Modification in the ascorbate-glutathione cycle leads to a better acclimation to high light in the rose Bengal resistant mutant of Nannochloropsis oceanica.

Understanding how to adapt outdoor cultures of Nannochloropsis oceanica to high light (HL) is vital for boosting productivity. The N. oceanica RB2 mutant, obtained via ethyl methanesulfonate mutagenesis, was chosen for its tolerance to Rose Bengal (RB), a singlet oxygen (1O2) generator. Compared to the wild type (WT), the RB2 mutant showed higher resilience to excess light conditions. Analyzing the ascorbate-glutathione cycle (AGC), involving ascorbate peroxidases (APX, EC 1.11.1.11), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (GR, EC 1.8.1.7), in the RB2 mutant under HL stress provided valuable insights. At 250 µmol photon m-2 s-1 (HL), the WT strain displayed superoxide anion radicals (O2▪-) and hydrogen peroxide (H2O2) accumulation, increased lipid peroxidation, and cell death compared to normal light (NL) conditions (50 µmol photon m-2 s-1). The RB2 mutant didn't accumulate O2▪- and H2O2 after HL exposure, and exhibited increased APX, DHAR, and GR activities and transcript levels compared to WT and remained consistent after HL treatment. Although the RB2 mutant had a smaller ascorbate (AsA) pool than the WT, its ability to regenerate dehydroascorbate (DHA) increased post HL exposure, indicated by a higher AsA/DHA ratio. Additionally, under HL conditions, the RB2 mutant displayed an improved glutathione (GSH) regeneration rate (GSH/GSSG ratio) without changing the GSH pool size. Remarkably, H2O2 or menadione (a O2▪- donor) treatment induced cell death in the WT strain but not in the RB2 mutant. These findings emphasize the essential role of AGC in the RB2 mutant of Nannochloropsis in handling photo-oxidative stress.

An efficient grafting method for phytoplasma transmission in Catharanthus roseus.

BACKGROUND: Phytoplasmas are parasitic plant pathogens that reside intracellularly within the sieve tube cells. Phytoplasmas induce various symptoms, including floral virescence, phyllody, leaf yellowing, and witches'-broom. Currently, it is challenging to culture phytoplasma in vitro. In the laboratory, phytoplasmas are generally maintained in alternative host plants, such as Catharanthus roseus. Grafting is used to transmit phytoplasmas among the alternative hosts. During the experiment, scions from infected plants are grafted onto healthy plants using a side grafting method. However, the practice has certain limitations, including its inability to be applied to small plants and its irregular disease incidence. RESULTS: Here, we demonstrate a new approach, penetration grafting, to overcome the limitations of side grafting. This grafting method allows phytoplasma to be efficiently and uniformly transmitted into the inoculated plants. No significant difference was observed in phytoplasma accumulation between both grafting techniques. However, penetration grafting allows rapid symptom development, saving waiting time and reducing space usage. CONCLUSIONS: This study provides a reliable and stable method for experiments that require grafting transmission.

Streptococcus gordonii empyema: A case report and literature review.

Empyema is often caused by Streptococcus anginous species, followed by Streptococcus pneumoniae. The organism Streptococcus gordonii belongs to the Streptococcus mitis group, which rarely causes empyema. We report the case of a 59-year-old man who presented with exertional dyspnea and chest pain on the right side. The image obtained showed effusion on the right side. Streptococcus gordonii was recovered from purulent pleural effusion culture. The patient underwent video-assisted thoracoscopic surgery with decortication, pneumolysis and received antibiotics for 13 days. A total of seven cases were analyzed after combining six cases in the literature and our presented case. The majority of Streptococcus gordonii empyema patients were male (six patients, 86%) and empyema on the right side (five patients, 71%). Common risk factors included poor dental hygiene or recent dental procedure (three patients, 43%), diabetes mellitus (three patients, 43%), and smoking (three patients, 43%). Only a few cases developed empyema-related complications, including bacteremia (one patient, 14%) and spleen abscesses (one patient, 14%). Most patients underwent chest tube insertion (seven patients, 100%) and survived without recurrent empyema (six patients, 86%).

Transcriptomic Analysis Suggests Auxin Regulation in Dorsal-Ventral Petal Asymmetry of Wild Progenitor Sinningia speciosa.

The establishment of dorsal-ventral (DV) petal asymmetry is accompanied by differential growth of DV petal size, shape, and color differences, which enhance ornamental values. Genes involved in flower symmetry in Sinningia speciosa have been identified as CYCLOIDEA (SsCYC), but which gene regulatory network (GRN) is associated with SsCYC to establish DV petal asymmetry is still unknown. To uncover the GRN of DV petal asymmetry, we identified 630 DV differentially expressed genes (DV-DEGs) from the RNA-Seq of dorsal and ventral petals in the wild progenitor, S. speciosa 'ES'. Validated by qRT-PCR, genes in the auxin signaling transduction pathway, SsCYC, and a major regulator of anthocyanin biosynthesis were upregulated in dorsal petals. These genes correlated with a higher endogenous auxin level in dorsal petals, with longer tube length growth through cell expansion and a purple dorsal color. Over-expression of SsCYC in Nicotiana reduced petal size by regulating cell growth, suggesting that SsCYC also controls cell expansion. This suggests that auxin and SsCYC both regulate DV petal asymmetry. Transiently over-expressed SsCYC, however, could not activate most major auxin signaling genes, suggesting that SsCYC may not trigger auxin regulation. Whether auxin can activate SsCYC or whether they act independently to regulate DV petal asymmetry remains to be explored in the future.

The Photosynthetic Bacterium Rhodopseudomonas palustris Strain PS3 Exerts Plant Growth-Promoting Effects by Stimulating Nitrogen Uptake and Elevating Auxin Levels in Expanding Leaves.

Rhodopseudomonas palustris strain PS3, a phototrophic bacterium, was originally isolated from a paddy field located in Taipei city, Taiwan, and showed positive effects on the growth of leafy vegetables. The aim of this study was to clarify the mechanism of the beneficial effects exerted by PS3 on plants. An ineffective R. palustris strain, YSC3, isolated from a paddy field located in Yilan County, was used as the negative control for comparative analyses. We cultivated non-heading Chinese cabbage (Brassica rapa var. chinensis) in 1/2 strength Hoagland hydroponic solution, in which nitrate is the main nitrogen source. We evaluated various plant physiological responses to inoculation with different bacterial inoculants. The N use efficiency (NUE) of PS3-inoculated plants was dramatically higher than that of YSC3-inoculated plants. The nitrate uptake efficiency (NUpE) was significantly elevated in plants treated with PS3; however, no excess nitrate accumulation was observed in leaves. We also noticed that the endogenous indole-3-acetic acid (IAA) levels as well as the cell division rate in the leaves of PS3-inoculated plants were significantly higher than those in the leaves of YSC3-inoculated plants. We examined the bacterial transcription of some genes during root colonization, and found that the expression level of IAA synthesis related gene MAO was almost the same between these two strains. It suggests that the elevated endogenous IAA in the PS3-inoculated plants was not directly derived from the exogenous IAA produced by this bacterium. Taken together, we deduced that PS3 inoculation could promote plant growth by enhancing nitrate uptake and stimulating the accumulation of endogenous auxin in young expanding leaves to increase the proliferation of leaf cells during leaf development.

Silencing of PhLA, a CIN-TCP gene, causes defected petal conical epidermal cell formation and results in reflexed corolla lobes in petunia.

BACKGROUND: TCP-domain proteins, plant specific transcription factors, play important roles in various developmental processes. CIN-TCPs control leaf curvature in simple leaf species while regulate leaf complexity in compound leaf species. However, the knowledge was largely based on findings in few model species. To extend our knowledge on this group of proteins in Solanaceae species, we identified a CIN-TCP gene from petunia, and studied its functions using virus-induced gene silencing (VIGS). RESULTS: Consistently, silencing of CIN-TCPs increases complexity of tomato leaves, and enhances leaf curvature in Nicotiana benthamiana. However, in petunia (Petunia hybrida), silencing of petunia LA, a CIN-TCP, through VIGS did not obviously affect leaf shape. The silencing, however, enhanced petal curvature. The event was associated with petal expansion at the distal portion where epidermal cell size along the midribs was also increased. The enlarged epidermal cells became flattened. Although shapes of PhLA-silenced flowers largely resemble phmyb1 mutant phenotype, PhMYB1 expression was not affected when PhLA was specifically silenced. Therefore, both PhLA and PhMYB1 are required to regulate flower morphology. In corolla, PhLA and miR319 deferentially express in different regions with strong expressions in limb and tube region respectively. CONCLUSIONS: In conclusion, unlike LA-like genes in tomato and N. benthamiana, PhLA plays a more defined role in flower morphogenesis, including petal curvature and epidermal cell differentiation.

Silencing of CrNPR1 and CrNPR3 Alters Plant Susceptibility to Periwinkle Leaf Yellowing Phytoplasma.

Phytoplasmas are prokaryotic plant pathogens that cause considerable loss in many economically important crops, and an increasing number of phytoplasma diseases are being reported on new hosts. Knowledge of plant defense mechanisms against such pathogens should help to improve strategies for controlling these diseases. Salicylic acid (SA)-mediated defense may play an important role in defense against phytoplasmas. Here, we report that SA accumulated in Madagascar periwinkle (Catharanthus roseus) infected with periwinkle leaf yellowing (PLY) phytoplasma. CrPR1a expression was induced in both symptomatic and non-symptomatic tissues of plants exhibiting PLY. NPR1 plays a central role in SA signaling, and two NPR1 homologs, CrNPR1 and CrNPR3, were identified from a periwinkle transcriptome database. Similar to CrPR1a, CrNPR1 expression was also induced in both symptomatic and non-symptomatic tissues of plants exhibiting PLY. Silencing of CrNPR1, but not CrNPR3, significantly repressed CrPR1a induction in Tobacco rattle virus-infected periwinkle plants. In addition, symptoms of PLY progressed fastest in CrNPR1-silenced plants and slowest in CrNPR3-silenced plants. Consistently, expression of CrNPR1, but not CrNPR3, was induced by phytoplasma infection as well as SA treatment. This study highlights the importance of NPR1- and SA-mediated defense against phytoplasma in periwinkle and offers insight into plant-phytoplasma interactions to improve disease control strategies.

Transcriptome profiling reveals regulatory mechanisms underlying corolla senescence in petunia.

The genetic regulatory mechanisms that govern natural corolla senescence in petunia are not well understood. To identify key genes and pathways that regulate the process, we performed a transcriptome analysis in petunia corolla at four developmental stages, including corolla fully opening without anther dehiscence (D0), corolla expansion, 2 days after anthesis (D2), corolla with initial signs of senescence (D4), and wilting corolla (D7). We identified large numbers of differentially expressed genes (DEGs), ranging from 4626 between the transition from D0 and D2, 1116 between D2 and D4, a transition to the onset of flower senescence, and 327 between D4 and D7, a developmental stage representing flower senescence. KEGG analysis showed that the auxin- and ethylene-related hormone biosynthesis and signaling transduction pathways were significantly activated during the flower development and highly upregulated at onset of flower senescence. Ethylene emission was detected at the D2 to D4 transition, followed by a large eruption at the D4 to D7 transition. Furthermore, large numbers of transcription factors (TFs) were activated over the course of senescence. Functional analysis by virus-induced gene silencing (VIGS) experiments demonstrated that inhibition of the expression of TFs, such as ethylene-related ERF, auxin-related ARF, bHLH, HB, and MADS-box, significantly extended or shortened flower longevity. Our data suggest that hormonal interaction between auxin and ethylene may play critical regulatory roles in the onset of natural corolla senescence in petunia.

Active extracellular substances of Bacillus thuringiensis ITRI-G1 induce microalgae self-disruption for microalgal biofuel.

Microalgal cultures are a clean and sustainable means to use solar energy for CO2 fixation and fuel production. Microalgae grow efficiently and are rich in oil, but recovering that oil is typically expensive and consumes much energy. Therefore, effective and low-cost techniques for microalgal disruption and oil or lipid extraction are required by the algal biofuel industry. This study introduces a novel technique that uses active extracellular substances to induce microalgal cell disruption. A bacterium indigenous to Taiwan, Bacillus thuringiensis, was used to produce the active extracellular substances, which were volatile compounds with high thermal stability. Approximately 74% of fresh microalgal cells were disrupted after a 12-h treatment with the active extracellular substances. Algal lipid extraction efficiency was improved and the oil extraction time was decreased by approximately 37.5% compared with the control treatment. The substances effectively disrupted fresh microalgal cells but not dehydrated microalgal cells. An analysis of microalgal DNA from fresh cells after disruption treatment demonstrated typical DNA laddering, indicating that disruption may have resulted from programmed cell death. This study revealed that biological treatments are environmentally friendly methods for increasing microalgal lipid extraction efficiency, and introduced a microalgal cell self-disruption mechanism.

Complex regional pain syndrome following the Nuss procedure for severe pectus excavatum.

Complex regional pain syndrome (CRPS) is not an uncommon complication after surgery, but has never been reported after the Nuss procedure for repairing pectus excavatum. A 22-year-old man with pectus excavatum had type I CRPS that developed 2 weeks after the Nuss procedure. He complained of persistent pain, hyperalgesia, weakness, edema, and color and temperature changes on right upper extremity. Following intensive rehabilitation, the degree of pain, weakness and edema were ameliorated. He recovered 6 months after surgery and the pectus bars were removed uneventfully 3 years after the repair.

Genotyping of human neutrophil antigens (HNA) from whole genome sequencing data.

BACKGROUND: Neutrophil antigens are involved in a variety of clinical conditions including transfusion-related acute lung injury (TRALI) and other transfusion-related diseases. Recently, there are five characterized groups of human neutrophil antigen (HNA) systems, the HNA1 to 5. Characterization of all neutrophil antigens from whole genome sequencing (WGS) data may be accomplished for revealing complete genotyping formats of neutrophil antigens collectively at genome level with molecular variations which may respectively be revealed with available genotyping techniques for neutrophil antigens conventionally. RESULTS: We developed a computing method for the genotyping of human neutrophil antigens. Six samples from two families, available from the 1000 Genomes projects, were used for a HNA typing test. There are 500 ~ 3000 reads per sample filtered from the adopted human WGS datasets in order for identifying single nucleotide polymorphisms (SNPs) of neutrophil antigens. The visualization of read alignment shows that the yield reads from WGS dataset are enough to cover all of the SNP loci for the antigen system: HNA1, HNA3, HNA4 and HNA5. Consequently, our implemented Bioinformatics tool successfully revealed HNA types on all of the six samples including sequence-based typing (SBT) as well as PCR sequence-specific oligonucleotide probes (SSOP), PCR sequence-specific primers (SSP) and PCR restriction fragment length polymorphism (RFLP) along with parentage possibility. CONCLUSIONS: The next-generation sequencing technology strives to deliver affordable and non-biased sequencing results, hence the complete genotyping formats of HNA may be reported collectively from mining the output data of WGS. The study shows the feasibility of HNA genotyping through new WGS technologies. Our proposed algorithmic methodology is implemented in a HNATyping software package with user's guide available to the public at http://sourceforge.net/projects/hnatyping/.

EBARDenovo: highly accurate de novo assembly of RNA-Seq with efficient chimera-detection.

MOTIVATION: High-accuracy de novo assembly of the short sequencing reads from RNA-Seq technology is very challenging. We introduce a de novo assembly algorithm, EBARDenovo, which stands for Extension, Bridging And Repeat-sensing Denovo. This algorithm uses an efficient chimera-detection function to abrogate the effect of aberrant chimeric reads in RNA-Seq data. RESULTS: EBARDenovo resolves the complications of RNA-Seq assembly arising from sequencing errors, repetitive sequences and aberrant chimeric amplicons. In a series of assembly experiments, our algorithm is the most accurate among the examined programs, including de Bruijn graph assemblers, Trinity and Oases. AVAILABILITY AND IMPLEMENTATION: EBARDenovo is available at http://ebardenovo.sourceforge.net/. This software package (with patent pending) is free of charge for academic use only. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The NPR1 ortholog PhaNPR1 is required for the induction of PhaPR1 in Phalaenopsis aphrodite.

BACKGROUND: Systematic acquired resistance (SAR) is an effective broad-spectrum defense mechanism that confers long-lasting protection against biotrophic pathogens trough defense related salicylic acid (SA) signaling. Gene(s) involved in SAR have been extensively studied in dicot plants; however, remains largely unresolved in monocot plants. NPR1, an evolutionary conserved gene, plays a central role in SAR, and PR-1 is widely used as a marker for effective SA signaling. RESULTS: We identified NPR1 and PR-1 homologous genes, PhaNPR1 and PhaPR1, from an economically important orchid, Phalaenopsis aphrodite, and characterized their roles in SA signaling and Cymbidium mosaic virus (CymMV) resistance. A phylogenetic analysis of NPR1 homologs showed that these genes appear to have evolved before angiospermy. Similar to Arabidopsis NPR1, PhaNPR1 was only moderately induced upon SA treatment and CymMV infection. Although PhaPR1 shows only 36% identity with AtPR1, its promoter shared conserved elements with those of other PR-1 genes, and it was induced upon SA treatment and CymMV infection. After CymMV infection, silencing on PhaNPR1 also reduced PhaPR1 expression; however, CymMV accumulation was not affected. CONCLUSIONS: In conclusion, after virus infection, PhaNPR1 is required for PhaPR1 induction, but plays little role in defense against CymMV.

Quantitative assessment of mitochondrial DNA copies from whole genome sequencing.

BACKGROUND: Mitochondrial dysfunction is associated with various aging diseases. The copy number of mtDNA in human cells may therefore be a potential biomarker for diagnostics of aging. Here we propose a new computational method for the accurate assessment of mtDNA copies from whole genome sequencing data. RESULTS: Two families of the human whole genome sequencing datasets from the HapMap and the 1000 Genomes projects were used for the accurate counting of mitochondrial DNA copy numbers. The results revealed the parental mitochondrial DNA copy numbers are significantly lower than that of their children in these samples. There are 8%~21% more copies of mtDNA in samples from the children than from their parents. The experiment demonstrated the possible correlations between the quantity of mitochondrial DNA and aging-related diseases. CONCLUSIONS: Since the next-generation sequencing technology strives to deliver affordable and non-biased sequencing results, accurate assessment of mtDNA copy numbers can be achieved effectively from the output of whole genome sequencing. We implemented the method as a software package MitoCounter with the source code and user's guide available to the public at http://sourceforge.net/projects/mitocounter/.

Phytoplasma-induced floral abnormalities in Catharanthus roseus are associated with phytoplasma accumulation and transcript repression of floral organ identity genes.

Floral symptoms caused by phytoplasma largely resemble floral reversion in other plants. Periwinkle leaf yellowing (PLY) phytoplasma and peanut witches'-broom (PnWB) phytoplasma caused different degrees of floral abnormalities on infected periwinkle plants. The PLY phytoplasma-infected plants exhibited floral discoloration, virescence, small flowers, and only occasionally full floral reversion. In contrast, PnWB phytoplasma frequently induced complete floral reversion and resulted in a witches'-broom symptom from the floral reversion. Although different degrees of floral symptoms were induced by these two phytoplasmas, the morphological disorders were similar to those of other plants carrying SEPALLATA mutations or gene silencing. Here, we compared expression levels of organ-identity-related genes and pigmentation genes during floral symptom development. Accumulation of phytoplasmas in malformed flowers and their closely surrounding leaves was also compared. In infected plants, transcript abundance of all examined organ identity genes and pigmentation genes was suppressed. Indeed, CrSEP3, a SEPALLALA3 ortholog, showed the greatest suppression among genes examined. Of the pigmentation genes, transcript reduction of chalcone synthase was most highly correlated with the loss in floral pigmentation. Floral symptom severities were associated with the accumulation of either phytoplasmas. Interestingly, both phytoplasmas accumulated to higher levels in malformed flowers than in their surrounding leaves. Many plant pathogens manipulate host plant development to their advantage. It is intriguing to see whether phytoplasmas alter floral development to increase their population.

Virus-induced gene silencing in ornamental plants.

Virus-induced gene silencing (VIGS) provides an attractive tool for high-throughput analysis of the functional effects of gene knockdown. Virus genomes are engineered to include fragments of target host genes, and the infected plant recognizes and silences the target genes as part of its viral defense mechanism. The consequences of gene inactivation, even of key metabolic, regulatory, or embryo-lethal genes, can thus be readily analyzed. A number of viral vectors have been developed for VIGS; one of the most frequently employed is based on tobacco rattle virus (TRV) due to its wide host range, efficiency, ease of application, and limited disease symptoms. TRV-based VIGS comprises two vectors. One (RNA2) includes a multiple cloning site into which fragments of target genes can be inserted. We have shown that the TRV/VIGS system can simultaneously silence as many as five independent genes. TRV is a mosaic-type virus, and silencing also occurs in a mosaic pattern. It is therefore desirable to have a reporter that can show where target genes have been silenced. The photobleaching induced by silencing phytoene desaturase (PDS) and the loss of purple pigmentation induced by silencing chalcone synthase (CHS) have successfully been used to indicate the location of coordinate silencing of other target genes. In this chapter, we outline our protocols for the use of VIGS for analysis of gene function, focusing particularly on the use of TRV with petunia and tomato.

Video-assisted thoracoscopic surgery using single-lumen endotracheal tube anaesthesia in primary spontaneous pneumothorax.

BACKGROUND AND OBJECTIVE: Primary spontaneous pneumothorax (PSP) is a common condition that typically affects young adults. With recent advances in techniques, VATS is now a safe and accepted procedure for treating PSP. Lung isolation techniques have been commonly used to facilitate surgical procedures in the past. The purpose of this study was to evaluate the feasibility of using a single-lumen endotracheal tube for thoracoscopic surgery in patients with PSP. METHODS: A series of 121 consecutive patients with PSP, who underwent VATS using a double-lumen or single-lumen endotracheal tube between January 2000 and December 2002, were assessed retrospectively. The clinical features, operation times, complications, hospital stays and recurrences of PSP in these patients were recorded and analysed. RESULTS: There were no significant differences in gender, BMI, smoking habits, blebs/bullae on CT, duration of surgery or recurrence of PSP between the two groups. Patients in the single-lumen endotracheal tube group had a shorter duration of anaesthesia (15.4 +/- 2.6 vs 25.6 +/- 3.2 min, P < 0.001), lower early complication rates, lower costs and shorter hospital stays (3.6 +/- 3.0 vs 4.5 +/- 2.8 days, P = 0.02) compared with those in the double-lumen endotracheal tube group. The follow-up period was 40-68 months (mean 54 months). There were two recurrences in each group (3.1% vs 3.4%). CONCLUSIONS: VATS for the treatment of PSP was easily performed using a single-lumen endotracheal tube, and resulted in lower intubation-related costs, fewer complications and equivalent outcomes, compared with procedures performed using double-lumen endotracheal tube anaesthesia.

The impact of smoking in primary spontaneous pneumothorax.

BACKGROUND: The crucial role of cigarette smoking in the development of pneumothorax is unclear because nonsmokers can also develop primary spontaneous pneumothorax. The purpose of this study was to clarify the pathophysiologic effects of cigarette smoking and its clinical correlations in primary spontaneous pneumothorax. METHODS: Included were 115 specimens of lung tissue from patients with primary spontaneous pneumothorax who underwent video-assisted thoracoscopic surgery from January 2001 to December 2002. We reviewed the clinical features of 56 smokers and 59 nonsmokers with an average follow-up of 67 months. The pathologic findings of resected lung specimens were analyzed retrospectively. RESULTS: There were no statistical differences in sex, age, body height, body weight, body mass index, or the presence of blebs/bullae on computed tomography scans of the lung or under thoracoscopy between the 2 groups. In the smoking group, patients had more extensive respiratory bronchiolitis (P < .001), a high prevalence of tobacco pigmentation (P < .001), and a higher recurrence rate without or after surgery than the nonsmoking group (57% vs 22%, P = .001 and 8.9% vs 1.7%, P = .02, respectively). Patients with extensive respiratory bronchiolitis had significantly higher nonoperative and postoperative recurrences than patients with nonextensive respiratory bronchiolitis (P = .004 and P < .001, respectively). CONCLUSION: Cigarette smoking is associated with the pathophysiologic consequences of extensive respiratory bronchiolitis, which had a significant impact on the recurrence rates of primary spontaneous pneumothorax.