[Thoughts on post-marketing evaluation of classical Chinese patent medicines based on clinical value].

Classical Chinese patent medicines(CPMs) are a kind of modern preparation developed from the experience of compatibility and application about ancient prescriptions. Its rich history of human use and reliable clinical efficacy imply the unique theoretical essence and precious value of traditional Chinese medicine(TCM). With the development of evidence-based medicine and the improvement of medical policy, it is particularly urgent to evaluate the clinical values of post-marketing classical CPMs. In this paper, some problems on the clinical value evaluation of CPMs would be described, and it is considered that the simplified evaluation procedures can lead to the lack of evidence for evaluating clinical value of CPMs, causing the difficulty in evaluating the quality of CPM, lack of R&D motivation of enterprises, low content of science and technology, and poor international development. Based on this background, it points out that the clinical value evaluation is the core of the post-marketing evaluation of the classical CPMs, and the eva-luation should be based on the direction of literature research and the latest practice. We should adhere to the research mode of combination disease with syndrome, and select the appropriate type of trials, with clinical efficacy, health economic benefits and safety eva-luation as the main content of the studies, in order to refine the indications and standardize the clinical positioning. Clinical value eva-luation is the basis and main content of post-marketing comprehensive researches on classic and famous CPMs to clarify their clinical value, obtain the conditions for continued marketingand standardize their clinical application, so as to optimize the evidence and quality service of classic and famous CPMs and inherit the core value concept of Chinese medicine.

Trans-splicing enhances translational efficiency in C. elegans.

Translational efficiency is subject to extensive regulation. However, the factors influencing such regulation are poorly understood. In Caenorhabditis elegans, 62% of genes are trans-spliced to a specific spliced leader (SL1), which replaces part of the native 5' untranslated region (5' UTR). Given the pivotal role the 5' UTR plays in the regulation of translational efficiency, we hypothesized that SL1 trans-splicing functions to regulate translational efficiency. With genome-wide analysis on Ribo-seq data, polysome profiling experiments, and CRISPR-Cas9-based genetic manipulation of trans-splicing sites, we found four lines of evidence in support of this hypothesis. First, SL1 trans-spliced genes have higher translational efficiencies than non-trans-spliced genes. Second, SL1 trans-spliced genes have higher translational efficiencies than non-trans-spliced orthologous genes in other nematode species. Third, an SL1 trans-spliced isoform has higher translational efficiency than the non-trans-spliced isoform of the same gene. Fourth, deletion of trans-splicing sites of endogenous genes leads to reduced translational efficiency. Importantly, we demonstrated that SL1 trans-splicing plays a key role in enhancing translational efficiencies of essential genes. We further discovered that SL1 trans-splicing likely enhances translational efficiency by shortening the native 5' UTRs, hence reducing the presence of upstream start codons (uAUG) and weakening mRNA secondary structures. Taken together, our study elucidates the global function of trans-splicing in enhancing translational efficiency in nematodes, paving the way for further understanding the genomic mechanisms of translational regulation.

Genetic Interaction Network as an Important Determinant of Gene Order in Genome Evolution.

Although it is generally accepted that eukaryotic gene order is not random, the basic principles of gene arrangement on a chromosome remain poorly understood. Here, we extended existing population genetics theories that were based on two-locus models and proposed a hypothesis that genetic interaction networks drive the evolution of eukaryotic gene order. We predicted that genes with positive epistasis would move toward each other in evolution, during which a negative correlation between epistasis and gene distance formed. We tested and confirmed our prediction with computational simulations and empirical data analyses. Importantly, we demonstrated that gene order in the budding yeast could be successfully predicted from the genetic interaction network. Taken together, our study reveals the role of the genetic interaction network in the evolution of gene order, extends our understanding of the encoding principles in genomes, and potentially offers new strategies to improve synthetic biology.

Codon-Resolution Analysis Reveals a Direct and Context-Dependent Impact of Individual Synonymous Mutations on mRNA Level.

Codon usage bias (CUB) refers to the observation that synonymous codons are not used equally frequently in a genome. CUB is stronger in more highly expressed genes, a phenomenon commonly explained by stronger natural selection on translational accuracy and/or efficiency among these genes. Nevertheless, this phenomenon could also occur if CUB regulates gene expression at the mRNA level, a hypothesis that has not been tested until recently. Here, we attempt to quantify the impact of synonymous mutations on mRNA level in yeast using 3,556 synonymous variants of a heterologous gene encoding green fluorescent protein (GFP) and 523 synonymous variants of an endogenous gene TDH3. We found that mRNA level was positively correlated with CUB among these synonymous variants, demonstrating a direct role of CUB in regulating transcript concentration, likely via regulating mRNA degradation rate, as our additional experiments suggested. More importantly, we quantified the effects of individual synonymous mutations on mRNA level and found them dependent on 1) CUB and 2) mRNA secondary structure, both in proximal sequence contexts. Our study reveals the pleiotropic effects of synonymous codon usage and provides an additional explanation for the well-known correlation between CUB and gene expression level.

Independent regulation of gene expression level and noise by histone modifications.

The inherent stochasticity generates substantial gene expression variation among isogenic cells under identical conditions, which is frequently referred to as gene expression noise or cell-to-cell expression variability. Similar to (average) expression level, expression noise is also subject to natural selection. Yet it has been observed that noise is negatively correlated with expression level, which manifests as a potential constraint for simultaneous optimization of both. Here, we studied expression noise in human embryonic cells with computational analysis on single-cell RNA-seq data and in yeast with flow cytometry experiments. We showed that this coupling is overcome, to a certain degree, by a histone modification strategy in multiple embryonic developmental stages in human, as well as in yeast. Importantly, this epigenetic strategy could fit into a burst-like gene expression model: promoter-localized histone modifications (such as H3K4 methylation) are associated with both burst size and burst frequency, which together influence expression level, while gene-body-localized ones (such as H3K79 methylation) are more associated with burst frequency, which influences both expression level and noise. We further knocked out the only "writer" of H3K79 methylation in yeast, and observed that expression noise is indeed increased. Consistently, dosage sensitive genes, such as genes in the Wnt signaling pathway, tend to be marked with gene-body-localized histone modifications, while stress responding genes, such as genes regulating autophagy, tend to be marked with promoter-localized ones. Our findings elucidate that the "division of labor" among histone modifications facilitates the independent regulation of expression level and noise, extend the "histone code" hypothesis to include expression noise, and shed light on the optimization of transcriptome in evolution.

[Correlation Research between Syndrome Elements of Chinese Medicine and KRAS, BRAF, NRAS Gene Mutations in Advanced Colorectal Cancer Patients].

Objective To study the correlation between syndrome elements of Chinese medicine (CM) and KRAS, BRAF, NRAS gene mutations in advanced colorectal cancer patients. Methods Syn- drome elements of CM, KRAS, BRAF, NRAS gene mutations (mutation " +" & wild type/non-mutation " --") were collected in 33 advanced colorectal cancer patients. The correlation between gene mutations and syndrome elements of CM was analyzed using binary Logistic regression. Results Wild type KRAS (KRAS-) was negatively correlated with Gan depression syndrome (P =0. 043, P <0. 05), while KRAS mutation (KRAS + ) was positively correlated with Gan depression syndrome (P =0. 043, P <0. 05). KRAS-and KRAS + had no significant relation with Pi deficiency, Fei deficiency, Shen deficiency, yang deficiency, yin deficiency, qi deficiency, blood deficiency, phlegm turbidity, qi stagnation, and blood sta- sis (P>0. 05). BRAF-and NRAS-had no significant relation with Gan stagnation, Pi deficiency, Fei defi- ciency, Shen deficiency, yang deficiency, yin deficiency, qi deficiency, blood deficiency, phlegm turbidi- ty, qi stagnation, and blood stasis (P >0. 05). Positive BRAF and NRAS were seldom found in this study, so they were not statistically analyzed. Conclusions There existed correlations between gene mutations and Gan stagnation. KRAS-was negatively correlated with Gan depression, while KRAS+ was positively correlated with Gan depression. Whether there exists mutation of BRAF or NRAS was not correlated with syndrome elements of CM.

Effect of thermal treatments on anti-nutritional factors and antioxidant capabilities in yellow soybeans and green-cotyledon small black soybeans.

BACKGROUND: The effects of different thermal processes on the anti-nutritional factors (tannins, phytic acids and trypsin inhibitors) and antioxidant capacity of yellow soybeans (YS) and green-cotyledon small black soybeans (GCSBS) were investigated. The soybean samples were subjected to roasting, microwaving and boiling as the thermal treatments. RESULTS: Sixty-minute boiling was most effective for eliminating the tannins in both YS and GCSBS, and also resulted in the maximal reduction of phytic acids. Trypsin inhibitors were most effectively reduced (76.92-97.91%) by microwaving, and boiling was least favourable for retaining total phenolic content. The total flavonoid content was significantly (P < 0.05) decreased by all thermal processes, and particularly by employing 60-min boiling. The total anthocyanin content of GCSBS was higher than that of YS, but significantly declined upon boiling (P < 0.05). Roasting did not significantly affect the DPPH free radical scavenging activity of GCSBS (P > 0.05). Microwaving increased the FRAP (ferric reducing antioxidant power) of GCSBS up to 6.07%. CONCLUSION: Our raw GCSBS possesses much better antioxidant capacity than raw YS. Microwaving resulted in a substantial reduction of the trypsin inhibitors of our soybean samples. Dry thermal treatments caused slight increase of FRAP in GCSBS.

Dynamic Treatment Strategy of Chinese Medicine for Metastatic Colorectal Cancer Based on Machine Learning Algorithm.

OBJECTIVE: To establish the dynamic treatment strategy of Chinese medicine (CM) for metastatic colorectal cancer (mCRC) by machine learning algorithm, in order to provide a reference for the selection of CM treatment strategies for mCRC. METHODS: From the outpatient cases of mCRC in the Department of Oncology at Xiyuan Hospital, China Academy of Chinese Medical Sciences, 197 cases that met the inclusion criteria were screened. According to different CM intervention strategies, the patients were divided into 3 groups: CM treatment alone, equal emphasis on Chinese and Western medicine treatment (CM combined with local treatment of tumors, oral chemotherapy, or targeted drugs), and CM assisted Western medicine treatment (CM combined with intravenous regimen of Western medicine). The survival time of patients undergoing CM intervention was taken as the final evaluation index. Factors affecting the choice of CM intervention scheme were screened as decision variables. The dynamic CM intervention and treatment strategy for mCRC was explored based on the cost-sensitive classification learning algorithm for survival (CSCLSurv). Patients' survival was estimated using the Kaplan-Meier method, and the survival time of patients who received the model-recommended treatment plan were compared with those who received actual treatment plan. RESULTS: Using the survival time of patients undergoing CM intervention as the evaluation index, a dynamic CM intervention therapy strategy for mCRC was established based on CSCLSurv. Different CM intervention strategies for mCRC can be selected according to dynamic decision variables, such as gender, age, Eastern Cooperative Oncology Group score, tumor site, metastatic site, genotyping, and the stage of Western medicine treatment at the patient's first visit. The median survival time of patients who received the model-recommended treatment plan was 35 months, while those who receive the actual treatment plan was 26.0 months (P=0.06). CONCLUSIONS: The dynamic treatment strategy of CM, based on CSCLSurv for mCRC, plays a certain role in providing clinical hints in CM. It can be further improved in future prospective studies with larger sample sizes.

Chinese Medicine Combined with Adjuvant Chemotherapy for Improving Myelosuppression in Colorectal Cancer Patients: A Systematic Review and Network Meta-Analysis.

OBJECTIVE: To assess the effectiveness of Chinese herbal medicine (CHM) combined with adjuvant chemotherapy on myelosuppression for colorectal cancer (CRC) patients using network meta-analysis (NMA). METHODS: Literature searches in both international (PubMed, Embase, Web of Science, and Cochrane Library) and Chinese (China Science and Technology Journal Database, Wanfang Data, China National Knowledge Infrastructure) databases for relevant randomized controlled trials (RCTs) were conducted from inception until October 10, 2022. We included RCTs of patients who received CHM combined with chemotherapy, including FOLFOX, XELOX, FOLFIRI, and other relevant regimens in the CHM treatment group. The outcomes included the incidence of myelosuppression, leukopenia, hemoglobin reduction, and thrombocytopenia. Two reviewers independently screened the databases, extracted the data, and assessed the risk of bias and credibility of evidence. RevMan 5.4.1 software and STATA 14.0 were used to perform the NMA. RESULTS: A total of 31 RCTs were included, published from 2008 to 2021 in Chinese. Among these, 2,314 participants comparing the following 9 CHMs were identified: Shengbai Recipe (SBR), Bazhen Decoction (BZD), Jianpi Jiedu Recipe (JJR), Jianpi Recipe (JR), Compound Cantharis Capsule (CCC), Zaofan Pill (ZFP), Guilu Erxian Gel (GL), Buzhong Tiaogan Decoction (BZ), and Qiamagu Capsule (QM). The results of NMA found an indirect comparison. Based on the surface under the cumulative ranking curve (SUCRA), the ZFP+ chemotherapy group had the lowest incidence of myelosuppression, with an odds ratio (OR) of 0.08 [95% confidence interval (CI): 0.01, 0.76], whereas the GL+ chemotherapy group had the lowest incidence of leukopenia, hemoglobin reduction, and thrombocytopenia, with an OR of 5.25 (95% CI: 2.41, 11.43), 4.66 (95% CI: 2.23, 9.72), and 0.27 (95% CI: 0.13, 0.54), respectively. Moreover, BZD + chemotherapy could alleviate leukopenia, hemoglobin reduction, and thrombocytopenia (P<0.01). Pairwise comparison showed that there was no difference in the efficacy among the 8 CHMs+ chemotherapy group. The comparison and adjustment funnel plot indicated that small-study effect had no impact on these outcomes. CONCLUSIONS: This NMA provided evidence to support that patients with CRC benefit from receiving different combination of CHM chemotherapies. Among these, GL plus chemotherapy and BZD plus chemotherapy were the more effective for myelosuppression in patients; however, as the qualtiy of evidence is insufficient, further research is needed. (PROSPERO, No. CRD42022369025).

Characterization and tissue expression analysis of mitochondrial creatine kinases (types I and II) from Pelodiscus sinensis.

The aim of this study was to characterize the functions of the mitochondrial creatine kinases in the Chinese soft-shelled turtle Pelodiscus sinensis (PSCK-MT1 and PSCK-MT2) to characterize function in relation to hibernation. Computational prediction via molecular dynamics simulations showed that PSCK-MT1 had stronger kinase- and creatine-binding affinity than PSCK-MT2. We measured PSCK-MT1 and PSCK-MT2 levels in the myocardium, liver, spleen, lung, kidney, and ovary of P. sinensis before and after hibernation and found that the expression of these enzymes was the most significantly upregulated in the ovary. We enumerated the ovarian follicles and evaluated the physiological indices of P. sinensis and discovered that fat was the main form of energy storage in P. sinensis. Moreover, both PSCK-MTs promoted follicular development during hibernation. Immunohistochemistry was used to study follicular development and revealed that both PSCK-MTs were expressed primarily in the follicular fluid and granulosa layer before and after hibernation. We found that PSCK-MT1 and PSCK-MT2 could play important roles in ovarian follicular development under hibernation. Hence, both PSCK-MTs probably function effectively under the conditions of low temperature and oxygen during hibernation. Communicated by Ramaswamy H. Sarma.

Study on the pattern of spermatogenesis during the breeding season of the Chinese soft-shelled turtle, Pelodiscus sinensis (Reptilia: Trionychidae).

The Chinese soft-shelled turtle, Pelodiscus sinensis (Reptilia: Trionychidae) is a typical seasonal breeding species and its spermatogenesis pattern is complex. In this study, the process of sperm cell development was studied using histology. The process of sperm cell development may be divided into six stages based on a combination of different cell types in the seminiferous epithelium. A close examination revealed two patterns of sperm cell development in the seminiferous tubules during the breeding season. The first is a normal sperm cell development pattern, in which the process of sperm cell development and maturation are completed in the seminiferous epithelium without round spermatozoa in the lumen. The second is rapid sperm cell development, in which the first batches of round spermatozoa fall off the seminiferous epithelium before they mature, thus beginning a second batch of sperm cell development. The round sperm cells are shed into the lumen and further mature in the seminiferous tubules and epididymis. This rapid sperm cell development process of the Chinese soft-shelled turtle is rare in other vertebrate species and may be an adaptation to cope with seasonal breeding. The results of this study provide insight into the theory of seasonal reproduction in reptiles.

Seasonal spermatogenesis, epididymal storage, and creatine kinase expression in Pelodiscus sinensis.

The soft-shelled turtle, Pelodiscus sinensis, is an important economic aquaculture species. Its reproduction exhibits seasonality; however, there is a lack of systematic studies focused on sperm maturation and epididymal storage. The testes and epididymides of P. sinensis were sampled from March to December. The seasonal reproduction and maturation of the spermatozoa were examined by anatomy, hematoxylin and eosin staining, AB-PAS staining, and immunohistochemistry. Spermatogenesis exhibited obvious seasonality in P. sinensis. It was found that the spermatogenic epithelium was most active during June to September, whereas the diameter of the epididymal tubules was smallest during June to October. As key enzymes of ATP metabolism, creatine kinases were highly expressed in the epididymal tubule epithelium during the breeding season, which may be important for the regulation of sperm maturation. In addition, the epididymal tubule epithelium changed with the season in June to September, the epididymal tubule epithelium proliferated to form villous structures, and secreted a large number of glycoproteins, which may be related to the rapid maturation of sperm during the breeding season. In conclusion, this study provided insights into the spermatogenesis of P. sinensis through histological analysis and enriched our understanding of reproduction in reptiles.

Structure and property changes of whey protein isolate in response to the chemical modification mediated by horseradish peroxidase, glucose oxidase and d-glucose.

Whey protein isolate (WPI) was modified by a ternary system containing horseradish peroxidase, glucose oxidase and d-glucose through the one- and two-step protocols, yielding two respective crosslinked products MWPI-1 and MWPI-2 with the enhanced relative dityrosine contents (127.4 and 101.0). Compared with WPI, both MWPI-1 and MWPI-2 had much ordered secondary structure, increased disulfide-bond contents, average particle sizes, surface hydrophobicity, oil-binding capacity, emulsification and thermal stability, but reduced free sulfhydryl groups contents and in vitro digestibility. Moreover, both MWPI-1 and MWPI-2 in dispersions showed higher apparent viscosity, larger viscoelastic moduli than WPI, together with the lower gelling temperatures (67.1 °C and 70.1 °C versus 73.6 °C). Overall, MWPI-1 with a higher crosslinking extent consistently exhibited more remarkable property alteration. It is concluded that the ternary system is an effective approach when aiming to modify secondary structure especially these properties of WPI, such as aggregation, emulsification, gelation, rheology and thermal stability.

Efficacy of Quxie Capsule in Metastatic Colorectal Cancer: Long-Term Survival Update of A Double-Blind, Randomized, Placebo Controlled Trial.

OBJECTIVE: To verify the efficacy of Quxie Capsule in patients with metastatic colorectal cancer (mCRC). METHODS: It was a block randomized, double-blind, placebo-controlled trial. Sixty patients with mCRC were randomized into 2 groups at a 1:1 ratio. The patients in the treatment group received conventional therapy including chemotherapy, radiotherapy, targeted therapy and supportive care, and Chinese herbal medicine combined with Quxie Capsule (each capsule of 0.4 g was orally administered at 50 mg/kg, twice daily, day 1-20, in a 30-day course) for 3 months. The patients in the control group received conventional therapy and Chinese herbal medicine combined with placebo for 3 months. Main outcome measures were overall survival (OS) and progression-free survival (PFS). Subgroup analysis was performed according to therapy lines, right or left-sided colon, targeted therapy and RAS gene status to determine the differences in PFS and OS between the two groups. Patients were followed up every 3 months until December 31st, 2018. RESULTS: Median follow-up time was 19.4 months. The median OS was 23.9 months in the treatment group [95% confidence interval (CI) 15.9-28.5] vs. 14.3 months in the control group (95% CI 11.3-21.4, P<;0.05). Hazard ratio (95% CI) was 0.55 (0.31-0.95, P=0.040). There were no significant differences between the two groups in PFS (P>0.05). In the subgroups of ⩾second-line therapy, non-targeted therapy, RAS gene wild type and left-sided colon, the treatment group showed a significant survival benefit compared with the control group (P<;0.05 or P<;0.01), respectively. CONCLUSION: Quxie Capsule can reduce the risk of death and prolong the OS of patients with mCRC. (Registration No. ChiCTR-IOR-16009733).

Patients with Metastatic Colorectal Cancer after Failure of Second-Line Treatment May Benefit from Low-Dose Apatinib and S-1 Combined with Jianpi Bushen Jiedu Decoction.

OBJECTIVE: To evaluate the effect and safety of low-dose of apatinib and S-1 combined with Jianpi Bushen Jiedu Decoction (JBJD) in patients with metastatic colorectal cancer (mCRC) who have failed second or above lines treatment, in order to provide more treatment option for mCRC patients by integrated medicine. METHODS: Thirteen patients were selected from a single-arm, open-label clinical study from April 2019 to September 2020. The patients were treated with low-dose apatinib (250 mg, once a day) and S-1 (20 mg, twice a day) combined with JBJD for at least one cycle and were followed up to August 2021. The primary endpoint was disease progression-free survival (PFS). Disease control rate (DCR), objective response rate (ORR), and overall survival (OS) of patients were observed as the secondary endpoints. Adverse events were recorded as well. RESULTS: The average age of the 13 patients was 56.5 ±13.0 years and 76.9% were male. The median PFS and median OS were 4.6 and 8.3 months, respectively. The ORR was 7.7% (1/13) while the DCR was 61.5% (8/13). The common adverse events were hypertension, proteinuria, elevated transaminase, and thrombocytopenia. One patient experienced thrombocytopenia of grade 3. CONCLUSIONS: Patients with mCRC after failure of the second or above lines of treatment may potentially benefit from the treatment of low-dose apatinib and S-1 combined with JBJD because of its similar effect as the standard dose of target therapy and relatively better safety. (Registration No. ChiCTR1900022673).

Effect of Jianpi Bushen Sequential Formula on Adjuvant Chemotherapy of Colon Cancer: Study Protocol for a Randomized Controlled Trial.

BACKGROUND: The side effects of chemotherapy-induced nausea and vomiting (CINV) and myelosuppression reduce the cancer patients' adherence to chemotherapy. Many Chinese patients choose Chinese medicine (CM) during chemotherapy to reduce side effects; however, the evidence is lacking. The efficacy of a CM herbal treatment protocol, Jianpi Bushen Sequential Formula (, JBSF) will be evaluated on chemotherapy completion rate among patients with colon cancer. METHODS: A multi-center double-blind randomized controlled trial (RCT) will be conducted on 400 patients with colon cancer who will receive 8 cycles of adjuvant chemotherapy with oxaliplatin and capecitabine (CAPEOX). Patients will be randomized 1:1 to receive the JBSF or placebo formula. The primary outcome is the overall chemotherapy completion rate. The secondary outcomes include individual chemotherapy completion rate, 4-cycle completion rate of chemotherapy, time to treatment failure, relative dose intensity and treatment toxicity. Follow-up visits will be scheduled before every and after last chemotherapy. DISCUSSION: This study will provide evidence on whether JBSF can improve the chemotherapy completion rate and reduce side effects among patients with colon cancer. (Trial registration: ClinicalTrials.gov, No. NCT03716518).

Effectiveness of Herbal Medicine for Leukopenia/Neutropenia Induced by Chemotherapy in Adults with Colorectal Cancer: A Systematic Review and Meta-analysis.

OBJECTIVE: To evaluate the effectiveness of Chinese Herbal Medicine (CHM) on leukopenia/neutropenia induced by chemotherapy in adults with colorectal cancer (CRC). METHODS: Eight electronic databases were searched from their inception to June 2020. Randomized controlled trials with clarified sequence generation were qualified. Two reviewers independently conducted the screening and data extraction. Methodological quality was assessed using the Risk of Bias tool. RevMan 5.4 was applied to the meta-analysis. RESULTS: Twenty-seven studies involving 1867 participants were qualified, of which 26 were included in the quantitative synthesis. Meta-analysis showed that CHM significantly reduced the incidence of leukopenia induced by chemotherapy (RR = 0.69; 95% CI 0.59-0.82), as well as the grade 3/4 leukopenia (RR = 0.71; 95% CI 0.55-0.90). Meanwhile,CHM decreased the occurrence of neutropenia (RR = 0.52, 95% CI 0.35-0.77), especially for the grades 3/4 neutropenia (RR = 0.42, 95% CI 0.27-0.64). Twenty-six of the included studies focused on the adverse events related to CHM. CONCLUSION: CHM may relieve neutropenia/leukopenia induced by chemotherapy in adults with colorectal cancer.

Evaluation of models of the mechanisms underlying intron loss and gain in Aspergillus fungi.

Although intron loss and gain have been widely observed, their mechanisms are still to be determined. In four Aspergillus genomes, we found 204 cases of intron loss and 84 cases of intron gain. Using this data, we tested common hypotheses of intron loss or gain. Statistical analysis showed that adjacent introns tend to be lost simultaneously and small introns were preferentially lost, supporting the model of mRNA-mediated intron loss. The lost introns reside in internal regions of genes, which is inconsistent with the traditional version of the model (partial length cDNAs are reverse transcribed from 3' ends of mRNAs), but consistent with an alternate version (partial length cDNAs are produced by self-primed reverse transcription). The latter version was not supported by examination of the abundance of T-rich segments in mRNAs. Preferential loss of internal introns might be explained by highly efficient recombination at internal regions of genes. Among the 84 cases of intron gain, we found a significantly higher frequency of short direct repeats near exon-intron boundary than in conserved introns, supporting the double-strand break repair model. We also found possible source sequences for two cases of intron gain, one by gene conversion and one by insertion of a mitochondrial sequence during double-strand break repair. Source sequences for most gained introns could not be identified and the possible reasons were discussed. In the four Aspergillus genomes studied, we did not find evidence of frequent parallel intron gains.

Characterization of Starch in Cucurbita moschata Germplasms throughout Fruit Development.

Pumpkins (Cucurbita moschata; Cucurbitaceae) are the rich source of nutrients and valued for their biologically active substances to be used for the treatment of several diseases. The contents, composition, and conformation of starch are the significant quality traits of C. moschata. Two germplasms were targeted for analysis regarding the taste difference. Results indicated that the total starch contents and amylose/amylopectin ratio were high in CMO-X as compared to CMO-E during each fruit development stage. Scanning electron microscopy and transmission electron microscopy observations revealed that smooth surface starch granules fused together to enhance the starch accumulation. For a comparison of fruit development in CMO-E and CMO-X, the putative pathway for starch metabolism was developed and homologs were identified for each key gene involved in the pathway. GBSS and SBE were correlated with the difference in the amylose/amylopectin ratio of CMO-E and CMO-X. Conclusively, the developmental regulation of genes associated with starch accumulation can be considered as an important factor for the determination of fruit quality.