The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no “gold standard” for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study. A large variety and number of microbes can coexist with animals, including humans . For exaDNA extraction is one of the key steps for metagenomic analysis by NGS. For DNA extraction, the purity, yield, level of contamination, and scalability (including automation and cost) must be considered. In addition, low bias is desirable for DNA extraction from diverse flora. To date, silica column based methods, such as PowerSoil (MO Bio Laboratories)  or the QA series of studies have evaluated the performance of these different DNA extraction methods , 14–16. The oral microbiome is related to carious or periodontal disease, and more recently has been linked to systemic diseases, such as atherosclerotic cardiovascular disease  and AlzhThe aim of this study was to perform a comparative analysis of typical DNA extraction methods  on human oral samples. The methods were validated with 16S ribosomal DNA sequencing. Our observations suggest no major drawbacks among the three methods in terms of bias; however, small differences in DNA extraction efficiency were observed for some bacterial species.All relevant research protocols and procedures were approved by the Ethics Committee of Tohoku University School of Medicine, Sendai, Japan. All adult subjects provided written informed consent.In this study, we collected two sets of specimen, oral plaque samples provided from three healthy Japanese male volunteers (A–C) for proof-of-concept initial evaluation and following 12 samples for optimization and detailed evaluation.In the former case, subjects were confirmed by a dentist to have no severe caries, periodontal disease, or any other dental disorders. The ages of the subjects ranged from 35 to 41 years. The Decayed-Missing-Filled Teeth (DMFT) index of the subjects was 7.7, including wisdom teeth (max = 32). The detailed clinical data at the time of sampling was as follows: volunteer A, DMFT = 14 , number of teeth = 29 , and active caries = 1; volunteer B, DMFT = 9 , number of teeth = 29 , and active caries = 0; volunteer C, DMFT = 0 , number of teeth = 29 , and active caries = 0. The supragingival plaques were taken from all teeth by the subjects themselves using a sterilized plastic toothpick and dissolved with 0.5 mL of Tris-EDTA . Volunteer A provided three independent samples taken over four sequential days.In the latter case, 12 specimen including the volunteers A to C and additional nine volunteers consist of eight Japanese males and one Japanese female, provided supragingival plaque samples collected in the same manner as described above. The pooled plaque samples were mixed by vortex, subdivided into daughter tubes corresponding to the series of DNA extraction methods, and kept at −30°C until the total DNA was extracted.For the initial evaluation with specimens from three volunteers, the following three kits or methods were used for DNA extraction: the PowerSoil DNA Isolation Kit , the QIAsymphony SP instrument and QIAsymphony DSP Virus/Pathogen Mini Kit, and the boiling method. DNA extraction with the PowerSoil DNA Isolation Kit was performed according to the manufacturer’s protocol. Extraction using the QIAsymphony SP instrument and the QIAsymphony DSP Virus/Pathogen Mini Kit was executed according to the Complex200_V6_DSP protocol with the following modifications in plaque sample pretreatment: after brief centrifugation of the collected plaque sample , pellets were resuspended in 300 μL of Buffer ATL (QIAGEN) containing 2 μL of Reagent DX (QIAGEN) and incubated for 15 min at 56°C  and the Mixer Mill MM 400 for 10 min at 20 Hz. For comparison, we also incubated the plaque samples with 0.5% Tween 20, mutanolysin (0.3 U/μL), and lysostaphin (24 mU/μL) at 37°C for 60 min.Using the extracted oral plaque DNA as a template, a partial 16S rRNA gene of the bacterial community was amplified by two PCR steps as described in our previous study of the oral microbiome . In brieThe obtained raw sequences of bacterial 16S rRNA V4 amplicons were subjected to primary data processing, including low-quality tail base trimming, paired-end read assembly, and primer sequence removal, as described in our previous study . In brieThe assembled amplicon sequences were applied to sequence similarity-based taxonomic assignments to conduct species composition and comparative diversity analyses of oral bacteria based on our previous study . In brieIn the first sample set, a total 25 DNA samples were extracted from dental plaque from three volunteers using the three methods . The numThe purpose of this study is a fine comparison of the DNA extraction methods rather than a fine characterization of each flora. Therefore, we chose species-level exact annotation provided by Blast rather than flexible one by QIIME which is more conservative and finishes in higher taxon in absence of highly homologous references . In partr) were high (0.96–0.98) between the triplicates as emphasized by a surrounding solid line . In the second sample set, a total of 72 DNA samples from dental plaque were obtained from 12 volunteers and using the following six methods: PowerSoil, boiling, boiling with bead beating, tween, mutanolysin, or lysostaphin treatment. The number of average reads number obtained from the sequencer was 121,322 ± 25,973, whereas the number of average reads number that passed the QC standard was 117,862 ± 25,369. Most of these reads (69.3%) were successfully assigned to one of the reference bacterial 16S sequence contained in HOMD database . There wUsing PCoA analysis, 12 obvious clusters appeared to correspond to the 12 individual volunteers, consisting of six points that correspond to each DNA extraction method . This obDespite the concordance among the DNA extraction methods, the assigned OTUs derived from each method were not identical. To determine whether there were any specific OTUs whose extraction efficiency was high or low compared with others, we examined deviation by the methods in each OTU and each specimen whether it was five times more than the median (high extraction efficiency group) or was one fifth times less than the median (low extraction efficiency group). To qualify the data, OTUs were exempted from the analysis if all of the read popuration by each DNA extraction methods in an OTU were less than 100 ppm for high extraction efficiency group or if median was less than 100 ppm for low extraction efficiency group. The number of high/low cases from the 12 samples was tallied for each OTU . To findEnterococcus italicus (HOMD ID 803), TM7[G-1] sp. (HOMD ID 347) and TM7[G-1] sp. (HOMD ID 353) as an example , were species that were never missed in our study by all three methods. Therefore, all three methods we examined fulfilled the demands of a 16S metagenomics for an oral specimen. Although the bead beating method demonstrated the best performance in this study, it only showed a minimal advantage. Therefore, the choice of the DNA extraction method should be balanced with the additional cost and effort, especially in a passive parallel analysis with NGS.Among the six methods, boiling with bead beating showed a slightly better performance than the others, consistent with some previous studies –11. Howe example . DespiteWe evaluated three different DNA extraction methods for human oral plaques, each with their own advantages. Among them, the boiling method is not only superior in its simplicity, cost, and short handling time, but it is also reliable, based on its low bias. Performance of the boiling method has been also demonstrated for fecal microbes , suggestS1 Table(XLSX)Click here for additional data file.S2 TableThe number of volunteers  that showed high or low extraction efficiency for each OTU is shown. The definitions of high and low extraction efficiency are described in the main text.(XLSX)Click here for additional data file.


Introduction. Although many previous studies reported detection of chicken anemia virus (CAV) in Egypt since 1990, genomic characterization of this circulating CAV has not been published. In the present study, four nucleotide sequences of detected CAV were genetically characterized. Methods. These nucleotide sequences were obtained from commercial chicken flocks in two different locations of Egypt during 2010. The target region for sequencing was 675 bp nucleotide of partial coding region of VP1 protein. The nucleotide and deduced amino acid sequences of the detected CAV were aligned and compared to worldwide CAV isolates including commonly used vaccine strains. Phylogenetic analysis of these sequences was also carried out. Results. Our results showed that all the Egyptian CAV sequences were grouped in one group with viruses from diverse geographic regions. This group is characterized by amino acids profile 75I, 97L, 139Q, and 144Q in VP1. The phylogenetic and amino acid analyses of deduced amino acid indicated that the detected CAV sequences differ from CAV vaccine strains. Conclusion. This is the first report that describes molecular characterization of circulating CAV in Egypt. The study showed that the detected CAV, in Egypt are field viruses and unrelated to vaccine strains. Gyrovirus of family Circoviridae [Chicken anemia virus (CAV) is an economically important pathogen with a worldwide distribution. CAV is a small DNA virus with a closed circular, negative, single stranded DNA genome. It belongs to genusoviridae . The genoviridae  that alsoviridae , and VP3oviridae . VP1 shooviridae , 6.Infection with CAV constitutes a serious economic threat, especially to the broiler industry and the producers of specific pathogen free (SPF) eggs. The clinical signs are mainly noticed in young chicks of 10–14 days of age, which acquire the infection vertically. Chickens older than 2-3 weeks of age are also susceptible to infection but only develop a subclinical disease evidenced by poor vaccine response . The disIn Egypt, many previous publications have reported detection of CAV in chicken population (cited in –11); howTissue homogenates were collected from Sharkia and Kafr El Sheikh provinces. The homogenates contained thymus loops, bone marrow, bursa of Fabricius, liver, spleen, and intestines samples. Tissue homogenates of Sharkia province were collected from three commercial broiler flocks. The flocks ranged in age from 12 to 35 days representing different breeds and localities in the province. Tissue homogenate of Kafr El Sheikh Province was only collected from one commercial broiler flock aged 25 days. Five liver samples were also collected from a commercial broiler chicken flock aged 30 days from Minoufiya province. All flocks of collected samples were showing clinical signs and lesions indicative of CAV infection . The plaTissues were homogenized in a mortar with sterile sand and phosphate-buffered saline. Cell debris and sand were eliminated by centrifugation, and the supernatants were collected and stored at −70°C.μL Reddy-Mix, 18 μL PCR grade water, 1 μL of each primer, and 5 μL template. The amplification was performed under the following conditions: one cycle of initial denaturation step at 95°C for 15 min followed by 30 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 1 min representing denaturation, annealing, and extension steps, respectively, and finally one cycle of final extension step at 72°C for 5 min. The amplified products were analyzed using electrophoresis unit. They were loaded to 1% agarose stained by ethidium bromide, visualized under 304 nm ultraviolet , and photographed by a gel documentation system using Canon power-shot camera.DNA was extracted from the supernatant of liver and tissue homogenates by QIAamp DNA mini kit  according to the manufacturer instructions. The oligonucleotide primers 5′-GAC TGT AAG ATG GCA AGA CGA GCT C-3′ and 5′-GGC TGA AGG ATC CCT CAT TC-3′ were used to amplify a 675 bp DNA fragment of Vp1 ; from nuSequencing was performed in both directions with virus specific primers. Sequences were analyzed using BioEdit program . This prμL/egg). After 14 days, all embryos tissues were homogenized. DNA extraction and PCR amplification were carried out from the supernatant of these tissue homogenates in the same condition as described above. The whole isolation attempt was repeated once.Isolation of CAV from the tissue homogenate of the PCR-positive samples was attempted in embryonated SPF eggs through yolk sac inoculation . Tissue All affected birds were at age of 12 to 35 days. All affected flocks showed signs of anemia, generalized weakness, depression, pale comb and wattles, growth retardation, and high mortality rates. The postmortem examination revealed pale and enlarged liver and spleen, mild to severe thymus atrophy, and atrophied bursa of Fabricius.Analysis of PCR amplification of the extracted DNA from tissue samples by agarose gel electrophoresis indicated DNA bands of corrected size as expected with a length of 675 bp. The authenticity of PCR amplification was confirmed by the nucleotide sequencing. All of the liver samples from the flock in Minoufiya province showed negative results. Also, all SPF embryo homogenates were PCR negative.The four nucleotide sequences of detected CAV displayed a limited diversity. Total nucleotide variation among the sequences ranged from 1 to 29 nucleotides . They shThe four detected CAV sequences showed 98.9% to 100% identity with each other at the level of amino acid sequence . Also, oThe deduced amino acid sequences of the partial vp1 sequence of Egyptian CAV were compared with those of other sequences deposited in the GenBank. All detected CAVs in Egypt were grouped together in one group . The topIt was reported that CAV spread among chicken in Egypt since the early 1980s when several outbreaks occurred in many breeds . However75I, 97L, 139Q, and 144Q. Furthermore, the phylogenetic analysis indicated that all detected Egyptian CAV sequences belong to group II based on Islam et al. classification [According to a previous study , CAV isofication . The memInterestingly, Egy-3 CAV has a unique amino acid substitution at position 22 which is similar to CIA-1 and AN-China 23 isolates. It had N instead of H at this position. van Santen et al.  consider139Q and 144Q could affect the rate of replication or spread of infection in cultured cells. They have proved that presence of these two amino acids is associated with decreased rate of spread in cell culture. Based on this finding, all detected Egyptian CAVs could have a reduced ability to spread in cell culture.It was suggested that the genetic grouping of CAV strains might be related to different biological properties of these strains. Renshaw et al.  suggeste75I, 125L, 141L, and 144E to produce attenuation. No one of Egyptian detected CAV sequences displayed this combination.Also, all detected Egyptian CAVs had threonine in position 89 instead of alanine. It was suggested that this substitution is associated with attenuation . Others Although the evidence of use of live attenuated CAV vaccines occurring in the Egyptian poultry industry is not readily available, the topology of the phylogenetic tree showed that the CAV Egyptian sequences are in a distant relationship with different vaccine strains commonly used, for example, Nobilis P4, Del-Ros, and Cux-1. All Egyptian CAV sequences are included in group II, whereas vaccine strains are located in groups I and III.Failure of isolation of CAV in SPF eggs may be due to the low virus concentration in the inoculum or due to the presence of CAV antibodies in the inoculated SPF eggs. It was reported that SPF eggs might contain CAV antibodies . Also, tThere are some CAV sequences from Egypt (unpublished report) available in the GenBank with accession numbers HM460879.1 to HM460883.1. However, these sequences shared only around 124 nucleotides of VP1 with our sequence. So, the alignment with our sequences was difficult.This is the first report that describes the molecular characterization of circulating CAV in Egyptian poultry farms. Although the number of samples was limited, our phylogenetic analysis revealed that CAV strains detected in Egypt were in one group that showed similarity to several strains all over the world, particularly to those circulating in African and South American continents. The results presented in this study clearly showed that the detected CAV sequences belong to field viruses and are not vaccine strains.


P < 0.01), as well as lower neuropsychological test scores including verbal and nonverbal memory  compared with normal control. Spearman rank correlation analyses did not disclose significant correlations between AN diffusion metrics and neuropsychological test scores in the patient group, whereas ranked scatter plots clearly demonstrated a dichotic sample distribution between patient and control samples. In summary, our study highlighted the potential compensatory role of the AN by increasing thalamocortical connectivity within the Papez circuit because memory function declines in early iNPH when early shunt treatment may potentially reverse the memory deficits.In this study, we aimed to investigate the reactive changes in diffusion tensor imaging (DTI)-derived diffusion metrics of the anterior thalamic nucleus (AN), a relaying center for the Papez circuit, in early idiopathic normal pressure hydrocephalus (iNPH) patients with memory impairment, as well as its correlation with the patients’ neuropsychological performances. In total, 28 probable iNPH patients with symptom onset within 1 year and 17 control subjects were prospectively recruited between 2010 and 2013 for this institutional review board-approved study. Imaging studies including DTI and a neuropsychological assessment battery were performed in all subjects. Diffusion metrics were measured from the region of the AN using tract-deterministic seeding method by reconstructing the mammillo–thalamo–cingulate connections within the Papez circuit. Differences in diffusion metrics and memory assessment scores between the patient and control group were examined via the Mann–Whitney U test. Spearman correlation analyses were performed to examine associations between diffusion metrics of AN and neuropsychological tests within the patient group. We discovered that early iNPH patients exhibited marked elevations in fractional anisotropy, pure diffusion anisotropy, and axial diffusivity (all  The AN has received considerable attention in recent investigations, particularly in studies regarding memory formation and processing, because of its unique morphological and clinical implications.10 Here, memory deficit in iNPH may alter the microstructural properties of AN; these alterations are a manifestation of neural plasticity modulated by compensatory recruitment via the Papez circuit in response to declined memory function. Such changes can be quantitatively measured by assessing diffusion metrics derived from diffusion tensor imaging (DTI). DTI is an magnetic resonance imaging (MRI) technique that enables noninvasive measurements of microstructural changes within the brain by characterizing their tensor magnitude, orientation, and anisotropy; thus, DTI offers insights into the microstructural integrity of both gray and white matter.13 Clinically, diffusion metrics such as fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (Da), and radial diffusivity (Dr) have been applied in the quantitative analyses of various brain diseases and neuropathological conditions, such as ischemic stroke, brain tumor, neuroinflammation, and neurodegenerative diseases; among these metrics, DTI may play a supportive role in diagnostic assessment, treatment planning, and prognostic evaluation.14 In particular, diffusion analysis in iNPH has provided valuable information in terms of differential diagnosis and clinical assessment of symptoms.15 Moreover, FA and MD have been used to evaluate cytoarchitectural changes within subcortical regions related to motor function by the pathogenesis of iNPH.19 In this study, we aimed to assess the changes in diffusion metrics within the AN of early iNPH patients with memory impairment, as well as determine whether such changes are correlated with their neuropsychological performances in terms of memory function.A central component of the Papez circuit is the anterior thalamic nucleus (AN), which is a specific relay nucleus of the thalamus that plays a pivotal role in modulating emotion and memory function.20 Patient data were excluded from analysis because of (a) incomplete neuropsychological tests; (b) none or incomplete imaging examinations; (c) history of strokes, head trauma, or other neurological diseases; and (d) severe motion-related imaging artifacts. In conclusion, data from 24 patients were included for analysis. For healthy control subjects, the exclusion criteria were as follows: history of neurologic or psychiatric disorders, head trauma or neurosurgery, and history of significant systemic diseases, including cardiovascular disease, diabetes mellitus, and renal function impairment. All subjects underwent neurological testing and clinical evaluation prior to MR imaging and neuropsychological assessments. Detailed demographic and clinical information of participants are summarized in Table This prospective study (conducted between 2008 and 2013) was approved by a local institutional review board  with written informed consent obtained from all participants. A total of 28 iNPH patients  and 17 healthy control subjects  matched for age, sex, and years of education were recruited for this study in Tri-Service General Hospital, Taipei. Years of education (mean years ± standard deviation) were 8.54 ± 4.9 years for iNPH patients and 9.03 ± 5.29 years for control subjects. Patients were diagnosed as probable iNPH within 1 year of onset of symptoms by a clinical neurologist (G.S.P.) on the basis of the diagnostic criteria as per the Japanese guidelines for management of iNPH.b value of 0 and 1000 s/mm2, 40 sections, 4 mm-thickness without intersection gaps, field of view of 240 × 240 mm2, 128 × 128 matrix zero-filled to 256 × 256, and 2 signals acquired. The resulting DTI voxel size was 0.9375 × 0.9375 × 4.00021 mm3. The total acquisition time on each subject was around 15 minutes. All image data were examined by 2 neuroradiologists independently  for quality control.MRI sessions were performed with a 1.5 T MR unit . Conventional sequences included transverse T1-weighted , sagittal T1-weighted , transverse T2-weighted fast spin-echo , and transverse fast fluid-attenuated inversion-recovery . The slice thickness for transverse sequences was 5 mm with an intersection gap of 1.5 mm and a 256 × 256 matrix. DTI data were acquired using pulsed gradient spin-echo diffusion-weighted echo-planar sequence with 15 noncollinear diffusion directions plus one B0 image. Sensitivity-encoding parallel imaging with an acceleration factor of 3 was applied to reduce susceptibility distortions. Other parameters were as follows: TR/TE 10,000/85.3 ms, flip angle of 90°, 21 The Wechsler Adult Intelligence Scale III (WAIS-III) was used to assess verbal intelligence quotient (IQ). Visuoconstructive ability, naming, and verbal fluency were assessed with the Chinese version of controlled oral word association test (COWAT). The test results were reported for WMS-WL in terms of standardized scale scores , RCFT and COWAT using standardized Z scores , and WAIS-III via conventional computation procedures.Standardized neuropsychological tests were performed after each MRI examination session within the same day by a clinical psychologist  in the Department of Psychiatry, Tri-Service General Hospital. Neuropsychological measures implemented in this study were focused on evaluating memory function, including both verbal and nonverbal aspects, as well as visuoconstructive ability, naming, and verbal fluency. We evaluated 10 neuropsychological scores. All tests were administered according to the published guidelines. The Chinese version of word list subtest of the Wechsler Memory Scale III (WMS-WL) was used to assess verbal memory in terms of immediate recall, 20 minutes delayed recall, and recognition memory. The Rey complex figure test (RCFT) was used to assess nonverbal memory by means of immediate recall, 20 minutes delayed recall, and recognition memory.22 and then placed in reference to each subject's T1-weighted images. With the proper placement of regions of interest (ROIs) for MB as seed masks and those of ATR as inclusion ROIs, the entire mammillo–thalamo–cingulate projection pathway was produced with high intersubject consistency without the use of exclusion ROIs. The delineation of AN is based on both the tractography results and basic knowledge on human thalamic anatomy. Given that the AN is located at the anterior–superior region of the thalamus, the region of AN (4 × 5 pixels) can be defined by tracking the course of tractography to the corresponding level of the thalamus. After this step, diffusion metrics were then extracted from the region of AN, including FA, pure diffusion anisotropy (q), pure diffusion magnitude (L), MD, Da, and Dr. q and L are mathematical indices calculated from eigenvalues, and they constitute the numerator and denominator of FA, respectively. The final output values of each diffusion metric were averaged between both cerebral hemispheres, as laterality in terms of neuropathological involvement and disease severity are generally not expected nor commonly reported among patients with iNPH. A similar analytical approach was adopted by a previous study in iNPH.23DTI data were extracted and analyzed  on an independent workstation. Eddy current correction and skull-striping procedures were conducted with FSL 5.0.6 . Diffusion tensor was then calculated to derive whole-brain pixel-wise FA maps. Fiber tracking was performed using MRtrix software package  on the basis of standard deterministic streamline tractography  to identify the region of the AN on each subject's original DTI image space. The level of thalamus was determined by both its fixed location within the diencephalon and relative position with adjacent structures, most notably the internal capsule, basal ganglia, lateral and third ventricles, and the midbrain of the brain stem. Two deterministic seeds Figure , namely,U test. Spearman's rank correlation analyses were performed to evaluate correlations between neuropsychological test scores and diffusion metrics derived from the AN within the patient group only. Statistical significance was set at P < 0.05 for all analyses.Nonparametric statistical methods were implemented by a clinical statistician . All statistical computations were performed using Statistical Package for the Social Sciences, Version 20.0 . Between-group differences in diffusion metrics and neuropsychological test scores were examined using Mann–Whitney t-test .The degree of change in diffusion metrics within the AN of both hemispheres was consistent in both groups of subjects. No significant differences were found by statistical analyses with student U = 88, P = 0.002), q value , and Da . No significant between-group differences were found among the other diffusion metrics, including MD (P = 0.459),L value (P = 0.195), and Dr (P = 0.731).Marked differences were found in diffusion metrics of the AN between the patient and control groups Figure . CompareP < 0.01)  Table . OverallP > 0.05). However, ranked correlation scatterplot containing data from the patient and control groups revealed dichotic sample distributions  has been demonstrated to significantly improve patient memory performance.41 Our patient group also exhibited marked impairments in recognition memory. This finding may correspond the study by Saito and colleagues, who suggested that recognition memory is more involved in iNPH than previously documented,4 but further investigations are warranted because our neuropsychological battery design did not incorporate tests that specifically evaluated recognition memory.42 Although significant differences in memory performances were found between the 2 groups, correlation analyses between the diffusion metrics within the AN of the patient group and their corresponding neuropsychological test scores did not reach statistical significance. Such incongruities could be partly explained by the limited sample size of our patient group, their individual variations in memory performance, and the neuropsychological tests we adopted to evaluate our subjects. At present, no general consensus has been reached by the international community in the recommendation of specific neuropsychological tests that can best assess the severity of memory deficits among patients with iNPH.41 Although several commercially available neuropsychological tests were implemented in our study for the assessment of memory, the lack of wider and finer discrepancies in memory performance scoring, presumably caused by the design of the scoring scales, may have led to the suboptimal ranking of memory performance and potentially hindered the outcome of our study analyses. Such considerations should be taken by future studies aimed at evaluating similar features among patients with iNPH. Nevertheless, the overall distribution of data points from both the patient and control groups, when examined collectively on the ranked correlation scatterplot, allowed for discrete visual separation of patients from the normal controls. This plot feature may assist in the visualization and gross evaluation of the severity of memory impairment among future patients.Memory impairment in iNPH patients was previously reported in terms of verbal and nonverbal memory recall, fluency, and visuoconstructive ability, with the deficits in recognition memory being less severely affected.44 however, the role of the thalamus has not been well explored. The thalamus is known to play a role in reactive upregulation and reorganization of its associated circuits in various diseases. For example, Liu et al found that changes in thalamic diffusivity, notably elevated FA, can reflect increased neuronal activity of thalamocortical pathways in MDMA (or ecstasy) drug abusers.45 Tang et al also reported that increases in functional thalamocortical connectivity are correlated with exacerbations of neurocognitive functions in patients with mild traumatic brain injury, which may be associated with thalamic functional reorganization and thalamic network compensatory mechanisms.46 From the perspective of the AN, in addition to its capacity to propagate memory-related hippocampal theta rhythms to cortical structures for higher processing,47 it modulates neuroplastic properties of its associated projection fibers.48 Given the cardinal location of the AN within the Papez circuit and its extensive reciprocal interconnections with other cortical and subcortical structures involved in memory processing, the integrity of the AN is crucial in maintaining synaptic transmission and synaptic plasticity within the anterior thalamic circuitry, as well as supporting the network via dynamic modification of afferent neuronal inputs in reference to preceding activities within the circuit.50 These notions provide valuable insight into the potential role of the AN in the disease process of iNPH underlying memory impairment. Considering that the integrity of many subcortical structures within the Papez circuit, including the hippocampus,48 fornix,51 and the ATR,52 was previously found to be impaired among patients with iNPH, the AN may undergo reactive microstructural changes at the synaptic level to compensate for the decline in memory performance among iNPH patients and ultimately result in elevated FA and q value. This form of neural compensatory mechanism was previously observed in the red nucleus of the midbrain, where increased FA suggests synaptic reorganization during compensatory mechanisms within the corticorubrospinal tract during functional motor recovery among stroke patients; moreover, increased FA is significantly correlated with patients’ motor function score.31 Although our current correlation analyses were inconclusive, our preliminary findings may shed light on the potential compensatory role of AN in the disease process of iNPH. Thus, our findings deserve further investigations by future studies.The underlying neuroanatomical bases of memory impairment in iNPH are currently unclear. Impairments of the frontal-subcortical projection fibers have been suggested to be involved in the disease process;Several limitations are present in this study and should be considered while interpreting the results. The current technique of delineating the AN within the Papez circuitry on the basis of DTT involves manual operation, which may be prone to possible operator bias. Moreover, this study enrolled a limited number of patients, which could affect the statistical power of our analyses. Therefore, generalization of our results for clinical application may warrant future studies incorporating large patient sample sizes. In addition, this study only evaluated early iNPH patients that had not yet undergone surgical treatment, such as the placement of a ventriculoperitoneal shunt, as well as long-term follow up. Assessment of the evolution of diffusion tensors within the AN following the course of iNPH treatment would be an important extension of the present study in the future.q value within the AN among early iNPH patients, along with marked deteriorations in patients’ neuropsychological performance in memory and verbal fluency. Our results may shed light on the possible compensatory role of the AN in the modulation of memory impairment in iNPH, as well as the prospective diagnostic value of diffusion metrics to assess memory functions in early iNPH patients.In summary, this study revealed significant elevations in FA and


The distribution of the Chinese Glyptosternoid catfish is limited to the rivers of the Tibetan Plateau and peripheral regions, especially the drainage areas of southeastern Tibet. Therefore, Glyptosternoid fishes are ideal for reconstructing the geological history of the southeastern Tibet drainage patterns and mitochondrial genetic adaptions to high elevations.pss was estimated to be 10.73, which is significantly higher than 1 , in COX1, which indicates positive selection in the common ancestral branch of Glyptosternoid fishes in China. We also found other signatures of positive selection in the branch of specialized species. These results imply mitochondrial genetic adaptation to high elevations in the Glyptosternoids.Our phylogenetic results support the monophyly of the Sisoridae and the Glyptosternoid fishes. The reconstructed ancestral geographical distribution suggests that the ancestral Glyptosternoids was widely distributed throughout the Brahmaputra drainage in the eastern Himalayas and Tibetan area during the Late Miocene (c. 5.5 Ma). We found that the Glyptosternoid fishes lineage had a higher ratio of nonsynonymous to synonymous substitutions than those found in non-Glyptosternoids. In addition, ωWe reconstructed a possible scenario for the southeastern Tibetan drainage patterns based on the adaptive geographical distribution of the Chinese Glyptosternoids in this drainage. The Glyptosternoids may have experienced accelerated evolutionary rates in mitochondrial genes that were driven by positive selection to better adapt to the high-elevation environment of the Tibetan Plateau.The online version of this article (doi:10.1186/s12862-015-0516-9) contains supplementary material, which is available to authorized users. The Tibetan Plateau (the “Roof of the World”) is the highest plateau on earth, with an average elevation of more than 4000 m. The plateau, which covers more than 2,500,000 km of plateaus and mountains in central Asia and is surrounded by towering mountain ranges, has been designated as a global hotspot of biodiversity . The envhttp://www.calacademy.org/scientists/projects/catalog-of-fishes). Chinese Glyptosternoids are found in the rivers around the Tibetan Plateau and eastern Himalayas, e.g., the Yaluzangbujiang (Brahmaputra River), Irrawaddy, Nujiang , Lancangjiang (Upper Mekong River), Jinshajiang (Upper Yangtze), Yuanjiang (Red River), Nanpanjiang (Upper Pearl River) and the Brahmaputra basin [Triplophysa) commonly found on the Tibetan Plateau. Habitat is thought to play a crucial role in diversification, and changes in habitat likely affect the distribution and diversification of biota in a particular region [Glyptosternoids refer to catfishes in the family Sisoridae subfamily Glyptosterninae tribe Glyptosternina. Currently, there are around 10 genera and 71 species of glyptosternoids, which 9 genera and 31 species distributed in China , the clr region . Chineser region –35. Due r region , 36–38.Bagarius yarrelli, Chu inferred that the Glyptosternoids originated in southeastern Tibet during the late Pliocene [Glyptosternum-like species then expanded eastward to western Sichuan and northern Yunnan after the formation of the Jinshajiang River. Diversification at the genus level was proposed to have occurred during the Pleistocene, with species then expanding into the rivers of Yunnan and Sichuan during the most recent uplift of the Himalayan area. (3) Other authors have suggested that the ancestor of the Glyptosternoids was widely distributed throughout the Tibetan Plateau in the early Pleistocene [Glyptosternum-like species maintained this distribution during the initial uplift of the Tibetan Plateau. The ancestor of the Euchiloglanis-like fish subsequently originated in the eastern Himalayan area during the second uplift of the Tibetan Plateau. Euchiloglanis-like species were then isolated to the Jinsha, Lancang, Nujiang, Yuanjiang, Pearl and Irrawaddy Rivers by the third uplift of the Tibetan Plateau. The specialized Glyptosternoids achieved their present distribution pattern due to the isolation of the rivers.Three different explanations have been suggested for the extant distribution patterns of the Chinese Glyptosternoids. (1) Hora and Silas suggested that the Glyptosternoids originated in the eastern Himalayan area of Yunnan province, southwestern China, but the exact origin and route of expansion were not clear . (2) BasPliocene . Accordiistocene , 40 and In this study, we aimed to reconstruct the ancestral distribution of the Glyptosternoids to test hypotheses concerning speciation with respect to southeastern Tibetan drainage patterns following the uplift of the Tibetan Plateau. Molecular clock approaches were used to infer divergence dates for this molecular phylogeny; to test whether the speciation, diversification and evolution of the Chinese Glyptosternoids are associated with the uplift of the Tibetan Plateau; and to examine high-elevation adaptive mitochondrial evolution in this lineage.Liobagrus nigricauda (Siluriformes: Amblycipitidae), Cranoglanis bouderius (Siluriformes: Cranoglanididae) and Ictalurus punctatus  included, putative close relatives to Sisoridae according to a recent study [The experiments were performed in accordance with the Ethics Committee of the Institute of Hydrobiology, Chinese Academy of Sciences. The Ethics Committee has also given ethics approval for our study. The policies were enacted according to Chinese Association for Laboratory Animal Sciences, and coordinated with the Institutional Animal Care and Use Committee (IACUC) protocols , 42. Thent study . VoucherThe complete mitochondrial genomes were amplified from the genomic DNA of the Sisoridae fishes using four overlapping amplification primers by long PCR methods: L9752 AGTACRAGTGACTTCCAATCACC, H2627 GTCCTGATCCAACATCGAGG, L295 GTAAAATTCGTGCCAGCCACC, and H10174 TCTGAGCCGAAATCAGAGGTC. The PCR reactions were prepared in total volumes of 50 μL as follows: 5x LongAmp buffer, 10 μL of each 2.5 mM dNTP, 6 μL of each 0.4 μM primer, 2.0 U LongAmp polymerase, and 20–50 ng of genomic DNA. The PCR conditions included an initial denaturation step at 94 °C for 30 s followed by 30–35 cycles at 94 °C for 30 s, 61–68 °C for 1 min, and 65 °C for 10 min, with a final extension of 10 min at 65 °C. Annealing temperatures were varied within these ranges in order to optimize the efficiency of different primers and samples.Library preparation was conducted using the “With-Bead” Method  with a sWe used the LightCycler 480 SYBR Green I Master  and qPCR Primers 1.1 and 2.1 according to the Illumina protocol . We pooled 40 indexed samples in equimolar ratios after they were quantified.http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc). The adapter sequences and sites with lower qualities were trimmed using Cutadapt [http://hannonlab.cshl.edu/fastx_toolkit/). Contigs were assembled de novo for each species using Trinity [http://www.bx.psu.edu/miller_lab/). Additional file We used 600 μL of 16 pM samples for paired-end 300 bp sequencing on a MiSeq sequencer . Sequence reads were sorted into each sample by the indices. As a first step, quality control checks were run on raw sequence data via fastqc  method  and MrBaTo trace the historical biogeography during the evolution of the Glyptosternoids, the ancestral distribution of the internal nodes were reconstructed with the Dispersal-Extinction-Cladogenesis (DEC) model  using thBagarius yarrelli from the Pliocene (5.3–1.8 Ma ago) of the Siwalik Hills in India [8 Markov Chain Monte Carlo (MCMC) iterations. Convergence of the chains to a stationary distribution was checked by visual inspection using TRACER v1.4 [The divergence times of the Chinese Glyptosternoids were determined using the protein-coding genes with the Bayesian molecular dating program Beast , accordiin India . C2 reprin India . These tCER v1.4 . We repeTo estimate lineage-specific evolutionary rates for each branch of the Glyptosternoids and their closest relatives, the CODEML program in the PAML package  with thepss) were significantly higher than 1, we performed the likelihood ratio test (LRT) with the null hypothesis that the ωpss value was 1. The candidates of the positively selected sites were predicted by the Empirical Bayes method [In molecular evolutionary biology, the natural selection acting on protein-coding genes is often characterized by comparison of synonymous and nonsynonymous substitution rates . The nons method .It has long been recognized that paleo-drainages of major continental East Asian Rivers, draining the southeastern Tibet plateau margin, differed markedly from their current drainage patterns , 63–69. Saturation tests  that incPseudecheneis, Bagarius, Glyptothorax and Glyptosternoids) with very high support values  are recovered with great posterior probability  form a sister group with high support values ; the second included the Creteuchiloglanis from Nujiang, Pareuchiloglanis gracilicaudata and Pseudexostoma; and the third lineage included the Oreoglanis, Pareuchiloglanis longicauda and P. macrotrem. The latter two lineages form sister groups separate from the Upper Yangtze lineage. Pareuchiloglanis was not resolved as monophyletic.The results of the Bayesian and ML nucleotide analyses produced by MrBayes and RAxML for the 12 protein-coding gene sequence datasets showed a marked consistency in topological congruence, differing only in the support values for certain nodes .Our newly estimated divergence times based on the mitochondrial genomes are shown in Fig. The optimal distributions at each ancestral node are given in Fig. Averaged across all 12 protein-coding genes, the ratio of nonsynonymous to synonymous substitutions is significantly higher in most of the Glyptosternoids lineages than observed in other non-Glyptosternoid lineages . Accordingly, the likelihood of positive selection in these eight branches was negligible. In contrast, AIC preferred the alternative hypothesis (ωpss = maximum likelihood estimation) for the other seven branches . Positively selected sites occurred in the COX1 gene. The candidates of the positively selected sites are shown in Additional file Using the branch-site model, positively selected signals were detected in fifteen branches. Among them, the signals of eight branches  were weak, and AIC preferred the null hypothesis  agrees best with our phylogeny . A rapid uplift of the Tibetan Plateau occurred at approximately 3.6 Ma, and the Yangtze, Nujiang, Lancang and Yuanjiang Rivers formed at this time. The specialized species, Pareuchiloglanis, Creteuchiloglanis, Euchiloglanis, Oreoglanis and Pseudexostoma, originated during this phase. These species then spread to the downstream portions of the Nujiang, Lancangjiang, Yaluzangbujiang (Brahmaputra) and Irrawaddy Rivers to form the current day distribution pattern, yielding a great species diversity. The process of speciation among Chinese Glyptosternoids resulted from dispersal and vicariance events associated with the uplift of the Tibetan area and the newly formed river systems.Based on our biogeographic results, we believe that the third explanation , for the gene COX1 under positive selection. These findings are similar to previous studies on native high-elevation animals that found that the COX1 gene experienced positive selection in Tibetan antelope [c oxidase has been identified as the mitochondrial enzyme that reduces NO-2 to NO, which induces expression of nuclear hypoxic genes, possibly via a pathway that involves protein nitration [c oxidase or complex IV of the respiratory chain may contribute to hypoxia adaptation. Furthermore, because oxygen is the ultimate electron acceptor, which results in the production of H2O in a process catalyzed by cytochrome c oxidase, modifications of the cytochrome c oxidase activities are expected to facilitate coping with a reduced oxygen supply. These modifications would affect mitochondrial NO production and, consequently, hypoxic signaling. Considering the reconstructed ancestral geographical distribution areas, this positive selection likely occurred during the process of high-elevation adaptation.We identified signatures of positive selection in the branch of the Chinese Glyptosternoid fish, and these signatures may indicate adaptation to physiological hypoxia and cold stress. Branch 1 Fig.  represenantelope  and platantelope . In colditration . TherefoPareuchiloglanis kamengensis, is found in the Brahmaputra/Tsangpo drainages and experienced positive selection. In this branch, the proportion of positively selected sites was 0.97 %, which corresponds to 35 codon sites in different genes. These codon sites are composed of Complex I, Complex III, Complex IV and Complex V. Branches 10, 16, 22, and 25, which were distributed in Irrawaddy, Salween, Upper Yangtze, and Pearl Rivers, were also under positive selection and correspond with many codon sites. However, these codon sites do not contain the genes associated with Complex V. Polypeptides are all subunits of the oxidative phosphorylation (OXPHOS) enzyme complexes. In aerobic organisms, OXPHOS supplies most of the ATP needed for cell metabolism. During this process, electrons from NADH or FADH2 are transferred to O2 via a series of electron carriers, which pump protons through the inner mitochondrial membrane, generating a proton gradient that drives ATP synthesis via ATP synthase (Complex V). Complex V consists of two main structural domains: an intrinsic membrane domain (F0) and an extrinsic globular domain (F1), linked by a central and a peripheral stalk [Pareuchiloglanis kamengensis), there are one and three positively selected candidate sites corresponding to ATP 6 and ATP 8, respectively (Additional file The other branches’ candidates for positively selected sites were predicted by the Empirical Bayes method  and are al stalk . The mamal stalk , among wal stalk . In branTaking into account the reconstructed ancestral states of the geographical distribution Fig.  and posiWe reconstructed a possible scenario for the southeastern Tibetan drainage patterns based on the adaptive geographical distribution of the Chinese Glyptosternoids in this drainage. In addition, the Glyptosternoid fishes lineage had a higher ratio of nonsynonymous to synonymous substitutions than those found in non-Glyptosternoids. They may have experienced accelerated evolutionary rates in mitochondrial genes that were driven by positive selection to better adapt to the high-elevation environment of the Tibetan Plateau.The data set supporting the results of this article is available in the GenBank under KP872682, KP872683, KP872690-KP872697 and provided as supplementary data.


Anopheles spp. and consequently, with malaria transmission. In this study, we analyzed the spatial and temporal dynamics of malaria in the state of Acre (and more closely within a single county) to investigate the potential links between aquaculture and malaria transmission in this region. At the state level, we classified the 22 counties into three malaria endemicity patterns, based on the correlation between notification time series. Furthermore, the study period (2003–2013) was divided into two phases (epidemic and post-epidemic). Higher fish pond construction coincided both spatially and temporally with increased rate of malaria notification. Within one malaria endemic county, we investigated the relationship between the geolocation of malaria cases (2011–2012) and their distance to fish ponds. Entomological surveys carried out in these ponds provided measurements of anopheline abundance that were significantly associated with the abundance of malaria cases within 100 m of the ponds . These results taken together suggest that fish farming contributes to the maintenance of high transmission levels of malaria in this region.Fish farming in the Amazon has been stimulated as a solution to increase economic development. However, poorly managed fish ponds have been sometimes associated with the presence of  Malaria has reemerged in the Amazon region following the introduction of large-scale agricultural colonization projects , 2. SincAn average of 310,390 malaria cases have been reported annually in the Brazilian Amazon during the last 14 years, but with a clear reduction in the last 4 years. Between 2000 and 2014, the number of reported cases dropped from 615,247 to 138,338, despite the population growth [Anopheles darlingi, the most important Neotropical malaria vector, seems to have adapted well to fish ponds, despite predation by fish juveniles [In recent years, as a measure to reduce deforestation and improve the local economy, fish farming, mostly performed in already deforested and abandoned areas, near or within small dammed rivers was stimulated as a sustainable alternative to cattle-farming. However, there has been a growing concern that fish ponds may increase malaria incidence, since uveniles –9. This uveniles  found hiuveniles  found evuveniles  observedIn 2005–2006 a large malaria epidemic struck Acre, with over 80,000 cases reported in two years. In subsequent years, the number of cases was reduced to a new baseline, higher than previously observed . In thisThe study was conducted at two scales: the state of Acre  and the county of Mâncio Lima . The Brazilian state of Acre, bordering Bolivia (Southeast), Peru (West and North) and the Brazilian States of Amazonas (North) and Rondônia (East) , is divi2) (Mauritia flexuosa). This environment is favorable for the development of aquaculture, and Mâncio Lima is one of the main targets of fish pond construction in the state.Mâncio Lima  , the wesc <1 indicates poor conditions for local transmission[Malaria notifications per county from 2003 to 2013 were obtained from the Brazilian Official Malaria Notification System . In Acre, Community health agents regularly perform thick blood smears to verify the presence of malaria parasite species in residents presenting non-specific acute febrile symptoms and their household contacts. Results were not confirmed with Polymerase Chain Reaction (PCR). The Annual (and Monthly) Parasite Index  were calculated as the number of positive malaria exams per year per 1,000 inhabitants. An index of malaria vulnerability, Rc, was calculated as the ratio between the number of autochthonous cases and the number of imported cases per year. Rnsmission.For the analysis, we split the study period into two parts (2003–2006 and 2007–2013), representing the epidemic and post- epidemic phases, since changes were anticipated following the major control initiatives implemented from 2006 onwards. In 2006, at the peak of epidemic, most cases were identified by active surveillance (65%), and 71% patients received treatment within 48 hours of symptom onset . MalariaAnopheles (Nyssorhynchus) larvae are distributed in the margins of water collections and are rarely found away from them [http://www.google.com/earth/).With the aid of satellite imagery from OpenStreetMap , an open source product that provides free world-wide geographic datasets , of the rom them –23 and crom them . We usedrom them  and thisTo visualize the spatial and temporal distribution of malaria incidence in Acre, maps and time series plots were created using GIS (ArcView). To aggregate counties in strata, we applied a hierarchical clustering technique to the monthly time series of MPI per county. To partition the counties into clusters, we used the package ClustOfVar  in softw = b0 exp (b1xdistance) was fitted to the data using the least squares technique. Statistical analyzes were performed using StatSoft Inc. (2011). Data is available in In Mâncio Lima, where primary data was collected, a map with the location of all malaria cases in 2011 and 2012, as well as the fish ponds, was created. Buffers 100 to 1000m around fish ponds were constructed and the number of malaria cases per buffer, calculated. An exponential regression model following the expression The study was approved by the ethical committee (CEP Fiocruz n.402.039). Entomological collections were carried out in private and public spaces. Access to private spaces was requested to each land owner, and collections carried out only after their oral consent. No personal information was recorded or used in this study. Access to public spaces did not require permission but before taking place, the overall study was presented and approved by local Health and Environmental Secretariats. The only request was that the results were presented to the population, which occurred in the form of talks in the city's conference room and in schools. The field studies did not involve endangered or protected species.From 2003 to 2013, 401,312 malaria cases were notified in Acre, 94% of which were classified as autochthonous. The majority of autochthonous cases (79%) occurred in the rural areas. Acre malaria cases from 2003–2013 are shown in Correlations between times series of reported autochthonous malaria cases per county from 2003 to 2013 are shown in the form of a dendogram . A stronIn the middle of the state, 5 remaining counties were grouped into the Central stratum. They share low malaria activity, low connectivity to other regions and mostly rural populations. They are Assis Brasil, Manoel Urbano, Feijó, Epitaciolândia and Santa Rosa do Purus. Although this last county is grouped with the Southeast stratum counties in the cluster dendogram, it shows low malaria activity like the Central stratum counties and we opted to assign it to the Central cluster .Considering the 2003–2013 period as a whole, API presented significant differences between the 3 strata: while the Northwest stratum (n = 6) had a median MPI of 10.8 cases per 1,000 inhabitants (min-max = 0 to 188), the Central stratum (n = 5) had a median of 0 cases/1,000 inhabitants (min-max = 0 to 2.3) and the Southeast stratum (n = 11) 0.44 cases/1,000 inhabitants (min-max = 0–23) per month.Regarding Rc, the Northwest stratum presented Rc > 1 in 31% of the months from 2003 until 2013, contrasting with the Southeast stratum, with only 12% of months with Rc >1. Transmission was lowest in the Central stratum, with Rc > 1 in only 5% of the months.A sequence of maps shows how the API varied from 2003 to 2013 in Acre counties . BetweenFish farming initiatives began in Cruzeiro do Sul before 2004, but the greatest number of ponds were built in 2005 . In MâncP < 0.005): while counties with high construction rates (n = 6) were associated with a mean of 833.8 (SE = 177.1) cases of malaria/1000 inhabitants/year, counties with the construction of fewer ponds (n = 16) had a mean malaria incidence of only 76.2 cases/1000 inhabitants-year (SE = 76.2).Counties with the construction rate higher than 80 fish ponds per year during the 2003–2006 study period differed significantly in malaria rates from those with lower construction rates  = 13.32; y = -14223 + 290*x ; where x is the number of estimated aquaculture in Mâncio Lima and y is the number of malaria cases per year in the county, as shown in The number of aquaculture hectares between 2003 and 2006 was directly correlated with malaria incidence in Mâncio Lima through the equation: A map of fish ponds and malaria cases in the Mâncio Lima town in 2011 and 2012 is shown in An. darlingi larvae was 74.1%, twice as high as the other water collection types (n = 37)  = 8.24; P << 0001). During the four collection periods, a mean of 1.94 times (SE = 0.17), fish ponds were found positive while the second most positive habitat, riverine areas, were positive only a mean of 0.67 times (SE = 0.73). The abundance of An. darlingi larvae was higher in fish ponds than in any other water collection types  = 4.37, P < 0.005); whereas the mean larval density in other water collections were at a maximum of 0.67 larvae/dip (SE = 4.60), fish ponds had a mean density of 7.16 (SE = 1.05). The density of An. darlingi larvae found in each positive fish pond was linearly associated with the number of malaria cases within 100 m from the ponds . Other anophelines were also found in these ponds but in lower abundance than An darlingi. A more detailed description of the chemical and environmental factors associated with anopheline abundance will be presented elsewhere. For the present study, the focus is the association between anopheline abundance and malaria.The proportion of fish ponds (n = 55) positive for Large investments have been made to reduce the burden of malaria in Brazil. Since 2000, large scale distribution of free treatment, supervised treatment administration within 48 hours of symptoms onset, distribution of impregnated bednets, among other strategies have been implemented in Acre, which has received successive prizes by OPAS, suggesting these are winning strategies . HoweverMalaria notifications in Acre were highest in the Northwest stratum. At a more regional scale, Braz et al.  identifiAn. darlingi larval habitat, the most constantly positive habitats and the ones with the highest An. darlingi larval densities; 6- larval densities in fish ponds were associated with the number of malaria cases within 100 m of ponds. Therefore, we believe that fish ponds played a major role in the 2003–2006 epidemics and continued to be an important factor in maintaining higher malaria incidence rates during subsequent years.Our results suggest that fish-farming has contributed to the receptivity of the Northwest stratum to malaria. This is supported by the following findings: 1- while contemplating all the counties in Acre from 2003 onwards, the Fish farming Incentive Program concentrated most of its efforts in the Northwest stratum, and higher fish pond construction rates per stratum coincided with higher malaria rates; 2- counties with higher fish pond construction rates were associated with higher mean annual malaria incidences during the epidemic period (2003–2006); 3- the number of hectares of aquaculture in Mâncio Lima was related to malaria incidence in the epidemic period; 4- although we lack fine-scale spatial data during the 2006 epidemic, increased transmission in the post-epidemic period coincides precisely with the occurrence of ponds, since cases decrease exponentially with increasing distance from ponds; 5- fish ponds were the most positive type of A few reports have demonstrated increased malaria transmission in proximity to farming ponds in South America and Africa ,11,31,32An. darlingi larvae abundance in fish ponds at distances of less than 100m. Studies of An. darlingi flight range in inhabited deforested areas have found that the numbers of mosquitoes flying a given distance from the larval habitat decreases exponentially following the regression equation: y = 4.43*exp(-0.003 x) , where x is the distance from the larval habitat in meters and y the percentage of emerging adult An. darlingi females [2 = 0.87), where y is the proportion (%) of An. darlingi adult females and x is the number of malaria cases. These results support the hypothesis that malaria incidence in Mâncio Lima in 2011 and 2012 was closely linked to fish farming ponds.We observed a direct association between malaria prevalence and  females . Using tAn. darlingi landing catches, as well as larval occurrence, in deforested areas. However, neither of these entomological studies adequately controlled for the presence of fish ponds, although these habitats were recognized as the most frequently positive habitat in the given study area. The association of malaria incidence with proximity to fish ponds and pond density, suggests that the presence of ponds may have biased the entomological data [We hypothesize that fish ponds are important anthropogenic modifications that account for increased malaria transmission. Using health district-specific malaria incidence data from Mâncio Lima, in 2006, higher malaria risk in areas with greater deforestation percentage change in the 1997–2000 period were interpreted by Olson et al. as an incal data ,11. Withcal data .An. darlingi bionomics. The importance of fish ponds’ size, density and bordering vegetation must be better evaluated to derive adequate economic and ecological management of fish ponds and guidelines for malaria surveillance and control. The way different types of secondary growth affect mosquito bionomics is also of great importance and merits further investigation.Aquaculture is an important driver of development in the Amazon, and has been advocated as a solution to alleviate poverty and foster economic growth. Fish production in Mâncio Lima may have actually improved the local socioeconomic conditions, not only by introducing a new source of income, diversifying food supplies, but also by creating a chain of secondary production and consumption items. S1 Fig(TIF)Click here for additional data file.S1 FileAn. darling in fish ponds and other water bodies, hectar_aqua.csv contains the hectares of aquaculture data in Mâncio Lima, ponds_constructed contains the ponds constructed per year data per each municipalities.It consists of six.csv files, API.csv contains the API each municipalities per year, cluster.csv contains the MPI of each municipalities per year, buffer_cases.csv contains the number of malaria cases per distance zones, entomological.csv contains mean of (RAR)Click here for additional data file.


US Latinos are growing at the fastest rate of any racial/ethnic group in the United States and have the highest lifetime risk of diabetes. Acculturation may increase the risk of diabetes among all Latinos, but this hypothesis has not been studied in a nationally representative sample. The objective of this study was to test the hypothesis that acculturation was associated with an increased risk of diabetes in such a sample.1C) values of 6.5% or higher were classified as having diabetes. An acculturation score, ranging from 0 (lowest) to 3 (highest), was calculated by giving 1 point for each of 3 characteristics: being born in the United States, speaking predominantly English, and living in the United States for 20 years or more. Logistic regression was used to determine the association between acculturation and diabetes.We conducted a cross-sectional analysis including 3,165 Latino participants in the 2007–2010 National Health and Nutrition Examination Survey. Participants with doctor-diagnosed diabetes and participants without diagnosed diabetes who had glycated hemoglobin  increased with level of acculturation— 1.71 , 1.63 , and 2.05  for scores of 1, 2, and 3, respectively. This association persisted after further adjustment for body mass index (BMI), total dietary calories, and physical inactivity.Acculturation was associated with a higher risk of diabetes among US Latinos, and this risk was only partly explained by BMI and weight-related behaviors. Future research should examine the bio-behavioral mechanisms that underlie the relationship between acculturation and diabetes in Latinos. Latinos in the United States have a higher lifetime risk of diabetes than those in other racial or ethnic groups . The ageAcculturation is the process whereby individuals from one culture adopt the behaviors, attitudes, and values of the prevailing culture . AccultuBy using data from the 2007–2010 National Health and Nutrition Examination Survey (NHANES), we examined the association between acculturation and diabetes. We used an acculturation score based on country of birth, predominant spoken language, and years of US residence. We also tested whether this association was mediated by body mass index, diet, and physical activity — factors that are influenced by acculturation and known to affect diabetes risk. Our study is the first to include a nationally representative sample of all US Latinos, whereas previous nationally representative studies have included only Mexican-Americans. Studying this novel population may provide further insight into the relationship between acculturation and diabetes in Latinos.Our analysis of the NHANES data was deemed exempt from review by the Temple University Institutional Review Board. NHANES, which is conducted by the National Center for Health Statistics, uses a complex multistage probability sample designed to be nationally representative of the US civilian, noninstitutionalized population. Beginning in 2007, the NHANES sampling strategy was modified to oversample all Latinos, rather than only Mexican Americans, as in previous cycles . This mo1c) , fasting plasma glucose (FPG) , and oral glucose tolerance test (OGTT) .NHANES participants complete a home-based interview and a subsequent physical and laboratory examination in a mobile exam center. Procedures for blood collection and processing are described elsewhere . Of the 1c . We excluded participants in whom diabetes prevalence could not be determined — those without previously diagnosed diabetes who were missing HbA1c data (n = 253). This accounted for 7.3% of the total Latino sample during this time period (unweighted). Pregnant women were also excluded (n = 53), leaving a final analytic sample of 3,165 Latino adults.We included adult Latinos who reported having received a doctor’s diagnosis of diabetes (n = 444), and those without doctor-diagnosed diabetes who had available data for HbADiabetes prevalence. Our binary dependent variable for diabetes included both diagnosed and undiagnosed diabetes. Participants were classified as having previously diagnosed diabetes if they answered affirmatively to the question of whether a doctor or health professional had ever told them that they have diabetes, other than during pregnancy. For those who answered the same question negatively, the presence of undiagnosed diabetes was determined on the basis of their HbA1c value. We chose to use HbA1c as the diagnostic criterion for undiagnosed diabetes because many more participants underwent HbA1c testing than FPG or OGTT. A diagnosis of diabetes was therefore defined by either of the following scenarios: 1) having received a doctor’s diagnosis of diabetes with any HbA1c value or missing data for HbA1c ; or 2) never having received a diagnosis of diabetes but having an HbA1c value at or greater than 6.5% . We considered all other participants to not have diabetes .Acculturation score. Following the example of a recent study, an acculturation score was developed from 3 commonly used proxies for acculturation — nativity, language spoken at home, and duration of US residence , age, sex, educational attainment, household income, marital status, insurance status, and having a usual source of medical care. Household income was assessed by the income-to-poverty ratio, calculated by dividing participants’ annual household income by the federal poverty level, and categorized into equal quartiles for those with available data . A fifth income category was created for participants missing income data (N = 446) and was used in dummy variable regression to estimate subclass means for these individuals. Educational attainment was grouped into the following categories based on common educational milestones and their distribution in the study population: less than 9th grade, 9th to 11th grade, high school, and more than high school. Participants self-reported whether they had insurance coverage and a usual source of medical care. Three other covariates — body mass index (BMI), total dietary calories, and physical inactivity — were considered potential mediators of the relationship between acculturation and diabetes. Participants’ BMI was calculated on the basis of their measured weight and height (kg/m2). Total dietary calories  were estimated from 24-hour dietary recall interviews and reported as a continuous measure of total energy intake. Those who reported doing no leisure-time physical activity were classified as being physically inactive.2 tests to examine the bivariate association between 11 covariates and both the prevalence of diabetes and level of acculturation (0–3). By using logistic regression, we calculated the odds of participants’ having diabetes at each level of the acculturation score , version 12.1. For all statistical testing, we used Stata survey commands, which adjust variance estimates to account for the complex sample design of NHANES. We first used descriptive statistics to characterize the study cohort with respect to the outcome (diabetes), the primary predictor variable (acculturation score), and the covariates. We used χon score .BMI, total dietary calories, and physical inactivity were included in model 4 to examine whether these factors mediated the primary relationship under study. In addition, generalized structural equation modeling  was usedOverall, the prevalence of diabetes in our cohort was 12.4%. The prevalence at each acculturation level was as follows: lowest (35.2%), low (21.6%), high (12.4%), and highest (30.9%). Nearly two-thirds of study participants were of Mexican origin, more than half were aged less than 40 years, and nearly one-third achieved more than a high school education . There wThe unadjusted prevalence of diabetes by acculturation level was as follows: lowest (6.7%), low (20.5%), high (14.5%), and highest (12.5%). After adjusting for Hispanic origin, age, sex, insurance status, usual source of care, educational attainment, household income, and marital status, the likelihood of diabetes increased with the level of acculturation. Compared with those with an acculturation score of 0, the odds of having diabetes were 1.71 , 1.63 , and 2.05  for those with acculturation scores of 1, 2, and 3, respectively. Adding BMI, total dietary calories, and physical inactivity to the fully adjusted model reduced the magnitude of the association between acculturation and diabetes only marginally, with this association remaining significant , Model 4We conducted additional regression analyses for which results are not shown. When each proxy measure of acculturation was examined in a regression model without the other 2, each was associated with a significantly increased risk of diabetes after controlling for covariates.In a nationally representative sample of US Latinos, the risk of diabetes was higher in those who were more acculturated, even after adjusting for demographic factors and socioeconomic status. The association between acculturation and increased diabetes risk remained significant after adjusting for BMI and weight-related behaviors in multivariable models. Mediation testing revealed that BMI only partially mediated this association between acculturation and diabetes.This is the first study to examine the relationship between acculturation and diabetes in a nationally representative sample of all US Latinos. Previous NHANES studies on this topic did not involve a nationally representative sample of all Latinos because such a sample was assembled for the first time during the 2007–2010 waves of data collection. Combining all Latinos into 1 group presents both strengths and weaknesses when compared with the existing literature on acculturation and diabetes. Previous studies have documented differences in diabetes rates by Latino subgroup .1c value of 6.5% or higher was used to classify those with undiagnosed diabetes — an approach that has been formally recommended for diagnosing diabetes by both national and international authorities and is commonly used for that purpose in clinical practice (1c cut-off may have been biased by misclassification (Another strength of our study is that we examined undiagnosed diabetes, rather than only including those with doctor-diagnosed diabetes. An HbApractice . Becausefication ,12.1c as the diagnostic criterion for undiagnosed diabetes limits direct comparison of these findings to other studies that used different diagnostic tests. Although Latinos have higher HbA1c levels than non–Hispanic whites (The cross-sectional nature of this analysis precludes drawing causal inferences about the relationship between acculturation and diabetes. NHANES does not collect data on whether individuals have type 1 or type 2 diabetes. However, on the basis of other studies, more than 90% of cases in NHANES are likely to be type 2 diabetes . Using Hc whites , this dic whites , but theOur findings are consistent with those of 4 other studies showing that the risk of diabetes increases with acculturation among Latinos –12. Only1c, among US-born versus foreign-born Mexican Americans (A novel finding in this study is that BMI and weight-related diet and physical activity behaviors explain only a small proportion of the relationship between acculturation and diabetes risk in Latinos. Like other covariates in our analysis, the association between BMI and acculturation was significant but nonlinear, which may partly explain its small effect on our multivariate mediation model. However, this relatively small contribution of BMI and weight-related behaviors to the relationship between acculturation and diabetes prompts consideration of other bio-behavioral mechanisms underlying this association. The stressful impact of acculturation on immigrants, or “acculturative stress” , may caumericans .A recent study revealed significant heterogeneity in diabetes risk among Latino subgroups , which m


However, despite our interrogation of the most exhaustive tephra leachate dataset available, it has become clear in this effort that more detailed insights into gas-tephra interaction mechanisms are prevented by the prevalent poor temporal and spatial representativeness of the collated data and the limited characterisation of the tephra deposits. Future leachate studies should aim to extensively sample across tephra deposit limits whilst simultaneously characterising deposit stratigraphy and tephra chemistry, mineralogy and granulometry, taking steps to ensure the quality and comparability of collected leachate datasets.Tephra particles in physically and chemically evolving volcanic plumes and clouds carry soluble sulphate and halide salts to the Earth’s surface, ultimately depositing volcanogenic compounds into terrestrial or aquatic environments. Upon leaching of tephra in water, these salts dissolve rapidly. Previous studies have investigated the spatial and temporal variability of tephra leachate compositions during an eruption in order to gain insight into the mechanisms of gas-tephra interaction which emplace those salts. However, the leachate datasets analysed are typically small and may poorly represent the natural variability and complexity of tephra deposits. Here, we have conducted a retrospective analysis of published leachate analyses from the 18 May 1980 eruption of Mount St. Helens, Washington, analysing the spatial structure of the concentrations and relative abundances of soluble Ca, Cl, Na and S across the deposits. We have identified two spatial features: (1) concentrated tephra leachate compositions in blast deposits to the north of the volcano and (2) low S/Cl and Na/Cl ratios around the Washington-Idaho border. By reference to the bulk chemistry and granulometry of the deposit and to current knowledge of gas-tephra interactions, we suggest that the proximal enrichments are the product of pre-eruptive gas uptake during cryptodome emplacement. We speculate that the low S/Cl and Na/Cl ratios reflect a combination of compositional dependences on high-temperature SO Tephra releases significant quantities of soluble elements upon leaching in water. These are generally accepted to originate from the dissolution of soluble sulphate and halide salts on particle surfaces , in Washington (WA), USA. The eruption datasets comprise more than 300 leachate analyses from 185 tephra samples, reported in ten different studies. Although, in our evaluation, differing leaching protocols render the majority of these incomparable  and fluorite  a ‘true’ concentration value at that point and (ii) a random error following a normal distribution of 0 mean and  via Eq. .1\documeThe final collated MSH dataset utilised in our study comprises 56 leachate compositions from the MSH tephra deposit. This represents only 20 % of the original 302 sample dataset, but nevertheless remains the largest for any single studied eruption. Tephra leachates are distributed across 32 locations across the deposit, although these locations are reported with varying precision. For example, Hinkley et al.  report s2 g−1) of the tephra sample. No SSA analyses were undertaken in the collated studies; we therefore calculated geometric specific surface area (SSAgeo) values, calculated via Eq. (pd is particle diameter (m) and ρ is individual particle density (g cm−3), from 37 particle size distributions taken from across the MSH deposit  theory  according to its geographic position. Where field duplicate data exists, we calculate and plot the mean concentration or value for that location. As no field duplicate data were available for SSABased on the mean values for each location, we utilised the Geostatistical Analyst extension of the ArcGIS 10.1 software ESRI  to generThere are no clear spatial patterns in the maps of soluble Ca, Cl, Na and S Fig. ; high angeo map  was assigned to leachate compositions from the Mossyrock area, all but one mean soluble Cl concentration, and two for mean soluble S, would still fall in the 90th percentiles of their respective datasets. A lower assigned SSAgeo would further increase these values.Although we attempt no interpretation of broad trends in SSAThe maps of soluble S/Cl and Na/Cl ratios are displayed in Fig. 2O and SiO2 content, and for derived data, i.e. soluble Ca/S, Na/Cl and S/Cl ratios. We assume that if field duplicate variance is greater than 50 % of the total variance of the whole dataset, mapping of data, and accordingly, interpretation of any spatial features, is not justified. The contribution of field duplicate variance for all datasets is less than 50 %, with the exception of Na2O (71 %) and Ca/S (64 %); hence, the latter two datasets were excluded in our analysis.Mapping and interpretation of leachate and bulk chemical composition data is only appropriate if the variability of field duplicate data at individual sampling locations is less than the total variability of the dataset. If this is not the case, any inferred spatial trends could be ultimately the product of tephra sampling strategies that do not adequately represent local deposit variability, or of differences in leaching protocols. The statistical formula for pooled variance was used to calculate the variance of the field duplicate data for leachate and tephra bulk chemical composition data. Table Tephra samples leached for 25 h may release proportionally more soluble Ca, Cl, Na and S than those leached for 4 h or fewer. This is demonstrated at eight locations within the collated dataset, which contain leachate compositions derived from one or more tephra samples which were leached for (i) a period of between 1 and 4 h  high mean soluble Ca, Cl, Na and S concentrations per unit mass Fig.  and per 2-rich and HCl-poor, in contrast to that erupted in the later stages of the eruption. However, the proximal enrichments in soluble S and Cl can also be attributed to a prolonged period of pre-eruptive gas-rock interactions within the cryptodome. Rock samples taken from the dome formed at MSH in 2004 showed extensive cubic and bleb-like surface deposits on internal surfaces  exhibited comparable reactivity with SO2. Under the assumption of the same comparable reactivity, high-temperature SO2 uptake, and accordingly CaSO4 formation, may be broadly correlated with bulk tephra CaO content, irrespective of mineralogy.Compositional dependences on leachate chemistry across the tephra deposit have been noted by Hinkley et al. ; solubley et al.  stated t2. Ayris et al. , Textor et al. , principally comprised of silicic pumice and glass shards  and Moscow and St. Maries (ID). Proximal enrichments may be the result of prolonged pre-eruptive exposure to magmatic gases during cryptodome growth. Conversely, the low S/Cl and Na/Cl ratios in the RSSM region may be due to the variable reactivity of ash to SO


Current commercial polymer membranes have shown high performance and durability in water treatment, converting poor quality waters to higher quality suitable for drinking, agriculture and recycling. However, to extend the treatment into more challenging water sources containing abrasive particles, micro and ultrafiltration membranes with enhanced physical durability are highly desirable. This review summarises the current limits of the existing polymeric membranes to treat harsh water sources, followed by the development of nanocomposite poly(vinylidene fluoride) (PVDF) membranes for improved physical durability. Various types of nanofillers including nanoparticles, carbon nanotubes (CNT) and nanoclays were evaluated for their effect on flux, fouling resistance, mechanical strength and abrasion resistance on PVDF membranes. The mechanisms of abrasive wear and how the more durable materials provide resistance was also explored. Water is becoming scarcer due to droughts brought on by climate change, as well as increased pressures on water sources due to increased urbanization and population growth. Water is essential for human life and culture, and is thus a critical resource for our sustainable future. To address the shortages and ensure water security, there is a rapid rise in the demand for new sources  and the use of recycled water to replace more valuable potable water. A key technology in our ability to access new water sources is membranes .The development of membrane technology has occurred over 150 years ,3. It stThe most common applications include producing valuable water from seawater and brackish water , as well as treating industrial wastewaters . Membrane systems are used to replace processes, such as secondary sedimentation, flocculation, settling tanks, and granular filtration , that arThe general classification of membrane types in order of decreasing pore size is microfiltration (MF), ultrafiltration (UF), nanofiltration (NF) and RO. MF and UF membranes are used as an advanced water treatment process for removing particles including silt and pathogens , while R2O3), titanium dioxide (TiO2), zirconium dioxide (ZrO2), silicon dioxide (SiO2), or their combinations, to achieve the desired filtration mode and performance  nanoclay loading. Other than the energy-dissipation mechanism induced by the PVDF β-phase, another reason for the improved mechanical properties can be attributed to the suppression of the macrovoids in the membranes with higher nanoclay loading.Lai et al.  also notet al. [® 20A into PVDF hollow fibre for direct contact membrane distillation (DCMD) applications. Cloisite® 20A was mixed with PVDF in NMP and ethylene glycol (EG) and the membranes were fabricated using the dry-jet wet phase inversion mechanism by using water as both internal and external coagulants. The ratio of dope was PVDF/NMP/Cloisite® 20A/EG (10.0/74.7/3.3/12.0). The PVDF/20A membranes had lower ductility (extension at break) and stretch resistance (tensile stress), but increased stiffness (Young’s modulus) compared to the unmodified membranes. Unlike neat PVDF fibres which collapsed under long operation time, nanocomposite membrane was able to withstand the desalination test over 220 h with stable vapour flux. This study also showed the inclusion of clay particles enhanced long-term mechanical stability.Wang et al.  studied et al. [® I34TCN  was melt extruded with PVDF at 190 °C, 180 rpm. It was observed that low nanoclay loading (1–2 wt %) had the highest ductility and impact strength as nanoclay can act as a temporary crosslinker to the polymer chains given their size and mobility are comparable. This provides localized regions of increased strength and inhibits the development of cracks and cavities. Nanocomposite at low nanoclay loading also had the lowest friction coefficient and wear rate. The author postulated that the shifting of the PVDF crystal phase induced by nanoclay addition increased the binding energy between macromolecules and improved abrasion resistance, as the material was less likely to peel off.Peng et al.  studied It was observed that nanoclay changed PVDF crystal phase and the PVDF/nanoclay nanocomposites demonstrated improvements to properties such as increases in toughness, strength and abrasion resistance. The incorporation into PVDF had benefits not only in providing physical reinforcement to the polymer network, but also acting as morphology directors by stabilising a metastable or conventionally inaccessible polymer phase, or introducing new energy dissipation mechanisms ,87. Thiset al. [® I.44P had improved abrasion resistance, lasting three times longer than the control membrane with no nanoclay addition. A model was proposed for the enhanced abrasion resistance of the nanocomposite membrane materials as shown in Using an in-house developed setup involving shaking hollow fibres in an abrasive slurry and periodically measuring bubble point for skin layer breakthrough, Lai et al.  evaluate2·h and 84 kg/m2·h, respectively. The drop in flux could be due to increases in tortuosity and thermal conductivity that acts to lower the thermal efficiency of the DCMD process. Despite this drawback, the composite membrane demonstrated enhanced mechanical performance especially for long operation times as stated in PVDF/nanoclay membrane showed slightly lower water vapour flux than neat PVDF membrane during DCMD operation . At inle2 particles in the PVDF/TiO2 1 wt % membrane was 25 nm and the average pore size was 0.18 μm [In MF/UF application, PVDF/nanoclay membranes also demonstrated lower pure water permeability compared to the control membrane . One pos 0.18 μm . For theN-vinlpyrrolidone modified MMT improved the anti-fouling properties of PVDF membrane when tested with BSA fouling solution due to changes of surface hydrophilicity and morphologies [A laboratory hologies . The ant2 and SBA-15. While fouling is one of the durability issues mentioned in 2/Al2O3 [2/Al2O3, a much higher loading was often required compared to nanoclay. ZnO used by Liang et al. [et al. [® nanoclay (by weight of PVDF) used by Wang et al. [A summary of the major findings of selected PVDF nanocomposite flat sheet and hollow fibre membranes is listed in  2/Al2O3  also shog et al.  was 6.7% [et al.  used 3% g et al. . Cost wig et al. . While net al. [Despite the identified progress in the use of nanofillers for improved membrane properties, there is little mentioned about the nanofillers becoming dislodged from the polymer matrix during filtration operation. Lai et al.  observedIn this review, the need for physical durability and the state of art in the literature on PVDF nanocomposite membranes for durability and performance was reviewed. Various types of nanofillers including nanoparticles, CNT and nanoclays were evaluated for their effect on the membrane properties. While limited work has investigated the effect of CNT on PVDF membrane, other types of nanofillers have demonstrated improvement in flux, fouling resistance, mechanical strength and abrasion resistance with the correct choice of fillers and loadings. However, few studies have considered nanoparticles in the presence of additives such as PEG or PVP which may act to negate the influence of nanoparticles on surface properties. Overall, PVDF reinforced with commercially available nanoclay was fabricated as hollow fibre membranes for low pressure filtration applications showed a shift of the PVDF crystalline phase from α- to β-phase, and brought about a more efficient energy-dissipation mechanism in the nanocomposite membrane. With enhancement in both mechanical strength and abrasion resistance, nanoclay is the most promising nanoparticle for improving the physical durability of membrane.