Triphasic regulation of ZmMs13 encoding an ABCG transporter is sequentially required for callose dissolution, pollen exine and anther cuticle formation in maize.

INTRODUCTION: ATP Binding Cassette G (ABCG) transporters are associated with plant male reproduction, while their regulatory mechanisms underlying anther and pollen development remain largely unknown. OBJECTIVES: Identify and characterize a male-sterility gene ZmMs13 encoding an ABCG transporter in modulating anther and pollen development in maize. METHODS: Phenotypic, cytological observations, and histochemistry staining were performed to characterize the ms13-6060 mutant. Map-based cloning and CRISPR/Cas9 gene editing were used to identify ZmMs13 gene. RNA-seq data and qPCR analyses, phylogenetic and microsynteny analyses, transient dual-luciferase reporter and EMSA assays, subcellular localization, and ATPase activity and lipidomic analyses were carried out to determine the regulatory mechanisms of ZmMs13 gene. RESULTS: Maize ms13-6060 mutant displays complete male sterility with delayed callose degradation, premature tapetal programmed cell death (PCD), and defective pollen exine and anther cuticle formation. ZmMs13 encodes a plasm membrane (PM)- and endoplasmic reticulum (ER)-localized half-size ABCG transporter (ZmABCG2a). The allele of ZmMs13 in ms13-6060 mutant has one amino acid (I311) deletion due to a 3-bp deletion in its fourth exon. The I311 and other conserved amino acid K99 are essential for the ATPase and lipid binding activities of ZmMS13. ZmMs13 is specifically expressed in anthers with three peaks at stages S5, S8b, and S10, which are successively regulated by transcription factors ZmbHLH122, ZmMYB84, and ZmMYB33-1/-2 at these three stages. The triphasic regulation of ZmMs13 is sequentially required for callose dissolution, tapetal PCD and pollen exine development, and anther cuticle formation, corresponding to transcription alterations of callose-, ROS-, PCD-, sporopollenin-, and anther cuticle-related genes in ms13-6060 anthers. CONCLUSION: ms13-6060 mutation with one key amino acid (I311) deletion greatly reduces ZmMS13 ATPase and lipid binding activities and displays multiple effects during maize male reproduction. Our findings provide new insights into molecular mechanisms of ABCG transporters controlling anther and pollen development and male fertility in plants.

ATP-Binding Cassette G Transporters and Their Multiple Roles Especially for Male Fertility in Arabidopsis, Rice and Maize.

ATP-binding cassette subfamily G (ABCG) transporters are extensive in plants and play essential roles in various processes influencing plant fitness, but the research progress varies greatly among Arabidopsis, rice and maize. In this review, we present a consolidated nomenclature and characterization of the whole 51 ABCG transporters in maize, perform a phylogenetic analysis and classification of the ABCG subfamily members in maize, and summarize the latest research advances in ABCG transporters for these three plant species. ABCG transporters are involved in diverse processes in Arabidopsis and rice, such as anther and pollen development, vegetative and female organ development, abiotic and biotic stress response, and phytohormone transport, which provide useful clues for the functional investigation of ABCG transporters in maize. Finally, we discuss the current challenges and future perspectives for the identification and mechanism analysis of substrates for plant ABCG transporters. This review provides a basic framework for functional research and the potential application of ABCG transporters in multiple plants, including maize.

Research on diagnosis-related group grouping of inpatient medical expenditure in colorectal cancer patients based on a decision tree model.

BACKGROUND: In 2018, the diagnosis-related groups prospective payment system (DRGs-PPS) was introduced in a trial operation in Beijing according to the requirements of medical and health reform. The implementation of the system requires that more than 300 disease types pay through the DRGs-PPS for medical insurance. Colorectal cancer (CRC), as a common malignant tumor with high prevalence in recent years, was among the 300 disease types. AIM: To investigate the composition and factors related to inpatient medical expenditure in CRC patients based on disease DRGs, and to provide a basis for the rational economic control of hospitalization expenses for the diagnosis and treatment of CRC. METHODS: The basic material and cost data for 1026 CRC inpatients in a Grade-A tertiary hospital in Beijing during 2014-2018 were collected using the medical record system. A variance analysis of the composition of medical expenditure was carried out, and a multivariate linear regression model was used to select influencing factors with the greatest statistical significance. A decision tree model based on the exhaustive χ 2 automatic interaction detector (E-CHAID) algorithm for DRG grouping was built by setting chosen factors as separation nodes, and the payment standard of each diagnostic group and upper limit cost were calculated. The correctness and rationality of the data were re-evaluated and verified by clinical practice. RESULTS: The average hospital stay of the 1026 CRC patients investigated was 18.5 d, and the average hospitalization cost was 57872.4 RMB yuan. Factors including age, gender, length of hospital stay, diagnosis and treatment, as well as clinical operations had significant influence on inpatient expenditure (P < 0.05). By adopting age, diagnosis, treatment, and surgery as the grouping nodes, a decision tree model based on the E-CHAID algorithm was established, and the CRC patients were divided into 12 DRG cost groups. Among these 12 groups, the number of patients aged ≤ 67 years, and underwent surgery and chemotherapy or radiotherapy was largest; while patients aged > 67 years, and underwent surgery and chemotherapy or radiotherapy had the highest medical cost. In addition, the standard cost and upper limit cost in the 12 groups were calculated and re-evaluated. CONCLUSION: It is important to strengthen the control over the use of drugs and management of the hospitalization process, surgery, diagnosis and treatment to reduce the economic burden on patients. Tailored adjustments to medical payment standards should be made according to the characteristics and treatment of disease types to improve the comprehensiveness and practicability of the DRGs-PPS.

Cost Analysis of Cervical Cancer Patients with Different Medical Payment Modes Based on Gamma Model within a Grade A Tertiary Hospital.

BACKGROUND: Cervical cancer shows a growing incidence and medical cost in recent years that has increased severe financial pressure on patients and medical insurance institutions. This study aimed to investigate the medical economic characteristics of cervical cancer patients with different payment modes within a Grade A tertiary hospital to provide evidence and suggestions for inpatient cost control and to verify the application of Gamma model in medical cost analysis. METHODS: The basic and cost information of cervical cancer cases within a Grade A tertiary hospital in the year 2011-2016 were collected. The Gamma model was adopted to analyze the differences in each cost item between medical insured patient and uninsured patients. Meanwhile, the marginal means of different cost items were calculated to estimate the influence of payment modes toward different medical cost items among cervical cancer patients in the study. RESULTS:: A total of 1321 inpatients with cervical cancer between the 2011 and 2016 were collected through the medical records system. Of the 1321 cases, 65.9% accounted for medical insured patients and 34.1% were uninsured patients. The total inpatient medical expenditure of insured patients was RMB 29,509.1 Yuan and uninsured patients was RMB 22,114.3 Yuan, respectively. Payment modes, therapeutic options as well as the recurrence and metastasis of tumor toward the inpatient medical expenditures between the two groups were statistically significant. To the specifics, drug costs accounted for 37.7% and 33.8% of the total, surgery costs accounted for 21.5% and 25.5%, treatment costs accounted for 18.7% and 16.4%, whereas the costs of imaging and laboratory examinations accounted for 16.4% and 15.2% for the insured patient and uninsured patients, respectively. As the effects of covariates were controlled, the total hospitalization costs, drug costs, treatment costs as well as imaging and laboratory examination costs showed statistical significance. The total hospitalization costs, drug costs, treatment costs as well as imaging and laboratory examination costs of insured patient were 1.33, 1.42, 1.52, and 1.44 times of uninsured patients. CONCLUSIONS: The analysis of different payment modes toward the medical economic characteristics based on Gamma model is basically rational. Medical payment modes are having certain influence toward the hospitalization expenses of cervical cancer patients in an extent, as drug costs, treatment costs, and examination costs appear to be the main causes.

Research of Medical Expenditure among Inpatients with Unstable Angina Pectoris in a Single Center.

BACKGROUND: With the rising incidence as well as the medical expenditure among patients with unstable angina pectoris, the research aimed to investigate the inpatient medical expenditure through the combination of diagnosis-related groups (DRGs) among patients with unstable angina pectoris in a Grade A tertiary hospital to conduct the referential standards of medical costs for the diagnosis. METHODS: Single-factor analysis and multiple linear stepwise regression method were used to investigate 3933 cases between 2014 and 2016 in Beijing Hospital (China) whose main diagnosis was defined as unstable angina pectoris to determine the main factors influencing the inpatient medical expenditure, and decision tree method was adopted to establish the model of DRGs grouping combinations. RESULTS: The major influential factors of inpatient medical expenditure included age, operative method, therapeutic effects as well as comorbidity and complications (CCs) of the disease, and the 3933 cases were divided into ten DRGs by four factors: age, CCs, therapeutic effects, and the type of surgery with corresponding inpatient medical expenditure standards setup. Data of nonparametric test on medical costs among different groups were all significant (P < 0.001, by Kruskal-Wallis test), with R2 = 0.53 and coefficient of variation (CV) = 0.524. CONCLUSIONS: The classification of DRGs by adopting the type of surgery as the main branch node to develop cost control standards in inpatient treatment of unstable angina pectoris is conducive in standardizing the diagnosis and treatment behaviors of the hospital and reducing economic burdens among patients.

Research on 2041 Cases of High Inpatient Expenditure and Influence Factors during 3 Years in a Single Center.

BACKGROUND: The study was to explore the causes of high inpatient expenditure through analyzing the distribution characteristics as well as the influence factors of high inpatient expenditure cases during 3 years within a Grade-A tertiary hospital through various aspects and multiple angles, thus identifying the major influence factors for high medical expenditure to develop further research. METHODS: We retrospectively studied 2041 inpatient cases which cost more than RMB 100,000 Yuan per case in a Grade-A tertiary hospital from 2013 to 2015. We analyzed the compositions of the medical cost to evaluate the major factors that cause the high inpatient expenditure. All the data and materials were collected from medical record system, and the statistical methods included t-test, variance of analysis, and multivariate linear regression. RESULTS: The average cost of the 2,041 cases was RMB 152,173 Yuan for medicines and materials of medical costs, which respectively accounted for 33.03% and 32.32% of the total cost; and the average length of hospital stay was 28.39 days/person. Diseases of skeletal and muscular system, circulatory system, and tumor were the top three disease categories of high inpatient expenditure, which accounted for 39.00%, 33.46%, and 18.03%, respectively. Complications, criticality of the disease, gender of the patients, the occurrence of death, and the excessive length of hospital stay all had great impacts on average medical expenditure, while age, hospital infection, and surgery showed no significant impact on average medical cost. CONCLUSIONS: The main factors for high inpatient expenditure included the inadequate use of high-value medicines and materials, lacking cost control measures within the hospital, the excessive length of hospital stay for inpatients, and the unnecessary treatment for the patients.

Identification and characterization of a novel adenine phosphoribosyltransferase gene (ZmAPT2) from maize (Zea mays L.).

Adenine phosphoribosyltransferase (APRT) is the key enzyme that converts adenine to adenosine monophosphate (AMP) in the purine salvage pathway. It was found that several different forms of APRT gene exist in plants, but no APRT gene in maize has been reported up to now. In this study, a novel maize APRT gene was cloned and characterized through a combination of bioinformatic, RT-PCR and RACE strategies. The full length of APRT cDNA sequence is 1202 nucleotides, with an ORF encoding 214 amino acid residues. Alignment of the deduced protein with that of other plant APRT genes indicates that the new gene is the form 2 of maize APRT, thus it was named ZmAPT2. Through basic local alignment search tool, search in the genomic survey sequence database of MaizeGDB, the putative genomic sequence of ZmAPT2 was obtained. Comparison of the cDNA and genomic sequence of the ZmAPT2 gene revealed that it contained seven exons and six introns. The locations of the introns within the maize ZmAPT2 coding region were consistent with those in the previously isolated APRTs of arabidopsis and rice. RT-PCR analysis showed that ZmAPRT was constitutively expressing in different organs under high temperature and salt stresses. Southern blot analysis indicated that at least three APRT genes existed in maize genome. These results confirmed that the novel maize ZmAPT2 gene was truly identified, and its potential role in maize growth and development was discussed.

Cloning, characterization, and transformation of the phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) in maize (Zea mays L.).

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-L-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering.

Cloning, sequencing and salt induced expression of PEAMT and BADH in oilseed rape (Brassica napus).

Agriculture productivity is severely hampered by soil salinity, drought and other environmental stresses. Studies on stress-resistant plants (halophytes, xerophytes, accumulating plants for specific toxic ions) have illuminated some mechanisms of stress tolerance in plants at metabolic or molecular levels, which gave some clues on how to genetically engineer stress-tolerant crops. With the isolation of more stress-responsive genes, genetic engineering with modified expression of stress responsive genes may be an effective way to produce stress-tolerant crops. In the present report, two genes (PEAMT and BADH) encoding the corresponding key enzymes for choline and glycine betaine (an important osmoprotectant) biosynthesis in plants were isolated in oilseed rape, an important oil crop in the world. Effects of salt stress on their expression were studied with quantitative PCR and their potential use in the genetic engineering of oilseed rape was discussed.