Comparing genetic diversity and demographic history in co-distributed wild South American camelids.

Vicuñas and guanacos are two species of wild South American camelids that are key ruminants in the ecosystems where they occur. Although closely related, these species feature differing ecologies and life history characters, which are expected to influence both their genetic diversity and population differentiation at different spatial scales. Here, using mitochondrial and microsatellite genetic markers, we show that vicuña display lower genetic diversity within populations than guanaco but exhibit more structure across their Peruvian range, which may reflect a combination of natural genetic differentiation linked to geographic isolation and recent anthropogenic population declines. Coalescent-based demographic analyses indicate that both species have passed through a strong bottleneck, reducing their effective population sizes from over 20,000 to less than 1000 individuals. For vicuña, this bottleneck is inferred to have taken place ~3300 years ago, but to have occurred more recently for guanaco at ~2000 years ago. These inferred dates are considerably later than the onset of domestication (when the alpaca was domesticated from the vicuña while the llama was domesticated from the guanaco), coinciding instead with a major human population expansion following the mid-Holocene cold period. As importantly, they imply earlier declines than the well-documented Spanish conquest, where major mass mortality events were recorded for Andean human and camelid populations. We argue that underlying species' differences and recent demographic perturbations have influenced genetic diversity in modern vicuña and guanaco populations, and these processes should be carefully evaluated in the development and implementation of management strategies for these important genetic resources.

Flunarizine in migraine-related headache prevention: results from 200 patients treated in the UK.

BACKGROUND AND PURPOSE: For over 20 years, as a group we have been using flunarizine in primary headache disorders. Flunarizine is widely used in Europe, but not licensed in the UK. In September 2014, the National Institute for Clinical Excellence published supportive guidelines for flunarizine use in migraine, based on randomized controlled evidence that it is as effective as propranolol and topiramate in adults. METHODS: We reviewed a cohort of adult patients (n = 200) treated with flunarizine from our practice. The clinical information of these patients, i.e. diagnosis, dose, efficacy, side effects and duration of treatment, was collected. RESULTS: The most common indication for flunarizine use was chronic migraine, followed by migraine with aura, sporadic hemiplegic migraine, familial hemiplegic migraine and new daily persistent headache with migrainous features. Flunarizine was generally effective, with only 24% (n = 47) of patients reporting no clinical effect. The most common dose used was 10 mg per day. Duration of treatment information was available for 39% (n = 78) of patients. Of these patients, 64% (n = 50) continued treatment for more than 1 year. Doses up to 15 mg were generally well tolerated, with only 10.5% (n = 21) of patients stopping treatment due to adverse effects. The most common adverse events were tiredness, mood change and weight gain. CONCLUSION: The data provide supportive evidence from tertiary headache practice in the UK for the use of flunarizine in migraine. The data encourages development of future guidance regarding flunarizine use in headache centres in countries where its use is not routine.

Y-chromosome and mtDNA variation confirms independent domestications and directional hybridization in South American camelids.

Investigations of genetic diversity and domestication in South American camelids (SAC) have relied on autosomal microsatellite and maternally-inherited mitochondrial data. We present the first integrated analysis of domestic and wild SAC combining male and female sex-specific markers (male specific Y-chromosome and female-specific mtDNA sequence variation) to assess: (i) hypotheses about the origin of domestic camelids, (ii) directionality of introgression among domestic and/or wild taxa as evidence of hybridization and (iii) currently recognized subspecies patterns. Three male-specific Y-chromosome markers and control region sequences of mitochondrial DNA are studied here. Although no sequence variation was found in SRY and ZFY, there were seven variable sites in DBY generating five haplotypes on the Y-chromosome. The haplotype network showed clear separation between haplogroups of guanaco-llama and vicuña-alpaca, indicating two genetically distinct patrilineages with near absence of shared haplotypes between guanacos and vicuñas. Although we document some examples of directional hybridization, the patterns strongly support the hypothesis that llama (Lama glama) is derived from guanaco (Lama guanicoe) and the alpaca (Vicugna pacos) from vicuña (Vicugna vicugna). Within male guanacos we identified a haplogroup formed by three haplotypes with different geographical distributions, the northernmost of which (Peru and northern Chile) was also observed in llamas, supporting the commonly held hypothesis that llamas were domesticated from the northernmost populations of guanacos (L. g. cacilensis). Southern guanacos shared the other two haplotypes. A second haplogroup, consisting of two haplotypes, was mostly present in vicuñas and alpacas. However, Y-chromosome variation did not distinguish the two subspecies of vicuñas.

Nociception-specific blink reflex: pharmacology in healthy volunteers.

BACKGROUND: The physiology and pharmacology of activation or perception of activation of pain-coding trigeminovascular afferents in humans is fundamental to understanding the biology of headache and developing new treatments. METHODS: The blink reflex was elicited using a concentric electrode and recorded in four separate sessions, at baseline and two minutes after administration of ramped doses of diazepam (final dose 0.07 mg/kg), fentanyl (final dose 1.11 µg/kg), ketamine (final dose 0.084 mg/kg) and 0.9 % saline solution. The AUC (area under the curve, µV*ms) and the latency (ms) of the ipsi- and contralateral R2 component of the blink reflex were calculated by PC-based offline analysis. Immediately after each block of blink reflex recordings certain psychometric parameters were assessed. RESULTS: There was an effect due to DRUG on the ipsilateral (F 3,60 = 7.3, P < 0.001) AUC as well as on the contralateral (F 3,60 = 6.02, P < 0.001) AUC across the study. A significant decrement in comparison to placebo was observed only for diazepam, affecting the ipsilateral AUC. The scores of alertness, calmness, contentedness, reaction time and precision were not affected by the DRUG across the sessions. CONCLUSION: Previous studies suggest central, rather than peripheral changes in nociceptive trigeminal transmission in migraine. This study demonstrates a robust effect of benzodiazepine receptor modulation of the nociception specific blink reflex (nBR) without any µ-opiate or glutamate NMDA receptor component. The nociception specific blink reflex offers a reproducible, quantifiable method of assessment of trigeminal nociceptive system in humans that can be used to dissect pharmacology relevant to primary headache disorders.

Out-of-focus background subtraction for fast structured illumination super-resolution microscopy of optically thick samples.

We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three-dimensional (3D) samples that allows the use of two-dimensional (2D) data processing. Indeed, obtaining super-resolution images of thick samples is a difficult task if low spatial frequencies are present in the in-focus section of the sample, as these frequencies have to be distinguished from the out-of-focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high-resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out-of-focus content from the raw images. After this cleaning step, we can obtain super-resolution images of optical sections in thick samples using a two-beam harmonic illumination pattern and a limited number of raw images. This two-step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two-dimensional.

Cross-amplification of nonspecific microsatellites markers: a useful tool to study endangered/vulnerable species of southern Andes deer.

Thirty-nine microsatellite loci that are highly conserved in red deer, sika deer, reindeer, Soay sheep, and other artiodactyls were tested in two vulnerable and endangered Neotropical deer (pudu: Pudu puda and huemul: Hippocamelus bisulcus) with the aim of producing a standardized set of markers that can be used successfully in noninvasive samples from these species. We also compared these nonspecific loci against eight polymorphic loci that were recently developed for huemul to determine whether the nonspecific markers could reflect the huemul's genetic variation that was observed with the specific loci. We identified 10 suitable loci, six of which constitute a standardized set for the two species and can be used to identify them in the absence of phenotypic data. The expected heterozygosity per locus for the panel of six loci ranged from 0.461 to 0.889 (average 0.665), and the maximum probability of identity value was 6.9x10(-6) and 3.2x10(-4) in pudu and huemul, respectively. This set of loci has potential applications in evolutionary, ecological, forensic, and conservation studies in pudu and huemul.

Manejo endovascular con stent recubierto de PTFE de lesión de arteria axilar producto de herida con proyectil de arma de fuego. Reporte de un caso / Successful endovascular management of shotgun injury with axillary artery lesion and arteriovenous fistula. Case report

Mundialmente el trauma vascular es un problema de salud pública. Las lesiones penetrantes de la arteria axilar son relativamente infrecuentes y solamente se han reportado muy pocos casos en la literatura mundial en relación al manejo endovascular de estas. Se hace reporte de un caso del manejo exitoso endovascular con stent recubierto de una lesión penetrante con proyectil de arma de fuego de la arteria axilar con la presencia de una fístula arteriovenosa (AU)

Vascular trauma is a public health problem worldwide. Penetrating injuries of axillar artery are relatively uncommon. Only a few cases have been reported in the literature of endovascular management of these lesions. We report a case of a successful endovascular management of an axillar artery lesion with arteriovenous fistula with a stent graft (AU)

Kidney graft outcome and quality (after transplantation) from uncontrolled deceased donors after cardiac arrest.

The use of uncontrolled deceased donors after cardiac arrest (uDDCA) has been developed in France to compensate for organ shortage. The quality of these kidneys remains unclear. We analyzed kidney graft function and histology from 27 uDDCA and compared them with kidneys from 30 extended criteria donors (ECD) and from 24 simultaneous pancreas kidney (SPK) donors as a control group of optimal deceased donors. Kidneys from ECD and SPK donors were preserved by static cold storage while kidneys from uDDCA were preserved by pulsatile perfusion. The uDDCA graft function at 3 years posttransplantation (estimated with MDRD and measured with inulin clearance) did not differ from that of the ECD group (eGFR 44.1 vs. 37.4 mL/min/1.73 m(2) , p = 0.13; mGFR 44.6 vs. 36.1 mL/min/1.73 m(2) , p = 0.07 in the uDDCA and ECD groups, respectively). The histological assessment of 3-month and 1-year protocol biopsies did not show differences for interstitial lesions between the uDDCA and ECD grafts (IF score at M3 was 30 vs. 28% and at M12 36 vs. 33%, p = NS). In conclusion, the results at 3 years with carefully selected and machine-perfused uDDCA kidneys have been comparable to ECD kidneys and encourage continuation of this program and development of similar programs.

Interstitial fibrosis evolution on early sequential screening renal allograft biopsies using quantitative image analysis.

Screening renal biopsies (RB) may assess early changes of interstitial fibrosis (IF) after transplantation. The aim of this study was to quantify IF by automatic color image analysis on sequential RB. We analyzed RB performed at day (D) 0, month (M) 3 and M12 from 140 renal transplant recipients with a program of color segmentation imaging. The mean IF score was 19 ± 9% at D0, 27 ± 11% at M3 and 32 ± 11% at M12 with a 8% progression during the first 3 months and 5% between M3 and M12. IF at M3 was correlated with estimated glomerular rate (eGFR) at M3, 12 and 24 (p < 0.02) and IF at M12 with eGFR at M12 and 48 (p < 0.05). Furthermore, IF evolution between D0 and M3 (ΔIFM3-D0) was correlated with eGFR at M24, 36 and 48 (p < 0.03). IF at M12 was significantly associated with male donor gender and tacrolimus dose (p = 0.03). ΔIFM3-D0 was significantly associated with male donor gender, acute rejection episodes (p = 0.04) and diabetes mellitus (p = 0.02). Thus, significant IF is already present before transplantation. IF evolution is more important during the first 3 months and has some predictive ability for change in GFR. Intervention to decrease IF should be applied early, i.e. before 3 months, after transplantation.

Specific targeting of the GABA-A receptor α5 subtype by a selective inverse agonist restores cognitive deficits in Down syndrome mice.

An imbalance between inhibitory and excitatory neurotransmission has been proposed to contribute to altered brain function in individuals with Down syndrome (DS). Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system and accordingly treatment with GABA-A antagonists can efficiently restore cognitive functions of Ts65Dn mice, a genetic model for DS. However, GABA-A antagonists are also convulsant which preclude their use for therapeutic intervention in DS individuals. Here, we have evaluated safer strategies to release GABAergic inhibition using a GABA-A-benzodiazepine receptor inverse agonist selective for the α5-subtype (α5IA). We demonstrate that α5IA restores learning and memory functions of Ts65Dn mice in the novel-object recognition and in the Morris water maze tasks. Furthermore, we show that following behavioural stimulation, α5IA enhances learning-evoked immediate early gene products in specific brain regions involved in cognition. Importantly, acute and chronic treatments with α5IA do not induce any convulsant or anxiogenic effects that are associated with GABA-A antagonists or non-selective inverse agonists of the GABA-A-benzodiazepine receptors. Finally, chronic treatment with α5IA did not induce histological alterations in the brain, liver and kidney of mice. Our results suggest that non-convulsant α5-selective GABA-A inverse agonists could improve learning and memory deficits in DS individuals.

Prevalence of Sarcocystis spp. in Argentinean cattle.

Sarcocystis cruzi, S. hirsuta and S. hominis are apicomplexan parasites that affect cattle worldwide with variable prevalence. The aim of the present study was to evaluate the prevalence of Sarcocystis spp. in Argentinean cattle comparing microscopic fresh examination and molecular methods. Blood, myocardium and loin samples were collected in five slaughterhouses from a total of 380 bovines. Origin of animals was representative of the major beef cattle production area of Argentina. Samples were analyzed by fresh microscopical examination, transmission electron microscopy (TEM), IFAT and PCR-RFLP. Thin walled sarcocysts corresponding with S. cruzi were found in 99.5% of heart samples. Sarcocysts were detected in 73.1% of loin samples; 71.5% had S. cruzi cysts and 23.1% had thick walled sarcocysts (S. hirsuta or S. hominis). TEM observation revealed the presence of characteristic S. hominis and S. hirsuta cyst walls in 7 and 1 loin samples respectively. Using IFAT, 379/380 animals had titers 25 or higher, showing a full agreement with fresh examination. Amplification products were detected in 35.5% (135/380) of loin samples; however Sarcocystis species could only be determined by RFLP in 29 samples. Agreement between fresh examination and PCR was low (Kappa value=0.262). This is the first report of S. hominis and S. hirsuta in Argentina. Further studies are needed to improve the sensitivity of molecular methods for species identification, especially for differentiation of S. cruzi and S. hirsuta from the zoonotic species S. hominis. The results of the present study and others focusing on sensitivity and specificity of Sarcocystis spp. diagnostic methods should contribute to improve food safety.

Interstitial fibrosis quantification in renal transplant recipients randomized to continue cyclosporine or convert to sirolimus.

Conversion from cyclosporine (CsA) to sirolimus at week 12 after kidney transplantation is associated with a significant improvement in renal function. The aim of this analysis was to investigate the effect of this conversion on interstitial fibrosis (IF), a hallmark of chronic allograft injury, in patients taking part in the CONCEPT trial. This multicenter, prospective, trial included 193 renal recipients randomized at week 12 to switch from CsA to sirolimus or to continue CsA, with mycophenolate mofetil. Routine biopsy with automated, quantified assessment of IF by a program of color segmentation was performed at 1 year in 121 patients. At 1 year, renal function was significantly improved in the conversion group as assessed by estimated GFR (MDRD) and measured GFR. Biopsy results, however, showed no between-group difference in percentage of IF. Calculated GFR at 1 year was significantly associated with the percentage of IF (p = 0.004, R(2)= 0.07). By multivariate analysis diabetic patients had more fibrosis than non-diabetic patients. In conclusion, although kidney transplant patients converted from CsA to sirolimus showed significant improvement in renal function, we found no difference of IF on 1-year biopsies.

Surgical management of hereditary colorectal cancer: surgery based on molecular analysis and family history.

The importance of colorectal cancer (CRC) is increasing. A proportion show a hereditary component, as in Lynch syndrome and Familial Adenomatous Polyposis, and a recently defined entity as well, namely, Familial Colorectal Cancer type X. The high probability to develop CRC in these groups may, at the time of recognition, change surgical management, including its timing or even the surgical technique. In some cases prophylactic surgery can play an important role. The possibility of using tools that allow recognition of the aforementioned syndromes, including microsatellite instability, immunohistochemistry for DNA mismatch repair system proteins, and especially their mutations, is on the basis of therapeutic strategies that differ from those employed in sporadic CRC cases.

Surgical management of hereditary colorectal cancer: surgery based on molecular analysis and family history

The importance of colorectal cancer (CRC) is increasing. Aproportion show a hereditary component, as in Lynch syndromeand Familial Adenomatous Polyposis, and a recently defined entityas well, namely, Familial Colorectal Cancer type X. The highprobability to develop CRC in these groups may, at the time ofrecognition, change surgical management, including its timing oreven the surgical technique. In some cases prophylactic surgerycan play an important role. The possibility of using tools that allowrecognition of the aforementioned syndromes, including microsatelliteinstability, immunohistochemistry for DNA mismatchrepair system proteins, and especially their mutations, is on thebasis of therapeutic strategies that differ from those employed insporadic CRC cases(AU)

Quantification of interstitial fibrosis by image analysis on routine renal biopsy 1 year after transplantation in patients managed by C2 monitoring of cyclosporine microemulsion.

BACKGROUND: Renal interstitial fibrosis (IF), the main histopathologic feature of chronic allograft nephropathy (CAN), may be an important surrogate endpoint for patient follow-up. IF is currently assessed by semiquantitative analysis, but automatic color image analysis may be a more reliable, reproducible method to evaluate IF. We performed a retrospective analysis to calculate IF on routine renal biopsies 1 year after transplantation. METHODS: Data were obtained from MO2ART, a prospective multicenter trial in which the cyclosporine microemulsion dose was adjusted based on C(2) levels. We included 26 patients in whom routine renal biopsy at 1 year was available from two centers. For each biopsy, a section was analyzed by a program of color segmentation image that automatically extracted green-colored areas characteristic of IF. Results were expressed as percent IF and grade namely grade I, <25%; grade II, 25% to 50%; and grade III, >50%. The results were compared according to clinical and biological data. RESULTS: The 26 patients had a mean IF score of 0.35 +/- 0.04. We observed 34.6% CAN grade I; 46.1%, grade II; and 19.2%, grade III. Serum creatinine at 3 years was greater in the higher grade of automated IF by repeated ANOVA. CONCLUSION: Automatic quantification of IF on routine biopsy at 1 year after transplantation was predictive of renal outcome. This technique may provide an interesting tool for the early diagnosis of CAN after renal transplantation.

An automatic image analysis approach to quantify stained cell cultures.

Counting cells in culture is a common task in biotechnology research and production. This process should be automated to provide fast and objective quantification. Flow cytometry is adapted to count cells in suspension. However, the morphological information and the spatial organisation of adherent cells are lost when cells are removed from culture. This paper proposes a methodology based on image analysis to quantify stained nuclei in culture. The protocol is composed of several steps: cell staining, automatic microscopy imaging, segmentation by an automatic algorithm including a classification approach, and computation of quantitative data that characterizes the growth of cells. An evaluation shows that the automatic process of counting provides results similar to human manual counting. The major interests of the proposed approach are the fully automated processing and preservation of cell shapes and positions in culture. More than two thousand culture conditions have been measured by this tool for various applications including optimization of cell culture media, improvement of the culture processes and measurement of drug toxicity.

Mitochondrial phylogeography and demographic history of the vicuña: implications for conservation.

The vicuña (Vicugna vicugna; Miller, 1924) is a conservation success story, having recovered from near extinction in the 1960s to current population levels estimated at 275,000. However, lack of information about its demographic history and genetic diversity has limited both our understanding of its recovery and the development of science-based conservation measures. To examine the evolution and recent demographic history of the vicuña across its current range and to assess its genetic variation and population structure, we sequenced mitochondrial DNA from the control region (CR) for 261 individuals from 29 populations across Peru, Chile and Argentina. Our results suggest that populations currently designated as Vicugna vicugna vicugna and Vicugna vicugna mensalis comprise separate mitochondrial lineages. The current population distribution appears to be the result of a recent demographic expansion associated with the last major glacial event of the Pleistocene in the northern (18 to 22 degrees S) dry Andes 14-12,000 years ago and the establishment of an extremely arid belt known as the 'Dry Diagonal' to 29 degrees S. Within the Dry Diagonal, small populations of V. v. vicugna appear to have survived showing the genetic signature of demographic isolation, whereas to the north V. v. mensalis populations underwent a rapid demographic expansion before recent anthropogenic impacts.