The Ability of Clinically Relevant Chemotherapeutics to Induce Immunogenic Cell Death in Squamous Cell Carcinoma.

BACKGROUND: Immunogenic cell death (ICD) is a crucial mechanism for triggering the adaptive immune response in cancer patients. Damage-associated molecular patterns (DAMPs) are critical factors in the detection of ICD. Chemotherapeutic drugs can cause ICD and the release of DAMPs. The aim of this study was to assess the potential for paclitaxel and platinum-based chemotherapy regimens to induce ICD in squamous cell carcinoma (SCC) cell lines. In addition, we examined the immunostimulatory effects of clinically relevant chemotherapeutic regimens utilized in the treatment of SCC. METHODS: We screened for differentially expressed ICD markers in the supernatants of three SCC cell lines following treatment with various chemotherapeutic agents. The ICD markers included Adenosine Triphosphate (ATP), Calreticulin (CRT), Annexin A1 (ANXA 1), High Mobility Group Protein B1 (HMGB1), and Heat Shock Protein 70 (HSP70). A vaccination assay was also employed in C57BL/6J mice to validate our in vitro findings. Lastly, the levels of CRT and HMGB1 were evaluated in Serum samples from SCC patients. RESULTS: Addition of the chemotherapy drugs cisplatin (DDP), carboplatin (CBP), nedaplatin (NDP), oxaliplatin (OXA) and docetaxel (DOC) increased the release of ICD markers in two of the SCC cell lines. Furthermore, mice that received vaccinations with cervical cancer cells treated with DDP, CBP, NDP, OXA, or DOC remained tumor-free. Although CBP induced the release of ICD-associated molecules in vitro, it did not prevent tumor growth at the vaccination site in 40% of mice. In addition, both in vitro and in vivo results showed that paclitaxel (TAX) and LBP did not induce ICD in SCC cells. CONCLUSION: The present findings suggest that chemotherapeutic agents can induce an adjuvant effect leading to the extracellular release of DAMPs. Of the agents tested here, DDP, CBP, NDP, OXA and DOC had the ability to act as inducers of ICD.

Non-Volatile Component and Antioxidant Activity: A Comparative Analysis between Litsea cubeba Branches and Leaves.

Litsea cubeba, which is found widely distributed across the Asian region, functions as both an economic tree and a medicinal plant with a rich historical background. Previous investigations into its chemical composition and biological activity have predominantly centered on volatile components, leaving the study of non-volatile components relatively unexplored. In this study, we employed UPLC-HRMS technology to analyze the non-volatile components of L. cubeba branches and leaves, which successfully resulted in identifying 72 constituents. Comparative analysis between branches and leaves unveiled alkaloids, organic acids, and flavonoids as the major components. However, noteworthy differences in the distribution of these components between branches and leaves were observed, with only eight shared constituents, indicating substantial chemical variations in different parts of L. cubeba. Particularly, 24 compounds were identified for the first time from this plant. The assessment of antioxidant activity using four methods (ABTS, DPPH, FRAP, and CUPRAC) demonstrated remarkable antioxidant capabilities in both branches and leaves, with slightly higher efficacy observed in branches. This suggests that L. cubeba may act as a potential natural antioxidant with applications in health and therapeutic interventions. In conclusion, the chemical composition and antioxidant activity of L. cubeba provides a scientific foundation for its development and utilization in medicine and health products, offering promising avenues for the rational exploitation of L. cubeba resources in the future.

Hyperfractionated Accelerated Radiotherapy Versus Stereotactic Body Radiotherapy in the Treatment of Limited-Stage Small Cell Lung Cancer: A Matched-Pair Analysis.

BACKGROUND: Concurrent chemoradiotherapy based on hyperfractionated accelerated radiotherapy (HART) is the first-line recommended regimen for the treatment of small-cell lung cancer (SCLC). However, Stereotactic Body Radiotherapy (SBRT) is also regarded as an effective treatment for limited-stage (LS) SCLC, and the efficacy and safety of HART versus SBRT stay controversial. METHODS: In this study, 188 LS-SCLC patients were retrospectively divided into two groups receiving chemotherapy combined with either HART or SBRT. In HART group, patients received 4500 cGy in 30 fractions, administered twice daily for 3 weeks. Whereas in the SBRT group, a total radiation dose of 4000-4500 cGy was delivered in 10 fractions over 2 weeks. Thirty-three pairs of patients were finally included for next analysis. RESULTS: The estimated objective response rates were 63.6 % (21/33) and 78.8 % (26/33) in HART group and SBRT group, respectively (P = 0.269). Furthermore, there was no significant difference between HART and SBRT groups in overall survival (26 months vs. 29 months, P = 0.362) and progression free survival (11 months vs. 15 months, P = 0.223). As for the adverse events, toxicity of both groups is similar and slight that no grade 4 event was observed. Grade 3 pneumonitis cases were all occurred in the HART group (9.1%, 3/33, P = 0.238), and grade 3 esophagitis cases were all occurred in the SBRT group (6.1%, 2/33, P = 0.492). CONCLUSION: Compared with HART, SBRT could be another effective treatment with satisfactory safety for the concurrent chemoradiotherapy in patients with LS-SCLC.

Selection of Suitable Reference Genes for RT-qPCR Gene Expression Analysis in Centipedegrass under Different Abiotic Stress.

As a C4 warm-season turfgrass, centipedegrass (Eremochloa ophiuroides (Munro) Hack.) is known for its exceptional resilience to intensive maintenance practices. In this research, the most stably expressed reference genes in the leaves of centipedegrass under different stress treatments, including salt, cold, drought, aluminum (Al), and herbicide, were screened by the quantitative real-time PCR (RT-qPCR) technique. The stability of 13 candidate reference genes was evaluated by software GeNorm V3.4, NormFinder V20, BestKeeper V1.0, and ReFinder V1.0. The results of this experiment demonstrated that the expression of the UBC (ubiquitin-conjugating enzyme) remained the most stable under cold and Al stress conditions. On the other hand, the MD (malate dehydrogenase) gene exhibited the best performance in leaf tissues subjected to salt and drought stresses. Under herbicide stress, the expression level of the RIP (60S ribosomal protein L2) gene ranked the highest. The expression levels of abiotic stress-associated genes such as PIP1, PAL, COR413, ALMT9, and BAR were assessed to validate the reliability of the selected reference genes. This study provides valuable information and reference points for gene expression under abiotic stress conditions in centipedegrass.

Identification and Validation of Reference Genes for RT-qPCR Analysis in Reed Canary Grass during Abiotic Stress.

Reed canary grass (Phalaris arundinacea L.) is known for its tolerance to drought, heavy metals, and waterlogging, making it a popular choice for forage production and wetland restoration in the Qinghai-Tibet Plateau (QTP). To accurately assess gene expression in reed canary grass under different abiotic stresses, suitable reference genes need to be identified and validated. Thirteen candidate reference gene sequences were selected and screened using RT-qPCR to detect their expression levels in reed canary grass leaves under drought, salt, cadmium, and waterlogging stresses. Four algorithms were used to assess the stability of the expression levels of the candidate reference genes. The most stably expressed genes were UBC and H3 under drought Cd, ETF and CYT under salt stress, and ETF and TUB under waterlogging stress. GAPDH was found to be less stable under abiotic stresses. PIP-1, PAL, NAC 90, and WRKY 72A were selected as response genes for quantitative expression assessment under drought, salt, Cd, and waterlogging stresses to confirm the accuracy of the selected stable reference genes. These results provide a theoretical reference for assessing gene expression in reed canary grass under abiotic stresses.

Complete chloroplast genomes of three wild perennial Hordeum species from Central Asia: genome structure, mutation hotspot, phylogenetic relationships, and comparative analysis.

Hordeum L. is widely distributed in mountain or plateau of subtropical and warm temperate regions around the world. Three wild perennial Hordeum species, including H. bogdanii, H. brevisubulatum, and H. violaceum, have been used as forage and for grassland ecological restoration in high-altitude areas in recent years. To date, the degree of interspecies sequence variation in the three Hordeum species within existing gene pools is still not well-defined. Herein, we sequenced and assembled chloroplast (cp) genomes of the three species. The results revealed that the cp genome of H. bogdanii showed certain sequence variations compared with the cp genomes of the other two species (H. brevisubulatum and H. violaceum), and the latter two were characterized by a higher relative affinity. Parity rule 2 plot (PR2) analysis illuminated that most genes of all ten Hordeum species were concentrated in nucleotide T and G. Numerous single nucleotide polymorphism (SNP) and insertion/deletion (In/Del) events were detected in the three Hordeum species. A series of hotspots regions (tRNA-GGU ~ tRNA-GCA, tRNA-UGU ~ ndhJ, psbE ~ rps18, ndhF ~ tRNA-UAG, etc.) were identified by mVISTA procedures, and the five highly polymorphic genes (tRNA-UGC, tRNA-UAA, tRNA-UUU, tRNA-UAC, and ndhA) were proved by the nucleotide diversity (Pi). Although the distribution and existence of cp simple sequence repeats (cpSSRs) were predicted in the three Hordeum cp genomes, no rearrangement was found between them. A similar phenomenon has been found in the cp genome of the other seven Hordeum species, which has been published so far. In addition, evolutionary relationships were reappraised based on the currently reported cp genome of Hordeum L. This study offers a framework for gaining a better understanding of the evolutionary history of Hordeum species through the re-examination of their cp genomes, and by identifying highly polymorphic genes and hotspot regions that could provide important insights into the genetic diversity and differentiation of these species.

Genetic diversity analysis and molecular characteristics of wild centipedegrass using sequence-related amplified polymorphism (SRAP) markers.

Centipedegrass (Eremochloa ophiuroides (Munro) Hack.) is commonly used as a low-maintenance warm-season turfgrass owing to its excellent adaptation to various soil types. A better understanding of the genetic diversity pattern of centipedegrass is essential for the efficient development and utilization of accessions. This study used fifty-five pairs of primers to detect the genetic variation and genetic structure of twenty-three wild centipedegrass accessions by Sequence-related amplified polymorphism (SRAP) markers. A total of 919 reliable bands were amplified, among which 606 (65.80%) were polymorphic and 160 (2.91%) were the monomorphic loci. The average polymorphic information content (PIC) value was 0.228. The unweighted pair group method with arithmetic mean (UPGMA) clustering analysis grouped the twenty-three accessions into two clusters. Meanwhile, the structure analysis showed that the tested accessions possessed two main genetic memberships (K = 2). The Mantel test significantly correlated the genetic and geographic distance matrices (r = 0.3854, p = 0.000140). Furthermore, geographical groups showed moderate genetic differentiation, and the highest intragroup genetic diversity was found in the Sichuan group (He = 0.201). Overall, the present research findings could promote the protection and collection of centipedegrass and provide comprehensive information to develop novel breeding strategies.

Integrating Full-Length Transcriptome and RNA Sequencing of Siberian Wildrye (Elymus sibiricus) to Reveal Molecular Mechanisms in Response to Drought Stress.

Drought is one of the most significant limiting factors affecting plant growth and development on the Qinghai-Tibet Plateau (QTP). Mining the drought-tolerant genes of the endemic perennial grass of the QTP, Siberian wildrye (Elymus sibiricus), is of great significance to creating new drought-resistant varieties which can be used in the development of grassland livestock and restoring natural grassland projects in the QTP. To investigate the transcriptomic responsiveness of E. sibiricus to drought stress, PEG-induced short- and long-term drought stress was applied to two Siberian wildrye genotypes (drought-tolerant and drought-sensitive accessions), followed by third- and second-generation transcriptome sequencing analysis. A total of 40,708 isoforms were detected, of which 10,659 differentially expressed genes (DEGs) were common to both genotypes. There were 2107 and 2498 unique DEGs in the drought-tolerant and drought-sensitive genotypes, respectively. Additionally, 2798 and 1850 DEGs were identified in the drought-tolerant genotype only under short- and long-term conditions, respectively. DEGs numbering 1641 and 1330 were identified in the drought-sensitive genotype only under short- and long-term conditions, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that all the DEGs responding to drought stress in E. sibiricus were mainly associated with the mitogen-activated protein kinase (MAKP) signaling pathway, plant hormone signal transduction, the linoleic acid metabolism pathway, the ribosome pathway, and plant circadian rhythms. In addition, Nitrate transporter 1/Peptide transporter family protein 3.1 (NPF3.1) and Auxin/Indole-3-Acetic Acid (Aux/IAA) family protein 31(IAA31) also played an important role in helping E. sibiricus resist drought. This study used transcriptomics to investigate how E. sibiricus responds to drought stress, and may provide genetic resources and references for research into the molecular mechanisms of drought resistance in native perennial grasses and for breeding drought-tolerant varieties.

Distinct circulating cytokine/chemokine profiles correlate with clinical benefit of immune checkpoint inhibitor monotherapy and combination therapy in advanced non-small cell lung cancer.

BACKGROUND: An ever-increasing number of efforts are focused on identifying effective biomarkers for immune checkpoint inhibitors (ICIs). Cytokines and chemokines are critical to tumor growth, metastasis, tumor angiogenesis, and the immune response against tumor cells. In the study here, we determined the correlation between circulating cytokines/chemokines and the clinical benefit of ICIs for non-small cell lung cancer (NSCLC) patients. METHODS: Peripheral blood samples were collected before and during treatment (at 12th week). Plasma levels of cytokines/chemokines and specific stress response markers were measured using the Bio-Plex Pro Human Cytokines Grp I Panel (27-plex), an APEX1 detection kit, and a human LAP(TGF-ß1) immunoassay kit. A Mann-Whitney U-test or Wilcoxon signed-rank test and a Cox proportional hazards model were employed for statistical analysis. RESULTS: In the ICI monotherapy cohort, a high level of IL-6 at pretreatment or an elevation of IL-6, IL-8, FGF2, CXCL10, CCR1, PDFGB, TNF, and APEX1 posttreatment was associated with poor progress-free survival (PFS). A posttreatment elevation (defined herein as change rate) of CXCL10 was also associated with poor overall survival (OS). In the combinational therapy group, a high level of IL-12, IL-17A, FGF2, VEGF, and APEX1 at pretreatment and an elevation of CCL2 posttreatment were associated with poor PFS. A high level of IL-9, FGF2, PDFGB, CCL4, TFGB, and APEX1 at pretreatment and an elevation of IL-13, CSF2, and CCL2 at posttreatment were associated with poor OS of patients receiving combination therapy. CONCLUSIONS: The study here suggests that circulating cytokines/chemokines are feasible, noninvasive biomarkers for predicting clinical benefit of ICI treatment for NSCLC. Distinct circulating factor profiles were observed in individuals receiving ICI monotherapy or combination therapy.

Pan-cancer analysis of chromothripsis-related gene expression patterns indicates an association with tumor immune and therapeutic agent responses.

Chromothripsis is a catastrophic event involving numerous chromosomal rearrangements in confined genomic regions of one or a few chromosomes, causing complex effects on cells via the extensive structural variation. The development of whole-genome sequencing (WGS) has promoted great progress in exploring the mechanism and effect of chromothripsis. However, the gene expression characteristics of tumors undergone chromothripsis have not been well characterized. In this study, we found that the transcriptional profile of five tumor types experiencing chromothripsis is associated with an immune evasion phenotype. A gene set variation analysis (GSVA) was used to develop a CHP score, which is based on differentially expressed gene sets in the TCGA database, revealing that chromothripsis status in multiple cancers is consistent with an abnormal tumor immune microenvironment and immune cell cytotoxicity. Evaluation using four immunotherapy datasets uncovered the ability of the CHP score to predict immunotherapy response in diverse tumor types. In addition, the CHP score was found to be related to resistance against a variety of anti-tumor drugs, including anti-angiogenesis inhibitors and platinum genotoxins, while EGFR pathway inhibitors were found to possibly be sensitizers for high CHP score tumors. Univariate COX regression analysis indicated that the CHP score can be prognostic for several types of tumors. Our study has defined gene expression characteristics of tumors with chromothripsis, supporting the controversial link between chromothripsis and tumor immunity. We also describe the potential value of the CHP score in predicting the efficacy of immunotherapy and other treatments, elevating chromothripsis as a tool in clinical practice.

Analysis of Complete Chloroplast Genome: Structure, Phylogenetic Relationships of Galega orientalis and Evolutionary Inference of Galegeae.

Galega orientalis, a leguminous herb in the Fabaceae family, is an ecologically and economically important species widely cultivated for its strong stress resistance and high protein content. However, genomic information of Galega orientalis has not been reported, which limiting its evolutionary analysis. The small genome size makes chloroplast relatively easy to obtain genomic sequence for phylogenetic studies and molecular marker development. Here, the chloroplast genome of Galega orientalis was sequenced and annotated. The results showed that the chloroplast genome of G. orientalis is 125,280 bp in length with GC content of 34.11%. A total of 107 genes were identified, including 74 protein-coding genes, 29 tRNAs and four rRNAs. One inverted repeat (IR) region was lost in the chloroplast genome of G. orientalis. In addition, five genes (rpl22, ycf2, rps16, trnE-UUC and pbf1) were lost compared with the chloroplast genome of its related species G. officinalis. A total of 84 long repeats and 68 simple sequence repeats were detected, which could be used as potential markers in the genetic studies of G. orientalis and related species. We found that the Ka/Ks values of three genes petL, rpl20, and ycf4 were higher than one in the pairwise comparation of G. officinalis and other three Galegeae species (Calophaca sinica, Caragana jubata, Caragana korshinskii), which indicated those three genes were under positive selection. A comparative genomic analysis of 15 Galegeae species showed that most conserved non-coding sequence regions and two genic regions (ycf1 and clpP) were highly divergent, which could be used as DNA barcodes for rapid and accurate species identification. Phylogenetic trees constructed based on the ycf1 and clpP genes confirmed the evolutionary relationships among Galegeae species. In addition, among the 15 Galegeae species analyzed, Galega orientalis had a unique 30-bp intron in the ycf1 gene and Tibetia liangshanensis lacked two introns in the clpP gene, which is contrary to existing conclusion that only Glycyrrhiza species in the IR lacking clade (IRLC) lack two introns. In conclusion, for the first time, the complete chloroplast genome of G. orientalis was determined and annotated, which could provide insights into the unsolved evolutionary relationships within the genus Galegeae.

A subset of VEGFR-TKIs activates AMPK in LKB1-mutant lung cancer.

The mutation of tumor suppressor gene liver kinase B1 (LKB1) has a prevalence of about 20% in non-small cell lung cancer (NSCLC). LKB1-mutant lung cancer is characterized by enhanced aggressiveness and immune escape and is associated with poor prognosis. Therefore, it is urgent to develop effective therapeutic methods for LKB1-mutant NSCLC. Recently, apatinib, a VEGFR-TKI, was found to significantly improve the outcome of LKB1-mutant NSCLC, but the mechanism is not completely clear. In this study, AMP-activated protein kinase (AMPK), the crucial downstream kinase of LKB1 was excavated as the potential target of apatinib. Biochemical experiments verified that apatinib is a direct AMPK activator. Moreover, clinically available VEGFR-TKIs were found to regulate AMPK differently: Apatinib and anlotinib can directly activate AMPK, while axitinib and sunitinib can directly inhibit AMPK. Activation of AMPK by apatinib leads to the phosphorylation of acetyl-CoA carboxylase (ACC) and inhibition of de novo fatty acid synthesis (FAsyn), which is upregulated in LKB1-null cancers. Moreover, the killing effect of apatinib was obviously enhanced under delipidated condition, and the combination of exogenous FA restriction with apatinib treatment can be a promising method for treating LKB1-mutant NSCLC. This study discovered AMPK as an important off-target of apatinib and elucidated different effects of this cluster of VEGFR-TKIs on AMPK. This finding can be the basis for the accurate and combined application of these drugs in clinic and highlights that the subset of VEGFR-TKIs including apatinib and anlotinib are potentially valuable in the treatment of LKB1-mutant NSCLC.

Complete Chloroplast Genomes of 14 Subspecies of D. glomerata: Phylogenetic and Comparative Genomic Analyses.

Orchardgrass (Dactylis glomerata L.) is a species in the Gramineae family that is highly important economically and valued for its role in ecology. However, the phylogeny and taxonomy of D. glomerata are still controversial based on current morphological and molecular evidence. The study of chloroplast (cp) genomes has developed into a powerful tool to develop molecular markers for related species and reveal the relationships between plant evolution and phylogenetics. In this study, we conducted comparative genomic analyses and phylogenetic inferences on 14 cp genomes of D. glomerata originating from the Mediterranean and Eurasia. The genome size ranged from 134,375 bp to 134,993 bp and exhibited synteny of gene organization and order. A total of 129-131 genes were identified, including 85-87 protein coding genes, 38 tRNA genes and 8 rRNA genes. The cp sequences were highly conserved, and key sequence variations were detected at the junctions of inverted repeats (IRs)/small single-copy (SSC) regions. Moreover, nine highly variable regions were identified among the subspecies based on a sequence divergence analysis. A total of 285 RNA editing sites were detected that were relevant to 52 genes, where rpoB exhibited the most abundant RNA editing sites. The phylogenetic analysis revealed that all Dactylis subspecies clustered into a monophyletic group and most branches provided a high support bootstrap. The main divergence time of D. glomerata was dated to the Miocene era, and this could have been due to changes in the climate. These findings will provide useful insights for further studies on phylogeny, the identification of subspecies and the development of hypotheses for the evolutionary history of the genus Dactylis and of the Gramineae family.

Full-length transcriptome sequencing analysis and characterization, development and validation of microsatellite markers in Kengyilia melanthera.

As a typical psammophyte of the Triticeae, Kengyilia melanthera possesses high feeding potential and great utilization values in desertification control in the Qinghai-Tibet Plateau. However, few gene function and genetic studies have been performed in K. melanthera. In this study, single-molecule real-time sequencing technology was used to obtain the full-length transcriptome sequence of K. melanthera, following the functional annotation of transcripts and prediction of coding sequences (CDSs), transcription factors (TFs), and long noncoding RNA (lncRNA) sequences. Meanwhile, a total of 42,433 SSR loci were detected, with 5'-UTRs having the most SSR loci and trinucleotide being the most abundant type. In total, 108,399 SSR markers were designed, and 300 SSR markers were randomly selected for diversity verification of K. melanthera. A total of 49 polymorphic SSR markers were used to construct the genetic relationships of 56 K. melanthera accessions, among which 21 SSR markers showed good cross-species transferability among the related species. In conclusion, the full-length transcriptome sequence of the K. melanthera will assist gene prediction and promote molecular biology and genomics research, and the polymorphic SSR markers will promote molecular-assisted breeding and related research of K. melanthera and its relatives.

Selection and Validation of Reference Genes for qRT-PCR Gene Expression Analysis in Kengyilia melanthera.

Kengyilia is a newly established genus. Most species in this genus survive in hash environment, which might be an indicator of an acquirement of stress resistance genes and the potential for molecular breeding in Triticeae species. Quantitative real-time PCR (qRT-PCR) is a widely used technique with varied sensitivity heavily dependent on the optimal level of the reference genes. K. melanthera is a typical psammophyte species which has high drought resistance. The reference genes of K. melanthera are not yet reported. This study aims to evaluate the expression stability of 14 candidate reference genes (EF1A, GAPDH, ACT1, UBI, TUBB3, TIPRL, CACS, PPP2R1B, TUBA1A, EIF4A1, CYPA3, TCTP, ABCG11L, and FBXO6L) under five treatments (drought, heat, cold, salt, and ABA) and find the most stable and suitable one even upon stressed conditions. The software NormFinder, GeNorm, BestKeeper, and RefFinder were used for data analysis. In general, the genes CACS and PPP2R1B are concluded to have the best overall performance under the various treatments. With the ABA treatment, TCTP and TIPRL show the best stability. CACS and TCTP, as well as TIPRL and CYPA3, were most stable under the treatments of cold and salt, respectively. CACS and FBXO6L were ranked the highest with the heat treatment and drought treatment, respectively. Finally, the Catalase-1 (CAT1) gene was used to verify the reliability of the above reference genes. Accordingly, CAT1's expression pattern remained unchanged after normalization with stable reference genes. This study provides beneficial information about the stability and reliability of potential reference genes for qRT-PCR in K. melanthera.

Molecular Phylogeography and Intraspecific Divergences in Siberian Wildrye (Elymus sibiricus L.) Wild Populations in China, Inferred From Chloroplast DNA Sequence and cpSSR Markers.

A detailed understanding of the distribution and degree of genetic variation within a species is important for determining their evolutionary potential, which in return facilitates the development of efficient conservation strategies aimed at preserving adaptive genetic variation. As an important perennial, cool-season grass in temperate Eurasia, increasing attention has been paid to Siberian wildrye (Elymus sibiricus) due to its excellent ecological utilization value and forage production potential in China, particularly in the Qinghai-Tibet Plateau (QTP) regions. In this study, we applied two chloroplast (cp) genes (matK and rbcL), three cp spacer regions (trnY-GUA∼trnD-GUC, atpH∼atpF, and rps4∼trnT-UGU), and six cpSSR markers to the genetic and phylogenetic analysis of 137 wild E. sibiricus accessions from 23 natural populations that represent the main distribution regions in China. The results show the highest genetic diversity (h = 0.913) and haplotype richness (10 haplotypes) for the QTP population, which indicates QTP as the probable diversity center and geographic origin of E. sibiricus in China. Population divergence was high, indicating a significant phylogeographic structure together with a significantly higher Nst value (Nst > Gst, P < 0.05) at the species level, QTP+XJ (combined populations from QTP and Xinjiang), QTP+NC (combined populations from QTP and North China), and XJ+NC (combined populations from Xinjiang and North China) group levels, respectively. An expansion was revealed in the distributional range of E. sibiricus in China from paleo times up to the recent past, while a dramatic range of contraction was predicted for the near future. The predicted main limiting factor for the further spread of E. sibiricus is an increasing global mean temperature. We recommend that the combination of Es-cpDNA1 and Es-cpDNA3+4+5 can be used as effective markers for phylogenetic analysis and phylogeographical history analysis of E. sibiricus. These findings shed new light on the historical population dynamics of cold-season herbs in the QTP region and the north of China and are of great significance for the future establishment of protection and collection strategies for wild E. sibiricus germplasm.

RAD-seq as an effective strategy for heterogenous variety identification in plants-a case study in Italian Ryegrass (Lolium multiflorum).

The primary approach for variety distinction in Italian ryegrass is currently the DUS (distinctness, uniformity and stability) test based on phenotypic traits. Considering the diverse genetic background within the population and the complexity of the environment, however, it is challenging to accurately distinguish varieties based on DUS criteria alone. In this study, we proposed the application of high-throughput RAD-seq to distinguish 11 Italian ryegrass varieties with three bulks of 50 individuals per variety. Our findings revealed significant differences among the 11 tested varieties. The PCA, DAPC and STRUCTURE analysis indicated a heterogeneous genetic background for all of them, and the AMOVA analysis also showed large genetic variance among these varieties (ΦST = 0.373), which were clearly distinguished based on phylogenetic analysis. Further nucleotide diversity (Pi) analysis showed that the variety 'Changjiang No.2' had the best intra-variety consistency among 11 tested varieties. Our findings suggest that the RAD-seq could be an effectively alternative method for the variety distinction of Italian ryegrass, as well as a potential tool for open-pollinated varieties (OPVs) of other allogamous species.

Elevated tumor markers for monitoring tumor response to immunotherapy.

Background: As the immune-related response evaluation criteria in solid tumors (irRECIST) by imaging greatly underestimated the objective response to immunotherapy, we established the response evaluation criteria in solid tumors based on tumor markers (RecistTM) to explore whether RecistTM can compensate for the deficiencies of the irRECIST criteria. Methods: This was an observational study, which consisted of two parts. The first part (Group A) was a retrospective study including the patients with malignant solid tumors. The second part (Group B) was a prospective study, which were EGFR-negative and ALK-negative patients with stage IIIB-IV non-small cell lung cancer receiving first-line treatment. From January 2017 to September 2020, one hundred and ten patients with a three-time increase in tumor markers receiving immunotherapy were recruited. The treatment response to immunotherapy was evaluated by irRECIST and RecistTM. Efficacy, overall survival (OS), first evaluation time and earliest response time under the different evaluation criteria were compared by statistics. Findings: The treatment response evaluated by the RecistTM criteria was not consistent with that evaluated by the irRECIST criteria (Kappa = 0.386, p < 0.001). RecistTM had a higher completed response (CR) rate compared to irRECIST criteria (20.9% vs 1.8%, p < 0.001). The earliest response time under the RecistTM criteria was 3.42 weeks earlier than that under the irRECIST criteria (u = -5.233, p < 0.001). There were significant differences in median OS between tumor marker-related complete response (tmCR) and tumor marker-related partial response (tmPR), as well as between tmPR and tumor marker-related stable disease (tmSD) (χ2 = 15.572, p < 0.001; χ2 = 7.720, p = 0.005), but not between tmSD and tumor marker-related progressive disease (tmPD) (χ2 = 1.596, p = 0.206). When applying both criteria together, for patients with immune-related CR / immune-related PR (irCR/irPR) (n = 54) under irRECIST criteria, there was a significant difference in median OS between achieving tmCR (n = 22) and tmPR (n = 32) (χ2 = 14.011, p < 0.001). RecistTM criteria can predict 1-year and 2-year OS more accurately than irRECIST criteria (AUCs:0.862 vs 0.552, 0.649 vs 0.521, respectively;both p < 0.001). In RecistTM, 4 patients had been observed with pseudoprogression in tumor markers. Interpretation: The RecistTM criteria could effectively distinguish CR, PR, and SD, which may help resolve the shortcomings of the RECIST criteria in evaluating the treatment response to immunotherapy, especially in assessing whether patients can achieve deep or even complete response as soon as possible. Funding: This work was supported by the Key projects of Chongqing Health and Family Planning Commission (to Xueqin Yang, 2019ZDXM011).

Risk Factors on the Incidence and Prognostic Effects of Colorectal Cancer With Brain Metastasis: A SEER-Based Study.

Background: Colorectal cancer (CRC) with brain metastases (BM) is uncommon and often diagnosed at a late stage. The aims of this study were to identify the clinical factors that can influence the incidence of CRC patients with BM (CRCBM) and to investigate the impact of clinical factors and therapies on the outcomes of CRCBM. Methods: Between 2010 and 2018, patients with CRCBM were enrolled under the Surveillance, Epidemiology, and End Results (SEER) program. Multivariable logistic and Cox regression models were used to identify risk factors and prognostic factors of BM. Kaplan-Meier curve and log-rank test were used to evaluate overall survival (OS) and tumor-specific survival (CSS) of CRCBM patients. Results: A total of 195 (0.34%) CRC patients initially diagnosed with BM were included for analysis. The positive level of CEA, pN2a-b, and additional organ metastases were positively associated with developing BM from the CRC cohort (p < 0.05). The median OS and CSS of the BM patients were both 4.0 months, while the corresponding survival time in CRC patients without BM was 14.0 and 16.0 months, respectively (HR = 2.621, 95% CI = 2.061-3.333 for CSS; HR = 2.556, 95% CI = 2.026-3.225 for OS; log rank p < 0.001, each). Only systematic treatment was independently associated with better survival (p < 0.05, each). Conclusions: Although the overall prognosis of CRCBM patients was extremely poor, the positive level of CEA, pN2a-b, and distant metastases could be bad risk factors for the incidence of CRCBM. In addition, only systematic treatment was found to be a negative prognostic factor for CRCBM patients. These related factors can provide more valuable reference for clinical individualized treatments.

Comparative Metabolomic Studies of Siberian Wildrye (Elymus sibiricus L.): A New Look at the Mechanism of Plant Drought Resistance.

Drought is one of the most important factors affecting plant growth and production due to ongoing global climate change. Elymus sibiricus has been widely applied for ecological restoration and reseeding of degraded grassland in the Qinghai-Tibetan Plateau (QTP) because of its strong adaptability to barren, salted, and drought soils. To explore the mechanism of drought resistance in E. sibiricus, drought-tolerant and drought-sensitive genotypes of E. sibiricus were used in metabolomic studies under simulated long-term and short-term drought stress. A total of 1091 metabolites were detected, among which, 27 DMs were considered to be the key metabolites for drought resistance of E. sibiricus in weighted gene co-expression network analysis (WGCNA). Ten metabolites, including 3-amino-2-methylpropanoic acid, coniferin, R-aminobutyrate, and so on, and 12 metabolites, including L-Proline, L-histidine, N-acetylglycine, and so on, showed differential accumulation patterns under short-term and long-term drought stress, respectively, and thus, could be used as biomarkers for drought-tolerant and drought-sensitive E. sibiricus. In addition, different metabolic accumulation patterns and different drought response mechanisms were also found in drought-tolerant and drought-sensitive genotypes of E. sibiricus. Finally, we constructed metabolic pathways and metabolic patterns for the two genotypes. This metabolomic study on the drought stress response of E. sibiricus can provide resources and a reference for the breeding of new drought-tolerant cultivars of E. sibiricus.