Despite numerous interventions promulgated by the Surgical Care Improve Project (SCIP) and other organizations, surgical site infection (SSI) continues to be a significant medical problem. DFA-02 is a novel bioresorbable modified-release gel consisting of both gentamicin (16.8 mg/mL) and vancomycin (18.8 mg/mL) to be applied during surgical incision closure for the prevention of SSIs. The following double-blind phase 2a trial was designed to test the safety and tolerability of DFA-02.At six US sites, the study planned to randomize 40 subjects undergoing colorectal surgery  in four ascending dose cohorts . Safety was ascertained and serum pharmacokinetics (PK) was determined.Study enrollment was discontinued after the first three dose cohorts  as even very large incisions could not accommodate more than 20 mL of gel, leaving no scientific justification for the 40-mL cohort. DFA-02 was well tolerated and showed no evidence of local tissue reaction or impairment of wound healing. No serious AEs were deemed related to study drug. Systemic exposure to gentamicin and vancomycin remained well below levels considered to be at higher risk for oto- or nephrotoxicity. The maximal gentamicin and vancomycin levels observed were 2.36 and 0.684 μg/mL at 6 h, which were well below the prespecified stopping criteria of 12 and 20 μg/mL, respectively.In this small phase 2a study, the study drug was well tolerated and appeared to be free of serious adverse effects. Consistent with these findings, the PK values were consistent with gradual release of the antibiotics from the gel in the surgical site.NCT01496352ClinicalTrials.gov,  Surgical site infections (SSIs) are a common nosocomial infection in the USA and a significant cause of mortality and morbidity  and coordinated by the Duke Clinical Research Institute , and was conducted at six US sites. It was registered at clinicaltrials.gov (NCT01496352).Ethics, consent, permissions, and competing interests—the study was approved by the Duke Health System Institutional Review Board as well as the IRBs overseeing research at each of the sites as follows: The Ohio State University Medical Center , Tampa General Hospital, and University of South Florida South Tampa Center , Scott & White Healthcare , Eliza Coffee Memorial Hospital , Memorial Hermann Memorial City Medical Center . All subjects provided written informed consent. Safety was reviewed on an ongoing basis by a colorectal surgeon  not otherwise involved in the trial. This trial was funded by the sponsor (Dr. Reddy’s Laboratories); however, the DCRI controlled the performance of all data analysis and drafted the manuscript.Adult patients undergoing colorectal surgery were enrolled in this trial.Inclusion criteria were as follows: (1) males and non-pregnant females 18 years of age or older; (2) body mass index (BMI) 25 to 40; and (3) scheduled to undergo non-emergent colorectal surgery involving a laparotomy incision of 7 cm or greater . Eligible procedures included left, right, or transverse colectomy; segmental/sleeve left colon resection; total abdominal colectomy with ileorectal anastomosis; total abdominal colectomy with ileostomy; total abdominal proctocolectomy; low anterior resection; sigmoid resection; non-emergent Hartmann’s procedure; colotomy with polypectomy distal to hepatic flexure; colostomy takedown through laparotomy  incision; ileo-pouch anal anastomosis; and abdominal perineal resection of the rectum.Exclusion criteria were as follows. (1) Known history of hypersensitivity to gentamicin or vancomycin, other aminoglycoside antibiotics, or the excipients of the study products (soy bean products or sesame oil). (2) Emergency surgery . (3) Significant concomitant surgical procedure . (4) Prior laparotomy within 60 days of this planned procedure. (5) Planned second laparotomy or colorectal surgical procedure  within 30 days of this planned first procedure. (6) Expectation that a surgical drain would be placed. (7) Preoperative sepsis, severe sepsis, or septic shock. (8) Abdominal wall infection/SSI from previous laparotomy/laparoscopy or for any reason. (9) Active systemic infection or systemic  antibiotic therapy within 1 week before the date of surgery other than specified preoperative antimicrobial prophylaxis . (10) Requirement for gentamicin or vancomycin preoperative antimicrobial prophylaxis . (11) Requirement for concomitant use or use during the 30 days before day 1 of any prescription or over-the-counter drug that would interfere with the study or place the patient at undue risk. Concurrent systemic or topical use of other potentially neurotoxic, nephrotoxic, and/or ototoxic drugs, such as gentamicin, cisplatin, cephaloridine, kanamycin, amikacin, polymyxin B, colistin, paromomycin, streptomycin, tobramycin, vancomycin, ethacrynic acid, furosemide, and viomycin, was to be avoided. (12) Preoperative evaluation suggested an intra-abdominal process that might preclude full closure of the skin. (13) Ongoing treatment  for non-colorectal cancer. (14) History of significant drug or alcohol abuse within the past year. (15) Serum creatinine >1.3 mg/dL. (16) Serum bilirubin >2.5 times upper limit of normal. (17) History of uncontrolled diabetes mellitus (controlled diabetic patients whose hemoglobin A1c was ≤9.0 % could be included). (18) Patients who were immunocompromised, including, but not limited to, systemic corticosteroid use or chemotherapy/radiation during the 30 days before surgery, organ transplantation, or human immunodeficiency virus infection . (19) Any clinically meaningful hearing loss . (20) Clinically exclusionary results on clinical laboratory, electrocardiogram, or physical examination, including, but not limited to, positive hepatitis B or C or human immunodeficiency virus. (21) Pregnant or lactating or if of childbearing potential and not practicing a birth control method with a high degree of reliability. (22) Refusal to accept medically indicated blood products. (23) Participation within 30 days before the start of this study in any experimental drug or device study or currently participating in a study in which the administration of investigational drug or device within 60 days was anticipated. (24) Patients with anterior abdominal wall mesh that was not planned to be completely removed during the planned procedure. (25) Unable to participate in the study for any reason in the opinion of the PI. (26) Postsurgical life expectancy of less than 30 days, in the investigator’s or sponsor’s opinion. (27) Expected discharge from the hospital less than 3 days after surgery.DFA-02 is a novel bioresorbable modified-release gel consisting of both gentamicin (16.8 mg/mL) and vancomycin (18.8 mg/mL). This sterile, viscous, clear gel contains sesame oil, soy lecithin, and dehydrated alcohol as excipients. The placebo gel was exactly the same as DFA-02 except for the active ingredients. The products were manufactured by NextPharma, Inc. . DFA-02 and matching placebo were supplied as 10 mL in 20-mL glass vials. The product was stored at −25 to −10 °C. It was thawed at room temperature until clear and used within 7 days of thawing. It was not refrozen and reused.Patients were randomized using a computer generated randomization scheme prepared by the trial’s statistician at the DCRI. DFA DFA-02 10, 20, or 30 mL or matching placebo was administered once at the conclusion of surgery in the operating room. The gel from one or two vials (10 or 20 mL) was drawn into a 30-mL syringe (provided by the sponsor) from the vial with a 14-gauge needle  or, alternatively, the gel from the vial could be decanted into a sterile container/cup and drawn into the syringe without a needle. For the 10- and 20-mL cohorts, a single 30-mL syringe was used, and for the 30-mL cohort, two 30-mL syringes were used. After closure of the fascia, the gel was administered from the syringe, evenly covering the subcutaneous tissue of the index incision (the longest incision if more than one incision was made). The subcutaneous tissue was then approximated and the incision closed with either sutures or staples. Any extruded gel after closure was collected in a syringe, the volume recorded by the surgeon, and then discarded. Only the longest (index) laparotomy incision was treated with study drug. If a patient required reopening of the surgical incision for any reason, no additional study drug was applied.Consistent with the Surgical Care Improve Project (SCIP) guidelines, all patients received standard preoperative antimicrobial prophylaxis antibiotics within 60 min of initial skin incision. Antibiotic prophylaxis was not continued for more than 24 h. Antibiotic prophylaxis could be redosed during surgery for long procedures based on SCIP guidelines. The use of oral nonabsorbable preoperative antibiotic treatment was at the discretion of the surgeon. Adherence to other SCIP guidelines, e.g., maintenance of normothermia, method of hair removal, was recorded. Prohibited interventions included administration of topical antimicrobials , systemic gentamicin or vancomycin preoperatively or within 72 h of surgery, and prophylactic use of negative pressure dressings .The main objectives of the study were to (1) evaluate the safety and tolerability of ascending doses of DFA-02 in patients undergoing colorectal surgery and (2) evaluate the systemic PK of gentamicin and vancomycin after single doses of 10 to 40 mL of DFA-02 in patients undergoing colorectal surgery.Safety and tolerability were assessed by (1) physical examination; (2) surgical incision examination on days 2, 3, and 5  and day 14; (3) vital signs: screening (baseline), day of surgery (pre- and postoperatively), days 2, 3, 5, and 14; (4) clinical laboratory tests : screening, days 5 and 14; (5) ECG: screening (baseline), day 5; (6) adverse event recording on days 1, 2, 3, 5, 14, and 30; (7) ASEPSIS score on day 5 (96 h) postoperatively; and (8) wound questionnaire on day 30.Additional recorded variables included (1) evaluation of postoperative renal function as manifested by serum creatinine levels on days 5 and 14 compared with baseline and (2) evaluation of the incidence of antibiotic resistance based on baseline and postoperative rectal/stool and nasal culture and sensitivities for vancomycin-resistant enterococcus (VRE) and MRSA, respectively, and antibiotic sensitivity of any culture positive SSIs. Molecular typing was also utilized to identify MRSA and VRE in lieu of culture and sensitivity.Systemic PK of gentamicin and vancomycin were determined from serial blood draws obtained at 1, 6, 24, 48, and 96 h after study drug administration. These assays were performed at MicroConstants, Inc .n, mean, standard deviation (SD), and minimum, median, and maximum values. Categorical variables were summarized using the number and percentage of patients in each category. Gentamicin and vancomycin plasma levels were obtained 1 and 6 h after study drug application and on days 2, 3, and 5 . Cmax, AUCt0–, AUC0–∞, Tmax, t1/2, CL/F, and V/F were calculated from the plasma concentrations using standard methods.The sample size and study design were typical for phase 2 safety, tolerability, and PK studies, and the study was not statistically powered. This study planned to enroll 40 patients (ten per cohort) undergoing colorectal surgery with a planned incision of 7 cm or greater. Four dose cohorts were planned: 10, 20, 30, and 40 mL of DFA-02 administered as a single dose into the surgical incision at closure. All study data were summarized using descriptive statistics at each assessment time for each treatment group based on actual values and change from baseline/screening values. Continuous variables were summarized using Study enrollment was discontinued after the first three dose cohorts  as even very large incisions could not accommodate more than 20 mL of gel, leaving no scientific justification for the 40 mL cohort. Each cohort contained ten subjects (two patients receiving placebo and eight patients receiving study drug). It was anticipated that not all of the study drug would be retained in the wound, e.g., smaller incisions, so we recorded the net volume of study drug retained. Therefore, results  were those commonly seen with colorectal surgery. No serious AEs were considered to be related to study drug. Serum creatinine measured on postoperative days 4 (or discharge) and 14 were not significantly increased compared with baseline values. No patients showed MRSA in preoperative nasal cultures or VRE in preoperative rectal cultures. One patient in the DFA-02 group showed nasal MRSA and one patient in the placebo group showed rectal VRE at discharge.Tmax values of 6.00, 6.09, 6.15, and 6.01 h for cohorts 1, 2, 3, and all cohorts combined, respectively. The Tmax values appeared to be dose-independent. The mean Cmax values were 0.693, 0.626, 0.608, and 0.642 μg/mL for cohorts 1, 2, 3, and all cohorts combined, respectively. Corresponding mean AUC0–t values were 15.30, 15.20, 12.10, and 14.20 μg∙h/mL for cohorts 1, 2, 3, and all cohorts combined, respectively. Vancomycin was absorbed into the systemic circulation with median Tmax values of 6.57, 23.9, 24.1, and 22.2 h for cohorts 1, 2, 3, and all cohorts combined, respectively. The median Tmax in cohorts 2 and 3 was longer than that of cohort 1. The mean Cmax values were 0.157, 0.168, 0.166, and 0.164 μg/mL for cohorts 1, 2, 3, and all cohorts combined, respectively. Corresponding mean AUC0–t values were 6.41, 6.28, 6.71, and 6.47 μg h/mL in cohorts 1, 2, 3, and all cohorts combined, respectively.Systemic exposure to gentamicin and vancomycin remained well below levels considered to be at higher risk for oto- or nephrotoxicity. The maximal gentamicin level for any patient was 2.36 μg/mL at 6 h, which was well below the prespecified stopping criterion of 12 μg/mL at 6 h. The maximal vancomycin level for any patient was 0.684 μg/mL at 6 h, well below the prespecified stopping criterion of 20 μg/mL. Pharmacokinetic data are shown in Table The study was not designed or powered to rigorously assess SSIs; however, no significant differences were observed in this endpoint .This phase 2 dose-ascending tolerability, safety, and pharmacokinetic study met its objectives. The inclusion of a placebo group in each dosing cohort enabled us to maintain a double-blind study, avoiding possible bias with regard to assessments of safety and tolerability. Consistent with preclinical animal studies conducted by the sponsor, DFA-02 was well tolerated and showed no evidence of local tissue reaction or impairment of wound healing. Safety was assessed by collection of adverse events and by recording safety events of interest, e.g., related to renal function and serum levels of the antibiotics. No serious adverse events were deemed related to study drug.Cmax)/DFA-02 dose correlations observed in preclinical in vivo studies were 0.69 μg/mL for gentamicin and 0.33 μg/mL for vancomycin, observed in a pig model of wound healing . Extrapolating from the Cmax/dose correlations in pigs, the expected human Cmax for a 60-kg person receiving 40 mL of DFA-02 was estimated to be 7.8 μg/mL for gentamicin and 4.2 μg/mL for vancomycin, both within the clinically accepted range. In our study, the maximal levels of gentamicin and vancomycin for any patient were 2.36 and 0.684 μg/mL at 6 h, respectively, well below the prespecified stopping criteria for the study. In fact, the maximum dose actually tested was 30 mL since as even very large incisions could not accommodate more than 20 mL of gel, leaving no scientific justification for advancing dose escalation to the 40 mL cohort. The systemic PK suggested slow release of the antibiotics from the gel in the incision site.Postoperative renal function, assessed by mean serum creatinine levels, showed no adverse effect for DFA-02 compared with placebo gel. A single patient had a creatinine elevation 2 days after surgery that returned to normal within 3 days. The maximum recommended daily intravenous dose for gentamicin in patients with normal renal function is 1.7 mg/kg every 8 h or 306 mg/day for a 60-kg patient with a goal of avoiding peak systemic levels greater than 12 μg/mL and trough levels greater than 2 μg/mL (Gentamicin Dosage - Drugs.com. Available at: http://www.drugs.com/dosage/gentamicin.html#Usual_Adult_Dose_for_Surgical_Prophylaxis. Accessed July 2 Our study has several limitations. As is typical for most “first in patient” studies, in order to optimize safety of study subjects, there was an extensive list of exclusion criteria, which can limit the generalizability of these results to all surgical patients. In particular, while preclinical models showed no adverse effects of the study drug on renal function, to be conservative, we excluded patients with serum creatinine >1.3 mg/dL preoperatively. While no adverse effect was shown on renal function, we cannot rule out different results in patients with preexisting renal dysfunction. As expected, and consistent with the small sample size, we observed imbalances between study arms in several patient characteristics. It is important to note, however, that this should not affect many of the study’s findings given the nature of the pharmacokinetic and other analyses. Finally, our study was not powered to draw any conclusions as to the efficacy of DFA-02 in preventing SSIs in colorectal surgery. A larger multicenter study is designed to evaluate the efficacy of DFA-02 with respect to prevention of SSI.In this small phase 2a study, the study drug was well tolerated and appeared to be free of serious adverse effects. Consistent with these findings, the PK values were consistent with gradual release of the antibiotics from the gel in the surgical site.Staphylococcus aureus; PD, pharmacodynamics; PK, pharmacokinetics; SCIP, Surgical Care Improvement Project; SD, standard deviation; SSI, surgical site infection; VRE, vancomycin-resistant enterococcusAUC, area under curve; BMI, body mass index; MIC, minimum inhibitory concentration; MRSA, methicillin resistant


Kocuria sp. BRI 35 isolated from Antarctic water sample. Amongst 10 variables evaluated, magnesium chloride, protease peptone, glucose, and temperature were significant. Response surface methodology consisting of a central composite design was developed to study the interactions between the variables and to determine optimal values of significant variables. A quadratic model  was built. The contour plots indicated that the isolate produced maximum PUFA at lower concentrations of magnesium sulfate (0.9 g/L) and higher concentrations of protease peptone (5 g/L) and glucose (10 g/L) at 15°C. MgSO4 and glucose exhibited quadratic as well as interactive effect on PUFA production whereas protease peptone and temperature showed interactive effects only. After optimization, PUFA production per unit biomass increased from 0.94 mg/g to 11.12 mg/g. This represented an increase from 3% to 58.62% of the total fatty acids. Among PUFAs, the yield of ω-6 fatty acids increased from 9.66 mg/L to 107.71 mg/L with significant increase in linoleic acid (20.36 mg/L) whereas ω-3 fatty acids increased up to 12.37 mg/L with DHA being the major ω-3 fatty acid produced.In this study, Plackett-Burman design was used to identify the most influential parameters affecting PUFA production by  Amongst these ω-3 fatty acids , eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA)) and ω-6 fatty acids (linoleic acid (LA), gamma linolenic acid (GLA), and arachidonic acid (AA)) are of importance in human health [ω-3 and ω-6 fatty acids are known to adversely affect the development and proper functioning of brain and central nervous system. Lack of these acids may lead to neurological disorders like attention deficit hyperactivity disorder (ADHD), dyslexia, dyspraxia, and autism [ω-3 fatty acids are also known for their anti-inflammatory properties [Research on production and purification of polyunsaturated fatty acids (PUFAs) has gained a lot of significance due to their role in human health and nutrition. PUFAs are fatty acids that have more than one double bond in their back bone structure. Based on the position of their double bonds they are grouped as n health . Moreoven health . Hence td autism . ω-3 fatoperties . WidesprFish oil is the most widely used source for obtaining PUFAs. However, this oil gets easily oxidized and hence is often associated with unpleasant taste and odor . Besides Moritella (25 mg/g) [ Pythium (90 mg/L) [ Thraustochytrium [ω-6 fatty acids, Klempova et al. [ Umbelopsis isabellina CCF 2412. On the other hand Mortierella alpina produces AA  [ Shewanella species  [ Aureispira sp. (21.5 mg/L of AA) [ Photobacterium sp. [Several fungi and bacteria are known to produce commercially important PUFAs. EPA production has been reported from25 mg/g)  and Pyth90 mg/L) , whereaschytrium  producesa et al.  have repy acids) . Bacteriy acids) , AureispL of AA) , and Phorium sp.  are the rium sp.  have rev Schizochytrium sp., Elrazak et al. [Considering importance of PUFA in human health and suitability of microbial sources, many researchers have published work on optimization of PUFA production from microorganisms , 19, 20. Kocuria sp. BRI 35. The most significant variables were identified using Plackett-Burman design. These variables were further optimized using RSM. To our knowledge this is the first report on optimization studies for PUFA production from Kocuria species.In this paper we report optimization of PUFA production fromAll the media components and chemicals were purchased from Sigma Aldrich, Hi Media, and Merck  and were of analytical grade.4, 7.0 g MgCl2, 5.0 g protease peptone, 2.0 g KCl, 1.0 g glucose, 0.36 g CaCl2, 0.06 g NaHCO3, 0.026 g NaBr, and 15 g agar with pH adjusted to 7.0  ±  0.2). BRI 35 was identified using 16S rRNA gene sequencing.BRI 35 was isolated from marine water sample (latitude S 51°06′34.4′′ and longitude E 57°35′40.2′′). The isolate was maintained on marine salt medium (MSM)  and was further used for inoculation in all the experiments at 10% concentration. For measurement of microbial biomass (g/L) the cell pellets were lyophilized. For determination of lipid content, lipids in the cells were extracted, dried, and weighed according to method described by Bligh and Dyer . EstimatFor all the experiments cell mass was obtained by centrifugation at 10,000 rpm for 10 minutes. 1 g of cell mass was boiled for 20 min in 0.5 N methanolic sodium hydroxide solution. Further 5 mL of boron trifluoride (methanolic) was added to the mixture. This was followed by vortexing and 15 minutes of incubation. The FAMEs thus prepared were extracted in 1 mL heptane .μm × 0.25 μm gas capillary column and a flame ionization detector was employed for FAMEs analyses. Nitrogen was used as a carrier gas at a pressure of 19.865 psi. 1 μL of sample was injected using split mode. Initial temperature of the column was 110°C which was increased up to 250°C at a rate of 3°C per minute. The column was maintained at 250°C for 2 minutes. The fatty acids present were detected by comparison of the retention time with those of standards.A gas chromatograph equipped with a 60 m × 250  Kocuria sp. BRI 35 were identified using the Plackett-Burman design. In all, 8 nutritional factors, namely, sodium chloride (NaCl), yeast extract (YE), magnesium sulfate (MgSO4), magnesium chloride (MgCl2), protease peptone (P.Pep), potassium chloride (KCl), glucose, and calcium chloride (CaCl2), along with 2 physical parameters, namely, temperature and pH, were evaluated. Each factor was studied at two levels. The variables studied and their high (+1) and low levels (−1) are presented in  P value < 0.05 were considered to be significant.The significant variables affecting PUFA production fromk + 2K + n0 . The matrix consisted of 6 central points and included experiments where one variable was set at extreme ±2 level and the other variables were maintained at central point level. In experimental runs where the software generated negative values, that component was not added in the medium. yc is coded level, Yc is actual value, Ycp is real value of central point, and Δyc is the step change. The amount of PUFAs produced can be expressed by the quadratic equationX is predicted response; β0 is the intercept; βc, βcc, and βcb are linear, quadratic, and interactive coefficients, respectively.The significant variables identified using the Plackett-Burman design were optimized using response surface methodology. A central composite design (CCD) matrix for 4 significant variables was generated. The total number of experiments generated was 2F value and a good multiple correlation coefficient (R). The fermentation conditions to obtain maximum PUFA production from BRI 35 were predicted using numerical optimization in the software. The factors under study were varied and the remaining variables were kept at level equal to those in MSM.The responses generated were analyzed using Design-Expert version 8.0. They were subjected to multiple regression analysis for calculation of the coefficients. The significance of the model was determined for testing its efficiency. The model under study was considered to be fit for optimization if it had a significant  Kocuria  (GenBank accession number: KF366396) [BRI 35 was maintained on MSM slants. The DNA was extracted using standard protocols and the isolate was identified using 16S rRNA gene sequencing. The results indicated it belonged to the genusF366396) . Kocuria sp. BRI 35. Eight nutritional and two physical parameters were studied at high and low levels. X is the effect of the tested variable, R+ and R− are the responses for high and low values, respectively, and N is the total number of experiments. The final equation generated in terms of actual factors wasP value at the confidence level of 95%  and those with P value <0.05 were considered to be significant. 4 (P = 0.0406), protease peptone (P = 0.0388), glucose (P = 0.0261), and temperature (P = 0.0211) were identified as variables that significantly affect PUFA production from Kocuria sp. BRI 35. On the other hand, variables like NaCl, YE, MgCl2, CaCl2, and pH exhibited P values >0.05 and hence were considered to be insignificant for PUFA production. Literature review suggests that amount of PUFA produced by microorganisms is greatly influenced by the temperature at which the microorganism is cultivated. Generally, microorganisms tend to produce more amounts of PUFAs at lower temperatures (5–25°C) [4, CaCl2, and so forth in the medium are also known to affect the type and amount of PUFAs produced [Plackett-Burman design was used to identify the significant variables affecting PUFA production from(5–25°C) , and theF-test and analysis of variance (ANNOVA)  . The effd values , 31. In d values . The R2A and C had not only quadratic  but also interactive effect on PUFA production  was found to be the most significant followed by AC (P < 0.0021) and BC (P < 0.0321). The optimal values of the variables were determined by constructing three-dimensional plots. The shapes of the plots are suggestive of the significance of the interaction between variables with respect to the response obtained. Elliptical plots indicate a significant interaction whereas circular plots indicate that the interaction between the variables is not a considerable contributor to the response obtained [4 (A) with protease peptone (B), glucose (C), and temperature (D) on PUFA production are demonstrated in Figures 4 are beneficial for PUFA production from Kocuria sp. BRI 35 with maximum production at 0.9 g/L MgSO4. Kang et al. [4 from Thraustochytrium aureum ATCC 34304. However, the same bacterium produces DHA optimally at 4.5 g/L of MgSO4 [4 plays a critical role in overall PUFA production as well as synthesis of individual fatty acids. This may be attributed to its function as a cofactor for enzymes involved in fatty acid synthesis. Hence its addition at proper concentration may increase the efficiency of the enzymes involved in fatty acid synthesis [Our results indicated that variables oduction . On the C P < 0.01. The opC P < 0.021. The og et al.  have repD) with MgSO4 (A) and protease peptone (B) are shown in Figures  Physcomitrella patens.The interactive effects of temperature (B) and glucose (C) on PUFA synthesis from BRI 35. The isolate produced PUFAs optimally in presence of protease peptone and glucose at 5 g/L and 10 g/L concentration, respectively. Similarly, Jang et al. [ Mortierella alpina. However authors have used starch as carbon source and KNO3 and yeast extract as nitrogen sources. C : N ratio of 25 : 7 was found to be optimum for production of ARA from Mortierella alpina CBS 754.68 [ Schizochytrium sp. S31 [g et al.  have repS 754.68 . Higher  sp. S31 . Kocuria sp. BRI 35 under the following fermentation conditions: MgSO4 0.94 g/L; protease peptone 5 g/L; glucose 10 g/L; temperature 15°C. The optimization strategies resulted in significant increase in the amount of PUFAs produced. In original medium PUFAs represented only 3% of total fatty acids produced. This corresponded to 0.94 mg/g of biomass. In the optimized medium more than 50% of fatty acids produced were PUFAs which accounted for 11.12 mg/g of biomass. Thus, the amount of PUFA's increased from 9.66 mg/L to 120.08 mg/L  in optimized medium is a unique finding since saturated and monounsaturated fatty acids have been mainly reported from the genus Kocuria [Thus, analysis of RSM plots indicated that maximum PUFAs are obtained from.08 mg/L . Kocurianditions . Presenc Kocuria , 41.ω-6 fatty acids increased from 9.66 mg/L  to 120.08 mg/L . Specifically, yield of linoleic acid increased from 1.14 mg/L to 20.36 mg/L under optimized conditions. Along with ω-6 fatty acids, our isolate exhibited the ability to produce ω-3 fatty acids particularly DHA (11.28 mg/L) under optimal conditions. Similarly, Morita et al. have reported maximum DHA production of 13.4 mg/L from Moritella sp. [ Kocuria sp. BRI 35 also produced several beneficial ω-6  and ω-3  PUFAs (ella sp. . Along wA) PUFAs . Kocuria sp. BRI 35 was accomplished using Plackett-Burman design and response surface methodology. MgSO4, protease peptone, glucose, and temperature were found to be the most significant parameters. Under optimized conditions, BRI 35 produced nutritionally important PUFAs like EPA, DHA, ARA, LA, GLA, and ALA. Thus BRI 35 exhibits the potential for commercial production of PUFAs.Optimization of culture conditions for production of PUFAs from


Herein, a dual-emissive conjugated polyelectrolyte P1 containing phosphorescent iridium(III) complex was designed and synthesized, which can form ultrasmall polymer dots (Pdots) in aqueous media. The F--responsive tert-butyldiphenylsilyl moiety was introduced into iridium(III) complex as the signaling unit for sensing F− with the quenched phosphorescence. Thus, the dual-emissive Pdots can rapidly and accurately detect F− in aqueous media and live cells as a ratiometric probe by measuring the change in the ratio of the F−-sensitive red phosphorescence from iridium(III) complex to the F−-insensitive blue fluorescence from polyfluorene. Moreover, the interaction of Pdots with F− also changes its emission lifetime, and the lifetime-based detection of F− in live cells has been realized through photoluminescence lifetime imaging microscopy for the first time. Both the ratiometric luminescence and lifetime imaging have been demonstrated to be resistant to external influences, such as the probe’s concentration and excitation power. This study provides a new perspective for the design of promising Pdots-based probes for biological applications.It is of paramount importance to develop new probes that can selectively, sensitively, accurately and rapidly detect fluoride in aqueous media and biological systems, because F In particular, reasonable water fluoridation and addition of fluoride to toothpaste have become a widespread practice due to the beneficial effects of fluoride on dental health and osteoporosis treatment123−1 (4 ppm or 211 μM) in drinking waterAs one of the important inorganic anions, fluoride anion  of fluoride− in aqueous media is based on the unique chemical reactivity between silicon and fluoride2223242526Currently, two standard fluoride-detecting methodologies have been recommended by WHO, including ion-selective electrode and ion chromatography. Both the techniques, however, require professional equipments and well-trained staff, and are difficult to be performed in those poor or even middle-income sufferers. Hence, a convenient and selective detecting methodology is highly desirable for the practical purpose. Fluorescent probes for F− detection especially in intracellular and in vivo detection, because they are endowed with a built-in correction for environmental effects28293032333435To solve the above problems, we hope to develop ratiometric luminescence and lifetime-based probes. Ratiometric luminescence detection, measuring the ratio changes of the emission intensity at two different wavelengths, allows more accurate F−-responsive tert-butyldiphenylsilyl (TBDPS) moiety into the ligand of red-emitting phosphorescent iridium(III) complex  complex to the F−-insensitive blue fluorescence from polyfluorene, the dual-emissive Pdots can rapidly and accurately detect F− in aqueous media and live cells as a ratiometric probe. Furthermore, the lifetime-based detection of F− in live cells has been realized through PLIM for the first time, and the advantages of lifetime detection have been demonstrated successfully.Herein, we introduced Fmplex 1, , which wx 1, P1, . CPE is 1 was shown in P1, two routes have been adopted (P1. The iridium(III) complex monomer 1’ was synthesized, which was then copolymerized with fluorene-based monomers by Suzuki coupling reaction. However, almost no target polymer P1 was generated, maybe due to the steric hindrance of two bulky TBDPS moieties in the ligands of 1’, which made the Suzuki coupling reaction difficult to take place. Then, route II was carried out. The macromolecular ligand Pr1 was synthesized first through Suzuki coupling reaction, which then reacted with cyclometalated iridium(III) chloro-bridged dimer. Thus, the target polymer P1 was obtained successfully. The detailed synthetic procedue of P1 was shown in Scheme S2. The chemical structures of 1 and P1 were characterized by 1H and 13C NMR. The weight-average molecular weight of P1 was 6400 with polydispersity index of 1.20 as determined by gel permeation chromatography. The actual content of iridium(III) complex in P1 was estimated to be 11.4 mol% via 1H NMR. Due to its amphiphilic structure, P1 can form the well-dispersed and ultrasmall Pdots in the phosphate buffer solution (PBS) with a diameter of <10 nm from transmission electron microscopy (TEM) via self-assembly  expected from the tert-butyl and two phenyl groups of the TBDPS moiety were visible. These signals disappeared in the presence of 5.0 equiv of F−. The results suggest that in the presence of F−, the Si-O bond-breaking of complex 1 takes place to afford complex 2  from the electron-rich oxygen atom to the excited metal complex.To evaluate the feasibility of our design strategy, the Fe minute . To furtce of F− . Before omplex 2 . This pron of F− . In orde1, the selectivity was tested by addition of 40 equivalents of common anions, including Cl−, Br−, CH3COO−, NO3−, NO2−, HSO4−, HCO32−, CO32−, HSO3− and F−. As shown in − induced the decrease of emission intensity and all other anions did not cause evident changes of the emission spectra. This result indicated that 1 is a promising probe for recognizing fluoride ions over other common anions.For further evaluation of the fluoride sensing ability of 1 to F− endowed P1 with excellent sensing properties by introducing it into the hydrophilic CPE mainchain. Using the F−-insensitive blue fluorescence from polyfluorene as the reference signal, P1 fulfilled the features of ratiometric probe for detecting F− in aqueous media. The F− sensing peformance of P1 was investigated in PBS (pH 7.4 at 25 °C) using NaF as F− source. The concentration of P1 was calculated based on the repeating units. As shown in −, the intensity of red emission band decreased gradually, while that of blue emission is almost unchanged. The difference between two emission wavelengths was large (180 nm) and there was almost no overlap between the two emission bands, which led to accurate measurement of two emission intensities to yield the ratiometric value. While the F− concentration achieved 13 μM, the quantum yields value became 0.03. As shown in I600/I420) exhibited a dramatic change from 2.03 to 0.66  have also been carried out. As shown in P1 exhibits excellent sensing performance under both pH values. Hence, we think that polymer P1 can work in the pH range of 4.0~9.0.Furthermore, to investigate the pH range of probe worked, the F− sensing in biological systems, such as live cells, the cytotoxicity of P1 was evaluated by the MTT  assay. As shown in P1 has excellent biocompatibility and low cytotoxicity. Therefore, P1 is preferable for live cell imaging and intracellular F− detection. Next, the intracellular F− sensing performance of P1 was investigated. We first incubated the cells with P1 for 60 min at 37 °C. After cellular uptake, we washed the cells with PBS buffer to remove the free P1 in solution and on the cell surface. Then the experimental group was further incubated with F− (20 μM) for 30 min at 37 °C, and the control group was directly incubated for 90 min. P1 in the presence or absence of F− at 37 °C. The excitation wavelength was 405 nm. It can be seen that for the live cells before and after treated with F−, no evident change was observed for the images collected at 410 to 480 nm (blue channel), which corresponded to the fluorescence from polyfluorene. However, the emission intensity of images recorded at 560–650 nm (red channel), which corresponded to the phosphorescence from iridium(III) complex in P1, decreased evidently in the presence of F− compared with that without F−. Thus, the intensity ratio of the red channel over the blue channel  and blue fluorescence from polyfluorene (Iblue) were disturbed by the changes in experimental conditions, such as that (1) the incubation duration was increased to 4 h; (2) the dose of P1 was increased from 1.0 × 10−2 mg/mL to 2.0 × 10−2 mg/mL; (3) the 405 nm laser power was randomly increased. Although the blue- or red-channel emission intensity (Iblue or Ired) measured at different experimental conditions showed the evident change, the Ired/Iblue values exhibited little fluctuation  of phosphorescence signal from iridium(III) complex, the photoluminescence lifetime imaging of different concentration of intracellular F30 μM F− . This inP1 has been designed and synthesized. This class of nanoprobe can solve the key issues during the F− sensing process. Firstly, the hydrophilic characteristic of CPE enables the probe to be soluble in water. Secondly, the reaction-based sensing mechanism ensures the detection in aqueous media and biological systems with high selectivity and rapid response time. Thirdly, both the ratiometric luminescence and lifetime detection have been demonstrated to be resistant to external influences, effectively improving the sensing reliability and accuracy. It is noteworthy that photoluminescence lifetime imaging of intracellular F− has been realized for the first time. Importantly, we believe that the design strategy for ratiometric and lifetime probes based on CPE in this work is not limited to fluoride sensing; the incorporation of responsive phosphorescent complex as reporters and insensitive polymer backbone as internal standard into the CPE will lead to new ratiometric luminescence and lifetime probes for specific analytes.In summary, a reaction-based fluorescent/phosphorescent dual-emissive nanoprobe 3∙3H2O was an industrial product and used without further purification. Photoluminescence (PL) spectra were measured on a Perkin Elmer LS-55 with Xe lamp excitation source and a Hamamtsu (Japan) 928 PMT, using a 90 degree angle for solution samples. UV-vis absorption spectra were recorded on a UV-1700 Shimadzu UV-vis spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded on Bruker ACF400 (400 MHz) instrument. Mass spectra were obtained on a Bruker autoflex matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF) mass spectrometer (MS3) and a Shimadzu GCMS-QP2010. The gel permeation chromatography (GPC) analysis of the polymers was conducted on a Shimadzu 10 Å with THF as the eluent and poly(styrene) as standard. The data were analyzed by using the software package provided by Shimadzu Instruments. Photographs of the solution samples were taken with a Cannon EOC 400D digital camera under a hand-held UV lamp. Average particle size was measured by laser light scattering (LLS) with particle sizing software  at a fixed angle of 90° at room temperature. Transmission electron microscopy (TEM) was conducted on a JEOL JEM-2100 Transmission electron microscopy at an acceleration voltage of 150 kV.All chemical reagents, unless otherwise specified, were purchased from Sigma Aldrich Chemical Company. All solvents for reaction and photophysical investigation were of HPLC grade. IrCl2.The human cervical cancer HeLa cell line was obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin . Cells were cultured at 37 °C in a humidified chamber containing 5% CO− for another 30 min. Then the cells were immediately transferred into Live Cell Imaging System  for confocal luminescence imaging. The emission was collected at 410–480 nm and 560–650 nm for the HeLa cells incubated with Pdots nanoprobes.Confocal luminescence imaging was carried out on an Olympus IX81 laser scanning confocal microscope equipped with a 40 immersion objective len. A semiconductor laser at 405 nm was served as excitation of the HeLa cells incubated with Pdots nanoprobes. The HeLa cells were incubated with the Pdots (10 μg/mL) nanoprobes at 37 °C for 3 h, then incubated with and without F− for another 30 min. The PLIM setup is integrated with Olympus IX81 laser scanning confocal microscope. The luminescence signals were detected by confocal microscope system and the correlative calculation of the data was performed using professional software which was provided by PicoQuant GmbH. The excitation light of 405 nm with a frequency of 0.5 MHz from the pulse diode laser  was focused onto the sample with a 40 X objective lens (NA 0.95) for single-photon excitation. The luminescence signals were collected in the range of 410–650 nm.The HeLa cells were incubated with the Pdots nanoprobe (10 μg/mL) at 37 °C for 3 h, and then with different concentration of FM1 , M2 , M3 , [Pd(PPh3)4] (5 mg) and tetrabutylammonium bromide (TBAB) were placed in a reaction tube. A mixed solvent of toluene (3.3  mL) and K2CO3 aqueous solution  were added. The reaction vessel was degassed and the reaction was stirred vigorously at 85 °C for 48 h under the protection of nitrogen-atmosphere at dark environment. Then, the reaction was cooled down to room temperature and precipitated in methanol. The polymer was filtered and washed with methanol and acetone, and then dried under vacuum at RT for 24 h to obtain the polymer Pr1 in the yield of 75%. 1H NMR : 9.06 , 8.59 , 8.16 , 7.39–7.86 , 3.30 , 1.65 . 13C NMR : 151.50, 140.52, 140.12, 128.85, 127.21, 126.37, 121.32, 121.00, 120.19, 83.81, 55.33, 40.30, 34.02, 33.35, 32.62, 29.71, 29.08, 27.77, 25.00, 23.72.The monomer Pr1 (40 mg) in THF (8 mL) at RT. The reaction was stirred at RT for 12 h. Then, the precipitate was redissolved by methanol (6 mL). Additional trimethylamine in tetrahydrofuran  was added, and the reaction was stirred at RT for 24 h. After removal of solvent, acetone was added to the precipitate. The polymer was dried under vacuum for 24 h to obtain the light yellow powder with the yield of 87%. 1H NMR : 9.12 , 8.59 , 7.73 , 3.05 , 2.28 , 0.77–1.61. 13C NMR 2SO, δ): 126.52, 121.25, 72.22, 65.96, 65.59, 60.71, 52.51, 35.49, 32.43, 29.34, 28.57, 27.46, 25.97, 22.48, 15.62.Trimethylamine in tetrahydrofuran  was added dropwise to a solution of polymer Pr2 and cyclometalated Ir(III) chloro-bridged dimer L3 were added to a 150 mL flash. A mixed solvent of methanol (6 mL) and dichloromethane (6 mL) were added. The reaction vessel was degassed and the reaction was stirred vigorously at 50 °C for 8 h under the protection of nitrogen-atmosphere at dark environment. When finished, the solution was cooled down to room temperature and then a 5-fold excess of potassium hexafluorophosphate was added. The suspension was stirred for another 2 h and then was filtered to remove insoluble inorganic salts. The product was concentrated and precipitated in acetone. The polymer was dried under vacuum for 24 h to obtain the light red powder. 1H NMR 2SO, δ): 7.45–8.48 , 7.37 , 7.18 , 6.89 , 3.06 , 2.19 , 0.76–1.48 . 13C NMR : 136.82, 134.94, 129.65, 127.98, 52.51, 30.89, 29.47, 28.96, 27.01, 26.43, 25.95, 24.21, 22.47, 19.14.Polymer The synthetic routes of the CPEs were shown in How to cite this article: Zhao, Q. et al. Dual-emissive Polymer Dots for Rapid Detection of Fluoride in Pure Water and Biological Systems with Improved Reliability and Accuracy. Sci. Rep.5, 16420; doi: 10.1038/srep16420 (2015).


Rubus occidentalis) fruit extract (ROE) in a rat model of chronic muscle pain and examine the mechanisms involved.The purpose of this study was to evaluate the antinociceptive effect of black raspberry . For the second experiment, 70 rats were randomly assigned to seven groups. Rats were injected intraperitoneally with saline, yohimbine, dexmedetomidine, prazosin, atropine, mecamylamine, or naloxone after the development of hyperalgesia. Ten minutes later, ROE (300 mg/kg) was administered intraperitoneally. For both experiments, the mechanical withdrawal threshold (MWT) was evaluated with von Frey filaments before the first acidic saline injection, 24 h after the second injection, and at 15, 30, 45, 60, 80, 100, and 120 min, 24 and 48 h after the drug administration.Compared with the control group, the MWT significantly increased up to 45 min after injection of ROE 100 mg/kg and up to 60 min after injection of ROE 300 mg/kg, respectively. Injection of ROE together with yohimbine or mecamylamine significantly decreased the MWT compared with the effect of ROE alone, while ROE together with dexmedetomidine significantly increased the MWT.ROE showed antinociceptive activity against induced chronic muscle pain, which may be mediated by α2-adrenergic and nicotinic cholinergic receptors.The online version of this article (doi:10.1186/s12906-016-1192-z) contains supplementary material, which is available to authorized users. Rubus spp.), has increasingly drawn attention owing to their high content of anthocyanins and other phenolic compounds [Rubus fruits have a high potential for medical application.Musculoskeletal pain is a significant health issue with considerable costs and impact for individuals and society. Chronic pain and hyperalgesia induced by musculoskeletal injuries are managed using a multidisciplinary approach, but can be disabling and difficult to treat . Recentlompounds . Anthocyompounds –5, whichRubus occidentalis, commonly called black raspberry, has one of the highest anthocyanin contents among Rubus spp. and a potent antioxidant capacity [R. occidentalis indicates its therapeutic potential. In addition, studies have reported antinociceptive effects of other Rubus species [capacity , 7. Oxidcapacity , and the species , 10.R. occidentalis extract (ROE) would be beneficial for chronic musculoskeletal pain. To test this hypothesis, we used a rat model of long-lasting hyperalgesia induced by two intramuscular injections of acidic saline, which produces bilateral muscle and cutaneous mechanical hypersensitivity [We hypothesized that sitivity , 12.R. occidentalis in chronic muscle pain. The secondary outcome was to assess the mechanisms involved in its analgesic activity.The primary outcome of this study was to evaluate the antinociceptive effect of The present study was performed and described according to the Animal Research: Reporting In Vivo Experiments (ARRIVE) statement .R. occidentalis were collected from Gochang-gun in South Korea and stored at −20 °C. In brief, the fruits were extracted twice with 50 % ethanol at 80 °C by using a reflux condenser, and extracts were filtered and concentrated. The concentrate was lyophilized in a freeze-dryer and stored at −20 °C until use. All procedures were conducted under sterilized condition, and stored ROE was used just before intraperitoneal injection.The ROE was provided by Gochang Berry & Bio Food Research Institute . Mature fruits of The experiment was approved by the Institutional Animal Care and Use Committee of Chung-Ang University (No. 2015–00009). All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult male Sprague–Dawley rats  were single-housed in cages in a temperature-controlled room (22 °C) and fed a standard laboratory diet and tap water. They were kept under a 12 h light/dark cycle (lights on from 8:00 a.m. to 8:00 p.m.) and acclimated to the housing facilities for 1 week prior to commencing experimental procedures. Rats showing any abnormalities were excluded.All procedures were performed under sterile conditions by an investigator who was unaware of the group allocations of the individual rats. Immediately after baseline behavioral measurements as described below, rats were anesthetized briefly with 1–4 % isoflurane in 100 % oxygen and injected with 100 μL of pH 4.0 preservative-free sterile saline into one lateral gastrocnemius muscle on day 0 and again on day 3. The pH was adjusted to within 3.9 to 4.1 with hydrochloric acid using a SevenEasy Mettler Toledo pH meter  .One researcher, who was not involved in this study, prepared syringes containing various doses of ROE which were dissolved in 2 mL of normal saline for ROE groups. Syringes containing 2 mL of normal saline were prepared for the control group. The syringes were covered with opaque tape and numbered sequentially according to a randomization list generated using PASS™ 11 software . As intraperitoneal injection is the most frequently used parenteral route of administration in rats, drugs were intraperitoneally administered according to the study protocol. All experimental procedures were conducted by researchers who were blinded to the group allocation of each animal.Rubus spp. [The purpose of experiment 1 was to evaluate the anti-nociceptive effect of ROE on induced chronic muscle pain. Fifty rats were randomly assigned to one of five groups of ten rats . The dose level of ROE was referred to the amounts used in other experimental studies on the pharmacological effect of bus spp. , 15, andIn addition, ketorolac of 10 mg/kg was injected intraperitoneally in ten rats for the positive control . We usedThe purpose of experiment 2 was to examine whether the effects of ROE on mechanical hyperalgesia induced by repeated intramuscular injections of acidic saline were mediated by α (1 and 2)-adrenergic, cholinergic (nicotinic and muscarinic), and/or opioid receptors. The use of drugs administered to elucidate the possible involvement of those receptor systems was based on previous studies –20. The For experiments 1 and 2, individual rats were placed on an elevated plastic mesh floor (8 × 8 mm perforations) under an overturned clear plastic cage (21 × 27 × 15 cm) and allowed to acclimate for 15 min. The rats were then tested to determine their withdrawal thresholds to mechanical stimulation using von Frey filaments . The filaments were applied vertically to the plantar aspect of the hindpaw by administering pressure sufficient to gently bend the filament. Filaments with bending forces of 4, 9, 20, 59, 78, 98, 147, and 254 mN were progressively applied until the hindpaw was withdrawn or a bending force of 254 mN  was reached. Each filament was applied three times at intervals of 3 min. The lowest bending force that caused paw withdrawal was recorded as the mechanical withdrawal threshold (MWT). After a response was observed, filaments with higher and lower bending forces were tested to confirm the MWT. In all groups, the MWT was assessed before first injection (BI), 24 h after second injection (AI), and 15, 30, 45, 60, 80, 100, and 120 min, 24 and 48 h after the injection of the test drugs.In order to identify the effect of ROE on motor function or the sedative effect of ROE, we used an accelerating Rota-Rod treadmill . Fifteen rats were randomly assigned to one of five groups of three rats . In addition, six rats were used in Naive and Ketorolac 10 mg/kg groups as positive and negative controls, respectively. The Rota-Rod test was performed 30 min after the intraperitoneal administration of ROE. The rats were placed on the Rota-Rod treadmill and its speed was gradually increased from one to 18 rotations per minute (rpm) for 120 s and maintained for another 30 s at 18 rpm . The timThe primary outcome measure of this study was MWT as assessed using von Frey filaments. To estimate the required group size for experiment 1, a pilot study was conducted to measure MWT in 6 hyperalgesia-induced rats (control group). The averages of the MWT at BI, AI, 15, 30, 45, 60, 80, 100, and 120 min, and 24 and 48 h after injection of normal saline were 91.5, 20.1, 20.1, 27.4, 27.4, 27.4, 33.0, 27.4, 33.0, 33.0, and 33.0 mN, respectively. The standard deviations of MWT ranged from 5.9–20.6 mN, and autocorrelation between adjacent measurements on the same subject was approximately 0.7. For our power calculation, we assumed that first-order autocorrelation adequately represented the autocorrelation pattern. We wanted to detect 10, 20, 30, and 40 % increases in MWT in the ROE 10, 30, 100, and 300 mg/kg groups, respectively, compared with that in the control group. With an α of 0.05 and a power of 80 %, we calculated that we needed nine rats per group. Allowing for a drop-out rate of 10 %, we included ten rats per group.For experiment 2, sample size estimation was performed using the results of the study assessing the antinociceptive effect of ROE. The average MWT in the 300 mg/kg ROE group at BI, AI, 15, 30, 45, 60, 80, 100, and 120 min, and 24 and 48 h after injection of normal saline were 94.1, 28.8, 59.4, 64.2, 63.9, 53.3, 50.0, 43.3, 39.4, 35.5, 32.1, and 45.5 mN, respectively. The standard deviations of MWT ranged from 10.5–30.6 mN, and autocorrelation between adjacent measurements on the same subject was approximately 0.7. For our power calculation, we assumed that first-order autocorrelation adequately represented the autocorrelation pattern. We aimed to detect a 20 % decrease of MWT in the ROE with yohimbine, mecamylamine, and dexmedetomidine groups and a 10 % decrease in the ROE with naloxone, atropine, and prazosin groups. With an α of 0.05 and a power of 80 %, we calculated that we needed eight rats per group. Allowing for a drop-out rate of 10 %, we included ten rats per group. PASS 11™ software (NCSS) was used to calculate the sample size.The Shapiro-Wilk test and q-q plot was used to test the normality of variables. MWT were analyzed using repeated measures of ANOVA followed by Tukey test, and Rota-Rod test were compared using the Kruskal-Wallis test followed by Bonferroni correction. Individual measurements were expressed as mean ± standard error and were analyzed using SPSS 20.0 . A p value of less than 0.05 was considered statistically significant.Throughout the experimental period, all rats remained well-groomed and seemed to maintain normal food and water intake, and their gait appeared unaffected. Two injections of pH 4.0 saline into one gastrocnemius muscle 2 days apart resulted in a bilateral decrease in the MWT Fig. . All ratp = 0.001, <0.001, 0.001, and 0.017 at ipsilateral site, Fig. p = 0.001, <0.001, 0.001 and 0.019 at contralateral site; Fig. p = 0.013, 0.008 and 0.030 at ipsilateral site, Fig. p = 0.010, 0.017, and 0.042 at contralateral site; Fig. An intraperitoneal injection of 300 mg/kg of ROE resulted in a significant increase in the MWT at 15, 30, 45, and 60 min bilaterally, compared with control group . The times to falling during the Rota-Rod test were 116.67 ± 30.55, 113.00 ± 16.09, 98.00 ± 29.86, 115.33 ± 18.56, 101.00 ± 22.65, 117.33 ± 19.55, and 107.33 ± 17.56 s in the control and 10, 30, 100, 300 mg/kg ROE, Ketorolac 10 mg/kg, and Naive groups, respectively.The administration of ROE showed no significant effect on motor performance as measured by the Rota-Rod test 30 min after intraperitoneal injection of ROE compared with the control group (In the present study, ROE administrated intraperitoneally was found to have antinociceptive activity against hyperalgesia induced by repeated intramuscular injections of acidic saline, and the strength of the antinociceptive effect was proportional to the dosage of ROE. Furthermore, the antinociceptive effect of ROE was significantly reduced by mecamylamine and yohimbine, and potentiated by dexmedetomidine, suggesting that the nicotinic cholinergic and α2-adrenergic receptors are linked to the analgesic effect of ROE.Rubus fruits and found that they contain antioxidants such as anthocyanins, phenolic acid, and ellagitannin [Rubus occidentalis is particularly rich in anthocyanins [R. occidentalis might be linked. Previous studies have reported antinociceptive effects of other Rubus spp., strengthening our hypothesis [Scientists have elucidated the active components in gitannin , 21. Rubocyanins . This procyanins , 6. Sincocyanins . Furtherocyanins , 24. Henpothesis , 10. Ourpothesis . It has pothesis –28. ThusChronic musculoskeletal pain poses great therapeutic challenges for clinicians and imposes high healthcare costs on a national scale, while reducing working time and productivity . AdditioAlthough the specific mechanisms of chronic musculoskeletal pain are not well understood, the participation of α-adrenergic and cholinergic systems in several painful processes is well established –33. To dSeveral studies have demonstrated an antinociceptive effect of cholinergic receptor activation. Rowley et al. reported the antinociceptive actions of a nicotinic cholinergic agonist , which iR. occidentalis fruit in our experiment. Several studies have investigated the fruits of Rubus spp. according to their degree of maturity [R. occidentalis fruit at varying stages of maturity would be helpful in the future.There were some limitations of the present study. First, we used a non-inflammatory pain model, but several studies have reported an anti-inflammatory effect of ROE , 37. Givmaturity , 42. FurOur study had the strength, notwithstanding these limitations, of being the first experimental study on the effect of ROE on chronic musculoskeletal pain model in rats using a rigorous study protocol.R. occidentalis showed antinociceptive activity against chronic muscle-induced pain, which may be mediated by α2-adrenergic and nicotinic cholinergic receptors.Rubus occidentalis extract; rpm, rotation per minute; spp, speciesAI, after second injection; ANOVA, analysis of variance; BI, before first injection; MWT, mechanical withdrawal threshold; ROE,


Physocarpus amurensis Maxim and the imported Physocarpus opulifolius “Diabolo” from North America. We aim to discuss the responses and the adaptive mechanism of these two cultivars of Physocarpus to a low light environment. The results show that the specific leaf area (SLA) and the chlorophyll content were significantly increased in the leaves of both Physocarpus cultivars in response to a low light intensity, and the SLA and chlorophyll content were higher in the leaves of low light-treated P. opulifolius “Diabolo” compared with the leaves of low light-treated P. amurensis Maxim. Moreover, the content of anthocyanin was markedly reduced in the leaves of P. opulifolius “Diabolo” under low light intensity, which allowed for a greater capacity of photon capture under the low light condition. Under natural light, the photosynthetic carbon assimilation capacity was greater in the leaves of P. amurensis Maxim compared with the leaves of P. opulifolius “Diabolo” that were rich with anthocyanin. However, in response to low light, AQY, Pmax, LCP and LSP decreased to a lesser extent in the leaves of P. opulifolius “Diabolo” compared with the leaves of P. amurensis Maxim. These results suggest that P. opulifolius “Diabolo” exhibits a greater ability in adaption to low light, and it is probably related to the relatively higher chlorophyll content and the smaller SLA in the leaves of P. opulifolius “Diabolo.” In addition, the low light intensity resulted in a reduced photochemical activity of photosystem (PS) II in the leaves of both Physocarpus, as evidenced by increased values of the relative variable fluorescence at point J and point I on the OJIP curve. This result suggests that the electron acceptor in PS II was the major responsive site to the low light stress in the leaves of both Physocarpus cultivars, and that the low light intensity significantly inhibited electron transfer on the acceptor side of PS II and reduced the activity of the oxygen-evolving complex (OEC) in the leaves of both Physocarpus cultivars. The PS II function in P. opulifolius “Diabolo” was higher than that in P. amurensis Maxim in response to low light. Under low light, the composition of photosynthetic pigments was altered in the leaves of P. opulifolius “Diabolo” in order to maintain a relatively high activity of primary photochemical reactions, and this is the basis of the greater photosynthetic carbon assimilation capacity and one of the main reasons for the better shade-tolerance in P. opulifolius “Diabolo.”The present study aims to investigate the differences in leaf pigment content and the photosynthetic characteristics under natural and low light intensities between the Chinese native  Physocarpus amurensis Maxim is a deciduous shrub belonging to the family Rosaceae, and is an endangered plant species in China  II is one of the most important protein complexes in the photosynthetic apparatus of higher plants. As a fast and non-invasive probe for PS II function, fast chlorophyll florescence dynamics can detect absorption and utilization of light, distribution of excessive energy and electron transfer by PS II during the process of chlorophyll fluorescence quenching . MoreoveP. amurensis Maxim and P. opulifolius “Diabolo” were provided by the Forest Botanic Garden of Heilongjiang province. In the spring of 2012, the live cutting seedlings of P. amurensis Maxim and P. opulifolius “Diabolo” were planted in peat soil in individual plastic pots with an upper diameter of 28 cm, a lower diameter of 15 cm and a height of 20 cm. The seedlings were planted outdoor in natural condition, with regular watering and weeding. The seedlings were thoroughly watered before winter, and grew outdoor through the winter. In early 2014, the seedlings were about 0.5 m in height and with 8–10 branches. After thawing in the spring of 2014, routine watering and weeding were resumed, and the two cultivars of Physocarpus in the vigorous growth stage was subjected to experiment in June 2014.Three-year old cutting (delete) seedlings of P. amurensis Maxim and P. opulifolius “Diabolo” seedlings (10 each) of similar size were selected. Five cuttings of each species were cultivated indoors under an artificial low light  with a lighting intensity of 100 μmol · m−2 · s−1 . The other seedlings remained under an outdoor natural light with a lighting intensity between 1,000 μmol · m−2 · s−1 and 1,500 μmol · m−2 · s−1 at noontime. Plants under low light or natural light were watered in the same manner, and fertilizers were not applied. In later 45 d, the indoor seedlings of both species showed signs of adaptation to low light. Measurements for leaf pigments, light responsive curve and chlorophyll fluorescence parameter were conducted at this 45 d stage for both species.P. amurensis Maxim and P. opulifolius “Diabolo.” A fresh leaf sample without main vein was sliced and incubated in pigment extraction solution containing acetone, anhydrous ethanol and distilled water . Contents of Chla, Chlb and Car were calculated according to −1 HCl extraction solution. Ant concentrations and Ant/Chl were calculated with the equations proposed by Pirie and Mullins.The concentrations of chlorophyll a (Chla), chlorophyll b (Chlb) and carotenoids (Car) were determined on fresh fully expanded leaves of Pn–PFD) response curves were made at leaf chamber temperature of 30 °C, and at 350 μmol · m−2 · s−1 CO2 with a portable photosynthetic system . PFD was fixed every 10 min in a sequence of 2,000, 1,600, 1,200, 800, 600, 400, 300, 200, 150, 100, 0 μmol · m−2 · s−1. Light intensity, CO2 concentration and leaf chamber temperature were controlled by automatic control device of the CIRAS-3 photosynthetic system. Photosynthetic rate measured at two O2 concentrations (21% O2 + 350 μmol · m−2 · s−1 CO2 and 2% O2 + 350 μmol · m−2 · s−1 CO2) under 1,200 μmol · m−2 · s−1 PFD was used to calculate photorespiration.Photosynthetic rate–Photosynthetic photon flux density (−2 · s−1 PFD) for 2 s after heat treatment in the dark at different times. Each transient obtained from the dark-adapted samples was analyzed according to the JIP-test ; (2) the maximum fluorescence intensity  and (3) the fluorescence intensities at 300 ms (K-step), 2 ms (J-step) and 30 ms (I-step). The maximum quantum yield of PSII photochemistry (Fv/Fm) was calculated as: Fv/Fm = (Fm − oF)/Fm, in this study, Fm = PF. The relative variable fluorescence intensity at J-step (VJ) and I-step (VI) were calculated as: Vt = (Ft − oF)/(Fm − oF). VK and VL were the relative variable fluorescence on the VO-J and VO-K point at 0.3 and 0.15 ms.Chlorophyll a fluorescence transient was measured with a Handy-PEA fluorometer . Seedlings leaf having been dark adapted for 1 h before heat treatment, all the leaves were immediately exposed to a saturating light pulse , Performance index on absorption basis (PIABS), Probability that a trapped exciton moves an electron into the electron transport chain beyond QA- (at t = 0) (Ψo), Quantum yield for electron transport (at t = 0) (φEo), quantum yield of absorption flux to dissipated energy (φDo), Absorption flux per RC (ABS/RC), Trapped energy flux per RC (at t = 0) (TRo/RC), Electron transport flux per RC (at t = 0) (ETo/RC) and Dissipated energy flux per RC (at t = 0) (DIo/RC).According to the JIP-test  could ob2)) was measured. Then, the leaf was heated  and dried  until it reached a constant weight, then its biomass (M (g)) was weighed. The surface area (S) was divided by the mass (M) to obtain the SLA (specific leaf area).Five fully expanded leaves were randomly selected from each plant. The surface area of each leaf  higher Chlb and 20.66% higher Chl(a + b) (p < 0.01) compared to P. amurensis Maxim. However, the ratio of Chla to Chlb (Chla/b) in the leaves of P. amurensis Maxim was 48.96% (p < 0.05) higher than P. opulifolius “Diabolo”  under low light conditions, and the content of Chla in P. opulifolius “Diabolo” was not different between the two conditions. The contents of Chlb and Chl(a + b) were significantly elevated in P. amurensis Maxim and P. opulifolius “Diabolo” under low light conditions (p < 0.01). The Chla/b ratio was reduced by 54.41% (p < 0.01) in the leaves of P. amurensis Maxim and 38.35% (p < 0.05) in P. opulifolius “Diabolo” under low light conditions  in P. amurensis Maxim and P. opulifolius “Diabolo” were lower in low light conditions, but also not statistically significant (P. opulifolius “Diabolo” was 3.52 times higher than P. amurensis Maxim. Ant content in P. opulifolius “Diabolo” under low light condition was 65.33% (p < 0.01) lower compared to natural light conditions. The Ant content was relatively low in P. amurensis Maxim, and it remained same under different light intensities (P. amurensis Maxim was 50.23% (p < 0.05) higher compared to natural light, which appeared to be caused by the high production of chlorophyll in the leaves of P. amurensis Maxim under low light condition. By contrast, the Ant/Chl ratio in P. opulifolius “Diabolo” under low light condition was 44.33% (p < 0.01) lower compared to the natural light.Based on the change in color we observed in the leaves, we measured the levels of carotenoids and anthocyanin in the leaves. The contents of carotenoids (Car) in the leaves of nditions . The leanificant . Under nensities . Under lP. amurensis Maxim was 15.03% (p > 0.05) higher than that of P. opulifolius “Diabolo,” yet the difference was not statistically significant. The SLA was 58.79% (p < 0.05) higher in P. amurensis Maxim and 55.17% (p < 0.05) higher P. opulifolius “Diabolo” under low light compared to natural light conditions. These results indicate that the leaves of these two cultivars became thinner in response to the low light. Under low light, the SLA of P. amurensis Maxim was 17.72% (p > 0.05) higher than that of P. opulifolius “Diabolo,” but the difference was not statistically significant , Pmax and the light saturation point (LSP) in P. amurensis Maxim were 3.84, 16.88 and 5.14% higher than that in P. opulifolius “Diabolo” under natural light respectively. The light compensation point (LCP) in P. amurensis Maxim was 7.73% lower than that in P. opulifolius “Diabolo,” indicating that the photosynthetic capacity in the leaves of P. amurensis Maxim was greater than that of P. opulifolius “Diabolo”  in the leaves of P. opulifolius “Diabolo” was lower than that of P. amurensis Maxim at all time points under natural light (Fo) in P. amurensis Maxim under low light condition was significantly higher compared to natural light, whereas the relative fluorescence intensity at time P (Fm) was lower, resulting in a flatter OJIP curve in P. amurensis Maxim in response to low light   .The relative fluorescence intensity  –6E, wher > 0.05)  and 6D. Fv/Fm and PIABS values were significantly lower in the leaves of both cultivars of Physocarpus under low light (Fv/Fm was decreased by 17.31% (p < 0.05) P. amurensis Maxim under low light condition compared to natural light, while Fv/Fm was only 2.84% (p > 0.05) lower in P. opulifolius “Diabolo” under low light condition. The changes in PIABS in the leaves of both Physocarpus were markedly reduced in response to low light, with a decreased of 59.40% (p < 0.05) in P. amurensis Maxim and 48.13% (p < 0.05) in P. opulifolius “Diabolo,” compared to natural light. Moreover, P. opulifolius “Diabolo” had 19.51% (p < 0.05) higher Fv/Fm and 169.11% (p < 0.05) higher PIABS values compared to P. amurensis Maxim.ow light  and 7B. Fo as 0, and Fm as 1 (Vt) under low light condition was higher at all time points in both cultivars of Physocarpus compared to natural conditions. The degree of change in the relative variable fluorescence at time point I (VI) was greater than that at time point J (VJ). P. amurensis Maxim had significantly higher VI and VJ than P. opulifolius “Diabolo” under natural light. In response to low light, VJ in P. amurensis Maxim and P. opulifolius “Diabolo” was increased by 12.43% (p < 0.05) and 18.41% (p < 0.05), respectively, while VI displayed a higher difference of 25.22% (p < 0.05) and 43.80% (p < 0.05), respectively (We next standardized the OJIP curves by defining  Fm as 1 . The relectively  and 8C.VK) and the relative variable fluorescence at 0.15 ms (time point L) on the standardized O-K curve (VL) were significantly different in low light compared to natural light conditions. VK in the leaves of P. amurensis Maxim and P. opulifolius “Diabolo” under low light condition was 8.61% (p > 0.05) and 14.78% (p > 0.05) higher, respectively, and VL in these two cultivars of Physocarpus under low light was increased by 10.84% (p > 0.05) and 3.73% (p > 0.05), respectively. The changes of VK and VL were not statistically significant  lower in P. amurensis Maxim and 7.86% (p < 0.05) lower in P. opulifolius “Diabolo.” By contrast, φEo was reduced by 22.89% (p < 0.05) in P. amurensis Maxim and 10.57% (p < 0.05) in P. opulifolius “Diabolo” when exposed to low light, while φDo increased by 73.64% (p < 0.05) and 13.27% (p < 0.05), respectively, with a greater degree of change in P. amurensis Maxim.Ψncreased –10C. Ψo ABS/RC) per reaction center was significantly increased in both cultivars of Physocarpus in response to low light conditions, which led to increased values of all parameters of energy flux per reaction center  and 150.54% (p < 0.05) higher, respectively. TRo/RC and DIo/RC in the leaves of P. opulifolius “Diabolo” under low light condition were increased by 15.81% (p < 0.05) and 34.68% (p < 0.05), respectively. Hence, DIo/RC increased to a greater degree in P. amurensis Maxim compared to P. opulifolius “Diabolo” under low light conditions.The absorption of luminous energy  content increased significantly under the low light, and chlorophyll a (chl a) changed non-significantly, which can result in two kinds of experimental materials energy captured on PSII, adjust the excitation energy distribution between PSI and PSII under the low light  in the leaves of P. opulifolius “Diabolo” was lower than that of P. amurensis Maxim at all time points. This is probably because the great amount of anthocyanin in the leaves of P. opulifolius “Diabolo” dampening the light intensity. In response to low light, however, the synthesis of anthocyanin in the leaves of P. opulifolius “Diabolo” was inhibited, turning leaves from purple to green, resulting in a significantly increased Ft in the OJIP curve at all points. This observation further corroborates with the fact that the presence of anthocyanin can reduce the fluorescence quenching in the leaves to a certain degree, suggesting that anthocyanin may exhibit a photoprotective property (Physocarpus showed an increase in the relative variable fluorescence at point J (VJ) and point I (VI), indicating an accumulative quantity of equation QA−  on the standardized O-J curve (VK) and the relative variable fluorescence at 0.15 ms (time point L) on the standardized O-K curve (VL) were higher in both Physocarpus cultivars under low light. It has been reported that the increasing of VK may be affected by the state of OEC and the link between PS II units  in the PS II reaction center was reduced, whereas the quantum yield of the absorbed light energy used for dissipation (φDo) was increased. Moreover, the energy flux parameters per PSII reaction center, including ABS/RC, TRo/RC, ETo/RC and DIo/RC, all showed an increasing trend in the leaves of both Physocarpus cultivars under low light. A decrease of φEo and an increase of ETo/RC indicated that there was higher utilization of absorbed light energy on electron transfer in a single PS II reaction center, although there was lower quantum yield of the absorbed light energy used for electron transfer in all PS II reaction centers. This is indirect evidence for the reduction in the number of active PS II reaction centers in the leaves of both Physocarpus cultivars in response to low light. Studies have found that in the situation that a number of PS II reaction centers were inactivated under stress, the function of the antenna pigments in the remaining active reaction centers increases in order to guarantee energy supply  was markedly increased in response to low light, suggesting that the electron transfer from pheophytin (Pheo) to QA  during photosynthesis in the leaves of both Physocarpus was minimally affected by the low light intensity. However, the ratio of energy flux to the electron transfer downstream of QA− (Ψo) in the PS II reaction centers was significantly decreased in low light, further demonstrating the blockade of electron transfer on the electron acceptor side of PS II occurred after QA− in the leaves of both Physocarpus under low light. By contrast, low light conditions had little effect on the electron transfer from Pheo to QA, indicating that low light resulted in an accumulation of QA− in the electron transport chain. This is consistent with the results of VJ and VI increasing as described above. In short, the PS II electron acceptor side appears to be the target site of the effect of low light on plants. In addition, φDo and DIo/RC increased in the leaves of both Physocarpus cultivars under low light, indicating that low light led to reduced utilization of optical energy in the leaves of both Physocarpus cultivars, and that the optical energy was dissipated mainly via heat and fluorescence when the activity of PS II reaction centers was dampened. Moreover, the extent of the decrease of φEo and the increases of φDo and DIo/RC was significantly less in P. opulifolius “Diabolo” than that in P. amurensis Maxim, suggesting that the utilization of optical energy and the activity of PS II reaction centers were higher in P. opulifolius “Diabolo.” Thus, the function of PS II in P. amurensis Maxim was more sensitive to light than that in P. opulifolius “Diabolo.”The low light intensity altered light utilization by the PS II reaction centers and the energy flux per PS II reaction center in the leaves of both y supply . Our stuP. opulifolius “Diabolo” is higher than that of P. amurensis Maxim under natural light, P. opulifolius “Diabolo” has purple leaves caused by the presence of high anthocyanin in the leaves. Moreover, the presence of anthocyanin reduces the capacity of photosynthetic carbon assimilation in P. opulifolius “Diabolo.” In response to low light, the content of chlorophyll in the leaves of both P. amurensis Maxim and P. opulifolius “Diabolo” increased, yet the capacity of photosynthetic carbon assimilation and the photochemical activity of PS II are significantly reduced. The relatively higher chlorophyll content and the smaller SLA in the leaves of P. opulifolius “Diabolo” provided this cultivar with a greater photosynthetic capacity as compared to P. amurensis Maxim. Our study indicates that the ornamental value of P. opulifolius “Diabolo” is higher than that of P. amurensis Maxim under natural light, and under low light, the effect of growth inhibition was lower in P. opulifolius “Diabolo.” Hence, the imported P. opulifolius “Diabolo” exhibits an advantage in shade tolerance, but the ornamental value of P. opulifolius “Diabolo” is lower under shaded conditions. Under a low light intensity, P. opulifolius “Diabolo” maintains a relatively high activity of primary photochemical reaction in PS II by altering the composition of photosynthetic pigments in the leaves, which is an important mechanism for its better shade-tolerant property over P. amurensis Maxim.Although the content of chlorophyll in the leaves of 10.7717/peerj.2125/supp-1Supplemental Information 1Click here for additional data file.


Ixodes ricinus males, females, and nymphs and of 582 questing Dermacentor reticulatus males and females were collected from vegetation on the territory of the Lublin province (eastern Poland). The ticks were examined for the presence of Babesia by PCR detecting part of 18S ribosomal RNA (rRNA) gene and nuclear small subunit rRNA (SS-rDNA) for determining of Babesia spp. and Babesia microti, respectively. The overall incidence of Babesia strains in I. ricinus ticks was 4.6 %. Three species of Babesia were identified. The prevalent species was B. microti which occurred in 2.8 % of ticks, while Babesia venatorum, Babesia divergens, and unidentified Babesia species were found at the frequency of 1.2, 0.2, and 0.3 %, respectively. Altogether, B. microti constituted 61.5 % of the total strains detected in I. ricinus, B. venatorum—25.7 %, B. divergens—5.1 %, and unidentified Babesia species—7.7 %. The prevalence of Babesia species in I. ricinus did not depend significantly on locality  nor on the tick stage . The incidence of Babesia strains in D. reticulatus ticks was 2.7 %. Two species of Babesia were identified. Again, the prevalent species was B. microti which occurred in 2.1 % of ticks, while B. canis was found in 0.7 % of ticks. In one D. reticulatus female, B. canis and B. microti co-infection was found. Altogether, B. microti constituted 75 % of the total strains detected in D. reticulatus while B. canis formed 25 % of the total strains. The frequency of the occurrence of Babesia species in D. reticulatus did not depend significantly on locality . The difference between the prevalence of Babesia in males and females of D. reticulatus was insignificant (P = 0.0954); nymphs were not found. The dominance of B. microti in the species composition of tick-borne Babesia found in this study was typical for eastern Europe. In conclusion, the results revealed that the population inhabiting the forested area of eastern Poland could be exposed to Babesia parasites, especially to those from the species B. microti, by a bite of I. ricinus, a competent vector of human babesiosis, and probably also by a bite of D. reticulatus whose role in the transmission of human babesiosis needs to be clarified.A total of 853 questing  Babesia Starcovici, 1893 (Apicomplexa: Piroplasmida: Babesiidae) comprises intraerythrocytic parasites of mammals and birds which are transmitted by hard ticks (Ixodidae) from the genera Amblyomma, Boophilus, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, and Rhipicephalus. These hemoprotozoans cause babesiosis, a disease of animals and humans manifested in severe cases by fever and hemolysis leading to anemia, hyperbilirubinuria, hemoglobinuria, and possible organ failure  in dogs. Babesiosis in humans is regarded as an emerging disease with the greatest number of cases (above 1000 per annum) caused by Babesia microti in North America  and B. microti. The disease may also develop in the immunocompetent individuals  and a total of 853 questing I. ricinus ticks  were collected during spring/summer season in the years 2011–2012 on the areas of six localities situated in the Lublin province (eastern Poland). D. reticulatus ticks were collected on the territory of three localities: Ostrów Lubelski , Suchawa , and Parczew . I. ricinus ticks were collected on the territory of Wilków , Suchawa, and Dąbrowa (51° 17′ N 22° 57′ E). Ticks were collected by dragging a woolen flag over the lower vegetation and litter along the paths and edges of deciduous and mixed forests, including suburban localities and recreational areas.A total of 582 questing D. reticulatus and from 300 to 500 ng/μl for females, from 180 to 330 ng/μl for males, and from 20 to 80 ng/μl for nymphs of I. ricinus.Total DNA was isolated from the adult ticks separately and from nymphs in pools of five specimens  was used, while nuclease-free water was used as a negative control. The amplifications were carried out in a C1000 Thermal Cycler .All tick lysates were examined for the presence of Babesia spp. including bovine Babesia: B. divergens, B. bigemina, B. major; B. venatorum; B. canis; B. odocoilei; B. ovata; B. motasi, and B. crassa—and primers for identification of B. divergens and B. venatorum were described previously by Hilpertshauser et al. , 1 × PCR buffer containing 15 mM MgCl2, 2.5 μl 2 mM dNTP  , 1.25 μl 10 μM each of primer , 2 μl of matrix DNA, and nuclease-free water . Tick lysates confirmed as positive for B. divergens and B. venatorum were used as positive and nuclease-free water as negative controls. The amplification was carried out in C1000 Thermal Cycler  under the following conditions: preincubation at 95 °C for 3 min, 45 cycles, each of 30 s at 94 °C (denaturation), 30 s at 61 °C , and 45 s at 72 °C (elongation). Final elongation was performed for 10 min at 72 °C. Products of amplification were identified in 2 % agarose gel , after electrophoresis in standard conditions and staining with ethidium bromide solution (2 μg/ml).Each PCR reaction was carried out in a 25-μl reaction volume which contained the following mix of reagents: 0.625 U Babesia spp. positive samples was performed with ABI PRISM 310 Genetic Analyzer  using ABI PRISM Big Dye Terminator v. 3.1. Cycle Sequencing Kits and Big Dye XTerminator Purification Kit (Applied Biosystems). For sequencing of B. microti positive samples, the tenfold dilution of amplified DNA was used, and fivefold for Babesia spp. The results were compared with sequences in GenBank database using the BLAST software at the National Center for Biotechnology Information .DNA sequencing of all χ2 test and Student’s t test, using the STATISTICA v. 6.0 package . The value p < 0.05 was considered significant.The obtained results were analyzed by Babesia strains in I. ricinus ticks collected in eastern Poland was 4.6 %  nor on the tick stage .The overall incidence of  % Table . Three sBabesia strains in D. reticulatus ticks collected in eastern Poland was 2.7 % . The difference between the prevalence of Babesia in males and females of D. reticulatus was insignificant (P = 0.0954); nymphs were not found. However, the difference between the diversity of Babesia species among I. ricinus and D. reticulatus ticks proved to be significant (P < 0.05).The incidence of I. ricinus ticks showed a high level of similarity to B. microti . B. venatorum was confirmed in ten samples . Two samples  were identified as B. divergens. In three cases, the sequencing failed and Babesia species remained unidentified.A total of 54 positive samples were sequenced (one sample was co-infected). Twenty-four samples from D. reticulatus ticks showed 100 % similarity to B. microti . Three samples were defined as B. canis , and in one isolate, two sequences of B. canis and B. microti (accession numbers: AY072926.1 and AB085191.1) were found.In most cases, isolates obtained from I. ricinus ticks collected on the territory of the Lublin region (eastern Poland) are infected with Babesia hemoprotozoans, which confirms that in Poland, there is a potential risk of babesiosis from exposure to the bite of this very common tick species, a competent vector of the disease. Values similar to the presented study, ranging from 3.5 to 4.1 % were recorded in ticks of this species in Germany by Eshoo et al.   examined in the presented study harbored B. canis, an important causative agent of babesiosis in dogs. D. reticulatus is a known vector of this pathogen, and its presence in ticks from eastern Poland is in accordance with the results of Adaszek et al. (The repeatedly found occurrence of k et al.  that canBabesia spp., as a tick-borne protozoan parasite developing in erythrocytes, could led to rare but potentially life-threatening parasitic disease, which is confirmed by reported clinical cases of babesiosis. The first two cases of this disease in Poland was described by Welc-Falęciak et al. (B. divergens or B. venatorum. In Europe, before the aforementioned study, only three clinical cases caused by B. microti were described, which is a minority of the circa. Fifty cases of human babesiosis recorded in continental Europe, caused mostly by B. divergens and, to a less extent, by B. venatorum in immunocompromised individuals (Hildebrandt and Hunfeld B. microti in Europe forms only a small fraction of those reported from North America (Yabsley and Shock Genus k et al.  as a co-I. ricinus, a competent vector of human babesiosis occurring on the territory of eastern Poland, is infected with a relatively marked frequency with three species of Babesia pathogenic for humans, which creates the risk of babesiosis in persons exposed to tick bite. The population of D. reticulatus, another tick species inhabiting this territory, is also infected with B. microti, and its potential role in spreading the disease should be considered and further investigated by experimental studies.In conclusion, the current study reveals that the population of


Using toothed (cf. circular) apertures minimizes diffraction by inducing destructive interference within the diffracted signal. Since diffraction effects can be quite complicated, their over-all reduction may help limit uncertainties in, say calibrations. Our analysis yields three principles to guide design of nonlimiting (baffle) apertures which minimize diffrac tion. We performed detailed diffraction calculations within scalar (Kirchoff) diffraction theory, using parallel-computing resources at the National Institute of Standards and Technology.We model diffraction errors found when using toothed apertures [L. P. Boivin, Reduction of diffraction errors in radiometry by means of toothed apertures, Appl. Opt.  Therefore, we ignore this dependence. When using ε⟩, or to determine a diffraction pattern for other purposes, one must perform many independent evaluations of BΨ  and ΨG  for different values of rS and rD. Use of parallel computing technology can easily accelerate calculations, and we have exploited this fact.Such cancellation allows us to use an effective-wavelength approximation, and the particular approximation used here—which differs slightly from earlier effective-wavelength approximations—provides a rS, rD, and rA. Ideally, one would conduct only the coarsest sampling necessary. Numerical convergence is improved at the cost of further computation. From calculations which included either coarser or finer samplings than were used to obtain the results presented, we estimate uncertainties, arising from controlled errors in numerical integration, as follows. For each ⟨ε⟩, call its expanded uncertainty δ⟨ε⟩. Then δ⟨ε⟩/⟨ε⟩ is around 0.03 for the results in Geometrical and other considerations indicate the required detail in sampling rD at 0.01 mm radial and 0.25° angular intervals, respectively. We improved sampling of rS incrementally, stopping when we could use numerical interpolation to estimate diffracted flux for rS everywhere on the source. Consequently, the radial coordinate of rS was sampled at intervals of 0.05 mm or 0.1 mm. We never found substantial dependence of the diffracted flux on the angular coordinate of rS. Symmetry was extensively exploited to accelerate calculations.We sampled ε⟩’s were sums of strictly positive numbers. Second, distances between various optical components were large compared to components’ dimensions, which were transverse to the optical axes. So phases of emitted or diffracted radiation exhibited only gradual spatial variations over the aperture and detector areas. This facilitated sampling rA at intervals up to 200 times λ. Integration about the aperture perimeter in Two factors assisted convergence of results with respect to detail of integration. First, the ⟨λ⟩ for this source-detector combination was 0.58 μm. When modeling the data, we shall also analyze the effective-wavelength approximation. Besides that approximation, deviation of our results from experiment can be attributed to Kirchoff theory and/or nonideal experimental circumstances. Experimental difficulties could include misalignments of optical components, even by 0.1 mm (based on our results), or could involve irregularities in aperture perimeters, an effect stressed in Ref. , ⟨k⟩ being 2π/⟨λ⟩. For diffracted rays reaching the detector, rays originating from one tooth will be similar to, except differing by a 180° phase-shift from, rays originating from an adjacent tooth (cf. Whereas diffracted rays from any single tooth might not be sufficiently weak, diffracted flux on a small, central detector may also be reduced by radially displacing half of the teeth by the distance ooth cf. .The 180° phase-shift depends on the wavelength and above, designed path-length difference. So this third principle works best when one’s source and detector are smaller than an aperture, and the effective bandwidth of radiation transferred for the source-detector combination is small compared to the radiation’s effective central frequency. The 180° phase-shift being exploited would vary as the wave number of radiation from a broad-band source, somewhat degrading the level of destructive interference. The wavelength range having substantial interference would presumably encompass many oscillations with respect to λ of the relative signs o the geometrical and boundary-diffraction waves.One might wish to constrain tooth depth. Very deep teeth may be difficult to realize and, obviously, undermine the functioning of a baffle. Given such a constraint, we propose a third principle for design of minimally diffracting apertures:a = b = 50 mm, r = 1.25 mm, R = 7.5 mm, and either an extended source with ρ = 0.5 mm or an axial, point source. We vary the number of teeth, N, from 120 to 240, 480 and 960, and use several effective wavelengths. Without application of the third principle, we set Δ = 0.2 mm (cf N teeth, divide an aperture into N/4 equal angular wedges. Further, divide each wedge equally into 8 smaller wedges. The aperture perimeter contains one straight segment within each of these smallest wedges. At the nine, equally spaced angles, which collectively define both edges of all eight smallest wedges, the aperture radius has the following values: 7.5 mm, 7.704 84 mm, 7.509 67 mm, 7.714 50 mm, 7.519 34 mm, 7.714 50 mm, 7.509 67 mm, 7.704 84 mm, 7.5 mm. All apertures reflecting the third principle are designed for λ = 0.58 mm. So we radially displace alternate pairs of teeth. In ε⟩’s with different combinations of N, ⟨λ⟩, source, and whether or not the third principle is applied. The results in both Tables demonstrate all three principles, and the third principle is robust with respect to small changes in λ.To illustrate these three principles further, we consider a set of optics like the others used in this work. We use .2 mm cf . Applica.2 mm cf . For theε⟩’s and those reported in this work.One should critically assess Kirchoff theory and the feasibility for manufacturing and using toothed apertures. This is particularly true for the apertures with the third principle applied, whose design relies on very small structure. One should also consider effects of edge roughness in real apertures. Within Kirchoff theory, sufficiently small features should not have large effects. However, since careful radial displacements of teeth by about 10 mm dramatically influence diffraction, similar irregularities in the apertures of Ref.  might coWe have studied the approach of toothing apertures to reduce diffraction. We modeled diffraction effects found by Boivin  for 25 oWe carried out detailed diffraction calculations using the Kirchoff formulation of scalar diffraction theory, with the work being facilitated by the boundary-diffraction-wave technique. We estimated the level of numerical convergence of these calculations. An effective-wavelength approximation was used, and its validity was assessed. Calculations were accelerated by use of parallel processing, and acceleration of calculations should continue to scale well up to networks of large numbers of processors.Future research considered includes application of toothed apertures in radiometry, the role of aperture irregularities, further assessments of the effective-wave-length approximation, the validity of the Kirchoff model, and extensions of this work to optics having limiting apertures. One might also consider further simplifying physical and mathematical approximations in the diffraction theory used, as these can reduce computational resources needed for various applications [9].