Transcriptomic and metabolic signatures of diatom plasticity to light fluctuations.

Unlike in terrestrial and freshwater ecosystems, light fields in oceans fluctuate due to both horizontal current and vertical mixing. Diatoms thrive and dominate the phytoplankton community in these fluctuating light fields. However, the molecular mechanisms that regulate diatom acclimation and adaptation to light fluctuations are poorly understood. Here, we performed transcriptome sequencing, metabolome profiling, and 13C-tracer labeling on the model diatom Phaeodactylum tricornutum. The diatom acclimated to constant light conditions was transferred to six different light conditions, including constant light (CL5d), short-term (1 h) high light (sHL1h), and short-term (1 h) and long-term (5 days) mild or severe light fluctuation conditions (mFL1h, sFL1h, mFL5d, and sFL5d) that mimicked land and ocean light levels. We identified 2,673 transcripts (25% of the total expressed genes) expressed differentially under different fluctuating light regimes. We also identified 497 transcription factors, 228 not reported previously, which exhibited higher expression under light fluctuations, including 7 with a light-sensitive PAS domain (Per-period circadian protein, Arnt-aryl hydrocarbon receptor nuclear translocator protein, Sim-single-minded protein) and 10 predicted to regulate genes related to light-harvesting complex proteins. Our data showed that prolonged preconditioning in severe light fluctuation enhanced photosynthesis in P. tricornutum under this condition, as evidenced by increased oxygen evolution accompanied by the upregulation of Rubisco and light-harvesting proteins. Furthermore, severe light fluctuation diverted the metabolic flux of assimilated carbon preferentially toward fatty acid storage over sugar and protein. Our results suggest that P. tricornutum use a series of complex and different responsive schemes in photosynthesis and carbon metabolism to optimize their growth under mild and severe light fluctuations. These insights underscore the importance of using more intense conditions when investigating the resilience of phytoplankton to light fluctuations.

Chlorophyll fluorescence as a light signal enhances iron uptake by the marine diatom Phaeodactylum tricornutum under high-cell density conditions.

BACKGROUND: Diatoms usually dominate phytoplankton blooms in open oceans, exhibiting extremely high population densities. Although the iron uptake rate of diatoms largely determines the magnitude and longevity of diatom blooms, the underlying mechanisms regulating iron uptake remain unclear. RESULTS: The transcription of two iron uptake proteins, ISIP2a and ISIP1, in the marine diatom Phaeodactylum tricornutum was enhanced with increasing cell density, whereas the cellular iron content showed the opposite trend. When compared with the wild-type strain, knockdown of ISIP2a resulted in 43% decrease in cellular iron content, implying the involvement of ISIP2a in iron uptake under high-cell density conditions. Incubation of the diatom cells with sonicated cell lysate conditioned by different cell densities did not affect ISIP2a and ISIP1 expression, ruling out regulation via chemical cues. In contrast, ISIP2a and ISIP1 transcription were strongly induced by red light. Besides, chlorophyll fluorescence excited from the blue light was also positively correlated with population density. Subsequently, a "sandwich" illumination incubator was designed to filter out stray light and ensure that the inner layer cells only receive the emitted chlorophyll fluorescence from outer layers, and the results showed that the increase in outer cell density significantly elevated ISIP2a and ISIP1 transcription in inner layer cells. In situ evidence from Tara oceans also showed positively correlated between diatom ISIP transcripts and chlorophyll content. CONCLUSIONS: This study shows that chlorophyll fluorescence derived from neighboring cells is able to upregulate ISIP2a and ISIP1 expression to facilitate iron assimilation under high-cell density. These results provide novel insights into biotic signal sensing in phytoplankton, which can help to elucidate the underlying mechanisms of marine diatom blooms.

Co-suppression in Pyropia yezoensis (Rhodophyta) Reveals the Role of PyLHCI in Light Harvesting and Generation Switch.

The red macroalga Pyropia yezoensis is an economically important seaweed widely cultured in Asian countries and is a model organism for molecular biological and commercial research. This species is unique in that it utilizes both phycobilisomes and transmembrane light-harvesting proteins as its antenna system. Here, one of the genes of P. yezoensis (PyLHCI) was selected for introduction into its genome to overexpress PyLHCI. However, the co-suppression phenomenon occurred. This is the first documentation of co-suppression in algae, in which it exhibits a different mechanism from that in higher plants. The transformant (T1) was demonstrated to have higher phycobilisomes and lower LHC binding pigments, resulting in a redder color, higher sensitivity to salt stress, smaller in size, and slower growth rate than the wildtype (WT). The photosynthetic performances of T1 and WT showed similar characteristics; however, P700 reduction was slower in T1. Most importantly, T1 could release a high percentage of carpospores in young blades to switch generation during its life cycle, which was rarely seen in WT. The co-suppression of PyLHCI revealed its key roles in light harvesting, stress resistance, and generation alternation (generation switch from gametophytes to sporophytes, and reproduction from asexual to sexual).

RNA-seq between asexual archeospores and meiosis-related conchospores in Neopyropia yezoensis using Smart-seq2.

In the life cycle of Neopyropia yezoensis, a potential model system for marine macroalgae, both asexual archeospores and meiosis-related conchospores develop into thalli (gametophyte). To understand this special life phenomenon in macroalgae, we picked out the two kinds of spores (10-30 cells in each sample) and conducted RNA-seq using Smart-seq2. Comparative analysis showed that light capture and carbon fixation associated differentially expressed genes (DEGs) were upregulated in archeospores, thus indicating that archeospores are in a state of rapid vegetative growth. In conchospores, protein synthesis and degradation, especially molecular chaperone, associated DEGs were up-regulated, indicating that complex life activities might be occurring in conchospores. There were 68 genes related to DNA replication and repair expressed in conchospores, showing that active DNA replication might occur in conchospores. Moreover, we found that one conchospore specifically expressed DEG (py04595: DNA helicase) only in diploid stages (conchocelis, sporangial filament) and three archeospores specifically expressed DEGs only in haploid stages (thalli). These molecular level results indicated that conchospores were closer to diploid, and might be the meiotic mother cells of N. yezoensis. In addition, we found that the knotted-like homeobox gene (PyKNOX), which might relate to the transition of gametophyte from sporophyte, was only expressed in sporophyte generation but not expressed in conchospores, archeospores and thalli, indicating the morphogenesis of gametophyte sin N. yezoensis might require the inactivation of PyKNOX.

The carbonate concentration mechanism of Pyropia yezoensis (Rhodophyta): evidence from transcriptomics and biochemical data.

BACKGROUND: Pyropia yezoensis (Rhodophyta) is widely cultivated in East Asia and plays important economic, ecological and research roles. Although inorganic carbon utilization of P. yezoensis has been investigated from a physiological aspect, the carbon concentration mechanism (CCM) of P. yezoensis remains unclear. To explore the CCM of P. yezoensis, especially during its different life stages, we tracked changes in the transcriptome, photosynthetic efficiency and in key enzyme activities under different inorganic carbon concentrations. RESULTS: Photosynthetic efficiency demonstrated that sporophytes were more sensitive to low carbon (LC) than gametophytes, with increased photosynthesis rate during both life stages under high carbon (HC) compared to normal carbon (NC) conditions. The amount of starch and number of plastoglobuli in cells corresponded with the growth reaction to different inorganic carbon (Ci) concentrations. We constructed 18 cDNA libraries from 18 samples (three biological replicates per Ci treatment at two life cycles stages) and sequenced these using the Illumina platform. De novo assembly generated 182,564 unigenes, including approximately 275 unigenes related to CCM. Most genes encoding internal carbonic anhydrase (CA) and bicarbonate transporters involved in the biophysical CCM pathway were induced under LC in comparison with NC, with transcript abundance of some PyCAs in gametophytes typically higher than that in sporophytes. We identified all key genes participating in the C4 pathway and showed that their RNA abundances changed with varying Ci conditions. High decarboxylating activity of PEPCKase and low PEPCase activity were observed in P. yezoensis. Activities of other key enzymes involved in the C4-like pathway were higher under HC than under the other two conditions. Pyruvate carboxylase (PYC) showed higher carboxylation activity than PEPC under these Ci conditions. Isocitrate lyase (ICL) showed high activity, but the activity of malate synthase (MS) was very low. CONCLUSION: We elucidated the CCM of P. yezoensis from transcriptome and enzyme activity levels. All results indicated at least two types of CCM in P. yezoensis, one involving CA and an anion exchanger (transporter), and a second, C4-like pathway belonging to the PEPCK subtype. PYC may play the main carboxylation role in this C4-like pathway, which functions in both the sporophyte and gametophyte life cycles.

Provision of carbon skeleton for lipid synthesis from the breakdown of intracellular protein and soluble sugar in Phaeodactylum tricornutum under high CO2.

BACKGROUND: Increasing CO2 emissions have resulted in ocean acidification, affecting marine plant photosynthesis and changing the nutrient composition of marine ecosystems. The physiological and biochemical processes of marine phytoplankton in response to ocean acidification have been reported, but have been mainly focused on growth and photosynthetic physiology. To acquire a thorough knowledge of the molecular regulation mechanisms, model species with clear genetic background should be selected for systematic study. Phaeodactylum tricornutum is a pennate diatom with the characteristics of small genome size, short generation cycle, and easy to transform. Furthermore, the genome of P. tricornutum has been completely sequenced. RESULTS AND DISCUSSION: In this study, P. tricornutum was cultured at high and normal CO2 concentrations. Cell composition changes during culture time were investigated. The 13C isotope tracing technique was used to determine fractional labeling enrichments for the main cellular components. The results suggested that when lipid content increased significantly under high CO2 conditions, total protein and soluble sugar contents decreased. The 13C labeling experiment indicated that the C skeleton needed for fatty acid C chain elongation in lipid synthesis under high CO2 conditions is not mainly derived from NaHCO3 (carbon fixed by photosynthesis). CONCLUSION: This study indicated that breakdown of intracellular protein and soluble sugar provide C skeleton for lipid synthesis under high CO2 concentration.

Effect of iron on the growth of Phaeodactylum tricornutum via photosynthesis.

Iron is a limiting factor that controls the phytoplankton biomass in the modern ocean, and iron fertilization of the ocean could lead to blooms dominated by diatoms. Thus, iron plays an important role in controlling the distribution of diatoms. In this study, we measured the growth rate and photosynthetic activity of the model diatom Phaeodactylum tricornutum cultured under different iron concentrations and found that it grew more rapidly and had a much higher photosynthetic efficiency under higher iron concentrations. In order to explore the unique mechanism of the response of diatoms to iron, a proteomic analysis was carried out, and the results indicated that iron promotes the Calvin cycle of P. tricornutum. Diatoms can tolerate the pressure of iron limitation by replacing iron-rich proteins with flavodoxin, and so on. Moreover, we found that the photosystem I (PSI) activity of iron-limited algae that were treated by N',N',N',N'-tetramethyl-p-phenylenediamine (TMPD) was increased significantly. As TMPD plays the role of a cytochrome b6 /f complex that transfers electrons from photosystem II to PSI, the cytochrome b6 /f complex is the key to photosynthesis regulation. Iron could influence the growth of P. tricornutum by regulating its biosynthesis. All of the results suggest that iron might affect the growth of diatoms through the Calvin cycle and the cytochrome b6 /f complex.

Photorespiration participates in the assimilation of acetate in Chlorella sorokiniana under high light.

The development of microalgae on an industrial scale largely depends on the economic feasibility of mass production. High light induces productive suspensions during cultivation in a tubular photobioreactor. Herein, we report that high light, which inhibited the growth of Chlorella sorokiniana under autotrophic conditions, enhanced the growth of this alga in the presence of acetate. We compared pigments, proteomics and the metabolic flux ratio in C. sorokiniana cultivated under high light (HL) and under low light (LL) in the presence of acetate. Our results showed that high light induced the synthesis of xanthophyll and suppressed the synthesis of chlorophylls. Acetate in the medium was exhausted much more rapidly in HL than in LL. The data obtained from LC-MS/MS indicated that high light enhanced photorespiration, the Calvin cycle and the glyoxylate cycle of mixotrophic C. sorokiniana. The results of metabolic flux ratio analysis showed that the majority of the assimilated carbon derived from supplemented acetate, and photorespiratory glyoxylate could enter the glyoxylate cycle. Based on these data, we conclude that photorespiration provides glyoxylate to speed up the glyoxylate cycle, and releases acetate-derived CO2 for the Calvin cycle. Thus, photorespiration connects the glyoxylate cycle and the Calvin cycle, and participates in the assimilation of supplemented acetate in C. sorokiniana under high light.

Desiccation enhances phosphorylation of PSII and affects the distribution of protein complexes in the thylakoid membrane.

Desiccation has significant effects on photosynthetic processes in intertidal macro-algae. We studied an intertidal macro-alga, Ulva sp., which can tolerate desiccation, to investigate changes in photosynthetic performance and the components and structure of thylakoid membrane proteins in response to desiccation. Our results demonstrate that photosystem II (PSII) is more sensitive to desiccation than photosystem I (PSI) in Ulva sp. Comparative proteomics of the thylakoid membrane proteins at different levels of desiccation suggested that there were few changes in the content of proteins involved in photosynthesis during desiccation. Interestingly, we found that both the PSII subunit, PsbS (Photosystem II S subunit) (a four-helix protein in the LHC superfamily), and light-harvesting complex stress-related (LHCSR) proteins, which are required for non-photochemical quenching in land plants and algae, respectively, were present under both normal and desiccation conditions and both increased slightly during desiccation. In addition, the results of immunoblot analysis suggested that the phosphorylation of PSII and LHCII increases during desiccation. To investigate further, we separated out a supercomplex formed during desiccation by blue native-polyacrylamide gel electrophoresis and identified the components by mass spectrometry analysis. Our results show that phosphorylation of the complex increases slightly with decreased water content. All the results suggest that during the course of desiccation, few changes occur in the content of thylakoid membrane proteins, but a rearrangement of the protein complex occurs in the intertidal macro-alga Ulva sp.

Positive correlation between PSI response and oxidative pentose phosphate pathway activity during salt stress in an intertidal macroalga.

Studies have demonstrated that photosynthetic limitations and starch degradation are responses to stress; however, the relationship between the two is seldom described in detail. In this article, the effects of salt stress on photosynthesis, the levels of NADPH and total RNA, the starch content and the activities of glucose-6-phosphate dehydrogenase (G6PDH) and ribulose-5-phosphate kinase (RPK) were evaluated. In thalli that underwent salt treatments, the cyclic electron flow through PSI showed greater stress tolerance than the flow through PSII. Even though the linear electron flow was suppressed by DCMU, the cyclic electron flow still operated. The electron transport rate I (ETRI) increased as the salinity increased when the thalli recovered in seawater containing DCMU. These results suggested that PSI receives electrons from a source other than PSII. Furthermore, the starch content and RPK activity decreased, while the content of NADPH and total RNA, and the activity of G6PDH increased under salt stress. Soluble sugar from starch degradation may enter the oxidative pentose phosphate pathway (OPPP) to produce NADPH and ribose 5-phosphate. Data analysis suggests that NADPH provides electrons for PSI in Ulva prolifera during salt stress, the OPPP participates in the stress response and total RNA is synthesized in excess to assist recovery.

Photosystem I shows a higher tolerance to sorbitol-induced osmotic stress than photosystem II in the intertidal macro-algae Ulva prolifera (Chlorophyta).

The photosynthetic performance of the desiccation-tolerant, intertidal macro-algae Ulva prolifera was significantly affected by sorbitol-induced osmotic stress. Our results showed that photosynthetic activity decreased significantly with increases in sorbitol concentration. Although the partial activity of both photosystem I (PS I) and photosystem II (PS II) was able to recover after 30 min of rehydration, the activity of PS II decreased more rapidly than PS I. At 4 M sorbitol concentration, the activity of PS II was almost 0 while that of PS I was still at about one third of normal levels. Following prolonged treatment with 1 and 2 M sorbitol, the activity of PS I and PS II decreased slowly, suggesting that the effects of moderate concentrations of sorbitol on PS I and PS II were gradual. Interestingly, an increase in non-photochemical quenching occurred under these conditions in response to moderate osmotic stress, whereas it declined significantly under severe osmotic stress. These results suggest that photoprotection in U. prolifera could also be induced by moderate osmotic stress. In addition, the oxidation of PS I was significantly affected by osmotic stress. P700(+) in the thalli treated with high concentrations of sorbitol could still be reduced, as PS II was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but it could not be fully oxidized. This observation may be caused by the higher quantum yield of non-photochemical energy dissipation in PS I due to acceptor-side limitation (Y(NA)) during rehydration in seawater containing DCMU.

SLC24A-mediated calcium exchange as an indispensable component of the diatom cell density-driven signaling pathway.

Diatom bloom is characterized by a rapid increase of population density. Perception of population density and physiological responses can significantly influence their survival strategies, subsequently impacting bloom fate. The population density itself can serve as a signal, which is perceived through chemical signals or chlorophyll fluorescence signals triggered by high cell density, and their intracellular signaling mechanisms remain to be elucidated. In this study, we focused on the model diatom, Phaeodactylum tricornutum, and designed an orthogonal experiment involving varying cell densities and light conditions, to stimulate the release of chemical signals and light-induced chlorophyll fluorescence signals. Utilizing RNA-Seq and Weighted Gene Co-expression Network Analysis, we identified four gene clusters displaying density-dependent expression patterns. Within these, a potential hub gene, PtSLC24A, encoding a Na+/Ca2+ exchanger, was identified. Based on molecular genetics, cellular physiology, computational structural biology, and in situ oceanic data, we propose a potential intracellular signaling mechanism related to cell density in marine diatoms using Ca2+: upon sensing population density signals mediated by chemical cues, the membrane-bound PtSLC24A facilitates the efflux of Ca2+ to maintain specific intracellular calcium levels, allowing the transduction of intracellular density signals, subsequently regulating physiological responses, including cell apoptosis, ultimately affecting algal blooms fate. These findings shed light on the calcium-mediated intracellular signaling mechanism of marine diatoms to changing population densities, and enhances our understanding of diatom bloom dynamics and their ecological implications.

The physiological links of the increased photosystem II activity in moderately desiccated Porphyra haitanensis (Bangiales, Rhodophyta) to the cyclic electron flow during desiccation and re-hydration.

Photosynthetic electron flow changed considerably during desiccation and re-hydration of the intertidal macroalgae Porphyra haitanensis. Activities of both photosystem (PSI) and photosystem (PSII) increased significantly at moderate desiccation levels. Whereas PSII activity was abolished at an absolute water content (AWC) <24 %, PSI remained active with progressive decreases in AWC to values as low as 16 %. This result suggested that cyclic electron flow around PSI was still active after inactivation of linear electron flow following severe desiccation. Moreover, the PSI activity was restored more rapidly than that of PSII upon re-hydration. Pretreatment of the blades with 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) suppressed PSII activity following desiccation to an AWC of ~16 % AWC. Cyclic electron flow around PSI decreased markedly in blades pretreated with DCMU than in blades without pretreatment of DCMU during re-hydration in seawater containing DCMU. All results suggested that the activity of PSII under desiccation conditions plays an important role in the operation of cyclic electron flow during desiccation and its recovery during re-hydration. Therefore, we proposed the PSII activity during desiccation could eventually lead to the accumulation of NADPH, which could serve as electron donor for P700(+) and promote its recovery during re-hydration, thereby favoring the operation of cyclic electron flow.

[Heat-sensitive moxibustion combined with western medication for low back pain of osteoporosis with kidney-yang deficiency: a randomized controlled trial].

OBJECTIVE: To compare the clinical therapeutic effect between heat-sensitive moxibustion combined with western medication and simple western medication for low back pain of osteoporosis with kidney-yang deficiency. METHODS: A total of 60 patients with osteoporosis were randomized into an observation group (32 cases, 2 cases dropped off) and a control group (32 cases, 3 cases dropped off). In the control group, alendronate sodium tablet and calcium carbonate and vitamin D3 tablet were taken orally. On the basis of the control group, heat-sensitive moxibustion was applied at Mingmen (GV 4), Yaoyangguan (GV 3), Guanyuan (CV 4), Shenshu (BL 23), Zusanli (ST 36) in the observation group, once a day, 5 times a week for 8 weeks. Before and after treatment，the visual analogue scale (VAS) score, Oswestry disability index (ODI) score, bone mineral density (BMD) and TCM clinical symptom score were compared in the two groups. RESULTS: The VAS scores, ODI scores and TCM clinical symptom scores after treatment were reduced in the two groups (P<0.05, P<0.01), and those in the observation group were lower than the control group (P<0.05, P<0.01). The BMD after treatment was increased in the two groups (P<0.01), and that in the observation group was higher than the control group (P<0.05). CONCLUSION: Heat-sensitive moxibustion combined with western medication could relieve low back pain, improve BMD in patients of osteoporosis with kidney-yang deficiency, and its clinical effect is superior to simple western medication.

Variations in morphology and PSII photosynthetic capabilities during the early development of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariales, Rhodophyta).

BACKGROUND: Red algae are primitive photosynthetic eukaryotes, whose spores are ideal subjects for studies of photosynthesis and development. Although the development of red alga spores has received considerable research attention, few studies have focused on the detailed morphological and photosynthetic changes that occur during the early development of tetraspores of Gracilaria vermiculophylla (Ohmi) Papenfuss (Gracilariales, Rhodophyta). Herein, we documented these changes in this species of red algae. RESULTS: In the tetraspores, we observed two types of division, cruciate and zonate, and both could develop into multicellular bodies (disks). During the first 84 hours, tetraspores divided several times, but the diameter of the disks changed very little; thereafter, the diameter increased significantly. Scanning electron microscopy observations and analysis of histological sections revealed that the natural shape of the disk remains tapered over time, and the erect frond grows from the central protrusion of the disk. Cultivation of tissue from excised disks demonstrated that the central protrusion of the disk is essential for initiation of the erect frond. Photosynthetic (i.e., PSII) activities were measured using chlorophyll fluorescence analysis. The results indicated that freshly released tetraspores retained limited PSII photosynthetic capabilities; when the tetraspores attached to a substrate, those capabilities increased significantly. In the disk, the PSII activity of both marginal and central cells was similar, although some degree of morphological polarity was present; the PSII photosynthetic capabilities in young germling exhibited an apico-basal gradient. CONCLUSIONS: Attachment of tetraspores to a substrate significantly enhanced their PSII photosynthetic capabilities, and triggered further development. The central protrusion of the disk is the growth point, may have transfer of nutritive material with the marginal cells. Within the young germling, the hetero-distribution of PSII photosynthetic capabilities might be due to the differences in cell functions.

Regulation of Ferredoxin-NADP+ Oxidoreductase to Cyclic Electron Transport in High Salinity Stressed Pyropia yezoensis.

Pyropia yezoensis can survive the severe water loss that occurs during low tide, making it an ideal species to investigate the acclimation mechanism of intertidal seaweed to special extreme environments. In this study, we determined the effects of high salinity on photosynthesis using increasing salinity around algal tissues. Both electron transport rates, ETR (I) and ETR (II), showed continuous decreases as the salinity increased. However, the difference between these factors remained relatively stable, similar to the control. Inhibitor experiments illustrated that there were at least three different cyclic electron transport pathways. Under conditions of severe salinity, NAD(P)H could be exploited as an endogenous electron donor to reduce the plastoquinone pool in Py. yezoensis. Based on these findings, we next examined how these different cyclic electron transport (CETs) pathways were coordinated by cloning the gene (HM370553) for ferredoxin-NADP+ oxidoreductase (FNR). A phylogenetic tree was constructed, and the evolutionary relationships among different FNRs were evaluated. The results indicated that the Py. yezoensis FNR showed a closer relationship with cyanobacterial FNR. The results of both real-time polymerase chain reaction and western blotting showed that the enzyme was upregulated under 90-120‰ salinity. Due to the structure-function correlations in organism, Py. yezoensis FNR was proposed to be involved in NAD(P)H-dependent Fd+ reduction under severe salinity conditions. Thus, through the connection between different donors bridged by FNR, electrons were channeled toward distinct routes according to the different metabolic demands. This was expected to make the electron transfer in the chloroplasts become more flexible and to contribute greatly to acclimation of Py. yezoensis to the extreme variable environments in the intertidal zone.

[Exploration on sensation positioning method of heat-sensitive moxibustion: the inheritance and development of ashi method].

From original concept and literature of acupoint, the concept and clinical significance of ashi method is discussed, which clarifies that the essence of ashi method is to locate the acupoints by patients' sensitivity on force. The clinical application of heat-sensitive moxibustion has illustrated that positioning method of this therapy is based on the appearance of heat-sensitive moxibustion sensation. Although both types are based on patients' feeling, positioning method of heat-sensitive moxibustion stands on a new angle and uses a new method to locate acupoint. Therefore, it is believed that the positioning method of heat-sensitive moxibustion is the inheritance and development of ashi method.

Quantitative proteomic analysis of thylakoid from two microalgae (Haematococcus pluvialis and Dunaliella salina) reveals two different high light-responsive strategies.

Under high light (HL) stress, astaxanthin-accumulating Haematococcus pluvialis and ß-carotene-accumulating Dunaliella salina showed different responsive patterns. To elucidate cellular-regulating strategies photosynthetically and metabolically, thylakoid membrane proteins in H. pluvialis and D. salina were extracted and relatively quantified after 0 h, 24 h and 48 h of HL stress. Proteomic analysis showed that three subunits of the cytochrome b6/f complex were greatly reduced under HL stress in H. pluvialis, while they were increased in D. salina. Additionally, the major subunits of both photosystem (PS) II and PSI reaction center proteins were first reduced and subsequently recovered in H. pluvialis, while they were gradually reduced in D. salina. D. salina also showed a greater ability to function using the xanthophyll-cycle and the cyclic photosynthetic electron transfer pathway compared to H. pluvialis. We propose a reoriented and effective HL-responsive strategy in H. pluvialis, enabling it to acclimate under HL. The promising metabolic pathway described here contains a reorganized pentose phosphate pathway, Calvin cycle and glycolysis pathway participating in carbon sink formation under HL in H. pluvialis. Additionally, the efficient carbon reorientation strategy in H. pluvialis was verified by elevated extracellular carbon assimilation and rapid conversion into astaxanthin.

[Clinical effect of different schemes of mild moxibustion for treatment of knee osteoarthritis].

OBJECTIVE: To observe the therapeutic effect of different schemes of mild moxibustion for treatment of (yang-deficiency induced cold-accumulation type)knee osteoarthritis (KOA). METHODS: Fifty-nine KOA patients were chosen and randomly divided into control group (n =30) and test group (n =29). Patients of the control group were treated by mild moxi- bustion of Neixiyan (EX-HE4) and Waixiyan (ST 35) for 30 min, once daily for 14 days (two courses), and those of the test group were treated by mild moxibustion of EX-HE 4 and ST 35 for 30 mini once daily for 7 days (the same to control group), followed by moxibustion of Yaoyangguan (GV 3) and Mingmen (GV 4) once daily for next 7 days more. In addition, patients of the two groups were also treated by routine acupuncture stimulation of EX-HE 4, ST 35, Yanglingquan (GB 34), Kunlun (BL 60), etc. The interval between two therapeutic courses was one day. The Lysholm Knee Score Scale (LKSS) was used to evaluate the therapeutic effect. Visual analogue scale (VAS) was employed to assess the patient's knee-joint pain severity (arthralgia), and scores of morning stiffness, arthrocele, and walking restraint degree of the knee-joint were also evaluated before and after the treatment. RESULTS: After the treatment, the scores of VAS, morning stiffness, arthrocele and walking restraint degree of the knee-joint of both groups were significantly decreased (P<0. 05), and the scores of the test group were obviously lower than those of the con- trol group (P<0. 05). The effective rate of the test group was 89.66% (26/29)which was obviously higher than that (70.00%, 21/30) of the control group (P<0. 05). CONCLUSION: Mild moxibustion of Neixiyan (EX-HE 4) and Waixiyan (ST 35, local acu- points), and Yaoyangguan (GV 3) and Mingmen (GV 4) has a better therapeutic effect for KOA patients than moxibustion of local acupoints only.

[Efficacy comparison of lumber disc herniation treated with mild moxibustion at Yaoyangguan (GV 3) under different conditions].

OBJECTIVE: To compare the difference in clinical efficacy on lumber disc herniation (LDH) treated with Yaoyangguan (GV 3) between mild moxibustion under thermosensitive condition and that under non-thermo-sensitive condition. METHODS: Fifty-seven LDH patients were selected as the study objects. Mild moxibustion at Yaoyangguan (GV 3) was applied for 45 min each time. Additionally, the conventional acupuncture was given, once a day, for 20 days. At the end of treatment, two groups were classified. A thermosensitive acupoint group (thermosensitive group) was composed of the cases with thermo-sensitization at Yaoyangguan (GV 3) and presenting for > or =4 times in the entire treatment. A non-thermosensitive acupoint group (tranquilization group) was composed of the cases without thermo-sensitization or the frequency of thermo-sensitization <4 times in the entire treatment. The modified Japanese orthopedics association scoring system (M-JOA) was adopted to observe the cases before and after treatment and 6 months after treatment in the two groups. The efficacy was compared between the two groups. RESULTS: After treatment and in 6 months after treatment, the score of M-JOA was apparently reduced as compared with that before treatment in the two groups (all P<0.05). The score reducing in the thermosensitization group was more obvious than that in the tranquilization group (both P<0.01). After treatment and in 6 months after treatment, the curative rate and remarkably effective rate were 89.7% (26/29) and 79.3% (23/29), which was better than 71.4% (20/28) and 60.7% (17/28, both P<0.05) in the tranquilization group separately. CONCLUSION: Mild moxibustion at acupoint under thermosensitive condition achieves the better short-term and long-term effects as compared with that under non-thermosensitive condition.