Ureaplasma spp. are associated with various infectious diseases in females, but there is still limited evidence regarding whether they are related to nonspecific cervicitis. The aim of this study was to develop and evaluate a digital droplet PCR (ddPCR) assay for the detection and quantification of Ureaplasma spp. in cervical swabs.Ureaplasma spp. to evaluate the analytical performance of the ddPCR assay. Then, we detected and quantified the bacterial load of Ureaplasma spp. in cervical swabs.A total of 267 non-specific cervicitis (NSC) patients and 195 asymptomatic females were included in this study. We produced standard curves for U. parvum were 37.8% (101/267) and 29.7% (58/195),  U. urealyticum were 9.0% (24/267) and 8.7% (17/195) in the NSC group and control group, respectively. In addition, the median copy number of U. parvum was 2.5 × 104 copies/ml (n = 101) in the NSC group and 9.2 × 103 copies/ml (n = 58) in the control group. The U. parvum load in the NSC group was significantly higher than that in the asymptomatic individuals (P < 0.001). whereas the median load of U. urealyticum was 8.4 × 103 copies/ml (n = 24) and 1.4 × 103 (n = 17) copies/ml in the two groups, respectively, , the difference was not statistically significant (P = 0.450).The prevalences of Ureaplasma spp. in clinical samples, and the method has excellent analytical performance and a wide range of clinical application prospects.Our study is the first to develop a droplet digital PCR (ddPCR) method for the detection and quantification of The online version contains supplementary material available at 10.1186/s12879-021-06355-6. Ureaplasma spp. has 14 known serotypes that were divided into two biovars: Ureaplasma parvum and Ureaplasma urealyticum. This pathogen is suspected to be the causative agents of non-gonococcal urethritis [Ureaplasma spp., such as idiopathic hyperammonemia [ethritis , cerviciethritis , and mulethritis , and preethritis . In addimmonemia , spontanmmonemia , multifommonemia , and ostmmonemia , especiaU. parvum and U. urealyticum are sometimes controversial. A meta-analysis included seven eligible case-control studies, including 1507 NGU patients and 1223 controls. The results suggested that U. urealyticum was significantly more prevalent inNGU patients, but U. parvum was found more oftenin controls [U. parvum induces adverse outcomes by causing a severe inflammatory response [U. parvum and U. urealyticum based on culture or PCR assays. These reasons make the interpretation of many previous studies extremely difficult. Moreover, some scholars have proposed that the host genetic background and bacteria load have an impacte on the disease outcomes with Ureaplasma spp. [However, the roles of controls . Neverthresponse , 12, 13.response , attribusma spp. .Ureaplasma spp. load should be considered for treatment [Of note, The European STI Guidelines Editorial Board issued a position statement that only people with a high reatment . The quareatment , 19; undreatment , 21. Sevreatment , 23. A rreatment , indicatUreaplasma spp. in non-specific cervicitis patients and an asymptomatic physical examination population (control group).Hence, the purpose of this study was to develop ddPCR for the detection and accurate quantification of Patients attending the gynaecology outpatient or health clinic in Minhang Hospital, Fudan University were recruited for this study from July 2019 to November 2020. All women 1) were aged ≥18 years without co-infection with HIV or autoimmune diseases, 2) had exhibited symptoms of cervical discharge  or cervical bleeding , 3) had Trichomonas vaginalis and then subjected to HPV, C. trachomatis, N. gonorrhoea, M. hominis, and M. genitalium, U. parvum, and U. urealyticum testing by qPCR in septuplicate. Finally, a total of 462 samples were ultimately enrolled in this study, including 267 non-specific cervicitis patients [A total of 537 cervical samples were obtained according to protocols performed by experienced clinicians. All specimens were examined microscopically to rule out fungi and patients  and 195 C. trachomatis, N. gonorrhoea, M. hominis, and M. genitalium were confirmed by commercial kits based on TaqMan probes  according to the manufacturer’s instructions. U. parvum serovar 1 (ATCC 27813) and U. urealyticum serovar 4 (ATCC 27816) were used as standard strains.Total DNA from 200 μl fresh cervical swab samples was extracted using MiniBest Universal Genomic DNA Extraction Kit (Takara) following the manufacturer’s instructions. The extracts were resuspended in 100 μl of nuclease-free water and  stored at − 20 °C or − 80 °C until use. The concentration of nucleic acids was determined with a NanoDrop 2000 Spectrophotometer . Human papillomavirus (HPV), U. parvum and U. urealyticum, respectively. In addition, the quality of PCR extraction was monitored by amplifying human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cervical swabs. CT values of > 37 suggest the failure of DNA extraction or qPCR amplification. Standard curves were generated by plotting the CT of the qPCR performed on a ten fold dilution series of purified DNA from U. parvumand U. urealyticum.QPCR amplification was performed in a total volume of 20 μl, including the 10 μl of Probe qPCR Mix (with UNG) (Takara), 500 nM target/reference primers, 250 nM target/reference probes, and 9 μl of each template DNA. The qPCR conditions were as follows: 25 °C for 10 min, followed by 45 cycles of 15 s at 95 °C and 34 s at 60 °C. Fluorescent accumulation data were analysed using ABI 7500 Software Version 2.3 (Applied Biosystems). Threshold cycle (CT) values of < 40 were defined as a positive result for the U. parvum and U. urealyticum, respectively . The Mastermix for ddPCR included 1× ddPCR Supermix for Probes (no dUTP), 900 nM target/reference primers, 250 nM target/reference probes, and 9 μl sample DNA. The generation of droplets was performed by the QX200 Droplet Generator according to the manufacturer’s protocols. PCR amplification was carried out using an Applied Biosystems Veriti 96-Well Thermal Cycler with the following PCR conditions: 95 °C for 10 min followed by 40 cycles of denaturation at 94 °C for 30 s, 60 °C for 1 min and the enzyme was deactivated at 98 °C for 10 min. The plate was stored at 4 °C until the droplets were analysed with QX200 Droplet Reader and QuantaSoft software version 1.7.4 (Bio-Rad). The threshold between positive and negative droplet populations was set manually using per-plate positive and no-template controls as a guide.U. parvum and U. urealyticum were evaluated with 12 kinds of common microorganisms of the genitourinary tract, including M. hominis, M. genitalium, C. trachomatis, Human papillomavirus (HPV), Herpes simplex virus (HSV), N. gonorrhoea, S. agalactiae, S. aureus, E. faecium, E. coli, P. aeruginosa, C. albicans. Second, the linear dynamic range of the ddPCR assay was assessed using the serial 10-fold dilutions of the standard DNA containing the target region as described previously [U. parvum and U. urealyticum were tested using the same primers/probe sets for both ddPCR and qPCR. Octuplicate results were used for intra-assay and inter-assay calculations.First, the specificity of the primers and probes for eviously . Each ditest. The medians were compared using Wilcoxon rank sum test. The Pearson correlation coefficient was performed to estimate the correlation betweenqPCR and ddPCR results of clinical samples. A P-value < 0.05 was considered a significant difference. The data were calculated in Stata 13.0  and plots of linear regression were generated with GraphPad Prism 8.0 Software, Inc. Continuous variables are expressed as “the mean  ±  standard deviation”. Intra- and interassay coefficients of variability (CV) were calculated for methodological evaluation. Continuous variables between the two groups were compared using Student's t-U. parvum and U. urealyticum with channel amplitude signals above 6000 and 2000, respectively  and a lower inter-assay CV (1.43–1.48%) for the U. urealyticum, respectively . The prevalence of U. parvum was 37.8% (101/267) in the NSC group and 29.7% (58/195) in the control group, respectively. The prevalence of U. urealyticum was  9.0%24/267) in the NSC group and 8.7%17/195) in the control group , and 195 asymptomatic females in the control group, with a mean age of 43.37 . There were no differences in age between the two groups   Fig. .Fig. 4CU. parvum was 2.5 × 104 copies/ml in the NSC group (n = 101) and 9.2 × 103 copies/ml in the control group (n = 58). However, the median load for U. urealyticum was 8.44 × 103 copies/ml in the NSC group (n = 24) and 1.38 × 103 copies/ml in the control group (n =17 ) .Based on ddPCR, the median bacterial load for Ureaplasma spp.. According to the standard curve of the ddPCR assay for DNA from ATCC reference strains of Ureaplasma spp., the assays showed excellent analytical performance. Our data suggested that the precision of ddPCR is higher than that of qPCR, especially for the low-copy specimens, similar to the report of Mahendran P[Ureaplasma spp. was higher between ddPCR and qPCR methods, . In contrast, the difference in U. urealyticum copy number between the two groups was not statistically significant (P = 0.450).The purpose of this study was to develop a ddPCR assay for the detection and quantification of hendran P. It can ds, Fig.  but very studies , 30. AccUreaplasma spp. can be isolated from blood, amniotic fluid, cerebrospinal fluid, sputum, bronchoalveolar lavage, pleural fluid, and semen [U. parvum and U. urealyticum using ddPCR technology. It is generally considered that Ureaplasma spp. load ≥104 CCU/ml is a sign of active infection and may require treatment with antibiotics. The establishment of this reference value is liquid culture based. However, traditional liquid culture is only a qualitative method with relatively poor sensitivity, with false positive and false negative, as well as the inability to distinguish U. parvum and U. urealyticum [nd semen . Here, walyticum . Real-tiUreaplasma spp. as a pathogen in multiple diseases with increasing clinical observation studies [that Ureaplasma spp. modulate cytokine [U. parvum (107 CFU/ml) can significantly increase prothrombin/thrombin mRNA expression to promote the onset of PPROM but that a lower load (105 CFU/ml) had no significant effect [U. parvum infection. Therefore, it is vital to detect the load of Ureaplasma spp. through more precisely quantitative technology. At this time, ddPCR, an emerging technology, is partitioned the PCR reaction into about 20,000 nm-sized droplets. Then, the relative concentration of targets, primers and probes are higher, while the inhibitor is relatively lower, which perfectly overcoming the shortcomings of conventional qPCR [It’s becoming gradually accepted that role of  studies , 33. In cytokine , 35 and cytokine . Interest effect . It indinal qPCR .U. urealyticum is relatively lower and the number of populations recruited for the study was relatively small. Finally, this was a single-centre cross-sectional study, and multicenter prospective studies with a large population should be carried out to provide more evidence.Our study had several limitations. First, due to the specialized instruments and consumables, ddPCR has a higher cost than qPCR, which limits its clinical application to a large extent. In addition, the prevalence of Ureaplasma spp. than qPCR in both asymptomatic and symptomatic individuals, and monitor dynamic changes in bacterial load after the use of antibiotics. Moreover, based on its high sensitivity and high tolerance to PCR inhibitors of ddPCR, it can be used for detection of samples with low bacterial content that are readily available . Further, it can be used with chorionic amniotic tissue or lung tissue which may contain potential PCR inhibitors, promoting analysis of multiple potential diseases, such as infertility, miscarriage, chorioamnionitis, bronchopulmonary dysplasia, bacteremia, and rare invasive infections of Ureaplasma spp.The ddPCR assay that we established has great application prospects. It can more accurately quantify the burden of Ureaplasma spp. with excellent analytical performance, which may be a promising technology.In conclusion, this study is the first to report the absolute quantitative analysis of Additional file 1. Multiple sequence alignment of the ParC genes of 14 serotypes of Ureaplasma spp. (a) Sequence of the U. parvum primes/probe; (b) Sequence of the U. ureaplasma primers/probe.


The maximum adsorption site and dissociation constant evaluated by the Langmuir model were 87.8 µg and 1.37 µmol/L, respectively. The fiber sheet withstood triplicate use. Thus, our NF exhibited high potential as a material for membrane chromatography.Nanofibers (NFs) are potential candidates as filter materials for affinity separation owing to their high liquid permeability based on their high porosity. Multiple and complex processes were conventionally performed to immobilize proteins for modifying NF surfaces. A simple method must be developed to immobilize proteins without impairing their biological activity. Herein, we succeeded in fabricating NFs with a core of cellulose acetate and a shell of hydrophilic polyvinyl alcohol immobilized with staphylococcal recombinant protein A by a one-step process based on core-shell electrospinning. A total of 12.9 mg/cm Staphylococcus aureus and consists of a single polypeptide chain with a molecular weight of 42 kDa. It has a binding domain with the Fc region of the IgG antibody. In addition, the bound antibody is readily desorbed under acidic conditions [Affinity chromatography is used to separate and purify target substances in a sample based on the affinity between the ligands and the target substances . BiologiVarious protein immobilization strategies reported, including covalent bond, physisorption, bioaffinity interaction, and their combinations, are summarized in excellent reviews ,6,7,8. EPolymer gels, beads, and porous membranes have been widely used as immobilization carriers. The flow velocity and amount used depends on the flow rate and pressure resistance . In addiIn this study, we designed a novel approach, where core-shell electrospinning was used to immobilize PA on the fiber surface. Via electrospinning NFs can be fabricated by extruding a polymer solution while applying a voltage to the grounded collector ,18. In cA core solution, 25% CA  dissolved in dimethylacetamide (DMAc) and acetone (2:1), was prepared. PVA  was added to dimethyl sulfoxide to completely dissolve while heating to 150 °C on a hot stirrer to obtain a 15% PVA solution. Then, 1,1’-Carbonyldiimidazole  was added at a final concentration of 0.05%, and the mixture was stirred uniformly. Purified staphylococcal recombinant PA  was added at a final concentration of 0.1% as a shell solution. Each polymer solution was immediately electrospun with a coaxial nozzle  under an applied voltage of 2.5 kV/cm, the core flow rate of 0.4 mL/h, the shell flow rate of 0.1 mL/h, and collector rotation speed of 900 rpm.To observe the fine surface structure, samples were Pt/Pd-coated  with an ion sputter  and observed with a scanning electron microscope  at an acceleration voltage of 15 kV. The samples spun on the carbon support membrane were observed using a transmission electron microscope  at an acceleration voltage of 100–200 kV to study the internal structure of the fibers. The adsorbed antibody was visualized by immersing the samples in a 1:100 diluted fluorescent-labeled antibody  and/or 20 µg/mL FITC-bovine albumin (Sigma-Aldrich) for 3 h at 25 °C and observing them with a fluorescence microscope.−1 with a resolution of 4 cm−1 and an average of 32 scans using a KBr detector.The surface chemistry was analyzed using a single-reflection attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectrometer on a Nicolet 6700 system  in 800–2000 cmA mouse IgG ELISA Quantitation Kit  was used according to the manufacturer’s instructions. In brief, the immobilized antibody  10 µg/mL diluted with a carbonate buffer  was added to a 96-well ELISA plate at 100 µL/well and incubated at 25 °C for 1 h. It was then washed five times with ELISA buffer  and blocked with 1% bovine serum albumin for 1 h. Next, 100 µL of the target antibody  was diluted with Blocking One  and added after washing five times with ELISA buffer for 1 h. A concentration of 0.0273 to 3.50 µg/mL was used as the standard solution for the calibration curve. After washing five times, 100 µL of labeled antibody  10 ng/mL diluted with ELISA buffer was added for 1 h. After washing five times, the samples were developed using the ELISA POD Substrate TMB kit  at 25 °C for 30 min. Subsequently, quenching with sulfuric acid (0.18 M), the absorbance was measured at 450 nm.2) and weighed. Mouse anti-goat IgG antibody was used as the target antibody. The samples were immersed in 100 µL of diluted target antibody, incubated at 25 °C for 1 h, and washed three times with 200 µL of ELISA buffer. This process is referred to as adsorption. Next, 600 µL of McIlvaine buffer (pH 3.0) was added to the sample, and the entire solution was recovered, in a process referred to as elution. The solution thus recovered (600 µL) was immediately neutralized by adding 300 µL of aqueous sodium hydroxide solution  for the ELISA assay.The fiber sample to be tested was cut into 5 × 5 mm pieces  was adsorbed onto the CA/PVA + PA NF and then observed under a fluorescence microscope, resulting in a uniformly stained fiber surface C. This dTo investigate the effect of the non-specific adsorption, the adsorption of IgG on the core-shell CA/PVA NF without the immobilization of PA was evaluated. For comparison, the same evaluation was also performed for the CA monolithic NF. As a result, non-specific IgG adsorption was barely observed in both of them D,E. As a−1 [ν(C=O), 1735 cm−1; ν(CO), 1220 cm−1; ν(CO), ν(CC), ν(CCO), 1035 cm−1) [The fabricated CA/PVA + PA NFs were studied using a single-reflection ATR-FTIR to investigate the immobilization of PA. PVA single-layered fiber (PVA NF), CA single-layered fiber (CA NF), PA-immobilized PVA single-layered fiber (PVA + PA NF), and CA/PVA core-shell fiber without PA (CA/PVA NF) were measured as controls . Single-−1 ,26 in PV−1 . In addi35 cm−1) ,28 were 2 and 37 ± 3 µm, respectively. Considering the sheet to be a thick and porous body, the amounts of antibody per area and volume were calculated to be 47.6 µg/cm2 and 12.9 mg/cm3.We obtained 0.22 mg of CA/PVA + PA NF sheet with an average thickness of 37 ± 3 µm by electrospinning for 90 min. The amount of loaded PA was estimated as 0.87 mg per g of NF sheet. We then cut the sheet and tested adsorption. First, the amount of the target antibody (mouse anti-goat IgG) adsorbed was quantified via sandwich ELISA. Then, 300 µg/mL of the target antibody solution was added to the fiber for adsorption, and the amount of unadsorbed target antibody was measured. After the first elution, the amount of the eluted antibody was measured. The elution was repeated and the quantity of eluted antibodies was measured. The amount of antibody added and unadsorbed, initial elution, and final elution amounts were obtained as 31.2 ± 2.1, 17.4 ± 0.5, 11.7 ± 0.2, and 0.222 ± 0.009 µg, respectively A. The toThe presence of free PAs as vacant sites explains the adsorption of only about 60% of the added antibody. Therefore, to increase the amount of adsorption onto vacant PA, repetitive adsorption was performed. Likewise, in the previous experiment, the first adsorption was performed with 300 µg/mL of the target antibody. The amount of unadsorbed target antibodies was measured. Subsequently, the second adsorption was performed by adding the same concentration of the antibody. The amount of unadsorbed antibodies was also measured. The targeting antibody added in the first adsorption and the amount of unadsorbed antibody were 31.3 ± 1.5 and 17.2 ± 0.9 µg, respectively. The added and unadsorbed amounts in the second adsorption were 31.3 ± 1.3 and 31.1 ± 0.3 µg, respectively B. This sC is the concentration of the antibody solution added, q is the amount of adsorbed antibody calculated from the concentration of the eluted antibody solution, maxq is the concentration of the PA adsorption site on the fiber, and K is the dissociation constant between PA and antibody. Fitting the experimental results to Equation (1), maxq, the adsorption site of PA, and K, the dissociation constant, in PA-antibody adsorption were obtained. The maxq of the PA adsorption site and K were 87.8 µg and 1.37 µmol/L, respectively. The dissociation constant obtained in this study was higher than the value reported previously (0.43 µmol/L) [To evaluate the maximum adsorption, adsorption-elution experiments were performed at different concentrations.  µmol/L) ,34, possThe adsorption-elution was repeated three times in succession to demonstrate the reusability as an affinity carrier. The antibody concentration at each adsorption was 300 µg/mL, as in the previous experiments, and the elution amount was measured at each elution. Thus, 30.7 µg of antibody was added each time, and the amounts eluted at the first, second, and third times were 12.7, 12.0, and 12.0 µg, respectively A. AssumiIn this study, it was shown that the core-shell fiber with a hydrogel layer on the surface captured the antibody with high efficiency. The dissociation constant of the antibody immobilized was higher than the previously reported ones. This would be because multiple IgG molecules bind to one PA , which l2 (2.1 mL) per device that adsorbs 0.08 mg/cm2 of antibody IgG [The amount of antibody loaded on an affinity carrier depends on the flow rate of the sample. The shapes of the affinity matrix, such as gel bead carriers and porous membrane carriers, affect the flow rate. Membrane carriers can also improve the flow-through performance with the high flow at a maximum rate of 10 mL/min. One commercially available membrane carrier  uses a membrane of 75 cmbody IgG . This me2. If a device of the same size as a commercially available membrane carrier is constructed, the amount of antibody that can be captured per device is 3 mg. Assuming the same flow as conventional membrane chromatography (10 mL/min), it would be able to capture 14 mg/min of antibody. Moreover, owing to the nanometer-sized fibers, it has higher flow-through performance than commercially available porous membranes. It was previously reported that microporous poly(caprolactam) hollow NFs have a flow rate of 25 mL/min [The amount of antibody captured by the CA/PVA + PA NF fabricated in this study was 0.04 mg/cm5 mL/min . Therefo3 of antibody was adsorbed on the protein-immobilized fiber. The maximum adsorption site evaluated by the Langmuir model was 87.8 µg. This high activity originated from the hydrophilic environment of the PVA hydrogel, where the ligands were immobilized. The fiber sheet withstood triplicate use. Thus, our NF showed high potential as a material for membrane chromatography.In this study, we successfully electrospun protein-immobilized core-shell NFs in which the core of CA was covered with a shell of hydrophilic PVA hydrogel immobilized using PA. Conventional immobilization of proteins on the NF surface requires some steps after NF preparation, including activation of the surface, the addition of a cross-linking agent, and immobilization of proteins. Our method enabled one-step electrospinning, which would speed up the production. The bioactivity evaluation revealed that 12.9 mg/cm


This study aimed to explore the level and changes in handgrip strength among preclinical Alzheimer’s disease (AD) and AD patients and to evaluate the association between handgrip strength and cognitive function.A total of 1431 participants from the memory clinic of Shanghai JiaoTong University Affiliated Sixth People’s Hospital and community were enrolled in the final analysis, including 596 AD, 288 mild cognitive impairment (MCI), and 547 normal individuals (NC). All participants received a comprehensive neuropsychological assessment. Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment-Basic (MoCA-BC), and the Chinese version of Addenbrooke’s Cognitive Examination III (ACE-III-CV) were used as cognitive tests. The receiver operating characteristic curve (ROC) was plotted to assess the power of handgrip strength as a screening measure to discriminate AD and MCI.P < 0.01). Multivariate logistic regression was performed with the handgrip strength quartiles, and the results showed that the ORs of AD for increasing levels of handgrip strength were 1.00, 0.58 (0.46–0.78), 0.51 (0.36–0.73), and 0.50 (0.35–0.68), showing a decreasing trend (Pfor trend < 0.01). The ROC curve demonstrated that the handgrip strength cutoff points for the identification of AD were 16.8 and 20.7 kg among the female participants above and under 70 yrs and 24.4 and 33.3 kg for the male participants above and under 70 yrs, respectively. Similarly, for the identification of MCI, cutoff points were 17.5 and 21.9 kg for females above 70 yrs and under 70 yrs, and 25.8 and 36.2 kg for males above 70 yrs and under 70 yrs, respectively.The results showed that handgrip strength in the MCI group was significantly lower than that of NC group, and the AD group had a further decline (both Our study provided the further knowledge on the relationship between noncognitive features and cognition in populations with differing cognitive status, revealed that the stronger handgrip strength was associated with better performances on cognitive function. It can be speculated that handgrip strength can help early recognition of Chinese AD patients. Alzheimer’s disease (AD) is the most common type of dementia, with typical clinical symptoms appearing probably decades after progressive cognitive function loss with the initial phase of subtle changes . IdentifPreviously, studies have shown that in addition to cognitive impairment, AD can also be accompanied by the performance of other systems of the organism, such as fatigue, slowed gait, and decreased muscle strength. These noncognitive features may become early predictors of cognitive impairment. Grip strength is one of the main indicators of body muscle strength and physical state, and it is also a relatively simple evaluation method . SeveralA total of 1431 participants were recruited from the memory clinic of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital and the community in Shanghai. This study was approved by the Institutional Ethics Reviewing Board of Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. Each subject had a uniform structured evaluation performed by a neurologist, which included a medical history inquiry and neurological examination. The study population was restricted to those without diseases that may have pathological effects on cognitive function. The inclusion criteria were normal vision and hearing to complete cognitive tests and no history of alcoholism, drug abuse, or head trauma. The exclusion criteria of this cohort included age ≤ 40 or above 90 years, low education level (≤ 5 years), presence of neurological or psychiatric antecedents, diseases involving the central nervous system, and severe diseases such as cancer, kidney disease, and chronic liver disease. Relevant laboratory tests were carried out to exclude metabolic disorders, nutritional deficiencies and infectious diseases that may adversely affect cognitive function, such as abnormalities in folic acid, vitamin B12, thyroid function, and rapid plasma regain or treponema pallidum particle agglutination. Cranial magnetic resonance (MR) imaging scanning was performed routinely to exclude any potential causes of cognitive decline, such as cerebral infarction, subdural hematomas, hydrocephalus, intracranial tumors and infections. All Subjects underwent comprehensive physical examination and positron emission tomography (PET) imaging.All participants received a comprehensive neuropsychological assessment, which was carried out by trained raters who were blind to the diagnosis. Six neuropsychological tests in three cognitive domains were examined: AVLT 30-minute delayed free recall auditory verbal learning test (AVLT-N5) and AVLT recognition (AVLT-N7) for memory domain ; animal AD was diagnosed based on the recommendations from the National Institute on Aging-Alzheimer’s Association (NIA-AA) workgroups , which i2 (kg/m2). Handgrip strength (kg) was estimated using a dynamometer . Participants were asked to squeeze the dynamometer for a practice trial using submaximal effort to determine their understandings of the procedure and the grip size adjustments. They were randomly assigned to start the test with their dominant or nondominant hand. To complete the test, participants were asked to use one hand to squeeze the dynamometer as hard as possible and repeat using the other hand for a total of three alternating hands. Similar to previous studies using this measure, we extracted the maximum value achieved using either hand as the summary measure [Height and body weight were measured, and body mass index (BMI) was calculated as weight/height measure , 30.t-tests and Wilcoxon signed rank sum tests. Intergroup comparisons of skewed data were conducted using the Kruskal-Wallis test. Spearman correlation analysis and logistic regression analysis were conducted to identify independent factors of cognitive status. Multivariable-adjusted linear regression analysis were conducted to identify the associations between handgrip strength and MMSE scores. The receiver operating characteristic curve (ROC) was plotted to assess the power of handgrip strength as a screening measure to discriminate AD and MCI. A two-tailed P value of less than 0.05 was considered to be statistically significant.SPSS, version 23.0  was used for statistical analysis. In this study, all continuous variables are presented as the mean ± standard deviation (SD), and categorical data are presented as numbers (percentages). Pair analyses were carried out using paired Student’s P < 0.01), education years (P < 0.01), and BMI than females (P = 0.034). There were no statistically significant differences in terms of MMSE, MoCA-BC, or ACE-III-CV scores between females and males , while both ADL and FAQ performed better in males (both P < 0.01). Females had significantly lower handgrip strength  than males.Overall, 1431 participants were enrolled in the final analysis, with 48.8 % men and 51.2 % women, and the mean age was 69.8 ± 9.9 years. Of the 1,431 participants, 596 (41.6 %) had AD, 288 (20.2 %) had MCI, and 547 (38.2 %) were NC. We observed no statistically significant differences between males and females for most characteristics (Table Pfor trend<0.01). The level of handgrip strength in the MCI group was significantly lower than that in the NC group, and the AD group had a further decline (both P < 0.01).The handgrip strength among cognitive functions was compared in both the male and female groups. As shown in Fig. all P < 0.01) and positively associated with BMI, MMSE, MoCA-BC, and ACE-III-CV scores (Table P < 0.01). Multivariate logistic regression was performed with the handgrip strength quartiles, and the independent variables were different stages of cognition. After adjusting for age and BMI, the results showed that handgrip strength tended to decrease with the decline of cognition in subjects with AD and MCI (Table Pfor trend < 0.01). Multivariable-adjusted ORs of AD and MCI for increasing levels of handgrip strength were 1.00, 0.58 (0.46–0.78), 0.51 (0.36–0.73), and 0.50 (0.36–0.68) and 1.00, 0.36 (0.26–0.49), 0.29 (0.21–0.40), and 0.16 (0.10–0.21), respectively, showing a decreasing trend (both Pfor trend < 0.01). The fitting curve of handgrip strength and MMSE scores is shown in Fig. 95 % CI: 0.214 to 0.357) and 0.450 (95 % CI: 0.422 to 0.528) higher MMSE scores in males and females, respectively (both P < 0.01).After dividing the population into sex subgroups of different sexes, spearman correlation analysis revealed that handgrip strength was negatively associated with age, FAQ, and ADL scores , 77.3 % (95 % CI: 68.1–83.8 %), and 0.732 (95 % CI: 0.686–0.775), respectively. The cutoff point of AD was 20.7 kg in females < 70 years old, with a sensitivity, specificity, and AUC of 66.8 % (CI: 62.2–73.8 %), 71.2 % (95 % CI: 64.3–78.5 %), and 0.700 (95 % CI: 0.617–0.734), respectively. For males above and under 70, the cutoff points were 24.4 and 33.3 kg, with a sensitivity, specificity, and AUC of 69.1 % (CI: 65.6–74.2 %), 73.0 % (95 % CI: 67.1–79.4 %), and 0.634 (95 % CI: 0.602–0.673), respectively, and 66.1 % (CI: 53.4–77.8 %), 72.5 % (95 % CI: 65.0–78.9 %), and 0.695 (95 % CI: 0.632–0.754), respectively. Similarly, for the identification of MCI, cutoff points were 17.5 and 21.9 kg for female participants above 70 yrs and under 70 yrs  and 0.450 (95 % CI: 0.422 to 0.528) higher MMSE scores in males and females, respectively. Finally, we proposed sex-specific and age-specific cutoff points for the initial identification of AD and MCI using grip strength, which provides evidence for the early detection of AD in the Chinese population.This study was the first to analyze and compare the levels of handgrip strength in Chinese preclinical AD and AD participants and to explore the association between handgrip strength and cognitive function. The results showed that the level of grip strength in the MCI group was significantly lower than that in the NC group, and the AD group had a further decline. We found that stronger handgrip strength was associated with better cognitive performance, and each increase in kg of handgrip strength was associated with 0.348 (95 % CI: 0.214 to 0.357) and 0.450 (95 % CI: 0.422 to 0.528) higher MMSE scores in the male and female groups, respectively, with a U-shaped association curve, which is similar to previous studies [Handgrip strength is a noninvasive measure of physical health that has been widely used in research and clinical settings. It can represent total body muscle strength and can also be an overall indicator of the integrity of the central nervous system . Weak grMechanistically, our findings are in accordance with the most notable hypotheses known as the ‘common cause hypothesis’, which demonstrates that cognition and muscle strength may share the same brain regions and networks . This foThere are some limitations in this study. First, this was a single-center study, and data from multiple centers are needed to further confirm the findings. Second, a longitudinal study needs to be conducted to investigate the association between handgrip strength and cognitive function in these participants at follow-up. Further studies need to be performed with a larger volume of samples to investigate the difference among different cognitive domain impairments.This study showed that the level of grip strength in the MCI group was significantly lower than that in the NC group, and the AD group had a further decline. We confirmed that stronger handgrip strength was associated with better cognitive function and proposed that grip strength can identify early AD patients in the Chinese population. The current study brings further knowledge on the relationship between non-cognitive features and cognition in populations with differing cognitive status. In the future, it is necessary to conduct developed research on the association between frailty and cognition.


The purpose of this study is to dynamically monitor the myocardial structure and function changes in diabetic mini-pigs by 1.5 T cardiac magnetic resonance.Three male mini-pigs underwent cardiac magnet resonance (CMR) imaging, and histologic examination. T1-mapping was acquired at basal, mid and apical segments. CMR feature-tracking (CMR-FT) is used to quantify left ventricle global longitudinal (LVGLS), circumferential (LVGCS) and radial strain (LVGRS). Epicardial adipose tissue (EAT) was evaluated using a commercially available software.2, p < 0.001; Native T1: 1005.5  vs. 1028.7  ms, p = 0.041; EAT: 16.1  vs. 24.6  mL, p = 0.020; ECV: 21.4  vs. 28.9  %, p = 0.011; LVGLS: − 22.8  vs. − 17.4 %, p = 0.008). The diffuse myocardial interstitial fibrosis was found in histology samples.Left ventricular mass (LVM), myocardial native T1 value, extracellular volume (ECV) value and EAT were increased gradually after 6 months of modeling, while LVGLS decreased gradually after 6 months of modeling  vs. 42.7  g/mThe progressive impairments in LV structure and myocardial deformation occurs in diabetic mini-pigs. T1 mapping and CMR-FT technology are promising to monitor abnormal changes of diabetic myocardium in the early stage of diabetic cardiomyopathy. In 2010, the prevalence of diabetes among adults (20–79 years old) was 6.4%, reaching 285 million people, but the prevalence rate will increase to 7.7%, and the number will reach 439 million in 2030 [Cardiac magnetic resonance (CMR) cine imaging is the gold standard for evaluating myocardial structure and function in many cardiac diseases. In recent years, as a new and sensitive technique of measuring myocardial deformation and function, CMR tissue tracking (CMR-FT) method based on the cine images has been widely used in myocardial strain analysis of different types of cardiomyopathy , 7, and So far, the researches on diabetic cardiomyopathy mainly focus on patients with type 2 diabetes . HoweverAll animal experiments were approved by Institutional Animal Care and Use Committee, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China and were executed conforming to the “Guide for the Care and Use of Laboratory Animals” and in compliance with the ARRIVE guidelines. Three male mini-pigs, aged 5–6 months, were provided by the Laboratory Animal Center of Tongji Medical College. Type 1.5 DM animal models were induced by intravenous injection of streptozotocin at a dose of 150 mg/kg . FastingThe mini-pigs were anesthetized through intravenous injection of pentobarbital sodium (0.1 mg/kg min). All three pigs have a pre-DM scan in our study, and all pigs have a post-DM scan at 1.5, 3, 4.5 and 6 months. Hematocrit level (HCT) was measured by collecting blood from ear vein before MR scanning was performed.2; and matrix, 256 × 205. T1 mapping technique was performed at the basal, mid and apical slices of the left ventricular short axis before and 15 min after the administration of gadolinium-diethylenetriamine pentaacetic acid (DTPA)  using a prototype-modifed Look-Locker inversion recovery (MOLLI) sequence. The following parameters were used: repetition time, 3.89 ms; echo time, 1.12 ms; flip angle, 35°; slice thickness, 8 mm; field of view, 360 × 270 mm2; matrix, 256 × 192; iPAT factor, 2; and acquisition scheme, 5b(3b)3b. The ECV mapping image is generated automatically by inputting the value of HCT through a prototype inline processing function from Siemens, based on native and post-contrast T1 mapping at the same slice and the subject-specific HCT value.All three pigs underwent a standard CMR examination with a 1.5 T scanner . A balanced steady-state free procession (b-SSFP) sequence was performed to acquire left ventricular long-axis and short-axis (coverage from the base to the apex segment) cines. The parameters included: repetition time, 2.93 ms; echo time, 1.16 ms; flip angle, 80°; slice thickness, 6 mm; field of view, 340 × 255 mmArgus software  was used to analyze cardiac structure and function. The basic parameters of cardiac function, including left ventricular end-diastolic volume (LVEDV); Left ventricular end-systolic volume (LVESV);Left ventricular ejection fraction (LVEF); Left ventricular mass (LVM). In order to avoid the influence of partial volume effect, the native T1 and ECV values were measured by manually sketching the region of interest (ROI) in the interventricular septum myocardium of the left ventricular basal, middle and apical segments Fig. . EAT meaMyocardial systolic strains were analyzed and processed using commercial cardiovascular postprocessing software  to obtain global measurements of LV strain. All the continuous short axis films and two-,three-, and four-chamber long-axis images were imported into Medis 3.0 software offline. At the end of diastole, the left ventricular endocardial and epicardial contours were manually delineated on the short axis and the long axis respectively. The trabecular and papillary muscles were included within the heart cavity . Continuous variable are presented as median and range (lowest and highest). One way analysis of variance (ANOVA) was used to compare baseline and the value of the relevant index at 6 months. For all comparisons, statistical significance was considered at a The LV strain of three mini-pigs were analyzed independently by two experienced observers. One observer made another analysis after 14 days. The repeatability of parameters within and between observers were calculated respectively. By calculating the inter class correlation coefficient (ICC), the repeatability of multiple measurements was analyzed.2, p < 0.001; LV remodeling index: 0.8  vs. 1.7 , p < 0.001; Native T1: 1005.5  vs. 1028.7  ms, p = 0.041; EAT: 16.1  vs. 24.6  mL, p = 0.020; ECV: 21.4  vs. 28.9  %, p = 0.011; LVGLS: − 22.8  vs. − 17.4 %, p = 0.008)  values of nativeT1, ECV, EAT, LVGRS, LVGCS and LVGLS measured by the same physician were 0.974, 0.957, 0.945, 0.985, 0.869 and 0.934 respectively. The ICC values of nativeT1, ECV, EAT, LVGRS, LVGCS and LVGLS were 0.969, 0.973, 0.948, 0.981, 0.893 and 0.926 respectively, measured by two different physicians.This animal model simulates the type 1.5 DM model of human disease, the advantages of this model not only avoid the effect of various confounding factors in patients on diabetic cardiomyopathy, such as age, the course of the disease, and coexisting diseases, but also obtain the imaging indicators of myocardial fibrosis by non-invasive imaging method and compare them with myocardial histological alterations to insight into the pathogenesis of DCM. The main findings of this study were that (1) the increases of myocardial T1 value, ECV and LVM and the decrease of left ventricular GLS were found at the 3rd months of modeling, and (2) EAT abnormalities were observed at the 6th months of modeling compared to the base line.T1 mapping can early monitor myocardial focal and diffuse fibrosis by quantifying myocardial T1 and extracellular volume values, and it has been confirmed in previous histological studies . Our resIn this study, it was found that the LV remodeling index (LV mass/volume ratio) was increased after the modeling of 4.5 months. We also found that LVM was increased after the modeling of 1.5 months. Similar results were reported by Sorensen et al. , 19 in aEAT comprises adipocytes, stromo-vascular cells, neurons, and immune cells , 24, whiIn this study, three limitations should be considered. First, our animal sample size was small, which greatly reduced the power of the study and did not allow us to draw generalized conclusions. We will continue to expand our sample size in our future studies on this topic. Second, our study was unable to carry out serial histological validation evaluation at various timepoints in type 1.5 DM animal models because pigs are large animals and limited by many experimental conditions. Third, impaired myocardial perfusion is one of the key characteristics of diabetic cardiomyopathy. However, due to technical reasons, we failed to do stress myocardial perfusion after many attempts. In the next experiment, we'll explore this myocardial perfusion imaging technique.In summary, our study suggests that increases in LVM, myocardial native T1, ECV, and EAT can be detected prior to a decline in left ventricular systolic function as diabetes progresses in diabetic mini-pigs. LVGLS is impaired gradually with the prolonged course of diabetes. Whether LV systolic dysfunction can be ascribed to the true effect of DM or to a confounding effect of DM and EAT still requires further study. T1 mapping and myocardial strain analysis are promising method in detecting subtle myocardial dysfunction in the early stages of DM.


Water retention capacity of HPMC varied linearly with Mv. The formation and curation of gels were monitored by rheology employing consecutive steps such as heating ramp (25–90°C), time sweep (90°C), cooling ramp (90–25°C), time sweep (25°C), and frequency sweep. Additionally, creep-recovery tests were performed. HPMC above 1.5% w/w delayed the range of gelatinization temperature of starch up to 2°C. Viscoelasticity and stiffness of corn starch gels with HPMC depend on both the amount of polymer added and Mv of the HPMC. Finally, to achieve corn gels with mimetic viscoelastic properties to wheat gel , HPMC with relatively low viscosity (low Mv) is necessary to be added at certain content.The objective of this study is to determine the effect of the addition of hydroxypropyl methylcellulose (HPMC)  with three different viscosities  to corn starch  gels. Average viscosimetric molecular weights (M Nowadays, gluten-free products are increasingly in demand due to more people are diagnosed as having celiac disease. Most bakery products are made with wheat flour that contains gluten, but there are flours (some of them underexploited) such as corn, rice, chestnut, and acorn, among others, useful to produce gluten-free starchy food materials . HoweverHydrocolloids are capable to control the swelling power, solubility, and rheology of starch aqueous systems, throughout the stabilization of emulsions, suspensions, and foams. In fact, they are widely used in the manufacturing of starchy foodstuffs improving the properties of starch gels .A common hydrocolloid used for cited purposes is the hydroxypropyl methylcellulose (HPMC) that belongs to the group of cellulose ethers and has a wide range of applications in food, cosmetics, adhesives, agriculture, and textiles . HPMC is5 g/mol) and, at constant guar gum content  the peak viscosity and setback increased with the lowest molecular weight guar gum and the addition of guar gum delayed the pasting temperature from 75.7°C (for CS) up to 79.6°C.In relation to the effect of the addition of hydrocolloids with different sizes to corn starch (CS), Funami et al.  studied In these previous studies, the effect of the addition of some hydrocolloids, such as xanthan gum, guar gum, and HPMC, among others, was investigated but without considering the effect of molecular size of HPMC. Likewise, considering the specific interaction depending on the starch source, it was hypothesized that the molecular size of HPMC added to CS, commonly used in gluten-free products, might affect starch interactions and in consequence viscoelasticity of CS gels. Therefore, the aim of this study is to determine the effect of the addition of three different HPMC with different molecular weights on CS gels by means of the evaluation of the viscoelastic behavior.2O at 20°C, . All the materials were provided by Sigma Aldrich.Corn starch  and wheat starch . HPMC, with constant methoxyl and hydroxypropyl content, 28.7 and 9.1%, respectively, of three different apparent viscosities, 40–60 (HPMC L), 80–120 (HPMC M), and 2,600–5,600 cP (HPMC H), at 2% in H0) and HPMC (μ) solutions, the relative viscosity (μμ = μ/0) and the specific viscosity (μsp = μr− 1) were calculated. Using the values of μr and μsp, the intrinsic viscosity (μ) was calculated by the Huggins (Eq. 1) and Kraemer (Eq. 2) Equations . Solutions were prepared with a concentration of 0.1%  in distilled water. For each HPMC, three dilutions were performed . All measurements (at least 5 replicates) were performed at 25°C (± 0.1°C). At these low hydrocolloid content, density was constant and equal to the density of solvent (water). With the absolute viscosities of solvent /C, and KH and KK are the Huggins and Kraemer constants, respectively. The values of viscosity average molecular weight, Mv, were determined by the Mark–Houwink Equation (Eq. 3):where μwhere (μ) (dl/g) is the intrinsic viscosity and K and α values depended on the solute–solvent system at a constant temperature.–1) was calculated by Eq. (4).Water retention capacity (WRC) values were determined to characterize the CS and HPMC-CS mixtures. WRC was calculated following the protocol established by Robertson et al. . BrieflyFRW is the fresh residue weight and DRW is the dry residue weight. WRC of HPMC was not directly determined due to water forms a viscous gel :where cous gel , but it WRCCS is the WRC of CS, wdHPMC and wdCS are the dry weights of HPMC and starch in the mixture, respectively.where Dispersions with constant solids content (30% w/w) were prepared by mixing CS and HPMC (by substitution) at different concentrations  and distilled water. First, the solids were gently blended and later distilled water was added. The slurry was mildly stirred (100 rpm) using a magnetic stirrer, for 10 min at room temperature. Aqueous HPMC solutions (2.0% w/w) were prepared by mixing HPMC and distilled water. These mixtures were kept in agitation until completely dissolved. The rheological characterization was performed with a stress-controlled rheometer  using a plate–plate geometry . Samples (1.2 ml) were loaded between the parallel plates and compressed up to obtain a gap of 0.5 mm. In the case of HPMC solutions, the same geometry was used with a smaller gap (0.3 mm) with a volume of sample of 0.9 ml. The measurements were performed at different temperatures [from 25 up to 90°C (± 0.1°C)], controlled by a Peltier system. All the samples were covered with light paraffin oil to prevent water evaporation. Tests were carried out at least in triplicate.–1, 1 Hz, 100 Pa), to accomplish starch gelatinization; (ii) time sweep ; (iii) temperature sweep ; (iv) time sweep ; and finally (v) frequency sweep  inside LVR of formed gels (preliminary tests were performed to determine the corresponding LVR). For HPMC solutions (2.0% w/w in water) a strain sweep from 0.1 to 100% at a constant frequency (1 Hz) was made to analyze the LVR. Then, a temperature sweep was carried out from 35 to 80°C at a constant strain of 10% and a constant frequency of 1 Hz (inside LVR). Strain sweep tests from 0.1 to 10% at a constant frequency (1 Hz) were made on dispersions and gels to define the corresponding linear viscoelastic regions (LVRs). Second, dispersions were subjected to the procedure previously reported  with min–1) = γ/σ, where γ is the strain experimentally measured. The Burgers model , of 100 or 400 Pa within the LVR during the creep phase. Before the measurements, the gels were rested for 15 min in the rheometer to allow the sample equilibrium. During the creep period, the selected constant stress was applied for 120 s and the recovery (stress zero) period lasted 120 s . The resrs model  was empl0J (Pa–1) is the instantaneous compliance, mJ (Pa–1) is the viscoelastic compliance, λ (s) is the mean retardation time, t (s) is the phase time, η0 (Pa⋅s) is the zero-shear viscosity, and Jmax (Pa–1) is the maximum creep compliance. The goodness of fitting was evaluated by means of the corresponding coefficients of determination [R2 and the root mean square error (RMSE)].where p-values ≤ 0.05 . All the experimental results were expressed as mean ± SD from at least triplicate experiments.Experimental data were analyzed through one-factor analysis of variance (ANOVA), followed by the Duncan test and considering significant H and KK constants, intrinsic viscosity (μ) values, and the average viscosimetric molecular weight (Mv) for each HPMC. Mv was calculated by the Mark–Houwink equation, Eq. (3), where the values of K and α parameters were 3.39 × 10–4 dl/g and 0.88, respectively, previously determined by Vázquez et al.  and obtained values from 3.78 to 14.98 dl/g and Mv values from 39.7 × 103 to 190.0 × 103 g/mol. Bustamante et al. z et al.  studied e et al.  studied H and KK obtained were from 0.21 to 0.63 and from –0.26 to –0.04, respectively. As the nominal viscosity of the HPMC increased, the KH, KK, (μ), and Mv significantly (p < 0.05) increased as well. HPMC solubility can be related to intrinsic viscosity (v) causes a decrease in the solubility of the biopolymer in water. The values of KH and KK could be analyzed to study the interactions between the polymers and solvents. In fact, KH < 0.5 and KK < 0 indicate good solvents (strong interactions polymer–solvent). An additional criterium given by KH – KK close to 0.5 based on the solvent goodness (solvent θ), defined a condition in which neither inter- or intramolecular polymer aggregation is produced and particulates behave as non-perturbed units and polymers adopt an extended conformation (flexible coil) in the solvent used (H = 0.63 > 0.5 and KH – KK close to 0.7) from theoretical considerations for good solvent–polymer interactions and this fact could be tested out in its low water solubility and slow solubilization.The values of Kiscosity . This beCS was 0.95 ± 0.01 (g water/g d.b.) whereas WRC values of HPMC L, M, and H, were 8.54 ± 0.28, 8.92 ± 0.22, and 12.97 ± 0.45 (g water/g d.b.), respectively. Yılmaz et al.  are more accessible for water and therefore, more water amount could be retained. Additionally, a linear correlation was found between intrinsic viscosity (μ), of HPMC and WRCHPMC (R2 > 0.99). As (μ) was proportional to Mv, WRCHPMC also varied linearly with Mv of HPMC (R2 > 0.99).In order to calculate the WRC of the different HPMC samples, mixtures of starch and HPMC were made. By means of the Eq. (4) the WRC of CS and mixtures were calculated. Then, using Eq. (5) the WRC of HPMC was evaluated. WRCz et al.  reported0) was evaluated from the first inflection point of elastic modulus (G′) and the final gelatinization temperature (Tf) was determined from the point in which the slope of G′ changes after the peak  to 62.8 ± 1.1°C (for 2.0%), while with HPMC M was 61.8 ± 1.1°C (for 0.5 and 1%) and 62.8 ± 1.1°C (for 1.5 and 2.0%). However, T0 value for HPMC H hardly changed, 62.8 ± 0.8°C, independently of its content. In addition, the Tf was delayed in a similar way, from 74.2 ± 0.8°C for CS gels to 75.3 and 76.4 ± 0.8°C with the presence of HPMC. This delay of the gelatinization process could be related to the water absorption of the added hydrocolloid (2O at 20°C) by substitution. Moreover, Zhang et al. (Dispersions were subjected to a temperature sweep (step i) where the onset gelatinization temperature   . For theerature) .As it can be observed in Analyzing in detail the results shown in 90′ = G′90f – G′90i, were evaluated where G′90f is the G′ after 30 min and G′90i is the initial value. For CS gel, without the addition of HPMC, ΔG90′ was −970 Pa, starch gels with HMPC L and M showed lower changes, but always G′90f < G′90i. These variations decreased with the amount of HPMC added. However, with the addition of HPMC H, two different trends were observed depending on the amount of polymer used. Negative values of ΔG90′ were also determined at low content (0.5% w/w), but increasing HPMC H content (>1.0% w/w) positive ΔG90′ values were determined achieving 1,560 Pa at 2.0% w/w. These results showed that Mv of HPMC clearly modifies the formation rate of gels and their final characteristics. In our tests, despite tested polymers had the same substitution degree, when they were added at the same content, the gel features were significantly different. The addition of HPMC, independently of Mv, dampened the decay of G′ observed in CS gels due to the presence of hydroxyl groups that are capable to form additional hydrogen bonds and therefore stronger structures   to evaluructures . The pos25′ = G′25f – G′25i. 25i and ΔG25′ values for tested gels. Globally, G′25i values decreased with increasing polymer addition of HPMC L and M. This result indicated that short-time starch retrogradation given by the rapid recrystallization of amylose molecules was slowed down by these HPMC additions due to the increase of G′ during cooling (step iii) was significantly lower. However, this decrease of G′25i only was observed at the lowest content of HPMC H (0.5% w/w) because beyond that level G′25i showed a steady increase with the level of HPMC addition (2.0% w/w) maintaining the trend observed at 90°C. In fact, the addition of hydrocolloids can increase, decrease, or have no effect on the extent of (short- and long-time) starch retrogradation, depending on the gel preparation method, temperature, time, added amount of hydrocolloid, and measurement techniques of starch retrogradation  decreased (up to 865 Pa with HPMC L at 2.0% w/w). No significant differences among ΔG25′ values of gels with different amounts of HPMC H added were observed, and these changes were close to that determined in CS gel (p > 0.05).In the next stage, the temperature sweep from 90 to 25°C (step iii), radation . The afoThe previous time sweep (step iv) showed that the gels have reached a stable state and are ready to analyze their structures. For this reason, frequency sweeps (step v)  were carried out to characterize the viscoelastic properties of gels . For allFor comparative purposes, the frequency sweep for a WS gel  formed under the same conditions that tested CS gels included in v) a positive linear trend was found (R2 > 0.98) with HPMC content. The firmness of gels with HPMC L and M decreased linearly with the amount of polymer added and no significant differences (p > 0.05) between both polymers were found. In samples with HPMC H, it was observed that the previous trend is maintained only at the low addition (0.5% w/w) level. At higher content (>1.0% w/w), the opposite effect is observed, and consequently, the change of G′ diminished  due to the increase of G′ with additional amounts of polymer added. This fact is given by the high viscosity of HPMC in the continuous phase . Parameters obtained using the Burgers model (Eqs 6 and 7) are shown in 0 values varied from 43.0 10–6 Pa–1 for CS gel up to 56.5, 57.7, and 40.2 10–6 Pa–1 for gels at maximum polymer addition (2.0% w/w) of HPMC L, M and H, respectively. For WS gel, J0 was 49.6 × 10–6 Pa–1. The Jm values obtained varied in a restricted range between 3.3 and 5.3 × 10–6 Pa–1, excepting for CS gels with HPMC L and M (at 2.0% w/w) which were noticeably higher, 13.92 and 44.70 × 10–6 Pa–1, respectively. In general, λ and μ0 varied between 4.1 and 5.7 s and 286 and 597 Pa⋅s, respectively. The Jmax values varied from 46.2 to 56.1 × 10–6 Pa–1, excepting for CS gels with HPMC L and M (at 2.0% w/w), 73.6 and 108.4 × 10–6 Pa–1, respectively. These results were also in agreement with the obtained results by oscillatory tests where G′ and tan δ values varied sharply showing lower G′ values and higher tan δ than remaining samples. J0 varied inversely with respect to G′ (from frequency sweep at 0.1 Hz and 25°C) and a successful linear correlation between both the parameters could be established (R2 > 0.96). Consequently, the trends found for G′ with HPMC amount in the oscillatory tests are also valid here. Similarly, Onyango et al.  values increased when the HPMC content increased in gluten-free doughs based on chestnut flour.The Burgers model satisfactorily fitted the experimental creep and recovery data  of HPMC. The temperature of gel point was invariant with Mv (when substitution degree is maintained constant), but HPMC with high Mv showed an aggregation step at temperatures near (below) gel point. The HPMC addition to CS gels causes significant changes in viscoelasticity and stiffness of samples. These effects depend on the amount of polymer added and the Mv of the HPMC.Water retention capacity of tested HPMC varied linearly with the average viscosimetric molecular weight  of HPMC of 27.2⋅103 g/mol or 1.5% (w/w) of HPMC of 32.7⋅103 g/mol must be added. Nevertheless, the use of HPMC with high Mv is not adequate for this purpose.Based on the viscoelastic response and creep-recovery tests, maintaining constant solid content (30% w/w), CS gels plus HPMC with mimetic features to WS gels can be only obtained under certain circumstances. HPMC with relatively low MThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.LM: methodology, data curation, and writing—original draft preparation. CR: supervision, validation, and writing—reviewing, and editing. RM: conceptualization, data curation, supervision, validation, and writing—reviewing and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Streptococcus pneumoniae have become one of the greatest challenges to global public health today and inappropriate use of antibiotics and high level of antibiotic use is probably the main factor driving the emergence of resistance worldwide. The aim of this study is, therefore, to assess the antimicrobial resistance profiles and multidrug resistance patterns of S. pneumoniae isolates from patients suspected of pneumococcal infections in Ethiopia.Antimicrobial-resistant strains of S. pneumoniae that were collected from pediatric and adult patients. Samples  from all collection sites were initially cultured on 5% sheep blood agar plates and incubated overnight at 37 °C in a 5% CO2 atmosphere. Streptococcus pneumoniae was identified and confirmed by typical colony morphology, alpha-hemolysis, Gram staining, optochin susceptibility, and bile solubility test. Drug resistance testing was performed using the E-test method according to recommendations of the Clinical and Laboratory Standards Institute.A hospital-based prospective study was conducted from January 2018 to December 2019 at Addis Ababa city and Amhara National Region State Referral Hospitals. Antimicrobial resistance tests were performed from isolates of Of the 57 isolates, 17.5% were fully resistant to penicillin. The corresponding value for both cefotaxime and ceftriaxone was 1.8%. Resistance rates to erythromycin, clindamycin, tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole were 59.6%, 17.5%, 38.6%, 17.5 and 24.6%, respectively. Multidrug resistance (MDR) was seen in 33.3% isolates. The most common pattern was co-resistance to penicillin, erythromycin, clindamycin, and tetracycline.S. pneumoniae isolates were susceptible to ceftriaxone and cefotaxime. Penicillin has been used as a drug of choice for treating S. pneumoniae infection. However, antimicrobial resistance including multidrug resistance was observed to several commonly used antibiotics including penicillin. Hence, it is important to periodically monitor the antimicrobial resistance patterns to select empirical treatments for better management of pneumococcal infection.Most  Streptococcus pneumoniae is one of the leading causes of bacterial infections, ranging from self-limiting respiratory tract infections to severe invasive infections. It is a major public health concern, being responsible for an estimated 3.7 million episodes (2.7 million to 4.3 million) in children globally and approximately 50% of all pneumococcal deaths in 2015 occurred in four countries in Africa and Asia [Antimicrobial resistance has been detected in all parts of the world; it is one of the greatest challenges to global public health today. The problem is increasing resistance to commonly used antimicrobial drugs which have elevated multidrug resistance (MDR). The fight against pneumococcal infections is based on curative treatment with antibiotics and preventive treatment using vaccination . HoweverS. pneumoniae has been described [In Asia, an alarming prevalence of penicillin and erythromycin-resistant escribed . In 1995escribed . In Soutescribed .S. pneumoniae have been described in central Africa [S. pneumoniae antimicrobial-resistance and the emergence of MDR isolates, which may result from inappropriate use of antibiotics and high level of antibiotic use is probably the main factor driving the emergence of resistance worldwide [Data on antimicrobial resistance from the African region are limited, but high-level penicillin-resistant l Africa . The incorldwide , 10.S. pneumoniae and to produce a structured data on drug resistance profile of this isolates in a regular base.In Ethiopia, there is lack of literatures on streptococcal drug resistance. A few studies have reported nasopharyngeal carriage rates , 12 and S. pneumoniae isolated from patients suspected of pneumococcal infections in Ethiopia using an E-test, where minimum inhibitory concentrations (MICs) determination is not routinely available.The present study aimed to investigate the antimicrobial resistance profile and multidrug resistance patterns of S. pneumoniae from clinical suspected patients. Suspected S. pneumoniae case means any reported case lacking confirmation of isolation from samples but has been diagnosed with S. pneumoniae by a clinician or other medical practitioner using sign and symptoms of the disease. The clinical sources of isolates were cerebrospinal fluid (CSF), blood, eye discharge, sputum, pleural fluid, ear discharge and peritoneal fluid. A hospital-based prospective study was conducted between January 2018 to December 2019 at Addis Ababa city  and Amhara National Regional State (ANRS)  Hospitals, Ethiopia. During the study period, 57 isolates were further confirmed as 2 atmosphere. Streptococcus pneumoniae was identified and confirmed by typical colony morphology, alpha-hemolysis, Gram staining, optochin susceptibility, and bile solubility test. After confirmation, the isolates were stocked in Skimmed Milk–Trypticase Soy–Glucose–Glycerol (STGG) medium and preserved at − 80 °C.As part of routine service, samples  were initially cultured on 5% sheep blood agar plates. All growing isolates were stored at − 20 °C and shipped to the University of Gondar Microbiology Laboratory for identification through ice box. All isolates received from the collection site were frozen at − 80 °C until further testing. Isolates were recovered by plating onto 5% sheep blood and incubated overnight at 37 °C in a 5% COS. pneumoniae were applied to categorize isolates as susceptible, intermediate, or resistant [The strains were shipped in dry ice to the Norwegian Reference laboratory for pneumococci at the Norwegian Institute of Public Health where antimicrobial susceptibility testing was performed. Minimum inhibitory concentrations (MICs) were determined by E-test on Mueller–Hinton agar supplemented with 5% sheep blood by the recommendation of the manufacturer (Biomerieux®). The antimicrobials tested were penicillin G, cefotaxime, ceftriaxone, erythromycin, clindamycin, tetracycline, chloramphenicol, trimethoprim/sulfamethoxazole, norfloxacin, and oxacillin. The Clinical and Laboratory Standards Institute (CLSI) 2020 clinical breakpoints for esistant . The CLSThe CLSI breakpoints for penicillin, cefotaxime, and ceftriaxone, differ between meningeal and non-meningeal infections. For penicillin, the breakpoints for meningitis is susceptible ≤ 0.06 µg/ml and resistant ≥ 0.12 µg/ml, and for non-meningitis ≤ 2 µg/ml susceptible, 4 µg/ml intermediate and ≥ 8 µg/ml resistance. For Cefotaxime and ceftriaxone, the non-meningitis breakpoint was used to classify isolates as susceptible (MIC ≤ 1 µg/ml), intermediate (MIC = 2 µg/ml) or resistant (MIC ≥ 4 µg/ml) and for meningitis susceptible (MIC ≤ 0.5 µg/ml), intermediate (MIC = 1 µg/ml) or resistant (MIC ≥ 2 µg/ml).S. pneumoniae ATCC 49619 was used as the quality control strain and was included in each set of tests to ensure accurate results.Interpretive criteria for norfloxacin are not given by the CLSI 2020. Thus, we used the breakpoints for norfloxacin obtained from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) . NorfloxData were entered and analyzed using the Statistical Package for the Social Science . Discrete variables were expressed as percentages and proportions.S. pneumoniae, 33 (57.9%) were males. The age of the patients ranged from 38 days to 71 years; 37 (67.9%) were children less than 18 years old and 20 (35.1%) were adults. Among 57 S. pneumoniae strains recovered from different clinical specimens, the most common clinical sources were CSF 36.4% (n = 20), blood 20% (n = 12), eye discharge 18.2% (n = 10), sputum 15.8% (n = 10), pleural fluid 3.5% (n = 2), ear discharge 3.5% (n = 2) and peritoneal fluid 1.7% (n = 1). Thus, 35 (61.4%) of the isolates were from cases of invasive pneumococcal disease (IPD), recovered from a sterile body site  and 22 (38.6%) were non-IPD strains, recovered from sputum, eye discharge and ear discharge (Table Of the 57 patients positive for Majority (75.4%) of isolates were resistant to at least one of the antibiotics tested. Intermediately resistant and fully resistant rates to penicillin were 26.3 and 17.5%, respectively. The corresponding values for both cefotaxime and ceftriaxone were 3.5 and 1.8%, respectively. Resistance rates to erythromycin, clindamycin, tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole were 59.6%, 17.5%, 38.6%, 17.5 and 24.6%, respectively  were resistant to only one class of antimicrobial, 14 (24.6%) were resistant to two classes of antimicrobials, while 19 33.3%) were resistant to three or more classes of antimicrobials; eight (14%) isolates were susceptible to all antimicrobials tested. Penicillin, erythromycin, clindamycin and tetracycline-resistant pneumococcus were usually also resistant to other antimicrobials. The most common MDR patterns were ERY + CD + TET (17.5%), PEN + ERY + TET (10.5%), followed by PEN + ERY + CD (7%), PEN + CD + TET (7%) and PEN + CTX + CRO + TET (7%)  was higher than other beta-lactam antibiotics such as cefotaxime 1.8%) and ceftriaxone (1.8%). Cefotaxime and ceftriaxone have a similar group of the spectrum. The resistance rate to penicillin in the present study was higher than studies in Central Africa (< 6%) % and cef and the nicillin .S. pneumoniae to erythromycin, clindamycin, tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole was 59.6%, 17.5%, 38.6%, 17.5%, and 24.6%, respectively. The rate of resistance to erythromycin was lower than studies done in Canada (100%) [The overall resistance rate of a (100%)  and China (100%) , but higa (100%) , Mozambia (100%)  and Pakia (100%) . Seventea (100%)  and China (100%) . In addia (100%)  and Nigea (100%) , China (a (100%) , Russia a (100%) , Nigeriaa (100%) . In addiThese differences in antimicrobial resistance may be explained by differences in the source of the isolates, geographical variability, high prevalence of HIV infection, and antibiotics usage. Higher rates of antibiotic resistance have been linked to high antimicrobial consuming countries . In EthiS. pneumoniae isolates has been a worldwide public health concern for several years. In this study, we demonstrated that penicillin-resistant S. pneumoniae strains were also resistant to erythromycin, tetracycline, and clindamycin . Erythromycin resistant S. pneumoniae strains were resistant to tetracycline, clindamycin, and chloramphenicol .The emergence of MDR The most common MDR phenotype was resistance to penicillin, erythromycin, tetracycline, and clindamycin, detected in 33.3% of the total isolates. Such MDR rates were higher than those observed in China (21.4%) , Russia S. pneumoniae infection. However, antimicrobial resistance including multidrug resistance was observed to several commonly used antibiotics including penicillin. Hence, it is important to periodically monitor the antibiotic resistance patterns to choose empirical treatments for better management of pneumococcal infection. We recommend developing an action plan for the promotion of rational use of antimicrobials, strengthening of antimicrobial resistance surveillance, and implementation of an antimicrobial stewardship program.In the present study, most bacterial isolates were susceptible to ceftriaxone and cefotaxime. Penicillin has been used as a drug of choice for treating


Gloeothece sp.–using Generally Recognized as Safe (GRAS) solvents like ethanol, acetone, hexane:isopropanol (3:2) (HI) and ethyl lactate. The bioactive potential of extracts was assessed in terms of antioxidant , anti-inflammatory (HRBC membrane stabilization and Cox-2 screening assay), and antitumor capacity ; while its composition was characterized in terms of carotenoids and fatty acids, by HPLC-DAD and GC-FID methods, respectively. Results revealed a chemopreventive potential of the HI extract owing to its ability to: (I) scavenge -NO• radical ; (II) inhibit 50% of COX-2 expression at 130.2 ± 7.4 µg·mL−1; (III) protect 61.6 ± 9.2% of lysosomes from heat damage, and (IV) induce AGS cell death by 4.2-fold and avoid its proliferation up to 40% in a concentration of 23.2 ± 1.9 µg·mL−1. Hence, Gloeothece sp. extracts, namely HI, were revealed to have the potential to be used for nutraceutical purposes.Bioactive lipidic compounds of microalgae, such as polyunsaturated fatty acids (PUFA) and carotenoids, can avoid or treat oxidation-associated conditions and diseases like inflammation or cancer. This study aimed to assess the bioactive potential of lipidic extracts obtained from  Spirulina  as food [The first reports on cyanobacteria date back to the time of Aztecs who used  as food . Nowaday as food ,2,3. The as food ,4.Nevertheless, the exploitation of prokaryotic and eukaryotic microalgae is restricted to a few strains and most species remain largely unexplored. So far, till 2019, 260 families of bioactive compounds were identified in cyanobacteria with a wide range of applications, e.g., agriculture, pharmacology, cosmetology, or in the food industry; belonging to 10 different classes: alkaloids, depsipeptides, lipopeptides, macrolides/lactones, peptides, terpenes, polysaccharides, lipids, polyketides, and others . AdditioExtensive research efforts during the last decades revealed that continued oxidative stress may activate mechanisms that lead to chronic inflammation—which, in turn, could mediate chronic diseases like cancer. Oxidative stress occurs due to an imbalance between the production of free radicals, such as reactive oxygen species (ROS), and their elimination by natural protective mechanisms, such as antioxidants molecules. This imbalance may lead to injury of vital biomolecules, cells, and eventually the whole organism . TherefoHelicobacter pylori [Cancer is nowadays the 6th leading single cause of death worldwide . This dir pylori .•, are produced during the inflammatory process [It should be emphasized that it was found long ago that oxidative stress, chronic inflammation, and cancer development are closely related, particularly in what concerns their activation pathways—which entail the production of several inflammatory signaling molecules, like prostaglandins (PGs) as well as oxygen- and nitrogen-derived free radicals, as schematized in  process  in respo process .2•−. This radical triggers, in turn, NF-κB phosphorylation, by activating several enzymes—namely cyclooxygenase 2 (COX-2), and iNOS which induce the release of PGE2, free radicals like O2•− and NO, and the chemokine MCP-1. Other activation products of NF-κB include anti-apoptotic factors, cell cycle regulators, and adhesion molecules—which may be related to cancer cells’ survival, proliferation, adhesion, invasion and metastasis, and angiogenesis [The triggering of the inflammatory pathway by lipopolysaccharides (LPS) causes rapid activation of NOX2 and NADPH oxidase, and release of internal Oogenesis . Of noteogenesis , see FigAnother common strategy followed in the formulation of anti-inflammatory agents is based on suppressing of production of inflammatory mediators, such as COX-2 inhibitors, that interfere with the initiation and progression of inflammation-associated diseases . PGs werIn practice, the synthetic drugs used to treat these disorders may bring about severe side effects; hence is important to find compounds from biological sources, such as cyanobacteria, lacking adverse effects . CarotenIn the particular case of cancer, some strategies of chemoprevention can be accomplished by incorporating antioxidant compounds in the diet, which would block or delay cancer development, either in the initial phase of carcinogenesis or at the stage of progression of neoplastic cells to cancer . A clearGloeothece sp., with promising bioactive lipidic composition [Gloeothece sp. [For this study, a scarcely studied prokaryotic colonial microalga was selected, position . This sthece sp. ,43.Gloeothece sp. extracts may have the potential of application in the nutraceutical industry, due to their content in bioactive compounds as carotenoids, polyunsaturated fatty acids (PUFA), or phenolic compounds. First, a crude characterization of extracts composition in terms of each family of compounds  was done, as depicted in C/mE), followed by HI extract . The contents of carotenoids were ca. 4% in all extracts, except for E, which reaches 6.5%.It can be observed that A and E extracts are mainly composed of fatty acids, ca. 60 and 66%, respectively, most of them PUFA (more than 40%). Extract A also exhibited the highest percent composition in phenolic compounds  and content .A detailed fatty acids composition, available in Fatty Acid·mgExtract−1—i.e., approximately half of concentration in E, and one quarter in HI and EL.Concerning the MUFA C18:1 n9 c+t , this is the one present in higher content and the 3rd in terms of all fatty acids. Its content in all extracts ranges between 14.4 (E) and 17.4% (EL), having a higher concentration in extract A, 53.796 ± 2.918 μgFA/mTFA), respectively. In another way, HI and EL accounted for 80 and 71.3 , respectively, in saturated fatty acids (data not shown).In terms of PUFA, E and A exhibited a higher content, 40.7 and 46.0% (mFA/mTFA); but with a higher concentration in A (96.765 ± 5.713 μgFA·mgE−1); followed by E (37.233 ± 0.685 μgFA·mgE−1), HI (13.216 ± 0.225 μgFA·mgE−1), and EL (4.575 ± 1.437 μgFA·mgE−1). In other way, the PUFA C18:2 n6 t  attained the highest concentration, 6.240 ± 1.510 μgFA·mgE−1, in the EL extracts. Note that conjugated linoleic acid , also in high content  and concentration (μgFA·mgE−1) in E, , followed by EL , HI , and A . Furthermore, C20:5 n3,  was detected in EL and HI extracts, in concentration of 0.462 ± 0.071 and 0.283 ± 0.105 μgFA·mgE−1, respectively.Among PUFA, C18:3 n3  attained the highest content in all extracts, between 23.4 (E) and 26.4 (EL) % (mcarot·mgE−1) than in E and HI, and 69% more than in EL, 3.19 ± 0.22 µgcarot·mgE−1. Neoxanthin is the second most abundant xanthophyll, with 3.21 ± 0.23 µgcarot·mgE−1 in A, i.e., 1.5-fold that of E, 2.1-fold of HI, and 4.1-fold of EL. Moreover, A is the only extract than contains zeaxanthin 1.07 ± 0.12 µgcarot·mgE−1, and the highest concentration of α-carotene, i.e., 0.53 ± 0.04 µgcarot·mgE−1, and β-carotene, i.e., 1.60 ± 0.03 µgcarot·mgE−1.Observing the carotenoid profile and concentration see , extract•+ and DPPH• methods), and specific radical antioxidant capacity for radicals O2•− and •NO.The extracts were tested for their total antioxidant capacity  in ABTS•+ assay, and A extract exhibited the lowest IC50 in DPPH• and •NO− assays. Although the IC50 for EL extract at •NO− assay could not be calculated in the range of concentrations tested, it was revealed to have antioxidant capacity.No significant differences were found between E and A extracts  uses the protein-binding dye SRB to indirectly assess cell growth ,46.v/v) was innocuous to AGS cells (data not shown).Despite DMSO being widely described to be cytotoxic depending on its concentration—yet, it was used to suspend extracts at low and non-cytotoxic concentrations. DMSO was thus titrated in these cell lines and, it was found that a concentration of 0.25% , yet a stronger effect was observed for HI extract—which increased AGS cells death by c.a. of 4-fold.TUNEL is a common method for detecting DNA fragmentation that may result from cell death, either by apoptosis or necrosis . Inductiproduced  show thaAssessment of cell proliferation by BrdU assay is based on the incorporation of BrdU into their replicating DNA, which can further be detected by immunofluorescence. For a quantitative approach, samples were analyzed by flow cytometry. Results revealed an anti-proliferative effect of the HI and EL extracts upon AGS, via 40% of inhibition of proliferation in ca., while cells treated with the E or A extracts behaved no differently from the negative control with DMSO , i.e., eGloeothece sp. were screened for their potential anti-inflammatory features, by resorting to two different assays, one reflecting the stabilization of extracts on Human red blood cell (HRBC) membrane induced by heat, and another that ascertains the capacity of such extracts to inhibit the human enzyme COX-2.The mechanism of inflammation can be partially triggered via the release of ROS, from activated neutrophils and macrophages, thus leading to damage in macromolecules causing, namely, lipid peroxidation of membranes. ROS spread inflammation by stimulating the release of cytokines, regulated by lysosomes, which in turn stimulate the recruitment of additional neutrophils and macrophages. Lysosome structure conveys a physical and functional interface among cell organelles, as it plays a role in negative or positive modulation of the production of inflammatory cytokines ,48. FurtGloeothece sp. extracts to protect erythrocytes from hemolysis when heat is supplied. Since the erythrocyte membrane is quite similar to the lysosomal one, indirectly is possible to conclude if any Gloeothece sp. extract holds any capacity in the stabilization of lysosomal membranes [This assay allows the characterization of the capacity of embranes , and so,v/v) extract is the most promising as it exhibits a protection capacity of 61.6 ± 9.6%; nonetheless, EL extract also appears to hold some potential in protecting HRBC membranes. Conversely, the E and A extracts did not show significant protective capacity (see Results show that the HI 3:2 (city see .Cyclooxygenases (COXs) catalyze reactions that lead to the formation of pro-inflammatory prostaglandins (PG), thromboxanes, and prostacyclins. Hence, the ability of extracts to inhibit the conversion of AA to Prostaglandin H2 (PGH2) via inhibition of COX-2 was determined. All concentrations tested exhibit anti-inflammatory activity in vitro, by inhibiting PG production in a dose-dependent manner. However, the extracts exhibited different behaviors within the range of concentrations tested, data not shown.p ˂ 0.05); however, the corresponding IC50 values (see p ˂ 0.05).While A and EL at lower extract concentration induces a higher inhibition, a linear percent of inhibition is of E concentration was observed, whereas a non-significantly percentage of inhibition variation was detected with HI concentration. In terms of total inhibition capacity of COX-2 enzymatic activity, one notices that A, E, and HI performed equally well beyond 50% with no significant differences between them  between composition (carotenoids and PUFA) and bioactive features, however possible synergetic effects among the molecules, that were not possible to measure, may contribute to its bioactive potential. Hence, these features will be discussed separately, and then in an integrated manner.Drugs commonly used to treat inflammation and cancer raise severe side effects, such as toxicity and decreased life quality ,52. In t50 values at all antioxidant assays. As observed previously, lutein probably contributes the most to said bioactivity, owing to its higher concentration [50 values of Gloeothece sp. extracts (A > E > HI > EL). Particularly, a correlation was found with C18:2 n6 .The antioxidant capacity of cyanobacterial carotenoids is well established—particularly concerning lutein and β-carotene ,29,30,53ntration . However• assay, in which the lowest IC50 was again observed in the A extract. The high concentration of total carotenoids and PUFA, namely lutein and C18:2 n6, may account for their important antioxidant role , as reported before [Concerning the specific radical’s scavenger capacity, results reveal the same trend, particularly in NOd before ,56,57.50 values for the O2•− assay could not be found at the tested concentrations, some scavenging effects were detected at E and EL extracts—data not shown.Although the IC• and O2•− radicals in vitro, a similar capacity is expected in vivo—with a preventive role of chronic inflammatory diseases, cancer, or neurodegenerative disorders [Hence, owing to the antioxidant scavenging capacity of A and E extracts against NOisorders ,59.Unlike observed with antioxidant capacity, the most promising extracts, in terms of inducing AGS cell death and cell proliferation, are HI and EL extracts; where it cannot be established a clear correlation of antitumor capacity and high content in carotenoids and fatty acids.Gloeothece sp. extracts, such as phenolic compounds. Although these compounds have not been characterized, the content in aromatic compounds is described to exert effects in bioactivities, particularly in antitumor and anti-inflammatory agents [Despite a possible interaction of all extracts’ compounds, some evidence relate such bioactivities with some compounds identified in y agents .From a nutraceutical point of view, dietary supplementation of β-carotene in animal models of colon carcinogenesis has revealed anticancer capacities for that compound , as wellr = 1, p < 0.083).Some PUFA, particularly ω-3, have been reported to possess in vitro and in vivo anticancer effects, via modulation of tumor growth or increase of cell death rate ,63, this50 value for the HI extract (23.2 ± 1.9 µg·mL−1) is lower than other hexanoic extracts reported before for human colon carcinoma cell line (HCT116), for example for Chlorella ellipsoidea and C. vulgaris which IC50 value was ca. 41µg·mL−1 and equivalent to the one obtained with pure lutein (21.02 ± 0.85 µg·mL−1) [r = 1, p < 0.083).It should be noted that the antitumoral ICµg·mL−1) . Also, cGloeothece sp. extracts was assessed by two assays. In the HRBC assay, HI extracts stood out in terms of inhibition capacity of 61%; hence, this HI extract may potentially stabilize cell membrane and thus prevent stress-induced decay, as well as stabilize the lysosomal membrane. This feature is crucial in the prevention of an anti-inflammatory response induced by the release of lysosomal constituents, which cause further tissue inflammation and damage upon extracellular release [The anti-inflammatory potential of  release .Gloeothece sp. exhibited that ability, some of them having a dose-dependent response, like E extracts. However, extract A exhibited the best performance at a concentration of 75 µg·mL−1, inhibiting in ca. 57% of COX-2 enzymatic activity; however, the possible application of A extracts use must be discarded due to its cytotoxicity to HCMEC cells. Nonetheless, HI extract follows as most promising due to ca. 48% of inactivation capacity and with no cytotoxicity associated.As seen before, the ability to inactivate COX-2 is indicative of the potential of an extract to be used as an anti-inflammatory drug. All extracts of C. ellipsoidea showed anti-inflammatory activity when it was tested on LPS-stimulated RAW 264.7 mouse macrophages, by inhibiting NF-κB activation and NO and prostaglandin E2 (PGE2) production [A number of anti-inflammatory molecules obtained from microalgae have been shown to display high antioxidant capacity, that is in the composition of A and HI, such as β-carotene, lutein, zeaxanthin, and ω3 PUFA . Some ofoduction .Chemoprevention consists of the use of pharmaceutical drugs, or nutritional supplements to reduce the risk of developing or having a recurrence of cancer. Several in vitro and animal studies showed the chemopreventive properties of a few metabolites from microalgae , namely against colon and skin cancer .Gloeothece sp. extracts, particularly the A and HI shows that they are a promising source in the eventual formulation of some nutraceutical products bearing antioxidant, anticancer, and anti-inflammatory capacities. But despite the notable antioxidant features of the A extract, particularly its ability to inhibit the radical NO•, its potential application as a nutraceutical is limited due to its cytotoxicity.Performance recorded for Experimental and epidemiological evidence reported before suggests that anti-inflammatory drugs may also decrease the incidence of some types of cancer, as well as tumor burden and volume ,69. An aGloethece sp. appeared to be the most promising as a chemo-preventive agent in the nutraceutical industry because of their features as (1) antioxidant namely high total antioxidant capacity and scavenging capacity against -NO• radical; (2) antitumor induction of cell death upon AGS cells, along with anti-proliferative effects; and (3) anti-inflammatory, namely inability to inhibit COX-2 expression while protecting lysosomes.Hence, the HI extracts of Gloeothece sp. (ATCC 27152) was purchased from ATCC—American Type Culture Collection (USA), and kept at 25 °C, using Blue Green (BG11) as culture medium [photon·m−2·s−1, and air bubbling at a flow rate of 0.5 L·min−1. Biomass was then collected by centrifugation at 18× g for 10 min, the supernatant was rejected and pellet freeze-dried, and stored under gaseous nitrogen until analyses were performed.e medium . For bioGloeothece sp. were obtained from 200 mg of lyophilized biomass, using four alternative food-grade solvents : ethanol (E), acetone (A), a mixture (3:2) of hexane/isopropanol (HI), and ethyl lactate (EL), as previously tested [Extracts from y tested .Gloeothece sp. extract, as detailed below.Fatty acids and carotenoids are among the most widely known bioactive compounds found in microalgae, which possess a high interest in the nutraceutical and pharmaceutical markets; hence, solvent extracts were evaporated and residue composition was determined for each The weighted residue was submitted to direct transesterification to produce fatty acid methyl esters according to the acidic method described by Lepage and Roy , after mTo determine the content in carotenoids of the extracts, high-performance liquid chromatography (HPLC) was applied as an analytical technique as detailed before . The res+•, DPPH•); while the other two were more specific for two biological radicals, superoxide (O2•−) and nitric oxide (•NO−)—with the later be known to be correlated with inflammation processes.The antioxidant capacity of each extract was evaluated via four spectrophotometric assays: two assessed total antioxidant capacity  containing 5% of DMSO. Each antioxidant assay was performed in triplicate, as described in the following sub-sections.A positive control, Trolox, was used to validate the antioxidant capacity of extracts and putatively establish a calibration curve but comparing the antioxidant capacity of the extracts, their IC•+) . Absorbance was accordingly determined at 734 nm, upon the reaction of the extract with ABTS•+ for 6 min—as previously optimized by Guedes et al. [The total antioxidant capacity was determined as the capacity to decrease the absorbance of blue/green chromophore 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)  , after an incubation period of 30 min at room temperature in dark. The scavenging reaction was monitored at 515 nm, as implemented before by Amaro et al. [The antioxidant capacity was determined, in triplicate, by reacting each extract with 2,2-diphenyl-1-picrylhydrazyl  derived from fragments of a tumor resected from a patient who had received no prior therapy, were maintained in RPMI1640  supplemented with 10% FBS  and kept at 37 °C, in a humidified 5% CO−1, for acetone, ethanol, HI and ethyl lactate extracts, respectively.Solvents of each extract were evaporated by rotavapor and extracts resuspended with the minimum amount of dimethyl sulfoxide (DMSO) , thus producing in concentrations of 130, 150, 120, and 450 mg·mL4 were seeded in 96-wells plates and treated for 48 h with different concentrations of microalgal extracts (0 to 550 µg·mL−1 whenever possible) or DMSO  as negative treatment control (0.05% v/v). As a positive control, DMSO 100%, was used to validate the antitumoral capacity of extracts. Then cells were fixed by the addition of 50 μL of cold 50% trichloroacetic acid  to each well, and incubating the plates at 4 °C for 1 h. Next the fixation step, the plates were washed three times with deionized water and dried at room temperature. The cells were then stained with 50 μL of 4% sulforhodamine B (SRB)  in 1% acetic acid  for 30 min and then washed three times with deionized water. After the plates were dry, the cells were solubilized with 100 μL of 10 mM unbuffered Tris Base , and the optical density at 510 nm was measured using the fluorimeter SynergyTM 4 Multi-Mode Microplate Reader . Results were plotted as dose-response curves, and the IC50 for each extract was found and expressed as µgE·mL−1.AGS cells in a concentration of 1 × 105, and treated for 48 h with the microalgal extracts at the IC50 found at the SRB assay, for 48 h. DMSO  was used as a positive control treatment. Cells were washed and trypsinized and the pellet obtained was fixed in 3 mL of ice-cold methanol for 15 min. Then, cells were washed and resuspended in 500 μL of PBS. Incubation with TUNEL reaction mix  was done for 1 h, at 37 °C, in the dark. Then, data were acquired using a BD Accuri C6 flow cytometer .AGS cells were cultured in 6-well plates in a concentration of 7.5 × 105 and treated with the extracts at the IC50 found at the SRB assay, for 48 h, using DMSO  as positive control treatment. 5-Bromo-2′-deoxyuridine (BrdU)  was incorporated in the cell culture medium at the ratio of 1:1000, and underwent incubation for 1 h, at 37 °C. Straightaway the following incubation, the cells were harvested, washed with PBS, fixed in 1 mL of ice-cold methanol for 30 min, washed again, and resuspended in 500 μL of PBS. This was followed by the incubation with 1 mL of HCl 4 M , for 20 min, two washing steps with PBS, a blocking step (PBS containing 0.5% Tween 20 and 0.05% BSA), and finally 1 h incubation at room temperature with the primary antibody against BrdU . Next, the cells were further washed with PBS and incubated with the secondary antibody labeled with FITC , for 30 min at room temperature washed two times and resuspended in 500 μL of PBS. Data acquisition was performed with a BD Accuri C6 flow cytometer .AGS cells were cultured in 6-well plates containing a concentration of 7.5 × 10To assess the anti-inflammatory potential of the lipidic extracts, two assays were performed. The Human red blood cell (HRBC) membrane stabilization assay, induced by heat, was used first; it allowed to observe if any extract holds the potential to stabilize lysosomal membranes. The second assay is specific to a prostaglandin-endoperoxide synthase, human COX-2 enzymatic activity inhibition—and helps conclusion on whether any extract has the potential to be used as a non-steroidal anti-inflammatory agent. The study was conducted according to the guidelines of the Declaration of Helsinki, and ap-proved by the Institutional Ethics Committee of CIIMAR .g for 10 min and supernatant (plasma) was removed. Hence human red blood cells (HRBC) were washed three times with an equal volume of isotonic PBS  pH 7.4) and then reconstituted at 40% (v/v) suspension. Salicylic acid at 500 µg mL−1 was used for positive control and PBS with 20% of DMSO  for negative control.Human fresh blood was collected intravenously to heparinized tubes, from a healthy volunteer that was not taking any non-steroidal anti-inflammatory drugs (NSAIDs) for 2 weeks before the experiment. Blood was centrifuged at 700× −1, for A, E, HI, and EL, respectively, were resuspended in PBS containing 20% of DMSO, and then mixed in 1:1 (v/v) with a solution of HRBC in 2% in PBS. Samples were incubated at 56 °C for 20 min, cooled in tap water, and centrifuged at 700× g for 5 min, and the supernatant was collected. The absorbance of the supernatant was measured spectrophotometrically at 560 nm using a microplate reader  [E denotes supernatant absorbance after reaction with extract; AbsEB denotes extract absorbance at 560 nm; and AbsC denotes the control absorbance of PBS with 20% of DMSO.Each extract, prepared as explained in re, EUA) . The per−1.The anti-inflammatory potential of the extracts was assessed via an enzyme inhibitory assay—inhibition of COX-2 enzymatic activity, using the COX-2 Enzyme Activity Assay Kit , according to the manufacturer’s instructions. Dried lipidic extracts were diluted in DMSO, and assayed at different concentrations—75, 125, and 250 µg·mLIn this assay, arachidonic acid (AA) served as a substrate for the human recombinant COX-2 enzyme, thus leading to the production of prostaglandin. The assay measures PGF2α produced by SnCl2 reduction of COX-derived PGH2. The PGF2α levels produced in the presence versus absence of test products were quantified through an enzyme immunoassay—using an antibody that binds to all major prostaglandin compounds, results are expressed in percent of inhibition, calculated according to kit instructions.4 cells mL−1 with Dulbecco’s Modified Eagle Medium (DMEM)  for 24 h.Cytotoxicity of the extracts was evaluated by measuring the viability of Human Cardiac Microvascular Endothelial Cells (HCMEC) obtained from the American Type Culture Collection (ATCC). Cells were seeded in a 96-well plate with a final concentration of 10 × 10−1—using DMSO 1% as negative control and DMSO 20% as the positive control. The assay was independently repeated four times, with duplicate extracts. Cytotoxicity was expressed as a percentage of cell viability, considering the values of the negative control as 100% viability.The cellular viability was assessed by the mitochondrial-dependent reduction of 3--2,5-diphenyltetrazolium bromide (MTT)  to formazan, quantified by optical density measurement at 510 nm, as described by Lopes et al. . Several