Dysregulated Lipid Metabolism Networks Modulate T-cell Function in People with Relapsing Remitting Multiple Sclerosis.

Altered cholesterol, oxysterol, sphingolipid, and fatty acid concentrations are reported in blood, cerebrospinal fluid, and brain tissue of people with relapsing remitting multiple sclerosis (RRMS) and are linked to disease progression and treatment responses. CD4+ T cells are pathogenic in RRMS, and defective T cell function could be mediated in part by liver X receptors (LXRs) - nuclear receptors that regulate lipid homeostasis and immunity. RNA-sequencing and pathway analysis identified that genes within the 'lipid metabolism' and 'signalling of nuclear receptors' pathways were dysregulated in CD4+ T cells isolated from RRMS patients compared with healthy donors. While LXRB and genes associated with cholesterol metabolism were upregulated, other T cell LXR-target genes, including genes involved in cellular lipid uptake (inducible degrader of the LDL receptor, IDOL), and the rate-limiting enzyme for glycosphingolipid biosynthesis (UDP-glucosylceramide synthase, UGCG) were downregulated in T cells from patients with RRMS compared to healthy donors. Correspondingly, plasma membrane glycosphingolipids were reduced, and cholesterol levels increased in RRMS CD4+ T cells, an effect partially recapitulated in healthy T cells by in vitro culture with T cell receptor stimulation in the presence of serum from RRMS patients. Notably, stimulation with LXR-agonist GW3965 normalised membrane cholesterol levels, and reduced proliferation and IL17 cytokine production in RRMS CD4+ T-cells. Thus, LXR-mediated lipid metabolism pathways were dysregulated in T cells from patients with RRMS and could contribute to RRMS pathogenesis. Therapies that modify lipid metabolism could help restore immune cell function.

Characterizing nascent transcription patterns of PROMPTs, eRNAs, and readthrough transcripts in the ENCODE4 deeply profiled cell lines.

Arising as co-products of canonical gene expression, transcription-associated lincRNAs, such as promoter upstream transcripts (PROMPTs), enhancer RNAs (eRNAs), and readthrough (RT) transcripts, are often regarded as byproducts of transcription, although they may be important for the expression of nearby genes. We identified regions of nascent expression of these lincRNA in 16 human cell lines using Bru-seq techniques, and found distinctly regulated patterns of PROMPT, eRNA, and RT transcription using the diverse biochemical approaches in the ENCODE4 deeply profiled cell lines collection. Transcription of these lincRNAs was influenced by sequence-specific features and the local or 3D chromatin landscape. However, these sequence and chromatin features do not describe the full spectrum of lincRNA expression variability we identify, highlighting the complexity of their regulation. This may suggest that transcription-associated lincRNAs are not merely byproducts, but rather that the transcript itself, or the act of its transcription, is important for genomic function.

Proteomics analysis reveals differential acclimation of coastal and oceanic Synechococcus to climate warming and iron limitation.

In many oceanic regions, anthropogenic warming will coincide with iron (Fe) limitation. Interactive effects between warming and Fe limitation on phytoplankton physiology and biochemical function are likely, as temperature and Fe availability affect many of the same essential cellular pathways. However, we lack a clear understanding of how globally significant phytoplankton such as the picocyanobacteria Synechococcus will respond to these co-occurring stressors, and what underlying molecular mechanisms will drive this response. Moreover, ecotype-specific adaptations can lead to nuanced differences in responses between strains. In this study, Synechococcus isolates YX04-1 (oceanic) and XM-24 (coastal) from the South China Sea were acclimated to Fe limitation at two temperatures, and their physiological and proteomic responses were compared. Both strains exhibited reduced growth due to warming and Fe limitation. However, coastal XM-24 maintained relatively higher growth rates in response to warming under replete Fe, while its growth was notably more compromised under Fe limitation at both temperatures compared with YX04-1. In response to concurrent heat and Fe stress, oceanic YX04-1 was better able to adjust its photosynthetic proteins and minimize the generation of reactive oxygen species while reducing proteome Fe demand. Its intricate proteomic response likely enabled oceanic YX04-1 to mitigate some of the negative impact of warming on its growth during Fe limitation. Our study highlights how ecologically-shaped adaptations in Synechococcus strains even from proximate oceanic regions can lead to differing physiological and proteomic responses to these climate stressors.

A Phase 1/2 multicenter trial of DKN-01 as monotherapy or in combination with docetaxel for the treatment of metastatic castration-resistant prostate cancer (mCRPC).

BACKGROUND: Dickkopf-related protein 1 (DKK1) is a Wingless-related integrate site (Wnt) signaling modulator that is upregulated in prostate cancers (PCa) with low androgen receptor expression. DKN-01, an IgG4 that neutralizes DKK1, delays PCa growth in pre-clinical DKK1-expressing models. These data provided the rationale for a clinical trial testing DKN-01 in patients with metastatic castration-resistant PCa (mCRPC). METHODS: This was an investigator-initiated parallel-arm phase 1/2 clinical trial testing DKN-01 alone (monotherapy) or in combination with docetaxel 75 mg/m2 (combination) for men with mCRPC who progressed on ≥1 AR signaling inhibitors. DKK1 status was determined by RNA in-situ expression. The primary endpoint of the phase 1 dose escalation cohorts was the determination of the recommended phase 2 dose (RP2D). The primary endpoint of the phase 2 expansion cohorts was objective response rate by iRECIST criteria in patients treated with the combination. RESULTS: 18 pts were enrolled into the study-10 patients in the monotherapy cohorts and 8 patients in the combination cohorts. No DLTs were observed and DKN-01 600 mg was determined as the RP2D. A best overall response of stable disease occurred in two out of seven (29%) evaluable patients in the monotherapy cohort. In the combination cohort, five out of seven (71%) evaluable patients had a partial response (PR). A median rPFS of 5.7 months was observed in the combination cohort. In the combination cohort, the median tumoral DKK1 expression H-score was 0.75 and the rPFS observed was similar between patients with DKK1 H-score ≥1 versus H-score = 0. CONCLUSION: DKN-01 600 mg was well tolerated. DKK1 blockade has modest anti-tumor activity as a monotherapy for mCRPC. Anti-tumor activity was observed in the combination cohorts, but the response duration was limited. DKK1 expression in the majority of mCRPC is low and did not clearly correlate with anti-tumor activity of DKN-01 plus docetaxel.

Establishing mouse and human oral esophageal organoids to investigate the tumor immune response.

Organoid culture systems are very powerful models that recapitulate in vivo organ development and disease pathogenesis, offering great promise in basic research, drug screening and precision medicine. However, the application of organoids derived from patients with cancer to immunotherapeutic research is a relatively untapped area. Esophageal cancer is one of the most lethal malignancies worldwide, including two major pathological subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma. ESCC shares many biological and genomic features with oral squamous cell cancers. Herein, we provide a versatile protocol for the establishment and maintenance of oral and esophageal organoid cultures derived from both murine and human samples. We describe culture conditions for organoids derived from normal tongue, esophagus and gastroesophageal junction, esophageal cancer and Barrett's esophagus. In addition, we establish an ex vivo model by co-culturing patient tumor-derived organoids and autologous CD8+ T lymphocytes to assess CD8+ T cell-mediated tumor killing. Our protocol can also be modified for organoid establishment from other squamous epithelia and carcinomas. The co-culture model can serve as a template for studies of other tumor-immune cell interactions and the efficacy of immune checkpoint blockade therapy.

Highly effective remediation of high arsenic-bearing wastewater using aluminum-containing waste residue.

Wastewater from non-ferrous metal smelting is known as one of the most dangerous sources of arsenic (As) due to its high acidity and high arsenic content. Herein, we propose a new environmental protection process for the efficient purification and removal of arsenic from wastewater by the formation of an AlAsO4@silicate core-shell structure based on the characteristics of aluminum-containing waste residue (AWR). At room temperature, the investigation with AWR almost achieved 100% As removal efficiency from wastewater, reducing the arsenic concentration from 5500 mg/L to 52 µg/L. With Al/As molar ratio of 3.5, the structural properties of AWR provided good adsorption sites for arsenic adsorption, leading to the formation of arsenate and insoluble aluminum arsenate with As. As-containing AWR silicate shells were produced under alkaline conditions, resulting in an arsenic leaching concentration of 1.32 mg/L in the TCLP test. AWR, as an efficient As removal and fixation agent, shows great potential in the treatment of copper smelting wastewater, and is expected to achieve large-scale industrial As removal.

Integrating the impact of global change on the niche and physiology of marine nitrogen-fixing cyanobacteria.

Marine nitrogen fixation is a major source of new nitrogen to the ocean, which interacts with climate driven changes to physical nutrient supply to regulate the response of ocean primary production in the oligotrophic tropical ocean. Warming and changes in nutrient supply may alter the ecological niche of nitrogen-fixing organisms, or 'diazotrophs', however, impacts of warming on diazotroph physiology may also be important. Lab-based studies reveal that warming increases the nitrogen fixation-specific elemental use efficiency (EUE) of two prevalent marine diazotrophs, Crocosphaera and Trichodesmium, thus reducing their requirements for the limiting nutrients iron and phosphorus. Here, we coupled a new diazotroph model based upon observed diazotroph energetics of growth and resource limitation to a state-of-the-art global model of phytoplankton physiology and ocean biogeochemistry. Our model is able to address the integrated response of nitrogen fixation by Trichodesmium and Crocosphaera to warming under the IPCC high emission RCP8.5 scenario for the first time. Our results project a global decline in nitrogen fixation over the coming century. However, the regional response of nitrogen fixation to climate change is modulated by the diazotroph-specific thermal performance curves and EUE, particularly in the Pacific Ocean, which shapes global trends. Spatially, the response of both diazotrophs is similar with expansion towards higher latitudes and reduced rates of nitrogen fixation in the lower latitudes. Overall, 95%-97% of the nitrogen fixation climate signal can be attributed to the combined effect of temperature on the niche and physiology of marine diazotrophs, with decreases being associated with a reduced niche and increases resulting due to a combination of expanding niche and temperature driven changes to EUE. Climate change impacts on both the niche and physiology of marine diazotrophs interact to shape patterns of marine nitrogen fixation, which will have important implications for ocean productivity in the future.

Molecular mechanisms underlying iron and phosphorus co-limitation responses in the nitrogen-fixing cyanobacterium Crocosphaera.

In the nitrogen-limited subtropical gyres, diazotrophic cyanobacteria, including Crocosphaera, provide an essential ecosystem service by converting dinitrogen (N2) gas into ammonia to support primary production in these oligotrophic regimes. Natural gradients of phosphorus (P) and iron (Fe) availability in the low-latitude oceans constrain the biogeography and activity of diazotrophs with important implications for marine biogeochemical cycling. Much remains unknown regarding Crocosphaera's physiological and molecular responses to multiple nutrient limitations. We cultured C. watsonii under Fe, P, and Fe/P (co)-limiting scenarios to link cellular physiology with diel gene expression and observed unique physiological and transcriptional profiles for each treatment. Counterintuitively, reduced growth and N2 fixation resource use efficiencies (RUEs) for Fe or P under P limitation were alleviated under Fe/P co-limitation. Differential gene expression analyses show that Fe/P co-limited cells employ the same responses as single-nutrient limited cells that reduce cellular nutrient requirements and increase responsiveness to environmental change including smaller cell size, protein turnover (Fe-limited), and upregulation of environmental sense-and-respond systems (P-limited). Combined, these mechanisms enhance growth and RUEs in Fe/P co-limited cells. These findings are important to our understanding of nutrient controls on N2 fixation and the implications for primary productivity and microbial dynamics in a changing ocean.

Removal of arsenic in acidic wastewater using Lead-Zinc smelting slag: From waste solid to As-stabilized mineral.

High-arsenic wastewater has long been considered a major threat to ecological balance and human health because of its strong toxicity and high mobility. Herein, an environmentally friendly process was proposed for As removal and fixation in the form of As-stabilized mineral, using Lead-Zinc smelting (LZS) slag as the in situ Fe donor, neutralizer, and crystal seed. The slag was dissolved in the wastewater and released Fe and Ca ions, while simultaneously increasing the pH value of the solution to help scorodite synthesis. The dissolved Ca2+ ion preferentially reacted with SO42- ion in the form of CaSO4·2H2O precipitate as in situ "seeds" for As precipitation. The dissolved Fe(II) and As(III) ions were oxidized to Fe(III) and As(V) ions by H2O2, and later reacted with each other to generated amorphous ferric arsenate on the surface of CaSO4·2H2O, and then evolved into scorodite crystals with high stability. With a Fe/As molar ratio of 2, a reaction temperature of 90 °C, and a reaction time of 12 h, 98.42% of As was effectively precipitated from the wastewater with an initial As concentration of 7530.00 mg/L. Moreover, the leached As concentration of the As-bearing precipitate in the TCLP test was 3.46 mg/L. The concentration of the residual As and heavy metals ions in the final filtrate was lower than local wastewater discharge standards, successfully realizing the treatment of smelting wastewater. In summary, a prospective process successfully shows a great potential for co-treatment of LZS wastewater and slag, which could advance the large-scale disposal of LZS plants.

Novel iron-supported ZSM-5 molecular sieve remove arsenic from wastewater by heterogeneous nucleation with pH limit breaking.

Molecular sieves have also been used for arsenic adsorption in recent years because of their special structure. In order to solve the problem of arsenic pollution in drinking water and/or industrial wastewater, ZSM-5/Fe adsorbent was prepared by loading iron on ZSM-5 molecular sieve. It is also used as an excellent adsorbent for removing arsenic and other heavy metal ions from industrial wastewater. At room temperature, the concentration of arsenic was reduced from 100 mg/L to 0.006 mg/L after the solution pH was adjusted to the range of weak acid to weak base (4-10) and 0.5 g of ZSM-5/Fe adsorbent was added for reacting 2 h. The adsorption capacity reached 40.00 mg/g, the adsorption efficiency reached 99.99%, reaching the national standard of drinking water. Adsorption thermodynamics, kinetics and isotherms showed that the adsorption mechanism of arsenic is heterogeneous nucleation adsorption (including electrostatic attraction and chemical precipitation). Moreover, ZSM-5/Fe adsorbent can adjust pH spontaneously by using non-skeleton Si-Al phase to achieve effective adsorption from weak acid to weak base. At the same time, ZSM-5/Fe adsorbent showed good reusability and stability in five cycles. This study provides an important idea for the application of ZSM-5 molecular sieve in many fields and the efficient removal of arsenic from drinking water and industrial wastewater.

Two co-dominant nitrogen-fixing cyanobacteria demonstrate distinct acclimation and adaptation responses to cope with ocean warming.

The globally dominant N2 -fixing cyanobacteria Trichodesmium and Crocosphaera provide vital nitrogen supplies to subtropical and tropical oceans, but little is known about how they will be affected by long-term ocean warming. We tested their thermal responses using experimental evolution methods during 2 years of selection at optimal (28°C), supra-optimal (32°C) and suboptimal (22°C) temperatures. After several hundred generations under thermal selection, changes in growth parameters, as well as N and C fixation rates, suggested that Trichodesmium did not adapt to the three selection temperature regimes during the 2-year evolution experiment, but could instead rapidly and reversibly acclimate to temperature shifts from 20°C to 34°C. In contrast, over the same timeframe apparent thermal adaptation was observed in Crocosphaera, as evidenced by irreversible phenotypic changes as well as whole-genome sequencing and variant analysis. Especially under stressful warming conditions (34°C), 32°C-selected Crocosphaera cells had an advantage in survival and nitrogen fixation over cell lines selected at 22°C and 28°C. The distinct strategies of phenotypic plasticity versus irreversible adaptation in these two sympatric diazotrophs are both viable ways to maintain fitness despite long-term temperature changes, and so could help to stabilize key ocean nitrogen cycle functions under future warming scenarios.

Diversity and Biocontrol Potential of Culturable Endophytic Fungi in Cotton.

Healthy cotton samples were collected and 93 endophytic fungal strains were isolated: 23 strains from the roots and 70 strains from the stems. Morphological characterization and ITS sequence analysis were used for the identification of these isolates. The results showed that the 93 strains including 20 species were highly diverse in terms of their taxonomy. Simpson's and Shannon's diversity indices were 0.915 and 3.848, respectively. Fusarium and Alternaria were the two dominant genera, constituting 19.4% of the total strains. Then, 72 spore-producing strains were tested for the suppression of cotton Verticillium wilt (CVW) caused by Verticillium dahliae in a greenhouse. Five strains exhibited effective suppression of CVW with average efficacy values higher than 50%. One of the effective strains, namely, Fusarium proliferatum 10R-7, was selected for the investigation of the role of fusaric acid, a secondary metabolite of strain 10R-7, in the suppression of V. dahliae and CVW. The results showed that F. proliferatum 10R-7 could produce fusaric acid, and this metabolite exhibited 100% inhibition of mycelial growth of V. dahliae at concentrations higher than 20 µg/ml. However, fusaric acid at 2.5 to 80 µg/ml was not effective in the suppression of CVW, compared with the control treatment with V. dahliae alone. F. proliferatum 10R-7 was labeled with green fluorescent protein (GFP), and the GFP-tagged strain was found to be able to colonize inside the taproots of cotton, suggesting that F. proliferatum 10R-7 is a true endophyte of cotton and endophytic colonization may play a role in the suppression of infection of cotton by V. dahliae.

Independent iron and light limitation in a low-light-adapted Prochlorococcus from the deep chlorophyll maximum.

Throughout the open ocean, a minimum in dissolved iron concentration (dFe) overlaps with the deep chlorophyll maximum (DCM), which marks the lower limit of the euphotic zone. Maximizing light capture in these dim waters is expected to require upregulation of Fe-bearing photosystems, further depleting dFe and possibly leading to co-limitation by both iron and light. However, this effect has not been quantified for important phytoplankton groups like Prochlorococcus, which contributes most of the productivity in the oligotrophic DCM. Here, we present culture experiments with Prochlorococcus strain MIT1214, a member of the Low Light 1 ecotype isolated from the DCM in the North Pacific subtropical gyre. Under a matrix of iron and irradiance matching those found at the DCM, the ratio of Fe to carbon in Prochlorococcus MIT1214 cells ranged from 10-40 × 10-6 mol Fe:mol C and increased with light intensity and growth rate. These results challenge theoretical models predicting highest Fe:C at lowest light intensity, and are best explained by a large photosynthetic Fe demand that is not downregulated at higher light. To sustain primary production in the DCM with the rigid Fe requirements of low-light-adapted Prochlorococcus, dFe must be recycled rapidly and at high efficiency.

Outer Membrane Iron Uptake Pathways in the Model Cyanobacterium Synechocystis sp. Strain PCC 6803.

Cyanobacteria are foundational drivers of global nutrient cycling, with high intracellular iron (Fe) requirements. Fe is found at extremely low concentrations in aquatic systems, however, and the ways in which cyanobacteria take up Fe are largely unknown, especially the initial step in Fe transport across the outer membrane. Here, we identified one TonB protein and four TonB-dependent transporters (TBDTs) of the energy-requiring Fe acquisition system and six porins of the passive diffusion Fe uptake system in the model cyanobacterium Synechocystis sp. strain PCC 6803. The results experimentally demonstrated that TBDTs not only participated in organic ferri-siderophore uptake but also in inorganic free Fe (Fe') acquisition. 55Fe uptake rate measurements showed that a TBDT quadruple mutant acquired Fe at a lower rate than the wild type and lost nearly all ability to take up ferri-siderophores, indicating that TBDTs are critical for siderophore uptake. However, the mutant retained the ability to take up Fe' at 42% of the wild-type Fe' uptake rate, suggesting additional pathways of Fe' acquisition besides TBDTs, likely by porins. Mutations in four of the six porin-encoding genes produced a low-Fe-sensitive phenotype, while a mutation in all six genes was lethal to cell survival. These diverse outer membrane Fe uptake pathways reflect cyanobacterial evolution and adaptation under a range of Fe regimes across aquatic systems.IMPORTANCE Cyanobacteria are globally important primary producers and contribute about 25% of global CO2 fixation. Low Fe bioavailability in surface waters is thought to limit the primary productivity in as much as 40% of the global ocean. The Fe acquisition strategies that cyanobacteria have evolved to overcome Fe deficiency remain poorly characterized. We experimentally characterized the key players and the cooperative work mode of two Fe uptake pathways, including an active uptake pathway and a passive diffusion pathway in the model cyanobacterium Synechocystis sp. PCC 6803. Our finding proved that cyanobacteria use ferri-siderophore transporters to take up Fe', and they shed light on the adaptive mechanisms of cyanobacteria to cope with widespread Fe deficiency across aquatic environments.

RNA interference-mediated knockdown of the hydroxyacid-oxoacid transhydrogenase gene decreases thiamethoxam resistance in adults of the whitefly Bemisia tabaci.

Bemisia tabaci has developed a high level of resistance to thiamethoxam, a second generation neonicotinoid insecticide that has been widely used to control this pest. In this study, we investigated whether hydroxyacid-oxoacid transhydrogenase (HOT) is involved in resistance to the neonicotinoid insecticide thiamethoxam in the whitefly. We cloned the full-length gene that encodes HOT in B. tabaci. Its cDNA contains a 1428-bp open reading frame encoding 475 amino acid residues. Then we evaluated the mRNA expression level of HOT in different developmental stages, and found HOT expression was significantly greater in thiamethoxam resistance adults than in thiamethoxam susceptible adults. Subsequently, seven field populations of B. tabaci adults were sampled, the expression of mRNA level of HOT significant positive correlated with thiamethoxam resistance level. At last, we used a modified gene silencing system to knock-down HOT expression in B. tabaci adults. The results showed that the HOT mRNA levels decreased by 57% and thiamethoxam resistance decreased significantly after 2 days of feeding on a diet containing HOT dsRNA. The results indicated that down-regulation of HOT expression decreases thiamethoxam resistance in B. tabaci adults.

[Application of FTIR and Active Ingredients Quantitative Analysis on Quality Control of Dai Medicine Alstonia scholaris (L.) R. Br.].

FTIR fingerprint of the leaves and immature stems of Alstonia scholaris (L.) R. Br. was established as a content determination method for the detection of picrinine, ursolic acid and oleanolic acid. Different medicinal parts were identified based on principal component analysis, while exploring the influence of immature stems for the leaves and the application of FTIR and HPLC in the Dai quality control in order to speed up the pace of Dai medicine modernization. Infrared spectroscopy of different batches samples were collected and the data was preprocessed as to automatic baseline correction, smooth, ordinate normalization, second order derivative, and then to PCA, all the datum in triplicate. For content determination of picrinine, mobile phase was acetonitrile (40) water (contain 0.1% ammonia water) (60) and the wavelength was set at 287 nm. For ursolic acid and oleanolic acid, the mobile phase was mixture (12∶88) of 0.1% formic acid in water (A) and methanol (B). Wavelength was 210 nm. As the results, the original spectrum difference was not obvious for leaves and stems. Pretreatment spectroscopy had a significant variation on absorption peak number and intensity in 3 000～2 800 and 1 800～500 cm(-1). The results of PCA showed that, the leaves and stems were separated; in addition the difference of different batches leaves was bigger than the stems. The mean contents of picrinine, ursolic acid and oleanolic acid in leaves were 0.79，8.47，7.51 and 0.21，1.78，1.67 mg·g(-1) in stems, respectively. The content of ursolic acid and oleanolic acid is higher than picrinine, but ursolic acid and oleanolic acid content had no obvious difference. Mean content of three ingredients in leaves is much higher than in stems. Picrinine content in leaves was 3.8 times of immature stems, ursolic acid and oleanolic acid content were 5.1 and 4.2 times of immature stems, respectively. The variety of picrinine content in different batches samples was biggest, ursolic acid and oleanolic acid content was relatively stable. The overall quality of leaves has an obvious difference compared with the immature stems, so the leaves of A. scholaris mix with immature stems could not be as Dai medicine in Dai clinic. Infrared spectroscopy combined with chromatography can quickly identify different medicinal parts and evaluate overall quality of Dai medicine, which can apply to quality control of Dai medicine.

[Distinguish and Quality Estimation of the Leaves of Alstonia scholaris (L.) R. Br. from Different Harvest Time Based on the UV-Vis·FP and HPLC·FP].

UV-Vis and HPLC fingerprint of different harvest time of the leaves of Alstonia scholaris (L.) R. Br. were establish the for identification and quality evaluation to promote the development of Dai Medicine modernization. The optimal extraction condition was used to obtain UV - vis data of different harvest time which were deducted background and eight spot smooth, were collected to make the principal component analysis in SIMCA-P(+)11.5, identifying the samples quickly with the first three principal component three-dimensional diagram. The HPLC fingerprint were obtained with Agilent ZORBAX Eclipse XDB C18 (250×4.6 mm, 5 µm) chromatographic column with the mobile phase of acetonitrile (B) - water (contain 0.1% formic acid) (A) for gradient elution (0～5 min, 5% B; 5～35 min, 5% B→26% B; 35～40 min, 26% B→56% B). The wavelength was set at 287 nm and the column temperature was maintained at 30 ℃. The flow rate was 1.0 mL·min-1 and the injection volume was 7 µL. The HPLC fingerprint of different harvest time of the leaves of Alstonia scholaris (L.) R. Br. was analysised by cluster analysis to quality evaluation. Research findings showing: (1) The UV-Vis spectrogram of different harvest time of the leaves of Alstonia scholaris (L.) R. Br. were divided into three parts according to the absorption peak position and amplitude of variation. The first was 235 to 400 nm, the second was 400 to 500 nm, and the third was 500 to 800 nm. In the first part, absorption peak were focused on 270, 287 and 325 nm, which can reflect the fingerprint character for the high absorbance and amplitude of variation. Absorption peak were distributed in 410 and 464 nm in the second part, absorbance and amplitude of variation were lower than the first part. There was a bigger absorption peak at 665 nm in the third part, but the absorbance had no difference. The UV-Vis data of different harvest time were gathered to make the principal component analysis, the result was that the samples of same month were concentrated distribution, but different month samples were dispersed distribution. (2) HPLC fingerprint were divided into three categories through hierarchical cluster analysis, 3, 4, 5 and 7 month were the first category, 6, 8, 9 month samples were second category, the others were third category. Chemical composition and content of the same category samples were similar, but the different category samples had a obvious difference, more important is that the third category samples content was the highest. Combining UV-Vis FP and HPLC FP can identify and evaluate quickly the samples of different harvest time of the leaves of Alstonia scholaris (L.) R. Br. The optimal harvest time of Alstonia scholaris (L.) R. Br. was from October to next February, which was the coldest season in the Dai calendar.

Characterization of LpGPAT gene in Lilium pensylvanicum and response to cold stress.

LpGPAT was obtained from L. pensylvanicum using RT-PCR and rapid amplification of cDNA ends. The cloned full-length cDNA was 1544 bp; it encoded 410 amino acids and had a molecular size of 46 KDa. The nucleic acid sequence analysis showed that it shared high homology with other known GPATs. SMAT result suggests that there is a PlsC that exists in 176-322 amino acid sequence of LpGAPT; it means LpGPAT protein is a member of the family of acyltransferase and has acyltransferase enzymatic activity. Result of real-time quantitative PCR and semiquantitative PCR support LpGPAT gene is definitely induced by low temperature stress.

Transcriptomic dissection of sexual differences in Bemisia tabaci, an invasive agricultural pest worldwide.

Sex difference involving chromosomes and gene expression has been extensively documented. In this study, the gender difference in the sweetpotato whitefly Bemisia tabaci was investigated using Illumina-based transcriptomic analysis. Gender-based RNAseq data produced 27 Gb reads, and subsequent de novo assembly generated 93,948 transcripts with a N50 of 1,853 bp. A total of 1,351 differentially expressed genes were identified between male and female B. tabaci, and majority of them were female-biased. Pathway and GO enrichment experiments exhibited a gender-specific expression, including enriched translation in females, and enhanced structural constituent of cuticle in male whiteflies. In addition, a putative transformer2 gene (tra2) was cloned, and the structural feature and expression profile of tra2 were investigated. Sexually dimorphic transcriptome is an uncharted territory for the agricultural insect pests. Molecular understanding of sex determination in B. tabaci, an emerging invasive insect pest worldwide, will provide potential molecular target(s) for genetic pest control alternatives.

Detoxification enzymes of Bemisia tabaci B and Q: biochemical characteristics and gene expression profiles.

BACKGROUND: The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is one of the most invasive and destructive pests of field crops worldwide. The sibling species B and Q are the two most damaging members of the B. tabaci species complex. That Q is more resistant than B to many insecticides has been well documented. Over the last decade, Q has gradually displaced B and has become the dominant form of B. tabaci in field agricultural systems in most parts of China. To help understand the differences in insecticide resistance, the activities and gene expression profiles of detoxification enzymes in B. tabaci B and Q were investigated. RESULTS: The activity of P450 towards 7-ethoxycoumarin was significantly higher (1.46-fold higher) in Q than in B. The expression of 43 of 65 P450 genes was higher (>1-fold) in Q than in B, and expression for eight P450 genes was more than 50-fold greater in Q than in B. The increased expression of selected P450 genes in Q relative to B was confirmed with two other B strains and two other Q strains. On the other hand, carboxylesterase (CarE) activity was significantly lower (0.71-fold lower) in Q than in B; the Km value of CarE was significantly lower in B than in Q, but the opposite was true for the Vmax value of CarE. Glutathione S-transferase activity and values of Km and Vmax did not differ between B and Q. CONCLUSION: Enhanced metabolic detoxification of insecticides by P450s may be an important reason why B. tabaci Q is more resistant than B. tabaci B to insecticides.