For several observational studies that have reported the factors related to gastroschisis, the target population in these studies was mainly residents of Europe or the US, and there is little data on the Asian population. In this study, we summarised characteristics of Japanese women who delivered infants with gastroschisis, particularly focusing on the pre-pregnancy body mass index (BMI), which was found to be inversely associated with gastroschisis in past studies, because the distribution of BMI is clearly different in Asia and the West.2) was 16.2%, reference weight (18.5–24.9 kg/m2) 73.1%, and overweight (≥ 25.0 kg/m2) 10.6%. We identified only 9 infants with gastroschisis, 2 of whose women were underweight (frequency of gastroschisis = 0.01%), 5 were in the reference group (0.01%), and 2 were overweight (0.02%). Of these 9 women, none were aged < 20 years, 2 were aged 20–29 years (frequency = 0.01%), and 7 were aged 30–39 years (0.01%). No reduction in the occurrence of gastroschisis was apparent among Japanese women who were overweight before pregnancy.We used data from a nationwide birth cohort study which recruited pregnant women between 2011 and 2014. Among 92,796 women who delivered singleton live births, the frequency of underweight (pre-pregnancy BMI < 18.5 kg/m Gastroschisis is a congenital abdominal wall birth defect, usually occurring to the right of the umbilicus . Its obsThough the pathogenesis of gastroschisis remains unclear, factors associated with the defect have been investigated through observational studies. However, the target populations in these studies were mainly residents of Europe or the US, and there is little data on the Asian population. Moreover, background characteristics differ between Asians and Westerners. Although a high frequency of gastroschisis has been observed among women in their teens , the perThe aim of this study was to summarise characteristics of Japanese women who delivered infants with gastroschisis, with particular focus on the frequency of gastroschisis based on the strata of pre-pregnancy BMI, which is distributed to the lower side in Japan.We used data from an ongoing nationwide birth cohort study, the Japan Environment and Children’s Study (JECS) , 15. TheIn this study, we restricted the study participants to 95,170 unique women (first JECS pregnancy), among the total of 100,778 pregnancies, which included women registered multiple times for sibling births. Of the 95,170 pregnancies, we excluded 947 twin or triplet pregnancies, and 1427 miscarriages or stillbirths. As a result, a total of 92,796 women who delivered singleton live births were included in our analysis.We collected self-reported information, such as demographic, lifestyle, and physical and mental health factors, through questionnaires, and clinical information from medical record transcriptions. One self-administered questionnaire was distributed to the women during their first trimester, and another during their second or third trimester. Medical record transcriptions adhering to the JECS in-house standard operating procedures were performed three times by physicians, midwives or nurses, and/or Research Co-ordinators: first during the first trimester, second after delivery, and finally at the first-month health check-up after delivery.2). As we included a question about pre-pregnancy height and weight in the first questionnaire, such questionnaire-based information was used as a fallback measure. The Pearson’s correlation coefficients between the medical record and questionnaire were 0.99 for height and 0.98 for weight before pregnancy. Based on the WHO criteria [2 (overweight). Other characteristics we summarised were maternal age at delivery , occupation in early pregnancy , smoking habits , alcohol consumption , current history of diabetes or gestational diabetes , parity , infertility treatment , routine use of folic acid supplement ), week of pregnancy at delivery , and infant sex.The pre-pregnancy maternal height and weight, obtained via the medical record, were used to calculate the pre-pregnancy BMI as weight (kg)/height squared .2, SD = 3.3), 16.2% were categorised as underweight, 73.1% as reference, and 10.6% as overweight. The percentage of obesity (a BMI of ≥ 30 kg/m2) was 2.5%. In this population, we identified 9 infants with gastroschisis . Among these 9, 2 were born from underweight women (frequency of gastroschisis = 0.01%), 4 from those in the reference group (0.01%), 2 from overweight women (0.02%), and in one case data on the mother’s pre-pregnancy height and weight was lacking. Women aged < 20 years accounted for 0.9%, those aged 20–29 years 36.6%, those aged 30–39 years 57.9%, and those aged ≥ 40 years 4.6%, of the total number of women. Two women aged 20–29 years delivered infants with gastroschisis (frequency of gastroschisis = 0.01%), and 7 women aged 30–39 years (0.01%). There were no cases in the < 20 or ≥ 40 groups. Also, no cases were observed in the current history of diabetes or gestational diabetes group, or in the infertility treatment group. The frequency of gastroschisis tended to be higher in the preterm (< 37 weeks of pregnancy) birth group. No noteworthy difference based on the strata of other characteristics, such as smoking status, alcohol consumption, or parity, was found.The baseline characteristics of the 92,796 women  = 5.1) are presented in Table 2. When she was categorised into either the < 18.5 kg/m2 or the 18.5–24.9 kg/m2 group, the OR point estimates for the overweight group were above unity, in comparison with the reference . In the case of two women in the overweight group (one in her 20 s and another one in her 30 s), since their BMI values were 28, it was unlikely that BMI category misclassification occurred.The association between pre-pregnancy BMI and gastroschisis is summarised in Table 2) [In this study, overweight women did not show a reduced likelihood of giving birth to infants with gastroschisis. This result would appear to contradict those of a majority of other related studies, which reported that a high BMI before pregnancy was protectively associated with gastroschisis –11. Howe2) , 21, 22.2) , whereas2) , had no A high frequency of gastroschisis births has been observed among teenage women . One casAs in past studies , the frWe found that no decrease in the occurrence of gastroschisis was apparent among Japanese women who were overweight before pregnancy.We identified a small number of cases of gastroschisis (n = 9), although this was a large-scale birth cohort, and the frequency of gastroschisis here observed  was within the range of past reports in Japan , 4. TherAdditional file 1: Association between pre-pregnancy body mass index and gastroschisis. Odds ratios were adjusted for maternal age at delivery. Error bars indicate 95% confidence intervals.


Acanthamoeba; however other giant viruses have been isolated in Vermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated.After the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genus In this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays.V. vermiformis cells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant.We observed, through optical and IF microscopy, morphological changes in The results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus. Acanthamoeba [Acanthamoeba; however, other giant viruses have been isolated in Vermamoeba vermiformis, such as Faustovirus and Kaumoebavirus [Giant viruses belong to a complex group of viruses commonly referred to as nucleocytoplasmic large DNA viruses (NCLDVs). The group gained notoriety after the isolation of Acanthamoeba polyphaga mimivirus (APMV), which has large particles ~ 750 nm) capable of infecting amoebae of the genus thamoeba . The stuthamoeba , 3. Mostebavirus , 5.0 nm capaV. vermiformis, which was isolated from rat faecal samples collected in France. These viruses have ovoid-shaped particles, as observed for Cedratvirus, Pandoravirus, and Pithovirus, ranging from 900 to 1100 nm in length and approximately 500 nm in diameter. These viruses are also marked by the presence of a circular double-stranded DNA genome of 1,473,573 bp, encoding 1512 predicted genes of which 57.5% are ORFans [In 2018, a new virus, Orpheovirus IHUMI-LCC2 , was described that is capable of infecting e ORFans . Regardie ORFans .V. vermiformis cells. After particle entry, the genome would be released into the cell cytoplasm through an ostiole located at the apex of the virion. An eclipse phase is established, and then viral factories (VFs) are formed, where new viral particles are assembled. In the final steps of the cycle, the cell cytoplasm is completely filled by new synthesized particles, which are released from the host cell by lysis [Due to its large size, it was proposed that the replication cycle of Orpheovirus was initiated by phagocytosis of viral particles by by lysis . DespiteV. vermiformis, including increased cell motility at some time points of infection. We also provide details about virus entry, VFs formation, organelle recruitment and particle morphogenesis. We also observe that viral particles are released from infected cells both by exocytosis and cell lysis. The data presented here reveal several peculiar characteristics of the life cycle, structure and host interaction of this new giant virus.In the present work, we present an in-depth investigation of the steps of the replication cycle of Orpheovirus. Our data revealed that Orpheovirus induces profound changes in the morphology of V. vermiformis (ATCC CDC19) were cultivated in Peptone Yeast Extract Glucose (PYG) medium supplemented with 0.14 mg/mL penicillin , 50 mg/mL gentamicin , and 2.5 mg/mL amphotericin  at 32 °C. For Orpheovirus production and purification, ten T175 cm2 flasks  containing 20 × 106 cells in PYG medium were infected with Orpheovirus at a multiplicity of infection (M.O.I.) of 0.01 and incubated for 4 days at 32 °C. The lysate was centrifuged at 1200 x g to remove cell debris. Then, the supernatant was collected, added over a 40% sucrose  cushion and centrifuged at 36,000 x g for 1 h. The pellet was re-suspended in PBS and stored at − 20 °C. Three aliquots of the virus stock were titrated to the 50% end-point and calculated by the Reed-Muench method [h method .V. vermiformis cells by optical microscopy, 25 cm2 cell culture flasks containing 3 × 106V. vermiformis cells were infected with Orpheovirus at an M.O.I. of 10, incubated at 32 °C and observed at different hours post infection (h.p.i)  for 24 h. Uninfected V. vermiformis cells (control) were also observed. A one-step growth curve was constructed using 25 cm2 flasks in duplicate at an M.O.I of 10. At different time points , the infected V. vermiformis cells and supernatants were collected, titred and calculated using the end point method. We also performed a quantitative polymerase chain reaction (qPCR) assay to quantify the viral genome load targeting the DNA polymerase gene, using oligonucleotide primer sequences Forward 5′- ATGGCGAAATATGCGGAAGGG-3′ and Reverse 5′-TCTTGTGCTCCTAACGCACC-3′. The thermal cycling conditions used were: one cycle at 95 °C for 10 min and 40 cycles at 95 °C for 10 s and 60 °C for 40 s; a melting curve analysis at 95 °C for 15 s, 58 °C for 15 s and a final cycle at 95 °C for 15 s was completed.To investigate the cytopathic effect (CPE) of Orpheovirus in 6V. vermiformis cells were infected with Orpheovirus at an M.O.I. of 5 and analyses were carried out at the infection times of 3, 6, 9, 12 and 24 h.p.i. Thirty minutes after infection, the monolayer of cells was washed once with PBS and the flasks were filled with 4 mL of PYG medium. After each time point, we separated 12 μL of the supernatant to count the number of released Orpheovirus particles during infection. The particles were observed by light microscopy  under 1000x magnification using a cell counting chamber .To investigate if the particles were released from the host cell by exocytosis, 3 × 10g. The pellet was washed twice with 0.1 M sodium phosphate buffer (pH 7.4) and fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer for at least 1 h at room temperature. The pellet was then washed twice with 0.1 M sodium phosphate buffer and suspended in the same solution. After, the amoebae were embedded in EPON resin using a standard method, as follows: 2 h of fixation in 2% osmium tetroxide, five washes in distilled water, overnight incubation in uranyl acetate 2% at 2–8 °C, two washes in distilled water, 10 min dehydration in increasing ethanol concentrations , 20 min incubation in acetone and embedding in EPON resin. Ultrathin sections were subsequently analysed under TEM (Spirit Biotwin FEI-120 kV) at the Center of Microscopy of UFMG, Brazil.For the transmission electron microscopy (TEM) assays, the cells were infected at an M.O.I. of 0.01, and when the CPE was observed, we centrifuged the flask content for 10 min at 800 x 2 point, transferred onto stubs and metalized with a 5 nm gold layer. The analyses were completed with SEM (FEG Quanta 200 FEI) at the Center of Microscopy of UFMG, Brazil.For the scanning electron microscopy (SEM) assays, the cells infected at an M.O.I. of 0.01 were added to round glass blades covered by poly-L-lysine and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 1 h at room temperature. Samples were then washed three times with 0.1 M cacodylate buffer and post-fixed with 1.0% osmium tetroxide for 1 h at room temperature. After a second fixation, the samples were washed three times with 0.1 M cacodylate buffer and immersed in 0.1% tannic acid for 20 min. Samples were then washed in cacodylate buffer and dehydrated by serial passages in ethanol solutions with concentrations ranging from 35 to 100%. They were dried at the critical CO5V. vermiformis cells were added to round glass blades and after incubation during 24 h at 32 °C, the cells were infected at an M.O.I. of 5. After 1, 3, 6, 8, 12 and 24 h.p.i., we fixed with acetone for 10 min at − 20 °C. After fixation, cells were stained with polyclonal anti-orpheovirus whole particle antibodies produced in mouse (1:400 diluted in PBS) for 1 h at 37 °C, followed by three rinses with PBS. Cells were then incubated with 3% bovine serum albumin (BSA)-PAS for 30 min, followed by three rinses with PBS. After a 1 h of incubation with anti-mouse secondary antibodies (1:400 diluted in PBS), followed by three rinses with PBS, the cells were also incubated with DAPI Sigma (1:1000 diluted in PBS) for 1 h at room temperature. After three rinses with PBS, the cells were incubated with rodamine-phalloidine (Invitrogen) (1:1000 diluted in PBS) for 1 h at room temperature. Uninfected cells (control) were also fixed and prepared as described above. Fluorescently labelled cells were observed using an Axio Imager Z2-Apotome 2 microscope (Zeiss). The Zen Lite software from Zeiss microscopy was used for image processing at the Center of Microscopy of UFMG, Brazil.For immunofluorescence (IF) analysis, 3 × 105V. vermiformis cells were infected with Orpheovirus at an M.O.I. of 5. Thirty minutes post-infection, the amoebae were washed with PAS and then we added 15 mL of PYG medium maintained at 32 °C. After 1 h, Brefeldin A (BFA), an inhibitor of membrane traffic, was added at a final concentration of 10 μM, and at 8 h post-infection, the amoebae were collected for TEM analysis. Samples were prepared for microscopy as previously described.To evaluate the role of cell membranes in the viral replication cycle, 14 × 10V. vermiformis cells were infected at M.O.I. of 10 and observed up to 24 h.p.i.. Using optical microscopy, we observed that the cells became stretched into a fusiform shape at 3 h.p.i., and this effect became more evident at 9 h.p.i. and 12 h.p.i., respectively Different time points during infection (M.O.I. of 5) were selected for IF assay. IF assays using anti-orpheovirus primary antibodies at 1 h.p.i. revealed particles being endocytosed by amoebae Fig. . At 3 h.miformis , 10. FurAdditional file 1: Video S1 Orpheovirus infection induces an increase in the motility of V. vermiformis cells, especially those with a fusiform shape. This effect starts at 6 h.p.i. and became more evident at 12 h.p.i.. This movie is not accelerated.V. vermiformis cells would occur by phagocytosis, as previously described for other giant viruses, such as Pandoravirus, Mimivirus and Cedratvirus [Due to the large size of Orpheovirus particles (~ 1.1 μm), it was proposed that their entry into ratvirus , 11, 12.ratvirus . IF analA. castellanii cells, but this event requires further investigation [As previously described in other giant viruses, Orpheovirus morphogenesis occurs in subcellular microenvironments called VFs, which are located in the host cell’s cytoplasm. Similar to Cedratvirus and Pandoravirus VFs, Orpheovirus VFs are large electron-lucent areas, which occupy a large part of the host cell Fig. a and b, tigation .Fig. 4EMitochondrial recruitment was also observed in peripheral regions and inside VFs Fig. c, as welWith analysis of TEM images of asynchronous infection of Orpheovirus (M.O.I. of 0.01), the formation of VFs that presented particles in different maturation stages was visualized Fig. a, which V. vermiformis is slower compared to other giant viruses, during approximately 30 h.p.i.In order to chronologically analyse viral genome replication and infectious particle formation, one-step growth assays were performed. An increase in genome amplification was observed at 9 h.p.i., and infectious particle detection increased at 12 h.p.i. Fig. i–j. At 4In addition, it was observed that Orpheovirus particles have a smaller fibril layer compared to those observed in mimiviruses Fig. a and b . The anaAfter VF formation, expansion of the crescents, and complete maturation, new particles were visualized in peripheral regions in the host cell’s cytoplasm Fig. a, and soV. vermiformis, triggering amoebae aggregates called bunches, as described for Tupanvirus [V. vermiformis cell motility never described before. Although speculative, one hypothesis is that this increased motility would be a strategy that would facilitate the infection of neighbouring cells, leading to an increased spread of viral particles.Giant viruses have notable differences in their replication cycles and structures of their virions. After the discovery of this group of viruses, studies have been conducted to increase the knowledge of their biology and host interaction through investigations related to the life cycle of these organisms , 16, 17.panvirus , and forpanvirus , as wellThe strategy of phagocytosis entry has often been described for other giant viruses, demonstrated through biological assays with pharmacological inhibitors for APMV and Cedratvirus getuliensis, which showed a considerable decrease in viral titres , 12. ConUnlike VFs described for mimiviruses, which present well-defined zones , VFs visAlthough Orpheovirus particles present an ovoid-shape, similar to pandoraviruses, cedratviruses, and pithoviruses, they have a distinct structure. They have a fibril layer that is smaller compared to other giant viruses, such as mimiviruses, that have fibrils, and despite scarce information about their constitution, they may be related to host cell adhesion, as demonstrated for mimiviruses . Unlike Moreover, it was observed that, in addition to fibrils, the particles have an outer layer to the capsid layer and an inner membrane surrounding the core. The presence of one or more particles involved by membranes is described for Cedratvirus getiliensis and Pithovirus sibericum, suggesting that they would be released from the host cell by exocytosis , 14. AltV. vermiformis have also been shown to have a longer duration multiplication cycle [Acanthamoeba. This suggests that the replication cycle duration profile of these viruses might be related to their host. Altogether, this work brings valuable information concerning the replication cycle, structure and CPE triggered by Orpheovirus, therefore contributing to enhance our understanding about this new giant virus. Future studies applying alternative methods will help to fill some gaps, especially concerning the virus-host interaction.One-step growth assays showed an increase in viral titres at 12 h.p.i. and increased viral genome amplification by qPCR at 9 h.p.i., reaching a plateau at 48 h.p.i. by both qPCR analysis and titration. These data corroborate those previously proposed , which son cycle , 10 compIn summary, the data presented here reveal several peculiar characteristics of the life cycle, structure, CPE and host interaction contributing to improve our understanding about this new giant virus.


Clostridium acetobutylicum strain expressing the engineered aldehyde/alcohol dehydrogenase gene was capable of producing butanol at a high level of selectivity.Renewable biofuel represents one of the answers to solving the energy crisis and climate change problems. Butanol produced naturally by clostridia has superior liquid fuel characteristics and thus has the potential to replace gasoline. Due to the lack of efficient genetic manipulation tools, however, clostridial strain improvement has been slower than improvement of other microorganisms. Furthermore, fermentation coproducing various by-products requires costly downstream processing for butanol purification. Here, we report the results of enzyme engineering of aldehyde/alcohol dehydrogenase (AAD) to increase butanol selectivity. A metabolically engineered  Clostridium acetobutylicum is accompanied by coproduction of acetone and ethanol, which reduces the yield of butanol and increases the production cost. Here, we report development of several clostridial aldehyde/alcohol dehydrogenase (AAD) variants showing increased butanol selectivity by a series of design and analysis procedures, including random mutagenesis, substrate specificity feature analysis, and structure-based butanol selectivity design. The butanol/ethanol ratios (B/E ratios) were dramatically increased to 17.47 and 15.91 g butanol/g ethanol for AADF716L and AADN655H, respectively, which are 5.8-fold and 5.3-fold higher than the ratios obtained with the wild-type AAD. The much-increased B/E ratio obtained was due to the dramatic reduction in ethanol production (0.59 ± 0.01 g/liter) that resulted from engineering the substrate binding chamber and the active site of AAD. This protein design strategy can be applied generally for engineering enzymes to alter substrate selectivity.Butanol production by  Clostridium acetobutylicum has been extensively studied as a model organism for ABE fermentation  ratio of 4.34 g/g without acetone production  of C. acetobutylicum DSM 1731 were evaluated with respect to butanol and ethanol production through inactivation of the corresponding genes (adhE2 gene resulted in a B/E ratio (8.15 g/g) that was higher than that seen with the wild-type strain (6.65 g/g). Furthermore, both the SMB_P058 mutant and yqhD mutant produced less ethanol without loss of butanol formation, which led to higher B/E ratios of 10.12 g/g and 10.17 g/g, respectively . This enzyme comprises an aldehyde dehydrogenase (ALDH) and an alcohol dehydrogenase (ADH), which converts butyryl-coenzyme A (butyryl-CoA) to butyraldehyde and butyraldehyde to butanol, respectively , which is encoded by the ectively . The metpropanol , 15–17. ing site . InspireZ. mobilis ADH2 responsible for higher butanol activity. Using a pTHL1-Cm expression plasmid allowing gene expression under the control of the strong constitutive thiolase promoter, plasmids pTHL1-MU613, pTHL1-MU619, and pTHL1-MU623  were transformed into the C. acetobutylicum HKW strain, in which the native adhE1 gene was inactivated  obtained with the M5 strain harboring the wild-type AAD in batch fermentation  The loop is placed at the passage to the active sites, which was found by the use of CAVER 3.0 . In addition, there is another shorter passage to the active sites in the C. acetobutylicum ADH. (E and F) The obstruction of a longer passage was not found in Z. mobilis ADH2 (PDB ID = 3OX4). (G and H) A shorter passage was found in another enzyme with NADH, namely, carbonyl reductase (PDB ID = 4C4O). Due to large structural differences, the structures of NAD were superimposed on that of Z. mobilis ADH2. Download FIG S1, TIF file, 48.9 MB.Substrate passage of ADHs. (A and B) The structure of Copyright © 2019 Cho et al.2019Cho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02683-18.3FIG S2Z. mobilis ADH2. (B) Horse liver ADH. (C) Yeast ADH. (D) Mutations reducing enzymatic activity for 2-butanol and 2-pentanol in Sulfolobus solfataricus ADH. (E) Stereo-specificity mutations in Thermoanaerobacter brockii ADH. (F) Stereo-specificity mutations in Rhodococcus ruber ADH. (G) Enzymatic activity increases in mutation for 2-methylcyclohexanone in Candida parapsilosis carbonyl reductase. Download FIG S2, TIF file, 49.2 MB.Substrate specificity mutations. (A) Substrate specificity of Copyright © 2019 Cho et al.2019Cho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02683-18.7TABLE S1Table S1, DOCX file, 0.02 MB.Sequence alignment for structure modeling. Download Copyright © 2019 Cho et al.2019Cho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M619A and AADM619G as a large nonpolar amino acid at the substrate binding chamber replaced by small amino acids. Intriguingly, similar structural change was observed in AADY623L despite the mutation site being located outside of the chamber. Still, the mutation could affect the chamber structure as it is located on the loop that forms the lining of the chamber. On the other hand, AADN613K induced only a slight change in the chamber structure although slightly greater numbers of water molecules were placed near the active site. Docking simulations performed with acetaldehyde and butyraldehyde indicated that the mutations can increase the volume of substrate binding sites  and decreased (blue) water density near ADHs induced by the mutations: (A) M619A. (B) M619G. (C) N613K. (D) Y623L. (E) Change in water occupancy at substrate binding chamber or water occupancy change determined by analysis within of the area within a 4-Å radius of the center of NADH O7N and N836 OE1 atoms. Download Copyright © 2019 Cho et al.2019Cho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the On the basis of these observations, two hypotheses could be proposed for the higher butanol production capacity enabled by these mutations. The first hypothesis is that the widened substrate binding chamber would increase hydrophobic lining of the substrate chamber to allow a disproportionally longer retention time for butyraldehyde than for acetaldehyde and would consequently increase the relative levels of butanol production. The second hypothesis is that widening of the structure near the active site would allow acetaldehyde and butyraldehyde to assume more-diverse orientations at the active site. This would reduce the enzymatic activity, but the longer aliphatic chain of butyraldehyde would be less affected, leading to the retention of activity with respect to butyraldehyde.F716L and AADM572V  database using BLAST , and their profiles were displayed with WebLogo . Residues with exceptional conservation can be found among ADHs, including the residues forming a Zn2+ binding site (highlighted in yellow shading). In addition, two residues near the active sites were well conserved for an unknown reason and are highlighted in blue shading. (B) The residues interacting with the Zn2+ ion in C. acetobutylicum AAD. (C) The active site of a typical ADH, G. thermoglucosidasius ADH (PDB ID = 3ZDR). Download FIG S4, TIF file, 53.3 MB.Conserved sites in ADHs. (A) The sequences similar to that of Copyright © 2019 Cho et al.2019Cho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the F716L and AADN655H, respectively, which are 5.8-fold and 5.3-fold higher than the ratios obtained with the wild-type AAD (C. acetobutylicum strain M5 itself produces a similar amount of ethanol (0.60 ± 0.02 g/liter) without any plasmid, the level of ethanol produced by the strains expressing AADF716L and AADN655H can be considered negligible. The recombinant M5 strains expressing AADF716L and AADN655H produced 10.31 ± 0.02 and 9.71 ± 0.01 g/liter of butanol, respectively, which are amounts similar to that (10.01 ± 0.12 g/liter) produced by the control strain expressing the wild-type AAD. Thus, it was possible to achieve higher butanol selectivity by engineering the substrate binding chamber and the active site of AAD without sacrificing butanol production.Batch fermentations of the M5 mutant strains expressing these newly designed AADs were performed, and the butanol selectivities were examined . Use of the AADions see . Howeverions see , but the10.1128/mBio.02683-18.6FIG S5FIG S5, TIF file, 35.9 MB.Change of water density in combinations of single mutations. The changes of water occupancy corresponding to combinatorial mutations were evaluated for highly butanol-selective mutants F716L, N655H, M619G, and N613K. The combinatorial mutations increased the volume of the substrate binding chambers, but the combinatorial effect was small. Download Copyright © 2019 Cho et al.2019Cho et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the groESL genes produced 17.1 g/liter of butanol and 0.97 g/liter of ethanol, resulting in a B/E ratio of 17.69 g/g  in C. acetobutylicum. In the future, it is expected that combining systems metabolic engineering and enzyme engineering in clostridia will result in production of butanol to a high titer with high yield, productivity, and butanol selectivity.There have been several studies on altering butanol selectivity by metabolic engineering of clostridia –13. Most–7.69 g/g . Also, t9.45 g/g . C. acet10.5 g/g , while C8.83 g/g . The str E. coli , 25, Sacrevisiae , Lactobas brevis , Pseudom E. coli , 25, pat E. coli , 30. In inducers . To reaconducted . This gradhE1 gene is the key enzyme in butanol production by C. acetobutylicum but is also directly involved in ethanol production. In this study, the substrate binding chamber and the active site of AAD were engineered to modulate the butanol and ethanol selectivities of AAD. The resultant enzymes showed remarkably increased in vivo selectivity of butanol versus ethanol. The high butanol selectivity of the newly engineered enzyme was due to the drastic reduction in ethanol production , which contains the following (per liter): 0.75 g KH2PO4, 0.75 g K2HPO4, 1.0 g NaCl, 0.017 g MnSO4 · 5H2O, 0.70 g MgSO4 · 7H2O, 0.01 g FeSO4 · 7H2O, 2.0 g l-asparagine, 5.0 g yeast extract, 2.0 g (NH4)2SO4, and 80 g glucose. For growth on solid medium, cells were cultured anaerobically at 37°C on 2× YTG (pH 5.8)  agar plates. For the screening of transformants, erythromycin and thiamphenicol were used at final concentrations of 40 mg/liter and 5 mg/liter, respectively.The bacterial strains used in this study are listed in Table A in Pfu DNA polymerase  was used for PCR.The plasmids and primers used in this study are listed in Table A in C. acetobutylicumadhE1 gene was amplified by PCR with primers AdhE1-F and AdhE1-R using genomic DNA of C. acetobutylicum ATCC 824 as a template. The resulting fragment and pTHL1-Cm were digested with PstI and AvaI and were ligated to make pTHL1-AdhE1.The C. acetobutylicum AAD) were determined by alignment of C. acetobutylicum AAD and Z. mobilis ADH2  .The amino acid residues at positions 613, 619, and 623  containing 2 ml of CGM supplemented with 5 mg/liter thiamphenicol as the first screening  of 2.0. After that, cells were inoculated into the bioreactor. The pH was adjusted to 5.5 with 14% (vol/vol) ammonia solution and was not controlled when it a level above 5.5 during the fermentation. The bioreactor was operated at 37°C and 200 rpm, and oxygen-free nitrogen gas was supplied at a flow rate of 0.5 liters/min after passage through an oxygen trap .Batch fermentation was performed in a 5.0-liter LiFlus GX bioreactor  containing 1.8 liters of CGM supplemented with 80 g/liter glucose. To prepare a preculture, a single colony was inoculated into a test tube containing 10 ml CGM supplemented with 80 g/liter glucose. This preculture was transferred into a 500-ml flask containing 200 ml CGM supplemented with 80 g/liter glucose until the cell density reached an optical density at 600 nm (OD600) using an Ultraspec 3100 Pro spectrophotometer . The relative concentrations of solvent and butanol were analyzed by the use of a gas chromatograph  equipped with an 80/120 Carbopack B AW packed glass column  and a flame ionization detector . The concentrations of glucose and organic acids were assessed with using a high-performance liquid chromatograph . A MetaCarb 87H column  (300 by 7.8 mm) was eluted isocratically with 0.01 N H2SO4 at 35°C at a flow rate of 0.5 ml/min.Cell growth was monitored by measuring the optical density at 600 nm (ODC. acetobutylicum AAD were retrieved from the NCBI nonredundant database (G. thermoglucosidasius ADH (PDB ID = 3ZDR) showed the highest (48%) sequence similarity to the C. acetobutylicum ADH. The structure of C. acetobutylicum ADH was modeled using MODELLER 9v11 . The system was stabilized with 5,000 steps of steep-descent energy minimization followed by 50 ps of equilibration simulation under protein heavy-atom position constraints. A total of 5 ns of main simulation was performed at 298 K using a Berendsen thermostat and LINCS algorithm constraint. For each structure model, five independent simulations were executed to reduce simulation bias. The parameters used in the simulation can be found in Table B in The structure models of .1 suite . An AmbeWater occupancies around AADs were calculated for the wild types and mutants, and their differences were evaluated. To calculate water occupancy, 5,000 structure snapshots were retrieved from each simulation trajectory, and their protein parts were superimposed. The coordinates of water molecules were transformed accordingly. Three-dimensional grids with 0.7-Å intervals were applied around the protein molecule, and the grids within a 1.4-Å radius of water oxygen atoms were assumed to be occupied by the water molecules. The grid occupancy data were averaged for all snapshots to calculate water occupancy fields.The modified site-directed mutagenesis protocol was applied to construct the mutants . All pri600 of 1.0 and washed with 10 ml lysis buffer . Pellets were resuspended with 3 ml lysis buffer, anaerobically lysed by the use of a Bioruptor sonicator , and centrifuged at 14,000 × g for 25 min at 4°C. After measurement of total protein concentrations by the Bradford method, the assay was performed with a GeneQuant 1300 spectrophotometer , monitoring decreases in absorbance at 340 nm. The reaction mixture contained 0.1 M Tris-HCl (pH 7.6), 5 mM DTT, 300 μM NADH, and 50 mM butyraldehyde or 150 mM acetaldehyde. The reaction was initiated by the addition of 100 μl of extract to 1 ml of reaction mixture. One unit of alcohol dehydrogenase activity was defined as the amount of enzyme necessary for the consumption of 1 μmol NADH per min. All experiments were carried out in at least triplicate under anaerobic conditions.To measure ethanol dehydrogenase (EDH) and butanol dehydrogenase (BDH) activities, cells cultured in static flasks were harvested at an ODThe nucleotide sequences of mutant AADs have been deposited at GenBank under the following accession numbers: MK307997 to MK308006.


Cardiovascular mortality in Lithuania is extremely high and abnormal lipid levels are very common among Lithuanian adults. Dyslipidemia is one of the main independent risk factors for cardiovascular diseases (CVD) leading to high absolute CVD risk. The aim of this study was to assess CVD risk in dyslipidemic middle-aged subjects.During the period of 2009–2016 a total of 92,373 people (58.4% women and 41.6% men) were evaluated. This study included men aged 40–54 and women aged 50–64 without overt CVD.p < 0.001), diabetes , abdominal obesity , BMI ≥30 kg/m2 , metabolic syndrome , family history of coronary heart disease , unbalanced diet  and insufficient physical activity . The prevalence of all evaluated risk factors, except smoking, increased with age. Average SCORE index was 1.87 in all study population, while dyslipidemic subjects had higher SCORE compared to control group .Any type of dyslipidemia was present in 89.7% of all study population. 7.5% of dyslipidemic patients did not have any other conventional risk factors. Three and more risk factors were detected in 60.1% of dyslipidemic subjects. All analyzed risk factors, except smoking, were more common in dyslipidemic adults compared to subjects without dyslipidemia: arterial hypertension (55.8% vs. 43.3%, Almost two thirds of dyslipidemic middle-aged Lithuanian adults without overt cardiovascular disease had three or more other CVD risk factors, which synergistically increase absolute risk of CVD. The average 10-year risk of CVD death in patients with dyslipidemia was 1.95%. The importance of managing dyslipidemia as well as other risk factors in order to reduce burden of cardiovascular disease in Lithuania is evident. Cardiovascular disease (CVD) is a major cause of premature death in Lithuania as more than a half of all deaths 56.2%) were caused by CVD in 2016 % were ca. The maiDyslipidemia is one of the most important modifiable risk factors described more than half a century ago . The assIt is settled that prevention is effective in reducing the impact of CVD and one of the main measures is lowering increased levels of modifiable cardiovascular risk factors . DespiteThe Lithuanian High Cardiovascular Risk (LitHiR) primary prevention programme, funded by the Ministry of Health, was started in 2006. The Local Research Ethics Committee’s approval was obtained. During the period of 2009–2016 a total of 92,373 people (58.4% women and 41.6% men) were evaluated. This study included men aged 40–54 and women aged 50–64 without overt cardiovascular disease. Subjects were divided into two groups by their dyslipidemia status – dyslipidemic and non-dyslipidemic. Their risk factors and lipid profile assessment were obtained and used for statistical analysis. Study data has been further analyzed by dividing all subjects into appropriate groups by the age, men: 40–44 years, 45–49 years, 50–54 years and women: 50–54 years, 55–59 years, 60–64 years.Dyslipidemia was considered if serum total cholesterol (TC) > 5 mmol/L, or low-density lipoprotein-cholesterol (LDL-C) > 3 mmol/L, or high-density lipoprotein cholesterol (HDL-C) < 1.0 mmol/L in men and < 1.2 mmol/L in women, or triglycerides (TG) > 1.7 mmol/L. Metabolic syndrome (MS) was assessed according to the National Cholesterol Education Program III modified criteria . Arteriap-values are two-tailed. The level of significance was set to 0.05.For continuous variables, the following descriptive statistics are reported: means, standard deviations (SD) and 95% confidence interval (CI). For categorical data frequencies are reported. In the case of dichotomous categorical variables, we also provide confidence intervals for proportions of interest . These intervals were obtained using the relationship between beta and binomial distributions. Categorical variables were compared with the help of the chi-square test. All reported p < 0.001)  women and 38,412 (41.6%) men. The average age of the sample group was 52.15 (±6.21) years. Women and men varied in age because of different study enrollment criteria. Baseline characteristics and prevalence of cardiovascular risk factors of the whole study population are shown in Table p < 0.001), DM , abdominal obesity , MS  and obesity . The prevalence of DM, AH, abdominal obesity, MS and obesity increased with age in both dyslipidemic and non-dyslipidemic men groups. Males with dyslipidemia had higher prevalence of DM, AH, abdominal obesity, MS and obesity in all age groups in comparison with control group . The highest frequency of smoking in males without dyslipidemia was observed in 45–49-year-olds (43.8%) followed by 50-55y (42.4%) and 40-44y (39.6%) groups. Prevalence of smoking was lower in men older than 50 years compared to younger males in both dyslipidemic and non-dyslipidemic groups .This study included 38,412 men aged 40–54: 33403 (87.0%) with dyslipidemia and 5009 (13.0%) without dyslipidemia. Mean value of TC in men was 6.07 ± 1.10 mmol/l, LDL-C – 3.92 ± 0.98 mmol/l, HDL-C – 1.39 ± 0.47 mmol/l and TG – 1.86 ± 1.45 mmol/l. Prevalence of different cardiovascular risk factors in dyslipidemic and non-dyslipidemic men is presented in Fig. p < 0.001), AH , abdominal obesity , smoking , MS  and obesity ). The prevalence of DM, AH, abdominal obesity, MS and obesity increased with age in both dyslipidemic and non-dyslipidemic groups. Subjects with dyslipidemia had higher prevalence of DM, AH, abdominal obesity, MS and obesity in all age groups in comparison with control group . Frequency of smoking was lower in older women in both dyslipidemic and non-dyslipidemic groups compared to younger females  with dyslipidemia and 4471 (8.3%) without dyslipidemia. Mean laboratory values of women were: TC – 6.41 ± 1.11 mmol/l, LDL-C – 4.12 ± 1.01 mmol/l HDL-C – 1.63 ± 0.45 mmol/l and TG – 1.53 ± 0.96 mmol/l. Prevalence of different cardiovascular risk factors in dyslipidemic and non-dyslipidemic women of different age is shown on Fig. p < 0.001). Across the study population adults without dyslipidemia tended to have less risk factors while women and men with dyslipidemia had bigger number of cardiovascular risk factors . This finding was not compatible with the EURIKA study results where average 10-year risk of CVD death was 8.2% across Europe . This reTo overcome the existing situation, it is essential to improve the control of multiple risk factors, which have an important role in cardiovascular pathology. There is good quality evidence of early and integrated approach to correction of dyslipidemia and we need to raise awareness about the benefit of timely detection and treatment of abnormal lipid levels.The present study examined a sample of men aged 40–54 years and women aged 50–64 years. A future study is needed to examine the younger and the older samples. Some risk factors, which are important in risk assessment, such as psychosocial factors, social class and others, were not taken into account. Also, dyslipidemia as defined in this study, cover broad spectrum of lipid abnormalities, so very high prevalence of this disorder was detected. Further studies are needed to evaluate prevalence of different types of dyslipidemia in this population.Dyslipidemia is very common in middle-aged Lithuanians without overt cardiovascular disease. Although prevalence of cardiovascular risk factors increases with age, dyslipidemia is associated with greater probability of having diabetes mellitus, arterial hypertension, abdominal obesity, metabolic syndrome and obesity, except for smoking, in all age groups compared to adults without dyslipidemia. Men and women with dyslipidemia have a higher total number of cardiovascular risk factors which synergistically increases absolute risk of CVD. The average 10-year risk of CVD death in patients with dyslipidemia was 1.95%. Our observations emphasize the importance of properly diagnosing and treating dyslipidemia as well as other concomitant risk factors in order to reduce burden of cardiovascular disease in Lithuania.


Thus, Soluplus achieved the purpose of improving the flowability and solubility of baicalein phospholipid complexes. The application of Soluplus to phospholipid complexes has great potential.In this study, a novel ternary complex system (TCS) composed of baicalein, phospholipids, and Soluplus was prepared to improve the flowability and dissolution for baicalein phospholipid complex (BPC). TCS was characterized using differential scanning calorimetry (DSC), infrared spectroscopy (IR), powder X-ray diffraction (PXRD), and scanning electron microscopy (SEM). The flowability, solubility, oil–water partition coefficient, in vitro dissolution, and in vivo pharmacokinetics of the system were also evaluated. DSC, IR, PXRD, and SEM data confirmed that the crystal form of baicalein disappeared in BPC and TCS. Furthermore, the angle of repose of TCS of 35° indicated an improvement in flowability, and solubility increased by approximately eight-fold in distilled water when TCS was compared with BPC (41.00 ± 4.89 μg/mL vs. 5.00 ± 0.16 μg/mL). Approximately 91.24% of TCS was released at the end of 60 min in 0.5% SDS (pH = 6.8), which suggested that TCS could improve the dissolution velocity and extent. Moreover, TCS exhibited a considerable enhancement in bioavailability with higher peak plasma concentration (25.55 μg/mL vs. 6.05 μg/mL) and increased AUC The poon-octanol solubility of baicalein was improved in baicalein phospholipid complex (BPC) [Given the phospholipid’s excellent biocompatibility and unique amphiphilicity, a drug–phospholipid complex has been employed as a technique to improve the oral absorption of the drugs which belong to BCS class III and IV ,10,11. Dex (BPC) . NeverthSoluplus B has beeIn the present study, a novel ternary complex system (TCS) composed of baicalein, phospholipids, and Soluplus was prepared to improve the flowability and dissolution in vitro of BPC. Due to ameliorative flowability, the successful development of TCS has created favorable conditions for large-scale production of baicalein in industrial production. Likewise, the improvement in dissolution is also advantageous for the improvement of bioavailability. Based on these findings, we conclude that Soluplus achieved the purpose of improving the flowability and solubility of BPC. The safe and effective excipients incorporation of phospholipid complex provides a new idea for its better development. In our design, TCS was prepared via solvent evaporation with baicalein, phospholipids, and Soluplus. First, TCS was characterized using differential scanning calorimetry (DSC), infrared spectroscopy (IR), powder X-ray diffraction (PXRD), and scanning electron microscopy (SEM). Second, flowability, solubility, oil–water partition coefficient, and dissolution in vitro were evaluated. Finally, the baicalin content was detected in rats after oral administration to further assess bioavailability via High Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry/Mass Spectrometry (HPLC-ESI-MS/MS) .Baicalein and baicalin were purchased from Sichuan Weikeqi Biology Technique Co., Ltd. Soluplus was kindly gifted by BASF SE . Icariin was purchased from the China Institute of Pharmaceutical and Biological Products and employed as an internal standard. All other chemical reagents used in the experiments were analytical grade or better. Purified Milli-Q water was utilized in the experiments .The drug and phospholipid (1:2 mass ratio) were used to prepare BPC. In brief, baicalein and phospholipid (phosphatidylcholine) were dissolved in 10 mL of absolute ethanol. The solution was magnetically stirred for 0.5 h and then vacuum dried for 24 h to obtain the solid product, which was stored in a desiccator.The drug and phospholipid (BPC) with Soluplus were weighed at a mass ratio of 1:2:2 and dissolved in 10 mL of absolute ethanol. The first two were mixed and dissolved by magnetic stirring for 0.5 h. Then, the weighed Soluplus was added to the above mixed solution, stirring to dissolve completely. Further stirring for 0.5 h resulted in a Soluplus complex with mass ratios of 1:2:2. The complex was vacuum dried for 24 h to obtain the solid powder, which was then stored in a desiccator.PM was prepared by thoroughly mixing baicalein, phospholipid, and Soluplus (1:2:2 mass ratio) in a mortar for 10 min until a homogenous mixture was obtained. The sample was stored in a desiccator.The thermal properties of Baicalein, BPC, TCS, and PM of TCS were studied using a differential scanning calorimeter  equipped with a thermal analysis system. Dry nitrogen was used as the purge gas (purge 40 mL/min). The right amount of samples of baicalein, BPC, TCS, and PM were weighed into an aluminum pan. The probes were heated at a temperature of 0–300 °C at a rate of 20 °C/min .−1.The IR spectra of baicalein, BPC, TCS, and PM of TCS were obtained using an IR spectrophotometer . Samples were scanned over the wavenumber range of 4000–400 cmThe crystalline state of prepared baicalein, BPC, TCS, and PM of TCS was acquired at room temperature with Cu Kα radiation source at 40 kV and 25 mA via XRD .The granule morphology of baicalein, BPC, TCS, and PM of TCS were determined using a SEM . The samples were sputter-coated  with gold–palladium and then observed at different magnifications.After TCS was added into the hopper, it was allowed to flow uniformly through the funnel until the highest point of the cone. The height (h) and radius (r) of the cone were then measured to calculate the angle of repose (θ).n-octanol (5.0 mL) was added to excess baicalein, BPC, and TCS to determine solubility in the swing bed at 37 °C for 24 h [Water or for 24 h . The liqn-octanol (water saturation) was shaken to balance, 2.0 mL of water (n-octanol saturation) was added. The miscible liquid was then agitated for 24 h, and the concentration in each phase was determined using HPLC after standing for layering. Experiments for each sample were performed in triplicate.After 2.0 mL of baicalein, BPC, or TCS-saturated v/v), and the flow rate was 1.0 mL/min. The injection volume was 10 μL. The method was optimized from the protocol described by Zhang [All samples were analyzed using Waters HPLC with a quaternary pump  on a Diamonsil C18 column (200 mm × 4.6 mm × 5 μm) at 276 nm, which was maintained at 35 °C. The mobile phase was methanol-0.1% formic acid  was used according to appendix XC of Chinese Pharmacopoeia 2010 edition. The samples, which were equivalent to 10 mg of baicalein, were filled in hard gelatin capsules and placed in the dissolution vessel containing 900 mL of 0.5% SDS with phosphate buffer (pH 2.0 or 6.8). The vessel was maintained at 37 °C ± 0.5 °C and stirred at 100 rpm. Approximately 2.0 mL of sample was withdrawn from the dissolution medium and replaced by an equivalent volume of fresh medium at 5, 10, 20, 30, 45, and 60 min. The baicalein content was determined using HPLC after the sample was filtered through a 0.45 μm membrane. Experiments for each sample were performed in triplicate.A total of 24 male SD rats (SPF grade) weighing 220 ± 10 g were purchased from Benbu Yinuogui Biology Technique Ltd. The rats were kept in the Animal Research Center to acclimatize to a new environment. The rats were then randomly divided into four groups—namely, baicalein, BPC, TCS, and PM of TCS.All animal experimental protocols were approved by the Animal Care Committee of the Institute of Chinese Medicine in Jiangsu Province. The rats were fasted for 12 h and provided free access to water prior to the experiment. Each group was orally administered with a single dose equivalent to 40 mg/kg baicalein . An aliq2PO4 were added and vortexed for another 3 min. The mixture was centrifuged at 15,000 rpm for 5 min prior to evaporation under nitrogen. The dried residue was dissolved in 100 μL of methanol–distilled water . After being vortexed at 15,000 rpm for 5 min, the supernatant was injected for HPLC-ESI-MS/MS analysis. The MS/MS system  was operated under positive mode and multiple reaction monitoring mode. The MS conditions were as follows: ion spray voltage of +5.5 kV; nitrogen as nebulizer gas, auxiliary gas, and curtain gas at 30, 60, and 10 psi, respectively; collision gas set at medium; and auxiliary gas temperature of 400 °C.Baicalein, baicalin (corresponding to 5.75 and 5.06 μg/mL), 100 μL of plasma, 100 μL of mixed standard solution, and 10 μL of internal standard solution (5.60 μg/mL) were mixed and vortexed for 15 s. Approximately 0.5 mL of methanol and 100 μL of 1 M KHmax) and maximum time (Tmax) were directly determined from the concentration–time data. Other pharmaceutical parameters (AUC0–24 h and t1/2) were analyzed using pharmacokinetic program DAS 2.1.1. ANOVA was performed using SPSS 16.0 software.The maximum concentration  in distilled water was higher than that of BPC (5.02 ± 0.09 μg/mL) (p < 0.01). The solubility of TCS (230 ± 8.78 μg/mL) in n-octanol was slightly lower than that of BPC (260 ± 7.52 μg/mL). Moreover, TCS had lower log p values than BPC (2.01 vs. 2.04). The increase in solubility in distilled water and decrease in n-octanol may be caused by the natural hydrophilic structure of Soluplus. Therefore, TCS could enhance the water solubility of BPC.p < 0.01) compared with BPC, which peaked at 1.01 h (6.05 μg/mL). TCS exhibited a marked enhancement compared with BPC in oral bioavailability, with an increase in AUC0–24 h (53.16 μg·h/mL vs. 38.40 μg·h/mL) (p < 0.05), and AUC0–∞ (62.47 μg·h/mL vs. 50.48 μg·h/mL) (p < 0.05). The relative bioavailability of TCS was approximately 123.75% compared with BPC, confirming the enhanced bioavailability in the complex. Similarly, a four-fold increase in Cmax (25.55 μg/mL vs. 6.05 μg/mL) was observed.Plasma concentration–time profiles are presented in max and AUC0–∞ of BPC. All these results demonstrated that TCS may be applied to baicalein’s oral solid preparation.Soluplus was adopted as a hydrophilic pharmaceutical excipient to improve solubility, in vitro dissolution, or in vivo bioavailability in previous studies, such as solid dispersion , nanosusmax and AUC0–24 h, compared with BPC. The preparation method is simple and convenient, but also for Soluplus as a safe and effective drug excipient to explore a new pharmaceutical application. In conclusion, TCS is a promising method to improve the flowability and dissolution for drug–phospholipid complex.In our study, a novel TCS composed of baicalein, phospholipids, and Soluplus was successfully developed. The 35° angle of repose of TCS indicated an improvement in flowability, which met the industrial demand (θ < 40°). Moreover, TCS exhibited a marked enhancement in both the rate and extent of dissolution in vitro, as well as the bioavailability parameters C


Antrodia cinnamomea (AC) is a unique fungus found inhabiting the rotten wood of Cinnamomum kanehirai. A submerged liquid culture of AC has been developed and its bioproducts have been used to meet the market demand for natural fruiting bodies. AC exhibits anti-inflammatory, antitumor, antioxidant, and immunomodulatory effects. Previously, we isolated polysaccharide AC-2 from AC mycelia by means of alkali extraction with subsequent acid precipitation and found it had a pronounced anti-inflammatory effect. In this study, a novel polysaccharide named “antrodan” was obtained by further purification of AC-2 using Sepharose CL-6B column chromatography. Antrodan exhibited a molecular weight of 442 kD and contained a particularly high content of uronic acid (152.6 ± 0.8 mg/g). The protein content was 71.0%, apparently, higher than the carbohydrate content (14.1%), and thus antrodan was characterized as a glycoprotein. Its total glucan content was 15.65%, in which β-glucan (14.20%) was prominently higher than α-glucan (1.45%). Its FTIR confirmed the presence of β-linkages between sugars, and intramolecular amide bonds between sugars and amino acids. Its 1H-NMR spectrum showed that antrodan was a complex union of α- and β-glucans, which had (1→4)-linked α-Glcp and (1→3)-linked β-Glcp linkages to the carbohydrate chains via asparagine linked to protein site. Biologically, antrodan was confirmed to be totally non-detrimental to RAW 264.7 cell line even at dose as high as 400 μg/mL. It showed potent suppressing effect on the lipopolysaccharide-induced inflammatory responses in RAW 264.7 cell line. Moreover, antrodan significantly reduced the nitrogen oxide production at doses as low as 18.75 μg/mL. Antrodia cinnamomea  is a novel basidiomycete growing indigenously and uniquely in Taiwan. Its habit is to grow inside the empty rotten trunks of Cinnamomum kanehirai Hay  test. In brief, to the culture medium MTT  was added and left to react for 2 h. The culture medium was centrifuged at 12,000 ×g and the supernatant was removed. DMSO (1 mL) was added to dissolve the formazan crystal. The optical density was read at 570 nm with an ELISA reader.The method of Mosmann et al.  was follRAW264.7 cell line was seeded at 6,000 cells/mL to 96 well plate and incubated with DMEM for 24 h. LPS was added at concentration range 0.0–5.0 μg/mL as indicated. The incubation was continued for 72 h. The MTT assays were conducted at 24, 48 and 72 h after addition of LPS.et al. [3 solution  was transferred onto a 96-well plate, then serum (100 μL), Griess reagent  were added sequentially. The mixture was kept away from the direct sunlight and left to react for 15 min. The optical density was read with ELISA reader at 540 nm. A calibration curve was established by using authentic NaNO2, from which the nitrite and nitrate contents were estimated.The method of Green et al.  was adopFor determination of the NO production in cell line, RAW264.7 cell line was seeded at 6,000 cells/mL to 96 well plate and incubated with DMEM for 24 h. LPS was added at concentration range 0.0–5.0 μg/mL as indicated. The incubation was continued for 72 h. The culture medium (100 μL) was analyzed at 24, 38 and 72 h, respectively for NO production using the similar protocol mentioned in the above.p < 0.05 was considered to be significantly difference between groups.Data obtained were statistically treated with One-Way Analysis of Variance (ANOVA) analysis. Tukey’s test or least significant difference test (LSD) was used to analyze the difference of significance. The in vitro antiinflammatory effects, suggesting antrodan could be a candidate for further development of functional foods.Antrodan, a unique glycoprotein, has been isolated from AC mycelia. Antrodan has revealed promising


An elevated incidence of type 2 diabetes and cardiovascular disease (CVD) has been reported in patients with nonfunctional pituitary macroadenoma (NFPMA). There is no information about metabolic syndrome and cardiovascular risk in patients with NFPMA in our population.  Analyze the metabolic syndrome (MetS) components and estimate cardiovascular risk in patients with NFPMA.  Retrospective study, at the tertiary care center.  71 patients with NFPMA treated according to a preestablished multimodal protocol.  Prevalence of diabetes, hypertension, high cholesterol, obesity, and cardiovascular risk and its relationship with the clinical and biochemical characteristics. p = 0.001). NFPMA patients showed a significant increase risk for high total cholesterol  and diabetes . According to Globorisk, the male gender was an evidence of high CVD before  and after  multimodal treatment.  The prevalence of diabetes, hypertension, high cholesterol, and obesity at diagnosis was 30%, 27%, 48%, and 85% and did not change upon the last visit. The prevalence of MetS changes from 54 to 48% ( A high prevalence of cardiovascular and metabolic disease and a high cardiovascular risk were evidenced in patients with NFPMA, especially in men. Risk factors such as the personal history of hypertension and dyslipidemia could explain the foregoing, so the control and treatment of metabolic parameters and cardiovascular risk should be an integral part of the follow-up of these patients. Nonfunctional pituitary macroadenoma (NFPMA) is a benign lesion of the pituitary gland with a diameter larger than 1 cm, which represents approximately 30% of all pituitary tumors. NFPMA do not secrete pituitary hormones that leads to a clinical syndrome; however, they are related to headache, visual field defects with or without decreased visual acuity (mass effects of the tumor), and hypopituitarism. The transsphenoidal surgery is the treatment of choice in these patients; nevertheless, some cases require adjuvant therapy with postoperative radiotherapy , as long as the triglyceride concentration was not greater than 400 mg/dL. The anterior pituitary hormones such as testosterone, estradiol, cortisol, and free T4 were quantified with commercially available tests.t-test, Mann–Whitney U test, or Wilcoxon signed-rank test were used, and for the categorical variables, the χ2 test was used. We made a multivariate logistic analysis to establish the magnitude of association and calculate SMRs. To establish a statistically significant association, the p value < 0.05 was considered. The statistical package SPSS Statistics version 17 and STATA version 11 were used.The continuous variables are described as mean ± standard deviation (SD) or median and interquartile range (IQR) according to their distribution. For the categorical variables, proportions were used . To establish the association between the continuous variables, the Student The studied population consisted of 71 patients . The median of follow-up from baseline diagnosis of NFPMA to the last visit was 10.2 ± 4.8 years. Fifty-five patients (78%) had a suprasellar tumor with cavernous sinus invasion. Thirty patients (44%) had a single surgery with a transsphenoidal approach, 22 patients (31%) had two surgeries, and 18 patients (25%) had three or more surgeries; most of which were performed through a transcranial approach. All patients had progression of the remnant on follow-up magnetic resonance and received fractionated radiotherapy as an adjuvant treatment. The mean of waiting time between surgery and radiotherapy application was 10 years (interquartile range [IQR] 5–12).3 (IQR 5233–24031). Before radiotherapy, the volume was 1601 mm3 (IQR 697–538). Tumor volume decreased to 1124 mm3 (IQR 382–2638) in 3 years (p = 0.002) and to 816 mm3 (IQR 92–1866) in 5 years after radiotherapy (p = 0.01). A Δ of 785 mm3 in tumor volume reduction at 5 years was observed compared to preradiotherapy volume .The initial tumor volume at diagnosis of NFPMA was 14130 mmp = 0.001); for hypocortisolism, 62% versus 78% (p = 0.001); for hypogonadism, 67% versus 76% (p = 0.001); for hyposomatotropism, 89% versus 94% (p = 0.002); and for panhypopituitarism, 67.8% versus 78% (p = 0.001)  .All patients with a hormone deficit had an appropriate hormonal replacement with levothyroxine, prednisone, testosterone, or estrogen/progestin during all the clinical follow-up, according to the case. The growth hormone was never replaced in patients with hyposomatotropism.At the last visit, no cases of optic neuritis or central nervous system tumors were evidenced.2 (IQR 26.5–31.4), respectively; after multimodal treatment, a weight of 74.7 ± 13.6 kg and a BMI of 29 kg/m2 (IQR 26.4–32) were evidenced at the last visit, which was not statistically significant. The 47% of patients had obesity at NFPMA diagnosis versus 40% after multimodal treatment (p = 0.001). At diagnosis, the mean of WC was 92.1 ± 12.6 cm in men and 89.6 ± 10.3 in women; at the last visit, WC was 91.9 ± 13.5 in men and 90.3 ± 11.0 in women, which was not statistically significant.The weight and BMI at diagnosis of NFPMA were 76.2 ± 13.4 kg and 29.3 kg/mAt NFPMA diagnosis, the fasting glucose was 94 mg/dL (IQR 86–99), fasting insulin 13.7 UI/dL (IQR 8.1–25), HOMA-IR 3.0 (IQR 1.8–6.4), HbA1c 6.3% (IQR 5.8–6.5), systolic blood pressure 117 ± 14 mmHg, and diastolic blood pressure 74.6 ± 9.4 mmHg. The total cholesterol was 210 ± 46 mg/dL, HDL-C 43 mg/dL (IQR 35–52), LDL-C 121.8 ± 50 mg/dL, and triglyceride 212 ± 126 mg/dL.p = 0.02) , fasting insulin 16 UI/dL (IQR 7.5–31.5), HOMA-IR 4.9 (IQR 1.9–16.6), HbA1c 6% (IQR 5.7–7.4), systolic blood pressure 114 ± 14.5 mmHg, and diastolic blood pressure 70 ± 15.5 mmHg. The total cholesterol was 199 ± 35 mg/dL, HDL-C 45 mg/dL (IQR 38–60), LDL-C 120 ± 31 mg/dL, and triglyceride 171 ± 67 mg/dL; triglycerides were the only variable with a statistical significance after multimodal treatment ( = 0.02) .p = 0.003). MetS was evidenced in 52% of patients at diagnosis. The proportion of MetS decreased from 54% to 48% at the last visit (p = 0.001). There was no difference in the presence of MetS between patients with hyposomatotropism and patients without hyposomatotropism .At the diagnosis of NFPMA, arterial hypertension was found in 27% of patients, CVD in 28%, type 2 diabetes in 30%, and mixed dyslipidemia in 65%. After multimodal treatment, these comorbidities remained in the same proportions. In the group of patients with diabetes, women were more affected than men  for the woman gender, an OR of 4.9  for personal history of hypertension, an OR of 5.37  for personal history of dyslipidemia, and an OR of 0.74  for hyposomatotropism.The multivariate analysis for diabetes risk showed an OR of 4.2  , NFPMA pOnly one patient (with diabetes and hypertension) had a stroke at five years of follow-up (patient received radiotherapy 2 years before CVD).p = 0.01) and after multimodal treatment   at diagnosis and continued with the same score during the follow-up. In gender analysis, there was evidence of high CVD (risk ≥ 10%) in men versus women, before  .In this study, we report the characteristics of metabolic syndrome and the cardiovascular risk (according to the Globorisk score) in patients with NFPMA at diagnosis and after multimodal treatment.Many studies have demonstrated a high prevalence of metabolic syndrome, a decrease in HDL cholesterol levels, and hypertriglyceridemia in patients with NFPMA; hypertension, hyperglycemia, or increased waist-hip ratio had not been reported –5.The increased prevalence of metabolic disorders and CVD could be associated with the extension of large suprasellar tumors to adjacent structures, like the hypothalamus, conditioning a dysregulation in energy balance, disturbed circadian rhythm, sleep irregularities, imbalances in the regulation of body temperature, obesity, heart rate and/or blood pressure imbalance, and feeding alterations due to hypothalamic dysfunction –9.Other related variables could have imperfections of hormone replacement strategies or the absence of hyposomatotropism replacement. The mortality in patients with NFPMA is greater than that in the general population and is related to the increase in cardiovascular, respiratory, and infectious diseases, mainly in women. There is no consensus of variables that predict mortality in NFPMA; however, the advanced age at the moment of the diagnosis and the elevated doses of long-term glucocorticoid replacement therapy could be associated with it. .In our country, the National Health Survey of 2016 reported a prevalence of 9.4% for type 2 diabetes and 25.5% for hypertension in the general population .We observed that the prevalence of diabetes among patients with NFPMA remains higher than that in the general population (30% versus 9.4%), which could increase the presence of CVD. The high prevalence of diabetes could be related to the size of tumor at diagnosis as well as to the delay on diagnosis (approximately 3 years), with the subsequent delay of initial surgery treatment.A suprasellar extension of tumor with visual field defects was observed in 78% cases; we do not discard the possibility of hypothalamic dysfunction due to suprachiasmatic/paraventricular nucleus damage secondary to tumor invasion or compression.Hypopituitarism was evidenced in more than 2/3 of patients, which could result in an increased risk and/or worsening of metabolic diseases.p ≤ 0.05), especially in women; this was not evident for hypertension .There are no studies in the Mexican population about metabolic syndrome or cardiovascular risk in patients with NFPMA. It would be expected that, in the absence of hormonal hypersecretion, there would be no clinical syndromes or metabolic disorders; however, we demonstrated an increased prevalence of diabetes in patients with NFPMA compared to the general population  and a decrease in triglycerides (p = 0.02) were evidenced, probably associated with lifestyle changes and nutritional control during the clinical follow-up.We found a similar prevalence of MetS at the diagnosis of NFPMA with respect to other series (44%); however, after multimodal treatment, a decrease in MetS  among patients with NFPMA remains higher than that in the general population, both at diagnosis and after multimodal treatment, especially in the male gender. Risk factors such as the personal history of hypertension and dyslipidemia could explain the foregoing, so the control and treatment of metabolic parameters and cardiovascular risk should be an integral part of the follow-up of these patients.


Saccharomyces boulardii and its tolerance to in vitro gastrointestinal conditions for probiotic alcoholic beverage development. Placket-Burman screening was used to select only statistically significant variables, and the polynomial mathematical model for yeast growth was obtained by central composite rotatable design. Confirmation experiments to determine the kinetic parameters for yeast growth were carried out by controlling the temperature and pH. Soon after, the survivability of yeast was tested under in vitro conditions mimicking the human upper gastrointestinal transit. S. boulardii had suitable resistance to alcohol and gastrointestinal conditions for probiotic alcoholic beverage development.The aim of this research was to optimize the growth parameters  for  Eating habits have changed over the decades in pursuit of a healthier diet. Therefore, food and drink are being consumed with the purpose of disease prevention or treatment instead of only basic nutrition. Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host . ProperlSaccharomyces sp., Escherichia coli and Bacillus sp. have also been employed  and response surface methodology (RSM) are important issues because its takes less time, effort, and resources as well as facilitates the collection of a large amount of information with minimizing the number of experiments . RSM hasOn the other hand, alcoholic fermented beverages are consumed extensively worldwide, mainly for the welfare . AlthougS. boulardii  in synthetic must and its tolerance under conditions that mimic in vivo human upper gastrointestinal transit, thereby facilitating probiotic alcoholic beverage development.For the reason above mentioned, the aim of this research was to applying RSM to optimize the growth parameters for Saccharomyces boulardii – 17  was obtained by streaking on Sabouraud agar media  followed by incubation at 25°C for 48 h. A single colony was picked with a sterile loop and transferred to a tube containing yeast-extract-peptone-dextrose (YPD) broth. After incubation overnight at 25°C, the working inoculum was obtained in YPD at the same temperature under gentle shaking (160 min–1). After growth, the cells were harvested by centrifugation at 6000 × g for 5 min. The liquid fraction was discarded, and the remaining cell pellet was washed twice with phosphate buffer pH 7.2 and resuspended in the same buffer up to an optical density at 620 nm (OD620nm) measurement corresponding to ca. 108 cfu mL–1.S. boulardii were expressed as dry weight (Xdw) and viable cells (Xvc).Cell concentration determinations performed during the culture of 10 g dry weight L–1) were determined from centrifuged samples by discarding the supernatant and resuspending the pellet in 100 mL of distilled water. Subsequently, the cell suspension was diluted 1:100 to quantify the optical density at 620 nm (OD620nm) in a spectrophotometer  within the linearity range (0.050 to 0.500). The OD620nm values were related to the dry weight of the cell suspensions filtered on cellulose acetate membranes with 0.45 μm pore diameter and dehydrated in a chamber with infrared radiation to construct a calibration curve and obtain the conversion factor of OD620nm in Dry weight.Dry weight (Log10 viable cells mL–1) was determined by mixing equal parts of the appropriately diluted cell suspension with methylene blue solution (0.1% m v–1). The solution was observed in a Neubauer chamber using an optical microscope .Viable cells (LogSaccharomyces boulardii metabolic sugar consumption (glucose) and product formation  were analyzed using a high-performance liquid chromatography system equipped with an infrared detector, binary pump, control module, and LabSolution chromatography software . The chromatography column used was an Aminex® HPX-87H 300 mm × 7.8 mm . The mobile phase was prepared in 5 mM H2SO4 at a flow rate of 0.8 mL min–1. The injection volume was set to 20 μL, and the temperature was set to 60°C.®, France) and pH 7.0 buffer , was used for pH measurements.A benchtop pH meter  with a digital microprocessor equipped with automatic temperature compensation, calibrated with pH 4.0 buffer  could be the 2k full factorial (k = 4). In this case, the total number of treatments will be 16 and, considering three repetitions, it will totalize 48 runs. Another disadvantage of full 2k factorial design would be that it only have two levels, and it will be impossible to detect the possible curvature effect. A 3k full factorial (k = 4) is able to evaluate the curvature effect but it would have 81 treatments totalizing 243 runs with 3 repetitions. On the other hand, a Plackett and Burman design with central points for four variables only need 11 runs with the advantage in detecting the possible curvature effect. Thus, four independent variables  were evaluated for screening performance at three different levels. Eight factorial point combinations (±1) and three central points, for a total of 11 trials (dw), measured by OD620nm every 24 h, was the dependent variable.One possible design to evaluated the four independent variables , six axial points (±α), and five central points to estimate the pure error were evaluated, for a total of 19 trials . The matWhere Y is predicted response; i, ii, and ij are linear, quadratic, and interaction coefficients, respectively; β are regression coefficient; and k is the number of factors.Yi) into an individual desirability (di) varying from 0 to 1, and then obtain the overall desirability (D) applying the geometric median according Eq. 3.Additionally, desirability functions was performed to optimization of simultaneous multiple responses. According to di is the individual desirability, and m are the number of responses used to carried out the overall desirability (D) and w is the weight attributed for each response.Were Each desirability is calculating according the objective for each variable, thus it could target for the response to be at minimum (Eq. 4).r is a weight used to determine the scale of desirability, if r = 1, the desirability function is linear, if r > 1 places more emphasis on being close to the target value, and choosing 0 ≤ r ≤ 1 makes this less important. y is the response value, L is the minimum acceptable response, and T is the maximum acceptable response  with the working volume filled (5 L) with YPD containing added alcohol (5.0°GL) and the pH adjusted to 5.5 was incubated at 24°C to confirm the adequacy of the developed polynomial mathematical model to maximize yeast growth.g, specific growth rate – μX, rate of substrate utilization – μS, and conversion factor of substrate into biomass – YX/S) for S. boulardii growth (X) and substrate consumption (S), in accordance with Eqs. 5–8.A polynomial mathematical model validation assay was developed to determine the kinetic parameters , μm – specific speed for maximum growth, from log phase (h–1), X0 – initial concentration of viable cells (cells L–1), X – concentration of viable cells (cells L–1), S0 – initial concentration of glucose (g L–1), S – concentration of glucose (g L–1), t0 – initial time of log phase development (h), and t – final time of log phase (h).Here, tS. boulardii survivability under in vitro gastrointestinal conditions, 10 mL aliquots from the polynomial mathematical model validation experiment samples were collected as described above. In vitro GI treatment was carried out as reported by –1 calcium chloride, 1.12 g L–1 potassium chloride, 2.0 g L–1 sodium chloride, and 0.4 g L–1 potassium dihydrogen phosphate. This solution was sterilized at 121°C for 15 min. Artificial gastric juice (GJ) was freshly prepared (75 mL) by adding 3.5 g L–1 swine mucin and 0.26 g L–1 swine pepsin . The pH was adjusted to 2.0 with 1.0 M HCl. Sample aliquots (1 mL) was transferred into GJ and anaerobically incubated at 36°C for 45 min, with gentle shaking. Subsequently, artificial intestinal juice was obtained by adding 3.0 g L–1 bile salt, 1.95 g L–1 pancreatin, and 0.1 g L–1 egg white lysozyme  to the GJ. Sterile distilled water was used to top up 100 mL final volume. The pH was adjusted to 7.0 with 1.0 M sodium bicarbonate solution and anaerobically incubated at 36°C for 180 min, with gentle shaking. 12-wells microtiter plate with WL agar  was used to count viable cells by drop plate technique both after growing in YPD medium and gastric and GI treatment, so that each well was seeded by only a drop from each dilution level – until 10–6. Plates were incubated at 30°C for 48 h, under aerobic condition. Colonies were quantified with the aid of colony counter. The results were expressed as weighted (Log10 cfu mL–1), using at least two successive dilutions level, according to To evaluate n = 3) and analyzed by a variance test (ANOVA) followed by Fischer’s or Dunnett’s test. Statistica software version 10.0  was used for the central composite rotatable design analyses. A desirability function was used to obtain the preferred conditions of the dependent variables as a function of cellular concentration in dry weight and viable cells. Action Stat software version 3.1.43.724.694  was used for other statistical analyses. All statistical analyses were performed at 95% confidence.The results were reported as the mean ± standard deviation (SD) (S. boulardii concentration was not significant (p < 0.05), indicating that the optimal point was among the levels tested. Three independent variables  were defined as significant for the central composite rotatable design optimization.The screening results revealed that alcohol content had a negative effect on dry weight, whereas temperature and pH had a positive effect on dry weight. Initial nificant . The PlaS. boulardii concentration effect was lower than that of other variables  and had no significant influence on dry weight. Based on the Plackett and Burman results, temperature (16.8 up to 25.2°C) and pH (4.66 up to 6.34°C) were adjusted to maximize the growth of Saccharomyces boulardii, and alcohol content (0.0 up to 14.8°GL) was extended to determine its growth ability in the presence of alcohol to maximize cellular concentration in dry weight and viable cells in central composite rotatable design performance. All coefficients (p ≤ 0.05), determination coefficient (R2) values (0.9154 and 0.9347) and adjusted R2 (0.8308 and 0.8693). A p (lack of fit) <0.0001 indicates the adequacy of the models, Eqs (9) and (10).The initial ficients  from thedi) (0.00 ≤ di ≤ 1.00), cellular concentration in dry weight and viable cells were maximized with alcohol content (0.0°GL), pH (5.92), and temperature (25.2°C) (The surface response graphs  show tha(25.2°C) .10 g dry weight L–1. The predicted value from polynomial mathematical models was −0.37 Log10 g dry weight L–1; therefore, a large relative deviation (63.74%) was observed. However, the viable cell concentration values in the laboratory experiment (7.63 Log10 viable cells mL–1) and the polynomial mathematical model (8.43 Log10 viable cells mL–1) were closer, with a relative deviation of 9.54%.For polynomial mathematical model validation, the stationary phase was considered at the first time point (37.5 h), where dry weight was −0.61 LogS. boulardii. The growth acceleration phase was from 3 to 14.5 h, followed by the exponential growth phase up to 35.76 h (R2 = 0.9957 e p < 0.0001). The values of tg (3.50 h) and μX (0.20 h–1) were calculated from the log phase.The adaptation phase (lag) for polynomial mathematical model validation was up to 3 h after inoculating  35.76 h , in acco–1), glycerol production (0.21 g L–1), and an increase in S. boulardii concentration (5.47 to 7.63 Log10 cells mL–1) occurred, in addition to reductions in glucose (5.35 up to 4.53 g L–1), dissolved oxygen (6.63 up to 0.16 ppm), and pH (5.35 up to 4.53). At the end of the exponential growth phase, all the glucose was consumed, and the acetic acid production had increased to 0.81 g L–1.During the exponential growth phase, acetic acid production  and the confirmation test (5 L bioreactor) can be explained by differences such as the volume . Inconsistent environmental conditions sometimes lead to different and unsatisfactory results at the laboratory or pilot scale due to physiological changes in the microorganism activity . When thS. boulardii. The models showed that the optimal pH for microorganism growth was between 4.5 and 5.5 with specific growth rate (μX) equal to 0.17 h–1. The experiment was carried out in medium without ethanol incubated at 30°C. Thus, the value obtained by calculating rate of substrate utilization (μS) indicates that in the exponential phase, 3.13 × 10–12 g glucose was consumed per cell of S. boulardii per hour. Similarly, the value obtained for conversion factor of substrate into biomass (YX/S) indicates that for S. boulardii, the growth of 1.42 × 1010 cells occurred per g of glucose consumption.S. boulardii growth in air lift and shaker bioreactors, obtaining values of specific growth rate between 0.350 and 0.382 h–1 for with an initial pH of 6.0 and incubation at 30°C in the absence of ethanol in the culture medium. The values reported by 10 cell) dose of S. boulardii to confer health benefits on humans. Thus, probiotic alcoholic beverage benefits may be obtained from the intake of approximately 100 mL of the drink, but this value depends on its alcohol content. In this work, S. boulardii survivability showed a slight reduction of 0.13 Log10 cfu mL–1 after gastric conditions and 0.97 Log10 cfu mL–1 after intestinal conditions , with a survivability of 6.85 Log10 cfu mL–1 after gastrointestinal conditions in culture medium with 5.0°GL.S. boulardii survivability showed a slight reduction of 0.13 and 0.97 Log10 cfu mL–1 after gastric conditions and intestinal conditions, respectively, with a survivability of 6.85 Log10 cfu mL–1 after gastrointestinal transit in culture medium with 5.0°GL.Enough viable probiotics are necessary to play a beneficial role on the host . GastroiS. boulardii, allowing the development of probiotic alcoholic beverage to obtain additional benefits. According to the results obtained using the polynomial mathematical model, it is possible to produce a probiotic wine with alcohol content equal 11.9°GL, obtaining viable cell concentration 6.82 Log10 viable cells mL–1. Following the criteria adopted by 10 viable cells, which corresponds to the minimum value indicated by 10 viable cell mL–1) with alcohol content equal 5°GL, corresponding to a Class III beer, it would be necessary to ingest only 23.44 mL, well below 330 mL (According to w 330 mL , to meet–1 (–1. At the beginning of the stationary phase, the glycerol concentration was 0.18 g L–1. According to –1.The minimum sensory limit for acetic acid perception is 0.07 g L–1 . TherefoSaccharomyces boulardii may have good potential for probiotic alcoholic beverage development. Slight microbial susceptibility to gastrointestinal conditions was observed, and alcohol exhibited no severe stress on S. boulardii survivability. The results provide new insight into the management of probiotic beverages and suggest new prospects for a more integrated strategy for increasing beverage quality.All datasets generated for this study are included in the manuscript and/or the supplementary files.BP participated in the planning, conducted the experiments, and wrote and revised the manuscript. DC assisted in the statistical analysis and revised the manuscript. WL and AG assisted in the microbiological analyses and reviewed the manuscript. MCor performed the chromatographic analyses and revised the manuscript. KP and MCoe planned and coordinated the experiments, and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To study nocturia in patients with lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH) after medical or placebo treatment.Patients with LUTS suggestive of BPH from several community clinics were included. Patients completed the International Prostate Symptom Score (I-PSS) questionnaire and a 3-day voiding diary. Urinalysis, prostate-specific antigen (PSA) measurement, and prostate ultrasonography were performed. Nocturnal polyuria (NP) was defined as a nocturnal urine fraction exceeding one third of the daily urine output in elderly men. A total of 148 outpatients were randomized to drug treatment (tamsulosin) or placebo treatment. After 8 weeks of treatment, they were re-evaluated using a 3-day voiding diary, PSA measurement, prostate volume (PV), I-PSS, etc.r = 0.419,P = 0.002; r = 0.302,P = 0.031). Eighty patients were randomized to drug treatment (tamsulosin) and 68 patients were randomized to placebo treatment. The I-PSS score was 16.8 ± 4.9 to 19.3 ± 5.0 (p = 0.002), the storage symptom score was 10.3 ± 3.4 to 10.7 ± 3.4 (p = 0.007), and the voiding symptom score was 7.5 ± 2.4 to 8.6 ± 2.3 (p = 0.003). The frequency of daytime urination was 7.5 ± 2.6 to 8.1 ± 2.6 (p = 0.002), maximum urine volume (ml) was 372.8 ± 103.3 to 302.8 ± 119.3 (p = 0.007), and morning urine volume (ml) was 280.5 ± 111.7 to 259.5 ± 100.7 (p = 0.003). However, the frequency of nocturia score was 2.8 ± 0.7 to 3.0 ± 0.6 (p = 0.306) and the nocturnal urine volume (ml) was 800.7 ± 323.0 to 845.7 ± 303.5 (p = 0.056), which did not change significantly. There were significant differences between the NP and non-NP groups in the duration of LUTS, first voided urine volume, daytime urination frequency, and the amount of water intake at night and 4 h before sleep.The average I-PSS score was 20.3, storage symptom score was 11.7, voiding symptom score was 8.6, quality of life (QoL) score was 3.7, PV was 40.4 ± 19.4 ml, and nocturnal urine volume (NUV) was 845.7 ± 339.0 ml. The mean frequency of nocturia was 2.3 ± 1.1 per day, and 94% of the patients had a nocturia frequency of more than two times per day. Of these patients, 76.5% had NP. A significant correlation was found between NUV and the amount of water intake at night and 4 h before sleep :  The medical therapy have not shown a significant reduction in frequency of nocturia so far. There are also not equal amounts of studies showing positive or negative results on nocturia [Nocturia, is perceived as one single symptoms of all lower urinary tract symptoms (LUTS) by most men, which is highly prevalent and the most bothersome component . It has nocturia . For allFrom January 2015 to May 2016, 148 patients were admitted to Beijing Huairou Community Health Service Center, with a median age of 69.0 years, a mean ± standard deviation (SD) age of 62.1 ± 13.6 years, LUTS/BPH duration of 5.0 ± 3.1 years, and absence of treatment using α-blockers and/or 5α-reductase inhibitors. The common inclusion criteria for all participants were age ≥ 50 years and diagnosis of BPH. All patients provided their written informed consent and the program was accepted by the hospital ethics committee. The exclusion criteria were prostate cancer, PSA > 10 ng/ml, urinary tract infection, nervous system disease, urolithiasis, medical therapy that could affect the function of urination, prostatic surgery, or pelvic surgery.Based on the literature , we estiThe patients included in the study were randomly divided (1:1) into two groups (drug treatment and placebo treatment) by use of a computer generated random number table. But some patients of the two groups had lost in follow-up because of financial status, phone lost, language barriers, individual patient factors, etc.At last all 148 patients with LUTS suggestive of BPH were divided into two random groups, which either received α-adrenoceptor antagonist (tamsulosin 2 mg qd po) or placebo for 8 weeks. IPSS, QoL index questionnaire, 3-day frequency volume charts (FVCs), PSA, urinalysis, PV, and uroflowmetry (Qmax) were assessed before and after medical therapy. Serum PSA and urinalysis were evaluated at Beijing Huairou Hospital. The specific instrument for the evaluation of nocturia was a 1000 ml graduated glass. This study was approved by the ethics committee of Beijing Huairou Hospital. Each patient was informed of the study and signed an informed consent form and had the right to withdraw from the study at any time.Nocturia, specifically, was the number of voids recorded during the nighttime . The firt-test. An analysis of the difference in the number of patients with nocturia before and after treatment was performed using McNemar’s test. A multivariate analysis was performed. A P-value of 0.05 or less was considered significant.SPSS 22.0 statistical software was used for data analysis. All variables were expressed as the mean ± SD values. The numerical data were compared using the unpaired r = 0.419, p = 0.002) and 4 h before bedtime drinking volume  were related. NPi and maximum urination volume  and morning urine volume  were related, but there was no correlation between NPi and prostate volume, age, LUTS duration, or I-PSS score.The relationship between NUV and evening drinking volume (Eighty patients were randomized to the drug treatment (tamsulosin) and 68 patients were randomized to the placebo treatment. The I-PSS score, storage symptom score, voiding symptom score, quality of life score, frequency of daytime urination, maximum urine volume, and morning urine volume were statistically significant between both groups. However, the frequency of nocturia score and nocturnal urine volume did not change significantly.In the indices of age, 24-h drinking volume, bladder function, PV, and PSA level, there were no significant differences between the groups. While in LUTS duration, nocturnal urine volume, morning urine volume, and daytime voiding frequency, the differences between the two groups were statistically significant.Nocturia, one of the most bothersome symptoms of LUTS, has been the focus of a high volume of rapidly evolving research. The purpose of this study was to describe the relevant recent research in the field of nocturia in China, with particular emphasis on its evaluation and management. Nocturia is a complicated clinical entity that is often multifactorial in etiology. Experts of the International Continence Society (ICS) define nocturia as the general complaint when an individual  and associated bother) must wake up at night one or more times to void , 2.Epidemiological studies showed that the prevalence of nocturia in the < 30-year-old population was approximately 3%, in the 60- to 69-year-old population was 30%, and in the > 70-year-old population was 40% . SchatzlNocturia has been identified as the leading cause for sleep disturbance and sleep fragmentation; it causes daytime fatigue, impacts daily activities, and deteriorates psychomotor performance, cognitive function, and mood –12. NoctIn these analyses, LUTS/BPH patients with nocturia had some urinary system diseases, such as lower urinary tract obstruction and overactive bladder, as well as cardiovascular diseases, diabetes, diabetes insipidus, and other awakening factors of urination (such as anxiety and sleep disorders). In this study, patients had hypertension, coronary heart disease, diabetes, and other diseases. Thus, nocturia may be caused by the combined effects of a variety of conditions. Kupelian et al. demonstrated that while increased odds of the metabolic syndrome were observed with mild to moderate degrees of nocturia . In thisBPH is a common disease in elderly men, not only with voiding symptoms such as dysuria but also with storage symptoms such as urinary frequency, urgency, and nocturia. Clinical studies showed that the application of α-blockers and 5α-reductase inhibitors could effectively relieve lower urinary tract symptoms caused by BPH, but the efficacy in improving nocturia was not obvious , 18.Tacklind thought that doxazosin and terazosin were significantly more effective than finasteride in improving nocturia, while no difference was found with tamsulosin . This laIn our study, 80 patients were randomized to drug treatment (tamsulosin) and 68 patients were randomized to placebo treatment. The scores and the data  were all statistically significant. However, the frequency of nocturia score and the nocturnal urine volume did not change significantly. The traditional tool for evaluating nocturia was the I-PSS in nocturia subtype score, which was not described in detail . WhatsmoWe used a 3-day urinary frequency voiding diary, including a 24-h urination, nocturnal urine volume, urine volume, total sleeping time, and amount of water intake for a detailed assessment of patients with nocturia to understand the correlation between nocturia and other factors. Therefore, the data that was obtained was in detail.r = 0.419, p = 0.002) and before falling asleep 4-h drinking water . Hence, we thought that the improvement of α-blockers treatment for nocturia was poor.Koseoglu et al. reported 58 cases of patients with BPH, of whom 95% had NP. The frequency of nocturia was 2.73 ± 1.44 times/day, which confirmed that patients with BPH and NP had a relationship between LUTS duration and 3-h drinking of water before falling asleep . In thisIn this study, between the NP group and the non-NP group, there was a significant difference in LUTS duration, maximum urine volume, morning urine volume, nocturnal urine volume, daytime urination, and 4-h before falling asleep drinking water volume. The frequency of daytime micturition and LUTS duration were the indices between the NP group and the non-NP group. A long disease history and more frequent daytime urination were considered the non-NP type, and α-blocker drug treatment could improve this type of nocturia. Nocturia could also be alleviated by treating the other causes, such as increased water drinking before going to bed.This study had some limitations, including a small sample size and the assessment with regard to short-term use of medication. Future studies of patients with LUTS/BPH and nocturia should, therefore, also include nocturia-specific instruments to correctly classify the underlying pathologies for nighttime voiding, and hence, provide a rationale for the treatment of all LUTS/BPH causes.In conclusion, patients with lower urinary tract symptoms improved, to some extent, after receiving medication. However, the nocturia was not obviously improved and the incidence was still high. The patients could not achieve satisfactory results, and NP was highly prevalent among patients. The correlation between the frequency of nocturia and nighttime water intake and drinking water before going to bed were significant. We could control water intake to ease nocturnal urination.