DNA and RNA samples from blood are the common examination target for non-invasive physical tests and/or biomedical studies. Since high-quality DNA and RNA samples guarantee the correctness of these tests and/or studies, we investigated the effects of storage temperature and storage duration of whole blood on DNA and RNA qualities. Subjects were enrolled to donate blood samples which were stored for different durations and at different temperatures, followed by the examinations on RNA quality, qPCR, DNA quality and DNA methylation. For RNA, we observed obvious quality decline with storage duration longer than 24 hours. Storage at low temperature does not keep RNA samples from degradation. And, storing whole blood samples in freezer dramatically damage RNA. For DNA, quality decline was not observed even with storage duration for 15 days. However, DNA methylation significantly altered with storage duration longer than three days. Storage duration within 24 hours is critical for collecting high-quality RNA samples for next-generation sequencing (NGS) assays (RIN≧8). If microarray assays are expected (RIN≧7), storage duration within 32 hours is acceptable. Although DNA is resistant within 15 days when kept in whole blood, DNA quantity dramatically decreases owing to WBC lysis. In addition, duration for more than three days significantly alter DNA methylation status, globally and locally. Our result provides a reference for dealing with blood samples. Circulation system is responsible for the transportation of oxygen, nutrition, water, waste and metabolite around human body. In animal, the concentration of these transported chemicals within circulation system tend to keep a balanced homeostatic state. Therefore, examining the concentration of these chemicals in blood may indicate one's physical conditions, especially for metabolic conditions. In animal, lymphocytes in circulation system function as protectors, conducting many immune activities to protect animals from external damage, chemical or biological. Therefore, examining the patterns of lymphocytes in blood may reflect one's immune conditions, especially for inflammatory conditions.et al identified susceptibility genes associated with coronary artery aneurysm formation in Kawasaki disease by analyzing WBC DNA [Because of high correlation with physical and immune conditions, DNA, RNA and protein samples from blood, especially from white blood cell (WBC) in most cases, are the common examination targets for non-invasive physical tests and/or biomedical studies. Margaritelis and colleagues determined tissue oxidative stress by measuring redox biomarkers  in blood . Kuo et  WBC DNA . Tang an WBC DNA . Moreove WBC DNA –9. Low-qSince the qualities of DAN and RNA samples from blood are critical to the result of physical test and biomedical study, we are interested in how to collect high-quality DNA and RNA samples from blood. In most cases, blood sample collection and further nucleic acid extraction from blood are conducted by different people at different places. So, the storage duration and storage temperature, from blood collection to nucleic acid extraction, are critical to high-quality DNA and RNA samples. Although the fresher the better is the golden rule when dealing with clinical samples, including liquid biopsies, we are interested in how much storage duration and storage temperature of blood affect the qualities of DNA and RNA. Therefore, we enrolled subjects and collected blood samples. These blood samples were stored for different durations and at different temperatures, followed by extracting nucleic acids and evaluating the qualities and quantities of DNA and RNA samples.® One-Use holder and Vacutainer® Blood Collection Tubes . All samples were categorized according to storage duration and storage temperature. After specified storage duration at specific temperature, blood samples were treated with dextran to induce RBC aggregation. The supernatant was then harvested and RBC lysis buffer was added into supernatant. After centrifugation, total WBCs were harvested from the pellets.This study has been approved by the Institutional Review Board of Chang Gung Memorial Hospital. All subjects signed the informed consent. Blood was collected using scalp vein set , vacutainerTM miRNA Isolation Kit , according to the manufacturer’s protocol. Next, the isolated RNA samples were eluted with 50 μl elution buffer. For DNA extraction, WBCs from 2ml blood were suspend with 1ml PBS and DNA isolation was conducted with QIAamp® DNA Blood Mini Kit , according to the manufacturer’s protocol. Next, the isolated DNA samples were eluted with 100 μl elution buffer. The extracted DNA and RNA samples were quantified with NanoDrop 2000 spectrophotometer .For RNA extraction, the harvested WBCs from 5ml blood were lysed with lysis/binding buffer and RNA isolation was conducted with mirVanaRNA quality was assessed with the RNA integrity number (RIN) by Agilent 2100 Bioanalyzer  with RNA 6000 Nano Kit, according to the manufacturer’s protocol. DNA quality was assessed by Agilent 2100 Bioanalyzer  with DNA 1000 Kit.® Green Master Mix and StepOnePlus TM System (Applied Biosystems). The sequences of the primers used were as follow:For 18S rRNA, GAPDH and IP-10 quantification, 0.5 μg RNAs were reverse transcribed to cDNA by High-Capacity cDNA Reverse Transcription Kit . The cDNA reverse transcription master mix was prepared as follow: 2 μl 10X RT buffer, 0.8 μl 25X dNTP mix (100mM), 2 μl 10X RT random primers, 1 μl reverse transcriptase, 1 μl RNase inhibitor, 4.2 μl nuclease-free water and 10 μl RNA sample. The thermal-cycling condition was as follow: 10 min at 25°C, 120 min at 37°C, 5 min at 85°C and hold at 4°C. Next, the quantification job was conducted with Fast SYBR5’- GTAACCCGTTGAACCCCATT -3’) and reverse primer (5’- CCATCCAATCGGTAGTAGCG -3’);18S: forward primer (5'-ATGGGGAAGGTGAAGGTCG-3') and reverse primer (5'-GGGGTCATTGATGGCAACAAT-3');GAPDH: forward primer (5’-TCTAGAACTGTACGCTGTACC-3’) and reverse primer (5’-GATTCAGACATCTCTTCTCACC-3’). The real-time PCR master mix was prepared as follow: 10 μl 2X fast SYBR green master mix, 1 μl forward primer (10 μM), 1 μl reverse primer (10 μM), 1 μl cDNA and 7 μl nuclease-free water. The default PCR thermal-cycling condition was as follow: 20 sec at 95°C and 40 cycles of 3 sec at 95°C and 30 sec at 60°C.IP-10: forward primer . The RT master mix was prepared as follow: 0.3 μl 100Mm dNTPs, 3 μl reverse transcriptase, 1.5 μl reverse transcription buffer, 0.19 μl RNase inhibitor, 6 μl primer, 1.01 μl nuclease-free water and 3 μl RNA. The RNU6B expression was quantified using TaqMan® Fast Universal PCR Master Mix (2✕), No AmpErase® UNG (Applied Biosystems) and StepOnePlus TM System. The real-time PCR master mix was prepared as follow: 7.5 μl 2X master mix, 0.8 μl probe, 6.7 μl nuclease-free water and 5 μl cDNA. The default PCR thermal-cycling condition was as follow: 20 sec at 95°C and 40 cycles of 3 sec at 95°C and 30 sec at 60°C.For RNU6B (U6) quantification, the RNAs were reverse transcribed to cDNA by TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) and TaqManTM Kit . Next, the bisulfite modified DNAs were eluted with 10 μl elution buffer, followed by PCR amplification with PyroMark PCR Kit (Qiagen). The PCR reagent was prepared as follow: 12.5 μl 2X master mix, 0.5 μl forward primer (10 μM), 0.5 μl reverse primer (10 μM), 5 μl 5X Q solution, 2.5 μl 10X coralload concentrate, 1.5 μl MgCl2, 0.5 μl nuclease-free water and 2 μl bisulfite modified DNA. After PCR reaction, we ran gel assay to make sure the success of PCR reaction and the correctness of the size of PCR product. Following amplification, the methylation percentage of CpG marker cg08298591 was assessed with PyroMark Q24 (Qiagen). The primer sequences are as follow: forward primer (5'-GTAGTAGTTAGAGTGAGTGGTTTAGAGTT-3'), reverse primer (Biotin 5'-TACCAAAAACATAACATTTCAAACAACTT-3') and sequencing primer (5'-GGTTTTTTATTTATTTAAAGGTAGG-3').DNA methylation was assessed by pyrosequencing. As suggested by the manufacturer’s protocol, 0.5 μg genomic DNA was bisulfite modified using EZ DNA Methylation-Lightning DNA samples (100ug) were added to denaturation buffer (0.4mM NaOH and 10mM EDTA) and denatured at 100°C for 10min. Then, samples were chilled on ice for 5min and applied to a positive charged nylon membrane . The membrane was UV cross-linked and dried for 1hr at 70°C. Membranes were probed with anti-5mC antibody  at 4°C overnight. Subsequently, membranes were probed with either a rabbit or mouse IgG antibody conjugated to HRP for 1 hour at room temperature. After three times of wash with PBS-Tween, immunoreactive dot were detected using an ECL kit (Amersham Pharmacia Biotech). Finally, the membranes were stained with Methylene Blue as loading control.In this study, statistical analyses were conducted with Microsoft Excel. All quatitative raw data for plotting each figure and sub-figure is available in Materials and Methodssection). The collected RNA samples were then subject to RNA integrity number (RIN) and 260/280 examination with Bioanalyzer and NanoDrop spectrophotometer, respectively. As shown in To examine how much time can blood samples be stored without a significant decline in RNA quality, we first drew blood samples from one single subject and stored the blood samples at room temperature  with different storage durations, followed by WBC enrichment and RNA extraction  and at three temperatures , where RT, LT and FR individually denoted room temperature (24°C), low temperature (4°C) and frozen (-80°C). As a result, 270 blood tubes were collected (3 replications * 3 temperatures * 3 durations * 10 subjects)., RNA samples are usually used for gene expression profiling with real-time quantitative polymerase chain reaction (qPCR). Therefore, in addition to RNA quality examination, we also investigated the expression abundances on three common internal control genes with qPCR assay. The same RNA samples presented in qPCR assay on WBCs samples is often conducted to observe the expression profiles of immune-related genes. In addition to internal control genes, we also examined one chemokine gene, Interferon gamma-induced protein 10 . IP-10 is induced by interferon gamma and involved in many inflammatory diseases such as chronic inflammatory arthritis, autoimmune diseases, type 1 diabetes, cardiovascular disease, psoriasis…etc –16. Fig qPCR assay was conducted to determine the relative abundances of gene expression transcripts. Therefore, the less degraded the RNA samples are, the more consistent Ct values the RNA samples have. Although In addition to RNA, DNA from blood is also usually examined for SNP calling , InDel iSince it seemed that DNA quality did not significantly decline with long storage duration, we turned to investigate DNA quantity, measuring the DNA concentration on the samples. Further, we investigated DNA concentration. As shown in P<0.05). In addition, with longer duration, the methylation status of cg08298591 gradually increased. So, storage duration DID affect DNA methylation at consistent temperature.DNA samples from WBC are also usually used for evaluating DNA methylation , 20. TheThe conclusion of et al conducted a study to evaluate mRNA change with different time courses, they worked on rat's spleen tissue [Previous studies tried to develop the protocol for dry DNA storage and shipment . In addin tissue . Blood nn tissue  but alson tissue . Therefo® Blood Collection Tubes; REF367835, BD, New Jersey, USA). Next, we conducted the same RNA enrichment procedures immediately after blood drawing, followed by qPCR reactions. For 18S, the heparin and EDTA qPCR Ct values were 9.65±0.70 (mean±S.D.) and 10.06±0.61, respectively; For GAPDH, they were individually 17.27±0.86 and 18.97±1.64; For IP-10, 25.41±0.95 and 26.42±0.92 were detected; For U6, the two qPCR Ct values were 25.47±0.11 vs. 25.54±0.18, respectively. In summary, none of the four genes had significantly different (p<0.05) Ct values by heparin or EDTA. So, heparin did not contaminate the RNA samples from WBC and did not interfere with PCR reaction according to our protocols.In this study, we used heparin-containing tubes to collect blood samples. It was reported that heparin was usually used as an anticoagulant and could interfere with PCR reaction, producing aberrant results in qPCR or PCR assays , 28. HowThe fresher the better is the golden rule when dealing with clinical samples, including liquid biopsies. In most hospitals, blood samples were collected by the specially-assigned staff rather than the people conducting physical tests and/or biomedical studies. So, the storage duration and storage temperature, from blood collection to nucleic acid extraction, are critical to high-quality DNA and RNA samples. As a result, storage duration and storage temperature of blood samples largely influence the correctness of physical test and the success of biomedical study.IP-10 is a protein-coding gene and usually participates in inflammatory diseases, served as a pro-inflammatory chemokine. It has been reported that hypoxia conditions in murine lung and brain caused higher expression level of IP-10 , 32. StoAccording to our result, although DNA is still resistant within 15 days when kept in whole blood, DNA quantity dramatically decreases owing to WBC lysis. In addition, DNA methylation status may alter with storage duration of whole blood. Actually, few studies have focused this issue. Vilahur and colleagues found that storing placenta tissues at room temperature for one, two, six and 24 hours brought small changes on global DNA methylation status . In thisIn this study, we investigated how much storage duration and storage temperature of whole blood affect the qualities of DNA and RNA. For RNA, storage duration within 24 hours is critical for collecting high-quality RNA samples for NGS assays (RIN≧8). If microarray assays are expected (RIN≧7), storage duration within 32 hours is acceptable. In addition, storage at low temperature does not keeping RNA samples from degradation. Most important of all, storing whole blood samples in freezer dramatically damage RNA samples. Although DNA is resistant within 15 days when kept in whole blood, DNA quantity dramatically decreases owing to WBC lysis. In addition, duration for more than three days significantly alter DNA methylation status, globally and locally. Our findings provide a reference for collecting high-quality DNA and RNA from blood samples.S1 FigWe conducted Bioanalyzer RNA 6000 Nano assay to examine RNA quality. Bioanalyzer calculated RIN value according the relative intensities of 18S and 28S to marker. As shown in this Fig, the samples with high RIN value have relatively strong and clear 18S and 28S peaks. And, the samples with low RIN value have relatively weak and ambiguous 18S and 28S peaks.(TIF)Click here for additional data file.S2 FigWe conducted DNA electrophoresis with 0.5% argarose gel. The gel illustration showed that DNA samples were resistant without breaking down into small fragments even with the storage duration of 15 days.(TIF)Click here for additional data file.S1 TableThe raw data for plotting each figure and sub–figure is available in S1 Table. These digits are RIN value, 260/280 value, qPCR Ct value, WBC count, WBC concentration or methylation percentage. Empty columns denote failed experiments.(XLS)Click here for additional data file.


Bursaridium, a relatively large ciliate species found in such lakes. The populations from 3 highly UV transparent lakes revealed the presence of 7 MAAs  in total concentrations of 3.6–52.4 10−5 μg μg−1 dry weight (DW) per individual, whereas in one glacially turbid and less UV transparent lake, no MAAs were detected in the Bursaridium population. The MAAs in the ciliates generally reflected the composition and relative amounts of the lakes’ seston MAAs, assuming that the ciliates fed on MAA-rich plankton. We experimentally found that naturally acquired MAAs prevented ciliate mortality under simulated UVR and photosynthetically active radiation (PAR) conditions. We further tested the dietary regulation of the MAAs-content in the ciliates under artificial UVR and PAR exposure and found an increase in MAAs concentrations in all treatments. Our assumption was that several stress factors other than irradiation were involved in the synthesis or up-regulation of MAAs.Ciliates in shallow alpine lakes are exposed to high levels of incident solar ultraviolet radiation (UVR). We observed the presence of specific sunscreen compounds, the mycosporine-like amino acids (MAAs), in several populations of  Zmax~15 m) and transparent lakes  is an essential factor influencing the life of planktonic communities. In the Alps, the ultraviolet-B radiation  increases by ~19% per 1000 m altitude  that absorb UVR between wavelengths of 309 and 362 nm (Shick and Dunlap Bursaridium may accumulate MAAs from phytoplankton (seston) that protect it against natural levels of UVR and photosynthetically active radiation (PAR). Bursaridium is a large planktonic ciliate ~150 μm in length commonly found in lakes located in the Central Alps  addressed the overall UV sensitivity of the ciliates under the assumption that they were well protected by naturally acquired MAAs, and (2) intended to prove a possible regulation of the MAA-content in the ciliate itself in a feeding experiment in which we offered algal food with and without MAAs.Our hypothesis was that the algivorous heterotrophic ciliate We sampled the 3 UV transparent alpine lakes Hairlachersee (HAI), Oberer Plenderlesee (OPL), and Schwarzsee ob Sölden (SOS), and the glacier-fed turbid and less UV transparent lake Rifflsee (RIF). All 4 lakes are located within a radius of ~20 km Table . HAI is a (Chl-a) were assessed on each sampling occasion for several years between 2002 and 2013. Water samples were taken at the deepest point of each lake from an inflated boat using a 5 L Schindler-Patalas sampler at depths of 0, 2, 4, and 6 m in HAI; 0, 3, 5, and 7 m in OPL; 0, 1.5, 3, 6, 9, 12, and 15 m in SOS; and 0, 1, 2, 4, 6, 10, 12, 14, 16, 18, and 20 m in RIF. Temperature was read directly from a thermometer attached inside the water sampler. Subsamples were collected to determine the water chemistry (1 L), Chl-a (1 L), seston MAAs , and ciliate abundance . Seston MAAs were determined for HAI and RIF only because values for SOS and OPL are available from literature , concentrated on glass-fiber filters (Whatman GF/F), and extracted with 90% acetone in the dark . After sonication , the extracts were cleared by filtration through glass-fiber filters (Whatman GF/F) and scanned in a spectrophotometer  at 400–750 nm against an acetone reference. Chl-a concentrations were calculated following Lorenzen , and nutrients Table . AnalyseLorenzen .in situ profiling UV radiometer . The profiles were made within 2 h of solar noon. The diffuse attenuation coefficients (Kd) of the downwelling UVR and PAR were calculated as the slope of an exponential regression between radiation and depth (r² > 0.99).The incident downwelling irradiance at 305, 320, 340, and 380 nm and the downwelling PAR (400–700 nm) were measured with an Ciliates were identified from life observations as well as from specimens preserved and stained using the quantitative protargol staining method Skibbe  followinBursaridium cells analyzed ranged from 5 to 76 individuals. Once, during a dinoflagellate bloom in OPL (2004), we extracted the MAAs of these dominant algae from a bulk sample to identify a possible MAAs source.Starved individuals from HAI , SOS , OPL , and RIF (2010) were screened for the amount and composition of MAAs acquired in the ciliates cytoplasm. The extraction procedure and HPLC analysis followed the protocol of Sonntag et al. . BecauseBursaridium, all the numerous approaches failed (data not shown), and we therefore performed our experiments and analyses with individuals directly collected by hand from the original water samples. Before starting the MAAs extraction and experiments, the ciliates were cleaned individually over 5 drops of sterile filtered lake water. To ensure the complete digestion of ingested algae, they were kept in well plates overnight in a light/temperature controlled chamber at ambient lake water temperatures (5 °C) and a light/dark cycle of 16:8 h . Experiments were conducted within 1–2 days after lake sampling.Although we attempted to cultivate −2 UV-A, 2.47 W m−2 UV-B; Q-Lab, Saarbrücken, Germany) and 2 visible fluorescence lamps  at a constant temperature of 5 °C. The ciliates were exposed for 6 h to simulated natural irradiation conditions  were placed into 12-well plates in sterile-filtered lake water and tested for their overall survival under simulated UVR+PAR (full spectrum), PAR only , and a dark control . Experiments were conducted in a walk-in climatic chamber equipped with 4 UV-A-340 fluorescent (8.60 W mBursaridium from SOS were placed into 12-well plates filled with sterile-filtered lake water and fed with (1) an MAA-negative Cryptomonas sp. , (2) an MAA-positive Peridinium inconspicuum strain , and (3) a mixture of both algae. Peridinium inconspicuum and cryptomonads are natural phytoplankton species found in SOS  and exposed them under the experimental conditions and approaches discussed earlier. Under the stereomicroscope, after 6 h of exposure on day 1, all ciliates were individually cleaned from remaining algae in the medium as described, transferred to fresh medium without food, and kept in the dark overnight. On day 2, the same individuals were again exposed under the same irradiation and dark conditions without food and finally collected for MAAs analysis . MAAs samples were stored at −80 °C until analysis .To statistically test for differences in MAAs concentrations in the ciliates, Kruskal-Wallis one-way analyses of variance (ANOVA) were performed. Spearman rank order correlations were conducted among ciliates abundance, abiotic lake parameters, seston MAAs, and Chl-−1) in HAI and 32.0–86.5 μS cm−1 in the other 3 lakes; pH in HAI and SOS was 5.9–6.6 and around neutral in OPL and RIF (7.0–7.2); sulfate was lowest in HAI (0.98–1.66 mg L−1) and highest in OPL (24.46–27.12 mg L−1); chloride was 0.0–0.15 mg L−1 in the 4 lakes; sodium was 0.14–0.27 mg L−1 in HAI and 0.61–0.88 mg L−1 in the other 3 lakes; potassium was lowest in HAI and SOS (0.13–0.20 mg L−1) and 0.90–1.11 mg L−1 in OPL and RIF; magnesium was lowest in HAI (0.10–0.20 mg L−1) and 0.65–1.66 mg L−1 in the other 3 lakes; calcium was lowest in HAI and SOS (0.55–3.65 mg L−1) and 8.28–10.40 mg L−1 in OPL and RIF; ammonium was 1–6 μg L−1 in HAI, OPL, and RIF, and the max was detected in SOS (1–15 μg L−1); min and max dissolved nitrogen were detected in OPL (10–966 μg L−1); total phosphorus was low in the clear lakes (0.9–9.1 μg L−1) and 23.2–47.4 μg L−1 in RIF; DOC was low in all lakes, (196–569 μg L−1); min and max dissolved reactive silica were found in OPL (113–2714 μg L−1); Chl-a was 0.67–1.40 μg L−1 in HAI, 1.22–2.51 μg L−1 in SOS, and lowest in RIF with 0.06–0.79 μg L−1 . In HAI, another as-yet undescribed Urotricha species was identified. Numbers of Bursaridium were low , and the abundance of the Urotricha (counted in 2009 and 2010 only) were high .Identifications of the 4 Bursaridium populations from the 3 clear lakes  in the seston (0.13 ± 0.1 μg [μg Chl-a]−1) were found. In HAI, we identified the same 2 MAAs in Bursaridium over all sampling years: 89 ± 4% SH and 11 ± 4% AS in concentrations of 33.9 ± 21.0 10−5 μg μg−1 DW; seston MAAs were detected in concentrations of 20.0 ± 4.5 μg [μg Chl-a]−1, and proportions of MAAs were 86 ± 0% SH, 13 ± 0% AS, 1 ± 0% PI, <1% mycosporine-glycine (MG), and traces of PR, PI, US, and palythene (PE). In SOS, we identified 7 MAAs in Bursaridium over all sampling years: 11 ± 1% MG, 39 ± 16% SH, 15 ± 17% PR, 30 ± 6% PI, 3 ± 4% AS, and 1 ± 2% PE and US, respectively, in total concentrations of 9.0 ± 1.6 10−5 μg μg−1 DW. In OPL, we identified 4 MAAs in Bursaridium in the 2 sampling years, respectively: 4% and 0% MG, 72% and 33% SH, 20% and 9% PI, and 4% PE, in total concentrations of 21.8 and 7.0 10−5 μg μg−1 DW; the bulk MAAs  detected in the dinoflagellate bloom were the same as analyzed in the individuals of Bursaridium , palythine (PI), porphyra-334 (PR), and asterina-330 (AS) in the kes Fig. . In BursP = 0.735 in 2011, P = 0.815 in 2012).No significant mortality was observed among treatments after 6 h of irradiation (Bursaridium were found after both exposure days (P < 0.050). The food alga P. inconspicuum synthesized 40% SH, 37% MG, 22% AS, and, 1% PI; in Cryptomonas sp., no MAAs were detected. Overall, the individual MAAs content per ciliate increased in all approaches: UVR+PAR (112.8–127.7 10−5 μg μg−1 DW), PAR (45.1–126.7 10−5 μg μg−1 DW), and controls (20.0–55.2 10−5 μg μg−1 DW) compared to the natural population  or in the Peridinium+Cryptomonas treatment .No significant cell losses of Bursaridium that we isolated from several Austrian alpine lakes occurs at water temperatures <12 °C in low numbers (Table Bursaridium pseudobursaria without investigating living cells (B.S. pers. observ.). Further, disregarding our multitudinous and finally unsuccessful cultivation approaches, we collected each individual Bursaridium by hand directly out of the original lake water sample to achieve MAAs analyses and experimental approaches. Irrespective of our difficulties and efforts, we are able to report valuable information on the occurrence, environmental conditions, and the presence of sunscreen compounds for this delicate and prominent protist. Most important, this is the first report on the presence of MAAs in a heterotrophic ciliate species, although several other ciliates such as Cyclidium spp., Glaucoma sp., Spirostomum teres, and Balanion planctonicum have been previously tested  into other MAAs Shick . For exaBursaridium from SOS escapes high solar irradiation by persisting in greater depths (Table Surface avoidance by daily vertical migration is one well-known photoprotective strategy of zooplankton to escape high solar radiation around midday (e.g., Rautio and Tartarotti hs Table , Fig. 2.hs Table .Bursaridium populations from the transparent alpine lakes observed here were equipped with a set of MAAs and well adapted to potentially damaging UVR levels. This study illustrates that Bursaridium from alpine lakes can acquire MAAs from algal food, and evidence indicates that these ciliates may also regulate their MAAs content. We cannot rule out that in our feeding experiment, diverse stress factors including the experimental handling were responsible for an MAA increase in all treatments, but future studies need to address this issue. A physiological adaptation to elevated incident solar radiation, as shown previously for A. chlorelligera, implies an ecological advantage not only to survive in UV transparent high mountain lakes, but also to explore food resources over the whole water column. Our results support the hypothesis that MAAs with high absorption efficiencies over a wide range of UVR wavelengths play a major role in minimizing damaging effects of solar UVR on ciliates.In conclusion, the This work was supported by Austrian Academy of Science [grant number DOC-fForte 22883], Austrian Science Fund [grant number P16559-B06], [grant number P21013-B03], [grant number I2238-B25].


Haemophilus quentini.” The isolates were recovered from blood cultures of a newborn neonate and an elderly patient with septicemia in Ontario, Canada.Here, we present the draft genome sequences of two strains (K068 and C860) of the genospecies “ Haemophilus influenzae strains are often limited to the upper respiratory tract; however, there have been reports of infections in the urogenital tracts. Most of these urogenital infections are caused by nontypeable (NT) H. influenzae biotype IV strains  for H. quentini-specific testing. The NML performed H. quentini-specific molecular testing to confirm their identification.In humans,  strains , 2. Invaterature , 4. Moleuentini” . H. quenH. quentini isolates K068 and C860 was extracted and purified using a QIAamp DNA minikit  from an overnight culture on a blood agar plate. The samples were indexed during library preparation using the Nextera XT DNA sample preparation kit . The sequencing library was quantified using Qubit 2.0 , and concentration and quality were analyzed by Bioanalyzer . The pooled libraries were sequenced using MiSeq Illumina with the V2 kit (2 × 150 bp), according to the manufacturer’s instructions, generating 2,735,812 and 1,308,728 high-quality reads corresponding to 407,844,516 and 190,672,282 detected bases for H. quentini strains K068 and C860, respectively.Genomic DNA of de novo assembler in CLC Genomics Workbench version 8.0.1 . The number of contigs per assembly was 63 and 78 for strains K068 and C860, respectively. All contigs less than 500 bp were filtered, and the remaining 59 contigs  (K068) and 71 contigs  (C860) were used for annotation. Using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (http://www.ncbi.nlm.nih.gov/genome/annotation_prok), 1,989 predicted protein-coding sequences (CDSs), 7 rRNAs, and 48 tRNAs were annotated for H. quentini strain K068, while 1,983 CDSs, 5 rRNAs, and 49 tRNAs were annotated for H. quentini strain C860.The raw Illumina reads were trimmed and assembled using the H. quentini isolates to be H. influenzae R2866 , followed by H. influenzae PittGG   server revealed the closest neighbor of our score, 50, followeMDJB00000000 (K068) and MDJC00000000 (C860).The draft genome sequences for both isolates have been deposited at GenBank under the accession numbers


Rhodiola rosea displayed specific and potent inhibition against cell entry of both Ebola virus and Marburg virus. In addition, twenty commercial compounds that were isolated from Rhodiola rosea were evaluated using the pseudotyped Ebola virus entry assay, and it was found that ellagic acid and gallic acid, which are two structurally related compounds, are the most effective ones. The activity of the extract and the two pure compounds were validated using infectious Ebola virus. The time-of-addition experiments suggest that, mechanistically, the Rhodiola rosea extract and the effective compounds act at an early step in the infection cycle following initial cell attachment, but prior to viral/cell membrane fusion. Our findings provide evidence that Rhodiola rosea has potent anti-filovirus properties that may be developed as a novel anti-Ebola treatment.The recent 2014–2016 West African Ebola virus epidemic underscores the need for the development of novel anti-Ebola therapeutics, due to the high mortality rates of Ebola virus infections and the lack of FDA-approved vaccine or therapy that is available for the prevention and treatment. Traditional Chinese medicines (TCMs) represent a huge reservoir of bioactive chemicals and many TCMs have been shown to have antiviral activities. 373 extracts from 128 TCMs were evaluated using a high throughput assay to screen for inhibitors of Ebola virus cell entry. Extract of  Ebola virus (EBOV) and Marburg virus (MARV) belong to the Filoviridae family and are enveloped, single-stranded, negative-sense RNA viruses with filamentous morphology. Infections by these viruses can cause severe hemorrhagic fevers in humans and nonhuman primates with mortality rates up to 90% . The recNatural products are considered to be a natural combinatorial chemical source because they contain structurally diversified bioactive chemicals, thus making them a valuable reservoir for new drug discovery. Natural products or their direct derivatives account for 34% of new medicines approved by the US Food and Drug Administration (FDA) between 1981 and 2010 [To identify new drug candidates from TCMs, we previously established a high throughput screen (HTS) platform to identify entry inhibitors for highly-pathogenic viruses . This scRhodiola rosea and two isolated, chemically related compounds effectively inhibit Ebola virus entry. These findings have important implications in exploring and developing TCMs as potential antivirals against Ebola and Marburg viral infections.In this report, we evaluated 373 TCM extracts and demonstrated that extracts of 2.Human lung epithelial cell line A549, human embryonic kidney cell line 239T, and Hela cell line were cultured in DMEM  supplemented with 10% fetal bovine serum , 100 μg/mL of streptomycin, and 100 units of penicillin  at 37 °C and 5% CO−E−) into 293T cells using a polyethylenimine based transfection protocol. Cells were washed with phosphate-buffered saline (PBS) 6 h after transfection, and 20 mL fresh media was added to each Falcon® 150 mm TC-treated cell culture dish. The supernatants were collected and filtered through 0.45 μm pore size filter  24 h post-transfection and the pseudovirion stocks were stored at 4 °C prior to use. The H7N1 pseudoviruses were activated with 10 µg/mL TPCK-trypsin before infecting A549 cellsHIV/MARV, HIV/EBOV, HIV/H5N1, and HIV/H7N1 pseudovirions were produced by transient co-transfection of a corresponding GP encoding plasmid (Influenza A—hemagglutinin (HA) and neuraminidase (NA) from A/Goose/Qinghai/59/05 (H5N1) strain or A/Netherlands/219/03 (H7N7) strain; Marburgvirus—GP; Ebolavirus Zaire—GP) and the HIV core plasmid .One hundred and twenty-eight traditional Chinese herbs were purchased from the Chinese herbal medicine market in China. 373 extracts were prepared from the plants using extraction methods including aqueous extraction and organic-solvent extraction. They were dried in vacuum at 50 °C and dissolved in DMSO. Extracts were arrayed in a 384-well plate at a 20 mg/mL stock concentration in solvent. All of the sample plates were stored at −20 °C until use. Twenty chemical components (>98% purity) from v/v). Low-passage A549 cells were seeded in 384-well plates at a density of 1000 cells/well 24 h before infection. In the presence of TCM extracts, A549 cells were challenged with HIV/EBOV, HIV/H5N1, or HIV/H7N1 pseudotyped virions in JANUS robotic workstation . Infection was quantified after 48 h incubation by measuring the luciferase activity with the neolite Reporter Gene Assay System (PerkinElmer). Data was normalized to signals from the negative controls  and an average >90% inhibition for duplicates was applied for picking hits.The TCM extract library was screened in 384-well plates with a final concentration of 12.5 µg/mL in 0.0625% DMSO (v/v) to confirm the primary results. Cell cytotoxicity was examined 48 h post treatment using the CellTiter-Glo® Luminescent Cell Viability Assay , treated as for the antiviral screen. The selected active extracts were then reformatted into new 96-well plates and tested against, HIV/EBOV, HIV/MARV, HIV/H5N1, or HIV/H7N1 at 12.5 µg/mL in 0.0625% DMSO .The hit extract was two-fold serially that was diluted for dose-response titration, and the ICR. rosea extract (4 µg/mL), gallic acid (30 µM), ellagic acid (30 µM), benztropine , heparin , zidovudine , CA-074 , or drug vehicle DMSO was introduced to assess their impact on virus entry (triplicate wells for each treatment at each time point). Virus infection was quantified 48 h post infection as described above.HIV/EBOV virion was incubated with A549 cells at 4 °C for 1 h and then the virus was removed. The cells were washed with ice cold PBS twice before fresh media was added and virus entry was initiated by shifting the temperature to 37 °C. At different time points of virus entry, g for 2 h at 4 °C. The virus pellet was suspended in PBS and stored in aliquots at −80 °C until use. For infection assays, 4000 HeLa cells per well were grown overnight in 384-well tissue culture plates in DMEM culture medium that was supplemented with 10% FBS; the cells were infected by EBOV-GFP virus (MOI of 0.05 to 0.15) in the presence of TCM extract or effective compounds at various concentrations. Bafilomycin at a final concentration of 10 nM was used as a positive control treatment. All of the treatments were done in duplicates. After 24 h incubation, post-infection cells were fixed by immersing the plates in formalin for 24 h at 4 °C. Fixed plates were inactivated using formalin and brought out of the BSL-4. Formalin was then decanted and plates were washed twice with PBS. Cell nuclei were stained using Hoechst at 1:50,000 dilution and plates were imaged. Nuclei (blue) and infected cells expressing GFP (green) were counted using CellProfiler software version 2.1.1 . Total number of nuclei (blue) was used as a check of cell function, with >30% reduction, indicating a growth arrest or toxicity and were noted during analysis.Experiments using recombinant infectious Zaire Ebola virus were performed in biosafety level 4 (BSL-4) facilities at the Texas Biomedical Research Institute. The infectious EBOV and MARV were produced according to previously described methods . The ZaiRhodiola rosea) was further evaluated for cell toxicity and antiviral activity against HIV/EBOV, HIV/MARV, HIV/H5N1, and HIV/H7N1 pseudovirions at 12.5 µg/mL. The extract exhibited specific antiviral activity against both pseudotyped EBOV and MARV entry, but little inhibitory activity against the entry of the two pseudotyped influenza virus subtypes , represent a rich source of structurally diversified bioactive compounds in drug discovery. To find potential antivirals from TCMs, we first made a library of 373 crude extracts from 128 TCMs by aqueous extraction or organic-solvent extraction. With a previously established cell-based HTS platform , we perfsubtypes b, indicaR. rosea by various extraction methods  of 0.25 µg/mL against EBOV pseudovirion, and an IC50 value of 4.0 µg/mL against the MARV pseudovirion, respectively  of 136 µg/mL , ellagic, and gallic acid were evaluated in HeLa cells against infectious EBOV infection in a BSL-4 facility. As shown in R. rosea extract, ellagic acid, and gallic acid displayed good inhibition against infectious EBOV in HeLa cells. R. rosea extract showed an IC50 value of 3.9 µg/mL, ellagic acid, and gallic acid showed an IC50 value of 10.5 µM and 25.4 µM, respectively. The cell toxicity of R. rosea extract, ellagic acid, and gallic acid were also evaluated in HeLa cells, with CC50s at 64 µg/mL, 141 µM, 241 µM, and selectivity indices at 16.4, 13.4, and 9.5, respectively. The antiviral activities were also examined against infectious MARV in HeLa cells, but only R. rosea extract exhibited ~75% inhibition at 25 µg/mL, the highest concentration tested , ellagic acid (30 µM) or gallic acid (30 µM), and several control compounds. HIV/EBOV virions were allowed to attach to A549 cells at 4 °C and unattached virus was washed free. Cell uptake of virus was then initiated by shifting the incubation temperature to 37 °C. TCM extract or compounds were introduced at various time points to evaluate their antiviral effects. As shown in R. rosea extract showed a similar trend as the cathepsin B inhibitor CA-074 (100 µM), but not that of heparin or zidovudine, suggesting that it may act at the same site as the cathepsin B inhibitor in the endosome. R. rosea extract and the two active components were also evaluated with benztropine. Benztropine started to lose its inhibitory effect when it was added at 1 h post entry and beyond, as consistent with its reported inhibition of EBOV-GP mediated fusion [R. rosea extract, ellagic, and gallic acid each showed a similar time of loss of antiviral activity trend as benztropine, indicating that they may act at a similar post-attachment step, as benztropine, which blocks the infection of EBOV prior to viral/endosome membrane fusion  in an effort to identify potential antiviral agents against filoviruses and other emerging viral pathogens, and showed that extracts of the plant Since the glycoprotein (GP) of Ebola (and Marburg) virus is the only viral surface protein that is responsible for binding to the surface receptor(s) and mediating the fusion of viral/cell membranes in the endosomal compartments , it has R. rosea extract is an effective anti-filoviral agent, particularly against Ebola virus. R. rosea extract had an IC50 value of 0.25 µg/mL in the pseudovirus infection assay and an IC50 value of 3.9 µg/mL in an infectious EBOV infection assay, respectively, while it inhibited Marburg virus with an IC50 value of 4.0 µg/mL in pseudovirus assay and an IC50 value of ~25 µg/mL in infectious virus assay, respectively (R. rosea is better than that (IC50 ~6.25 µg/mL) of the best reported TCM Prunella vulgaris extract so far [In contrast to synthetic small molecules, TCMs  have better coverage of biologically relevant chemical space and are more likely to reach the intracellular site of action ,37. In fectively . The antt so far .R. rosea has been extensively characterized and more than 140 chemicals have been identified, and roughly 50 polar compounds can be isolated from the water alcoholic extracts, including glycosides, organic acids, and phenolics [R. rosea  in the extract are involved in blocking Ebola virus entry in an additive or synergistic manner with ellagic acid and gallic acid. In addition, the much less anti-MARV activities of both the acids than the R. rosea extract also suggest that other component(s) may target glycoprotein in a virus-specific way. Further R. rosea fractionation studies are needed to identify and characterize those components.henolics . Here, wR. rosea . In addiactivity a. Ellagiectively . When coR. rosea, ellagic acid and gallic acid block Ebola virus entry is not clear at present. Gallic acid has been reported to inhibit the entry of human rhinoviruses and the Herpes Simplex Virus type 1 virus by preventing virion attachment to the cell surface [R. rosea extract after EBOV binding to the cell surface (after time point 0 h) still strongly inhibited EBOV entry, suggesting that the binding step is not the primary target by gallic acid or R. rosea extract [R. rosea target endosomal calcium channels, called two pore channels (TPCs), like tetrandine, which is a bio-benzylisoquinoline alkaloid isolated from a TCM plant, Stephania tetrandra [Mechanistically, how the extracts of  surface ,43. Howe extract . The tim extract ,28. Furtetrandra . These fR. rosea as a potent EBOV entry agent. This TCM has potent anti-Ebola activity and proven safety profile, making it a promising antiviral for further development. Though gallic and ellagic acids have been identified as active components, further detailed fractionation and chemical research is needed to purify other active ingredients and to elucidate how these chemicals act synergically in inhibiting filovirus entry.In conclusion, we have identified


Spinal cord injury (SCI) is highly related to the devastating sensory and motor dysfunction.The GSE45006 gene expression profile dataset was downloaded from Gene Expression Omnibus, which was collected from 24 rats including 20 animals with injured T7 spinal cords using an aneurysm clip impact-compression injury model and killed after 1 day, 3 days, 1 week, 2 weeks, and 8 weeks and four sham-operated rats. Differentially expressed genes (DEGs) between the injured rats at each time point and the sham-operated rats were screened. DEGs commonly detected throughout different time points were further identified, followed by comparing the expression level of these DEGs at each time point between the injured spinal cord samples and controls. Pathway enrichment analysis of the common DEGs was performed.P < 0.05), with the most significant difference 1 day after SCI. The common DEGs were enriched in three pathways, namely Fcγ R-mediated phagocytosis, mitogen-activated protein kinase (MAPK) signaling pathway, and chemokine signaling pathway. AKT3 and RAC2 were enriched in all the three pathways; RAP1B in both MAPK signaling pathway and chemokine signaling pathway; and VAV1, LYN, and HCK in both Fcγ R-mediated phagocytosis and chemokine signaling pathway.The difference in the expression level of 416 common DEGs was significant between the injured spinal cord samples and the controls at each time point (AKT3, RAC2, VAV1, RAP18, LYN, and HCK may have critical roles in the pathological responses to SCI.This study has confirmed the occurrence of neuronal death, inflammation, and neuronal regeneration after SCI.  Spinal cord injury (SCI) can lead to sensory and motor dysfunction, manifesting a wide range of functional deficits depending on the level of injury , 2. FollIn the present study, the GSE45006 microarray data submitted by Chamankhah et al. that were collected from rats at different time points after SCI were downloaded and further analyzed. Here, we identified differentially expressed genes (DEGs) that were common throughout different time points. Then, based on the common DEGs, pathway enrichment analysis was performed and an interaction network was also constructed. These common DEGs may be essential for the pathological responses in rats after SCI, thus can be alternatives for screening new therapeutic targets for spinal cord-injured patients.http://www.ncbi.nlm.nih.gov/geo/) database. The annotation platform was GPL1355 [Rat230_2] Affymetrix Rat Genome 230 2.0 Array. As described by Chamankhah et al., a total of 24 animals were used and divided into six groups of four animals each. Twenty female Wistar rats in five groups underwent a T6-T8 laminectomy and then received a 35-g  moderate to severe aneurysm clip impact-compression injury at T7 for 1 min, which were killed after 1 day, 3 days, 1 week, 2 weeks, and 8 weeks following the surgery. For each operated animal, a tissue sample was collected from the T7 spinal cord epicenter for extraction of total RNA; meanwhile, the samples were also collected from the T7 spinal cord tissue of sham-operated rats in the control group.Gene expression profile dataset GSE45006 was downloaded from the Gene Expression Omnibus  method [Genes with |log2FC (fold change)| >1 and a false discovery rate (FDR) value <0.05 were selected as DEGs . The FDR) method .t test was used to determine whether there was significant difference in the expression level of these common DEGs between the injured spinal cord samples and the controls at each time point (P < 0.05) [The common DEGs throughout different time points were further screened, which were defined as successive DEGs. Then,  < 0.05) .https://david.ncifcrf.gov/home.jsp) bioinformatics resources consist of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene or protein lists [P < 0.05) [Database for Annotation, Visualization and Integrated Discovery  , 20.Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) can provide uniquely comprehensive coverage and ease of access to both experimental as well as predicted interaction information that is denoted with a confidence score . In the In total, 19,763 genes were obtained for DEG screening after removing null probes. A total of 3405, 2165, 1262, 1391, and 1841 DEGs were obtained at day 1, day 3, week 1, week 2, and week 8 following SCI, respectively.RAC2, RAP1B, VAV1, LYN, HCK, JUN, and the down-regulated ones, AKT3 , with the most significant difference 1 day after SCI (P = 1.42e-05), followed by less significant difference, 3 days (P = 0.000115), 1 week (P = 0.0001974), 8 weeks (P = 0.006143), and 2 weeks (P = 0.005989), after SCI successively , mitogen-activated protein kinase (MAPK) signaling pathway (P = 0.001434), and chemokine signaling pathway (P = 0.001592).According to the pathway enrichment analysis, the 416 common DEGs were enriched in three pathways (Table AKT3 (serine/threonine protein kinase 3) and RAC2 (Ras-related C3 botulinum toxin substrate 2) were enriched in all the three pathways  was involved in both the MAPK signaling pathway and the chemokine signaling pathway; VAV1 (guanine nucleotide exchange factor) and two Src family members, LYN (tyrosine protein kinase) and HCK (hemopoietic cell kinase), were involved in both the Fcγ R-mediated phagocytosis and the chemokine signaling pathway  and p38 MAPK , mediate inducible nitric oxide synthase (iNOS)-induced spinal neuron degeneration, which is a general way to cause neuronal apoptosis by generating nitric oxide (NO) after acute traumatic SCI [Chamankhah et al. have performed both time point and time-series analyses of the identified DEGs, which were further analyzed using Gene Ontology (GO) . In the atic SCI .AKT3 and RAC2 were enriched in all the three pathways, indicating their important role after SCI. The AKT  family consists of three members, Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ, which share a high degree of structural similarity [AKT3 is significantly down-regulated across all the time points overall in our study further validates their proposal.milarity . Pevianimilarity . The finRAC2 was up-regulated after SCI in our study, which is consistent with the finding that Rho activity is raised after SCI [The Rho guanosine triphosphatases (GTPases) comprising RAC1 and RAC2 are key regulators of cytoskeletal dynamics and affect many cellular processes, including cell polarity, migration, vesicle trafficking, and cytokinesis . Overallfter SCI . Activatfter SCI , 32. Furfter SCI . c-JUN, fter SCI , 35, whiVAV1, LYN, and HCK were involved in both the Fcγ R-mediated phagocytosis and the chemokine signaling pathway. VAV1 is engaged in the regulation of T cell migration [HCK and LYN, with similar roles in a signal pathway, were both up-regulated in our study, which is consistent with the up-regulation of LYN observed by Yang et al., who also studied the peripheral nerve injury-induced modification of genes in Sprague-Dawley rat dorsal spinal cords using both microarray analysis and immunohistochemistry [RAP1B (Ras-related protein Rap-1B) was also observed to participate in both the MAPK signaling pathway and the chemokine signaling pathway. Schwamborn and Püschel have discovered that localization of the GTPase RAP1B (Ras-related protein Rap-1B) to the tip of a single neurite is a decisive step in determining which neurite becomes the axon [Additionally, igration , and thehemistry . Meanwhithe axon ; thus, tAKT3, RAC2, VAV1, RAP1B, HCK, and LYN, that are involved in the above three pathways may be essential for the pathological responses to SCI, which may be considered as candidate genes for the targeted therapy of SCI. Our work has deepened our understandings into the molecular mechanisms underlying SCI. However, since our findings here were obtained by bioinformatics methods, they should be taken prudently and further validated by experimental proofs before any clinical use.In summary, we identified genes that were differentially expressed at different time points after SCI in rats, as well as three pathways, Fcγ R-mediated phagocytosis pathway, MAPK signaling pathway, and chemokine signaling pathway, through which these DEGs were speculated to respond to SCI. This further proves that neuronal death, inflammation, and neuronal regeneration occur after SCI, especially inflammation. And, DEGs,


We tested ‘HOSUT’, a winter wheat line expressing a grain-targeted sucrose transporter of barley in response to combinations of CO2 enrichment, a heat wave, and high nitrogen fertilization. Compared with wild-type Certo, HOSUT had a superior performance for grain yield, aboveground biomass, and ears per plant, obviously due to transgene activity in developing grains and young vegetative sinks. HOSUT grains were larger and contained more endosperm cells. HOSUT and high CO2 effects similarly improved phenological and yield-related traits. Significant HOSUT–CO2 interactions for biomass of stems, ears, grain yield, nitrogen yield, and grain number revealed that Certo was promoted by CO2 enrichment, whereas HOSUT responded weakly. CO2 enrichment strongly reduced and HOSUT effects weakly reduced grain nitrogen, storage proteins, and free amino acids. In contrast to CO2 enrichment, HOSUT effects did not impair grain micronutrient concentrations. Significant HOSUT–nitrogen fertilization interactions for ear biomass, grain yield, grain number per plant, and harvest index indicated that HOSUT benefited more from additional nitrogen. The heat wave decreased aboveground and ear biomass, grain yield, harvest index, grain size, and starch and water use, but increased grain sucrose concentration.Crop yield stability must be ensured under future climate conditions such as elevated CO We have shown earlier that in these lines sucrose uptake capacity and storage protein synthesis are stimulated  HOSUT24 transgenic line (‘HOSUT’) (2 (550 ppm); (iii) 200 kg N ha–1 versus 300 kg N ha–1 fertilization; and (iv) no heat wave (0) versus application of a ‘typical’ heat wave according to www.originlab.de/) and the statistical software program R (www.r-project.org/).A full factorial experiment was performed under controlled conditions in phytochambers with four factors, each with two levels. The factors were: (i) winter wheat (www.klasmann-deilmann.com/), and sand (2:2:1) for 2 weeks, followed by 8 weeks of incubation in vernalization chambers . For the four-factorial experiment, plants were transferred to a CO2-equipped climate chamber system  under controlled temperature, humidity, and photon flux density (PFD) involving two CO2 levels. Chamber conditions simulated the typical seasonal increments of day length, temperature, and light in South-Western Germany (Seeds of winter wheat cv. Certo and HOSUT were germinated in greenhouses (20 °C) in trays with substrate mix compost, substrate 2 (–1. Exposure in the climate chambers was performed at ambient (380 ppm) and elevated (550 ppm) CO2, in combination with normal (200 kg N ha–1) and increased N (300 kg N ha–1) by adding nutrient solution using calcium nitrate tetrahydrate, under both standard climate and application of a heat wave. The heat wave was modulated with respect to duration and daytime air temperature and humidity according to typical conditions in Western Europe , with 2 min at 50 °C, 40 cycles of 10 min at 95 °C, 15 s at 95 °C, and 1 min at 60 °C following a dissociation stage of 15 s at 95 °C, 15 s at 60 °C, and 15 s at 95 °C. Amplification efficiency was assessed with the LinRegPCR program. The wheat actin gene (accession no. AB181991) was used for normalization; actin primers were forward, 5'-GTG GAG GTT CTA CCA TGT TTC CTG-3'; and reverse, 5'-GCT AAG AGA GGC CAA AAT AGA GCC-3'.Wheat plants were grown in greenhouses . As each cell harbours one nucleus, the number of counted nuclei directly reflects the cell number.Endosperms from the greenhouse plants, 4–18 d after fertilization, were prepared by hand dissection, and nuclei of five endosperms each of Certo and HOSUT were isolated by chopping with razor blades in 1 ml of nuclei isolation buffer (www.gta-sensorik.com/). Mature grains were photographed using the Keyence VX 100K microscope unit (www.keyence.com/). A 5 g aliquot of grains per sample was ground using a ball mill to a fine powder. Sucrose and starch were determined by coupled enzyme assays in duplicate as described (http://www.elementar.de/).TGW, grain width, length, and area of mature dry grains from the plants grown in the climate chambers were determined using the digital seed analyser MARVIN . A 50 mg aliquot of dried and ground samples was digested with 2 ml of 69% nitric acid (HNO3)  using the high performance microwave reactor UltraClave IV from MLS GmbH (Germany). Digested samples were filled up to 15 ml final volume with deionized water from the Milli-Q® Reference System, Merck (Germany). Element standards  Mo, Na, Mg, P, S, Ca, Mn, Fe, Cu, Zn, and K were used as external standards, and Yttrium (ICP Standard Certipur® Merck Germany) was used as an internal standard for matrix correction.Macro- and microelements in mature grains were measured by inductively coupled plasma optical emission spectrometry  combined with the CETAC ASXPRESS™ ® system (www.waters.com/). Extraction and analysis were performed as described ; temperature, 60 °C; injection volume, 5 µl of albumin/globulin, 10 µl of gliadin, and 20 µl of glutenin extracts; elution solvents, (A) water/trifluoroacetic acid (TFA) , (B) acetonitrile/TFA ; gradient for albumins/globulins, 0 min 0% B, 0.5 min 20% B, 7 min 60% B, 7.1–11 min 90% B, 11.1–17 min 0% B; gradient for gliadins and glutenins, 0 min 0% B, 0.5 min 24% B, 20 min 56% B, 20.1–24.1 min 90% B, 24.2–30 min 0% B; flow rate, 0.2 ml min–1; detection, UV absorbance at 210 nm. PWG-gliadin (Free amino acid concentrations were measured by ultra performance liquid chromatography (UPLC) using a Waters Acquity UPLCThe date of flowering of the main stem was registered daily using the BBCH decimal code  and is pHvSUT1 transgene was strongly expressed in caryopses at 25 d after fertilization but also in young shoots, roots, and apices . The flowering start date for HOSUT, under both ambient and elevated CO2, was 90 d after planting, indicating that in contrast to Certo the high CO2 treatment did not shift the start of flowering of HOSUT. Furthermore, HOSUT under ambient and elevated CO2 started flowering 2.5 d and 1 d earlier than Certo, respectively (P<0.0001).The flowering start date for Certo was 92.5 d after planting under ambient CO2 similarly affected plant performance by increasing canopy height and organ biomass, especially that of stems and ears. The number of tillers was clearly lower for HOSUT compared with Certo, but was higher under elevated CO2 compared with ambient conditions. High N fertilization led to higher aboveground biomass, particularly for leaves. Heat wave treatment during early grain filling decreased aboveground biomass, especially that of ears  did not differ between lines, was lower at elevated COlication . A line×or Certo .2 increased TGW and grain width, whereas the heat wave application strongly decreased TGW, grain width, and length .There is no conflict of interest concerning this manuscript.Supplementary_Tables_Figure_S1Click here for additional data file.


Irpex lacteus is one of the most potent white rot fungi for biological pretreatment of lignocellulose for second biofuel production. To elucidate the underlying molecular mechanism involved in lignocellulose deconstruction, genomic and transcriptomic analyses were carried out for I. lacteus CD2 grown in submerged fermentation using ball-milled corn stover as the carbon source.Irpex lacteus CD2 efficiently decomposed 74.9% lignin, 86.3% cellulose, and 83.5% hemicellulose in corn stover within 9 days. Manganese peroxidases were rapidly induced, followed by accumulation of cellulase and hemicellulase. Genomic analysis revealed that I. lacteus CD2 possessed a complete set of lignocellulose-degrading enzyme system composed mainly of class II peroxidases, dye-decolorizing peroxidases, auxiliary enzymes, and 182 glycoside hydrolases. Comparative transcriptomic analysis substantiated the notion of a selection mode of degradation. These analyses also suggested that free radicals, derived either from MnP-organic acid interplay or from Fenton reaction involving Fe2+ and H2O2, could play an important role in lignocellulose degradation.I. lacteus CD2, in combination with low extracellular glycosidases cleaving plant cell wall polysaccharides into fermentable sugars, may account for high pretreatment efficiency of I. lacteus. Our study also hints the importance of free radicals for future designing of novel, robust lignocellulose-degrading enzyme cocktails.The selective strategy employed by The online version of this article (10.1186/s13068-018-1060-9) contains supplementary material, which is available to authorized users. The white rots are a large group of fungi that can efficiently decompose plant cell wall and utilize all its components including lignin, cellulose, and hemicellulose. The white rot fungi depend mainly on oxidoreductases and glycoside hydrolases for lignin and polysaccharides degradation, respectively . In contPhanerochaete chrysosporium and Ceriporiopsis subvermispora, have been sequenced and are publicly available [2O2) production collectively constitute the ligninolytic enzyme system in white rots [P. chrysosporium grown on aspen, pine, and spruce [C. subvermispora on aspen [Pycnoporus coccineus on aspen and pine [Phlebia radiata on spruce [Dichomitus squalens on aspen, spruce, wheat bran, and cotton seed hulls [With the advancement in next generation sequencing, whole genome sequencing is widely used to explore the underlying molecular mechanisms of lignocellulose degradation by white rot fungi , 7. To dvailable . Comparaite rots . With thd spruce –12, C. son aspen , Pycnopoand pine , Phlebian spruce , and Diced hulls . These sP. chrysosporium, simultaneously removes cellulose, hemicellulose, and lignin. The selective degraders, typified by C. subvermispora, instead preferentially destroy lignin rather than cellulose and hemicellulose at the early stage [P. chrysosporium confronting lignocellulose [C. subvermispora secrets mainly peroxidases oxidizing lignin initially, then switches to carbohydrate active enzymes (CAZymes) acting on cellulose and hemicellulose at the advanced stage [White rot fungi are further grouped into simultaneous and selective lignocellulose degraders . The forly stage . Correspellulose . In conted stage .Irpex lacteus CD2, a white rot basidiomycete with potent lignocellulose-degrading ability [I. lacteus CD2 preferentially degrades lignin at the early stage, followed by sharply elevated cellulose degradation rate at the advanced stage [I. lacteus strain Fr. 238 617/93 [I. lacteus strain F17 was reported [I. lacteus strain Fr. 238 617/93 grown on wheat straw has also been studied [I. lacteus. However, with these achievements, the molecular mechanism underlying the selective-like mode of lignocellulose degradation by I. lacteus and its implication remain unknown. The genomic information of I. lacteus has not yet been linked to transcription of genes relevant to lignocellulose degradation. There are no systematic analyses available for I. lacteus CD2 (and other strains) grown on corn stover, which is an agricultural residue produced in large amounts and can be used as a feedstock for second-generation biofuels [I. lacteus CD2 was sequenced and analyzed, combing with biochemical and transcriptomic analyses to elucidate the molecular mechanism on its efficient deconstruction of lignocellulose in corn stover.The simultaneous and selective paradigms of white rots are defined primarily based on their degradation patterns associated with woody lignocellulose. Interestingly,  ability , displayed stage . The sam8 617/93 . Recentlreported . The sec studied . These iI. lacteus CD2 to a 79-fold coverage using a combination of Illumina HiSeq 2000 platform and the PacBio RS 3rd generation technology. The genome of I. lacteus CD2 was distributed in 280 contigs with an assembly of 43.16 Mb. More than half of the total sequence and 69% of the 10,853 predicted genes were in the six largest contigs. Above 92% of the genes could be assigned with a definitive function through homology search against the KEGG, KOG, GO, NR, UniProt, and Tremble databases. The summaries of functional annotations of the genome and the top 50 PFAM domains are shown in Additional files I. lacteus CD2 , which is able to cleave various kinds of linkage in lignin in the presence of appropriate mediators [I. lacteus CD2 had DyPs  oxidoreductases (AA3) and seven copper radical oxidases (AA5) involved in extracellular H2O2 generation , and oxalate were involved in stimulating ligninolytic enzymes production and depolymerizing lignin . Heme warogenase . Besides enzymes , 34. FouI. lacteus CD2 could be grouped into 8 clans containing 17 families with a large portion of the members originating from gene duplication [I. lacteus CD2 showed an expansion of the CYP53 clan P450 members involved in lignin metabolism, compared with that in model white rot fungus P. chrysosporium [In addition, P450s are indispensable in intracellular metabolism of lignin metabolites and related compounds, and widely distributed in the white rots . The 130lication . I. lactosporium , 38.Irpex lacteus CD2 encoded a total of 273 CAZymes, which could be grouped into 182 glycoside hydrolases (GHs), 66 glycosyltransferases (GTs), 5 polysaccharide lyases (PLs), and 20 carbohydrate esterases (CEs), respectively. The cellulose-degrading GHs in I. lacteus CD2 consisted of cellobiohydrolases (GH6 and GH7), endoglucanases , and β-glucosidases (GH1 and GH3). The hemicellulose-degrading GHs were composed of xylanases (GH10), xylosidase (GH43), arabinofuranosidase (GH51), and acetylxylan esterase (CE1).I. lacteus CD2 encoded 17 LPMOs potentially involved in stimulating cellulose and/or hemicellulose degradation [Except for enzymatic hydrolysis, the oxidative cleavage by lytic polysaccharide monooxygenases (LPMOs) was another important route to promote polysaccharide degradation , 40. I. radation . Fifteenradation .I. lacteus CD2 genome. Instead, they existed in cluster, particularly for those enzymes belonging to the same CAZyme family. These included a triplet of GH10, a triplet of GH43, a pair of GH5, and a pair of GH18 genes , which are important enzymes involved in lignin degradation by white rots [I. lacteus CD2 [ncreased , I. lactite rots , 13, 48,ite rots , 50. Minite rots , was simteus CD2 .Fig. 2TiI. lacteus CD2 cultured in media containing either lignocellulose or glucose on day 3 and day 6, respectively, and used for RNA-seq transcriptomic analysis. Corresponding to the temporal expression of ligninolytic enzymes and polysaccharides degrading enzymes, the expression levels of a large number of genes were significantly altered (≥ 2-fold)  Fig. . Comparild) Fig. . KEGG anld) Fig. .Fig. 3DiI. lacteus CD2 was mainly through MnPs rather than DyPs and LiPs at the early stage. As for H2O2 generating oxidases, two glyoxal oxidase (GLOX) genes largely accumulated in LC3d but then quickly diminished in LC6d. AOX1 was expressed to a comparably high level in LC3d and LC6d  were highly induced in LC3d but rapidly decreased in LC6d Fig. . In compI. lacteus CD2 [Low molecular weight compounds such as heme, aromatic compounds, and oxalate play important roles in efficient lignin degradation. Five key genes in the heme biosynthesis pathway were up-regulated in LC3d and the interfering siroheme biosynthesis pathway was repressed . Despite this similarity, however, I. lacteus CD2 encodes higher numbers of plant cell wall polysaccharide-degrading enzymes, with 182 GHs compared to 161 GHs in I. lacteus F17 [I. lacteus CD2 does not have a laccase, but have 7 MnPs, 2 LiPs, and 4 DyPs, while I. lacteus F17 bears one laccase, one LiP, 13 MnPs but no DyP. The study of strain F17 focused on detoxification of synthetic dyes but not in pretreatment of lignocellulose [I. lacteus F17 is closest to C. subvermispora, a selective lignin degrader [I. lacteus CD2 is very close to the selective white rot fungus P. carnosa.Previous to our study, there was only one brief report on the genome sequence of an rain F17 . In thisteus F17 . In addiellulose , 57. Thedegrader . Taxonomdegrader –60, whilI. lacteus CD2 presented here provides an excellent platform for subsequent biochemical and transcriptomic analyses of this fungus grown on lignocellulose. The carbon source used for submerged shake flask fermentation in this study was ball-milled corn stover, whose natural physical structure has been disrupted to some extent. However, the expression pattern of extracellular lignocellulose-degrading enzymes was still very similar to that of this fungus grown in solid-state fermentation (SSF) on corn stover [The sequenced and annotated genome of n stover . Both thI. lacteus CD2 exhibited Mn2+-dependent oxidation ability for phenolic lignin model compounds [2+ greatly improved the pretreatment efficiency of corn stover by I. lacteus CD2 [I. lacteus to generate free radicals, which can mediate oxidation of non-phenolic lignin compounds [I. lacteus CD2 growth on corn stover.The large accumulation of MnP transcripts and enzymatic activities at the early stage highlights their importance involved in lignin degradation. Indeed, two recombinant MnPs from ompounds . These fteus CD2 . MnPs arteus CD2 . Howeverompounds . As a suI. lacteus CD2 [3+-reducing activity was detected during all the culture periods. It was noteworthy that the peak of Fe3+-reducing activity appeared at the very early stage (day 2) even before the MnPs (day 3). Fe2+ can react with H2O2 via Fenton reaction and produce hydroxyl radical, attacking both lignin and plant cell wall polysaccharides [3+-reducing activity rapidly decreased to normal state from day 3 to day 9 in lignocellulose medium, which was at a level similar to that in glucose medium , a range of AA3 and AA5 auxiliary enzymes were highly enriched (Table 2O2 for both MnPs and Fe2+. At the late stage (LC6d), part of AA3 and AA5 enzymes was still highly expressed , when MnPs are to be expressed, I. lacteus CD2 up-regulates the Fe3+-reducing power. Fe2+ reacts with low level of H2O2 to produce hydroxyl radical for an initial attack and breakage of interlinking lignocellulose network. At the early stage (days 3–4), MnPs are predominantly expressed and directly oxidize phenolic lignin compound. With assistance of mediators such as oxalate, non-phenolic lignin components are also oxidized by MnPs. Cellulase and xylanase are beginning to accumulate, but still not to the highest level. At the late stage , MnPs disappear, but part of enzymes responsible for H2O2 generation are highly expressed, which allow production of H2O2 and subsequent hydroxyl radical. Cellulase and xylanase are abundantly expressed at this stage, decomposing cellulose and hemicellulose at the maximal speed. In a model described previously [I. lacteus pretreatment. Indeed, our findings support this hypothesis, because in LC3d, the transcription of β-glucosidase and the enzymatic activity were very low. With this in mind, our findings corroborate the idea that selective expression of lignin-degrading enzymes at the early stage could be an additional key factor for high saccharification efficiency of lignocellulose after I. lacteus CD2 biopretreatment. Selective removal of lignin at the early stage exposes cellulose ready for enzymatic hydrolysis, while low expression of extracellular β-glucosidase keeps cellulose in long-chain, non-fermentable form. These two characters enable I. lacteus a suitable white rot for pretreating grass feedstock for biofuel production with high efficiency. We also notice that the regulation of Fe3+-reducing activity, as well as hydroxyl radical generation, is complex. Since high concentrations of free radicals and H2O2 are harmful for both enzymes and microbial hosts, this implies that, any attempts to harness the power of free radicals for formulating novel, robust lignocellulose-degrading enzyme cocktails should be delicately designed.Taken together, based on biochemical, genomic, and transcriptomic analyses, we could propose a model for lignocellulose degradation by eviously , the lacI. lacteus CD2. The genome annotation indicated that I. lacteus CD2 has a full array of enzymes for lignin, cellulose, and hemicellulose degradation. Both biochemical and transcriptomic analyses indicated that the fungus employed a selective strategy for degradation of lignocellulose in this submerged fermentation, a phenomenon reminiscent to that in solid-state fermentation on corn stover. This strategy, in combination with low extracellular glycosidase activity, forms the basis for its high efficiency in biological pretreatment of corn stover for saccharification.In this study, we sought to decipher the molecular mechanism underlying efficient lignocellulose degradation by Irpex lacteus CD2 was isolated from Shennongjia Nature Reserve  and preserved in the Institute of Environment and Resource Microbiology, Huazhong University of Science and Technology, Wuhan, China. I. lacteus CD2 was maintained at 4 °C on potato-dextrose agar plates. The inoculum was pre-cultured in potato-dextrose broth for 7 days at 28 °C, transferred into the modified Kirk’s medium as 10% (v/v) inoculum, and shaken at 150 rpm. The Kirk’s medium contained: ball-milled corn stover (or glucose) as carbon source, 10 g/L; ammonium tartrate, 0.2 g/L; KH2PO4, 2 g/L; MgSO4·7H2O, 0.71 g/L; CaCl2, 0.1 g/L; and 70 mL trace element solution. The trace element solution contains NaCl, 1 g/L; CoCl2·6H2O, 0.184 g/L; FeSO4·7H2O, 0.1 g/L; ZnSO4·7H2O, 0.1 g/L; CuSO4, 0.1 g/L; H3BO3, 0.01 g/L; Na2MoO4·2H2O, 0.01 g/L; KAl(SO4)2·12H2O, 0.01 g/L; and nitrilotriacetic acid, 1.5 g/L [ 1.5 g/L .I. lacteus CD2 was extracted using an improved cetyltrimethylammonium bromide (CTAB) method [The genomic DNA of ) method  from the) method .I. lacteus CD2 genome, the genome assembly was first analyzed for simple sequence repeat using the MISA software and masked by RepeatMasker, RepeatProteinMasker, and TRF using the RepBase library. Using the repeat-masked assembly, gene models were predicted using de novo prediction tools  and homology prediction tool (GeneWise) with default parameters. The de novo and homology-based gene models were merged to form a comprehensive and non-redundant reference gene models by EVM [To identify gene models in the s by EVM . The pres by EVM . The rRNs by EVM .I. lacteus CD2 and eight other Polyporales fungi including B. adusta, C. subvermispora, D. squalens, Ganoderma sp., P. carnosa, P. chrysosporium, P. placenta, and T. versicolor were collected, and a phylogenetic tree was constructed by using the MAFFT and FastTree software based on the deduced amino acid sequences of orthologous genes [The genome sequences of us genes , 73.I. lacteus CD2 protein models were subjected to a procedure combing BLAST and HMMer3 searches against sequence libraries and HMM profiles derived from the families of GHs, PLs, CEs, GTs, auxiliary activities (AAs), and CBMs featured in the CAZy database. The Class II peroxidases and DyPs were further confirmed by BLAST search against the PeroxiBase database [All database .I. lacteus CD2  were collected by centrifugation and then dried at 60 °C for 3 days. Lignin, cellulose, and hemicellulose components of corn stover were determined according to the procedure described by the National Renewable Energy Laboratory [For compositional analysis, the non-treated corn stover and that treated by boratory . For eac4  at 270 nm, in a buffer containing 50-mM pH 5.0 malonate, 1-mM MnSO4, and 0.1-mM H2O2. One unit of MnP activity was defined as the amount of enzyme that oxidized 1 μmol of Mn2+ per min at 25 °C [2+-independent activity was measured by monitoring the oxidation of 1-mM ABTS  at 420 nm, in a buffer containing 50-mM pH 5.0 malonate and 0.1-mM H2O2. One unit of Mn2+-independent activity was defined as the amount of enzyme that oxidized 1 μmol of ABTS per min at 25 °C [2+–ferrozine complex in the 50-mM pH 4.8 acetate buffer containing 0.3-mM FeCl3 and 4-mM ferrozine. One unit of iron-reducing activity was defined as the rate of absorbance increase at 562 nm/min. The CDH activity was determined by oxidation of 0.3-mM 2,6-dichloroindophenol sodium salt  at 520 nm in the presence of 30-mM lactose in the 50-mM pH 4.8 acetate buffer. One unit of CDH activity was defined as the amount of enzyme that oxidized 1 μmol of DCPIP per min at 25 °C [p-nitrophenyl butyrate (ε348 = 8321/M/cm) at 348 nm in the 50-mM pH 6.0 sodium phosphate buffer. One unit of esterase activity was defined as the amount of enzyme that released 1 μmol of p-nitrophenol per min at 25 °C [For lignin-degrading enzymes, MnP activity was measured by monitoring the oxidation of 1-mM MnSOat 25 °C . The Mn2at 25 °C . Iron-reat 25 °C . For celat 25 °C . The estat 25 °C .Irpex lacteus CD2 was grown in the modified Kirk’s medium containing corn stover or glucose as carbon source. The total RNA was extracted from mycelia on days 3 and 6 using the TRIZOL reagent  according to the manufacturer’s instructions. The total RNA was sent to Annoroad Genomics  for sample preparation and sequencing. All samples were in duplicate. The cDNA library was synthesized and prepared for sequencing using the Illumina mRNA-Seq Sample Prep Kit . Briefly, first, the poly(A) containing mRNA was purified from total RNA with poly(T) oligo-attached magnetic beads and fragmented. Next, the cleaved RNA fragments were reversely transcribed into first-strand cDNA using reverse transcriptase and random primers, followed by second-strand cDNA synthesis. Then, the DNA fragments were further subjected to end repair, addition of “A” bases, and adapters’ ligation. Finally, cDNA templates were purified and enriched by PCR amplification. The cDNA libraries were run in independent lanes and paired-end sequences of 125 bp were obtained with at least 4-Gb clean data for each sample using the Illumina Hiseq 2500.I. lacteus CD2 genome as a Ref. [p ≤ 0.05 and a false discovery rate (FDR) ≤ 0.01.The raw reads were trimmed and filtered using Trimmomatic software to remove adapters and low-quality bases . Then, cs a Ref. . All seqs a Ref. , and reas a Ref.  with a p−ΔΔCT method with the glycerol 3-phosphate dehydrogenase-normalized Ct values [Reverse transcription-quantitative PCR (RT-qPCR) of selected ligninolytic genes was used for confirmation of RNA-seq data, which was conducted using TransStart Green RCR SuperMix (TransGen) with gene-specific primers , LC6d versus LC3d (b).Additional file 9. KEGG enrichment analysis of differently expressed genes in comparisons of LC3d versus Glu3d (a), LC6d versus LC3d (b).Additional file 10. Verifying the differential expression as revealed by RNA-seq for selected lignocellulose-degrading genes by RT-qPCR.Additional file 11. Primers used for RT-qPCR.


ZT of BiCuSeO was pushed to a higher level. It motivates us to systematically summarize the recent reports on the BiCuSeO system. In this short review, we start with several attempts to optimize thermoelectric properties of BiCuSeO. Then, we introduce several opinions to explore the origins of low thermal conductivity for BiCuSeO. Several approaches to enhance thermoelectric performance are also summarized, including modulation doping, introducing dual-vacancies, and dual-doping, etc. At last, we propose some possible strategies for enhancing thermoelectric performance of BiCuSeO in future research.A BiCuSeO system has been reported as a promising thermoelectric material and has attracted great attention in the thermoelectric community since 2010. Recently, several remarkable studies have been reported and the  Thermoelectric (TE) materials have been widely developed to provide a direct solution for energy conversion between electricity and heat. As one of the most promising thermoelectric systems, the BiCuSeO family was first reported in 2010 ,7,8,9,10P4/nmm [2O2)2+ layers and anti-fluorite (Cu2O2)2− layers which stacking along the c-axis. (Bi2O2)2+ layers act as carrier reservoir while (Cu2O2)2− layers are responsible for transporting carriers [p-type semiconductor, in which holes are generated from either Cu- or Bi-vacancies which participate in transportation in the conductive (Cu2O2)2− layers. Due to its layered structure, BiCuSeO shows anisotropic transport behaviors along in-plane and out-plane directions for both electrical and thermal conductivity. The electrical and the thermal transport properties along the in-plane direction outperform those along the out-plane direction. However, the Seebeck coefficient is isotropic [BiCuSeO crystallizes in a layered ZrCuSiAs structure type with space group P4/nmm . The crycarriers . Pristinsotropic .2Te3-based systems [ZT) value of the BiCuSeO system has boosted to the maximum ZT of 1.5 [ZT subverts the general opinions that thermoelectric oxides usually have low ZT values due to poor electrical conductivity and high thermal conductivity.Compared with the state-of-the-art TE materials including lead and tin chalcogenides ,16,17,18 systems ,21,22,23 systems ,27,28,29T of 1.5 , from thSince the review paper summarized by Zhao et al. in 2014 , a lot o18 cm−3 [2+  doping [0.8Sr0.2CoO3 [2Se [It is well known that the thermal conductivity of BiCuSeO is impressively low. Thus, most recent attention has focused on improving the electrical transport properties through various attempting approaches. For instance, considering the carrier concentration of undoped BiCuSeO is as low as ~1 × 1018 cm−3 , the mos) doping ,30. Besi) doping ,36,37,38r0.2CoO3 , Cu2Se [oO3 [2Se ) could sx1−PbxCuSeO through an ultrafast self-propagating high-temperature synthesis (SHS) method. In addition, Yang et al. [0.94Pb0.06CuSeO and further explored the thermodynamic reaction mechanism in details. It was confirmed that the entire reaction process consists of four parts. External heating initiated two rapid SHS reactions  which emitted heat and drove the other two reactions . Through optimizing reaction parameters such as sample density and reaction atmosphere, etc. the ZT value could be enhanced to 1.2 at 923 K for Bi0.94Pb0.06CuSeO [Initially, it was suggested that polycrystalline BiCuSeO compounds should be synthesized through a multi-step solid-state reaction (SSR), which is complicated and time-consuming. Therefore, any facile or time-saving methods are advanced for future scale-up of resultant products. Ren et al.  exploredg et al.  adopted .06CuSeO .2O3 impurity. Compared with solid-state reaction, mechanical alloying is simple, less time-consuming, and much more convenient for large-scale productions. Taking into account these advantages, Wu et al. systematically investigated the mechanical alloying mechanism for BiCuSeO [2O3, Cu, and Se) powders used in the solid-state reaction, Bi, CuO, and Se were selected as starting powders to avoid the Bi2O3 second phase. Encouragingly, pure single phase BiCuSeO was obtained through ball milling at 500 rpm for 7 h. Apparently, the above results confirmed that mechanical alloying is an effective and promising method to synthesize BiCuSeO [Although the mechanical alloying (MA) method was applied to BiCuSeO systems in 2013 , it was  BiCuSeO , instead BiCuSeO ,24.2·V−1·s−1 obtained in polycrystalline BiCuSeO [ZT value. To date, it is still a challenge to synthesize large size BiCuSeO single crystal. Dong et al. [3 were obtained [x1−PbxCuSeO film with high texture orientation through the pulsed laser deposition technique on the SrTiO3 (001) substrates, a high power factor of ~1.2 mW·m−1·K−2 at 673 K was realized. Therefore, a higher ZT can be expected in BiCuSeO single crystals. However, the challenge still lies in obtaining sufficiently large single crystals to investigate the thermal transport properties.In BiCuSeO systems, due to the layered structure, carrier scattering along the in-plane direction is much smaller than that along the out-plane direction. Therefore, we can expect a much higher carrier mobility in the single crystal than that of 22 cm BiCuSeO , hence ag et al.  made a sobtained . Meanwhiobtained  synthesiApart from various attempts to enhance thermoelectric performance mentioned above, the interested readers are encouraged to refer to these extensive works that have been done on BiCuSeO systems, such as ultrathin BiCuSeO nanosheets , elasticZT value of BiCuSeO is derived from its low thermal conductivity (κ) in temperatures ranging from 300 K (~0.9 W·m−1·K−1) to 923 K (~0.45 W·m−1·K−1) [D ~ 243 K) [The large m−1·K−1) . The lowm−1·K−1) , weak ch~ 243 K) . Thus, aDing et al.  systematThe mode Grüneisen parameters were calculated by quasi-harmonic approximation. As shown in Saha  carried Eu  ~ 53 cm−1) and LaCuSeO  ~ 62 cm−1). The larger displacement in heavy Bi atoms is observed, which leads to higher anharmonic effects. Eu  in BiCuSeO behaves as a quasi-acoustic mode [tic mode , the acotic mode  used firVaqueiro et al.  believe 2  in the aFe4Sb12  is compaaFe4Sb12  in BiCuSaFe4Sb12  contribu−1) optical phonons contributed more than one-third to the lattice thermal conductivity through first-principles calculations. Kumar et al. obtained a similar result that optical phonons can dramatically reduce lattice thermal conductivity κlat by a first principle phonon analysis [Generally, the contribution of high-frequency optical phonons to total lattice thermal conductivity is so small that it can be ignored. However, in the BiCuSeO system, Shao et al.  found thanalysis . Both rex1−MxCuSeO  [0.875Ba0.125CuSeO using scanning transmission electron microscopy (STEM) [3 second phase in BiCuSeO matrix which played a positive role for further reduction of the thermal conductivity. The solubility limit of Ba in BiCuSeO was found to be less than 5% [ZT value was obtained in Bi0.875Ba0.125CuSeO, where as high as 12.5% Ba was dissolved in the matrix. The visual observation of the second phase BaSeO3 through STEM was a reasonable explanation of low thermal conductivity. As shown in 3 which widely exist in the Bi0.875Ba0.125CuSeO, but there are no precipitates in Bi0.95Ba0.05CuSeO.Extensive experiments confirmed that alkaline-earth metals doping is effective to enhance the thermoelectric performance of Bi Sr, Ba) ,30,63,64y (STEM) , and fou than 5% . However3 second phase, the lattice thermal conductivity of Bi0.875Ba0.125CuSeO is lower than that of pristine BiCuSeO and Bi0.95Ba0.05CuSeO in the entire temperature range, as shown in Benefitting from the all-scale phonons scattering from the interface of the layered structure, the Ba-Bi point defects and the BaSeO2·V−1·s−1) and low carrier concentration (~1.1 × 1018 cm−3) [0.875Ba0.125CuSeO with high carrier concentration (~1.2 × 1021 cm−3) and low carrier mobility (~2.1 cm2·V−1) [0.875Ba0.125CuSeO each accounting for 50 percent. Meanwhile, the Fermi level of modulation doping is higher than that of uniformly doping and lower than that of the undoped sample. There is a proceeding that holes flow from doped phase to undoped phase due to the successively small difference of chemical potential. Thus, the heavy doped phase sustains the high carrier concentration while the undoped phase provides a path that is easy to conduct, and both of these are responsible for the high electrical conductivity.The electrical conductivity is determined by carrier concentration (n) and carrier mobility (μ). On the one hand, heavy doping can improve the carrier concentration significantly. On the other hand, the excessive dopants decrease the carrier mobility obviously because of the intensive ionized impurity scattering . Therefo18 cm−3) . In cont0.875Ba0.125CuSeO, undoped BiCuSeO, uniformly doped Bi0.875Ba0.125CuSeO and heavily doped Bi0.75Ba0.25CuSeO. Benefited from the high electrical concentration and mobility, the electrical conductivity of modulation doped Bi0.875Ba0.125CuSeO is higher than that of both undoped BiCuSeO and uniformly doped Bi0.875Ba0.125CuSeO at 300–923 K, as shown in −1·K−2 at 923 K, as shown in ZT value 2− layers where Cu vacancies located being responsible for the carrier transmission in BiCuSeO, the increasing hole concentration can effectively enhance the electrical conductivity. Theoretically, the negatively charged centers of positive vacancies can annihilate positrons. As shown in 0.975CuSeO and BiCu0.975SeO, respectively, while positrons are mostly distributed around Bi vacancies in Bi/Cu dual vacancy BiCuSeO (Li et al.  confirme BiCuSeO d.ZT value of dual vacancy Bi0.975Cu0.975SeO is higher than those of monovacancy Bi0.975CuSeO and BiCu0.975SeO in the whole temperature range, resulting in a relatively high ZT of 0.84 was reached at 750 K for the dual vacancy sample (The projection of the positron density distribution proves the existence of carrier transmission from Bi vacancies to Cu vacancies in Bi/Cu dual vacancies BiCuSeO. It is easy to understand why the electrical conductivity of the dual vacancy sample is larger than that of monovacancy samples a. In addy sample f.n-type BiCuSeO, all of these are eagerly expected in the BiCuSeO system.In this review, we introduced the latest achievements and progress in BiCuSeO system, including enhancing thermoelectric properties through doping and compositing, developing less time-consuming methods, attempting to synthesize single crystals, exploring the origins of low thermal conductivity, and typical examples to enhance thermoelectric performance of BiCuSeO systems. Aside from the progress mentioned above, there is still room left for further focusing, such as the synthesis of BiCuSeO single crystal and a high performance


Eremostachys azerbaijanica Rech.f., were screened for their antimalarial properties by cell free 𝛽-hematin formation assay. Dichloromethane (DCM) extracts of both parts of plant showed significant antimalarial activities with IC50 values of 0.949 ± 0.061 mg/mL in aerial parts and 0.382 ± 0.011 mg/mL in rhizomes. Bioactivity-guided fractionation of the most potent part (DCM extract of rhizomes) by vacuum liquid chromatography (VLC) afforded seven fractions. Two fractions [100% Ethyl acetate (EtOAC) and 100% Methatol (MeOH)] showed considerable antimalarial activity with IC50 values of 0.335 ± 0.033 mg/mL and 0.403 ± 0.037 mg/mL, respectively.Six extracts with different polarity from aerial parts and rhizomes of According to GC-MS analysis, the sesquiterpene, steroid and coumarin derivatives are the main constituents of the most potent fractions; therefore, it seems that the anti malarial activity of these fractions may be related to the presence of these types of compounds. Plasmodium and it is transmitted via the bite of an infected female Anopheles sp mosquito  against the parasite  and 9. Nparasite , 10.Artemisia annua by Chinese scientists in 1960 have provided a new class of highly effective antimalarial drugs and the global demand for artemisinin-based combination therapy (ACT) that has developed since its recommendation by the World Health Organization in 2002  that occur mainly in central Asian countries. Some Eremostachys species such as E. glabra, E. laciniata, E. lanata and E. labiosa are well distributed in Iran , Hematin porcine, Chloroquine diphosphate, Sodium dodecyl sulfate (SDS), Sodium acetate, Magnesium sulfate, Sodium hydrogen phosphate, Sodium chloride, Potassium chloride, Sodium hydroxide, Glucose, and Sodium bicarbonate were purchased from Sigma-Aldrich, Chemical Company (United kingdom), Oleic acid from Fluka (India), Dimethylsulfoxide (DMSO), Hydrochloric acid (HCl), and silica gel 60 (0.040–0.063 mm) from Merck (Germany).Plant material:E. azerbaijanica Rech.f. were collected respectively during July and September 2012 from Sahand mountains in East Azarbaijan province in Iran 37.759 (37° 45ʹ 32.4″ N) latitude 45.9783 (45° 58ʹ 41.9″ E) longitude and altitude 1850 m above sea level. A voucher specimen (No.738) has been retained in the herbarium of the Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.The aerial parts and rhizomes of ExtractionE. azerbaijanica (100 g each) were Soxhlet-extracted successively with n-hexane, DCM and MeOH (1.1 L each). All these extracts were separately concentrated using a rotary evaporator at a maximum temperature of 45 °C  with solvent mixtures of increasing polarities: EtOAC/n-Hexan (10: 90), EtOAC/n-Hexan (20 : 80), EtOAC/n-Hexan (40 : 60), EtOAC/n-Hexan (60 : 40), EtOAC/n-Hexan (80 : 20), EtOAC/n-Hexan (100 : 0), and methanol (100). All the fractions were fully dried using a rotary evaporator at a maximum temperature of 45 °C.1.7 g of DCM extract obtained from rhizomes of Cell free 𝛽-Hematin Formation Assay.et al.  and the hemozoin pellet was repeatedly washed with incubation (15min at 37 °C with regular shaking) in 2.5% (w/v) SDS in phosphate buffered saline followed by a final wash in 0.1 M sodium bicarbonate until the supernatant was clear . After the final wash, the supernatant was removed and the pellets were dissolved in 1mL of 0.1M NaOH before determining the hemozoin content by measuring the absorbance at 400 nm (Spectronic Genesys spectrophotometer). The results were recorded as % inhibition (I%) of heme crystallization compared to negative control (DMSO) using the following equation:𝐼% = [(AN−AS)/AN] ×100, where AN is absorbance of negative control and AS is absorbance of test samples.GC-MS Analysis of potent FractionsGC–MS analyses were carried out on a Shimadzu QP-5050A GC–MS system equipped with a DB-1 fused silica column ; oven temperature, rising from 50 °C to 310 °C at a rate of 3 °C/min; injector temperature, 280 °C; carrier gas, helium at a flow rate of 1.3 mL/min ; split ratio, 1:10; ionization energy, 70 eV; scan time, 1 s; mass range, 30–600 amu.Identification of ComponentsIdentification of the constituents was based on direct comparison of the retention times and mass spectral data with those for standard compounds, and computer matching with the NIST 21, NIST 107 and WILEY229 library, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature (Adams 2004).Statistical Analysis50 and IC90 values were calculated from nonlinear regression analysis.All experiments were done in triplicate measurements and presented as the Mean ± SD. Data were analyzed by Excel 2010 Microsoft. The ICPlasmodium parasite uses host erythrocyte hemoglobin as a major nutrient source of amino acids. Degradation of hemoglobin produces brown heme crystals which are toxic for parasite  but obtained results revealed that the n-hexane and the MeOH extracts of both parts of E. azerbaijanica did not show any significant antimalarial activities. The inhibition of 𝛽-hematin formation expressed as percentage (I%) and standard deviations (𝑛 = 3) are given for each fraction. IC50 and IC90 values were measured graphically by plotting concentrations versus percentage of inhibition.50 values of DCM extracts from aerial parts and rhizomes were 0.949 ± 0.061 and 0.382 ± 0.011 mg/mL, and were 1.836 ± 0.071 and 0.800 ± 0.019mg/mL for IC90 values of these extracts, respectively.ICExperiment was performed in triplicate and expressed as Mean ± SD.50 values of 0.335 ± 0.033 and 0.403 ± 0.037 mg/mL and IC90 values of 0.410 ± 0.130 and 0.949 ± 0.035 mg/mL, respectively. Two other fractions of VLC  demonstrated remarkable anti-malarial effects with close IC50 and IC90 values (After fractionation of the most potent extract (DCM extract of rhizomes) by VLC method over silica gel, the results showed that among the seven different polarity fractions, 100% EtOAC and 100% MeOH fractions had considerable anti-malarial activity with IC0 values .GC-MS analysis of the most potent fractions showed various compounds with different functional groups. The results of GC-MS analysis of seven VLC fractions from DCM extract of rhizomes have given in The main groups of compound identified by GC-MS analysis of fractions were steroide derivatives, sesquiterpene derivatives, coumarins, fatty acid derivatives, polycyclic aromatic hydrocarbons and some linear structures with functional groups such as aldehyde, ketone, ether and alcohol.50 and IC90 values could be reduced in 20% EtOAC/n-Hexane fraction by entirely removing the lipids and more purification of potent constituents. These results are in agreement with previous research that showed the synergistic effect of lipids and other fatty acids in the mixtures of extracts with oleic acid in the assay  persuade us to focus on purifying the active components of these extracts and investigating further on animal models for in vivo evaluation.From the 6 extracts with different polarity of aerial parts and rhizomes of