Biology subjects

Pan, X.

Publications and source records attributed to Pan, X..

At least 163 records · Page 9Linked to original sources

bioRxiv2021

Paired heavy and light chain signatures contribute to potent SARS-CoV-2 neutralization in public antibody responses

Understanding protective mechanisms of antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We discovered a new antibody, 910-30, that targets the SARS-CoV-2 ACE2 receptor binding site as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. We performed sequence and structural analyses to explore how antibody features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer revealed its binding interactions and ability to disassemble spike. Despite heavy chain sequence similarity, biophysical analyses of IGHV3-53/3-66 antibodies highlighted the importance of native heavy:light pairings for ACE2 binding competition and for SARS-CoV-2 neutralization. We defined paired heavy:light sequence signatures and determined antibody precursor prevalence to be ~1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These data reveal key structural and functional neutralization features in the IGHV3-53/3-66 public antibody class to accelerate antibody-based medical interventions against SARS-CoV-2. HighlightsO_LIA molecular study of IGHV3-53/3-66 public antibody responses reveals critical heavy and light chain features for potent neutralization C_LIO_LICryo-EM analyses detail the structure of a novel public antibody class member, antibody 910-30, in complex with SARS-CoV-2 spike trimer C_LIO_LICryo-EM data reveal that 910-30 can both bind assembled trimer and can disassemble the SARS-CoV-2 spike C_LIO_LISequence-structure-function signatures defined for IGHV3-53/3-66 class antibodies including both heavy and light chains C_LIO_LIIGHV3-53/3-66 class precursors have a prevalence of 1:44,000 B cells in healthy human antibody repertoires C_LI

immunology↗

bioRxiv2020

Automatic Recognition of Auditory Brainstem Response Characteristic Waveform based on BiLSTM

BackgroundAuditory brainstem response (ABR) test is widely used in newborn hearing screening and hearing disease diagnosis. Identifying and marking are challenging and repetitive tasks because of complex rules of ABR characteristic waveform and interference of background noise. MethodsThis study proposes an automatic method to recognize ABR characteristic waveform. First, binarization is created to mark 1024 sampling points accordingly. The selected characteristic area of ABR data is 0-8ms. The marking area is enlarged to expand feature information and reduce marking error. Second, a bi-directional long short-term memory (BiLSTM) network structure is established to improve relevance of sampling points, and an ABR sampling point classifier is obtained by training. Finally, mark points are obtained through thresholding. ResultsSpecific structure, related parameters, recognition effect, and noise resistance of network were explored in 614 sets of ABR clinical data, and recognition accuracy of waves I, III, and V can reach 92.91%. DiscussionThus, the proposed method can reduce the repetitive work of doctors and meet accuracy effectively. Therefore, this method has clinical potential.

bioinformatics↗

bioRxiv2020

Transferrin receptor (Tfr1) ablation in satellite cells impacts skeletal muscle regeneration through the activation of ferroptosis

Satellite cells (SCs) are critical to the postnatal development and skeletal muscle regeneration. Inactivation of SCs is linked with the skeletal muscle loss. Leveraging on the RNAseq screening, transferrin receptor (Tfr1) is identified to be associated with muscle/SC ageing and the declined regeneration potential. Muscle-specific deletion of Tfr1 results in the growth retardation, metabolic disorder and lethality, shedding light on the importance of Tfr1 in skeletal muscle physiology. Here, our investigation reported that conditional SC-ablation of Tfr1 leads to the SCs inactivation and skeletal muscle regeneration defects, followed by the labile iron accumulation, de novo lipogenesis via fibroadipogenic progenitors (FAPs) and Gpx4/Nrf2-mediated ROS-scavenger defects. These abnormal phenomena, such as Hmox1-mediated myoglobin degradation, Tfr1-Slc39a14 functional switch and the activation of unsaturated fatty acid biosynthesis pathway are orchestrated with the occurrence of ferroptosis in skeletal muscle. Ferroptosis may further prevent SC proliferation and skeletal muscle regeneration. Ferrostatin-1, a ferroptosis inhibitor could not rescue Tfr1-ablation induced ferroptosis. However, intramuscular administration of lentivirus expressing Tfr1 could partially reduce labile iron accumulation, decrease de novo lipogenesis and promote skeletal muscle regeneration. Most importantly, Tfr1/Slc39a14 functional switch, labile iron accumulation and fatty acid biosynthesis are recapitulated in aged skeletal muscle of rodents, indicating that ferroptosis occurs in the skeletal muscles of aged rodents. The present study also bridges the gap between pathogenesis of iron and functional defects in the skeletal muscle, providing mechanistic information to develop anti-aging strategies. One Sentence SummaryConditional ablation of Tfr1 in satellite cells (SCs) results in the SC inactivation, skeletal muscle regeneration defects, labile iron accumulation, and unsaturated fatty acid biosynthesis, leading to the activation of ferroptosis, which is recapitulated in skeletal muscles of aged rodents to be a new cell death form identified in skeletal muscle and sheds light on the development of novel anti-ageing strategies.

cell biology↗

bioRxiv2020

Necrotizing enterocolitis-induced systemic immune suppression in neonatal preterm pigs

ObjectivesPreterm infants are at high risks of sepsis and necrotising enterocolitis (NEC). Some develop sepsis shortly after suspected or confirmed NEC, implying that NEC may predispose to sepsis but the underlying mechanisms are unknown. Using NEC-sensitive preterm pigs as models, we investigated the immune status in animals with and without NEC. MethodsPreterm pigs (n=113, caesarean delivered at day 106) were reared until day 5 or 9. Blood was analyzed for T cell subsets, neutrophil phagocytosis, trans criptomics and immune responses to LPS challenge. Gut tissues were used for histology and cytokine analyses. Pigs with/without macroscopic NEC lesions were scored as healthy, mild or severe NEC. ResultsOverall NEC incidence was similar on days 5 and 9 (61-62%) with less severe lesions on day 9, implying gradual mucosal repair following the early phase of NEC on day 5. Pigs with NEC, especially severe NEC, showed decreased goblet cell density and increased MPO+ and CD3+ cell density in the distal intestine or colon. Circulating parameters were minimally affected by NEC on day 5, but widely altered on day 9 in pigs with NEC, especially severe NEC, to the direction of immune suppression. These included elevated Treg frequency, impaired neutrophil phagocytosis, diminished LPS-induced cytokine secretions and immune gene responses, and consistently low expressions of genes related to innate immune signalling and Th1 polarization. ConclusionWe shows evidence for NEC-induced systemic immune suppression, even with mild and sub-clinical NEC lesions, thereby suggesting mechanisms for increased secondary infections in infants with previous NEC diagnosis.

pathology↗

bioRxiv2020

Early blood transcriptomic markers of necrotizing enterocolitis in preterm pigs

Preterm infants frequently develop necrotizing enterocolitis (NEC), a severe intestinal disorder associated with high mortality. Early detection of NEC is difficult due to poor specificity and sensitivity of clinical signs. We hypothesized that early development of NEC, before clear clinical symptoms appear, might affect expression of blood genes, potentially related to early systemic immune responses. Using preterm pigs as models for preterm infants, a retrospective analysis was performed on 129 infant formula fed pigs that had NEC diagnosis at necropsy on day 5. Clinical data including growth, activity, hematology, gastric residuals, incidence of diarrhea, bloody stool and abdominal distention were retrospectively reviewed. During this early postnatal period, except that bloody stool was observed in 19% of NEC pigs and absent in healthy pigs, no other clinical outcomes showed difference between NEC and healthy pigs. Whole blood transcriptome was compared between NEC pigs (NEC, n=20) and their litter-mate healthy controls (CON, n=19) on day 5, and revealed 344 differentially expressed genes (DEGs). PubMed literature search identified 123 genes that co-occurred with at least one of 9 NEC-related keywords (NEC, colitis, necrotic, hemorrhage, epithelial apoptosis, intestinal inflammation, inflammatory bowel disease, Crohns disease, ulcerative colitis). Co-expression network analysis suggested PAK2 as one of the hub genes. Using whole blood and dried blood spots (DBS) from another group of preterm pigs for validation, up-regulation of PAK2 and genes that co-occurred with NEC and other keywords in PubMed literatures (AOAH, ARG2, FKBP5 and STAT3) was confirmed in severe NEC cases. Specifically, ex vivo stimulation of cord blood with S.epidermidis increased ARG2. Our results show that whole blood gene expressions are affected in preterm pigs at an early stage when NEC is suspicious. Expression of target genes may be used to indicate NEC severity and associated bacterial infection. Routinely collected neonatal DBS may be used to develop early biomarkers for identifying infants with severe NEC lesions, thus providing better intervention strategy.

molecular biology↗

bioRxiv2020

Ceftazidime Is a Potential Drug to Inhibit SARS-CoV-2 Infection In Vitro by Blocking Spike Protein-ACE2 Interaction

Coronavirus Disease 2019 (COVID-19) spreads globally as a sever pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Cell entry of SARS-CoV-2 mainly depends on binding of the viral spike (S) proteins to angiotensin converting enzyme 2 (ACE2) on host cells. Therefore, repurposing of known drugs to inhibit S protein-ACE2 interaction could be a quick way to develop effective therapy for COVID-19. Using a high-throughput screening system to investigate the interaction between spike receptor binding domain (S-RBD) and ACE2 extracellular domain, we screened 3581 FDA-approved drugs and natural small molecules and identified ceftazidime as a potent compound to inhibit S-RBD-ACE2 interaction by binding to S-RBD. In addition to significantly inhibit S-RBD binding to HPAEpiC cells, ceftazidime efficiently prevented SARS-CoV-2 pseudovirus to infect ACE2-expressing 293T cells. The inhibitory concentration (IC50) was 113.2 M, which is far below the blood concentration (over 300 M) of ceftazidime in patients when clinically treated with recommended dose. Notably, ceftazidime is a drug clinically used for the treatment of pneumonia with minimal side effects compared with other antiviral drugs. Thus, ceftazidime has both anti-bacterial and anti-SARS-CoV-2 effects, which should be the first-line antibiotics used for the clinical treatment of COVID-19.

immunology↗

bioRxiv2020

Coordination Among Multiple Receptor Tyrosine Kinase Signals Controls Drosophila Developmental Timing and Body Size

Body size and the timing of metamorphosis are two important interlinked life-history traits that define holometabolous insect development. Metamorphic timing is largely controlled by a neuroendocrine signaling axis composed of the prothoracic gland (PG) and its presynaptic neurons (PGNs). The PGNs produce prothoracicotropic hormone (PTTH) that stimulates the PG to produce the metamorphosis inducing hormone ecdysone (E) through activation of Torso a Receptor tyrosine kinase the Receptor Tyrosine kinase and its downstream Ras/Erk signal transducers. Here we identify two additional timing signals produced by the RTKs Anaplastic lymphoma kinase (Alk) and the PDGF/VEGF-receptor related (PvR), Similar to Torso, both Alk and PvR trigger Ras/Erk signaling in the PG to up regulate expression of E biosynthetic enzymes, while Alk also suppresses autophagy induction after critical weight by activating Pi3K/Akt. When overexpressed, both RTKs hyperactivate an endogenous low-level Jak/Stat signal in the PG resulting in developmental delay or arrest. The Alk ligand Jelly belly (Jeb) is produced by the PGNs, and together with PTTH serves as a second PGN derived tropic factor that stimulates E production by the PG. In addition, we find that Pvf3, a PvR ligand, is also produced by the PGNs, but we show that the activation of PvR primarily relies on autocrine signaling by PG-derived Pvf2 and Pvf3. These findings illustrate that a multitude of juxtracrine and autocrine signaling systems have evolved to regulate the timing of metamorphosis, the defining event of holometabolous development.

developmental biology↗

bioRxiv2020

Gluconeogenic Enzyme PCK1 Deficiency Is Critical for CHK2 O-GlcNAcylation and Hepatocellular Carcinoma Growth upon Glucose Deprivation

Elevated hexosamine-biosynthesis pathway (HBP) activity and O-GlcNAcylation are emerging hallmarks of hepatocellular carcinoma (HCC). Inhibiting O-GlcNAcylation could be a promising anti-cancer strategy. Here, we investigate this possibility and demonstrate that deficiency of phosphoenolpyruvate carboxykinase 1 (PCK1), a rate-limiting enzyme in gluconeogenesis, promotes O-GlcNAcylation and hepatoma cell proliferation under low-glucose conditions. PCK1 loss results in oxaloacetate accumulation and AMPK inactivation, promoting uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) synthesis and CHK2 threonine 378 O-GlcNAcylation and counteracting its ubiquitination and degradation. O-GlcNAcylation also promotes CHK2-dependent Rb phosphorylation and HCC cell proliferation. Therefore, blocking HBP-mediated O-GlcNAcylation suppresses tumor progression in liver-specific Pck1-knockout mice. We reveal a link between PCK1 depletion and hyper-O-GlcNAcylation that underlies HCC oncogenesis and suggest therapeutic targets for HCC that act by inhibiting O-GlcNAcylation.

cancer biology↗

bioRxiv2020

Oxidations and amino acid substitutions in urinary proteins are the distinguishing characteristics of aging

Aging is an inevitable course of life. Additionally, the risk of chronic diseases or cancer increases with age. The comprehensive identification of signs related to aging can be beneficial for the prevention and early diagnosis of geriatric diseases. The comparison of global modifications in the urine proteome is a means of multidimensional information mining. This approach is based on urine, in which changes from whole-body metabolism can accumulate. This study used the urine of healthy people at different ages (22 children, 10 young people, 6 senior people) as the research object and using high-resolution tandem mass spectrometry, label-free quantitation combined with non-limiting modification identification algorithms and random group test, compared the differences in protein chemical modifications among three groups. The results show that multi-sites oxidative modifications and amino acid substitutions are noticeable features that distinguish these three age groups of people. The proportion of multi-site oxidations in urine proteins of senior (29.76%) is significantly higher than the young group (13.71% and 12.97%), which affect the biological processes of various proteins. This study could provide a reference for studies of aging mechanisms and biomarkers of age-related disease.

biochemistry↗

bioRxiv2020

Early defects in lysosomal storage diseases disrupt excitatory synaptic transmission

At least two thirds of patients affected with lysosomal storage disorders (LSD) exhibit neurological symptoms. For mucopolysaccharidosis type IIIC (MPS IIIC, Sanfilippo disease type C) caused by mutations in the HGSNAT gene and lysosomal storage of heparan sulfate the major burden is progressive and severe neuropsychiatric problems, mental retardation, and dementia though to be mainly mediated by neurodegeneration. HGSNAT knockout mice match human clinical phenotype and develop hyperactivity followed by memory impairment and death. In order to understand whether early clinical symptoms in MPS IIIC mice occurring before the onset of massive neurodegeneration are caused by neuronal dysfunction we studied synaptic transmission and morphology in cultured hippocampal and CA1 pyramidal neurons of MPSIIIC mice. Synaptic spines were also studied in other mouse LSD models and postsynaptic densities in post-mortem cortices of human neurological MPS patients. Cultured hippocampal and CA1 pyramidal neurons of MPS IIIC mice showed a drastic decrease or abnormal distribution of multiple pre- and postsynaptic proteins that could be rescued in vitro and in vivo by virus-mediated gene correction. Dendritic spine densities were immature in cultured hippocampal MPS IIIC mouse neurons and reduced in pyramidal neurons of mouse models of MPS IIIC and other (Tay-Sachs, sialidosis) LSD starting from postnatal day 10. MPS IIIC neurons presented alterations in frequency and amplitude of miniature excitatory and inhibitory postsynaptic currents, sparse synaptic vesicles, reduced postsynaptic densities, disorganised microtubule network and partially impaired axonal transport of synaptic proteins. Postsynaptic densities were also reduced in post-mortem cotrees of human MPS I, II, IIIA, C and D patients suggesting that the pathology is common for these neurological LSD. Together, our results demonstrate that lysosomal storage causes alterations in synaptic structure and abnormalities in neurotransmission originating from disrupted vesicular transport and preceding the first cognitive symptoms and suggest drugs known to affect synaptic transmission can be potentially applied to treat behavioral and cognitive defects in neurological LSD patients.

neuroscience↗

bioRxiv2020

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damage and constitutes an antiviral target

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of the virus excessively damaging abilities remains unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is sufficient to cause acute respiratory distress syndrome (ARDS)-like damage in vitro and in vivo. Overexpression of 2-E protein induced rapid pyroptosis-like cell death in various susceptible cells and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damage in lung and spleen. Overexpressed 2-E protein formed cation channels in host cell membranes, eventually leading to membrane rupture. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent protective effects against the 2-E-induced damage both in vitro and in vivo. Importantly, their channel inhibition, cell protection and antiviral activities were positively correlated with each other, supporting 2-E is a promising drug target against SARS-CoV-2.

microbiology↗

bioRxiv2020

Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes

Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from [~]0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.

immunology↗

bioRxiv2020

Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of COVID-19 Spike psedotype virus

BackgroundThe novel coronavirus disease (2019-nCoV) has been affecting global health since the end of 2019 and there is no sign that the epidemic is abating. The major issue for controlling the infectious is lacking efficient prevention and therapeutic approaches. Chloroquine (CQ) and Hydroxychloroquine (HCQ) have been reported to treat the disease, but the underlying mechanism remains controversial. PurposeThe objective of this study is to investigate whether CQ and HCQ could be ACE2 blockers and used to inhibit 2019-nCoV virus infection. MethodsIn our study, we used CCK-8 staining, flow cytometry and immunofluorescent staining to evaluate the toxicity and autophagy of CQ and HCQ, respectively, on ACE2 high-expressing HEK293T cells (ACE2h cells). We further analyzed the binding character of CQ and HCQ to ACE2 by molecular docking and surface plasmon resonance (SPR) assays, 2019-nCoV spike pseudotyped virus was also used to observe the viropexis effect of CQ and HCQ in ACE2h cells. ResultsResults showed that HCQ is slightly more toxic to ACE2h cells than CQ. Both CQ and HCQ could bind to ACE2 with KD =(7.31{+/-}0.62)e-7 M and (4.82{+/-}0.87)e-7 M, respectively. They exhibit equivalent suppression effect for the entrance of 2019-nCoV spike pseudotyped virus into ACE2h cells. ConclusionsCQ and HCQ both inhibit the entrance 2019-nCoV into cells by blocking the binding of the virus with ACE2. Our findings provide novel insights into the molecular mechanism of CQ and HCQ treatment effect on virus infection.

biochemistry↗

bioRxiv2020

Thiamine pyrophosphokinase deficiency induces Alzheimer's pathology

BackgroundThiamine diphosphate (TDP) reduction plays an important role in cerebral glucose hypometabolism, the neurodegenerative indicator, in Alzheimers disease (AD). The mechanism underlying TDP reduction remains elusive. Thus, it is critical to define the mechanism and its effect on neurodegeneration, the pathological basis of the disease occurrence and progression. MethodsThe mRNA levels of all known genes associated with thiamine metabolism, including thiamine pyrophosphokinase (TPK), Solute Carrier Family 19 Member 2 (SLC19A2), SLC19A3, and SLC25A19, in brain samples of patients with AD and other neurodegenerative disorders in multiple independent datasets were analyzed. TPK protein levels were further examined in the brain tissues of AD patients and control subjects. A mouse model with conditional knockout (cKO) of TPK gene in the excitatory neurons of adult brain was established. ResultsThe brain TPK mRNA level was markedly lower in AD patients, but not in other neurodegenerative disorders. The brain TPK protein level was also significantly decreased in AD patients. TPK gene knockout in the mice caused cerebral glucose hypometabolism, {beta}-amyloid deposition, Tau hyperphosphorylation, neuroinflammation, and neuronal loss and brain atrophy. Cross-species correlation analysis revealed the similar changes of gene profiling between the cKO mice and AD patients. ConclusionsThe deficiency of brain TPK, a key enzyme for TDP synthesis, is specific to AD. The cKO mice show AD-associated phenotypes and could serve as a new mouse model for AD studies. Our study provides a novel insight into the critical role of TPK in AD pathogenesis and its potential for the disease treatment.

neuroscience↗

bioRxiv2020

Fecal filtrate transfer protects against necrotizing enterocolitis in preterm pigs

Background and aimsNecrotizing enterocolitis (NEC) is an acute and life-threatening gastrointestinal disorder afflicting preterm infants, which is currently unpreventable. Fecal microbiota transplantation (FMT) is a promising preventative therapy, but potential side effects raise concern. Removal of bacteria from donor fecal water may reduce side effects while maintaining wanted effects. We aimed to assess preclinical efficacy and safety of bacteria-free fecal filtrate transfer (FFT). MethodsUsing fecal material from healthy suckling piglets, we administered rectal FMT or cognate FFT by either rectal or oro-gastric administration to formula-fed preterm, cesarean piglets, and compared gut pathology and related safety parameters with saline controls. We then analyzed mucosa and luminal bacterial and viral composition using 16S rRNA gene amplicon and metavirome sequencing, respectively. Finally, we used isolated ileal mucosa, coupled with RNA-Seq, to gauge the host response to the different treatments. ResultsOro-gastric FFT eliminated NEC, which was confirmed by microscopy, whereas FMT did not perform better than control. Moreover, FFT but not FMT reduced intestinal permeability, whereas FMT animals had reduced body weight increase and intestinal growth. Oro-gastric FFT increased viral diversity and reduced Proteobacteria abundance in ileal mucosa relative to control. Global gene expression of host mucosa responded to FMT but not FFT with increased and decreased bacterial and viral defense mechanisms, respectively. ConclusionsAs preterm infants are extremely vulnerable, rational therapies need incontestable safety profiles. Here we show in a clinically relevant animal model that FFT, as opposed to FMT, efficiently prevents NEC without any recognizable side effects. If translatable to preterm infants, this could lead to a change of practice and in turn a reduction in NEC burden.

microbiology↗

bioRxiv2020

Massively parallel quantification of CRISPR editing in cells by TRAP-seq enables better design of Cas9, ABE, CBE gRNAs of high efficiency and accuracy

The CRISPR RNA-guided endonucleases Cas9, and Cas9-derived adenine/cytosine base editors (ABE/CBE), have been used in both research and therapeutic applications. However, broader use of this gene editing toolbox is hampered by the great variability of efficiency among different target sites. Here we present TRAP-seq, a versatile and scalable approach in which the CRISPR gRNA expression cassette and the corresponding surrogate site are captured by Targeted Reporter Anchored Positional Sequencing in cells. TRAP-seq can faithfully recapitulate the CRISPR gene editing outcomes introduced to the corresponding endogenous genome site and most importantly enables massively parallel quantification of CRISPR gene editing in cells. We demonstrate the utility of this technology for high-throughput quantification of SpCas9 editing efficiency and indel outcomes for 12,000 gRNAs in human embryonic kidney cells. Using this approach, we also showed that TRAP-seq enables high throughput quantification of both ABE and CBE efficiency at 12,000 sites in cells. This rich amount of ABE/CBE outcome data enable us to reveal several novel nucleotide features (e.g. preference of flanking bases, nucleotide motifs, STOP recoding types) affecting base editing efficiency, as well as designing improved machine learning-based prediction tools for designing SpCas9, ABE and CBE gRNAs of high efficiency and accuracy (>70%). We have integrated all the 12,000 CRISPR gene editing outcomes for SpCas9, ABE and CBE into a CRISPR-centered portal: The Human CRISPR Atlas. This study extends our knowledge on CRISPR gene and base editing, and will facilitate the application and development of CRISPR in both research and therapy.

systems biology↗

bioRxiv2020

Broad-spectrum antivirals of protoporphyrins inhibit the entry of highly pathogenic emerging viruses

Severe emerging and re-emerging viral infections such as Lassa fever, Avian influenza (AI), and COVID-19 caused by SARS-CoV-2 urgently call for new strategies for the development of broad-spectrum antivirals targeting conserved components in the virus life cycle. Viral lipids are essential components, and viral-cell membrane fusion is the required entry step for most unrelated enveloped viruses. In this paper, we identified a porphyrin derivative of protoporphyrin IX (PPIX) that showed broad antiviral activities in vitro against a panel of enveloped pathogenic viruses including Lassa virus (LASV), Machupo virus (MACV), and SARS-CoV-2 as well as various subtypes of influenza A viral strains with IC50 values ranging from 0.91{+/-}0.25 M to 1.88{+/-}0.34 M. A mechanistic study using influenza A/Puerto Rico/8/34 (H1N1) as a testing strain showed that PPIX inhibits the infection in the early stage of virus entry through biophysically interacting with the hydrophobic lipids of enveloped virions, thereby inhibiting the formation of the negative curvature required for fusion and blocking the entry of enveloped viruses into host cells. In addition, the preliminary antiviral activities of PPIX were further assessed by testing mice infected with the influenza A/Puerto Rico/8/34 (H1N1) virus. The results showed that compared with the control group without drug treatment, the survival rate and mean survival time of the mice treated with PPIX were apparently prolonged. These data encourage us to conduct further investigations using PPIX as a lead compound for the rational design of lipid-targeting antivirals for the treatment of infection with enveloped viruses.

pharmacology and toxicology↗

bioRxiv2020

Expanding the space of protein geometries by computational design of de novo fold families

Naturally occurring proteins use a limited set of fold topologies, but vary the precise geometries of structural elements to create distinct shapes optimal for function. Here we present a computational design method termed LUCS that mimics natures ability to create families of proteins with the same overall fold but precisely tunable geometries. Through near-exhaustive sampling of loop-helix-loop elements, LUCS generates highly diverse geometries encompassing those found in nature but also surpassing known structure space. Biophysical characterization shows that 17 (38%) out of 45 tested LUCS designs were well folded, including 16 with designed non-native geometries. Four experimentally solved structures closely match the designs. LUCS greatly expands the designable structure space and provides a new paradigm for designing proteins with tunable geometries customizable for novel functions. One Sentence SummaryA computational method to systematically sample loop-helix-loop geometries expands the structure space of designer proteins.

bioengineering↗