Pneumocystis pneumonia (PCP) is one of the common opportunistic infection among HIV and non-HIV immunocompromised patients. The lack of a rapid and specific diagnostic test necessitates a more reliable laboratory diagnostic test for PCP. In the present study, the loop-mediated isothermal amplification (LAMP) assay was evaluated for the detection of Pneumocystis jirovecii. 185 clinical respiratory samples, including both BALF and IS, were subjected to GMS staining, nested PCR, and LAMP assay. Of 185 respiratory samples, 12/185 (6.5%), 41/185 (22.2%), and 49/185 (26.5%) samples were positive by GMS staining, nested PCR, and LAMP assay, respectively. As compared to nested PCR, additional 8 samples were positive by LAMP assay and found to be statistically significant (p < 0.05) with the detection limit of 1 pg. Thus, the LAMP assay may serve as a better diagnostic tool for the detection of P. jirovecii with high sensitivity and specificity, less turn-around time, operational simplicity, single-step amplification, and immediate visual detection. Pneumocystis pneumonia (PCP) is an opportunistic fungal infection caused by Pneumocystis jirovecii. It causes life threatening pneumonia among HIV as well as non-HIV immunocompromised patients. Clinical manifestation of PCP found to be different among HIV and non-HIV immunocompromised patients. PCP is a diagnostic challenge due to nonspecific signs and symptoms. Laboratory diagnosis by microscopy is less sensitive and often inconclusive. In 1990, Wakefield and colleagues [ P. jirovecii. Since then, several molecular assays directed at various gene targets employed for the detection of this organism included the dihydropteroate synthase (DHPS), dihydrofolate reductase (DHFR), internal transcribed spacer (ITS) regions of the rRNA gene, and mt LSU rRNA. Among all the available PCR platforms based assays, nested PCR assay targeting mitochondrial large subunit ribosomal RNA (mt LSU rRNA) was found to be one of the widely employed assays for the detection of P. jirovecii [irovecii , 3.However, the nested PCR assays are bit cumbersome and carryover contamination prone. To overcome such limitations more advanced quantitative real time PCR (qPCR) assays with the added advantage of simultaneous amplification and detection have also been employed in developed countries. Furthermore, the qPCR based assays have additional advantage of higher specificity lesser turnaround time and chances of contamination but require costly equipment. Therefore, they are not feasible in general clinical laboratories settings, especially in developing countries. Bst DNA polymerase. LAMP primers consisted of six sets of primers comprising two outer (F3 and B3), two inner (FIP and BIP), and two loop primers (FL and BL). These sets of primers recognize eight distinct regions of the target sequence. The FIP primer consisted of F2 and the complementary strand of F1 (F1c). The BIP primer consisted of B2 and the complementary strand of B1 (B1c) and thus ensures high specificity of the assay. Additionally, LAMP ensures higher yield of target gene, that is, 109, in less than an hour in a simple incubator. Further, one-step amplification reaction followed by visual detection based on turbidity has contributed to LAMP assay as an attractive alternative to PCR/qPCR platforms based assays for routine microbiological diagnostic tool [Loop-mediated isothermal amplification (LAMP) recently developed by Notomi et al. is a rapid, simple, and cost effective method of DNA amplification . It is atic tool . An adde Legionella spp. [ Mycobacterium spp. [ Paracoccidioides [ Salmonella spp. [ Staphylococcus aureus [ Listeria monocytogenes [ Plasmodium spp. [ Entamoeba histolytica [Since the introduction of LAMP assay, it has been extensively used and evaluated with other nucleic acid amplification techniques for the detection of various pathogens, namely,lla spp. , Mycobacium spp. , Paracocidioides , dengue idioides , Salmonella spp. , Methicis aureus , Listeriytogenes , Plasmodium spp. , and Enttolytica . Pneumocystis jirovecii.Thus, the aim of the present study was to evaluate the applicability of a LAMP assay for the detection of Pneumocystis jirovecii in immunocompromised patients.Prospective study was designed to evaluate loop-mediated isothermal amplification (LAMP) assay for the detection of+ T cells count below 200 cells/mm3 and 121 non-HIV immunocompromised patients (immunosuppressive drugs and chemotherapy). Among 121 non-HIV immunocompromised patients, 66 were postrenal transplant recipients, 20 with malignant disorders, and 35 with other immunocompromised states.One hundred eighty-five immunocompromised adult (>18 years) patients with the high index of clinical suspicion of PCP were enrolled in the study. These 185 patients consisted of 64 HIV seropositive patients with CD4The study was conducted in Department of Microbiology, All India Institute of Medical Sciences (AIIMS), New Delhi.Study was conducted over a period of 3 years (2010–2013).A total of 185 respiratory samples (nonduplicate) were obtained, including 135 bronchoalveolar lavage fluids (BALF) and 50 induced sputa (IS) . All theImmunocompromised patients above 18 years of age with high index of clinical suspicion of PCP were included in the study.Individuals not fulfilling the inclusion criteria and/or not willing to participate in the study were excluded.Informed consent was taken from all the patients enrolled under study. The demographic profile and clinical and radiological characteristics of all the patients were taken into account prospectively (data not shown). Ethical approval for this study was obtained from the Institutional Ethics Committee.μL of the pellet was used for preparing a smear for GMS staining. DNA extraction was done using a Qiagen DNeasy tissue kit  following manufacturer's instructions with some modifications. 200 μL of pellet was lysed in 200 μL of lysis buffer containing 50 mM KCl, 15 mM Tris-HCl (pH-8.3), 0.5% NP-40, and 500 μg of proteinase K followed by incubation at 56°C for 45 minutes [μL of buffer AL (provided with the kit) was added and the contents were mixed immediately and incubated at 70°C for 10 minutes. After incubation, 200 μL of ethanol (96%) was added and mixed thoroughly by vortexing. The above mixture was applied to the DNeasy minispin columns (provided with the kit) and was centrifuged at 8000 rpm for 1 minute and the step was repeated until the whole mixture was used up. Thereafter, 500 μL of buffer AW1 was added to the column and centrifugation was carried out again at 8000 rpm for 1 minute. This was followed by a second washing with buffer AW2 and centrifugation. To elute the DNA, the column was placed in a microcentrifuge (1.5 mL) tube and elution buffer AE provided with the kit was pipetted into the DNeasy membrane. It was further incubated for 1 minute at room temperature and centrifuged at 8000 rpm for 1 minute. The eluted DNA was stored at −20°C for further studies. DNA extracted from a previously microscopically positive clinical sample was used as positive control and double distilled water was used as a negative control in the study.For extracting DNA induced sputum samples were initially treated with 6.5 mM dithiothreitol (DTT), a mucolytic agent, and then both BALF and IS samples were centrifuged. 50 The DNA extracted from all clinical samples was subjected to nested PCR targeting mt LSU rRNA gene, as described previously by Gupta et al. . The PCR P. jirovecii as described previously by Uemura et al. [μL reaction mixture (containing 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 8 mM MgSO4, 10 mM (NH4)2SO4, 0.1% (v/v) Tween 20, 0.8 M betaine, and 1.4 mM of each deoxynucleoside triphosphate (dNTP), 1.6 μM of each inner primer (FIP and BIP), 0.2 μM of each outer primer (F3 and B3), 2.5 μL primer mix (6.25 μL), 1 μL Bst DNA polymerase, and 3 μL DNA), and final volume was made up with double distilled water for 25 μL reaction. The reaction mixture was incubated in a water bath at 65°C for 60 minutes, followed by heating at 85°C for 3 min to terminate the reaction. Positive reaction was detected as turbidity of the reaction mixture observable with the naked eye. Besides this, the results were assessed by adding propidium iodide to the reaction tube after amplification reaction takes place and observed under ultraviolet light. The amplicons were also analyzed by running on 2%  (wt/vol) agarose gel electrophoresis for typical ladder like pattern. To prevent crossover contamination, all the necessary precautions were taken into consideration like different sets of pipettes and different work areas were designated for the extraction of genomic DNA and performing the LAMP assay.LAMP assay was performed using primers specific for 18S rRNA genes ofa et al. . The spe Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, Cytomegalovirus (CMV), and Mycobacterium tuberculosis.To determine the detection limit of the assay, the genomic DNA extracted from the previously microscopically PCP positive sample (positive control) was diluted 10-fold from 100 ng to 100 fg and the amplified products were analyzed visually as well as by 2% (w/v) agarose gel electrophoresis. The specificity of the LAMP assay was assessed using genomic DNA extracted from other respiratory pathogens, includingp value < 0.05 was considered statistically significant. All statistical analyses were carried out using Statistical Package for the Social Sciences (SPSS) software version 20 . The McNemar test was used to compare the sensitivity of the LAMP assay with nested PCR. “Receiver operating characteristic (ROC) curve analysis and area under curve (AUC)” were made to determine the clinical sensitivity and specificity of the LAMP assay.A Of 185 patients, 12/185 (6.5%) were GMS staining positive and 41/185 (22.2%) were nested PCR positive. However, 49/185 (22.2%) were LAMP positive. These LAMP assay positive samples consisted of 16 patients from HIV seropositive and 33 from non-HIV immunocompromised patients  recipients, 4 with malignant disorders, and 8 among other immunocompromised states ). P. jirovecii positive samples produced luminescence with dye and showed a typical ladder like pattern when amplicons were run on the 2% agarose gel electrophoresis. There was no discordance between the results of microscopy and molecular assay (nested PCR and LAMP assay); that is, all the GMS positives were also positive by nested PCR and LAMP assay.Among 8 additional positive samples by LAMP assay 2 were BALF samples from HIV seropositive patients and 6 were sputa from non-HIV immunocompromised patients . All P. p = 0.008).The detection limit of the LAMP assay  was found to be 1 pg of DNA and that is ten times more than that of nested PCR (previously established in the laboratory) in tenfold serial dilution of the 100 ng DNA , thus, s P. jirovecii in both BALF and sputum samples [Loop-mediated isothermal amplification (LAMP) assay is the boon of the 21st century that amplifies nucleic acid with high specificity, sensitivity, and less turnaround time. In addition, it is a simple, rapid, and cost-effective method, which requires only water bath or heating blocks to perform the assay . Hence,  samples . Subsequ P. jirovecii in both BALF and IS samples. The LAMP assay was compared with nested PCR, targeting 18S rRNA gene and mt LSU rRNA gene, respectively, and microscopic examination using GMS staining. In our study, GMS staining was used as it provides a sharp contrast between the background of host cells and organism [p ≤ 0.05). Furthermore, high specificity of the LAMP assay was also demonstrated by no nonspecific genomic amplification when tested against other respiratory pathogens. Thus, the results of the present study demonstrated that the LAMP assay did not show any cross-reactivity with DNA of other respiratory pathogens and negative controls.Thus, the present study was designed to evaluate the performance of the LAMP assay for the detection oforganism . Of 185  Toxoplasma gondii [ Mycobacterium avium [Similar reports of the higher sensitivity of LAMP assay over nested PCR have been reported by Lau et al. and Fallahi et al. in case ofa gondii , 19. In um avium .+ T cells count more than 100 cells/mm3. Both of these patients had received anti-PCP prophylaxis and thus the number of organisms might have decreased [In the present study, 8 patients were additional positive by LAMP assay, with clinical and radiological features suggestive of PCP. From these 8 patients, 8 samples were obtained that comprised 2 and 6 BALF and IS, respectively. BAL fluid samples were obtained from HIV seropositive patients with CD4ecreased . This ag Bst DNA polymerase used in LAMP assay is not sensitive to such inhibitors. Thus, it may be speculated that the rate of detection of P. jirovecii in both BALF and IS was higher in the case of LAMP assay.Among the remaining 6 LAMP assay positive patients, induced sputum samples were obtained. Taking into account that sputum samples contain PCR inhibitors, the same might be encountered in the present study. However, M. tuberculosis and the COPD patients were having Candida albicans as a copathogen. This reflects that these patients might be colonized with P. jirovecii. This was an important finding and was in concordance with that of Probst et al. 2000 and Vidal et al. 2006 [Furthermore, two of these six were having Wagner's diseases with CMV as one of the primary respiratory pathogens; after administration of ganciclovir as anti-CMV drug the signs and symptom of PCP were also resolved. The remaining 4 patients included two PRT recipients, one with sarcoidosis, and one with chronic obstructive pulmonary disease (COPD). The other microbiological investigation demonstrated that the PRT patients were infected with CMV as the primary pathogen. The patient with sarcoidosis was sputum smear positive foral. 2006 , 23. The Pneumocystis jirovecii with the detection limit of 1 pg of DNA. Moreover, the LAMP assay and interpretation of its results require minimal laboratory facilities. Therefore, it could be best employed as a diagnostic tool in resource limiting countries and point of care settings as well.To conclude, the LAMP assay is a novel promising technique which is sensitive, specific, rapid, simple, and cost-effective for the detection of P. jirovecii from this Indian subcontinent.To the best of our knowledge, this is the first report evaluating LAMP assay with respect to nested PCR for the detection of


We investigated whether routine elective irradiation of a clinically negative inguinal node (IGN) is necessary for patients with locally advanced distal rectal cancer and anal canal invasion (ACI).We reviewed retrospectively 1,246 patients with locally advanced rectal adenocarcinoma managed using preoperative or postoperative chemoradiotherapy and radical surgery between 2001 and 2011. The patients’ IGN was clinically negative at presentation and IGN irradiation was not performed. ACI was defined as the lower edge of the tumor being within 3 cm of the anal verge. Patients were divided into two groups, those with ACI  and without ACI .p < 0.001). Isolated IGN recurrence occurred in three patients, all of whom had ACI tumors. These three patients received curative intent local treatments, and one was alive with no evidence of disease 10 years after IGN recurrence. Salvage treatments in the other two patients controlled successfully the IGN recurrence for >5 years, but they developed second malignancy or pelvic and distant recurrences. Seven patients with non-isolated IGN recurrence died of disease at 5–22 months after IGN recurrence.The follow-up period was a median of 66 months . Among the 1,246 patients, 10 developed IGN recurrence; 7 with ACI and 3 without ACI. The actuarial IGN recurrence rate at 5 years was 0.7%; 3.5% and 0.2% in patients with and without ACI, respectively (The low IGN recurrence rate even with ACI and the feasibility of salvage of isolated IGN recurrence indicated that routine elective IGN irradiation is not necessary for rectal cancer with ACI. The lymphatic drainage from the rectum occurs preferentially in a proximal direction. The lymphatic system supplies the mesenteric and para-aortic nodes through the perirectal and pelvic lymphatic vessels. However, when a distal rectal tumor invades the anal canal, the lymphatic system drains in another direction to the inguinal nodes (IGN), as observed in primary anal cancer, through the perirectal, perianal and pudendal lymphatics .Radiation therapy (RT) is an important constituent of multi-modality management recommended for locally advanced (stage II or III) rectal cancer . PreoperIn our institution, the treatment policy for rectal cancer with clinically negative IGN at presentation has been no irradiation of IGN, regardless of tumor location. To determine the requirement for routine elective IGN irradiation for patients with distal rectal cancer and ACI, we investigated the failure patterns and salvage feasibility regarding IGN recurrence and conducted a comparison between rectal tumors with and without ACI.A total of 1,246 patients with locally advanced rectal cancer treated with preoperative or postoperative chemoradiotherapy (CRT) and radical surgery between 2001 and 2011 at the National Cancer Center  were reviewed retrospectively. The patients had histologically confirmed rectal adenocarcinoma and no evidence of distant metastasis, including IGN, at the time of initial workup and surgery. Concurrent CRT was administered preoperatively (cT3–4) or postoperatively (pT3–4 or pN+) in 877 (70.4%) and 369 (29.6%) patients, respectively. Since the introduction of preoperative CRT at our institution in October 2001, routine treatment for clinically staged T3–4 rectal cancer located at the mid-to-low rectum  has gradually changed from postoperative to preoperative CRT. During and after the transition period, upfront surgery with postoperative CRT was performed routinely for upper rectal cancer, and for mid-to-low rectal cancer, it was determined by the preferences of patients or attending physicians.18F-deoxyfluoroglucose positron emission tomography-CT was performed as required. Clinically positive lymph node involvement was defined as a lymph node with the smallest diameter of 0.5 cm, observed on CT or MRI. All stages were determined according to the American Joint Committee on Cancer Staging System, 7th edition [Pretreatment workups for clinical staging included digital rectal examination, complete blood count, liver function tests, serum carcinoembryonic antigen tests, video colonoscopy, chest radiography and computed tomography (CT) scanning of the abdomen and pelvis with or without transrectal ultrasonography and pelvic magnetic resonance imaging (MRI).  edition . Anal caRadiotherapy was delivered to the whole pelvis at a dose of 45 Gy in 25 fractions, followed by a 5.4-Gy boost in three fractions within 6 weeks. All patients underwent CT simulation for three-dimensional conformal planning, and a three-field treatment plan used a 6-MV photon posterior-anterior field and 15-MV photon-opposed lateral beams. The prescription dose was specified at the isocenter of the planning target volume. The initial radiation field encompassed the gross tumor and mesorectum (preoperative CRT) or tumor bed (postoperative CRT), presacral space, the entire sacral hollow and the regional lymphatics, including the perirectal, internal iliac, presacral and distal common iliac lymphatics. The superior border was placed at L5/S1 and the inferior border at > 3 cm caudal to the gross tumor or tumor bed. The boost field included the gross tumor volume and mesorectum (preoperative CRT) or tumor bed (postoperative CRT), with ≥ 2 cm margin in all directions.Chemotherapy administered concurrently with RT used one of the following regimens: fluoropyrimidine, irinotecan, or an oxaliplatin-based regimen. Patients underwent radical proctectomy, including high ligation of the inferior mesenteric vessels and total mesorectal excision. The interval between preoperative CRT and surgery was 4–8 weeks, and 3–8 weeks between surgery and postoperative CRT. Postoperative chemotherapy was initiated 3–6 weeks after surgery or postoperative CRT, using fluoropyrimidine or an oxaliplatin-based regimen.18F-deoxyfluoroglucose positron emission tomography.All patients underwent standardized follow-up, consisting of physical examination, complete blood count, liver function tests, serum carcinoembryonic antigen tests and chest radiography every 3 months for the first 2 years and every 6 months thereafter, as well as abdominopelvic CT every 6 months. Colonoscopic examinations were performed 1 year postoperatively and then at 2-year intervals. Recurrence was determined based on clinical, radiological or histological findings. Radiological evidence involved serial radiological examinations showing progressive mass growth, including abnormally high uptake on t-test, depending on the nature of the data. Multivariate logistic regression was conducted to determine parameters that are related with IGN recurrence. Actuarial IGN recurrence rate was estimated using the Kaplan-Meier method and the significance of differences was assessed with the log-rank test. The level of statistical significance was set at p < 0.05 and all reported p-values were two-tailed. Statistical analyses were performed using the SPSS software .Intergroup comparisons regarding patient, tumor and treatment characteristics were conducted using the chi-square test, Fisher’s exact test, linear-by-linear association, or vs. 1,057 without ACI; 71.4 ± 35.4 vs. 68.8 ± 33.1 months, respectively (p = 0.333).Patient, tumor and treatment characteristics according to existence of ACI in all 1,246 patients are listed in Table vs. > 3 cm; odds ratio 7.287, 95% confidence interval 1.102 – 48.171, p = 0.039).Of 1,246 patients, 10 developed IGN recurrence; 7 with ACI and 3 without ACI. Characteristics of the 10 patients are listed in Table p < 0.001). The disease-free interval until IGN recurrence was a median of 29 months  from initial management; 27.9 ± 16.5 vs. 50.3 ± 49.1 months with and without ACI, respectively (p = 0.513).Actuarial IGN recurrence at 5 years was 0.7%. When estimated separately in patients with and without ACI, the rates were 3.5% and 0.2%, respectively  developed IGN recurrence and peritoneal seeding.Salvage outcomes of the 10 patients with IGN recurrence are described in Table Non-isolated IGN recurrence occurred in all three patients without ACI. They received palliative CRT, chemotherapy, pelvic exenteration or supportive care, but died of rectal cancer at 8, 11, and 20 months after IGN recurrence.et al. [In the 2009 consensus report of the Radiation Therapy Oncology Group on the definition of clinical target volume for anorectal cancer, the panel group was divided as to whether elective irradiation of the IGN region in rectal adenocarcinomas with ACI is necessary . So far,et al.  assessedet al. [In contrast, Lee et al.  reportedet al. ,9. When et al. . If theset al. [et al. [et al. [vs. 12 months from IGN recurrence; p = 0.045). In a case series, Bardia et al. [et al. [Pelvic recurrence and/or distant metastasis was detected synchronously with IGN recurrence in rectal tumors without ACI, whereas isolated IGN recurrence developed only in rectal tumors with ACI, both in the present study and that by Taylor et al. . Salvage [et al.  treated  [et al.  reported [et al.  reported [et al. . Our gro [et al. . If isol18F-deoxyfluoroglucose positron emission tomography-CT [Enlargement of the RT field to encompass the groin region is associated with a variety of acute and late morbidities, such as desquamation and fibrosis of the inguinal skin, wound and bowel complications, and edema of the extremities and femoral neck fractures . To avoiraphy-CT , and theraphy-CT .Although in the present study IGN recurrence from rectal cancer developed more frequently in the presence of ACI, the 5-year rate was 3.5% despite the fact that the IGN region was not irradiated. Isolated IGN recurrence occurred only from rectal tumors with ACI and increased the IGN recurrence rate of these rectal tumors; however, salvage of isolated IGN recurrence appeared feasible. Our results indicated that routine elective IGN irradiation is unnecessary for distal rectal cancer with ACI.


Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 have demonstrated this direct conversion of carbon dioxide into FFAs; however, FFA yields in these hosts are limited by the negative impact of FFA production on the host cell physiology. This work investigates the use of Synechococcus sp. PCC 7002 as a cyanobacterial host for FFA production. In comparison to S. elongatus PCC 7942, Synechococcus sp. PCC 7002 strains produced and excreted FFAs at similar concentrations but without the detrimental effects on host physiology. The enhanced tolerance to FFA production with Synechococcus sp. PCC 7002 was found to be temperature-dependent, with physiological effects such as reduced photosynthetic yield and decreased photosynthetic pigments observed at higher temperatures. Additional genetic manipulations were targeted for increased FFA production, including thioesterases and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Overexpression of non-native RuBisCO subunits (rbcLS) from a psbAI promoter resulted in more than a threefold increase in FFA production, with excreted FFA concentrations reaching >130 mg/L. This work illustrates the importance of host strain selection for cyanobacterial biofuel production and demonstrates that the FFA tolerance of Synechococcus sp. PCC 7002 can allow for high yields of excreted FFA.Microbial free fatty acids (FFAs) have been proposed as a potential feedstock for renewable energy. The ability to directly convert carbon dioxide into FFAs makes cyanobacteria ideal hosts for renewable FFA production. Previous metabolic engineering efforts using the cyanobacterial hosts  Escherichia coli, which began nearly four decades ago  into FFAs. Cyanobacteria are photosynthetic prokaryotes, which are particularly amenable to genetic manipulation, and as such, several cyanobacterial strains have been engineered for FFA production. Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942, two model freshwater strains of cyanobacteria, were engineered for FFA production by gene knockout of the FFA-recycling acyl-acyl carrier protein (ACP) synthetase/long-chain-fatty-acid CoA ligase and expression of a thioesterase for fatty acid (FA) cleavage from the ACP, resulting in extracellular FFA concentrations of 83.6 and 32 mg/L, respectively , expression of a chloroplastic acetyl-CoA carboxylase, and improved gene expression using strong native promoters, yet these modifications did not improve the concentration of excreted FFAs in engineered S. elongatus PCC 7942  has recently garnered much attention as a potential feedstock for renewable energy production  , BD (agar), MP Biomedicals (CuSO4⋅5H2O), Fisher Scientific , and Sigma-Aldrich . Primers were synthesized by Integrated DNA Technologies (IDT). DNA purifications were performed using the Plasmid Miniprep, DNA Clean and Concentrator, and Gel DNA Recovery kits from Zymo Research. Restriction enzymes, DNA polymerases, and ligases were purchased from New England Biolabs. Suppliers of all other materials used in this study are described below.Chemicals used in this study were purchased from Acros Organics , responsible for FFA recycling  and BglII/XhoI (3′ fragment) restriction enzyme sites, generating the fadD knockout plasmid, pS12. Transformation of pS12 into Synechococcus sp. PCC 7002, followed by spectinomycin selection and PCR screening of fadD, resulted in the construction of strain S01. Transformation of Synechococcus sp. PCC 7002 was performed using a modified protocol based on that proposed by Stevens and Porter  in a 16 mm glass test tube with ventilated cap. The transformation culture was incubated at 30°C with 150 rpm and 60 μmol photons m−2 s−1. After 24 h of incubation, the transformation culture was concentrated to 100 μL using centrifugation  and spread on medium A+/agar plates with 1 mM of sodium thiosulfate and 40 μg/mL of spectinomycin dihydrochloride pentahydrate and 50 μg/mL of kanamycin monosulfate, as required. Colonies were re-streaked a minimum of three times to obtain complete segregants. After antibiotic selection and PCR screening, two positive transformants were tested for FFA production in 400 mL cultures (see Culture Conditions).Strains used and constructed in this study are listed in Table d Porter . SynechoE. coli thioesterase, ‘tesA, from pSE16  was constructed by redesigning the forward primer to eliminate the predicted chloroplast-targeting signal, determined using ChloroP 1.1  from S. elongatus PCC 7942 were cloned from pSE18  and insertion into pS20 at the BsrGI site produced pS21. Transformation of pS21 into Synechococcus sp. PCC 7002 yielded strain S07.To insert another restriction enzyme site for Ruffing,  . Agar plates of medium A+ contained 1 mM of sodium thiosulfate. Single transformed colonies were re-streaked at least three times or until full segregants were obtained, as confirmed by PCR. Synechococcus sp. PCC 7002 strains grown on solid media were inoculated into 4 mL of medium A+ with antibiotics as necessary and grown at 30 or 38°C and 150 rpm in a New Brunswick Innova 42R shaking incubator with photosynthetic light bank. Alternating cool white and plant fluorescent lights provided ~60 μmol photons m−2 s−1 of continuous illumination. After 5–7 days of growth, 1 mL of the test tube inoculum was transferred to a 500 mL baffled Erlenmeyer flask containing 100 mL of medium A+ with appropriate antibiotics. After 4 days of growth under the aforementioned conditions, the 100 mL culture was used to inoculate 400 mL of medium A+ in a 1 L glass media bottle with a three-port lid for sampling, bubbling of 1% CO2 in air, and ventilation through a 0.22 μm filter. The initial cell concentration of the 400 mL culture vessels was approximately an OD730 of 0.1. No antibiotics were added to the large culture vessels to eliminate any potential effects associated with antibiotic supplementation. The large cultures were sampled every 2 days for ~3 weeks. IPTG was added at 100 h to induce expression from the trc and LlacO-1 promoters.Wild-type and engineered strains of Fv) is the difference between the maximum fluorescence (Fm) under saturated light and the initial fluorescence (F0) under actinic light; the apostrophe indicates that the samples were not dark adapted prior to measurement. Photosynthetic yield Cell growth was measured using optical density (OD) readings at 730 nm from a PerkinElmer Lambda Bio spectrophotometer. Photosynthetic yield measurements were obtained using a Waltz mini-PAM photosynthetic yield analyzer; for each sample, triplicate technical measurements were averaged due to variability associated with these measurements. Photosynthetic yield or efficiency measurements Synechococcus sp. PCC 7002 was genetically engineered for FFA production and excretion by targeting the long-chain-fatty-acid CoA ligase  for gene knockout and overexpression of a truncated thioesterase from E. coli (‘tesA). The resulting engineered strains, S01 (ΔfadD) and S02 , are analogous to the previously constructed, FFA-producing strains of S. elongatus PCC 7942, SE01 (Δaas) and SE02 . A schematic of the engineered pathway for FFA production can be found in the Supplemental Material . The FFA concentrations, cell growth profiles, and photosynthetic yields of Synechococcus sp. PCC 7002, S01, and S02 are shown in Figure S. elongatus PCC 7942, SE01, and SE02  did not show improved FFA production in comparison to the S. elongatus PCC 7942 strains (SE01 and SE02), there were notable differences in the physiological responses of the hosts. The late exponential growth rates of S01 and S02 showed a slight decrease in comparison to the wild-type 7002 after induction at 100 h (4.17 days)  may also influence the optimal growth temperature, and a more rigorous, multi-factorial investigation of Synechococcus sp. PCC 7002 growth is required to evaluate the optimal growth temperature of this strain. In addition to this unexpected growth response, there were significant changes in photosynthetic yields at 38°C in comparison to 30°C. At the higher temperature, the wild-type (7002) had reduced photosynthetic yields with values up to 50% lower compared to the 30°C temperature condition  and the phycobiliproteins (635 nm) at 38°C . This suggests that DesB is not involved in FFA tolerance at this temperature, but it does not rule out a role for membrane saturation in the mechanism of temperature-dependent FFA tolerance in Synechococcus sp. PCC 7002.At high temperatures, bacteria have been shown to change the degree of saturation of their cell membrane lipids, with increasing saturation accompanying increasing temperatures Koga, . The incSynechococcus sp. PCC 7002 compared to S. elongatus PCC 7942, additional strategies for improving FFA biosynthesis were investigated. The acyl-ACP thioesterase from C. reinhardtii (fat1) was previously shown to have activity similar to ‘tesA in S. elongatus PCC 7942 . Expression of tfat1 in S05 led to a nearly threefold increase FFA production and excretion compared to the fat1-expressing S03 strain  did not yield an improvement in FFA production in the Synechococcus sp. PCC 7002 strain S06 [, overexpression of the truncated E. coli thioesterase (‘tesA), and overexpression of a heterologous RuBisCO (rbcLS), an engineered strain of Synechococcus sp. PCC 7002 was constructed, which produced FFAs at concentrations as high as 131 mg/L. The high levels of FFA achieved in this study demonstrate not only a potential for large-scale production applications, but also a potential separation mechanism, as the FFAs precipitated from the culture medium and floated to the surface upon reaching saturation  which are more desirable, as these temperatures are readily achieved in open outdoor cultivation systems without additional heating . The enhanced membrane viscosity associated with a lower degree of membrane saturation may allow for increased FFA diffusion across the cell membrane and therefore higher extracellular FFA concentrations. This proposed mechanism of FFA excretion also supports the aforementioned hypothesis for improved FFA tolerance at lower temperatures. The change in photosynthetic pigments in response to high FFA production also differed for the Synechococcus sp. PCC 7002 strains in comparison with the S. elongatus PCC 7942 strains. FFA-producing strains of S. elongatus PCC 7942 showed selective degradation of chlorophyll-a pigment in response to FFA production . When a cytotoxic unsaturated FFA, α-linolenic acid, was exogenously added to cultures of Synechococcus sp. PCC 7002 also displayed different responses to the genetic manipulations of aas/fadD knockout, thioesterase expression, and RuBisCO expression. While Aas from S. elongatus PCC 7942 and FadD from Synechococcus sp. PCC 7002 are homologs (58.4% amino acid identity), aas knockout in SE01 led to the accumulation of 43 mg/L of excreted FFAs, yet fadD knockout in S01 yielded only 5 mg/L of excreted FFAs. These results may indicate that S. elongatus PCC 7942 has a naturally higher rate of membrane degradation and FFA recycling compared to Synechococcus sp. PCC 7002. In the S. elongatus PCC 7942 strains SE02 and SE03, both ‘TesA’ and ‘Fat1’ thioesterases had similar activities, generating nearly equivalent levels of excreted FFA , suggesting that further improvement may be achieved with codon optimization. Regardless of the underlying mechanisms for improved FFA tolerance and production, Synechococcus sp. PCC 7002 was shown to be a superior host for FFA biosynthesis compared to S. elongatus PCC 7942. This work highlights the importance of host selection in metabolic engineering efforts, and since host responses are difficult to predict a priori, screening and testing of potential hosts remains a critical step in cyanobacterial strain development.In addition to differences in FFA stress response, Ruffing, . In the Synechococcus sp. PCC 7002 (this study), S. elongatus PCC 7942  may weaken the integrity of the host strain and its ability to survive under adverse conditions, which may be experienced in large-scale production systems. Moreover, as the genetic manipulations differ between the engineered strains of Synechocystis sp. PCC 6803 and those of Synechococcus sp. PCC 7002 and S. elongatus PCC 7942, this direct comparison must be viewed with caution. Synechocystis sp. PCC 6803 also has the highest tolerance of exogenous FFA, yet the results of this study clearly demonstrate that exogenous FFA tolerance does not directly correlate with tolerance to intracellular FFA biosynthesis. When comparing inherent strain properties of doubling time, high light tolerance, high salt tolerance, and temperature tolerance, Synechococcus sp. PCC 7002 is superior to the other two cyanobacteria. These basic strain traits, which can be difficult to engineer into a host, make Synechococcus sp. PCC 7002 a beneficial host for cyanobacterial biofuel production, and this study provides direct evidence of the advantages afforded by this host, encouraging further investigation and development of Synechococcus sp. PCC 7002 for biofuel production.To date, three model cyanobacterial strains have been genetically engineered for FFA production: Anne M. Ruffing conceived the study, conducted all experimental work and data analysis, and drafted the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fbioe.2014.00017/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.


The ability of cancer cells to metastasize is dependent on the interactions between their cell-surface molecules and the microenvironment. However, the tumor microenvironment, especially the cancer-associated stroma, is poorly understood. To identify proteins present in the stroma, we focused on phyllodes tumors, rare breast tumors that contain breast stromal cells. We compared the expression of proteins between phyllodes tumor and normal tissues using an iTRAQ-based quantitative proteomic approach. Decorin was expressed at reduced levels in phyllodes tumor tissues, whereas periostin was upregulated; this result was validated by immunohistochemical analysis of phyllodes tumors from 35 patients. Additionally, by immunoprecipitation and mass spectrometry, we confirmed that decorin forms a complex with periostin in both phyllodes tumors and BT-20 breast cancer cells. Following siRNA-mediated knockdown of periostin in T-47D cells, secreted decorin in the culture medium could be detected by multiple reaction monitoring (MRM). Furthermore, periostin knockdown in BT-20 cells and overexpression of decorin in MDA-MB-231 cells inhibited cell motility and invasion. Our results reveal the molecular details of the periostin–decorin complex in both phyllodes tumor tissues and breast cancer cells; this interaction may represent a novel target for anti-cancer therapy. The tumor microenvironment plays a critical role in cancer progression. The stromal and epithelial cells that constitute the tumor microenvironment strongly influence tumor proliferation, invasion, and metastasis, and the phenotypes of tumors are largely determined by interactions between cancer cells and their microenvironment12et al. reported that periostin interacts with integrin receptorsRecent studies have shown that periostin and decorin are components of the extracellular matrix that affect the biology of various types of cancer4615Small leucine-rich proteoglycans (SLRPs) are components of the extracellular matrix, which is altered in the environment surrounding a tumor. SLRPs such as decorin, lumican, and biglycan are expressed in the vicinity of colon, pancreas, breast, and prostate cancers202124Decorin has recently emerged as a potential natural anticancer agent produced by normal cells2831In this study, we focused on phyllodes tumors, which are composed of epithelial and cellular stromal components of the breast. We compared tissue-specific protein expression in phyllodes tumor and normal tissues by iTRAQ (isobaric tag for relative and absolute quantitation) and tandem mass spectroscopy. These analyses revealed that decorin was expressed at lower levels, whereas periostin expression was upregulated, in phyllodes tumor tissues and cancer cells. Furthermore, we characterized the periostin–decorin complex. In particular, we found that knockdown of periostin results in translocation of decorin from the cytoplasm to the extracellular space, leading to the inhibition of cancer cell migration and invasion.Cancer stroma consists mainly of cancer-associated fibroblasts (CAFs), which affect aspects of the tumor microenvironment such as angiogenesis, invasion, and metastasis. CAFs promote tumor progression in breast cancer, but the details of their role remain unclear, primarily because the collection of CAFs from cancer tissue is technically difficult. Therefore, in this study we focused on phyllodes tumors, which consist of breast stromal and epithelial cells. We used the iTRAQ-based quantitative proteomic approach to identify proteins that were differentially expressed between phyllodes tumor and normal tissues. As shown in 10173617To validate the accuracy of the results described above, we performed immunohistochemical analysis to examine the levels of decorin and periostin in tumor and normal tissues from 35 phyllodes tumor patients. Decorin expression in normal tissues was higher than in tumor tissues  , whereasNext, we verified the expression of both proteins in phyllodes tumors (n = 35) and breast fibroadenomas (n = 37) by immunohistochemical analysis. Fibroadenomas, the most common benign breast tumors, arise from intralobular fibrous tissue. Decorin was present at higher levels in fibroadenomas than in phyllodes tumors  , whereasTo identify decorin- or periostin-binding proteins in normal and phyllodes tumor tissues, we immunoprecipitated both proteins, and subjected the immunoprecipitates to SDS-PAGE and silver staining. Gel bands representing differences between normal and phyllodes tumor tissue were cut out and subjected to in-gel trypsin digestion and mass spectrometry . Thirtee13C6, 15N2) or DLPPDTTLLDLQNNK ] was separated by nano-LC, and the MRM transitions were monitored. The peptides were delivered in 5% (v/v) acetonitrile at a concentration of 5 pmol/μl. We next sought to investigate the functional significance of the interaction between decorin and periostin, both of which are secreted proteins. First, we confirmed that both proteins were expressed in the breast cancer cell lines BT-20 and T-47D; in other cell lines we examined , periostin was present but decorin was not . By co-i4The data described above demonstrate that decorin is secreted from periostin-knockdown BT-20 cells, as well as from decorin-overexpressing BT-20 and MDA-MB-231 cells. We next investigated whether these cells could inhibit cell motility and invasion. To this end, we treated BT-20 cells with siRNA-periostin for 48 h. Each sample was subjected to SDS-PAGE, followed by immunoblot analysis with anti-decorin or anti-periostin antibody. β-actin was used as a loading control . We thenIn this study, we characterized the interaction between cellular decorin and periostin not only in phyllodes tumors but also in BT-20 breast cancer cells. We detected secreted decorin in the culture medium of periostin-knockdown T-47D cells . LikewisOur findings demonstrate that periostin is more abundant in phyllodes tumors than in normal tissues , and tha39As a component of the extracellular matrix, decorin prevents migration and invasion. Consistent with this, stromal decorin expression adjacent to malignant cells in invasive breast cancer tumors is significantly weaker than that in pure ductal carcinoma in situ (DCIS)Information about immunoblotting analysis, immunoprecipitation, knockdown of gene expression, total RNA extraction, RT-PCR, immunofluorescence and cell proliferation assay can be found in the All human experiments were performed in accordance with the guidelines approved by the Ethics Committee of Tokyo Medical and Dental University (TMDU). The Institutional Review Board of TMDU approved the study, and written informed consent was obtained from each patient before surgery.Tissue specimens analyzed in this study were obtained from 35 patients with phyllodes tumors and 37 patients with fibroadenomas. All patients underwent surgical resection in the Department of Breast Surgery at Tokyo Medical and Dental University, Japan, between March 2003 and August 2012. The clinical characteristics of the tumors we examined are summarized in 2. All cell lines were authenticated in December 2012.BT-20, T-47D, MDA-MB-231, and HeLa S3 cells were generously provided by the Japanese Foundation for Cancer Research. The media for each cell line are summarized in g at 4°C for 1 hour. Albumin and IgG were removed from the supernatants using the ProteoSeek™ Albumin/IgG Removal Kit , followed by concentration of the sample using Nanosep centrifugal devices . For mass spectrometry, 100 μg of protein lysate was reduced in 25 mM TCEP and 0.05% SDS for 60 min at 60°C, alkylated with methyl methanethiosulfonate for 10 min at RT, and then digested with trypsin at 37°C for 12–16 h. Desalted tryptic peptides were labeled with isobaric tags for relative quantification using iTRAQ reagents . Briefly, peptides were dried and resuspended in 20 μl iTRAQ dissolution buffer. Trypsin-digested peptides isolated from tumor and normal tissue of three patients with phyllodes tumors were labeled with 2-plex iTRAQ tags; isobaric tags with m/z of 114 were added to normal tissue, and isobaric tags with m/z of 117 were added to tumor tissue . The stained gel bands were cut out and treated with dithiothreitol  dissolved in ammonium hydrogen carbonate , followed by treatment with iodoacetamide . After the gels were dried, 20 μl of 0.05 pmol/μl trypsin (AB SCIEX) solution was applied to each gel piece and incubated for 12–16 h at 37°C to digest proteins. Digested peptides were extracted by washing the gel pieces twice with 50% trifluoroacetic acid , followed by washing with 80% TFA. The purified peptide samples were injected onto a reversed-phase C18 column  and separated by nanoflow liquid chromatography (300 nL/min) on a nano LC Dina-A system (KYA TECH Corp.) in line with a Q-TRAP 5500 instrument (AB SCIEX) using a 75-min gradient of 5–100% acetonitrile in 0.1% formic acid.13C6, 15N2) and DLPPDTTLLDLQNNK , were purchased from Thermo Fisher Scientific . The peptides were delivered in 5% (v/v) acetonitrile at a concentration of 5 pmol/μl.Standard peptides, VSPGAFTPLVK (Conditioned medium (CM) was concentrated 100-fold using a Vivaspin 20 . The protein concentration in CM was determined using the Bradford protein assay . Peptides obtained from in-solution digestion of raw CM were analyzed by multiple reaction monitoring (MRM). The doubly charged precursor ion was chosen as the Q1 mass, and the most intense fragment ion from the precursor was chosen as the Q3 mass. The optimized instrument parameters and selected MRM transitions were tested by analyzing the endogenous protein and standard peptides. The samples were separated by nanoflow liquid chromatography (300 nl/min) on a nano LC Dina-A system in line with a Q-TRAP 5500 instrument using a 45-min gradient of 5–100% acetonitrile in 0.1% formic acid. Absolute quantitation was performed using the MultiQuant Software (AB SCIEX). To improve the accuracy of the quantitation, the “heavy” peptide was added into each biological sample at a fixed amount to act as the internal standard for these samples. The actual concentration of peptide in the biological sample was computed from the ratio of the endogenous peptide (light) to the added internal standard (heavy). The concentration C of the targeted endogenous peptide was calculated as Immunohistochemical staining was carried out by the streptavidin–biotin method using the Histofine SAB-PO kit . Sections (4 μm thick) were cut from each FFPE tissue block. After deparaffinization and rehydration, antigen retrieval treatment was carried out in a temperature-controllable microwave processor  at 98°C for 20 min (decorin) or 30 min (periostin) in 10 mM sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubating the sample in a solution of 3% hydrogen peroxide in absolute methanol for 15 min. Nonspecific binding was blocked by treating the slides with 5% EzBlock  for 10 min at RT. For detection of decorin, sections were incubated with anti-decorin antibodies (1:1000 dilution), and then beam-irradiated with the MW processor at 27°C for 15 min. The Histofine SAB-PO kit was used for visualization. For detection of periostin, sections were incubated for 90 min at RT with anti-periostin antibodies (1:2000 dilution). The Histofine Simple Stain MAX PO (MULTI) kit (Nichirei Corp.) was used for visualization. Color development was carried out with DAB  for 10 min at RT. The sections were then counterstained with 1% Mayer's hematoxylin.Immunostaining of decorin and periostin was analyzed under a light microscope. Evaluations of stromal decorin and periostin expression were performed around normal gland and tumor tissue. Digital images were analyzed semiquantitatively. The intensities of decorin and periostin signals were determined using the ImageJ software, according to the method described by Augoff et alConfluent cell monolayers were wounded (lightly scratched) with a pipet tip. After careful washing to remove detached cells, the cells were cultured for 24 h. Phase-contrast images were taken every 30 min for 24 h. The width of the wound was monitored using an FW4000-TZ time-lapse microscope (Leica).5) were seeded in serum-free media onto polycarbonate membrane inserts (8 μm thick) whose upper surfaces were coated with a uniform layer of dried Bovine Type I Collagen matrix (Cell Biolabs). Inserts were then submerged in media containing 10% fetal bovine serum (FBS), and the cells were cultured for 24 h. Invading cells were stained with cell stain solution, followed by measurement using a Fluoroskan Ascent plate reader (Thermo Scientific) at 560 nm.Cell invasion was measured using the CytoSelect 96-Well Collagen I Cell Invasion Assay (Cell Biolabs). Cells .All the authors contributing to this work have made the following declarations: Y.M., A.N. and T.I. conceived and designed the experiments. A.N. and T.I. performed the experiments. A.N. and T.I. analyzed the data. T.I. contributed reagents and materials tools. Y.M., A.N. and T.I. wrote the manuscript. M.N., T.N., T.S., H.U. and K.S. reviewed the manuscript.Supplementary Information


Pygospio elegans is one of few species polymorphic in developmental mode, with multiple larval phenotypes, providing a possibility to examine the potential mechanisms allowing transitions in developmental mode. We investigated the presence of DNA methylation in P. elegans, and, since maternal provisioning is a key factor determining eventual larval phenotype, we compared patterns of DNA methylation in females during oogenesis in this species. We demonstrate that intragenic CpG site DNA methylation and many relevant genes necessary for DNA methylation occur in P. elegans. Methylation-sensitive AFLP analysis showed that gravid females with offspring differing in larval developmental mode have significantly different methylation profiles and that the females with benthic larvae and non-reproductive females from the same location also differ in their epigenetic profiles. Analysis of CpG sites in transcriptome data supported our findings of DNA methylation in this species and showed that CpG observed/expected ratios differ among females gravid with embryos destined to different developmental modes. The differences in CpG site DNA methylation patterns seen among the samples suggest a potential for epigenetic regulation of gene expression (through DNA methylation) in this species.Transitions in developmental mode are common evolutionarily, but how and why they occur is not understood. Developmental mode describes larval phenotypes, including morphology, ecology and behavior of larvae, which typically are generalized across different species. The polychaete worm  Epigenetic changes can affect the regulation of gene expression and increase variation at the phenotypic level without changing the underlying DNA sequence. The most commonly studied mechanism of epigenetic change is DNA methylation, which involves addition of a methyl group to a cytosine nucleotide (predominantly at CpG dinucleotide sites), altering its structure to 5-methylcytosine (5mC). DNA methylation has significant roles in many biological processes, including cell type differentiation during embryonic development, X-chromosome inactivation, regulation of genome stability and silencing of transposable elements (reviewed by –4). DNA Arabidopsis thaliana, Dubin and colleagues [DNA methylation  as a mechanism for regulating gene expression has been suggested to be a particularly important means for organisms to adapt to new or varying environments or to maintain phenotypic plasticity in some traits (reviewed by ). This illeagues  found a lleagues .Gasterosteus aculeatus, have differences in their genome-wide methylation patterns, and epigenetic regulation (through DNA methylation) is suggested to be associated with the phenotypic differences seen between the morphs [Several experiments with plants have shown a connection between methylation or demethylation patterns and phenotypic differences . Marine invertebrate species normally produce only one type of larva, but P. elegans is one of the rare poecilogonous species, having benthic, planktonic and intermediate larval modes. Females brood their larvae in egg capsules within the sand tubes they inhabit, and differential maternal provisioning is associated with different larval developmental modes. The types of larvae differ in e.g. brood size, larval size, and larval dispersal ability, i.e. length of pelagic period. Benthic larvae develop when mothers lay only a few embryos, but abundant nutritional resources (yolky nurse eggs) in their egg capsules. These larvae have a long brooding period and settle soon after they emerge from the egg capsules. Planktonic larvae, on the other hand, emerge from the egg capsules early in development to feed and grow in the plankton before settlement as juveniles. Mothers of planktonic larvae lay many embryos and fewer nurse eggs in the capsules. Intermediates between these two extremes also exist [Our study species, the spionid polychaete so exist , 19.P. elegans is not known, but epigenetic regulation (e.g. through DNA methylation) could be one contributing factor. Although some genetic structuring is present among populations, it does not appear to correlate with developmental mode and there is no evidence for cryptic species or lineages with different developmental modes [The underlying cause of the observed polymorphism in developmental mode in al modes , 20, 21.al modes , unpubliP. elegans, we studied DNA methylation in this species. First, we confirmed that DNA methylation at CpG sites occurs in P. elegans using bisulfite treatment and DNA sequencing of a single locus. Secondly, genome-wide methylation patterns were compared in gravid females producing different types of larvae using restriction enzymes with different sensitivity to methylation. Additionally, we searched for genes suggested to be involved in the methylation machinery from the transcriptome of P. elegans. Finally, we performed an analysis of the observed/expected ratio of CpG sites in the transcriptome in order to investigate the evolutionary signature of methylation in P. elegans and to compare levels of intragenic germline DNA methylation in different functional groups of genes.To begin to address the question of whether epigenetic processes could be involved in the developmental polymorphism observed in P. elegans, we used bisulfite treatment and DNA sequencing of a specific gene fragment. This method can be used to detect methylation at single base resolution. Bisulfite treatment of DNA converts unmethylated cytosines to uracil while methylated cytosines remain unchanged. After sequencing, the converted sites can be detected as thymines in the sequences. P. elegans samples were collected in March 2014 from Vellerup, Denmark . Two adult females and five samples of larvae at different developmental stages  were used. DNA was extracted using the DNeasy blood and tissue kit spin column protocol (Qiagen). Portions of the same DNA samples were bisulfite treated using the EZ DNA Methylation kit (Zymo Research) according to the manufacturer’s instructions. A fragment of the Histone H3 gene was amplified and sequenced from both untreated and bisulfite treated DNA samples. For amplification of untreated DNA, we used primers published previously . For amplification, the following PCR program was used: 94°C for 2 min, 34 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec, with a final elongation of 72°C for 3 min. PCR products were treated with Exonuclease I and Shrimp alkaline phosphatase (Fermentas), cycle sequenced in both directions using the BigDye v.3.1 kit, and visualized with an ABI 3130xl Genetic Analyzer and Sequencing Analysis v.6 software (Applied Biosystems). Sequences were checked and alignments of sequences obtained from both untreated and bisulfite treated DNA were made using Sequencer v.5.2.4 (Gene Codes Corporation) to investigate the methylation status of the cytosines.To investigate the presence of CpG site DNA methylation in and H3R, ). Due toP. elegans, methylation patterns and epigenetic variation was examined using the methylation-sensitive AFLP (MS-AFLP) technique [MspI and HpaII, which have the same recognition site (5’- CCGG -3’) but different sensitivity to methylation. MspI is insensitive to methylation of the CpG site in the recognition sequence, but HpaII activity is inhibited if the inner cytosine is methylated. With this method, no genome sequence information is needed, and the resulting banding patterns indicate differentially methylated loci in the genome.After confirming the presence of CpG methylation in echnique , modifieechnique . This meEcoRI and either MspI or HpaII enzyme, which, as described earlier, are isoschizomers that differ in their sensitivity to CpG site methylation.We used the MS-AFLP method to estimate levels of methylation and epigenetic differentiation of females producing different types of larvae. We sampled gravid and non-reproductive females in May 2014 from populations that produce either benthic larvae or planktonic larvae: Herslev, Denmark , and Rømø, Denmark (Wadden Sea), respectively. Each genomic DNA sample was digested in parallel reactions with EcoRI and 1 μl of either MspI or HpaII . Samples were digested at 37°C for 15 min, followed by 5 min both at 65°C and then 80°C.Individual worms were first macerated in 180 μl of ATL Buffer and 20 μl of Proteinase K (Qiagen) and then incubated at 56°C for three hours, after which DNA extraction was completed using the Genomic DNA Clean and Concentrator kit (Zymo Research) according to the manufacturer’s instructions. For MS-AFLP, 300 ng of genomic DNA was used per reaction. Parallel digestions were done in 20 μl reactions containing 1 μl of EcoRI: 5’- AATTGGTACGCAGTCTAC -3’ and 5’- CTCGTAGACTGCGTACC -3’ [HpaII/MspI: 5’- GACGATGAGTCTAGAA -3’ and 5’- CGTTCTAGACTCATC -3’ [EcoRI adaptor, 15 pmol of the HpaII/MspI adaptor, and 2 U of T4 DNA ligase (Thermo Scientific) was first prepared, and 5 μl was added to each 20 μl digestion sample (described above) and incubated at 18°C overnight (16 hours). Afterwards, the ligase enzyme was inactivated at 65°C for 10 min.Double stranded ligation adaptors were prepared separately by mixing equal amounts of the oligonucleotides  and hea2) in 10 μl volume. The following PCR program was used: 72°C for 2 min, 30 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 2 min, with a final extension at 60°C for 30 min. Six different primer combinations (Table C in 2 concentration of 2 mM). PCR cycling conditions were as follows: 72°C for 2 min, 13 cycles of 95°C for 30 s, 65°C for 30 s (decreasing temperature 0.7°C/cycle), and 72°C for 2 min, followed by 23 cycles of 95°C for 30 s, 57°C for 30 s and 72°C for 1 min (with an extension of time by 2 sec/cycle), ending with 72°C for 20 min. The resulting PCR products were separated on an ABI PRISM 3130xl (using GeneScan -500 LIZ as size standard), and fragments between 50 and 500 bp were scored using GeneMapper v.5 software (Applied Biosystems).Preselective PCR contained 2 μl of the prepared ligation mix as template, 0.5 μM of both preselective primers , 200 μMEcoRI-MspI and EcoRI-HpaII), with four possible resulting patterns: 1/1, 1/0, 0/1 and 0/0. In an unmethylated state, the fragment is seen in both reactions (pattern 1/1), whereas absence of both fragments  is considered an uninformative state, which could be due to either hypermethylation or absence of the restriction site [MspI band present, HpaII blocked by methylation, band absent). The interpretation of the 0/1 pattern  remains ambiguous, and in some cases [e.g. EcoRI-MspI and EcoRI-HpaII profiles) into one line of binary data/individual, and two different scoring approaches were used (described below).Presence (1) and absence (0) of fragments was scored for each individual in the two parallel reactions , condition II indicates methylation of the internal cytosine(s) (m: band present in MspI reaction only) and condition III represents either hemimethylation of the external cytosine or unspecific products (h: band present in HpaII reaction only). These three conditions are respectively, the 1/1, 1/0 and 0/1 patterns described above. Absence of fragments in both reactions indicates an uninformative state. During data transformation, one to three subloci (or epiloci) for each locus (restriction site) of the raw data are generated, i.e. one original locus can contain three epiloci . This data was used to measure epigenetic diversity within the samples by calculating number and percentage of polymorphic epiloci of each type in the samples, percentage of private epiloci (pattern seen in just one sample) and mean Shannon’s information index  in GenAlEx [Epigenetic diversity was also examined by transforming the data with another scoring method using the R script MSAP_calc . Since tdex (see ). Differ GenAlEx , using tP. elegans  was utilized. The data are pooled from multiple Illumina RNAseq paired-end libraries  obtained from: gravid females that produce planktonic larvae , gravid females that produce benthic larvae  and non-reproductive females from populations that produce both planktonic and benthic larvae . The transcriptome is expected to be suitable for evaluation of the methylation status of P. elegans’ DNA because earlier studies with other invertebrate species have shown that methylation is typically seen in coding regions [DNA methylation at CpG sites leads to gradual depletion of CpG dinucleotides in genomes over evolutionary time , 32. Thi regions , 33, and regions , 35, 36.P. elegans contains 80853 contigs presenting predicted open reading frames (ORFs). GO terms were linked to ORFs according to their best BLAST hit in Swiss-Prot or TrEMBL database  and PfamA domain hits. In order get an as reliable as possible set of transcripts for evaluation of the difference in CpG o/e ratio among gene functional classes, we chose 8658 ORF containing contigs annotated with a biological process GO term and expressed with a minimum average of 10 fragments per kilobase of exon per million fragments mapped (FPKM) in at least one of the studied conditions . The CpG observed/expected ratio (CpG o/e) was calculated for each contig in this set using the following equation :In total, the draft transcriptome of http://www.informatics.jax.org). Because multiple GO terms are typically associated to each contig, the same contig might have been assigned to many GO Slim bins. However, any single contig was allowed to be assigned only once to a specific GO Slim bin. For each GO Slim category, we calculated the mean CpG o/e and standard error of the mean. The differences among the category means were analyzed with one way ANOVA followed by pairwise comparison using t-tests with pooled standard deviation. Division to GO Slim categories was applied separately for the subgroups of contigs expressed in the three study groups. The differences between mean CpG o/e in different GO Slim categories were analyzed with t-tests . All statistical testing was conducted in R .Additionally, each of the ORF containing contigs were assigned to a functional group based on the mgi GO Slim Categories biological process . Transcriptome data were obtained from multiple individuals . We attempted to find homologs of genes from the relevant protein families: DNA methyltranseferases  and Methyl-CpG Binding Domain proteins (MBD). DNA methyltransferases are a well characterized family of proteins involved in establishing and maintaining DNA methylation of cytosines . Protein translations of the matched P. elegans contigs were also blasted  to confirm gene identity (http://blast.ncbi.nlm.nih.gov/Blast.cgi). In addition, phylogenetic reconstructions were used to give further support for the orthology of the gene sequences. Sequence alignments with known representative sequences and best blast hits of the contigs were done in MEGA6 [We searched for genes expected to be involved in the molecular machinery of DNA methylation from a draft transcriptome of nes see , 37). Me. MeP. elnes , by, byP. eluery see . Once main MEGA6 , and NeiP. elegans. A 367 bp fragment of the gene was sequenced from genomic DNA and the sequences of all seven samples were identical . After bisulfite conversion and sequencing with newly designed primers, the resulting fragments (191–230 bp), were aligned with the data from the untreated DNA , but different sensitivity to methylation. Methylation profiles for each individual can be constructed by comparing the fragments produced by the different enzymes.Methylation-sensitive AFLP (MS-AFLP) protocol was used for analyses of genome wide methylation levels and to compare methylation profiles between females producing embryos destined to different larval developmental modes. In the MS-AFLP protocol, each DNA sample is digested in separate parallel reactions using ST = 0.057, P < 0.001). Most pairwise comparisons of the methylation profiles showed significant differences , whereas in Rømø, gravid females did not differ from non-reproductive females . Gravid females producing different larval types (Herslev: benthic & Rømø: planktonic) also showed significant differences in their MSL loci . In addition, also the non-reproductive females from the two sites were significantly differentiated in their methylation profiles . Using msap , and metferences . Within profiles .An alternative scoring method, the mixed scoring transformation  of the P. elegans draft transcriptome to look for signatures of intragenic DNA methylation in this species. Low CpG o/e ratio is interpreted as a sign of high persistence of (germline) methylation, whereas a high CpG o/e ratio is expected for genome regions or genes with low historical methylation , indicating that depletion of CpG dinucleotides is associated with an increase in TpG dinucleotides (mean for distribution of TpG o/e ratio was 1.318). Since the mean CpG o/e value seen in P. elegans transcripts is less than 1, we can conclude that methylation is present and has left an evolutionary footprint in the genome.The observed/expected ratio of CpG sites was calculated from the on e.g. , 44). Sp. SpP. elof 0.669 . For comP. elegans involved in discrete biological processes, each of the 8658 ORF containing contigs were assigned to a GO Slim category. In the whole set of transcripts, significant differences between CpG o/e ratios of genes in different biological processes were for those involved in ‘cell adhesion’ compared to ‘cell cycle’ (P = 0.029) and for those involved in ‘death’ compared to those involved in ‘protein metabolism’ (P = 0.003), ‘RNA metabolism’ (P = 0.021), 'cell adhesion' (P = 0.001), and 'stress response' (P = 0.033) . In addition, PYGEL76073 contained three different methyltransferase specific domains  located at the end of the N-terminal region. These two P. elegans sequences do not overlap, but are likely two partial transcripts from the same gene  and phylogenetic analyses support a conclusion that the contig is Dnmt1. In the NJ tree, sequences from vertebrates form a separate, well supported group that does not include any sequences from invertebrates. The sequence found in P. elegans  shows similarity to Dnmt1 from other marine invertebrates  which contains a domain from the AdoMet_MTase super family . With blastp searches, this sequence showed a good match to invertebrate tRNA cytosine-5-methyltransferase (Dnmt2). When using Dnmt3 sequences as query, we found sequences in the transcriptome of P. elegans containing both of the conserved domains found in the Dnmt3a and Dnmt3b regulatory region . PYGEL1MBD2/3, combining the functions of MBD2 and MBD3, e.g. [H. sapiens MBD protein sequences  as query, MBD domain containing sequences were also found in the P. elegans transcriptome. Sequence PYGEL94139 contained two domains from a MBD enzyme . Using this sequence as query in a blastp search, the best hits are to invertebrate methyl-CpG-binding domain protein 2 or 2/3. Phylogenetic reconstructions using NJ supported orthology of this P. elegans MBD sequence to methyl-CpG-binding domain proteins. Vertebrate MBD2 and MBD3 proteins form their own well supported clade separated from invertebrate MBD2/3 sequences. PYGEL94139 sequence was most similar to a predicted MBD like protein sequence from another annelid Capitella teleta. Furthermore, sequences from the Lophotrochozoa  grouped separately from those from Arthropoda used in the phylogenetic analyses  were found using H. sapiens TETs as query. Several contigs, e.g. PYGEL82188 and PYGEL4276 contained the conservative oxygenase domain of the 2OGFeDO superfamily (Tet_JBP), and blastp search showed that they matched well to TET methylcytosine dioxygenase proteins from other species. Phylogenetic NJ analysis groups the P. elegans sequence (PYGEL82188) with predicted TET protein sequences of mollusks, whereas insect samples group together, and mammal TET sequences form their own well supported clade, as expected  are found , 39, wheD3, e.g. ). Using Fig D in . AnotherFig E in .Chaetopterus variopedatus embryos [Spiophanes tcherniai [Polydora cornuta [Epigenetic variation, especially DNA methylation and its functions, has been widely studied in vertebrate model species , 37, 45. embryos  and Spiocherniai  have CpG cornuta . InformaPygospio elegans, a poecilogonous species with females that can produce different types of larvae [P. elegans is an important first step in determining whether epigenetic regulation via changes in DNA methylation, possibly leading to changes in gene expression, could have a role in the observed differences in maternal provisioning and larval phenotype evident in the different larval developmental modes.We examined the presence of DNA methylation in the spionid polychaete f larvae –20. How f larvae ). InvestP. elegans, a pattern commonly seen in other invertebrates [Nasonia; [Bisulfite treatment and sequencing of a fragment of Histone H3 gene revealed the presence of intragenic CpG site methylation in tebrates , 35,46. tebrates , 8, 33. tebrates , 8, 35, Methylation-sensitive AFLP (MS-AFLP) analysis showed differences in genome-wide CpG site DNA methylation patterns and therefore suggests epigenetic differences among our samples. Most interestingly, MS-AFLP analysis showed significantly different methylation profiles for the gravid females with different developmental modes, i.e. different loci, or genes, were methylated. In Herslev, methylation profiles between gravid females with benthic larvae and non-reproductive females were also significantly different and methylation level was higher in the gravid females (using both scoring approaches for the MS-AFLP data). However, no differences were seen in Rømø, despite reproductive status of the individuals. In addition, the non-reproductive females from the two sites (Herslev & Rømø) also showed differentiation in their methylation profiles, which could be associated with possible genetic differences between the sampled populations.P. elegans exhibits methylation in regions of the genome other than at CpG sites  between mature and immature Atlantic salmon  and betwebrates: ).Dnmt1 is the maintenance enzyme copying methylation status of the genome during DNA replication (patterns inherited through mitosis), whereas two of the three enzymes in the Dnmt3 family (Dnmt3a and Dnmt3b) are involved in the de novo methylation of previously unmethylated sites. A third Dnmt3 protein (Dnmt3L) and Dnmt2 do not have methyltransferase activity, but Dnmt3L interacts with the other Dnmt3 enzymes and Dnmt2 is involved in tRNA methylation , whereas others have multiple copies of the Dnmt1 gene (e.g. the wasp Nasonia vitripennis). In molluscs, for example, a Dnmt3 homologue is found in Crassostrea gigas [Biomphalaria glabrata [C. variopedatus [C. teleta genome (see [Dnmt1 (and Dnmt2) in the transcriptome of P. elegans. The presence of genes from the Dnmt3 family could not be verified in this study. Dnmt3 family enzymes are particularly relevant since it is suggested that Dnmt3s are involved in establishing new methylation patterns in response to environmental triggers [Dnmt3 genes in P. elegans.Additionally, we used transcriptome data to search for genes suggested to be involved in processes of DNA methylation. DNA methyltransferases are enzymes that establish and maintain DNA methylation of cytosines (by attaching the methyl group). In human and many other vertebrates, three subfamilies are found: ion see , 37). In. InDnmt1iewed in ), some sea gigas , but notglabrata . DNA metopedatus , 57 and ome (see ). We foutriggers . More trDnmt1 to affect gene expression [MBD2/3) is often found [P. elegans. Also, indication of the presence of a TET enzyme (methylcytosine oxygenase) is found from the P. elegans transcriptome. TETs are suggested to be involved in demethylation  and regulation of DNA methylation, at least in mammals [TET orthologue is found in many invertebrate species, and a recent study has shown that the single TET orthologue found in honeybee A. mellifera has oxidation activity and is capable of converting methylated cytosine to 5hmC [P. elegans transcriptome data, suggesting that some crucial elements of DNA methylation machinery are present and that this species is capable of DNA methylation. Given these findings, it is possible that DNA methylation could be one mechanism for epigenetic regulation of gene expression in this species.Methyl-CpG binding proteins (MBDs) recognize and bind to methylated CpG sites and affect gene expression by blocking the binding of transcription factors or forming complexes with other proteins, such as histone modifying enzymes, to change chromatin structure and the shape of active sites , 6, 9. Mpression . Multiplpression , 39, wheen found , 44, and mammals , 41. One to 5hmC . HoweverP. elegans transcriptome also provided an estimation of DNA methylation patterns in this species. Low CpG o/e ratio indicates higher levels of consistent historical methylation of germline leading to depletion of CpG over time due to the higher mutation vulnerability (deamination to thymines) of methylated cytosines [D. melanogaster and Anopheles gambiae genomes show high CpG o/e ratios close to 1, indicating no CpG depletion [P. elegans, the distribution of CpG o/e in the predicted ORFs was unimodal  with a mean value much lower than 1, a signature of CpG site methylation leading to depletion of CpG sites in the genome of this species.An analysis of observed/expected ratios of CpG sites in the ytosines , 43, 44.Apis mellifera, a bimodal distribution of CpG o/e ratios has been observed [C. gigas: 0.70 & 0.40 [B. glabrata: 0.62 & 0.21 [A. mellifera studies and other similar analyses of CpG o/e ratios in addition to bisulfite sequencing of other invertebrate species have shown that housekeeping genes and conserved proteins with constant expression are commonly found in the group of genes with low CpG o/e ratio, indicating depletion of CpG sites  [P. elegans: ORF containing contigs divided into groups based on discrete biological processes did not significantly differ in CpG o/e ratios, possibly due to the lack of bimodality in the distribution curve.In the honeybee, observed , 59, wit0 & 0.40  and B. g2 & 0.21 , both wiylation) , 44, 59.ylation) . Additioylation) , 43, 59.ylation) , 56. We P. elegans would reveal more directly the degree to which different genes are methylated.We did not find many significant differences in CpG o/e ratios in the transcriptomes of gravid females producing different types of larvae, however, in most gene categories, females producing planktonic larvae were expressing transcripts with higher CpG o/e ratio (indicating gene transcripts with lower germline methylation). This is interesting, since the MS-AFLP analysis indicated similar overall levels of methylation in the genomic DNA of both types of gravid females (those producing planktonic larvae and those producing benthic larvae). However, differences in the methylation profiles between the types were also observed, and this could explain the different pattern seen at the gene expression level (different genes or transcripts with different CpG content were expressed). In the future, it would be interesting to compare the different gravid females by comparing differentially expressed genes only, to clarify if genes associated with developmental mode differences also differ in CpG o/e ratios and methylation levels. Epigenetic regulation of differentially expressed genes might allow for higher phenotypic plasticity if epigenetic patterns are changed (transiently) in response to external factors . AdditioP. elegans genome, as has been observed for some other marine invertebrates. However, more studies are needed, for example some genes involved in the methylation pathway may not have been captured by our transcriptomic data set, and these could be identified if genome data becomes available. Also, other epigenetic changes not studied here, such as histone modifications or microRNAs could be used in epigenetic regulation of gene expression in this species. In addition, we found differences in CpG site methylation patterns between most of our samples: MS-AFLP method showed significant differences between methylation profiles of females producing larvae destined to different developmental modes, and gravid females from Herslev producing benthic offspring show a different methylation profile in comparison to non-reproductive females from the same population. Additionally, CpG o/e ratios of expressed genes give indications that gravid females producing the different larval types are expressing transcripts with differences in their CpG site content (i.e. differences in gene expression profiles are suggested). These indications of differences in DNA methylation patterns suggest a potential for epigenetic regulation of developmental mode.To conclude, we found that intragenic CpG site DNA methylation and some of the genes of methylation machinery are present in the S1 File(PDF)Click here for additional data file.S2 FileThis file contains detailed results of the blast searches and conserved domains search for the genes involved in DNA methylation machinery.(XLSX)Click here for additional data file.


CYP19A1 single-nucleotide polymorphisms (SNPs) are associated with clinical response (CR) and adverse effects (AEs) among BC patients treated with AIs. The aim of this study was to analyze the association between CYP19A1 polymorphisms and AI treatment in BC patients.Many clinical trials have shown the efficacy of aromatase inhibitors (AIs) in the management of breast cancer (BC). There is growing evidence that CYP19A1 variants and treatment response among BC patients.A systematic review was performed in MEDLINE, EMBASE, and LILACS. A meta-analysis was conducted to compare the association between P = 0.002). Seven studies analyzed the association between AEs with different polymorphisms of CYP19A1. Although there was a statistically significant association with musculoskeletal adverse events  and with vasomotor symptoms  in individual studies, similar associations were not observed in further studies. No statistically significant association between musculoskeletal AEs and SNPs rs4646, rs10046, rs727479, and rs1062033 was found.A total of 12 studies were included in the final analysis. There was significant variation among the populations studied and the SNPs and outcomes investigated. A meta-analysis was only possible for the evaluation of SNP rs4646 vs. the wild-type variant with respect to time to progression (TTP) among metastatic BC patients treated with AI. TTP was significantly increased in patients with the rs4646 variant compared with the wild-type gene (hazard ratio (HR) = 0.51 , CYP19A1 polymorphisms on clinical outcomes were most often detected in individual studies, suggesting that longer-term studies will better clarify these associations. Additional studies are needed to clarify the predictive value of other SNPs and whether CYP19A1 genotyping should be used to guide AI treatment.These findings suggest that the presence of the rs4646 variant may be a predictive factor of the benefit of AI treatment for BC. The effects of  Breast cancer (BC) is the most common cancer among women, and it accounts for the majority of cancer-related deaths among women worldwide, representing 23 % of all cancer diagnoses and 14 % of cancer-related deaths. In developing countries, BC has also replaced cervical cancer as the current leading cause of cancer deaths among women ; P = 0.044). In addition, for rs7176005, the homozygous variant genotypes (TT) were associated with a higher odds of VMS . Finally, the rs16964189 SNP was associated with the occurrence of VMSs for the homozygous TT genotype . Park et al. ; P = 0.007. However, this association was not found in any other of the 29 SNPs analyzed. Furthermore, Henry et al. [CYP19A1 SNPs) in 432 BC patients and observed a non-significant increase in MS-AEs when at least eight rs60271534 repeat alleles were present . No other associations were identified.In relation to treatment-related MS-AEs, Liu et al.  found thy et al.  analyzedP = 0.01), but no other significant associations were reported. Finally, Mao et al. [P = 0.57), rs727479 (P = 0.94), rs10046 (P = 0.20), or rs11575899 (P = 0.80). For SNP rs60271534, subjects with at least one TTTA 7-repeat allele had a non-significant 1.7-fold increase in odds of AIAA  after correcting for multiple testing, whereas patients with at least one TTTA 8-repeat allele had a lower risk of aromatase inhibitor-associated arthralgia (AIAA) . In addition, Napoli et al. [P = 0.03) and hip (P = 0.03) compared with other genotypes (AG + GG).Park et al.  describeo et al.  analyzedi et al.  analyzedCYP19A1 genotypes and clinical outcomes following treatment with AIs in BC patients. A total of 12 studies were included in this systematic review. To our knowledge, this is the first systematic review and meta-analysis published on the association between CYP19A1 polymorphisms and AI treatment in BC patients.There is growing evidence that polymorphic gene variants can contribute to differences in complex traits between individuals, and the assessment of genes involved in drug metabolism will provide valuable information for treatment planning. Although AI efficacy among BC patients has been proven, there is significant variability in response rate and AE frequency. Therefore, tests that are able to predict treatment response and prognosis would be valuable in the management of these patients. We investigated evidence of the association between TTP was analyzed as an outcome in four studies , 24, 40.P = 0.007), but the same was not observed for SNP rs10046 (P >0.05). In contrast, Miron et al. [P >0.05) but did identify an association between OS and rs10046 (P = 0.003). These conflicting data suggest that CYP19A1 genotypes may be associated with OS in BC patients treated with AIs. However, the magnitude of this association appears highly variable between patients.Liu et al.  reportedn et al.  found noP = 0.011), TTF and PFS (no significance association with any SNP) were quite limited in terms of number and heterogeneity of studies, but the overall lack of effect observed may indicate that there are in fact no differences or that the follow-up times used were insufficient to detect relapse/recurrence.The variations in the definitions of DFS, PFS and TTF among studies limited pooling in our analysis. The results involving DFS  that could influence the outcomes analyzed.Furthermore, it is important to mention that this systematic review/meta-analysis might have some publication bias, because we prioritized the inclusion of English publications. Moreover, it is well known that often studies that find negative results, i.e., with no statistically significant correlation between genetic polymorphisms and clinical outcomes, are more rarely published, which may cause a possible limitation in this kind of study. This meta-analysis did not use single patient data, which although more challenging, may have certain advantages over aggregate-level analyses . FurtherCYP19A1 polymorphisms and clinical outcomes and AEs in BC patients receiving AIs. The effects of CYP19A1 polymorphisms on clinical outcomes were most often detected in individual studies, suggesting that longer-term studies will better clarify these associations. Results, such as the association of SNP rs4646 with longer TTP as well as the association of rs934635 and M_5_3 haplotype with VMS and the genetic variants M_3_5, rs700518 and rs60271534 with osteoarticular symptoms, are significant outcomes indicating the impact of these variants on treatment with AIs in BC. Hence, CYP19A1 polymorphisms are potentially useful biomarkers for predicting prognosis and AE profiles in BC patients and may become a promising tool to aid physicians in making therapeutic decisions in the future, although the exact role of CYP19A1 has yet to be clarified when taking the different clinical settings and disease stagings into account. Additional studies must be conducted with larger sample sizes, more homogeneous patient populations , and using standardized outcomes and genotyping strategies to allow for a comprehensive understanding of these associations.This systematic review of the literature revealed associations between


This study was oriented to investigate the benefit of anti-Müllerian hormone (AMH) level in the management of polycystic ovary syndrome (PCOS). To assess the impact of metformin and oral contraceptives (OC) on serum AMH levels in a cohort of adolescents with PCOS.Forty-nine adolescents with PCOS were recruited to the study. Twenty-nine patients without insulin resistance were treated with OC (group 1), and 20 patients with insulin resistance were treated with metformin and OC (group 2). AMH and androgen levels were measured prior to and 6 months after the initiation of treatment.AMH levels were significantly decreased with treatment in both group 1 (p=0.006) and group 2 (p=0.0048). There was a significant correlation between pre- and post-treatment AMH and left ovarian volume .This study investigated two different treatment regimens in adolescents with PCOS and revealed that AMH levels decreased with treatment. AMH levels were correlated with ovarian volume. Anti-Müllerian hormone (AMH) levels decreased with treatment in adult patients with polycystic ovary syndrome (PCOS).This study revealed that AMH levels decreased with treatment in adolescent patients with PCOS.Polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age . PCOS isAMH is produced in the granulosa cells of early developing follicles. AMH expression in the ovary starts at the end of the third trimester of pregnancy, although its levels at birth can be almost undetectable. Serum levels of AMH increase after puberty, probably as a result of follicular growth, and it remains detectable until the end of ovarian activity. Serum levels of AMH were higher in patients with PCOS than in women with normal cycles in several studies ,4. This The objective of this study was to evaluate whether treatment with OC and OC plus metformin would reduce AMH levels in adolescents with PCOS.The study was conducted with 49 adolescent patients (aged 13-17.5 years) who were being followed-up for PCOS in Göztepe Education and Research Hospital, Pediatric Endocrinology Clinic patients who had been recently diagnosed as having PCOS were divided into two groups, those receiving OCs (group 1) and those receiving OCs plus metformin (group 2), and their treatments were evaluated. G power 3.1.4 was used for the sample size, which was calculated as 54 to give a power of 0.95 (1-β probability of error), effect size f: 0.5, and α probability of error: 0.05.Thirty patients were recruited for each group at the start of the study but 9 were excluded due to treatment incompliance, and 2 due to failure to report for follow-up visits. Thus, group 1 included only 29 patients and group 2 comprised 20. This was planned as a two-stage prospective study; the study compared AMH, androgen levels, and pelvic ultrasonography (USG) before and after treatment, as well as between the two groups.PCOS diagnosis was based on the Rotterdam  criteriaApproval from the hospital’s Scientific Research Evaluation Commission  and written consents from the patients and their relatives were received for the study.Patients who presented to our clinic who had previously been diagnosed as having PCOS were measured pre-treatment and at least 6 months post-treatment (Pt) for AMH, total testosterone, 1,4 androstenedione (AS), dehydroepiandrostenedione sulfate (DHEAS), sex-hormone binding globulin (SHBG), insulin, glucose, prolactin (PRL), luteinizing hormone (LH), and follicle stimulating hormone (FSH) levels. Each patient was evaluated for age, menarche age, body weight, height, body mass index (BMI), hirsutism and menstruation disorders. From these measurements, BMI was calculated using the body weight (kg)/height (m2) formula. Based on the resulting BMI, patients with values above the 95th percentile of the age- and sex-matched curve were considered as obese in accordance with Bundak et al’s  to investigate IR and to establish whether the patient had type 2 diabetes mellitus (DM). The test was performed by administering 1.75 g/kg (75 g) oral glucose following a 12-hour overnight fast.OGTT-based diagnoses of IR, glucose tolerance, and type 2 DM were established based on the American Diabetes Association (ADA) criteria . PatientLH (IU/L), FSH (IU/L), total testosterone , DHEAS , insulin, and PRL were analyzed at the central laboratory of our hospital using an immunoenzymatic method . AS (ng/mL), 17-OHP (ng/mL) were studied using radioimmunoassay with the Immunotech (Beckman Coulter) kit and an ICN ISO DATA Gamma Counter, SHBG (nmol/L) was studied using a chemiluminescence assay with a Siemens kit and Siemens Immulite 2000XPI analyzer at the Gelişim Laboratory.AMH (ng/mL) was studied by the Gelişim Laboratory using enzyme-linked immunosorbent assay (ELISA) with an Immunotech Gen II (A73818) Beckman Coulter kit and a Pasteur ELISA reader. The minimum sensitivity of the kit was 0.08 ng/mL. The intra-assay variability coefficient was 5.4% for 4.42 ng/mL and 3.6% for 14.03 ng/mL, and the inter-assay variability coefficient was 5.6% for 4.42 ng/mL and 4.5% for 14.03 ng/mL.The results were transferred to ‘Statistical Package for the Social Sciences 15.0 for Windows’ software, which was used for the descriptive analyses of the study group. The Shapiro-Wilk test was used to establish whether the data were normally distributed. Insulin, Pt-insulin, Pt-LH, Pt-FSH, Pt-E2, Pt-SHBG, Pt-right ovary, and Pt-left ovary without normal distribution were expressed as median (IQR) and other data with normal distribution were expressed as mean ± standard deviation values.The pre- and Pt data differences between groups 1 and 2 were studied using the independent samples t-test for data with normal distribution and Mann-Whitney U test for data without normal distribution.The differences between pre- and Pt measurements of patients in groups 1 and 2 were calculated separately using the Paired Sample test for data with normal distribution and Wilcoxon’s signed-rank test for data without normal distribution. The difference in AMH values before and after treatment was designated as delta AMH. Correlations between the data were calculated using Spearman’s correlation (rho and p provided). The results were expressed with 95% confidence interval and statistical significance was set at p<0.05.The clinical characteristics of the study population are summarized in Patients’ pre- and Pt AMH and other androgen levels were compared between the groups , and sigAMH serum levels were significantly decreased in group 1 from 6.7±3.7 ng/mL at baseline to 4.3±3.1 ng/mL after 6 months of treatment (p=0.006). In group 2, AMH serum levels ranged from 4.7±3.07 ng/mL at baseline to 3.1±2.05 ng/mL after 6 months of treatment (p=0.048).Delta AMH was used to determine whether AMH reductions were significant between the groups; no significant difference was found . SHBG increased significantly in both groups. TT levels decreased in both groups but without statistical significance. AS decreased significantly in group 2 (p=0.02).Evaluation of correlations between AMH, androgens, insulin, FGS and ovarian volumes did not yield any correlations between AMH, FGS, and androgens. There was only a significant correlation between AMH and left ovarian volume  . DHEAS wEvaluation of correlations between Pt FGS, AMH, androgens, insulin, and ovarian volume demonstrated a correlation between AMH and left ovarian volume . There was no correlation between Pt AMH, FGS, and androgens.This study aimed to investigate whether AMH was a good indicator for treatment and monitoring of adolescent patients with PCOS. For this purpose, levels of AMH and other androgens were studied in patients who were newly diagnosed as having PCOS, after which the patients were divided into two groups to investigate changes in AMH levels in groups and treated with OC or OC plus metformin.Several studies have demonstrated increased levels of AMH in adult women and adolescent girls with PCOS ,14,15,16Studies on the relationship of AMH with insulin in PCOS are contradictory. Some studies found no correlation between insulin and AMH levels , whereasThe relationship between AMH and ovarian volume has been investigated in several studies because attempts are being made to ascribe AMH elevations in patients with PCOS to increased numbers of small follicles. A positive relation between AMH and mean ovarian volume was found in the study of Li et al . HealthyPiltonen et al  were theWe did not measure free testosterone because free testosterone measurements are not very reliable, thus we preferred to measure total testosterone. TT levels decreased in both groups but without statistical significance. In our study, we found no correlation between AMH level and FGS or testosterone levels. In group 1, where FGS was not statistically significant, there was a decline on AMH and ovarian volumes. This finding indicates that the decline in AMH was independent of hyperandrogenism and was mostly related with ovarian volumes. Rosenfield et al  showed tOne of the weaknesses of our study was that it did not incorporate a patient group treated with metformin alone because it was not possible to distinguish which agent was more effective in reducing AMH levels in group 2, although it is likely that AMH levels were decreased with OC only. A second weakness is the lack of a control group. This was because the approval of the local ethics board could not be obtained for healthy controls. Another limitation of our study was the inadequate number of patients.In conclusion, the present study demonstrated that treatment reduced AMH levels in adolescents with PCOS and it was not associated with hyperandrogenism. AMH correlated with ovarian volume and both AMH and ovarian volume decreased after treatment. We think that AMH could be used instead of transabdominal pelvic USG, independent of hyperandrogenism. AMH seems to be a good parameter for monitoring adolescent patients with PCOS because it is easy to measure at any period during the cycle. However, in the absence of adequate relevant studies, larger studies are needed.Ethics Committee Approval: Medeniyet University Göztepe Training and Research Hospital ,Informed Consent: It was taken.Peer-review: External peer-reviewed.