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Biology subjects

Lu, G.

Publications and source records attributed to Lu, G..

At least 55 records · Page 3Linked to original sources

Single Cell RNA-Seq Analysis of Regenerative Drug-Treated Human Pancreatic Islets Identifies A Cycling Alpha Cell Population As Key Beta Cell Progenitors

Diabetes ultimately results from an inadequate number of functional, insulin-producing human beta cells. Although current attempts to replenish the remaining beta cell pool in people with diabetes are encouraging, scalability and cost limit access for the millions of people with diabetes. The small molecule DYRK1A inhibitor class of beta cell regenerative drugs, either alone or in combination with GLP1 receptor agonists or TGF{beta} superfamily inhibitors, are capable of inducing beta cell replication in vitro and increasing beta cell mass in vivo. Despite these advances, the precise mechanisms of action of DYRK1A inhibitors remain incompletely understood. To address the mechanisms more deeply, we performed single cell RNA sequencing on human pancreatic islets treated with a DYRK1A inhibitor, either alone, or in combination with a GLP1 receptor agonist or a TGF{beta} superfamily inhibitor. We identify a cluster of Cycling Alpha Cells as the cells most responsive to DYRK1A inhibition. Velocity and pseudotime lineage trajectory analyses suggest that Cycling Alpha Cells serve as the primary target cell type for of DYRK1A inhibitors, and may serve as precursor cells that transdifferentiate into functional human beta cells in response to the DYRK1A inhibition. In addition to providing a novel mechanism of action for DYRK1A inhibitors, our findings suggest that efforts to target regenerative drugs to human beta cells may be mis-directed: the proper target may be Cycling Alpha Cells.

cell biology↗

Systematic enhancement of protein crystallization efficiency by bulk lysine-to-arginine (KR) substitution

Structural genomics consortia established that protein crystallization is the primary obstacle to structure determination using x-ray crystallography. We previously demonstrated that crystallization propensity is systematically related to primary sequence, and we subsequently performed computational analyses showing that arginine is the most overrepresented amino acid in crystal-packing interfaces in the Protein Data Bank. Given the similar physicochemical characteristics of arginine and lysine, we hypothesized that multiple lysine-to-arginine (KR) substitutions should improve crystallization. To test this hypothesis, we developed software that ranks lysine sites in a target protein based on the redundancy-corrected KR substitution frequency in homologs. We demonstrate that three unrelated single-domain proteins can tolerate 5-11 KR substitutions with at most minor destabilization and that these substitutions consistently enhance crystallization propensity. This approach rapidly produced a 1.9 [A] crystal structure of a human protein domain refractory to crystallization with its native sequence. Structures from bulk-KR-substituted domains show the engineered arginine residues frequently make high-quality hydrogen-bonds across crystal-packing interfaces. We thus demonstrate that bulk KR substitution represents a rational and efficient method for probabilistic engineering of protein surface properties to improve protein crystallization.

biophysics↗

Transposon elements shape population divergence of rice blast fungus Magnaporthe oryzae

Dynamic transposition of transposable elements (TEs) in fungal pathogens have significant impact on genome stability, gene expression, and virulence to the host. In Magnaporthe oryzae, genome plasticity resulting from TE insertion is a major driving force leading to the rapid evolution and diversification of this fungus. Despite their importance in M. oryzae population evolution and divergence, our understanding of TEs in this context remains limited. Here we conducted a genome-wide analysis of TE transposition dynamics in the 11 most abundant TE families in M. oryzae populations. Our results show that these TEs have specifically expanded in recently isolated M. oryzae rice populations, with the presence/absence polymorphism of TE insertions highly concordant with population divergence on Geng/Japonica and Xian/Indica rice cultivars. Notably, the genes targeted by clade-specific TEs showed clade-specific expression patterns and are involved in the pathogenic process, suggesting a transcriptional regulation of TEs on targeted genes. Our study provides a comprehensive analysis of TEs in M. oryzae populations and demonstrates a crucial role of recent TE bursts in adaptive evolution and diversification of the M. oryzae rice-infecting lineage. IMPORTANCEM. oryzae is the causal agent of the destructive blast disease, which caused massive loss of yield annually worldwide. The fungus diverged into distinct clades during adaptation toward two rice subspecies, Xian/indica and Geng/japonica. Although the role of TEs in the adaptive evolution was well established, mechanisms underlying how TEs promote the population divergence of M. oryzae remains largely unknown. In this study, we reported that TEs shape the population divergence of M. oryzae by differentially regulating gene expression between Xian/Indica-infecting and Geng/Japonica-infecting populations. Our results revealed a TE insertion mediated gene expression adaption that led to the divergence of M. oryzae population infecting different rice subspecies.

genomics↗

LSM14B is essential for mitochondrial clustering in the oocyte meiosis

As oocyte meiotic maturation, they undergo two successive meiotic M phases, notably lacking an intervening interphase phase. During these M phases, oocytes remain transcriptionally quiescent, and we now know that "translational repressed mRNAs" are stored in a structure called the mitochondria associated ribonucleoprotein domain (MARDO). LSM14B is one of the abundant proteins of MARDO, and is predicted to bind mRNA, but its function(s) remain elusive. Here, we demonstrate that LSM14B functions to promote MARDO assembly in mouse oocytes. We also found that LSM14B knockout female mice are infertile, and show that the knockout oocytes fail to enter meiosis II, instead entering an aberrant interphase-like stage. Finally, we show that the failure of oocyte maturation results from decreased expression of Cyclin B1. Our study has revealed that the RNA-binding protein LSM14B modulates MARDO assembly and is essential for oocyte meiotic maturation.

developmental biology↗

Superhydrophobic Rotation-Chip for Computer-Vision Identification of Drug-Resistant Bacteria

The transport, distribution, and mixing of microfluidics often require additional instruments, such as pumps and valves, which are not feasible when operated in point-of-care (POC) settings. Here, we present a simple microfluidic pathogen detection system known as Rotation-Chip that transfers the reagents between wells by manually rotating two concentric layers without using external instruments. The Rotation-Chip is fabricated by a simple computer numerical control (CNC) machining process and is capable of carrying out 60 multiplexed reactions with a simple 30-degree or 60-degree rotation. Leveraging superhydrophobic coating, a high fluid transport efficiency of 92.78% is achieved without observable leaking. Integrated with an intracellular fluorescent assay, an on-chip detection limit of 1.8x106 CFU/mL is achieved for ampicillin-resistant Escherichia coli (E. coli), which is similar to our off-chip results. We also develop a computer vision method to automatically distinguish positive and negative samples on the chip, showing 100% accuracy. Our Rotation-Chip is simple, low-cost, high-throughput, and can display test results with a single chip image, ideal for various multiplexing POC applications in resource-limited settings.

bioengineering↗

Huntingtin expression influences spontaneous seizure disorder susceptibility in FVN/B mice

Huntington disease (HD) is an adult-onset neurodegenerative disorder that is caused by a trinucleotide CAG repeat expansion in the HTT gene that codes for the protein huntingtin (HTT or Htt in mice). HTT is a multi-functional, ubiquitously expressed protein that is essential for embryonic survival, normal neurodevelopment, and adult brain function. The ability of wild-type HTT to protect neurons against various forms of death raises the possibility that loss of normal HTT function may worsen disease progression in HD. Huntingtin-lowering therapeutics are being evaluated in clinical trials for HD, but concerns have been raised that decreasing wild-type HTT levels may have adverse effects. Here we show that Htt levels modulate the occurrence of an idiopathic seizure disorder that spontaneously occurs in FVB/N mice. These abnormal FVB/N mice demonstrate various cardinal features of mouse models of epilepsy including spontaneous seizures, astrocytosis, neuronal hypertrophy, upregulation of brain-derived neurotrophic factor (BDNF), and sudden seizure-related death. Interestingly, decreasing wild-type Htt levels increased the frequency of this disorder, while over-expression of HTT completely prevented it. Examination of the mechanism underlying huntingtins ability to modulate the frequency of this seizure disorder indicated that over-expression of full length HTT can promote neuronal survival following seizures. Overall, our results demonstrate a protective role for huntingtin in this form of epilepsy and provide a plausible explanation for the observation of seizures in the juvenile form of HD, Lopes-Maciel-Rodan syndrome, and Wolf-Hirschhorn syndrome. Adverse effects caused by altering huntingtin levels has ramifications related to Huntingtin-lowering therapies in development to treat HD.

neuroscience↗

PHF1 compartmentalizes PRC2 at target loci via phase separation

Polycomb repressive complex 2 (PRC2) is central to polycomb repression as it trimethylates lysine 27 on histone H3 (H3K27me3). How PRC2 is recruited to its targets to deposit H3K27me3 remains an open question. Polycomb-like (PCL) proteins, a group of conserved PRC2 accessory proteins, direct PRC2 to its targets. In this report, we demonstrate that a PCL protein named PHF1 forms phase-separated condensates at H3K27me3 loci that recruit PRC2. Combining cellular observation and biochemical reconstitution, we show that the N-terminal domains of PHF1 cooperatively mediate target recognition, the chromo-like domain recruits PRC2, and the intrinsically disordered region (IDR) drives phase separation. Moreover, we reveal that the condensates compartmentalize PRC2, DNA, and nucleosome arrays by phase separation. Luciferase reporter assays confirm that PHF1 phase separation promotes transcription repression, further supporting a role of the condensates in polycomb repression. Based on our findings, we propose that these condensates create favorable microenvironments at the target loci for PRC2 to function.

molecular biology↗

The Slingshot phosphatase 2 is required for acrosome biogenesis during spermatogenesis in mice

The acrosome is a membranous organelle positioned in the anterior portion of sperm head and is essential for male fertility. Acrosome biogenesis requires the dynamic cytoskeletal shuttling of vesicles towards nascent acrosome which is regulated by a series of accessory proteins. However, much remains unknown about the molecular basis underlying this process. Here, we generated Ssh2 knock-out (KO) mice and show that Slingshot phosphatase 2 (SSH2), a regulator of actin remodeling, is essential for acrosome biogenesis and male fertility. In Ssh2 KO males, spermatogenesis was arrested at the early spermatid stage with enhanced germ cell apoptosis and the impaired acrosome biogenesis was characterized by defective transport/fusion of proacrosomal vesicles. Moreover, disorganized F-actin structures accompanied by excessive phosphorylation of COFILIN were observed in testes of Ssh2 KO mice. Collectively, our data reveal a modulatory role for SSH2 in acrosome biogenesis through COFILIN-mediated actin remodeling and the indispensability of this phosphatase in male fertility in mice.

developmental biology↗

Huntingtin Over-Expression Does Not Alter Overall Survival in Murine Cancer Models

A reduced incidence of various forms of cancer has been reported in Huntingtons disease (HD) patients and may be due to pro-apoptotic effects of mutant huntingtin. We tested this hypothesis by assessing the effects of huntingtin protein over-expression on survival in two murine cancer models. We generated yeast artificial chromosome (YAC) HD mice containing human huntingtin transgenes with various CAG tract lengths (YAC18, YAC72, YAC128) on either an Msh2 or p53 null background, which have increased cancer incidence. In both mouse models of cancer, the over-expression of either mutant or wild-type huntingtin had no significant effect on overall survival. These results do not support the hypothesis that mutant huntingtin expression is protective against cancer.

cancer biology↗

A spatial map of human macrophage niches links tissue location with function

Macrophages are the most abundant immune cell type in the tumor microenvironment (TME). Yet the spatial distribution and cell interactions that shape macrophage function are incompletely understood. Here we use single-cell RNA sequencing data and multiplex imaging to discriminate and spatially resolve macrophage niches within benign and malignant breast and colon tissue. We discover four distinct tissue-resident macrophage (TRM) layers within benign bowel, two TRM niches within benign breast, and three tumor-associated macrophage (TAM) populations within breast and colon cancer. We demonstrate that IL4I1 marks phagocytosing macrophages, SPP1 TAMs are enriched in hypoxic and necrotic tumor regions, and a novel subset of FOLR2 TRMs localizes within the plasma cell niche. Furthermore, NLRP3 TAMs that colocalize with neutrophils activate an inflammasome in the TME and in Crohns disease and are associated with poor outcomes in breast cancer patients. This work suggests novel macrophage therapy targets and provides a framework to study human macrophage function in clinical samples.

cancer biology↗

The Piks allele of the NLR immune receptor Pik breaks the recognition of AvrPik effectors of the rice blast fungus

Arms race co-evolution of plant-pathogen interactions evolved sophisticated recognition mechanisms between host immune receptors and pathogen effectors. Different allelic haplotypes of an immune receptor in host mount distinct recognition against sequence or non-sequence related effectors in pathogens. We report the molecular characterization of the Piks allele of the rice immune receptor Pik against rice blast pathogen, which requires two head-to-head arrayed nucleotide binding site and leucine-rich repeat proteins. Like other Pik genes, both Piks-1 and Piks-2 are necessary and sufficient for Piks-mediated resistance. However, unlike other Pik alleles, Piks does not recognize any known AvrPik variants of M. oryzae. Sequence analysis of the genome of an avirulent isolate V86010 further revealed that its cognate avirulence (Avr) gene most likely has no significant sequence similarity to known AvrPik variants. We conclude that Piks breaks the canonical Pik/AvrPik recognition pattern. Piks-1 and Pikm-1 have only two amino acid differences within the integrated heavy metal-associated (HMA) domain. Pikm-1-HMA interact with AvrPik-A, -D and -E in vitro and in vivo, whereas Piks-1-HMA does not bind any AvrPik variants. Reciprocal exchanges of single amino acid residues between Piks-1 and Pikm-1 further reveal a dynamic recognition mechanism between Piks/Pikm alleles and their respective effectors. Piks-1E229Q/Pikm-1V261A can only activate immunity to AvrPik-D but not to other effectors, indicating that the amino acid change of E to Q at position 229 leads to its gain of a partial recognition spectrum of Pikm. By contrast, Piks-1A261V/Pikm-1Q229E confers immunity to the Piks cognate effector, indicating that the amino acid change of Q to E at position 229 leads to its shifts of the recognition from Pikm to Piks. Intriguingly, binding activities in both Y2H and analytical gel filtration assays are illustrated between Piks-1A261V/Pikm-1Q229E and AvrPik-D. However, it is unable to mount immunity against AvrPik-D, suggesting that biochemical activities based on in vitro and in vivo assays could be insufficient for sustaining biological function of receptor and effector pairs.

plant biology↗

Rab7/Retromer-based endolysosomal trafficking facilitates effector secretion and host invasion in rice blast

Secretion is a fundamental process in all living organisms. Using conventional secretion pathways, many plant pathogens release effectors into the host plants to downregulate immunity and promote infection. However, this does not always constitute the only way that effectors are sorted and tracked to their final destination such as the biotrophic interfacial complex-associated effectors produced by the blast fungus Magnaporthe oryzae. Here, we uncover a novel unconventional route originating from fungal vacuolar membrane to the host interface and plasma membrane. We found that a GFP-MoRab7 labeled vacuole is closely associated with the interface structure throughout M. oryzae invasive growth. Conditional inactivation of MoRab7 impaired the establishment of the biotrophy interface and secretion of Pwl2 effector. To perform the vacuolar trafficking pathway, MoRab7 first recruits the retromer complex to the vacuole membrane, enabling it recognizes a batch of SNARE proteins, including the v-SNARE MoSnc1. Live-cell imaging supports both retromer complex component and MoSnc1 protein labeled vesicles showing the trafficking dynamics toward the interface or plasma membrane, and then fusion with target membranes. Lastly, disruption of the MoRab7/Retromer/MoSnc1-based endolysosomal cascade affects effector secretion and fungal pathogenicity. Taken together, we discovered an unconventional protein and membrane trafficking route starting from the fungal endolysosomes to the M. oryzae-rice interaction interface, and dissect the role of MoRab7/Retromer/MoSnc1 constituent sorting machinery in effector secretion during invasive growth in M. oryzae.

plant biology↗

Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET

Gas vesicles (GVs) are gas-filled protein nanostructures employed by several species of bacteria and archaea as flotation devices to enable access to optimal light and nutrients. The unique physical properties of GVs have led to their use as genetically-encodable contrast agents for ultrasound and MRI. Currently, however, the structure and assembly mechanism of GVs remain unknown. Here we employ cryo-electron tomography to reveal how the GV shell is formed by a helical filament of highly conserved GvpA subunits. This filament changes polarity at the center of the GV cylinder--a site that may act as an elongation center. High-resolution subtomogram averaging reveals a corrugated pattern of the shell arising from polymerization of GvpA into a {beta}-sheet. The accessory protein GvpC forms a helical cage around the GvpA shell, providing structural reinforcement. Together, our results help explain the remarkable mechanical properties of GVs and their ability to adopt different diameters and shapes.

molecular biology↗

Single Nucleus RNA Sequencing of Human Pancreatic Islets In Vitro and In Vivo Identifies New Gene Sets and Three β-Cell Subpopulations with Different Transcriptional Profile

Single-cell RNA sequencing (scRNA-seq) has provided valuable insights into human islet cell types and their corresponding stable gene expression profiles. However, this approach requires cell dissociation that complicates its utility in vivo and provides limited information on the active transcriptional status of islet cells. On the other hand, single-nucleus RNA sequencing (snRNA-seq) does not require cell dissociation and affords enhanced information from intronic sequences that can be leveraged to identify actively transcribing genes in islet cell populations. Here, we first sought to compare scRNA-seq and snRNA-seq analysis of human islets in vitro using exon reads or combined exon and intron reads, respectively. Datasets reveal similar human islet cell clusters using both approaches. In the snRNA-seq data, however, the top differentially expressed genes in human islet endocrine cells are not the canonical genes but a new set of non-canonical gene markers including ZNF385D, TRPM3, LRFN2, PLUT ({beta} cells), PTPRT, FAP, PDK4, LOXL4 ( cells), LRFN5, ADARB2, ERBB4, KCNT2 ({delta} cells) and CACNA2D3, THSD7A, CNTNAP5, RBFOX3 ({gamma} cells). Notably, these markers also accurately define endocrine cell populations in human islet grafts in vivo. Further, by integrating the information from nuclear and cytoplasmic transcriptomes, we identify three {beta}-cell sub-clusters: an active INS mRNA transcribing cluster ({beta}1), an intermediate INS mRNA-transcribing cluster ({beta}2), and a mature INS mRNA rich cluster ({beta}3). These display distinct gene expression patterns representing different biological dynamic states both in vitro and in vivo. Interestingly, the INS mRNA rich cluster ({beta}3) becomes the predominant sub-cluster in vivo. In summary, snRNA-seq analysis of human islet cells is a previously unrecognized tool that can be accurately employed for improved identification of human islet cell types and their transcriptional status in vivo.

cell biology↗

Preclinical evaluation of RQ3013, a broad-spectrum mRNA vaccine against SARS-CoV-2 variants

The global emergence of SARS-CoV-2 variants has led to increasing breakthrough infections in vaccinated populations, calling for an urgent need to develop more effective and broad-spectrum vaccines to combat COVID-19. Here we report the preclinical development of RQ3013, an mRNA vaccine candidate intended to bring broad protection against SARS-CoV-2 variants of concern (VOCs). RQ3013, which contains pseudouridine-modified mRNAs formulated in lipid nanoparticles, encodes the spike(S) protein harboring a combination of mutations responsible for immune evasion of VOCs. Here we characterized the expressed S immunogen and evaluated the immunogenicity, efficacy, and safety of RQ3013 in various animal models. RQ3013 elicited robust immune responses in mice, hamsters, and nonhuman primates (NHP). It can induce high titers of antibodies with broad cross-neutralizing ability against the Wild-type, B.1.1.7, B.1.351, B.1.617.2, and the omicron B.1.1.529 variants. In mice and NHP, two doses of RQ3013 protected the upper and lower respiratory tract against infection by SARS-CoV-2 and its variants. We also proved the safety of RQ3013 in NHP models. Our results provided key support for the evaluation of RQ3013 in clinical trials.

immunology↗

Structure and mechanism of a mitochondrial AAA+ disaggregase CLBP

The human AAA+ ATPase CLPB (SKD3) is a protein disaggregase in the mitochondrial intermembrane space and functions to promote the solubilization of various mitochondrial proteins. CLPB deficiency by mutations is associated with a few human diseases with neutropenia and neurological disorders. Unlike canonical AAA+ proteins, CLPB contains a unique ankyrin repeat domain (ANK) at its N-terminus. The mechanism of CLPB functions as a disaggregase and the role of its ANK domain are currently unclear. Herein, we report a comprehensive structural characterization of human CLPB in both the apo- and substrate-bound states. CLPB assembles into homo- tetradecamers in apo-state and is remodeled into homo-dodecamers upon binding to substrates. Conserved pore- loops on the ATPase domains form a spiral staircase to grip and translocate the substrate in a step-size of two amino acid residues. The ANK domain is not only responsible for maintaining the higher-order assembly but also essential for the disaggregase activity. Interactome analysis suggests that the ANK domain may directly interact with a variety of mitochondrial substrates. These results reveal unique properties of CLPB as a general disaggregase in mitochondria and highlight its potential as a target for the treatment of various mitochondria-related diseases.

molecular biology↗