Biology subjects

Kobayashi, M.

Publications and source records attributed to Kobayashi, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Maternal regulation of the vertebrate oocyte-to-egg transition

Maternally-loaded factors in the egg accumulate during oogenesis and are essential for the oocyte and egg to acquire developmental competence and ensure the production of viable embryos. The oocyte-to-egg transition consists of the regulation of multiple molecular processes both cytoplasmic and nuclear acting in the late oocyte during a process called oocyte maturation. However, the molecular nature and functional importance of factors acting at this stage remain poorly understood. Here, we present a collection of 5 recessive maternal-effect mutants identified in a zebrafish forward genetic screen that reveal unique molecular insights into the mechanisms controlling the vertebrate oviparous oocyte-to-egg transition. We identified critical cytoplasmic regulators of yolk globule formation and maturation that are essential for egg development and embryogenesis. Specifically, the maternal-effect genes, over easy, poached, p33bjta, and black caviar control yolk globule sizing and/or protein cleavage during oogenesis, likely through endolysosomal organization independent of nuclear oocyte maturation. Furthermore, we cloned one of the mutant genes, identifying a subunit of the Adaptor Protein complex 5, which regulates intracellular trafficking, and yolk vesicle formation. Together, these mutant genes represent novel genetic entry points to decipher the molecular mechanisms functioning in the oocyte-to-egg transition, fertility, and human disease. Additionally, our genetic screen provides valuable functional tools for exploring the evolutionary fates of maternal factors and their contribution to developmental strategies for reproductive success in metazoans. Author SummaryThe oocyte-to-egg transition consists of the coordinated regulation of multiple molecular processes acting in the late oocyte. This transcriptionally silent period requires the precisely timed function of maternally-supplied gene products during oogenesis. However, knowledge of their molecular nature and in vivo function remains incomplete. The mutants reported here provide access to maternal factors regulating the processes that prepare an oocyte for reproductive competence and embryogenesis. We have identified essential regulators of yolk granule formation and protein processing. Specifically, we found that the highly conserved maternal Ap5m1 protein regulates yolk granule maturation, which generate essential nutrients and immunity for growth and development in oviparous animals. The mutants presented here represent attractive genetic models to investigate the molecular and cell biological mechanisms that control the oocyte-to-egg transition, as well as reveal a collection of genetic factors indispensable for reproduction and survival. Importantly, knowledge of their genetic underpinnings and biological importance in reproduction will also pave the way to investigate the evolution of maternal genes during vertebrate development.

developmental biology↗

bioRxiv2023

Discovery of the apiosyltransferase, celery UGT94AX1 that catalyzes the biosynthesis of a flavone glycoside, apiin

Apiose is a unique branched-chain pentose found in plant glycosides and a key component of the cell wall-polysaccharide pectin and other specialized metabolites. More than 1,200 plant-specialized metabolites contain apiose residues, represented by apiin, a distinctive flavone glycoside found in celery and parsley in the family Apiaceae. The physiological functions of apiin remain obscure, partly due to our lack of knowledge on apiosyltransferase during apiin biosynthesis. Here, we identified celery UGT94AX1 (AgApiT) as a novel apiosyltransferase, responsible for catalyzing the last sugar-modification step in apiin biosynthesis. AgApiT showed strict substrate specificity for the sugar donor, UDP-apiose, and moderate specificity for acceptor substrates, thereby producing various apiose-containing flavone glycosides in celery. Homology modeling of AgApiT with UDP-apiose, followed by site-directed mutagenesis experiments, identified unique Ile139, Phe140, and Leu356 residues in AgApiT, which are seemingly crucial for the recognition of UDP-apiose in the sugar donor pocket. Sequence comparison and molecular phylogenetic analysis of celery glycosyltransferases paralogous to AgApiT suggested that AgApiT is the sole apiosyltransferase-encoding gene in the celery genome. This is the first report on the identification of a plant apiosyltransferase gene that will enhance our understanding of the physio-ecological functions of apiose and apiose-containing compounds.

biochemistry↗

bioRxiv2022

Crosstalk between AQP4-dependent ATP/Adenosine release and dopamine neurotransmission in depressive behavior during cocaine withdrawal

Our previous study has demonstrated the AQP4-dependent ATP release and following elevation of extracellular adenosine in hippocampal slices during hypo-osmotic treatment. In the present study, we examined the crosstalk between AQP4-dependent adenosine elevation and dopaminergic neurotransmissions, especially focusing on the mouse depressive behavior during cocaine withdrawal. The depressive behavior was mitigated in AQP4 knockout mice, and was sensitive to adenosine A1 receptor antagonists. Thus, the influence of cocaine withdrawal on the adenosine or dopamine elevation in the slices from the striatum or medial prefrontal cortex, which is the focus of depression was analyzed by the originally-developed biosensor cells, which elevates intracellular Ca2+ in response to adenosine or dopamine. We have found that the evoked-dopamine release in the striatum is tonically suppressed via A1 receptor in a AQP4 dependent manner, and this suppression is augmented by the cocaine withdrawal. In contrast, the evoked dopamine elevation in the medial prefrontal cortex was suppressed by treating slices with adenosine or an A1 receptor antagonist, and the cocaine withdrawal abolished the suppression by A1 receptor antagonist alone in an AQP4-dependent manner. Since a GABAA or group II metabotropic glutamate receptor antagonist occluded the suppression by A1 receptor antagonist, it has been suggested that the glutamatergic and GABAergic terminals suppressing dopamine releases have A1 receptors, which the cocaine withdrawal down-regulates in a AQP4-dependent manner. These results indicate that astrocytes modulate dopaminergic neurotransmissions via A1 receptor in an AQP4-dependent manner, and suggest that cocaine causes depression by changing this modulation.

neuroscience↗

bioRxiv2022

Viral proteins and virus-like particles of the LTR5_Hs endogenous retrovirus in human primordial germ cell-like cells

The hominoid-specific endogenous retrovirus LTR5_Hs is transcriptionally activated in human primordial germ cell-like cells (hPGCLCs), a pluripotent stem cell-derived cell culture model of PGCs. Here, taking the unique advantage of our novel cell culture method to obtain large amounts of pure hPGCLCs, we performed proteomics profiling of hPGCLCs and detected various viral proteins produced from the LTR5_Hs RNA via ribosomal frameshifting. We also present transmission electron microscopy images of 100-nm diameter virus-like particles (VLPs) assembled at the surface of hPGCLCs. Compared to hPGCLCs, expression of LTR5_Hs RNA is far weaker in human seminomas, the germ cell tumors resembling PGCs. Re-analysis of published single cell RNA-seq data of human embryos revealed strong activation of LTR5_Hs in migrating PGCs but suppressed in PGCs upon they reach the gonadal anlagen. In the microfluidics-supported polarized embryoids mimicking peri-implantation stages of human embryos, LTR5_Hs RNA was detected by RNA in situ hybridization in NANOG+/TFAP2C+/SOX17+ cells resembling freshly emerged PGCs. These results support that human germ cells produce LTR5_Hs proteins and VLPs during their earliest stages of normal development until their settlement in the gonadal anlagen. SUMMARY STATEMENTThe hominoid-specific endogenous retrovirus LTR5_Hs is activated in a cell culture model resembling early-stage human primordial germ cells, producing not only viral RNA but also retrovirus proteins and virus-like particles.

developmental biology↗

bioRxiv2022

Spring dynamics and regional variation in two different root developmental types in seedlings of a boreal evergreen conifer species

Premise of researchIn boreal forests with various snow cover conditions, local adaptation can be expected in spring root exploration due to soil freezing or competition for the fundamental niche. However, few studies have investigated the adaptive strategies for acquiring soil space and resources after snow thawing. To get insights for the eco-evolutional significance of root growth dynamics in boreal regions, this study evaluated the intraspecific variation of morphology and structures in pioneer roots and fibrous roots. MethodologyValidation model specie was Abies sachalinensis, a representative boreal conifer species with local adaptation. Five-year-old seedlings derived from an eastern provenance with low snow cover and a northern provenance with high snow cover were grown for two years in a common garden of high snow cover region. To predict spring growth patterns of roots, seedlings were dug up in early spring just after snow thawing and late spring after one month has passed. Pioneer roots exploring soil space and fibrous roots acquiring soil resource were respectively collected, and their functions were evaluated by morphology and structures indicating its construction costs and maturity. Pivotal resultsIn pioneer roots, morphological and structural difference between sampling months were significant only in northern provenance, and high construction cost and well-developed structure were observed in early spring. This represents the intraspecific variation in reaction norms of pioneer roots to spring soil environment, indicating the soil space exploration in northern provenance may start in early spring or earlier. In fibrous roots, differences of specific root length and area between sampling months were significant only in eastern provenance, and low construction cost were observed in early spring. This may reflect the plastic responses of fibrous roots to the condition of high snow cover region. ConclusionsThis study indicated that local adaptation to contrasting snow cover condition may lead to the differentiation of spring root growth dynamics for the competitive or conservative strategies. Our results address better understandings of the mechanisms of niche acquisition in evergreen conifer.

ecology↗

bioRxiv2022

MicroRNA-218 instructs proper assembly of hippocampal networks

The assembly of the mammalian brain is orchestrated by temporally coordinated waves of gene expression. A key aspect of this developmental program is mediated at the post-transcriptional level by microRNAs (miRNAs). Deletion of neuronal enriched miRNAs induces strong developmental phenotypes, and multiple reports have found altered levels of miRNAs in patients with neurodevelopmental disorders. However, cellular and molecular mechanisms used by miRNAs to instruct proper brain development remain largely unexplored. Here, through multiple screens, we identified miR-218 as a critical regulator of hippocampal assembly in mice. MiR-218 is highly expressed in the hippocampus and enriched in both excitatory principal neurons and GABAergic inhibitory interneurons. Transient inhibition of miR-218 in early life results in an adult brain with heightened network activity and a predisposition to seizures. We used RNA-seq and FACS-seq (fluorescence-activated cell sorting followed by RNA-seq) to identify global and cell type-specific changes in gene expression in the absence of miR-218 and narrow down which altered developmental processes would lead to long-term network instability. We find that miR-218 inhibition results in the disruption of early depolarizing GABAergic signaling, structural defects in dendritic spines, and altered intrinsic membrane excitability. Finally, conditional knockout of miR-218 in interneurons, but not pyramidal neurons is sufficient to recapitulate the effects on long-term stability. Taken together, the data suggest that miR-218 orchestrates hippocampal network assembly to produce a stable network in the adult, primarily by regulating interneuron function in early postnatal life.

neuroscience↗

bioRxiv2022

Tangled gene-for-gene interactions mediate co-evolution of the rice NLR immune receptor Pik and blast fungus effector proteins

Studies focused solely on single organisms can fail to identify the networks underlying host-pathogen gene-for-gene interactions. Here, we integrate genetic analyses of rice (Oryza sativa, host) and rice blast fungus (Magnaporthe oryzae, pathogen) and uncover a new pathogen recognition specificity of the rice nucleotide-binding domain and leucine-rich repeat protein (NLR) immune receptor Pik, which mediates resistance to M. oryzae expressing the avirulence effector gene AVR-Pik. Rice Piks-1, encoded by an allele of Pik-1, recognizes a previously unidentified effector encoded by the M. oryzae avirulence gene AVR-Mgk1, which is found on a mini-chromosome. AVR-Mgk1 has no sequence similarity to known AVR-Pik effectors, and is prone to deletion from the mini-chromosome mediated by repeated Inago2 retrotransposon sequences. AVR-Mgk1 is detected by Piks-1 and by other Pik-1 alleles known to recognize AVR-Pik effectors; recognition is mediated by AVR-Mgk1 binding to the integrated heavy metal-associated (HMA) domain of Piks-1 and other Pik-1 alleles. Our findings highlight how complex gene-for-gene interaction networks can be disentangled by applying forward genetics approaches simultaneously to the host and pathogen. We demonstrate dynamic co-evolution between an NLR integrated domain and multiple families of effector proteins.

plant biology↗

bioRxiv2022

Efficient Human Germ Cell Specification from Stem Cells via Combinatorial Expression of Transcription Factors

The generation of germline cell types from human induced pluripotent stem cells (hiPSCs) represents a key milestone toward in vitro gametogenesis, which has the potential to transform reproductive modeling and medicine. Methods to recapitulate advanced germline cell specification in vitro have relied on extensive, long term culture methods, the most notable of which is a four-month culture protocol employing xenogeneic reconstituted ovaries with mouse embryonic ovarian somatic cells. Recently, transcription factor (TF)-based methods have demonstrated the feasibility of exogenous factor expression to directly differentiate hiPSCs into cell types of interest, including various ovarian cell types. The protocols leveraged in these studies, however, utilize more local methods of factor selection, such as basic differential gene expression analysis, and lower-throughput screening strategies via iterative testing of a small set of TFs. In this work, we integrate our recently-described graph theory pipeline and highly-parallelized screening protocols to globally identify and screen 46 oogenesis-regulating TFs for their role in human germline formation. We identify ZNF281, LHX8, SOHLH1, ZGLP1, and ANHX whose combinatorial overexpression drives DDX4+ induced oogonia-like cell (iOLC) formation from hiPSCs. In contrast to previous methods, our protocol employs a simple four-day, feeder-free monolayer culture condition. We additionally demonstrate a method of post-isolation, feeder-free expansion of DDX4+ iOLCs that shows retained cell identity in vitro. We additionally identify DLX5, HHEX, and FIGLA whose individual overexpression enhances hPGCLC formation from hiPSCs. We characterize these TF-based iOLCs and hPGCLCs via gene and protein expression analyses and demonstrate their broad similarity to in vivo and in vitro-derived oogonia and primordial germ cells. Together, these results identify new regulatory factors that enhance in vitro human germ cell specification and further establish unique computational and experimental tools for human in vitro oogenesis research.

developmental biology↗

bioRxiv2022

Directed Differentiation of Human iPSCs to Functional Ovarian Granulosa-Like Cells via Transcription Factor Overexpression

An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and their supporting somatic cells, known as granulosa cells. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human iPSCs, a method of generating granulosa cells has been elusive. Here we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of human iPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs, and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is necessary and sufficient to generate granulosa-like cells. Our granulosa-like cells form ovary-like organoids (ovaroids) when aggregated with hPGCLCs, and recapitulate key ovarian phenotypes including support of germ cell maturation, follicle formation, and steroidogenesis. This model system will provide unique opportunities for studying human ovarian biology, and may enable the development of therapies for female reproductive health.

developmental biology↗

bioRxiv2022

AT-hook DNA-binding motif-containing protein one knockdown downregulates EWS-FLI1 transcriptional activity in Ewings sarcoma cells

Ewings sarcoma is the second most common bone malignancy in children or young adults and is caused by an oncogenic transcription factor by a chromosomal translocation between the EWSR1 gene and the ETS transcription factor family. However, the transcriptional mechanism of EWS-ETS fusion proteins is still unclear. To identify the transcriptional complexes of EWS-ETS fusion transcription factors, we applied a proximal labeling system called BioID in Ewings sarcoma cells. We identified AHDC1 as a proximal protein of EWS-ETS fusion proteins. AHDC1 knockdown showed a reduced cell growth and transcriptional activity of EWS-FLI1. AHDC1 knockdown also reduced BRD4 and BRG1 protein levels, both known as interacting proteins of EWS-FLI1. In addition, AHDC1 co-localized with BRD4. Our results suggest that AHDC1 supports cell growth through EWS-FLI1.

cancer biology↗

bioRxiv2021

HSC-independent definitive hematopoietic cells persist into adult life

The stem cell theory that all blood cells are derived from hematopoietic stem cell (HSC) is a central dogma in hematology. However, various types of blood cells are already produced from hemogenic endothelial cells (HECs) before the first HSCs appear at embryonic day (E)11 in the mouse embryo. This early blood cell production from HECs, called HSC-independent hematopoiesis, includes primitive and definitive erythromyeloid progenitors that transiently support fetal blood homeostasis until HSC-derived hematopoiesis is established. Lymphoid potential has traditionally been detected in the extra-embryonic yolk sac (YS) and/or embryos before HSC emergence, but the actual presence of lymphoid progenitors at this stage remains unknown. In addition, whether HSCs in the fetal liver are the main source of innate-like B-1a cells has been controversial. Here, using complementary lineage tracing mouse models, we show that HSC-independent multipotent progenitors (MPPs) and HSC-independent adoptive B-lymphoid progenitors persist into adult life. Furthermore, HSCs minimally contribute to the peritoneal B-1a cell pool; most B-1a cells are originated directly from ECs in the YS and embryo and HSC-independent for life. Our discovery of extensive HSC-independent MPP and B-lymphoid progenitors in adults attests to the complex blood developmental dynamics through embryo to adult that underpin the immune system and challenges the paradigm of HSC theory in hematology.

developmental biology↗

bioRxiv2021

Dominant Awn Inhibitor, which encodes an ALOG protein in sorghum, suppresses awn formation in rice

The awn, a needle-like structure extending from the tip of the lemma in grass species, plays a role in environmental adaptation and fitness. In some crops, awns appear to have been eliminated during domestication. Although numerous genes involved in awn development have been identified, several dominant genes that eliminate awns are also known to exist. For example, in sorghum (Sorghum bicolor), the dominant awn inhibiting gene has been known since 1921; however, its molecular features remain uncharacterized. In this study, we conducted quantitative trait locus analysis and a genome-wide association study of awn-related traits in sorghum and identified DOMINANT AWN INHIBITOR (DAI), which encodes the ALOG family protein on chromosome 3. DAI appeared to be present in most awnless sorghum cultivars, likely because of its effectiveness. Detailed analysis of the ALOG protein family in cereals revealed that DAI originated from duplication of its twin paralog (DAIori) on chromosome 10. Observations of immature awns in near-isogenic lines revealed that DAI inhibits awn elongation by suppressing both cell proliferation and elongation. We also found that only DAI gained a novel function to inhibit awn elongation through an awn-specific expression pattern distinct from that of DAIori. Interestingly, heterologous expression of DAI with its own promoter in rice inhibited awn elongation in the awned cultivar Kasalath. We found that DAI originated from gene duplication, providing an interesting example of gain-of-function that occurs only in sorghum but shares its functionality with rice and sorghum.

plant biology↗

bioRxiv2021

Milk casein prevents inactivation effect of black tea galloylated theaflavins on SARS-CoV-2 in vitro

Repeated emergence of highly contagious and potentially immune-evading variant SARS-CoV-2 is posing global health and socioeconomical threats. For suppression of the spread of the virus infection among people, a procedure to inactivate virus in saliva may be useful, because saliva of infected persons is the major origin of droplets and aerosols that mediate viral transmission to nearby persons. We previously reported that SARS-CoV-2 is rapidly and remarkably inactivated by treatment in vitro with tea including green tea, roasted green tea, oolong tea and black tea. Tea catechin-derived compounds including theaflavins (TFs) with (a) galloyl moiety(ies) showed this activity. Although black tea is popularly consumed worldwide, a lot of people consume it with sugar, milk, lemon juice, and so on. But it has not been determined whether these ingredients may influence the inactivation effect of black tea against SARS-CoV-2. Moreover, it has not been revealed whether black tea is capable of inactivating variant viruses such as delta variant. Here we examined the effect of black tea on some variants in the presence or absence of sugar, milk, and lemon juice in vitro. Black tea and galloylated TFs remarkably inactivated alpha, gamma, delta and kappa variants. Intriguingly, an addition of milk but not sugar and lemon juice totally prevented black tea from inactivating alpha and delta variant viruses. The suppressive effect was also exerted by milk casein. These results suggest the possibility that intake of black tea without milk by infected persons may result in inactivation of the virus in saliva and attenuation of spread of SARS-CoV-2 to nearby persons through droplets. Clinical studies are required to investigate this possibility.

microbiology↗

bioRxiv2021

A genetically linked pair of NLR immune receptors show contrasting patterns of evolution

Throughout their evolution, plant nucleotide-binding leucine-rich-repeat receptors (NLRs) have acquired widely divergent unconventional integrated domains that enhance their ability to detect pathogen effectors. However, the functional dynamics that drive the evolution of NLRs with integrated domains (NLR-IDs) remain poorly understood. Here, we reconstructed the evolutionary history of an NLR locus prone to unconventional domain integration and experimentally tested hypotheses about the evolution of NLR-IDs. We show that the rice (Oryza sativa) NLR Pias recognizes the effector AVR-Pias of the blast fungal pathogen Magnaporthe oryzae. Pias consists of a functionally specialized NLR pair, the helper Pias-1 and the sensor Pias-2, that is allelic to the previously characterized Pia pair of NLRs: the helper RGA4 and the sensor RGA5. Remarkably, Pias-2 carries a C-terminal DUF761 domain at a similar position to the heavy metal-associated (HMA) domain of RGA5. Phylogenomic analysis showed that Pias-2/RGA5 sensor NLRs have undergone recurrent genomic recombination within the genus Oryza, resulting in up to six sequence-divergent domain integrations. Allelic NLRs with divergent functions have been maintained trans-species in different Oryza lineages to detect sequence-divergent pathogen effectors. By contrast, Pias-1 has retained its NLR helper activity throughout evolution and is capable of functioning together with the divergent sensor-NLR RGA5 to responds to AVR-Pia. These results suggest that opposite selective forces have driven the evolution of paired NLRs: highly dynamic domain integration events maintained by balancing selection for sensor NLRs, in sharp contrast to purifying selection and functional conservation of immune signaling for helper NLRs. Significance statementPlants have evolved sophisticated defense mechanisms to fend off pathogens. Plant nucleotide-binding leucine-rich repeat receptor (NLR) proteins play crucial roles in detecting pathogen molecules inside plant cells and mounting defense responses. Here, we identified the Pias gene from rice, which encodes the NLR pair Pias-1 "helper" and Pias-2 "sensor." These proteins function together to detect the pathogen molecule AVR-Pias of Magnaporthe oryzae and defend against rice blast disease. Pias is allelic to the previously reported Pia gene. A comparison of Pias/Pia alleles among Oryza species showed that Pias/Pia helper is evolutionarily and functionally conserved, whereas the Pias/Pia sensor shows highly dynamic evolution, with various host domains integrated into similar positions, allowing it to detect a wide variety of pathogen molecules.

plant biology↗

bioRxiv2021

A Jacalin-like lectin-domain-containing protein of Sclerospora graminicola act as an apoplastic virulence effector in plant-oomycete interactions

The plant extracellular space, including the apoplast and plasma membrane, is the initial site of plant- pathogen interactions. Pathogens deliver numerous secreted proteins, called effectors, into this region to suppress plant immunity and establish infection. Downy mildew caused by the oomycete pathogen Sclerospora graminicola (Sg) is an economically important disease of Poaceae crops including foxtail millet (Setaria italica). We previously reported the genome sequence of Sg and showed that the Jacalin-related lectin (JRL) gene family has significantly expanded in this lineage. However, the biological functions of JRL proteins remained unknown. Here, we show that JRL from S. graminicola (SgJRL) functions as an apoplastic virulence effector. We identified eight SgJRLs via protein mass spectrometry analysis of extracellular fluid from S. graminicola-inoculated foxtail millet leaves. SgJRLs consist of a Jacalin-like lectin domain and an N-terminal putative secretion signal, and SgJRL expression is induced by Sg infection. Heterologous expression of three SgJRLs with N-terminal secretion signal peptides in Nicotiana benthamiana enhanced the virulence of the pathogen Phytophthora palmivora inoculated onto the same leaves. Of the three SgJRLs, SG06536 fused with GFP localized to the apoplastic space in N. benthamiana leaves. INF1-mediated induction of defense-related genes was suppressed by co-expression of SG06536-GFP. These findings suggest that JRLs are novel apoplastic effectors that contribute to pathogenicity by suppressing plant defense responses.

plant biology↗

bioRxiv2021

Radiolabeling of PSMA-617 with 89Zr: A Novel Use of DMSO for Radiochemical Yield Enhancement and Preliminary Small-Animal PET Results

IntroductionProstate-specific membrane antigen (PSMA)-targeted ligands, including PSMA-617, have been developed for theranostics of prostate cancer. 68Ga-PSMA-617 is the de facto standard of PSMA Positron Emission Tomography (PET) for imaging in prostate cancer patients prior to radioligand therapy (RLT) with 177Lu-PSMA-617. The dose-limiting toxicity for PSMA-RLT is damage to the kidney. PET scans using 68Ga-PSMA-617 have to be performed within a few hours of injection due to its short half-life (68 min). However, the presence of radioactivity in urine at the PET imaging timepoint hampers the dose optimization of 177Lu (half-life 6.6 d)-labeled PSMA-617. Thus, the long-lived positron emitter 89Zr (half-life 3.3 d) is suited for optimizing the doses of 177Lu-PSMA-617 because PET scans can be performed after excretion of radioactive urine. Although 89Zr has great potential for PET imaging, its inadequate incorporation into 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), limits its applications. Here, we report the radiolabeling of PSMA-617 with 89Zr and preliminary PET imaging studies using 89Zr-PSMA-617. MethodsDMSO and HEPES buffer were used to label PSMA-617 with 89Zr. The dissociation constant (Kd) of 89Zr-PSMA-617 was determined using a cell-binding assay. Delayed-PET scans using 89Zr-PSMA-617 were performed at 24 h (N = 5). Results89Zr-PSMA-617 was prepared with a radiochemical yield of 70 {+/-} 9%. The Kd value was 6.8 nM. In PET imaging, standardized uptake value (SUV) was highest in LNCaP tumors (SUVmax = 0.98 {+/-} 0.32), whereas it was low in kidney (SUVmax = 0.18 {+/-} 0.7). ConclusionThe preparation of 89Zr-PSMA-617 was achieved by using the DMSO and HEPES buffer. 89Zr-PSMA-617 visualize the PSMA positive LNCaP tumors without accumulation in bladder. Advances in knowledge and implications for patient careThe use of 89Zr-PSMA-617 to predict the radiation doses in normal tissues lead to safe and effective RLT with 177Lu-PSMA-617.

cancer biology↗

bioRxiv2021

SMURF2 phosphorylation at Thr249 modifies the stemness and tumorigenicity of glioma stem cells by regulating TGF-β receptor stability

Glioma stem cells (GSCs) contribute to the pathogenesis of glioblastoma, the most malignant form of glioma. The implication and underlying mechanisms of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) on the GSC phenotypes remain unknown. We previously demonstrated that SMURF2 phosphorylation at Thr249 (SMURF2Thr249) activates its E3 ubiquitin ligase activity. Here, we demonstrate that SMURF2Thr249 phosphorylation plays an essential role in maintaining GSC stemness and tumorigenicity. SMURF2 silencing augmented the self-renewal potential and tumorgenicity of patient-derived GSCs. The SMURF2Thr249 phosphorylation level was low in human glioblastoma pathology specimens. Introduction of the SMURF2T249A mutant resulted in increased stemness and tumorgenicity of GSCs, recapitulating the SMURF2 silencing. Moreover, the inactivation of SMURF2Thr249 phosphorylation increases TGF-{beta} receptor (TGFBR) protein stability. Indeed, TGFBR1 knockdown markedly counteracted the GSC phenotypes by SMURF2T249A mutant. These findings highlight the importance of SMURF2Thr249 phosphorylation in maintaining GSC phenotypes, thereby demonstrating a potential target for GSC-directed therapy.

cancer biology↗

bioRxiv2021

Effect of luminal surface structure of decellularized aorta on thrombus formation and cell behavior

As the number of cardiovascular diseases increases, artificial heart valves and blood vessels have been developed. Although cardiovascular application using decellularized tissue have been studied, the mechanism of their functionality is still unknown. To find the important factor for preparing decellularized cardiovascular prothesis which shows good in vivo performance, the effect of luminal surface structure of decellularized aorta on thrombus formation and cell behavior was investigated. Various luminal surface structures of decellularized aorta were prepared by heating, drying and peeling. The luminal surface structure and collagen denaturation was evaluated by immunohistological staining, collagen hybridizing peptide (CHP) staining and scanning electron microscopy (SEM) analysis. To evaluate the effect of luminal surface structure of decellularized aorta on thrombus formation and cell behavior, blood clotting test and recellularization of endothelial cells and smooth muscular cells were performed. The results of blood clotting test showed that the closer the luminal surface structure is to native aorta, the higher the anti-coagulant property. From the result of cell seeding-test, it was suggested that vascular cells recognize the luminal surface structure and regulate adhesion, proliferation and functional expression. These results will provide important factor for preparing decellularized cardiovascular prothesis and lead to future development on decellularized cardiovascular applications.

bioengineering↗