A rapid quarantine approach for the pathogenic and invasive Phytophthora species associated with imported fruits in China.

BACKGROUND: Phytophthora spp. represent a pivotal genus of plant pathogens with a global distribution, exerting significant deleterious effects on food safety and forestry ecosystems. Numerous pathogenic and invasive Phytophthora species, introduced through imported fruits, have been frequently detected at Chinese ports. With the rise in global trade activities, the plant quarantine of imported fruits is becoming increasingly important but challenging. Fast, simple, and labor-saving techniques are necessary and anticipated. RESUITS: A polymerase chain reaction restriction fragment length polymorphism capillary electrophoresis (PCR-RFLP-CE) technology-based quarantine approach was developed for 16 Phytophthora species associated with the imported fruits in China. The Ypt1 gene, exhibiting abundant interspecific variations, was selected as the marker gene for PCR. The restriction endonuclease AluI was proven to be capable and compatible in simultaneously separating different Phytophthora species during CE. By combining with a fast and efficient DNA extraction kit, the developed PCR-RFLP-CE technique was successfully applied to identify Phytophthora species in artificially infested fruits. CONCLUSION: We provide a quick, practical, and high-throughput detection approach for hazardous and invasive Phytophthora species associated with imported fruits in China. This strategy can give good convenience and technological support for carrying out massive quarantine activities at Chinese ports. © 2024 Society of Chemical Industry.

The nuclear receptor THRB facilitates differentiation of human PSCs into more mature hepatocytes.

To understand the mechanisms regulating the in vitro maturation of hPSC-derived hepatocytes, we developed a 3D differentiation system and compared gene regulatory elements in human primary hepatocytes with those in hPSC-hepatocytes that were differentiated in 2D or 3D conditions by RNA-seq, ATAC-seq, and H3K27Ac ChIP-seq. Regulome comparisons showed a reduced enrichment of thyroid receptor THRB motifs in accessible chromatin and active enhancers without a reduced transcription of THRB. The addition of thyroid hormone T3 increased the binding of THRB to the CYP3A4 proximal enhancer, restored the super-enhancer status and gene expression of NFIC, and reduced the expression of AFP. The resultant hPSC-hepatocytes showed gene expression, epigenetic status, and super-enhancer landscape closer to primary hepatocytes and activated regulatory regions including non-coding SNPs associated with liver-related diseases. Transplanting the hPSC-hepatocytes resulted in the engraftment of human hepatocytes into the mouse liver without disrupting normal liver histology. This work implicates the environmental factor-nuclear receptor axis in regulating the maturation of hPSC-hepatocytes.

SARS-CoV-2 RNA reverse-transcribed and integrated into the human genome.

Prolonged SARS-CoV-2 RNA shedding and recurrence of PCR-positive tests have been widely reported in patients after recovery, yet these patients most commonly are non-infectious. Here we investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the human genome and that transcription of the integrated sequences might account for PCR-positive tests. In support of this hypothesis, we found chimeric transcripts consisting of viral fused to cellular sequences in published data sets of SARS-CoV-2 infected cultured cells and primary cells of patients, consistent with the transcription of viral sequences integrated into the genome. To experimentally corroborate the possibility of viral retro-integration, we describe evidence that SARS-CoV-2 RNAs can be reverse transcribed in human cells by reverse transcriptase (RT) from LINE-1 elements or by HIV-1 RT, and that these DNA sequences can be integrated into the cell genome and subsequently be transcribed. Human endogenous LINE-1 expression was induced upon SARS-CoV-2 infection or by cytokine exposure in cultured cells, suggesting a molecular mechanism for SARS-CoV-2 retro-integration in patients. This novel feature of SARS-CoV-2 infection may explain why patients can continue to produce viral RNA after recovery and suggests a new aspect of RNA virus replication.

Human T Cells Expressing a CD19 CAR-T Receptor Provide Insights into Mechanisms of Human CD19-Positive ß Cell Destruction.

Autoimmune destruction of pancreatic ß cells underlies type 1 diabetes (T1D). To understand T cell-mediated immune effects on human pancreatic ß cells, we combine ß cell-specific expression of a model antigen, CD19, and anti-CD19 chimeric antigen receptor T (CAR-T) cells. Coculturing CD19-expressing ß-like cells and CD19 CAR-T cells results in T cell-mediated ß-like cell death with release of activated T cell cytokines. Transcriptome analysis of ß-like cells and human islets treated with conditioned medium of the immune reaction identifies upregulation of immune reaction genes and the pyroptosis mediator GSDMD as well as its activator CASP4. Caspase-4-mediated cleaved GSDMD is detected in ß-like cells under inflammation and endoplasmic reticulum (ER) stress conditions. Among immune-regulatory genes, PDL1 is one of the most upregulated, and PDL1 overexpression partially protects human ß-like cells transplanted into mice. This experimental platform identifies potential mechanisms of ß cell destruction and may allow testing of therapeutic strategies.

Formation of Human Neuroblastoma in Mouse-Human Neural Crest Chimeras.

Neuroblastoma (NB), derived from the neural crest (NC), is the most common pediatric extracranial solid tumor. Here, we establish a platform that allows the study of human NBs in mouse-human NC chimeras. Chimeric mice were produced by injecting human NC cells carrying NB relevant oncogenes in utero into gastrulating mouse embryos. The mice developed tumors composed of a heterogenous cell population that resembled that seen in primary NBs of patients but were significantly different from homogeneous tumors formed in xenotransplantation models. The human tumors emerged in immunocompetent hosts and were extensively infiltrated by mouse cytotoxic T cells, reflecting a vigorous host anti-tumor immune response. However, the tumors blunted the immune response by inducing infiltration of regulatory T cells and expression of immune-suppressive molecules similar to escape mechanisms seen in human cancer patients. Thus, this experimental platform allows the study of human tumor initiation, progression, manifestation, and tumor-immune-system interactions in an animal model system.

Establishment of human pluripotent stem cell-derived pancreatic ß-like cells in the mouse pancreas.

Type 1 diabetes is characterized by autoimmune destruction of ß cells located in pancreatic islets. However, tractable in vivo models of human pancreatic ß cells have been limited. Here, we generated xenogeneic human pancreatic ß-like cells in the mouse pancreas by orthotopic transplantation of stem cell-derived ß (SC-ß) cells into the pancreas of neonatal mice. The engrafted ß-like cells expressed ß cell transcription factors and markers associated with functional maturity. Engrafted human cells recruited mouse endothelial cells, suggesting functional integration. Human insulin was detected in the blood circulation of transplanted mice for months after transplantation and increased upon glucose stimulation. In addition to ß-like cells, human cells expressing markers for other endocrine pancreas cell types, acinar cells, and pancreatic ductal cells were identified in the pancreata of transplanted mice, indicating that this approach allows studying other human pancreatic cell types in the mouse pancreas. Our results demonstrate that orthotopic transplantation of human SC-ß cells into neonatal mice is an experimental platform that allows the generation of mice with human pancreatic ß-like cells in the endogenous niche.

A Chimeric Egfr Protein Reporter Mouse Reveals Egfr Localization and Trafficking In Vivo.

EGF receptor (EGFR) is a critical signaling node throughout life. However, it has not been possible to directly visualize endogenous Egfr in mice. Using CRISPR/Cas9 genome editing, we appended a fluorescent reporter to the C terminus of the Egfr. Homozygous reporter mice appear normal and EGFR signaling is intact in vitro and in vivo. We detect distinct patterns of Egfr expression in progenitor and differentiated compartments in embryonic and adult mice. Systemic delivery of EGF or amphiregulin results in markedly different patterns of Egfr internalization and trafficking in hepatocytes. In the normal intestine, Egfr localizes to the crypt rather than villus compartment, expression is higher in adjacent epithelium than in intestinal tumors, and following colonic injury expression appears in distinct cell populations in the stroma. This reporter, under control of its endogenous regulatory elements, enables in vivo monitoring of the dynamics of Egfr localization and trafficking in normal and disease states.

Three-dimensional culture system identifies a new mode of cetuximab resistance and disease-relevant genes in colorectal cancer.

We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.

Excess PLAC8 promotes an unconventional ERK2-dependent EMT in colon cancer.

The epithelial-to-mesenchymal transition (EMT) transcriptional program is characterized by repression of E-cadherin (CDH1) and induction of N-cadherin (CDH2), and mesenchymal genes like vimentin (VIM). Placenta-specific 8 (PLAC8) has been implicated in colon cancer; however, how PLAC8 contributes to disease is unknown, and endogenous PLAC8 protein has not been studied. We analyzed zebrafish and human tissues and found that endogenous PLAC8 localizes to the apical domain of differentiated intestinal epithelium. Colon cancer cells with elevated PLAC8 levels exhibited EMT features, including increased expression of VIM and zinc finger E-box binding homeobox 1 (ZEB1), aberrant cell motility, and increased invasiveness. In contrast to classical EMT, PLAC8 overexpression reduced cell surface CDH1 and upregulated P-cadherin (CDH3) without affecting CDH2 expression. PLAC8-induced EMT was linked to increased phosphorylated ERK2 (p-ERK2), and ERK2 knockdown restored cell surface CDH1 and suppressed CDH3, VIM, and ZEB1 upregulation. In vitro, PLAC8 directly bound and inactivated the ERK2 phosphatase DUSP6, thereby increasing p-ERK2. In a murine xenograft model, knockdown of endogenous PLAC8 in colon cancer cells resulted in smaller tumors, reduced local invasion, and decreased p-ERK2. Using MultiOmyx, a multiplex immunofluorescence-based methodology, we observed coexpression of cytosolic PLAC8, CDH3, and VIM at the leading edge of a human colorectal tumor, supporting a role for PLAC8 in cancer invasion in vivo.

Naked1 antagonizes Wnt signaling by preventing nuclear accumulation of ß-catenin.

Cyto-nuclear shuttling of ß-catenin is at the epicenter of the canonical Wnt pathway and mutations in genes that result in excessive nuclear accumulation of ß-catenin are the driving force behind the initiation of many cancers. Recently, Naked Cuticle homolog 1 (Nkd1) has been identified as a Wnt-induced intracellular negative regulator of canonical Wnt signaling. The current model suggests that Nkd1 acts between Disheveled (Dvl) and ß-catenin. Here, we employ the zebrafish embryo to characterize the cellular and biochemical role of Nkd1 in vivo. We demonstrate that Nkd1 binds to ß-catenin and prevents its nuclear accumulation. We also show that this interaction is conserved in mammalian cultured cells. Further, we demonstrate that Nkd1 function is dependent on its interaction with the cell membrane. Given the conserved nature of Nkd1, our results shed light on the negative feedback regulation of Wnt signaling through the Nkd1-mediated negative control of nuclear accumulation of ß-catenin.

[Cloning and heterologous expression of a novel delta6 -desaturase gene from Rhizopus arrhizus NK030037].

A 593 bp DNA fragment was amplified from Rhizopus arrhizus NK030037 with degenerate oligonucleotide primers designed based on the sequences information for fungi delta6-fatty acid desaturase genes by RT-PCR and sequenced. Gene specific primers derived from this partial sequence were used for the amplification of the 3'- and 5'-ends of this cDNA by RACE method, and this lead to a full-length cDNA sequence of 1 482 bp was amplified. Sequence analysis showed this cDNA sequence had an open reading frame(ORF) of 1 377 bp coding 458 amino acids of 52 kD. The deduced amino-acid sequence of the ORF showed similarity to those of the above delta6-fatty acid desaturases which comprised the characteristics of membrane-bound desaturases, including three conserved histidine-rich boxes and hydropathy profile. A cytochrome b5-like domain was observed at the N-terminus. The full-length cDNA sequence is a putative novel delta6-fatty acid desaturase gene. To elucidate the function of the protein, two specific primers corresponding to the nucleotide sequences of start and stop codons were used to amplify the coding sequence. The amplified cDNA RAD6 was subcloned into the expression vector pYES2.0 to generate a recombinant plasmid pYRAD6, which was subsequently transformed into Saccharomyces cerevisiae strain INVScl for heterologous expression by lithium acetate method. Grown to logarithmic phase at 30 degrees C, the transformed cells were supplemented with 0.5 mmol/L. Linoleic acid and induced by 2% galactose for a further 48 h of cultivation at 20 degrees. Total fatty acids were extracted from the induced cells and subjected to methyl-esterification. The resultant fatty acid methyl esters(FAME) were analyzed by gas chromatography(GC). A novel peak corresponding to gamma-linolenic acid (GLA) methyl ester standards was detected with the same retention time, which was absent in the cell transformed with empty vector. The percentage of this new fatty acid to total fatty acids was 3.85%. Gas chromatography-mass spectrometry( GC-MS) analysis of this fatty acid methyl derivative demonstrated that the novel peak was GLA methyl ester. These results showed that the transgenic product exhibited delta6-fatty acid desaurase activity, converting LA to GLA specifically.

Identification and characterization of a novel Delta6-fatty acid desaturase gene from Rhizopus arrhizus.

A cDNA sequence putatively encoding a Delta(6)-fatty acid desaturase was isolated from Rhizopus arrhizus using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends methods. Sequence analysis indicated that this cDNA sequence had an open reading frame of 1377 bp encoding 458 amino acids of 52 kDa. The deduced amino acid sequence showed high similarity to those of fungal Delta(6)-fatty acid desaturases which comprised the characteristics of membrane-bound desaturases, including three conserved histidine-rich motifs and hydropathy profile. A cytochrome b(5)-like domain was observed at the N-terminus. To elucidate the function of this novel putative desaturase, the coding sequence was expressed heterologously in Saccharomyces cerevisiae strain INVScl. The result demonstrated that the coding product of the sequence exhibited Delta(6)-fatty acid desaturase activity by the accumulation of gamma-linolenic acid.

[Progress on molecular biology of delta6-fatty acid desaturases].

Polyunsaturated fatty acids (PUFAs) including gamma-linolenic acid are valuable products because of their involvement in several aspects of human health care. GLA has been claimed to play a crucial role in development and prevention of some skin diseases, diabetes, reproductive disorder and others. At present, market demand for most gamma-linolenic acid is growing continually and current sources are inadequate for satisfying this demand due to the significant problems of low productivity, complex and expensive downstream process and unstable quality. Therefore, seeking for alternative sources are demanding. delta6-fatty acid desaturase is the rate-limiting enzyme for the biosynthesis of PUFAs, which catalyses the conversion of linoleic acid and alpha-linolenic acid to gamma-linolenic acid and stearidonic acid respectively. Unfortunately, the structure information on membrane desaturases is scarce because of the technical limitations in obtaining quantities of purified protein and the intrinsic difficulties in obtaining crystals from membrane proteins. With the isolation of the genes coding for delta6-fatty acid desaturase from various organisms, its characteristics will be elucidated gradually. Here we concisely reviewed the recent progress on studies of molecular biology including the cloning of delta6-fatty acid desaturase gene, structure and function, phylogeny and prospects of gene engineering application.