Biology subjects

Huang, P.

Publications and source records attributed to Huang, P..

At least 145 records · Page 8Linked to original sources

bioRxiv2020

Light-inducible Deformation of Mitochondria in Live Cells

Mitochondria, the powerhouse of the cell, are dynamic organelles that undergo constant morphological changes. Increasing evidence indicates that mitochondria morphologies and functions can be modulated by mechanical cues. However, the mechano-sensing and -responding properties of mitochondria and the correlation between mitochondrial morphologies and functions are unclear due to the lack of methods to precisely exert mechano-stimulation on and deform mitochondria inside live cells. Here we present an optogenetic approach that uses light to induce deformation of mitochondria by recruiting molecular motors to the outer mitochondrial membrane via light-activated protein-protein hetero-dimerization. Mechanical forces generated by motor proteins distort the outer membrane, during which the inner mitochondrial membrane can also be deformed. Moreover, this optical method can achieve subcellular spatial precision and be combined with other optical dimerizers and molecular motors. This method presents a novel mitochondria-specific mechano-stimulator for studying mitochondria mechanobiology and the interplay between mitochondria shapes and functions.

synthetic biology↗

bioRxiv2020

Computational design of two new soluble epoxide hydrolase (sEH) inhibitors

Inhibitors of soluble epoxide hydrolase (sEH) enzymes have shown great potential for the treatment of neuropathic pain. However, current sEH inhibitors have poor physicochemical properties and has not been proven to be safe for human treatments yet. New inhibitor designs could have the potential to improve current drugs efficacy, and so in this work, chemical intuition and bioisosteric replacement were used to computationally design two novel sEH inhibitors. These new candidates showed good pharmacokinetic properties and presented better docking scores compared to a known sEH inhibitor, t-TUCB, used in the treatment of pain in horses. Homology analysis revealed that Mus musculus may not be suitable organism for preclinical trials studies of these novel inhibitors.

pharmacology and toxicology↗

bioRxiv2020

Theoretical basis for stabilizing messenger RNA through secondary structure design

RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery, and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Here, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall hydrolysis rate. To characterize the stabilization achievable through structure design, we compare AUP optimization by conventional mRNA design methods to results from more computationally sophisticated algorithms and crowdsourcing through the OpenVaccine challenge on the Eterna platform. These computational tests were carried out on both model mRNAs and COVID-19 mRNA vaccine candidates. We find that rational design on Eterna and the more sophisticated algorithms lead to constructs with low AUP, which we term superfolder mRNAs. These designs exhibit wide diversity of sequence and structure features that may be desirable for translation, biophysical size, and immunogenicity, and their folding is robust to temperature, choice of flanking untranslated regions, and changes in target protein sequence, as illustrated by rapid redesign of superfolder mRNAs for B.1.351, P.1, and B.1.1.7 variants of the prefusion-stabilized SARS-CoV-2 spike protein. Increases in in vitro mRNA half-life by at least two-fold appear immediately achievable. Significance statementMessenger RNA (mRNA) medicines that encode and promote translation of a target protein have shown promising use as vaccines in the current SARS-CoV-2 pandemic as well as infectious diseases due to their speed of design and manufacturing. However, these molecules are intrinsically prone to hydrolysis, leading to poor stability in aqueous buffer and major challenges in distribution. Here, we present a principled biophysical model for predicting RNA degradation, and demonstrate that the stability of any mRNA can be increased at least two-fold over conventional design techniques. Furthermore, the predicted stabilization is robust to post-design modifications. This conceptual framework and accompanying algorithm can be immediately deployed to guide re-design of mRNA vaccines and therapeutics to increase in vitro stability.

biophysics↗

bioRxiv2020

Computationally Guided Design of Novel Selective Serotonin Reuptake Inhibitors

Current selective serotonin reuptake inhibitor (SSRI) drugs such as paroxetine, fluoxetine and escitalopram are prescribed for mental health conditions such as anxiety and depression. However, there are still several unpleasant side effects which fuels the need for new designs. In this work, computational studies were conducted to design two novel drug candidates with improved docking scores than paroxetine, an existing SSRI drug. Homology analysis was carried out to determine the suitability of an organism for preclinical trials and revealed laboratory mouse (Mus musculus) to be a good candidate.

pharmacology and toxicology↗

bioRxiv2020

Dietary Protein on Growth Performance, Serum Biochemical Index and offspring of Hu ewes in late pregnancy

The purpose of this study was to explore the effects of ewe growth performance, serum biochemical indicators and lamb growth and development at different protein levels in late pregnancy. A total of 15 pregnant ewes (46.4 {+/-} 1.38kg initial BW) were assigned to 3 groups with 5 ewes in each group in a randomized block design. P1, P2 and P3 of three groups were fed diets with different levels of crude protein at 11.25%, 12.5% and 13.75% (low, medium and high) respectively, while the dietary energy levels remained unchanged. Choose ewes with the same body condition from 90 days of pregnancy to different protein diets during delivery, and feed the same diets during lactation. New-born lambs were left to suckle their dams freely for the first three days and were kept with their dams till weaning at 60 days of age. Dietary protein levels during late gestation showed no significant effect on the weight gain of ewes and their offspring in the late pregnancy (P > 0.05) but had significant effects on blood urea nitrogen (BUN), glucose (GLU), C-reactive protein (CRPL3) and ammonia nitrogen (NH3L) before parturition, and on triglyceride (TG) in lactating ewes. Dietary protein levels during late gestation had significant effects on birth height, body length, chest depth, chest circumference, straight crown hip length and curved crown hip length of lambs (P < 0.05) as well as on weaning body length, chest circumference, head width and head length (P < 0.05). The results showed that although different dietary protein levels during late gestation have no significant effects on growth performance of ewes, singleton, twins and triplets, it affects lambs body shape and ewes metabolism. Therefore, the optimum protein concentration for the growth of ewes and subsequent lambs in this experiment is 11.25%.

physiology↗

bioRxiv2020

Microbubble-Mediated Cavitation Promotes Apoptosis and Suppresses Invasion in AsPC-1 Cells

This study aimed to identify the potential and mechanisms of microbubble-mediated cavitation in promoting apoptosis and suppressing invasion in cancer cells. AsPC-1 cells were used and divided into four groups: control group, microbubble-only (MB) group, ultrasound-only (US) group, and ultrasound combined with microbubbles (US+MB) group. Pulse ultrasound was used with a frequency of 360 kHz and an intensity of 0.7 W/cm2 for 1 min (duty rate=50%). Then cells in four groups were cultured for 24 h. Cell counting kit-8 illustrated that US+MB could decrease cell viability. Western blot confirmed that US+MB increased cleaved Caspase-3, Bcl-2-associated X protein (BAX), and decreased the B-cell lymphoma-2 (Bcl-2) levels. Besides, US+MB increased intracellular calcium ions and down-regulated cleaved Caspase-8. For proliferation ability, cells in US+MB group had a lower expression of Ki67 and the weakened colony formation ability. The transwell invasion assay showed that US+MB could decrease invasion ability in AsPC-1 cells. Further evidence showed that cells conducted with US+MB had the lower level of hypoxia-inducible factor-1 (HIF-1) and Vimentin and the higher expression of E-cadherin than the other three groups. Finally, cells conducted with US+MB had less invadopodia formation. In conclusion, these results suggested that microbubble-mediated cavitation promoted apoptosis and suppressed invasion in AsPC-1 cells. SignificanceCancer cells have a high rate of cell division and a high rate of cell division can speed up the development of hypoxia which lead to promote cancer invasion and metastasis. Here we used microbubble-mediated cavitation to generate a mechanical shock wave which could effectively kill cancer cells through physical damage and intrinsic signaling pathway. Furthermore, effective killing of cancer cells could restrain the development of hypoxia and prevent invasion ability of cancer cells.

bioengineering↗

bioRxiv2020

Dual function of perivascular fibroblasts in vascular stabilization in zebrafish

Blood vessels are vital to sustain life in all vertebrates. While it is known that mural cells (pericytes and smooth muscle cells) regulate vascular integrity, the contribution of other cell types to vascular stabilization has been largely unexplored. Using zebrafish, we identified sclerotome-derived perivascular fibroblasts as a novel population of blood vessel associated cells. In contrast to pericytes, perivascular fibroblasts emerge early during development, express the extracellular matrix (ECM) genes col1a2 and col5a1, and display distinct morphology and distribution. Time-lapse imaging reveals that perivascular fibroblasts serve as pericyte precursors. Genetic ablation of perivascular fibroblasts results in dysmorphic blood vessels with variable diameters. Strikingly, col5a1 mutants show spontaneous hemorrhage, and the penetrance of the phenotype is strongly enhanced by the additional loss of col1a2. Together, our work reveals dual roles of perivascular fibroblasts in vascular stabilization where they establish the ECM around nascent vessels and function as pericyte progenitors. AUTHOR SUMMARYBlood vessels are essential to sustain life in humans. Defects in blood vessels can lead to serious diseases, such as hemorrhage, tissue ischemia, and stroke. However, how blood vessel stability is maintained by surrounding support cells is still poorly understood. Using the zebrafish model, we identify a new population of blood vessel associated cells termed perivascular fibroblasts, which originate from the sclerotome, an embryonic structure that is previously known to generate the skeleton of the animal. Perivascular fibroblasts are distinct from pericytes, a known population of blood vessel support cells. They become associated with blood vessels much earlier than pericytes and express several collagen genes, encoding main components of the extracellular matrix. Loss of perivascular fibroblasts or mutations in collagen genes result in fragile blood vessels prone to damage. Using cell tracing in live animals, we find that a subset of perivascular fibroblasts can differentiate into pericytes. Together, our work shows that perivascular fibroblasts play two important roles in maintaining blood vessel integrity. Perivascular fibroblasts secrete collagens to stabilize newly formed blood vessels and a sub-population of these cells also functions as precursors to generate pericytes to provide additional vascular support.

developmental biology↗

bioRxiv2020

The Forensic Landscape and the Population Genetic Analyses of Hainan Li Based on Massively Parallel Sequencing DNA Profiling

Due to the formation of the Qiongzhou Strait by climate change and marine transition, Hainan island isolated from the mainland southern China during the Last Glacial Maximum. Hainan island, located at the southernmost part of China and separated from the Leizhou Peninsula by the Qiongzhou Strait, laid on one of the modern human northward migration routes from Southeast Asia to East Asia. The Hlai-language speaking Li minority, the second largest population after Han Chinese in Hainan island, is the direct descendants of the initial migrants in Hainan island and has unique ethnic properties and derived characteristics, however, the forensic associated studies on Hainan Li population are still insufficient. Hence, 136 Hainan Li individuals were genotyped in this study using the MPS-based ForenSeq DNA Signature Prep Kit (DNA Primer Set A) to characterize the forensic genetic polymorphism landscape, and DNA profiles were obtained from 152 different molecular genetic markers (27 autosomal STRs, 24 Y-STRs, 7 X-STRs, and 94 iiSNPs). A total of 419 distinct length variants and 586 repeat sequence sub-variants, with 31 novel alleles (at 17 loci), were identified across the 58 STR loci from the DNA profiles of Hainan Li population. We evaluated the forensic characteristics and efficiencies of DAPA, it demonstrated that the STRs and iiSNPs in DAPA were highly polymorphic in Hainan Li population and could be employed in forensic applications. In addition, we set up three Datasets, which included the genetic data of (I). iiSNPs (27 populations, 2640 individuals), (II). Y-STRs (42 populations, 8281 individuals), and (III). Y-haplogroups (123 populations, 4837 individuals) along with the population ancestries and language families, to perform population genetic analyses separately from different perspectives. In conclusion, the phylogenetic analyses indicated that Hainan Li, with a southern East Asia origin and Tai-Kadai language-speaking language, is an isolated population relatively. But the genetic pool of Hainan Li influenced by the limited gene flows from other Tai-Kadai populations and Hainan populations. Furthermore, the establishment of isolated population models will be beneficial to clarify the exquisite population structures and develop specific genetic markers for subpopulations in forensic genetic fields.

genetics↗

bioRxiv2020

ProGen: Language Modeling for Protein Generation

Generative modeling for protein engineering is key to solving fundamental problems in synthetic biology, medicine, and material science. We pose protein engineering as an unsupervised sequence generation problem in order to leverage the exponentially growing set of proteins that lack costly, structural annotations. We train a 1.2B-parameter language model, ProGen, on [~]280M protein sequences conditioned on taxonomic and keyword tags such as molecular function and cellular component. This provides ProGen with an unprecedented range of evolutionary sequence diversity and allows it to generate with fine-grained control as demonstrated by metrics based on primary sequence similarity, secondary structure accuracy, and conformational energy.

synthetic biology↗

bioRxiv2020

A novel protein complex that regulates active DNA demethylation in Arabidopsis

Active DNA demethylation is critical for altering DNA methylation patterns and regulating gene expression. The 5-methylcytosine DNA glycosylase/lyase ROS1 initiates a base excision repair pathway for active DNA demethylation and is required for the prevention of DNA hypermethylation at thousands of genomic regions in Arabidopsis. How ROS1 is regulated and targeted to specific genomic regions is not well understood. Here, we report the discovery of an Arabidopsis protein complex that contains ROS1, regulates ROS1 gene expression, and likely targets the ROS1 protein to specific genomic regions. ROS1 physically interacts with a WD40 domain protein (RWD40), which in turn interacts with a methyl-DNA binding protein (RMB1) as well as with a zinc finger and homeobox domain protein (RHD1). RMB1 binds to DNA that is methylated in any sequence context, and this binding is necessary for its function in vivo.Loss-of-function mutations in RWD40, RMB1, or RHD1 cause DNA hypermethylation at several tested genomic regions independently of the known ROS1 regulator IDM1. Because the hypermethylated genomic regions include the DNA methylation monitoring sequence in the ROS1 promoter, plants mutated in RWD40, RMB1, or RHD1 show increased ROS1 expression. Importantly, ROS1 binding to the ROS1 promoter requires RWD40, RMB1, and RHD1, suggesting that this complex dictates ROS1 targeting to this locus. Our results demonstrate that ROS1 forms a protein complex with RWD40, RMB1, and RHD1, and that this novel complex regulates active DNA demethylation at several endogenous loci in Arabidopsis.

plant biology↗

bioRxiv2020

Modulation of metabolic functions through Cas13d-mediated gene knockdown in liver

RNA knockdown in vivo carries significant potential for disease modelings and therapies. Despite the emerging approaches of CRISPR/Cas9-mediated permanent knock out of targeted genes, strategies targeting RNA for disruption are advantageous in the treatment of acquired metabolic disorders when permanent modification of the genome DNA is not appropriate, and RNA virus infection diseases when pathogenic DNA is not available (such as SARS-Cov-2 and MERS infections). Recently, Cas13d, a family of RNA-targeting CRISPR effectors, has been shown to accomplish robust down-regulation of cellular RNAs in mammalian cells in vitro. Among the various Cas13d subtypes, CasRx (RfxCas13d) showed the most potent RNA knockdown efficiency in HEK293T cells. However, the RNA-targeting activity of Cas13d still needs to be verified in vivo. In this study, the CasRx system was demonstrated to efficiently and functionally knock down genes related to metabolism functions, including Pten, Pcsk9 and lncLstr, in mouse hepatocytes. CasRx-mediated simultaneous knockdown of multiple genes was also achieved by sgRNA arrays, providing a useful strategy to modulate complex metabolism networks. Moreover, the AAV (adeno-associated virus)-mediated delivery of CasRx and Pcsk9 sgRNAs into mouse liver successfully decreased serum PCSK9, resulting in significant reduction of serum cholesterol levels. Importantly, CasRx-mediated knockdown of Pcsk9 is reversible and Pcsk9 could be repeatedly down-regulated, providing an effective strategy to reversibly modulate metabolic genes. The present work supplies a successful proof-of-concept trial that suggests efficient and regulatory knockdown of target metabolic genes for a designed metabolism modulation in the liver.

genomics↗

bioRxiv2020

Protein sequence design with a learned potential

AO_SCPLOWBSTRACTC_SCPLOWThe task of protein sequence design is central to nearly all rational protein engineering problems, and enormous effort has gone into the development of energy functions to guide design. We investigate the capability of a deep neural network model to automate design of sequences onto protein backbones, having learned directly from crystal structure data and without any human-specified priors. The model generalizes to native topologies not seen during training, producing experimentally stable designs. We evaluate the generalizability of our method to a de novo TIM-barrel scaffold. The model produces novel sequences, and high-resolution crystal structures of two designs show excellent agreement with the in silico models. Our findings demonstrate the tractability of an entirely learned method for protein sequence design.

bioinformatics↗

bioRxiv2019

Design to Data for mutants of β-glucosidase B from Paenibacillus polymyxa: Q22T, W123R, F155G, Y169M, W438D, V401A

A key goal of protein engineering is to accurately model the stability and catalytic activity of enzymes. However, the limitations of functional predictive abilities pose a major challenge for modeling algorithm design, and can be attributed to the lack of large data sets quantifying the functional properties of enzymes. Here, the thermal stability (TM) and Michaelis-Menten constants (kcat, KM, and kcat/KM) of six new variants of the {beta}-glucosidase B (BglB) protein are quantitatively characterized. Molecular stability of the enzyme variants were hypothesized using the Foldit software and BglB was synthesized in E. coli cells. Testing was done through a colorimetric kinetic assay and thermal stability fluorescence-based protein unfolding assay. Results from the assays suggest that all mutations, with the exception of variant Y169M, all experienced reduced catalytic efficiency compared to the wildtype. Assay results indicate that variant W123R is more thermally stable compared to the wildtype, while the differences in thermal stability between the other variants, and the wildtype are negligible. The collected thermal stability and catalytic efficiency data has been added to a data set with the aim of improving Rosetta algorithms for modeling and predicting the functional interactions between biomolecules through a machine learning algorithm and facilitate the precise engineering of protein catalysts.

biochemistry↗

bioRxiv2019

Optical activation of TrkB receptors

Brain-derived neurotrophic factor (BDNF), via activation of tropomyosin receptor kinase B (TrkB), plays a critical role in neuronal proliferation, differentiation, survival, and death. Dysregulation of TrkB signaling is implicated in neurodegenerative disorders and cancers. Precise activation of TrkB receptors with spatial and temporal resolution is greatly desired to study the dynamic nature of TrkB signaling and its role in related diseases. Here we develop different optogenetic approaches that use light to activate TrkB receptors. Utilizing the photosensitive protein Arabidopsis thaliana cryptochrome 2 (CRY2), the light-inducible homo-interaction of the intracellular domain of TrkB (iTrkB) in the cytosol or on the plasma membrane is able to induce the activation of downstream MAPK/ERK and PI3K/Akt signaling as well as the neurite outgrowth of PC12 cells. Moreover, we prove that such strategies are generalizable to other optical homo-dimerizers by demonstrating the optical TrkB activation based on the light-oxygen-voltage domain of aureochrome 1 from Vaucheria frigida. The results open up new possibilities of many other optical platforms to activate TrkB receptors to fulfill customized needs. By comparing all the different strategies, we find that the CRY2-integrated approach to achieve light-induced cell membrane recruitment and homo-interaction of iTrkB is most efficient in activating TrkB receptors. The optogenetic strategies presented are promising tools to investigate BDNF/TrkB signaling with tight spatial and temporal control.

synthetic biology↗

bioRxiv2019

An optimized acetylcholine sensor for monitoring in vivo cholinergic activity

The ability to directly measure acetylcholine (ACh) release is an essential first step towards understanding its physiological function. Here we optimized the GRABACh (GPCR-Activation-Based-ACh) sensor with significantly improved sensitivity and minimal downstream coupling. Using this sensor, we measured in-vivo cholinergic activity in both Drosophila and mice, revealing compartmental ACh signals in fly olfactory center and single-trial ACh dynamics in multiple regions of the mice brain under a variety of different behaviors

neuroscience↗

bioRxiv2019

Evaluating molecular modeling tools for thermal stability using an independently generated dataset

Engineering proteins to enhance thermal stability is a widely utilized approach for creating industrially relevant biocatalysts. Computational tools that guide these engineering efforts remain an active area of research with new data sets and develop algorithms. To aid in these efforts, we are reporting an expansion of our previously published data set of mutants for a {beta}-glucosidase to include both measures of TM and {Delta}{Delta}G, to complement the previously reported measures of T50 and kinetic constants (kcat and KM). For a set of 51 mutants, we found that T50 and TM are moderately correlated with a Pearson correlation coefficient (PCC) of 0.58, indicated the two methods capture different physical features. The performance of predicted stability using five computational tools are also evaluated on the 51 mutants dataset, none of which are found to be strong predictors of the observed changes in T50, TM, or {Delta}{Delta}G. Furthermore, the ability of the five algorithms to predict the production of isolatable soluble protein is examined, which revealed that Rosetta {Delta}{Delta}G, ELASPIC, and DeepDDG are capable of predicting if a mutant could be produced and isolated as a soluble protein. These results further highlight the need for new algorithms for predicting modest, yet important, changes in thermal stability as well as a new utility for current algorithms for prescreening designs for the production of soluble mutants.

biophysics↗

bioRxiv2019

Design to Data for mutants of β-glucosidase B from Paenibacillus polymyxa: M319C, T431I, and K337D

The use of computational tools has become an increasingly popular tool for engineering protein function. While there are numerous examples of computational tools enabling the design of novel protein functions, there remains room for improvement in both prediction accuracy and success. To improve algorithms for functional and stability predictions, we have initiated the development of a data set designed to be used for training new computational algorithms for enzyme design. To date our dataset is composed of over 129 mutants with associated expression levels, kinetic data, and thermal stability for the enzyme {beta}-glucosidase B (BglB) from Paenibacillus polymyxa. In this study, we introduced three new variants (M319C, T431I, and K337D) to our existing dataset with the goal of cultivating a larger dataset to train new design algorithms and more broadly explore structure-function relationships in BglB.

biochemistry↗

bioRxiv2019

Global kinome silencing combined with 3D invasion screening of the tumor microenvironment identifies fibroblast-expressed PIK3Cδ involvement in triple-negative breast cancer progression

As there is growing evidence for the tumor microenvironments (TME) role in tumorigenesis, we sought to investigate the role of fibroblast-expressed kinases in triple negative breast cancer (TNBC). Using a high-throughput kinome screen combined with 3D invasion assays, we identified fibroblast-expressed PIK3C{delta} (f-PIK3C{delta}) as a key regulator of progression. Although PIK3C{delta} has been mainly described in leucocytes, we detected high expression in primary fibroblasts derived from TNBC patients, while PIK3C{delta} was undetectable in cancer epithelial cell lines. Genetic and pharmacologic gain- and loss-of functions experiments verified the contribution of f-PIK3C{delta} in TNBC cell invasion. By employing an integrated secretomics and transcriptomics analysis, we revealed a paracrine mechanism via which f-PIK3C{delta} confers its pro-tumorigenic effects. Inhibition of f-PIK3C{delta} promoted the secretion of factors, including PLGF and BDNF, which subsequently led to upregulation of NR4A1 in TNBC cells where it acts as a tumor suppressor. Inhibition of PIK3C{delta} in an orthotopic BC mouse model reduced tumor growth only after inoculation with fibroblasts, indicating a role of f-PIK3C{delta} in cancer progression. Similar results were observed in the MMTV-PyMT transgenic BC mouse model, in addition to a decrease on tumor metastasis emphasizing the potential immune-independent effects of PIK3C{delta} inhibition. Finally, analysis of BC patient cohorts and TCGA datasets identified f-PIK3C{delta} (protein and mRNA levels) as an independent prognostic factor for overall and disease free survival, highlighting it as a therapeutic target for TNBC.

cancer biology↗