Pneumonia is the most common cause of death in patients with dementia, but the outcomes of patients with dementia hospitalized with pneumonia are poorly understood. We sought to illuminate the association between dementia and in-hospital mortality and discharge status in patients hospitalized with pneumonia.We used the Diagnosis Procedure Combination database, a national inpatient database in Japan, to identify retrospectively patients aged ≥60 years admitted to hospital with pneumonia during the study period of May 1, 2010 to March 31, 2014. We recorded their sex, age, body mass index, severity of pneumonia and comorbidities (including dementia). The outcomes were in-hospital mortality and discharge home. Multivariable Cox regression analysis was performed to analyze factors influencing discharge home.P = 0.63). The proportions of patients discharged home were 52.9% and 71.3% in patients with and without dementia, respectively (P < 0.001). The adjusted hazard ratio for discharge home for patients with dementia was 0.68 .We identified 470,829 patients hospitalized with pneumonia; 45,031 were recorded as having dementia (9.6%). In-hospital mortality was 13.1% and 13.4% in patients with and without dementia, respectively (In-hospital mortality from pneumonia did not differ significantly between patients with and without dementia; however, those with dementia were less likely to be discharged home. Pneumonia and dementia are major concerns as societies age worldwide , 2. TheyPatients with dementia frequently have impaired ability to engage with activities of daily living, and may be bedridden before or after the diagnosis of pneumonia, potentially resulting in failure to be discharged home after hospitalization. Factors associated with discharge home has been intensively studied in stroke patients –15. DyspWe used a national inpatient database in Japan to establish whether a diagnosis of dementia influences in-hospital prognosis or discharge status in elderly patients with pneumonia.2) while breathing a fraction of inspired oxygen <0.35 or ≥0.35, level of consciousness and systolic blood pressure. Level of consciousness was assessed using the Japan Coma Scale. Japan Coma Scale score 0 denoted alert consciousness; scores 1–3 denote delirium; scores 10–30 denote somnolence; and scores 100–300 denote coma [Ours was a retrospective cohort study using the Japanese Diagnosis Procedure Combination (DPC) database , 17, whiote coma . In the We identified patients aged ≥60 years hospitalized with pneumonia (ICD-10 code J10 − J18 or J69) between May 1, 2010 and March 31, 2014. Among these patients, those with dementia were identified by the ICD codes for ‘dementia’ (F00–F03 or G30) or the prescription of donepezil, galantamine, rivastigmine or memantine. Patients with cancer and dysphagia was identified by ICD codes for ‘Neoplasm’ (C) and ‘dysphagia’ (R13), respectively.Conduct of the study was approved by the Institutional Review Board of The University of Tokyo, which waived the requirement for informed consent owing to the anonymity of the data.The study outcome was discharge status .2 [2 < 90%, impaired consciousness, systolic blood pressure < 90 mmHg), cancer, hemodialysis and intensive care unit admission. We classified A-DROP score as follows; mild: 0, moderate: 1-2, severe: 3, most severe: 4-5 or systolic blood pressure < 90 mmHg. Multiple imputation method was utilized to impute missing values in BMI and A-DROP items [Body mass index (BMI) was grouped into the following six categories according to the World Health Organization body mass index (BMI) classification: <15.9, 16.0–16.9, 17.0–18.4, 18.5–22.9, 23.0–24.9, 25–29.9, 30.0–34.9 and ≥35.0 kg/mWe identified a total of 470,829 patients aged ≥60 years hospitalized with pneumonia during the study period: of these, 45,031 (9.6%) had dementia and 100,198 (21.3%) were admitted more than once. Dysphagia and nasogastric tube feeding was observed in 4953 (11.0%) and 4120 (9.1%) patients with dementia, respectively, and 19,980 (4.7%) and 22,123 (5.2%) patients without dementia, respectively. Of those admitted more than once, 9118 (9.1%) had dementia and 91,080 (90.9%) did not.2, consciousness and systolic blood pressure were missing in 1.8% and 2.2%, 1.9% and 2.3%, 3.2% and 2.3% and 1.6% and 2.1% in patients with and without dementia, respectively.Table P < 0.001). There was no significant difference in all-cause in-hospital mortality between the groups .Discharge status is presented in Table P < 0.001) for those with dementia with reference to those without. Age, A-DROP items , cancer, hemodialysis and intensive care unit admission was significantly associated with reduction in discharge home, while higher BMI was significantly associated with increase in discharge home (Table In the Cox proportional hazards regression model, the adjusted hazard ratio for discharge home was 0.68 (95% confidence interval 0.67–0.69, me Table . The proWe found that all-cause in-hospital mortality after hospitalization for pneumonia was not significantly different between elderly patients with or without dementia, but those with dementia were significantly less likely to return home.Bronchopneumonia is reportedly the most common cause of death in patients with dementia disorders . PreviouAs with the rest of the world, the importance of discharge home is increasingly recognized in Japan, allowing patients to benefit from home care in familiar surroundings. Home care is an increasingly important part of continuous care for seniors. Home care may reduce the risk of infection with antimicrobial-resistant micro-organisms  and deliA diagnosis of dementia appears to condemn hospitalized patients to be discharged to another medical facility rather than home , 34. In Our study had some limitations. First, the diagnosis of dementia was not necessarily certified by clinicians, so may have been prone to inaccuracy. Secondly, the etiology and severity of dementia, and the etiology of pneumonia, were not taken into consideration. Thirdly, family environment and support from the community were unable to be assessed.In conclusion, all-cause in-hospital mortality of patients hospitalized with pneumonia did not appear to be significantly influenced by dementia. However, patients with dementia hospitalized with pneumonia were less likely to return home than those without dementia, even after adjustment for other factors such as age, BMI and severity of pneumonia.


Spirulina subsalsa as feedstock, using monosodium glutamate residue (MSGR) produced by the glutamate extraction process as an inexpensive nutrient source.To overcome the daunting technical and economic barriers of algal biofuels, we evaluated whether seawater can be a viable medium for economically producing filamentous Spirulina subsalsa cannot grow in pure seawater, but exhibited faster biomass accumulation in seawater supplemented with MSGR than in freshwater medium (modified Zarrouk medium). Introducing seawater into media ensured this cyanobacterium obtained high lipid productivity (120 mg/L/day) and suffered limited bacterial infections during growth. Moreover, the yields of protein, carotenoids and phytols were also improved in seawater mixed with MSGR. S. subsalsa exhibited high biomass and lipid productivity in bag bioreactors with 5- and 10-L medium, demonstrating the potential of this cultivation method for scaling up. Moreover, seawater can produce more biomass through medium reuse. Reused seawater medium yielded 72% of lipid content compared to pristine medium. The reason that S. subsalsa grew well in seawater with MSGR is its proficient adaptation to salinity, which included elongation and desaturation of fatty acids, accumulation of lysine and methionine, and secretion of sodium. The nutrients provided by MSGR, like organic materials, played an important role in these responses.Spirulina subsalsa has an efficient system to adapt to saline ambiance in seawater. When supplemented with MSGR, seawater is a great potential medium to produce S. subsalsa in large scale as biofuel feedstock. Meanwhile, value-added products can be derived from the ample protein and pigments that can broaden the range of biomass application and improve this biorefinery economics.The online version of this article (10.1186/s13068-019-1391-1) contains supplementary material, which is available to authorized users. Scenedesmus, Chlorella, and Nannochloropsis etc., due to their adaptation in wastewater and high lipid content [Many studies have characterized the potential of microalgae as a feedstock for renewable biofuel and bioproducts, noting benefits such as high growth rate, high oil content, and little demand of arable land , 2. Amon content . However content , 5. Fila content .To commercially produce cyanobacteria-based biofuel, it is necessary to reduce the demand of the cultivation on freshwater supply . To addrSpirulina subsalsa reserved by Institute of Hydrobiology  is a good candidate. Spirulina subsalsa grow to a size of approximately 200 µm, facilitating harvest compared to smaller microalgae. Moreover, it was isolated from an alkaline hot spring and can tolerate 0–5 wt/vol% salinity [S. subsalsa aiding its adaptation to seawater. As of yet, there are few publications focusing on biofuel applications for S. subsalsa, due to the lower lipid content in this cyanobacterial cell. This would be an exciting development to demonstrate the feasibility of applying S. subsalsa biomass to produce biofuel using seawater and wastewater as a promising cultivation method.Limnetic filamentous cyanobacterial species are better suited to obtain increased lipid productivity from this new method than marine species that may not experience salt stress in seawater. Based on this, salinity , which iWastewater containing luxury macronutrients and trace heavy metals can benefit cyanobacterial growth and improve the production system. Hence, residue or byproducts derived from industrial food production can obtain a high score in terms of sanitation and nutrients.Spirulina biomass production from seawater.Monosodium glutamate (MSG) as a flavor enhancer is extensively used in food products. China produces 75% of the world’s supply of MSG since 2011, according to data from the Chinese Condiments Association. During MSG production, there is residue generated after glutamate extraction and sterilization that is rich in nitrogen and phosphorus, with total nitrogen (TN) of 62–72 g/L and total phosphorus (TP) of 657–686 mg/L. Moreover, it contained trace heavy metals . The residue (MSGR) may be an economical and acceptable nutrient source for S. subsalsa. The main goals in this study comprise: (1) observing the growth and lipid accumulation of S. subsalsa in seawater media; (2) validating the potential of scaling up this new cultivation system; (3) improving the efficiency of seawater to produce cyanobacterial biomass as a biofuel feedstock; and (4) analyzing the regulation of limnetic S. subsalsa in response to seawater.Based on this information, we chose seawater as our source of water and minerals for algal biomass production, and MSGR as the source of macroelements  to culture limnetic Spirulina subsalsa was purchased from the Freshwater Algae Culture Collection of the Institute of Hydrobiology (FACHB-Collection), and cultured as FACHB recommended.S. subsalsa. This medium, as shown in previous study [3; 0.5 NaCl; 0.15 MgSO4·7H2O; 0.04 CaCl2·2H2O; 1.25 single super phosphate; 0.898 KCl; 16.8 NaHCO3.In the experiment, modified Zarrouk medium (mZM) was used as the control freshwater medium to culture us study , include3-N, 3.67 ± 0.14 mg/L TN, 0.003 ± 0.000 mg/L TP, 13.92 ± 0.14 g/L Na, 346.6 ± 7.2 mg/L K, 988.8 ± 13.6 mg/L Ca, 1295 ± 3 mg/L Mg, 24.10 ± 3.58 mg/L Al, 5.60 ± 0.08 mg/L Fe, 5.20 ± 0.06 mg/L Cu, 2.61 ± 0.08 mg/L Zn; Mn and Co were not detected.The seawater was collected from Qingdao, China, and filtered through 0.22 μm membranes before use. It contained: 76.38 ± 7.16 mg/L TOC, 0.60 ± 0.00 mg/L NHCr 18–22 g/L, BOD5 10–11 g/L, TOC 6.9–7.0 g/L SO42− 190–210 mg/L, NH3-N 550–575 mg/L, TN 62–71 g/L, and TP 657–686 mg/L, pH 3.8-4.3.The nutrient MSGR is a residue remaining after glutamate extraction and bacteria removal during MSG production, and was supplied by Liangshan Linghua Gourmet Powder factory . The brown acidic liquid MSGR was used directly for algal cultivation and presented the following characteristics: CODThis study was designed as below to check the feasibility of seawater to produce biomass as biofuel feedstock in low cost, which is schematically shown in Additional file VMSGR/Vseawater) were made up as the seawater media. Pure seawater, mZM and MSGR were employed as controls  sealed with very day . All theS. subsalsa in bag reactor was a preliminary step to scale up this cultivation system .Culturing S. subsalsa in reused medium was also observed, as the schematic figure shown  and chlorophyll a content. Measurement of dry cell mass was carried out as Jiang et al. [The growth of g et al.  did, accPA) and volumetric productivity (PV) were calculated. The former was based on the place occupied by the reactor, while the latter was considered the input of medium:A is the area of baggy reactor, V is the volume of medium, and T stands for the period of cultivation.For cyanobacteria in baggy reactor, areal productivity (A665.2 and A652.4) of the extract, chl a concentration can be calculated as:Chlorophyll a (chl a) was detected using the same method from our previous report . BrieflyS. subsalsa was harvested by filtration with 200-mesh bolting silk at the point of maximum biomass concentration. Collected cyanobacterial biomass was dried to constant mass at − 50 °C in a lyophilizer , and then ground to homogeneous powder in preparation for nutritional materials analysis.Based on the growth curve, Total crude lipid of cyanobacterial biomass was quantified gravimetrically following the extraction. About 0.1 g dried biomass powder was transferred into a 5 mL chloroform/methanol  mixture and homogenized for 10 min under a sonication; this was followed by centrifugation at 4000 rpm at 4 °C for 10 min to collect supernatant in a separation funnel; the entire extraction process was repeated twice; sodium chloride solution (0.9%) was then added at a proportion of 1:5 (v/v) of lipid extract; the extract was shaken vigorously for 1 min and allowed to undergo phase separation for 15 min; the lower phase containing essentially all the extracted lipids was transferred into a clean weighed glass tube and dried under nitrogen flow at 60 °C to get the weight of lipids for calculating total lipid content in cells.S. subsalsa were in situ esterified with methanol for GC–MS detection, which was accomplished through a pre-esterification process-catalyzed fatty acids in cells by Amberlyst-15 and the following transesterification base catalyzed by 4 wt% KOH/methanol in a 60 °C water bath. Then, fatty acid methanol ester (FAME) was collected in 1 mL hexane in the upper layer and separated from water and other residues staying in the lower layer. The upper layer solution (100 μL) was transferred into a GC vial with fused-in insert, and 25 μL heptadecanoic acid methyl ester  was added as internal standard of methyl ester. The FAME profile was analyzed by gas chromatography–mass spectrometry  equipped with an automatic sampler  and a capillary column of VF–23 ms . The column started at 150 °C and held for 1 min, raised to 165 °C at a rate of 1 °C/min. The FAME composition was expressed as its percentage compared with the total transesterifiable lipids of biomass.The fatty acids and phytols in M is the number of carbon–carbon double bonds in each FA composition, Yi is the mass fraction of the corresponding FA constitution, y1–6 represent kinematic viscosity, specific gravity, cloud point, cetane number, iodine value and higher heating value successively, and x is the value of ADU.To evaluate the biodiesel property, the average degree of unsaturation (ADU), kinematic viscosity, specific gravity, cloud point, cetane number (CN), iodine value and higher heating value (HHV) were calculated to follow the Eqs. –10 in th2SO4). Then, the vial was placed into ice bath for instantly cooling down. The last step was colorimetrically determining the absorbance in a Multiskan FC. Solutions containing different level of glucose were used to set a relationship between sugar concentration and absorbance.The carbohydrate in biomass was quantified by colorimetric assay on a 96-well plate in a Multiskan FC  at 620 nm wavelength, following extraction from about 100 mg biomass using hydrochloric acid, as we previously did [4·5H2O, K2SO4 and H2SO4, and then N concentration was measured for the calculation of protein content.Characterization of protein content in algal biomass involved elemental analysis of total N , and theS. subsalsa was obtained with an amino acid analyzer .The amino acid composition of A450 is the absorbance at wavelength of 450 nm, 3 is the ratio between the volume of ethanol addition and culture used or carotenoids detection, and 250 represents the conversion factor calculating absorbance to CC.As described by Shao et al. , the car3 at 95 °C for 75 min, after which organic matter was dissolved with the addition of H2O2 (30% v/v). Samples were then incubated at 95 °C for 1 h in concentrated HCl, and made up to the required volume with ultrapure water, awaiting detection.The measurement of mineral and trace elements was conducted with an atomic absorption spectrophotometer . The pretreatment of algal biomass was similar to that used by Tibbetts et al. . About 1p < 0.05.Results are presented in the form of mean values ± standard deviation from three independent experiments and analyzed using one-way analysis of variance in Duncan’s test. A difference was considered statistically significant when S. subsalsa was compared between those grown in cultures supplied with a range of MSGR concentrations and those grown in three control cultures, which composed solely of seawater, MSGR, or modified Zarrouk medium (mZM), respectively . Conversely, cells in 1/100 and 1/200 MSGR media obtained a higher chl a level than in the mZM control.As chlorophyll is the main element related to the photosynthetic system in cyanobacteria, Fig.  biomass . In the The chlorophyll concentration suggested the capability of growing photoautotrophic microalgae for biomass production Fig. . After 5S. subsalsa in mineral-enriched seawater and sulfate-enriched MSGR always resulted in the formation of white precipitates, impending the passage of light to the algal cells. This phenomenon occurred severely for high ratios of MSGR in seawater, such as 1/25, 1/10 and 1/5, whereas it was slight in treatments with the other, lower MSGR concentrations. These results are consistent with the growth profiles of S. subsalsa in the respective MSGR concentrations, indicating that the precipitate formation inhibited the biomass production through decreasing photo-availability for S. subsalsa.Extracellular particles in culture could also have an impact on the photosynthesis system. Cultivation of S. subsalsa growth. S. subsalsa usually has a low lipid production (around 15%) in mZM, similar to our previous result [Lipid yield was analyzed from 1/1000 to 1/50 MSGR and mZM control  were only approximately half of that in 1/100 MSGR . After culturing S. subsalsa, the ammonia and TP were exhausted, and the remaining TN was 143 mg/L and 142 mg/L in 1/100 and 1/200 MSGR, respectively. These results suggest that the remaining nitrogen might not be bioavailable to S. subsalsa, and nitrogen and phosphorus limitation likely occurred in 1/200 MSGR. This difference in nutrient availability altered the energy storage of S. subsalsa. For S. subsalsa grown in 1/200 MSGR, energy was mainly stored as lipid (35% of lipid and 8% carbohydrate), but cells grown in 1/100 MSGR stored energy as carbohydrates (18% of lipid and 16% carbohydrate) . In the 1/500 and 1/100 groups, the protein content matched with the standard of more than 55% [Protein is the main macromolecular component in most cyanobacterial cells and has very high nutritional value for animal feed or pharmacy. Total concentrations of protein, expressed as percentages of DCM, in the biomass harvested after 5 days of growth are presented in Fig. than 55% . The maxCarotenoids and phytols are important components of chlorophyll, and play crucial roles in photosynthesis, antibacterial activity , and cheCarotenoids are used in the food industry and are unique constituents of a healthy diet, playing an important role in improving antioxidant status and preventing cancers . As showS. subsalsa. Biomass in 1/100 MSGR and seawater had the highest phytol productivity of 12.9 mg/L/day, followed by 5.6 mg/L/day in the 1/200 group, which all outweighed the 1.3 mg/L/day level in mZM.These two ratios of MSGR in seawater also improved the phytol production from S. subsalsa biomass to explore its application in livestock and agriculture from both the nutritional and the toxicological point of view.Minerals and trace elements are of pivotal importance for animals and plants because they cannot synthesize the materials and must depend on uptake from feeds or fertilizers. We evaluated the inorganic materials in Minerals assayed include calcium (Ca), magnesium (Mg) and potassium (K). Trace elements assayed include cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), selenium (Se), vanadium (V) and zinc (Zn). Heavy metals assayed include arsenic (As), cadmium (Cd), chromium (Cr) and lead (Pb). The results obtained for the minerals and trace elements analyzed in all the cyanobacterial samples from different media are presented in Table S. subsalsa in other seawater media accumulated less Mg than that in mZM. Regarding trace elements, the highest levels of Cu and Fe occurred in 1/100 MSGR medium, Co in 1/50 medium, and Zn in 1/200 medium.The maximum contents of individual minerals and trace elements occurred at different MSGR concentrations. The calcium content ranged from 1.14% (1/1000 MSGR) to 4.58% (1/200 MSGR), which was higher than the content of 1% for mZM. Besides the 2.92% level of magnesium in the 1/1000 run, 5(PO4)3(OH)), where the Ca:P ratio is 2.15:1. Nutritional experts recommended 2:1 as the most suitable dietary Ca:P ratio for young rapidly growing farmed livestock and fish, even optimally 1:1 [Spirulina biomass with well-balanced Ca:P ratios (1.2 ± 0.06), which are consistent with reports for commercially established Spirulina species (1.0–1.7:1) summarized by Tibbetts et al. [The Ca:P ratio is an important index of nutritional value. The principle component of animal bone is hydroxyapatite  [As feed or fertilizer, toxic elements must be a priori consideration for nutritional and safety evaluations. Fortunately, as given in Table  2 µg/g) .The above trials show that seawater plus MSGR is an efficient cultivation system to produce biofuel feedstock in laboratory. However, more analysis and trials are needed to confirm if this method can be economically viable large scale to realize the goal of applying algal technology to combat the global energy crisis.To construct an offshore cyanobacteria site, infrastructure such as water piping, reactor and material transportation is an inevitable and undifferentiated investment independent of medium. However, in terms of water and nutrient input, seawater-MSGR would cost less compared to mZM. Seawater costs significantly less than the fresh water used in mZM due to its huge reserves and simple pretreatment. In terms of nutrients, it costs approximately 1 CNY to prepare one liter of mZM media. Considering total nutrients of 22.138 g/L in mZM and 5 mL/L in 1/200 MSGR, using MSGR is as feasible as using the compounds needed for mZM media. The generation of MSGR, the nutrients in seawater medium, did not cause any negative effect on economy. Seawater added with MSGR could be granted as a low-cost medium and possibly environmentally friendly system for producing cyanobacteria biomass.VMSGR/Vseawater).To consider the potential of scaleup, we used a bag bioreactor to limit evaporation and contamination and ensure the sustainable operation of this system, as we discussed in our previous report . Based oIn large-scale production, areal and/or volumetric productivity are the main parameters to consider for space and cost efficiency , 24. TheS. subsalsa, limited phosphorus (< 0.05 mg/L) remained in spent medium, so 1/200 MSGR was supplemented to supply nutrients before reuse. The growth and lipid accumulation of S. subsalsa in different times of reused medium are shown in Fig. S. subsalsa to survive in the third-use medium, but the second use of seawater obtained around 81% of biomass yield  in its membrane, compared with that in mZM. Moreover, the MUFA proportion increased from 32 to 50% with an increase in the ratio of MSGR to seawater from 1/1000 to 1/50, which indicated that the supplement of this wastewater could help S. subsalsa deal with salinity in seawater.The fluidity of the cell membrane is changed through its fatty acid tails to deal with abiotic stress . Fatty a systems , 30. AltSpirulina subsalsa accumulates more glutamic acid than any other amino acid, making up approximately 15% of total amino acids (TAA). Cysteine accumulated in the lowest concentration, ranging from 0.3 to 0.4% of TAA  –33. The TAA Fig. a. BecausTAA Fig. b.Fig. 7ASpirulina subsalsa involved increased lysine and methionine content responding to salinity in seawater media.Among amino acids and proteins that are induced by salt or drought stress, proline has been studied most extensively . CompareCraterostigma plantagineum promoted phosphatidic acid binding and protected enzymes from adverse effects of desiccation [Spirulina subsalsa might also protect itself from salinity by accumulating lysine-related protein or other organic substances.Lysine is an important metabolite that has stress-associated functions. It protects cells from salt or desiccation stress both as an osmolyte and via enzymatic protection. Betaine substances, derived from lysine, acts as an efficient osmolyte in plant cells to adjust the osmotic pressure and stabilize other macromolecular materials . The lysiccation . Wang eticcation  also manMethionine (Met) is also involved in abiotic response as a compatible osmolyte . In thisSpirulina cells must possess some suitable mechanisms to exclude sodium. All runs in seawater media (except in the 1/500 media) had the same or lower amount of sodium content in the cells as the mZM control  and H+ ATPase(s) [Organisms secrete sodium out through NaTPase(s) . These t+/K+ channel in Fig. +/K+ ratios in saline media. However, aside from 1/100 MSGR medium, cyanobacterial cells in most seawater treatments exhibited the Na+/K+ ratios ranging from 4.3 to 7.3, which were higher than the ratio of 2.3 for mZM. S. subsalsa may not depend a lot on this process coupled with enhanced K uptake due to the increased average degree of unsaturation (ADU) in seawater media  and H+ ATPase in Fig. + ATPase might work well. Indeed, this process can only be created and maintained at the expense of energy that would be provided by photosynthesis and respiration. The production and distribution of energy in cyanobacterial cells likely influenced the intracellular ion activity in seawater media at various MSGR concentrations. For S. subsalsa cultivated in seawater and MSGR media, the energy from photosynthesis could be reflected by the chlorophyll a content and that from respiration was tied up in the organic carbon mainly provided by MSGR.With respect to unsaturated fatty acids, the second regulation method worked harmoniously with membrane protection, because the increased ADU can trigger the synthesis of the NaS. subsalsa suggested that the energy flowing into the Na-excluding process was sufficient, although chl a content decreased in 1/1000, 1/500 and 1/50 MSGR media  has been added. MSGR added in seawater in ratios (1/1000–1/50) obtained equal or higher levels of biomass concentration compared to freshwater medium mZM, and the ratios of 1/200 and 1/100 performed best. Besides biomass production, S. subsalsa obtained two-fold higher lipid content (35%) in 1/200 MSGR than that in mZM. MSGR-supplemented seawater in baggy reactors also produced S. subsalsa with a high biomass productivity (253 mg/L/day) and lipid generation (92 mg/L/day). Moreover, this medium can be reused once to achieve another 81% of biomass yield and 72% of lipid yield. S. subsalsa exhibits a powerful ability to tolerate high salinity in seawater through elongation and desaturation of fatty acids, accumulation of lysine and methionine, and secretion of sodium. The exclusion of sodium might be associated with Na+/H+ antiporter and H+ ATPase that depend on the energy from photosynthesis and respiration of organic carbon. This cultivation method using both seawater and MSGR could be a cost-effective way to produce S. subsalsa as the source of biofuel feedstock and has the potential to be amplified with large-scale baggy reactors in practice.This work indicates the feasibility of Additional file 1: Fig. S1. Schematic figure of Spirulina subsalsa production with seawater and MSGR: (a) flask bench to optimize the addition of MSGR for cell growth; (b) baggy reactor trial with reused seawater as a preliminary step of scaling up; (c) photo of baggy reactor. Fig. S2. Carbohydrate accumulation for Spirulina subsalsa grown in mZM and in seawater supplemented with MSGR in different volume ratios . All data are averages of biological triplicates ± standard deviation. Fig. S3. Microscopy images of Spirulina subsalsa grown in seawater supplemented with MSGR in different volume ratio  1/1000, (b) 1/500, (c) 1/200, (d) 1/100, (e) 1/50, (f) 1/25, (g) 1/10), in (f) mZM. The blue round cycled bacterium. Scale bar, 20 µm. Fig. S4. Microscopy images of Spirulina subsalsa grown in seawater supplemented with 1/50 MSGR (VMSGR/Vseawater). Scale bar, 20 µm. Fig. S5. The pH value of Spirulina subsalsa culture from mZM, and from seawater supplemented with MSGR in different volume ratios . All data are averages of biological triplicates ± standard deviation. Fig. S6. Total organic carbon in mZM, in seawater supplemented with MSGR in different volume ratios  for cultivating Spirulina subsalsa. All data are averages of biological triplicates ± standard deviation.


The degradation of caffeic acid under ultrasound treatment in a model system was investigated. The type of solvent and temperature were important factors in determining the outcome of the degradation reactions. Liquid height, ultrasonic intensity and duty cycle only affected degradation rate, but did not change the nature of the degradation. The degradation rate of caffeic acid decreased with increasing temperature. Degradation kinetics of caffeic acid under ultrasound fitted a zero-order reaction from −5 to 25 °C. Caffeic acid underwent decomposition and oligomerization reactions under ultrasound. The degradation products were tentatively identified by FT-IR and HPLC-UV-ESIMS to include the corresponding decarboxylation products and their dimers. Caffeic acid and its analogues are widely distributed in the plant kingdom and are found in coffee beans, olives, propolis, fruits, and vegetables ,2,3. Thep-hydroxybenzoic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid and sinapic acid are stable up to 100 °C, whereas at 125 °C there was significant degradation during microwave-assisted extraction − peak at m/z 491.1 and [M+Na]+ at m/z 514.9. The molecular weight of Peak 4 was 220 based on a [M–H]− at m/z 219.1, [M+H]+ at m/z 220.8 and [M+Na]+ at m/z 243.0. However, a reasonable explanation of Peaks 3-4 could not be given based on the molecular weights and our knowledge of caffeic acid, which needs further investigation. With evidence of four molecular ions [M–H]− at m/z 357.1, [M+Cl]− at m/z 393.1, [M+H]+ at m/z 359.0 and [M+Na]+ at m/z 381.0, the molecular weight of Peak 5 was 358, which is twice the molecular weight of caffeic acid (180 × 2 − 2H = 358), thus it was provisionally assumed to be a caffeic acid dimer. In terms of Peak 6, 8, 9, 10 and 11, the ESIMS spectra responses were too low to distinguish them clearly from the background. The ESIMS spectrum of Peak 1 showed the [M–H]+ molecular ion at m/z 161.0 and the [M+Na]+ one at m/z 183.0, but a reasonable structure cannot be proposed at this time. The FT-IR spectra of untreated caffeic acid treated and caffeic acid treated by ultrasound at −5 to 25 °C were different (−1 corresponding to ν(CO2) was observed in FT-IR spectra of caffeic acid treated by ultrasound  and oligomerization reactions occurred. The proposed degradation mechanism of caffeic acid under ultrasound is demonstrated in HPLC-grade methanol was purchased from Tedia Company, Inc. . Ethanol, acetone and acetic acid  were purchased from Sinopharm Chemical Reagent Co. . The standard caffeic acid (purity ≥ 98.0%) was purchased from Sigma Chemical Co. .2. The sample macerated under the same conditions was used as control check (CK). The US and CK solution were filtered through a 0.45 μm polyvinylidene fluoride microfiltration membrane , then stored at −18 °C for further HPLC analysis.Ultrasound treatments (US) were carried out with a probe ultrasonic processor . Some parameters of the probe ultrasonic processor are as follows: the highest power is 900 W, frequency is 20 kHz, and the diameter of horn microtip is 6 mm. The 8.0 μg/mL solution of standard phenolic acid was prepared using volumetric flask. The solution was added to brown glass tubes (3 cm diameter × 10–20 cm height), and then the tubes with solution were immersed in low-temperature thermostatic ethanol (T ≤ 0 °C) or water (T ≥ 5 °C) bath  to maintain a constant temperature. The solution was treated with ultrasound. Apart from the special ultrasound conditions mentioned in the results, the general ultrasound conditions were: the probe was placed 1 cm from the top surface of the extraction cell, the liquid height measured the distance from the horn microtip to tube bottom was 4 cm, and the temperature of −5 °C , the treatment time of 60 min, electrical ultrasonic intensity of 796.18 W/cm2, respectively.The electrical acoustic intensity dissipated from the probe microtip was calculated according to the following formula :2where2 > 0.997 for caffeic acid. The repeatability of intraday analysis ranged from RSD 0.13% to 1.23% (n = 3). The detection limit was 0.035 μg/mL, and the quantification limit was 0.117 μg/mL.Phenolic acids UFLC analyses were carried out on an LC-20 (Shimadzu) instrument linked simultaneously to a SPD-M20A. One μL treated phenolic acid solution was injected on a reversed phase 2.0 × 100 mm, 2.9 μm CAPCELL PAK C18 MG S3 column . The column thermostat was set at 40 °C. Solvent A was 4% acetic acid/water, solvent B was methanol (A:B = 80:20) and flow rate was 0.2 mL/min, which was in accordance with Xu  with som−1 against time (second-order) and concentration−2 against time (third-order)] should be linear. The result showing the best correlation coefficient (R) was selected. The zero-order, first-order, second-order, third-order models are as follows in turn:c is the concentration of reactant at given time, 0c is the initial concentration of reactant, k is the rate constant, t is treatment time.The reaction order of degradation of caffeic acid was obtained using the integral method . This me−1. The spectral resolution was 1 cm−1 and the collection time was about 1 min. The peaks analysis of FT-IR was performed with Origin-lab 7.5.The functional groups of the degradation products at −5 to 25 °C were analyzed by FT-IR spectroscopy. FT-IR spectra of degradation products were obtained on a Nicolet 5700 . The wavenumber range covered was from 400–4,000 cmm/z 50–1,000. Fragmentor was set at 135 V, gas temperature was set at 325 °C and gas flow was 5 L/min. Sheath gas temperature was 250 °C and sheath gas flow was 11 L/min. The nozzle voltage was 500 V. Capillary voltage was 4,000 V for positive ion mode and −3,500 V for negative ion mode.The HPLC-UV-ESIMS system analyses were carried out on an Agilent-6460 Triple Quad LC/MS fitted with an ESI source. The HPLC conditions were in accordance with the description above with some revisions. Solvent A was changed to water containing 0.1% formic acid. Data acquisition and processing were performed using the Mass Hunter software. Positive and negative ion mass spectra of the column eluate were recorded in the range p < 0.05.Each treatment was replicated in triplicate. The results were expressed as mean ± SD. All the data were subjected to statistical analyses using SPSS16.0. The main effect of each factor of ultrasound treatment was subjected to analysis of variance and Duncan’s multiple range tests using One-Way ANOVA procedure. Mean values were considered significantly different when To obtain greater knowledge about the stability of caffeic acid during ultrasound-assisted extraction, the factors effecting degradation, the degradation kinetics and degradation products were investigated in a model system. The results indicated that caffeic acid was degraded under ultrasound treatment, and the type of solvents and temperature were important factors in determining the degradation reaction outcome and other factors such as liquid height, ultrasonic intensity and duty cycle of ultrasound exposure only affected the rate of degradation but did not change the nature of degradation. Degradation rate of caffeic acid decreased with increasing temperature. Degradation kinetics of caffeic acid under ultrasound fitted a zero–order reaction equation at −5 to 25 °C. Caffeic acid underwent decomposition and polymerization reactions under ultrasound. This study provides very useful information for the application of the ultrasound technique for the extraction of caffeic acid.


Autism spectrum disorder (ASD) is defined as a group of genetically and clinically heterogeneous neurodevelopmental disorders. Interplay between de novo and inherited rare variants has been suspected in the development of ASD.Here, we applied 750K oligonucleotide microarray analysis and whole‐exome sequencing (WES) to five trios from Taiwanese families with ASD.JMJD1C, TCF12, BIRC6, and NHS have not been previously reported. A novel de novo variant was identified in NHS (p.I7T). Additionally, seven pathogenic variants, including SMPD1, FUT2, BCHE, MYBPC3, DUOX2, EYS, and FLG, were detected in four probands. One of the involved genes, SMPD1, had previously been reported to be mutated in patients with Parkinson's disease.The chromosomal microarray analysis revealed three representative known diagnostic copy number variants that contributed to the clinical presentation: the chromosome locations 2q13, 1q21.1q21.2, and 9q33.1. WES detected 22 rare variants in all trios, including four that were newly discovered, one of which is a de novo variant. Sequencing variants of These findings suggest that de novo or inherited rare variants and copy number variants may be double or multiple hits of the probands that lead to ASD. WES could be useful in identifying possible causative ASD variants. We applied 750K oligonucleotide microarray analysis and whole‐exome sequencing to five trios from Taiwanese families with ASD. A novel de novo variant was identified in NHS . de novo or inherited rare variants and copy number variants may be double or multiple hits of the probands that lead to ASD. The most common microarray abnormalities in ASD involve the chromosome regions 15q11‐q13, 16p11.2, and 22q11.2  have focused on protein‐coding regions and analyzed trio information to identify sequence‐level de novo mutations , which was modified from the third edition of the Autism Behavior Checklist of Autism Screening Instrument for Education Planning  were hybridized to the Affymetrix CytoScan 750K array according to the manufacturer's instructions. The 750K array contained greater than 750,000 markers for copy number analysis and 200,000 SNP probes for genotyping. The following standard experimental procedures were performed: digestion, ligation, polymerase chain reaction (PCR), PCR purification, fragmentation, labeling, hybridization, washing, staining, and scanning. After hybridization, GeneChip Scanner 3000 7G, Affymetrix GeneChip Command Console software, and Affymetrix ChAS 2.0 software were used for scanning the arrays, extracting the images, and performing the analysis, respectively. All data had to pass quality control (QC) metrics including the median of the absolute values of all pairwise differences ≤ 0.30, SNPQC ≥ 15, and a waviness standard deviation ≤ 0.12.2.3In total, 100 ng of genomic DNA based on Qubit quantification was mechanically fragmented on a M220 focused ultrasonicator Covaris , and QC was performed using an Agilent Bioanalyzer 4200  to ensure an average fragment size of 150–200 bp. End repair, A‐tailing, adaptor ligation, and enrichment of DNA fragments were then performed. A 200–400 bp band was gel‐selected, and exome capture was performed using a TruSeq Exome Library Preparation Kit . The DNA library was quantified in the Qubit 3.0 Fluorometer (Invitrogen) and Agilent 4200 Bioanalyzer (Agilent Technologies). Samples were sequenced on an Illumina NextSeq500 platform and 150‐bp paired‐end reads were generated.2.4http://gnomad-old.broadinstitute.org/), Denovo‐db (http://denovo-db.gs.washington.edu/denovo-db/), ClinVar, Polyphen‐2, SIFT, and CADD . Additionally, ASD‐related genes reported in the public databases OMIM and AutDB were selected  using the Burrows–Wheeler alignment . Table We used PCR and Sanger sequencing to validate candidate variants from WES. Segregation analysis was carried out on family members. PCR primers were designed using Primer3 . WES revealed four rare variants in SHANK3 , DNAH10 , ESR2 , and NAALADL2  (Table SMPD1 (p.P186L), FUT2 (p.R202X), and BCHE (p.T343fs).Following QC of the WES data, five probands were analyzed further and confirmed by Sanger sequencing. For these, a mean coverage depth of 141X was achieved Table . The 750DLGAP3 , SLC1A2 , and CLTCL1  (Table MYBPC3 (p.E334K) and DUOX2 (p.K530X).Patient 2, an 8‐year‐old male, had an ABC‐T score of 27 Table . The 750HYDIN2, PRKAB2, FMO5, CHD1L, BCL9, ACP6, GJA5, GJA8, GPR89B, and NBPF11). WES revealed seven rare variants in WFS1 , TNN , JMJD1C  , SYNE1 , MPP6 , and MCC  (Table EYS gene (p.C2139Y) was observed.Patient 3, a 15‐year‐old male, had an ABC‐T score of 20 , SETBP1 , TCF12  , BIRC6  , and ASMT  . WES revealed one de novo variant in the NHS  gene  was observed.Patient 5, a 10‐year‐old male, had an ABC‐T score of 36  . The p.F2115C mutation is in the JmjC domain, a domain family that is part of the cupin metalloenzyme superfamily. Mutations in this gene are associated with Rett syndrome and intellectual disability  CANNTG. This study identified a variant, c.770G > A, which results in substitution of an arginine by histidine (p.R257H) in TCF12. BIRC6 encodes an inhibitor of apoptosis protein with baculoviral inhibition of apoptosis protein repeat (BIR) and ubiquitin‐conjugating enzyme E2, catalytic (UBCc) domains. This study found a variant of BIRC6, c.6600G > T, which results in substitution of a glutamine by histidine (p.Q2200H). NHS encodes a protein with four conserved nuclear localization signals that function in brain development. This study identified a variant, c.20T > C, which results in substitution of isoleucine by threonine (p.I7T) in NHS. Mutations in this gene are associated with Nance–Horan syndrome  were found to carry a novel missense variant of four genes . The pathogenic mutations were detected in ts Table . SMPD1 eIn conclusion, we report on five ASD patients with rare variants and one patient with a de novo variant. However, this association study was performed with only a small number of cases; therefore, further studies with larger sample sizes are needed.The authors declare that they have no conflict of interest. Click here for additional data file.


Panax quinquefolius (PQ) and Acorus gramineus (AG) are drug target pairs in traditional Chinese medicine (TCM), which are used to treat age-related diseases. In the present study, we simultaneously determined the contents of four main bioactive ginsenosides  in rat plasma using an ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method. Plasma specimens were purified by using the solid-phase extraction procedure, and separation was performed on Waters ACQUITY UPLC BEH C18  in multiple reaction monitoring (MRM) mode and negative electrospray ionization (ESI) mode. The established UPLC-MS/MS method showed good linear correlation (r ≥ 0.9978), stability (−11.93 to 12.11%), precision (RSD < 14.63%), and recovery (76.43%–95.20%). The lower limit of quantification was 3.6 ng/mL for Rb1, 1.6 ng/mL for Rb2, 1.2 ng/mL for Rd, and 2.5 ng/mL for Re. This validated method was successfully employed to investigate the pharmacokinetics of the four ginsenosides in rat plasma after oral administration of PQ-AG and PQ extracts. The results revealed the pharmacokinetic profiles of PQ-AG drug pair and clarified that AG played a critical role in stimulating the absorption of active ginsenosides in PQ. Collectively, our findings provided valid and reliable evidence for the rational use of PQ-AG in clinical practice. Panax quinquefolius (PQ) is used to restore vital energy and treat diabetes − at m/z 1, 107.5 for Rb1, and the product ion peak was at m/z 459.6, which correspond to the loss of four molecules of Glc [M-4Glc-H]−. The precursor ion was [M-H]− at m/z 1, 077.7 for Rb2, and the product ion peak was at m/z 459.5, which correspond to the loss of three molecules of Glc and one molecule of Ara [M-3Glc-Ara-H]−. The precursor ion was [M-H]− at m/z 945.5 for Rd, and the product ion peak at m/z 621.5, which correspond to the loss of two molecules of Glc [M-2Glc-H]−. The precursor ion was [M-H]− at m/z 945.5 for Re, and the product ion peak was at m/z 475.5, which correspond to the loss of two molecules of Glc and one molecule of Rha [M-2Glc-Rha-H]−. The precursor ion was [M-H]− at m/z 867.6 for internal standard (IS), and the product ion peak was at m/z 721.0, which correspond to the loss of one molecule of Rha [M-Rha-H]−. In the optimization process, we found that acetonitrile/H1, Rb2, Rd, Re, and dioscin. Dioscin was used as the IS. The chemical structure of IS was found to be similar to analytes, and it exhibited a stable response, indicating effective separation.Under the above-mentioned chromatographic conditions, saponin components showed more intensive deprotoned ions in the negative ion mode than those of protonated or sodiated precursors in the positive mode. Therefore, an UPLC-MRM-MS with an ESI interface in the negative mode was used for separation and detection of all compounds tested. Reference standards showed good peak shape and excellent resolution. 1, Rb2, Rd, and Re.r) for each calibration curve was higher than 0.9978. The result indicated that the ratio of peak area strongly correlated with the concentration of each compound within the acceptable linearity ranges. The LLOQs of the four analytes are shown in The precision and accuracy of the assay were validated for the samples at LLOQ and three QC levels analyzed on the same day or during three consecutive days. The results are presented in The recoveries were obtained at three concentration levels, ranging from 76.43% to 95.20%. The ME of all the analytes in blank plasma were within the acceptable range of 90.44%–107.11% . The ME The sample stability during storage and processing procedures was evaluated by the analysis of QC samples , showing1, Rb2, Rd, and Re after oral administration of PQ-AG or PQ. Cmax), time to reach the maximum concentrations (Tmax), half-time (t1/2), and area under concentration-time curve (AUCt0⟶ and AUC0⟶∞). These results showed that UPLC-MS/MS could be used to detect Rb1, Rb2, Rd, and Re, revealing good absorption of four compounds. The significant differences among Rb1, Rb2, Rd, and Re were observed between PQ-AG and PQ groups after oral administration. For Rb1 with certain bioavailability [Cmax was 600.2 ± 53.6 ng/mL in the PQ-AG group, which was significantly higher than that (461.2 ± 15.9 ng/mL) of the PQ group (P < 0.05). Meanwhile, the Tmax of Rb1 was 6.67 ± 1.03 h for the PQ-AG group and 7.00 ± 1.10 h for the PQ group, illustrating Rb1 with high uptake in rat plasma for the PQ-AG group. The reason might be attributed to those other components in the PQ-AG group which promoted the absorption and metabolism of Rb1. The Cmax and Tmax values of Rb2 in the PQ-AG group were 264.1 ± 24.5 ng/mL and 7.67 ± 0.82 h, respectively. The Cmax and Tmax values of Rb2 in the PQ group were 160.1 ± 18.8 ng/mL and 7.67 ± 0.82 h, respectively. Rd showed a good absorption in the PQ-AG group, indicating that Cmax was 283.4 ± 11.1 ng/mL and Tmax was lower than that in the PQ group (P < 0.05), in which the Cmax was 200.9 ± 7.3 ng/mL and Tmax was 7.67 ± 0.82 h. The results also showed that the Tmax of Rd and Re in the PQ-AG group was shorter than those in the PQ group (P < 0.05). Moreover, as shown from Tmax value of Re, which was different from the other three compounds between the two groups, was 0.58 ± 0.20 and 1.08 ± 0.20 h. Although the entire hypothesis needed to be further validated, Rd and Re had different absorptions compared with Rb1 and Rb2, and the double-peak phenomenon was detected at the same time, which could be probably attributed to the enterohepatic circulation in drug metabolism or the interactivity of PQ-AG drug pair [Cmax, AUCt0⟶, and AUC0⟶∞ of the four active ingredients in the PQ-AG group were higher than those in the PQ group. It suggested that four compounds in the PQ-AG group had synergistic effects with other compounds, leading to increased plasma contents of four analytes by promoting absorption. Compared with the PQ group (P < 0.05), the t1/2 of four analytes in the PQ-AG group was 18.52 ± 2.61, 15.61 ± 5.21, 14.21 ± 1.98, and 9.81 ± 2.15 h, respectively, indicating slow elimination. Moreover, the absorption was enhanced after compatibility, and the duration of drug action was prolonged for t1/2 of the four compounds in the PQ-AG group. Furthermore, the bioavailability of bioactive components in the PQ-AG group showed an upward trend, indicating that the four bioactive compounds of Rb1, Rb2, Rd, and Re were well absorbed and gradually removed in the PQ-AG group, which might be attributed to the synergic action between PQ and AG. According to TCM theory, the compatibility mechanisms of drug pairs are not only arbitrary plus of two drugs but also the regularity of active components in vivo [lability , the plarug pair , 22. In  in vivo . In the  in vivo , 26. In The results showed that AG enhanced the absorption and delayed the elimination of the four analytes in PQ. Moreover, it could result in a better bioavailability of ginsenosides. Some previous literatures have shown that certain compounds in AG may suppress P-glycoprotein functions in the intestinal membrane and the activity of enzyme CYP1A2 . These f1, Rb2, Rd, and Re in PQ-AG after oral administration. Our data showed that the four analytes had a better absorption and slower elimination after PQ and AG were combined. The changes of four main active substances in vivo provided valuable insights into the compatibility principles of PQ-AG. In addition, our current findings clarified the rational compatibility of TCM, suggesting better clinical application and research potentials.In the present study, we successfully applied a simple, rapid, and sensitive UPLC-MS/MS method to simultaneously determine four bioactive ginsenosides in PQ and the combination of PQ and AG. We, for the first time, reported pharmacokinetic parameters of Rb


Senna alata, S. alexandrina, and S. occidentalis) as effective remedies against gastrointestinal trematode parasite (Paramphistomum gracile) of ruminants. Live adult parasites were collected in 0.1 M phosphate-buffered saline (PBS) from fresh autopsied goat’s rumen. Parasites were incubated in leaf extracts of S. alata, S. alexandrina and, S. occidentalis individually and in combination (1:1) ratio at 37 ± 1°C. Treatment media contain extracts at different concentrations  in 10 mL of 0.1 M PBS with 1% dimethylsulphoxide (DMSO). Parasites in control group were incubated in PBS without extract. The efficacy of three Senna extracts was evaluated on the basis of dose-dependent motility and mortality of the trematode. Immediately after paralysis, all treated parasites were collected for histology, SEM and biochemical study.Paramphistomosis is a pathogenic disease of domesticated ruminants, causing great economic loss in dairy industry and meat production. It is considered as a neglected tropical disease with highest prevalence throughout tropical and subtropical regions, particularly in Africa, Asia, Europe, and Australia. There are few trematocidal drugs available in the market. Most are resistant and have elevated side effects. Therefore, alternative trematocidal drugs need to discover. This study was conducted to evaluate three plants leaf extracts (from Senna plant extracts. S. occidentalis and S. alexandrina showed better efficacy in combination than comparing with individual treatment groups. Histological study and scanning electron microscopic observations revealed conspicuous deformity of surface architecture in all treated parasites. Scanning electron micrographs also revealed shrinkage, vacuolization, infoldings and blebbings on the body surface of treated worms. Activities of tegumental enzymes were inhibited in all treatment groups compared to control.Dose-dependent efficacy was observed in terms of motility and time of mortality in all treated parasites after exposure in various concentrations of the Senna leaf extracts individually and in combination showed potential antitrematocidal activity against Paramphistomum gracile by damaging body tegument and neural propagation. Thus, this study confirmed that all three Senna extracts can be considered as a potential drug-like candidate in indigenous system of traditional medicine against trematode infections in livestock.The overall findings from this study revealed that all three  Paramphistomum sp. It has a worldwide geographical distribution with the highest prevalence in different regions of tropical and subtropical regions, particularly in Africa, Asia, Australia and Europe [Paramphistomum sp. [Paramphistomum sp. infection [Paramphistomum daubneyi in Europe can cause significant gastrointestinal disease, which drops in yield or even death in ruminants [Paramphistomosis is a neglected parasitic disease of ruminants caused by one of the most common trematode parasites d Europe –4. The dd Europe –10. Slauomum sp. . It has nfection , 13. Parnfection . Mortalinfection –17. In snfection , 19. Nevuminants . Most inuminants . MigratiParamphistomum sp. infection in the small intestine of young (less than 2 years of age) and immune deficiency goats, which effects on the organs, is characterized by listlessness, strangulation, haemorrhage, and anorexia [Acute paramphistomosis is caused by anorexia –24. Rastanorexia  showed tanorexia . Higher anorexia .Paramphistomosis has also recently emerged as a major cause of productivity loss in livestock . The disSenna comprises 580 species of herbs, shrubs, and trees, which are widely distributed throughout the world, of which only 20 species are indigenous to India [Senna species has been reported of medicinal properties e.g. - S. alata has been recognized for centuries in traditional medicine for its antimicrobial [S. alexandrina is known for its purgative properties and for regularization of bowel movements [S. occidentalis has been traditionally used for the treatment of constipation, skin diseases, and as laxative and diuretic [Medicinal plants used by ethnic groups worldwide have been shown to possess anthelmintic activity using modern scientific tools. As per the World Health Organization, 80% of the world’s population relies on traditional medicines for their primary health care. Medicinal plants have been used as valuable drugs, either as crude extracts or in the form of molecules. The use of medicinal plants in developing countries has grown considerably in the second half of twentieth century  and ofteto India . Many oficrobial , antifunicrobial , purgatiicrobial , anti-inicrobial , analgesicrobial , antitumicrobial , and hypicrobial . S. alexovements  while S.diuretic . All thrdiuretic –47. HoweHymenolepis diminuta, Raillietina tetragona) and nematodes  [Fasciola hepatica in the animal husbandry industry. Despite few observations on the fine surface features of Paramphistomum parasites, is closely associated with Fasciola hepatica [Senna plants  ethanolic leaves extracts on Paramphistomum gracile in terms of paralysis and death of parasites, (2) Investigation of alteration or damage in tegument architecture of Paramphistomum gracile by histomorphological and scanning electron microscopy (SEM) due to action of Senna treatments, (3) The effects of three Senna plants extracts on tegument enzymatic activity of Paramphistomum gracile.Though anthelmintic studies have been reported in cestodes (linarum) –46, but hepatica –50. BaseSenna extracts with high significant value (p < 0.001)  of treatment, paralysis (PT) occurred after 0.6 ± 0.15 h, 0.61 ± 0.2 h and 1.52 ± 0.7 h in S. alata, S. alexandrina, and S. occidentalis incubation respectively. Similarly, mortality (TM) or death of parasite occurred at 2.54 ± 0.1 h, 3.03 ± 0.26 h, and 4.28 ± 0.12 h, after treated with S. alata, S. alexandrina, and S. occidentalis respectively at 40 mg/ml concentration. While the control parasites stay alive in control medium till 43.53 ± 2.29 h. The post-paralytic time in all the three Senna extracts tested was comparatively shorter. In the three combinations treatment worms get paralysed at 1.03 ± 0.33 h, 1.0 ± 0.25 h, and 1.42 ± 0.52 h after treatment with ,  and  respectively in 40 mg/mL concentration. The same pattern was revealed in mortality S. alata  get thine and the parenchyma layered (PL) as well as vitelline cells (VC) loss their integrity while treated with S. occidentalis + S. alexandrina combination treatment parenchyma layered (PL) as well as vitelline cells (VC) were found totally distorted  and an ovary (O) was seen situated close to the posterior sucker Fig. a. Prominata Fig. b followeina Fig. c, d. Mosina Fig. a. After ite Fig. b followeina Fig. c, d. In Senna extracts, parasites body size reduced and shrinkage occurred through body length. It was observed that the texture of tegument was disrupted in S. alata treated parasite and highly shrinkage throughout the body , bound by a muscular rim around the opening along with scattered papillae. Small gonopore was seen at a position, one third from the anterior end of the body. The tegument contained numerous elevated papillae arranged in transverse rows and intensity is more at the anterior region of the body surface Fig. a. After ody Fig. b. Similaina Fig. c. Howeveite Fig. d. At higata Fig. c, d. In ina Fig. e and f pina Fig. a, b. Minent Fig. g, h. SEMent Fig. a, b. Butred Fig. c, d. In ted Fig. e, f. Howted Fig. g, h. Theted Fig. a, b. Aftata Fig. c and d, ina Fig. e, f and cts Fig. g, h. Numcts Fig. a, b. Howent Fig. g, h.FigSenna extracts on tegumental enzymatic activity were given detailed in Table P. gracile to the different Senna extracts. Among the three Senna extracts treatment, the highest reduction of AcPase activity was observed in S. alata treated parasites, where the AcPase unit activity was 10.35 ± 0.69 unit/g wet wt. tissue and specific activity was 0.30 ± 0.01 unit/mg protein. It has calculated about 53.9% reduction occurred in AcPase activity in comparison with control. Similarly in S. occidentalis and S. alexandrina treated parasites AcPase total unit activity was 12.51 ± 2.94 and 14.23 ± 3.35 unit/g wet wt. tissue and specific activities were 0.36 ± 0.08 and 0.43 ± 0.09 unit/mg protein respectively. So, it has calculated that 44.6% inhibition occurred in specific activity in S. occidentalis treatment and 33.9% in S. alexandrina treated parasite with comparable with control , Alkaline phosphatase (AlkPase), ATPase and 5′-Nucleotidase (5′Nu) when compared to control. The particular effects of Senna extracts. From the obtained result it was observed that the highest decline in AlkPase activity was clearly remarkable in S. alata treated parasites followed by S. occidentalis and S. alexandrina. It was found that S. alata treated parasite AlkPase unit activity 175.55 ± 2.12 unit/g wet wt. tissue and specific activity 0.54 ± 0.01 unit/mg protein. Thereby, it was showing about 50.5% inhibition occurred due to S. alata treatment. Similarly, significant decreases were also noticeable in S. occidentalis and S. alexandrina, where unit activity was 187.78 ± 2.98 and 203.63 ± 6.9 unit/g wet wt. tissue, as well as specific activity was 0.55 ± 0.02 and 0.62 ± 0.01 unit/mg protein respectively. So, it was calculated that about 49.54 and 43.12% depletion occurred in AlkPase specific activity in parasites after treatment with S. occidentalis and S. alexandrina respectively, comparable with control  as well as specific activity (4.76 ± 0.01 unit/mg protein) comparable with control. From the obtained result it was observed that highest decline in 5′Nu activity was clearly remarkable in S. alata treated parasites. In the case of AChE activity, control parasite AChE unit activity was observed 473.82 ± 21.55 unit/g wet wt. tissue and specific activity were 11.66 ± 1.11 unit/mg proteins. The AChE activity in the parasite tissue decreases significantly about 20% after treatments with S. alata and S. occidentalis extracts, while only a little decrease in activity  was observed in treatment with S. alexandrina. Here also S. alata showed highest inhibitory effects among the three plants  showed dose-dependent trematocidal activity on Moniezia spp, Thysaniezia spp, Schistosoma mansoni, Fasciola hepatica, and Echinostoma caproni parasites in vitro. Amongst the three species of Senna plants, S. alata caused earliest paralysis followed by S. alexandrina and S. occidentalis respectively. However, S. occidentalis treatment has taken longer time to paralyse the worms by itself, but in combination with S. alata and with S. alexandrina it reduced the time of paralysis (PT) and time of mortality (TM) significantly (P < 0.001). This may be suggested that in combination the two plants may possess a synergistic effect and the combined action may have an efficacious intervention on the target organisms, like that observation also noticed by Kundu et al., in Hymenolepis diminuta treated with different Senna plants [Senna extracts [Hymenolepis diminuta was treated with Senna leaf extracts [Senna extracts treatment, still there was a considerable significant paralysis occurred. It may therefore, be suggested that all Senna extracts singly or in combination possibly exert a reversible action on the neuromuscular system of the parasite, and though it did not cause early mortality, but once paralyzed, it took very short time for mortality to commence. Thus, it may be suggested that it possesses a vermifugal activity in nature and the inactiveness caused would last long enough for the parasites to be swept out of host’s body [Senna extracts have the potential effects of P. gracile in motility and mortality.The present investigation illustrates that the three species of a et al. , 52 thata plants . These vextracts . Similarextracts , 45. Thot’s body –45. TherSenna extracts treated parasites revealed that remarkable changes occurred on tegumental surface, with extensive shrinkage and consisted of swelling, blebbing, which was later ruptured, leading to erosion, perforation, and desquamation of the tegument, resulting in the lesion, and finally the exposure and disruption of basal lamina. A similar type of disruptions noticeable in Paramphistomum cervi after treatment with Plumbagin is a compound of Plumbago indica/rosea [Senna extracts treated parasites may be attributed on the ionic homeostasis and neural impulse conductance of worm muscle membrane producing hyperpolarization and reducing excitability that could lead to muscle relaxation and flaccid paralysis, which caused would lead the treated parasite to be swept out of the host’s tissue [P. gracile after treatment with the three Senna extracts treatment. Disorientation and loss of density of sensory papillae are indications of disruption in sensory system of the parasite [Senna leaves extracts treatment which could cause the loss of sensory functions. Besides, the changes were found at the oral sucker and acetabulum, which exhibited the swollen appearance and scattered blebs along their rims. Such pronounced destruction affects the attachment ability with the host tissues [For evaluating anthelmintic action, histopathology and scanning electron microscopy has proved the first and most obvious pathological effects of plant treatment on the parasite. The tegument is an important structure of trematode parasite because it provides covering and protection of the parasite’s body, and supports internal organs. It also controls the secretion, synthesis, perception of sensory stimuli and osmoregulation. Therefore, histomorphology and electron microscopic observations of ca/rosea . Depletica/rosea . Pronouns tissue , 51, 54.s tissue . Once thparasite . And disparasite –60. The  tissues , 62.P. gracile by three Senna plants may affect in absorption, intracellular digestion of food items, and membrane transportation in the parasite tissue. Similar finding also observed in tegumental enzymes activities in Raillietina echinobothrida after treatment with Acacia oxyphylla, Securinega virosa and synthetic drug praziquental [P. gracile due to Senna extracts treatment, it denotes the less production of ATP and neuromuscular disability in the parasite, which promotes early paralysis and death of parasite. The decrease of the enzyme’s activity may be occurred due to disruption of the absorptive surface. Similar observations have been reported in a trematode after treatment with mebendazole and levamisole [P. gracile affects the metabolism of nucleotides in the parasite body, which affects egg production in the parasite [Tegumental enzymes  have an important role in maintaining ionic homeostasis, active transport and also regulation in metabolic processes within the parasite . Alkaliniquental . Furtheriquental . Thus, 5vamisole . In helmvamisole , 67. Theparasite . Therefoparasite .S. alata, S. alexandrina, and S. occidentalis can be used as an antitrematodal drug, which possesses strong antitrematodal activity against ruminant parasite P. gracile based on in vitro screening assay. Furthermore, histomorphological and ultrastructural observation showed that Senna extracts inflict structural alteration on the tegument and also showed inhibitory effects on tegumental enzymes activity. Taken in conjunction with our present study, it was announced that three Senna plants have strong potentiality as a drug like candidate for the treatment of paramphistomum infection.In conclusion, from our present study, we discover that ethanolic leaf extracts of Senna alata (L.) Roxb., Senna occidentalis (L.) Link., and Senna. alexandrina Mill. grown widely surrounding the Visva-Bharati University campus, were collected during the winter season. The herbariums were prepared for identification of these three plants species and voucher specimens have been deposited in the Central National Herbarium, Botanical Survey of India, Kolkata, India, with voucher No. VBSL-1, VBSL-2, and VBSL-3 for Senna alata (L.) Roxb, Senna occidentalis (L.) Link, and Senna alexandrina Mill. respectively.Fresh leaves of Senna were collected separately. The leaves were washed thoroughly with deionized water, allow it to air-dry. For total drying, the leaves were kept under hot-air oven at 45°C temperature for 1 hour (h). Dried leaves were crushed into powder and kept in airtight containers as labeled separately for further use for extract preparation. For the ethanolic extract preparation, about 250 g of every type powder sample was added in 1 L of 90% ethanol in Soxhlet apparatus  for 8 h. The decoction was filtered through Whatman filter paper. The filtrate was then concentrated as a semisolid mass under vacuum at 40°C using a rotary evaporator . The extract  was stored at 4°C until use for evaluation of antitrematocidal activity.Fresh leaves of three plants species of (Paramphistomum gracile) were collected from different goats rumen from various local slaughterhouses in the Santiniketan campus  throughout the year around and were washed thoroughly with 0.9% phosphate-buffered saline (PBS) at pH 7.4. The parasites were deposited in the Zoological Survey of India with Accession No. W0772/1.Live parasites n = 5) were incubated with different concentration  of alcoholic leaf extracts of S. alata, S. occidentalis, and S. alexandrina dissolves in 10 mL of PBS (pH -7.4) with 1% dimethylsulfoxide (DMSO) in a covered glass Petri plates respectively. For combination treatment, assessment was done according to the Kundu et al. [n = 5) were incubated with different concentrated  of the combined extracts of Senna  in (1:1) ratio dissolved in 10 ml of PBS with 1% dimethylsulfoxide (DMSO) in a covered glass Petri plates separately. One group of parasites (n = 5) were maintained only in 10 mL of PBS with 100 μL of 1% DMSO as a control group. The experiments were carried out at 37°C temperature with 60% humidity in a humidity incubator. Motility and mortality time was recorded during this experiment. Time for paralysis (PT) was observed at different time intervals when no movement of worms could be observed except when shaken vigorously or kept in warmer PBS (40°C temperature). Time of mortality (TM) or death was recorded at the time when worms showed no movement, even when shaken vigorously or put into slightly hot PBS.In vitro anthelmintic assessment was performed according to the Kundu et al.  with sliu et al. , where pImmediately after paralysis, treated parasites from the various treatments groups (single and in combination treatment) were fixed in Bouin’s fixative as a prospective type of treatments for 24 h. Subsequently processed for histological study after proper fixation following the method of Drury & Wallington . In brieImmediately after paralysis, all the treated and control parasites were fixed for scanning electron microscope (SEM) studies following standard method Roy and Tandon . In brieFor assaying the activity of tegumental enzymes, immediately after paralysis single leaf extract at 40 mg/ml dose treated parasites and control parasites were collected and kept into − 20°C for further biochemical estimations.Acid phosphatase (AcPase) and Alkaline phosphatase (AlkPase) activity were assayed by estimating the p-nitrophenol product following the method abducted from Plummer  with sli2 (100 μM), Na-ATP (100 μM) and enzyme source (0.05 mL). One milliliter of 15% trichloroacetic acid (TCA) was added into the reaction mixture, continuation with centrifugation at 3000–4000 rpm for 10 min. After completion of the centrifuge, 0.25 mL of parasite tissue supernatant was added with a mixture of 0.75 ml distilled water, 0.5 ml 5 N H2SO4, 0.5 mL ammonium molybdate (2.5% w/v) and 0.1 ml freshly prepared Fiske and Subbarao reducer. The reaction mixture was incubated for 10 min at 37°C and the absorbance was taken at 700 nm.Adenosine triphosphatase (ATPase) activity was assayed by estimating the free phosphate released following the method of Kaplan  with sli4 (12 μM), AMP (5 μM) and 0.1 ml of 10% tissue homogenate as enzyme source. After that 1.5 ml of 10% (w/v) (PCA) was added to the reaction mixture for 1 h. Precipitated protein was separated out by centrifugation at 3000 rpm for 10 min at room temperature and the supernatant was collected. Then 0.5 ml water, 0.5 ml 5 N H2SO4, 0.5 mL ammonium molybdate (2.5% w/v) and 0.1 mL freshly prepared Fiske and Subbarao reducer were added to the supernatant. The reaction mixture was incubated for 10 min at 37°C temperature and absorbance was taken at 700 nm.The 5′ Nu activity was assayed by estimating the free phosphate released following the method described by Bunitian  with sliAcetylcholine esterase (AChE) activity was estimated following the method of Ellman et al.  with sliProtein was estimated following the method of Lowry et al. , using bn = 5). For determining statistical significance, standard error and analysis of variance (ANOVA) at 95% level of significance were employed considering P < 0.001 as significant. Evaluation of statistical significance between the control and experimental data set was carried out by Duncan’s Multiple Range Test (DMRT). Different small letters on the error bars indicate significant (P < 0.001) differences in the values of a particular variable between the different treated group and control. Same letters indicate no significant differences within these groups.Data are expressed as mean values ± standard error (SE) for each group (


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.Increasing COP. 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).In this study, 2 concentration.This study indicated that breakdown of intracellular protein and soluble sugar provide C skeleton for lipid synthesis under high CO Ocean acidification has not only increased photosynthetic and respiration rates [2 conditions. For systematic research, it is important to select model species with a clear genetic background, that are easy to transform, and perform in metabolic flow analysis.Increased COon rates , and dowon rates , 4. SincPhaeodactylum tricornutum is a pennate diatom with the characteristics of small genome size, short generation cycle, and easy to transform [P. tricornutum is rich in polyunsaturated fatty acids and can therefore be used as food for aquaculture animals [P. tricornutum under high CO2 has not only fundamental research significance but also important application value.As the main components of marine phytoplankton, marine diatoms produce approximately 20% of global primary productivity and play an important role in inorganic C fixation and material cycles , 6. Phaeransform . Since iransform , it has  animals . It has  animals . Its mai animals . TherefoP. tricornutum under high CO2 have been reported. Wu et al. (2010) found that under short term exposure to high CO2 concentration, the growth, photosynthetic, and respiration rates of P. tricornutum increased, while carbon concentrating mechanism activity decreased [2 had no effect on the growth of P. tricornutum [P. triconutom [P. tricornutum to high CO2, which are important for revealing the physiological and ecological processes of marine diatoms under high CO2. However, since this research is limited to growth and photosynthetic physiology only, the molecular mechanism of the response process is not fully understood. Based on genomic data, Levering et al. (2017) integrated the metabolic network and regulation mode of P. tricornutum, and predicted its global regulation mechanism under different CO2 concentrations  at the transcriptome level [The physiological and biochemical responses of ecreased . After lecreased . Li et acornutum . These iiconutom , 16. Theet al. 20 integratP. tricornutum were significantly increased in short-term culture under high CO2 . The key enzymes of the Calvin cycle and pentose phosphate pathway were elevated at both transcriptional and enzymatic levels [P. tricornutum readjusts central C metabolism under high CO2, and supplements its growth and lipid synthesis by high-speed operation of pentose phosphate pathway. These results provide theoretical and technical support for the study the response of P. tricornutum to high CO2. However, the molecular mechanism of its response process has not been comprehensively analyzed, and further research is needed.In a previous study, the current authors found that the growth rate, lipid content, and fucoxanthin content of c levels . This suP. tricornutum was cultured at different CO2 concentrations. Changes in cell composition with culture time were determined. The 13C isotope tracing technique was used to detect the origin of C skeletons. This study reveals the C flow distribution mechanism of P. tricornutum in response to high CO2 concentration.In this study, P. tricornutum showed significant differences under ~ 400 ppm of CO2 (NC) or high CO2 of ~ 2,000 ppm (HC)  Fig. . Under Hus study .Fig. 1GrTotal lipid content changed relatively little at the beginning of cultivation (within 6 days after inoculation) under both HC and NC. After 8 days of cultivation, total lipid content increased a little under NC, but was significantly enhanced under HC Fig. a. This wUnder NC, soluble sugar content increased gradually within 8 days after inoculation. While under HC, soluble sugar content increased sharply within 6 days after inoculation, then decreased significantly on D8. There was little difference between soluble sugar content under HC and NC during the entire cultivation, except on D6, when soluble sugar content under HC was significantly higher than that under NC Fig. b.Under NC, total protein content changed little within 8 days after inoculation. While under HC, total protein content increased a little on D4, then decreased gradually on D6. On D8, total protein content decreased significantly under HC. On D2, total protein content under HC was higher than that under NC. While on D6 and D8, it was significantly lower than that under NC  FLs of major cellular components under HC and NC were analyzed. In general, for the same substance, the FL was higher under NC than under HC.To determine the main C flow of photosynthesis and to track the origin (de novo synthesis through photosynthesis or degradation of intracellular substances) of the C skeleton of the major cellular components, the To analysis the FLs of total lipids, total lipids were derivatized to butyl amides and the FLs of fatty acids were measured. Based on GC-MS, five fatty acids were detected. Among these, stearic acid was detected throughout all samples, and its abundance was much higher than that of the other fatty acids. Therefore, its FL was used to represent the FLs of lipids. Cells on D1, D3, D5, and D7 were labelled and sampled on D2, D4, D6, and D8 (1 day after labelling), respectively. Under HC, the FL of stearic acid increased a little on D4. After that, the FL decreased with increasing culture time. The FL on D8 (~0.045) was significantly lower than that on D2 (~ 0.131). Under NC, the FL increased on D4 and D6. On D8 The FL decreased to ~ 0.083  trifluoroacetamide. The FL of glucose was measured and used to represent the FL of soluble sugar. Under HC, the FL was ~ 0.176 on D2, increased significantly on D4 (~ 0.217), then decreased rapidly on D6 0.103). On D8, FL was ~ 0.083. The changing trend of FL under NC was similar to that under HC, except that the level was higher under NC at the same time point. On D8, the FL was still ~ 0.229 under NC , then decreased significantly to ~ 0.036 on D4. On D6 and D8, the values were ~ 0.026 and ~ 0.022, respectively, which was similar to the value of unlabeled cells. This indicated that under HC, de novo synthesized amino acids through photosynthetic C fixation decreased after D4. Under NC, protein content changed relatively little. Accordingly, the labeling degree of amino acids was maintained above 0.04 at the beginning 6 days after cultivation. On D8, the value decreased to ~ 0.036. This indicated that on D8, de novo synthesized amino acids through photosynthetic C fixation decreased.Under HC, the soluble sugar content increased within 6 days after inoculation and then decreased on D8. Accordingly, the labeling degree of sugar was highest on D4 (0.217) and then decreased to 0.104 on D6. This indicated that de novo synthesized sugar through photosynthetic C fixation had decreased on D6. On D8, the value was ~ 0.083, indicating that there was still a small part of sugar was de novo synthesized through photosynthetic C fixation. Under NC, the soluble sugar content increased rapidly initially and then stabilized. Correspondingly, the labeling degree of sugar increased rapidly on D4 (from ~ 0.185 to ~ 0.463), then decreased slowly. On D8, the value was still 0.231, indicating that sugar de novo synthesis remained in progress.3 (C fixed by photosynthesis), but from the degradation of intracellular substances (such as proteins and soluble sugars).This was not the case for lipids. Under NC, total lipid content did not change much within 6 days of culture. Consistently, the labeling degree did not change much. However, on D8, when total lipid content increased slightly, the labeling degree did not increase but decreased significantly to ~ 0.083. Under HC, total lipid content increased significantly on D8, yet the labeling degree decreased to ~ 0.045. This indicated that the C skeleton needed for fatty acid C chain elongation in lipid synthesis under HC is not mainly derived from NaHCOThis study indicated that the degradation of intracellular substances (such as proteins and soluble sugars) provide the C skeleton needed for fatty acid C chain elongation in lipid synthesis under HC.P. tricornutum (IOCAS-001) were maintained in the laboratory [− 2 s− 1 cool white fluorescent light, 12 /12 h light-dark cycle) in artificial seawater (NaHCO3 was not contained) [− 1 NaNO3, 5 mg L− 1 NaH2PO4·2H2O, 3.2 mg L− 1 FeCl3·6H2O, 1.8 mg L− 1 EDTANa2, 20 mg L− 1 Na2SiO3·9H2O), trace elements, and vitamins (filter-sterilized). The initial pH was ~ 8.8 and was not controlled in the process of cultivation. Cell growth was monitored as described [2 treatment, cultures were cultivated with 1.5-L medium in 2-L flasks, continuously aerated with air  or with a mixture gas of air and CO2  at a constant flow rate of ~ 150 ml min− 1. There were three replicates for each treatment. For cell composition analysis, cells were harvested at 2 days, 4 days, 6 days, and 8 days after inoculation . After 5,000 g centrifugation for 4 min at 20 °C, the cell pellet washed with steam-sterilized artificial seawater, and then frozen immediately in liquid nitrogen and stored at − 80 °C.Axenic cultures of boratory . Cells wboratory    inorganiescribed , 19. For13C labeling, 13C-NaHCO3 (Cambridge Isotope Laboratories) was added to a final concentration of 0.174 g l− 1 to flasks No.1, No.2, No.3, and No.4 at D1, D3, D5, and D7, respectively. Samples were collected at 1 day after labeling  as described above.Cultures were grown in four replicates in 2-L flasks containing 1.5-L medium. For GC-MS was performed with an Agilent 6890–5973 GC-MS system. Agilent HP-5MS column (30 m × 0.25 mm × 0.25 μm) was equipped and He was the carrier gas.− 1 HCl at 110 °C for 3 h and dried at ~ 80 °C overnight, dissolved in 100 μL of anhydrous pyridine, and then derivatized with 50 μL of Bis(trimethylsilyl) trifluoroacetamide. The reaction was conducted at 70 °C for 3 h. After centrifuged at 10,000 g for 5 min, the supernatant was collected and filtered with 0.22 μm pore size filters. GC-MS was performed as previously described [Analysis of sugar labelling enrichment was conducted as described previously . Brieflyescribed .− 1 HCl at 110 °C for 12 h, dried overnight under ~ 80 °C, and dissolved in 100 μL of anhydrous pyridine, and then derivatized with 50 μL of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide. The reaction was conducted at 85 °C for 60 min and centrifuged and filtered as described above. GC-MS was performed as previously described [For labelling enrichment analysis of protein, samples were hydrolyzed with 6 mol lescribed , 23.g for 5 min. The supernatant was recovered and the process repeated three times. Hexane aliquots were combined and evaporated at ~ 20 °C. Butyl amide reactions were performed as previously described [For labelling enrichment analysis of lipids, 1 ml hexane was added to ~ 5 mg freeze-dried algal powder, stirred vigorously for 5 min, and centrifugated at 8,000 escribed . 20 μL oescribed .The GC-MS data were analyzed as described previously , 25–27. 3PO4 (containing 1 M KCl). The mixture was centrifugated for 5 min at 5,000 g and the solvent phase was recovered. After three times of extraction, the solvent phases were combined, washed with distilled water for three times, and then evaporated at ~ 20 °C within a fume hood. The total lipid content was weighed and expressed as % cell dry weight (CDW).Total lipids content was determined as described previously , 29. Brig. The supernatant was collected and merged after three times of extraction, and evaporated at 85 °C to ~ 0.3 ml, made up to a total volume of 1 ml with distilled water and used for the soluble sugar analysis. 0.2 ml sample was mixed with 1 ml of anthrone solution (2 g anthrone in 1 L 72% (v/v) H2SO4)and incubated at 100 °C for 8 min, and the absorbance under 625 nm was detected. Glucose was used as standard.Total soluble sugar content was analyzed as described previously , 31. Bri2O was added to the hydrolysate to bring the volume to 1 mL. The mixture was centrifuged for 15 min at 12,000 g and the supernatant was recovered. After three times of extraction, all the resulted supernatants were merged, and analyzed using a bicinchoninic acid protein assay kit (cat no. P0010S). Bovine serum albumin was used as the standard.Total protein content was determined as previously described . Briefly− 1. The temperature of the combustion tube was controlled at 800 °C, and the maximum integration time was 200 s.The concentration of nitrate in the medium was determined with a Multi N/C 2100S Analyzer  equipped with a solid-state electrochemical detector (ChD). Sodium nitrate was used as the standard. The injection volume was 250 μL and the linear range was 0.2–5 mg LP-value < 0.05.Each treatment was conducted in 3 replications. Student t-test were used to detect the significance of differences among samples, and the statistical significance level was set at


CYP21A2 gene variant. In males, the excess androgens produce varying degrees of penile enlargement and small testes. CHARGE syndrome (CS) has a broad spectrum of symptoms. In males, genital features such as micropenis and cryptorchidism are found in 48% of CS. There are no reports of patients with combined 21‐OHD and CS; therefore, it is unknown whether the external genitalia shows penile enlargement or micropenis with/without cryptorchidism.21‐hydroxylase deficiency (21‐OHD) is caused due to A boy, born at 37 weeks and 5 days of gestational age with no consanguineous marriage, was admitted to our hospital due to congenital cleft lip, cleft palate, micropenis, cryptorchidism, and a ventricular septal defect. He had severe hyponatremia and hyperkalemia on day 10. He was diagnosed to have 21‐OHD and CS. His external genitalia demonstrated both cryptorchidism and micropenis, but not penile enlargement.CYP21A2. Exome sequence was performed, and then, Sanger sequence was performed around variant in CHD7.DNA was extracted from peripheral leukocytes using standard procedures. Sanger sequence was performed in CYP21A2 gene was performed and compound heterozygous variants of c.293‐13A/C>G (IVS2‐13A/C>G) and c.518T>A (p.I172N) were detected in chromosome 6p21.3. His mother had been heterozygous variant of c.293‐13A/C>G, and his father had been heterozygous variant of c.518T>A. Simultaneously, a de novo splicing acceptor alteration in c.7165–4 A>G, in chromodomain helicase DNA binding protein‐7 (CHD7), located in chromosome 8q12 was detected, and the patient was diagnosed with 21‐OHD and CS.Genetic screening for Although these two disorders exhibit different modes of inheritance and their co‐morbidity is extremely rare, we encountered one male patient who suffered from both 21‐OHD and CS. The symptoms of SV in boys are progressive penile enlargement, small testes, and rapid growth. The symptoms of NC are basically null. The typical symptoms of SW, the most severe type of 21‐OHD, include vomiting, failure to thrive, and skin pigmentation during the neonatal period  (OMIM 201910) is caused due to CHD7) located in 8q12.1. CS usually occurs in an autosomal dominant manner. Family histories of CS are rare, and 97% of CHD7 variants are de novo  (OMIM 214800) has a broad spectrum of symptoms, such as coloboma, heart defects, atresia of choanae, retarded growth and development, genital abnormalities, ear anomalies, and/or hearing loss defects  and birth height was 46.8 cm (−0.52 SD). At the time of birth, a cleft lip, cleft palate, micropenis, cryptorchidism, and ventricular septal defect (VSD) were detected in the patient. Seven days after birth, heart failure developed due to VSD, and diuretic agents were started; furthermore, the patient exhibited severe hyponatremia, hyperkalemia, and hypoglycemia. Neonatal mass screening revealed that his 17‐OHP was elevated to 8.9 ng/ml  at 1 day after birth and 18.3 ng/ml at 5 days after birth. He was clinically diagnosed with 21‐OHD. Because his elder brother had previously been diagnosed with 21‐OHD, with compound heterozygous variants of c.293‐13A/C>G (IVS2‐13A/C>G) and c.518T>A (p.I172N) in CYP21A2  (Katoh‐Fukui et al., two novel gene variants of 21‐OHD and CS.The male patient was born at 37 weeks and 5 days of gestation to nonconsanguineous healthy parents. His birth weight was 2,712 g (−0.45 This study was approved by the Institutional Review Board Committees of the National Research Institute for Child Health and Development, National Hospital Organization Kyoto Medical Center, and Kurume University. Written and informed consent was obtained from the family of the patient before reporting this case.2Our case was genetically diagnosed with 21‐OHD and CS. In Japan, the prevalence of 21‐OHD is 1 in 18,000 (Tsuji et al., According to previous reports of comorbidities associated with 21‐OHD, only two females were reported with Turner's syndrome (Larizza et al., Male patients with 21‐OHD often show increased penis length due to excess adrenal androgen exposure. (El‐Maouche, Arlt, & Merke, Three forms of 21‐OHD are SW, SV, and NC. We diagnosed this case as SW from the symptoms of hyponatremia, hyperkalemia, hypoglycemia, increased ACTH. The poor feeding, weight loss, failure to thrive, vomiting, dehydration, and hypotension might not be shown due to cardiac failure and treatment of intravenous infusion, diuretic medication, hydrocortisone, and fludrocortisone. Penile enlargement was not seen. The symptoms of CS are coloboma, heart defects, atresia of choanae, retarded growth and development, genital abnormalities, ear anomalies, and/or hearing loss defects, which were fully shown in this case. This case had symptoms of both 21‐OHD and CS; however, micropenis and cryptorchidism appeared as the feature of CS.3It is extremely rare to observe overlapping genetically rare diseases, 21‐OHD and CS, in the same individual. For this case, the fetal androgen level may have been less‐than‐normal, although he suffered from 21‐OHD. Untypical clinical features resulted in a definitive diagnosis, which hopefully will benefit patients and families during the treatment and follow‐up periods.None declared.