.939–1006.752; p < 0.001), which will help remind physicians of the risk of clinical deterioration in HIV/TB patients after antiretroviral therapy in China.

Review history. (DOCX 53 kb)

Figure S1. Carrier RNAs sequences and gel images of in vitro digestions. Figure S2. Holo-mRNA-Seq flowchart. Figure S3. RPKM scatterplots of expressed genes after Not I and Cas9 digestion. Figure S4. RPKM scatterplots of expressed genes. Figure S5. Hierarchical clustering of mRNA transcriptomes. Figure S6. Hierarchical clustering heatmap of mESC bulk-mRNA-Seq, mESC Holo-Seq (1Â ng total RNA and single-cell total RNA), and mESC Smart-Seq2 (1Â ng total RNA and single-cell total RNA). Figure S7. Comparison of Holo-mRNA-Seq with Smart-Seq2 coupled with Nextera XT workflow. Figure S8. The saturation curves of Holo-Seq, Smart-Seq2, SUPeR-Seq and MATQ-Seq. Figure S9. Holo-Seq flowchart for total RNA with a complete strand of origin information. Figure S10. Hierarchical clustering of expressed genes and antisense transcripts. Figure S11. Signal plot of the Rpe locus by Holo-Seq. Figure S12. Comparison of the diversity of antisense transcripts and coding transcripts at similar expression level. Figure S13. RPKMs of mRNAs and introns of selected core genes and housekeeping genes. Figure S14. Holo-Seq flowchart for profiling small RNAs. Figure S15. The saturation curves of miRNA. Figure S16. RPM scatterplots of expressed small RNAs. Figure S17. Relative expression heat maps of super-enhancer-regulated master miRNAs and mRNAs. Figure S18. Hematoxylin and Eosin (HE) staining of the HCC tissue. Figure S19. Relative expression levels of gene groups between HCC Exp-subpopulations. Figure S20. mRNA capture sequencing of the Holo-Seq total RNA library. Figure S21. mRNA and miRNA solo transcriptome analyses of hepatocellular carcinoma (HCC) single cells. (DOCX 5908 kb)

Step-by-step Holo-Seq protocols. (DOCX 29 kb)