Comparative transcriptome analysis and simple sequence repeat marker development for two closely related Isodon species used as ‘Xihuangcao’ herbs

Purpose: To facilitate the molecular identification of original plants, resolve taxonomic problems and identify standards for ‘Xihuangcao’-based products on the market.Methods: A transcriptomic analysis of two closely related species, i.e., Isodon serra (Maxim.) (IS) and I. lophanthoides (Buch.-Ham. ex D. Don) Hara, was conducted by using the Illumina HiSeq 2500 platform, and expressed sequence tag-derived simple sequence repeat (EST-SSR) markers were developed based on these transcriptomes.Results: In total, 149,650 and 103,221 contigs were obtained, with N50 values of 1,400 and 1,516, from the IS and I. lophanthoides RNA-Seq datasets, respectively. These contigs were clustered into 107,777 and 68,220 unigenes, which were functionally annotated to identify the genes involved in therapeutic components. In total, 14,138 and 11,756 EST-SSR motifs were identified, and of these motifs, 7,453 and 6,428 were used to design primers for IS and I. lophanthoides, respectively. After PCR verification and fluorescence-based genotyping, 24 SSR markers with bright bands, high polymorphism, and single amplification were obtained and used to identify closely related Isodon species/varieties.Conclusion: These data could help herbal scientists identify high-quality herbal plants and provide a reference for genetic improvement and population genetic and phylogenetic studies investigating ‘Xihuangcao’ herbs.Keywords: Xihuangcao, Transcriptome, EST-SSR, Molecular marker

Combining the Tyrosine Kinase Inhibitor Cabozantinib and the mTORC1/2 Inhibitor Sapanisertib Blocks ERK Pathway Activity and Suppresses Tumor Growth in Renal Cell Carcinoma.

UNLABELLED: Current treatment approaches for renal cell carcinoma (RCC) face challenges in achieving durable tumor responses due to tumor heterogeneity and drug resistance. Combination therapies that leverage tumor molecular profiles could offer an avenue for enhancing treatment efficacy and addressing the limitations of current therapies. To identify effective strategies for treating RCC, we selected ten drugs guided by tumor biology to test in six RCC patient-derived xenograft (PDX) models. The multitargeted tyrosine kinase inhibitor (TKI) cabozantinib and mTORC1/2 inhibitor sapanisertib emerged as the most effective drugs, particularly when combined. The combination demonstrated favorable tolerability and inhibited tumor growth or induced tumor regression in all models, including two from patients who experienced treatment failure with FDA-approved TKI and immunotherapy combinations. In cabozantinib-treated samples, imaging analysis revealed a significant reduction in vascular density, and single-nucleus RNA sequencing (snRNA-seq) analysis indicated a decreased proportion of endothelial cells in the tumors. SnRNA-seq data further identified a tumor subpopulation enriched with cell-cycle activity that exhibited heightened sensitivity to the cabozantinib and sapanisertib combination. Conversely, activation of the epithelial-mesenchymal transition pathway, detected at the protein level, was associated with drug resistance in residual tumors following combination treatment. The combination effectively restrained ERK phosphorylation and reduced expression of ERK downstream transcription factors and their target genes implicated in cell-cycle control and apoptosis. This study highlights the potential of the cabozantinib plus sapanisertib combination as a promising treatment approach for patients with RCC, particularly those whose tumors progressed on immune checkpoint inhibitors and other TKIs. SIGNIFICANCE: The molecular-guided therapeutic strategy of combining cabozantinib and sapanisertib restrains ERK activity to effectively suppress growth of renal cell carcinomas, including those unresponsive to immune checkpoint inhibitors

Spatially restricted drivers and transitional cell populations cooperate with the microenvironment in untreated and chemo-resistant pancreatic cancer

Pancreatic ductal adenocarcinoma is a lethal disease with limited treatment options and poor survival. We studied 83 spatial samples from 31 patients (11 treatment-naïve and 20 treated) using single-cell/nucleus RNA sequencing, bulk-proteogenomics, spatial transcriptomics and cellular imaging. Subpopulations of tumor cells exhibited signatures of proliferation, KRAS signaling, cell stress and epithelial-to-mesenchymal transition. Mapping mutations and copy number events distinguished tumor populations from normal and transitional cells, including acinar-to-ductal metaplasia and pancreatic intraepithelial neoplasia. Pathology-assisted deconvolution of spatial transcriptomic data identified tumor and transitional subpopulations with distinct histological features. We showed coordinated expression of TIGIT in exhausted and regulatory T cells and Nectin in tumor cells. Chemo-resistant samples contain a threefold enrichment of inflammatory cancer-associated fibroblasts that upregulate metallothioneins. Our study reveals a deeper understanding of the intricate substructure of pancreatic ductal adenocarcinoma tumors that could help improve therapy for patients with this disease

Enhancing negative messages broadcasting with Meet-Table and TTL in VANET

Abstract In VANET (vehicular ad hoc network), a negative message has an objective vehicle and describes the negative attributes of the vehicle. Broadcasting negative messages in VANET is essential to make the VANET secure for these messages are about the untrustworthy vehicles. The epidemic model distributes negative messages rapidly with unlimited broadcasting, but uncontrolled epidemic may cause flooding storm. TTL (time-to-live) can be used to control broadcasting, but it may heavily decrease the coverage of negative messages in VANET. Meet-Table is used with TTL to solve the problem. A Meet-Table is a data structure held by a vehicle to record vehicles it met. When a vehicle receives a message, it checks whether the message’s objective vehicle is in its Meet-Table. If it is, the vehicle resets the TTL of the message to the maximum TTL to extend the transmission of the message. The negative message, coverage percentage and accurate coverage percentage of the negative message, and the Meet-Table are formally defined. The algorithm for broadcasting negative messages with Meet-Table and TTL is given. The simulation results show that Meet-Table can increase the coverage and decrease the delay of negative messages. More importantly, the simulation results show that the coverage increased by Meet-Table is accurate

Development and Characterization of High-Throughput EST-Based SSR Markers for Pogostemon cablin Using Transcriptome Sequencing

Simple sequence repeats (SSRs) or microsatellite markers derived from expressed sequence tags (ESTs) are routinely used for molecular assisted-selection breeding, comparative genomic analysis, and genetic diversity studies. In this study, we investigated 54,546 ESTs for the identification and development of SSR markers in Pogostemon cablin (Patchouli). In total, 1219 SSRs were identified from 1144 SSR-containing ESTs. Trinucleotides (80.8%) were the most abundant SSRs, followed by di- (10.8%), mono- (7.1%), and hexa-nucleotides (1.3%). The top six motifs were CCG/CGG (15.3%), AAG/CTT (15.0%), ACC/GGT (13.5%), AGG/CCT (12.4%), ATC/ATG (9.9%), and AG/CT (9.8%). On the basis of these SSR-containing ESTs, a total of 192 primer pairs were randomly designed and used for polymorphism analysis in 38 accessions collected from different geographical regions of Guangdong, China. Of the SSR markers, 45 were polymorphic and had allele variations from two to four. Furthermore, a transferability analysis of these primer pairs revealed a 10–40% cross-species transferability in 10 related species. This report is the first comprehensive study on the development and analysis of a large set of SSR markers in P. cablin. These markers have the potential to be used in quantitative trait loci mapping, genetic diversity studies, and the fingerprinting of cultivars of P. cablin

The plastid genome and its implications in barcoding specific-chemotypes of the medicinal herb Pogostemon cablin in China

Pogostemon cablin (Blanco) Benth. (Patchouli) is not only an important essential oil plant, but also a valuable medicinal plant in China. P. cablin in China can be divided into three cultivars (Shipai, Gaoyao, and Hainan) and two chemotypes (pogostone-type and patchoulol-type). The pogostone-type and patchoulol-type are, respectively, used for medicinals and perfumes. In this study, we sequenced and characterized the plastid genomes for all three Chinese cultivars and aimed to develop a chemotype-specific barcode for future quality control. The plastid genomes of P. cablin cultivars ranged from 152,461 to 152,462 bp in length and comprise 114 genes including 80 protein coding genes, 30 tRNA genes, and four rRNA genes. Phylogenetic analyses suggested that P. cablin cultivars clustered with the other two Pogostemon species with strong support. Although extremely conserved in P. cablin plastid genomes, 58 cpSSRs were filtered out among the three cultivars. One single variable locus, cpSSR, was discovered. The cpSSR genotypes successfully matched the chemotypes of Chinese patchouli, which was further supported by PCR-based Sanger sequences in more Chinese patchouli samples. The barcode developed in this study is thought to be a simple and reliable quality control method for Chinese P. cablin on the market

Reconstituting Arabidopsis CRY2 Signaling Pathway in Mammalian Cells Reveals Regulation of Transcription by Direct Binding of CRY2 to DNA.

In response to blue light, cryptochromes photoexcite and interact with signal partners to transduce signal almost synchronously in plants. The detailed mechanism of CRY-mediated light signaling remains unclear: the photobiochemical reactions of cryptochrome are transient and synchronous, thus making the monitoring and analysis of each step difficult in plant cells. In this study, we reconstituted the Arabidopsis CRY2 signaling pathway in mammalian cells and investigated the biological role of Arabidopsis CRY2 in this heterologous system, eliminating the interferences of other plant proteins. Our results demonstrated that, besides being the light receptor, Arabidopsis CRY2 binds to DNA directly and acts as a transcriptional activator in a blue-light-enhanced manner. Similar to classic transcription factors, we found that the transcriptional activity of CRY2 is regulated by its dimerization and phosphorylation. In addition, CRY2 cooperates with CIB1 to regulate transcription by enhancing the DNA affinity and transcriptional activity of CIB1 under blue light

Genomic epidemiology reveals early transmission of SARS-CoV-2 and mutational dynamics in Nanning, China

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are a fatal pathogen resulting in substantial morbidity and mortality, and posing a great threat to human health with epidemics and pandemics. Methods: Next-generation sequencing (NGS) was performed to investigate the SARS-CoV-2 genomic characterization. Phylogenetic analysis of SARS-CoV-2 genomes was used to probe the evolutionary. Homology protein structure modelling was done to explore potential effect of the mutations. Results: The eighty genome sequences of SARS-CoV-2 obtained from the thirty-nine patients with COVID-19. A novel variant with mutation H625R concomitant with S50L in spike glycoprotein had been identified. Phylogenetic analysis revealed that SARS-CoV-2 variants belong to several distinct lineages. Homology modelling indicated that variant with mutation H625R and S50L increases flexibility of S1 subunit. Conclusions: SARS-CoV-2 genomes are constantly evolving by accumulation of point mutations. The amino acid H625R in combination with S50L may have a significant impact on the interaction between spike glycoprotein and ACE2