Biology subjects

Tan, Y.

Publications and source records attributed to Tan, Y..

At least 163 records · Page 9Linked to original sources

bioRxiv2021

A recombinant selective drug-resistant M. bovis BCG enhances the bactericidal activity of a second-line tuberculosis regimen

Drug-resistant tuberculosis (DR-TB) results from infection by Mycobacterium tuberculosis strains resistant to at least rifampin or isoniazid. To improve the treatment outcome in DR-TB, therapeutic vaccines are considered an ideal choice as they can enhance pathogen clearance and minimize disease sequelae. To date, there is no therapeutic vaccine reported to be effective when combined with a chemotherapy regimen against DR-TB. The only available TB vaccine, the M. bovis BCG (BCG) is susceptible to several anti-TB drugs hence not a perfect option for therapeutic vaccination. Herein, we developed a recombinant BCG (RdrBCG) overexpressing Ag85B and Rv2628 with resistance to selected anti-TB drugs. When administered three times adjunct to a second-line anti-TB regimen in a classical murine model of DR-TB, the RdrBCG lowered lung M. tuberculosis colony-forming units by 1 log10. Furthermore, vaccination with the RdrBCG adjunct to TB chemotherapy minimized lung tissue pathology in mice. Most importantly, the RdrBCG maintained the exogenously inserted genes and showed almost the same virulence as its parent BCG Tice strain in severe combined immune-deficient mice. All these suggested that the RdrBCG was stable, safe and effective. Hence, the "recombinant" plus "drug-resistant" BCG strategy could be a useful concept for developing therapeutic vaccines against DR-TB.

microbiology↗

bioRxiv2021

Dendritic membrane resistance modulates activity-induced Ca2+ influx in oxytocinergic magnocellular neurons of mouse PVN

Hypothalamic oxytocinergic magnocellular neurons have a fascinating ability to release peptide from both their axon terminals and from their dendrites. Existing data indicates there is a flexible relationship between somatic activity and dendritic release, but the mechanisms governing this relationship are not completely understood. Here we use a combination of electrical and optical recording techniques to quantify activity-dependent calcium influx in proximal vs. distal dendrites of oxytocinergic magnocellular neurons located in the paraventricular nucleus of the hypothalamus (OT-MCNs). Results reveal that the dendrites of OT-MCNs are weak conductors of somatic voltage changes, and yet activity-induced dendritic calcium influx can be robustly regulated by a diverse set of stimuli that open or close ionophores located along the dendritic membrane. Overall, this study reveals that dendritic membrane resistance is a dynamic and endogenously regulated feature of OT-MCNs that is likely to have substantial functional impact on central oxytocin release. IMPACT STATEMENTActivity-induced dendritic calcium influx in oxytocinergic magnocellular neurons can be robustly modulated by a highly diverse set of stimuli acting on distinct types of ionophores expressed along the dendritic membrane.

neuroscience↗

bioRxiv2020

Unification of the M/ORF3-related proteins points to a diversified role for ion conductance in pathogenesis of coronaviruses and other nidoviruses

The new coronavirus, SARS-CoV-2, responsible for the COVID-19 pandemic has emphasized the need for a better understanding of the evolution of virus-host conflicts. ORF3a in both SARS-CoV-1 and SARS-CoV-2 are ion channels (viroporins) and involved in virion assembly and membrane budding. Using sensitive profile-based homology detection methods, we unify the SARS-CoV ORF3a family with several families of viral proteins, including ORF5 from MERS-CoVs, proteins from beta-CoVs (ORF3c), alpha-CoVs (ORF3b), most importantly, the Matrix (M) proteins from CoVs, and more distant homologs from other nidoviruses. By sequence analysis and structural modeling, we show that these viral families utilize specific conserved polar residues to constitute an ion-conducting pore in the membrane. We reconstruct the evolutionary history of these families, objectively establish the common origin of the M proteins of CoVs and Toroviruses. We show that the divergent ORF3a/ORF3b/ORF5 families represent a duplication stemming from the M protein in alpha- and beta-CoVs. By phyletic profiling of major structural components of primary nidoviruses, we present a model for their role in virion assembly of CoVs, ToroVs and Arteriviruses. The unification of diverse M/ORF3 ion channel families in a wide range of nidoviruses, especially the typical M protein in CoVs, reveal a conserved, previously under-appreciated role of ion channels in virion assembly, membrane fusion and budding. We show that the M and ORF3 are under differential evolutionary pressures; in contrast to the slow evolution of M as core structural component, the CoV-ORF3 clade is under selection for diversification, which indicates it is likely at the interface with host molecules and/or immune attack. IMPORTANCECoronaviruses (CoVs) have become a major threat to human welfare as the causative agents of several severe infectious diseases, namely Severe Acute Respiratory Syndrome (SARS), Middle Eastern Respiratory Syndrome (MERS), and the recently emerging human coronavirus disease 2019 (COVID-19). The rapid spread, severity of these diseases, as well as the potential re-emergence of other CoV-associated diseases have imposed a strong need for a thorough understanding of function and evolution of these CoVs. By utilizing robust domain-centric computational strategies, we have established homologous relationships between many divergent families of CoV proteins, including SARS-CoV/SARS-CoV-2 ORF3a, MERS-CoV ORF5, proteins from both beta-CoVs (ORF3c) and alpha-CoVs (ORF3b), the typical CoV Matrix proteins, and many distant homologs from other nidoviruses. We present evidence that they are active ion channel proteins, and the Cov-specific ORF3 clade proteins are under selection for rapid diversification, suggesting they might have been involved in interfering host molecules and/or immune attack.

microbiology↗

bioRxiv2020

iTRAQ-Based Proteomic Profiling of PLC-Mediated Expression of 20E-Induced Protein in Apolygus lucorum (Meyer-Dür)

The polyphagous pest Apolygus lucorum has become the dominant insect in Bacillus thuringiensis (Bt) cotton fields. The hormone 20-hydroxyecdysone (20E) regulates multiple events in insect development and physiology. 20E responses are controlled by pathways triggered by phospholipase C (PLC)-associated proteins. However, 20E-modulated genes whose expression is affected by PLC remain unknown. Here, isobaric tag for relative and absolute quantitation (iTRAQ) and immunoblot were carried out for comparing differentially expressed proteins (DEPs) in A. lucorum in response to 20E and the PLC inhibitor U73122, respectively. Totally 1624 DEPs were, respectively, found in the 20E/control, U73122/control, and 20E+U73122/control groups. Venn diagram analysis further revealed 8 DEPs that were shared among the three groups. Immunoblot validated these findings, which corroborated and highlighted the reliability of proteomics. KEGG enrichment analysis showed that the DEPs were included in diverse signaling pathways. The largest portion of DEPs among the three groups were categorized in metabolic pathways. In addition, DEPs among the three groups were also found to regulate the Ras-MAPK and PI3K-AKT pathways. This is the first time that iTRAQ was carried out to assess proteome alteration in A. lucorum nymphs in response to 20E and a PLC inhibitor. These findings provide novel insights into protein expression in A. lucorum in response to 20E, and a more comprehensive understanding of the function of PLC in 20E signal transduction.

biochemistry↗

bioRxiv2020

Telomere-to-telomere assembly of the genome of an individual Oikopleura dioica from Okinawa using Nanopore-based sequencing

BackgroundThe larvacean Oikopleura dioica is an abundant tunicate plankton with the smallest (65-70 Mbp) non-parasitic, non-extremophile animal genome identified to date. Currently, there are two genomes available for the Bergen (OdB3) and Osaka (OSKA2016) O. dioica laboratory strains. Both assemblies have full genome coverage and high sequence accuracy. However, a chromosome-scale assembly has not yet been achieved. ResultsHere, we present a chromosome-scale genome assembly (OKI2018_I69) of the Okinawan O. dioica produced using long-read Nanopore and short-read Illumina sequencing data from a single male, combined with Hi-C chromosomal conformation capture data for scaffolding. The OKI2018_I69 assembly has a total length of 64.3 Mbp distributed among 19 scaffolds. 99% of the assembly is in five megabase-scale scaffolds. We found telomeres on both ends of the two largest scaffolds, which represent assemblies of two fully contiguous autosomal chromosomes. Each of the other three large scaffolds have telomeres at one end only and we propose that they correspond to sex chromosomes split into a pseudo-autosomal region and X-specific or Y-specific regions. Indeed, these five scaffolds mostly correspond to equivalent linkage groups of OdB3, suggesting overall agreement in chromosomal organization between the two populations. At a more detailed level, the OKI2018_I69 assembly possesses similar genomic features in gene content and repetitive elements reported for OdB3. The Hi-C map suggests few reciprocal interactions between chromosome arms. At the sequence level, multiple genomic features such as GC content and repetitive elements are distributed differently along the short and long arms of the same chromosome. ConclusionsWe show that a hybrid approach of integrating multiple sequencing technologies with chromosome conformation information results in an accurate de novo chromosome-scale assembly of O. dioicas highly polymorphic genome. This assembly will be a useful resource for genome-wide comparative studies between O. dioica and other species, as well as studies of chromosomal evolution in this lineage.

genomics↗

bioRxiv2020

A Human Multi-Lineage Hepatic Organoid Model for Liver Fibrosis

BackgroundTo characterize fibrogenic mechanisms, genome engineering and a human hepatic organoid system was used to produce an in vitro model for human liver fibrosis. Methods and resultsHuman hepatic organoids that were engineered to express the most common causative mutation for Autosomal Recessive Polycystic Kidney Disease (ARPKD) developed the key features of ARPKD liver pathology (abnormal bile ducts and hepatic fibrosis) in only 21 days. Second harmonic generation microscopy confirmed that the ARPKD mutation increased collagen abundance and thick collagen fiber production in hepatic organoids; and we demonstrated that these changes mirrored that occurring in ARPKD liver tissue. Transcriptomic and other analyses indicated that the ARPKD mutation generates cholangiocytes with increased TGF{beta}-associated pathway activation, which are actively involved in collagen fiber generation. The abnormal cholangiocytes promote the expansion of collagen-producing myofibroblasts with markedly increased PDGFR{beta} protein expression and an activated STAT3 signaling pathway. Moreover, the transcriptome of ARPKD organoid myofibroblasts resembled that of myofibroblasts in liver tissue obtained from patients with commonly occurring acquired forms of liver fibrosis. The involvement of the PDGFRB pathway was confirmed by the anti-fibrotic effect observed when ARPKD organoids were treated with PDGFRB inhibitors. ConclusionsBesides providing mechanistic insight into the pathogenesis of congenital (and possibly acquired) forms of liver fibrosis, ARPKD organoids could also be used to test the anti-fibrotic efficacy of potential anti-fibrotic therapies.

cell biology↗

bioRxiv2020

MicroRNA-1 promotes the development of and prolongs engorgement time in Hyalomma anatolicum anatolicum (Acari: Ixodidae) ticks

MicroRNAs act as mRNA posttranscriptional regulators, playing important roles in cell differentiation, transcriptional regulation, growth and development. In this study, microRNA expression profiles of Hyalomma anatolicum anatolicum ticks at different developmental stages were detected by high-throughput sequencing and functionally assessed. In total, 2,585,169, 1,252,678, 1,558,217 and 1,155,283 unique reads were obtained from eggs, larvae, nymphs and adults, respectively, with 42, 46, 45 and 41 conserved microRNAs in these stages, respectively. Using eggs as a control, 48, 43 and 39 microRNAs were upregulated and 3, 10 and 9 downregulated in larvae, nymphs and adults, respectively. microRNA-1 (miR-1) was expressed in high abundance throughout Ha. anatolicum development, with an average of nearly one million transcripts, and it is highly conserved among tick species. Quantitative real-time PCR (qPCR) showed that miR-1 expression gradually increased with tick development, reaching the highest level at engorgement. Differential tissue expression was detected, with significantly higher levels in the salivary glands and epidermis than in the midgut. Inhibition assays showed no significant change in body weight or spawning time or amount between experimental and control groups, but there was a significant difference (p<0.01) in engorgement time. With miR-1 inhibition, ticks displayed obvious deformities during later development. To more fully explain the microRNA mechanism of action, the miR-1 family was analyzed regarding target gene; members acting on Hsp60 include miR-5, miR-994, miR-969, and miR-1011, which jointly play a role. Therefore, microRNAs are critical for normal tick development, and the primary structure of the mature sequence of miR-1 is highly conserved. Nonetheless, different developmental stages and tissues show different expression patterns, with a certain role in prolonging feeding. miR-1, together with other family members, regulates mRNA function and may be used as a molecular marker for species origin and evolution analysis and internal reference gene selection.

microbiology↗

bioRxiv2020

Inactivation of p21-Activated Kinase 2 (Pak2) Inhibits the Development of Nf2-Deficient Malignant Mesothelioma

Malignant mesotheliomas (MM) show frequent somatic loss of the NF2 tumor suppressor gene. The NF2 product, Merlin, is implicated in several tumor-related pathways, including p21-activated kinase (PAK) signaling. Merlin is both a phosphorylation target for PAK and a negative regulator of this oncogenic kinase. Merlin loss results in PAK activation, and PAK inhibitors hold promise for the treatment of NF2-deficient tumors. To test this possibility in an in vivo genetic system, Nf2f/f;Cdkn2af/f mice were crossed to mice with conditional knockout of Pak2, a highly expressed group I Pak member. Cohorts of these animals were injected in either the thoracic or peritoneal cavities with adeno-Cre virus to delete floxed alleles in the mesothelial lining. Loss of Pak2 resulted in a markedly decreased incidence and delayed onset and progression of pleural and peritoneal MMs in Nf2;Cdkn2a-deficient (NC) mice, as documented by Kaplan-Meier survival curves and in vivo bioluminescent imaging. RNA-seq revealed that MMs from NC;Pak2-/- mice showed downregulated expression of genes involved in several oncogenic pathways (Wnt, Akt) when compared to MMs from mice retaining Pak2. Kinome profiling showed that, as compared to NC MM cells, NC;Pak2-/- MM cells had multiple kinase changes indicative of an epithelial to mesenchymal transition. Collectively, these findings suggest that NC;Pak2-/- MMs adapt by reprogramming their kinome and gene signature profiles to bypass the need for PAK activity via the activation of other compensatory oncogenic kinase pathways. The identification of such secondary pathways offers opportunities for rational combination therapies to circumvent resistance to anti-PAK drugs.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2020

Therapeutic role of recurrent ESR1-CCDC170 gene fusions in breast cancer endocrine resistance

Background Endocrine therapy is the most common treatment for estrogen receptor (ER)-positive breast cancer, but its effectiveness is limited by high rates of primary and acquired resistance. There are likely many genetic causes and recent studies suggest the important role of ESR1 mutations and fusions in endocrine resistance. Previously we reported a recurrent ESR1 fusion called ESR1-CCDC170 in 6-8% of the luminal B breast cancers that has a worse clinical outcome after endocrine therapy. Despite being the most frequent ESR1 fusion, its functional role in endocrine resistance have not been studied in vivo, and the engaged mechanism and therapeutic relevance remain uncharacterized.Methods The endocrine sensitivities of HCC1428 or T47D breast cancer cells following genetic perturbations of ESR1-CCDC170 were assessed using clonogenic assays and/or xenograft mouse models. The underlying mechanisms were investigated by reverse phase protein array, western blotting, immunoprecipitation, and bimolecular fluorescence complementation assays. The sensitivity of ESR1-CCDC170 expressing breast cancer cells to concomitant treatments of tamoxifen and HER/SRC inhibitors was assessed by clonogenic assays.Results Our results suggested that different ESR1-CCDC170 fusions endow different levels of reduced endocrine sensitivity in vivo, resulting in significant survival disadvantages. Further investigation revealed a novel mechanism that ESR1-CCDC170 binds to HER2/HER3/SRC and activates SRC/PI3K/AKT signaling. Silencing of ESR1-CCDC170 in the fusion-positive cell line, HCC1428, downregulates HER2/HER3, represses pSRC/pAKT, and improves endocrine sensitivity. More important, breast cancer cells expressing ectopic or endogenous ESR1-CCDC170 are highly sensitive to treatment regimens combining endocrine agents with the HER2 inhibitor lapatinib and/or the SRC inhibitor dasatinib.Conclusion ESR1-CCDC170 may endow breast cancer cell survival under endocrine therapy via maintaining/activating HER2/HER3/SRC/AKT signaling which implies a potential therapeutic strategy for managing these fusion positive tumors.Competing Interest StatementDr. Rachel Schiff receives research funding from AstraZeneca, GlaxoSmithKline, Gilead Sciences, and PUMA Biotechnology, outside of this project; and is a consulting/advisory committee member for Macrogenics, and Eli Lilly. The other coauthors do not have competing financial interests.AbbreviationsABCATP-binding cassetteAIsaromatase inhibitorsBiFCBimolecular Fluorescence ComplementationCCDC170coiled-coil domain containing 170CSScharcoal-stripped serumΔCCDC170truncated CCDC170 proteinsE2estradiolERestrogen receptorFDAFood and Drug Administration4-OHT4-hydroxytamoxifenORFopen reading frameRPPAReverse phase protein array analysisSERDsselective ER down-regulatorsSERMsselective ER modulatorssiRNAsmall interfering RNASMCstructural maintenance of chromosomesTPERTissue Protein Extraction ReagentView Full Text

cancer biology↗

bioRxiv2020

Centromere-specific antibody-mediated karyotyping of Okinawan Oikopleura dioica suggests the presence of three chromosomes.

Oikopleura dioica is a ubiquitous marine tunicate of biological interest due to features that include dioecious reproduction, short life cycle, and vertebrate-like dorsal notochord while possessing a relatively compact genome. The use of tunicates as model organisms, particularly with these characteristics, offers the advantage of facilitating studies in evolutionary development and furthering understanding of enduring attributes found in the more complex vertebrates. At present, we are undertaking an initiative to sequence the genomes of Oikopleura individuals in populations found among the seas surrounding the Ryukyu Islands in southern Japan. To facilitate and validate genome assemblies, karyotyping was employed to count individual animals chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3. New imaging data of embryos and oocytes stained with two different antibodies were obtained; interpretation of these data lead us to conclude that the Okinawan Oikopleura dioica has three pairs of chromosomes, akin to previous results from genomic assemblies in Atlantic populations. The imaging data have been deposited to the open-access EBI BioImage Archive for reuse while additionally providing representative images of two commercially available anti-H3S28P antibodies staining properties for use in epifluorescent and confocal based fluorescent microscopy.

cell biology↗

bioRxiv2020

Insight into vaccine development for Alpha-coronaviruses based on structural and immunological analyses of spike proteins

Coronaviruses that infect humans belong to the Alpha-coronavirus (including HCoV-229E) and Beta-coronavirus (including SARS-CoV and SARS-CoV-2) genera. In particular, SARS-CoV-2 is currently a major threat to public health worldwide. However, no commercial vaccines against the coronaviruses that can infect humans are available. The spike (S) homotrimers bind to their receptors through the receptor-binding domain (RBD), which is believed to be a major target to block viral entry. In this study, we selected Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) as models. Their RBDs were observed to adopt two different conformational states (lying or standing). Then, structural and immunological analyses were used to explore differences in the immune response with RBDs among these coronaviruses. Our results showed that more RBD-specific antibodies were induced by the S trimer with the RBD in the "standing" state (SARS-CoV and SARS-CoV-2) than the S trimer with the RBD in the "lying" state (HCoV-229E), and the affinity between the RBD-specific antibodies and S trimer was also higher in the SARS-CoV and SARS-CoV-2. In addition, we found that the ability of the HCoV-229E RBD to induce neutralizing antibodies was much lower and the intact and stable S1 subunit was essential for producing efficient neutralizing antibodies against HCoV-229E. Importantly, our results reveal different vaccine strategies for coronaviruses, and S-trimer is better than RBD as a target for vaccine development in Alpha-coronavirus. Our findings will provide important implications for future development of coronavirus vaccines. ImportanceOutbreak of coronaviruses, especially SARS-CoV-2, poses a serious threat to global public health. Development of vaccines to prevent the coronaviruses that can infect humans has always been a top priority. Coronavirus spike (S) protein is considered as a major target for vaccine development. Currently, structural studies have shown that Alpha-coronavirus (HCoV-229E) and Beta-coronavirus (SARS-CoV and SARS-CoV-2) RBDs are in lying and standing state, respectively. Here, we tested the ability of S-trimer and RBD to induce neutralizing antibodies among these coronaviruses. Our results showed that Beta-CoVs RBDs are in a standing state, and their S proteins can induce more neutralizing antibodies targeting RBD. However, HCoV-229E RBD is in a lying state, and its S protein induces a low level of neutralizing antibody targeting RBD. Our results indicate that Alpha-coronavirus is more conducive to escape host immune recognition, and also provide novel ideas for the development of vaccines targeting S protein.

microbiology↗

bioRxiv2020

Limbal Niche Cells in Three-Dimensional Matrigel Induced Dedifferentiation of Mature Corneal Epithelial Cells toward a Progenitor State

PurposeTo investigate the possibility and the key factors of stably committed mature corneal epithelial cells dedifferentiate into corneal epithelial stem cells in vitro. MethodsMature cornea epithelia cell (MCEC) sheets or limbal epithelial progenitor cell (LEPC) sheets were isolated from central corneas or limbal segments by Dispase II and further digested with 0.25% trypsin/1 mM EDTA (T/E) to yield single cells. Limbal niche cells (LNC) were isolated from the limbal stroma by collagenase A and expanded on 5% Matrigel coated plastic. Single MCECs were seeded on 50% Matrigel with or without LNC culturing for 10 days, regarding as three-dimensional MCEC (3D-MCEC) group or three-dimensional MCEC+LNC (3D-MCEC+LNC) group. Expression of CK12, p63, PCK, Vimentin were analyzed with immunofluorescence staining. ResultsThe expression of mature cornea epithelial marker (CK12) in MCEC was higher than that in LEPC (P=0.020) but epithelial stem cell marker (p63) was lower than that in LEPC (P=0.000). When seeded in 3D Matrigel, single MCEC cells could form spheres within 72 hours, and the expression of CK12 reduced (P=0.005) and the expression of p63 also reduced to zero (P=0.000) compared to MCEC. Serial passages of LNC which were expanded in coated Matrigel could form spheres in 3D Matrigel. After mixing MCECs with LNC, rounder spheres emerged within 24 hours which consisted of both epithelia cells (PCK+/Vim-) and LNC (PCK-/Vim+). Moreover, epithelia cells in 3D-MCEC+LNC group expressed less CK12 and more p63 than those in MCEC group (P=0.043, 0.000). Besides, the diameter of spheres in 3D-MCEC+LNC group were larger than that in 3D-MCEC group (P=0.000). ConclusionHuman LNC and three-dimensional Matrigel could induce the dedifferentiation of mature corneal epithelial cells into corneal epithelial stem cells.

bioengineering↗

bioRxiv2020

Frizzled-7 Identifies Platinum Tolerant Ovarian Cancer Cells Susceptible to Ferroptosis

Defining traits of platinum tolerant cancer cells could expose new treatment vulnerabilities. Here, new markers associated with platinum tolerant cells and tumors were identified by using in vitro and in vivo ovarian cancer (OC) models treated repetitively with carboplatin and validated in human specimens. Platinum-tolerant cells and tumors were found to be enriched in ALDH (+) cells, formed more spheroids, and expressed increased levels of stemness-related transcription factors compared to parental cells. Additionally, platinum-tolerant cells and tumors highly expressed the Wnt receptor, Frizzled 7 (FZD7). FZD7 knock down improved sensitivity to platinum, decreased spheroid formation, and delayed tumor initiation. The molecular signature distinguishing FZD7(+) from FZD7(-) cells included epithelial-to-mesenchymal (EMT), stemness, and oxidative phosphorylation enriched gene sets. Overexpression of FZD7 activated the oncogenic factor Tp63, driving upregulation of glutathione metabolism pathways, including glutathione peroxidase 4 (GPX4), which protects cells from chemotherapy-induced oxidative stress. FZD7(+) platinum-tolerant OC cells were more sensitive and underwent ferroptosis after treatment with GPX4 inhibitors. FZD7, Tp63 and glutathione metabolism gene sets were strongly correlated in the OC Tumor Cancer Genome Atlas (TCGA) database and in human OC specimens residual after chemotherapy. These results support the existence of a platinum-tolerant cell population with partial stem cell features, characterized by FZD7 expression and dependent on FZD7-{beta}-catenin-Tp63-GPX4 pathway for survival. The findings reveal a novel therapeutic vulnerability of platinum tolerant cancer cells and provide new insight into a potential "persister cancer cell" phenotype.

cancer biology↗

bioRxiv2020

PALLAS: Penalized mAximum LikeLihood and pArticle Swarms for inference of gene regulatory networks from time series data

We present PALLAS, a practical method for gene regulatory network (GRN) inference from time series data, which employs penalized maximum likelihood and particle swarms for optimization. PALLAS is based on the Partially-Observable Boolean Dynamical System (POBDS) model and thus does not require ad-hoc binarization of the data. The penalty in the likelihood is a LASSO regularization term, which encourages the resulting network to be sparse. PALLAS is able to scale to large networks under no prior knowledge, by virtue of a novel continuous-discrete Fish School Search particle swarm algorithm for efficient simultaneous maximization of the penalized likelihood over the discrete space of networks and the continuous space of observational parameters. The performance of PALLAS is demonstrated by a comprehensive set of experiments using synthetic data generated from real and artificial networks, as well as real time series microarray and RNA-seq data, where it is compared to several other well-known methods for gene regulatory network inference. The results show that PALLAS can infer GRNs efficiently and accurately. PALLAS is a fully-fledged program with a commandline user interface, written in python, and available on GitHub (https://github.com/yukuntan92/PALLAS).

bioinformatics↗

bioRxiv2020

Synthetic Maturation of Multilineage Human Liver Organoids via Genetically Guided Engineering

Pluripotent stem cell (PSC)-derived organoids are emerging as novel human-based microphysiological models but display immature phenotypes with limited subsets of endothelial or stromal cells. Here we demonstrate that in vitro manipulation of gene regulatory networks (GRNs) in PSC-derived liver organoids selected either through computational analysis or targeted tissue design can advance tissue maturation in vitro. Through an unbiased comparison with the genetic signature of mature livers, we identify downregulated GRNs in fetal liver organoids compared to adult livers. We demonstrate that overexpression of PROX1 and ATF5, together with targeted CRISPR-based transcriptional activation of endogenous CYP3A4, drives maturation in vitro. Single cell analyses reveal hepatobiliary-, endothelial-, and stellate-like cell populations. The engineered organoids demonstrate enhanced vasculogenesis, capture native liver characteristics (e.g. FXR signaling, CYP3A4 activity), and exhibit therapeutic potential in mice. Collectively, our approach provides a genetically guided framework for engineering developmentally advanced multilineage tissues from hiPSCs. HIGHLIGHTSO_LIIn vitro tissue maturation via genetically encoded molecular programs C_LIO_LIComputational analysis to identify maturation transcription factors in liver organoids C_LIO_LIPromoting vascularization of organoids via genetically encoded molecular programs C_LIO_LISingle cell analysis of parenchymal and non-parenchymal cells C_LIO_LIModeling of native liver functions and in vivo therapeutic potential C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=168 HEIGHT=200 SRC="FIGDIR/small/087445v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@6ec32aorg.highwire.dtl.DTLVardef@1cf2eeforg.highwire.dtl.DTLVardef@1128748org.highwire.dtl.DTLVardef@16fb5a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Evaluating rehabilitation progress using motion features identified by machine learning

Evaluating progress throughout a patients rehabilitation episode is critical for determining effectiveness of the selected treatments and contributing to the evidence-based practice. The evaluation process is complex due to the inherent large human variations in motor recovery and the limitations of commonly used clinical measurement tools. Information recorded during a robot-assisted rehabilitation process can provide an effective means to continuously quantitatively assess movement performance and rehabilitation progress. However, selecting appropriate motion features for rehabilitation evaluation has always been challenging. This paper exploits unsupervised feature learning techniques to reduce the complexity of building the evaluation model of patients progress. A new feature learning technique is developed to select the most significant features from a large amount of kinematic features measured from robotics, providing clinically useful information to health practitioners with reduction of modeling complexity. A novel indicator that can reflect monotonicity and trendability is proposed to evaluate the suitability of kinematic features, which are derived from the collected data of a population of stroke patients participating in robot-aided rehabilitation. The selected kinematic features allow for human variations across a population of patients as well as over the sequence of rehabilitation sessions. The study is based on data records pertaining to 41 stroke patients using three different robot assisted exercises for upper limb rehabilitation. Consistent with the literature, the results indicate that features based on movement smoothness are the best measures among 17 kinematic features used to evaluate rehabilitation progress.

bioengineering↗

bioRxiv2020

Hypoxia-related radiomics predict immunotherapy response: A multi-cohort study of NSCLC

Checkpoint blockade immunotherapy provides improved long-term survival in a subset of advanced stage non-small cell lung cancer (NSCLC) patients. However, highly predictive biomarkers of immunotherapy response are unmet clinical need. In this study, we utilized pre-treatment clinical factors and quantitative image-based biomarkers (radiomics) to identify a parsimonious model that predicts survival outcomes among NSCLC patients treated with immunotherapy. The NSCLC patients treated with single or double agent immunotherapy were included in three different cohorts: Training (N = 180), test (N = 90) and validation (N = 62) cohorts. The models were created based on overall survival (OS) and were additionally assessed for progression-free survival (PFS). Including most predictive radiomic features and clinical covariates, Classification and Regression Tree analysis was applied to stratify patients into survival risk-groups in the training cohort. The risk groups were later generated in the test and validation cohorts. Four independent NSCLC cohorts (total N = 446) were utilized for further validation of the radiomic signature. The biological underpinnings of the most informative radiomics were assessed using gene expression data from a radiogenomics dataset and validated by immunohistochemistry data (IHC). A parsimonious clinical-radiomics model was found to be significantly associated with OS and PFS after stratifying patients into groups of low-, moderate-, high-, and very-high risk of death and progression. This trained model was further tested and validated in two independent cohorts. When the extreme phenotypes were compared, the very-high risk group was found to be associated with extremely poor OS in both the test (hazard ratio [HR] = 5.35, 95% confidence interval [CI]: 2.14 - 13.36; 1-year OS = 11.1%) and validation (HR = 13.81, 95% CI: 2.58 - 73.93; 1-year OS = %47.6) cohorts when compared to the low risk group (HRs = 1.00; 1-year OS = 85.0% & 80.2%). Similar findings were observed for PFS. The final radiomic feature (GLCM inverse difference) was associated with OS in four independent NSCLC cohorts and was found to be positively associated with the hypoxia-related carbonic anhydrase, CAIX, by gene expression profiling and immunohistochemistry. We validated a novel clinical-radiomics model that is associated with OS and PFS among NSCLC patients treated with immunotherapy and identified a highly vulnerable subset of patients that are unlikely to respond to immunotherapy. The most informative radiomic feature was associated with CAIX, a marker of tumor hypoxia, tumor acidosis, and treatment resistance.

bioinformatics↗

bioRxiv2020

FPfilter: A false-positive-specific filter for whole-genome sequencing variant calling from GATK

1.MotivationAs whole genome sequencing (WGS) is becoming cost-effective progressivelly, it has been applied increasingly in medical and scientific fields. Although the traditional variant-calling pipeline (BWA+GATK) has very high accuracy, false positives (FPs) are still an unavoidable problem that might lead to unfavorable outcomes, especially in clinical applications. As a result, filtering out FPs is recommended after variant calling. However, loss of true positives (TPs) is inevitable in FP-filtering methods, such as GATK hard filtering (GATK-HF). Therefore, to minimize the loss of TPs and maximize the filtration of FPs, a comprehensive understanding of the features of TPs and FPs, and building an improved model of classification are necessary. To obtain information about TPs and FPs, we used Platinum Genome (PT) as the mutation reference and its 300x deep sequenced dataset NA12878 as the simulation template. Then random sampling across depth gradients from NA12878 was performed to study the depth effect. ResultsFPs among heterozygous mutations were found to have pattern distinct from that of FPs among homozygous mutations. FPfilter makes use of this model to filter out FPs specifically. We evaluated FPfilter on a training dataset with depth gradients from NA12878 and a test dataset from NA12877 and NA24385. Compared with GATK-HF, FPfilter showed a significantly higher FP/TP filtration ratio and F-measure score. Our results indicate that FPfilter provides an improved model for distinguishing FPs from TPs and filters FPs with high specificity. AvailabilityFPfilter is freely available for download on GitHub (https://github.com/yuxiangtan/FPfilter). Users can easily install it from anaconda.

bioinformatics↗