To evaluate the risk of cancer among Taiwanese female registered nurses (RNs) using a nationwide population-based dataset.We recruited female RNs without antecedent cancer from the Taiwan National Health Insurance Research database during 2000–2010. Standardized incidence ratios (SIRs) of cancer were calculated. We also compared rates of Papanicolaou (Pap) smear use between the RNs and the general population matched by age and sex.p = 0.009).A total of 2,077 cancers developed among 184,809 female RNs, with a follow-up of 1,371,910 person-years (median follow-up of 7.86 years), leading to an increased SIR of 1.10 [95% confidence interval (CI) 1.05–1.15]. RNs aged between 40–59 years also had a significantly increased SIR . For specific cancer types, RNs had an increased SIR for breast , thyroid , lung and mediastinum , and uterine cancers . A decreased SIR was found for cervix  and liver and biliary tract cancers . Pap smear use averaged 5.80 times per person among female RNs aged 35 years or older and 5.50 times per person in the age-matched control group (This study found that overall cancer risk was higher among female RNs than general population. For individual cancers, the risks of breast, lung, thyroid and uterine cancer were higher and the risks of cervix and liver cancer were lower than general population. The lower risk of cervical cancer might be partially explained by the increased use of Pap smears in the RNs group. Further large, unbiased population-based prospective studies are needed to investigate the association between nurses and cancer risk and identify the risk factors of cancer in nurses. Nursing is a profession within the health care system focused on the care of individuals, families and communities so they may attain, maintain, or recover optimal health and quality of life. It is also one of the oldest female occupations in modern society, providing work for a large number of women. Being a nurse implies the possibility of exposure to over 100 agents in their work environment that are associated with increased risks of cancer, including biological, chemical, physical, and psychological health hazards. The aim of the current study was to determine whether female registered nurses (RNs) in Asia are associated with a higher risk of cancer than general population. This study was a population-based retrospective cohort study using a database derived from the National Health Insurance (NHI) system in Taiwan.The Institutional Review Board of Taipei Veterans General Hospital approved this study (2012-12-002AC). Written consent from study objects was not obtained, as the NHI dataset consists of de-identified secondary data for research purposes and the Institutional Review Board of Taipei Veterans General Hospital issued a formal written waiver for the need for consent.The NHI program, which began in 1995, is a mandatory universal health insurance program, offering comprehensive medical care coverage to all Taiwanese residents with a coverage rate of up to 98%. We conducted a retrospective cohort study from January 1, 1996 to December 31, 2010. Female RNs were selected from the Registry for medical personnel (PER) and the Longitudinal Health Insurance Database 2000 (LHID 2000) from January 1, 2000 to December 31, 2010. The PER contains basic data of all medical staff in Taiwan, such as birthday and sex. The LHID 2000, which contains all original claims data for 1,000,000 subjects, is a representative database randomly selected from the 2000 Registry of Beneficiaries under the NHI program. This study recruited female RNs aged 20 years or older who had no prior malignancies from January 1, 1996 to December 31, 1999.The main dependent variable was occurrence of cancer, as reported in the Registry for Catastrophic Illness. For a diagnosis of cancer to be reported in the Registry, histological confirmation is required. Cancer was defined according to the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM). The risk of cancer in the female RN cohort was determined using a standardized incidence ratio (SIR), which is defined as the observed number of cancer occurrences divided by the expected number. The expected number of cancers was calculated by multiplying the national incidence rate of cancers, stratified by gender, calendar year, and age in 5-year intervals, by the corresponding stratum-specific person-time accrued in the cohort. The incidence rates of cancers among the general population were obtained from the Taiwan Cancer Registry. The 95% confidence intervals (CIs) for the SIRs were estimated under the assumption that the number of cancers followed a Poisson probability distribution. We determined the SIRs for subgroups based on age. SIRs were also estimated for each cancer type. Finally, in order to compare the use of Pap smears by both the RNs and the general population, we selected female RNs in the LHID 2000 from January 1, 2000 to December 31, 2010 and recruited an age-matched control group from the LHID 2000 at a ratio of 1∶4. The average number of Pap smears in the two cohorts was compared using the paired t test.p value of less than 0.05.Data extraction and computations were performed using the Perl programming language (version 5.12.2). Microsoft SQL Server 2005  was used for data linkage, processing, and sampling. All statistical analyses were performed using SPSS statistical software version 17.0 for Windows . Statistical significance was defined as a Overall, the cohort was observed for 1,371,910 person-years from 2000–2010. A total of 184,809 female RNs were identified. The median age at enrollment was 25.05 years , and the median follow-up period was 7.86 years . Nurse demographics are shown in A total of 2,077 cancers occurred within the observation period. Compared with the general population, female RNs had a higher overall cancer risk, with an SIR of 1.10 (95% CI 1.05–1.15). A subgroup analysis by age showed that an increased SIR was observed for nurses aged between 40–59 years . The results of the subgroup analysis are summarized in The cancer types most commonly observed in the female registered nurse cohort were breast (n = 827), followed by thyroid (n = 239), then colon and rectum (n = 174). An increased SIR was observed for breast , thyroid , lung and mediastinum , and uterine cancers  in the nurses. However, a decreased SIR was found for cervix  and liver and biliary tract cancers . SIRs for specific types of cancers are presented in p = 0.009).In all, 7,778 female RNs and 31,112 age- and sex-matched controls were selected from the LHID 2000. During the follow-up period, the Pap smear was used 5.80 times per person among the female RNs aged 35 years or older and 5.50 times per person among the controls aged 35 years or older  and a lower risk of liver and biliary tract cancer  were noted in our work. However, when taking into account multiple comparisons, the p-values of the above results did not have statistical significance  and the values may be chance findings.A higher risk of uterine cancer (SIR 1.23, 95% CI 1.01–1.49, The current study also found an increased SIR in female RNs aged 40–59 years. This is in agreement with the findings of two studies in which higher breast cancer risk was limited to postmenopausal nurses. There are several limitations to this large population-based study. First, many demographic variables were not available, such as socioeconomic status, family history of cancer, reproductive patterns including early age at first pregnancy and multiparity, diet, cigarette smoking, and alcohol use. Second, actual occupational information, such as nursing specialty, workplace, cumulative number of lifetime night shifts, lifetime average number of night shifts per month, and so on, could not be obtained. All the above factors have been linked with cancer risks in nurses In conclusion, this population-based retrospective cohort study found a higher cancer risk among female RNs compared with the general population. The risks of breast, lung, thyroid and uterine cancers specifically were higher and the risks of cervix and liver cancers were lower among female RNs. Further large, unbiased population-based prospective studies are needed to investigate the association between nurses and cancer risk.


Introduction. Visceral artery aneurysms are uncommon. Among them, splenic artery is the most common (46–60%). Most splenic artery aneurysms are asymptomatic and diagnosed incidentally, but its rupture, potentially fatal, occurs in up to 8% of cases. Presentation of Case. A female patient, 64 years old, diagnosed with a giant aneurysm of the splenic artery (approximately 6.5 cm in diameter) was successfully submitted to endovascular treatment by stent graft implantation. Discussion. Symptomatic aneurysms and those larger than 2 cm represent some of the main indications for intervention. The treatment may be by laparotomy, laparoscopy, or endovascular techniques. Among the various endovascular methods discussed in this paper, there is stent graft implantation, a method still few reported in the literature. Conclusion. Although some authors still consider the endovascular approach as an exception to the treatment of SAA, in major specialized centers these techniques have been consolidated as the preferred choice, reserving the surgical approach in cases where this cannot be used. For being a less aggressive approach, it offers an opportunity of treatment to patients considered “high risk” for surgical treatment by laparotomy/laparoscopy. Visceral artery aneurysms (VAAs) are uncommon. Among them, splenic artery is the most common 46–60%), followed by hepatic artery (20%) and superior mesenteric artery (5-6%) –6.–60%, fol Seventy-five percent of VAAs are asymptomatic. The most common symptom is pain in the upper left quadrant of the abdomen or in the epigastrium, radiating to left shoulder, nausea, and vomiting. , 2, 7, 8 The splenic artery aneurysms can be approached by laparotomy, laparoscopy, or endovascular techniques. The endovascular option, less invasive, has less morbidity and faster postoperative recovery –4, 8. Among the various endovascular techniques, covered stent implantation has been little reported in the literature. The authors present a case of splenic artery aneurysm treated by this method.A female patient, 64 years old, controlled hypertension, and 2 previous pregnancies, presented as main complaint episodes of mild pain in the epigastrium and left hypochondrium, evolving for about 12 months. Physical examination of the abdomen was nonspecific, with ill-defined pain on palpation of the mesogastrium and left hypochondrium. In the hospital of origin she was submitted to computerized tomography (CT) revealing a mixed density lesion with heterogeneous enhancement after injection of intravenous contrast (IV), relating to the pancreatic tail, measuring 6.5 cm in diameter . Considering the appearance of the lesion on CT, the diagnosis of splenic artery aneurysm was suggested and the patient referred to our institution to be submitted to an angiography for further diagnostic evaluation and possible endovascular treatment. The examination, performed by puncture of the right common femoral artery, with angiographic series of the abdominal aorta, celiac trunk, and splenic artery identified a patent and tortuous splenic artery, presenting a voluminous saccular aneurysm, with large neck, in the transition between their proximal and medium segments .Endovascular treatment was indicated and executed in a second moment. The procedure was carried out under sedation and local anesthesia. Initially a puncture of the right common femoral artery was performed and a 5F sheath was installed. The celiac trunk and then splenic artery were catheterized with a C2 5F catheter. With the aid of road map, a hydrophilic guide wire 0.035′′ × 260 cm was positioned beyond the aneurysm, until a terminal branch of the splenic artery.The angiographic catheter was advanced over the hydrophilic guide wire, which was replaced by an extra-rigid one, that once positioned, and allowed to exchange the catheter and the 5F sheath for an angiographic 9F sheath, which had its distal extremity positioned at the origin of the splenic artery. The patient received heparin intravenously. After a new road map, a 6 × 50 mm Viabahn stent graft (Gore) was released on the topography of the aneurysm neck. An 6 × 40 mm angioplasty balloon was used for expansion and stent accommodation . Intraoperative control arteriography showed patency of the celiac trunk and splenic artery and absence of aneurysm opacification .A manual compression of the puncture site was performed for 30 minutes and then compressive dressing. Immediately after the procedure the patient received 300 mg of clopidogrel. In the following days the patient received clopidogrel 75 mg/day and acetylsalicylic acid 200 mg/day and progressed without complaints or complications until discharge from hospital on the second postoperative day (POD).  On 30° POD, the patient remained without complaints or signs of complications. A CT with intravenous contrast was made on the 32nd postoperative day and confirmed thrombosis of the aneurysm sac and patency of the implanted stent, without evidence of splenic ischemia . The patient continues to be followed, with clopidogrel prescription until the sixth postoperative month and permanent use of aspirin. The episodes of abdominal pain ceased since the intervention.Visceral artery aneurysms (VAAs) have an incidence of 0.1% to 2% in the general population . The splenic artery originates from the celiac trunk and is responsible for vascularization of the spleen, pancreas, and the stomach . The fir The disease is more prevalent in women 75–87%) , 4, 5, 7–87% , 4, Most aneurysms are smaller than 2 cm and located in the middle or distal vessel segment , 3, 5–7. Seventy-five percent are asymptomatic , 5, 7, 8 Usually, SAAs are incidental findings. Due to the development of radiological methods, the frequency of the diagnosis is increasing , 3, 5–7. The high-resolution CT and arteriography can confirm the diagnosis of small SAA, but this last one locates more precisely, defines the size and the presence of other aneurysms, and still offers the possibility of therapeutic intervention. Generally, an arteriography is performed for planning of surgery intervention , 7, 9. The intervention is indicated for symptomatic aneurysms despite its diameter, to those larger than 2 cm, expanding aneurysms, women of childbearing age, pregnant women before the third quarter, in the presence of portal hypertension and if there is possibility of liver transplantation , 7, 10.  Treatment can be performed by laparotomy, laparoscopy, or by endovascular technique. The surgical procedure consists of ligation of the aneurysm neck or aneurysmectomy with or without reconstruction of the artery and may require splenectomy. The mortality of the endovascular treatment varies between 0.5 and 5% in elective situations and can reach 25% in emergency procedures [ The endovascular options include transcatheter embolization with coils , 3–11, a Transcatheter embolization with coil is indicated in cases with favorable anatomy, for narrow-necked saccular aneurysms and in the presence of adequate collateral circulation, once there is distal embolization risk , 8. This technique has been successfull in 85% of cases , 8. Some The injection of thrombin directly into the aneurysm, through percutaneous puncture, is a possibility when transcatheter embolization is not feasible or was not successful . Covered stents represent an option for patients with the proximal and distal splenic artery preserved . Aneurys The use of endoprosthesis reduces the incidence of ischemic complications caused by embolization of coils. Even so, splenic infarction is the main complication of this approach, although it occurs in up to 40%, normally does not require splenectomy , 5. The use of covered stents is considered a good alternative and promising technique, but so far few cases have been reported , 8, 10.  In addition to specific complications of each endovascular method described above, postembolization syndrome, contrast nephropathy, hematoma at the puncture site, and transient elevation of pancreatic enzymes may occur. The paralytic ileus, infectious complications, bleeding, and acute pancreatitis, most common in conventional surgical treatment, rarely occur. After endovascular treatment, periodic CT or Doppler ultrasound follow-up is recommended , 3.  Although some authors still consider the endovascular approach as an exception to the treatment of SAA , in majo For being a less aggressive approach, it offers an opportunity of treatment to patients considered “high risk” for surgical treatment by laparotomy/laparoscopy.


Corynebacterium pseudotuberculosis is a facultative intracellular pathogen and the causative agent of several infectious and contagious chronic diseases, including caseous lymphadenitis, ulcerative lymphangitis, mastitis, and edematous skin disease, in a broad spectrum of hosts. In addition, Corynebacterium pseudotuberculosis infections pose a rising worldwide economic problem in ruminants. The complete genome sequences of 15 C. pseudotuberculosis strains isolated from different hosts and countries were comparatively analyzed using a pan-genomic strategy. Phylogenomic, pan-genomic, core genomic, and singleton analyses revealed close relationships among pathogenic corynebacteria, the clonal-like behavior of C. pseudotuberculosis and slow increases in the sizes of pan-genomes. According to extrapolations based on the pan-genomes, core genomes and singletons, the C. pseudotuberculosis biovar ovis shows a more clonal-like behavior than the C. pseudotuberculosis biovar equi. Most of the variable genes of the biovar ovis strains were acquired in a block through horizontal gene transfer and are highly conserved, whereas the biovar equi strains contain great variability, both intra- and inter-biovar, in the 16 detected pathogenicity islands (PAIs). With respect to the gene content of the PAIs, the most interesting finding is the high similarity of the pilus genes in the biovar ovis strains compared with the great variability of these genes in the biovar equi strains. Concluding, the polymerization of complete pilus structures in biovar ovis could be responsible for a remarkable ability of these strains to spread throughout host tissues and penetrate cells to live intracellularly, in contrast with the biovar equi, which rarely attacks visceral organs. Intracellularly, the biovar ovis strains are expected to have less contact with other organisms than the biovar equi strains, thereby explaining the significant clonal-like behavior of the biovar ovis strains. Corynebacterium belongs to the CMNR group from the supra-generic group of Actinomycetes, which includes genera of great medical, veterinary, and biotechnological importance, such as Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus. These genera have specific features in common, such as a high DNA G+C content and a specific organization of the cell wall, which is mainly composed of peptidoglycans, arabinogalactans, and mycolic acids Corynebacterium was originally created to include Corynebacterium diphtheriae and other pathogenic species Corynebacterium diphtheriae, the causative agent of diphtheria Corynebacterium jeikeium, which is responsible for some nosocomial infections in humans Corynebacterium glutamicum, which is highly utilized in industrial amino acid production The genus Corynebacterium pseudotuberculosis is a facultative intracellular and pleomorphic member of the genus Corynebacterium. This bacterium is non-motile, although it does possess fimbriae, and it is the causative agent of caseous lymphadenitis (CLA) in sheep and goats C. pseudotuberculosis and Corynebacterium ulcerans has been suggested because these organisms are the only corynebacteria that produce the exotoxin phospholipase D C. pseudotuberculosis and C. ulcerans express the diphtheria toxin, which indicates a relationship between both species and C. diphtheriaerpoB gene C. pseudotuberculosis was based on morphological and biochemical characteristics ovis  from the biovar equi  C. pseudotuberculosis biovar ovis strains are responsible for causing the aforementioned infectious, contagious, chronic disease CLA, which is mainly characterized by the presence of caseous necrosis on the lymphatic glands or abscess formation in superficial lymph nodes and subcutaneous tissues C. pseudotuberculosis are also associated with the following: a high resistance to environmental conditions In sheep and goats, C. pseudotuberculosis was initially identified as causing CLA in sheep and goats, this bacterium has also been isolated from other species that exhibit different symptoms, including horses, cows, camels, buffalo, and even humans C. pseudotuberculosis between small ruminants and cattle does not appear to occur Although C. pseudotuberculosis infections in horses can display three different disease patterns: external abscesses (pigeon fever), ulcerative lymphangitis of the limbs, and a visceral form that affects the internal organs C. pseudotuberculosis have been described in cattle: pyogranulomatous reactions, abscess formation, mastitis, visceral commitment, and necrotic and ulcerative dermatitis on the heel of the foot, which is accompanied by edematous swelling and lameness C. pseudotuberculosis by houseﬂies or other diptera, in addition to transmission via skin contact between animals C. pseudotuberculosis and the pathogenic mechanisms of the other diseases caused by this bacterium et al.C. pseudotuberculosis genetic factors, the fagABC operon and the fagD gene, that play an important role in virulence; they are involved in iron acquirement and, therefore, enable the bacterium to survive in environments where iron is scarce. The fagABC operon and the fagD gene are found in a pathogenicity island along with the pld gene, which encodes phospholipase D (PLD) C. pseudotuberculosis; it promotes the hydrolysis and degradation of sphingomyelin in endothelial cell membranes, which increases vascular permeability and contributes to the spread and persistence of the bacterium in the host et al.C. pseudotuberculosis FRC41 strain, which is in agreement with the previously reported visualization of pilin structures in other strains of C. pseudotuberculosisAlthough the pathogenic mechanism of CLA is well understood, there remains a lack of information about the virulence factors of C. pseudotuberculosis infections in the broad spectrum of hosts and how genome plasticity is related to the symptom patterns, we performed pan-genomic comparative analyses of 15 C. pseudotuberculosis strains. In the following sections, we present the phylogenomic correlations between C. pseudotuberculosis and other corynebacteria. Furthermore, we describe the content and extrapolations of the following gene subsets from C. pseudotuberculosis: the “pan-genome”, which is the complete inventory of genes found in any member of the species; the “core genome”, which is composed of the genes that are present in all the species strains and that are thus important for basic life processes; and the “singletons”, which represent genes found only in a given strain. Finally, we provide insights into the specific subsets (singletons and the pan- and core genomes) of both biovars of C. pseudotuberculosis, ovis and equi, and we correlate these subsets with the plasticity of pathogenicity islands, virulence genes, and biovar-specific diseases.To better understand the different symptoms of C. pseudotuberculosis strains were retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/genbank/): 9 biovar ovis strains, which were isolated from sheep, goats, humans, llamas, antelopes, and cows, and 6 biovar equi strains, which were isolated from horses, camels, and buffalo  to green (high similarity) The Gegenees (version 1.1.4) software was used to perform the phylogenomic analyses at the genus level and to retrieve the GenBank sequences of all the complete C. pseudotuberculosis strain 1002 as a reference; B) the biovar ovis strains, using C. pseudotuberculosis strain 1002 as a reference; and C) the biovar equi strains, using C. pseudotuberculosis strain CIP52.97 as a reference. To calculate the pan-genome, core genome and singletons of the C. pseudotuberculosis species, we used EDGAR (version 1.2), multiple-strain genome comparison software that performs homology analyses based on a specific cutoff that is automatically adjusted to the query dataset C. pseudotuberculosis were retrieved from GenBank, and a new project was created on the annotation platform GenDB (version 2.4) to homogenize the genome annotations This section describes the analyses that were performed for all of the following three datasets: A) all strains, using C. pseudotuberculosis sequenced strain.The core genome was calculated as the subset of genes presenting orthologs in all the selected strains. The gene set of subject strain A was compared with the gene set of query strain B, and only genes with orthologs in both strains were members of core AB. The resulting subset was then compared with the gene set of query strain C, and the comparisons continued in a reductive manner. The pan-genome was calculated in the same way, but in an additive manner: the initial pan-genome was composed of strain A, and the non-orthologous genes of strain B were added to pan-genome A to create the pan-genome AB. The resulting set of genes was then compared with strain C, and the comparisons continued in the same manner. Finally, the singletons were calculated as genes that were present in only one strain and thus did not present orthologs in any other C. pseudotuberculosis were calculated based on permutations of all the sequenced genomes. The developments of the core genome and singletons were calculated using the least-squares fit of the exponential regression decay to the mean values. In contrast, the statistical computing language R was used to calculate the pan-genome extrapolation using Heaps’ Law by estimating the parameters κ and γ using the nonlinear least-squares curve fit to the mean values The developments of the core genome, pan-genome and singletons of ovis strains and the biovar equi strains, were classified by their Cluster of Orthologous Genes (COG) functional category as the following: 1. information storage and processing; 2. cellular processes and signaling; 3. metabolism; and 4. poorly characterized. To perform this analysis, the query sets of core genes were submitted to BLAST protein (blastp) similarity searches against the COG database, the proteins with E-values higher than 10−6 were discarded, and the best BLAST results for each protein were considered for the COG functional category information retrieval. Finally, the whole-genome comparison maps were visualized using the software CGView Comparison Tool (CCT) C. pseudotuberculosis strains 1002 and CIP52.97 to generate two genome comparison maps.The core genes of all the strains, including the biovar C. glutamicum strain ATCC 13032 was selected as the non-pathogenic organism of the same genus The plasticity of the 15 genomes was assessed using PIPS: Pathogenicity Island Prediction Software (version 1.1.2). PIPS is a multi-pronged approach that predicts pathogenicity islands (PAIs) based on common features, such as G+C content, codon usage deviation, high concentrations of virulence factors and hypothetical proteins, the presence of transposases and tRNA flanking sequences, and the absence of the query region in non-pathogenic organisms of the same genus or related species C. pseudotuberculosis strains and other species of the genus Corynebacterium, the Corynebacterium shared gene content was automatically determined using Gegenees. Then, the shared gene content was subtracted from all genomes and the resulting variable content of each genome sequence was cross-compared to generate a phylogenomic tree and to plot a heatmap  and non-pathogens (C. glutamicum and C. efficiens). Finally, the opportunistic bacteria C. jeikeium, Corynebacterium urealyticum and Corynebacterium resistensCorynebacterium variabileCorynebacterium aurimucosum formed a new branch To evaluate the phylogenomic relationships between  heatmap . AccordiC. pseudotuberculosis genomes clustered in two separate groups representing the two biovars of the species: biovar ovis, with more than 99% similarity according to the heatmap; and biovar equi, with a similarity ranging from 95% to 100%. Moreover, the heatmap indicated an almost clonal-like behavior of C. pseudotuberculosis compared with the C. diphtheriae species, which presented similarities raging from 82% to 100%.At the species level, the C. pseudotuberculosis strain 1002 (bv. ovis) and C. pseudotuberculosis strain CIP52.97 (bv. equi) as references. equi strains compared with C. pseudotuberculosis 1002. In C. pseudotuberculosis CIP52.97 are not specific to particular biovars, but rather are generalized. In both cases, the genomes that were classified as having the same biovar as the reference strain were clustered together in the outer circles, whereas the other strains were clustered in the inner circles.An alternative to assess the clonal-like behavior of species is the use of a circular genome comparison, which was performed with the software CCT. The results reveal regions of plasticity based on a chosen reference and, interestingly, plot the genomes from outer to inner circles by order of decreasing similarity. As shown in C. pseudotuberculosis, the pan-genome  was calculated using the abovementioned SRV method with the software EDGAR . However, when the pan-genomes of the biovars were calculated separately, a slightly different scenario emerged, in which the biovar ovis had a pan-genome of 2,403 genes, 1.14-fold the average total number of genes in each biovar ovis strain , and the biovar equi had a pan-genome with 2,521 genes, 1.23-fold the average total number of genes in each biovar equi strain .To achieve a global view of the genome repertoire of re EDGAR . The resC. pseudotuberculosis pan-genome was calculated by curve fitting based on Heaps’ Law, as represented by the formula −αn = κ * N, where n is the expected number of genes for a given number of genomes, N is the number of genomes, and the other terms are constants defined to fit the specific curve κ and γ were determined to be 2,043.06 and 0.11, respectively, by using the statistical computing language R. According to Heaps’ Law,1) an α≤1 is representative of an open pan-genome, meaning that each added genome will contribute some new genes and the pan-genome will increase, and 2) an α>1 represents a closed pan-genome, in which the addition of new genomes will not significantly affect the pan-genome. Using the formula α = 1−γ, we inferred that the pan-genome of C. pseudotuberculosis is increasing with an α of 0.89, indicating that it has an open pan-genome. The extrapolation of the pan-genome was also separately calculated for both biovars, ovis and equi. Although the biovar equi had the same α as the entire pan-genome (0.89), the biovar ovis had a much-higher α of 0.94.Additionally, the extrapolation of the C. pseudotuberculosis was calculated with the software EDGAR by defining the subset of genes that presented orthologs in all the strains using the SRV method. The subset of core genes of C. pseudotuberculosis contained 1,504 genes, which represented 54% of the entire pan-genome of the species . This subset may decrease with the addition of new genomes, as shown by the tendency of the core genes in the blue curve (n = κ * exp[−x/τ]+tg(θ), where n is the expected subset of genes for a given number of genomes, x is the number of genomes, exp is Euler’s number, and the other terms are constants defined to fit the specific curve. Interestingly, that formula can be used to predict that with a high number of genomes (x), the κ * exp[−x/τ] term will tend toward 0, where tg(θ) represents the convergence of the genome subset. Based on this observation, the core genome of C. pseudotuberculosis tended to converge to 1,347 genes, which represented 48% of the pan-genome of the species .The core genome of a species is defined as the subset of genes from the pan-genome that are shared by all strains. Here, the core genome of ue curve . Howeverovis and equi , we calculated the tg(θ)  the biovar equi genomes. The tg(θ) for all the genomes was 18.805, meaning that each sequenced genome added approximately 19 genes to the total gene pool of the species C. pseudotuberculosis, i.e., the pan-genome. However, the individual analysis of each biovar revealed a scenario in which each sequenced biovar ovis strain contributed ∼16 genes, but each sequenced biovar equi strain contributed ∼34 genes.The singletons of a strain are defined as the subset of genes that are absent from all the other strains and are thus responsible for increases in the number of genes in the pan-genome. We used the SRV method and EDGAR to calculate the subset of he tg(θ)  for the Intraspecies genome plasticity may result from several events, of which horizontal gene transfer is particularly important because it can cause the acquisition of blocks of genes , producing evolution by quantum leaps C. pseudotuberculosis biovar ovis strains 1002 and C231 (PiCps 1–7) C. pseudotuberculosis strain 1002 by further comparisons with C. pseudotuberculosis strains 316 and 258 (PiCps 8–11) C. pseudotuberculosis strains. Briefly, in addition to the previously identified 11 PAIs, we found 5 new PAIs, identified as PiCps 12–16. Although the 16 PAIs are present in all strains, they have different patterns of deletions, especially in the biovar equi strains  in C. pseudotuberculosis strain 31, and PiCps 7 and 15 harbor the spaD and spaA pilus gene clusters, respectively.In previous studies, seven PAIs were identified in  strains . PiCp1, ovis and equi biovar strains, similar to the phylogenomic tree created using Gegenees  present in each of the other strains. Using these data, we generated a phylogenomic tree of the strains with SplitsTree . The phyGegenees . A furthovis strains with respect to the PAI content (82–100%). Although biovar ovis showed a lower level of similarity to biovar equi with respect to the PAI content (78–91%), the former tended to present a similar deletion pattern in the same PAIs, independent of the strain. The biovar equi strains, however, contained large deletions and a lower level of similarity intra-biovar (77–88%) and also compared with the biovar ovis PAIs (62–74%)  .C. pseudotuberculosis is phospholipase D (PLD), which is encoded by the pld gene and is strongly associated with the spread of bacteria throughout the host cells fag operon, which also encodes important virulence factors that are responsible for iron acquisition in environments where this element is scarce pld gene was present in 14 of 15 strains, with similarities ranging from 98–100%. This finding was expected due to the important role of PLD during the disease course; pld mutants present a diminished ability to spread throughout the host As described previously, the major toxin of pld gene plays a pivotal role in pathogenesis, C. pseudotuberculosis strain 31 contains a frameshift mutation near the 3′-end of this gene that could decrease the ability of this strain to spread throughout the host. However, C. pseudotuberculosis strain 31 was the only strain in our dataset to present another important virulence factor, the diphtheria toxin gene (tox) (Cp31_0135). The diphtheria toxin (DT) is an important virulence factor in C. diphtheriae, in which the gene was acquired through lysogenization by corynephages, meaning that the tox gene is also present in a PAI in this species and can be horizontally transferred to other organisms. Briefly, the tox gene is regulated by the chromosomal iron-dependent repressor DtxR tox operator Although the C. ulcerans strains, where it causes diphtheria-like illness C. pseudotuberculosis strains isolated from buffalo in Egypt tox gene from C. pseudotuberculosis 31 has 560 amino acids in length, does not present any frameshift and has ∼96–97% similarity to the tox genes from several C. diphtheriae strains and from corynephage β, as well as ∼94–95% similarity to the tox gene from C. ulcerans 0102 (data not shown). Given the absence of the pld gene, the similarity of the tox gene from C. pseudotuberculosis to those from the C. diphtheriae strains, the conservation of all the domains and the presence of the gene in other strains isolated from buffalo in Egypt, the following question can be raised: is DT required for C. pseudotuberculosis to infect buffalo or is this feature more closely related to the geographical location (Egypt) than to the host?The exotoxin catalizes the transfer of adenosine diphosphate ribose (ADP-ribosylation) from nicotinamide adenine dinucleotide (NAD) to a histidine residue of elongation factor 2 (EF-2), called diphthamide. This process leads to inactivation of EF-2 and inhibits chain elongation during protein synthesis C. pseudotuberculosis CIP52.97, 316 and 258 (biovar equi strains) have been demonstrated equi strains, which indicates that these deletion events were specific to the mentioned biovar  and spaD (srtC-spaD-spaY-spaE-spaF) clusters, where srtA and srtB are the specific sortases of the spaA cluster; spaA, spaB and spaC encode the major, base and tip pilin proteins, respectively, of the spaA cluster; srtC is the specific sortase of the spaD cluster; spaD, spaE and spaF encode the major, base and tip pilin proteins, respectively, of the spaD cluster; and spaX and spaY have currently unknown functions. Additionally, a housekeeping sortase (srtD) is likely responsible for anchoring the pili to the cellwall According to work performed by Yanagawa and Honda in 1976 spaA and spaD gene clusters were located in PAIs  . Furthermore, the entire srtA-spaB-spaX-spaC region presented a low similarity to the same region in the biovar ovis strains, which was caused by small deletions, frameshift mutations and nucleotide substitutions  , which iitutions .spaD cluster of the biovar ovis strains, the major pilin gene spaD contains a frameshift in C. pseudotuberculosis P54B96 and PAT10; and in C. pseudotuberculosis 267, the tip pilin gene spaF also contains a frameshift. In biovar equi strains, the spaD gene of all the strains had 99% similarity to the spaD gene of the biovar ovis strains. However, C. pseudotuberculosis CIP52.97 contains a frameshift mutation in the specific sortase gene srtC. Furthermore, the base and tip pilin genes, spaE and spaF, respectively, of C. pseudotuberculosis strains 258, 316, 1/06-A and Cp162 are merged into the same reading frame.With respect to the rpoB gene tree generated by Khamis et al.C. jeikeium, C. urealyticum, C. kroppenstedtii and C. variabile cluster together in group 3, and C. aurimucosum appears in group 1. Moreover, C. glutamicum and C. efficiens cluster together in one branch, whereas C. pseudotuberculosis, C. diphtheriae and C. ulcerans appear closely related in another branch. Furthermore, C. ulcerans appears closer to C. pseudotuberculosis than to C. diphtheriae. Based on our results, we can deduce that although many variable regions exist between the pathogenic members of the genus Corynebacterium, these species tend to cluster together because they most likely share some core virulence determinants. Finally, although C. kroppenstedtii did not cluster with group 3, the other species were in perfect agreement with the rpoB analysis of Khamis et al.According to the C. kroppenstedtii are the absence of mycolic acids in the cell wall (due to the losses of a condensase gene cluster and a mycolate reductase gene) and a lipophilic phenotype  C. kroppenstedtii between the pathogenic and non-pathogenic species was in agreement with the lack of important virulence genes and the low pathogenic potential characteristic of C. kroppenstedtiiTwo striking characteristics of C. pseudotuberculosis compared with the C. diphtheriae species. Trost et al.C. diphtheriae genome, which is mainly related to the 57 genomic islands identified in this species. With respect to the clonal-like behavior of C. pseudotuberculosis, Bolt C. pseudotuberculosis typed by MLST, where 7 and 4 STs were associated with 64 and 9 strains of biovar ovis and equi, respectively. The few number of STs identified by MLST was in agreement with previous typing studies C. pseudotuberculosis are clonally related. Moreover, although there were 7 STs identified for biovar ovis strains, 6 and 7 of them were clustered in one sole eBURST group when considering single locus variants (SLVs) and double locus variants (DLVs), respectively; and, all the STs identified for biovar equi shared two alleles with the biovar ovis strains ovis and equi strains share a common evolutionary origin, although they are now relatively distinct genotypic clusters; and, 2) biovar ovis is a clonal-like organism. Our results with respect to this clonal-like behavior of C. pseudotuberculosis are also in agreement with PFGE data from Connor et al.C. pseudotuberculosis had a slightly higher α value of 0.89 compared with the C. diphtheriae α value of 0.69; and, from the total number of genes in the pan-genome of C. pseudotuberculosis , which is compact compared with that of the closely related species C. diphtheriae, which contains 4,786 genes At the species level, the heatmap indicated a clonal-like behavior of C. pseudotuberculosis displays some clonal-like behavior, the resulting α of 0.89 from the extrapolation of the pan-genome indicates that it has an open pan-genome. Moreover, considering that α is inversely proportional to the pan-genome increasing rate, in contrast to the C. diphtheriae α of 0.69, the α of 0.89 of the C. pseudotuberculosis pan-genome indicates that the latter is increasing at a slower rate. This slow increase is related to the low number of singletons (∼19) added to the pan-genome of C. pseudotuberculosis by each newly sequenced strain, whereas each strain of C. diphtheriae added ∼65 genes to the entire pan-genome α value are in agreement with the intracellular facultative behavior of this species. Because strictly intracellular organisms tend to have closed pan-genomes due to their limited contact with potential gene donors, an intracellular facultative organism such as C. pseudotuberculosis, even when it has different hosts, can be expected to have an α that is closer to 1 than that of C. diphtheriaeAlthough Aggregatibacter actinomycetemcomitans, in which one-third of the genes were categorized as “Poorly characterized” and approximately one-third were classified under “Metabolism” With respect to the core genome of all the strains, a large number of genes are related to the categories “Metabolism” and “Information storage and processing”. The “Information storage and processing” category contains genes involved in translation, ribosomal structure and biogenesis, RNA processing and modification, transcription, replication, recombination and repair, and other important functions; the “Metabolism” category contains genes involved in the production and conversion of energy, as well as the transport and metabolism of carbohydrates, amino acids, nucleotides, coenzymes, lipids, inorganic ions and secondary metabolites. Given the importance of the core genome, these two functional categories are expected to be highly represented in the analyzed subset. Finally, although a large number of “Poorly characterized” genes were identified in the core gene subset, this result is in agreement with previous core genome analyses of et al.C. pseudotuberculosis using PFGE and MLST, respectively, which enabled them to differentiate the equi and ovis biovars. On the phylogenomic tree, the C. pseudotuberculosis genomes also clustered in two separate groups representing the two biovars of the species: biovar ovis, with more than 99% similarity according to the heatmap; and biovar equi, with a similarity ranging from 95% to almost 100%. This result highlights the higher plasticity of C. pseudotuberculosis biovar equi compared with the biovar ovis strains, although this plasticity is not as high as that described for C. diphtheriae strains. Moreover, the same conclusion (regarding the relative plasticity of the two biovars) may be drawn from the number of singletons, in which the biovar equi strains presented higher levels of variability in the number of singletons, compared with the biovar ovis strains  was slightly higher than that of the biovar equi strains , most of the variable genes of the biovar ovis strains were acquired in blocks through horizontal gene transfer and are highly conserved throughout the entire biovar, as shown in equi strains presented great variability, both intra- and inter-biovar, in the content of the detected pathogenicity islands  and glycine (G) amino acids, capturing the cleaved polypeptides, polymerizing the monomers, and transferring the final product to the housekeeping sortase of the bacterium for its final incorporation into the cell wall With respect to the gene content of the PAIs, the most interesting finding is the high similarity of the pilus genes in the biovar ovis strains present a complete spaA cluster, the biovar equi were shown to present large deletions in this cluster. Because of the deletion of the major pilin SpaA in the biovar equi, the base and tip pilin monomers would be expected to be the only pilin structures that could attach to the cell wall in a non-polymerized manner. Moreover, the deletion of one of the specific sortase genes in biovar equi, srtB, could also interfere in the efficient cell wall-anchoring of these monomers, causing them to be secreted equi strains because these proteins contain small deletions and frameshift mutations. Altogether, the differences in the spaA cluster of the biovar equi strains could account for the different levels of host cell attachment compared with the biovar ovis strains and even among the biovar equi strains, as found in the C. diphtheriae species Although the biovar spaA clusters of the biovar ovis strains, the spaD clusters presented differences in three strains of this biovar. In C. pseudotuberculosis P54B96 and PAT10, a frameshit in the major pilin gene spaD impairs the coding of the entire protein and, thus, the polymerization of the pilin structure; and, in C. pseudotuberculosis 267, the tip pilin gene spaF also contains a frameshift. Although the tip pilin is not required for the polymerization of the pilin structure and adhesion to the host cell wall, its absence can slightly decrease the degree of adherence, which could reduce the spread of C. pseudotuberculosis strain 267 spaD cluster of the biovar equi strains, a frameshift mutation in the specific sortase gene srtC of C. pseudotuberculosis CIP52.97 prevents the polymerization of the pilin structure. Moreover, the base and tip pilin genes, spaE and spaF, respectively, of C. pseudotuberculosis strains 258, 316, 1/06-A and Cp162 are merged into the same reading frame. Overall, these results suggest that although C. pseudotuberculosis 258, 316, 1/06-A and Cp162 can polymerize the major pilin, C. pseudotuberculosis strain 31 is most likely the only biovar equi strain able to polymerize an entire pilin structure from the spaD cluster, whereas all the biovar ovis strains are likely capable of producing one or two types of pilin structures (spaA and spaD).In contrast to the high similarity found between the C. pseudotuberculosis biovar ovis strains likely contain a functional spaA cluster of pilus genes; only three strains  are unable to polymerize an entire spaD pilin structure . In contrast, all the biovar equi strains contain deletions, which render them unable to polymerize any spaA pilin structures; within this biovar, only C. pseudotuberculosis 31 appears to be able to polymerize an entire spaD pilin structure. Given the pivotal role played by pili in the processes of adhesion and internalization, the polymerization of complete pilin structures in the biovar ovis strains could be responsible for the great ability of these strains to spread throughout host tissues and penetrate cells to grow intracellularly ovis strains are expected to have less contact with other organisms than the biovar equi strains and to therefore show more clonal-like behavior. Finally, these results could also explain the distinct pattern of the diseases caused by C. pseudotuberculosis in horses, which involves ulcerative lymphangitis that rarely evolves to a visceral form C. pseudotuberculosis biovars equi and ovis truly present different patterns of pilin formation and, thus, variable degrees of host tissue adhesion, spreading and cell internalization.Summarizing, all the Figure S1C. pseudotuberculosis strains were aligned using C. pseudotuberculosis strain 1002 as a reference. From the inner to outer circle on A1, A2 and A3: the biovar equi strains Cp31, Cp1/06-A, CpCp162, Cp258, Cp316, CpCIP52.97; and, the biovar ovis strains CpC231, CpP54B96, Cp267, CpPAT10, CpI19, Cp42/02-A, Cp3/99-5, CpFRC41 and Cp1002. B, all the C. pseudotuberculosis strains were aligned using C. pseudotuberculosis strain CIP52.97 as a reference. From the inner to outer circle on B1, B2 and B3: the biovar ovis strains CpC231, Cp1002, CpPAT10, Cp267, CpP54B96, CpI19, Cp42/02-A, CpFRC41, Cp3/99-5, Cp1/06-A; and, the biovar equi strains Cp31, CpCp162, Cp316, Cp258 and CpCIP52.97. CDS, coding sequences; tRNA, transfer RNA; rRNA, ribosomal RNA; and PAI, pathogenicity island.Plasticity of PiCps 4, 5 and 9. A1 and B1, PiCp9; A2 and B2, PiCp4; A3 and B3, PiCp5. A, all the (TIFF)Click here for additional data file.


Giant sigmoid diverticulum (GSD) is a rare complication of diverticulosis. These lesions arise from herniations of the mucosa through the muscle wall which progressively enlarge with colonic gas to become large air-filled cysts evident on plain X-ray and CT scans. We present a rare case of a 72-year-old female presenting with abdominal distention, abdominal tenderness, and fever who developed a type 1 giant sigmoid diverticulum (pseudodiverticulum) that subsequently formed an intra-abdominal abscess and an accompanying type 2 diverticulum as well. The patient was treated with surgical resection of the diverticulum with a primary anastomosis and abscess drainage. The patient's postoperative course was uneventful. This case helps to support the need for the consideration of GSD in patients aged 60 and older with a history of diverticulosis and presenting with abdominal discomfort and distension. Diverticulosis of the colon is a common clinical entity affecting 35% of individuals over the age of 65 in the western world , 2. DiveA 72-year-old female with hypertension presented with complaints of abdominal distention, fever, decreased appetite, fatigue, and weight loss for 2 months. The patient denied any abdominal pain, cramping, vomiting, constipation, or dysphagia. On physical exam, the patient's abdomen was distended, soft, and diffusely tender with presence of normal bowel sounds. Laboratory results revealed a low hemoglobin value (7.7 g%) with a normal mean corpuscular volume, white blood cell count of 38,200 cells/mL with 21% bands, The patient was started on normal liver function, amylase, and lipase. Patient was started on ciprofloxacin, flagyl, and intravenous fluids. Plain X-ray film of the thorax was normal. A plain abdominal X-ray showed a complete filling of the upper abdomen by a giant radiolucent mass with an air-fluid level . ComputeIn 1943, Bonvin and Bonte reported the first case of a solitary air cyst of the peritoneal cavity attached to the sigmoid colon, subsequently called a giant diverticulum of the colon . Hughes All patients with giant sigmoid diverticula present a common clinical profile. They are generally elderly, with an age range of 40 to 90 years and most occurring after the age of 60. The few reported cases of a true colonic diverticulum occurred at a slightly younger age suggesting a possible congenital component , 12. SexA giant colonic diverticulum (GCD) is defined as a colonic diverticulum measuring 4 cm or larger, with a few described spanning more than 25 cm , 7, 10. McNutt et al. divide giant diverticula into 3 types based on their pathology , 7, 12. Type 1 22%) (pseudodiverticulum) is a type of preexisting pulsion diverticulum that arises gradually, without perforation, and its wall consists of chronic granulation and fibrous tissue with chronic inflammatory cells and remnants of muscularis mucosa [2% (pseudType 2 (66%) (inflammatory diverticulum) is secondary to a local perforation of the mucosa and submucosa which leads to a walled-off abscess cavity that communicates with the bowel lumen and acts as a one-way valve that allows the diverticulum to enlarge. It is lined by fibrous scar tissue without any normal intestinal layer , 7, 12.Type III 12%) (true diverticulum) contains all layers of normal bowel wall and is in continuity with the gut lumen, likely representing a communicating bowel duplication cyst [%  or with more indolent features. The most consistent physical findings of giant colonic diverticula are abdominal tenderness 87%)  or an ab7%  or anComplications of GSD occur in 15–35% of cases such as perforation, intra-abdominal abscess formation, diverticulitis, volvulus, small bowel obstruction (secondary to adhesion to the large bowel), adhesion with the bladder, and lower gastrointestinal bleeding , 15, 19.The investigations of choice for diagnosing GCD include a plain abdominal X-ray and an abdominal CT scan; both can accurately demonstrate the classical “balloon sign” of GCD . Plain aBarium enema can confirm the diagnosis when there is a communication between the diverticulum and colon , 3, 7. CComputed tomography is useful when barium enema fails to make the diagnosis. However, given several reported cases of perforation precipitated by barium enemas, CT scans have been shown to be adequate for the diagnosis of suspected GSD . CT scanThe differential diagnosis for giant colonic diverticulum includes volvulus , bowel duplication, Meckel and duodenal diverticula, infected pancreatic pseudocyst, emphysematous cholecystitis, emphysematous cystitis, vesicoenteric fistula, and intra-abdominal abscess , 12, 15.Resection of the diverticulum and adjacent sigmoid colon is the preferred treatment in uncomplicated GCD –10, 15. Giant sigmoid diverticulum is a rare manifestation of colonic diverticulosis. We present a rare case of a patient who developed a type 1 giant sigmoid diverticulum (pseudodiverticulum), which further developed an accompanying type 2 diverticulum (inflammatory). An intra-abdominal abscess formed as the sequelae of the inflammatory diverticula. Perforation is a known complication of GCD, as evidenced in the patient described above. It is likely that more than one pseudodiverticulum was present, one found incidentally and the other forming an abscess leading to the patients physical findings of abdominal distention.This case helps to support the need for the consideration of GCD in patients aged 60 and older with a history of diverticulosis and presenting with abdominal pain and distension. This case is unique in that it is a rare presentation of a GSD that presented as a type 1 diverticulum with accompanying type 2 diverticula. A left hemicolectomy, rectal Hartman's pouch, and colostomy were performed, and the patient was subsequently discharged without complications.


Nanomaterials with particle sizes <100 nm have been already applied in various applications such as cosmetics, medicines, and foods. Therefore, ensuring the safety of nanomaterials is becoming increasingly important. Here we examined the localization and biological responses of intranasally administered amorphous nanosilica particles in mice, focusing on the coagulation system.in vivo transmission electron microscopy analysis, hematological examination and coagulation tests.We used nanosilica particles with diameters of 30, 70, or 100 nm , and conventional microscale silica particles with diameters of 300 or 1000 nm . BALB/c mice were intranasally exposed to nSP30, nSP70, nSP100, mSP300, or mSP1000 at concentrations of 500 μg/mouse for 7 days. After 24 hours of last administration, we performed the In vivo transmission electron microscopy analysis showed that nanosilica particles with a diameter <100 nm were absorbed through the nasal cavity and were distributed into liver and brain. Hematological examination and coagulation tests showed that platelet counts decreased and that the activated partial thromboplastin time was prolonged in nSP30 or nSP70-treated groups of mice, indicating that nanosilica particles might have activated a coagulation cascade. In addition, in in vitro activation tests of human plasma, nanosilica particles had greater potential than did conventional microscale silica particles to activate coagulation factor XII. In nanosilica-particle-treated groups, the levels of soluble CD40 ligand, and von Willebrand factor which are involved in stimulating platelets tended to slightly increase with decreasing particle size.These results suggest that intranasally administered nanosilica particles with diameters of 30 and 70 nm could induce abnormal activation of the coagulation system through the activation of an intrinsic coagulation cascade. This study provides information to advance the development of safe and effective nanosilica particles. Over the past decade, the field of nanotechnology has developed remarkably, and nanomaterials (NMs), which are defined as objects with a diameter less than 100 nm, have been created for various applications. For example, NMs have already been included in many consumer products, such as cosmetics, food, and medicine, to improve their stability and efficacy . In partConcerns about the potential health risks of NMs have caused international organizations, such as the World Health Organization and the Organization for Economic Co-operation and Development, to call for an urgent and detailed evaluation of NMs’ safety. To create effective and safer NMs, information about the biodistribution and biological effects of NMs must be acquired. Recent studies have reported that NMs may have unpredicted biological effects that conventional-sized materials do not possess ,7. For ein vivo localization and biological effects of various sizes of nanosilica particles following intranasal administration in mice. In addition, we examined whether nanosilica particles could influence the coagulation system of mice. We expect that our results will contribute to the creation of safer NMs.Here, we have investigated the in vivo localization or biological effects, we used nanosilica particles with diameters of 30, 70, or 100 nm , and conventional microscale silica particles with diameters of 300 or 1000 nm . In a previous study, we confirmed that the mean secondary particle diameters of each of these types of silica particles are 39, 76, 106, 264, and 1136 nm  by dynamic laser scattering analysis [In this study, to assess the influence of the size of silica particles on analysis ,14, and in vivo localization of silica particles after intranasal administration  remained unchanged in all groups Figure a–d. The Previously, we revealed that a drastic decrease in platelets after systemic exposure to nSP70 could result from consumptive coagulopathy . TherefoNext, we examined the mechanism of abnormal activation of the coagulation system induced by nanosilica particles after intranasal administration. The blood coagulation system can be initiated by two pathways: an extrinsic cascade pathway, which is triggered by the release of tissue factor (TF) from the site of injury, and an intrinsic cascade pathway, which is triggered by activation of coagulant factor contacted with a negatively charged substance or accumulation of activated platelets to the collagen layer under the vascular endothelium . We examin vitro activation tests of coagulation factor XII using human plasma to confirm the presence of an intrinsic cascade pathway. One of our overall research goals is to contribute to the development of NMs that will be effective as well as safe for human exposure. To conduct a preliminary evaluation of the effects of nanosilica particles on humans, we used human plasma in the experiment, rather than mouse plasma. The in vitro activation tests showed that all sizes of the silica particles had the potential to activate coagulation factor XII, with activation apparently increasing as the size of the particles decreased  . Plateled Figure a. In addd Figure b,c. Theset al. suggested that the inhalation of amorphous silica particles in the workplace could induce silicosis [via intranasal administration [Merget ilicosis . In addistration ,24. Howeet al. and Lee et al. reported that the no-observable-effect level (NOEL) of colloidal silica particles is 489 μg/lung  [Warheit 0 mg/m3) ,26. Accoin vitro. In this study, we examined only the nasal cavity, lung, and liver, so we cannot comment as to whether nanosilica particles are localized in other tissues. However, other groups have reported that titanium dioxide nanoparticles with a diameter of 80 nm were localized in the brain after intranasal administration [et al. showed that copper nanoparticles with a diameter of 23.5 nm enter the olfactory bulb in the brain [in vitro studies have suggested that silica particles could be degraded in humans and animals after absorption [et al. showed that approximately 50% to 80% of a sample of microsilica particles was dissolved within 36 h in a solution of phosphate-buffered saline with 10% bovine serum, which is a simulated body fluid [et al. showed that hollow mesoporous nanosilica particles degraded when injected into human umbilical vein endothelial cells [We found that nSP30, nSP70, and nSP100 were located not only in the nasal cavity and lung but also in the liver , the number of opportunities for contact between the nanosilica particles and coagulation factor XII would have increased with decreasing particle size, thus ultimately leading to the activation of coagulation factor XII. In addition, we need to take into account not only the number of silica particles but also the surface area. The intrinsic cascade pathway involves various factors, such as factor XI and prekallikrein. Therefore, to reveal the mechanism of abnormal activation of the coagulation cascade by nanosilica particles, we need to examine the effects of nanosilica particles on other factors in intrinsic cascade pathways. Increases in the levels of sCD40L and vWF were observed in plasma from the nSP30- and nSP70-treated groups , which is similar to our silica nanoparticles, and organically modified silica (ORMOSIL) nanoparticles induce significant abnormal activation of the coagulation system via a different mechanism in in vitro studies [Contact activation of coagulation factor XII is one of the major factors of blood coagulation ,40, and,s Figure , meaning studies . More spIn summary, we revealed that intranasally administered nanosilica particles have the potential to induce abnormal activation of a coagulation cascade in mice. Recently, nanosilica particles have been used in food additives and cosmetics, and thus opportunities for such particles to be inhaled by workers during manufacturing are increasing ,42. FurtWe have shown that nanosilica particles with diameters of 30, 70, and 100 nm intranasally administered to mice were absorbed into the bloodstream and distributed into certain organs, such as the liver. The obtained results suggest that the activation of an intrinsic cascade pathway induced by nanosilica particles with diameters of 30 and 70 nm, both of which activated coagulation factor XII and platelets, could result in abnormal activation of the coagulation system. We expect that the findings of this study will contribute to the ongoing development of NMs that are safe for use in humans and animals.13, 2.8 × 1012, 9.5 × 1011, 3.5 × 1010, and 9.5 × 108 particles/mg for nSP30, nSP70, nSP100, mSP300, and mSP1000, respectively. Each type of silica particles was sonicated for 5 min and vortexed for 1 min before use.Amorphous nanosilica particles with diameters of 30, 70, and 100 nm, as well as microscale silica particles with diameters of 300 and 1000 nm  were used in this study. The particle numbers of the silica particles were 3.5 × 10BALB/c mice  were purchased from Japan SLC, Inc. . Mice were housed in a ventilated animal room maintained at 20 ± 2°C with a 12-h light/12-h dark cycle. Mice had free access to water and alfalfa-free forage . All of the animal experimental procedures in this study were performed in accordance with the National Institute of Biomedical Innovation and Osaka University Guidelines for the Welfare of Animals.Five BALB/c mice were intranasally exposed to a 20 μL aliquot (10 μL per nostril) of nSP30, nSP70, nSP100, mSP300, or mSP1000 at a concentration of 500 μg/mouse for 7 days. Twenty-four hours after the final intranasal administration, the nasal cavity, lung, and liver from two mice were excised and fixed in 2.5% glutaraldehyde for 2 h. Then, small pieces of tissue sample were washed with phosphate buffer 3 times and postfixed in sodium cacodylate-buffered 1.5% osmium tetroxide for 60 min at 4°C, block-stained in 0.5% uranyl acetate, dehydrated by dipping each sample through a series of ethanol solutions containing increasing concentration of ethanol, and embedded in Epon resin (TAAB). Ultrathin sections were stained with uranyl acetate and lead citrate. The stained samples were subsequently observed under an electron microscope .Twenty-four hours after the final intranasal administration of each type of silica particles, the nasal cavity, lung, and liver from three mice were excised and fixed immediately in 4% paraformaldehyde. These tissues were embedded in paraffin blocks and then sliced, and the slices were placed on glass slides. After hematoxylin–eosin staining, the slides were observed, and cell aggregation in the nasal cavity, microglial aggregation in the brain, and cell aggregation and rarefaction in the liver were classified into one of five grades .g for 15 min. The levels of ALT, ALB and BUN were determined in the plasma using a biochemical auto-analyzer .Twenty-four hours after the final intranasal administration of each type of silica particles, blood samples were collected from the heart using plastic syringes  containing 5 IU/mL heparin sodium. Plasma was harvested by centrifuging the blood at 1750 × Twenty-four hours after the final intranasal administration of each type of amorphous silica particles, blood samples were collected from the heart using plastic syringes (Terumo) containing 0.1 mM EDTA. Whole blood samples were analyzed with a VetScan HMII Hematology System  to determine the number of white blood cells, lymphocytes, monocytes, and platelets.g for 15 min. APTT and PT levels were determined at 37°C in a Clotek dry-block bath system  with APTT and PT reagents , respectively.Twenty-four hours after the final intranasal administration of each type of silica particles, the bleeding time for each mouse was measured by Duke’s method . Briefly2 and 0.1 mg/mL bovine serum albumin, pH 8.0) containing 2 mM t-butyloxycarbonyl-l-glutaminyl-glycyl-l-arginine-4-methyl-courmaryl-7-amide  in dimethyl sulfoxide. The initial rate of reaction was calculated by measuring the fluorescence intensity  every 5 min. The initial rate is given by Initial rate = (relative fluorescent unit of each measurement time – blank) / measurement time.One hundred microliters of human health plasma  and 100 μL of various sized silica particles (3.13 mg/mL) were mixed for 1 min at room temperature with an enzyme reaction solution  kits ; sCD40L: Mouse sCD40L ELISA ; vWF: Mouse von Willebrand Factor ELISA kit ).Differences among each group were compared by using Williams’s or Dunnett’s method after analysis of variance (ANOVA).The authors declare that they have no competing interests.TY and YY designed the study. TY, ST, TH, MU, and KI performed the experiments. TY and YY collected and analyzed the data. TY and YY wrote the manuscript. KN, YA, HK, ST, HN, KH, and TY provided technical support and conceptual advice. YT supervised the project. All authors discussed the results and commented on the manuscript. All authors read and approved the final manuscript.


Iberolacerta monticola, an endemism considered to be facing a high risk of extinction in several populations.The study of the factors that influence population connectivity and spatial distribution of genetic variation is crucial for understanding speciation and for predicting the effects of landscape modification and habitat fragmentation, which are considered severe threats to global biodiversity. This dual perspective is obtained from analyses of subalpine mountain species, whose present distribution may have been shaped both by cyclical climate changes over ice ages and anthropogenic perturbations of their habitats. Here, we examine the phylogeography, population structure and genetic diversity of the lacertid lizard Northwestern quadrant of the Iberian Peninsula.b (598 bp) loci, as well as at 10 nuclear microsatellite loci from 17 populations throughout the distribution range of the species.We analyzed the mtDNA variation at the control region (454 bp) and the cytochrome According to nuclear markers, most sampling sites are defined as distinct, genetically differentiated populations, and many of them show traces of recent bottlenecks. Mitochondrial data identify a relatively old, geographically restricted lineage, and four to six younger geographically vicariant sister clades, whose origin may be traced back to the mid-Pleistocene revolution, with several subclades possibly associated to the mid-Bruhnes transition. Geographic range fragmentation of one of these clades, which includes lowland sites, is very recent, and most likely due to the accelerated loss of Atlantic forests by human intervention.Iberolacerta. However, the changes operated during the Holocene severely compromise the long-term survival of those genetic lineages more exposed to the anthropogenic perturbations of their habitats.Altogether, the data fit a “refugia within refugia” model, some lack of pattern uniformity notwithstanding, and suggest that these mountains might be the cradles of new species of  The mean height of these mountains falls off considerably after the so-called Picos de Europa massif, and Sierras de Peña Prieta, Peña Sagra and Híjar, on entering Cantabria, which turns out to be a determinant feature of the eastern limit of the I. m. cantabrica distribution  must have been unsuitable for the species during the last ice age, a situation that most likely took place repeatedly during the cyclical climate changes of the Pleistocene ribution . WesternI. monticola, which may have been shaped both by Pleistocene glacial cycles and Holocene habitat fragmentation. But then we would like to address several issues concerned with the conservation of this species, by assessing the effects of population decline on the levels of deleterious mutation load and evolutionary potential to respond to an ever-changing environment.Molecular data openly question the subspecific rank of the population from Serra da Estrela and, conversely, point out an unexpectedly high degree of differentiation between the populations of Galicia and the Cantabrian Range, but fail to produce conclusive evidence on the location of putative glacial refugia, or the eventual dispersal routes from them I. monticola was designed to include a full representation of the known distribution of this species. Ethics approval of all procedures involving vertebrate animals is legally required under the Spanish legislation , which is a transposition of the European Directive 86/609/EEC. In agreement with article 18 and annexes VII and XI of the said Royal Decree, all animal procedures performed as part of the experimental work described in this paper have received prior and explicit approval from the competent authorities, defined in article 3e of the Law, and substantiated in the corresponding regulations of the Spanish autonomous communities. Thus, permissions for fieldwork and the concomitant experimental procedures were issued by the Xunta de Galicia, Junta de Castilla-León, and Principado de Asturias, in Spain, and, in application of an analogous transposition of the European Directive, by the Instituto da Conservação da Natureza in Portugal. Altogether, 316 lizards were genotyped, from 17 populations and 24 sites covering most of the species range , corresponding to position intervals 14,193–14,763 and 16,311–16,773, respectively, of the Lacerta viridis mitochondrial genome (GenBank acc. no. AM176577); for amplification details, see I. monticola populations, and then to 15 representative localities (http://dx.doi.org/s2479). This is made explicit in this section, since two of the sampling sites happened to harbor not I. monticola, but a different, so far undescribed, new Iberolacerta taxon (see Results). DNA extractions from tissue samples and genotyping were carried out as described elsewhere odonCode Alignerv. 3.5.7 (CodonCode Corporation). Newly reported sequences have been deposited in GenBank . A sequence from I. cyreni (constructed from EF121834 and AY267232) was used as outgroup to root the phylogenies.Sampling of es range . In evericrosatelliteToolKithobs) and expected under Hardy-Weinberg equilibrium (hHW), and numbers of alleles for each locus and population (an), together with the corresponding averages across loci for each population  statv. 2.9.3 ISf , and possible non-random allelic associations between pairs of polymorphic loci (2100 permutations). The significance level of all these tests was adjusted by the sequential Bonferroni procedure enepopv. 4.0.The Mottleneckv. 1.2.02 M-ratio software ottleneck, a two-phase mutation model with 5% of multi-step changes was assumed, using 10,000 iterations, and Wilcoxon signed rank tests of significance. For the M-ratio test, the bottleneck hypothesis was tested under three different population scenarios, corresponding to different values of the mutation parameter θ (4Neµ), as in θ = value estimated from the data, θ = 10, and θ = 0.5. See footnote at We checked the analyzed populations for evidence of recent bottlenecks using both Bi.e., with no a priori assumptions about populations) were taken to visualize the similarities among multilocus genotypes. First, by constructing a UPGMA tree of individuals microsatelliteanalyser (MSA) v. 4.05 eighbor program in PHYLIP v. 3.6 ega 4 enetixv. 4.05.2 Two geographically “blind” approaches  within I. monticola, we used both the method suggested in the original Structure paper, based on scoring mean log likelihoods penalized by one-half of their variance ), and the approach developed by Evanno et al. k values (Δk). We used an admixture model of genetic clustering with correlated allele frequencies, run for 50,000 generations after a burn-in of 100,000 generations, assuming that there were up to 14 possible partitions of the data (k = 2 to k = 16), and ran 10 parallel chains to estimate what number of genetic clusters had the highest probability. Membership coefficients of individuals to each of the clusters were plotted with Distructv. 1.1 At the population level, we first constructed a neighbor-joining tree of the populations, based on Cavalli-Sforza & Edwards chord distance, v. 4.0 ega 5 p-distances between sequences). MP trees were obtained using the Close-Neighbor-Interchange algorithm, with search level 3, and 100 random addition sequence replicates. The NJ tree was based on distances obtained by the Tamura-Nei model, allowing for a heterogeneous pattern among lineages and gamma-distributed rates among sites. The value of the gamma shape parameter (0.20) was obtained using a combination of ancestral sequence inference and maximum likelihood estimation amma , based on 10,000 bootstrap replicates. For ML, we first determined the model of sequence evolution that best fitted each of the four partitions of the data, namely (i) control region, (ii) 1st, (iii) 2nd, and (iv) 3rd codon positions of the cytb, by the Bayesian Information Criterion (BIC), using the metaPIGA v.2.1.3 development (http://www.metapiga.org) jModelTestmetaPIGA, choosing the best-fit model selected by BIC for each partition . We used loose Neighbor-Joining (20% range) to generate the starting trees, based on a HKY85 distance matrix, with rate heterogeneity across sites (shape parameter of the Γ distribution = 0.20). A ML test of the molecular clock hypothesis for the consensus topology thus obtained was carried out with Mega 5. As for the Bayesian phylogenetic inference, we used MrBayesv. 3.2 rBayes default priors until the standard deviation of split frequencies dropped below 0.01, and the potential scale reduction factor for all parameters lied close to 1.0. Two simultaneous, completely independent analyses starting from different random trees were run. For the Markov chain Monte Carlo (MCMC) sampling of the target distribution, three heated chains and one cold chain were used. The first 25% samples from the cold chain were discarded as burn-in. We used Bayes factor comparisons to test several topological hypotheses. Marginal model likelihoods were estimated by the stepping-stone method; strength of the evidence in favor of the better model was then assessed by the magnitude of the log-difference, following Kass and Raftery cyreni, and from 1.9 to 2.9 My on the monticola-galani-martinezricai split. These “hard” bounds are based on Carranza et al. (2004) Iberolacerta) fall well within the 95% confidence intervals produced by nearly all the methods and models tested by Hipsley et al. (2009) igTreev. 1.3.1 The topological congruence between the phylogenies obtained with the two markers was assessed by the incongruence length difference (ILD) test tcsv. 1.21 Since tree-building methods tend to resolve intraspecific gene genealogies poorly when the different mitochondrial types are separated by few mutations, and ancestral haplotypes are still present in the populations lignment Transformation EnviRonment (ALTER) The Aa priori continuous or fragmented distributions of the species  or DC) against geographic distance (linear or log-transformed) Π), obtained with Megav. 4, considered as a proxy of historical population connectivity r2. Geographical distance matrices were generated with GDMG v. 1.2.3 Genetic samples were separated into two groups, corresponding to cies see . Pairwisrlequinv. 3.5 FST over all loci) were used to compute the distance matrix. Significance tests were based on 1,000 permutations.Using the same two groups of populations, an analysis of molecular variance (AMOVA) in Acytb) produced essentially similar phylogenetic signals , so that they were concatenated to produce a two-gene data set . The null hypothesis of equal evolutionary rate throughout the ML tree was not rejected at a 5% significance level (P<0.878), and Bayes factor comparisons rendered very strong evidence in favor of a strict molecular clock (log-difference of 10 units), which was accordingly assumed for all the phylogenetic reconstructions. Two of the samples, a priori from I. monticola, showed an exceptionally large number of nucleotide differences as compared with the others . They correspond to individuals from the Sierra de Gistredo, at the SW of the Cantabrian Mountain Range, which is part of those largely independent lesser mountain groups found in the province of León, to the south of the Minho-Sil river basin, and east from the Ancares-Courel groups  and I. monticola (to the northwest). As shown at ca. 1.6 Million years ago , thus probably meriting the consideration of its full species or subspecies status (see discussion).The sequences of the two mitochondrial markers (CR and I. monticola sensu stricto (i.e. excluding Gistredo) is concerned, the mitochondrial results indicate that the most recent common ancestor (MRCA) of their different haplotypes dates back to ca. 0.9 Mya (0.67–1.25). The samples from the Cantabrian Mountain Range (CAMOR) give rise to two basal haplotype clusters, with strong statistical support, namely Xistral-Courel-Ancares in Galicia, on the one hand . The high diversity of the system formed by these mountains stands in sharp contrast with the unexpectedly low diversity and close similarity of the haplotypes observed in the main (eastern) part of the Cantabrian Range (clade II), in spite of the high census size and the large extension occupied by the corresponding populations, without apparent geographic discontinuity among them (ca. 0.4 Mya (0.19–0.62). The single haplotypes found at Pindo and Queixa-1, the two remaining northwestern populations, also began to diverge approximately 0.4 Mya (0.10–0.75). They are joined in a separate clade (VI) both by the MP and the Bayesian reconstruction, but its statistical support is relatively low , which is most closely related to clade V, although the statistical support for this topology (favored by a very strong log-difference of 23 units in Bayes factors comparisons) is not conclusive , which might be due to segregating null alleles, namely A5 at Queixa-L6A, C113 at Courel-L9A, and PB20 at Courel-L9B and Leitariegos. Applying a conservative criterion, these data were not taken into account for estimating genetic distances among populations, nor for evaluating the evidence of past bottlenecks (see later). However, it should be mentioned that the number of positive fIS estimates for each locus and sample nearly doubles the number of negative estimates, and that only two negative global FIS scores were obtained for the different samples . On the other hand, individuals from each locality generally group together in a UPGMA tree based on allele-sharing distances (tructure unveils a clear hierarchy in the partition of population samples among clusters (L(k) or Δk as optimization criterion , CAMOR+E and NWP groups become apparent , all the populations except Eume, Xistral, Ancares, Estrela and Leitariegos, show traces of recent bottlenecks.As regards the demographic history of these populations, the results of BST-based distances. This finding, together with the clear geographic subdivision of the genetic diversity of I. monticola (and the ensuing reduction of sample sizes), also hindered any attempt to infer the demographic history of its different lineages by mismatch-distribution analyses of pairwise mtDNA differences.In principle, all CAMOR populations should be connected by gene flow, since no apparent geographical or ecological barrier has been found within their range see , whereasP<0.000001). Lower values are related to differences among populations within groups , and still lower to differences among groups . Results from several one-way AMOVAs, together with different measures of genetic diversity in continuous and fragmented populations are shown in FST score (0.215 vs. 0.107), whereas the mean number of alleles per locus and expected heterozygosis at HWE are considerably lower in them . Actually, OCCIDENTAL_R shows lower average numbers of alleles in 10 out of the 11 loci analyzed, and lower heterozygosis in nine of them . In addition, on average, large populations show an excess of homozygotes over HW expectations in a significant proportion of loci ; this observation is consistent with mean FIS scores for these categories, which can be easily obtained from θ estimates derived from mean expected heterozygosis point out a reduction of 48% in the scores obtained from reduced populations (1.62), as compared with large populations (3.10), which, assuming equal mutation rates in both classes, amounts to an equivalent descent in their corresponding mean effective population sizes.Results from a two-way AMOVA indicate that most of the observed microsatellite variation is explained by differences within populations , karyology and osteology, will be necessary to establish the definitive taxonomic status of this population, together with microsatellite data to investigate its genetic structure (in preparation).Comparative phylogeographic analyses have shown strong genetic subdivisions, indicative of extended periods of population isolation, for many species and species complexes in the Iberian Peninsula, and especially for those with limited effective dispersal that live in mountains. This is most likely due to their survival throughout the Pleistocene ice ages in multiple refugia or sanctuaries, which tend to occur in mountain ranges icai see  and I. gions see . But theI. monticola into I. m. monticola at Serra da Estrela, and I. m. cantabrica elsewhere, falls too short to describe the actual pattern of diversification within this species. With the conspicuous exception of Serra de Ancares, which shows extensive mitochondrial admixture, most other populations are characterized by parapatrically distributed clades and subclades. According to their estimated coalescence times, this pattern could be the outcome of the relatively recent (re)colonization of these areas from different refugial populations, which, as in other well documented cases vs. Xistral (clade III) in the CAMOR group, or the low-altitude Artabrian Gulf vs. the moderate-high altitude populations of Serra da Queixa (clade V) in the OCCIDENTAL group, whose dating (mean node ages of 0.4–0.5 Mya) agrees quite well with the second distinct climate change of the Quaternary, the mid-Bruhnes event, about 0.43 Mya The mitochondrial results are consistent with the simultaneous vicariance of not less than four, and a maximum of six populations after the mid-Pleistocene revolution (about 0.9 Mya), one of the two major climate transitions of the Quaternary period, with drastic effects on the geographical distributions of many species I. monticola populations, might also be explained by a recent selective sweep, as suggested in other cases Lacerta schreiberi), will be necessary for a more thorough understanding of the evolution of these populations, which undoubtedly have experienced multiple episodes of admixture.The primarily east-west orientation of the Cantabrian Range may have facilitated the survival of populations simply by moving up or down mountains, as the general climate worsens or ameliorates Iberolacerta bonnali, which inhabits rocky habitats at 1,600–3,300 m asl, and whose current distribution is most likely a consequence of post-glacial range fragmentation after the end of the Last Glacial Maximum, would perfectly fit into the first category Iberolacerta monticola should be expected to lie somewhere in-between the two extremes. Its mitochondrial lineages seem to have Pleistocene origins, with diversification times estimated to be younger than 1.5 Mya, in contrast to other Iberian species characterized by the persistence of generally much older lineages through glacial cycles Iberolacerta do in fact exist, but they are nowadays associated to different, closely related species. In terms of the phylogeographic models of “S” (sanctuary) and “R” (refugia) species I. monticola  is actually similar to the genetic signature of “R” species .On general grounds, perhaps the main factor determining the level of genetic and regional subdivision of species inhabiting European mountains is the duration of the cycles of range contraction and expansion triggered by climate changes. For species dwelling at upper elevation  habitats, vicariance events are most likely associated to warm, relatively short interglacials, thus giving rise to shallow phylogenies. On the contrary, according to the displacement refugia model Iberolacerta, except I. monticola, are confined to mountains in the centre and NE of the Iberian Peninsula, probably because of the low thermal quality and scarcity of refuges of lower elevation environments at those regions I. monticolaI. monticola populations, which can be traced through the results of all our analyses of microsatellite variation, but particularly of FCA and Mantel tests. The significant association of Cavalli-Sforza chord distance (DC), but not FST-based indices, both with connectivity and with log-distance between populations, probably reflects the better performance of DC in recovering the topology of microsatellite trees I. monticola populations have low statistical support. This still on-going process of fragmentation has produced a patchy distribution of I. monticola in the NW of the Iberian Peninsula, which includes large, moderate and small sized populations, and both montane and lowland habitats (OCCIDENTAL group).In addition to the large effects of glaciations on the diversification of this species, the combined evidence from microsatellites and mitochondrial markers clearly shows that its current fragmented distribution at most nearby lowland populations has a very recent origin. All the species of I. monticola is so far not conclusive, and mostly based on differences in home-range sizes of the closely related species I. cyreniI. monticola, so that it is probably a contributing factor to the phylogenetic pattern observed at Estrela. But there are other, not mutually excluding possibilities as well. One plausible scenario is that after the few hundred thousand years that followed the vicariance episodes, during which the different mitochondrial lineages of I. monticola evolved in isolation, there would have been a colonization of Serra da Estrela from northeastern populations (Courel-Ancares). A mitochondrial “occidental” allele may have drifted by chance to high frequency at the population of Estrela, even though the nuclear genomes bear the trace of that secondary contact. If the above scenario is correct then there may be alleles from the Cantabrian lineage III still present elsewhere in the Serra da Estrela.Although mtDNA data suggest that the Estrela population is most closely related to other occidental populations (at Serra da Queixa and the Artabrian Gulf), thus implying that it has been separated from the CAMOR populations for nearly 1 million years, the microsatellite data do not support this conclusion, and indicate that it has been last connected with the populations of Courel and Ancares, at the western part of the Cantabrian Range, instead. Sex-biased gene flow is the most common explanation for such discordant patterns of biparental nuclear and maternal mitochondrial markers I. monticola show footprints of genetic erosion, such as a reduction of standing levels of genetic variation and, insofar as FST is accepted as a valid index of drift load FST values exceeding 0.2 I. monticola is threatened by extinction in these isolated sites seems justified, particularly so if considering that there has been scarcely time for these populations to reach mutation-drift equilibrium, so that the observed STF value of 0.215  is most likely to be an underestimate. However, there are other considerations that must be taken into account on this respect.Small isolated populations of I. monticola at pre-fragmentation stages may have been much lower than for an undivided population. It all depends on whether the species is expected to largely fit a metapopulation model or not. In species displaying low natal and breeding dispersal, such as many lizards FST scores, such as those observed among “continuous” populations of I. monticola, the expected reduction in inbreeding depression and mutation load of the metapopulation as compared with an undivided population can be quite important First, the frequency of segregating deleterious mutations and the mutation load of I. monticola show no evidence of overall excess of homozygotes, not even Lambre, a very small population that may have gone extinct by now Second, small isolates of endless formsThe tempo and mode of the building-up of genetic differences among populations that ultimately become new species may assume The Holocene may represent an inflexion point for the long-term survival of those genetic lineages more exposed to the anthropogenic perturbations of their habitats. Thus, successful heirs of many thousand years of evolution, with unique genetic characteristics forged in the engine of the cyclic climate changes of the Pleistocene, might not get through the next glacial stage, but become extinct relatively soon.Figure S1Bayesian phylogenetic tree of I. monticola populations based on mitochondrial sequences. The tree is rooted using I. cyreni as outgroup, and it includes representative sequences from I. martinezricai and I. galani. See legend of (DOC)Click here for additional data file.Figure S2Different estimates of genetic variation at microsatellite loci, for the different geographical samples of I. monticola.(A) Expected heterozygosity in HW equilibrium (He); bars correspond to standard deviations. (B) Average allelic richness (blue bars) and private allelic richness (red bars), corrected for sample size.(DOC)Click here for additional data file.Figure S3tructure.Results of the analysis for genetic clustering using S Colors correspond to the different clusters inferred by the analysis. The log likelihood of each assumed number of clusters (lnL) is shown, together with the % of independent runs that correspond to the represented partition of populations among clusters.(DOC)Click here for additional data file.Figure S4Identification of the most likely number of I. monticolatructurev. 2.1 populations by the analysis of microsatellite data with S. (A) Estimated log probability of data for the different number of inferred clusters (K); bars correspond to standard deviation, after 10 independent runs. (B) Rate of change in the log probability of data between successive k values (Δk). Both figures were obtained with the aid of Structure Harvesterv. 0.56.4, http://taylor0.biology.ucla.edu/struct_harvest/.(DOC)Click here for additional data file.Table S1Information about the populations and the genetic samples of I. monticola used in this work.(DOC)Click here for additional data file.Table S2Information summary of the DNA markers used in this work.(DOC)Click here for additional data file.Table S3Genetic variation at microsatellite loci in populations of I. monticola .(DOC)Click here for additional data file.