About 3885 women are diagnosed with breast cancer and 1285 die from the disease each year in Bulgaria. However no genetic testing to identify the mutations in high-risk families has been provided so far.
We evaluated 200 Bulgarian women with primary invasive breast cancer and with personal/ family history of breast cancer for the presence of unequivocally damaging germline mutations in BRCA1/2 using Sanger sequencing.
Of the 200 patients, 39 (19.5 %) carried a disease predisposing mutation, including 28 (14 %) with a BRCA1 mutation and 11 (5.5 %) with a BRCA2 mutation. At BRCA1, 6 different mutations were identified, including 2 frameshifts, 1 nonsense and 1 missense that had been previously reported (c.5030_5033delCTAA, c.5263_5264insC, c.4603G > T, c.181 T > G), and 2 frameshifts, which were novel to this study (c.464delA, c.5397_5403delCCCTTGG). At BRCA2, 7 different frameshift mutations were identified, including 5 previously reported (5851_5854delAGTT, c.5946delT, c.5718_5719delCT, c.7910_7914delCCTTT,c.9098_9099insA) and 2 novel (c.8532_8533delAA, c.9682delA).
A BRCA1 mutation was found in 18.4 % of women diagnosed with breast cancer at/or under the age of 40 compared to 11.2 % of women diagnosed at a later age; a BRCA2 mutation was found in 4 % of women diagnosed at/or under the age of 40 compared to 6.5 % of women diagnosed at a later age. A mutation was present in 26.8 % patients with a positive family history and in 14.4 % of women with a negative family history.
The most prevalent mutation observed in 22 patients (11 %) was BRCA1 c.5263_5264insC, a known Slavic mutation with founder effect in Eastern European and AJ communities. Other recurrent mutations were BRCA2 c.9098-9099insA (2 %), BRCA1 c.181T > G (1 %) and BRCA2 c.5851_5854delAGTT (1 %). Notably, BRCA1 c.5263_5264insC represented 56 % of all mutations identified in this series. Of the 22 patients with BRCA1 c.5263_5264insC, 9 were diagnosed with early onset breast cancer, 11 with TNBCs, 4 with bilateral breast cancer, and 6 with both breast and ovarian cancer.
This is the first comprehensive study of the BRCA1/2 mutation spectrum in Bulgaria and will assist the establishment of efficient protocols for genetic testing and individualized risk assessment for Bulgarian breast/ovarian cancer patients and healthy individuals at a high-risk.
Breast cancer (BC) is the second most common cancer in the world and, by far, the most frequent malignant disease among women with an estimated 1.67 million new cases diagnosed in 2012 (25 % of all cancer cases) . Despite the advancement of diagnostic techniques and treatment in the last decade, BC is still the most frequent cause of cancer death in women in less developed regions (324.000 deaths, 14.3 % of total) and the second cause of cancer death in more developed regions (198.000 deaths, 15.4 %) after lung cancer .
The etiology of BC is multifactorial and includes both environmental and genetic factors, as well as genetic and epigenetic changes during progression. Up to 5–10 % of all BC cases and 10–15 % of all ovarian cancer (OC) cases are due to germline mutations in one of the two breast cancer susceptibility genes, BRCA1 [MIM#113705] and BRCA2 [MIM#600185] [2–4].
Germline mutations in BRCA1 or BRCA2 explain about 50 % of disease aggregation in severely affected BC and OC families while their prevalence is lower among BC and OC patients unselected for family history or age of diagnosis: BRCA1 mutations are found in <1–7 % cases and BRCA2 mutations in 1-3 % cases . However, as high a proportion as 84 % has been proposed for certain families . Higher prevalence is associated with a family history of BC and/or OC, early onset, male BC or multiple tumours such as bilateral breast cancer (BBC) or BC and OC in the same patient . Furthermore, some histopathologic features such as lack of expression of estrogen, progesterone, and HER2 that define the biologically aggressive and difficult to treat Triple Negative Breast Cancers (TNBC) subtype, have been also attributed to deleterious changes mainly in BRCA1 .
Pathogenic mutations in the BRCA1 and BRCA2 genes confer high risks of breast, ovarian, and contralateral BC. In a recent study the average cumulative risks by age 70 years for BRCA1 carriers were estimated to be 60 % (95 % confidence interval [CI] = 44 to 75 %) for BC, 59 % (95 % CI = 43 to 76 %) for OC, and 83 % (95 % CI = 69 to 94 %) for contralateral BC . For BRCA2 carriers, the corresponding risks were 55 % (95 % CI = 41 to 70 %) for BC, 16.5 % (95 % CI = 7.5 to 34 %) for OC, and 62 % (95 % CI = 44 % to 79.5 %) for contralateral BC .
The prevalence of BRCA1 and BRCA2 mutations varies between ethnic groups and geographical areas . At present, more than 1600 mutations in BRCA1 and more than 1900 mutations in BRCA2 have been described . The spectrum of disease-associated BRCA1 and BRCA2 alleles includes frameshift, nonsense, and missense mutations altering protein function, splice mutations leading to truncation, as well as large genomic rearrangements [11, 12]. The majority of germline mutations identified in BRCA1 and BRCA2 are "private" or family-specific . However, several examples of founder mutations have been described in certain geographical areas and ethnic communities where the BRCA1 and BRCA2 mutational spectra are limited to a few founders . In fact, founder mutations have been described in Ashkenazi Jews (AJ), Icelandic and Finnish populations, in certain Dutch and French-Canadian communities, and in countries like Turkey, Pakistan, India, etc. [12–14].
Identification of BRCA1/2 mutation carriers allows nondirective clinical decisions to be made in the management of high life time risk of BC/OC including follow-up, prophylactic mastectomy and salpingooophorectomy . Furthermore, mutations in BRCA1/2 have been shown to be predictive of good response to certain treatments, such as cisplatin and Poly (ADP)-Ribose Polymerase (PARP) inhibitors .
According to the National Cancer Registry, BC is the most common female malignancy in Bulgaria . Around 3885 women are diagnosed with BC and 1285 die from the disease each year . The first study of 20 Bulgarian familial BC patients was performed in 1998 aiming to develop a screening approach for the BRCA1 gene . No mutations but only benign polymorphisms were identified, most likely due to the small sample size, the low stringency of the selection criteria and the lack of complete analysis of BRCA2. Up to date no other surveys on BRCA1/2 mutations in Bulgarian patients with familial BC/OC have been published.
The lack of sufficient genetic studies for inherited mutations in BRCA1/2 genes in the Bulgarian population impedes the introduction of an effective mutation screening that would identify the individuals at high risk in BC/OC families. The aim of the present work was to conduct a genetic analysis for BRCA1/2 germline mutations in a cohort of 200 Bulgarian women with BC, fulfilling the recognized international criteria [17, 18]. Direct sequencing of all coding exons and intron-exon junctions of both genes was performed. This is the first comprehensive study in Bulgaria aiming to ascertain the contribution of BRCA1/2 germline mutations to familial BC in the Bulgarian population.
In the present study 200 unrelated female patients with primary BC, were selected from the Departments of Surgery and General and Clinical Pathology, University Hospital, "Alexandrovska", Medical University of Sofia, and the Clinic of Medical Oncology, Specialized Hospital for Active Treatment in Oncology, Sofia, during their treatment and follow up procedures for the period of 2007–2012. All patients were of Bulgarian ethnicity, except one of Jewish origin. In personal interviews all clinical information, histopathology reports and family history were obtained from the patients. Their clinical features are summarized in Table 1. Sixty-one firstdegree relatives (8 diagnosed with BC/OC and 53 healthy) of 30 probands possessing a strong family history of BC/OC have been also recruited. They were considered for testing of possibly inherited damaging mutations. The cancer diagnosis of the affected relatives was verified by their personal clinical records.
Probands were selected for BRCA1/2 genetic testing according to their age of diagnosis, family history and tumour characteristics following the recognized Breast Cancer Linkage Consortium (BCLC) and National Comprehensive Cancer Network (NCCN) Criteria, summarized in Additional file 1: Table S1 [17, 18].
The distribution by criteria was as follows: 61 % (n = 122) fulfilled the BCLC criteria, 25 % (n = 50) - the NCCN criteria and 14 % (n = 28) were TNBC. The predominant was the group with early onset (38 %, n = 76), followed by the group with family history of BC (37 %, n = 73) in at least one first or second-degree relative diagnosed under the age of 60 (Table 1). Fifteen % (n = 30) of the patients were diagnosed with BBC, 6 % (n = 12) had a personal history of OC, whereas 5 % (n = 11) – family history of OC in at least one first or second-degree relative diagnosed at any age (Table 1). In addition to the TNBC group (14 %, n = 28) (Table 1), 14 % (24/172) of the patients selected by the BCLC and 2 NCCN criteria were also TNBC.
All deleterious mutations and some of the variants with unknown clinical significance (VUSs) found in the patients were validated in a control group of 96 healthy women matched by age and ethnicity to the patients, without family history of BC/OC. Only VUSs associated with BC in previous studies were screened in the control group.
The control group was collected through the clinical specialists participating in the study and included women undergoing regular prophylactic breast examinations in the University Hospital "Alexandrovska", Medical University of Sofia, and Specialized Hospital for Active Treatment in Oncology, Sofia. They were defined as healthy based on the results of their last breast examinations signed by breast specialists and in addition were screened for lack of family history of BC/OC.
The study was approved by the Ethics Committee, Medical University of Sofia. All persons confirmed their agreement for participation upon signing an informed consent and donated 10 ml of blood for genetic analysis. The participants have also given consent for publishing their genetic results and relevant clinical data anonymously under a specific ID code assigned.
Total genomic DNA was isolated from peripheral blood using a Chemagic Magnetic Separation Module and the Chemagic DNA Blood kits according to the manufacturer's recommendations (Chemagen, Perkin Elmer).
In order to amplify all coding sequences and exon-intron junctions of the BRCA1/2 genes, we have used a set of 81 primer pairs (33 for BRCA1 and 48 for BRCA2) selected from the BIC database  or designed by ExonPrimer software  (Additional file 2: Table S2). PCR reactions were performed in 1X PCR buffer (Invitrogen), 0.4 mM of each dNTP, 1.5 mM MgCl2, 0.4 pmol of each primer and 1U of Taq DNA polymerase (Invitrogen) with 50 ng of genomic DNA in a final volume of 10 μl. Amplification conditions were identical for all amplicons of both genes, ranging in size from 190 to 720 bp, excluding the specific hybridization temperatures of the primer pairs given in Additional file 2: Table S2 initial denaturation at 94 °C (5 min); followed by 35 cycles of 94 °C (45 s), 51 °C - 63 °C (35 s), 72 °C (30 s) and 72 °C (10 min).
The mutation screening of BRCA1/2 genes was performed using Sanger sequencing. PCR products were purified with ExoSapIT (Affymetrix) according to the manufacturer's instructions – equal amounts of ExoSap and dH2O (0.4 μl each) were added to 1 μl of PCR product and incubated in a cycler for 30 min at 37 °C, followed by 15 min at 80 °C. Sequencing reactions were carried out with Big Dye® Terminator kit v3.1 (Life Technologies) in a final volume of 10 μl under the following conditions: 96 °C (5 min), 96 °C (20 s), 55 °C (20 s), 60 °C (2 min), 60 °C (5 min).
The fragments were analysed by automated Genetic Analyser ABI 3130xl (Applied Biosystems). Obtained sequences were examined for the presence of mutations by alignment with reference DNA sequence (GenBank U14680 for BRCA1; GenBank U43746 for BRCA2) using BLAST  and SeqScape TM v 2.0 (Applied Biosystems, USA) software packages. Any mutation found was confirmed in a second PCR reaction followed by sequencing in both forward and reverse directions.
Potential structural and functional effect of the missense VUSs was predicted by the following online tools: PolyPhen 2 (http://genetics.bwh.harvard.edu/pph/), SIFT (http://sift.jcvi.org/) and PROVEAN (http://provean.jcvi.org/index.php) [21–23].
In the current study 200 Bulgarian patients selected by the established genetic testing criteria were screened for mutations in the BRCA1/2 genes by direct sequencing. The mean age of the patients at diagnosis was 49.5 (25–74) years. Thirty eight percent (n = 76) of the patients were under 40, 49.5 % (n = 99) between 41 and 60, and 12.5 % (n = 25) between 61 and 80 years of age (Table 1).
Upon direct sequencing, 13 different damaging mutations were identified, 6 in BRCA1 (four frameshift, one nonsense, one missense) and 7 frameshift in BRCA2 (Table 2). Of those 84.61 % (11/13) were frameshift mutations seen in 18 % (36/200) of the patients. None of the pathogenic mutations was found in the healthy controls.
In the BRCA1 gene we have identified six unequivocally deleterious mutations of which four frameshift, one nonsense and one missense (Table 2). Among the frameshift mutations two: c.5030_5033delCTAA and c.5263_5264insC with frequencies of 0.5 % and 11 %, respectively, had been previously reported and two, namely c.464delA and c.5397_5403delCCCTTGG with frequencies of 0.5 % each were novel (Table 2). The mutation c.464delA, located in exon 8, was detected in a patient BC134 diagnosed with TNBC at the age of 54 with three cases of BC in her pedigree (Table 3). The second novel mutation c.5397_5403delCCCTTGG in exon 22 was also found in an individual with TNBC (BC205) diagnosed at the age of 51 with family history of BC and PC (Table 3).
The deletion c.5030_5033delCTAA in BRCA1 exon 17 was observed in one patient (BC194) without family history diagnosed with TNBC at the age of 63 (Table 3). Twenty-two of the patients (11 %) harboured the mutation c.5263_5264insC in BRCA1 exon 20 (Table 3). Of those six (BC6, BC21, BC99, BC143, BC152 and BC171) had developed both BC and OC, four were diagnosed with BBC (BC7, BC39, BC152 and BC204). Eleven of the cases were TNBC (BC73, BC111, BC121, BC142, BC143, BC155, BC161, BC164, BC171, BC175 and BC190) of which 5 (BC111, BC121, BC142, BC155 and BC190) developed the disease before the age of 40. Altogether in 9 of the patients, the c.5263_5264insC mutation correlated with early onset (BC3, BC39, BC140, BC111, BC121, BC142, BC155, BC190 and BC204).
In addition to the listed above indels we found two deleterious point mutations that had previously been reported (Table 2): one nonsense (c.4603G > T) with a frequency of 0.5 % and one missense (c.181T > G) with a frequency of 1 %. The mutation c.4603G > T was carried by a patient BC37 with early onset (Table 3) while c.181T > G was detected in two patients (BC28 and BC132), with early onset and BBC, respectively (Table 3).
Seven damaging frameshift mutations were found in BRCA2 (Table 2), of which five had previously been reported: c.5851_5854delAGTT, c.5946delT, c.5718_5719delCT, c.7910_7914delCCTTT, c.9098_9099insA; and two novel: c.8532_8533delAA and c.9682delA. The most frequent BRCA2 frameshift mutation c.9098_9099insA, located in exon 23, was observed in four patients (BC32, BC81, BC85 and BC88) with familial BC (2 %). In addition one of them (BC88) had TNBC (Table 4).
The second in frequency BRCA2 frameshift mutation c.5851_5854delAGTT, located in exon 11, was found in two patients (1 %): BC76 with a family history of BC and stomach cancer, diagnosed with TNBC at the age of 53, and BC58 with family history of BC and CRC, diagnosed with BC at the age of 48 (Table 4).
The rest of the BRCA2 frameshift mutations were seen only once in our study with a frequency of 0.5 % each (Table 2). Two deletions in exon 11: c.5718_5719delCT and c.5946delT were observed respectively in a patient BC90 with early onset (35 years) and in a Jewish patient BC52 with family history of BC, diagnosed with both BBC (at the age of 41/68) and OC (at the age of 59) (Table 4).
The deletion c.7910_7914delCCTTT, located in exon 17, was present in a patient BC19 with early onset, BBC (at the age of 37/41) and family history of BC/OC (Table 4).
One of the novel BRCA2 deletions c.8532_8533delAA, located in exon 20, was found in a patient BC87 with BBC and early onset (at the age of 30/37). Interestingly, the second new BRCA2 deletion c.9682delA, located in exon 27, was also observed in a patient (BC228) with BBC (Table 4).
Additional 50 sequence variants were identified at BRCA1 and BRCA2, with either benign or unknown pathogenic effect. All of them were named sequence variants due to inconsistency in their classification in the databases. The spectrum of unclassified variants included missense, synonymous and intronic variants, as well as one inframe deletion (Table 5).
In total 24 missense VUSs were identified, of which 14 in BRCA1and 10 in the BRCA2 (Table 5). According to their minor allele frequencies (MAFs) in the patients they can be divided into two groups: rare variants with MAFs between 0.0025 and 0.005, and frequent variants with MAFs > 0.005. We have compared the MAFs of the unclassified missense variants found among patients with other Europeans  and with healthy controls (only for the variants associated with BC in previous studies). The studied variants in the control group included VUSs in exons 5 (c.139 T > G); 7 (c.441 + 36_441 + 38delCTT); 8 (c.536A > G, c.442-34C > T); 9 (c.591C > T, c.548-58delT); 11 (c.1067A > G, c.1648A > C, c.2077G > A, c.2612C > T, c.3113A > G, c.3119G > A, c.3548A > G, c.3999 T > C, c.2082C > T); 16 (c.4837A > G, c.4956G > A); and 20 (c.5277 + 56delGTA) of BRCA1 and the variants in exons 11 (c.3515C > T, c.5744C > T); 17 (c.7806-14 T > C); and 23 (c.9104A > C) of BRCA2 (Table 5). Variants for which no information for association with BC was found in the databases were not screened in the healthy controls but were included in Table 5. In addition in silico analysis was conducted in order to predict potential deleterious effect of all detected missense VUSs (Table 6).
Six rare BRCA1 missense variants were seen in patients (Table 5): c.139 T > G, c.536A > G, c.736 T > G, c.1456 T > C, c.1648A > C and c.3999 T > C. Of those c.3999 T > C, causing a replacement of alanine to valine at codon1333 has never been reported. Even though c.3999 T > C was observed in a patient BC20 with BBC and early onset in the absence of other pathogenic mutations, the in silico analysis did not confirm its pathogenic effect (Table 6).
The missense variants c.139T > G had been predicted to be deleterious in previous functional studies . Inour study it was found in one patient (0.5 %) with TNBC BC10 and appeared to be clinically important according to wo prediction programs (POLYPHEN 2 and SIFT), while PROVEAN predicted it as neutral (Table 6).
One patient with family history of BC117 was the only carrier of three rare missense variants c.536A > G, c.1456T > C and c.1648A > C, which were not seen in the control group (Table 5). Another rare missense BRCA1 variant c.736T > G, listed as variant of unknown significance (VUS) in BIC  and neutral in UMD (Universal Mutation Database) , was found in patient BC30 with BBC (Table 5).
Eight missense variants were observed in BRCA1 with MAF>0.005 in both patients and controls (Table 5). They were designated as VUS in BIC and neutral in UMD databases (Table 5). Only two of them (c.1067A > G and c.3113A > G) appeared to be deleterious according to the conducted in silico analysis (Table 6).
Four synonymous and 6 intronic variants were also detected in the BRCA1 gene (Table 2). Of those c.5277 + 62_c.5277 + 64delGTA had not previously been reported and was seen in two patients (1 %) but not in the control group (Table 5). The variant c.591C > T was more frequent in patients (MAF = 0.007), but was not found in the control group and was rare in other Europeans (MAF = 0.001) (Table 5). All other variants had MAF > 0.005 in both patients and other Europeans, except c.441 + 36_441 + 38delCTT, for which data about MAF were not available in the databases (Table 2). Variants c.2082C > T, c.441 + 36_441 + 38delCTT, c.442-34C > T and c.548-58delT were also genotyped and found with high frequencies (MAF > 0.005) in the healthy controls (Table 5).
Five rare missense variants (MAF < 0.005) were identified in BRCA2 (Table 5). Three of them c.6322C > T, c.7469 T > C andc.9104A > C were classified as VUS in both BIC  and UMD  databases, while c.3515C > T and c.6101G > A were designated as VUS only in BIC (Table 5). In UMD c.3515C > T was suggested to be likely neutral but no information was available for c.6101G >A (Table 5). The in silico analysis predicted pathogenic effect of both c.3515C > T and c.6101G > A (Table 6).
We found four frequent (MAF > 0.005) missense variants in BRCA2 (Table 5). None of them was predicted to be pathogenic upon in silico analysis (Table 6). In addition we detected one in frame deletion of glutamate in exon 11- c.4146_4148delAGA (MAF = 0.0025), which was classified as VUS in both BIC  and LOVD (Leiden Open Variation Database) .
The other BRCA2 sequence variants were observed with high frequencies in patients (MAF > 0.005), similar to those found in other Europeans (Table 5). Among them seven were synonymous replacements; two variants –c.-26G > A and c.*105A > C, were localized in 5′-UTR- and 3′-UTR, respectively and 8 were intronic. One of the intronic variants c.7617 + 28C > A was novel (Table 5).
This is the first comprehensive study aiming to ascertain the contribution of BRCA1/2 germline mutations to BC development in the Bulgarian population, where 1285 women die from the disease each year . The thorough mutation screening of all coding sequences and exon-intron junctions of BRCA1/2 genes in a cohort of 200 Bulgarian women with primary invasive BC and with personal or family history of BC/OC, selected according to the recognised international criteria [17, 18] led to the identification of pathogenic mutations in 19.5 % of them (39/200).
We have found 13 unequivocally deleterious mutations of which 6 in BRCA1 and 7 in BRCA2 gene (Table 2). The predominant mutations seen in 18 % of the studied group of patients were frameshift mutations leading to truncated proteins with impaired function. In total 11 (84.6 %, 11/13) indels were seen, 4 in BRCA1 and 7 in BRCA2 The other two mutations identified were nonsense and missense mutation in BRCA1, each accounting for 7.7 % (1/13) of all disease causing mutations.
The median age of diagnosis was 46 (range 29–63 years) in the 28 BC with a BRCA1 mutation (Table 3) and 45.5 (range 30–61 years) in the 11 BC with a BRCA2 mutation (Table 4), compared with a median age of diagnosis of 49.6 years (range 25–74 years) for the 161 cases without a mutation. A BRCA1 mutation was found in 18.4 % (14 of 76) women diagnosed with BC at or under the age of 40 compared to 11.2 % (14 of 124) of women diagnosed at a later age (Table 3); a BRCA2 mutation was found in 4 % (3 of 76) women diagnosed with BC at or under the age of 40 compared to 6.5 % (8 of 124) of women diagnosed at a later age (Table 4). A mutation was present in 26.8 % (22 of 82) BC patients with a positive family history and in 14.4 % (17 of 118) of women with a negative family history.
The most prevalent mutation observed in 22 patients (11 %) was BRCA1 c.5263_5264insC, a known Slavic mutation with founder effect in Eastern European as well as AJ communities, (Table 2). This frameshift mutation is the second most frequently reported in BIC database . It has been found with various frequencies in high risk BC/OC families from Poland (34 %), Russia (14 %), Hungary (14 %), Slovenia (13 %), AJ (10 %), Greece (8 %), Germany (4 %), and Italy (3 %) . The c.5263_5264insC was not found in Spain and Portugal and it has been observed with a low frequency in the Netherlands, Belgium and Scandinavian countries . In Russia, Belarus, Poland, Latvia, Czech Republic, Greece and Lithuania it accounts for respectively 94 %, 73 %, 60 %, 55 %, 37–52 %, 46 % and 34 % of all BRCA1 mutations .
Being a founder mutation in Greece , c.5263_5264insC has been detected in other Balkan countries such as Romania, Turkey, Serbia, Croatia, and Macedonia but without a founder effect [29–33]. Apparently Bulgaria is the second Balkan country in which the most prevalent Slavic mutation c.5263_5264insC has a founder effect accounting for a very high proportion, about 76 % (22/29) from all detected BRCA1 mutations (Table 3), with high intermediate frequency (11 %) compared to the other Central and Eastern European countries .
Originally c.5263_5264insC was described as a founder mutation in AJ, but recent haplotype analysis suggested that it most likely originated in Northern Russia or possibly Scandinavia, between 1800 and 1500 years ago, and was subsequently spread to the various populations from East to West and nearly worldwide . A common ancestor for families with c.5263_5264insC mutation, reported from Europe, Brazil, North America and India is evident [34–36].
We suppose that in Bulgaria the mutation c.5263_5264insC also has a Slavic origin since the modern Bulgarian nation originated as a mixture of Slavic, Thracian and Bulgar tribes more than 1300 years ago. A detailed haplotype analysis is necessary to prove this hypothesis.
The increased risk of BC and OC in c.5263_5264insC mutation carriers has been estimated at 67 % (95 % CI, 36–83 %) and 33 % (95 % CI, 8–50 %), respectively . At a molecular level it leads to a stop codon at position 1829 and respectively to a truncated protein that lacks its C-terminal BRCT motif. In our study the presence of the c.5263_5264insC mutation in the patients was associated with early onset in 9 patients (BC3, BC39, BC140, BC111, BC121, BC142, BC155, BC190 and BC204), BBC in 4 patients (BC7, BC39, BC152 and BC204), two subsequent events of BC and OC in 6 patients (BC6, BC21, BC99, BC143, BC152 and BC171) and/or TNBC in 11 patients (BC73, BC111, BC121, BC142, BC143, BC155, BC161, BC164, BC171, BC175 and BC190) (Table 3). Several carriers were also identified among the relatives of the patients with c.5263_5264insC (one individual with OC, another with both BC and OC, as well as three healthy individuals).
The mutation c.5030_5033delCTAA located in BRCA1 exon 17 is a prevalent frameshift mutation reported in at least five French families with BC/OC and most likely originating from a common ancestor (13). It has been also detected in BC patients with early onset from USA (0.35 %, 1/282) and Taiwan (2.8 %, 1/36) [37, 38]. We observed the mutation in one patient (0.5 %, 1/200) with TNBC (BC194) diagnosed at the age of 63 (Table 4).
According to BIC database  the majority of the pathogenic mutations in BRCA1/2 are frameshift mutations (around 70 %), while nonsense and missense mutations contribute with around 10 % each. In the present study we have identified only two previously reported BRCA1 point mutations with clear pathogeniceffect:c.181T > G and c.4603G > T, located in exon 5 and 15, respectively (Table 2). The mutation c.4603G > T was reported several times in BIC as clinically important and it was found in Non-AJ patients from America and Venezuela [10, 39]. Among the studied Bulgarian patients c.4603G > T was observed only once (0.5 %) and was associated with early onset BC (BC37, Table 3).
Following the most prevalent mutation c.5263_5264insC, the missense variant c.181T > G has been recognised as the second most frequent mutation in Poland and other European countries among BC/OC families . These two founder mutations accounted for 70–90 % of the BRCA1 mutations found in the Polish population, 80 % in Hungary and 28 % in Germany, respectively . Located in the RING domain of BRCA1, c.181T > G has been found to impede the coordination of the zinc ions upon binding to the protein in functional studies .
In Austria, Slovenia, and Czech Republic the families with c.181T > G mutation represented 15 %, 18 % and 9 %, respectively, of all families carrying BRCA1 mutations . It has been also known as one of the three founder mutations in Byelorussian population with frequencies of 1 % in unselected BC families and 0.2 % in control individuals . The mutation was detected at a very low rate in our neighboring countries such as Greece, Serbia and Romania, as well as in Croatia, Austria, Slovakia, Ukraine, Latvia, Lithuania and Russia . In our studyc.181T > G was found as a disease causing mutation in two patients (1 %), with early onset (BC132) and BBC (BC28), respectively (Table 3).
We have identified two new frameshift mutations in the BRCA1 gene: c.464delA and c.5397_5403delCCCTTGG with 0.5 % frequency each (Table 2). The mutation c.464delA, located in exons 8, leads to a truncated protein, lacking its Coiled Coil and BRCT domains and was detected in a patient BC134 with TNBC diagnosed at the age of 54 with three cases of BC in her pedigree (Table 3). Three of her healthy first-degree relatives (sister, and 2 sons) were also carriers of the mutation (Additional file 3: Figure S1). The second novel mutation c.5397_5403delCCCTTGG in exon 22 residing in the BRCT motif leads to a truncated protein without C-terminal BRCA2/AD2 domain. It was found in an individual BC205 with TNBC diagnosed at the age of 51 with family history of BC (Table 3, Additional file 4: Figure S2).
The second in frequency (2 %) mutation observed in our study was the insertion c.9098_9099insA, located in exon 23 of BRCA2 gene. It has been reported as one of the most frequent BRCA1/2 mutations in Germany with a possible founder effect . Together with c.5946delT it also accounted for 50 % of all BRCA2 mutations in BC/OC families from Hungary . The consequence of this mutation is a premature stop codon at position 3042 that leads to a truncated protein losing its C terminal OB3 and NLC motifs and thus the ability to bind a DSS1 protein that regulates its repairing function (Table 2). All four carriers of the insertion c.9098_9099insA in our study (BC32, BC81, BC85 and BC88) had family history of BC, and were diagnosed with BC between 50 and 61 years of age. One of them (BC88) developed TNBC (Table 4).
Three of the observed known frameshift mutations were located in exon 11 of BRCA2 (Table 2): c.5851_5854delAGTT, c.5946delT, and c.5718_5719delCT. The deletion c.5851_5854delAGTT has been detected with a low frequency in cohorts of Italian, American Asian and Indian BC/OC patients [41, 42]. In the present investigation we have identified two (1 %) unrelated Bulgarian patients harbouring c.5851_5854delAGTT deletion. One of the patients (BC76) with a family history of BC and stomach cancer was diagnosed with TNBC at the age of 53, while the other (BC58) developed BC at the age of 48 and had a family history of BC and CRC (Table 4).
The c.5946delTwas found in one patient with Jewish origin and family history of BC (BC52), who had developed both BBC at the age of 41/68 and OC at the age of 59 (Table 4). A healthy daughter of the patient also harboured the mutation. The c.5946delT deletion has been recognised as one of the three founder mutations in AJ, together with the two BRCA1 indels c.68_69 delAG and c.5263_5264insC . The mutation c.5946delT has been also detected in non-Jewish populations such as the Hungarian BC/ OC families .
The third mutation, observed in exon 11 of BRCA2 c.5718_5719delCT, was reported several times in BIC database . It has been found with a very low frequency in patients with BC, OC and PC from Germany, UK and North America [10, 43]. The mutation was found with similar low frequency (0.5 %) in our study and correlated with early onset (BC90, Table 4).
Another recurrent mutation according to BIC database , c.7910_7914delCCTTT, located in BRCA2exon 17, was identified in a proband BC19 (0.5 %) with early onset BBC (at the age of 37/41) with family history of BC/OC (Table 4). A cousin of the patient also harboured the mutation and possessed identical clinical phenotype. Interestingly, c.7910_7914delCCTTT has been associated with BBC in other European populations as well . It was first reported in UK and was further observed with low frequencies in patients with BBC from Germany and Denmark and with a frequency of 3.6 % (1/27) among males with sporadic BC in the Polish population [44, 45].
Two novel frameshift mutations were found in BRCA2: c.8532_8533delAA and c.9682delA with frequency of 0.5 % each (Table 2). The mutation c.8532_8533delAA, located in exon 20, was found in a patient BC87 with BBC (at the age of 30 and 37) and early onset (Table 4). The patient's mother was also diagnosed with BBC and carried the mutation (Additional file 5: Figure S3). This mutation is located in the Tower domain of the protein and terminates the translation at the middle of OB2 domain as a result of which BRCA2 lacks its OB3 and NLC motifs at the C terminus. Similarly the second novel BRCA2 deletion located in exon 27 was observed in a patient BC24 with BBC (Table 4). The consequence of this mutation is a truncated protein without NLC2 and 3 motifs at the C-terminus.
In addition to the unequivocally damaging mutations, many VUSs have been reported in index cases of high-risk BC/OC families in the absence of pathogenic mutation, and their effect on the protein structure and function could not be immediately inferred . Classifying these variants of unknown clinical significance as neutral or disease causing is very important for the genetic counseling.
We have identified in total 50 VUSs in both genes: 24 in BRCA1 and 26 in BRCA2, of which 24 were missense variants (Table 5). The in silico analysis suggested a possible deleterious effect of only five missense variants with all prediction programs (c.1067A > G, c.1648A > C and c.3113A > G in BRCA1 and 3515C > T and c.6100C > T in BRCA2) (Table 6). Another two: 139 T > G and c.536A > G in BRCA1 appeared to be clinically important according to POLYPHEN 2 and SIFT only (Table 6). One new BRCA1 missense variant c.3999 T > C was observed in a patient BC20 with BBC and early onset in the absence of other pathogenic mutations, but the in silico analysis did not predict a possible pathogenic effect (Table 6).
The variant c.139 T > G that appeared to be clinically important upon in silico analysis is colocalized with the missense mutation c.181T > G in the RING finger domain of BRCA1 (exon 5) and has been recognized as one of the zinc-coordinating residues of the protein . It was reported once in BIC database as a VUS . Up to date there is no information available about its allele frequencies in different populations. Several functional studies have suggested a pathogenic effect of c.139T > G since in cell culture experiments it caused disability in coordination of the zinc ions in the RING domain and abolished the ubiquitin ligase activity of BRCA1 protein as well as its participation in the homologous recombination and the control of the centrosome number . In our study the missense variantc.139T > G was found in one patient (0.5 %) with TNBC and early onset of the disease BC10, in the absence of other pathogenic mutation. However in order to ascertain its pathogenicity, further analysis of large genomic rearrangements in BRCA1/2 genes, as well as segregation analysis in the family of the patient need to be performed.
For all other missense variants that were predicted pathogenic by the in silico analysis (Table 6), the existing information in the literature has been controversial. For example, in some studies no association with BC has been found for the BRCA1 VUS c.1067A > G (Q356R), although the authors claimed that being homozygous for 356R might protect against BC . According to other prediction models it demonstrated a possible harmful role . Similar results have been obtained for the c.536A > G, c.1648A > C, c.3113A > G in BRCA1 and 3515C > Tin BRCA2 . Functional studies suggested that c.536A > G but not c.1648A > C variant might be related to BRCA1-associated pathogenicity by affecting its function in nonhomologous end joining (NHEJ) .
In the present study c.536A > G and c.1648A > C in BRCA1, as well as c.3515C > T in BRCA2 (Table 5) were observed only in patients, with a low frequency (MAF < 0.005). In contrast 1067A > G and c.3113A > G in BRCA1 (Table 5) were detected in both patients and controls with a high frequency (MAF > 0.005).
The c.536A > G, c.1456 T > C and c.1648A > C have been often seen together and probably constitute a rare haplotype . One of these substitutions, c.1456T > C was classified as neutral in the in silico analysis, in contrast to the other two (Table 6). We have observed this haplotype in one patient BC117 with family history of BC diagnosed at the age of 48. Her sister also developed BC at the age of 42, and subsequently died of the disease. We genotyped the patient's healthy daughter and the two healthy daughters of her sister. One of the proband's nieces was also carrier of the three missense variants. Identical case was reported in a Sicilian family where the proband with early onset of BC, her mother with early onset of uterine cancer and her healthy sister harboured the haplotype 536A > G, c.1456T > C, c.1648A > C . It has been suggested that the three amino acid changes might alter the charge and stoichiometry of the protein and in consequence its function .
Even though the in silico analyses might assign deleterious function to some VUSs, they are not always consistent with the biological evidences. Further functional assays are necessary to prove the pathogenic effect of all missense variants predicted to be deleterious in current and previous studies.
As a result of the present study a mutation profile of the BRCA1/2 genes in Bulgarian BC/OC patients has been established with 13 (11 frameshift, 1 nonsense, 1 missense) unequivocally disease causing mutations. Mutations in BRCA1 gene were found in 14 % (28/200) and in BRCA2 in 5.5 % (11/200) of the Bulgarian patients selected by the recognized criteria for BRCA1/2 genetic testing. Four new frameshift (2 in BRCA1 and 2 in BRCA2 genes) have been found. Altogether inherited BC predisposition was identified in 39 (19.5 %) of the patients.
The most prevalent mutation in Eastern Europe c.5263_5264insC appeared to have a founder effect in the Bulgarian population with an overall frequency of 11 % in the studied cohort of familial BC/OC patients. It was also found in 14 % of the patients with TNBC without family history. Together with the other 3 recurrent mutations identified (c.181T > G in BRCA1; c.9098_9099insA and c.5851_5854delAGTT in BRCA2) they account for 77 % of all detected mutations. However, MLPA analysis is necessary to be performed in order to ascertain the contribution of the large genomic indels and rearrangements in BRCA1/2 genes for familial BC/OC development in the Bulgarian population.
Consequently, we suggest a mutation screening pipeline in which an initial test is performed to specifically detect BRCA1 c.5263_5264insC and the 3 additional recurrent mutations in BC patients from severely affected families to identify about two thirds of the carriers. In the remaining patients without mutations, complete sequencing of the coding regions of BRCA1 and BRCA2 is warranted, followed by MLPA screening. Such an approach would improve the effectiveness of the protocol and reduce the costs of mutation screening. This may have direct effect on the efficient molecular diagnostics of the genetic predisposition to BC/OC in Bulgaria.
The data sets supporting the results of this article are included within the article and its additional files. The distribution of patients by criteria is represented in Additional file 1: Table S1. The list of primers sequences used for PCR amplification of the entire exons and exon-intron junctions of the BRCA1 and BRCA2 genes is shown in Additional file 2: Table S2. The pedigrees of the patients harboring the novel damaging mutations c.464delA and 5397_5403delCCCTTGG in BRCA1, and c.8532_8533delAA in BRCA2 are represented in Additional file 3: Figure S1, Additional file 4: Figure S2 and Additional file 5: Figure S3, respectively.
Rumyana Ivanova Dodova and Atanaska Velichkova Mitkova contributed equally to this work.
An erratum to this article is available at http://0-dx.doi.org.brum.beds.ac.uk/10.1186/s12885-017-3188-6.
The authors would like to thank all the patients, their family members and healthy controls for participating in the study and for their support and patience. Special acknowledgements to Dr. Irena Bradinova, Assoc. Prof. Veneta Georgieva, and Dr. Radoslava Vazharova for genetic counseling of the patients. The work has been supported by grants DUNK01-2/2009 from the National Science Fund, Ministry of Education and Science, Republic of Bulgaria; Grants 56/2013, 47/2011, 54/2009 and 58/2007, as well as, PhD Grants 20-D/2012, 25-D/2011, 9-D/2010, all from the Medical Science Fund, Medical University of Sofia.
Additional file 1: Table S1. Total number of BC patients distributed by criteria.
Additional file 2: Table S2. List of primers sequences used for PCR amplification of the entire exons and exon-intron junctions of the BRCA1 and BRCA2 genes.
Additional file 3: Figure S1. A pedigree of the patient BC134 harboring the novel damaging mutation c.464delA in BRCA1. The patient was diagnosed with TNBC at the age of 54 with three cases of BC in her family. Three of her healthy first degree relatives (sister, and 2 sons) were also carriers of the mutation.
Additional file 4: Figure S2. A pedigree of the breast cancer (BC) patient BC205 harboring the novel damaging mutation c.5397_5403delCCCTTGG in BRCA1. The patient was diagnosed with TNBC at the age of 51 and had a personal history of brain papilloma (BP) at the age of 18 and thyroid adenoma (TA) around 30 years of age. Her mother and great-grandmother were also diagnosed with BC at the age of 51 and 30, respectively. The healthy (Ht) sister was screened for the presence of the mutation and was not a carrier (wt –wild type).
Additional file 5: Figure S3. A pedigree of the patient BC87 harboring the novel damaging mutation c.8532_8533delAA in BRCA2. The patient was diagnosed with bilateral breast cancer (BBC) and had early onset (y 30; y 37) of the disease. Similarly her mother was diagnosed with early onset BBC (y 37; y 38) and also carried the mutation.
RD carried out the major part of the molecular genetic analysis, participated in the sequence alignment, data analysis, interpretation of the results and drafting of the manuscript; AM participated in the design and coordination of the study, sequence alignment, data analysis and interpretation of the results, and drafted the manuscript; DD participated in the molecular genetic analysis, sequence alignment, data analysis and interpretation of the results; LH, MC and SP participated in the molecular genetic analysis; AV, MT-H, SV, TD, TS, AI, KT, and SC recruited the patients and revised the clinical and pathological data; IP performed the in silico analysis of sequence variants and helped with the data analysis; IK participated in patient recruitment and genetic counseling; VM participated in the design and coordination of the study, and critical revision of the manuscript; RK participated in the design and coordination of the study, data analysis and interpretation of the results, helped with drafting and performed the final critical revision of the manuscript. All authors read and approved the final manuscript.
Rumyana Ivanova Dodova and Atanaska Velichkova Mitkova are joined first authors.
GLOBOCAN 2012: Estimated Incidence, Mortality and Prevalence Worldwide in 2012. http://globocan.iarc.fr/Pages/fact_sheets_cancer.aspx. Accessed 23 Jan 2015.
Breast Cancer Information Core. http://www.nhgri.nih.gov/Intramural_research/Lab_transfer/BIC 1997. Accessed 23 Jan 2015.
Daly MB, Axilbund JE, Buys S, Crawford B, Farrell CD, Friedman S, et. al., National Comprehensive Cancer Network: NCCN Clinical Practice Guidelines in Oncology 2010: Genetic/ familial high risk assessment: breast and ovarian. Version I. http://www.nccn.org/professionals/physician_gls/PDF/genetics_screening.pdf. Accessed 22 Nov 2010.
ExonPrimer. http://ihg.gsf.de/ihg/ExonPrimer.html. Accessed 10 Dec 2008.
National Center for Biotechnology Information Blast. http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/BLAST/. Accessed 02 July 2013.
PolyPhen 2. http://genetics.bwh.harvard.edu/pph2/. Accessed 10 Jan 2015.
SIFT. http://sift.jcvi.org/. Accessed 15 Apr 2013.
PROVEAN. http://provean.jcvi.org/index.php. Accessed 15 Apr 2013.
National Center for Biotechnology Information SNP. http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/SNP/. Accessed 02 Apr 2015.
Universal Mutation Database. http://www.umd.be/LSDB.html. Accessed 15 Jan 2015.
Leiden Open Variation Database. http://chromium.liacs.nl/LOVD2/cancer/home.php. Accessed 15 Jan 2015.


Setting your home page in Firefox is easy. Can't decide on just one page? No problem. Firefox lets you set a group of websites as your home page. This article will give you some examples and step-by-step instructions for customizing your home page settings to best fit how you work.
If you like to keep things simple, here's how to set your home page in three easy steps.
1. Open the web site you want to set as your home page. If you want a blank home page, open a new tab.
2. Click the icon to the left of the web address, drag it to the Home button, and release.
3. Click Yes to set this as your home page.
Try it out: Click the Home button and your new home page will load in the current tab. It doesn't get much easier than that!
This is the way to have one-click access to all of your favorite websites. For example, you can set the Firefox home button to open your email, favorite news site, and Facebook all at once.
1. Open a new window and load the first web site you want in your home page.
2. Click the new tab button and open the next web site you want in your home page. Repeat this step until you've opened all desired sites in new tabs.
4. Select the General panel.
5. Click Use Current Pages.
Try it out: Close all of your tabs and then click the Home button. Watch all of your chosen pages open up in tabs. Do it again! You know you want to.


text:
declare class FormElement extends HTMLElement {
  reset(): void;
}

redpajama_set_name: RedPajamaGithub


Finding a great dataset is all very well, but the next step is working out how to get the data onto your computer so that you can start playing with it. Datasets come in many forms, and there are different ways of collecting the data. In this article I will use some examples from the list of datasets in this previous article on women composers.


text:
Questa pagina raccoglie i dati riguardanti l'Unione Sportiva Lucchese Libertas nelle competizioni ufficiali della stagione 1934-1935.

Stagione

Rosa

Statistiche

Statistiche di squadra

Statistiche dei giocatori

Note

Bibliografia

Collegamenti esterni
Campionato 1934-35, lucchese.100annidistoria.it.

1934-1935

redpajama_set_name: RedPajamaWikipedia


The CDC has added Fiji to its list of countries pregnant women should avoid because of the Zika virus.
The influx of Zika cases in South America has been associated with a spike in children born with microcephaly, a birth defect resulting in small heads, as well as a spike in cases of Guillain-Barre syndrome. While many people who contract Zika don't even experience symptoms, the potential side effects are severe enough that pregnant women have been advised to avoid countries with a great number of cases, ranging from Brazil to Haiti and now Fiji.


text:
2.2.3.3. Deforestation
Most of the definitions collected by Lund (1999) characterize deforestation as the long-term or permanent removal of forest cover and conversion to a non-forested land use. For example, deforestation is the "permanent removal of forest cover and withdrawal of land from forest use, whether deliberately or circumstantially." Similarly, the IPCC Guidelines emphasize the conversion of forests (to pasture, cropland, or other managed uses): "Conversion of forests is also referred to as 'deforestation,' and it is frequently accompanied by burning."
It is important to note that a common forestry definition of reforestation is not the antonym (mirror) to the common definition of deforestation. In forestry practice, reforestation commonly refers to any act involving reestablishment of trees regardless of whether it follows harvesting or a long period of deforestation, but harvesting is not equated with deforestation. For example: "Clear-cutting (even with stump removal), if shortly followed by reforestation, is not deforesting" (FAO, cited in Lund, 1999).
Nevertheless, many of the issues that arise with regard to certain definitions of reforestation also apply in defining deforestation. The period of changed land use required for reestablishment of trees to be treated as reforestation must be compatible with the period of changed land use that describes deforestation. Including a waiting period in the definition of deforestation (e.g., "the act of removing forest cover and not beginning to regenerate forest within X years") introduces accounting needs that can extend beyond a specific commitment period. For example, areas cleared of forest during the first commitment period would not be confirmed as deforested for some years after the clearing, which may extend beyond 2012.
Many definitions of deforestation include natural (non-anthropogenic) events such as landslides, volcanism, and so forth. Where these events are unambiguously not direct results of human activities, the Protocol and Decision 9/CP.4 are clear: If they do not meet the "direct human-induced LULUCF activities" requirement, they would not be included as deforestation under Article 3.3.
Not all natural events are so easily partitioned, however. In particular, several difficult definitional issues arise where significant forest losses occur as a result of fire (and, in some cases, landslides). Forest fires can be the result of natural events (notably lightning) or the direct (or indirect) result of human activities, including prescribed burning (and their escape), accidental fires, and arson. Even if the cause of the fire can be unambiguously attributed to prescribed burning, arson, or escape from deliberate fires (including camp fires and cigarettes), the act may or may not be deforestation in the sense defined above. In many-but not all-instances, forest cover loss is followed by natural (or aided) regeneration of new forest. To the extent that the regrowth is complete, the net release of carbon to the atmosphere may be completely recovered over time by the new forest. There is, however, an asymmetry in the rates (Kurz et al., 1995): The net release of carbon to the atmosphere is rapid, whereas the recapture takes decades or more. From a scientific perspective, however, asymmetrical accounting of changes in carbon stock over time arises if only one of the paired activities (forest loss and regrowth) is accounted as deforestation or reforestation, respectively.
Given that distinguishing between natural and anthropogenic factors that influence the vulnerability of the land to disturbance will be difficult, a key question for policymakers is whether the accounting should include only direct human activities (reforestation) or both the human activities and the event (fire) that makes the opportunity for reforestation possible.
In some cases, there are definitional and carbon accounting issues concerning drawing a clear boundary between natural phenomena and human-induced activities, when, for example, significant forest losses occur as a result of fires or disturbances such as pest outbreaks. In cases involving lands under Article 3.3 or 3.4 where fires or pest outbreaks occur in a forest, a question is whether accounting should, inter alia: (i) count neither the loss nor subsequent uptake of carbon (which reflects the actual net change in carbon stocks on those lands and exchange of carbon with the atmosphere in the long term, but creates problems in continuing to account for the area burnt/defoliated as lands under Article 3.3 or 3.4); (ii) count both the loss and subsequent uptake of carbon (which reflects the actual net change in carbon stocks on those lands and exchange of carbon with the atmosphere, but creates an initial carbon debit for the Party concerned); (iii) count only the loss of carbon (which would overestimate the actual losses of carbon stocks, not represent the exchanges of carbon with the atmosphere, and create future accounting problems), or (iv) count only the subsequent uptake (which would fail to reflect the actual changes in carbon stock and would not represent the exchanges of carbon with the atmosphere, and would provide carbon credits for the Party concerned).
In cases involving lands that do not fall under Articles 3.3 or 3.4, where fires or pest outbreaks trigger land-use change, the consequences are similar to deforestation. If similar vegetation cover is allowed to regenerate, such disturbances may not lead to a long-term change in carbon stocks.
Will forest protection practices (such as fire management systems that seek to prevent wildfires or ameliorate the damage they cause through suppression efforts) be rewarded (in a Protocol sense)? Will Parties be penalized (under the Protocol) for failing to manage wildfires? These issues remain to be resolved through negotiation by the Parties.
A further complication is the fact that fires in many ecosystems do not result in complete tree mortality, even though there are significant changes in the carbon balance of the affected areas. Individual trees and small patches of trees and understory can survive. In some forest types (e.g., eucalypt and some pine forests), even the most intense fires do not cause significant mortality of mature trees. Significant quantities of carbon may be released, but most trunks and large branches survive and regrow full canopies within a few years. Although measurement of these changes under the stock change method as recommended by Article 3.3 is possible, in practice the areas of forest meeting the deforestation requirement of Article 3.3 would be contentious and difficult to assess.
A less difficult situation arises when land-use change takes place subsequent to or as a result of fire. In many parts of the world, major forest fires (whether ignited by humans or naturally caused) are taken as the cue for land-use change to agricultural or pastoral uses. In these cases, the combination of fire and land-use change should be accounted as deforestation in a comprehensive and symmetric accounting system.
For several reasons, accounting procedures that always treat changes in carbon stocks that are attributable to fire as debits may be inconsistent with long-term objectives of increasing carbon stocks. First, fire has been used in some ecosystems as a forest management tool for enhancing forest growth over the long term, even though it might result in some carbon reductions over the short term (for example, during a commitment period). Second, fire is a natural part of many forest ecosystems, and management to eliminate fire can be futile or counterproductive. Natural or prescribed ground fires in such ecosystems may result in preservation of carbon stocks at the landscape scale by creating firebreaks-thus reducing the likelihood of widespread, intense fires.
In the long term and over large areas, there is a balance between carbon loss resulting from fires and carbon sequestered in regrowth, providing there is no change in the fire regime. This balance underlies the recommendation in the IPCC Guidelines to treat wildfires as carbon-neutral. Although this approach is pragmatic, there is some evidence that fire regimes are changing in parts of the world (e.g., Kurz and Apps, 1999). In some cases, these changes may be a result of deliberate human manipulation; in others, it is an indirect consequence of human actions-possibly including climate change. Moreover, because of asymmetry in the rates and timing of carbon loss from fire and its re-accumulation through regrowth, the complete balance is not achieved if fire regimes vary in response to changes in El Ni�o patterns or rapid climate change.

redpajama_set_name: RedPajamaCommonCrawl


Students often grapple with the rising costs of living, tuition and meeting basic needs. This can create barriers that undermine their ability to academically succeed. UW Tacoma has begun a partnership with Shared Housing Services to provide UW Tacoma Students with safe, stable, and affordable housing to assist in their ability to pursue their educational goals.
Introducing Husky2Husky (H2H), an innovative Homesharing Program that offers Tacoma Residents the opportunity to help a UW Tacoma student obtain affordable housing by utilizing that 'spare room' in their home. This program offers housemate matches based on lifestyles and personalities. This program was created through the partnership with Shared Housing Services.
H2H is a safe, low-cost screening and referral service that processes applications, completes background checks, and builds compatibility profiles for qualified Home Providers (those who wish to rent out rooms in their house in exchange for an agreed level of support such as assistance with household tasks and/or a financial exchange) with qualified Home Seekers (UW Tacoma students needing affordable housing). The H2H homesharing program benefits Home Providers and Home Seekers by facilitating a home share situation that is mutually helpful to each side. It's a win-win!
WHO IS SHARED HOUSING SERVICES?
Shared Housing Services is a non-discriminatory, equal opportunity 501(c)(3) non-profit, serving Tacoma and Pierce County since 1991. Their programs promote diversity, equity and inclusiveness while aiming to reduce and prevent homelessness through creating a culture of sharing resources for the good of our community as a whole.


Elusive brews get a geistly affair on Elm Street.
Cincinnati craft brewery Rhinegeist is hosting an inaugural ball of all that is sparse in beer. On October 28th, they will honor the extraordinary, the unobtainable, and the unexpected by curating hard-to-get-your-hands-on brews made by well known breweries from across the country at Rare Beer Fest.
Claim the Brew Studs reader discount for a time almost as limited as the beer.
50 hand-selected breweries will journey to Rhinegeist with their rarest brews (sour, wild, imperial, hoppy, barrel aged, fruitified, funky and similarly rarified) in tow for a magnificent day of craft beer celebration. Much of what will be poured at Rare Beer Fest beer is not available in Ohio under normal circumstances, but this will not be your normal beer festival.
Rare Beer Fest will kick-off at 12pm with a special early access session for VIP ticketholders, followed by General Admission entry at 1pm.
4 Hands Brewery (St Louis, MO), 21st Amendment (San Leandro, CA), Allagash Brewing Company (Portland, ME), Bell's Brewery (Kalamazoo, MI), Blackberry Farm Brewery (Walland, TN), BrewDog (Canal Winchester, OH), The Bruery (Placentia, CA), Burial Beer Co (Asheville, NC), Cigar City (Tampa, FL), Crooked Stave Artisan Beer Project (Denver, CO), Destihl Brewery (Normal, IL), Drake's Brewing (San Leandro, CA), Faction Brewing (Alameda, CA), Fat Head's (Middleburg Heights, OH), Fieldwork Brewing Company (Berkeley, CA), Firestone Walker (Paso Robles, CA), Fullsteam Brewery (Durham, NC), Lagunitas Brewing Co. (Petaluma, CA), Little Fish Brewing Company (Athens, OH), The Lost Abbey (San Diego, CA), Noble Ale Works (Anaheim, CA), Other Half Brewing Co (Brooklyn, NY), Pipeworks Brewing Co. (Chicago, IL), Sun King (Indianapolis, IN), Three Weavers (Inglewood, CA), TRVE (Denver, CO), Upland Brewing Co. (Bloomington, IN), Wiseacre Brewing Company (Memphis, TN), with additional breweries to be announced.
Festivalgoers will also enjoy some extremely rare samplings from Cincinnati-based breweries on tap at the Local Rare Beer Bar.
A selection of over 4000 specialty, craft-brewed, and imported beers.
General Admission (1pm-5pm) $40 in advance | $45 day-of | Includes 10 sample tickets + commemorative sample glassware.
VIP | Early Access (12pm-5pm) $60 in advance | Includes 10 sample tickets + commemorative sample glassware + early access + 5 additional VIP tastings *Sample 5 additional rare beers not available to GA festivalgoers, while chatting with brewers/reps from featured breweries.
Tickets on-sale Friday, September 8th at 10am.


I have a Kenmore front load washer that doesn't seem to clean whites well. All of my whites come out dingy. I have tried more bleach and less bleach. I have tried everything I can think of but nothing works. Please help.
My LG frontload washer (the "top of the line" one) does not seem to get my clothes clean. My whites are all dingy and I notice there are fabric softener stains on my colored clothes as if it never rinsed them out. What could I do differently, or is this just a piece of junk machine? Who do I contact to rectify the problem? This machine wasn't cheap! My front loader in my other house works great (it's a different brand). My LG frontload washer (the "top of the line" one) does not seem to get my clothes clean. My whites are all dingy and I notice there are fabric softener stains on my colored clothes as if it never rinsed them out. What could I do differently, or is this just a piece of junk machine? Who do I contact to rectify the problem? This machine wasn't cheap! My front loader in my other house works great (it's a different brand).
Have you checked your water for too much iron?
The water in my front-loaded kenmore-he washer is not draining and doesn't seem to spin very fast either.
Whites are dingy. Random stains appear on my husband's dress shirts that weren't there when I put them in the wash. We have a water softener; don't know if that plays into the problems. Please help!
Put washer through a clean cycle with bleach only. Check to be sure you are using HE soap. Check the bellows(door seal) make sure it is not jamming clothes.
Try nappysan. As you have found out bleach is no good. In the "day" we used to use a "Bluing" Agent, see if you can get those, My solution is to use just a dot or 2 of Dawn dish washing liquid on any spots I thought would stain-scrub them for just a minute with a fingernail brush. Then I would fill my washer about 1/4 of the way with straight hot water & add a 1/2 c. of automatic dishwasher soap to the tub--the cheaper the better because most cheaper brands have more phosphorous. I'd swish the water around, turn the temp. to warm, & put in my whites when the tub was about halfway full. Then I'd add about half the amount of detergent to the tub that I would normally use.
I wouldn't blame it on the washer but did you have the same problem with your previous washer? Are you using the same water source as previously. Do you use a chlorine bleach with it and did you use it before? Is the cheer a liquid or powder. You should us a powder.
I have an LG front load washer too and sometimes my whites don't come out as nicely as they should. Usually draining the machine and running several cleaning cycles solves the problem. I hope it works for you!
What is the answer for dingy clothes? I have the same problems and I'm getting ready to get rid of this washer because I'm tired of not getting clean clothes and having my towels smelly after taking them out of the washer.
First I would start by cleaning your washer system with vinegar. Ran your machine like you would without clothes and pour a cup of vinegar like you would with the soap for a whole cycle. If this doesn't work chances are your drain is clog or your pump isn't working properly.
Once the dingy look gets into them, it seems to be impossible to get out. It is caused by using too much soap and / or fabric softener. Remember, your front load LG is using only about 12 gallons of water to wash those towels. Your old machine used as much as 45. This means that all wash loads need to use less soaps and fabric softeners. Use 1 tablespoon of regular HE detergent for standard loads. (only a teaspoon if it is 3X HE) Only a teaspoon of fabric softener when you use it. Which brings me to towels. If washing bathtowels: You are clean and rinsed before you use the towel. The towel is not getting dirty, only soaking up the clean water off your body. Hang them on a towel rack and use them several times before washing, and when you do wash bath towels, most of the time you do not need any detergent, as they are really not dirty. Of course you should know that you never use fabric softener on loads of towels. Now, back to the dingyness. The dingy stuff is residual soap / fabric softener in the fabric, which once machine dried seems to be impossible to remove. If they are white towels, make sure to use liquid bleach and you might try to use a little Mrs. Stewart's concentrated liquid blueing in the fabric softener dispenser to see if if might help. Most likely you will have to try a few new towels, washing with no soap or fabric softener when laundered.
Please don't rate this, as it is really just informational, not what most people would call a solution, and any rating other than FixYa drops my score.
I too have a front load washer. The problem is that it's not washing off all the soap suds. Try this. After a complete cycle, soak the clean clothes in a container w/ water and you'll see there's still soap suds coming out of the clothes. I tried running it several wash cycles and there was still soap left on the clothes. I think, it's mfg defect. We should all complain to the maker of the machines.


text:
#region Disclaimer/License Info

/* *********************************************** */

// sBlog.Net

// sBlog.Net is a minimalistic blog engine software.

// Homepage: http://sblogproject.net
// Github: http://github.com/karthik25/sBlog.Net

// This project is licensed under the BSD license.
// See the License.txt file for more information.

/* *********************************************** */

#endregion
using System.Collections.Generic;
using System.Linq;
using sBlog.Net.Domain.Entities;
using sBlog.Net.Domain.Generators;
using sBlog.Net.Domain.Interfaces;

namespace sBlog.Net.FluentExtensions
{
    public static class TagExtensions
    {
        public static List<TagEntity> GetTagEntities(this ITag tagRepository, List<string> tags)
        {
            var newTags = new List<string>();
            var allTags = tagRepository.GetAllTags();

            var existingTags = allTags.Select(t => t.TagName.ToLower()).ToList();

            tags.ForEach(tag =>
                {
                    if (tag != null && tag.Trim() != string.Empty && !existingTags.Contains(tag.ToLower()) && newTags.SingleOrDefault(newtag => newtag.ToLower() == tag.ToLower()) == null)
                        newTags.Add(tag.Trim());
                });

            var allSlugs = allTags.Select(t => t.TagSlug).ToList();

            var newTagsList = new List<TagEntity>();
            newTags.ForEach(tag =>
                {
                    var slug = tag.GetUniqueSlug(allSlugs);
                    allSlugs.Add(slug);
                    newTagsList.Add(new TagEntity {TagName = tag, TagSlug = slug});
                });

            return newTagsList;
        }
    }
}


text:
August 24, 2016 Svalbard No comments
Coffins from Svalbard's biggest graveyard saved from coastal erosion, offer clues to life in 1600s
The remote and harsh setting often made life very hard indeed, but also meant Mother Nature took great care with the dead after they submitted to the elements.
Until recently.
The well-preserved remains of three men in coffins at Likneset were removed from Svalbard's largest graveyard earlier this month and are now in cold storage at Svalbard Museum. The graveyard is on a coastal cliffside at Likneset on northwestern Spitsbergen, described as the heartland of whaling in the archipelago during the 17th and 18th centuries, but erosion had put the coffins in immanent danger of being washed into the sea.
Snorre Haukalid, a cultural heritage advisor for The Governor of Svalbard, examines a grave on the edge of a cliffside at Likneset. Photo courtesy of The Governor of Svalbard.
"Previous studies have shown that it is whaler graves that can tell the most exciting stories," wrote Arild Skjæveland Vivås, an archeologist for Norark, in a blog about the project. "In the Arctic the climate is cold and dry, and where the tombs were built on top of permafrost and have been frozen most of the year, which has resulted in unique preservation conditions."
So while the wood on the first coffin removed was "soft and in some places completely pulverized" despite appearing fresh, many traits of the person inside were immediately obvious.
"Under the coffin lid lay a complete skeleton with dark blond hair on a bed of sawdust," Vivås wrote. "We were somewhat surprised that there was not visibly preserved textiles. Earlier excavations at Likneset, and elsewhere in the area have uncovered everything from socks to wigs and hats."
The preservation of such items for such a long period is considered largely unique to Svalbard and may offer clues to life throughout Europe during that era, according to project and museum officials.
There are at least 230 graves at Likneset, which was frequented by Dutch, English, Norwegian and other whalers. There were four archaeological investigations at the site between 1985 and 1990, and erosion on the five-meter-high cliffside has been monitored since 1998.
Most of the whalers apparently died of scurvy due to poor nutrition. While the modern visitors didn't face that problem, they did have to cope with some of the other deadly elements such as polar bears and hazardous weather.
"The first morning we were visited by a lean and hungry polar bear in Sallyhamna," Vivås wrote. "The next morning bear had wandered to the excavation field on Likneset and prevented us from going ashore. We we decided to keep our distance and let nature take its course. When we returned after four hours bear had wandered on."
Likneset, coffins, whaling


Blue Gal: Best Liberal Blog Nominee.
I sort of expected some technorati-based acknowledgement of my small traffic to frequent writing ratio, but this...so this...kinda blows me out of the water.
(Go Driftglass for best individual blogger and Jon Swift for best humor blog. Just sayin').
Wow, nice swag there, BG! Like, CONGRATS!
So cool and so very well deserved!!
That is so great. Can hardly wait to vote for you!
Congratulations, Blue Gal, you're the best; it will be a pleasure to vote early and often!
Where do you get a Publicist?
Way to go, BG, and so well deserve.
too grand!!! way ta go!
No surprise here. Well deserved recognition. Congrats.
I received the news as I was conversing with the bold and durable Cal Ripken Jr. we both agreed congratulations are in order, so please take this to be them. and also too, cha cha CHA!
By "liberal" do they mean politically leftish, or is this a reference to the amount of mayo you put in your tuna salad?
Congratulations, Blue Gal! I will be spreading the word.
Let me add my congratulations. Nice to know that hard work, perseverance and talent is noticed.
sweet! very much deserved! fingers are crossed for you, not that you need that kind of luck! congrats to you!
I cannot believe I did not post a comment: Congrats, BG!
Those other bloggers should be honored to be mentioned in the same breath as you!
Congrats to you!!! You Go!
A test for Hillary Clinton.
Another reason why Blago has to go and fast.
Stone cold sober, as a matter of fact.
Gentlemen, we have to talk.
There is video of this Hello Kitty Wedding.
What Bob Herbert said. And didn't say.
Oh my God! You killed Kitty!


▷ HOW LONG DO SNAKES LIVE? | FACTS and HABITAT | How Long Do Animals Live?
The life expectancy of snakes depends on their size. Larger snakes can live for up to 25-50 years while smaller ones have a lifespan of 5-10 years but can also live longer than this.
How long do snakes live in the wild?
Ball python or royal python: 20-30 years (with a good care, they can live more than 40 years).
Carpet python: lives between 20 and 30 years.
Corn snakes: 6-8 years (live between 15 and 20 years when they are kept in captivity).
Kingsnakes: between 15 and 25 years.
How long do snakes live in captivity?
Snakes can live for 15-50 years in captivity.
The Burmese python, a well-known type of snake, lives more than 25 years in the wild, but when it is kept in captivity it usually lives for ten years. This is a rare case when animals live shorter when they are kept as pets. The fact that they demand special treatment when kept at home is one of the reasons why they live shorter.
Those who are interested in keeping a snake as a pet should know that there are few factors that affect the lifespan of every snake.
One of the most important things is the snake cage. Snakes need a clean and dry cage even though there are some species that need access to water. Of course, this part should be separated and isolated from the others. The owner should also monitor the temperature and humidity in the place where the snakes live.
Needless to say, their diet plays a crucial role. By learning more about the diet of the specific snake you have, you can make sure that the snake gets all the nutrients and lives a long life.
Related posts: How long do king cobras live? | How long do crocodiles live?
According to official statistics, there are over 3000 different species of snakes on our planet.
They live on all continents (except Antarctica). Snakes are found in most tropical regions in different parts of the world, apart from regions like Iceland, Antarctica, Ireland, New Zealand and Greenland.
Their main habitation is forests, deserts, water and grasslands. Snakes also live in underground burrows and spaces under big rocks.
Many people find snakes scary and intimidating, while others keep them as pets in their homes.
Snakes are carnivores. Most snakes feed on eggs, insects, fish, rodents, lizards and small mammals. Big snakes like anacondas and pythons bigger animals like pigs and bears. Some of them poison their prey while others squeeze/suffocate their prey to death.
How much do snakes measure?
Different species of snakes have different sizes. According to National Geographic, the smallest snake, which is the thread snake, measures around 10 cm (3.9 inches). The reticulated python, which is believed to be the largest of snakes, can grow up to 30 feet (equivalent to 9 metres).
A snake's weight varies between different species. The giant anaconda, which is the largest snake in the world, can weigh up to 250-550 pounds.
Not all snakes lay eggs. Some snakes give birth much like mammals. These snakes give birth to live young broods 1-2 or new-born and the number can litter to 100. Snakes living in colder climates do not lay eggs because the weather is too cold for eggs to hatch.
King cobra is the only snake species known to build a nest for its young ones.
There are some rare species of snakes that lay eggs without mating.
Newly hatched snakes emerge using special teeth to break their leathery membrane shells. They must feed in order to survive.
Unlike humans and many other mammals, snakes do not use their nose to smell. They use split tongues to taste and smell chemical composition in the air.
Snakes do not have ears or eyelids, neither do they move their eyes. They rely on vibrations through the ground to hear.
Snakes shed their entire skin almost 3 times a year through a process called molting.
Yes. Snakes like paradise tree-snake of South East Asia can comfortably fly. It does this by swinging its body through the air and then flatten into a C-shape where it catches the airflow. It can change directions by flipping its body back and forth.
Snakes with diamond shaped pupils are poisonous. Non-poisonous snakes have round pupils. Their colours can also give a clue whether the snake is poisonous or not. Coral snakes are deadly while.


On 23 May 2018, the UK Government has made a statutory instrument 2018/630 to bring into effect arrangements between the UK and 77 other jurisdictions following the outcome of the OECD BEPS project (Base Erosion and Profit Shifting).
On 23 May 2018, the UK Government has made a statutory instrument 2018/630 to bring into effect arrangements between the UK and 77 other jurisdictions following the outcome of the OECD BEPS project (Base Erosion and Profit Shifting). Existing Double Tax Agreements (DTAs) between the UK and other jurisdictions have not been affected by this order. The DTAs would be modified only once the UK and the other jurisdiction give notice that they wish them to be "Covered Tax Agreements". A list of provisional notifications made by the UK has been published and contains those DTAs which the government wishes to be a Covered Tax Agreement. The statutory instrument will not modify the provisionally listed DTAs until the other jurisdiction either makes an equivalent notification, or signs and ratifies the arrangements and makes such notification.


text:
Chris Butler from AL wrote on October 8, 2018 at 12:22 am:
When are yall coming to the Birmingham area ?
Sean from Ontario/Canada wrote on September 8, 2018 at 8:37 pm:
Hey guys! You just are amazing and one of the motivators, besides the old man, why I picked up playing the banjo! I sure hope I find a way and time to see one of your shows!
Dana Michaud from NY wrote on September 8, 2018 at 1:34 pm:
Holy Crap!! You guys are GREAT!! This - from an old hippie, rocker, blues enthusiast. Raised in NY but my roots are in Virginia. My southern family, of course, listens to bluegrass and now my sons do. They turned me on to you. This is a first since Ive groomed THEM for years to love good music, helped them discover all the great music from the 60s and 70s (and on). Of course the key was always to appreciate talent and they learned well! When you find music that makes you exclaim, tear up, laugh with joy, well, theres nothing better. You all are a joy to behold. I am officially a fan!
Gail Rieger from Maryland wrote on September 5, 2018 at 1:25 am:
I love your music!!! You are above ALL bands in my book. Please come to Maryland!
Amy from New Zealand wrote on August 31, 2018 at 10:08 am:
My husband and I were in a place called Bareknuckle BBQ tonight in Hastings, New Zealand (I am American but live in NZ, my husband is a Kiwi) and they had one (or a couple) of your albums playing. It was awesome. I had not heard your music before. My husband is a big metal head and liked hearing some of the songs he knows played in a bluegrass way. I think I will look into buying an album or two. I have downloaded one already on iTunes. My husband suggest some Judas Priest for a future project. Any Chance of a tour of New Zealand?!?!?!
Josh from AL wrote on August 4, 2018 at 3:41 am:
Would love to see you guys do Pickin on Bowie.
Brad Odum from Tennessee wrote on August 2, 2018 at 2:23 am:
When are you playing in the East Tennessee area? Great sound! Music the way it's supposed to be.
Chloe from New Jersey wrote on August 1, 2018 at 5:21 pm:
Hi Iron Horse! I first discovered you guys because my dad showed me your Rocketman cover, and everyone Ive shown it to agrees its better than the original. Please come play in the Northeast United States soon (preferably NJ or NY, but Ill gladly go a little out of my way to see you guys live). Good luck on all your upcoming performances!
John Devine from CA wrote on July 25, 2018 at 6:01 pm:
PLEASE DO A PANTERA ALBUM STARTING WITH THE SONG TITLED DOMINATION! LOVE YOUR WORK. MANY THANKS
Chris Oxley from UK, Northumberland wrote on July 6, 2018 at 8:38 pm:
Im sure that there are people who write academic papers about the common threads in music, and how chord sequences and all that stuff travel from one culture to another. Nice work if you can get it. All I know is that when I hear you play, my gut (which was born and bred in the north east of England) tells me that Im home. So your music works magic that crosses cultures. Respect to you for that.
Andrew Sheils from Telluride, Colorado wrote on June 30, 2018 at 4:40 pm:
Huge thanks to Iron Horse for the show they put on at my rehearsal dinner at Gorrono Ranch! Hands down one of the best bluegrass groups out there. They played everything from classics to covers. Every guest was out there dancing to them, even my 74 year old father! Couldn't imagine a better night of grass. Thanks fellas! Andrew Sheils
DEREK LAWS from U.S.A. wrote on June 21, 2018 at 1:39 am:
Hey Iron Horse, I was talking with a friend of mine who lives in Odessa, Ukraine. We were talking about different styles of music and she mentioned that American Bluegrass was very popular in Ukraine and amongst her circle of friends, Iron Horse was their most favorite bluegrass band. I told her I had never heard of Iron Horse and thought maybe they were a bluegrass Ukrainian band. I told her I would check the band out. Well, what can I say, I checked you out on YouTube and your music absolutely blew me away and I can see why they love you in Ukraine!!! I was really stunned how rock music could sound so fantastic being played bluegrass style. I have been a big fan of bluegrass all my life, saw Bill Monroe back in the 70's, big fan of Lester Flat and Earl Scruggs. Iron Horse has opened a whole new door of sound and music for me. So Nataliya, if you read this, THANKS !!! And thank you Iron Horse !!!!!
Fabio from Italy wrote on June 7, 2018 at 8:42 am:
Hi, Im your fan. I live in Rome. I met you with youtube because I started playing the banjo. Unfortunately there are not your concerts in Italy and at least in Rome. If you were to come to Italy I would be very happy to come and listen to you. Keep it up so you are very strong. Fabio
Robert Bracey from Virginia/USA wrote on June 6, 2018 at 6:57 pm:
Love. It. Would love to see you guys in Southeast Virginia.
Brandon Hewitt from WA wrote on May 24, 2018 at 7:40 am:
So sorry I missed the show in Ellensburg, WA. Come back to the Pacific Northwest soon! Your music is amazing!!
tarey archer from CO wrote on May 11, 2018 at 2:37 am:
I could not resist getting tickets to go see you in Fort Collins June 22nd. Even though it is a six hour drive. You are my favorite band. I cant wait. Thank you!
Dan from California wrote on May 7, 2018 at 10:04 pm:
Just heard your rendition of Rocket Man and was completely blown away! Come to California soon, well be there!
David Price from NC wrote on May 6, 2018 at 2:26 am:
Just saw your show in Yadkinville N.C. absolutely the best performance of a truly unique band. Thank you so much for the music! David Price
erin from alaska wrote on May 3, 2018 at 8:43 am:
Come north! Your rocket man is the best rendition, ever!
Ed Boerner from Ohio wrote on April 26, 2018 at 5:17 pm:
You guys are great. My son and I saw you at Pennyroyal Opera House. Love the covers. Have you ever thought about doing the OJays Backstabber and/or For the love of money. Bluegrass just seems to make the songs better with much more human feelings and understanding. Its like...now I get it.


We are looking for post-docs and prospective PhD students interested in working with us.
We currently have projects for bachelor and master thesis as well as for short to long term stage periods for students and visiting researchers. Available projects include wet lab projects, purely computational projects and any combination of the two.
We are open to collaborations and to host research scholars interested in deepen their expertise in microbiome research (both wet and dry parts). Also, if you have a microbiome-related project we can support you possibly writing a Marie Curie fellowship.
The lab and our microbial friends as of March 2016.
Nicola Segata is an associate professor and principal investigator. Is background is in metagenomics, machine learning, microbiome research, and microbial genomics. He started and he is leading the Laboratory of Computational Metagenomics at the Centre for Integrative Biology (University of Trento).
Federica joined the Lab in November 2016 as a postdoctoral fellow. She is interested in microbial biodiversity, focusing mainly in viruses, in aquatic ecosystems. Her project (MetaVirAlp-Marie Skłodowska-Curie IF) focuses on the characterisation of viral and bacterial communities in Alpine Lakes.
Federica is a biologist working as a wet lab technician, she has joined the lab in March 2015. Currently she is processing the samples from different projects, preparing them for sequencing.
Francesco has joined the lab in March 2014. He is a PhD student participating in the interdisciplinary program on Computational Biology. His major research interests are metagenomic and phylogenetic analyses.
Serena has joined the lab in November 2014. She is a PhD Student in Biomolecular Sciences, currently working on microbial communities of Cystic Fibrosis lungs using a metatranscriptomic approach.
Graduated in Dentistry at the University of Verona in 2009. 3-year Post-graduate School at University of Milan, and awarded a certificate as Specialist in Oral Surgery in 2012. In 2015 he achieved the Master Diploma in Oral Implantology at the University of Padova. Currently he is PhD Candidate at CIBIO with a research project on peri-implant diseases.
Moreno is a PhD Student in Biomolecular Sciences with a background in Computer Science. He joined the lab in 2013 and is now working on the characterisation of viruses in the human microbiome from metagenomics.
Francesco is a PhD Student. He is currently working on the characterization of Blastocystis spp. in the human microbiome and on the characterization and the evaluation of the effects of tobacco exposure on the oral microbiome of New Yorkers enrolled in the New York City Health and Nutrition Examination Study.
Paolo has joined the lab in January 2017. His main research interests are bounded to the History & Geography of human being and to the machine-learning approach in Bioinformatics.
Nicolai obtained his Master's degree in Molecular Biology in the Zeller group at the EMBL in Heidelberg where he benchmarked ecological dissimilarity measures on microbial abundance data. He joined the lab in July 2017 to explore the global diversity of Eubacterium rectale strains using metagenomic assembly.


Experience the ultimate luxury of owning a condo completely renovated and decorated not only for today's exciting concepts in design, but for the timeless coastal d'cor guests visiting beach resorts worldwide have come to expect when booking their reservations. Renovated and updated in 2017 to include: wood look ceramic flooring, baths updated to include elegant, custom tile work with spacious jet tub and huge shower in master bath. The exquisite coastal furnishings are elegant as well as durable for the comfort of guests. Spacious balconies feature cheerfully seating areas with room for families to enjoy spectacular gulf views. All appliances have been updated including washer & dryer. Laundry room was renovated so that owners have TWO owners closets. As expected, unit has excellent rental numbers. The resort also includes world class ammenities with multipule pools, sauna & fitness facilities. Short walk to all the seasonal festivites Gulf Shores offers.
Listing provided courtesy of Kaiser Sotheby's International Realty - Orange Beach. Listing information © 2019 Baldwin County Association of REALTORS® MLS.


The alarm bells are ringing full mode. As you know it a very dark sign when instant payouts switch to manual payouts. Maybe it has something to do with the bitcoin crash.
Let's wait and see. I had a terrible day with Bitcoin crashing more than 20%. Another dying bot is such a small nail in the coffin of my good mood.


I need to use floating point maths with BoostBasic, but I get silly errors with the most trivial program.
I cannot see a missing right parenthesis and I have tried various permutations with no success.
Could it be that BoostBasic does not support the float data type ?
The float libraries seem to be only for C and C++ according to the reference manual.
Following help from Pavel, I can get the maths functions to work, but some of the comparison functions fail, namely: ge, gt, ge_quiet and gt_quiet.
"error: unresolved external function: 'float32_ge(float,float)' " followed by an exit code of -2.
Unfortunately the "ge" and "gt" functions are needed for the program which I am developing.
...Following help from Pavel, I can get the maths functions to work, but some of the comparison functions fail, namely: ge, gt, ge_quiet and gt_quiet.
There are no such functions as float32_gt or float32_ge in the library. If you look into float.h file you'll find that these two are defines based on float32_lt and float32_le functions. Use these two instead.
The new release 6.89 now supports float data type in BoostBasic and includes a sample project that shows how to use float math in basic.


ST. LOUIS, MO., August 28, 2015 –The St. Louis Cardinals and their Sr. Vice President/General Manager John Mozeliak announced today the team's decision to hire former Cardinals pitcher and 2006 World Series Champion Randy Flores as its Director of Scouting, beginning Tuesday, September 1.
Flores, 40, holds a master's degree in Postsecondary Administration/Sports Administration from the University of Southern California and has been involved in coaching, scouting, private business and broadcasting since his playing career ended.
Flores, a left-handed pitcher, spent eight seasons in the majors with Texas (2002), Colorado (2002, 2009-10), St. Louis (2004-08) and Minnesota (2010) and was a member of the Cardinals World Championship team in 2006.
Mozeliak plans to introduce Flores to the media next Tuesday, Sept. 1, prior to the Cardinals game with the Washington Nationals.


Valentine's Day gives us an opportunity to spread the love with encouraging cards and tasty morsels. This peyote stitch bracelet sends a warm and fuzzy message to anyone who receives it — even if you give it to yourself!
*The Delica beads were purchased from www.firemountaingems.com (unless otherwise noted) and reflect the numbering and naming conventions of the vendor.
2. Using 11/0 Delica cylinder beads, work the pattern in flat even-count peyote stitch, starting at the bottom left-hand corner. End and add thread as needed.
3. Hold the band horizontally so the working thread is pointing away from you. To work the first edging stitch: With the working thread exiting the end cylinder in the last row, pick up three medium pink cylinders, sew down through the next edge cylinder, and sew up through the following edge cylinder (FIGURE 1, a–b).
4. To work subsequent edging stitches: Pick up three medium pink cylinders, and sew through the next edge cylinder. Sew through the beadwork to exit the second edge cylinder from the one you just sewed through (b–c). Repeat this stitch for the length of the edge, ending and adding thread as needed.
5. Sew through the beadwork to exit the end cylinder along the other edge. Repeat steps 3 and 4 for this edge.
6. To make the toggle loop: Using the working thread, sew through the end row of the band to exit the sixth up-bead from one edge. Pick up 27 medium pink cylinders, and sew through the next up-bead. Sew back through all the cylinders just added and the sixth up-bead from the end (FIGURE 2). Continue through the first cylinder added in this step. Using medium pink cylinders, work a row of peyote stitch off the loop of cylinders. End the working thread.
7. To attach the toggle button: Remove the stop bead, and using the tail, sew through the end row of the band to exit the sixth up-bead from one edge. Pick up 12 medium pink cylinders and the shank of the heart button, and sew through the next up-bead. Sew back through all the cylinders just added and the first up-bead. End the tail.