There is presently no method of predicting meat quality based on a single examination of birds before slaughter. That could potentially be accomplished with a computer-assisted analysis of ultrasonographic images. Hence, this experiment was designed to draw correlations between various physical and chemical properties of chicken pectoral muscles and pixel values (brightness elements) measured in corresponding ultrasonograms (a.k.a. echotexture). Our study revealed that echotextural characteristics of pectoral muscles in live birds were predictive of several important physical parameters  as well as intramuscular fat and protein content. However, different dietary fats that caused changes in the chemical composition of chicken breast also affected the associations between muscle echotexture and its physicochemical properties. For example, no correlations with the chemical composition of the muscles could be found in birds fed with soybean oil. We concluded that ultrasonographic imaging combined with a computerized image analysis can offer significant benefits to the poultry industry and consumers. It can aid in livestock genetic selection and improvement programs as well as enhance the quality of poultry meat and meat products. However, more confirmatory studies are needed.p = 0.003), and between crude fat/protein content and MPI/MPH of pectoral the major muscles in Groups FO/BF . There exists a potential application of ultrasonographic imaging and computerized image analysis for predicting certain physicochemical properties of pectoralis major muscles in broiler chickens.This study examined the relationships among physicochemical properties and ultrasonographic image attributes of pectoralis major muscles in broiler chickens. Forty male Ross 308 chicks were randomly assigned to four equinumerous fat-supplementation groups . Ultrasonograms of birds’ pectoral muscles were obtained just before slaughter at 6 weeks of age and were subjected to digital image analyses to determine the mean pixel intensity (MPI) and pixel heterogeneity values . A total of 2, 4, 2, and 6 significant correlations were recorded in Groups SO, FO, SO + FO, and BF, respectively; there were no correlations with the chemical composition of the muscles in Groups SO and SO + FO. The strongest correlations were found between muscle lightness (L*) and MPH in Group BF (physical characteristic; r = −0.82,  In order to provide high quality products to consumers, it is necessary for the food industry to obtain reliable information on factors influencing meat quality throughout the entire production cycle . In factIt is feasible that all major physicochemical properties of muscles could be assessed using ultrasound technology. A promising new area of research that has not been fully explored yet is the potential for utilizing computer-assisted analyses of ultrasonographic images in situ for the prediction of tissues’ chemical compositions and histomorphological attributes ,6,7,8. UIn the field of medicine, numerous studies have attempted to analyze tissue microstructure and chemical composition with ultrasonographic techniques. For instance, Clague et al.  reportedWe hypothesized that echotextural attributes of the pectoralis major muscle  in broiler chickens in situ would be significant predictors of its physicochemical properties. The specific objectives of the present study were (i) to examine pectoralis major muscles for correlations between their quantitative echotextural attributes and physicochemical properties and (ii) to assess the influence of the dietary fat source  on the relationships between the echotexture, physical characteristics, and chemical composition of pectoralis major muscles in broiler chickens.The local Ethics Committee for Animal Experimentation approved all experimental procedures performed in the present study. This experiment utilized forty 42-day-old male broiler chicks (Ross 308). One-day-old chickens hatched in a commercial poultry hatchery were transported to an experimental farm of the National Research Institute of Animal Production in Aleksandrowice near Cracow, Poland. The birds were randomly assigned to four experimental groups based on the source of fat in the feed mixtures: soybean oil (Group SO), flaxseed oil (Group FO), mixed oil , or beef fat (Group BF). Broilers were kept in pens on deep litter in automatically controlled environmental conditions. An initial temperature was set at 30 °C and was decreased gradually to 20 °C until the end of the experiment. Lighting was provided for 23 h per day (1−7 days), 20 h per day (8−39 days), and 23 h per day (40−42 days). The birds were fed ad libitum with complete starter (1−21 days), grower (22−35 days), and finisher (36−42 days of age) mixtures containing 22%, 20.5%, and 20.5% crude protein and 2990, 3130, and 3130 kcal/kg metabolizable energy, respectively, and had unrestricted access to drinking water. Each group of birds received feed mixtures that differed only in the dietary fat source, and the net fat contents in the consecutive diets  were 2.9%, 4.8%, and 4.8%, respectively. The mixtures were composed according to the dietary requirements for meat-type chickens . The chiThe pH values of the pectoralis major muscles were determined 15 min post mortem and after 24 h of air-chilling at 4 °C to examine the rate and intensity of muscle acidification, using a portable CyberScan10 pH meter  equipped with a glass electrode and a temperature probe for automatic temperature adjustments. The pH meter had been calibrated using two calibration buffers (pH 4.01 and pH 7.00) at a temperature similar to the temperature of muscles, and then, measurements were taken by placing the electrode at a 45° angle mid-way through the body of the muscle.The color of the inner surface of each muscle was determined immediately after a dissection using the reflectance spectrophotometer Minolta CR 310 Chroma Meter  equipped with a 50-mm reading head. Whenever possible, the entire surface area of the muscle was analyzed; however, a special care was taken to avoid areas containing visible bruises, hemorrhages, or superficial blood vessels .The amount of exudate from the pectoralis major muscles was measured 48 h post mortem for the determination of their drip loss. Meat samples (weighing ~80 g) were placed in tightly sealed containers and stored in a refrigerator at 4 °C. Drip loss was expressed as the percentage of sample weight loss in relation to its weight recorded before refrigeration. The thawing weight loss was determined after 28 days of storage at ~18 °C. Samples of the muscles weighing ~80 g were stored in sealed containers and then moved to a cold storage room, where they were thawed for 24 h at 4 °C. After thawing, the samples were re-weighed, and the weight loss was expressed as the percentage of initial (pre-freezing) weight. The cooking weight loss was defined as the percentage loss of meat weight after cooking in a water bath at 100 °C until the attainment of a core temperature of 76 °C (measured in the thickest portion of the sample). All samples (~80 g each) were cooked individually in small plastic bags, then chilled at room temperature for 30 min, and finally kept at 4 °C for 45 min before determining the relative thermal loss .®, High Wycombe, UK) as the maximum force (N) perpendicular to the fibers. A texture analysis, which included hardness (N), springiness (mm), elasticity, cohesiveness, gumminess (N), and chewiness (MJ), was performed with the same instrument fitted with a 50-mm compression attachment using a Texture Profile Analysis (TPA) software . A double compression test with a 60% deformation was performed with a crosshead speed of 200 mm/min, an interval of 2 s between successive compressions, and detection threshold of 0.01 N. The samples were compressed perpendicularly to the muscle fiber orientation. All physicochemical analyses of the broiler chicken breast muscles were conducted in the National Research Institute of Animal Production.After weighing for the determination of thermal loss, the chilled meat samples were prepared for shear force and texture measurements. Samples measuring ~1.3 cm in diameter and ~3 cm (for shear force determination) or ~1.5 cm in length  were removed from each sample parallel to the fiber orientation through the thickest portion of the cooked muscle. The Warner–Bratzler shear force was determined using an Instron 5542 system  was cut out of the left pectoralis major; frozen in isopentane, which was precooled with liquid nitrogen; and then stored at ~80 °C until the histological examinations at a later date. The tissue samples were mounted on a cryostat chuck with a few drops of Tissue-Tek freezing medium . Transverse sections (10 µm in thickness) were cut at ~20 °C with the cryostat  and stained with hematoxylin and eosin. The sections were then dehydrated in a graded series of ethyl alcohol, cleared in xylene, and mounted onto the glass slides using DPX mounting medium . A minimum of 200 fibers was counted in each section using a Nikon E600  light microscope. The diameters of the muscle fibers were measured with the image analytical software MultiScan®14.02 . Samples of the breast  were collected from birds of each group in order to determine the chemical composition, i.e., the contents of total dry matter, total protein (Kjeldahl method), and crude fat (Soxhlet method) using the AOAC standards [Within 15 min post mortem, a block measuring ~20 mm in length and with a base area of ~100 mm®7.0 analytical software . Four identical, non-overlapping circular spot meters (33 pixels in diameter) were used to compute the mean pixel intensity (MPI) and mean pixel heterogeneity  of each ultrasonogram; the echotextural variables for all spot meters were then averaged to give the final echotextural value of a muscle.Ultrasonograms of the birds’ pectoralis major muscles were obtained just before slaughter, using the Aloka PS2 ultrasound scanner  connected to a hand-held 5.0-MHz linear-array transducer . Each mu®11.0; Systat Software Inc., San Jose, CA, USA). Echotextural differences between the groups and scanning planes  were analyzed by two-way ANOVA and the least significant difference (LSD) tests for comparisons of the individual means. A Pearson product moment analysis was used to examine the correlations among quantitative echotextural attributes and the physical properties/chemical composition of pectoralis major muscles for the four fat-supplementation groups of birds. Since there were no significant differences in echotextural variables between the lateral and transverse scanning planes, the data averaged on a per animal basis were used for correlation analyses. A p value ≥ 0.05 was considered statistically significant, and all results are given as the mean ± standard error of the mean (SEM).One-way analysis of variance (ANOVA) and Tukey tests were used to compare the weights of birds/carcasses and the physicochemical variables of pectoralis major muscles among the four groups of broiler chickens studied (SigmaPlotp < 0.05) in the mixed-oil group , and it was greater (p < 0.05) for Group BF (3012 ± 5 g) compared with Groups SO (2899 ± 6 g) and FO (2912 ± 4 g) of broiler chickens. The mean weight of chilled carcasses was greatest (p < 0.05) for Group SO + FO birds (2460 ± 12 g) followed by Groups BF (2349 ± 16 g) and FO (2346 ± 10 g), and it was the lowest (p < 0.05) for Group SO (2266 ± 12 g). There were no significant differences among the four fat-supplementation groups of chickens in the mean estimated weight of combined pectoral muscles (674 ± 14 g).The mean body weight of the chickens before slaughter was greatest (p > 0.05). The total protein content was greater (p < 0.05) in Group BF compared with Group SO, but both Groups SO and FO exceeded (p < 0.05) Group BF in crude fat content of the pectoralis major muscles.The pH of the muscles recorded 24 h post mortem were significantly lower in Group FO and Group SO + FO compared with Group BF . The colp < 0.05) Group BF animals in mean MPI values of the left pectoralis major muscle scanned in the transverse plane.There was a significant main effect of the fat-supplementation group for the mean pixel intensity (MPI) of pectoralis major muscles in the birds of the present study . The MPIp < 0.05) MPH than those of the right muscle obtained in both scanning planes. In addition, the MPH of the left pectoralis major muscle in the longitudinal plane was less (p < 0.05) in Group SO compared with Groups FO, SO + FO, and BF.There was no significant main effect of either dietary group or scanning plane for MPH values determined in the ultrasonograms of pectoralis major muscles, but the interaction of the two terms was statistically significant. Longitudinal scans of the left pectoralis major muscle in Group FO chickens exhibited a greater , and crude fat content exhibited two significant correlations each : with MPI in Group FO and with MPH in Group BF; and crude fat: with MPI in Group FO and with MPH in Group BF). With respect to the relative sensitivity of ultrasound imaging, muscle lightness (L*) was the variable with the strongest correlations to image attributes . The strongest correlations for each group of birds were as follows: Group SO—between MPH and pH15 min ; Group FO—between MPI and color saturation towards yellow (b*) ; Group SO+FO—between MPI and hardness ; and Group BF—between MPH and lightness (L*). When data were pooled and analyzed for all animals, there were only three significant overall correlations .Significant correlations between the echotextural variables and physicochemical characteristics of the pectoralis major muscle determined in the present study are summarized in 2 diffusion, which parallels the formation of carbonic acid, decreased muscle pH in carp over a 15-day period. Janisch et al. [The pectoralis major muscles of Group SO and Group FO birds had significantly higher crude fat and lower crude protein contents compared with Group BF. This result contrasts with the findings of a previous study by Cortinas et al. , which dh et al.  observedThere were differences among the four treatment groups of birds in both of the echotextural variables of the left pectoralis major muscle . In addition, the mean MPH of the left pectoralis major muscle in the transverse plane was greater than the pixel heterogeneity of the right muscle in either the transverse or longitudinal plane. Differences among the four fat-supplementation groups may be due to a summation of differences in their physicochemical properties that were more pronounced in the left compared with the right pectoral muscle. Similarly, the differences in MPH between the right and left muscles and scanning planes may be indicative of the varying chemical composition or histomorphological properties ,8. Niels15 min in Groups SO and BF). Moreover, even though correlations between MPI and cutting force were of identical magnitude, they were in opposite directions . A correlation between crude fat and MPI was recorded in Group FO, which had the highest crude fat content. Moreover, the threshold content may vary between proteins and lipids. In the present study, the crude fat content of pectoralis major muscles was approximately ~2%, while the crude protein content was ~23%. Lipids significantly increase tissue echointensity, and so, it is attractive to assume that a 10-fold lower amount of fat compared with proteins is required for the detection of quantitative correlations with numerical pixel values. As with the MPI values discussed above, ultrasound imaging may require a minimum degree of heterogeneity in the muscle tissues to detect correlations between MPH and chemical constituents. The pectoral muscles from Group BF chickens exhibited the highest crude protein and lowest crude fat content; this contrast is likely the reason why Group BF had the greatest MPH values and why MPH, in this subset of animals, could be large enough to identify correlations with crude protein and crude fat content.The pectoral muscles of broiler chickens in this study were examined with a 5.0-MHz transducer. Earlier ultrasonographic studies of skeletal muscles utilized ultrasound frequencies ranging from 5 to 8 MHz. High-frequency waves are suitable for imaging mainly superficial structures as they are more attenuated than lower-frequency waves for a given distance, but they offer images of higher resolution ,26. The To recapitulate, ultrasonographic image attributes were significantly correlated with certain physicochemical properties of broiler chicken pectoralis major muscles. Different dietary fats induced significant differences in physicochemical characteristics of the muscles among the four fat-supplementation groups of birds. The present study demonstrates the potential applicability of computerized analysis of ultrasonograms to study certain physicochemical characteristics of pectoral major muscles in broiler chickens. At present, however, due to the lack of consistent correlations across the dietary groups and considering significant differences in echotextural parameters between the scanning planes or right and left pectoralis major muscles, we remain skeptical of the immediate utility of this technique for predicting physiochemical properties of the muscles in commercial settings, at least until further confirmatory studies are complete.


Posterior reversible encephalopathy syndrome (PRES) is a complication that occurs during various diseases’ treatment. Imaging examination is the gold standard for diagnosis. PRES frequently occurrence in patients with hematological malignancies results in poorer prognosis and higher mortality. We aim to establish a practical and operable scale for early prediction, assessment of the severity of the Posterior Reversible Encephalopathy Syndrome, and timely intervention for better prognosis.The scale designed by reviewing the literature and by referring to clinical practice. We assessed the reliability and validity of the scale. Scale-based assessment of children undergoing chemotherapy for acute lymphoblastic leukemia conducted as early warning and intervention for those who may have PRES.Establishment of Posterior Reversible Encephalopathy Syndrome early warning scoring (PEWS) scale included three parts, as follows: (1) risk factors, including underlying disease, hypertension, Infection, and drug toxicity; (2) clinical features, including high cranial pressure, visual symptoms, seizure, and disturbance of consciousness; and (3) EEG features, including slow wave and epileptiform discharges. Utility assessment of PEWS scale showed that in 57 patients with acute lymphoblastic leukemia, 54 scored less than 10 and none of them detected as PRES. The other two had scores of 12 and 13 both diagnosed with PRES by brain MRI scan.PEWS scale can predict PRES early. PRES was highly suspected when the score was 10 points and more. Thus, prophylactic intervention can give to improve the prognosis of PRES. Posterior reversible encephalopathy syndrome (PRES) has drawn the attention of clinicians in recent years. PRES was first discovered by Hinchey in 1996 as a clinical imaging syndrome . Most stLarge-sample retrospective studies about PRES in the past 2 years mainly focused on disease spectrum, pathogenesis, atypical lesion, and complications . These sImaging examination is the gold standard for PRES diagnosis. Characteristic MRI results include symmetrical hypointense T1 signal, hyperintense T2 signal, and isointense or slightly hyperintense DWI signal in the posterior parietal lobe and occipital lobe. Studies in the past 2 years indicated that PRES can occur in areas beyond the posterior brain region, such as frontal lobe, temporal lobe, cerebellum, basal ganglia, brainstem, and the entire brain. PRES is not always reversible. Many sequelae, like cerebral hemorrhage, cerebral infarction, focal gliosis, brain atrophy, and cerebral necrosis, were observed when vasogenic brain edema develops into cytotoxic brain edema .Cohort studies showed the presence of PRES in patients with hematological malignancies resulted in poorer prognosis and higher mortality, especially after hematopoietic stem cell transplantation (HSCT) , 9. We nThirty-one children diagnosed with PRES from 1 January 2001 to 1 April 2013 in Chinese PLA General Hospital, PLA Army General Hospital, Peking Union Medical College Hospital, Xin Hua Hospital affiliated to Shanghai Jiao Tong University School of Medicine, and Yu Ying Children’s Hospital of Wenzhou Medical University enrolled in this study. This research performed by the Declaration of Helsinki, and told consent existed from each patient’s parents or caregivers before the trial-related procedures. The criteria include 1) a manifestation of acute neurological syndromes including headache, visual changes, seizures, and altered mental status; and 2) cerebral magnetic resonance imaging (MRI) showed angioedema in the posterior region of the brain.The clinical information of patients established by searching for “posterior reversible encephalopathy syndrome” and “PRES” in five cooperative medical centers. Clinical features of all the objects, including initial symptoms, risk factors , drugs, and prognosis assessed. Symptoms recorded including headache, seizure, and visual changes. MRI reconfirmed by radiologists to analyze lesion distribution and involvement.Statistical analysis: Data analyzed using SPSS 17.0. Chi-square test or Fisher’s exact test used to assess the classified variables. Mann-Whitney U test or Wilcoxon test used to assess continuous variables. Age described as median age (Q1–Q3).Electroencephalogram (EEG) imaging involved awake and complete sleep (2 h to 4 h) states. EEG performed in children diagnosed with acute lymphoblastic leukemia by the MICM criteria for the initial time-induced chemotherapy (CCLG-ALL2008 scheme), before chemotherapy (D1), 15th day of chemotherapy (D15), and at the end of chemotherapy. All the patients routinely underwent sleep deprivation the night before the EEG examination. One or two parents of each patient accompanied their children during hospital stay as observers. Clinical manifestations and laboratory test results recorded in a sheet (disease diary) with the help of health care providers. These data also logged into a medical record. Symptoms recorded headache, seizure, and visual changes. After each EEG, the patients would reassessed by a scale, and those with a score of 10 or more would undergo brain MRI for PRES diagnosis. Early preventive chemotherapy adjustment program included the following: 1) suspension of Methotrexate intrathecal injection and vincristine administration for one time; and 2) dexamethasone decrement earlier than scheduled. We recruited 31 patients diagnosed with PRES, which consisted of 16 males and 15 females. The median age of the patients was 7 years old (range 3–12 years), and 74.2% of the patients were younger than 10 years old. Only 4 of 29 patients experienced seizure once. Other patients experienced multiple episodes, and the maximum number of seizure attacks was > 10 times. After the onset of PRES, 9 patients presented notable confusion, and 4 patients were in a coma. The initial symptom for 23 patients (74.2%) was seizure. For 6 patients (19.4%), the first symptom was confusion. For 2 patients (6.5%), the initial symptom was visual impairment. The clinical symptoms and prognosis of patients with and without hematologic malignancies listed in Table By comparing the clinical characteristics and prognosis of children with PRES in hematologic malignancies and other diseases, we found no statistical difference in the results of the above-mentioned two groups of patients. However, partial remission and death patients in hematologic malignancies group were higher than that in the non-hematological malignancies group. The small sample size resulted in the lack of statistical difference. We concluded that children with hematological malignancies might present poor prognosis.Seventeen patients with PRES showed slow-wave activities in the background. Both δ and θ activities were found in 9 (52.9%) patients. In three cases, slow wave detected in the occipital region. The same finding found in four cases with epileptic discharges. The EEG of three patients (17.6%) suggested θ activity in the occipital region. Diffused δ activity observed in five patients (29.4%), among which one patient presented epileptic discharges.Among 17 patients, 6 (35.3%) presented abnormal EEG in the occipital area. Eleven patients showed a diffused slow wave in the EEG background, among which five (45.5%) cases confined to the occipital and parietal lobes by MRI, and others (54.5%) affected in the frontal and temporal lobes Table .Table 2cThe PEWS scale consists of the three following aspects: high-risk factors, clinical characteristics, and EEG features. All criteria derived from the literature or clinical experience. To reduce the influence of diagnostic criteria for the inappropriate performance of a patient in the literature on the whole scale, we restricted the scores for each part of the scale.The maximum scores for the risk factors, clinical features, and EEG features were 10, 10, and 6, respectively, for a maximum total score of 26 points. Table .Table 3PIn our study, two groups of patients evaluated using the PEWS scale. Seventeen PRES cases with a median age of 12 years old  and 15 NON-PRES cases with a median age of 9.5 years old , all with age-matched hospitalization, enrolled. All the patients completed radiology and EEG.The PEWS scale demonstrated excellent consistency compared with the gold standard. The median score for the PRES group was 15.5 (range 10–19). The median score for the other encephalopathy group was 8 (range 6–12). The scores of the two groups showed in Fig. Receiver operating characteristic (ROC) curve of PEWS scale for patients showed in Fig. The PEWS scale scores of the two groups presented a partial intersection within a certain range. Thus, we concluded that if the final score is 0–9 points, children are less likely to diagnose with PRES; if the score is 10–12 points, children may diagnosed with PRES; and children with 13 points or above are more likely to diagnose with PRES.Fifty seven patients with acute lymphoblastic leukemia treated with the VDLP/VDLD program enrolled in this study, including 39 males and 18 females with a median age of 8.7 years old (1–16 years old).EEG monitoring performed at the beginning of admission, at 2–3 weeks after chemotherapy, and at the end of chemotherapy. The EEG monitoring time of each patient was no less than 2 h. The record should include the awake period, as well as the complete sleep period.The EEG background activity was normal or nearly normal during initial checking in all patients. EEG examination of the 16 patients during the first 2–3 weeks of chemotherapy suggested a background of slow-wave activity, mainly involving the parieto-occipital lobes 12/16, 75%). Twelve patients exhibited focal δ activity, 3 showed diffused δ and θ activities, and 1 presented focal θ activity Table . PEWS sc%. Twelve54 of the 57 patients achieved a PEWS scale score of less than 10 points, and none of the 54 patients diagnosed with PRES. Of the three other patients, one patient with a PEWS score of 10 points presented no significant imaging abnormalities. One patient attained a PEWS scale score of 12, and existed a radiographic presentation of PRES for this patient. After adjusting chemotherapy regimens, the patient did not experience headache, hypertension, seizure, or disturbance of consciousness. One other patient achieved a PEWS scale score of 13 points, suggesting a great possibility of PRES diagnosis. Head MRI findings suggested bilateral parieto-occipital subcortical white matter long T1 signal, long T2 signal, and equal DWI signal, which confirmed to occur PRES. The following day, this patient experienced a single short grand mal seizure. He gave prophylactic intervention, such as antihypertensive treatment, adjustment of chemotherapy, and other intensive management treatments that used before the onset of seizure. The patient achieved complete clinical recovery with subsequent radiologic and EEG resolution in less than 6 months  confined to the occipital and parietal lobes, as detected by MRI. Other cases (54.5%) affected in the frontal and temporal lobes. This result was consistent with the findings of previous adult studies. EEG is a sensitive and rapid diagnostic technique. EEG results with slow-wave background performance can use for early diagnosis of PRES.We have demonstrated the PEWS scale was the first research that assessed the reliability of clinical presentation, risk factors, and EEG features for the early diagnosis of PRES.In the first part of this study, we summarize the risk factors and the clinical, imaging, and EEG features of PRES children. We developed a PEWS scale to help diagnose PRES. In the second part of our study, the scale validated to clarify the effect of the PEWS scale. Considering those different diseases that induce PRES present different prognoses, we specifically selected acute lymphoblastic leukemia patients receiving induction chemotherapy as subjects in the second part of our study. The patients in this period presented a variety of risk factors that induced PRES. The clinical and imaging features of concurrent PRES were often atypical and demonstrated poor prognosis. To address all the elements of this study and to avoid interfering different underlying diseases, we used these patients as our research subjects to be as representative as possible. In the second part, 57 children with leukemia evaluated using the PEWS scale. According to assessing of the patients’ condition and adjustment of treatments, only one patient with PRES presented mild symptoms and rapid recovery without leaving any sequelae.MRI is the gold standard for the diagnosis of PRES, and practitioners need to decide whether MRI scan should give. PEWS scale is an effective and operable tool for early warning of PRES and assessment of its severity. This scale has a wide range of applications. Clinicians should consider PRES when PEWS scale scores are more than 10 points, and they should perform MRI scan for diagnosis. This will help with selecting different levels of prophylactic treatments to reduce the incidence and to improve the prognosis of patients with PRES. We designed the flowchart of PRES early warning scoring scale to help clarify its use Fig. .Fig. 4ThThe low prevalence of PRES was the reason that we only found two patients with this condition. Now, in our center, PEWS has been used as a routine assessment of patients with acute encephalopathy and hematologic malignancies. After publishing of the PEWS scale, we hope that more pediatricians can apply it to diagnose and provide intensive intervention in patients with suspected PRES.PEWS scale successfully applied to our 57 patients, a larger sample size and a multi-center collaborative study. But our study required for further clinical validation.Seizures are the most common clinical features among children with PRES. Children with hematological malignancies present poor prognosis. Slow-wave background activity is a common EEG manifestation of PRES, and its appearance is a significant clue for occurs of this disease. PRES highly suspected when the PEWS scale score is 10 or higher, and pediatricians need to focus more on this condition.


Leishmania donovani MON 37. Confirmation of diagnosis is done through microscopy, either directly or after in vitro culture. Molecular diagnostic methods are sensitive, but require well established laboratories. Loop mediated isothermal amplification assay (LAMP) is rapid, specific for parasite species-specific DNA amplification, and requires only basic laboratory equipment. The aim of the study was to determine the potential utility of LAMP to diagnose leishmaniasis.Cutaneous leishmaniasis (CL) in Sri Lanka is caused by Thirty one patients clinically diagnosed as CL were enrolled in the study. Light microscopy, a widely used and universally accepted method was used as the reference standard for confirmation of diagnosis.LAMP was positive for 19/23 microscopically positive patients, yielding a sensitivity of 82.6%. Specificity of the LAMP assay was 100% and the positive and negative predictive values were 100% and 66% respectively. The average time taken for the LAMP assay was 1 hour and 40 minutes and the cost per sample was about SLR 2 000, which was approximately half the time and cost of a nested PCR (polymerase chain reaction).LAMP could be considered a potentially useful diagnostic tool for leishmaniasis. Leishmania donovani is the causative agent in Sri Lanka (L. donovani MON 37) [Leishmania spp. promastigotes are transmitted through female sandflies (Phlebotomus argentipes) during their blood meal. CL is the most prevalent clinical phenotype recorded in Sri Lanka, although a few cases of muco-cutaneous and visceral leishmaniasis have also been reported [Cutaneous leishmaniasis (CL) is considered as a neglected tropical disease . A genet MON 37) . Leishmareported . Accordiin vitro cultures, which increases the sensitivity [Diagnosis of cutaneous leishmaniasis is traditionally done by microscopy, which is the only established method in the endemic regions, though it requires services of skilled technicians and microscopists for preparation and examination of the slides . Furthersitivity . HoweverLoop-mediated isothermal amplification (LAMP) is a recently established tool for detection of many diseases including leishmaniasis . The LAMin vitro growth of parasites as previously described [A total of 31 patients with skin lesions that were suspected to be leishmaniasis who were referred to the leishmaniasis clinic at the Department of Parasitology, Faculty of Medicine from July 2014 to May 2015 for laboratory tests for confirmation of diagnosis, were enrolled in the study. The participants formed a random series. Slit-skin scrapings and/or lesion aspirates were collected from the lesions of the patients using a standard protocol. Giemsa-stained smears of lesion aspirates were examined under the microscope (x100 magnification). Microscopy, which is a universally accepted and widely used method for parasite detection, was taken as the reference standard for the comparison of results. The culture media were inoculated for escribed . CultureLeishmania genus. DNA with R 221(5’GGTTCCTTTCCTGATTTACG3’) R332.DNA was extracted from the lesion aspirates or from the parasite culture after two days of inoculation, with the use of QIAamp DNA blood mini kits . The extracted DNA was eluted in a final volume of 60 μl AE buffer . Nested PCR was performed using previously described methods that detect (5’GGCCGGTAAAGGCCGAATA3’) as primers for the first round; R223.2, 5 mM KCl, Tris-HCl (pH 9), 2 mM and 1U of Taq polymerase. For the second nested reaction 1 μl of the first PCR product was used as a template in the presence of 3 μl of each R223 and R333. Reaction conditions were same for both the first and second nested PCR cycles. Thermal conditions were 96°C for 5 minutes and 25 amplification cycles of 96°C for 30 seconds, 52°C for 30 seconds, 72°C for 5 minutes and termination at 4°C.The product was visualised on a 2% agarose gel.(5’TCCCATCGAACCTCGGTT3’) and R333 (5’AAAGCGGGCGCGGTGCTG3’) as primers for the nested PCR round . Two micL.donovani kinetoplast minicircle DNA were used (GenBank accession no.Y11401) [2SO4, 10 mMKCl, 2 mM MgSO4, 0.1% Triton X-100, pH set for 8.8 at final 1× and additional 6 m M MgSO4 and 8 units of bst polymerase large fragment . The reaction mixture was incubated at 62°Cfor 1 hour and 30 minutes and chilled on ice to visualize the precipitate. The tube was wrapped in paraffin prior to the LAMP assay to avoid contamination.LAMP was performed using the original protocol with minor modifications . A set o.Y11401) . The LAMSensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of each assay were calculated considering microscopy as the gold standard. Adjusted Walds method was used to calculate the confidence interval, which is considered as the most accurate method for small binomial samples . ApprovaTwenty three out of 31 patient samples were found to be positive for Leishmania parasites through microscopy (either direct or after culture). All the microscopy positive samples were also positive via nested PCR. The results of LAMP assay were interpreted depending on the presence or absence of turbidity through direct visualisation. Out of the 23 specimens that were positive through microscopy and PCR, 19 were positive for the LAMP assay.The LAMP assay yielded a sensitivity of 82.6%. The specificity of the LAMP assay was 100% while the positive and negative predictive values were 100% and 66% respectively. Nested PCR on the other hand showed 100% sensitivity, specificity, PPV and NPV values. The 95% 2-tailed confidence interval of a success rate for the LAMP assay as calculated by this test was 0.70–0.95, while the values for nested PCR and the microscopy were 0.87–1.02. The overlap between these values indicated that results were not significantly different.The average time taken for the LAMP assay was 1 hour and 40 minutes when compared to the nested PCR that took approximately 3 hours and 30 minutes. The cost analysis was done for the determination of the cost-effective method between LAMP and nested PCR. The cost of performing LAMP was SLR 2000 per sample and the cost of performing the nested PCR per sample was SLR 3560. The cost was calculated with all the reagents used for the methods including the shipping cost, but the cost calculation excluded the initial investment made for laboratory equipment, such as water bath, pipettes, eppendorf tubes, tips, PCR machine and the wages.Cutaneous leishmaniasis is a newly established disease in Sri Lanka, and still lacks a relatively simple, low cost and a reasonably sensitive diagnostic tool that can be performed with minimal technical expertise.bst polymerase that gets activated during higher temperatures minimises non-specific bands, and has the potential to resist inhibitors, which occur during nested PCR [LAMP is a technique used for the diagnosis of many infectious diseases, such as malaria and leishmaniasis ,10. The sted PCR . Unlike sted PCR .The LAMP assay has other advantages such as relative simplicity in performance, low cost, easy interpretation, high level of reproducibility, and relatively rapid performance time. The nested PCR assay, on the other hand, is only genus-specific (similar to microscopy) and requires expensive equipment, highly trained personnel, stringent laboratory conditions and longer performance time. LAMP technique could be considered particularly attractive in the local setting since it requires only a heat block or a simple water bath, in contrast to the expensive PCR thermo cyclers, gel documentation systems and UV transilluminator that are required for regular molecular assays. Furthermore, the assay is conducted under isothermal conditions for a shorter time, which is known to minimise inhibitions that generally occur during the latter stages of the PCR cycles, that could interfere with performance.The LAMP technique could be performed even in places without electricity, since the power supply to the water bath can be replaced with alternative methods such as batteries or solar systems. The result of the LAMP assay is interpreted through visualisation of turbidity, bypassing the need for post-PCR analysis involving gel electrophoresis, which is laborious and has the risk of product contamination, possible errors in interpretation, as well as potential exposure to toxic agents. Sensitivity and negative predictive value of LAMP assay, were less than with either microscopy or nested PCR. So, a negative result in the LAMP assay, particularly in a patient who is strongly suggestive of leishmaniasis based on clinical grounds, might require nested PCR as a second-line investigation to confirm or refute the diagnosis. The small sample size used in our study is a limitation.In conclusion, the LAMP assay optimised for use under local conditions, appears to be a useful tool for diagnostic laboratories in Sri Lanka for the confirmation of leishmaniasis.


CPT1A, SOCS3 and ABCG1) using real data made available through the GAW20.Transgenerational epigenetic inheritance has been posited as a possible contributor to the observed heritability of metabolic syndrome (MetS). Yet the extent to which estimates of epigenetic inheritance for DNA methylation sites are inflated by environmental and genetic covariance within families is still unclear. We applied current methods to quantify the environmental and genetic contributors to the observed heritability and familial correlations of four previously associated MetS methylation sites at three genes (Our findings support the role of both shared environment and genetic variation in explaining the heritability of MetS and the four MetS cytosine-phosphate-guanine (CpG) sites, although the resulting heritability estimates were indistinguishable from one another. Familial correlations by type of relative pair generally followed our expectation based on relatedness, but in the case of sister and parent pairs we observed nonsignificant trends toward greater correlation than expected, as would be consistent with the role of shared environmental factors in the inflation of our estimated correlations.Our work provides an interesting and flexible statistical framework for testing models of epigenetic inheritance in the context of human family studies. Future work should endeavor to replicate our findings and advance these methods to more robustly describe epigenetic inheritance patterns in human populations. Metabolic syndrome (MetS) is a widespread problem in the United States, with 35% of U.S. adults having MetS in 2012 [Given the heritability that remains unexplained by established genetic variants for the subcomponents of MetS, transgenerational epigenetic inheritance has been posited as a possible contributor to the observed heritability . AlthougCPT1A, SOCS3, and ABCG1 have been associated with MetS, or its subcomponents  [To date, CpG methylation sites at , ABCG1) –12. The , ABCG1) , extende, ABCG1) , 14, andCPT1A, SOCS3, and ABCG1), adjusting for demographic, environmental factors and genetic variation in a stepwise fashion using both fixed and random effects. Then we estimated familial correlations of methylation profiles at these CpG sites, both with and without adjustments, and across relative pair types.We aimed to apply current methods  to quantify the environmental and genetic contributors to the observed similarity within families at four specific MetS CpG sites. To do this, we leveraged data on 1105 adults made available through the Genetic Analysis Workshop (GAW20) to estimate the heritability at CpG sites near 3 genes  study . Our anaCPT1A, SOCS3), or with Type 2 diabetes, lipids, and obesity-related traits  including: cg00574958 and cg17058475 near CPT1A [SOCS3 [ABCG1 [From the literature we selected 4 CpG sites that were previously associated with MetS . Then using the fixed effects identified in the reduced model above, we added household variance components to account for siblings and half-siblings within 15 years of each other, who were the relative pair type most likely to have shared an ‘early life’ environment at some point during their childhood or adolescence (Model 3a). Separately we added a variance component for parent pairs , who were the relative pair type most likely to have shared ‘later life’ environmental exposures (Model 3b). Lastly, in a fourth modeling strategy that included the same fixed effects from the reduced model (Models 1 and 2), we screened at P value < 0.05 local cis-acting genetic variants at each locus. To select these variants, we used publicly available 1000 Genomes phase 3 CEU (Northern Europeans from Utah) reference data to query two independent sets  [n = 8, 19, and 21 local and n = 6, 7, and 13 distant variants screened in heritability models for CpG sites at CPT1A, SOCS3, and ABCG1, respectively.No fixed effect covariates were included in our crude heritability models (Model 0). Further analyses accounted for an individual’s age and sex , quadratic age effects, and their interactions with sex (Model 1). In all subsequent models, environmental covariates were added into the models in the following sequence: center , followed by cigarette smoking status . We then screened all these demographic and environmental fixed effects, including only the effects that remained suggestively significant in the heritability models (on 1.07)  of geneton 1.07) . This reS.A.G.E. version 6.4 package (http://darwin.cwru.edu/sage/) within various pair types, representing a quasi-independent subset of the family pedigrees. We contrasted our correlations before and after creating a residual of methylation to account for the fixed effects identified in multiple reduced heritability models, and among a subset of unrelated individual pairs.The expected intra−/interclass correlation for each relative pair is a function of the pairs’ expected relatedness and the CpG site-specific heritability. We estimated weighted correlations using the FCORR module of the P value = 1E-5, n = 1103) in a model where fixed effects  explained 13% of the variation in MetS. Separately, we included variance components for early life shared environment , or later life shared environment . Although the addition of these terms influenced the magnitude of the heritability estimates , the resulting heritability estimates did not differ significantly.The prevalence of MetS at the baseline examination of GOLDN was 38.4% and its heritability was 0.47  were strongly associated with MetS and another site  was suggestively associated with MetS. Retaining these 3 sites in the polygenic model decreased the heritability estimate slightly  and increased the variance explained (VE) by all the fixed covariates to 18%. The addition of random effects of early life shared environment  decreased the heritability estimate , resulting in a nonsignificant MetS heritability estimate, whereas accounting for shared late life environment  increased the heritability only slightly .When we added fixed effects for the 4 MetS CpG sites into the model without shared environment-related variance components, two of the CpG sites  variant, rs17601808, was significantly associated with cg00574958  and cg17058475 heritability estimates  after accounting for fixed effects from the reduced model . For the SOCS3 site, one local (±250 kb) genetic variant, rs7220979, and two distant variants, rs9908993 and rs17736494, were significantly associated with cg18181703 heritability after accounting for fixed effects , resulting in similar heritability estimates  as in previous models. Including these 3 SNPs resulted in nonsignificant estimates for center and former smoking, and dropping these nonsignificant fixed covariates also yielded similar heritability estimates . At the ABCG1 site, 2 local  and 1 distant genetic variant  were associated with cg06500161 heritability  after accounting for fixed effects.For the two 2, sex, center, and current smoking, which were retained in more than 1 reduced heritability model , and were 0.01 to 0.15 greater than our expected correlation.We then estimated familial coefficients across a number of relationship pairings, before and after creating a residual adjusting for age, ageP value ≥0.3), the correlations estimated for sister pairs were the largest across all sites  positive correlations at 3 CpG sites among parent pairs (65 independent pairs), which were between 0.02 and 0.12 greater than expected. Among unrelated pairs, we observed correlations that were closer to our expectation of no correlation , which supports the upward bias of shared household environments on familial correlations. When we further paired this unrelated with respect to MetS status, the correlation at the 2 CPT1A CpG sites were biased upwards among concordant pairs, and downwards from the null among discordant pairs.Although not statistically significantly different from other pairings  across various types of relative pairs. We observed that correlations generally followed our expectation based on relatedness, but in the case of sister and parent pairs we observed nonsignificant trends toward greater correlation than expected. We posit that shared social and environmental factors may make particular relative pairs appear more similar than we would expect based on their relatedness alone, which could lead to further inflation of heritability and familial correlation estimates.Previous research has not been able to address the extent of inflation of epigenetic inheritance estimates by shared environmental effects, even though the sharing of social or environmental exposures within households may be a key driver of the observed similarity of methylation profiles within families , 13, 14.


Historically, Iceland has been an iodine-sufficient nation due to notably high fish and milk consumption. Recent data suggest that the intake of these important dietary sources of iodine has decreased considerably.To evaluate the iodine status of pregnant women in Iceland and to determine dietary factors associated with risk for deficiency.n = 983; 73% of the eligible sample) attending their first ultrasound appointment in gestational weeks 11–14 in the period October 2017–March 2018. Spot urine samples were collected for assessment of urinary iodine concentration (UIC) and creatinine. The ratio of iodine to creatinine (I/Cr) was calculated. Median UIC was compared with the optimal range of 150–249 μg/L defined by the World Health Organization (WHO). Diet was assessed using a semiquantitative food frequency questionnaire (FFQ), which provided information on main dietary sources of iodine in the population studied (dairy and fish).Subjects were women (P for trend <0.001). A small group of women reporting complete avoidance of fish (n = 18) had UIC of 50  μg/L and significantly lower I/Cr compared with those who did not report avoidance of fish  μg/g vs. 100  μg/g, P = 0.041). Women taking supplements containing iodine  had significantly higher UIC compared with those who did not take supplements  μg/L vs. 87 , P = 0.037).The median UIC ) and I/Cr of the study population was 89 μg/L  and 100  μg/g, respectively. UIC increased with higher frequency of dairy intake, ranging from median UIC of 55  μg/L for women consuming dairy products <1 time per week to 124  μg/L in the group consuming dairy >2 times per day (For the first time, insufficient iodine status is being observed in an Icelandic population. There is an urgent need for a public health action aiming at improving iodine status of women of childbearing age in Iceland. Despite Iceland’s historically high iodine consumption, decreased fish and dairy consumption among women of reproductive age have been a concern.The median UIC of 89 μg/L in the present study indicates insufficient iodine status in the population of pregnant women in Iceland.Action needs to be taken on public health level to attend to the matter.Iodine (I) is a trace mineral necessary for the production of thyroid hormone, thereby essential for a wide range of physiological processes such as proper growth, development, and metabolic function , 2. The n = 162)  , which in = 162) . The UICn = 162) . Howevern = 162) , and resn = 162) . Given tn = 162) , 5. The n = 1,021) agreed to participate in the study. Spot urine samples were provided by 73% of eligible women (n = 983).All women who visited the Prenatal Diagnostic Unit at Landspitali National University Hospital, Reykjavik, Iceland, in their 11–14th weeks of pregnancy between 2 October 2017 and 28 March 2018 were invited to participate in the study. During the study period (6 months), 1,684 women were scheduled for their first ultrasound screening at Landspitali, corresponding to approximately 77% of the pregnant population in Iceland. Out of these 1,684 women, 244 women (15%) were excluded from the study because they did not speak Icelandic and could therefore not fill out the questionnaire. Other exclusion factors included falling short or exceeding the 11–14 week pregnancy range, missing the scheduled appointment time, or miscarriage, which in total excluded an additional 90 women. This left a remaining 1,350 women eligible to participate in the study. Of these, 128 women declined because of personal time constraints, and 201 declined without further explanation. Therefore, 76% of eligible women  and the Medical Directorate of Landspitali University Hospital (LSH 5-17). Written informed consent was obtained from the participants.Subjects were asked to complete a spot urine sample and instructed to collect the midstream of urine in a sterile screw-top container. Samples were stored in freezers at a temperature of −80°C until enough samples were collected for a group shipment. Concentration of iodine and creatinine in the urine samples was measured at the Biochemistry Laboratory at the National Institute for Health and Welfare (THL) in Helsinki (Finland). The laboratory (No.T077) has been accredited by Finnish Accreditation Service FINAS and it fulfills the requirement of the standard SFS-EN ISO/IEC 17025. The scope of accreditation covers the UIC method. The urine sampling and shipment was in accordance with EUthyroid project guidelines . UICs weAccording to WHO guidelines, a median UIC value between 150 and 249 μg/L is considered the optimal population range during pregnancy . The use2). BMI < 18.5 kg/m2 was defined as underweight, 18.5–24.9 kg/m2 as normal weight, 25–29.9 kg/m2 as overweight, and ≥30.0 kg/m2 as obese.Women who agreed to participate in the study answered a questionnaire in an electronic format on dietary intake (food frequency questionnaire [FFQ]) and information regarding maternal age, education, smoking habits, parity, nausea in pregnancy, prepregnancy weight, and height. Information on weight and height was used to calculate prepregnancy body mass index (BMI) . When answering this question, women were instructed to provide information on their diet in general, and not to include avoidance only due to nausea or avoidance of raw fish during pregnancy. For other questions, the instructions were to record intake, reflecting dietary intake in the past 3 months (approximately from the beginning of pregnancy). Frequency of dairy intake was defined as 250 mL portions in the FFQ and intake of fish defined as fish consumed as a main meal. Women selected between 10 potential frequency responses ranging from ‘less than once a month’ to ‘more than 5 times a day’. The development of the FFQ has previously been described in detail and the FFQ published . The freP < 0.05.Data are presented as median and 95% confidence interval (CI) for median. Median UIC and I/Cr ratio between groups are compared using either Kruskal–Wallis H test for several independent samples or Mann–Whitney U test for two independent samples. Spearman’s correlation was used to assess trend in UIC and I/Cr across categories of dairy and fish intakes. The data were analyzed using IBM Statistical Package for Social Sciences (SPSS) for Windows, Version 24.0 . The level of significance was accepted as P = 0.002) and I/Cr (P = 0.04) than nulliparous women, which could be explained by higher intake of dairy products by multiparous women compared with nulliparous women (data not shown). Maternal education, prepregnancy BMI, pregnancy-related nausea, and smoking during pregnancy were not related to iodine status. Median frequency of intake for dairy products (excluding cheese) was 1.1 times per day and for fish 1.3 times per week (lean fish 1 time per week and oily fish 0.3 times per week).The median UIC (95% CI) and I/Cr of the study population were 89 μg/L  and 100  μg/g, respectively. Characteristics of subjects are detailed in P for trend <0.001). Women were also asked how many portions of dairy they consumed yesterday (the day before collection of the urine sample), and clear trend toward higher UIC and I/Cr was seen with higher number of portions (Supplementary Table 1). Less than 20% of women consumed at least two portions of dairy daily (excluding cheese), and about 35% had two or more portions fish per week (P = 0.012). A trend toward higher I/Cr was seen with more frequent consumption of fish (n = 18) had a lower I/Cr compared to those who did not , and the difference was indicative for UIC (P = 0.07). It should be noted that intake of fish was significantly lower in the group avoiding dairy and vice versa (Supplementary Table 2).Iodine status in relation to intake of fish and dairy is shown in per week . Women w of fish . We repeOnly 17 women claimed that they were taking supplements containing iodine. However, after assessing the validity of answers (assessment of iodine content of the brand name provided), five out of these 17 women were not taking supplements containing iodine. Two women who claimed that they were taking supplements containing iodine wrote iron in the open answer box where women were asked to provide brand names. Altogether, 124 women did not know if the supplement they were taking contained iodine. Going through the list of brand names provided, 22 more women were labeled as taking supplements containing iodine. All women using iodine containing supplements claimed that they used the supplements at least five times per week and daily dose of most of the supplements contained 150 μg. As shown in The median UIC of 89 μg/L indicates insufficient iodine status in the population of pregnant women in Iceland, using the cutoff value of 150 μg/L , 17, 18.n = 162) was in accordance with WHO recommendations with a median value of 180 μg/L , and out of the 40 women who reported avoidance or abstinence of dairy products, 32 women reported either having milk allergy or lactose intolerance (data not shown). The results show that it is of great importance to provide information about iodine nutrition to women of reproductive age with diagnosed food allergies or intolerances for iodine rich foods and to those who consume very low amounts of fish and dairy. Part of the women who reported avoidance of dairy and fish might have been following a vegan or plant-based diet at the time of the study. It has been shown that vegan and plant-based diets during pregnancy can be safely adhered to, but adverse pregnancy outcomes are possible without educated meal planning to avoid nutrient deficiencies . NumerouWomen using iodine-containing supplements in the present study had UIC close to the optimal range defined by the WHO, and as most of the iodine-containing supplements recorded were prenatal supplements, it is likely that they started taking the supplements postconception. Current research findings regarding benefits and effectiveness of beginning iodine supplementation during pregnancy remain inconclusive , 32, andn = 520) found, for example, that only 32% of the participants correctly identified pregnancy as the most critical life stage for adequate iodine nutrition and 41% were unaware of adverse health effects associated with deficiency  responded ‘I don’t know’, and despite very low intake of fish and dairy in a large proportion of the population studied, only 3.5% reported taking supplements containing iodine. Interestingly, the median intake of fish and dairy was not different between those who were taking supplements and those who did not. Yet, when examining the filled in answers detailing the supposed iodine supplement being taken, two women responded iron (may have been confused between the difference between iron and iodine), and other three women who thought that they were using supplements containing iodine turned out to be using supplements that did not contain iodine . The results of the present study highlight a lack of nutrition literacy, which is reflected in the knowledge of iodine in the population studied. Low iodine knowledge has been linked as a major risk factor for iodine deficiency –36. A stficiency . In Norwficiency , 38.n = 983) and high participation rate (75%). According to Statistics Iceland, a total of 2,188 infants were born in Iceland from April to September 2018 (approximately covering the expected delivery dates of women attending 11–14 weeks ultrasound during the study period). The study therefore includes 45% of the total population of pregnant women in Iceland. It might be considered to be a limitation that recruitment was limited to the capital of Reykjavik. According to Statistics Iceland, about 70% of women in Iceland live in the capital area. However, we cannot exclude that iodine status of women living outside the capital area is different from that reported in the present study. Another limitation is that the study was conducted during the winter months. It is possible that seasonal variations could have influenced our results, but as studies have found that the iodine concentration in cow’s milk is lower in summer than during winter time (The main strengths of the present study are large sample size (ter time , 40, samInsufficient iodine status was observed for the first time in an Icelandic population, most likely related to a decline in fish and dairy intake. The reasons for the decrease in fish and dairy intake in Iceland , 12, 16 Click here for additional data file.


The USV method showed lower drying times at all temperatures. The rehydration values of the freeze-dried minced meat samples were higher than those obtained by the USV and VD techniques. The samples prepared using USV showed higher rehydration values than the vacuum dried samples for all temperatures. The effects of the different drying techniques and drying conditions on the microstructural properties of the minced meat samples were investigated using scanning electron microscope (SEM). The USV method resulted in higher porosity and a more open structure than the VD method. Total color differences (ΔE) for VD, USV, and FD were 8.27–20.81, 9.58–16.42, and 9.38, respectively, and were significantly affected by the drying methods and temperatures (p < 0.05). Higher drying temperature increased the ΔE value. Peroxide values (PV) significantly increased after the drying process, and samples treated with USV showed lower PV values than the VD treated samples. This study suggests that USV could be used as an alternative drying method for minced meat drying due to lower drying times and higher quality parameters.This study aimed to investigate the effect of different drying methods, namely ultrasound-assisted vacuum drying (USV), vacuum drying (VD), and freeze-drying (FD), on the drying kinetics and some quality parameters of dried minced meat. In this study, USV was for the first time applied to the drying of minced meat. The USV and VD methods were conducted at 25 °C, 35 °C, and 45 °C. The different drying methods and temperatures significantly affected the drying time ( Meat and meat products are valuable and perceived as high-quality foods because they contain dietary protein; vitamin B; minerals, especially zinc, iron, and phosphorus; and essential fatty acids in high amounts ,2. HowevThe drying process is one of the oldest and most widely used preservation methods because it is efficient and cheap. Drying extends the shelf-life of meat and meat products because of the reduction in water activity . In addiIn conventional thermal drying methods, flavor, color, and nutritional loss (vitamin degradation and loss of amino acids) occur because of thermal degradation, which decreases the drying rate and rehydration ratio . For thiTo shorten the drying time, ultrasonic treatment and vacuum drying are combined in the USV technique . Using tFD is one of the best food drying methods for biological materials sensitive to heat and oxidation. It does not cause protein denaturation or loss of vitamins, and therefore freeze-dried products retain their nutritional value . Using FBesides selecting the correct drying method, optimization of the drying temperature is another important parameter affecting the quality of dried food products. High temperatures provide low drying times due to more rapid heat and moisture transfer. However, high temperatures lead to the degradation of some heat sensitive components, such as protein and vitamins, as well as reduce color quality and rehydration ability, and increase shrinkage rates ,14,15. LThe main aim of this study was to determine the effects of different drying techniques on the quality of minced meat. For this purpose, the drying of minced meats was conducted using different drying methods at different temperatures and the effects on the quality of the minced meat were evaluated. In this study, VD , with and without ultrasound pretreatment, and FD methods were used to determine the physicochemical properties of the minced meat.Minced meat samples (from beef meat) were purchased from a local marketplace in Istanbul, Turkey and brought to the laboratory at 4 °C. The samples were stored in the refrigerator until analysis and drying process. Minced meat was spread on a silicon plate with a diameter of 9 cm and a thickness of 5 mm (21 ± 0.5 g) before the drying process.The research was carried out in two steps. First, the minced meat was dried by different drying techniques, and then the rehydration ratio, shrinkage, color measurement, and microstructure of dried minced meat were determined. All measurements were carried out in triplicate.The dry matter content of meat samples was determined by the procedure of AOAC 950.46 . The dryThe different drying techniques, namely ultrasonic vacuum (USV), vacuum (VD), and freeze dryer (FD), were used to perform the drying process of minced meat. The drying temperatures of 25 °C, 35 °C, and 45 °C were selected for USV and VD. w/w). The vacuum drying technique was carried out according to the method described by Tekin et al.  using a Mt, M0 and Me are, respectively, moisture content at each measurement time, initial moisture content, and equilibrium moisture content (kg water/kg dry matter). The relative moisture of the drying air (Equation (1)), which varied continuously during the drying experiments, is simplified into Equation (2) [The moisture ratio (MR) of minced meat samples was calculated according to Equation (1):tion (2) :(2)Mt−MeDeff) during the drying of most food materials. Equation (3):Deff is the effective moisture diffusivity (m2/s), L is the half-thickness of the slab in samples (m), and t is the drying time (s). Equation (3) uses an analytical solution and neglecting shrinkage, constant temperature, and diffusion contribution, as well as uniform starting moisture distribution. Equation (4) [t drying time, Deff effective diffusivity (m2/s), n is the number of terms of the Fourier series, and L slices are half slab thickness (m). For long drying times Equation (4) can be further simplified in Equation (5) [Fick’s second law of diffusion, Equation (3), is widely used to describe the effective spread of moisture (tion (4) :(4)MR=8πtion (5) :(5)MR=8πDeff values can be calculated by plotting the ln(MR) value as a function of time, and a straight line with a slope of (K) can be calculated from this plot, which can be written as Equation (6):The Deff values Equation (7): D0 represents the pre-exponential factor of the Arrhenius equation (m2/s), Ea is the activation energy (kJ/mol), R is the ideal gas constant (kJ/mol·K), and T is the drying temperature in Kelvin [The Arrhenius equation is generally used to determine the temperature dependency of the n Kelvin . The rehydration of the minced meat was performed according to a modified method described by . Two graSurface change% (shrinkage) is a common widely-known phenomenon during drying. Dimensional shrinkage of the dried minced meat samples was determined using the following Equation (9). The diameter and thickness of the fresh and dried minced meat samples were measured by using a steel Vernier caliper.L* (whiteness/darkness), a* (redness/greenness), and b* (yellowness/blueness).The effect of different drying methods and temperatures on color changes of dried minced meats were determined at room temperature using a chroma meter  at four different edge spots on the surface of each sample before and after the drying treatment. The color evaluation procedure was based on the determination of Hunter values E) value was calculated according to the following Equation (10):To describe the change in the color values of samples, the total color difference  . Dried minced meat samples were cut and fixed on the SEM stub and the samples were covered with a golden coat to provide a reflective surface for the electron beam. The gold-plated samples were then imaged under a microscope at 5 kV .p < 0.05) main effect was found, mean values were further analyzed using the Duncan’s Multiple Range Test comparison test. The measurements were repeated twice with three replications. Collected data were subjected to analysis of variance (ANOVA) using JMP 9 . When a significant ; this time was approximately 20% shorter than the time observed for VD at 45 °C (100 min). The constant rate periods for the USV and VD were 60 min and 70 min, respectively. The total drying times at 25 °C for the USV and VD were 165 min and 195 min, respectively, and the drying times at 35 °C were 115 min and 125 min for the USV and VD, respectively. Resulting from the ultrasound treatment, the diffusion boundary layer was changed, and this change is explained by the properties of the acoustic waves caused by the ultrasound ,26. Baslar, et al.  used an As shown in Deff), an important concept regarding physical and thermal properties, is defined as the transport of moisture at distant rates during the drying of food. As seen in Deff values increased with increasing temperature. The Deff values were calculated as 1.07 × 10−8–2.96 × 10−8 m2/s for the USV and 1.10 × 10−8–2.72 × 10−8 m2/s for the VD. These results were in agreement with previously published studies conducted on different food materials [Deff values of the USV and vacuum drying techniques showed that the Deff value for the USV was larger than the vacuum drying technique Deff value at 35 °C and 45 °C (p < 0.05).Effective moisture diffusivity . The rehydration ratios of the freeze-dried minced meat samples were higher than those obtained by the VD and USV techniques. The USV samples showed higher rehydration values than the VD samples. This is explained by the formation of a porous structure resulting from the ultrasonic process. The highest rehydration values for the VD and USV techniques were obtained at 35 °C. This result is explained by the food matrix having less deformation due to moderate drying temperature and drying time. Similar to our results, in a study [ a study  on the e a study . The pos a study ,34. As aThe shrinkage properties of the minced meats are presented in Low shrinkage values are very important for dried minced meat samples. As shown in The effect of different drying techniques and conditions on the microstructural properties of minced meat samples was observed using SEM . As showE value, which indicates the color difference between the fresh and dried samples. Moreover, this means that high temperatures over a long time period cause loss of color and a darker appearance. As shown in L* values of the FD dried samples were higher than the samples dried with other methods. A possible explanation is that high pore structures in the FD treated samples led to more uniform light reflection from the surface, and therefore a relatively translucent object was obtained [The Hunter color scale was used to evaluate the color characteristics of the minced meat samples dried by using different drying techniques. As shown in obtained . Therefoobtained  reportedobtained . In our In this study, the effects of different drying temperatures and drying methods on the drying characteristics and quality parameters of minced meat samples were investigated and a USV technique was used for drying minced meat for the first time. The results of the study indicate that the drying methods and parameters significantly affected the drying time and quality characteristics. Drying time significantly decreased with increased drying temperature. The FD technique resulted in higher rehydration ratios and lower shrinkage, oxidation (PV) rates, and color change. The USV technique resulted in lower drying times, less color change and shrinkage, and a high porosity structure as compared with conventional VD. This study suggests that the USV technique is a useful method for reducing the drying time and increasing the quality parameters of minced meat.


Among the pol sequences derived from a large sample dataset from the Comunitat Valenciana (Spain), we identified nine putative recombinant samples. We aimed at fully characterizing these samples and performing a detailed analysis of the corresponding recombination events. We obtained nearly full-genome sequences and used jpHMM and RDP4 to detect and characterize recombinant fragments. We assessed the confidence of these inferences by likelihood mapping and phylogenetic placement with topology congruence tests. Next, we performed a phylogenetic analysis of each putative recombinant fragment to determine its relationships to previously described recombinant forms. We found that two samples related to CRF44_BF whereas the rest corresponded to new URFs . These URFs have a complex recombination pattern that cannot be determined accurately. They seem to have arisen by successive recombination events among lineages, including other CRFs. Our results highlight the usefulness of routine surveillance analysis for the detection of new HIV-1 recombination forms and, at the same time, the need for full-genome sequencing and recombination detection guidelines to properly characterize this complex process.Recombination is one of the main processes shaping the evolution of HIV-1, with relevant consequences for its epidemiology. In fact, Circulating and Unique Recombinant Forms (CRFs and URFs) cause 23% of current infections. The routine analyses of antiretroviral resistance yield partial  CRFs are recombinant HIV-1 genomes that share the same recombination breakpoints (BPs) between the same parentals and have been found in at least three non-epidemiologically related individuals. If a recombinant form does not fulfill the requirements to be considered a CRF, it is called a unique recombinant form (URF). RFs can also be classified according to their mosaic structure: they are denoted as complex (cpx) if they are composed by three or more subtypes or second-generation recombinants (SGRs) if they result from a recombination event in which pre-existing RFs are involved. RFs account for 23% of the current infections and are responsible for almost half of total infections in areas such as Central Africa  and its reducing costs have led to a substantial increase in the availability of complete HIV-1 genomes. This is partially responsible for the increased reporting of RFs world-wide. This increase also results from the mixing of newly formed HIV-1 variants, resulting in a complex and dynamic epidemiology . Due to this lack of standardization and the intrinsic difficulties in identifying recombinant events, some authors have postulated that several methods should be used to obtain a precise picture of recombination  , we detepol gene was sequenced. The phylogenetic analysis of this region revealed nine samples with a suspected recombinant nature because they did not cluster with high enough bootstrap support with reference sequences of pure subtypes and representative CRFs (data not shown). These nine samples, all from Spanish patients that attended hospitals of the Comunitat Valenciana (Spain), were subjected to nearly-WGS.From 2010 to 2013, about 1,800 serum samples of individuals infected with HIV-1 were screened for antiretroviral resistance mutations in our laboratory . For thi® EasyMag® automated platform . Reverse transcription (RT) of RNA was performed in two adjacent genome regions to improve yield. The first hemigenome was obtained by priming with IN-T-A2 . The second hemigenome was obtained with UNINEF7′ , 10 mM DTT and 200 U of SuperScriptTM III reverse transcriptase . Reagents and RNA were incubated for cDNA synthesis at 50°C for 2 h, followed by 85°C for 5 min.RNA was extracted from sera using the NucliSENS IN-T-A2  in the vUNINEF7′ , close tpol . The second amplicon, of 2.1 kb, covered from the second half of pol to the start of vif . The third amplicon, of approximately 1.4 kb, covered until env gp160. Lastly, the fourth amplicon consisted of 3.9 kb from env gp160 to the end of nef . The reaction volume was 25 μL, containing 1x PCR buffer, 350 uM dNTP mixture, 0.4 uM of each primer and 2.5 U TaKaRa Ex Taq® . Cycling conditions were 94°C for 2 min and then 10 cycles of , and 20 cycles of  followed by 68°C for 10 min. Nested-PCRs were done with the same conditions as above but adding 1.5 μL of the previous PCR product with appropriate primers.Next, four overlapping regions were amplified using first-round PCRs and, if needed, nested-PCRs . The firPositive PCR products were purified with a High Pure PCR Product Purification Kit  and sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit  in an ABI 3730xl DNA sequencer . The set of primers used in this study is available upon request. We obtained the consensus nearly full-length genome (8.9 kb) by assembling the sequence readings that passed the default quality filter of Pregap4  along wiThe mosaic recombinant structure of the samples was screened using two different strategies. Firstly, breakpoints (BPs) were determined with the default jpHMM options . Secondl4) and each putative recombinant sample using the option -add of MAFFT v.7  with the RIP reference alignment detailed above. This partial alignment was used to reconstruct a reference maximum likelihood (ML) tree using IQ-TREE v.1.6.5  but adding, with MAFFT v.7, all the URFs identified as such . Then, new ML trees were built with 1,000 bootstrap replicates. The closest relatives (CRs) of each fragment were determined considering as CRs the minimum set of sequences that grouped with the fragment of interest with a bootstrap support value higher than 70%.https://github.com/BBeamud/PhyloRecomb/.Finally, the inferred mosaic structure from the above analyses was drawn using the ggplot2 library in R . These aenv gene. This is reflected in the proportion of undetermined bases of each sequence that were used for scaffolding  length was covered in our samples. The per base coverage ranged from 2.44 to 4.53. In four of these genomes, it was not possible to assemble all the sequencing reads into a single contig due to hypervariability in the ffolding . For theRecombinant mosaic structures were obtained by two strategies. Firstly, we considered the recombination events and their defining BPs supported by at least three of the methods tested in RDP4. Additionally, we considered the BPs reported by jpHMM. These two programs assign a subtype for each fragment. All the strategies revealed putative recombination events in all the samples, but the total number of such events differed among them. To facilitate the comparison between detection strategies, we retained as major parental (MP) the subtype with most genomic nucleotides assigned. In addition, we considered the same recombination event if the minor assigned subtype and the inferred BPs were similar ( ± 200 nt) in both sides for both strategies. A total of 42 recombination events were detected but only eight were shared by the two strategies. Of the 34 remaining events, 21 were detected exclusively by jpHMM and 13 by RDP4. There were substantial differences in the performance of the methods implemented in RDP4, with a minimum of 3 (GENECONV) and a maximum of 19 (MaxChi and 3Seq) detected events . Shared To further corroborate these events, we performed ELW congruence tests and likelihood mapping analyses for each fragment. A recombination event was considered verified only when the tree with the sample fragment grouped with the minor subtype identified had a relative support value, as determined by ELW, larger than 0.5 than that of the second-best tree. Additionally, we considered fragments with less than 0.25 unresolved quartets reliable for phylogenetic inferences. All the events jointly detected by jpHMM and RPD4 were verified by ELW tests and phylogenetic signal. However, 15 out of the 21 events detected only by jpHMM were corroborated, regardless of their phylogenetic signal, whereas only 3 of the 13 events detected only by RDP4 were verified. For the latter, the main discrepancies were observed in the assigned subtype whereas for jpHMM a high proportion of cases with low phylogenetic signal was observed . A summaDifferent mosaic structures were observed in the samples considering verified events. In particular, we observed two BF recombinants, two AD recombinants, three BG recombinants, and two complex recombinant forms. These mosaic structures were refined with the reconstruction of ML trees for each fragment based on the analysis of closest relatives (CRs) with 226 representative sequences of HIV-1. We performed these analyses for all the major and minor parental fragments although the results of the former analyses are not shown for simplification .5 to confirm it relatedness. The results of the ELW tests showed a lack of congruence for the three recombination events with their grouping with subtype B, thus corroborating them . This structure resembles that of CRF38_BF  althoughing them . Hence, Samples 678, 703, and 724 are BG recombinants, with G being the major parental with a single B recombination event. A similar situation to that in BF recombinants was found in this group, except for two recombination events detected only by RDP4, 703_2R (U|D) and 724_2R(H), which were not verified by ELW and phylogenetic signal tests. CRs analyses showed that the G fragment was related to CRF24_BG whereas the B fragment grouped consistently with pure B sequences or CRF51_01B . To confThe two AD recombinants corresponded to samples 2011 and 2014 with A subtype being the major parental and with four and three fragments of subtype D, respectively. Initially, more recombination events of other subtypes were identified by jpHMM and RDP4. However, they were discarded based on the results of the ELW congruence tests. RDP4 failed to detect three events that were detected by jpHMM and further verified by tree-topology and phylogenetic signal . CRs anaThe genome sequences derived from samples 3011 and 3164 were identified as complex forms, composed by more than two ‘pure’ subtypes: A, G, H, J, and K for 3011 and A, H, J, and K for 3164. For both samples, the A subtype was considered as the major parental although, in this case, there is little difference between the portion of the genome covered by the different subtypes. As before, the description of 3164 is limited because about only half of its genome sequence was obtained. These recombinant forms were very complex both in the detection and verification results . A totalMost of the events detected by jpHMM in sample 3011 (7/11) showed a low phylogenetic signal, with more than 25% of unresolved quartets  whereas In this study, we have described seven nearly-complete and two partial genomes of different HIV-1 recombinant forms, detected in infected individuals from Comunitat Valenciana (Spain). We have performed detailed analyses to characterize their recombination events. Firstly, we identified putative recombinant fragments using different methods, jpHMM and seven of those included in RDP4. In a second phase, we corroborated the recombinant fragments by evaluating their phylogenetic signal, testing their topological congruence and analyzing their closest relatives using a comprehensive dataset of HIV-1 sequences including representatives of all the subtypes, CRF and URFs.Two samples, 79 and 93, were initially identified as CRF38_BF based on the results of jpHMM and RDP4. However, our additional analyses of closest relatives and ELW congruence test revealed that both were related to CRF44_BF, a recombinant form first described in Chile in 2010 , but alrThe three BG recombinants shared a similar mosaic structure, which was further verified in our subsequent analyses. They might constitute a SGR resulting from a new recombination event between CRF24_BG and subtype B. CRF24_BG was firstly identified in Cuba in 2007 . HoweverSamples 2011 and 2014 might represent a new URF_AD, originated from several recombination events of ‘pure’ A3 and D sequences. In Spain, A and D subtypes are not frequent as they represent less than 1% of total infections  and no Cpol sequences of the 1,800 samples at the origin of this study revealed an important number of non-B infections (16.2%) but they did not include subtypes K, H, or J . Two of them are related to CRF44_BF but the rest correspond to new URFs. Some samples of this study revealed a complex recombination nature and origin. This might suggest that, apart from the endemic areas that exhibit the highest levels of genetic diversity and therefore represent recombination hotspots, other regions could be sources of recombinant progeny due to the increasing global gene flow . To bettThe present study highlights the utility of surveillance analyses of HIV-1 for additional goals to identify resistance mutations, such as studying genetic diversity and characterizing new recombinant forms. A thorough analysis of partial genome sequences obtained in the surveillance of antiretroviral resistance mutations may provide the best starting point for the discovery and characterization of new recombinant forms in HIV-1.Approval of surveillance studies by an ethics committee is not required as per local legislation and host institution (FISABIO) guidelines.The idea for this study was conceived by BB, MAB, and FG-C, who was also in charge of supervising the whole project. Data collection and reference searching were done by BB and MAB. The data analyses and drafting the manuscript were done by BB. The final manuscript was written, curated, and confirmed by BB, MAB and FG-C.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ovibos moschatus) are one of few large herbivores able to survive this harsh environment. Microbiome research of the gastrointestinal tract may hold clues as to how muskoxen exist in the Arctic, but also how this species may respond to rapid environmental changes. In this study, we investigated the effects of season (spring/summer/winter), year (2007–2016), and host genetic structure on population‐level microbiome variation in muskoxen from the Canadian Arctic. We utilized 16S rRNA gene sequencing to characterize the fecal microbial communities of 78 male muskoxen encompassing two population genetic clusters. These clusters are defined by Arctic Mainland and Island populations, including the following: (a) two mainland sampling locations of the Northwest Territories and Nunavut and (b) four locations of Victoria Island. Between these geographic populations, we found that differences in the microbiome reflected host‐associated genetic cluster with evidence of migration. Within populations, seasonality influenced bacterial diversity with no significant differences between years of sampling. We found evidence of pathogenic bacteria, with significantly higher presence in mainland samples. Our findings demonstrate the effects of seasonality and the role of host population‐level structure in driving fecal microbiome differences in a large Arctic mammal.The Canadian Arctic is an extreme environment with low floral and faunal diversity characterized by major seasonal shifts in temperature, moisture, and daylight. Muskoxen . Our results showed differences in the microbiome reflected host‐associated genetic cluster with evidence of migration between these two regions. We found evidence of pathogenic bacteria, with significantly higher presence in Mainland samples. They are highly adapted to surviving in arctic conditions with a natural range spanning the Canadian Arctic, from the Northwest Territories and Nunavut into the Arctic islands and western Greenland  are dispersing northwards into previously inhospitable environments  were obtained over a 9‐year period from six sampled regions and two genetically defined populations  and Victoria Island   Table . Samples]) Table  or fresh]) Table . In tota]) Table . Only sa2.2Total DNA was extracted from a 0.25 g subsample taken from one fecal pellet per individual and homogenized with the QIAamp PowerFecal DNA kit (QIAGEN). The V3‐V4 fragment of the 16S rRNA gene was amplified with the primers 341F and 805R following the 16S metagenomic Illumina protocol. Primers were modified by adding 0–3 Ns  v 2018.6  plots as metrics to detect bacterial diversity between different population groups Kohl, . The funWickham,  was used2.4adegenet  and assign individuals to their population of origin. The optimal number of clusters was predicted using the sequential K‐means clustering method, using the Bayesian Information Criterion (BIC) for choosing the best number of k clusters from one to ten. We also inferred the number of population clusters using the model‐based STRUCTURE algorithm, using the same parameters as set in Prewer et al.  database  had varying numbers of reads, ranging from 0 to 3,095 reads postquality control . Furthermore, the samples largely clustered by population of origin (Mainland vs. Victoria Island), while four samples clustered in between the two groups Figure . SimilarWe merged the two technical runs to describe the bacterial abundances in the muskox microbiome and estimate diversity indices. The final data library consisted of 9,958 ASVs and was rarefied to an equal sequencing depth of 39,059 reads, ensuring a high Good's coverage index (>99%) per sample. The total number of ASVs after rarefaction was 9,832.3.2dada2 wrapper, was dominated by two main bacterial phyla; Firmicutes  and Bacteroidetes (7%). Within these phyla, the two most dominant orders were Clostridiales (98%) and Bacteriodales (89%), respectively  and Lachnospiraceae (13%). Within the Ruminococcaceae family, the most common genera were Ruminococcaceae (70%) and Ruminiclostridum (4%), while 12% was uncultured. Within the family Lachnospiraceae, various Lachnospiraceae genera (47%) and Roseburia (7%) composed the assemblage, while 11% was uncultured. Within the Bacteroidetes phyla, Rikenellaceae (33%), Bacteroidaceae (29%), and Prevotellaceae (9%) were the dominant families. Within the Rikenellaceae family, the Rikenellaceae RC9 gut group (47%) and Alistipes (44%) were the dominant genera, while for Bacteroides, 100% of the total sequences were from the Bacteroidaceae family.The fecal microbiome derived from both sequencing runs, merged and processed using QIIME2 and the Ruminococcaceae UCG‐005, and one from each taxon Bacteroidaceae Bacteroides, Lachnospiraceae Roseburia, and Clostridiales Family XIII. Separating the samples by population showed that Victoria Island samples shared a core microbiome of 31 ASVs , while Mainland samples shared only three ASVs belonging to the bacterial genus Ruminococcaceae UCG‐005. The common microbiome  consisted of 184 shared ASVs .The core microbiome  of muskoxen was composed of only six shared ASVs: three of which belonging to the bacterial genus Pseudomonadales bacteria . One sample from the Mainland population collected during winter (406s2c) showed similar bacterial abundances as Arctic snow, with Pseudomonadales, Flavobacteriales, and Sphingobacteriales as the most abundant orders , and both samples were excluded from further analyses.We detected two samples (308s13134 and 406s2C) with possible environmental contamination. Unlike most of the samples, these were abundant for 3.3p < .05) and Shannon's diversity index .Of the 9,958 ASVs prerarefaction within the muskox fecal microbiome, 1,720 of those were shared between Victoria Island and Mainland populations when putative migrant samples were removed see “. The numA PCoA plot of all samples identified a clear separation between the Mainland and Victoria Island populations. Four individuals sampled from Kitikmeot Region, Nunavut, (Mainland) grouped more closely to individuals from Cambridge Bay (Victoria Island) Figure .adonis. The year of collection was defined as samples collected pre‐2009 and post‐2009 when disease pressures in our sampling area were proposed to be higher. The geographic separation between Mainland and Victoria Island had the greatest effect on the bacterial community , followed by seasonality  and year . Each variable was found to have a significant effect on the bacterial community, so AIC was used to compare and rank the models. When considering all pairwise interactions, the relationship between sampling region and seasonality had the overall strongest influence on the bacterial community  while the effect of sampling year had the least . No models, with the exception of region when variables were assessed individually , were able to surpass a 95% confidence set , summer , and winter . Seasonality is shown to affect the microbial community in abundance , richness , and diversity . Minor separation can be seen in the PCoA analysis of Victoria Island samples , while the abundance of Firmicutes (p = .147), Proteobacteria (p = .616), and Tenericutes (p = .757) bacteria stayed relatively similar across all seasons. Together, Firmicutes and Bacteriodetes represented over 90% of the total sequences across all seasons, with a mean increase of 28% in Bacteriodetes in summer months (p = .001).To test the effect of seasonality on the muskox microbiome, we analyzed samples from Victoria Island collected during spring , even when accounting for spatial variation . Likewise, there was a positive correlation between the host microbial community distance and geographical distance when controlling for genetic distance . Similar to the PCoA , and one from the genus Clostridium (C. orbiscindens). Of these, P. baetica and P. syringae are known plant pathogens while the other three identified ASVs were nonpathogenic bacteria, as per a literature review. The remaining 42 ASVs were unclassified at species‐level by QIIME2, and thus, a representative sequence from each was aligned to 16S rRNA gene sequences from the BLAST database at the highest maximum match (Table Arthrobacter koreensis/A. luteolus (100% BLAST identity match), Enterococcus casseliflavus (100%), Escherichia fergusonii (100%), Serratia fonticola (100%), Streptococcus gallolyticus (100%), Mycoplasma sp. clone A33 (98%), P. baetica (100%), P. fluorescens (100%), and P. syringae (100%). Among the shared ASVs, four out of eight pathogenic bacterial genera differed significantly in their abundance between Mainland and Victoria Island (p < .05), with all showing higher abundance in Mainland.A total of nine putative pathogenic genera were detected by QIIME2, including bacteria from the order Erysipelothrix, which is a bacterium of particular interest as it has been associated with mass die‐offs across Victoria and Banks Islands. However, the sequence isolated from our library was identified as an uncultured environmental sample, aligning to the reference E. rhusiopathiae genome at only 93% identity (GenBank accession number: AP012027). Another bacterium, Yersinia pseudotuberculosis, has also been responsible for deaths in Canadian muskoxen  . Mainland had a higher proportion of potentially pathogenic bacteria, which might be expected due to the more recent emergence and northern expansion of pathogens to Victoria Island  were found in common with muskoxen from Greenland  were exactly as expected for muskox fecal microbiome, and our controls allowed us to use a higher sample size to infer population microbial diversity and dynamics  have been a growing concern in modern microbial analyses (e.g., Goh, Wang, & Wong, dada2 wrapper due to this program's ability to handle large datasets and general acceptance within the microbial scientific community.Many bioinformatics pipelines and independent programs exist for microbiome data analyses, such as QIIME (Caporaso et al., 5The bacterial communities present within the gastrointestinal tract of muskoxen are integral to their survival in the arctic environment. Studying internal bacterial communities contributes to our understanding of how this iconic Canadian mammal interacts with its environment in terms of digestion, health, immunity, and adaptation; however, it has been unclear how the microbiome community varies within and between regions on temporal and spatial scales, and further how this variation links to population genetic structure. This study used a combined sequencing run of 16S rRNA gene markers in 78 individuals to explore the differences in abundance and diversity of bacteria within the gastrointestinal tract of muskoxen in an attempt to better understand the drivers of microbiome variation. We found that geographic location was a strong driving pressure to microbial diversity. Within population, seasonality impacted microbial diversity, though time of sampling, described here as changing pressures of disease, showed little effect. Future work should consider sequencing the potential pathogens found in the muskox gastrointestinal tract to provide a greater context of health and host capacity to locally adapt to rapidly changing environments. Secondly, the role that diet plays in altering the bacterial component of the microbiome should be more clearly addressed by sequencing plant DNA using a chloroplast marker. Geographic distance shows to affect community diversity and it should be considered how changing vegetation in different landscapes contributes to bacterial variance in a wild mammal, as well as to elucidate the roles of bacteria in fiber and plant digestion for a large herbivore.None declared.STV and CJK designed the research, SK and LML provided samples, SB, STV, and EP performed the research and analyzed data, SB and STV wrote the paper with input from all authors. All authors edited and approved the final version. Click here for additional data file. Click here for additional data file. Click here for additional data file.