The Number of Intraoperative Intestinal Venous Circulating Tumor Cells Is a Prognostic Factor for Colorectal Cancer Patients.

Purpose: To assess the association between intestinal venous blood (IVB) circulating tumor cells (CTCs) and clinicopathological parameters in stage I-III colorectal cancer (CRC) patients. Methods: Participants were retrospectively retrieved, who were admitted to our hospital or took annual physical exams between December 1, 2015 and December 31, 2018. A negative enrichment-immunofluorescence in situ hybridization (NE-imFISH) technique was used to isolate and identify CTCs. Receiver operating characteristic (ROC) curves and Youden index values were used to determine the critical CTC cutoff value for the diagnosis of CRC. Kaplan-Meier and log-rank methods were used to conduct survival analyses, and multivariate Cox regression analyses were employed for multivariate corrections to comprehensively evaluate the value of CTCs in the diagnosis of CRC. Relationships between IVB CTCs, clinicopathological parameters, and prognosis were then analyzed based upon patient postoperative follow-up data. Results: In total, we retrieved 282 patients including 48 healthy controls, 72 patients with benign colorectal tumors, and 162 CRC patients. CRC patients exhibited significantly higher numbers of CTCs relative to control patients or those with benign disease. CTC numbers in CRC patient peripheral blood (PB) and IVB were closely associated with tumor node metastasis (TNM) staging (P < 0.01), carbohydrate antigen-125 (CA-125) levels (P < 0.001), and KRAS (Kirsten rat sarcoma virus oncogene) mutation status (P < 0.001). The disease-free survival (DFS) of patients in the CTC-negative group was significantly longer than that of patients in the CTC-positive group (24.60 ± 13.31 months vs. 18.70 ± 10.19 months, P < 0.05), with the same being true with respect to their overall survival (OS) (30.60 ± 12.44 months vs. 35.25 ± 11.57 months, P < 0.05). A multivariate analysis revealed that the detection ≥2 CTCs/3.2 ml was independently associated with poorer DFS and OS. CTC counts were independently predictive of CRC patients TNM staging, CA-125, and KRAS mutation status in both univariate and multivariate Cox proportional hazards regression analyses. Conclusion: CTCs are valuable biomarkers that can be monitored to predict CRC patient disease progression.

Rutaecarpine targets hERG channels and participates in regulating electrophysiological properties leading to ventricular arrhythmia.

Drug-mediated or medical condition-mediated disruption of hERG function accounts for the main cause of acquired long-QT syndrome (acLQTs), which predisposes affected individuals to ventricular arrhythmias (VA) and sudden death. Many Chinese herbal medicines, especially alkaloids, have risks of arrhythmia in clinical application. The characterized mechanisms behind this adverse effect are frequently associated with inhibition of cardiac hERG channels. The present study aimed to assess the potent effect of Rutaecarpine (Rut) on hERG channels. hERG-HEK293 cell was applied for evaluating the effect of Rut on hERG channels and the underlying mechanism. hERG current (IhERG ) was measured by patch-clamp technique. Protein levels were analysed by Western blot, and the phosphorylation of Sp1 was determined by immunoprecipitation. Optical mapping and programmed electrical stimulation were used to evaluate cardiac electrophysiological activities, such as APD, QT/QTc, occurrence of arrhythmia, phase singularities (PSs), and dominant frequency (DF). Our results demonstrated that Rut reduced the IhERG by binding to F656 and Y652 amino acid residues of hERG channel instantaneously, subsequently accelerating the channel inactivation, and being trapped in the channel. The level of hERG channels was reduced by incubating with Rut for 24 hours, and Sp1 in nucleus was inhibited simultaneously. Mechanismly, Rut reduced threonine (Thr)/ tyrosine (Tyr) phosphorylation of Sp1 through PI3K/Akt pathway to regulate hERG channels expression. Cell-based model unables to fully reveal the pathological process of arrhythmia. In vivo study, we found that Rut prolonged QT/QTc intervals and increased induction rate of ventricular fibrillation (VF) in guinea pig heart after being dosed Rut for 2 weeks. The critical reasons led to increased incidence of arrhythmias eventually were prolonged APD90 and APD50 and the increase of DF, numbers of PSs, incidence of early after-depolarizations (EADs). Collectively, the results of this study suggest that Rut could reduce the IhERG by binding to hERG channels through F656 and Y652 instantaneously. While, the PI3K/Akt/Sp1 axis may play an essential role in the regulation of hERG channels, from the perspective of the long-term effects of Rut (incubating for 24 hours). Importantly, the changes of electrophysiological properties by Rut were the main cause of VA.

FLOURY ENDOSPERM19 encoding a class I glutamine amidotransferase affects grain quality in rice.

As a staple food for more than half of the world's population, the importance of rice is self-evident. Compared with ordinary rice, rice cultivars with superior eating quality and appearance quality are more popular with consumers due to their unique taste and ornamental value, even if their price is much higher. Appearance quality and CEQ (cooking and eating quality) are two very important aspects in the evaluation of rice quality. Here, we performed a genome-wide association study on floury endosperm in a diverse panel of 533 cultivated rice accessions. We identified a batch of potential floury genes and prioritize one (LOC_Os03g48060) for functional analyses. Two floury outer endosperm mutants (flo19-1 and flo19-2) were generated through editing LOC_Os03g48060 (named as FLO19 in this study), which encodes a class I glutamine amidotransferase. The different performances of the two mutants in various storage substances directly led to completely different changes in CEQ. The mutation of FLO19 gene caused the damage of carbon and nitrogen metabolism in rice, which affected the normal growth and development of rice, including decreased plant height and yield loss by decreased grain filling rate. Through haplotype analysis, we identified a haplotype of FLO19 that can improve both CEQ and appearance quality of rice, Hap2, which provides a selection target for rice quality improvement, especially for high-yield indica rice varieties. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01226-z.

Grape Seed Proanthocyanidin Extract Ameliorates Streptozotocin-induced Cognitive and Synaptic Plasticity Deficits by Inhibiting Oxidative Stress and Preserving AKT and ERK Activities.

Progressive memory loss and cognitive impairment are the main clinical manifestations of Alzheimer's disease (AD). Currently, there is no effective drug available for the treatment of AD. Previous studies have demonstrated that the cognitive impairment of AD is associated with oxidative stress and the inhibition of AKT and ERK phosphorylation. Grape seed proanthocyanidin extract (GSPE) has been shown to have strong antioxidant effect and can protect the nervous system from oxidative stress damage. This study aimed to investigate the protective effect of GSPE on the cognitive and synaptic impairments of AD using a sporadic AD rat model induced by intracerebroventricular (ICV) injection of streptozotocin (STZ) (ICV-STZ). Rats were treated with GSPE (50, 100, or 200 mg/kg every day) by intragastrical (ig.) administration for continuous 7 weeks, and ICV-STZ (3 mg/kg) was performed on the first day and third day of week 5. Learning and memory abilities were assessed by the Morris water maze (MWM) test at week 8. After behavioral test, hippocampal long-term potentiation (LTP) was recorded, and the levels of malondialdehyde (MDA), superoxide dismutases (SOD), glutathione (GSH) and the protein expression of AKT and ERK were measured in the hippocampus and cerebral cortex of rats. Our study revealed that ICV-STZ significantly impaired the working learning ability and hippocampal LTP of rats, significantly increased the levels of MDA, and decreased the activity of SOD and GSH in the hippocampus and cerebral cortex. In contrast, GSPE treatment prevented the impairment of cognitive function and hippocampal LTP induced by ICV-STZ, decreased the level of MDA, and increased the level of SOD and GSH. Furthermore, Western blot results showed that GSPE treatment could prevent the loss of AKT and ERK activities in the hippocampus and cerebral cortex induced by ICV-STZ. Our findings demonstrate that GSPE treatment could ameliorate the impairment of cognitive ability and hippocampal synaptic plasticity in a rat model of sporadic AD by inhibiting oxidative stress and preserving AKT and ERK activities. Therefore, GSPE may be an effective agent for the treatment of cognitive deficits associated with sporadic AD.

The Chinese herb Prunella vulgaris promotes apoptosis in human well-differentiated thyroid carcinoma cells via the B-cell lymphoma-2/Bcl-2-associated X protein/caspase-3 signaling pathway.

Prunella vulgaris (PV), a traditional Chinese herb, has been shown to be rich in bioactive chemicals and possess anti-proliferative and pro-apoptotic effects on tumor cells. The effect of PV on human well-differentiated thyroid carcinoma (WDTC), which accounts for the majority of common endocrine malignancies, remains to be elucidated. The present study aimed to investigate the function of PV on WDTC cell lines and apoptosis-associated signaling pathway activity. Additional studies demonstrated that PV may induce apoptosis in WDTC TPC-1 and FTC-133 cell lines, using the Cell Counting Kit-8 assay. Morphological changes of apoptotic cells were observed by Hoechst 33342 and acridine orange/ethidium bromide staining. In addition, ladder pattern of fragmented DNA was observed by DNA gel electrophoresis. It was also observed that PV significantly increased Bcl-2-associated X protein and caspase-3 expression, and downregulated B-cell lymphoma-2 expression in TPC-1 and FTC-133 by reverse transcription-quantitative polymerase chain reaction (P<0.05). Thus, the present results indicated that PV has the potential to be a future WDTC therapeutic agent.

OsAP65, a rice aspartic protease, is essential for male fertility and plays a role in pollen germination and pollen tube growth.

Aspartic proteases (APs) comprise a large proteolytic enzyme family widely distributed in animals, microbes, viruses, and plants. The rice genome encodes 96 APs, of which only a few have been functionally characterized. Here, the identification and characterization of a novel AP gene, OsAP65, which plays an indispensable role in pollen tube growth in rice, is reported. The T-DNA insertion line of OsAP65 caused severe segregation distortion. In the progeny derived from an individual heterozygous for the T-DNA insertion, the wild type and T-DNA-carrying heterozygote segregated at a ratio close to 1:1, while homozygotes of disrupted OsAP65 (OsAP65-/-) were not recovered. Reciprocal crosses between heterozygotes and wild-type plants demonstrated that the mutant alleles could not be transmitted through the male gamete. Examination of the anthers from heterozygous plants revealed that the mutant pollen matured normally, but did not germinate or elongate. OsAP65 was expressed in various tissues and the transcript level in heterozygous plants was about half of the amount measured in the wild-type plants. The subcellular localization showed that OsAP65 is a pre-vacuolar compartment (PVC) protein. These results indicated that OsAP65 was essential for rice pollen germination and tube growth.

The rice RAD51C gene is required for the meiosis of both female and male gametocytes and the DNA repair of somatic cells.

The RecA/RAD51 family of rice (Oryza sativa) consists of at least 13 members. However, the functions of most of these members are unknown. Here the functional characterization of one member of this family, RAD51C, is reported. Knockout (KO) of RAD51C resulted in both female and male sterility in rice. Transferring RAD51C to the RAD51C-KO line restored fertility. Cytological analyses showed that the sterility of RAD51C-KO plants was associated with abnormal early meiotic processes in both megasporocytes and pollen mother cells (PMCs). PMCs had an absence of normal pachytene chromosomes and had abnormal chromosome fragments. The RAD51C-KO line showed no obvious difference from wild-type plants in mitosis in the anther wall cells, which was consistent with the observation that the RAD51C-KO line did not have obviously abnormal morphology during vegetative development. However, the RAD51C-KO line was sensitive to different DNA-damaging agents. These results suggest that RAD51C is essential for reproductive development by regulating meiosis as well as for DNA damage repair in somatic cells.

OsLIS-L1 encoding a lissencephaly type-1-like protein with WD40 repeats is required for plant height and male gametophyte formation in rice.

Although a large number of genes encoding the WD40 motif have been identified as being involved in various developmental processes in Arabidopsis, little is known about the function of these genes in rice (Oryza sativa). Here, we report the cloning and functional characterization of a novel rice gene OsLIS-L1 (Lissencephaly type-1-like 1), which is required for normal fertility and the first internode elongation. OsLIS-L1 encodes a lissencephaly type-1-like protein containing the WD40 motif that is required for brain development in human. SMART algorithm analysis indicated that OsLIS-L1 contains a LIS1 homology (LisH) domain, a C terminus to LisH (CTLH) domain, a five WD40-repeat domain in the middle, and a domain with four WD40 repeats which is homologous to the ß subunit of trimeric G-proteins (G(ß)). OsLIS-L1 transcript is relatively highly abundant in stem and panicle and has a dynamic expression pattern at different panicle developmental stages. Two independent alleles, designated oslis-l1-1 and oslis-l1-2, exhibited similar abnormal developmental phenotypes, including semi-dwarf, shorter panicle length, and reduced male fertility. Cytological examination confirmed that OsLIS-L1 does not affect the meiosis in pollen mother cells. Compared with wild type, the oslis-l1 mutant had abnormal male gametophyte formation, but anther cell wall and pollen wall development were not affected. Histological analysis revealed that OsLIS-L1 regulates the cell proliferation in the first internode under the panicle. Our results indicate that OsLIS-L1 plays an important role in male gametophyte formation and the first internode elongation in rice.

Effects of wuwei dilong decoction on inflammatory cells and cytokines in asthma model guinea pigs.

OBJECTIVE: To explore the effects and the mechanism of Wuwei Dilong Decoction (Schisandra Fruit and Earthworm Decoction) for treatment of asthma. METHODS: The asthma guinea pig model was established with spray of ovalbumin (OVA). Fifteen days later, the guinea pigs were administered by intra-gastric perfusion of Wuwei Dilong Decoction once a day for 8 consecutive days. Blood samples were taken for testing the total leucocytes, eosinophil (EOS), lymphocytes, interferon-gamma (IFN-gamma) and leukotriene B4 (LTB4). RESULTS: In the asthma model group, the total leucocytes, EOS and lymphocytes were all increased, with significant differences as compared with the different dosage Wuwei Dilong Decoction groups (P < 0.01 or P < 0.05). The serum LTB4 in the asthma model group was significantly increased and IFN-gamma decreased. After administration of Wuwei Dilong Decoction of the large, medium and small dosages, LTB4 decreased, while IFN-gamma increased (P < 0.05 or P < 0.01). CONCLUSION: Wuwei Dilong Decoction can inhibit infiltration and diffusion of the inflammatory cells in the asthma model guinea pigs, and regulate LTB4 and IFN-gamma, which is probably one of the important mechanisms of Wuwei Dilong Decoction for relieving asthma.

The expression profile of genes in rice roots under low phosphorus stress.

Phosphorus (P) is one of the most essential macronutrients required for plant growth. Although it is abundant in soil, P is often the limiting nutrient for crop yield potential because of the low concentration of soluble P that plants can absorb directly. The gene expression profile was investigated in rice roots at 6, 24 and 72 h under low P stress and compared with a control (normal P) profile, using a DNA chip of 60000 oligos (70 mer) that represented all putative genes of the rice genome. A total of 795 differentially expressed genes were identified in response to phosphate (Pi) starvation in at least one of the treatments. Based on the analysis, we found that: (i) The genes coding for the Pi transporter, acid phosphatase and RNase were up-regulated in rice roots; (ii) the genes involved in glycolysis were first up-regulated and then down-regulated; (iii) several genes involved in N metabolism and lipid metabolism changed their expression patterns; (iv) some genes involved in cell senescence and DNA or protein degradation were up-regulated; and (v) some transmembrane transporter genes were up-regulated. The results may provide useful information in the molecular process associated with Pi deficiency and thus facilitate research in improving Pi utilization in crop species.

Replication protein A (RPA1a) is required for meiotic and somatic DNA repair but is dispensable for DNA replication and homologous recombination in rice.

Replication protein A (RPA), a highly conserved single-stranded DNA-binding protein in eukaryotes, is a stable complex comprising three subunits termed RPA1, RPA2, and RPA3. RPA is required for multiple processes in DNA metabolism such as replication, repair, and homologous recombination in yeast (Saccharomyces cerevisiae) and human. Most eukaryotic organisms, including fungi, insects, and vertebrates, have only a single RPA gene that encodes each RPA subunit. Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), however, possess multiple copies of an RPA gene. Rice has three paralogs each of RPA1 and RPA2, and one for RPA3. Previous studies have established their biochemical interactions in vitro and in vivo, but little is known about their exact function in rice. We examined the function of OsRPA1a in rice using a T-DNA insertional mutant. The osrpa1a mutants had a normal phenotype during vegetative growth but were sterile at the reproductive stage. Cytological examination confirmed that no embryo sac formed in female meiocytes and that abnormal chromosomal fragmentation occurred in male meiocytes after anaphase I. Compared with wild type, the osrpa1a mutant showed no visible defects in mitosis and chromosome pairing and synapsis during meiosis. In addition, the osrpa1a mutant was hypersensitive to ultraviolet-C irradiation and the DNA-damaging agents mitomycin C and methyl methanesulfonate. Thus, our data suggest that OsRPA1a plays an essential role in DNA repair but may not participate in, or at least is dispensable for, DNA replication and homologous recombination in rice.

[Effect of earthworm decoction on airway inflammation of bronchial asthma in guinea pigs].

OBJECTIVE: To investigate the effect of Earthworm decoction on the airway inflammation of experimental bronchial asthma in guinea pigs and inquire into the mechanism in the decoction. METHOD: Forty-eight guinea pigs were randomly divided into six groups: the control group, the model group, the dexamethasone group, the Xiaoqinglong decoction group, the earthworm decoction large dosage group and the Earthworm decoction low dosage group, 8 guinea pigs in each group. Except the control group, the other groups were sensitized with ovalbumin (OVA) by a combination of intraperitional injection and repeated intranasal challenges to establish the guinea pigs asthma model. However, in the control group, normal saline was used. The morphological changes of bronchial tube, the lung tectology and the inflammation germ cell quantity of eosinophils (Eos), lymphocytes (Ly), neutrophils (Neu) and total blood cells in the blood and bronchoalveolar lavaga fluid (BALF) were examinated in each group respectively. RESULT: The levels of Eos, Ly, Neu and total cell quantity in the blood and BALF after the earthworm decoction treatment in the large dosage group were significantly lower than those in the model group (P <0.01), and in the low dosage group were lower too (P <0.05). The Earthworm decoction large dosage could obviously improve the bronchial tube epidermis damage, the mucous membrane gland proliferation and hydrops, asthma pathology change and basilar membrane accumulation. Eos apoptosis was obsered in the bronchoalveolar, blood and BALF. The Earthworm decoction small dosage had a similar effect but slightly to the large dosage. CONCLUSION: The Earthworm decoction can lighten the airway inflammation in asthmatic guinea pigs, its mechanism is related with the inhibition of Eos infiltration, acceleration of Eos apoptosis and improvement of the bronchial tube and the lung tectology changes. The effect of the decoction is dose-dependent.

Promoter mutations of an essential gene for pollen development result in disease resistance in rice.

Disease resistance and sexual reproductive development are generally considered as separate biological processes, regulated by different sets of genes. Here we show that xa13, a recessive allele conferring disease resistance against bacterial blight, one of the most devastating rice diseases worldwide, plays a key role in both disease resistance and pollen development. The dominant allele, Xa13, is required for both bacterial growth and pollen development. Promoter mutations in Xa13 cause down-regulation of expression during host-pathogen interaction, resulting in the fully recessive xa13 that confers race-specific resistance. The recessive xa13 allele represents a new type of plant disease resistance.

Dual function of rice OsDR8 gene in disease resistance and thiamine accumulation.

The function of OsDR8, a rice disease resistance-responsive gene, was studied. Silencing of OsDR8 using an RNA interference approach resulted in phenotypic alteration of the plants. The transgenic plants with repressed expression of OsDR8 showed reduced resistance or susceptibility to Xanthomonas oryzae pv. oryzae and Magnaporthe grisea causing bacterial blight and blast, which are two of the most devastating diseases in rice worldwide, respectively. The putative product of OsDR8 was highly homologous to an enzyme involved in the biosynthesis of the thiazole precursor of thiamine. Transgenic plants showing repressed expression of OsDR8 and reduced resistance had significantly lower levels of thiamine than the control plants. Exogenous application of thiamine could complement the compromised defense of the OsDR8-silenced plants. The expression level of several defense-responsive genes including the earlier functional genes of defense transduction pathway, OsPOX and OsPAL, and the downstream genes of the pathway, OsPR1a, OsPR1b, OsPR4, OsPR5 and OsPR10, was also decreased in the OsDR8-silenced plants. These results suggest that the impact of OsDR8 on disease resistance in rice may be through the regulation of expression of other defense-responsive genes and the site of OsDR8 function is on the upstream of the signal transduction pathway. In addition, the accumulation of thiamine may be essential for bacterial blight resistance and blast resistance.