Anopheles in Surat Thani province, Thailand.Ivermectin mass drug administration (MDA) could accelerate malaria elimination in the Greater Mekong Subregion. This study was performed to characterize the bionomics of Anopheles mosquitoes were morphologically identified to species. Primary Anopheles malaria vectors were dissected to assess parity status, and a subset were evaluated for molecular identification and Plasmodium detection.Mosquitoes were collected via human landing collections between February and October 2019. Anopheles mosquitoes. Morphological studies identified 15 Anopheles species, of which the most abundant were Anopheles minimus (s.l.) , An. dirus s.l.  and An. barbirostris s.l. . Molecular identification confirmed that of the An. minimus s.l. mosquitoes collected, 99.80% were An. minimus (s.s.) (n = 484) and 0.2% were An. aconitus (n = 1), of the An. dirus (s.l.) collected, 100% were An. baimaii (n = 348), and of the An. maculatus (s.l.) collected, 93.62% were An. maculatus (s.s.) (n = 44) and 6.38% were An. sawadwongporni (n = 3). No Anopheles mosquito tested was Plasmodium positive (0/879). An average of 11.46 Anopheles were captured per collector per night. There were differences between species in hour of collection , with more An. barbirostris (s.l.) and An. maculatus (s.l.) caught earlier compared to An. minimus (s.l.)  and An. dirus (s.l.) . The proportion of parous An. minimus (s.l.) captured by hour increased throughout the night . Overall, An. minimus (s.l.) parity was 67.68% (2375/3509) with an intra-cluster correlation of 0.0378. A power calculation determined that an An. minimus (s.l.) parity reduction treatment effect size = 34%, with four clusters per treatment arm and a minimum of 300 mosquitoes dissected per cluster, at an α = 0.05, will provide 82% power to detect a significant difference following ivermectin MDA.A total of 17,348 mosquitoes were collected during the study period; of these, 5777 were An. minimus parity.The study area in Surat Thani province is an ideal location to evaluate the impact of ivermectin MDA on  Anopheles species in the Dirus complex, Minimus complex and the Maculatus group having been recognized as the primary malaria vectors in the region. From the Minimus complex, Anopheles minimus (s.s.) is the primary malaria vector and can be found across Thailand and the GMS    ) were obtained from mosquito colonies maintained at the Armed Forces Research Institute of Medical Science Department of Entomology. Negative DNA/RNA-free distilled water controls were included.To identify anopheline sibling species, multiplex allele-specific PCR (AS-PCR) assays were used to examine the internal transcribed spacer 2 (ITS2) region of DNA and distinguish the members of the Dirus complex [philous] , the Macvidicus]  and the ampanai] . Previouampanai] –34 were Amplifications were performed in a T100 DNA thermal cycler  under the following PCR conditions. For the Maculatus group, the cycling program started with an initial denaturation at 95 °C, 10 min; then denaturation at 94 °C/1 min, primer annealing at 55 °C/30 s, extension at 72 °C/30 s for 35 cycles; and a final extension at 72 °C for 10 min. The amplification conditions for the Dirus complex were the same as those for the Maculatus group, except that the annealing time was 15 s instead of 30 s. For the Funestus group, which contains the Minimus complex, the cycling program started with an initial denaturation at 95 °C, 10 min; then amplification at 94 °C/30 s, 45 °C/30 s, 72 °C/40 s for 35 cycles; and a final extension at 72 °C for 10 min. The amplified PCR products were subjected to DNA fragment analysis using the QIAxcel Advanced System (Qiagen) according to the manufacturer’s instructions. Briefly, 10 μl of PCR product was analyzed with the QIAxcel DNA Fast Analysis Cartridge (Qiagen), using the DM190 method and QX 15-bp/1-kb alignment markers. Fragment sizes were calculated using the BioCalculator (Qiagen).2 and 0.2 µM of each primer. The PCR cycling program consisted of an initial denaturation step at 94 °C, 10 min; then 94 °C/30 s, 60 °C/1 min, 72 °C/1 min for 37 cycles; and a final extension step at 72 °C for 5 min. The size of the PCR product was determined using the QIAxcel Advanced System as described above. The PCR product was cleaned by the ExoSAP-IT™ PCR Products Clean-up kit: 2 µl of ExoSAP-IT™ was added directly to 5 µl of PCR reaction product, incubated at 37 °C for 15 min and then at 80 °C for 15 min. The ITS2 rDNA gene PCR product was sequenced using the Bigdye® Terminator v3.1 Cycle Sequencing Kit  according to Applied Biosystems’ protocol with forward and reverse universal ITS2A and ITS2B primers and run on a SeqStudio Genetic Analyzer (Life Technologies). Forward and reverse nucleotide sequence data for each sample were assembled using the Sequencher 5.1 software package . The Anopheles species were identified by phylogenetic analysis. Briefly, the MUSCLE algorithm was used for sequence alignment in Molecular Evolutionary Genetics Analysis (MEGA) 6.0 software   were included in the analyses . While all clusters showed an increasing trend in the proportion of parous An. minimus (s.l.) throughout the night  there was no significant trend in the proportion of parous mosquitoes by hour throughout the night    were included in the analysis.Overall, there was a significant increasing trend in the proportion of parous Plasmodium infection rates in Anopheles in the GMS are extremely low, either survival or parity become the ideal metrics to measure. However, capturing indoor-resting, blood-fed Anopheles in the GMS is very difficult, rendering assessment of mosquito survival as done previously in Africa , secondary malaria vectors [An. epiroticus] or suspected malaria vectors . An. minimus (s.l.) accounted for 87.16% (5035/5777) and An. dirus (s.l.) 7.15% (407/5777) of the total Anopheles collected., respectively. Molecular identification confirmed that of all An. minimus (s.l.) collected, 99.80% were An. minimus (s.s.) (n = 484) and 0.2% were An. aconitus (n = 1), and of all An. dirus (s.l.) collected, 100% were An. baimaii (n = 348). Since the secondary and suspected vectors were captured in such low numbers, it is likely that the primary malaria vectors, An. minimus (s.s.) and An. baimaii, are largely responsible for Plasmodium transmission in the study area. However, as no specimens were Plasmodium positive (0/879), it cannot be confirmed that these vectors are solely responsible for transmission. A previous study in Ubon Ratchathani province, in northeastern Thailand, reported that suspected malaria vectors An. barbirostris (s.l.), An. philippinensis and An. hyrcanus (s.l.) are highly zoophagic, feeding mostly on cattle [Plasmodium transmission in Surat Thani. One limitation of this study is the lack of assessment on the proximity of the mosquito collection sites to potential influential factors, such as the forest, larval habitat and livestock populations.Surat Thani is unique in that it is a malarious province in Thailand that is not located along an international border. It has some of the highest rainforest coverage in the GMS and the highest rubber plantation coverage in Thailand, making it an ideal habitat for malaria vectors in the GMS. Indeed, of the 15 species captured in these surveys, 11 were either primary malaria vectors [n cattle . Since wAnopheles as possible, all HLCs were performed outdoors. Results from a cross-sectional survey in Surat Thani in the same study area where HLCs were performed indicated that staying outdoors is a primary risk factor for asymptomatic Plasmodium carriage, suggesting that most of the transmission occurs outside the home [Anopheles malaria vector regardless of location or time.Primary malaria vectors in the GMS tend to be collected more frequently outdoors than indoors –11. Sincthe home . This fiAnopheles species collected, with only six species, and Anopheles species diversity was similar to that in Khiri Rat Nikhom, which were both lower than species diversity in Phanom  were captured later in the night compared to Phanom (P = 0.0275) and Khiri Rat Nikhom (P = 0.0002). This could be due to the production of durian in Vibhavadi as thermal fogging with malathion occurs at night-time for the control of several durian crop pests, in particular Scirtothrips dorsalis  and Allocaridara malayensis , and this fogging was observed at some of the mosquito collection sites during HLCs in Vibhavadi clusters. Vibhavadi has the fourth highest coverage (1.33%) of durian plantation of the 19 districts in Surat Thani, with Khiri Rat Nikhom having the eighth highest coverage (0.33%) and Phanom the twelfth highest coverage (0.18%) [Vibhavadi had the fewest number of om Table . This re (0.18%) .An. dirus (s.l.) varied among districts, with the greatest numbers captured in Phanom, likely due to geographical  proliferation [Anopheles dirus (s.l.) were captured later in the night in Phanom compared to Khiri Rat Nikhom (P = 0.0023) and Vibhavadi (P = 0.0088), but this may have been an artifact due to the smaller number of An. dirus (s.l.) captured in Khiri Rat Nikhom and Vibhavadi districts.The abundance of al Figs. , 2 and bferation , 22. AnoAn. hodgkini, An. donaldi and An. pollicaris. However, it is possible that these species may have been identified as An. barbirostris (s.l.) previously as they are difficult to distinguish morphologically [Anopleles sawadwongporni was also recorded for the first time in Surat Thani, but this species may not have been identified in previous surveys due to lack of molecular species identification [Anopheles baimaii is also a newly recorded species for Surat Thani, likely missed previously due to the lack of molecular species identification. It is somewhat surprising that more members of the Dirus complex were not identified because An. dirus (s.s.), An. cracens, An. nemophilous and An. scanloni have been observed in the adjacent provinces of Phang Nga, Krabi, Nakhon Si Thammarat and Ranong [These surveys recorded some members of the Barbirostris group in Surat Thani for the first time, including ogically . Future ogically . Anoplelfication . Anopheld Ranong .Plasmodium-positive Anopheles (0/879) specimens detected. However, this is not very surprising as the likelihood of finding sporozoite-infected Anopheles in the GMS is very low, typically lower than 1:1000 [Anopheles populations in Surat Thani. This study found an overall An. minimus (s.l.) parity of 67.68% (2375/3509), which is comparable with the results of other surveys in Thailand [An. minimus (s.l.) parity was observed from cluster to cluster, with an ICC value of 0.0378. For An. minimus (s.l.), there was a significant trend of increasing parity by hour of collection, suggesting that older An. minimus tend to feed later at night   parity reduction treatment effect size = 34%, with four clusters per treatment arm and a minimum of 300 mosquitoes dissected per cluster at an α = 0.05 will provide 82% power to detect a significant difference in the population age structure (i.e. parity). Due to reduced malaria cases and transmission in Surat Thani [Anopheles parity rate is expected to provide a valuable outcome to assess the impact of ivermectin MDA on malaria transmission.There were no n 1:1000 . This is 67.68% 275/3509, Thailand , 46, 47.at Thani , evaluatAnopheles primary malaria vectors were captured in Surat Thani. This study illustrates that Surat Thani will be an ideal field site for evaluating the impacts of ivermectin MDA on local Anopheles population age structure.An abundance of


Coturnix coturnix eggshell and to evaluate its abrasive, remineralizing, and antibacterial activities.  To formulate and develop a new experimental polishing paste based on S. aureus, E. coli, E. faecalis, C. albicans, and S. mutans were inoculated with experimental paste (Coturnix coturnix quail eggshell base) and control paste  consisting of n = 12 for each group.  The research was experimental, longitudinal, comparative, and prospective. To measure the antibacterial efficacy, analysis units consisted of wells were made. The microorganisms C. albicans and S. mutans with an average of 8.70 ± 0.14 and 11.65 ± 0.15 mm, respectively. On the other hand, the Universal Polishing paste only had an average of 7.00 ± 0.11 and 8.71 ± 0.11 mm for C. albicans and S. mutans, respectively. Significant differences were observed only in these two strains p < 0.001.  It was found that, among the strains inoculated, the quail paste presented with the highest antimicrobial effectiveness to C. albicans and S. mutans compared to control paste, Diamond Excel, and Universal Polishing according to the time and type of microorganism. The quail paste demonstrated antimicrobial efficacy against  Currently, tons of eggshells are discarded everyday causing environmental problems such as unpleasant odor and flies and other vectors of communicable diseases. Different mechanisms are being used worldwide to recycle eggshell wastes. Typically, eggshell wastes go to a landfill, fertilizer, feed additive, adsorbent, calcium supplement, paper making etc. Therefore, these mechanisms can be more sustainable techniques to manage the waste by potential use in dental science –3.3) to reduce the depletion of natural limestone reserves, which are irreplaceable [Eggshells are traditionally used in different industrial processes for agricultural engineering with the main objective of correcting the pH of some acidic soils. Currently, this method has an economic value. Similarly, mollusk shells can also be an alternative source of calcium carbonate  has been demonstrated since it contains approximately 39% elemental CaCodiseases –8.Another important point to consider is the roughness of the surface of the restorative materials because it is a primary parameter that can induce the accumulation of a biofilm that could subsequently negatively affect oral health , 10. EviCoturnix coturnix eggshell and to evaluate its antimicrobial efficacy.Thus, this study aimed to develop and formulate a novel experimental polishing dentifrice based on This research would be sent to the Ethics Committee of the Universidad Científica del Sur for review and for authorization (no: 174-CIEI-AB-CIENTÍFICA-2020). No ethical conflicts are anticipated because this is a purely experimental in vitro study.® 15.0 statistical software was used. For this sample calculation, a beta of 0.8 and an alpha of 0.05 were used. A total of 60 wells, divided among the 5 experimental groups, were considered, with n = 12 wells for each group.S. aureus ATCC® 25923 ™ compared to the 4 pastes   Group A: E. coli ATCC® 25922 ™ compared to the 4 pastes   Group B: E. faecalis ATCC® 29212 ™ compared to the 4 pastes   Group C: C. albicans ATCC® 10231 ™ compared to the 4 pastes   Group D: S. mutans ATCC® 25175 ™ compared to the 4 pastes   Group E: The sample size was calculated based on the means comparison formula. The STATAThe eggshells were collected from a poultry farm in Lima, Peru. They were then washed and disinfected for six hours in a diluted sodium hypochlorite solution. The eggshells were vacuum-dried at 37°C for 24 hours and were ground with a mill until a fine-textured powder was obtained. Sodium lauryl surfactant was mixed to the eggshell powder (4.5 kg) to improve its solubility in water. The eggshell powder was then placed in a 500 ml flask and was dry-ground at 400 rpm for forty minutes until about 0.3 mm fine grains were obtained.The quail eggshell was crushed until reaching 125 g. The other components described were weighed according to the proportions in S. mutans agar had 5% defibrinated sheep blood at 42°C in 10% CO2. The microorganisms were stored in the MH medium with 20% glycerol. The minimum inhibitory concentration (MIC) was determined using the broth microdilution method. A control medium was prepared; it was compared to a bacterial suspension approximation of 0.5 McFarland CFU/ml. Finally, the inhibition halos were measured by Kirby–Bauer method . Only the r method .U-test or Student's t-test will be used. The statistical analysis will be carried out in the STATA 15 program, establishing a level of significance of p < 0.05.For the elaboration of the descriptive analysis, the measures of central tendency and dispersion of the numeric variables will be obtained. In addition, normality will be determined using the Shapiro–Wilk test. Depending on this, for the bivariate analysis, Mann–Whitney . aureus, E. coli, E. faecalis, C. albicans, and S. mutans strains with an average of 12.63 ± 0.08; 12.68 ± 0.07; 12.71 ± 0.07; 8.70 ± 0.14; and 11.65 ± 0.15 mm, respectively. On the other hand, the Universal Polishing paste only had an average of 7.00 ± 0.11 and 8.71 ± 0.11 mm for C. albicans and S. mutans, respectively. Significant differences were seen in these two strains only (p < 0.001)  .3) from eggshells could provide substitute minerals used in paper treatment to improve its surface brightness, opacity, and strength. This chemical compound can also be used to enhance the texture and appearance of the paper among other functions such as abrasives on different textures [Calcium carbonate (CaCOtextures , 12, 13.Although there are currently some toothpaste formulations that have great antimicrobial activity , there iAlthough there is a diverse use of eggshell, there is limited evidence available on its potential as an abrasive powder on denture polishing. In addition, calcium could play an important role in the process of enamel remineralization and has very high bioavailable calcium , 18. HenC. albicans, S. mutans, S. aureus, E. coli, and E. faecalis. Similarly, the study by Verkaik et al. [S. oralis and A. naeslundii, which are two important colonizers of the surface enamel. They concluded that herbal toothpastes have immediate and continuous antibacterial properties like chlorhexidine gluconate. Another study by Smolarek et al. [Regarding antimicrobial activity, the present study found that the quail eggshell toothpaste had optimal antimicrobial activity against multiple oral cavity strains such as k et al.  discussek et al.  also staC. albicans and S. mutans. Although it was found that both formulations showed antimicrobial activity for some microbes, more studies are required.Guven et al.  investigThis study has some limitations. Scientific literature , 25 on tC. albicans and S. mutans.Statistically significant differences were only found in antimicrobial efficacy between quail paste and Diamond Excel, Universal Polishing, and control paste against


Etomidate (ETM) is a hydrophobic drug, and organic solvents must be added to an etomidate injectable solution (ETM-SOL) to aid dissolution, that causes various adverse reactions on injection. Lipid emulsions are a novel drug formulation that can improve drug loading and reduce adverse reactions. ETM-ILE was prepared using high-pressure homogenization. Univariate experiments were performed to select key conditions and variables. The proportion of oil, egg lecithin, and poloxamer 188 (F68) served as variables for the optimization of the ETM-ILE formulation by central composite design response surface methodology. The optimized formulation had the following characteristics: particle size, 168.0 ± 0.3 nm; polydispersity index, 0.108 ± 0.028; zeta potential, −36.4 ± 0.2 mV; drug loading, 2.00 ± 0.01 mg/mL; encapsulation efficiency, 97.65% ± 0.16%; osmotic pressure, 292 ± 2 mOsmol/kg and pH value, 7.63 ± 0.07. Transmission electron microscopy images showed that the particles were spherical or spheroidal, with a diameter of approximately 200 nm. The stability study suggested that ETM-ILE could store at 4 ± 2 °C or 25 ± 2 °C for 12 months. Safety tests showed that ETM-ILE did not cause hemolysis or serious vascular irritation. The results of the pharmacokinetic study found that ETM-ILE was bioequivalent to ETM-SOL. However, a higher concentration of ETM was attained in the liver, spleen, and lungs after administration of ETM-ILE than after administration of ETM-SOL. This study found that ETM-ILE had great potential for clinical applications.The aim of this investigation was to develop an etomidate intravenous lipid emulsion (ETM-ILE) and evaluate its properties  The formulation used 35% (v/v) propylene glycol as a co-solvent to dissolve ETM. However, transient venous pain was observed immediately after administration of ETM-SOL in approximately 20% of the patients (reported incidence was 1.2–42%) because of its strong acidity (pH = 6.0) and high osmotic pressure (OP) . This did not contain propylene glycol so its OP was close to that of human plasma. Therefore, the incidence of adverse reactions caused by transient venous pain was considerably reduced. However, there have been a few reports on this product.Currently, most formulations of ETM are injectable solutions and emulsions. In September 1982, an etomidate injectable solution (ETM-SOL) was first prepared by Hospira (Amidate®). This did not contain a pharmaceutical drug, and was meant to serve as a source of calories and essential fatty acids for patients lacking adequate nourishment  and Solutol HS 15, have also been used in intravenous emulsions.Although intravenous lipid emulsions—mainly used for the delivery of hydrophobic drugs—have been around for nearly 60 years, there are far fewer formulations of this type on the market than conventional dosage forms such as tablets and capsules. The present study aimed to optimize the formulation of ETM intravenous lipid emulsion (ETM-ILE) and evaluate its pharmacokinetic profile and tissue distribution in rats, using ETM-SOL as a reference.®), and sodium oleate were purchased from A.V.T. Pharmaceutical Co. Ltd. . Soybean oil (SO), corn oil, and castor oil were purchased from Zhejiang Tianyushan Medicinal Oil Co. Ltd. . Medium-chain triglycerides (MCT) and poloxamer 188 were obtained from Xi’an Tianzheng Medicinal Materials Co. Ltd. . All chemicals and reagents were of analytical grade, except for methanol, which was of high performance liquid chromatography (HPLC) grade. Ultrapure water was produced in the laboratory.ETM (> 99.5%) was purchased from Wuhan Dongkang Source Technology Co. Ltd. . Soybean phospholipid, egg lecithin  and male Japanese white rabbits (2.5 ± 0.5 kg) were obtained from Beijing HFK Bioscience Co. Ltd. . The animal license numbers were 2019–0004 and 2019–0008, respectively. The animal experiments were approved by the Institutional Animal Ethics Committee of North China University of Science and Technology . All animals were treated humanely and suffering was alleviated.Excess ETM powder was added to 5 mL of corn oil, castor oil, SO, MCT, SO-MCT mixture, and phosphate buffer solutions (PBS) of pH 3–10. The mixtures were vibrated for 36 h at 25 °C using the constant temperature gas bath oscillator. The supernatant was collected and filtered through a 0.45 μm membrane. The filtrate was subjected to HPLC to determine the concentration of ETM.Four kinds of oil  and three types of emulsifiers  were used to investigate the influence of these factors on the properties of ETM-ILE, including droplet size, polydispersity index (PDI), zeta potential (ZP), and drug loading (DL). Furthermore, the process of homogenization had a significant influence on particle size and PDI, so the effect of homogenization at 700, 1000, and 1300 bar was investigated.Central composite design response surface methodology (CCD-RSM) was used to design the experiments for the optimization of the ETM-ILE formulation. Based on the results of the single-factor experiments, the independent variables were the proportion of oil (5–30%), egg lecithin (0.6–1.8%), and F68 (0.1–0.5%) used. The primary aim was to minimize the droplet size of ETM-ILE. In the range of 20–200 nm, the small droplet size can resist the physical destabilization caused by gravitational separation, flocculation and/or coalescence  operated at 13,000 rpm for 10 min. Finally, ETM-ILE was prepared by M-110EH high-pressure homogenization . The emulsion was sealed in 10 mL penicillin bottles flushed with nitrogen, and sterilized by high-pressure steam at 121 °C for 15 min.ETM-ILE was prepared by high-pressure homogenization. ETM and egg lecithin were dissolved in the oil phase, while in the aqueous phase, sodium oleate, glycerol and F68 were dissolved in water for injection. For both phases, solubilization was ultrasound-assisted, and carried out at 50 °C. Subsequently, the oil phase was slowly added to the aqueous phase, with stirring at 500 rpm for 10 min, using a MYP11-2A magnetic stirrer . A coarse emulsion was prepared using a FlukoPhoton correlation spectroscopy  was used for the measurement of these three parameters. ETM-ILE was diluted 1:5000 (v/v) with purified water for the evaluation of droplet size, PDI, and ZP. The ZP of the emulsion is indicative of its stability: high surface charge results in stronger inter-particulate repulsion and less aggregation of particles  at a flow rate of 1.0 mL/min. ETM was detected at a wavelength of 247 nm. Quantification was performed using propyl paraben as an internal standard. The column temperature was maintained at 40 °C, and the injection volume was 20 μL. ETM-ILE (100 μL) was diluted 100-fold with methanol to break the emulsion. Took 300 μL added into 300 μL of internal standard solution, mixed until uniform. Then the content of ETM was analyzed by HPLC. The DL was calculated based on the The concentration of ETM was analyzed by HPLC using a Shimadzu LC-20AT system. The mobile phase consisting of 40% water and 60% methanol was pumped through an Agilent Cc is the concentration analyzed by HPLC, the units of c and DL are both mg/mL.Here, ETM in the dispersed and continuous phases was isolated using a Sephadex G-50 gel column (10 × 100 mm). ETM-ILE (300 μL) or ETM-SOL (300 μL) was added to a Sephadex G-50 gel column and eluted with distilled water. Absorbance was measured at 247 nm for every 2 mL of eluent, and the elution volume of the dispersed phase was determined. The drugs in the dispersed phase were collected and analyzed by HPLC , calibrated with 100, 290 and 1500 mOsmol/kg standard solutions. ETM-ILE was kept at room temperature for 10 min, and transferred into the sample tube for OP measurement.The pH meter  was used to measure the pH of ETM-ILE at room temperature. Each sample was determined three times.The long-term stability test (4 ± 2 °C) and accelerated stability test (25 ± 2 °C) were investigated for a period of 12 months. The samples were determined the changes of parameters such as droplet size, PDI, ZP, DL, EE and pH respectively.Blood was collected from the ear marginal veins of rabbits and centrifuged at 2500 rpm for 10 min. The plasma was discarded and the cells were washed with normal saline until the supernatant was clear. A 2% (v/v) suspension of erythrocytes in normal saline was then prepared.The erythrocyte suspension, 0.9% normal saline, distilled water, and ETM-ILE were filled into tubes labeled 1–7, respectively, as shown in n = 3). ETM-ILE or ETM-SOL (1.0 mg/kg) was injected into the right ear margin vein and the same volume of 0.9% normal saline was injected into the left ear margin vein. The rabbits were sacrificed 24 h after administration, and the ear tissues were cut off and fixed with 10% formaldehyde solution. Morphological changes in the blood vessels were observed by hematoxylin-eosin (HE) staining.The rabbits were randomly divided into two groups (n = 6) and were intravenously administered either ETM-ILE or ETM-SOL (5.0 mg/kg) via the tail vein. Blood samples (0.5 mL) were collected from the jugular vein at 2, 5, 10, 15, 25, 35, 45, 60, 120, and 180 min. The samples were centrifuged at 10,000 rpm for 10 min to separate plasma. This was collected and stored at −80 °C.The rats were fasted for 12 h with free access to water prior to the experiments . ETM-SOL or ETM-ILE was administered via the tail vein at a dose of 5.0 mg/kg. The rats were humanely sacrificed at 2, 5, 10, 30, 60, and 120 min after drug administration. The brain, heart, liver, spleen, lungs, and kidneys were collected immediately. Each tissue was washed with ice-cold saline, cut into pieces, weighed, and homogenized thrice with 0.9% normal saline (w/v) , S(MCT:SO 1:1), and S(MCT:SO 9:1) were found to be 30.26 ± 0.32 mg/mL, 166.83 ± 12.86 mg/mL, 166.86 ± 35.51 mg/mL, 146.22 ± 15.67 mg/mL, and 173.21 ± 15.13 mg/mL, respectively, confirming previous reports that MCT could improve DL  , could be predicted using the quadratic polynomial model expressed in 1, X2, and X3 represent the oil, egg lecithin, and F68, respectively.Modeling and statistical analyses were performed using Design-Expert 6.0.11 . Variance analysis results showed that the quadratic model adequately expressed the relationships between the variables (< .0001) . There w1, X2, X3, X1X2, X1X3, X2X3, X22, and X32 were significant model terms (p < .0001). The ‘Lack of Fit F-value’ of 2.29 implied that it was not significant relative to the pure error. The correlation coefficient 2R was 0.9985, implying that only 0.15% of the variations were not justified by the fitted model. The adjust R2 value was 0.9972, which was close to R2, implying that only 0.28% of the total variation was not explained by the model. This confirms the suitability of the polynomial regression model.In this case, XThe 3D plots depicted in The optimum formulation predicted by the software was as follows: ETM , oil , egg lecithin , F68 , sodium oleate , and glycerol .The predicted and observed particle sizes were 173.2 nm and 168.0 nm, respectively, for the ETM-ILE prepared according to the optimized formulation. A bias of 3.10%, was calculated according to The particle size, PDI, ZP, DL, EE, OP and pH were found to be 168.0 ± 0.3 nm, 0.108 ± 0.028, −36.4 ± 0.2 mV, 2.00 ± 0.01 mg/mL, 97.65% ± 0.16%, 292 ± 2 mOsmol/kg, and 7.63 ± 0.07 respectively. The particle size and ZP of ETM-ILE, measured by the Zetasizer are shown in t-test was used to analyze the results. No significant differences were found between the measured value and initial value at different time and temperature (p > 0.05). The appearance of ETM-ILE was the uniform milky white liquid without obvious oil-water separation and flocculation. The change of each parameter was within the acceptable range. Therefore, ETM-ILE was stable at 4 ± 2 °C or 25 ± 2 °C for at least 12 months.The results of long-term stability test at 4 ± 2 °C and accelerated stability test at 25 ± 2 °C were shown in Red blood cells were deposited at the bottom of test tubes number 1 to 5, that results were consistent with that of negative control tube 6. While in tube 7, there was no red blood cells at the bottom , indicatAfter administration of ETM-SOL, ETM-ILE, and 0.9% normal saline, no obvious redness or swelling were observed at the site of injection in the groups. Inflammatory stimulation observed after HE staining is shown in p > .05). The results indicated pharmacokinetic bioequivalence between ETM-ILE and ETM-SOL. It has been reported that lipophilic drugs are released rapidly from lipid emulsions, since it is a diffusion-controlled process, and the barrier effect of the oil-water interface is negligible  values in various tissues are shown in The mean concentration-time profiles of ETM in different tissues after intravenous administration of ETM-ILE and ETM-SOL are shown in 0-∞ for ETM-ILE was 2.56, 1.58, and 1.73-fold better in the liver, spleen, and lungs, respectively, compared to ETM-SOL. Interestingly, the AUC0-∞ of ETM-SOL in the brain was higher than that of ETM-ILE at 2 minutes point after administration. It is reasonable to assume that ETM is a small molecule with high lipophilic characteristics, which makes it can penetrate cross the blood-brain barrier (BBB) quickly. However, the concentration of ETM-ILE in the brain depended on the penetration speed of oil droplets with large volume in the lipid emulsion. It is reported that the BBB can prevent many water-soluble molecules and even lipid-soluble molecules with large molecular weight from entering the brain freely  , oil , egg lecithin , F68 , sodium oleate  and glycerol . The emulsion exhibited good physical and chemical properties. It could be stored stably for 12 months at 4 ± 2 °C or 25 ± 2 °C. Hemolysis and vascular irritation tests confirmed that ETM-ILE was safe for injection. The pharmacokinetic and tissue distribution studies were performed in rats, and the results showed that ETM-ILE exhibited a similar pharmacokinetic profile to ETM-SOL, but allowed greater drug distribution to the liver, spleen, and lungs. Consequently, this study suggests that ETM-ILE has great potential for clinical applications.


Escherichia coli responded similarly to both CAMPs and ceragenins by inducing a Cpx envelope stress response. However, whereas E. coli exposed to CAMPs increased expression of genes involved in colanic acid biosynthesis, bacteria exposed to ceragenins specifically modulated functions related to phosphate transport, indicating distinct mechanisms of action between these two classes of molecules. Although traditional genetic approaches failed to identify genes that confer high-level resistance to ceragenins, using a Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) approach we identified E. coli essential genes that when knocked down modify sensitivity to these molecules. Comparison of the essential gene-antibiotic interactions for each of the CAMPs and ceragenins identified both overlapping and distinct dependencies for their antimicrobial activities. Overall, this study indicated that, while some bacterial responses to ceragenins overlap those induced by naturally occurring CAMPs, these synthetic molecules target the bacterial envelope using a distinctive mode of action.Ceragenins are a family of synthetic amphipathic molecules designed to mimic the properties of naturally occurring cationic antimicrobial peptides (CAMPs). Although ceragenins have potent antimicrobial activity, whether their mode of action is similar to that of CAMPs has remained elusive. Here, we reported the results of a comparative study of the bacterial responses to two well-studied CAMPs, LL37 and colistin, and two ceragenins with related structures, CSA13 and CSA131. Using transcriptomic and proteomic analyses, we found that  Our current arsenal of antibiotics will soon be ineffective against the simplest bacterial infections due to the continued spread of antibiotic resistance (AR) . AR has 3–Antimicrobial peptides are structurally diverse molecules expressed in a wide array of organisms that directly kill microbes, including bacteria , 7. Many7–Staphylococcus aureus and identified only modest and unstable resistance in Gram-negative organisms   or opaque (mc22500D) colonies  . M. aviucolonies . The flucolonies , was act of colistin, LL37, CSA13, CSA131, CSA44, CSA144, and ciprofloxacin against Copyright © 2022 Mitchell et al.2022Mitchell et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli (L. monocytogenes (M. smegmatis (L. monocytogenes culture treated with a 2-fold higher concentration (4 μg/mL) of CSA131 (data not shown). The bactericidal activities of CAMPs, ceragenins, and ciprofloxacin are in contrast with the activity of the bacteriostatic antibiotic erythromycin against L. monocytogenes  of the molecules  to K. LiE. coli , L. monoytogenes , and M. megmatis , althougytogenes , which din vitro and unstable . These results confirmed that resistance to ceragenins is infrequent  between biological replicates for each of the conditions tested  following exposure of bacteria to colistin, LL37, CSA13, and CSA131 were measured  in genes upregulated by CAMPs but not ceragenins , which likely contributes to differences in antibacterial activities and bacterial responses. Overall, these results showed that transcriptional responses of E. coli to the naturally occurring CAMPs colistin and LL37 are similar but differ from the response to ceragenins, identifying for the first time that these CAMP mimics have a distinct function. These results also suggested that E. coli responds differently to the structurally related ceragenin compounds, CSA13 and CSA131.The global transcriptional responses of ch other . Pathwayragenins . Pathwayragenins . While tE. coli genes that had significant increases in mRNA abundance upon treatment with each of the molecules. In particular, 86 genes were induced in all 4 conditions, 68 were upregulated specifically during CAMP treatment while 57 were induced by the ceragenins  were among the most significantly downregulated genes by these antimicrobials  (E. coli exposed to CAMPs and ceragenins.To further confirm a role for CpxR and PurR in the response of e detail . The heand yebE)  and corrnd yebE)  and 4A. nd yebE) , again cnd yebE) , and 4B.E. coli exposed to CAMPs, but not ceragenins  and the phosphate starvation-inducible psiEF genes were specifically observed in bacteria exposed to ceragenins . Approximately 1800 unique proteins were detected for each biological replicate  were observed following exposure of E. coli to colistin  and adjusted P values  are shown. Genes were selected based on significant upregulation  at the protein level for the following categories: CpxR regulon proteins upregulated by colistin and CSA13, colanic acid or LPS biosynthesis proteins upregulated by colistin, PhoB regulon proteins upregulated by CSA13 and heat response proteins upregulated by CSA13. Download Table S2, PDF file, 0.3 MB.Comparison of the transcriptional and protein responses of Copyright © 2022 Mitchell et al.2022Mitchell et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02726-21.8DATA SET S5E. coli exposed to colistin and CSA13. This file includes fold changes and adjusted P values  as well as lists of proteins significantly up- or down-regulated following exposure to colistin and CSA13. Download Data Set S5, XLSX file, 0.2 MB.Proteomic responses of Copyright © 2022 Mitchell et al.2022Mitchell et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02726-21.9DATA SET S6E. coli exposed to colistin or CSA13. The analysis was performed with the DAVID Bioinformatics Resources using terms from UniProt keywords, COG ontology, GO, and the KEGG pathway. This dataset also includes Venn diagram lists and annotation term enrichment analysis for proteins significantly up- or down-regulated in E. coli by both colistin and CSA13, only colistin or only CSA13 . Download Data Set S6, XLSX file, 0.1 MB.Annotation term enrichment analysis for proteins up- or down-regulated in Copyright © 2022 Mitchell et al.2022Mitchell et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the H  in abundance in response to CAMPs and ceragenins were calculated in comparison to the control condition for each knockdown strain, and significantly resistant or sensitized strains  were identified for each of the compounds  influence the susceptibility of E. coli to ceragenins and directly contribute to the bacterial response.Profiling the responses of proteins , 41. Thiproteins , 43 and teins  of ceragenins may lead to the development of broad-spectrum therapeutics against bacterial diseases.CSA13 and CSA131  as well E. coli MG1655   plates or in cation-adjusted Mueller-Hinton (CAMH) (BD) broth and on Brain-heart infusion agar (BHIA)  plates or in Brain-heart infusion (BHI) (BD) broth, respectively. M. avium and M. tuberculosis were cultured in Middlebrook 7H9 (BD) broth containing 0.5% glycerol, 10% Oleic Albumin Dextrose Catalase (OADC) (Sigma-Aldrich), and 0.05% Tween 80. M. marinum was cultured in Middlebrook 7H9 broth containing 0.5% glycerol, 10% OADC, and 0.2% Tween 80. M. smegmatis was grown on Middlebrook 7H10 (BD) plates or Middlebrook 7H9 broth containing 0.5% glycerol, 0.5% dextrose, and 0.2% Tween 80 unless otherwise stated. All bacterial strains were grown at 37°C, except M. marinum, which was grown at 30°C. Liquid cultures were incubated with shaking unless otherwise stated.s 10403S , M. maristrain M , M. smegs mc2155 , and M. s Erdman  were as E. coli and L. monocytogenes were determined by a broth microdilution technique following the recommendations of the Clinical and Laboratory Standards Institute (CLSI) . A similar protocol using extended incubation periods was used to determine MICs against M. avium (10 days), M. marinum (5 days), M. smegmatis (3 days), and M. tuberculosis (14 days). For the determination of MICs using mycobacterial species, plates were placed in a vented container containing damp wipes to minimize evaporation.MICs of antimicrobial compounds against e (CLSI) , except 5 to 106 colony forming units (CFU)/mL in liquid media in the absence or presence of antibiotics at the following concentrations: E. coli, 0.5 μg/mL colistin, 64 μg/mL LL37, 4 μg/mL CSA13 and 4 μg/mL CSA131; L. monocytogenes, 2 μg/mL CSA13, 2 μg/mL CSA131, 2 μg/mL ciprofloxacin and 0.25 μg/mL erythromycin; M. smegmatis, 0.5 μg/mL CSA13 and 0.5 μg/mL ciprofloxacin. Bacterial cultures were grown at 37°C with shaking and the number of CFU/mL was determined at several time points. Plates without Tween 80 were used for the CFU determination of M. smegmatis cultures.Time-kill experiments were performed to characterize the effect of compounds on bacterial growth and survival. Bacteria were inoculated at 10E. coli and L. monocytogenes, respectively. In a few cases, bacteria growing at the two highest subinhibitory concentrations of antimicrobial had to be combined to get inoculums of 105 to 106 CFU/mL.Serial passage of bacteria in the presence of subinhibitory concentrations was performed as previously described , 60. ExpE. coli MG1655 was inoculated into CAMH broth and incubated for 16 to 18 h at 37°C with shaking. Cultures were diluted in fresh media to an absorbance at 600 nm (A600) of 0.1, incubated at 37°C with shaking until an A600 of 0.8 to 1.0 (∼2 h) and antibiotics were added to each culture, which was further incubated for 1 h at 37°C with shaking. Antibiotics were adjusted to concentrations having a similar impact on E. coli growth for that particular, higher bacterial density, culture format . Cultures samples were then mixed 1:2 with RNA Protect Bacteria Reagent , vortexed immediately for 5 s, and incubated for 5 min at room temperature. The bacterial suspensions were centrifuged for 10 min at 5,000 × g, supernatants were discarded, and pellets were stored a few days at −80°C before proceeding to RNA extraction.A single colony of g for 2 min at room temperature. Supernatants were transferred to new 1.5 mL RNase-free microcentrifuge tubes before proceeding to the washing and elution steps described in the PureLink RNA minikit (Ambion Life Technologies). Samples were treated with DNase (New England BioLabs) for 15 min at 37°C in a volume of 50 μL and 5 μL of 25 mM EDTA was added. Samples were further incubated for 10 min at 75°C and quickly placed on ice before being cleaned and reeluted using the RNA Clean & Concentrator kit  and stored at −80°C. The quality and the quantity of each RNA sample were analyzed by the UC Berkeley QB3 facility using a bioanalyzer and the Qubit technology. RNA samples were sequenced and preliminarily analyzed by the University of California (UC) Davis Genome Center and the UC Davis Bioinformatics Core.Bacterial pellets were resuspended in 100 μL of 10 mM Tris, 1 mM EDTA, pH 8.0 buffer containing 10 mg/mL lysozyme (Sigma-Aldrich). Proteinase K  (2.5 μL of 20 mg/mL) was added and samples were incubated at room temperature for 10 min with frequent mixing. Samples were combined with 0.5 μL of 10% SDS and 350 μL of lysis buffer  containing β-mercaptoethanol, vortexed and lysates were transferred into 1.5 mL RNase-free microcentrifuge tubes. Samples were then passed 5 times through an 18 to 21-gauge needle and centrifuged at 12,000 × cpxQ, csgC, cyaR, efeU, rprA, rseD), PurR  and PhoB  were not included in this analysis. The information related to the expected activity (induction and/or repression) of each transcription factor on specific genes was also retrieved from the RegulonDB Database.The differential expression analyses were conducted using the limma-voom Bioconductor pipeline  EdgeR v, 63. OnlE. coli MG1655 was inoculated into CAMH broth and incubated for 16 to 18 h at 37°C with shaking. Cultures were diluted in fresh media to an A600 of 0.1, incubated at 37°C with shaking until an A600 of 0.8 to 1.0 (∼2 h) and antibiotics (4 μg/mL colistin or 8 μg/mL CSA13) were then added to each culture, which was further incubated for 3 h at 37°C with shaking. Protein was extracted, digested, and desalted, as previously described . Clarified lysates (1 mg each) were reduced with 4 mM tris(2-carboxyethyl)phosphine for 30 min at room temperature, alkylated with 10 mM iodoacetamide for 30 min at room temperature in the dark, and quenched with 10 mM 1,4-dithiothreitol for 30 min at room temperature in the dark. Samples were diluted with three volumes of 100 mM ammonium bicarbonate, pH 8.0, and incubated with 10 μg of sequencing grade modified trypsin  while rotating at room temperature for 18 h. Trifluoroacetic acid (TCA) was then added to a final concentration of 0.3% to each sample, followed by 1:100 of 6 M HCl and the removal of insoluble material by centrifugation at ∼2,000 × g for 10 min. SepPak C18 solid-phase extraction cartridges  were activated with 1 mL of 80% acetonitrile (ACN), 0.1% TFA, and equilibrated with 3 mL of 0.1% TFA. Peptides were desalted by applying samples to equilibrated columns, followed by a washing step with 3 mL of 0.1% TFA and elution with 1.1 mL of 40% ACN, 0.1% TFA. The subsequent global protein analysis was performed using 10 μg of each desalted peptide sample.A single colony of escribed , with feE. coli sequences and MS1 intensities were extracted with MaxQuant (version 1.6.0.16) (E. coli (strain K-12) protein database . MaxQuant settings were left at the default except for that trypsin (KR|P) was selected, allowing for up to two missed cleavages. Data were then further analyzed with the artMS Bioconductor package  . Data we package , using t package  and the  samples , and P vA pooled CRISPRi library of ∼500 strains that allow the inducible knockdown of genes predicted to be essential was used to study the genetic determinants of resistance to CAMPs and ceragenins, as previously described , with feLog P values (partition coefficient) were determined using Chemicalize from ChemAxon .GraphPad Prism software (v.7.00) was used to generate graphs and perform statistical tests. The number of independent experiments is indicated in each figure legend.GSE160082. DNA sequencing data obtained with the pooled CRISPRi library were deposited in the Short Read Archive under accession number PRJNA669343. The mass spectroscopy proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE (PXD022149.RNAseq data were deposited in the GEO repository under the GEO accession number he PRIDE  partner


Lactose increased the glass transition temperature to over 70 °C, producing lyoprotective effects similar to those obtained with sucrose. Based on these results, formulations containing liposomes lyophilized with lactose meet the FDA’s requirements and can be used as a biocompatible and biodegradable vehicle for the pulmonary delivery of therapeutic doses of sildenafil citrate.The lyoprotective effects of mannitol and lactose have been evaluated in the production of sildenafil citrate liposomes. Liposomes were prepared by mixing the components under ultrasonic agitation, followed by a transmembrane pH gradient for remote drug loading. Mannitol and lactose, as compared to sucrose and trehalose, were used as the stabilizing agents, and different freeze-drying cycles were assayed. The remaining moisture and the thermal characteristics of the lyophilized samples were analyzed. Size, entrapment efficiency, biocompatibility, and cell internalization of original and rehydrated liposomes were compared. The type of additive did not affect the biocompatibility or cell internalization, but did influence other liposome attributes, including the thermal characteristics and the remaining moisture of the lyophilized samples. A cut-off of 5% ( Among colloidal carriers, liposomes are safe for pulmonary administration in humans and animals ,2,3,4, aL-α-phosphatidylcholine (EPC), lanolin cholesterol (Ch), and formic acid were purchased from Merck KGaA, Darmstadt, Germany. Sildenafil citrate (SC) was obtained from Acofarma, Madrid, Spain. Mannitol, lactose, and sucrose were from Guinama, Valencia, Spain. Trehalose was purchased from AppliChem GmbH, Darmstadt, Germany. Coumarin-6 was obtained from Acros Organics, Geel, Belgium. Acetonitrile HPLC reagent was purchased from Fisher Scientific, Madrid, Spain. Ultra-pure water was obtained using a Milli-Q A10 system from Merck Millipore, Darmstadt, Germany. The Chromafil® PET-45/25 and PET-20/25 (0.45 and 0.20 µm) syringe filters were purchased from Nacherey-Nagel, Panreac Química S.L.U., Barcelona, Spain. Karl Fischer Aqualine Complete 5 and dry methanol were purchased from Fisher Scientific, Madrid, Spain.Egg Normal human dermal fibroblasts (NHDF) were purchased from PromoCell, Labclinics, S.A., Barcelona, Spain. Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 Ham (DMEM)-F12, paraformaldehyde (PFA), and phosphate-buffered saline solution (PBS) were purchased from Sigma-Aldrich, Sintra, Portugal. L(+)-ascorbic acid was purchased from Panreac Química S.L.U., Barcelona, Spain. Sodium hydroxide (NaOH) was purchased from José Manuel Gomes dos Santos, Lda. Odivelas, Portugal. Fetal bovine serum (FBS) and bovine serum albumin (BSA) were purchased from Biowest, Nuaillé, France. Glucose was purchased from Labchem, Santo Antão do Tojal, Portugal.TM, St. Louis, MO, USA. Ethylenediamine tetraacetic acid (EDTA) was purchased from Guinama Valencia, Spain. MTT (thiazolyl blue tetrazolium bromide), dimethyl sulfoxide (DMSO), and ethanol absolute were purchased from VWR, Barcelona, Spain. Hoestcht 33342 and wheat germ agglutinin (WGA) were purchased from ThermoFisher, Carlsbad, CA, USA.Penicillin/streptomycin amphotericin B (100×) was purchased from Lonza, Barcelona, Spain; trypsin was purchased from SAFC Biosciencesw/v. The mixtures were placed in a Fisher Scientific FB 15061 ultrasonic bath (50 Hz) at 50 ± 2 °C for 30 min. The resulting suspensions were filtered 10 times across syringe filters (Cromafil® PET) of a predetermined pore size (0.45 µm or 0.20 µm). The filtrated samples were kept at room temperature for 60 min and stored at 4 °C for 1 h. A transmembrane pH gradient was then applied for remote drug loading as follows: NaOH 0.1 N was added to the liposome samples until pH = 7.0 ± 0.1 and maintained for 20 h at 4 ± 0.2 °C in a shaking water bath (Unitronic OR Selecta P) to facilitate the drug diffusion and accumulation in the water core. Liposomes with coumarin 6 as the fluorescent probe (C liposomes) were also prepared. In this case, 400 µL of coumarin solution in ethanol (0.01% w/v) was added to the lipid mixture before ultrasonic agitation, and the rest of the procedure was the same as previously described. Liposomes with additive were prepared following the above procedure, but either mannitol (M), lactose (L), sucrose (S), or trehalose (T) at 4% w/w was incorporated in the buffer solution.Liposomes with and without additive were prepared by mixing the components under ultrasonic agitation, followed by the application of a transmembrane pH gradient for remote drug loading . For lipThe size (Dh), polydispersity index (PDI), and zeta potential of liposomes, with and without additive, were analyzed via the dynamic light scattering (DLS) technique using a Zetasizer Nano ZS . The analysis was performed at 25 °C and a scattering angle of 173° after appropriate dilution (×100 or ×1000) with Milli-Q water to avoid the phenomenon of multiple scattering.v/v), adjusted to pH 4.2 using triethanolamine, were used at a flow rate of 1.5 mL/min. The UV detector was set at 292 nm . Standard solutions (25–500 µg/mL) were prepared with citrate buffer at pH 3.2 adjusted to pH 7 using 0.1 N NaOH. Standard solutions containing M, L, S or T were also prepared to test the selectivity of the analytical method (results in Entrapment efficiency (EE) and drug loading (DL) were determined as follows: liposome suspensions were centrifuged at 14,000 rpm for 45 min at 6 °C to separate the non-entrapped SC. The drug in the supernatant was quantified by HLPC. A Purosphere STAR rpE18 50 cm × 4.0 mm 3 µm column and a mixture of 0.1% formic acid in water and acetonitrile (70/30 The amount of SC in the liposomes (Qin) was estimated as follows:The entrapment efficiency was estimated using the following equation:The DL was determined as the Qin/Qlipid ratio, where Qlipid is the amount of lipid in the liposomes.2/s) and the viscometer constant  was estimated. The viscosity of the liposome suspensions was then calculated as follows:The kinematic viscosity of the suspensions (η) with and without the corresponding additive was determined using a capillary viscometer by measuring the time it took the sample to flow through the capillary under the influence of gravity at 25 ± 2 °C. The instrument was calibrated with Milli-Q water  using a Zeiss EVO HD259 microscope. Diluted samples were fixed with poly-L-lysine and osmium, and acetone was used as the desiccant product.2 containing 10 mL of DMEM-F12 medium supplemented with heat-inactivated FBS (10% v/v) and 1% antibiotic/antimycotic solution, and then incubated at 37 °C in a 5% CO2 humidified atmosphere. When cells reached confluence, they were released by incubation in 0.18% trypsin (1:250) and 5 mM EDTA. Cell growth was monitored using an Optika inverted light microscope equipped with an Opikam B5  digital camera. The MTT assay was then used, following ISO 10993–5, to evaluate cell viability in the presence of liposomes. Cells were seeded in 96-well plates with DMEM-F12, using a cellular density of 2 × 104 cells/well and incubated at 37 °C in a 5% CO2 humidified atmosphere for 24 h. After 24 h, the culture medium was removed and substituted with fresh culture medium mixed with sterile liposome suspension diluted to 1:100, which corresponds to 0.018% w/v. This concentration is higher than that reached during lung exposure, as estimated from the amount of liposomes that provide a suitable SC pulmonary dose. The mixture was maintained at 37 °C in a 5% CO2 humidified atmosphere for 24 h (n = 5). After 24 h the culture medium was removed and replaced with 50 µL of MTT. The cells were then incubated for 3 h at a 37 °C in a 5% CO2 humidified atmosphere. Thereafter, MTT was removed and 100 µL of dimethyl sulfoxide (DMSO) was added and gently agitated for 15 min at room temperature to dissolve the formazan crystals. The absorbance was then registered at 570 nm using a microplate reader (Multiskan GO—Thermo Scientific). Subsequently, 96% ethanol was added to the cells used as positive controls (K+) (dead cells), whereas untreated cells (without liposomes) were used as negative controls (K−). The mean cell viability values have been expressed as the percentage of K−.Normal human dermal fibroblasts (NHDF) were used to evaluate the effects of liposomes with and without additives on cellular growth. Cells were first seeded in T-flasks of 75 cm4 cells/well) on glass-bottomed coverslips and incubated for 24 h at 37 °C in a 5% CO2 humidified atmosphere [2 humidified atmosphere. Cells without liposomes were used as control. After 12 h of incubation, the samples were taken for labeling preparation. The fibroblast nucleus was labelled with Hoestcht 33342 (5 µg/mL) and the cytoplasm with WGA (5 µg /mL) then fixed with PFA (4% v/v) in PBS for 10 min at room temperature. Imaging analyses were performed with a Zeiss LSM710 laser scanning confocal microscope .Confocal laser scanning microscopy (CLSM) was used to study the cell internalization of liposomes. Cells were seeded (4 × 10mosphere . After 2w/w) M, L, S, or T was added to the liposome suspension (additive/EPC mass ratio = 2.6) before freezing, and the mixtures were softly agitated for 3 min. Glass vials were filled with 1.0 mL of mixture and frozen at −80 ± 3 °C . Annealing to induce the crystallization of the lyoprotectant agents was assayed. Since experimental values of T’g were not determined, information from the literature was used, and annealing was performed at −33 ± 2 °C for 4 h . A laboratory freeze-drying instrument , with a condenser temperature of −80 ± 4 °C and chamber pressure of 0.008 ± 0.002 mBar (Pirani gauge), was used. The temperature during primary drying was −45 ± 5 °C. Batches of 30–35 vials were employed for each freeze-drying cycle. Protocols with and without annealing, and with and without secondary drying (10–20 °C), were carried out with the same chamber pressure as was used in the primary drying.A total of 4%  in each freeze-dried cake was determined using the Karl Fisher method, transferring 0.1 g of sample to the titration cell. The mass of the analyzed sample was determined by analytical balance  and the volumetric water content was measured using a Metrohm 870 KF Titrino plus KF titrator. The results are shown as the dry product RM The thermal analyses of fresh and lyophilized samples were carried out by DSC. The gel-to-liquid crystalline phase transitions (Tm) of dry EPC (control), fully hydrated EPC (fresh liposomes), and freeze-dried EPC (lyophilized cakes) were determined. In addition, the glass transition temperatures (Tg) of the products lyophilized with M, L, S, or T were analyzed. Experiments were performed using a Mettler Toledo DSC-1. The samples (1.0 mg) were transferred to 40 μL aluminum pans, sealed, and scanned from −20 to 150 °C at 10 °C/min. The analyses of the thermograms and the transition temperatures were performed using the STARe software package. Tm was recorded as the peak temperature of the endotherm in the lipid gel-to-liquid crystalline phase transition and Tg as the onset temperature of the endotherm for glass transition, both during the heating scan .After a visual inspection and revision of cake appearance, 1 mL of ultra-pure water was added to the dry cake during manual shaking. The time required for the complete disappearance of solid content was determined by visual inspection and recorded as the reconstitution time. The EE, DL, Dh, PDI, and zeta potential in the rehydrated samples were determined as described for the original liposomes.p-values ≤ 0.05.Comparison of results was performed using one-way analysis of variance (ANOVA) with Tukey´s post hoc test. Statistical significance was considered at p = 0.11 and p = 0.18, respectively). Filtration through a 0.45 µm filter produced larger liposomes showing higher PDI values (Dh = 270.71 ± 6.70 nm and PDI = 0.28 ± 0.06 for B liposomes and Dh = 293.34 ± 13.69 nm and PDI = 0.32 ± 0.08 for SC liposomes), with statistically significant differences between B and SC liposomes for Dh (p < 0.05). Negative zeta potential values in the range of −11.23 ± 1.39 mV to −9.20 ± 1.11 mV were obtained for all liposomes without additives, regardless of cargo and size.The application of a transmembrane pH gradient to liposomes without any additive led to an EE% > 60%. In addition, the size of liposomes affected the EE%, with mean values of 83.10 ± 9.18% and 62.10 ± 9.32% for the larger and smaller liposomes, respectively. Filtration through a 0.20 µm filter produced liposomes with mean Dh and PDI values of 162.73 ± 15.01 nm and 0.17 ± 0.03, respectively, for B liposomes, and 175.40 ± 1.32 nm and 0.13 ± 0.02, respectively, for SC liposomes. The differences in Dh and PDI between B and SC liposomes did not show statistical significance (p < 0.05). In terms of PDI, differences between SC, SC + M, SC + L, and SC + S were found (p < 0.05). The zeta potentials of small and large liposomes were not affected by the additives, and all liposomes showed values in the range of −11.23 ± 1.39 mV to −7.84 ± 1.12 mV. In addition, higher ƞ values were found for liposome samples containing an additive when compared to those without additives, and the differences were statistically significant (p < 0.05). Among the additives, the differences in ƞ were not statistically significant (p = 0.81).For liposomes containing an additive, the EE% did not reach the values mentioned above. However, lower EE% values were obtained for both large and small liposomes, and the small liposomes with T presented the lowest EE% values (44.92 ± 7.10%). The additives also affected the Dh and PDI of small and large liposomes. A statistically significant increase in Dh was observed when SC liposomes without additive (SC) were compared to those containing mannitol or lactose (−).The microscopic images  revealedIn the positive control (K+), with cell death induced by the addition of ethanol, neither adhesion nor proliferation was observed; however, a spherical shape typical of dead cells was observed. The results of the MTT assay  show thap = 0.07).The results of RM show that annealing did not improve water removal during primary drying. Since the crystallization of lyoprotectants was not essential for our samples due to their low solid content, annealing at −33 °C ,36,37 wap > 0.05). Scanning electron microscopy (SEM) images of the lyophilized cakes . With respect to the EE, statistical differences between the original and rehydrated liposomes were not observed for the large liposomes (p = 0.37). For the small liposomes, however, the EE increased from 62.10 ± 9.32  to 97.96 ± 0.33%, 85.10 ± 3.10%, 82.56 ± 1.71%, and 84.91 ± 2.76% for the rehydrated samples containing M, L, S, and T, respectively. The SEM images of fresh and rehydrated liposomes were compared , while the small liposomes with T showed the lowest DL (26.94 ± 4.26 mg SC/g of lipid). Diffusion through the lipid bilayer is the underlying mechanism for remote loading when a transmembrane pH gradient is applied. The lower EE values registered for samples containing L, S, or T can be attributed to the higher viscosity of these samples, but the increase in ƞ did not affect the samples containing M. Therefore, the specific interaction between the phospholipid head groups and the sugar molecules is likely the mechanism hindering drug diffusion through the lipid bilayer for SC liposomes prepared with L, S, and T. The size and polydispersity of the liposomes obtained via the procedure described were indeed affected by the additives; however, the negative zeta potential was not affected, and the values were in the range of −11.23 ± 1.39 mV to −7.84 ± 1.12 mV. Based on the above, the SC liposomes obtained in this study showed the following critical attributes: (A) biocompatibility and biodegradability, since EPC and Ch are both components of the pulmonary surfactant; (B) no risk of solvent residuals, since liposomes were prepared in the absence of organic solvents; (C) the size and zeta potential accorded with those values recommended for particles used for pulmonary administration; (D) the DL values were high enough to enable a level of pulmonary drug exposure higher than that achieved with the current administration routes used for pulmonary hypertension. Moreover, the results from the biocompatibility and cell internalization experiments show that liposomes with or without additive did not produce cytotoxic effects and were able to enter the cells. Accordingly, these liposomes meet the regulatory requirements for pulmonary formulations and seem to be suitable for the inhalation of therapeutic SC doses.As previously mentioned, liposomal instability remains a major translational limitation in the development of liposome-based formulations. Due to the regulatory restrictions on additives used for pulmonary delivery, it was necessary to analyze the lyoprotective effects of mannitol and lactose considering that lyophilization is proposed for its stabilization. SC was selected as the prototype drug as there is interest in improving the benefit/risk ratio of this drug for its use in the clinical treatment of pulmonary hypertension. The RM, Tm, and Tg of the lyophilized products were selected as critical attributes because they are directly related to physical and chemical stability . Our resThe comparison of the additives revealed that M is beneficial to drying, but not to stabilizing, the liposomes. In addition, the thermograms proved that L and S are able to reduce the Tm to the value associated with fully hydrated EPC, while M and T only produced a slight change. Tm reduction enables the liposomal phospholipid membrane to retain a single phase during both drying and rehydration, avoiding structural transitions and drug leakage . Tg has Biocompatible and biodegradable liposomes with high DL values were prepared using a solvent-free procedure. In vitro assays showed that the liposomes had no cytotoxic effects and were able to enter the cells regardless of size and drug loading. In addition, these properties were not affected by the additives used. With regard to lyophilization, mannitol was useful as a bulking and drying agent, but not as a lyoprotectant. Lactose succeeded in producing cakes with Tm and Tg values associated with stability at room temperature and the avoidance of structural transitions. An RM cut-off value of 5% was found to be an indicator of primary drying completion, which would facilitate the scale-up and transfer from laboratory to large-scale production. Long-term stability and in vivo studies are necessary to confirm the potential of liposomes lyophilized with lactose as being suitable for the pulmonary delivery of SC to clinically treat pulmonary hypertension. Other drugs used for pulmonary diseases may benefit from this promising liposomal formulation.


Scorpion stings comprise a serious problem throughout the globe, especially in regions where they are more frequent. Despite a recent upsurge of interest in scorpion venoms by various research groups, there remain many challenges.Hottentotta saulcyi, Hottentotta schach and Androctonus crassicauda, three scorpion species collected in Iran.Therefore, in this study, we aimed to study the toxicity and protein composition of venoms of A. crassicauda, H. schach and H. saulcyi by electrical stimulation of 8 and 10 V. The toxicity of each venom was established by using four groups of male Swiss albino mice aged 2 months (weighting 18–20 g) for testing each dose of venom. One group was used as a control. Venom was injected into mice by subcutaneous route. Then, animals were monitored for 24 h and LD50 was estimated by the graphic method of Miller and Tainter. Thus, high‐performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was used to determine amino acids in the venom, and protein concentrations were determined by the Biuret method.Scorpion species were collected from Esfahan farm scorpion company and maintained in the laboratory in containers that mimic their natural habitat. Venom was extracted from 50 of scorpion venoms by subcutaneous route was found to be 1.70 mg/kg b.w (A. crassicauda), 1.47 mg/kg b.w (H. saulcyi) and 0.85 mg/kg b.w (H. schach). A. crassicauda, H. saulcyi and H. schach contain 26, 30, and 31 amino acids, respectively. A. crassicauda contains low concentrations of alpha‐aminoadipic acid, beta‐aminoisobutyric acid, beta‐alanine and citrulline. H. saulcyi contains a concentration of hydroxylysine, whereas H. schach has no such concentration. A. crassicauda also had the highest levels of tyrosine and threonine. Only A. crassicauda venom contains a low proportion of proteins (14.80%) compared with those of H. schach (16.26%) and H. saulcyi (16.20%). Albumin content in the venoms was 11.7% (H. saulcyi), 5.4% (H. schach) and 4.4% (A. crassicauda).LDScorpions venoms have a variable toxicity and an interesting composition in amino acids and proteins. Work on the development of anti‐venom is fundamental. Hottentotta saulcyi, Hottentotta schach and Androctonus crassicauda). The results from this study show that H. saulcyi and H. schach contain more amino acids and protein and have higher toxicity than A. crassicauda.This study could be useful for understanding the venom extraction and toxicity, the protein and amino acid diversities of three species of scorpions ( Based on morphological characteristics such as body colour, pedipalp shape and size, sternum shape, telson colour and several pectin teeth, collected scorpions were identified at the species level.2.3In the laboratory, scorpions were placed in a large glass aquarium  on top layer of soil about 3 in. in depth, above which proper size stones were kept, imitating their natural habitat.As a source of drinking water, a petri dish lid containing tap water was kept in the centre of the glass aquarium. The scorpions were fed with crickets and worms of flour. At 3‐day intervals, the water content of the petri dish was checked and, if necessary, filled.Scorpions were only handled on the day of venom extraction and during soil replacement, which was done once every 2 months. The venom was not removed the day immediately following the feed and no food was added up to 3 days after the venom was removed.A. crassicauda, H. schach and H. saulcyi were stimulated twice, at 8 and 10 V, respectively, for 2–3 s with short intervals between two stimulations.The tail was held firmly wing forceps and electrically stimulated by pointing two electrodes connected to a step‐down transformer at junctions between the tail segments, one next to the telson. The other at the junction between IV and V metasomal segment. The venom was allowed to secrete over a piece of parafilm placed at the base of its tail and then transferred to a vial. The venom obtained in a single episode from many scorpions of a species were pooled, mixed with excess double distilled water and centrifuged at 15,000 rpm for 20 min to remove the mucus; the supernatant was lyophilised and stored at 20°C until used for protein estimation and toxicity studies; venom was resuspended in phosphate‐buffered saline (PBS) PH7.2.4A. crassicauda, H. schach and H. saulcyi was studied by estimating the lethal dose 50 (LD50) value under in vivo conditions. Four groups of 2‐month‐old Swiss male albino mice (weighing 18–20 g), each group containing four mice, were used to test each dose of venom for toxicity, and one group of five was used as a control. The mice were kept at room temperature where they had libitum access to rodent food and surface water throughout the experiment.The toxicity of venom from A. crassicauda , H. schach  and H. saulcyi  scorpion venoms were dissolved in 0.2 ml of PBS. The control group was injected with an equivalent volume of PBS only. The animals were monitored for 24 h and the LD50 was calculated by the graphical method of Miller and Tainter  method is used to determine amino acids in the venom of three medically important scorpion species.The HPLC–MS/MS system consists of an Agilent 1200 high‐performance liquid chromatography coupled with an Agilent 6430 series triple quadrupole mass spectrometer with an electrospray ionisation source. The chromatographic separation was achieved on a CORTECS C18 column  and the column temperature was kept at 30°C. Mobile phases which consisted of 0.1% formic acid in water (A) and acetonitrile (B) were used in the following gradient elution method: 0–10 min, 10–85% B; 10–13 min, 85–95% B; 13–19 min, 95–95% B. The flow rate was set at 0.3 ml/min, and the injection volume was 10 μl. All the data were analysed by Mass Hunter workstation software .The mass spectrometer was carried out in both positive and negative ionisation multiple reaction monitoring mode. The source parameters were as follows: the capillary voltage set at 300 V for positive ionisation mode and −300 V for negative ionisation mode, the drying gas temperature was 320°C, the flow was 11 L/min and nebulising gas pressure was 30 psi.2.6All reagents were of analytical grade; the solvents were of chromatographic purity and the water was purified by deionisation . Most of the standards were supplied by Fluka–Sigma–Aldrich . A few specific standards were supplied by Extrasynthèse , Radian International  and pharmaceutical laboratories participating in the Brazilian government DST/AIDS program. Ampholyte solutions in the pH range 6–8 and 3–10 were supplied by Amersham Pharmacia Biotech  and a 4% methylcellulose stock solution was supplied by Applied Biosystems .2.7Protein concentrations were determined by the Biuret method. In this technique, peptide bonds in proteins produce a violet colour in the presence of alkaline copper, and the intensity of the colour is estimated with a spectrometer at 540 nm wavelength.3DCE from Agilent Technologies. The P/ACE unit was equipped with a filter‐carrousel UV detector, while the HP3DCE unit was equipped with a diode array detector. Both systems had temperature control devices, maintained at 25–30°C and data acquisition and treatment software supplied by the manufacturer. Uncoated fused‐silica capillaries  were used in all experiments except for the haemoglobin analyses, where a dimethylpolysiloxane coated fused‐silica capillary  with dimensions of 27 cm total length, 20 cm effective length, 50 cm i.d. and 0.20 cm coating thickness was used. At the beginning of the day, regular capillaries were typically washed with a 1 mol L–1 NaOH solution , followed by deionised water (5 min) and electrolyte (30 min). In between runs, the capillaries were usually reconditioned by a flush with the electrolyte solution . At the beginning of the day, the coated capillary was conditioned by a flush of methanol followed by deionised water . In between runs, the coated capillary was just replenished with fresh ampholyte working solution (2 min flush). At the end of the day, the coated capillary was washed with deionised water, methanol and dried with a flush of nitrogen .All experiments were conducted in capillary electrophoresis systems, either model P/ACE 5510 from Beckman Instruments  or model HP2.8No statistical analysis was performed.33.1A. crassicauda, H. schach and H. saulcyi were collected from Esfahan Farm Scorpion Company and maintained in the laboratory in containers that mimic their natural habitat. They were captured carefully using long forceps by holding the tip of their tail without harming the animal. Scorpions were then transferred into separate containers with the soil of about two inches in depth upon which small stones were placed. The venom was extracted from A. crassicauda, H. schach and H. saulcyi by electrical stimulation (8 and 10 V).3.2Based on morphological characteristics such as body colour, pedipalp shape and size, sternum shape, telson colour and several pectin teeth, identified scorpions were presented in Table 3.3H. saulcyi venom in mice. Four doses were used for the experiment . At the end of the analyses, the LD50 obtained was 1.47 mg/kg b.w.Table H. schach venom on mice. Four doses were used for the experiment . At the end of the analyses, the LD50 obtained was 0.85 mg/kg b.w.Table A. crassicauda on mice. Four doses were used for the experiment . At the end of the analyses, the LD50 obtained was 1.70 mg/kg b.w.Table 50 recorded for the tested venoms were 1.47 mg/kg for H. saulcyi, 0.85 mg/kg for H. schach and 1.70 mg/kg for A. crassicauda. The venom of A. crassicauda thus presented the lowest toxicity and the highest toxicity was recorded for H. schach.The LD3.4A. crassicauda has few amino acids compared with the venom of H. saulcyi and H. schach. A. crassicauda venom does not contain allo‐isoleucine, cystathionine, homocitrulline, homocystine, hydroxylysine and hydroxyproline. Similarly, unlike H. schach, H. saulcyi does not contain traces of cystathionine and homocystin at all. H. saulcyi contains a concentration of hydroxylysine, whereas H. schach has no such concentration.None of the venoms studied contain argininosuccinic acid and sulfocysteine. The venom of A. crassicauda, H. saulcyi and H. schach contain 26, 30 and 31 amino acids, respectively, of which 7, 11 and 12 are in low concentrations , 5.4% (H. schach) and 4.4% (A. crassicauda). A. crassicauda had the lowest level of albumin. It should be noted that the albumin content in the different venoms was lower than that of the control group (61.1%) (Table A. crassicauda (0.05) followed by H. schach (0.06) and the highest was obtained with the venom of H. saulcyi (0.13). The most protein‐rich venoms were H. saulcyi (16.2%) and H. schach (16.26). The venom of A. crassicauda contains less proportion of proteins (14.8%) compared with those of H. schach and H. saulcyi.Albumin content in the venom was 11.7% . This voltage variability was noted by Nagaraj et al.  found that the LD,b found  Bonnet, . Also, I Bonnet,  reported Ismail, . InducedH. saulcyi venom was 0.73 mg/kg in mice , 5.4% (H. schach) and 4.4% (A. crassicauda). A. crassicauda had the lowest level of albumin. The lower albumin level recorded for A. crassicauda indicates its higher level of purity compared with other venoms. He showed a higher level of purity followed by those of H. schach and H. saulcyi, respectively. The low presence or absence of albumin in the venom expresses its higher level of purity . The results from this study show that H. saulcyi and H. schach contain more amino acids and protein and have higher toxicity than A. crassicauda. Furthermore, the venom of A. crassicauda has a high level of purity compared with the others. Note that toxicity, number of amino acids, protein content and degree of purity of scorpion venoms vary from one scorpion to another.The method adopted in this study for the maintenance of scorpions in the laboratory is efficient. The claustration device and electrical stimulation are significantly effective for the extraction of venom from scorpions of different species. The constitution of this study is important for researchers. This study could be useful for understanding the venom extraction and toxicity, the protein and amino acid diversities of three species of scorpions (Formal analysis, investigation, methodology and writing‐original draft: Ani Boghozian. Methodology, resources, supervision, validation, visualisation, writing‐review and editing: Habibollah Nazem. Conceptualisation, data curation, supervision, validation, visualisation, writing‐review and editing: Mohammad Fazilati. Formal analysis, investigation, methodology, project administration, writing‐review and editing: Seyed Hossein Hejazi. Methodology, validation, visualisation, writing‐review and editing: Mohammadreza Sheikh Sajjadieh.


H type hypertension is defined as homocysteine (Hcy) ≥ 10 μmol/L in combination with primary hypertension. Studies demonstrated that the existence of hyperhomocysteine (HHcy) in hypertensive exacerbates the poor outcome of cardiocerebral incidents. This study was to investigate the current epidemic situation of H type hypertension and determine the risk factors in order to find intervention targets for H type hypertensives.We conducted a cross-sectional study using cluster sampling design in Shanghai, China from July 2019 and April 2020. 23,652 patients with primary hypertension were enrolled in this study. Their medical information was recorded, and the level of Hcy concentrations and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms were detected.p < 0.01). The risk for H type hypertension was higher in elderly people. Men had ~ 5.55-fold odds of H type hypertension compared with women. Patients with CT genotype and TT genotype had ~ 1.36- and ~ 2.76-fold odds of H type hypertension compared with those with CC genotype, respectively. Smoking and diabetes were not significantly associated with H type hypertension.In total, 22,731 of 23,652 patients were recorded. The mean age was 68.9 ± 8.6 y and 43% were men. 80.0% of the enrolled patients had H type hypertension. The frequency of allele T was 40.9%, and the proportions of the CC, CT, and TT genotypes were 36.1%, 46.0%, and 17.9%, respectively. Compared with the TT genotype, the plasma Hcy concentration levels were lower in patients with the CC/CT genotype (18.96 ± 13.48 μmol/L vs. 13.62 ± 5.20/14.28 ± 5.36, F = 75.04, The prevalence of H type hypertension in patients with primary hypertension was 80.0%, which was higher than the 75% found in prior report in China. Age, gender, and MTHFR C677T polymorphisms rather than smoking and diabetes were independently associated with H type hypertension. Homocysteine (Hcy), a sulfur-containing amino acid, was deemed to participate in the process of arteriosclerosis as early as 1969 –8. An inHyperhomocysteinemia (HHcy) is common in the general population of Western countries as well as Eastern countries, particularly in patients with kidney dysfunction and vitamin defects –14. The Methylenetetrahydrofolate reductase (MTHFR) is a primary regulatory enzyme in the Hcy and folate metabolisms, catalyzing the reduction of 5,10-methyleneterahydrofolate to 5-methyleterahydrofolate , 21. TheThe trial was a multisite cross-section population-based research with cluster sampling method. The design method was used because it provides a snapshot of the exposed variables across a wide population without manipulating or influencing the study population in any way. Subjects were classified according to their residential quarters from six towns of Qingpu district, the suburb of Shanghai city. . In total, there were 173 clusters being chosen from the six towns, including 18 clusters from Yingpu town, 42 clusters form Xiayang town, 12 clusters from Xianghuaqiao town, 47 clusters from Jinze town, 28 clusters from Xujing town, and 26 clusters from Zhujiajiao town. Subjects in accord with inclusion criteria were recruited. In total, 23,652 participants were recruited between July 2019 and April 2020 from Qingpu district. After excluding invalid information and abnormal data, 22,731 participants were included in the study.The study protocol was approved by the ethics committee of Zhongshan Hospital Fudan University . All methods were performed in accordance with the relevant guideline and regulations. All invited participants provided written informed consent. The inclusion criteria were (1) age ≥ 18 years old; (2) confirmed with primary hypertension; and (3) no history of stroke. The exclusion criteria were (1) refusal to take part in the trial; and (2) refusal to provide a personal medical history.The baseline demographic data, a medical history, and a physical and laboratory examination were collected at community health centers by trained research staff during the study entry process. Using a face-to-face interview, we collected information, including the name, gender, date of birth, history of smoking, medical history of diabetes mellitus, and hypertension and medication use. A history of hypertension was defined as patients who had hypertension prior to admission to the community health center, according to the question “Have you ever received a diagnosis of hypertension from any doctor or hospital?”; the use of antihypertension medication; or a systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg. Diabetes mellitus was defined by a self-reported physician diagnosis, a self-reported current medical therapy, or a fasting blood-glucose ≥ 7 mmol/L. Smoking was defined by a self-reported history of smoking 1 cigarettes per day over at least 6 months. The blood samples were taken from all enrolled subjects for the test of the plasma Hcy level and only participants from the Xiayang community health center and Yingpu community health center were tested for the MTHFR C677T genotype. Patients whose plasma Hcy levels over 10 μmol/L combined with hypertension were defined as H type hypertension. After collection of data, it was edited, coded and examined for errors and omissions then corrected appropriately. All missing data were excluded after scrutiny before statistical analysis.g for 10–15 min. The plasma and red blood cells were separated and frozen at − 80 °C, and stored for at most 2 weeks. All specimens were transported at 1–2 °C conditions by 1–2 day air transportation to a national qualified laboratory at Shenzhen Tailede enterprise for the test of the plasma concentration of Hcy and the MTHFR C677T genotype .Venous blood was drawn and collected at each community health center and allowed to stand for < 1 h and, then, centrifuged at 3000 × t tests were used to compare the mean values of the Hcy concentration between the two genders. Anova analysis was used to compare the mean values of the Hcy concentration among the CC, CT and TT genotypes. Plasma Hcy concentrations were divided into four groups . The χ2 tests were used to compare the genotype proportion among different Hcy concentration groups. Univariate and multivariate logistic regression models were used to assess the association between H type hypertension and potential risk factors . The odds ratios (ORs) and 95% confidence intervals (95% CIs) were used to evaluate the risk of H type hypertension after adjusting for important confounding factors. All p values were 2-tailed, and a significance level of 0.05 was used.Using IBM SPSS 20.0 for the primary analysis, we calculated the mean and standard deviation (SD) of the patients’ age and Hcy concentration. Student In total, 22,731 of 23,652 participants were recorded after eliminating missing data. For these 22,731 participants, 87.2% of them were over 60 years old, the mean age was 68.9 ± 8.6 years old and 43% were men. Patients with diabetes accounted for 31.0% , and 32.4%  were smokers. Male smokers accounted for 72.5% (7086/9767) of the men participants, whereas only 2.2%  female subjects had a habit of smoking. 2965 participants from Xiayang community health center and Yingpu community health center were recruited for the test of MTHFR C677T genotype at first, however only 2296 subjects were finally counted after excluding invalid data.2 = 1696, p < 0.01). 11.6% participants had plasma Hcy concentrations of ≥ 20 μmol/L, and men accounted for a larger percentage than women . The proportions of patients with H type hypertension in the Yingpu, Jinze, Xiayang, Xianghuaqiao, Xujing, and Zhujiajiao community health centers were 64.1%, 82.3%, 67.8%, 78.9%, 92.6%, and 80.2%, respectively. The mean plasma Hcy concentration was 14.72 ± 8.37 μmol/L, and men had higher Hcy concentrations than women  The demographic characteristics of H type hypertension in our study are shown in Table The proportion of H type hypertension was 80.0%  in our study. We found that 31.5% patients had plasma Hcy concentrations of ≥ 15 μmol/L and the proportion of men was higher than that of women (χp < 0.01) . Compared with the ≥ 70 years of age group, the mean Hcy concentration level was 21.58 μmol/L in subjects with the TT genotype, which was much higher than that of the < 70 years of age group, whose mean Hcy concentration level was only 17.27 μmol/L. However, the difference was not as clear in subjects with the CC/CT genotype . With age, area, smoking, and diabetes included in the logistic regression model, men had ~ 5.55-fold odds of H type hypertension compared with women . For age, the analysis demonstrated that, after age 40, the risk of H type hypertension increased as people grew older, and the risk remained unchanged when including gender, area, smoking, and diabetes in the multivariate logistic regression model , which had lowest prevalence of H type hypertension. The regional differences persisted after controlling for gender, age, smoking, and diabetes. In addition, cigarette smokers had ~ 3.67-fold odds of H type hypertension compared with non-smokers . However, in a multivariate model with further control for age, gender, area, and diabetes, this association was attenuated toward null. Diabetes was not associated with H type hypertension in either the univariate or multivariate analysis  was 51.6% .Another reason may be the changing lifestyles and dietary habits that resulted from the rapid industrialization in China, particularly in Shanghai, which is an international first-tier city. A systematic review, comprising 36 studies, explicated that the prevalence of HHcy (> 15 μmol/L) increased from 22.7% in 1990–2005 to 29.6% in 2006–2012 in Chinese people . AnotherWe suggest that factors not captured by our study might contribute to the high prevalence of H type hypertension, eg, impaired renal function. In China, the prevalence of hypertension among predialysis chronic kidney disease (CKD) was 78.4% , and theMen had higher Hcy concentrations than women (17.13 ± 10.32 μmol/L vs. 12.90 ± 5.91 μmol/L), and, compared to women, men had ~ 4.91-fold odds of H type hypertension, which was in agreement with previous results , 21, 28.Previous study demonstrated that the smoking rate was higher in male compared with female (58% vs 5%) . In our The prevalence of H type hypertension varied in different community health centers, and the difference remained significant after adjusting for gender, age, smoking, and diabetes. This may result from the uneven daily dose of folic acid and vitamin B-12 prescribed to the patients in different community health centers. However, more information would be required to prove this.Although 91.1% of male diabetic subjects had H type hypertension in our study, diabetes was not associated with H type hypertension in either the univariate or multivariate analysis. There may be some reasons accounting for the contradiction. As demonstrated before, men had higher risk of suffering H type hypertension than women, which inducing the high proportion of H type hypertension in diabetic subjects. Other mechanisms, eg, insulin resistance and/or impaired renal function, may also be responsible for the high blood pressure values observed in diabetic subjects. Previous research revealed that diabetes-related variables, eg, insulin resistance, metabolic control and oral hypoglycemic drugs, may specifically modulate the concentration of Hcy –42. LikeOverall, of the 2296 patients tested for the MTHFR C677T gene type, the frequency of allele T was 40.9% and the proportions of the CC, CT, and TT gene types were 36.1%, 46.0%, and 17.9%, respectively. McAndrew et al. analyzed 101 unselected Caucasians and 102 unselected African Americans and reported that the frequency of allele T was 30% in Caucasians and 10% in African Americans . Pollak p < 0.01). In the group with high Hcy concentration (> 20 μmol/L), the proportion of the TT genotype was much higher compared with the CC genotype (23.70% vs. 5.70%), while, in the lower Hcy concentration group (10 μmol/L ≤ Hcy < 15 μmol/L), the result was the opposite. The ratio of the CC genotype was higher than the ratio of the TT genotype (76.30% vs. 55.60%).The plasma Hcy concentration level was significantly higher in subjects with the TT genotype compared with those with the CC/CT genotype (18.96 ± 13.48 μmol/L vs.13.62 ± 5.20/14.28 ± 5.36, F = 75.04, The Hcy concentration levels were much higher in elders with the TT genotype. The average Hcy concentration in participants < 70 years of age with the CC/CT genotype was only 12.99 μmol/L; however, in participants < 70 years of age with the TT genotype, the mean Hcy concentration was 17.27 μmol/L, which was lower compared with participants > 70 years of age with the TT genotype whose mean Hcy concentration was 21.58 μmol/L. Our results are consistent with previous research , 46, 47.Of all the participants tested for the C677T gene type, compared with patients with the CC genotype, patients with the CT gene type and TT gene type had ~ 1.36- and ~ 2.76-fold higher odds of H type hypertension, respectively. After controlling for the ln-age and gender, the association remained unchanged. Yuan et al. selected 224 cases of ischemic stroke and H type hypertension and randomly divided them into two groups with and without treatment of mecobalamin to analyze the effect of mecobalamin on the early-functional outcomes of patients with ischemic stroke and H type hypertension. The results showed that mecobalamin could reduce the level of plasma Hcy, resulting in a more significant functional recovery . AnotherOur study had certain limitations. Parts of our information depended on self-reports of medical history, including hypertension and diabetes. The age of our participants mostly ranged from 50 to 90 years old, leading to an insufficient sample size in participants whose age ranged from 20 to 50 years old. This inevitably caused bias in the explanation of patients from 20 to 50 years old. In addition, the study was cross-sectional, limiting the interpretation of cause and effect of any associations found. Besides, we haven’t taken the folic acid and vitamin B-12 status into consideration, which could also influence the Hcy concentrations.The prevalence of H type hypertension in patients with primary hypertension was 80.0%, which was higher than the 75% found in prior report in China. Age, gender, and MTHFR C677T polymorphisms rather than smoking and diabetes were independently associated with H type hypertension. Increased attention should be paid to the prevention and treatment of H type hypertension in China.