Transcription Factors and Their Regulatory Roles in the Male Gametophyte Development of Flowering Plants.

Male gametophyte development in plants relies on the functions of numerous genes, whose expression is regulated by transcription factors (TFs), non-coding RNAs, hormones, and diverse environmental stresses. Several excellent reviews are available that address the genes and enzymes associated with male gametophyte development, especially pollen wall formation. Growing evidence from genetic studies, transcriptome analysis, and gene-by-gene studies suggests that TFs coordinate with epigenetic machinery to regulate the expression of these genes and enzymes for the sequential male gametophyte development. However, very little summarization has been performed to comprehensively review their intricate regulatory roles and discuss their downstream targets and upstream regulators in this unique process. In the present review, we highlight the research progress on the regulatory roles of TF families in the male gametophyte development of flowering plants. The transcriptional regulation, epigenetic control, and other regulators of TFs involved in male gametophyte development are also addressed.

FLA14 is required for pollen development and preventing premature pollen germination under high humidity in Arabidopsis.

BACKGROUND: As an important subfamily of arabinogalactan proteins (AGPs), fasciclin-like AGPs (FLAs) contribute to various aspects of growth, development and adaptation, yet their function remains largely elusive. Despite the diversity of FLAs, only two members, Arabidopsis FLA3 and rice MTR1, are reported to be involved in sexual reproduction. In this study, another Arabidopsis FLA-encoding gene, FLA14, was identified, and its role was investigated. RESULTS: Arabidopsis FLA14 was found to be a pollen grain-specific gene. Expression results from fusion with green fluorescent protein showed that FLA14 was localized along the cell membrane and in Hechtian strands. A loss-of-function mutant of FLA14 showed no discernible defects during male gametogenesis, but precocious pollen germination occurred inside the mature anthers under high moisture conditions. Overexpression of FLA14 caused 39.2% abnormal pollen grains with a shrunken and withered appearance, leading to largely reduced fertility with short mature siliques and lower seed set. Cytological and ultramicroscopic observation showed that ectopic expression of FLA14 caused disruption at the uninucleate stage, resulting in either collapsed pollen with absent intine or pollen of normal appearance but with a thickened intine. CONCLUSIONS: Taken together, our data suggest a role for FLA14 in pollen development and preventing premature pollen germination inside the anthers under high relative humidity in Arabidopsis.

Transcriptional profiling reveals molecular basis and the role of arginine in response to low-pH stress in Pichia kudriavzevii.

The non-conventional yeast Pichia kudriavzevii is considered to be a promising biotechnological host for the production of organic acids under low-pH conditions. However, little is known about the low-pH stress response in P. kudriavzevii, which significantly restricts its future development. In this study, P. kudriavzevii N-X showed great tolerance to low-pH stress, but the cell aggregation upon extremely acidic conditions might be unfavorable for low-pH fermentation. We therefore conducted RNA-Seq to compare global gene expression of P. kudriavzevii N-X in response to different pH stresses. Totally 434 genes were identified to be differentially expressed genes (DEGs), and annotation and enrichment analysis suggested that multiple genes associated with regulation of membrane lipid composition, filamentous growth and arginine metabolism were differentially expressed. The increased specific activity of arginase and intracellular ammonia concentration of P. kudriavzevii cultured at pH 2.0 further implied potential roles of arginine in response to extreme low-pH conditions. Extracellular supplementation of 5 mM arginine resulted in increased pHi and cell growth at pH 2.0, meanwhile the cell aggregation was partially suppressed. Additionally, overexpression of ARG J involving in arginine synthesis can also enhance the cell growth and reduce the aggregation effect. These results suggested that increasing arginine flux might be an alternative approach in the developing of P. kudriavzevii as a platform host for production of organic acids under low-pH conditions.

Comprehensive analysis of Ogura cytoplasmic male sterility-related genes in turnip (Brassica rapa ssp. rapifera) using RNA sequencing analysis and bioinformatics.

Ogura-type cytoplasmic male sterility (Ogura-CMS) has been widely used in the hybrid breeding industry for cruciferous vegetables. Turnip (Brassica rapa ssp. rapifera) is one of the most important local cruciferous vegetables in China, cultivated for its fleshy root as a flat disc. Here, morphological characteristics of an Ogura-CMS line 'BY10-2A' and its maintainer fertile (MF) line 'BY10-2B' of turnip were investigated. Ogura-CMS turnip showed a reduction in the size of the fleshy root, and had distinct defects in microspore development and tapetum degeneration during the transition from microspore mother cells to tetrads. Defective microspore production and premature tapetum degeneration during microgametogenesis resulted in short filaments and withered white anthers, leading to complete male sterility of the Ogura-CMS line. Additionally, the mechanism regulating Ogura-CMS in turnip was investigated using inflorescence transcriptome analyses of the Ogura-CMS and MF lines. The de novo assembly resulted in a total of 84,132 unigenes. Among them, 5,117 differentially expressed genes (DEGs) were identified, including 1,339 up- and 3,778 down-regulated genes in the Ogura-CMS line compared to the MF line. A number of functionally known members involved in anther development and microspore formation were addressed in our DEG pool, particularly genes regulating tapetum programmed cell death (PCD), and associated with pollen wall formation. Additionally, 185 novel genes were proposed to function in male organ development based on GO analyses, of which 26 DEGs were genotype-specifically expressed. Our research provides a comprehensive foundation for understanding anther development and the CMS mechanism in turnip.

Comparative transcriptome analysis and ChIP-sequencing reveals stage-specific gene expression and regulation profiles associated with pollen wall formation in Brassica rapa.

BACKGROUND: Genic male sterility (GMS) line is an important approach to utilize heterosis in Brassica rapa, one of the most widely cultivated vegetable crops in Northeast Asia. However, the molecular genetic mechanisms of GMS remain to be largely unknown. RESULTS: Detailed phenotypic observation of 'Bcajh97-01A/B', a B. rapa genic male sterile AB line in this study revealed that the aberrant meiotic cytokinesis and premature tapetal programmed cell death occurring in the sterile line ultimately resulted in microspore degeneration and pollen wall defect. Further gene expression profile of the sterile and fertile floral buds of 'Bcajh97-01A/B' at five typical developmental stages during pollen development supported the result of phenotypic observation and identified stage-specific genes associated with the main events associated with pollen wall development, including tapetum development or functioning, callose metabolism, pollen exine formation and cell wall modification. Additionally, by using ChIP-sequencing, the genomic and gene-level distribution of trimethylated histone H3 lysine 4 (H3K4) and H3K27 were mapped on the fertile floral buds, and a great deal of pollen development-associated genes that were covalently modified by H3K4me3 and H3K27me3 were identified. CONCLUSIONS: Our study provids a deeper understanding into the gene expression and regulation network during pollen development and pollen wall formation in B. rapa, and enabled the identification of a set of candidate genes for further functional annotation.

Systematic identification of long non-coding RNAs during pollen development and fertilization in Brassica rapa.

The importance of long non-coding RNAs (lncRNAs) in plant development has been established, but a systematic analysis of lncRNAs expressed during pollen development and fertilization has been elusive. We performed a time series of RNA-seq experiments at five developmental stages during pollen development and three different time points after pollination in Brassica rapa and identified 12 051 putative lncRNAs. A comprehensive view of dynamic lncRNA expression networks underpinning pollen development and fertilization was provided. B. rapa lncRNAs share many common characteristics of lncRNAs: relatively short length, low expression but specific in narrow time windows, and low evolutionary conservation. Gene modules and key lncRNAs regulating reproductive development such as exine formation were uncovered. Forty-seven cis-acting lncRNAs and 451 trans-acting lncRNAs were revealed to be highly coexpressed with their target protein-coding genes. Of particular importance are the discoveries of 14 lncRNAs that were highly coexpressed with 10 function-known pollen-associated coding genes. Fifteen lncRNAs were predicted as endogenous target mimics for 13 miRNAs, and two lncRNAs were proved to be functional target mimics for miR160 after experimental verification and shown to function in pollen development. Our study provides the systematic identification of lncRNAs during pollen development and fertilization in B. rapa and forms the foundation for future genetic, genomic, and evolutionary studies.

The distinct functions of two classical arabinogalactan proteins BcMF8 and BcMF18 during pollen wall development in Brassica campestris.

Arabinogalactan proteins (AGPs) are extensively glycosylated hydroxyproline-rich glycoproteins ubiquitous in all plant tissues and cells. AtAGP6 and AtAGP11, the only two functionally known pollen-specific classical AGP encoding genes in Arabidopsis, are reported to have redundant functions in microspore development. BcMF18 and BcMF8 isolated from Brassica campestris are the orthologues of AtAGP6 and AtAGP11, respectively. In contrast to the functional redundancy of AtAGP6 and AtAGP11, single-gene disruption of BcMF8 led to deformed pollen grains with abnormal intine development and ectopic aperture formation in B. campestris. Here, we further explored the action of BcMF18 and its relationship with BcMF8. BcMF18 was specifically expressed in pollen during the late stages of microspore development. Antisense RNA transgenic lines with BcMF18 reduction resulted in aberrant pollen grains with abnormal cellulose distribution, lacking intine, cytoplasm and nuclei. Transgenic plants with repressive expression of both BcMF8 and BcMF18 showed a hybrid phenotype, expressing a mixture of the phenotypes of the single gene knockdown plant lines. In addition, we identified functional diversity between BcMF18/BcMF8 and AtAGP6/AtAGP11, mainly reflected by the specific contribution of BcMF18 and BcMF8 to pollen wall formation. These results suggest that, unlike the orthologous genes AtAGP6 and AtAGP11 in Arabidopsis, BcMF18 and BcMF8 are both integral to pollen biogenesis in B. campestris, acting through independent pathways during microspore development.

Characterization of BcMF23a and BcMF23b, two putative pectin methylesterase genes related to pollen development in Brassica campestris ssp. chinensis.

Two homologous genes, Brassica campestris Male Fertility 23a (BcMF23a) and Brassica campestris Male Fertility 23b (BcMF23b), encoding putative pectin methylesterases (PMEs) were isolated from Brassica campestris ssp. chinensis (syn. Brassica rapa ssp. chinensis). These two genes sharing high sequence identity with each other were highly expressed in the fertile flower buds but silenced in the sterile ones of genic male sterile line system ('Bcajh97-01A/B'). Results of RT-PCR and in situ hybridization suggested that BcMF23a and BcMF23b were pollen-expressed genes, whose transcripts were first detected at the binucleate pollen and maintained throughout to the mature pollen grains. Western blot indicated that both of the putative BcMF23a and BcMF23b proteins are approximately 40 kDa, which exhibited extracellular localization revealed by transient expression analysis in the onion epidermal cells. The promoter of BcMF23a was active specifically in pollen during the late pollen developmental stages, while, in addition to the pollen, BcMF23b promoter drove an extra gene expression in the valve margins, abscission layer at the base of the first true leaves, taproot and lateral roots in seedlings.

Intratumoral heterogeneity: Role of differentiation in a potentially lethal phenotype of testicular cancer.

BACKGROUND: Intratumoral heterogeneity presents a major obstacle to the widespread implementation of precision medicine. The authors assessed the origin of intratumoral heterogeneity in nonseminomatous germ cell tumor of the testis (NSGCT) and identified distinct tumor subtypes and a potentially lethal phenotype. METHODS: In this retrospective study, all consecutive patients who had been diagnosed with an NSGCT between January 2000 and December 2010 were evaluated. The histologic makeup of primary tumors and the clinical course of disease were determined for each patient. A Fine and Gray proportional hazards regression analysis was used to determine the prognostic risk factors, and the Gray test was used to detect differences in the cumulative incidence of cancer death. In a separate prospective study, next-generation sequencing was performed on tumor samples from 9 patients to identify any actionable mutations. RESULTS: Six hundred fifteen patients were included in this study. Multivariate analysis revealed that the presence of yolk sac tumor in the primary tumor (P = .0003) was associated with an unfavorable prognosis. NSGCT could be divided into 5 subgroups. Patients in the yolk sac-seminoma subgroup had the poorest clinical outcome (P = .0015). These tumors tended to undergo somatic transformation (P < .0001). Among the 9 NSGCTs that had a yolk sac tumor phenotype, no consistent gene mutation was detected. CONCLUSIONS: The current data suggest that intratumoral heterogeneity is caused in part by differentiation of pluripotent progenitor cells. Integrated or multimodal therapy may be effective at addressing intratumoral heterogeneity and treating distinct subtypes as well as a potentially lethal phenotype of NSGCT. Cancer 2016;122:1836-43. © 2016 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Maintenance Therapy Containing Metformin and/or Zyflamend for Advanced Prostate Cancer: A Case Series.

Metformin is derived from galegine, a natural ingredient, and recent studies have suggested that metformin could enhance the antitumor effects of hormone ablative therapy or chemotherapy and reduce prostate cancer-specific mortality. Zyflamend is a combination of herbal extracts that reduces inflammation and comprises turmeric, holy basil, green tea, oregano, ginger, rosemary, Chinese goldthread, hu zhang, barberry, and basil skullcap. We propose a maintenance regimen with metformin and/or Zyflamend that targets cancer stem cells and the tumor microenvironment to keep the cancer dormant and prevent it from activation from dormancy. Herein, we report the clinical course of four patients who experienced a clinical response after treatment with metformin and/or Zyflamend.

BcMF8, a putative arabinogalactan protein-encoding gene, contributes to pollen wall development, aperture formation and pollen tube growth in Brassica campestris.

BACKGROUND AND AIMS: The arabinogalactan protein (AGP) gene family is involved in plant reproduction. However, little is known about the function of individual AGP genes in pollen development and pollen tube growth. In this study, Brassica campestris male fertility 8 (BcMF8), a putative AGP-encoding gene previously found to be pollen specific in Chinese cabbage (B. campestris ssp. chinensis), was investigated. METHODS: Real-time reverse transcription-PCR and in situ hybridization were used to analyse the expression pattern of BcMF8 in pistils. Prokaryotic expression and western blots were used to ensure that BcMF8 could encode a protein. Antisense RNA technology was applied to silence gene expression, and morphological and cytological approaches (e.g. scanning electron microscopy and transmission electron microscopy) were used to reveal abnormal phenotypes caused by gene silencing. KEY RESULTS: The BcMF8 gene encoded a putative AGP protein that was located in the cell wall, and was expressed in pollen grains and pollen tubes. The functional interruption of BcMF8 by antisense RNA technology resulted in slipper-shaped and bilaterally sunken pollen with abnormal intine development and aperture formation. The inhibition of BcMF8 led to a decrease in the percentage of in vitro pollen germination. In pollen that did germinate, the pollen tubes were unstable, abnormally shaped and burst more frequently relative to controls, which corresponded to an in vivo arrest of pollen germination at the stigma surface and retarded pollen tube growth in the stylar transmitting tissues. CONCLUSIONS: The phenotypic defects of antisense BcMF8 RNA lines (bcmf8) suggest a crucial function of BcMF8 in modulating the physical nature of the pollen wall and in helping in maintaining the integrity of the pollen tube wall matrix.

Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates.

BACKGROUND: Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. PRINCIPAL FINDING: In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. CONCLUSION: These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.

Folotyn (pralatrexate injection) for the treatment of patients with relapsed or refractory peripheral T-cell lymphoma: U.S. Food and Drug Administration drug approval summary.

PURPOSE: On September 24, 2009, the U.S. Food and Drug Administration granted accelerated approval for Folotyn (pralatrexate injection, Allos Therapeutics, Inc.) as a single agent for the treatment of patients with relapsed or refractory peripheral T-cell lymphoma (PTCL); it is the first drug approved for this indication. EXPERIMENTAL DESIGN: This review was based on study PDX-008, a phase II, single-arm, nonrandomized, open-label, international, multicenter trial, designed to evaluate the safety and efficacy of pralatrexate when administered concurrently with vitamin B(12) and folic acid supplementation in patients with relapsed or refractory PTCL. RESULTS: The overall response rate was 27% in 109 evaluable patients [95% confidence interval (CI), 19-36%]. Twelve percent of 109 evaluable patients (95% CI, 7-20%)] had a response duration of ≥14 weeks. Six of these 13 patients achieved a complete response, and one patient had complete response unconfirmed. The most common grade 3 and 4 toxicities were thrombocytopenia, mucositis, and neutropenia. CONCLUSION: This accelerated approval was based on a response rate that is reasonably likely to predict clinical benefit in this heavily pretreated patient population with this rare disease. The applicant has committed to conducting postmarketing clinical trials to assess clinical benefit. The recommended starting dose of pralatrexate in patients with relapsed or refractory PTCL is 30 mg/m(2) via intravenous push over 3 to 5 min weekly for 6 weeks followed by a one-week rest (one cycle). Intramuscular injection of 1 mg vitamin B(12) should be administered every 8 to 10 weeks along with 1.0 mg folic acid given orally once a day.

Stanniocalcin 1 and ovarian tumorigenesis.

BACKGROUND: Stanniocalcin 1 (STC1) is a secreted glycoprotein hormone. High expression of STC1 has been associated with several cancers including ovarian cancer, but its role in the development of ovarian cancer is not clear. METHODS: We used five human ovarian epithelial cancer cell lines (OVCA420, OVCA432, OVCA433, SKOV3, and HEY), immortalized human ovarian surface epithelial cells (T29 and T80), ovarian cancer tissues from 342 patients, serum from 73 ovarian cancer patients and from58 control subjects, and 116 mice, with six or eight per group. Protein expression was assessed. Cells overexpressing STC1 protein were generated by ectopic expression of human STC1 cDNA. STC1 expression was silenced by using small interfering RNA against STC1. Cell proliferation, migration, colony formation, and apoptosis were assessed. Xenograft tumor growth in mice was studied. Neutralizing anti-STC1 antibody was used to inhibit STC1 function. All statistical tests were two-sided. RESULTS: STC1 protein expression was higher in all human ovarian cancer cell lines examined than in immortalized human ovarian epithelial cell lines, higher in ovarian cancer tissue than in normal ovarian tissue (P < .001), and higher in serum from ovarian cancer patients than from control subjects (P = .021). Ovarian cancer cells with STC1 overexpression, compared with corresponding control cells, had increased cell proliferation, migration, and colony formation in cell culture and increased growth of xenograft tumors in mice. These activities in normal or malignant ovarian cells with STC1 overexpression, compared with control cells, were also accompanied by increased expression of cell cycle regulatory proteins and antiapoptotic proteins but decreased cleavage of several caspases. Within 24 hours of treatment, apoptosis in cultures of HEY ovarian cancer cells treated with neutralizing anti-STC1 monoclonal antibody was higher (17.3% apoptotic cells) than that in cultures treated with mouse IgG control cells (4.4%) (12.9% difference, 95% confidence interval = 11.6% to 14.2%). CONCLUSIONS: STC1 protein may be involved in ovarian tumorigenesis.

Reversal of optic disc cupping after trabeculotomy in primary congenital glaucoma.

BACKGROUND: The phenomenon of reversal of glaucomatous cupping of the optic disc following lowering of the intraocular pressure (IOP) was originally recognized in infants. We evaluated the change in optic disc cupping with normalization of the IOP after trabeculotomy in primary congenital glaucoma and assessed the factors associated with reversal of cupping. METHODS: We reviewed the records of 17 patients (24 eyes) who underwent trabeculotomy between July 1993 and June 1999 and who had been followed for at least 1 year. Surgical success was defined as IOP less than 22 mm Hg without anti-glaucoma medication, stable or reduced optic disc cupping, and lack of further corneal enlargement disproportionate to normal growth. Patients who required more than one surgical procedure to control the IOP and those with cloudy media that precluded documentation of cupping were excluded from analysis. Optic disc cupping was assessed independently before and after surgery by two clinicians. The cup:disc ratio was estimated as the percentage of surface area of the optic disc occupied by cupping in the vertical axis. We accepted a difference of 0.1 or 0.2 in the cup:disc ratio between the two observers in each subjective assessment and used the mean value of the two results for data analysis. If the difference was more than 0.2, the eye was excluded from further study. RESULTS: Of the 17 patients 4 were excluded: 2 because they received antiglaucoma medication to control the IOP postoperatively, 1 because he underwent more than one surgical procedure to control the IOP during follow-up, and 1 owing to disagreement in the assessment of the cup:disc ratio between the two observers. Eighteen eyes of 13 patients were thus included in the analysis. Twelve eyes were from boys and six, from girls. The patients were followed for a mean of 43.2 (standard deviation [SD] 30.4) months (range 12 to 90 months). The mean cup:disc ratios pre- and postoperatively were 0.74 (SD 0.20) and 0.60 (SD 0.21) respectively (p = 0.003). Of the 18 eyes 11 (61.1%) had documented reduction in optic disc cupping. The mean time to stabilization of cupping reversal was 4.8 (SD 2.8) months (range 2 to 12 months). In multivariable analysis the age of the patient at surgery was the only variable significantly associated with reversal of cupping (p = 0.027). The mean age at surgery for the 11 eyes with reduction in cupping was 6.9 (range 3 to 15) months, compared with 23.4 (range 12 to 42) months for the 7 eyes with unchanged cupping. The mean preoperative cup:disc ratio was 0.67 (SD 0.17) in the former group and 0.83 (SD 0.17) in the latter group. Six of the seven eyes with unchanged cupping had advanced cupping. INTERPRETATION: Optic disc cupping can be reversed at an early stage of primary congenital glaucoma following successful reduction of IOP. Younger age at surgery was associated with reversal of cupping.

Enhanced suppression of prostate tumor growth by combining C-CAM1 gene therapy and angiogenesis inhibitor TNP-470.

We have previously shown that C-CAM1-based gene therapy effectively suppressed prostate tumor growth in nude mice xenograft models. In this study, we examined the effects of combining C-CAM1-based therapy and TNP-470, a potent angiogenesis inhibitor, on prostate cancer in a xenografted tumor model. The direct cytotoxic effects of Ad-C-CAM1 (recombinant adenovirus containing C-CAM1 cDNA) and TNP-470 on DU145 cells in vitro were determined by microculture tetrazolium assay. The in vivo antitumor effects of either agent alone were studied in a DU145 xenografted tumor model. Cells were infected with Ad-C-CAM1 or the control virus at multiplicities of infection (m.o.i.) of 5 or 10 and then inoculated onto nude mice 48 h later. TNP-470 (0, 17 or 35 mg/kg) was given 15, 17 and 19 days after inoculation. Combined treatments in vivo were carried out to determine whether there were synergistic antitumor effects. Both Ad-C-CAM1 and the control virus were minimally toxic to DU145 in vitro. There was evident dose-dependent suppression of xenografted tumor growth by either Ad-C-CAM1 or TNP-470. By the median-effect analysis, combination of the two agents generated strong synergistic antitumor effects as shown by marked tumor suppression as compared to either treatment alone. The novel strategy may have clinical implications for the treatment of prostate cancer.