Six different homogeneous and heterogeneous catalysts were used to obtain maximum monomer yield. The MBAA was obtained with 95% yield by using Cu(II) catalyst containing carboxylate groups ligands.N,N′-Methylenebisacrylamide (MBAA), which is an important raw material in a widely industrial area, was synthesized using convenient catalysts. The synthesized monomer was fully characterized by the elemental analysis, fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA),  Acrylamide is one of the most common compounds that use MBAA during the polymerization reactions. The bisacrylamide part of the molecule polymerizes with acrylamide and creates a crosslinked structure as opposed to a linear structure. This crosslinking imparts higher strength and toughness to the polymer in its end-use applications. Being a crosslinking agent, MBAA is used in various polymerization reactions to produce polymers and copolymers . As a re2.2.1.−1 at room temperature. HPLC analysis was performed for the characterization and purity of MBAA. For the HPLC analysis a Shimadzu HPLC system equipped with a reversed phase C8 column  was used. CH3OH:CH3CN:H2O  was used for the separation of the mobile phase. The column temperature was 35 °C, the detection wavelength was 254 nm, the flow rate was 1.0 mL min−1 and the retention time was 15 min. TGA analysis was conducted in nitrogen atmosphere with Perkin Elmer Pyris Diamond TG/DTA equipment at a heating rate of 10 °C min−1. 1H-NMR and 13C-NMR analysis were recorded wit a Bruker Avance III HD 600 MHz Ultra Shield TM. GC/MS analysis was performed by using Thermo Brand chromatograph with TR5MS capillary columns.All chemicals were purchased from a major chemical supplier as high or highest purity grade and used without further purification. Catalysts were synthesized in the laboratory. Melting points were determined with a Digital Melting Point Gallenkamp. IR spectra were measured with Thermo Scientific Nicolet IS10 Model FT-IR Spectrophotometer using ATR method with a resolution of 4 cm2.2.4  instead of HCl refluxed 2 h, the yield was increased and 12.78 g yellow solid was obatined. Yield was 60%, M. P. > 300 °C.A solution of acrylamide , formaldehyde , copper(II) acetate , and concentrated HCl  were added succesively into the flask. The mixture was refluxed for 90 min and then cooled at room temperature. The solution mixture was separated from the solid phase and dried at room temperature. Yellow solid obtained was 11.20 g. Yield (52%), M. P. > 300 °C. It is interesting to note that when using NaHSO2.3.4  were added succesively into the flask. The mixture was refluxed for 2 h and then cooled at room temperature. The solution mixture was separated from the solid phase and dried at room temperature. Yellow solid obtained was 11.20 g. Yield (52%), M. P. > 300 °C.A solution of acrylamide , formaldehyde , Cu(II) glyoxime , and NaHSO2.4.The preparation of MBAA was similar to that of the synthesis 2.3 except that Pd(II) glyoxime  was used instead of copper(II)-glyoxime.No conversion was observed.2.5.The preparation of MBAA was similar to that of the synthesis 2.3 except that Fe(II) glyoxime  was used instead of copper(II) glyoxime. No conversion was observed.2.6.The preparation of MBAA was similar to that of the synthesis 2.3 except that Ni(II) glyoxime  was used instead of copper(II) glyoxime. No conversion was observed.2.7.°C) for about two hours then cooled, was filtered out and dried. Yellow solid obtained was 19.85 g. Yield (95%), M. P. > 300 °C. Anal. Calcd. for C7H10N2O2: C, 54.54; H, 6.54; N, 18.17. Found: C, 54.10; H, 6.14; N, 18.34%. IR data (cm−1): 3300(m), 3065(w), 2955(w), 1656(s), 1625(s), 1538(s), 1408(s), 1383(s), 1304(s), 1225(s), 1120(s), 989(s).The preparation of MBAA was similar to that of the synthesis 2.3 except that Cu(II) carboxylate  was used instead of copper(II) glyoxime. All ingredient were mixed as mentioned above. The mixture was refluxed  in the molecule. The absorption related to the υ(C=CH) and υ(C–H) stretching vibrations of the MBAA were observed at 3065 and 2955 cm−1, respectively .https://doi.org/10.1080/15685551.2017.1332138.The supplemental data for this article can be obtained online at No potential conflict of interest was reported by the authors.Click here for additional data file.


Streptococcus pneumoniae infection in mice. These data highlight the homeostatic role of ILC2s in setting the activation threshold in the lung and underline their implications in anti-bacterial defenses.From birth onward, the lungs are exposed to the external environment and therefore harbor a complex immunological milieu to protect this organ from damage and infection. We investigated the homeostatic role of the epithelium-derived alarmin interleukin-33 (IL-33) in newborn mice and discovered the immediate upregulation of IL-33 from the first day of life, closely followed by a wave of IL-13-producing type 2 innate lymphoid cells (ILC2s), which coincided with the appearance of alveolar macrophages (AMs) and their early polarization to an IL-13-dependent anti-inflammatory M2 phenotype. ILC2s contributed to lung quiescence in homeostasis by polarizing tissue resident AMs and induced an M2 phenotype in transplanted macrophage progenitors. ILC2s continued to maintain the M2 AM phenotype during adult life at the cost of a delayed response to   •The first breath triggers IL-33 induction by AEC2 in lungs of newborn mice•IL-33 promotes the perinatal expansion and activation of ST2-expressing ILC2s•ILC2-derived IL-13 polarizes newborn’s AMs into an M2 phenotype•This homeostatic type 2 pathway delays antibacterial effector responses The postnatal lung immune environment is largely unexplored. Saluzzo et al. find that birth is associated with the induction of an IL-33/ILC2/IL-13-driven immune response in mice. This type 2 response is preserved throughout life and maintains alveolar macrophages in an M2 phenotype at the expense of a delayed response to bacterial infections. M1 macrrophages .reg), dendritic cells (DCs), mast cells, group 2 innate lymphoid cells (ILC2s), natural killer (NK) cells, and AMs  was the most abundant cell population upregulating Il33 in the first few days after birth   H and S1IIl33Cit/Cit) mice. Il33Cit/Cit mice showed reduced expression of the type 2 cytokines IL-5 and IL-9 and of inflammatory mediators like IL-6, IFN-γ, IL-1α, IL-1β, CCL5, and CXCL10 (+ ILC2s are major producers of IL-5 and Il-9 and considered the primary targets of IL-33 in the lung (− CD127+ST2+ICOS+). We detected few ILC2s at E19 but markedly increased numbers by P7 that stabilized by week 6 (Il33Cit/Cit (Il1rl1−/−) mice at P7  and IL-33-deficient  reporter mice  consistently induced higher levels of CXCL1 than WT AMs  influx of neutrophils in bronchoalveolar lavage fluid (BALF) and lung -induced acute lung injury. Similar to our observations in pneumococcal pneumonia, we discovered an augmented early inflammatory response to LPS  produced IL-13  and adult Il7rCreRorasg/fl mice, congenitally deficient in lung resident ILC2s  and Car4, respectively , Il1rl1−/− (Il5−/− (Rag2−/− (Il7rCre (Rora+/fl (Rorasg/+ mice (Jackson Laboratories) were on a C57BL/6 background. We obtained Il7rCreRorasg/fl mice  or Il7rCreRora+/fl littermate controls by crossing Il7rCre with Rora+/fl and Rorasg/+. Il33Cit/+  with heat-inactivated S. pneumoniae at a MOI 100 or S. aureus LTA (10 μg/mL). In AMs from newborn mice were isolated by cell sorting using an FACSAria II (BD Biosciences) by gating on viable CD45Recombinant mouse IL-13 and IL-33 were purchased from BioLegend. Anesthetized mice were treated daily with rmIL-13 (6 ng/50 μL NaCl for 3 consecutive days) or rmIL-33 (0.5 μg/50 μL NaCl for 5 consecutive days). Mice were sacrificed 1 hr after the last administration.− , Thy1.2+ ST2+ ICOS+ and Tom+ from Il13Tom/+ mice that had been treated intranasally (i.n.) with rmIL-33 for 5 days. Cells were transferred intravenously to Il13−/− or WT mice recipients (1 × 105 cells per mouse) and assessed for localization in lungs 5 days later.Lung ILC2s were FACS purified as defined by lineageRorasg/sg or WT littermates served as bone marrow donors. CD45.1 recipients were irradiated (9 Gy) and reconstituted on the same day with 2 × 106 bone marrow cells per recipient by intravenous injection. Mice were analyzed for reconstitution and absence of lung resident ILC2s after 8 months.6-week-old CD45.2 5 CFUs S. pneumoniae serotype 3 (ATCC 6303) as described (7 CFUs S. aureus (USA300). Acute lung injury was induced by administration of 100 ng LPS i.n. (E. coli O55:B5). BALF was collected, cells were counted with an automated cell counter , and Giemsa-stained cytospin preparations were used for differential cell counts. Lung tissues were homogenized in sterile saline using a Precellys 24TM (Peqlab), and lung colony-forming units (CFUs) were determined by 10-fold serial dilutions of homogenates on blood agar plates. An aliquot of lung homogenates was incubated in RA1 buffer (Macherey-Nagel) containing 10% of beta-mercaptoethanol  and stored at −80° for RNA extraction. The remaining lung homogenates were incubated in Greenberger lysis buffer as described previously (Mice were infected i.n. with 10escribed , or witheviously , and supParaffin-embedded lung sections were stained with H&E and scored by a trained pathologist who was blinded to experimental groups. The final pneumonia score was the sum of the following parameters: severity of pleuritis, interstitial inflammation, edema, and thrombi formation were scored as 0 = absent, 1 = mild, 2 = moderately severe, 3 = severe; bronchitis was scored as 1 if present; endotheliitis was scored as 0 = absent, 2 = present, 3 = present with endothelial wall necrosis; the existence of a lobar confluent infiltrate was scored as 1, and a score of 0.5 was added for every infiltrate covering 10% of the lung area.U test was used for analysis of nonparametric data. For multivariable comparisons we performed a one-way ANOVA followed by Sidak’s multiple comparison test. Results were analyzed with Graph Pad Prism software version 6, and a p < 0.05 was regarded as statistically significant.Data are expressed as mean ± SEM. Statistical significance in two-group comparisons was assessed with an unpaired Student’s t test. When indicated, a Mann-Whitney S. Saluzzo and S.K. conceived the study. A.N.J.M. hosted S. Saluzzo, contributed to experimental design, and provided critical reagents. S. Saluzzo, A.-D.G., B.M.J.R., R.M., S. Scanlon, P.S., K.L., A.H., O.S., A.K., J.M.W., and H.J. performed the experiments or contributed to experimental design, reagents, and analysis. I.M. scored histological slides. S. Scanlon performed the immunofluorescence experiments. R.M. analyzed the newborns alveolarization. A.-D.G. and B.M.J.R. contributed equally to this work. S. Saluzzo and S.K. wrote the manuscript with contributions from A.N.J.M., B.M.J.R., O.S., and S. Scanlon. A.N.J.M and S.K. are joint senior authors.


Ly6) gene cluster) were proteins with cationic nature and rich in cysteine content, that are characteristic of antimicrobial peptides (AMPs) and their expression were all significantly up-regulated after challenge with lipopolysaccharide (LPS). These strongly suggested that Ly2.1–3 are potential AMPs, but firm evidence are lacking. Here, we clearly showed that the recombinant proteins of Ly2.1–3 were capable of killing Gram-negative bacteria Aeromonas hydrophila and Escherichia coli, while they had little bactericidal activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis. We also showed that recombinant proteins Ly2.1–3 (rLy2.1–3) were able to bind to the Gram-negative bacteria  A. hydrophila,  E. coli and the microbial signature molecule LPS, but not to the Gram-positive bacteria  S. aureus and B. subtilis as well as the microbial signature molecule LTA. Moreover, the Scatchard analysis revealed that rLy2.1–3 could specifically bind to LPS. Finally, we found that Ly2.1–3 were not cytotoxic to mammalian cells. All these together indicate that Ly2.1–3 can function as AMPs.The emergence of multidrug-resistant (MDR) microbes caused by overuse of antibiotics leads to urgent demands for novel antibiotics exploration. Our recent data showed that Ly2.1–3 (a novel lymphocyte antigen 6 ( AMPs Lymphocyte antigen 6 (Ly6) is a superfamily of proteins that includes a conserved LU domain with eight or ten cysteine residues, forming four to five conserved disulfide bonds. The biological functions of Ly6 superfamily members are diverse, and our knowledge as such has been increasingly accumulated. For example, in mammals, Ly6 proteins such as E48, ly6A.2, ly6C and urokinase-type plasminogen activator receptor (uPAR) have been shown to play roles in cell adhesion and migration through interaction with specific receptors –8, lynx1Escherichia coli and Staphylococcus aureus  ×100% (n=3). The control was processed similarly except that Tris/NaCl buffer or TBS was used instead of protein solution.A colony formation assay was used to test the bactericidal activity of Ly2.1–3 against u et al. . In brieA. hydrophila, E. coli, S. aureus and B. subtilis were cultured in TSB and LB media respectively. They were cultured to mid-exponential phase, and then they were collected by centrifugation at 6000 g at room temperature for 5 min. The bacterial pellets were resuspended in 25 mM Tris buffer containing 10 mM NaCl or TBS (pH 6.5), giving a concentration of 1 × 108 cells/ml and used for the following experiments.The strains of A. hydrophila, E. coli, S. aureus and B. subtilis suspension (1 × 108 cells/ml) were mixed with 300 μl of 100 μg/ml rLy2.1, rLy2.2, rLy2.3 or TRX-His-tag peptide in 25 mM Tris buffer with 10 mM NaCl (pH 6.5) or with the same volume of Tris/NaCl solution alone. The mixtures were incubated at 25°C for 2 h, and the bacteria were then collected by centrifugation at 6000 g at room temperature for 5 min. After washing in Tris/NaCl solution, the bacterial pellets were resuspended in Tris/NaCl solution (pH 6.5), giving a concentration of 2 × 107 cells/ml. Both recombinant protein-treated bacteria and non-treated bacteria as well as purified rLy2.1–3 or TRX-His-tag peptide were run on an SDS/PAGE (12% gel), followed by Western blotting analysis using anti-His tag antibody as described above.Aliquots of 150 μl of N-hydroxysuccinimide ester  as described by Zhang et al. [4)2SO4 was added to the mixture to inactivate the uncombined biotin. The uncombined biotin was removed by ultrafiltration. To test the binding of Ly2.1–3 to LPS and LTA, 50 μl of 40 μg/ml LPS or LTA in 10 mM PBS (pH 7.4) was applied to each well of a 96-well microplate and air-dried at 25°C overnight. The plate was incubated at 60°C for 30 min to further fix LPS or LTA, and the wells were each blocked with 200 μl of 40 mg/ml BSA in 10 mM PBS (pH 7.4) at 37°C for 2 h. After washing four times with 200 μl of 10 mM PBS supplemented with 0.5% Tween-20, 50 μl of biotinylated Ly2.1–3  was added to each well. After incubation at room temperature for 3 h , the wells were each rinsed four times with 200 μl of 10 mM PBS supplemented with 0.5% Tween-20 and 100 μl of streptavidin–HRP (CWBIO) diluted to 1:2000 with 10 mM PBS (pH 7.4) containing 1 μg/ml BSA was added to each well. After incubation at room temperature for 1 h, the wells were washed four times with 200 μl of 10 mM PBS supplemented with 0.5% Tween-20, added with 75 μl of 0.4 mg/ml O-phenylenediamine (Amresco) in the buffer consisting of 51.4 mM Na2HPO4, 24.3 mM citric acid and 0.045% H2O2 (pH 5.0) and reacted at 37°C for 5 to 30 min. Subsequently, 25 μl of 2 M H2SO4 was added to each well to terminate the reaction and absorbance at 492 nm was monitored by a microplate reader. For control, recombinant TRX-His-tag peptide at the same concentrations was treated similarly.Both rLy2.1–3 and TRX-His-tag peptide were individually biotinylated with biotinamidohexanoic acid g et al. . BrieflyKd) and apparent maximum number of bindings (Bmax) were determined according to the Scatchard plot using the software GraphPad Prism 5.01.For Scatchard analysis, LPS and LTA were individually biotinylated with NHS-LC-biotin  as described by Zhang et al. . Fifty m6 cells/ml) were sampled into a 96-well plate and cultured at 37°C under 5% CO2 for 2 h. Subsequently, aliquots of 200 μl of Ly2.1–3 in serum-free DMEM were added to each well, giving final concentrations of 25, 50, 100 and 150 μg/ml respectively, incubated for another 4 h, and then 20 μl of MTT solution (5 mg/ml in PBS) was added into each well. After incubation for 4 h, the medium was removed and 150 μl of DMSO were added. The absorbance at 492 nm was measured under a microplate reader. The cells treated with DMEM alone served as control. The percent of viability against the control was calculated as follows: (A of treated groups/A of control groups) × 100% (n=3).To test if Ly2.1–3 are cytotoxic to murine RAW264.7 cells, MTT assay was performed as described by Hu et al. . RAW264.g for 10 min. After washing three times with PBS (pH 7.4), RBCs were suspended in PBS to give a concentration of 4% (v/v). An aliquot of 200 μl RBCs suspension was mixed with 200 μl of Ly2.1–3 solution, giving final concentrations of 25, 50, 100 and 150 μg/ml. After incubation at 37°C for 1 h, the mixtures were centrifuged at room temperature at 1000 g for 10 min. The supernatants were collected and added into a 96-well plate. The absorbance was measured at 540 nm under a microplate reader . RBCs incubated with PBS alone, BSA solution (150 μg/ml), 0.1% Triton X-100 solution served as blank, negative and positive controls respectively. Each treatment was performed in triplicate.Human red blood cells (RBCs) were used to test the haemolytic activity of Ly2.1–3 as described by Hu et al. . HealthyP<0.05 was considered significant. All the data were expressed as mean ± S.E.M.All the experiments were conducted at least three times. Statistical analyses were performed using the GraphPad Prism 5. The significance of difference was determined by one-way ANOVA, and the difference at E. coli and purified by affinity chromatography on a Ni-nitrilotriacetic acid resin column. The purified rLy2.1–3 and TRX-His-tag peptide all yielded a single band of approximately 12, 12, 12 and 21 kDa respectively, on SDS/PAGE gel after Coomassie blue staining, matching the expected sizes . These indicated that rLy2.1, rLy2.2 and rLy2.3 were all able to specifically kill Gram-negative bacteria such as A. hydrophila and E. coli rather than Gram-positive bacteria. For control, recombinant TRX-His-tag peptide had no bactericidal activity against the Gram-negative bacteria A. hydrophila and E. coli and the Gram-positive bacteria  S. aureus and B. subtilis. By contrast, the bactericidal activity of standard antibiotic is very strong, the bactericidal rate can reach almost 100%.The colony formation assay was used to test if rLy2.1–3 showed any antibacterial activity against the Gram-negative bacteria A. hydrophila and E. coli and the Gram-positive bacteria  S. aureus and B. subtilis. As revealed by Western blotting, rLy2.1–3 were all capable of binding to both A. hydrophila  and B. subtilis . For control, recombinant TRX-His-tag peptide were not capable of binding to A. hydrophila, E. coli, S. aureus and B. subtilis .Next, we tested if rLy2.1–3 bind to the Gram-negative bacteria drophila A and E.  and the Kd values (μg/ml) of the affinity of rLy2.1–3 to LPS were approximately 1.078 and 2.125, 1.077 and 1.330 as well as 0.989 and 10.070. Here, Bmax value is the maximum saturation concentration of LPS bound to rLy2.1–3  superfamily of molecules was first identified in mouse, murine Ly-6-encoded molecules play an important role in the antigen-independent activation of lymphocytes . And Ly-A. hydrophila and E. coli and two Gram-positive bacteria S. aureus and B. subtilis to test the bactericidal activity of recombinant Ly2.1–3. The data indicated that Gram-negative strains are more susceptible to Ly2.1–3 than Gram-positive strains. This specific selection is similar to that of medaka β-defensin peptide and human β-defensin 2 (HBD-2) [A member of Ly6 superfamily has been identified in various metazoans, but functions of most Ly6 members remain poorly characterized. Recent study suggested that the members of Ly6 superfamily Ly2.1, Ly2.2 and Ly2.3 are potential AMPs . In the  (HBD-2) ,28. In a (HBD-2)  identifi (HBD-2)  have shoA. hydrophila and E. coli, but are non-cytotoxic to mammalian cells. All these together indicate that Ly2.1–3 can function as AMPs.The emergence of MDR microbes caused by overuse of antibiotics has caused the less efficacy of major antimicrobial drugs used in clinical settings  and thus


Vitamin D metabolites were measured by UPLC/APCI/HRMS method. Circulatory 27 cytokines/chemokines and 10 metabolic hormones were measured. Significantly decreased 25(OH)D and 1,25(OH)2D levels were observed in HDP. The levels of 25(OH)D were significantly lower in PE and EC, whereas the serum levels of 1,25(OH)2D significantly decreased only in EC subjects. Serum 25(OH)D and 1,25(OH)2D levels were negatively correlated with systolic- and diastolic blood pressure, creatinine, and uric acid levels. Serum interleukin (IL)-6 and IL-13 decreased, and GIP levels were increased in gestational hypertensive subjects. Platelet-derived growth factor-BB and IL-8 levels were increased and macrophage inflammatory protein-1beta levels were decreased in EC subjects. IL-8 and IL-10 increased, and rantes and GIP levels decreased in the EC group as compared with the GH group. Multivariate logistic regression analysis showed that eotaxin, monocyte chemotactic protein-1, 25(OH)D, and 1,25(OH)2D were predictors of HDP. Our analyses suggest that lower vitamin D metabolites are associated with altered cytokines/chemokines and metabolic hormones in HDP.The aim of this study was to investigate whether lower serum vitamin D metabolite levels were associated with altered cytokine/chemokine and metabolic hormone levels in three different hypertensive disorders in pregnancy (HDP). Healthy pregnancy ( Hypertension is the most common medical problem encountered during pregnancy, complicating 5–10% of pregnancies . HDP is Vitamin D is a secosteroid and plays pivotal role in bone and mineral metabolism. During pregnancy, vitamin D plays important role in implantation and placental function due to angiogenic, immunomodulatory, and anti-inflammatory effects . While sHypertensive disorders in pregnancy are characterized as excessive maternal inflammatory responses to pregnancy. Inflammation involves a complex network of cytokines released from stressed cells and damaged tissues . CytokinCross-sectional study consists of pregnant women  in their third trimester, and inclusion criteria of subjects age was 18–45 years. Blood samples collected from women admitted with HP, GH, PE, and EC at the maternity ward at Mediciti Institute of Medical Sciences outpatient clinic, Hyderabad, India, were used for the study. GH was defined as development of hypertension  for the first time after mid pregnancy after 20 weeks). PE was defined as blood pressure ≥ 140/90 and proteinuria ≥ +1, measured at least twice 4–6 h apart after gestational week 20 . All women gave written informed consent for this study. The study did not include any vulnerable population. During the study, participants were interviewed about maternal age, chronic diseases, medication, ethnical origin, smoking status, method of conception, any previous pregnancies affected by PE or GH, and family history of PE.g. A total of 20 μL of each serum sample is used for creatinine and uric acid assay (within 3 h). Rest of the serum sample is appropriately labeled and stored at −80°C until batch analysis for vitamin D metabolites, serum cytokine/chemokine, and metabolic hormones measurement.After collection, the blood samples are allowed to stand for complete clot formation at room temperature and subsequently centrifuged for 10 min at approximately 1,500 × A total of 10 mL of early morning midstream urine samples was collected into appropriately labeled sterilized containers and analyzed immediately for urine protein.Blood pressure was measured using oscillometric blood pressure monitor (model–omron HEM-780N3), calibrated prior to and once during the study period. Calibration of the instrument was checked periodically. Two measurements were taken after sitting comfortably on a chair for more than 5 min with left arm at the level of heart resting on a table. Serum creatinine levels were measured by modified kinetic Jaffe’s reaction in Dade Behring dimension Xpand plus system (creatinine flex reagent cartridge) according to the manufacturer’s instructions. Serum uric acid levels were measured by modified uricase method in Dade Behring dimension Xpand plus system (uric acid flex reagent cartridge) according to manufacturer’s instructions. Urinary protein levels have been measured by the pyrogallol red test. In brief, the concentration of total protein was determined by measuring the absorbance of colored solution at 600 nm by Systronics UV double beam spectrophotometry .Serum levels of 27 cytokines, i.e., interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-5, Il-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, eotaxin, basic FGF, G-CSF, GM-CSF, interferon (IFN)-gamma, IFN-gamma-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, platelet-derived growth factor (PDGF)-BB, rantes, tumor necrosis factor (TNF)-alpha, and vascular endothelial growth factor (VEGF), and 10 metabolic hormones, i.e., C-peptide, ghrelin, GIP, GLP-1, glucagon, insulin, leptin, plasminogen-activating factor (PAI)-1 , resistin, and visfatin were measured by using Bio-Plex Pro human cytokine Grp I panel 27-plex (Cat#M50-0KCAF0Y) and Bio-Plex Pro human diabetes panel 10-plex (Cat#171-A7001M), respectively. On the day of experiment, frozen serum samples were thawed, mixed by vortexing, and then centrifuged at 10,000 rpm for 5 min to isolate debris. All experiments were performed according to the manufacturer’s instructions using handheld magnetic separator bloc for 96-well flat bottom plates and analyzed using the Bio-Plex-200 system (Bio-Rad Corp.). All cytokine/chemokine and metabolic hormone standards were provided by the manufacturers. Acquisition gates were set at 8,000–15,000. Sample volume used for this analysis was 25 μl, and 50 events per bead were acquired. Mean fluoresce intensity was measured using Bio-Plex manager software version 5.0 (Bio-Rad) and compared to a standard curve to generate concentration values. Values below the range of the standard curve were set to the lower limit of detection. Three of the 27 cytokines analyzed  were excluded from further analyses, since approximately 95% sample concentration is below than the lowest standard or its maximum fluorescent intensity value near to background. The serum samples were measured in a single replicate, whereas cytokine standards and blank samples were measured in duplicate on each plate.g for 20 min, and supernatant was removed and filtered. The filtered serum was again centrifuged at 6,000 × g, and the supernatant was separated. Stripped serum was injected into the UPLC/APCI/HRMS method to confirm untraceable level of vitamin D3 (VitD3) and vitamin D2 (VitD2) and its respective metabolites such as 25(OH)D3, 1,25(OH)2 D3, 25(OH)D2, and 1,25(OH)2 D2.Human serum  was stripped and used as blank serum. Biocell serum was mixed with activated charcoal and agitated at room temperature overnight. The Biocell serum was then centrifuged at 9,000 × 3, VitD2, and its respective metabolites were prepared in ethanol. Serial dilution was done to prepare the primary aliquots for calibration curve samples ranging from 3 to 200 ng/mL for VitD3, 25(OH)D3, and VitD2 and 5–200 ng/mL for 1,25(OH)2 D3, 25(OH)D2, and 1,25(OH)2 D2. Similarly, low, middle, and high QC  samples at three different levels were prepared independently at concentrations of 3 ng/mL (LQC), 10 ng/mL (MQC), and 200 ng/mL (HQC) for VitD3, 25(OH)D3, and VitD2. Whereas, 5 ng/mL (LQC), 10 ng/mL (MQC), and 200 ng/mL (HQC) for 1,25(OH)2D3, 25(OH)D2, and 1,25(OH)2D2. All the stock solutions were stored at 0–4°C for further use. Stock solution of 1 mg/mL of dihydrotachysterol [Internal standard (IS)] was prepared in ethanol and diluted with methanol to prepare working solution containing a concentration of 50 ng/mL. To an aliquot of 100 μL of stripped serum, 10 μL of 50 ng/mL IS solution was added followed by 1 mL of hexane:heptane:acetone in the ratio of 45:40:15. This mixture was thoroughly mixed, shook, and centrifuged at 6,000 × g at 4°C. Evaporation of supernatant was done on ScanVac speed Vacuum Concentrator, and 100 μL of methanol was added and 10 μL aliquots of sample solution were injected into the UPLC/APCI/HRMS system for analysis.The stock solution of VitDmin/% proportion of solvent B): 0/5, 0–5/95, 5–6/5, and 6–8/5. The flow rate of the mobile phase was 1.00 mL/min, the column temperature 25°C, and the injection volume 10 μL. The typical operating source conditions for MS scan in positive ion APCI mode were optimized as follows sheath gas flow rate 65; Aux gas flow rate 20; discharge current 10.00 μA; capillary temperature 300°C; capillary voltage 50 V; tube lens voltage 85.0 V; skimmer voltage 18.00 V; and vaporizer temperature 380°C. For quantification, EICs of [M + H]+ at m/z 385.34649, [M + H-H2O]+ at m/z 383.33084, and [M + H-H2O]+ at m/z 399.32576 for VitD3, 25(OH)D3 and 1,25(OH)2D3, respectively with a 5 ppm range centered on the exact m/z value were generated. Similarly, EICs of [M + H-H2O]+ at m/z 379.33593, [M + H-H2O]+ at m/z 395.33084 and [M + H-H2O]+ at m/z 411.32576, and [M + H-H2O]+ at m/z 381.31519 for VitD2, 25(OH)D2 and 1,25(OH)2D2, and IS, respectively. The developed method was validated with respect to specificity, sensitivity, linearity, precision, accuracy, matrix effect, and stability. Details of the method were provided briefly in our previous publication  equipped with a quaternary pump, a degasser, a diode-array detector, an autosampler, and a column compartment. Mass spectrometric detection was carried out using an Orbitrap mass analyzer  equipped with an APCI source. The data acquisition was under the control of Xcalibur software. The separation of vitamin D and its metabolites and IS from endogenous substances was achieved using Water’s X select CSH phenyl hexyl column  and mobile phase consisting of a mixture of ammonium formate 10 mM in methanol (A) and acetonitrile:acetone:IPA  in an gradient program mode. The gradient solvent program was set as follows: (Tlication .p < 0.05). ORs with 95% CIs were used to report the results. Figures and tables were generated using GraphPad Prism v.5.0 and Matlab v.r2013b. SAS® V.9.4 software was used for all other statistical analyses.Descriptive statistics of non-normally distributed data was reported as median (interquartile range), while normally distributed data were represented as mean ± SD and categorical variables as number (percentages). Data were tested for normality using the Kolmogorov–Smirnov test. Non-normal data were analyzed by Kruskal–Wallis test and Dunn’s test for pairwise comparisons. Spearman’s rank correlation coefficient was used to test for correlations between serum vitamin D metabolite levels and serum cytokine/chemokine and metabolic hormones with Bonferroni adjustment of multiple analysis. Univariate and multivariable logistic regression analyses were performed to identify covariates as potential confounders on the basis of their significance (p < 0.05) increased in PE and EC subjects as compared with the HP group and GH group. Serum uric acid and creatinine levels significantly (p < 0.05) increased in PE and EC subjects as compared with the HP group and significantly increased (p < 0.05) in EC as compared with the gestational hypertensive disorders group D levels in all subjects is mentioned in Figure In the present study, 96.6% of normal healthy pregnant women and 46.6% of hypertension disorders of pregnant women have adequate (> 30 ng/mL) level of 25(OH)D. A total of 3.3% of HP group and 53.3% of hypertensive disorders in pregnant women have inadequate (<30 ng/mL) level of 25(OH)D Figure B.3, and total 1,25(OH)2D is evaluated by sum of 1,25(OH)2D2 + 1,25(OH)2D3. Total 25(OH)D and total 1,25(OH)2D levels were decreased significantly (p < 0.05) in HDP group as compared with healthy pregnant group D is evaluated by sum of 25(OH)D + 25(OH)D3, 25(OH)D3, 1, 25(OH)2D3, VitD2, and 1,25(OH)2D2 levels were significantly (p < 0.05) lower in the HDP group as compared with the HP group . However, 25(OH)D2 did not show any alteration between the study groups .Five vitamin D metabolites, i.e., VitDp < 0.05) reduction was observed only in PE and EC groups as compared with the HP group  significantly decreased only in EC as compared with the HP group D levels were gradually decreased in three hypertensive disorders, i.e., GH, PE, and EC. However, significant  decreased in GH, PE, and EC subjects as compared with the HP group . 25(OH)D3, VitD2, and 1,25(OH)2D2 levels significantly decreased in PE subjects as compared with the HP group . VitD2 levels significantly (p < 0.05) decreased in EC subjects as compared with the gestational hypertensive subjects . However, any alteration of 25(OH)D2 levels in hypertensive disorders in pregnant women was not found as compared with the HP group .Vitamin DAlteration of cytokines/chemokines and metabolic hormones has been compared among four groups. Serum levels of cytokine/chemokine and metabolic hormones are shown in Table p < 0.05) decreased in pregnant women with hypertensive disorders as compared with the HP group  and DBP  in overall subjects  and serum urea levels  were also negatively correlated with 25(OH)D. Also, significant negative correlations were observed between 1,25(OH)2D and parameters like SBP  and DBP  in overall subjects D shows positive correlation with MCP-1 , MIP-1α , MIP-1β , and TNF-α  levels. Significant (p < 0.05) negative correlations were observed between 1,25(OH)2D with serum creatinine , serum urea , and ghrelin  levels, while positive correlations were observed between 1,25(OH)2D and parameters such as IL-9 , IL-17 , INF-γ , and MIP-1β  levels  and not with DBP . However, there is no association found between 25(OH)D and SBP, DBP in healthy pregnant group .Correlation analysis, between 25(OH)D with various blood pressure parameters in hypertension disorder groups, reveals that only 25(OH)D is negatively associated with SBP  with Bonferroni adjustment for multiple testing. Similarly, in the hypertensive group, systolic blood pressure was positively associated with urinary microprotein , serum creatinine , and serum urea . The diastolic blood pressure of the disease group positively correlated with serum creatinine  and serum urea  levels , SBP is positively correlated with serum creatinine  and negatively correlated with INF-γ  and VEGF  in the HP group. DBP correlated with IL-1ra , IL-5 , TNF-α , and VEGF  in the HP group. In the hypertension disorders group, significantly SBP is negatively correlated with IL-6  and IL-10 . DBP is positively correlated with UMP , IL-10 , and GM-CSF  and negatively correlated with FGF-basic   and (ii) fold change should be ≥ 1.4. Both criteria were used to eliminate background noise to identify markers for specific disease condition. When we consider only fold change ≥ 1.4 as criteria, IL-6, IL-10, IL-13, MCP-1, VEGF, C-peptide, and GIP levels were altered in GH as compared with the HP group  alteration should be statistically significant p < 0.0 and (ii)p = 0.0344], MCP-1 [OR 0.87 (CI 0.77–0.98) p = 0.0237], 25(OH)D [OR 0.88 (CI 0.88–0.94) p = 0.0001], and 1,25(OH)2D [OR 0.62 (CI 0.44–0.86) p = 0.0044] were found to be significant. However, univariate adjusted analysis reveals that IL-17 [OR 0.93 (0.88–0.99) p = 0.0229], MCP-1 [OR 0.76 (0.628–0.927) p = 0.0066], 25(OH)D [OR 0.83 (0.80–0.95) p = 0.0016], and 1,25(OH)2D [OR 0.67 (0.46–0.98) p = 0.0370] were found to be significant when adjusted with gestational age and season of last menopausal period. Two multivariable-adjusted models are shown in Table p = 0.0092], MCP-1 , and 25(OH)D [OR 0.857 (0.758–0.969) p = 0.0141] were predictors of HDP, whereas model 2 confirms that eotaxin [OR 1.06 (1.01–1.11) p = 0.0154] and MCP-1  are predictors of HDP with 1, 25(OH)2D [OR 0.457 (0.239–0.876) p = 0.0023].Multivariable–multivariate logistic regression analyses were performed to test the association of the vitamin D metabolites and cytokine/chemokine and metabolic hormones with the presence of HDP. Table 2. Total 25(OH)D decreased significantly in PE and EC as compared with the HP group. However, the active vitamin D metabolites, i.e., 1,25(OH)2D levels, significantly decreased only in EC subjects. Overall, our data confirming that vitamin D deficiency may contribute to the development of hypertensive disorders in pregnant women. Our results were consistent with previous results, which reported that VitD3 levels significantly decreased in PE and EC subjects  altered parameters with a ≥ 1.4 fold change, inflammatory markers such as IL-6, IL-13, MCP-1, and metabolic hormones such as GIP and C-peptide play important role in GH. However, we could not detect any markers that altered in PE. In case of EC, IL-8 and MIP-1β play major role for the pathogenesis. This clearly indicates that inflammation plays a crucial role in HDP and these alterations were more in early hypertension disorders and severity of EC conditions. In the present study, correlation of vitamin D metabolites with other parameters such as blood pressure, cytokine/chemokines, and metabolic hormones was calculated. Upon considering significance (p < 0.002) with Bonferroni adjustment, negative correlation was observed between vitamin D metabolites  and clinical characteristics such as systolic blood pressure and diastolic blood pressure. However, only 25(OH)D is negatively correlated with serum creatinine and serum urea levels. When significance (p < 0.05) was considered, 25(OH)D is negatively correlated with UMP and positively correlated with inflammatory markers MCP-1, MIP-1α, MIP-1β, and TNF-alpha. Similarly, 1,25(OH)2D is negatively correlated with creatinine, urea, and ghrelin and positively correlated with IL-9, IL-17 IFN-γ, and MIP-1β. These data confirm that correlation of lower vitamin D and its metabolite levels with clinical characteristics and laboratory parameters of GH, PE, and EC in Indian pregnant women. Data from the present study confirm that there is a strong association between cytokines and vitamin D metabolites. Recently, Zerofsky et al. reported that vitamin D supplementation with 2,000 IU/day associated with increased regulatory T cell immunity D and 1,25(OH)Although some good correlations have been found between vitamin D metabolites and cytokine levels, the present study had some limitations. It was a single-center one-point study among South Indian subjects. Thus, the results may not be generalizable to other population. Another limitation of this study is the relatively small number of cases, but these numbers were found to be comparable to previous case–control studies , 60. The2D levels were predictors of HDP as compared to the HP group. However, future studies with large number of subjects are needed to confirm these findings.Our data suggest that vitamin D deficiency may play major role in progression of HDP. Circulatory cytokine/chemokine levels were altered in the HDP group. Furthermore, our analyses suggest that lower vitamin D metabolites were associated with altered cytokines/chemokines and metabolic hormones in HDP. Eotaxin, MCP-1, 25(OH)D, and 1,25(OH)RA and SKB designed the research; wrote the manuscript. RA measured cytokine/chemokine and metabolic hormone levels; performed data analysis. RB, MB, and RS performed the vitamin D metabolites measurement. BV and NM were clinicians. GV helped in statistical analysis and statistical writing.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.


Listeria monocytogenes survival on fresh apples under various storage temperatures. The objective of this study was to comprehensively evaluate the effect of storage temperature on apple fruit decay and L. monocytogenes survival. Unwaxed apple fruits of selected varieties (Fuji and Granny Smith) were dip inoculated in a three-strain L. monocytogenes cocktail to establish ∼3.5 and 6.0 Log10 CFU/apple. Twenty-four hours post-inoculation, apples were subjected to 1, 4, 10, or 22°C storage for up to 3 months. Apples under the different storage treatments were sampled at 1-, 4-, 7- and 14-day for short-term storage under all four tested temperatures, and 2-, 4-, 8-, and 12-week for long-term storage at 1, 4, and 10°C. A set of uninoculated and unwaxed apples were simultaneously subjected to the previously mentioned storage temperatures and sampled biweekly for their total bacterial count (TPC) and yeasts/molds (Y/M) count. During the 2-week short-term storage, L. monocytogenes population on organic Granny Smith apples stored at 1, 4, or 10°C was reduced by 0.2–0.3 Log. When apples were stored at 22°C, there was a 0.5–1.2 Log10 CFU/apple reduction 14-day post storage dependent on the initial inoculation level. During the 12-week cold storage under 1, 4, and 10°C, L. monocytogenes count on organic Granny Smith apples decreased by 0.5–1.5 Log10 CFU/apple for both inoculation levels. L. monocytogenes had similar survival pattern on conventional Granny Smith and Fuji apples with 0.8–2.0 Log10 CFU/apple reduction over a 3-month cold storage period. Interestingly, both TPC and Y/M count were stable regardless of apple variety or cultivation practice during the 12-week storage at all tested temperatures. In summary, while L. monocytogenes did not proliferate on apple surfaces during 12 weeks of refrigerated storage, only a limited reduction of L. monocytogenes was observed in this study. Therefore, the apple industry cannot rely on cold storage alone to control this pathogen. Additional interventions are needed to eradicate Listeria on fresh apples during long-term cold storage.Fresh apples are typically stored for up to 1 year commercially; different apple varieties require different storage temperatures to maintain their quality characteristics. There is sparse information available about  Listeria monocytogenes is a major foodborne pathogen and causes about 1600 illnesses annually in the United States with a mortality rate of ∼16% (L. monocytogenes is listed by the U.S. Food and Drug Administration as a “pathogen of concern” and has been singled out on ready-to-eat (RTE) produce due to its nature as a environmental species, its frequent occurrence in many produce-associated environment and operations, and its high mortality rate  (L. monocytogenes in fresh apples stored at different temperatures during both short- and long-term storage. Granny Smith apples (GSA) were studied since it’s a cultivar involved in the L. monocytogenes outbreaks   requiresutbreaks . Both orutbreaks . ConventListeria monocytogenes NRRL B-57618 (1/2a), NRRL B-33053 (4b), and NRRL-33466 (1/2b) were obtained from USDA-ARS culture collection  and maintained at -80°C in Trypticase Soy Broth  supplemented with 0.6% Yeast Extract  (TSBYE) and 20% (v/v) glycerol. The frozen culture was first activated in TSBYE at 37°C for 24 h statically, and then 1:500 subcultured in TSBYE for additional 24 h at 37°C before use.g for 5 min at 4°C. The resulting bacteria pellets were washed once and then re-suspended in Phosphate Buffered Saline . Equal volume of each L. monocytogenes strain was combined to make a three-strain cocktail and, subsequently, diluted to ∼6 and 3 Log10 CFU/ml for apple inoculation. At the high inoculation level, there were 6.29 ± 0.04, 6.42 ± 0.04, and 6.01 ± 0.07 Log10 CFU/apple on organic GSA, conventional GSA, and Fuji apples, respectively, immediately after inoculation. At the low inoculation level, 3.01 ± 0.06, 3.04 ± 0.07, and 3.48 ± 0.03 Log10 CFU/apple on organic GSA, conventional GSA, and Fuji apples, respectively, immediately after inoculation.Twice-activated bacteria cultures were centrifuged at 8,000 × L. monocytogenes by submerging 15 apples each time in 5 L of three-strain L. monocytogenes cocktail inoculum solution at a selected inoculation level, and gently massaged by turning the apples in the inoculum for 10 min so that microorganisms were evenly distributed in the sample. For each variety at each inoculation level, four inocula were independently prepared with a total of ∼100 apples inoculated per inoculum. Inoculated apples were dried for 24 h at RT (∼22.5°C) before being subjected to storage. Twelve apples were sampled (three apples were randomly sampled from each of four inoculation batches) immediately following inoculation and 24 h post-inoculation for microbial enumeration to confirm inoculum uniformity, inoculation level, and the attachment of L. monocytogenes on fresh apples.Whole fresh unwaxed GSA  and Fuji apples  were donated by Allan Brothers Inc. and Stemilt Growers LLC. All fruit was of commercial maturity (the optimum maturity for the fresh market or storage) . Apples L. monocytogenes were sampled at 1-, 4-, 7- and 14-day intervals for short-term storage, and at 2-, 4-, 8-, and 12-week for long-term cold storage .Both uninoculated and 24 h post-inoculation apples were placed in cardboard boxes and stored at 1°C, 4°C, 10°C, and RT in lab-scale refrigerated incubators. Temperatures were monitored continuously by the built-in sensors, and double-checked weekly using two calibrated digital thermometers  to ensure precision. Actual storage temperatures were 0.93 ± 0.01, 4.17 ± 0.03, 9.76 ± 0.03, and 22.65 ± 0.03°C. Twelve apples (three apples from each of the four inoculation batches) were sampled for microbial analysis from each temperature of each inoculation level  at selected sampling points. Uninoculated apples were sampled at 2-, 4-, 8-, 12-week of storage for total plate count (TPC) and yeasts/molds (Y/M) enumeration. Apples inoculated with L. monocytogenes level higher than 4.0 Log10 CFU/apple where L. monocytogenes counts are much higher than the background flora (<3.0 Log10 CFU/apple). TSAYE overlaid with Modified Oxford agar  plates were used for the enumeration of L. monocytogenes when its counts are lower than 4.0 Log10 CFU/apple to discern from resident background flora of apples. The detection limit of L. monocytogenes is 10 CFU/apple. For uninoculated apples, rub solutions were plated onto TSAYE for TPC and Potato Dextrose Agar  plates for Y/M count. TSAYE plates were incubated at 35 ± 1°C for 48 h. PDA plates were incubated at RT for 5 days.Each apple was placed into a sterile stomacher bag with 10 ml sterile PBS and hand-rubbed for 1.5 min. Wash solutions from each bag were 10-fold serially diluted with sterile PBS, and 0.1 or 1 ml from appropriate dilutions was plated on duplicate appropriate plates. For inoculated apples, TSAYE (TSBYE with 1.5% agar) plates were used for apples with a P-values less than 0.05 were considered significant. Four batches of inocula were independently used to inoculate apples at each inoculation level for each variety. Each individual apple was an experimental unit. At each sampling point, three apples were randomly sampled from each batch (N = 12). Results were reported as mean ± SEM (standard error of mean).Data was analyzed by GLM from Statistical Analysis Systems . Mean values were compared by least significant difference (LSD) multiple-comparison test. 10 CFU/apple of L. monocytogenes, respectively . After 24 h attachment at RT, bacterial counts were 6.31 ± 0.06 and 3.51 ± 0.08 for their respective inoculation levels . There was a significant increase of 0.5 Log10 CFU/apple for the low inoculation level . During 2 weeks of cold storage at 1, 4, and 10°C, bacterial populations did not significantly change . On apples stored at 22°C, however, bacterial counts were reduced by 1.17 and 0.66 Log10 CFU/apple for the high and low inoculation levels, respectively . L. monocytogenes established on fresh apples at either high or low level remained stable during cold storage. At both inoculation levels, only a 0.5–1.5 Log10 CFU/apple reduction on organic GSA during the 3-month cold storage was achieved .Immediately after inoculation, organic GSA exhibited attachment of 6.29 ± 0.04 and 3.01 ± 0.06 LogL. monocytogenes established on conventional GSA during storage. At the high inoculation level, L. monocytogenes maintained the same population size 24 h post-inoculation . At the low inoculation level, the bacterial count was increased by ∼0.7 Log10 CFU/apple 24 h post-inoculation . After 2 weeks of cold storage, L. monocytogenes counts were slightly decreased for the high inoculation level, while no reduction was observed for the low inoculation level . L. monocytogenes counts on apples stored at RT were reduced by 1.1 and 0.6 Log10 CFU/apple for high and low inoculation level, respectively . After 3-month cold storage, a 0.9–2.0 Log10 CFU/apple reduction was achieved for both inoculation levels with 1°C showing the least reduction and 10°C the most . Given the similar behavior of L. monocytogenes on organic and conventional GSA, the fate of L. monocytogenes was only accessed on conventional Fuji apples. Similar results were observed on inoculated conventional Fuji apples . After 2 weeks of cold storage, L. monocytogenes population on fresh Fuji apples was slightly reduced. There was about ∼1.7 and 0.7 Log10 CFU/apple reduction of L. monocytogenes on Fuji apples with high and low inoculation level, respectively, after 2-week of RT storage , a slightly higher reduction than that on GSA. After 3-months, L. monocytogenes populations on Fuji apples were decreased by 0.8, 0.9, and 1.4 Log10 CFU/apple at 1, 4, and 10°C, respectively, for the high inoculation level . For the low inoculation level, L. monocytogenes counts on Fuji apples declined by 1.4, 1.5, and 1.8 Log10 CFU/apple at 1, 4, and 10°C, respectively .Apples from different agricultural practice or different varieties may behave differently in terms of bacterial adhesion and inactivation of the attached microorganisms to their surface. Therefore, we evaluated the fate of 10 CFU/apple for organic GSA, conventional GSA, and conventional Fuji apples, respectively . After 3-month storage, TPC was significantly reduced by 0.2–0.4 Log10 CFU/apple for organic GSA, and 0.5–0.6 Log10 CFU/apple for conventional GSA and Fuji apples . There was no difference in TPC on apples stored under different temperatures.Bacteria, mold, and yeast can cause postharvest decay of apples , reducin10 CFU/apple, respectively . For both GSA  and Fuji apples, Y/M count stayed the same throughout the cold storage with only a slight decline on apples stored at 22°C .The initial Y/M count of organic GSA, conventional GSA, and conventional Fuji apples were 4.74 ± 0.05, 3.67 ± 0.10, and 4.73 ± 0.07 Log10 CFU/apple, L. monocytogenes population on fresh apples did not change during the 24 h establishment on the apple surface at RT, regardless of apple varieties or agricultural practice. Our results were corroborated with other previously published studies where the population of L. monocytogenes inoculated on whole strawberries at ∼7.5 Log10 CFU/berry was not significantly changed 24 h post-inoculation at 21°C  or different varieties (GSA and Fuji apples) behaved similarly with 0.5–1.7 Log10 CFU/apple reduction regardless of the inoculation level after 2-week storage under RT. Consistent with our findings, L. monocytogenes inoculated onto a GSA surface at ∼5 Log10 CFU/apple was reduced by ∼1.5 Log10 CFU/apple after 15-day storage at 25°C  was inoculated onto whole Golden Delicious apples; there was no L. monocytogenes reduction during a 9-day storage at 4°C (L. monocytogenes inoculated on whole tomatoes at ∼3.5 Log10 CFU/g remained at the same level throughout the 21-day storage at 10°C (Fresh apples are commercially stored at ∼1°C for extended periods of time . Apples e at 4°C . Similar at 10°C .L. monocytogenes was similar among 1, 4, and 10°C, which was higher than that of RT storage. It might because apples stored at RT produced more volatile compounds including those with antimicrobial effects such as alcohols, aldehydes, ketones, and esters compared with apples at refrigerated storage (L. monocytogenes death under RT storage. On molecular level, cold storage might increase survival through altering fatty acid composition of the bacterial membrane (L. monocytogenes against other environmental stresses during storage (B, facilitating L. monocytogenes survival (L. monocytogenes inoculated on whole strawberries was reduced by ∼3 Log10 CFU/berry after 7-day storage at 4°C (L. monocytogenes survival at refrigerated temperature. The reduction of L. monocytogenes in strawberries might also be associated with the high moisture and low pH value of the fruit surface.Survival of  storage , which fmembrane , which p storage . Cold tesurvival . Howevere at 4°C . These dL. monocytogenes on apples was temperature-dependent with relatively more reduction at 10°C. In support of our data, less L. monocytogenes were recovered from stainless steel surfaces stored at 10°C than those at 4°C (L. monocytogenes survival on apple surfaces during cold storage.During 3 months of extended cold storage, reduction of e at 4°C . This mie at 4°C . The phee at 4°C  with more at 4°C . Inocula10 CFU/g, respectively, after the 7-day storage at 15°C (Resident bacteria, yeasts, and molds on apples contribute to fruit decay . Thus, t at 15°C . In anot at 15°C . The dif10 CFU/apple reduction of L. monocytogenes on fresh apples. Growers and packers in the apple industry often assume that L. monocytogenes on apples will die off during long-term cold storage. However, a limited reduction of L. monocytogenes on apple surfaces occurred during 12 weeks of refrigerated storage regardless of inoculation levels, apple varieties, or agricultural practices. This indicates that cold storage alone is not enough for eradicating L. monocytogenes from fresh apple surfaces. Additional intervention steps or hurdles for removal of L. monocytogenes are urgently needed during fruit storage or thereafter, such as during the packing of the fruit, in the apple industry.Short-term RT storage resulted in 0.5–1.7 LogLS is the main person who performed the experiment and wrote the manuscript. KE helped with the experiment and manuscript revision. H-CT helped with the experiment. IH provided the experimental apples and helped revise the manuscript. M-JZ guided the experimental design, experiment, as well as the manuscript revision.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.


Serotonin receptor 5-HT6 is involved in cognition and Alzheimer’s disease (AD) development. However, the mechanism of 5-HT6 in AD pathology is not clear.Since 5-HT6 is almost exclusively expressed in the primary cilia, using immunostaining we examined the number of cilia in the hippocampus of AD animal model APP/PS1 mice. By overexpressing and knocking down 5-HT6 in the primary cultured hippocampal neurons, we investigated the roles of 5-HT6 in alternating ciliary morphology. Furthermore, 5-HT6 antagonist was applied to confirm its roles in cognition using the Morris water maze test, Y maze, and fear conditioning.In the present study, we found that the primary cilia were elongated in the hippocampus of APP/PS1 mice compared with WT mice. 5-HT6 regulated cilia length, influenced cilia and axon initial segment (AIS) morphology, and affected localization of ARL13B and AnkG. We also found that, by changing cilia morphology, the AIS was elongated, branched, and more proximal to the cell body in both WT and APP/PS1 mouse neurons. Alterations of cilia also decreased the axonal length in WT and APP/PS1 neurons. Furthermore, in the water maze test, Y maze, and fear conditioning test, 5-HT6 antagonist SB271046 recovered the cognitive impairment of APP/PS1 mice.We suggest that 5-HT6 plays a critical role in AD development through regulating the morphology and function of neuronal primary cilia, which is possibly related to the AIS and axon alterations in AD development.The online version of this article (doi:10.1186/s13195-017-0304-4) contains supplementary material, which is available to authorized users. Alzheimer’s disease (AD) is the most common form of dementia in people aged 65 years and older. AD is associated with impairments in memory, cognition, language, behavior, and personality . The patSwe/London/PS1M146V mice, an established familial AD (FAD) transgenic mouse model [Previous studies have shown that 5-HT6 can influence dendritic growth and neuronal migration , 9, suggse model . Howeverse model . The hedse model . Therefo5-HT6 is highly expressed in the central nervous system (CNS) . High leThe mouse EGFP-tagged 5-HT6 plasmid was provided by Kirk Mykytyn . We constructed 13 mutagenesis plasmids. In each clone, the codons for amino acids 69, 70, 72, 106, 230, 234, 262, 265, 284, and 350 were replaced by triplet nucleotides. Mouse GFP-tagged ARL13B was amplified from cDNA from the mouse brain. 5-HT6-mcherry plasmid was constructed. PEGFP-N3 and mcherry-N3 were purchased from Addgene.SB271046 drug (Santa Cruz Biotechnology), 5-HT6R siRNA (QIAGEN), siRNAs of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3A, 5-HT4, 5-HT5A, 5-HT5B, and 5-HT7 (Santa Cruz Biotechnology), 5-HT6R antibody (Abcam), MAP2 antibody (Abcam), AC3 antibody (Santa Cruz Biotechnology), SSTR3 antibody (Santa Cruz Biotechnology), ARL13B antibody (NeuroMab), ankyrin G antibody (Invitrogen), neurofascin 186 antibody (Abcam), γ-tubulin antibody (Sigma), IFT88 antibody (Proteintech), calbindin antibody (Swant), CamkII antibody (Millipore), Gad antibody (Abcam), Nav1.1 antibody (NeuroMab), Nav1.6 antibody (Alomone), Nav1.2 antibody (NeuroMab), Kv1.1 antibody (Alomone), and AlexaFluor fluorescence antibodies (Invitrogen) were utilized.g for 2 minutes. The cells were resuspended in DMEM-F12 medium with 10% FBS and plated on a coverslip coated with poly-D-lysine (Sigma) in 5% circulating CO2. Neurobasal medium (Gibco) containing Pen-Strep (Invitrogen), B27 (Gibco), and GlutaMAX (Thermo Fisher) was added to the medium after 4 hours. Half of the medium was replaced with fresh medium every 3 days.Primary culture neurons were cultured from the hippocampus of newborn C57 and APP/PS1 mice. The use of mice was in accordance with the regulations of the Peking University Animal Care and Use Committee (LSC-ZhangY-1). Fetal hippocampal samples were dissociated from the brain and digested by 0.25% trypsin (Invitrogen). Digestion was stopped by adding DMEM-F12 medium (Gibco) with 10% FBS (Gibco), after which the tissue was dispersed by a pipettor. After 2 minutes of precipitation, the supernatant was collected and centrifuged at 500 × 2, H2O, and HBS (pH 7.1) was prepared and centrifuged. After adding the mixture to each well for 1–1.5 hours, HBS (pH 6.8) was added to each well to clear the precipitate. Finally, the original medium was added to every well and the cells were placed in a 37 °C incubator.Calcium phosphate transfection was used to transfer siRNA or a plasmid into hippocampal neurons. Neurons were transfected after 3–8 days. A mixed solution containing plasmids, CaClCells were washed in PBS and fixed in 4% PFA (Sigma) at room temperature. Next, cells were permeabilized in 0.1% Triton at 4 °C, after which they were blocked in 5% donkey serum at room temperature and incubated with primary antibodies at 4 °C for 24 hours. Secondary antibodies were added to the cells for 1 hour in the dark. Nuclei were stained by DAPI (Sigma) for 15 minutes. Finally, coverslips with cells were put onto slides for imaging.Each mouse was perfused with 0.9% saline and 4% PFA by myocardial perfusion. Brain slices were coated with OCT (Tissue-Tek) and sectioned by freezing microtomy into sections 35 μm thick. Next, the tissue samples were permeabilized in 0.3% Triton, blocked in 5% donkey serum, incubated in primary and secondary antibodies, and stained with DAPI. The brain slices were blocked in protection liquid.HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen) for 48 hours. Cells were collected using RIPA lysis buffer (R&D) and proteins were obtained by centrifugation. Proteins were incubated with protein A + G agarose beads (Beyotime) and antibodies overnight. Immunoprecipitated proteins were collected after washing three times with PBS (Sigma). The bicinchoninic acid assay (Pierce) was used to measure protein concentration. Proteins were denatured at 100 °C for 5 minutes and separated in 10% SDS-PAGE at 80 mA for 2 hours. They were then transferred to PVDF membrane (Millipore) at 100 mA for 2 hours. The membrane was blocked in 5% BSA (Sigma) in TBS (Sigma) with 0.1% Tween 20 (TBST) at room temperature for 1 hour. The antibodies were diluted at suitable concentration and added into TBST with 5% BSA. Primary antibodies were incubated overnight at 4 °C. HRP-conjugated second antibodies were added after washing three times for 10 minutes by TBST. Enhanced chemiluminescence was used to detect optical density of HRP after washing three times for 10 minutes. BioRad ChemiDox (BioRad) was used to analyze optical density of HRP.swe/PS1ΔE9 and C57 WT mice were subjected to the water maze test. Mice were kept at room temperature with food and water. Both APP/PS1 and WT mice were divided into two groups. Every group included 12 male APP/PS1 mice. The mice in the SB271046 group were given an intraperitoneal injection of SB271046 (10 mg/kg) 2 hours before water maze training and tested for 7 days. In the vehicle group, mice were injected with an equal amount of 0.9% NaCl for 7 continuous days.Twelve-month-old male APPThe water maze test consisted of a circular pool (60 cm in diameter and 50 cm high). The pool was filled with 21–22 °C water at a depth of 30 cm. The hidden platform was 11 cm in diameter and placed 1.5 cm under the surface of the water. The pool was divided into four quadrants. The hidden platform was in one of the four quadrants. Mice were tested in the four quadrants with a fixed order. The starting location was random in the four quadrants. Mice spent four sessions of 90 s in the pool to adapt to the water environment 1 day before the experiment. The test began when a mouse was placed on the quadrants facing the wall and ended when the mouse got onto the platform. If a mouse got onto the platform within 90 s, it remained for 10 s on the platform. If the mouse failed to get onto the platform within 90 s, it was guided to the platform and stayed there for 10 s. The mice were trained and recorded in four quadrants per day for 6 continuous days to measure the swimming speed, time to the platform, and swimming distance with the hidden platform. On day 7, the hidden platform was removed. Then, we tested the time spent in the target quadrant without the platform and the crossing times at the original location of the platform on the seventh day. The position of the mouse was traced by the automatic tracking system (CSI) in real time.swe/PS1ΔE9 and C57 WT mice were used to test in the Y maze. APP/PS1 mice were divided into two groups (n = 12). One group was injected with SB271046 (10 mg/kg) and the other was injected with equal vehicle (0.9% NaCl). C57 WT mice were also divided into SB271046 and vehicle groups (n = 12). Drug or vehicle was injected 2 hours before the Y maze. The apparatus consisted of three arms which were 34 cm long, 8 cm wide, and 14.5 cm high. There are different images pasted on the end of three inner arms, containing a red circle, a black square, and a white triangle. The test began when a mouse was placed on the apparatus facing the wall. After 10 minutes, the experiment was ended. The automatic tracking system (CSI) traced the position of the mouse in real time and calculated the times when mice chose the correct arm. The correct arm was the one which was different form the last and next arms which mice chose.Twelve-month-old male APPswe/PS1ΔE9 and C57 WT mice were used. Both APP/PS1 and C57 WT mice were divided into two groups (n = 12). One group was injected with SB271046 (10 mg/kg) and the other was injected with equal vehicle (0.9% NaCl). Drug or vehicle was injected 2 hours before training or testing for 2 continuous days. Mice were adapted on an operant box for 6 minutes on the day before the experiment. Training started for a period of 6 minutes on day 1. Mice received a 60-dB noise for a period of 30 s, followed by a 0.8-mA foot shock for a period of 5 s. This stimulus was repeated three times with an interval of 150 or 90 s. The mice were then placed in the chamber for 5 minutes on day 2. The device then recorded the duration of the freezing time of the mice.Twelve-month-old male APPt test was used in two groups and one-way ANOVA followed by Dunnett’s test were used in more than two groups. If data were nonnormally distributed, a Mann–Whitney U test was used in two groups and the Kruskal–Wallis test followed by Dunnett’s test were used in more than two groups. For multivariable data analysis, two-way ANOVA followed by Bonferroni test or three-way ANOVA followed by Holm–Sidak test were used.For univariate data analysis, data were detected whether they were normally distributed or not. If data were equally distributed, a 5-HT6 is mainly located at the primary cilia, where it plays an important role . Our resWe first measured the length of cilia in cultured APP/PS1 and WT mouse neurons. APP/PS1 neurons had increased ciliary length. siRNA of 5-HT6 decreased cilia length in hippocampal neurons of APP/PS1 and WT mice Fig. . To meas5-HT6 expression levels were detected by western blot analysis in different brain sections from 6-month-old APP/PS1 or WT mice Fig. . In the To identify the key sites of 5-HT6 involved in its regulation of ciliary length, 13 reported mutations of 5-HT6  were examined. The related sequence information is presented in Additional file In addition, we also found that 5-HT6 mutations altered cilia protein localization. ARL13B, normally located at the primary cilia, disappeared from the primary cilia with 5-HT6 overexpression Fig. . MutatioInterestingly, ankyrin G (AnkG), a typical marker for the AIS , was traThe AIS is the region initiating action potential at the axon. We measured the AIS length with 5-HT6 overexpression. The AIS length was calculated from the 1/3 brightest point near the soma to the 1/3 brightest point far from the soma . OverexpIn order to explore the function of changed AIS morphology in neurons, some ion channels specifically located at AIS were tested. Nav 1.2, Nav1.6, and Kv1.1 were located at the AIS. The position pattern of Nav1.2 at AIS was altered  Endogenous 5-HT6 located at the primary cilia of neurons. ARL13B and SSTR3 were markers of primary cilia. MAP2 was the neuronal marker. γ-tubulin was the marker of the centrosome from which primary cilia grow. (B) Exogenous 5-HT6, which was expressed from 5-HT6-GFP vector, located at the primary cilia of neurons. ARL13B and IFT88 were markers of primary cilia. Scale bars, 10 μm. (DOC 8716 kb)Showing 5-HT6 located at the primary cilia of hippocampal neurons.  Both 5-HT6 and 5-HT3A affected cilia length in hippocampal neurons. AC3 indicated cilia. (B) siRNA of 5-HT6 and 5-HT3A reduced cilia length. 5-HT3A-siRNA, **p < 0.01; 5-HT6-siRNA, *p < 0.05 . Scale bars, 10 μm. All data presented as mean ± SEM. (DOC 4600 kb)Showing siRNAs of 5-HT6 and 5-HT3A decreased cilia length.  5-HT6 regulated cilia length in excitatory neurons. 5-HT6 indicated cilia. CamkII was the marker of excitatory neuron. (B) Overexpression of 5-HT6 increased cilia length. siRNA of 5-HT6 reduced cilia length. 5-HT6, ***p < 0.001; 5-HT6 siRNA, *p < 0.05 . (C) 5-HT6 regulated cilia length in inhibitory neurons. Gad was the maker of inhibitory neuron. 5-HT6 indicated cilia. (D) Cilia length increased by overexpression of 5-HT6 and decreased by siRNA of 5-HT6. 5-HT6, ***p < 0.001; 5-HT6 siRNA, *p < 0.05 . Scale bars, 10 μm. All data presented as mean ± SEM. (DOC 4724 kb)Showing the effect of 5-HT6 on cilia length in excitatory and inhibitory neurons.  Mutations D72A, D106A, and F69L + T70I + D72A recovered the number of branches in cilia. Mutations F69L, D72A, D106A, F284A, A230F + Q234F, and F69L + T70I + D72A restored the percentage of neurons with branched cilia. 5-HT6-EGFP indicated primary cilia. (B) Sequence information of all overexpression, siRNA, and specific point mutations. Underline indicates sequence after the mutation;  indicates original sequence. Scale bars, 10 μm. (DOC 6514 kb)Showing 5-HT6 mutations influenced the number of ciliary branches and the percentage of neurons with branched cilia.  AnkG (purple) located at the primary cilia following overexpression of 5-HT6 in hippocampal neurons. Mutations of 5-HT6 did not recover the location of AnkG in primary cilia. 5-HT6-EGFP (green) indicated primary cilia. (B) 5-HT6 had no effect on total protein level of AnkG in HEK293T cells. Kruskal–Wallis test, p = 0.77. β-actin was the internal control. Scale bars, 10 μm. All data presented as mean ± SEM. (DOC 6089 kb)Showing 5-HT6 influenced the location of AnkG between AIS and cilia.  indicated primary cilia. NF186 (purple) was the marker of the AIS. Scale bars, 10 μm. (DOC 6200 kb)Additional file 7: Figure S7.Showing 5-HT6 mutations influenced the distance between the AIS and cell body in hippocampal neurons. Mutations D72A and K265A restored the distance between the AIS and cell body. 5-HT6-EGFP (green) indicated primary cilia. NF186 (purple) was a marker of the AIS. Scale bars, 10 μm. (DOC 6784 kb)Additional file 8: Figure S8.Showing 5-HT6 mutations influenced the percentage of cells with branched AIS in hippocampal neurons. Mutations F69L + T70I + D72A decreased the percentage of neurons with branched AIS, compared with that of 5-HT6 overexpression. 5-HT6-EGFP (green) indicated primary cilia. NF186 (purple) was the marker of the AIS. Scale bars, 10 μm. (DOC 7506 kb)Additional file 9: Figure S9.A) 5-HT6 affected the Nav1.2 positioning pattern in the AIS. Nav1.2 was located at the AIS of neurons specifically. (B) Overexpression of 5-HT6 increased AIS length and percentage of branched AIS marked by Nav1.2, and decreased distance from AIS to cell body. siRNA of 5-HT6 had the opposite results. AIS length: 5-HT6, *p < 0.05; 5-HT6 siRNA, *p < 0.05 . Distance to cell body: 5-HT6, *p < 0.05; 5-HT6 siRNA, *p < 0.05 . Branched AIS: 5-HT6, *p < 0.05; 5-HT6 siRNA, *p < 0.05 . (C) 5-HT6 had no effect on Nav1.6 positioning pattern in AIS. Nav1.6 was located at AIS of neuron specifically. (D) There was no difference in positioning pattern of Nav1.6 with regulation of 5-HT6. AIS length , distance to cell body , branched AIS . (E) 5-HT6 had no effect on Kv1.1 positioning pattern in AIS. (F) Positioning pattern of kv1.1 unaltered. AIS length , distance to cell body , branched AIS . Scale bars, 10 μm. All data presented as mean ± SEM. (DOC 6017 kb)Showing 5-HT6 regulated AIS morphology marker by Nav1.2.  SB271046 improved the percentage of correct times in APP/PS1 and WT mice in Y maze. APP/PS1 mice chose more wrong arms, compared with WT mice. Vehicle versus SB271046 in WT, *p < 0.05; vehicle versus SB271046 in APP/PS1, *p < 0.05; WT versus APP/PS1 in vehicle, *p < 0.05 . (B) SB271046 increased the duration of freezing in APP/PS1 and WT mice in fear conditioning. The duration of freezing of APP/PS1 mice was longer than that of WT mice. Vehicle versus SB271046 in WT, *p < 0.05; vehicle versus SB271046 in APP/PS1, *p < 0.05; WT versus APP/PS1 in vehicle, *p < 0.05 . (C) Concentration of SB271046 in serum in water maze test. HPLC-MS was used to measure concentration of SB271046 in serum after 2 hours of injecting drug. Concentration of SB271046 was increased gradually from 2 μM on day 1 to 12 μM ion day 7. Kruskal–Wallis test, p < 0.01. (DOC 190 kb)Showing SB271046 restored cognitive impairment of APP/PS1 mice.  5-HT6 influenced arborization of dendrites of excitatory neurons in vitro. CamKII was the maker of excitatory neurons. (B) siRNA of 5-HT6 increased the arborization of dendrites in cultured excitatory neuron APP/PS1 and WT mice. The arborization of dendrites was lower in neurons of APP/PS1 mice, compared with WT mice . (C) 5-HT6 influenced arborization of dendrites of excitatory neuron in vivo. (D) SB271046 enhanced arborization of dendrites of APP/PS1 and WT mice. Arborization of dendrites was lower in neurons of APP/PS1 mice, compared with WT mice . Scale bars, 10 μm. All data presented as mean ± SEM. (DOC 6890 kb)Showing 5-HT6 affected the arborization of dendrites. (


The incidence of nontuberculous mycobacteria (NTM) infections is increasing worldwide, however formal evaluations of the epidemiology of NTM infections are limited. Understanding the trends and true prevalence of NTM is a major priority for optimizing infection control programmes and resources. The purpose of this study was to investigate the epidemiology, clinical manifestations, and radiologic findings in NTM-infected patients at specialized Tuberculosis (Tb) treatment centre in South Korea, which is endemic to Tb, and find solutions to control NTM infections.A retrospective descriptive study was conducted among patients who were diagnosed with NTM from November 2011 to January 2016 at Seoul Metropolitan Government Seobuk hospital, Korea, using medical records and chest radiography results. Prevalence of NTM using national health insurance data was compared to the prevalence and incidence of Tb using National statistics data.Mycobacterium avium complex , and 4 with M. kansasii. Among 31 (48.4%) patients with rapidly growing nontuberculous mycobacteria (RGM) infection, 27 and 4 patients were detected with M. abscessus complex and M. fortuitum complex, respectively. RGM patients were more likely to have current Tb (P = 0.041), cough (P < 0.05), and sputum (P < 0.01) than SGM patients in the univariate analysis, but not in the multivariate analysis.The age- and sex- adjusted prevalence of NTM infection per 100,000 population increased between 2009 (9.4) and 2016 (36.1). However, the prevalence and incidence of Tb per 100,000 population decreased from 106.5 to 74.4, and 81.2 to 61.8, respectively. In total, 64 patients (37 [57.8%] men) were enrolled in the study. Among 33 (51.6%) patients with slowly growing nontuberculous mycobacteria (SGM) infection, 29 were detected with Given the increasing prevalence of NTM infections, precise epidemiological and surveillance data should be obtained by reporting NTM infections to public health authorities. Introducing nucleic acid amplification tests to differentiate between Tb and NTM in smear-positive specimens should be considered. The incidence of nontuberculous mycobacteria (NTM) infections is increasing worldwide, including in the United States . It is kP-value <0.05 was considered significant. The SPSS version 20.0  statistical software package for Windows was used for all statistical analyses. Ethical approval was obtained from Institutional Review Board of Seobuk Hospital for this study (16805–012-HR).Seobuk Hospital, run by the Seoul metropolitan government is a 450-bed specialized Tb treatment centre. We retrospectively reviewed the medical records and chest radiography results of 64 patients who were diagnosed with NTM infections from November 2011 to January 2016 at the Seobuk Hospital. Factors that were assessed in this study included patients’ clinical and epidemiologic characteristics. We used Korean data from the Health Insurance Review and Assessment Service (HIRA)  and is mThe age- and sex- adjusted prevalence of NTM infections per 100,000 population increased between 2009 (9.4) and 2016 (36.1) based on HIRA data ). The following symptoms were observed: chronic fatigue , cough , sputum , hemoptysis , dyspnoea on exertion , fever , chest pain , and weight loss . The mean BMI was 19.7 ± 3.4 kg/m2. The mean ± SD of laboratory findings were as follows; haemoglobin 12.3 ± 1.7 g/dL, white blood cell (WBC) count 6961.7 ± 2237.2 cells/mm3, protein 7.1 ± 0.7 g/dL, albumin 3.8 ± 0.5 g/dL, aspartate aminotransferase (AST) 24.4 ± 12.3 IU, alanine aminotransferase (ALT) 16.4 ± 14.3 IU, glucose 104.4 ± 24.2 mg/dL, C-reactive protein (CRP) 3.1 ± 3.5 mg/dL, creatinine 0.8 ± 0.2 mg/dL.In total, there were 64  NTM patients at the Seobuk hospital from November 2011 to January 2016. The mean age of the patients was 63.5 ±15.6 years. Approximately 40% of patients had initiated anti-Tb medications during the study period within a mean duration of 68 days. Among the 33 (51.6%) SGM patients, 29 and 4 patients had P = 0.041), cough (P < 0.05), and sputum (P < 0.01) than patients with SGM  to 2008 (n = 133) [In 1987, the US Centers for Disease Control and Prevention (CDC) estimated a population NTM disease rate of 1.8/100,000 between 1981 and 1983 in North America [n = 133) , 12. Onen = 133) , as was Notification of Tb but not of NTM, is mandatory in Taiwan. Partly due to the strict regulation of Tb notification, several patients infected with NTM were notified as Tb cases , imposing additional burdens on the public health system . NTM patM. kansasii and M. abscessus [M. abscessus were the most common SGM and RGM infections, respectively. These findings were similar to those from the study by Koh et al. in which M. abscessus was found to be the second most common pathogen responsible for lung diseases caused by NTM, after MAC in the Republic of Korea [The most often isolated cases in the USA are MAC cases, followed by bscessus . In Japabscessus . In thisof Korea , 19. Theof Korea .Pulmonary presentation of NTM was characterized by the presence of bronchiectasis, cavitation, pneumoconiosis, fibrosis, and nodules. We also observed these radiographic findings, such as bronchiectasis (44.6%), cavities (43.1%), destroyed lung (18.5%), nodules (52.3%), infiltration (78.5%), and fibrosis (70.8%). The cardinal symptoms of fever, chest pain, and unintentional weight loss were prevalent in the survey participants with NTM. We also observed cough (93.8%), sputum (93.8%), chronic fatigue (75.4%), dyspnoea on exertion (30.8%), hemoptysis (27.7%), weight loss (26.2%), chest pain (10.8%), and fever (9.2%) among patients enrolled in this study.Misdiagnosis or delayed diagnosis of NTM, due to similarity in clinical presentation to Tb, can result in inappropriate isolation with psychosocial stress  and seriTo the best of our knowledge, this was the first study conducted using epidemiologic NTM data from a specialized Tb treatment centre in Korea, a country that is endemic to Tb. As Tb and NTM show similar clinical manifestations, and relatively lower incidence of NTM, NTM has been neglected until now limiting the available epidemiologic data on NTM. Our study highlights the negative impact of NTM on Tb surveillance and control system. Understanding the epidemiology of NTM lung diseases remains crucial to addressing this burgeoning public health challenge . It is eGiven the increasing incidence of NTM infections worldwide, precise epidemiological and surveillance data should be obtained through the reporting of NTM infections to public health authorities. NAA tests should be considered to differentiate Tb and NTM in smear-positive specimens.


Alzheimer’s disease (AD) is a chronic neurodegenerative disease leading to dementia, which is characterized by progressive memory loss and other cognitive dysfunctions. Recent studies have attested that noninvasive repetitive transcranial magnetic stimulation (rTMS) may help improve cognitive function in patients with AD. However, the majority of these studies have focused on the effects of high-frequency rTMS on cognitive function, and little is known about low-frequency rTMS in AD treatment. Furthermore, the potential mechanisms of rTMS on the improvement of learning and memory also remain poorly understood. In the present study, we reported that severe deficits in spatial learning and memory were observed in APP23/PS45 double transgenic mice, a well known mouse model of AD. Furthermore, these behavioral changes were accompanied by the impairment of long-term potentiation (LTP) in the CA1 region of hippocampus, a brain region vital to spatial learning and memory. More importantly, 2-week low-frequency rTMS treatment markedly reversed the impairment of spatial learning and memory as well as hippocampal CA1 LTP. In addition, low-frequency rTMS dramatically reduced amyloid-β precursor protein (APP) and its C-terminal fragments (CTFs) including C99 and C89, as well as β-site APP-cleaving enzyme 1 (BACE1) in the hippocampus. These results indicate that low-frequency rTMS noninvasively and effectively ameliorates cognitive and synaptic functions in a mouse model of AD, and the potential mechanisms may be attributed to rTMS-induced reduction in Aβ neuropathology. PS45 transgenic mice carry human presenilin-1 cDNA with the M146V mutation. The genotype of the mice was confirmed by PCR using DNA from tail tissues  and APP23/PS45 double mutant transgenic mice (1.5-month old) were selected. All mice were housed in plastic cages in a temperature-controlled (21°C) colony room on a 12 h light/12 h dark cycle, and all electrophysiological and behavioral experiments were conducted during the light cycle. Food and water were available Both WT and AD mice are randomly divided into two subgroups: WT, WT+rTMS, AD and AD+rTMS. Both AD+rTMS and WT+rTMS groups were treated with one session of low-frequency rTMS daily (between 14:00 and 17:00) for 14 consecutive days . Similar to our previous report  and 28 AD (15 for sham treatment and 13 for rTMS treatment) mice were used to perform the Morris water maze task as described previously  prior to decapitation as described previously  and 12 AD (7 for sham treatment and 5 for rTMS treatment) mice at age of 3 months were deeply anesthetized with urethane  and transcardially perfused with artificial cerebral spinal fluid . Then slices were blocked with 5% non-fat milk and incubated overnight with mouse monoclonal 4G8 antibody (1:500) at 4°C to label Aβ. Every sixth slice with the same reference position was mounted onto slides for staining. Plaques were visualized by the avidin-biotin-peroxidase complex (ABC) and DAB  method. All visible plaques were counted by microscopy at ×40 magnification in a double-blind manner. The mean plaque count per slice was recorded for each mouse as described previously  and one-half of the brains was immediately frozen for protein extraction. The other half of the brains for immunocytochemical staining was fixed with 4% PFA for 24 h at 4°C. Then cryoprotected with 30% sucrose until the brain sank to the bottom. The brain was coronally sectioned into 20 μm slices. 3% Hg for 10 min at 4°C. Supernatant was collected, and protein concentration was determined by BCA protein assay kit . Equal amounts of protein samples were mixed with 4× sample buffer and boiled at 95°C for 5 min. Proteins were separated on 10% tris-glycine SDS-PAGE or 16% tris-tricine SDS-PAGE and then transferred to immobilon-PTM polyvinylidene fluoride (PVDF) membranes with an electrophoresis apparatus . The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature and then incubated overnight at 4°C with primary antibody. After TBST washing 3 × 5 min, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After another three times washing with TBST, the protein was visualized in the Bio-Rad Imager using ECL Western blotting substrate (Pierce). Immunoblotting with anti-β-actin  was used to control equal loading and protein quality. Anti-APP antibody (1:1000) C20 was used to detect APP and its C-terminal fragment (CTF) products. Both Anti-APP and Anti-BACE1 antibody (1:1000) were obtained from professor Weihong Song in the University of British Columbia, Vancouver, BC, Canada. The band intensity of each protein was quantified by the Bio-Rad Quantity One software.After behavioral testing, the hippocampal tissues from each mouse were collected for western blotting as described previously  as the between-subjects factor. For immunohistochemical and immunoblotting assays, all data were analyzed by the Student’s t-test and nonparametric Mann-Whitney U test, respectively. Significance level was set at p < 0.05.For behavioral and electrophysiological experiments, all data are presented as mean ± SEM. Spatial learning and swimming speed data were analyzed by a two-way ANOVA, with treatment (group) as the between-subjects factor and learning day as the within-subjects factor. All the other data were analyzed by a one-way ANOVA followed by n = 16, 70.3 ± 8.8 s for day 2, 48.14 ± 6.9 s for day 3, 36.7 ± 6.1 s for day 4, 18.1 ± 1.7 s for day 5; AD: n = 15, 93.8 ± 5.8 s for day 2, p < 0.05 vs. WT, 72.6 ± 8.4 s for day 3, p < 0.05 vs. WT, 57.8 ± 68.8 s for day 4, p < 0.05 vs. WT, 45.7 ± 5.1 s for day 5, p < 0.01 vs. WT; Figure n = 13, 64.9 ± 7.9 s for day 2, p < 0.05 vs. AD, p > 0.05 vs. WT; 43.8 ± 6.6 s for day 3, p < 0.05 vs. AD, p > 0.05 vs. WT; 32.6 ± 8.6 s for day 4, p < 0.05 vs. AD, p > 0.05 vs. WT; 22.8 ± 4.2 s for day 5, p < 0.01 vs. AD, p > 0.05 vs. WT; Figure n = 16, 45.7 ± 2.3 s in the target quadrant, 19.6 ± 1.5 s in the opposite quadrant; AD: n = 15, 36.1 ± 2.6 s in the target quadrant, p < 0.01 vs. WT; 28.4 ± 2.3 s in the opposite quadrant, p < 0.01 vs. WT; Figure n = 16, 5.4 ± 0.5; AD: n = 15, 1.4 ± 0.3, p < 0.01 vs. WT; Figure n = 13, 44.1 ± 2.8 s in the target quadrant, p < 0.05 vs. AD, p > 0.05 vs. WT; 20.8 ± 2.5 s in the opposite quadrant, p < 0.05 vs. AD, p > 0.05 vs. WT; Figure n = 13, 5.2 ± 0.9, p < 0.01 vs. AD, p > 0.05 vs. WT; Figure To test the potential rescue effect of rTMS on AD, we first examined the effects of low-frequency rTMS on spatial learning and memory by using a hippocampus-dependent learning and memory task, the Morris water maze, in APP23/PS45 double transgenic AD mice. The AD transgenic mice displayed a significant deficit in spatial learning, as reflected by taking much longer to find the hidden platform than WT control on day 2–5 . The results showed that rTMS treatment significantly decreased the levels of APP (AD+rTMS: 67.1 ± 10.0% relative to AD, p < 0.05 vs. AD; Figures p < 0.05 vs. AD; Figures p < 0.05 vs. AD; Figures p < 0.01 vs. AD; Figures We next want to determine whether rTMS-ameliorated cognitive function and synaptic plasticity in APP23/PS45 mice is attributed to a decrease in Aβ neuropathology such as neuritic plaque formation, APP processing and BACE1 expression. The results showed that the number of plaques was decreased in AD mice treated with rTMS compared with that in AD mice (AD: In the present study, we confirm that spatial learning and memory as well as hippocampal LTP are significantly impaired in APP23/PS45 double transgenic mice, and demonstrate that low-frequency rTMS treatment alleviates AD-related neuropathology, which may contribute to the amelioration of cognitive function and synaptic plasticity. We have therefore provided evidence that rTMS is an effective and non-invasive brain stimulation method to treat AD and related memory disorders.So far, no cure has been found for AD. Recently, non-invasive rTMS has been applied to patients with AD and displays beneficial effects on various cognitive functions (Cotelli et al., So far, the mechanism underlying cognitive improvement after rTMS treatment in patients with AD is still poorly understood. Preclinical studies suggested that inhibition of Aβ production by altering APP processing at the β- or γ-secretase site could potentially be avenues for AD drug development (Weggen et al., Alternatively, it has been well documented that neuronal hyperactivity has been observed in the hippocampus (Palop et al., Overall, our study demonstrates that low-frequency rTMS can ameliorate the deficits of cognitive and synaptic functions through reducing BACE1 and APP processing in APP23/PS45 double transgenic mice of AD, suggesting that low-frequency rTMS may serve as a highly effective anti-amyloid treatment in AD. However, the molecular mechanisms underlying the beneficial effects of low-frequency rTMS on AD need to be further investigated in the future.ZH, TT, YD, LC, MF, YY and LZ performed the research. ZD designed the research study. TT, WS and ZD contributed essential reagents or tools. ZH and ZD analyzed the data. ZH and ZD wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The catalyst retained its high catalytic power after recycling five times. Furthermore, the determined non-catalyzed activation energy was 76 kJ·mol−1 and 54 kJ·mol−1 with the D001 resin catalyst. The product’s chemical structure was investigated through FT-IR and 1H NMR. The viscosity, flash point, pour point, and anti-wear properties of the product were improved compared with those of epoxidized biodiesel.The optimization and kinetics of the ring-opening reaction of an epoxidized biodiesel (epoxidized rapeseed oil methyl ester) (EBD) with 2-ethyl hexanol (2-EH) were studied. The determined optimum conditions were 4:1 2-EH/oil molar ratio, 90 °C, 18 h, and 7 wt % of Amberlyst D001 (dry) catalyst; the product’s oxirane oxygen content was 0.081% with 38.32 mm Renewable resources are increasingly important for many fields including the development of environmentally friendly lubricants. Mineral oils are widely used in various lubricants. However, residual mineral oils are retained for a very long time in water or soil because of their poor biodegradability, creating serious environmental issues ,2. FurthMany strategies are applied to reduce unsaturation in vegetable oil, including selective hydrogenation ,8,9, epoet al. showed that the pour point and tribological property of rapeseed oil’s ring-opening product were improved compared with those of epoxidized vegetable oil  is the molar concentration of the epoxy group, mol·L−1; [2-EH] is the 2-EH molar concentration, mol·L−1; m and n are the reaction orders with respect to the epoxy group and 2-EH, and k is the rate constant of the ring-opened reaction.The epoxy group of the epoxidized biodiesel is a ternary ring, which reacts easily with alcohol and acid. Generally, the kinetic model of EBD with 2-EH can be expressed as follows:k′ is the rate constant when 2-EH is in large excess, i.e.,0/[Ep]) and the reaction time, indicating first-order kinetics for the oxirane concentration. First order kinetics is also observed for similar reactions with vegetable oils [k′  is 0.0164 h−1, 0.0469 h−1, 0.0925 h−1, and 0.1591 h−1. Applying the D001 resin catalyst, the rate constant k′  is 0.0437 h−1, 0.0873 h−1, 0.1478 h−1, and 0.2178 h−1.If the molar concentration of 2-EH in a set of experiments is in large excess as compared to that of the oxirane oxygen, the rate equations can be simplified to:ed by GC a. A lineble oils ,28. WithaE, as an important parameter in the reaction kinetic equation, is determined by the reaction rate constant and the temperature. In general, their relationship can be expressed by the Arrhenius formula:The activation energy k and 1/T (aE/R) of −9.116 × 103. Therefore, the activation energy (aE) is 76 (±7) kJ·mol−1. In the catalyzed reaction, the slope (−aE/R) is −6.479 × 103 with an aE of 54 (±4) kJ·mol−1 , and the activation energy was 78.56 (±1.63) kJ·mol−1 [Lin kJ·mol−1 . However−1 to 2855 cm−1 (methylene asymmetric stretching), 1741 cm−1 (triglycerides carbonyl stretching), 1464 cm−1 (CH2 bending vibration), 1377 cm−1 , and 724 cm−1 (CH2 rocking vibrations). The epoxide peaks between 823.4 cm−1 and 844.6 cm−1 do not appear in the product. Additionally, the broad and strong absorption peak of the alcoholic hydroxyl at 3453 cm−1 confirms the presence of the ring-opening product. Moreover, C-O absorption peaks of aliphatic alcohols and aliphatic ethers can be found between 1200 cm−1 and 1025 cm−1.The FTIR spectra of the product and the raw material are presented in 1H NMR spectra of EBD and 2-EHBD are presented in 1H NMR peaks at δ 2.25–2.5 ppm for CH2 protons α to >C=O, 1.68–1.85 ppm for CH2 protons connected with the epoxy groups, 1.15–1.28 ppm for all other CH2 protons, 0.8–1.0 ppm for terminal CH3 protons, and 2.8–3.2 ppm for CH protons of the epoxy group. The epoxy peaks at 2.8–3.2 ppm decrease, and a new CH2 proton peak on the side chain (–CH–O–CH2–CH(C2H5)(CH2)3CH3) appears at 3.95–4.01 ppm. The presence of a peak at δ 5.81 suggests a furan product in our system, which was previously reported by Bantchev et al. [et al. [The  [et al. .PB (maximum non-seizure load)  . The proSEM images of the stainless steel plate after the anti-wear experiment appear in The EBD was obtained from the laboratory . Oxirane oxygen 4.95% was used, equivalent to 0.3094 mol oxirane in 100 g of EBD. Amberlyst D001 (dry) (ionic from H+) was supplied by Xin Rui Technology Co. Ltd . The EBD was prepared through the epoxidation procedure for canola oil  using hy1H NMR spectroscopy. The FTIR spectra were obtained using a Nicolet Protege-460 FT-IR spectrometer  with KBr crystals in thin-film after 64 scans at a resolution of 4 cm−1. The 1H NMR spectra were obtained with a Bruker  Avance III-400 spectrometer. The weight loss of the sample was measured using a Diamond DSC Thermogravimetric Analysis  in the presence of air (flow rate: 20 mL/min) and a constant heating rate of 10 °C/min. The tribological tests were performed on a MRS-10A-four-ball tribometer, .The product’s oxirane oxygen content was determined according to the AOCS method Cd 9-57. The structure of the epoxidized biodiesel and the ring-opening product were identified through Fourier-transform infrared (FTIR) and 4, and 2-EH was removed by vacuum distillation. The reaction equation is presented in Epoxidized biodiesel (50.0 g), 2-EH  and Amberlyst D001 (dry)  were added to a 500 mL three-necked round-bottomed reaction flask (250 mL capacity), equipped with a stirrer, a thermometer, and a reflux condenser. The mixture was placed in a water bath and heated to 90 °C, while stirring for 18 h. After the reaction was completed, the mixture was poured into a separatory funnel. The sample was washed with 100 mL hot distilled water (50 °C), 50 mL hot dilute alkali , and 100 mL hot distilled water (50 °C) again. Finally, the product was dehydrated over MgSO1H NMR spectrometry. The ring-opening product exhibits good viscosity, high flash point, low pour point, small steel ball surface wear-scar diameter, and excellent tribological properties.The 2-EHBD is synthesized at high yield from the reaction of EBD with 2-EH in the presence of Amberlyst D001 (dry) catalyst. The optimum reaction conditions are 4:1 2-EH/oil molar ratio, 90 °C, 18 h reaction time, and 7 wt % catalyst. The catalyst is highly stable under the reaction conditions and can be easily recycled and reused. The kinetic model and activation energy are, furthermore, determined. The chemical structure of the product is determined by FTIR and