Anoectochilus roxburghii is a widespread herbaceous plant with high medicinal value. Wild A. roxburghii resources face extinction due to their slow growth rate and over exploitation. The growing market demand has led to advances in the field of artificial planting of A. roxburghii. Methods to increase the economic benefits of cultivation and the production of medicinal ingredients are very useful.A. roxburghii was exposed to red light, blue light (BL), yellow light (YL), green light, or white light as supplemental lighting at night (18:00–02:00) in a greenhouse or were left in darkness  to investigate the effects of various light qualities on growth indices, photosynthetic pigments, chlorophyll fluorescence, root vitality, stomatal density, soluble proteins, sugars, and the accumulation of secondary metabolites.A. roxburghii growth and secondary metabolite accumulation. Leaf number, stem diameter, fresh weight, dry weight, chlorophyll a content, and secondary metabolite  content increased significantly. YL treatment showed significantly higher soluble sugar and polysaccharide contents than the control.Supplementation of BL had a positive effect on Anoectochilus, so future research will focus on the combination of blue and YL.BL treatment was conducive to promoting the growth and accumulation of secondary metabolites ; YL treatment significantly increased the content of soluble sugar and polysaccharides more than the control. Polysaccharides and total flavonoids are important medicinal ingredients of  Anoectochilus roxburghii (Orchidaceae) is a valued perennial herb that is used for medicinal and ornamental purposes in China and many Asian countries. The chemical composition of A. roxburghii includes flavonoids, polysaccharides, alkaloids, amino acids, trace elements, organic acids, cardiac glycosides, and steroids   treatments (Rehmannia glutinosa (Gaertn.) DC. Maximal total flavonoid content and total phenolic content was observed when Prunella vulgaris was grown under BL (Many studies indicate that light has a positive effect on the growth and accumulation of plant secondary metabolites . Light itivities . Anoectovonoids) . In additabolism . In insteatments . Whereasunder BL . Howeverunder BL .Anoectochilus is sensitive to light, many studies about light intensity of Anoectochilus have been reported, but the effects of light-emitting diode (LED) light quality on the yield and bioactive substance content of A. roxburghii have not been studied. Thus, the hypothesis of this study was that supplemental lighting with different LED light qualities at night has a positive effect on the growth and secondary metabolism of A. roxburghii. This study aimed to use energy-saving LED lamps to explore the effects of supplemental lighting with different light qualities on growth, photosynthetic pigments, chlorophyll fluorescence, root vitality, stomatal density, soluble protein, sugars, and accumulation of secondary metabolites in the cultivation stage of A. roxburghii and to determine the optimal light quality to increase the medicinal value and economic benefits for Anoectochilus growers.A. roxburghii cv. Narrow-leaf.Test species: A. roxburghii plantlets were obtained from Diyuan Biotechnology Co., Ltd. . Tissue-culture bottles were opened to acclimate the plantlets to the environmental conditions of the greenhouse for 15 days. The average fresh weight of the acclimated plantlets was 0.55 g and leaf number was 3.8. These plantlets were transplanted to plastic trays (50 × 30 × 5 cm) at a density of 110 plants per tray. The cultivation soil was composed of peat moss and perlite in a 2:1 ratio by volume.The experiment was performed in December of 2016 in the greenhouse of the Fujian Institute of Subtropical Botany, China . The day (highest)/night (lowest) temperatures of the greenhouse were 23/12 °C. −1 (18:00–02:00). A. roxburghii grown under different light qualities at night are shown in In the supplemental lighting experiment, natural light was used during the day and LED lamps were used as supplemental lighting at night. LED lamps were provided by Xiamen Guangpu Electronics Co., Ltd. . Plantlets were subjected to five different light quality treatments: RL , BL , yellow light , green light , WL (420–780 nm) and a control without supplemental lighting (CK). The spectral characteristics of the lamps are shown in −2·s−1 ). Different treatments were insulated from one another by black shading materials.The LED lamps were placed above of the plantlets. The conclusion from After 40 days of treatment, 40 plantlets were chosen randomly from each repeat for subsequent testing. A total of nine of these 40 plantlets were selected randomly for biomass analysis within each treatment. And six of these 40 plantlets were selected randomly for root activity analysis and stomata observation within each treatment. The others were stored in a −80 °C refrigerator after flash freezing.The leaf numbers, length and width of each leaf, stem diameter, stem length, root length, root numbers, fresh weight, and dry weight were separately determined. The fresh weight of the plantlets were measured with an electronic balance , and the plantlets were dried to a constant weight at 80 °C to determine their dry weight. The stem diameter was measured with a vernier caliper . The stem length was measured from the top of the plantlet to the base of the first root. The root length was measured from the top of the root tip to the main stem base.Chlorophyll content was determined following the method from 0), and qN was calculated as (Fm′−F0′)/(Fm−F0).Chlorophyll fluorescence was measured with a MINIPAM fluorometer  . Leaves g for 5 min, the residues were re-extracted under the same conditions. The respective extracts and re-extracts were pooled before analysis. All samples were extracted in duplicate.Fresh leaves (0.2 g) were homogenized in liquid nitrogen and soluble sugar was extracted in boiling ultrapure water (15 ml) for 20 min. Following centrifugation at 15,000×−1.The soluble sugar content was evaluated using the anthrone colorimetric method , 194–197−1.The reducing sugar content was evaluated by 3,5-dinitrosalicylic acid (DNS) colorimetry , 197–1992). Stomatal density = Number/Field/Field area.Pretreatment of samples was performed based on the method of Root activity was determined using the triphenyltetrazolium chloride method , 119–120g for 10 min. The supernatant was saved and then one ml of extract and five ml of coomassie brilliant blue G-250  were thoroughly mixed. To generate a standard curve, 0, 0.2, 0.4, 0.6, 0.8, or one ml of 100 g l−1 of bovine serum albumin  was added to six volumetric flasks, and distilled water was added to reach a total volume of one ml. The optical density was measured at 595 nm.The soluble protein content was measured following the method of For total flavonoids estimation, the method of The total polyphenol content was determined with the Folin–Ciocalteu reagent according to the method of P set to 0.05).SPSS Version 13.0  was used for all statistical analysis. Data was analyzed by one factor, light quality. The results were analyzed by one-way analysis of variance. Duncan’s multiple range test was employed to detect differences between means  were observed under the BL treatment, at 17.8% higher than the control. However, the leaf numbers did not differ significantly between the RL (5.22 ± 1.09), YL (5.00 ± 0.87), GL (5.11 ± 0.60), and WL (5.33 ± 0.71) treatments and the control. The leaf length, leaf width, stem length, and root numbers did not differ significantly between all supplemental lighting treatments and the control. Stem diameter was significantly greater in the BL treatment  than in the control. Root length of the RL , BL , and YL  treatments was significantly higher than in the control. Fresh weight  and dry weight  of the BL treatment were significantly greater than in the control. However, the RL, YL, GL, and WL treatments had no significant effect on the yield of A. roxburghii.The growth indices of −1 FW, P < 0.05) was observed with the BL treatment, at 38.98% higher than in the Control. In this study, chlorophyll b content was not affected significantly by different light qualities. The chlorophyll a + b content showed a similar trend compared to chlorophyll a. However, different light qualities had no significant effect on the chlorophyll a + b content of A. roxburghii. The highest chlorophyll a + b content (1.19 ± 0.11 mg g−1 FW) was observed with the BL treatment, at 22.6% higher than in the control (0.97 ± 0.03 mg g−1 FW), but this increase was not statistically significant (P > 0.05).As shown in A. roxburghii (P < 0.05), at 31.9% higher than in the control. A significant decrease in yield was observed in RL  treatment, at 31.9% lower than in the control. However, the yield value did not differ significantly between the BL (0.56 ± 0.05), GL (0.38 ± 0.17), and WL (0.34 ± 0.11) treatments and the control.Different light qualities had variable effects on the chlorophyll fluorescence of xburghii . The YL There was no significant difference in the qP value between all of the supplemental lighting treatments and the control.P < 0.05), GL treatment , and WL treatment  than in the control. However, the qN value did not differ significantly between the BL (0.55 ± 0.20) and YL (0.32 ± 0.06) treatments and the control.The qN value was significantly higher in the RL treatment  than in the control. A significantly lower soluble sugar content was observed in the WL treatment  than in the control. The soluble sugar content did not differ significantly between the RL (30.52 ± 2.03 mg g−1 FW), BL (27.38 ± 1.45 mg g−1 FW), and GL (28.1 ± 1.32 mg g−1 FW) and control treatments (27.10 ± 4.21 mg g−1 FW) (The soluble sugar content was significantly higher in the YL treatment (27.10 ± 4.21 vs. 32.51 ± 3.32 mg g g−1 FW) .−1 FW), BL (5.17 ± 0.22 mg g−1 FW), YL (5.37 ± 0.13 mg g−1 FW), GL (5.04 ± 0.39 mg g−1 FW) or control (5.54 ± 0.38 mg g−1 FW) treatments, but a significantly lower reducing sugar content was observed in the WL treatment  (The reducing sugar content did not differ significantly between the RL (5.62 ± 0.26 mg g < 0.01) .−1 FW, P < 0.05), at 25.9% higher than in the control treatments. A significantly lower polysaccharide content was observed in the WL treatment . However, the polysaccharide content did not differ significantly between the RL (24.90 ± 1.80 mg g−1 FW), BL (22.21 ± 1.29 mg g−1 FW), GL (23.06 ± 1.18 mg g−1 FW) and control (21.55 ± 4.58 mg g−1 FW) treatments , BL (1016.76 ± 140.50 μm2), YL (921 ± 110.54 μm2), GL (961.44 ± 138.74 μm2), and WL (1051.58 ± 69.33 μm2) treatments and the control (1003.55 ± 99.48 μm2).The stomatal area of −2, P < 0.01) than in the control. However, the stomatal density did not differ significantly between the RL (17.4 ± 2.82 mm−2), BL (20.4 ± 3.07 mm−2), GL (20 ± 3.83 mm−2), WL (17.3 ± 2.42 mm−2) treatments and the control (17.9 ± 2.64 mm−2).As shown in −1·h−1, P < 0.05) than in the control. However, the root vitality did not differ significantly between the RL (0.33 ± 0.08 mg g−1·h−1), BL (0.35 ± 0.01 mg g−1·h−1), GL (0.46 ± 0.02 mg g−1·h−1), and WL (0.41 ± 0.02 mg g−1·h−1) treatments and the control (0.41 ± 0.10 mg g−1·h−1)  .−1 FW, P < 0.01) than in the control. However, soluble protein content did not differ significantly between the RL (2.83 ± 0.32 mg g−1 FW), YL (2.63 ± 0.09 mg g−1 FW), GL (3.13 ± 0.36 mg g−1 FW), and WL (2.72 ± 0.17 mg g−1 FW) treatments and the control (3.05 ± 0.30 mg g−1 FW) (A significantly lower soluble protein content was observed in the BL treatment (3.05 ± 0.30 vs 2.30 ± 0.28 mg g g−1 FW) .−1 FW, P < 0.01), at 24.2% higher than in the control treatment (−1 FW), YL (1.59 ± 0.08 mg g−1 FW), GL (1.72 ± 0.02 mg g−1 FW), WL (1.56 ± 0.14 mg g−1 FW) and control (1.57 ± 0.20 mg g−1 FW) treatments.A significantly higher total flavonoid content was observed in the BL treatment  than in control treatment. A significantly lower total polyphenol content was observed in the GL treatment  than in the control. The total polyphenol content did not differ significantly between the RL (4.38 ± 0.43 mg g−1 FW), YL (3.52 ± 0.04 mg g−1 FW), WL (4.12 ± 0.71 mg g−1 FW) and control (3.70 ± 0.12 mg g−1 FW) treatment  compared to controls. These results suggest that the supplementation of LED BL is a good choice for A. roxburghii growers to increase production and secondary metabolite content. This is conducive to meeting the increasing market demand while avoiding the extinction of wild resources of A. roxburghii. Considering the promotion of the soluble sugar and polysaccharide contents after the YL treatment, further research will focus on the combination of BL and YL.The supplementation of LED BL had a positive effect on 10.7717/peerj.5274/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.5274/supp-2Supplemental Information 2Click here for additional data file.


Almonds have shown to beneficially modify some cardiovascular risk factors in clinical trials conducted in diverse ethnic populations but this relationship has never been tested in Koreans. Thus, we tested the impact of almonds consumed as a snack within the context of a typical Korean diet on cardiovascular risk factors.2) consumed either 56 g of almonds or isocaloric cookies daily for 4 weeks.We conducted a randomized, crossover trial in a free-living setting with a 2-week run-in period, two 4-week intervention phases, and a 2-week washout period between interventions. Eighty four overweight/obese participants . Almonds increased plasma α-tocopherol by 8.5% (P ≤ 0.05) from the baseline and tended to increase its value as compared to cookies (P = 0.055). Neither the almonds nor cookies altered plasma protein carbonyls, MDA or oxLDL. Of serum inflammatory markers, IL-10 was decreased by almond intake (P ≤ 0.05), and ICAM-1, IL-1β, and IL-6 tended to be lower with almonds, compared to the cookies.Mean % daily energy intake at baseline was 64.8, 21.3, and 14.9% from carbohydrate, fat, and protein, respectively. The addition of 56 g of almonds daily decreased carbohydrate energy to 55.0%, increased fat to 32.0%, and maintained protein at 14.7%. Consuming the almonds increased intake of MUFA by 192.3%, PUFA by 84.5%, vitamin E by 102.7%, and dietary fiber by 11.8% and decreased % energy from carbohydrate by 14.1%. Total caloric intake was increased by the almonds, but body weight, waist circumference, and body composition were not affected. Almonds in overweight and obese Korean adults decreased TC, LDL-C, and non-HDL-C by 5.5, 4.6, and 6.4%, respectively, compared to the cookie control  contains supplementary material, which is available to authorized users. Cardiovascular disease (CVD) is the leading cause of mortality and morbidity in the world , includiThe traditional Korean diet is rich in carbohydrates and low in fats, with carbohydrate (CHO), fat, and protein contributing to 72.1, 13.4, and 14.5%, respectively, of total energy intake . FurtherNuts, including tree nuts and peanuts, contain a wide range of beneficial nutrients, such as fiber, protein, unsaturated fats, vitamins, minerals, and phytosterols and other phytochemicals , 14. Of The benefit to cardiovascular health of almond consumption has been demonstrated in people living in Canada, China, Taiwan, United States, and United Kingdom, but not in South Korea. However, the health benefit of any specific food is subject to the influence of many physiological, genetic, dietary, and environmental factors. As ethnicity and background diet may modulate the bioefficacy of almond nutrients, we tested the impact of almonds on CVD risk factors in overweight/obese Korean adults. We hypothesized that almonds could improve vitamin E status and CVD risk factors including lipid profile, oxidative stress, and inflammation.2 or waist circumference ≥85 cm for women, ≥90 cm for men, (3) free of any diagnosed chronic disorders or acute inflammatory diseases 2 year prior to the enrollment, (4) no known allergies to nuts, (5) nonsmoking (or ceased smoking for ≥1 year), (6) not taking any vitamin supplements, functional foods or hormone replacement therapy for the last 1 month prior to the enrollment, (7) not taking any medications known to affect lipid metabolism, such as statins, (8) consuming typical Korean diet. Subjects who had passed an initial phone screening were invited for on-site screening to confirm their eligibility. The study was approved by the Institute Review Board of the Korea National Open University, and informed consent was obtained from each participant before the conduct of any study element was performed. The trial was registered in the Institute Review Board of the Korea National Open University: KNOU IACF (ABN01-201412-11-28).Subjects were recruited in the Seoul metropolitan area by advertising on the website of Korea National Open University and the campus bulletin boards Fig. . The eliThe study was a 12-week randomized crossover intervention trial in a free-living setting  formula. Waist and hip circumferences were measured using a measuring tape. Body composition was assessed using bioelectrical impedance analysis . After ≥10 min of rest, blood pressure was determined using an automatic blood pressure monitor .Body weight, height, body composition, and blood pressure were measured before and after each intervention phase. Body weight and height were measured using an anthropometer . BMI was calculated using the weight/heightg for 10 min at 4 °C. Plasma and serum were separated and aliquots were stored in −80 °C until biochemical analyses were performed.After a 12-h fast, venous blood was collected from the brachial vein of one arm before and after each intervention phase. Whole blood was centrifuged at 1500×Serum triglyceride (TG), total choesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein choesterol (HDL-C), glucose, high-sensitive C-reactive protein (hs-CRP), apolipoprotein A (apo A), and apolipoprotein B (apo B) were analyzed using an automated biochemical analyzer . The intra- and inter-day coefficients of variation (CV) were 1.9 and 5.3% for TG, 0.5 and 8.4% for TC, 3.1 and 4.4% for LDL-C, 3.8 and 7.5% for HDL-C, 1.3 and 9.6% for glucose, 7.7 and 8.4% for hs-CRP, 1.6 and 12.6% for apo A, and 8.8 and 5.4% for apo B, respectively. Plasma protein carbonyls and oxidized low-density lipoprotein cholesterol (oxLDL) were measured using commercial kits  and a microplate reader . The intra- and inter-day CV for protein carbonyls were 6.8 and 12.2% and for oxLDL were 2.3 and 9.8%, respectively. Tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-10 in serum were determined using a cytometric cytokine bead array human enhanced sensitivity master buffer kit  and an Accuri C6 flow cytometer (BD Biosciences). Soluble vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were measured using commercial kits . The intra- and inter-day CV for VCAM-1 were 1.3 and 12.8% and for ICAM-1 were 5.2 and 11.0%, respectively. Plasma α- and γ-tocopherol were determined using a high-performance liquid chromatography (HPLC) with photo-diode array (PDA) method according to Liu et al. . The intt test was performed to compare nutrient intakes between the time points. Student’s t test was also used to assess the significance of differences in biochemical biomarkers between cookie baseline and almond baseline, as well as between before and after each intervention phase. The mixed model was performed (SAS: Proc Mixed) to assess the treatment effect to control variance in the outcome by accounting for the crossover nature of the study. In this model, subject was designated as the random effect and treatment and sequence were designated as the fixed effects. Significance was considered at P ≤ 0.05.The results are expressed as mean ± standard deviation (SD). The data were analyzed using SAS 9.4 for Windows . Student’s n = 3) and abdominal discomfort caused by almonds in the first week (n = 2). In addition, the data from one participant were not included in data analysis because of the elevated inflammatory status (hs-CPR and IL-6) that was three times of the SD. The percent of overweight (23≤ BMI <25) and obese (BMI ≥25) subjects was 39.3 and 51.2%, respectively (WHO obesity guideline for Asia–Pacific region)  completed the study with full compliance   was 19.0P ≤ 0.001), as well as 5.8% higher from fats (P ≤ 0.001). The addition of almonds also significantly increased the intake of PUFA and MUFA, dietary fiber, and vitamin E by 67.9, 107.8, 22.5, and 114.4%, respectively, as compared to the cookie control. Intake of energy, calcium, and magnesium were significantly increased by 6.2, 20.6, and 185.0%, and SFA and cholesterol were significantly decreased by 44.0 and 14.1% during the almond phase compared to the cookie phase.Compared with the values from the pre-almond phase (wk 0), almonds increased total energy by 12.3%, MUFA by 192.3%, PUFA by 84.5%, dietary fiber by 11.8%, vitamin E by 102.7%, calcium by 19.7%, and magnesium by 190.8% and decreased % energy from CHO by 14.1% . The overall mean body weight, waist circumference, body fat, and systolic and diastolic blood pressures were 66.2 ± 8.7, 89.5 ± 5.9, 43.1 ± 8.6, 122.1 ± 16.1, and 79.4 ± 10.2, respectively.P = 0.189) while the cookies had no impact. As compared to the end value of the cookie phase, almonds decreased TC, LDL-C, and non-HDL-C by 5.5, 4.6 and 6.4%, respectively (P ≤ 0.05), and tended to decrease serum apo B by 4.6% (P = 0.109). Neither almonds nor cookies affected blood glucose status.Mean TG and HDL-C values were not significantly changed during the intervention Table . HoweverP = 0.055) and γ-tocopherol was lower (P ≤ 0.001) than cookie consumption  and decreased γ-tocopherol by 18.1% (P ≤ 0.01). At the end of the intervention, almonds increased the ratio of α-tocopherol to TC by 10.4% (P ≤ 0.001) and decreased the ratio of γ-tocopherol to TC by 20.0% (P ≤ 0.001), as compared to cookies. Compared with the pre-intervention value, almonds increased the ratio of α-tocopherol to TC by 8.0% (P ≤ 0.05) and decreased the ratio of γ-tocopherol to TC by 17.2% (P ≤ 0.01).After 4 weeks of almond consumption, plasma α-tocopherol concentration tended to be higher . Serum ICAM-1 (P = 0.187), IL-1β (P = 0.137) and IL-6 (P = 0.142) tended to be lowered at the end of the almond phase than the cookie phase. Other biomarkers of inflammation, serum VCAM-1, TNF-α, and hs-CRP, showed no notable changes during the intervention.Neither almonds nor cookies affected the selected biomarkers of oxidative stress, i.e., plasma protein carbonyls, MDA, and serum oxLDL Table . AlmondsSeveral studies support a cardiometabolic benefit of almond consumption based on their association with improvements to lipid profile, blood glucose, inflammation, and oxidative stress –19. HoweAlmonds are a nutrient dense food because they are a particularly good source of unsaturated fatty acids, α-tocopherol, arginine, magnesium, copper, calcium, and potassium . The addThe addition of almonds (56 g/day) as a snack increased mean daily energy intake by 12%, but did not alter body weight. Despite the provision of 15 g fat and 169 kcal energy from a 28 g of serving, several clinical trials reveal that almonds show a beneficial or null effect on body weight , 30–33, Korean adults consume 17.7 mg of α-tocopherol equivalents (TE)/day which is larger than the adequate intake level of the KDRI at 12 mg TE/d . HoweverA recent meta-analysis of 18 randomized controlled trials showed that almonds improved TC, LDL-C, and TG statuses but had no effect on HDL-C . The favThe effects of almonds on oxidative stress and inflammation reported in the literature are mixed, likely due to variations in study design, subject inclusion criteria, etc. Jenkins et al.  found thWith regard to biomarkers of inflammation, Rajaram et al.  reportedDaily supplementation of almonds in typical Korean diet for 4 weeks improves nutrient intakes, circulating vitamin E status, and lipid profiles in overweight and obese Koreans. However, there are several limitations in our study. Due to a relatively short intervention duration, the positive effects of chronic almond consumption on the outcome measures remain to be examined. Nutrient intake may not be accurately captured as values were calculated from self-reported dietary information. The sample size of this study may not be powered to detect the effect of almonds on TG, apo B, ICAM-1, IL-1β, and IL-6.The impact of any food or nutrient on CVD risk must be evaluated against the background of ethnicity, genetics, diet, and lifestyle for the cohort studied. However, our almond intervention in a Korean population appears generally consistent with those found in other Asian as well as North American and European countries. This relationship may result from the positive nutrient attributes of almonds, i.e., their content of MUFA, PUFA, fiber, and vitamin E, and their combined association with heart health.Supplementary material 1 (DOCX 12 kb)Below is the link to the electronic supplementary material.


The antimicrobial activity of a wound dressing is a key factor for preventing and treating wound infection. The current study evaluated the physiochemical properties and antimicrobial activities of semi-IPNs (interpenetrating polymer networks) based on chitosan/polyvinyl alcohol (PVA) films and nanofibers as candidates for wound dressings and investigated the effects of morphologies (nanofibrous mats and films), crosslinking conditions of chitosan chains (uncrosslinked and crosslinked with genipin), and the presence of antibacterial drug (doxycycline) on their physicochemical and antibacterial properties. The morphology, chemical structure, fluid uptake, water vapor transmission rate, antimicrobial activity, and doxycycline release profile were assayed using SEM, FTIR spectroscopy, swelling test, permeation test, agar diffusion antibiogram, and dissolution test, respectively. The results demonstrated that crosslinking chitosan with genipin reduced the diameter of nanofibers, fluid uptake, and drug release from both nanofiber mats and film samples. According to the results, wound dressings with film morphology have better antimicrobial activity than those with nanofiber. The chitosan/PVA/Doxycycline 1% film has the potential for use as an antimicrobial wound dressing. An ideal wound dressing contains features like antimicrobial activity, sustained drug release into the wound, gaseous exchange capability, non-toxicity, biocompatibility, absorption of exudate, and ease of removal -3. BacteDoxycycline is a semisynthetic tetracycline. The antibacterial mechanism of doxycycline is the ability to inhibit aminoacyl-tRNA attachment to ribosome . In addiw 89000-98000, 99% hydrolyzed) were purchased from the Sigma Aldrich Co., USA. Acetic acid was purchased from Merck Co., Germany. Doxycycline hyclate was supplied by Sigma Co., USA. Genipin was purchased from Challenge Bioproducts Co., LTD, Taiwan. Low molecular weight chitosan with deacetylation degrees (DD) of 75–85% and poly  (PVA) (MFilm preparation1%/genipin (G) 0.05%, and f-C/P/D1%/G0.1% were fabricated using the casting method (Four different film (f) formulations: f-chitosan (C)/poly (P), f-C/P/doxycycline (D) 1%, f-C/P/ Dg method . ChitosaNanofiber preparation1%, n-C/P/D1%/G0.05%, and n-C/P/D1%/G0.1% (Chitosan powder 3% (w/v) was stirred at 250 rpm in acetic acid 70% (v/v) for 3 h at 25 °C. PVA powder 10% (w/v) was dispersed in distilled water at 120 °C with intense stirring for 3 h. After degassing the solutions, chitosan and PVA with a ratio of 20/80 (v/v) was mixed overnight for 18 h at 25 °C. In order to fabricate semi-IPN nanofibers, doxycycline 1% (w/w) was dissolved in distilled water, genipin 0.05% or 0.1% (w/w) was dissolved in 0.5 mL ethanol at 90 °, and ethanol was added to the chitosan/PVA solutions. Four different formulation solutions were prepared: nanofiber (n)-C/P, n-C/P/D1%/G0.1% . The polFT-IR and ATR analysis-Nicolet Nexus 670, USA) at a wavenumber range of 400-4000 cm-1, and attenuated total reflection (ATR) was performed for the film samples  with a wavenumber range of 650-4000 cm-1.The chemical structure of the nanofiber samples was recorded with FT-IR The surface morphology of film samples and the diameter of the nanofiber mats were determined using scanning electron microscopy (SEM)  at voltage of 20 kV. All samples were sputter coated with gold for 30 min. The nanofiber diameters were measured with an image processing software.Water uptake measurements1), and placed into the dishes with 50 mL PBS (pH 7.4) at room temperature. After 4 h, the samples were taken out and excess PBS was removed with filter paper and weighted (W2). For each sample, the experiment was repeated 3 times and the mean ± SD was reported. The water uptake was calculated using the following formula:To measure water uptake of the wound dressings, circular samples with a diameter of 15 mm were cut, weighted (W2 - W1)/W1) × 100                     (1)  Water uptake percentage = ((WWater vapor transmission rate (WVTR) measurement0) and inserted into a glass jar containing 1 kg fresh silica gel and placed in the incubator at 37 °C. At appropriate time intervals, the tubes were weighted (W1) and the WVTR was calculated from the line slope of weight changes where, according to Equation 2, area = exposure area of tubes and slope = weight changes relative to time (Circular samples of films and nanofibers were placed on the top of glass tubes containing PBS (pH 7.4) and fixed. Afterwards the glass tubes were weighted (W to time .2 × day] [slope × 24/area]                      (2)WVTR = ). Doxycycline hydrochloride may reduce the viscosity and increase the conductivity of the electrospinning solution, causing a reduction in the diameter of the nanofibers. The small size of the nanoscale fiber increases the cell-matrix interaction , 1066 cm-1 (C-O stretching), 1321 cm-1 (vibration of CH), 1540 cm-1 (NH bending of NH2), 1625 cm-1 (C=O of amide bond), 2916 cm-1 (CH vibration), and 3415 cm-1 (-OH hydrogen bonds and –NH stretching). PVA peaks were shown at 3265 cm-1 (OH hydrogen bonds), 2936 cm-1 (CH vibrations), 1739 (C=O of remaining acetate groups), and 1084 and 1416 cm-1 (C-O groups). -1 in the chitosan spectrum of NH and OH was shifted to 3266 cm-1. Also, the peak observed at1654 cm-1 in the chitosan spectrum was shifted to 1647 cm-1 after PVA was added to the formulation. These shifts show the formation of strong hydrogen bonds between PVA and the chitosan chains. The addition of doxycycline to the chitosan/PVA formulation shifted the peak observed at 1647 cm-1 to 1643 cm-1. This may be explained by the hydrogen binding of the C=O group of doxycycline with PVA and the chitosan chains. ATR-FTIR and FTIR were used to evaluate the chemical structure of the wound dressings prepared by film casting and electrospinning methods, respectively -31. FiguThe addition of genipin did not change the position of the peaks significantly. This may be explained by the overlaps of the new amide formed via the interaction between the NH2 groups of chitosan with genipin and those already existing in the chitosan structure. Genipin molecules interacted only with the NH2 groups and not with the OH groups of PVA. Water uptake1%/G0.05% [p ˂ 0.01], f-C/P/D1%/G0.1% [p ˂ 0.0001]), and nanofibers (n-C/P/D1%/G0.1% [p ˂ 0.05]). The addition of a cross-linking agent to the chitosan significantly reduced the water absorption and swelling properties of the polymer. Aldana et al. found that an increase in genipin  in chitosan/PVP films led to a reduction in swelling behaviors 1%) increased the film WVTR (719 ± 15.92 g/m2/24 h), and genipin (f-C/P/D1%/G0.1%) decreased WVTR (598 ± 13.63 g/m2/24 h). The WVTRs of the nanofibers were 2797 ± 6.48 (n-C/P), 2830 ± 14.97, and 2781 ± 12.76 g/m2/24 h in genipin 0.05% and 0.1%, respectively. Furthermore, the addition of doxycycline (n-C/P/D1%) to nanofiber mats decreased the WVTR to 2728 ± 10.20 g/m2/24 h (p ˂ 0.001). A suitable wound dressing must regulate the balance of moisture between the wound surface and its environment to avoid wound exudate accumulation or wound dehydration (34). The WVTRs for the carboxyl-modified, PVA-crosslinked chitosan hydrogel film  were 872, 858, and 772 g/m2/24 h respectively . Aldana et al. reported that cross-linking with genipin reduced the release of propranolol hydrochloride from the film containing chitosan and polyvinyl pyrrolidone  compared to uncrosslinked ones (C/P/D). This may be explained by the decrease in doxycycline release. Film and nanofiber samples of C/P/D1% showed the greatest antimicrobial effect. Nanofibers had no antibacterial effect on Pseudomonas aeruginosa bacteria. F-C/P/D1% showed more antibacterial effect on Pseudomonas aeruginosa, Staphylococcus aureus, and Acinetobacter baumannii compared to n-C/P/D1% (p ˂ 0.0001). The findings of the present study were in agreement with those of Hafsa et al. who also showed that pure chitosan film in agar disc diffusion test produced no inhibition zone on E. coli, S. aureus, P. aeruginosa, and Candida spp  could inhibit the pigment produced by Pseudomonas aeruginosa. These pigments, called pyocyanin and pyoverdin, are important virulence factors in the pathogenicity of Pseudomonas aeruginosa . Th. ThPseudion test , 43. In ruginosa , 45. Phage forms . The results of the current study showed the antimicrobial activity of films was more potent than that of the nanofiber mats. This may be explained by the increased rate of drug release to the agar medium and the consequent increase in antibacterial activity. The mechanism of chitosan’s antimicrobial activity is unclear, but it has been suggested that the interaction between the positive charge of chitosan and the negative charge of the bacterial cell membrane leads to destabilization of the cell membrane, permeation of intracellular components, and eventually cell death , 46. The1% is recommended for use as a suitable wound dressing.Many factors affect the properties of nanofibers produced by electrospinning methods, including viscosity, Mw, conductivity, voltage, type of polymer, surface tension, feed rate, and concentration . In the present study, electrospinning and casting methods were used to prepare films and nanofibrous mat of C/P/D and crosslink them with genipin in different concentrations. The results indicated that crosslinking with genipin decreased the diameter of nanofiber mats, water uptake, and the WVTR of films and increased the WVTR of nanofiber mats. Furthermore, the antimicrobial activities of films were more potent than those of nanofiber mats, and crosslinking with genipin decreased doxycycline release and antimicrobial activity. Genipin creates transverse bonds and reduces both the water absorption and the drug release rate from the wound dressing. Based on these results, f-C/P/D


Young women aged 16–24 are at high risk of common mental disorders (CMDs), but the risk during pregnancy is unclear.To compare the population prevalence of CMDs in pregnant women aged 16–24 with pregnant women ≥25 years in a representative cohort, hypothesising that younger women are at higher risk of CMDs , and that this is associated with low social support, higher rates of lifetime abuse and unemployment.Analysis of cross-sectional baseline data from a cohort of 545 women (of whom 57 were aged 16–24 years), attending a South London maternity service, with recruitment stratified by endorsement of questions on low mood, interviewed with the Structured Clinical Interview DSM-IV-TR.Population prevalence estimates of CMDs were 45.1% (95% CI 23.5–68.7) in young women and 15.5% (95% CI 12.0–19.8) in women ≥25, and for ‘any mental disorder’ 67.2% (95% CI 41.7–85.4) and 21.2% (95% CI 17.0–26.1), respectively. Young women had greater odds of having a CMD (adjusted odds ratio (aOR) = 5.8, 95% CI 1.8–18.6) and CMDs were associated with living alone  and abuse .Pregnant women between 16 and 24 years are at very high risk of mental disorders; services need to target resources for pregnant women under 25, including those in their early 20s. Interventions enhancing social networks, addressing abuse and providing adequate mental health treatment may minimise adverse outcomes for young women and their children.None. The same survey reported rates of post-traumatic stress disorder (PTSD) and self-harm in young women to be 12.6% and 20%, respectively; this was higher than reported in other age groups and in men of the same age, and the rate of self-harm in young women was more than double compared with a previous survey conducted in 2000 using the same methodology.Young people, defined by the United Nations as between 15 and 24 years, experience a significant burden of disease because of mental disorders globally.,It is well established that pregnant adolescents are at an increased risk of perinatal depression,compare the population prevalence estimates of mental disorders , history of self-harm, and recent thoughts of self-harm, in young pregnant women aged between 16 and 24 years against older women in a representative pregnancy cohort in South-East London;compare the correlates of mental disorders in young pregnant women with those in women aged ≥25 years.We therefore aimed to:a priori confounders.We hypothesised that young pregnant women (<25 years old) would be at higher risk of having CMDs  than pregnant women aged 25 years or older, and that this would be associated with low levels of social support , having experienced abuse and unemployment, after adjusting for et al,et al.This paper reports the results of a secondary data analysis of a pregnancy cohort recruited from an inner-city London maternity service, between 10 November 2014 and 30 June 2016. The cohort of pregnant women were recruited using sampling stratified according to responding positively or negatively to two depression screening questions (‘Whooley questions’), which are routinely asked by UK midwives at a woman's first antenatal (or ‘booking’) appointment.Researchers administered the Structured Clinical Interview for DSM-IV-TR Axis I Disorders (SCID-I-Research Version),Sociodemographic, obstetric and medical history details were collected at interview. Women who had their first antenatal appointment at ≥13 weeks of pregnancy were defined as ‘late bookers’.Self-reported immigration status was categorised as ‘secure legal status’  or ‘insecure legal status’ . Employment status was categorised: ‘employed, homemaker or student’ or ‘unemployed or unable to work due to disability, illness or immigration status’.Past year alcohol misuse was assessed using the Alcohol Use Disorders Identification Test (AUDIT),Measures for current thoughts of self-harm were dichotomised from question ten of the Edinburgh Postnatal Depression Scale (EPDS)A composite measure of abuse experienced across the lifetime was used for multivariate analysis; this included domestic abuse , child abuse and sexual abuse – including attempted or completed. The composite measure was derived from three questionnaires: the modified pregnancy version of the Composite Abuse Scale – Shortsvy command.et al,Analyses were carried out in Stata version 14. Sampling weights were used to account for bias induced by stratified sampling in the study design;a priori sociodemographic confounders  (model 1), with subsequent models additionally adjusting for employment status (model 2), social support (living status) (model 3) and abuse (model 4). To test for collinearity, chi-squared tests assessed the association between relationship and living status, qualifications and household income level, and unplanned pregnancy and experience of abuse.We used complete case analysis to first examine group differences in sociodemographic and clinical variables in young women compared with women ≥25 years. Second, we examined the association between age and CMDs followed by sensitivity analyses. We examined this association, unadjusted, and then initially adjusted for n<5), by comparing characteristics of included to excluded participants in the final regression model. Two (3.5%) young women, and nine women ≥25 years (1.8%) had missing data for CMD, and a further five women had missing data for sociodemographic or clinical variables . To investigate the potential impact of missing data, primarily because of 21 participants declining response to the PTSD module (SCID-I), we conducted a sensitivity analysis to examine the extent to which missing data on PTSD affected the results; first assuming all data missing for PTSD was positive, and second assuming missing data as negative. Because of the small number of young women aged <19 years (n = 6), multivariate analyses were also repeated including only women aged 19–24 years.We investigated the patterns of missing CMD data in the adjusted models using chi-squared tests .In total, 545 women participated in the study with a mean age of 32.8 (s.d. = 5.7) years. Of these, 57 women were aged between 16 and 24 years ; with only 6 aged under 19. The older age group comprised 488 pregnant women aged 25.1–47.5 years .v. 28.8% (95% CI 23.8–34.3); odds ratio (OR) = 6.9, 95% CI 2.0–24.1, P = 0.003; reference category ‘White’); to live alone  v. 9.5% (95% CI 6.6–13.3); OR = 6.6, 95% CI 1.7–25.7, P = 0.007; reference category ‘living with partner’); to live with parents  v. 6.0% (95% CI 3.8–9.3); OR = 13.7, 95% CI 3.7–50.5, P<0.001) or be homeless/living in emergency accommodation  v. 0.5% (95% CI 0.1–2.2); OR = 54.2, 95% CI 6.3–466.2, P<0.001); to be single  v. 7.4% (95% CI 5.0–11.0); OR = 6.4, 95% CI 2.2–19.0, P = 0.001); to have an unplanned pregnancy  v. 24.8% (95% 20.2–30.1); OR = 5.9, 95% CI 2.1–16.4, P = 0.001); be unemployed/unable to work  v. 9.1% (95% CI 6.3–12.9); OR 5.4, 95% CI 1.9–15.6, P = 0.002; reference category ‘employed, homemaker or student’) and to have a low household income  v. 11.9% (95% CI 8.4–16.6); OR = 21.9, 95% CI 3.7–130.5, P = 0.001). Young women were also less likely to have other children  v. 53.6% (95% CI 47.8–59.3); OR = 0.2, 95% CI 0.1–0.7, P = 0.008)  = 0.008) . All butThe population prevalence estimates for lifetime experience of abuse was 38.9% (95% CI 19.3–62.9) in young women and 22.9% (95% CI 18.5–28.1) in women ≥25 years. This included 19.5% (95% CI 7.0– 43.9) of young women and 6.2% (95% CI 4.0–9.7) of women ≥25 years having experienced intimate partner violence. A total of 20.6% (95% CI 7.8–44.4) of young women and 12.3% (95% CI 9.1–16.5) of women ≥25 years had experienced sexual violence; including rape or attempted rape (11.1% (95% CI 3.0–33.5) in young women; 4.6% (95% CI 2.7–7.5) in women ≥25 years). Additionally, 11.2% (95% CI 3.0–33.9) of young women and 5.0% (95% CI 3.0–8.1) of women ≥25 years reported experiencing physical or sexual abuse as a child (under 16 years).v. 6.7% (95% CI 4.5–9.9) in women ≥25 years; particularly social phobia, 22.7% (95% CI 8.1–49.5) v. 3.0% (95% CI 1.6–5.7), respectively. The population prevalence of generalised anxiety disorder was 9.6% (95% CI 2.2–33.8) in young women compared with 4.1% (95% CI 2.5–6.6) in women ≥25 years.The population prevalence estimate for any mental disorder was 67.2% (95% CI 41.7–85.4) in young women compared with 21.2% (95% CI 17.0–26.1) in women ≥25 years . For CMDOf the total number of women in the cohort diagnosed with any mental disorders at the research interview, only 13 (34.2%) young women and 47 (25.8%) older women self-reported a problem when asked by the researcher if they have a ‘current mental health issue’. 19.8% (95% CI 7.2– 44.0) of young women, and 6.7% (95% CI 4.4–9.9) of women ≥25 years had a history of self-harm. Also, 3.7% (95% CI 1.7–8.1) of young women and 2.1% (95% CI 1.0–4.2) of women ≥25 years were experiencing current thoughts of self-harm.P<0.001), educational qualifications and household income (P<0.001), and unplanned pregnancy and experience of abuse (P<0.001). We therefore did not include relationship status, household income and unplanned pregnancy in multivariate models.There was a strong relationship between living status and relationship status OR = 5.8, 95% CI 1.8–18.6, P = 0.003). CMD was associated with living alone . Lifetime experience of abuse was associated with CMD , with a stronger association  in the sensitivity analysis assuming missing PTSD data is indicative of PTSD (see below for more details). We did not find evidence that ethnicity, educational level, or unemployment were associated with increased risk of CMDs .In the unadjusted and adjusted models  young won = 16) were more likely than those included to have a current chronic physical condition (P = 0.019) and to have experienced abuse in their lifetime (P<0.001) (supplementary Table 1 available at https://doi.org/10.1192/bjo.2019.6). No other differences between participants' sociodemographic or clinical characteristics were found.Participants excluded from the complete case analysis (n = 540): aOR = 5.0, 95% CI 1.5–16.1, P = 0.007 and aOR =  6.0, 95% CI 1.9–19.2, P = 0.002, respectively), demonstrated comparable results with the complete case analysis (supplementary Table 2). When PTSD missing data was assumed to indicate PTSD, CMDs were more strongly associated with lifetime experience of abuse .The sensitivity analysis that assessed the extent to which missing data affected results by first assuming all missing data on PTSD to be positive (+ve) and then negative (−ve) (adjusted model (n = 6), analyses were repeated including only women aged 19–24 years. The average age of the young women aged between 19 and 24 years was 22.4 (s.d. = 1.5). Results from the adjusted model closely aligned with the full-cohort analysis (see supplementary Table 3): young women aged 19–24 years had greater odds of having a CMD during pregnancy than women ≥25 years . CMD was associated with living alone  and lifetime experience of abuse ; but was not significant at P = 0.05.As a result of the small number of young women aged <19 years (P = 0.003), with a population prevalence of 45.1% (95% CI 23.5–68.7) compared with 15.5% (95% CI 12.0–19.8) in women ≥25 years; prevalence of anxiety disorders were especially high in young women , particularly social phobia . Young pregnant women were also more likely to be Black and minority ethnic, single, live in poverty, homeless or living in emergency accommodation, unemployed or unable to work, and to have an unplanned pregnancy. Risk factors for CMDs included low social support  and being a victim of abuse .Young pregnant women under 25 in this maternity population had a very high prevalence of mental disorders, with population estimates of 67.2% (95% CI 41.7–85.4) compared with 21.2% (95% CI 17.0–26.1) in older women. There was a sixfold increased risk of having a CMD in early pregnancy in young women . This unexpected result may be explained by the insecure and discriminative nature of employment that is experienced by women who are employed during pregnancy – recent reports show that low paid and insecure work are major problems for young women.We hypothesised that unemployment would be a risk factor for CMDs as it has been reported elsewhere that antenatal anxiety and depression are more prevalent in unemployed compared with employed women.,,P = 0.020). Pregnant women similarly declining to answer trauma-related questions during clinical interviews could lead to a failure of healthcare professionals to identify symptoms of a mental disorder. Low rates of identification of abuse in pregnant women has been described elsewhere,We found that the rate of lifetime experience of abuse was very high in young pregnant women , with sexual abuse  and partner abuse rates  being particularly concerning; it is important to note that these rates may still underestimate the extent of abuse, as abuse is often underreported.Young people under the age of 25, whose needs occur while in the transition between health services for children and those for adults, are often neglected by service providers and policymakers. For example, the National Institute for Health and Care Excellence provides specific guidelines for mental disorders such as depression in children and young people, but this does not extend beyond 18 years. Similarly, there are specialist services available for pregnant teenagers but few target the under 25s. However, we demonstrate here that the increased risk for mental disorders is not limited to pregnant teenagers; indeed, only 6 of the 57 young women in this cohort were under the age of 19 . Our findings therefore indicate that the vulnerability of young pregnant women highlighted in this paper is not limited to those under 19 years, and is also relevant to those in their early 20s. Although there has been increasing concern about the mental health of young women aged 16–24Our findings suggest that universal services in high-income countries may need to target resources on pregnant women under 25. We also highlight the need to improve identification and interventions for women experiencing abuse, potentially through training of staff and integrated interventions addressing mental health alongside abuse.This study draws on data from a large and representative sample of women in inner-city London using probability weights to account for the bias introduced by the sampling strategy. A limitation of this study was the relatively small number of young women in this cohort, which led to wide confidence intervals; despite this, population prevalence estimates were derived that demonstrated clear associations between young age (<25 years) and CMDs. However, the study included only six women under the age of 19, which limits conclusions that can be drawn regarding teenage pregnant women (i.e. <19 years); further research, with larger cohorts of young women, would be beneficial.,In conclusion, although there is increasing traction for research and policy focusing on adolescent health


A critical prerequisite for studying rumen microbial community by high throughput molecular biology methods is good quality community DNA. Current methods of extraction use kits designed for samples from the different origin for rumen. This puts stress on the development of a relevant manual method for DNA extraction. The objective of this study was to modify the existing methods of community DNA extraction and thereby systematic comparison of their efficiency based on DNA yield, purity, 16S rRNA gene sequencing, and identification to determine the optimal DNA extraction methods whose DNA products reflect targeted bacterial communities special to rumen.Enzymatic method, Chemical method, Enzymatic + Chemical method, and Enzymatic + Chemical + Physical method were modified toward evaluation of community DNA extraction from solid, squeezed, and liquid fractions of goat rumen digesta. Each method was assessed critically for nucleic acid yield and its quality. The methods resulting in high nucleic acid yield, optimal purity ratios with intact band on agarose gel electrophoresis were optimized further. Optimized methods were studied using standard polymerase chain reaction (PCR) with universal bacterial primers and 16S rRNA primers of targeted rumen bacteria. Methods denoting the presence of targeted rumen bacteria were assessed further with 16S rRNA gene sequencing and identification studies. It led toward methods efficacy estimation for molecular biology applications. Effect of rumen sample preservation on community DNA extraction was also studied. Their mean standard deviation values were calculated to understand sampling criticality.260nm/A280 nm) and 2.3 (A260nm/A230 nm) respectively. Comparative analysis showed the better efficiency of ECP method and chemical method toward freshly squeezed rumen digesta and solid rumen digesta. However, sample preservation at −80°C for 1.5 months drastically affected the yield and purity ratios of community DNA. New protocol revealed targeted microbial community having Gram-positive as well as Gram-negative bacteria such as Prevotella ruminicola, Streptococcus lutetiensis, Ruminococcus flavefaciens, Fibrobacter succinogenes, and Selenomonas ruminantium.Modified Chemical method (Cetrimonium bromide) and Enzymatic+Chemical+Physical (ECP) method (Lysozyme-Cetrimonium bromide-Sodium Dodecyl Sulfate-freeze-thaw) could extract 835 ng/µl and 161 ng/µl community DNA from 1.5 g solid and 2 ml squeezed rumen digesta with purity ratios of 1.8 (ATo date, this is the first report of modified methods wherein least chemicals and steps lead toward PCR and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta. Detection of targeted rumen bacteria in solid and squeezed rumen digesta proves their strongest association with rumen fiber mat. It also marks the presence of distinct microbial communities in solid and squeezed rumen fractions that in turn differs the performance of each different method employed and yield of nucleic acid obtained. It also leaves a possibility of the presence of complex microbial consortia in squeezed rumen digesta whose DNA extraction methods need more attention. Finally, manual protocols of community DNA extraction may vary in different ruminant which suggests undertaking rigorous research in their establishment. There are approximately 10 viruses . To dateet al. [DNA quality and yields, even though a critical prerequisite are highly restricted since the extraction methods do not work equally efficiently for diverse microbial groups. To date, several studies have shown that the DNA extraction method used has an impact on the microbial community representation in samples from different habitats, including the rumen. The sampling technique used from a wide choice such as oral stomach tubing, sample collection through a rumen fistula, as well as fractionation of rumen sample , can also have an impact on microbial community parameters ,2. To miet al.  used PCQet al.  sufficieet al. [et al. [To enable a direct comparison of the rumen community structure from different individual samples, standardization of DNA extraction methods is crucial. Several DNA extraction methods, including commercial kits, have been tried for ruminal DNA extraction -7. Howevet al.  found ve [et al.  demonstrRuminococcus flavefaciens and Streptococcus bovis are Gram-positive, which make it difficult to extract their DNA effectively using the manual community DNA extraction protocols. Further, extracted DNA should carry good quality and its amenability for standard polymerase chain reaction (PCR) and 16S rRNA gene sequencing is most important prerequisite.Present study evaluates the performance of modified Chemical method (CTAB), Enzymatic method (EM) (lysozyme-SDS-proteinase K), Enzymatic+ Chemical method (lysozyme-SDS-proteinase K+ CTAB), and Enzymatic+ Chemical+ Physical method (lysozyme-SDS-CTAB-Polyvinylpyrrolidone-freeze-thaw) for community DNA extraction from goat rumen digesta. Targeted bacteria such as The current research was aimed to modify the existing methods of community DNA extraction and thereby systematic comparison of their efficiency based on DNA yield, purity, 16S rRNA gene sequencing, and identification to determine the optimal DNA extraction methods whose DNA products reflect targeted bacterial communities special to rumen.Not applicable as the sample was brought from Government approved slaughterhouse.3, 9.80 g/l; Na2HPO4, 4.97 g/l; KCl, 0.57 g/l; NaCl, 0.47 g/l; MgCl2, 0.123 g/l; and CaCl2, 0.04 g/l. Before use, saliva was heated at 39°C and infused with CO2 for anaerobicity. Squeezed and solid rumen digesta were processed separately for DNA extraction. Solid rumen digesta was centrifuged at 12,000 rpm/10 min, 4°C. The solid and liquid fractions were used separately. In the case of squeezed rumen digesta, the solid fraction was used after centrifugation. Both fractions were washed further with NaCl -EDTA (Sigma-Aldrich) (1.5 M) thrice, before DNA extraction.Fresh rumen sample was brought from Government approved slaughterhouse near Pune, Maharashtra, India, under controlled environmental conditions. The sample was either used immediately for DNA extraction or stored at −80°C for 24-48 h without any preservatives. To evaluate the effect of long-term storage, the sample was stored at −80°C for 1.5 months and was studied further. For processing, rumen digesta was squeezed and washed with the artificial saliva, which consists of NaHCOEM11.5 g of fresh solid rumen digesta was centrifuged at 12,000 rpm/10 min, 4°C. Pellet and liquid fractions were separated before DNA extraction and washed with 1 ml 0.03 M NaCl-0.002M EDTA (pH: 8) followed by centrifugation at 12,000 rpm/5 min, 4°C. After three washing cycles, fractions were suspended in 100 µl NaCl-EDTA solution and 100 µl freshly prepared lysozyme . It was mixed thoroughly and incubated at 37°C for 1 h. It was followed by addition of 50 µl , 10 µl proteinase K (Sigma-Aldrich), and 0.03 M NaCl-0.002M EDTA (pH: 8.0) to have total volume of 500 µl, followed by incubation at 55°C for 1 h.After incubation, an equal amount of chloroform: isoamyl alcohol (24:1) was added to both fractions, mixed thoroughly and incubated at room temperature for 10 min. Whole content was centrifuged at 12,000 rpm/15 min, 4°C. The supernatant containing crude DNA was processed twice in a similar manner. 400 µl isopropanol (Sigma-Aldrich) and 7.5M ammonium acetate  were added into the washed liquid and incubated overnight at −20°C. Pellet was seen visually after the incubation. Entire content was centrifuged at 10,000 rpm/20 min, 4°C. Pellet obtained was washed thrice with 70% ethanol (Fisher Scientific) by centrifugation at 12,000 rpm/20 min, 4°C. Ethanol was removed completely after washings, and purified DNA was dried in a vacuum dryer (Thermo Fischer Scientific) without heating. Finally, the pellet was suspended in chilled 1× TE buffer (Invitrogen) and its quality was checked on a NanoDrop spectrophotometer (Thermo Fischer Scientific).EM21.5 g of solid and 2 ml of squeezed rumen digesta were centrifuged at 12,000 rpm/10 min, 4°C. Their solid fractions were washed with 1 ml of 0.03M NaCl-0.002M EDTA (pH: 8) by centrifugation at 12,000 rpm/5 min, 4°C. They were suspended in 100µl 0.03M NaCl-0.002M EDTA (pH: 8) and a variable concentration of freshly prepared lysozyme  followed by thorough mixing and incubation at 37°C for 1 h. Remaining steps were carried out as mentioned above for method EM1.EM350 mg to 1000 mg of solid rumen digesta was centrifuged at 12,000 rpm/10 min, 4°C. Their solid fractions were washed with 1ml of 0.03M NaCl-0.002M EDTA (pH: 8) by centrifugation at 12,000 rpm/5 min, 4°C. After three washing cycles, they were suspended in 100 µl of 0.03M NaCl-0.002M EDTA (pH: 8) and 100 µl freshly prepared lysozyme . They were mixed thoroughly and incubated at 37°C for 1 h. Remaining steps were carried out as mentioned above for method EM1.CM11.5 g solid rumen digesta was centrifuged at 12,000 rpm/10 min, 4°C. Pellet and liquid fractions were processed separately for DNA extraction. 2 ml liquid fraction was used in this study. They were washed with 1 ml 0.03M NaCl-0.002M EDTA (pH: 8) by centrifugation at 12,000 rpm/5 min, 4°C. After three washing cycles, fractions were suspended in lysis solution containing 100 mM Tris HCl , 20 mM EDTA , 1.4M NaCl , and 2% w/v CTAB  and incubated at 65°C for 1 h. Once brought to room temperature, whole content was centrifuged at 12,000 rpm/10 min, 4°C. The supernatant containing crude DNA was suspended in equal volume of fresh and chilled Chloroform: isoamyl alcohol (24:1) and incubated at room temperature for 10 min followed by centrifugation at 12,000 rpm/15 min, 4°C. After three washing cycles, remaining steps were carried out as mentioned in the method EM1.CM21.5 g solid rumen digesta was processed as described in CM1. Their solid fraction was used for DNA extraction. It was added with 100 mM Tris HCl , 20 mM EDTA , 1.4M NaCl , and 0.2 to 1% w/v CTAB  lysis solution followed by incubation at 65°C for 1 h. After incubation, remaining steps were carried out as mentioned in the method EM1.CM31.5 g solid rumen digesta was processed as described in CM1. Its solid fraction was used for DNA extraction. It was added with 2% w/v CTAB  followed by incubation at 65°C for 30 min. After incubation, remaining steps were carried out as mentioned in the method EM1.CM41.5 g solid rumen digesta was processed as described in CM1. Its solid fraction was used for DNA extraction. It was added with 2% w/v CTAB  followed by incubation at 65°C for 2 h. After incubation, remaining steps were carried out as mentioned in the method EM1.ECM11.5 g solid and 2 ml of squeezed rumen digesta were processed as described in method EM1. Their solid fractions were used for DNA extraction. Fractions lysed with enzymatic method were treated with lysis solution containing 100 mM Tris HCl , 20 mM EDTA , 1.4M NaCl , and 2% w/v CTAB . It was incubated at 65°C for 10 min. Remaining steps were carried out as mentioned in the method EM1.ECPM11.5 g solid and 2 ml of squeezed rumen digesta were processed as described in method EM1. Their solid fractions were used for DNA extraction followed by enzymatic lysis using lysozyme as described earlier. They were added with lysis solution containing 100 mM Tris HCl , 20 mM EDTA , 1.4M NaCl , and 2% w/v CTAB . It was mixed with 50 µl  followed by quick freezing of the content at −80°C for 10 min and thawing at 65°C. After three freeze-thaw cycles, content was brought at room temperature. It was centrifuged at 12,000 rpm/10 min, 4°C. The supernatant containing crude DNA was suspended in equal volume of fresh and chilled Chloroform: isoamyl alcohol (24:1) and incubated at room temperature for 10 min followed by centrifugation at 12,000 rpm/15 min, 4°C. After three washing cycles, remaining steps were carried out as mentioned in the method EM1.ECPM2This was performed similarly as ECPM1 as described above. ECPM2 was performed differently where 2.0 % w/v PVP  was added to the lysis solution, before three freeze-thaw cycles. Remaining steps were carried out as mentioned in the method ECPM1.ECPM32 ml of squeezed rumen digesta was used for DNA extraction. This was performed similarly as ECPM1 as described above. ECPM3 was different where lysis solution was supplemented with 2.0 % w/v PVP , and the number of freeze-thaw cycles was increased from three to eight. Remaining steps were carried out as mentioned in the method ECPM1.200 mg of solid and 200 µl of squeezed rumen digesta were used for DNA extraction using QIAamp DNA Stool Mini Kit . Effect of lysis temperature (70°C and 90°C for 5 min) on community DNA extraction was also evaluated. Remaining steps were performed according to the instructions provided by the manufacturer.1.5 g of solid and 2 ml squeezed rumen digesta were used for statistical analysis of CM4 and ECPM2 methods, respectively. Details of the methods were as described above. Three independent trials were conducted with fresh solid and squeezed rumen digesta for assessing its reproducibility. Each trial was conducted in eight different vials for analyzing its reliability. Furthermore, an independent trial was conducted with −80°C stored, 1 day old, and 1.5-month-old rumen digesta using CM4 and ECPM2 methods. Statistical analysis was performed using mean and standard deviation.260nm/A280nm to A260nm/A230nm ratios to indicate the presence of buffer salts and organic compounds as a part of DNA contamination. Integrity was determined by agarose (2% w/v) gel electrophoresis  at 140 V using 1 Kb Plus DNA Ladder  as a molecular weight marker, post-staining with SYBR® Safe DNA gel stain (Invitrogen) and illumination under UV light.DNA concentrations were measured on NanoDrop spectrophotometer (Thermo Fisher Scientific). The purity of DNA was assessed spectrophotometrically from AThe purity of community DNA for downstream applications was assessed by amplification of 16S rRNA using the universal bacterial primers as (27F 5’ AGAGTTTGATCMTGGCTCAG 3’ and 1492R 5’ TACGGYTACCTTGTTACGACTT 3’). PCR amplification was done using a 20 µl reaction mixture containing 0.2 mM each dNTP , 0.5 µM of each forward and reverse primer, 100 ng DNA template, and 0.02 U/µl of Q5 Hot Start High Fidelity DNA polymerase (New England Biolabs) with 1× reaction buffer supplied by the manufacturer. Amplification was performed with a thermal cycler (Bio-Rad) using the following program: 98°C for 10 min; 35 cycles consisting of 98°C for 10 s; 55°C for 30 s; 72°C for 1 min; and a final extension step consisting of 72°C for 10 min. The amplification was determined by electrophoresis of reaction product in 1% agarose gel.® Hot Start High-Fidelity DNA Polymerase , 0.2 mM of each dNTPs , 100 ng template, and 10 nM of forward and reverse primer . The forward and reverse primers were used in sequencing of the eluted PCR products; the sequencing process was done in First Base Malaysia by Sanger sequencing technique. The obtained sequences were analyzed by a BLASTN search in NCBI GenBank. A phylogenetic tree was constructed using sequence distance method.PCR product of each sample was subjected to electrophoresis on 2% agarose gel. 418 bp, 862 bp, 835 bp, 513 bp, and 446 bp fragments were observed and eluted from the gel using Macherey Nagel NucleoSpin260nm/A280nm and A260nm/A230nm ratios were similar in the range of 1.3 and 0.5, respectively. EM1 processed with 10 mg/ml lysozyme, and solid-liquid fractions of rumen digesta could extract 1710 ng/µl and 30 ng/µl nucleic acid. Whereas solid and squeezed rumen digesta processed with 15 mg/ml, 20 mg/ml, and 25 mg/ml lysozyme in EM2 could extract successively higher nucleic acid with increasing concentration of lysozyme, where 25 mg/ml lysozyme could give 500 ng/µl and 180 ng/µl nucleic acid from solid and squeezed rumen digesta. In EM3, varying sample size from 50 mg to 1000 mg could extract highest nucleic acid of 1844 ng/µl from 200 mg. However, purity ratios of A260nm/A280nm and A260nm/A230nm obtained were poor for any set of modifications conducted in enzymatic method. Suspension of extracted community DNA was brownish, non-uniform. Furthermore, it failed to show community DNA on agarose gel electrophoresis This work was aimed at the development of PCR and 16S rRNA gene sequencing quality community DNA extraction protocols through modification in existing methods followed by their optimization. In this instance, details of four different methods of community DNA extraction modified in various ways are shown in 260nm/A280nm and A260nm/A230nm ratios. CM1 processed with solid and liquid fractions of rumen digesta with 2% CTAB could extract 644 ng/µl and 40 ng/µl nucleic acid. Community DNA extraction with solid fraction and increasing concentration of 0.2-1%, CTAB was performed with CM2 where; increasing concentration of CTAB could extract successively higher nucleic acid with better purity ratios and 1% CTAB was found to be the most promising concentration. Nonetheless, CM3 and CM4 with 2% CTAB application followed by 30 min and 2 h lysis buffer incubations resulted into nearest optimal ratios of A260nm/A280nm and A260nm/A230nm for the extracted community DNA. In the application of CM4, DNA suspension was white, translucent and it showed an intact band of >15 Kb on 1% agarose gel electrophoresis  but also Aphoresis .260nm/A280nm and A260nm/A230nm shown below optimum and it also failed to show community DNA on agarose gel electrophoresis  followed by 70°C and 90°C lysis incubation could give highly efficient cell lysis with optimal purity ratios at 90°C. Irrespective of lysis temperature used, solid fraction could extract higher nucleic acid than squeezed rumen digesta which was 40 ng/µl and 10 ng/µl, respectively .260nm/A280nm and A260nm/A230nm ratio of day 0 was observed to be 1.8± 0.06 and 2.3±0.83 for CM4 method. It was seen to be 1.8±0.075 and 2.3±0.18 for ECPM2 method. Whereas, A260nm/A280nm and A260nm/A230nm of 1.5 month stored rumen digesta seen reduced from 1.8± 0.06 to 1.72±0.15 and 1.43±0.23, 2.3±0.83 to 1.27±0.41 and 0.62±0.17 for CM4 and ECPM2 methods respectively. In common, nucleic acid concentration showed the highest mean standard deviation values irrespective of the storage period of solid rumen digesta.Irrespective of CM4 or ECPM2 methods application, statistical analysis  showed aBacteroides and Prevotella (418 bp), Streptococcus bovis (869 bp), Ruminococcus flavofaciens (835 bp), Fibrobacter succinogenes (446 bp), and Selenomonas ruminantium (513 bp) with targeted bacterial 16S rRNA gene primers  from solid fraction. This emphasizes on the strongest association of microbial community with solid rumen digesta, where they must be present in highest abundance. Increasing lysozyme concentration up to 25 mg/ml could not improve it further which marks its poor efficacy in conclusion. Furthermore, the increase in sample size showed decreased yield which is a sign of increasing concentration of inhibitors. Here, 200 mg sample size found most suitable.260nm/A230nm for carbohydrates, aromatic compounds, humic acids, and phenolics; A260nm/A280nm for protein) provide an indication of DNA purity and should ideally be 2.0 to 2.2 for A260nm/A230nm and 1.8 for A260nm/A280nm for most molecular biology applications [et al. [260nm/A280nm of 1.8 with community DNA extraction from Yak rumen digesta using SDS-lysozyme method. A260nm/A280nm obtained in our study could not exceed beyond 1.3. This might indicate variable efficacy of community DNA extraction method in each different ruminant and respective rumen fractions.In continuation to this; brownish, non-uniform DNA suspension followed by smearing in the agarose gel electrophoresis is indicative of non-feasibility of an enzymatic method for community DNA extraction. Rumen content samples contain many substances, such as tannins, that could inhibit the PCR. Relative absorbance readings  the only kit method studied here; could give highest nucleic acid in solid fraction than squeezed rumen digesta. Furthermore, lysis temperature of 90°C found most effective for efficient cell lysis. High temperature may help in efficient cell wall degradation of Gram-positive bacterial communities and thereby improve the yield.et al. [Thus, community DNA extraction performed using QIAamp DNA Stool Mini Kit , methods EM1 and CM4 found most effective with solid rumen digesta. Methods ECM1 and ECPM2 showed efficient performance with squeezed rumen digesta. Apart from this, 1.5 months stored rumen sample showed a statistically significant drop in purity ratios of community DNA. The choice of DNA extraction method followed by sampling and storage may have an impact on the revealed community structure . This iset al.  which shet al. . This emR. flavofaciens, F. succinogenes, S. ruminantium, and S. lutetiensis in goat rumen. Along with this, P. ruminicola was detected against targeted genus Bacteroides and Prevotella. PCR and 16S rRNA based culture independent analyses are thus very informative and necessary to understand population wide community patterns [M. multacida was not detected in this study. This is in agreement with Pers-Kamczyc et al. [et al. [Following this, community DNA extracted with CM4 and ECPM2 methods could give standard PCR amplification with universal bacterial primers and targeted bacterial 16S rRNA gene primers which prove its efficacy for a variety of molecular biology applications. Furthermore, PCR amplification of targeted bacteria with solid fraction and squeezed fraction of goat rumen digesta shows their strong association with rumen. It may denote different compatibility of each of the fraction for specific extraction method employed. 16S rRNA gene sequencing and identification of PCR amplified products confirmed the presence of patterns -27. In cc et al.  and Li e [et al.  which stP. ruminicola, S. lutetiensis, R. flavofaciens, S. ruminantium, and F. succinogenes in solid and squeezed rumen digesta proves their strongest association with rumen fiber mat. It also marks the presence of distinct microbial communities in solid and squeezed rumen fractions that, in turn, differs the performance of each different method employed and yield of nucleic acid obtained. It also leaves a possibility of the presence of difficult microbial consortia in squeezed rumen digesta whose DNA extraction methods need more attention. Finally, manual protocols of community DNA extraction may vary in different ruminant which suggests undertaking rigorous research in the development of new protocols.To the best of our knowledge, this is the first report of optimization of modified CTAB and CTAB-SDS-PVP-lysozyme-freeze-thaw methods for community DNA extraction from solid rumen digesta, its centrifuged supernatant and squeezed rumen digesta of a slaughtered goat. Furthermore, this study showed that short-term rumen sample preservation at −80°C without any external preservatives does not affect the quality of community DNA though long-term sample preservation for 1.5 months does. Methods optimized in our study do not recommend the use of phenol, RNAase, proteinase K or kit based DNA purifications which differentiate them from others. Possible extraction of >15 Kb community DNA from rumen confirms their efficacy for molecular biology applications. Consistent purity ratios, high nucleic acid yield, and PCR amenability stand out the reliability and reproducibility of these methods. Further, detection of DA and AK designed the research; DA collected the samples, performed the research, analyzed data and prepared the manuscript, AK guided the entire research, data analysis, and manuscript preparation. All authors read and approved the final manuscript.


PTPN2/22 (T-cell negative-regulators) occur in 78% of rheumatoid arthritis (RA), along with Mycobacterium avium paratuberculosis (MAP) infection in 33% of patients. In Crohn’s disease, we reported that SNPs in TNFα and receptors (TNFRSF1A/TNFRSF1B) benefited intracellular MAP-survival, increased infection, and elevated inflammatory response mimicking the poor response to anti-TNFα treatment in some patients. Here, we studied the frequency and effects of SNPs in TNFα/TNFRSF1A/TNFRSF1B in RA including gene expression, MAP infection, and osteoporosis marker levels in blood . TNFα:rs1800629 (GA) was detected in 19/48 (40%) RA and 8/54 (15%) controls  = 3.6, 95% CI: 1.37–9.54). TNFRS1B:rs3397 (CT) was detected in 21/48 (44%) RA and 10/54 (19%) controls . In RA, rs3397 downregulated TNFRSF1B expression (CC > CT (0.34 ± 0.14) and CC > TT (0.27 ± 0.12)), compared to wildtype CC (0.51 ± 0.17), p-value < 0.05. MAP DNA was detected significantly in 17/48 (35.4%) RA compared to 11/54 (20.4%) controls . The average osteocalcin level was significantly lower  in RA (2.70 ± 0.87 ng/mL), RA + MAP (0.60 ± 0.31 ng/mL), RA + TNFRSF1B:rs3397 (TT) (0.67 ± 0.35 ng/mL), compared to the healthy control (5.31 ± 1.39 ng/mL), and MAP-free RA (3.85 ± 1.31 ng/mL). Overall, rs3397 appears to downregulate TNFRSF1B, increase MAP infection, worsen inflammation, and cause osteocalcin deficiency and possibly osteoporosis in RA.We previously discovered that single nucleotide polymorphisms (SNPs) in  Rheumatoid arthritis (RA) is a chronic inflammatory disorder which affects the joints and results in tearing of articular cartilage . PatientMycobacterium avium subspecies paratuberculosis (MAP) [The elevated level of pro-inflammatory cytokines, mainly tumor necrosis factor alpha (TNFα), in RA blood has negative effects on osteoclast paracrine stimulation, which leads to bone resorption as seen in osteoporosis . Severalis (MAP) . Specifiis (MAP) . Most imTNFRSF1A and TNFRSF1B) have downregulated the expression of their corresponding genes significantly, which resulted in increased susceptibility to MAP infection in CD [SNPs in TNF Receptor Superfamily Member 1A/1B , MAP infection, and active osteocalcin levels (an osteoporosis marker) in blood samples from RA patients.Most recently, we discovered that MAP infection also occurred in RA patients who are associated with SNPs in gulators . Patientgulators . In this2-EDTA) from 102 study participants  were acquired from the University of Central Florida Health Center. All participants provided written informed consent prior to enrollment, and the study was approved by the University of Central Florida Institutional Review Board #SBE-16-12193 . RA diagnosis was performed and reported by Dr. Shazia Beg  based on approved standard American College of Rheumatology (ACR) criteria for RA diagnosis. This includes a physical exam, imaging and laboratory tests , erythrocyte sedimentation rate (ESR), and anti-cyclic citrullinated peptide (AntiCCP)).Peripheral blood  and P91 (GAGGTCGATCGCCCACGTGA) primers at the following conditions: 95 °C for 5 min, then 34 cycles of 95 °C for 1 min, 58 °C for 1.5 min, 72 °C for 1.5 min. Final extension of 10 min at 72 °C. The product size amplified from this round was 398 bp. A second round of amplification was followed, which involved using AV1 (ATGTGGTTGCTGTGTTGGATGG) and AV2 (CCGCCGCAATCAACTCCAG) primers at the following conditions: 95 °C for 5 min, then 34 cycles of 95 °C for 1 min, 60 °C for 0.5 min, 72 °C for 1.5 min. Final extension of 10 min at 72 °C. The final PCR amplified fragment was 298 bp and was analyzed on 2% agarose gel.Purified white blood cells separated from the blood samples were analyzed for the presence of MAP DNA. Genomic DNA extraction and nested PCR (nPCR) analysis were performed as described earlier ,14. BrieAn ELISA sandwich assay was used to measure the serum active osteocalcin level. A Life Technologies Osteocalcin Human ELISA Kit  was used following the manufacturer’s instructions. The absorbance readings were measured at 450 nm wavelength and osteocalcin concentration was calculated from the generated standard curve equation following the manufacturer’s instructions.TNFα:rs1800629, TNFRSF1A:rs767455, and TNFRSF1B:rs3397 were performed on purified DNA samples following the manufacturer’s protocol at the University of Florida Pharmacotherapy and Translational Research Department . Two solutions; 2 × TaqMan master mix and 20 × assay working stock (primers with VIC and FAM fluorophore attachment) made up the reaction mixture. Following reverse transcription polymerase chain reaction (RT-PCR) assay , isolated DNA samples in addition to the reaction mixture and controls were added into a 384-well microtiter plate using the Applied Biosystems QuantStudio RT-PCR System. The plate was analyzed for VIC and FAM fluorophores at 551 and 517 nm, respectively. Fluorescence of VIC or FAM alone determined that the sample had allele 1 or allele 2, while VIC and FAM together determined that the sample was heterozygous for each allele. Each selected SNP is described in Extraction and purification of genomic DNA for genotyping was performed using QIAamp DNA Blood Mini Kit  following the manufacturer’s instructions. TaqMan genotyping assays  for ® reagent (Invitrogen) and then subjected to RNA extraction as described earlier [2O, and finally heated at 60 °C for 10 min. Then, 600 ng of RNA from each sample was added to 0.25 mL tubes containing 0.2 mL of PCR reaction, 4 μL of iScript™ Reverse Transcription (Bio-Rad®), and up to 20 μL RNase free H2O for cDNA synthesis. All tubes were then transferred to a thermal cycler  and run for 5 min at 25 °C, 20 min at 46 °C, and 1 min at 95 °C. The final concentration of cDNA synthesized for each sample was 30 ng/μL. A total volume of 1 μL of cDNA, 10 μL of Fast SYBR Green Mastermix (Thermo Fisher Scientific®), 1 μL of either TNFα, TNFRSF1A, or TNFRSF1B PrimePCR SYBER Green Assay mix (Bio-Rad®), and 8 μL of molecular biological grade sterile H2O were mixed in a 96-well microamp RT-PCR reaction plate. Then, 18s RNA gene oligonucleotide primer controls  were used in order to acquire baseline CT readings. The RT-PCR reaction was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems®). The equation (2^ (−∆CT) × 1000), where ∆CT = Sample RT-PCR CT reading-18s CT baseline, was used to calculate relative mRNA expression levels.Leukocytes from 1.0 mL of blood were isolated in a 2.0 mL RNase free microcentrifuge tube, suspended in 1.0 mL of TRIzol earlier . RNA pel® 8.02. For the osteocalcin level, data are presented as mean ± SD, where a two-tailed t test was used to compare the level in RA patients vs. healthy controls and the presence vs. the absence of MAP infection. For genotype frequency, a two-tailed Z test and odds ratio analysis were used to compare SNPs in RA patients vs. healthy controls. At each gene examined, SNP genotypes were categorized into four groups , then each genotype group was tested for significance within its subcategory at p < 0.05 and a 95% confidence interval (CI). For gene expression analysis, we compared the average gene expression in RA vs. healthy control for each gene regardless of their genotype using an unpaired two-tailed t-test at p < 0.05 and a 95% confidence interval (CI), and then we compared individuals who carried two major alleles with others for each SNP tested using one-way ANOVA, where a Newman–Keuls post-hoc test was selected for multiple comparisons. For MAP infection susceptibility, infection proportions between SNP genotypes and major alleles in the RA group was compared to the healthy control group separately using a two-tailed Z test at p < 0.05. Age and gender were not included as covariates, as for all data sets no age or gender effects were observed.All statistical tests were performed using GraphPad Prismp-value < 0.05, odds ratio (OR) = 2.14, 95% CI: 1.12–5.20).Overall, MAP DNA was detected in the blood of 28 out of 102 (27.5%) of participating subjects. Specifically, MAP DNA was detected in 17/48 (35.4%) RA patients and 11/54 (20.4 %) healthy controls .As shown in p-value < 0.05) in blood samples that are MAP negative. Specifically, the average osteocalcin level in RA patients who are positive for MAP was 0.60 ± 0.31 ng/mL compared to 3.85 ± 1.31 ng/mL  in RA patients who are negative for MAP DNA, as shown in As shown in TNFα (rs1800629), two SNPs in TNFRSF1A (rs767455 and rs4149570), and one SNP in TNFRSF1B (rs3397). Genotype distribution of these SNPs conveyed the Hardy–Weinberg equilibrium and are all listed in TNFα:rs1800629 (GG to GA) was significant in RA patients compared to healthy controls . Specifically, TNFα:rs1800629 was detected in 19/48 RA patients (40%) compared to 8/54 healthy controls (15%) . TNFRSF1B:rs3397 (CC to CT) was detected in 21/48 RA patients (44%) compared to 10/54 (10%) healthy controls . TNFRSF1A:rs767455 (AA to AG or GG) was not significant in RA patients.We have assessed 102 subjects  for one SNP in TNFα was 3-fold higher in RA patients (2.68 ± 0.34), compared to healthy subjects (0.79 ± 0.23) . As shown in TNFRSF1A was 0.42 ± 0.11 in RA compared to 0.81 ± 0.14 in healthy controls . The relative gene expression of TNFRSF1B was 0.39 ± 0.15 in RA compared to 0.71 ± 0.13 in healthy controls . Most importantly, the average relative gene expression level of TNFRSF1B was significantly lower in RA patients with rs3397 (CT: 0.34 ± 0.14 or TT: 0.27 ± 0.12) compared to RA patients with the major genotype (CC: 0.51 ± 0.17) , as shown in rs767455 did not have any significant effect on TNFRSF1A expression, as shown in rs1800629 on TNFα expression was not significant, as shown in In general, and regardless of the presence or absence of SNPs, the average relative gene expression for TNFRSF1B:rs3397 was associated with a lower active osteocalcin level. Blood samples from patients with the CC genotype had a significantly higher level of active osteocalcin (1.86 ± 0.61 ng/mL) , compared to samples from patients who had the TNFRSF1B:rs3397 homozygous (TT) genotype (0.67 ± 0.35 ng/mL), as shown in Among RA patients, only TNFα:rs1800629 SNP (GG genotype) was 3.55 ± 0.61 ng/mL, compared to the heterozygous or homozygous SNPs (GA or AA genotype), which was 1.75 ± 0.36 ng/mL, as shown in TNFRSF1A:rs767455 (AA genotype) had an average active osteocalcin level of 3.43 ± 0.74 ng/mL, which is significantly higher than healthy controls who had a homozygous SNP (GG genotype), which was 0.94 ± 0.17 ng/mL , as shown in The average active osteocalcin level of healthy controls without the TNFRSF1B:rs3397 (CT or TT genotype) were significantly positive for MAP DNA , compared to blood from RA patients with the wildtype (CC) genotype . Similarly, MAP DNA was detected in the blood samples of 50% of the healthy controls with the TNFRSF1B:rs3397 (TT) genotype, compared to the wildtype (CC) genotype of healthy controls , as shown in TNFα:rs1800629 or TNFRSF1A:rs767455. Additionally, we categorized RA patients into four groups depending on the treatment they were receiving. There was no significant association between treatment option and MAP infection or osteocalcin level, as shown in Blood samples from RA patients with The development of osteoporosis and osteoporotic fractures are common among RA patients ,2,3,4,5.Mycobacterium avium subspecies avium (MAA) [M. tuberculosis infection in patients on anti-TNFα treatment [Our group has recently established that in vitro infection of macrophages with MAP caused more production of pro-inflammatory cytokines such as TNFα, IL-6, and IL-12 . Severalum (MAA) . Additioum (MAA) . In contum (MAA) . Interesum (MAA) . This isreatment . In thisSince MAP was associated with low active osteocalcin levels in CD in an earlier study , we wereα profiling and anti-TNFα treatment options [M. tuberculosis infection and subsequent impact on bone erosions and osteoporosis in these patients. As observed in CD, we determined that the relative expression level of TNFα was 2.7-fold higher, while the TNFRSF1A and TNFRSF1B expression level was 1-fold lower in RA patients compared to healthy controls. More importantly, both the CT and TT genotypes of TNFRSF1B:rs3397 have significantly reduced gene expression. Coincidentally, MAP was detected significantly higher in the blood of RA patients with TNFRSF1B:rs3397 compared to wildtype RA. The data strongly associates TNFRSF1B:rs3397 with MAP infection and low osteocalcin levels in RA. This confirms earlier findings in CD with unique SNPs in TNFα receptors (TNFRS1A:rs767455 and TNFRS1B:rs3397) [The shared similarities between CD and RA, especially with regards to TNF options ,32, have:rs3397) .TNFRSF1A and TNFRSF1B in MAP-infected cells. MAP infection resulted in lower expression of TNFRSF1A, while no changes in expression of TNFRSF1B were observed in infected macrophages [TNFRSF1A has been recognized as the more pro-apoptotic gene, while TNFRSF1B has been linked to apoptosis suppression [TNFRSF1A:rs767455 and TNFRSF1B:rs3397 altered expression of corresponding genes, leading to further downregulation, which may cause MAP-infected macrophages to persist with apoptosis and production of higher levels of TNFα. An elevated TNFα level plays a crucial role in the osteoclast paracrine stimulation, which influences bone resorption ultimately [A critical point to consider in the intersection of immune response and apoptosis is the expression of rophages . Interespression . TNFRSF1timately . The datFurther genetic and functional analyses should be done in order to establish whether these SNPs cause linkage disequilibrium with a nearby-localized gene responsible for the observed effect. It is worth mentioning that TNFα:rs1800629 was strongly associated with the resolution of hepatitis B virus (HBV) infection and reported in susceptibility to human papillomavirus (HPV) infection . The SNPTNFα receptors (TNFRS1A:rs767455 and TNFRS1B:rs3397). Collectively, the data supports a link between intracellular infection with MAP, SNPs in TNFα receptors, and active osteocalcin levels. The conclusion of this study should provoke scientists to consider inflammatory disorders and their complications as an interplay between genetic predisposition, effects of standard treatment options, and acquired infection. A personalized treatment option should be more effective to achieve a cure.Overall, MAP infection has significantly lowered sera levels of active osteocalcin, a major biomarker for osteoporosis in both CD and RA patients, which confirms our earlier data, where MAP infection decreased active osteocalcin levels in patients with CD . MAP inf


In this study, we examined the effects of these herbs on LPS-treated mouse macrophage RAW264.7 cells. Both shokyo and kankyo reduced LPS-induced PGE2 production in a concentration-dependent manner. Shokyo and kankyo did not inhibit cyclooxygenase (COX) activity, nor did they alter the expression of molecules in the arachidonic acid cascade. In addition, these herbs did not alter NF-κB p65 translocation into nucleus, or phosphorylation of p65 or ERK. These results suggest that shokyo and kankyo inhibit cPLA2 activity. Although 6-shogaol produced similar results to those of shokyo and kankyo, the concentration of 6-shogaol required for the reduction of PGE2 production were higher than those of 6-shogaol in shokyo and kankyo. Therefore, several gingerols and shogaols other than 6-shogaol may play a role in the reduction of LPS-induced PGE2 production. Thus, 6-shogaol, and other gingerols and shogaols inhibit cPLA2 activity and reduce LPS-induced PGE2 production via a different mechanism from traditional anti-inflammatory drugs. Moreover, kampo medicines that contain shokyo or kankyo are considered to be effective for inflammatory diseases.We previously reported that shokyo and kankyo, which are water-extracted fractions of ginger, reduced LPS-induced PGE We focused on the inflammatory effects of these kampo medicines, and found that lipopolysaccharide (LPS)-induced PGEo (TJ-9) , orento o (TJ-9) , hangesho (TJ-9) , kakkonto (TJ-9) , shinbuto (TJ-9) . Moreove Cortex) , and kanoduction . In partZingiber offinale Roscoe). As described in the recent our review . Arachidonic acid and 6-shogaol were purchased from Cayman Chemical . NF-κB inhibitor, BAY 11-7082, was purchased from Wako. Mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor, PD98059, were purchased from Sigma . Other reagents were purchased from Nacalai Tesque.Powders of shokyo and kankyo were provided by Tsumura & Co. 3-D HPLC profiles of shokyo and kankyo were shown in 2.The mouse macrophage cell line RAW264.7  was cultured in culture medium at 37 °C in a humidified atmosphere of 5% CO2 incubator.The numbers of viable cells were measured using WST-8  according to the manufacturer’s instructions. In brief, cells were seeded onto 96-well plates  , and treated with shokyo or kankyo for 24 h. Then, the media were removed by aspiration and the cells were treated with a 100 µl mixture of WST-8 with culture medium for 2 h at 37 °C in a COn = 4).The optical density was measured (measured wavelength at 450 nm and reference wavelength at 655 nm) using an iMark microplate reader , and the mean background value was subtracted from each value. Data are presented as mean ± SD  and incubated in culture medium at 37 °C overnight. For simultaneous treatment, cells were treated with varying concentrations of each herb in the absence or presence of LPS (100 ng/ml) for 24 h (200 µl per well) in triplicate or quadruplicate for each sample. For sequential treatment, cells were treated with medium or LPS for 30 min, and thereafter treated with medium or each herb for 24 h. After the culture supernatants were collected, viable cell numbers were measured using WST-8 as described above.2 in the culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions , and were adjusted by the number of viable cells. Data are presented as pg per 10,000 cells (mean ± SD).The concentrations of PGE2 activity was evaluated using cPLA2 Assay kit  according to the manufacturer’s instructions. RAW264.7 cells were cultured in 100-mm dishes, and treated with 100 ng/ml of PgLPS for 2 h. Then, the cells were washed twice with Tris-buffered saline (TBS), transferred into microcentrifuge tubes, and centrifuged at 6,000× g for 5 min at 4 °C. Supernatants were aspirated, and cells were resuspended in TBS with 1/100 volume of protease inhibitor cocktail  and 1/100 volume of phosphatase inhibitor cocktail , and were homogenized with Dounce tissue grinder , Then, cells were centrifuged at 12,000× g for 15 min at 4 °C. The supernatant was collected and used as sample. To detect only cPLA2 activity, samples were pretreated with 20 µM of bromoenol lactone  and 20 µM of thioetheramide-PC  for room temperature for 15 min. Bee venom PLA2 was used as positive control.cPLA2 in the supernatants were measured by ELISA. Data are presented as 100% at LPS alone (mean ± SD).COX-2 activity was indirectly evaluated as reported previously , with slRAW264.7 cells were cultured in 60-mm dishes, and treated with combinations of LPS and herbs for the indicated times. Then, the cells were washed twice with TBS, transferred into microcentrifuge tubes, and centrifuged at 6,000× g for 5 min at 4 °C. Supernatants were aspirated and cells were lysed on ice in lysis buffer  for 30 min at 4 °C. Samples were next centrifuged at 12,000× g for 15 min at 4 °C, and supernatants were collected. The protein concentration was measured using a BCA Protein Assay Reagent kit .The samples (50 µg of protein) were fractionated in a polyacrylamide gel under reducing conditions and transferred onto a polyvinylidene difluoride (PVDF) membrane . The membranes were blocked with 5% ovalbumin for 1 h at room temperature and incubated with the primary antibody for an additional 1 h. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized with an ECL kit . Protein levels were quantified using image analysis software ImageJ .2 , LOX-5 , annexin 1 , actin , NF-κB p65 , and phosphorylated p65 (Ser536)  were purchased from Santa Cruz Biotechnology . Antibodies against extracellular signal-regulated kinase  and phosphorylated ERK  were from Cell Signaling Technology . Horseradish peroxidase-conjugated anti-goat IgG  was from Santa Cruz, and anti-rabbit IgG  and anti-mouse IgG  were from DakoCytomation .Antibodies against COX-2 , cPLATo detect p65 localization RAW264.7 cells were directly cultured on Lab-Tek chamber slides  overnight. The cells were treated with shokyo, kankyo (100 µg/ml), 6-shogaol , or BAY 11-7082 (10 µM) for 2 h, and further treated with LPS (100 ng/ml) and shokyo, kankyo, 6-shogaol, or BAY 11-7082 for 30 min. Then, the cells were fixed with ice-cold methanol for 30 min at −20 °C. Subsequently, the cells were washed with PBS for 3 times, blocked with 1% BSA in PBS for 1 h, incubated with anti-p65 antibody (1:50 dilution) for 1 h at room temperature. After washing with PBS for three times, cells were incubated for 1 h at room temperature with Alexa Fluor488-conjugated secondary antibody . After washing with PBS for three times, cells were mounted with VECTASHIELD Antifade Mounting Medium with DAPI . Fluorescence was visualized using Axioplan2 imaging .The quantification of 6-shogaol in each herb was performed by the Nagano Prefecture Pharmaceutical Association Analytical Examination Center . In brief, one ml of samples (herbs in culture medium) was absorbed to a reverse-phase system cartridge . Columns were washed with two ml of 40% MeOH and one ml of 70% MeOH. Then, samples were eluted with 100% MeOH and concentrated to one ml. These samples were subjected to high-performance liquid chromatography (HPLC) with LC-20A . The conditions were as follows: column, X-Bridge 2.1 × 150 mm, three µm (Waters); solvent, 70% aqueous acetonitrile; flow rate, 0.15 min/ml; column oven, 40 °C; detection, 228 nm; and injection, 10 µl.50 value and its 95% confidence interval (CI) were calculated using the ‘drm’ function in the ‘drc’ package  strongly reduced LPS-induced PGE2 production . Next, we examined the effects of shokyo and kankyo on COX activity. In order to bypass PLA2, we added exogenous arachidonic acid to RAW264.7 cells treated with LPS alone or LPS plus herbs (simultaneous treatment experiment). Then, we measured the PGE2 level produced by COX. Both shokyo and kankyo increased LPS-induced PGE2 production -5 expression was not altered by LPS treatment. Moreover, LOX-5 expression was not affected by shokyo or kankyo . Shokyo as added  and S2C. of PGE2  and 3C.2 production by RAW264.7 cells at a similar concentration to shokyo and kankyo, we quantified the amount of 6-shogaol in shokyo and kankyo. HPLC analysis revealed that the 5 mg/ml shokyo and kankyo solutions used in this study contained 2.97 µM and 4.87 µM 6-shogaol, respectively  6-shogaol inhibited PGE2 production. The IC50 value of 6-shogaol was 105 nM (95% CI: 27.7–182 nM). However, comparing with the calculated 6-shogaol concentration in shokyo and kankyo based on the results in 2 production were weaker than those of shokyo and kankyo  is induced by the ingestion of acid nonsteroidal anti-inflammatory drugs (NSAIDs), and is considered to be caused by leukotrienes, which are increased by acid NSAIDs an contract the bronchus , which includes fresh ginger roots, demonstrated a protective potentiality against indomethacin-induced gastric ulcer in rats via increment of the indomethacin-declined PGE2 levels in the stomach (Next, we will discuss about protective effect on gastric ulcer. In general, ginger reduced PGE stomach . Therefo stomach . These r2 among PLA2 isotypes. Therefore, in the future, the evaluation of the effects of shokyo and kankyo on iPLA2 and sPLA2—expressions and activities—will be needed. Moreover, the evaluation of the anti-inflammatory effects of shokyo and kankyo on the inflammatory diseases such as periodontal disease or stomatitis by animal models will be needed.There are several limitations in this study. We evaluated only cPLA2 production in RAW264.7 cells. Their mechanisms of action were suggested to via the inhibition of cPLA2 activity because both herbs did not inhibit COX activity or suppress the expression of molecules in the arachidonic acid cascade.We demonstrated that shokyo and kankyo, which are water-extracted fractions of ginger, reduced LPS-induced PGE10.7717/peerj.7725/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.7725/supp-2Data S1Click here for additional data file.10.7717/peerj.7725/supp-3Data S2Click here for additional data file.10.7717/peerj.7725/supp-4Data S3Click here for additional data file.10.7717/peerj.7725/supp-5Data S4Click here for additional data file.10.7717/peerj.7725/supp-6Data S5Click here for additional data file.10.7717/peerj.7725/supp-7Data S6Click here for additional data file.10.7717/peerj.7725/supp-8Data S7Click here for additional data file.10.7717/peerj.7725/supp-9Data S8Click here for additional data file.10.7717/peerj.7725/supp-10Data S9Click here for additional data file.10.7717/peerj.7725/supp-11Data S10Click here for additional data file.10.7717/peerj.7725/supp-12Data S11Click here for additional data file.