Aedes albopictus, has spread from the Western Pacific and Southeast Asia to Europe, Africa, the Middle East, North and South America, and the Caribbean. This species of mosquito transmits arboviral infections, such as yellow fever, chikungunya, dengue, zika, and several encephalitides. The objective of this research was to provide a radiation dose inducing sterilization in adult male Ae. albopictus in the pupal stage. A cobalt-60 source of gamma radiation at a dose rate of 381 Gy/h was used. The pupae were irradiated with doses of 0 (control), 20, 30, 40, 50, and 60 Gy. Each treatment had a total of five replications using 60 pupae. After irradiation, the different phases of Ae. albopictus development  in the F1 generation were observed daily. Parameters such as viability, fertility, longevity, and mortality were recorded. The results from these studies showed that a dose of 60 Gy was necessary to sterilize 100% of the male Ae. albopictus pupae.The pathogen-carrying tiger mosquito,  Aedes albopictus, was described in Albania in 1979 [Ae. albopictus spread to Mexico by the early 1990s [The invasive nature of the Asian tiger mosquito,  in 1979 , the spely 1990s ,4, and aly 1990s , all couDirofilaria immitis, which causes heartworm parasitism. However, the introgressions suggest diverse geographic origins for this invasive mosquito species. In the New World, Costa Rica and Panamá have the same groups of population origins as in Latin America [In Italy, the Asian tiger mosquito has become the most serious pest mosquito species ,7 as it  America , whereasAe. albopictus was first detected in Brazil (São Paulo State) in 1986 and is currently in 24 of the 27 Brazilian Federal Units [Ae. albopictus have shown high vector competence for the chikungunya virus, reinforcing the importance of the entomological and epidemiological surveillance of this mosquito species in the country. In tropical zones, Ae. albopictus has developed a photoperiodic egg diapause and freezing tolerance [al Units . Althougal Units . The labolerance ,13,14. Tolerance .Several studies were designed to develop a vaccine against these arboviruses ,17; howeAe. albopictus in Latin America [Consequently, the intense use of these compounds by both governmental campaigns and citizens  has selected resistant populations to nearly all classes of insecticides available . Compoun America ,22.Anopheles quadrimaculatus. However, the result was not satisfactory due to the low quality of sterilization [Conventional methods do not produce satisfactory results; hence, to preserve the environment, new techniques must be applied. One such method is the sterile insect technique (SIT); this method involves the release of massive numbers of sterile males that compete with fertile wild males for mating opportunities with wild female insects ,10. The lization ,24. In 1Culex quinquefasciatus irradiated at 60–120 Gy was effectively released in India and Florida between 1967 and 1974 [Cx. quinquefasciatus, Ae. aegypti, and Anopheles stephensi pupae, funded by the World Health Organization (WHO) and by the United States Public Health Service (USPHS) in cooperation with the Indian Council of Medical Research (ICMR). The mass rearing facility in New Delhi produced up to one million males per day at a cost of USD $40 per million. The sterilization induced 99% sterility [and 1974 . These sterility ,27.Ae. aegypti population; however, fitness studies conducted in the field and laboratory showed that the translocation strains had low fertility, rate of larval development, larval and adult survival, and low mating competitiveness; consequently, none of the translocation strains became established in the field, and they rapidly disappeared [In Mombasa, Kenya, translocation homozygote strains were introduced to replace the wild appeared .Ae. aegypti strains [Ae. aegypti population in the test area when compared with the experimental control area (76–78%). In Brazil, the release of transgenic insects reduced the Ae. aegypti population density by 85% in areas located in the Juazeiro and Jacobina municipalities (Bahia State) [In 2011, a transgenic lineage termed RIDL  in  strains  was libea State) .Ae. albopictus. In the first experiment, adults of male pupae showed significant results for a high sterilization rate in comparison with control treatments; research is continuing on a large scale to improve rearing efficiency [In Italy, from 2004 until currently, research has been conducted to investigate the feasibility of introducing the SIT strategy to control ficiency ,32.Wolbachia to suppress a population. Although population replacement approaches can be performed with the simultaneous release of both male and female insects, population-suppression strategies should aim to release only males. Besides Ae. aegypti, other mosquito species, such as Ae. polynesiensis (South Pacific), Ae. albopictus , and Culex pipiens quinquefasciatus (Southwestern Indian Ocean), are also being tested in different countries to demonstrate the feasibility of using IIT for the population control of mosquito species [The incompatible insect technique (IIT) uses the cytoplasmic incompatibility characteristics of  species .Wolbachia to reduce dengue transmission is being tested in several countries [Wolbachia-infected mosquitoes are released in large numbers weekly, and the bacteria are transmitted to the wild Ae. aegypti population. Field trials have been conducted at four sites in Australia, one in Vietnam, and two in Indonesia; experiments in Brazil have been underway since September 2014 using Ae. aegypti infected with the wMel Wolbachia strain. Laboratory and field assays performed in Brazil showed that wMel infection has no detrimental effects on the Brazilian Ae. aegypti mosquito population. This technology can be used to control mosquito populations where local access is impossible  [The use of ountries . Wolbachn areas) ,35,36. LAe. aegypti pupae since 2015 [Ae. albopictus. [Ae. albopictus, which is also a potential vector of human diseases, is necessary. This species is particularly suitable for the application of the sterile insect technique (SIT) due to its urban-related distribution, recent introduction, vector potential, low population density, which can be maintained by conventional control measures, and ease of mass rearing.In Brazil, the SIT technique has been applied only to nce 2015 ,38. Thesopictus. . Thus, topictus. ,11,12. Dopictus. , steriliAe. albopictus is a potential candidate for SIT application. The objective of this research was to induce a sterilizing radiation dose for adult male Ae. albopictus irradiated in the pupal stage.Therefore, The research was performed in the Laboratory of Environment and Radiobiology, Center of Nuclear Energy in Agriculture (CENA/USP), at the Universidade de São Paulo, Piracicaba-São Paulo State, Brazil and Análise e Estudos Biológicos (AEB), Charqueda-São Paulo State, Brazil. The mosquitoes used in this experiment were obtained from the mass rearing colony at the Entomology Laboratory at LAFICAVE–IBEX–FIOCRUZ , Benfica, RJ and had been maintained in the laboratory for more than three years.Insects were reared under standard laboratory conditions , 12 h photoperiod). Adults were kept in Plexiglas cages (50 × 50 × 50 cm). The cages were supplied with deionized water and 10% honey solution and contained adult males and females aged 5 days. During this period, the mosquitoes were fed with sheep blood via a vacuum collector. Females laid eggs in Petri dishes containing deionized water and a strip of white filter paper. After oviposition, the filter paper was removed from the cage and left to dry in a closed plastic container.After four days, the eggs were counted and placed in a 1.0 L closed bottle with 750 mL of deionized water and 0.05 mg of food to stimulate hatching. Larvae were reared in white plastic trays (31 × 22 × 10 cm) at a density of 200 larvae/tray containing 2.5 L deionized water and provided with TetraMin fish food .To separate males from females in the pupal stage, a device comprising two overlapping acrylic plates with an adjustable, downward-pointing, and wedge-shaped space was used, into which the pupae could be inserted. Pupae were separated on the basis of size by regulating the thickness and angle of the wedge-shaped space using four control knobs, one for each angle. The lower opening was adjusted so that the larger female pupae were retained in a layer in the tapering space between the acrylic plates. The smaller male pupae were drained into a plastic tray placed below. The operation was completed by opening the wedge and flushing the female pupae into a second receptacle .Each male pupa was transferred separately to a Petri dish containing white filter paper with a 10-mm layer of deionized water to sustain them. Afterward, they were placed in a Gammacell-220 irradiator  located in then Center for Nuclear Energy in Agriculture—CENA/USP and exposed to cobalt-60 as a gamma radiation source at a dose rate of 381 Gy/h.Doses in the irradiator were mapped by six Gammachrome dosimeters  with a range of 0.1 to 3 kGy and read using a Genesy 20 spectrophotometer . Doses were certified by the Institute for Energy and Nuclear Research (IPEN), São Paulo, Brazil. The traceability of dose measurements was maintained by comparison with the international service assurance dose offered by the International Atomic Energy Agency, Vienna, Austria.The Petri dishes were centered in the irradiator to ensure the uniformity of the radiation was not disrupted. The six dosimeters were positioned as follows: one on top of the dish, one at the bottom, and four equally spaced at lateral positions. The uncertainty in each dish was ±1.6%. The variation in measured doses was ±1.5% in the Gammacell-220 source.Male pupae were irradiated at doses of 0 (control), 20, 30, 40, 50, and 60 Gy, with a total of five replications with 60 pupae per treatment.Ae. albopictus  in the F1 generation were evaluated daily for viability, fertility, longevity, cycle life duration of immature stages (duration stage), and mortality.After irradiation, the Petri dishes containing the adult males were transferred to new cages with 60 virgin females. The different phases of ®, Redmond, WA, USA) and R software [A completely randomized experimental design was used in each experiment with five replications per treatment. Data were initially submitted to the Shapiro–Wilk normality test at a 5% level of significance. Proportional data were analyzed using a generalized linear model with binomial distribution. The graphics were produced with Microsoft Excel 2013 . Higher doses of 40 or 50 Gy resulted in lower larvae and pupae numbers, whereas in doses of 60 Gy, no larval survival was observed. Thus, 60 Gy was the effective sterilizing dose.The results for the cycle life in each stage  in days showed that some irradiation treatments were overall slightly lower than in controls. However, at dose levels of 50 Gy, eggs developed to the larval L1-stage (L1). At doses of 40 and 50 Gy, however, the cycle duration time increased when there was an increase in age and dose, although no statistical difference was observed in comparison with the controls.p < 0.001). At the 50 Gy dose, approximately 3% transformation was observed compared with the 60 Gy dose, where there was no observed L1–L4 larvae stage . At the 60 Gy dose, the data obtained for male adult survival and male adult longevity were obtained from the P generation and compared with the other doses in the F1 generation. This was conducted because there were no pupae or adults in the F1 generation for this dose.In the first seven days, there was no observed decrease in the lifespan of pupae in any treatment group studied a–d. A slp > 0.001). At 0–48 h at the 40, 50, and 60 Gy doses, the longevity of adult males in the P and F1 generations showed a significant reduction compared with the controls (p > 0.001).When comparing the viable percentage of adult males in Overall, the radiation dose required to produce sterility was proportional to an increase in the dose, as expected. Some authors reported that increasing the radiation doses in pupae in mosquito species from the genus Aedes causes a large reduction in the number of male adults ,43,44,45Ae. aegypti male pupae aged >24 h resulted in a 99.9% sterility rate with a dose of 78 Gy [Ae. albopictus pupae with a 40 Gy dose; however, a 60 Gy dose was needed to achieve a 100% sterilization rate [Here, we showed that sterility was reached at the 60 Gy dose for all pupae ages (0–48 h). Other authors used larger doses than those reported in the present study ,41. For of 78 Gy . In anotion rate .The adult emergence rate is considered to be an important parameter for measuring pupal irradiation. The radiation effects on insect pupae are greater than on adults, with the latter generally leading to a sterile male competing almost equally with wild males ,49,50,51A significant reduction in male adult longevity was observed in radiation treatments, especially at doses of 40 Gy or higher. A slight reduction was also observed in other treatments when compared with controls, although there was no significant difference among the doses.Ae. albopictus at 40 Gy or higher [Anopheles pharoensis and Anopheles stephensi irradiated at more than 100 Gy [Other studies also reported a significantly negative effect on male adult longevity after the pupal irradiation of r higher ,50. Thisn 100 Gy ,52.An. pharoensis pupae  irradiated with low doses in the range of 4.8 to 68 Gy exhibited no reduction in longevity compared with controls (no irradiation).The possible causes of somatic injuries induced by radiation treatment that result in decreased longevity have been previously described ,52,53. TAnopheles arabiensis found that low doses, such as 4–15 Gy, could prolong the longevity of adult males [Other studies on lt males . The phelt males . HoweverAe. aegypti, which is a closely related species and may be taken as a useful example. Thus, the dose determined in this manuscript was used for the first time for the Brazilian strain of Ae. albopictus. Although positive, the results reported here need to be confirmed through a larger trial and with wild mosquitoes in the field, because laboratory strains of many generations could produce limited results in future pilot trials.The SIT methodology has not been attempted in Brazil, although studies of this nature have been conducted on several other mosquito species, including In future investigations, the main aims should be to evaluate the following: (1) the number of sterile males to be released, (2) a comparison of costs and benefits with conventional methods, (3) methods of improving the rearing efficiency by increasing larval density through introducing new larval diets and decreasing larvae numbers in plastic trays, (4) processes to increase pupal production by sieving at 30–36 h after the start of pupation instead of at 24 h, (5) the development of a larger greenhouse for colony maintenance, (6) testing for a lower sterilizing dose, (7) a pilot trial of population suppression and operational implementation in the field, (8) improvements in the quality of sterile males, and (9) the mass rearing of wild mosquitoes to study. Ae. albopictus at 60 Gy.The results presented here are important for mosquito species control programs that include SIT technology for efficient integrated pest management (IPM) in Brazil. From the results of longevity and gamma radiation doses, an effective sterilization dose was determined for male pupae of


Supplemental Digital Content is Available in the Text.This large-sample item response theory-based evaluation assessed the measurement properties of SF-36, EQ-5D, and hospital anxiety and depression scale for chronic pain patients in clinical settings. Recent research has highlighted a need for the psychometric evaluation of instruments targeting core domains of the pain experience in chronic pain populations. In this study, the measurement properties of Short Form-36 Health Survey (SF-36),EuroQol 5-dimensions (EQ-5D) and Hospital Anxiety and Depression Scale (HADS) were analyzed within the item response-theory framework based on data from 35,908 patients. To assess the structural validity of these instruments, the empirical representations of several conceptually substantiated latent structures were compared in a cross-validation procedure. The most structurally sound representations were selected from each questionnaire and their internal consistency reliability computed as a summary of their precision. Finally, questionnaire scores were correlated with each other to evaluate their convergent and discriminant validity. Our results supported that SF-36 is an acceptable measure of 2 independent constructs of physical and mental health. By contrast, although the approach to summarize the health-related quality of life construct of EQ-5D as a unidimensional score was valid, its low reliability rendered practical model implementation of doubtful utility. Finally, rather than being separated into 2 subscales of anxiety and depression, HADS was a valid and reliable measure of overall emotional distress. In support of convergent and discriminant validity, correlations between questionnaires showed that theoretically similar traits were highly associated, whereas unrelated traits were not. Our models can be applied to score SF-36 and HADS in chronic pain patients, but we recommend against using the EQ-5D model due to its low reliability. These results are useful for researchers and clinicians involved in chronic pain populations because questionnaires' properties determine their discriminating ability in patient status assessment. Considerable resources have therefore been invested into isolating core domains of the chronic pain experience.64 Health-related quality of life (HRQoL) and emotional distress are 2 central domains that consistently recur in some form; they reflect perceived functioning and well-being in physical, mental, and social dimensions of health, and feelings of depression, anxiety, and anger, respectively. Both are recommended as core outcome domains in pain intervention clinical trials to increase research consistency.64 Emotional distress has additionally been considered a critical psychosocial component in chronic pain patient profiling,35 and was recently included as a central feature of the chronic pain experience in ICD-11.62Chronic pain is a globally prevalent condition that can permeate all aspects of a person's life.4 Self-administered questionnaires are the most commonly used indicators, with the Short Form-36 Health Survey (SF-36), the EuroQol 5-Dimensions (EQ-5D) for HRQoL,66 and the Hospital Anxiety and Depression Scale (HADS) for emotional distress being the most predominant.70 The measurement properties of these questionnaires have been extensively studied in various populations, including the general population, and patients with mental health conditions, cardiovascular disease, and cancer.21 The combined evidence reveals that their properties vary widely in different populations,19 and recent research has highlighted the paucity of psychometric evaluations of these instruments in chronic pain patients.17Unlike objective characteristics such as weight and height, human experiences are unobservable latent traits that need to be inferred from indicators through statistical procedures.57 For a questionnaire to be valid, it needs to be both based on solid underlying theory and have statistically robust empirical properties.57 Rather than being an inherent questionnaire property, validity is a property of the test score, which, in turn, hinges on the population characteristics and the applied setting.57 To avoid biased conclusions about the latent trait status, it is therefore critical to establish validity for individual populations separately.57 Hence, psychometrically sound instruments are a prerequisite for the latent trait to be captured accurately, and the properties of SF-36, EQ-5D, and HADS have not yet been systematically addressed in chronic pain patients. We therefore evaluated their measurement properties in a large sample of this population.This is an important void to fill because the accuracy with which a latent trait is captured depends on a questionnaire's validity.43 It consists of self-report cross-sectional data from 35,908 patients who were consecutively recruited between 2009 and 2016 from 38 interdisciplinary pain specialist treatment clinics, distributed according to Swedish population density. These clinics target patients with particularly complex chronic pain conditions, characterized by psychological comorbidities, impaired work, and social ability, and a failure to respond to monodisciplinary interventions. Inclusion criteria were: aged 18 years or older with noncancer musculoskeletal pain for a minimum of 90 days. During their first visit, patients received information about the registry and signed a written informed consent form. They then provided information on demographics and pain characteristics, and completed the Swedish versions of HADS, EQ-5D, and SF-36 in this order. This study was approved by Uppsala's Medical Research Ethics Committee (DNR 2018/036).This psychometric study was based on data from the Swedish Quality Registry of Pain Rehabilitation, which is an extensive nationwide database of patients with chronic musculoskeletal pain who are eligible for specialist rehabilitation.http://links.lww.com/PAIN/A877), along with path diagrams of their conceptual frameworks, which are also described below.English translations of the questionnaires are provided in the supplementary materials , mediated through the 8 health concepts , drive the item responses.67 Although this theory is largely consistent across studies, its empirical representations vary, which has resulted in various scoring methods and disparate association schemes linking the constructs, subscales, and items together.68SF-36 was designed to measure physical and mental health based on 8 health concepts: physical and social functioning, role limitations due to physical and emotional problems, mental health, vitality, bodily pain, and general health perception.25 It contains 5 items with 3 ordered response categories each, which were selected to target different HRQoL dimensions. The original theory defined the items as independent causes of HRQoL and summarized them as a preference-based index.7 However, because the items represent different aspects of HRQoL, they could simultaneously be conceptualized as indicators of a unidimensional construct.EQ-5D is a standard instrument in health economic evaluations that was constructed as a generic multiattribute utility measure to reflect the multidimensionality of HRQoL.70 The original theory defined 2 constructs of anxiety and depression represented by 7 items each;70 however, alternative structures have since been proposed and evaluated in various populations.42 These include a single distress construct,47 variations of the 2 constructs of anxiety and depression,40 3 constructs of anxiety and depression combined with restlessness,10 agitation,28 or negative affectivity,23 and bifactor structures with a general factor of emotional distress and 2 or 3 specific factors for residual item dependencies.42The Hospital Anxiety and Depression Scale is a 14-item questionnaire, rated on a 4-point ordinal scale, which was designed to measure emotional distress in nonpsychiatric patient populations.52 The simplest structure is the unidimensional model, where all items measure a single latent trait. However, multidimensional models are required for questionnaires that measure more than one latent trait and can manage both between- and within-item multidimensionality. The correlated-traits model accommodates between-item multidimensionality and is implemented when subsets of items reflect different traits, which, in turn, are correlated with each other. By contrast, the two-tier model manages within-item multidimensionality in situations where items simultaneously measure some general traits of interest and specific features unique to item subsets, where the specific features can be thought of as residual dependencies not captured by the general trait.12 The two-tier model simplifies to both the bifactor model, when a single general trait is measured,30 and to the more widespread second-order model  consists of a set of such generalized models that were developed for categorical data; they subclassify according to the mathematical function they are based on. Conceptually, IRT considers the item responses to be caused by (reflect) the latent trait. Item response theory–based analyses describe the shape of the item–trait relationship by estimating the probability of observed response patterns across the latent trait. Two common IRT models are the logistic graded response model and its more constrained form, the 2-parameter logistic model; they are used for ordered and dichotomous item responses, respectively.54 Item response theory models are advantageous in psychometric evaluations due to their flexibility and the detailed information they provide on item characteristics.69 However, to be valid, they require that several assumptions are met: that the items are independent after accounting for the latent trait, that the item–trait relationship follows the specified mathematical function, and generally that the latent trait follows a predefined probability distribution.Functional models describe how the latent trait and items relate to each other mathematically.14 Four types of structural models were used to accommodate the underlying theories of the questionnaires .Statistical analyses were computed in R  using the package “mirt” (v1.30).32 Specifically, data were randomly split into 5 equal parts, models were fitted on 4 parts (training set), and model fit was evaluated using the parameter estimates from the training set on the remaining part . Subsequently, models were refitted on the validation set using the same procedure with no additional constraints, and the training and validation set parameter estimate differences were computed. The procedure was repeated 5 times so that each part of the data was used as a validation set once.Patients with complete missing data were excluded from the analyses. For each questionnaire, k-fold cross-validation was computed to identify the model with the best fit and to assess the robustness of that model's parameter estimates.2* statistic were used, as it generally is unrealistic to expect exact model-data fit in IRT applications due to the many degrees of freedom resulting from the possible response pattern combinations.52 The root mean square error of approximation (RMSEA) and the standardized root mean square residual (SRMSR) were the primary indicators, with estimates ≤ 0.05 considered an acceptable fit for both.52 However, to facilitate comparison with previous studies, the Tucker–Lewis index (TLI) and the comparative fit index (CFI) were also included, which indicate the model fit relative to the null model, with estimates ≥0.90 typically considered acceptable. Third, item-level model-data fit was assessed based on the residual correlation matrix, with estimates ≤0.05 considered negligible,52 and by comparing observed and expected S-X2-based category response probabilities.36 Fourth, person-level fit was assessed by the Zh-statistic, with estimates <|2.0| considered acceptable; negative and positive values suggest misfit (unpredictability) and overfit (redundancy), respectively.52 Finally, to determine whether the model warranted its concomitant increase in complexity, expected and maximum a posteriori scores (IRT scores) for up to 2 and 3 or more latent dimensions, respectively, and their standard errors were compared across models.52 For bifactor models, the explained common variance was also included as a measure of the general factor's strength relative to other factors, with estimates ≥0.85 suggesting that a unidimensional model would be sufficiently complex.52Model selection was guided by several criteria. Firstly, after confirming model convergence, Akaike's and Schwartz's Bayesian information criteria were calculated to compare the model–model fit, with lower values supporting a better fit. Second, to evaluate the scale-level model-data fit, approximate fit indices based on the limited-information M14 with estimates ≥0.70 generally considered acceptable.60 Finally, IRT scores were correlated with each other as well as the conventionally computed scores, as a measure of convergent and discriminant validity.Once the final model was selected, it was refitted on the complete data set to compute the final parameter estimates. The empirical internal consistency of each trait was also computed as a summary measure of precision,Of the 35,908 patients, data from 34,910 (97.2%), 35,262 (98.2%) and 35,545 (99.0%) were used in the analyses, with partial missingness for 5703 (15.9%), 760 (2.1%), and 1946 (5.4%) of SF-36, EQ-5D, and HADS, respectively. Nearly 72% of the patients were female, the average age was 44 years, 84% had a secondary school education or higher, and 59% were actively employed. Moreover, 75% had a history of persistent or recurrent pain for 2 years or more, with the most prevalent locations after widespread pain being the lower back and the neck/shoulders, or upper extremities. Table For all questionnaires, individual item distributions were either unimodal or monotone. The minimum number of observations per response category was 44 (followed by 171), 243, and 1239 for SF-36, EQ-5D, and HADS, respectively. No ceiling or floor effects were observed, with maximum and minimum test scores consistently being less than 0.1%.Sixteen, 1, and 9 confirmatory models were computed for SF-36, EQ-5D, and HADS, respectively; all converged. For SF-36, scale-level indices supported that 5 models roughly had an acceptable fit. A two-tier model with all items free to load on 2 orthogonal general factors displayed the best fit, closely followed by its corresponding structure with 2 oblique general factors, whereas 3 bifactor models of the complete questionnaire and of the physical or mental health questionnaire sections separately had a somewhat worse fit. Piecewise indices more distinctly favored the orthogonal two-tier model because nonnegligible amounts of residual dependencies remained for the other models. However, the relationships between the general trait scores and conventional scores of all questionnaires revealed some trait–subscale inconsistencies and illogically high associations between the physical health trait and mentally oriented scales. The orthogonal two-tier model was therefore refitted with all factor loadings limited to positive values, which both mitigated the inconsistency and improved construct validity with no decrease in statistical properties. For EQ-5D, a unidimensional model was fitted, which showed satisfactory properties. Finally, indices unanimously supported that HADS was best modeled with a bifactor structure with 2 specific factors; other models performed similarly to each other, with the exception of the unidimensional model, which was markedly worse. For the selected models, latent trait distribution proxies supported normality, observed and expected categorical probabilities were similar, and item residual correlations were mostly within an acceptable level, thereby supporting the fact that the model assumptions were roughly met.r ≤ 0.13), and between item 22 from the bodily pain subscale with items 13 to 16 from the physical role limitation subscale (r ≤ 0.13). In addition, individual residual correlations remained, including between items 4 and 5 from the physical functioning subscale (r = 0.15), and items 23 and 27 from the vitality subscale (r = 0.16). Meanwhile, observed and expected response category probabilities generally matched well for most items, with the exception of items 29 and 31 of the vitality subscale, which showed distinct differences for higher categories. At person level, 4.0% (n = 1389) and 5.2% (n = 1808) of patients misfitted and overfitted the model, respectively. In addition, the cross-validation supported that most item parameters were relatively stable ; however, thresholds varied considerably more for items 10, 11, and 31 (≤|1.4| logits). Nonetheless, the variation in person IRT scores was less than the size of their standard errors for both traits (≤|0.3| logits).Two dominant physical and mental health traits were observed, which accounted for 21.2% and 20.4% of the total variance, and constituted 30.3% and 29.3% of the variance common with the other latent traits, respectively. Scale-level indices unanimously supported an acceptable two-tier model fit {RMSEA: 0.041 ; SRMSR: 0.038; TLI: 0.971; CFI: 0.976}. However, although mean item residual correlations were below 0.05, 72 individual correlations surpassed 0.05, with the maximum correlation of 0.19. Positive correlation patterns were observed both between items 24 and 25 from the mental health subscale with items 17 to 19 from the emotional role limitation subscale , item residual correlations below 0.06, observed and expected response category proportions matching well, and only 2.1% (n = 752) of patients misfitting the model. The cross-validation further sustained that item parameters  and person IRT scores (≤|0.3| logits) were stable.EQ-5D was well targeted and provided most information in the central range of the latent trait Fig. ; howeverA strong emotional distress trait was identified, which represented 46.4% of the total variance, and constituted 74.1% of the variance common with the other latent traits. All indices supported an acceptable bifactor model fit, with scale-level indices within predefined thresholds , and piecewise indices limited to minor misfits. At item level, mean residual correlations were below 0.05, and although 12 individual correlations surpassed that threshold, the maximum correlation was 0.09. Observed and expected response category probabilities also matched well, with deviances for items 2 and 9 in particular. At person level, 3.1% (n = 1105) misfitted the model and 1.9% (n = 678) overfitted it. The cross-validation further supported that item parameters  and person IRT scores (≤|0.2| logits) were stable.The bifactor model was also motivated with respect to parsimony because less complex models, although showing similar IRT-test score relationships, tended to overestimate IRT score precision Fig. . In addir = |0.63-0.85|) and the weakest association with mental health scores . Likewise, mental health scores generally had the strongest association with each other , and the weakest association with physical health scores . The pattern was less obvious for EQ-5D, which was more equally associated to both physical  and mental measures . Finally, all IRT scores were strongly related to their conventionally calculated scores (r ≥ |0.80|), most noticeably for the EQ-5D index and the HADS anxiety subscale, which had nearly perfect associations (r ≥ |0.92|).The IRT score relationships were consistent with their underlying theories Fig. . PhysicaSF-36, EQ-5D, and HADS are widely used questionnaires for measuring HRQoL and emotional distress in the health sciences, and many theories pertaining to their latent structures have arisen over time. We compared the empirical representations of the most recognized theories, and evaluated their properties through a robust statistical procedure, in a large sample of chronic pain patients. For all 3 questionnaires, we identified conceptualizations that were structurally sound and logically associated, which provides evidence for their validity in the chronic pain population. However, because the internal consistency of our EQ-5D representation was unacceptably low, we recommend that our models be used to score SF-36 and HADS only.67 They were most accurately captured in a model that also accounted for unrelated item dependencies within the 8 SF-36 subscales. Although these constructs are generally agreed upon,68 2 conflicting perspectives, either viewing them as independent or correlated, have largely dominated the psychometric literature.67 Our results support the former view owing to a better data fit and higher parsimony. Summary scores based on the independent representation have received criticism for negative scoring weights that result in inconsistencies between summary scales and subscales.59 To a lesser extent, these concerns apply to our results because the physical and mental health scores were inversely associated with some SF-36 items. These inconsistencies were not eliminated by allowing the physical and mental traits to correlate, in line with previous findings.31 Instead, the problem was mitigated to a higher degree by restricting item loadings to positive values on all latent factors. In practice, the inconsistency is primarily a concern towards the trait score extremes and the subscale scores should therefore be considered when interpreting the overall scores in this range.59 However, although the selected model had the comparatively best properties of the fitted models, further refinements are likely possible. In agreement with previous reports,65 the physical and mental health constructs were most strongly associated with the physical functioning and mental health subscales, respectively, whereas the social functioning, vitality, and general health subscales were more evenly associated to both constructs. Largely, they were also logically correlated with other scales, particularly so between the mental health trait and HADS, which both measure mental state. However, given that the physical and mental health traits were uncorrelated by design, a small albeit seemingly contradictory association arose between the physical health construct and mentally oriented scales. A potential explanation for this is the importance of physical health perception for mental health, as previously motivated for the association between the physical health summary score and the mental health subscale of SF-36.49 Hence, in general agreement with most previous studies, our results support 2 summary scales and 8 subscales, but suggest that the summary scales are independent rather than correlated, which is still an open question in the scientific community.67Consistent with the original theory behind SF-36, we observed 2 general constructs of physical and mental health.51 However, from a causality perspective, it may be conceptually inappropriate to model EQ-5D within the IRT framework because the original theory defines the items as independent causes of HRQoL, whereas they are viewed as correlated effects of HRQoL in IRT. Nonetheless, EQ-5D's causal structure is an open question, with recent studies providing some support for that it contains both cause and effect items.29 Our results are coherent with other IRT evaluations in chronic pain and mental illness patients, which also assessed the properties of EQ-5D as a unidimensional scale and reported acceptable unidimensionality.56 Our IRT-based score was strongly associated with the preference-based EQ-5D index and other physical scales and, to a lesser extent, with mental scales. These findings support construct validity and are intuitive because 4 of 5 items in EQ-5D target physical health.7 They are also consistent with studies of other populations, which have reported similar associations.9 Regardless, we do not recommend the use of the IRT-based score because its internal consistency was too low to reliably discriminate between patients of different HRQoL levels. This is consistent with previous results,56 and is hardly surprising because items intentionally target different HRQoL aspects. Interestingly, the multidimensional 3-level version of EQ-5D is also known to have low discriminatory power, resulting in the development of a 5-level version.8 Our results are limited to the former, and it is possible that an IRT score of the latter would have acceptable reliability.Despite the conventional conceptualization of EQ-5D as a multidimensional scale, its HRQoL construct was acceptably represented by a unidimensional model. It is justifiable to model the scale both ways from the perspective of dimensionality alone because the unidimensional model only assumes that items share one common construct, not whether systematic components unique to each item remain within the residuals.20 our results supported that it is a valid and reliable questionnaire. However, rather than the 2 original constructs of anxiety and depression, we observed one strong emotional distress construct. Similar to SF-36, the construct was most accurately captured in a model that accounted for unrelated item dependencies within the anxiety and depression subscales. This result corresponds to those of another study that compared the same latent structures in a meta-confirmatory factor analysis of 28 samples from various populations.42 Conversely, 2 studies that evaluated the dimensionality of HADS in one sample of musculoskeletal pain patients came to somewhat different conclusions, which nonetheless are supported by our findings.45 The first found that 2 highly correlated (r = 0.80) constructs of anxiety and depression had an acceptable fit with item 7 excluded.44 Similarly, we observed a nearly acceptable scale-level fit for the original 2-factor model in a sample subgroup, and item 7 also behaved differently in our analysis by loading negatively on the specific factor. Their second study suggested that HADS was sufficiently unidimensional to be used as a global measure of emotional distress, but that residual patterns related to the anxiety and depression subscales of a considerable size remained.45 Our findings are largely in agreement because we found additional features not accounted for by the emotional distress construct that was necessary to factor into the model. The combined evidence thereby supports our selected structure for HADS.Although HADS has received considerable criticism in the past,49 and are unlikely to implicate consequences other than a slight overestimation of the latent trait reliability. In addition to model fit, cross-validation supported that the item parameters obtained were generally stable for all questionnaires, although a more pronounced instability was observed for 3 SF-36 items, which we attribute to data sparsity owing to the many possible response patterns. Nevertheless, the IRT scores remained stable, which supports the fact that our models capture the marginal relationships in this population and that the models provided can be used to score HRQoL and emotional distress in chronic pain patients. EQ-5D was unidimensionally modeled and its scoring is thus unequivocal, but to accurately score the multidimensionally modelled traits, it is necessary to estimate them jointly with all modeled factors. Trait scores can be derived in accordance with recommended methods,52 using the scripts provided in the supplementary materials (available at http://links.lww.com/PAIN/A877). Alternatively, the models can be included directly in statistical analyses through the structural equation modeling framework.For all questionnaires, the latent structures selected had the best statistical properties. However, inspecting the fit of individual SF-36 items revealed that small residual dependencies, not accounted for by the model, remained. Such dependencies are common and may reflect interpretation difficulties or response bias,Our results rest on a large population-representative patient sample obtained from a nearly complete database of Sweden's pain specialist treatment clinics, and should therefore be robust and generalizable. The more important limitations to consider include a possible response bias due to varying physical and social settings during data collection, and from the systematic completion of the 3 questionnaires in the same order, which partially explains the higher data attrition for the SF-36. Amounts of missing data were, however, acceptable and sensitivity analyses supported the fact that missingness did not result in any meaningful bias. Another limitation was the observational design, which necessitated the assumption of identical item–trait relationships irrespective of patient characteristics. This assumption should be tested in future measurement invariance studies. Finally, internal consistency estimates should be interpreted with caution because they summarize reliability into a single value, whereas in reality, it varies across the latent trait.This study evaluated the measurement properties of SF-36, EQ-5D, and HADS for chronic pain patients in clinical settings. Our results support that SF-36 is an acceptable measure of 2 independent constructs of physical and mental health. In contrast, although it was a valid approach to summarize the HRQoL construct of EQ-5D as a unidimensional score, its low reliability rendered practical model implementation of dubious value. Finally, rather than dividing into 2 subscales of anxiety and depression, HADS was a valid and reliable measure of overall emotional distress. Relationships between the measured constructs were consistent with their underlying theories, which further supports their construct validity. We recommend that the provided models be used to score SF-36 and HADS in chronic pain patients.The authors have no conflicts of interest to declare.http://links.lww.com/PAIN/A877.Supplemental digital content associated with this article can be found online at


Purpose: Radioiodine (131I) thyroid remnant ablation is an important treatment of differentiated thyroid carcinoma (DTC) and various factors affecting its efficacy have been reported but not well defined. The aim of our study was to evaluate the efficacy and the affecting factors of 131I ablation after total or near-total thyroidectomy in a relative large DTC cohort.Methods: 261 DTC patients with negative thyroglobulin antibody received 100–200 mCi 131I for thyroid remnant ablation after total or near-total thyroidectomy between January 2012 and October 2015 in our hospital. The efficacy and affecting factors of 131I ablation therapy were retrospectively investigated.Results: The success rate of the first 131I thyroid remnant ablation was 65.90%. Univariate analysis demonstrated that larger tumor size, higher level of pre-ablation stimulated thyroglobulin (sTg), intermediate to high risk stratification for recurrence, and lymph node and distant metastases were associated with a lower success rate of the first 131I ablation . Multivariate logistic regression analysis showed that tumor size, pre-ablation sTg, and lymph node and distant metastases were independent factors affecting the efficacy of the first 131I ablation. Areas under receiver operating characteristic curves for sTg, sTg/TSH ratio, and tumor size to predict unsuccessful ablation were 0.831, 0.824, and 0.648, respectively. The threshold values were 4.595 ng/ml, 0.046 mg/IU, and 1.350 cm, respectively. The sensitivities were 95.51, 96.63, and 73.03% and the specificities were 64.54, 61.63, and 49.41%, respectively. The excellent response (ER) ratio of the successful group was significantly higher than that of the unsuccessful group.Conclusions: The efficacy of the first 131I thyroid remnant ablation after surgical treatment of DTC is well demonstrated, and tumor size, pre-ablation sTg, lymph node, and distant metastases are independent factors affecting its efficacy. Primary site cancer recurrence, distant metastasis recurrence, and disease-specific patient death rates are significantly lower after 131I thyroid remnant ablation than those with TSH suppression alone or no postoperative medical therapy  patients with histological confirmation of DTC, (2) patients who received total or near-total thyroidectomy, and (3) patients who met the treatment criteria of 131I thyroid remnant ablation and received 131I ablation. Exclusion criteria were as follows: (1) patients with positive thyroglobulin antibody, (2) patients whose TSH level was <30 mIU/L before 131I ablation, and (3) patients who lost to follow-up and had inadequate information. Near-total thyroidectomy was as classically defined; i.e., the estimated residual thyroid tissue after thyroidectomy was <1.0 g. Total or near-total thyroidectomy was treated equally in our study, which is consistent with the recommendations of American Thyroid Association guidelines as well as Chinese Thyroid Association guidelines on the management of DTC  and 41 lost follow-up with inadequate information. None of the patient with TSH <30 mUI/L was included in the study. Therefore, 261 patients were finally enrolled in the study. The TgAb was negative in the DTC patients analyzed. The detection range of TSH and sTg were 0.005–100.00 mIU/L and 0.04–1000.00 ng/ml, using Electrochemiluminescence immunoassay . We collected the following information: surgery type, histological type, with or without Hashimoto thyroiditis (HT), unilateral or bilateral foci, number of foci, TNM stage, dosage of 131I, time interval between surgery and the first 131I ablation, tumor size, risk stratification for recurrence  DTC with lymph node metastasis or distant metastasis after thyroidectomy, highly invasive histological features such as tall cell variant, columnar cell variant, hobnail variant and papillary carcinoma with a focal undifferentiated component, and extrathyroidal extension; (2) tumor size >4 cm even in the absence of other higher risk features; and (3) for the patients having tumor size <4 cm with preoperative lymph node metastasis but absence of postoperative metastasis, the treatment decision will be made based on the physician's judgment and the patient's wishes. If the tumor diameter, preoperative lymph node status (site and number), and age are indicative for the increased risk of the future recurrence and metastasis, 131I ablation therapy is recommended. 131I treatment is not recommended for all low-risk DTCs with tumor lesions <1 cm and no extra-glandular infiltration, no lymph nodes, and absence of distant metastasis.According to the 2009 ATA guideline and 2012 Chinese guideline , 7, the 131I ablation. To achieve urinary iodine <100 ug/L before 131I ablation, all patients were required to maintain a low-iodine diet (<50 ug per day) for 1–2 weeks and to avoid drugs containing iodine as well as contrast agent. Following tests were completed within 1 week before 131I ablation, including TSH, free thyroxine, free triiodothyronine, thyroglobulin, TgAb, neck ultrasound, and chest CT scan to rule out contraindications for 131I ablation and to evaluate the size of residual thyroid tissue and the presence of pulmonary metastasis. On the day immediate before 131I ablation treatment, 99mTcO-4 thyroid static imaging and urine iodine determination were performed to assess the size of residual thyroid and the level of urine iodine for helping determination of the dose of 131I. Treatment dose of 131I  was given on an empty stomach. Subjects were routinely given a dose of 100 mCi  except for patients with suspected or confirmed microscopic residual lesions, lymph node metastasis, distant metastasis or unexplained serum Tg level elevation who were given a higher dose of 131I (100–200 mCi) according to the disease extent .All patients avoided taking levothyroxine (L-T4) after thyroid operation or stopped taking L-T4 for 3–4 weeks to raise the endogenous TSH level>30 mIU/L before 131I ablation, treatment effect was evaluated. 131I thyroid remnant ablation was considered to be successful when subjects met the following two conditions, as previously reported  131I treatment was analyzed according to the 2015 ATA guideline  curve analysis was constructed to determine the best cut-off values of sTg, sTg/TSH ratio and tumor diameter before 131I ablation for predicting the unsuccessful ablation and to assess the predict values of the affecting factors. Correlation analysis was performed via Spearman bivariate correlation analysis. A p < 0.05 was considered as statistically significant.Statistical analysis was performed using the SPSS 17.0 statistical software. The data were expressed as mean ± standard error (S.E.) or median. Student-131I thyroid remnant ablation, the first 131I thyroid remnant ablation succeeded in 172 patients and failed in the remaining 89 patients, with a success rate of 65.90%.Of the total of 261 DTC patients receiving X2 = 1.700, p = 0.182). Accordingly, there was no significant difference of the successful ablation between the patients <45 years old and those ≥ 45 years old  was 0.1 cm to 6 cm, with a median of 1.5 cm. The minimum, maximum, and median tumor sizes of the successful and unsuccessful ablation groups were 0.1, 5.0, 1.5 cm and 0.3, 6.0, 1.7 cm, respectively. The size of primary carcinomas in the successful group was smaller than that in the unsuccessful group, and the difference was statistically significant (p < 0.05)  . Tumor diameter > 1.680 cm, pre-ablation sTg > 5.450 ng/ml, sTg/TSH ratio > 0.066 mg/IU, higher dose of 131I, intermediate- to high-risk stratification for recurrence, and lymph node metastasis and distant metastasis were significantly associated with unsuccessful ablation , 3 cases were successfully ablated ; and 252 cases were >18 years old (adult), 169 cases were successfully ablated , there was no significant difference in the success rate of ablation between the two groups  no metastasis (absence of metastasis before and after surgery), (2) no postoperative metastasis (surgery had the metastatic lymph nodes cleaned. No lymph node and distant metastasis was found after surgery), (3) postoperative lymph node metastasis (metastatic lymph nodes were found but no distant metastasis after surgery), and (4) distant metastasis . Results showed that the difference between 4 groups was statistically significant (p = 0.000), and the pairwise comparison between the other three groups was statistically significant  except for non-metastasis group and postoperative non-metastasis group (p > 0.008). The ER rates in the non-metastasis group, the postoperative non-metastasis group, the postoperative lymph node metastasis group, and the distant metastasis group decreased gradually, while the SIR rate showed an upward trend, and no significant regularity were found for the IDR rate and BIR rates. Further comparison of the ER rates between the groups showed that the non-metastasis group and the postoperative non-metastasis group were higher than those in the postoperative lymph nodes metastatic group or distant metastasis group (both p < 0.008); postoperative lymph node metastasis group ER rate was higher than that of distant metastasis group (p < 0.008), except that there was no significant difference between the non-metastasis group and the postoperative non-metastasis group . Then we analyzed the success rate of 131I and ER rate, the ER ratio of the successful group was 74.42% (128/172), and the ER ratio of the unsuccessful group was 5.62% (5/89), the difference was statistically significant .The clinical efficacy of 131I therapy, which is a major adjuvant treatment for DTC after thyroid surgery, can reduce risk of recurrence or metastasis and improve the survival of patients with DTC  group and >18 years old (adults) group and between the group aged <45 years old and ≥45 years old group. Results show no significant difference, indicating the heterogeneity caused by age stratification is negligible. This is consistent with the ATA guideline /I(-) symporter (NIS) and therefore retain the function and the ability of iodine uptake in varying extent  is lower than in high-dose group (100 mCi), suggesting that a higher success rate of ablation could be achieved by applying higher dose of 131I (131I (100–200 mCi) according to the disease extent   .131I ablation and in monitoring recurrence or metastasis of thyroid carcinoma (131I ablation is associated with lower success rate of 131I ablation and sTg has an important predictive value for successful 131I ablation (131I ablation is clearly lower in the group with higher pre-ablation sTg level. When sTg is >4.595 ng/ml, its sensitivity, specificity, positive predictive value and negative predictive value to predict thyroid remnant ablation failure are 95.51, 64.54, 58.22, and 96.52% respectively. These indicate that up to 96.52% of the patients with DTC may have successful ablation for the first time if the sTg is <4.595 ng/ml before 131I ablation. Therefore, the pre-ablation sTg measurement is an important predictor of the efficacy of 131I thyroid remnant ablation. The threshold value of sTg level in our study is similar to that of the Tamilia's report but is different from that of the Zubair's study. This discrepancy may be related to different research approaches, amount of the residual thyroid tissue, baseline TSH level, and detection method of thyroglobulin.Thyroglobulin is a glycoprotein, synthesized in thyroid cells or well-differentiated thyroid carcinoma cells. It is secreted together with thyroid hormones under the stimulus of TSH. Thyroglobulin is a critical parameter in predicting therapeutic response to arcinoma , 33. Manablation –36. Tamiablation  and Zubaablation  report tablation , 38. Our131I ablation is likely to succeed in ablation. Thus, sTg/TSH ratio is a reliable predictor of the efficacy of 131I thyroid remnant ablation and is helpful to the clinical decision-making in the radioiodine remnant ablation of DTC patients.It is well known that the main factors affecting the thyroglobulin level include the amount of residual thyroid tissue or cancer lesion, TSH level, the time after the operation, and the thyroid hormone withdrawal time . These a131I ablation 6 months after treatment among no metastasis, no postoperative metastasis, postoperative lymph node metastasis, and distant metastasis groups, showing that the difference between the 4 groups is statistically significant. The ER rate of the non-metastasis group, the postoperative non-metastasis group, the postoperative lymph node metastasis group, and the distant metastasis group decreased gradually, while the SIR rate shows an upward trend. Further comparison of ER rates between the groups demonstrate that the non-metastasis group and the postoperative non-metastasis group are higher than those in the postoperative lymph nodes metastatic group or distant metastasis group and that the ER rates in postoperative lymph node metastasis group is higher than that of distant metastasis group. The correlation analysis shows that ER rate is negatively correlated with metastasis. The ER ratio of the successful group is significantly higher than that of the unsuccessful group. All of these results are consistent with previous studies (131I ablation depends on the presence and the severity of metastasis. On the other hand, the success rate of first 131I disinfection is an influencing factor of ER, that is, the higher the success rate of first ablation, the higher the possibility of ER and the better the prognosis. In view of the recurrence rate of ER patients is only 1–4% and the tumor related mortality risk is < 1% (131I treatment should be optimized as far as possible so that the success rate of 131I ablation can be improved and more patients can enter the ER state as early as possible with the improved prognosis.The 2015 ATA guidelines  propose  studies , 41. On  is < 1% –44, the 131I thyroid remnant ablation is high and that tumor size, pre-ablation sTg level, lymph node metastasis, and distant metastasis are the major factors affecting therapeutic success. Moreover, pre-ablation sTg and sTg/TSH ratio are reliable predictors of thyroid remnant ablation efficacy. When 131I ablation is performed in DTC patients, in order to optimize the ablation efficacy, to achieve the highest ER rate, and improve the prognosis, these factors should be taken into consideration in determining individualized 131I dosage and in anticipating the success potential of 131I ablation.In summary, our study demonstrates that the success rate of WZ developed the model, carried out the parameter estimations, and planned as well as performed the mass transfer experiments. CW wrote the main part of the manuscript and took part in the planning and execution of the fermentation experiments. HD took part in the development of the model, planned and carried out the main part of the fermentation experiments, analyzed the results, and assisted in the mass transfer experiments. PR helped analyze the results and assisted in the mass transfer experiments and wrote parts of the manuscript. XW and YW participated in the coordination of the study and reviewed the manuscript. All authors read and approved the final manuscript.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.


However, after four days, the hydrocarbonoclastic activity reached its maximum values and was accompanied by increases in the relative abundance of the well-known hydrocarbonoclastic Alteromonas. At the end of the experiment, the diversity was restored to similar values as those observed in the initial time, although the community structure and composition were clearly different, where Marivita, Pseudohongiella, and Oleibacter were detected to have differential abundances on days eight–14. These changes were related with total nitrogen  and polycyclic aromatic hydrocarbons , according to PERMANOVA. The results of this study contribute to the understanding of the potential response of the bacterioplankton from sGoM to crude oil spills.The southern Gulf of Mexico (sGoM) is highly susceptible to receiving environmental impacts due to the recent increase in oil-related activities. In this study, we assessed the changes in the bacterioplankton community structure caused by a simulated oil spill at mesocosms scale. The 16S rRNA gene sequencing analysis indicated that the initial bacterial community was mainly represented by Gamma-proteobacteria, Alpha-proteobacteria, Flavobacteriia, and Cyanobacteria. The hydrocarbon degradation activity, measured as the number of culturable hydrocarbonoclastic bacteria (CHB) and by the copy number of the  The Gulf of Mexico (GoM) is a hotspot of biological diversity in which several oil-related industries are hosted, representing a high risk for oil spill disasters ,2. Two oPseudomonas), while potential degraders of aromatic compounds  were observed in a second stage [Microbial communities have different responses to environmental perturbations ,7 dependnd stage ,11,13. Tnd stage .Cycloclasticus has been reported as one of the most important degraders of low molecular weight PAHs in the cold waters of Galicia, Spain [Alcanivorax as relevant microorganisms for cleaning oil-polluted marine systems, due to their high capability to use aliphatic compounds as carbon source [For over a decade, several simulations at mesocosm scale have been performed for the evaluation of the effect of hydrocarbons on planktonic microorganisms ,16. At ta, Spain . Moreoven source ,20,21. Fn source .The southern Gulf of Mexico is considered the largest base of the petroleum industry in Mexico, and explorations have been performed since 1970 and continue to increase until current times ,23. Desp−1), and no nutrient addition was performed. For the determination of the microbial community composition in the seawater acclimatized to the mesocosm before crude oil addition, a sample was collected and catalogued as “Reference” (R). At day zero, the tank was supplemented with light crude oil  at 80 mg−1 L.Superficial seawater (< 3 m depth) was collected during October 2017 at Progreso harbor, in the Yucatan Peninsula, Mexico . A single tank  was filled up to 2500 L with seawater and transported to the CINVESTAV-IPN facilities in Merida, Yucatan. The mesocosm was equipped with an electric thruster to homogenize seawater temperature, oxygen, and salinity . Before 3−+NO2−), nitrite (NO2-), orthophosphate (PO43−), and silicic acid (SiO44−) analyses were performed according to Strickland and Parsons [During the experiment, one-meter depth water samples were collected from the mesocosm on days zero, two, four, six, eight, 10, and 14 to measure oxygen, salinity, temperature, nutrients, and hydrocarbons. The Winkler method was approached according to the steps suggested by Carrit and Carpenter  and Brya Parsons , using aCrude oil concentration was measured as the total petroleum hydrocarbon (TPH). It was determined as the sum of aliphatic, aromatics (PAHs) and unresolved complex mixture (UCM) concentrations. Hydrocarbons were obtained by liquid–liquid extraction with 150 mL of dichloromethane and 4 L of one-meter depth seawater from the mesocosm. PAHs and UCM fractions were analyzed by high-resolution Gas Chromatography-Mass Spectroscopy (GC-MS) Perkin Elmer Clarus 500 and Gas Chromatography with Agilent 7890A  equipped with a flame ionization detector (GC-FID) for aliphatic fraction.−1) as the sole carbon source, and resazurin as an indicator of bacterial growth. The quantification of the alkB gene, related to first-step hydroxylases involved in the metabolism of alkanes, was performed on a Rotor-Gene Q System  with the primers and the protocol previously reported by Uribe-Flores et al. [r2) of 0.98 and PCR amplification efficiency of 98.3%.Water samples (15 mL centrifugate tubes) were collected specifically for assessing the culturable hydrocarbonoclastic bacteria (CHB). Estimates of the number of CHB were obtained using the most probable number (MPN) method reported by ,28. The s et al. , with co® 3.0 Fluorometer using the Qubit dsDNA HS Assay Kit . Amplicon size was verified by capillary electrophoresis at QIAxcel Advanced . Individual amplicons were diluted in 10 mM Tris (pH 8.5) and pulled at equimolar concentrations (4 nM). Sequencing was carried out in CINVESTAV-Mérida using an Illumina-Miseq platform , with the MiSeq reagent kit V3 (2 × 300), following the manufacturer’s recommendations.Changes in the microbial community were followed by 16S rRNA Illumina sequencing. DNA extraction was performed using the PowerWater Sterivex DNA Isolation kit , according to manufacturer’s instructions. Seawater from the mesocosm was filtered (5 L) through a 0.22 µm Sterivex cartridge filter  using a peristaltic pump. The quality of DNA extraction was evaluated by agarose gel (1%) electrophoresis. Amplification of hypervariable V3 and V4 region of 16S rRNA was performed using the primers and the conditions suggested by Klindworth et al. . Amplicop values were obtained using 999 permutations. Shannon-Weaver diversity index was estimated in vegan library. A Kruskal Wallis test was performed to detect ASVs with differential abundance among three groups of samples based on changes in the Shannon index: Group 1 (samples R and zero), Group 2 , and Group 3 . The taxonomic assignation of the differential ASVs, was improved, with the selection of the three best blast hits from the RefSeq NCBI database. Raw sequence data produced in this study was deposited in NCBI under the Bioproject accession number PRJNA560042The demultiplexed paired-end reads (2x300), in the fastq format were processed with the QIIME2 pipeline (2018.8) . The err−1, and the final concentration was 4.54 mg L−1 at day 14. Salinity ranged from 35.53–36.70 PSU. Inorganic nutrients , Total C, and Total N, had an increase from day zero to day 14, however, this increment was more conspicuous in the nitrogen concentration range (0.04 to 1.55%).All the physicochemical parameters measured through the 15 days of the mesocosms experiment are shown in alkB gene copies were 31.96 µg L−1, 70 CFU, and 2 × 105 copies µL−1, correspondingly. At day four, increases in these measurements were observed . However, at day six the TPH concentration (117.48 µg L−1) and alkB gene copies (5 × 104 copies µL−1) decreased considerably. At the end of the experiment the values of TPH concentration and the alkB gene copies remained low ; additionally, the CHB count decreased to 90 CFU  of TPH concentration, CHB count, and o 90 CFU .Alteromonas increased considerably at day four. By the last period of the experiment (days eight to 14), the values of the diversity index were similar to those found at the initial time , Prochlorococcus (96.37%), Angustibacter (96.80%), Ilumatobacter (97.2), and Candidatus Pelagibacter (96.78%)  . Marinobspicuous .The mesocosm design used for this research allowed to maintain similar environmental conditions to those previously reported for the superficial seawater from sGoM. The temperature, oxygen, salinity, and nutrient concentration measured during the mesocosm experiment were within the ranges previously reported for the water column of the Yucatan Peninsula ,42. The In agreement with previous studies significant changes in the bacterial community were observed were observed after 48 h of exposition to crude oil ,45,46. DPrevious studies have reported a potential recovery of the bacterioplankton diversity in late periods of simulated hydrocarbon spills . In concCandidatus Thiobio, Candidatus Actinomarina, Acinetobacter, and Synechococcus. Previous studies have reported to Synechococcus and Actinomarina as sensitive taxa for contaminated sites with heavy metals [In our study, some of the main changes in the bacterial composition after the simulated oil spill were related with decreases in the relative abundances of y metals . HoweverSeveral works have demonstrated that changes in the bacterial community can be largely attributed to specific physicochemical parameters and hydrocarbons ,54. In tPrevious mesocosm studies that have undertaken lower temperatures (≤18 °C) have found that the main crude oil removal occurred from 15 to 20 days ,59. UnliAlteromonas, Haroferula, Rugeria, and Alcanivorax were distinctive (During the period of highest crude oil degradation (day two to four), increases in the relative abundance of the tinctive b. These Marivita and Oleibacter presented differential abundance in this period of the experiment (Marivita belongs to the Roseobacter lineage (Alpha-proteobacteria) and has been reported to degrade aromatic and aliphatic compounds [Oleibacter has been reported with the capacity to degrade aliphatic hydrocarbons in tropical environments [Marivita, and Oleibacter had important participation related with the hydrocarbon degradation during the last days of our mesocosm system, however further research is required for a better understanding of its contribution in the microbial community.By the last stage of the experiment (days eight–14), increases in the relative abundances of Sphingobacteriia were observed a. Moreovperiment . Previouompounds ,70,71. Tronments ,73,74. BalkB gene copy number, the greatest hydrocarbon degradation activity performed by the bacterioplankton of sGoM occurred after four days from the disturbance. Alteromonas, Haloferula, Ruegiera, and Alcanivorax were found as the main groups putatively involved in the hydrocarbon degradation, based on increases in their relative abundances at this point of the experiment. Despite of the bacterial diversity being recovered at the end of the experiment (14 days), the community composition was different from the initial time. To our knowledge, this is the first mesocosm experiment to evaluate the effect of oil spills over the bacterioplankton community from sGoM. All the results presented here can be employed as a guideline for future studies aiming to evaluate bioremediation strategies for marine environments.The mesocosm experiment of this study evidenced a decrease in bacterioplankton diversity and changes in the community composition from sGoM after two days from the simulated oil spill. Increases in the relative abundances of Alpha-proteobacteria and decreases in Cyanobacteria and Gamma-proteobacteria were some of the main changes in the bacterial composition observed in this period of the experiment. According to the TPH concentration, counts of culturable hydrocarbonoclastic bacteria, and


Scaling and root planing (SRP) is of limited value in many cases, so adjunctive treatment was applied to augment its outcome. Adipose-derived stem/stromal cells (ADSCs) were investigated in periodontal regeneration with promising results. However, they have safety concerns. The exosomes (Exo.), which are extracellular vesicles mediating the action of stem/stromal cells, represent a new approach to overcome these concerns. Ligature-induced periodontitis was induced in 50 rats for 14 days, and they were divided into control , SRP group, ADSCs group, and Exo. group, with evaluation intervals at 2 days, and 2 and 4 weeks, including 5 rats in each interval for each group. The specimens were evaluated for histologic description (H&E), histochemical study (Masson trichrome), and histomorphometric study, to evaluate the area % of newly formed tissues. The Exo. group revealed the best results in all intervals with significantly higher area % of newly formed tissues, followed by ADSCs and, finally, SRP. Both Exo. and ADSCs showed organized newly formed tissues with the Exo. group obtaining comparable histologic results to the normal, healthy tissues by 4 weeks. Adipose-derived stem/stromal cells and their Exo. represent a promising adjunctive treatment to SRP. Periodontitis is a chronic immune inflammatory disease of supporting tooth structures initiated by dysbiotic polymicrobial dental biofilm in a susceptible host . The disRegeneration is defined as the reproduction or reconstitution of a lost or injured part of the body, in such a way that the architecture and function of the lost or injured tissues are completely restored. However, few techniques can be regarded as regenerative methods in periodontics, including biologics . To achiThe nonsurgical periodontal treatment includes scaling and root planing (SRP) as a basic procedure, which aimed at mechanical removal of the dental biofilm. However, it is of limited effect in the deeper periodontal pockets and inaccessible areas, where extra surgical interventions are indicated . The adjA tissue engineering approach for tissue regeneration implements four elements, including blood supply, progenitor cells, proper signaling molecules, and suitable scaffold . Among tIn attempts to overcome the safety considerations associated with biomaterials and cell-based therapy, exosomes (Exo.) have emerged as a cell-free therapeutic approach, which is suggested to be more safe as they are endogenous in origin . They arThe aim of the current study was to investigate the therapeutic potential of both ADSCs and their Exo. to SRP in nonsurgical periodontal treatment as an adjunctive therapy. The study was conducted in theanimal house, Kasr Alainy, Cairo University, in accordance with the ethical guidelines Ethical approval number: CU/III/73/18]. The study included 50 adult male Abino Wistar rats, as referred to in Du J. et al. , weighinPeriodontitis was induced in all rats in accordance with Ionel A. et al. , as the Fat tissue was obtained through subcutaneous incision from both the omentum and the inguinal fat pad. The procedures of ADSCs and their Exo. preparation had been carried out in the Molecular Biology and Tissue Engineering Unit, Department of Medical Biochemistry, Cairo University, School of Medicine.2 at 37 °C. Then, at 80–90% ADSC confluence, they were detached with 0.25% trypsin-ethylenediaminetetraacetic acid , and resuspended in other flasks. At fourth passage, ADSCs were used in all experiments. The surface marker expression of adipose MSCs had been characterized, in culture, by their morphological spindle shape-like cells. Furthermore, they had been identified by flow cytometry , in the fourth passage, to assess positivity of expression of CD29, CD90, and CD105, and lack of, or negativity for, CD45 expression. To this end, they were trypsinized and adjusted to 1 × 106 cells/mL cells, followed by incubation with 10 μL of monoclonal antibodies: CD45 fluorescein isothiocyanate FITC, CD29 phycoerythrin PE, CD105 PE, and CD90 PE  at 4 °C in the dark, and the same species isotypes served as a negative control .The excised fat tissue was hydrolyzed using collagenase type II  dissolved in phosphate-buffered saline  at 37 °C for 2 h. Strainers, 2 µm, were used to remove any tissue debris, followed by centrifugation at 1000 rpm for 5 min to form a cell pellet which was cultured with Roswell Park Memorial Institute RPMI medium , 10% fetal bovine serum , and humidified in a cell culture incubator containing 5% CO6 cells/mL) that were cultured in RPMI deprived of FBS and supplemented with 0.5% of bovine serum albumin (BSA) (Sigma). In order to remove the debris, the cell-free supernatant was first obtained by centrifugation at 2000× g for 20 min, then centrifuged at 100,000× g (Beckman Coulter Optima L 90K ultracentrifuge) for 1 h at 4 °C. Thereafter, the obtained pellet was washed in serum-free mediumcontaining HEPES 25 mM (Sigma), and submitted to a second ultracentrifugation under the same conditions. The quantification of protein content was done by the Bradford method . After that, the purified extracellular vesicles (EVs) were cultured overnight in the medium used for collection of EVs. Electron microscope analysis was done, where the images were obtained at a working distance of 15 to 25 mm, and an accelerating voltage of 20 and 30 kV, where the digital acquisition and analysis were performed using the JEOL T300 system . Flow cytometry analysis was performed using the following FITC-conjugated antibodies: CD83  and CD73 , and FITC mouse non-immune isotypic IgG  was used as a control [The Exo. had been obtained from the supernatants of the third passage ADSCs ; accordingly, they were not included in histochemical and histomorphometric studies.Scaling and root planing (SRP) group: 15 rats that had received scaling and root planing only.7) suspended in 200 µL PBS , and injected locally into the pocket using a disposable plastic syringe, as an adjunctive treatment.ADSC group: 15 rats that had received scaling and root planing and ADSCs , injected locally into pockets using a disposable plastic syringe, as an adjunctive treatment. The animals were allowed to heal for intervals of 2 days , 2 weeks , and 4 weeks , and then they were anesthetized and sacrificed by cervical dislocation. After the sacrifice at the assigned dates, the samples were extracted from animals and were preserved in 10% formalin for 72 h. Then, they were decalcified using 20% formic acid for a period that ranged from 2 to 4 weeks. Thereafter, the decalcified samples were embedded in paraffin, where the serial sections, 5 mm thickness in the mesiodistal direction, were prepared and stained with hematoxylin and eosin (H&E) for descriptive analysis, and Masson’s trichrome for histochemical analysis, where the newly formed collagen and osteoid were represented with blue or green reaction, and cellular cytoplasm represented by red reaction.p-value was calculated to determine significance, which was done using statistical package for the social science SPSS computer system version 9. All the histochemically stained sections were examined by a light microscope, using an image analyzer computer system with software , where the area containing the most uniform positive histochemically stained tissues were selected for evaluation of area %, using magnification 200× at five points, where one point in each slide for each group interval was selected. Then, they were calibrated automatically to convert the measurement units (pixels) produced by the image analyzing program into actual micrometer units, in order to be tabulated and statistically evaluated using one-way ANOVA test and the post hoc Tukey test, and the mean values of data ± standard deviation were expressed and a The two day interval reflects the early healing events, including the inflammatory reaction, while the two and fourweek intervals reflect the healing represented in newly formed cementum, periodontal fibers, and alveolar bone. After the two day interval, all groups showed infiltration of inflammatory cells to different degrees, where there was a mixture of neutrophils and mononuclear cells (lymphocytes and macrophages). In the SRP group, there was abundant inflammatory infiltration in the periodontal tissues a, while After the two week interval, all groups show formation of new periodontal tissues with different degrees of proliferation and organization. In the SRP group, there were multiple large blood vessels in periodontal space, with less evidence of proliferating periodontal cells, while the newly formed bone trabeculae were disorganized with a layer of osteoid tissue a. In theAfter the four week interval, in comparison to normal, healthy periodontal tissue of control group a, the SRThe Exo. group revealed the most dense reaction, indicating formation of new collagen, osteoid, and cellular proliferation in an organized pattern during all three intervals, followed by ADSCs group, while the SRP group showed the weaker reaction among the study groups, and these results were represented statistically.The ANOVA test showed significant difference between all groups, This is the first study to investigate the therapeutic effect of Exo. in periodontal treatment, generally. Also, it is the first study to investigate the effect of ADSCs in nonsurgical periodontal treatment.The animal study provides a valuable tool for examining the effect of new therapeutic regenerative techniques in periodontics, especially using small animals, with many advantages, including cost effectiveness and simplicity of handling , where tThe inflammatory phase represents the early wound healing events during the first 3 days, and it functions mainly in wound debridement during the early phase and, additionally, in the late phase, the macrophages contribute to wound healing via release of various growth factors targeting the involved cells in the tissues’ formation as fibroblasts . As bothThe SRP represents the basic procedure in periodontal treatment by elimination of the bacterial biofilm, leading to decrease in the bacterial load and creation of a more favorable environment for periodontal healing . HoweverThe ADSC group in the current study showed better results, with significantly higher area percentage of newly formed periodontal tissues than SPR, showing more proliferation and organization in all intervals. These results are similar to our previous study , and theThe Exo. group of the current studies showed a striking result by evidence of new periodontal tissue formation in the 2 day interval, that was represented in proliferating primitive periodontal fibroblast and osteoid tissues while, on the other hand, there was minimal evidence of inflammatory cells, that can be explained by the anti-inflammatory effects of exosomes . Also, tIn the current study, both ADSCs and Exo. represent promising regenerative techniques that can be used as an adjunctive treatment to SRP, enhancing the outcome of the nonsurgical periodontal treatment. Exo. showed superior results to ADSCs, with the additional advantages of safety and easy preparation. They can be applied as a suspension injected locally to periodontal defects, without the need or additional cost of preparation of a scaffold.


This study investigated the effect of sea and road transport on the physiological response of cattle. The animals were transported by sea (14 d) from Darwin Port, Australia, to Pasir Gudang Port, Johor, Malaysia. Thereafter, the animals were road transported (330 km) from Pasir Gudang Port to Universiti Putra Malaysia (UPM). From the results of the various blood stress indicators and brain activity of the animals, both sea and road journeys were found to be stressful to the animals. However, the animals recovered from the stressful transport journeys following 4 and 7 days post-transport.The objective of the current study was to evaluate the effects of sea and road transport on the acute phase proteins (APP), cortisol, metabolic, haematological and electroencephalographic (EEG) responses of Brahman crossbred heifers. Sixty Brahman crossbred heifers were subjected to 14 d of transportation by sea from Darwin Port, Australia, to Pasir Gudang Port, Johor, Malaysia, and 330 km of road transportation. Results revealed that the intensity of response for most blood biochemical parameters increased significantly and were different from the baseline values taken while the animals were in Darwin Port, Australia. Haematological results obtained also revealed a significant increase and were different from the baseline values. Cortisol and APP  values increased significantly and were different from the baseline values. Haematological parameters, APP, cortisol and EEG data  decreased significantly following 4 and 7 days post-transport, suggesting a recovery of the animals from the stressfulness of transport. In conclusion, the current results revealed that the concentrations of biochemical and haematological parameters, cortisol, APP and EEG data were affected by both sea and road transport as evidenced by the significant changes recorded from the parameters above. There is increasing transport of livestock through long distances as demand for meat products increases, particularly in Asia; freight opportunities grow and trade barriers are reduced . Most ofIn 2017, Malaysia imported 25,585 live cattle from Australia, of which 23,542 were beef cattle , in a joThe experimental protocol was approved by the institutional animal care and use committee (IACUC) of the Universiti Putra Malaysia (UPM/IACUC/R028/2016).g at 8 °C for 15 min, and subsequently stored at −80 °C until they were air freighted to Universiti Putra Malaysia (UPM). On 11 March, 2016, the animals were road transported 12 km to Darwin and loaded into a vessel. This livestock carrier, which was built in 2006  and operated out of Darwin by Karumba Livestock Holding, had a gross tonnage of 2964 t, dimensions 81 × 13.6 m, with two naturally ventilated decks and two artificially ventilated decks at or below sea level. Details of the housing condition in the vessel are not available.Sixty Brahman crossbred heifers of about 18 months of age (mean live weight 240 ± 50 kg) were sourced from Katherine, a town located in the Northern Territory of Australia. A total of 60 animals were imported from Australia to allow random blood sampling at each point and for a separate experiment. Brahman crossbred is the most common imported beef cattle breed in Malaysia because of its ability to withstand the hot environment. It normally takes two days for mustering, holding in yards and delivery to the pre-assembly depot. The animals were road transported for 306 km to the Berrimah Quarantine Inspection Centre on 29 February, 2016. Upon arrival, the animals were unloaded and held in pens with ad libitum feed and water. The mean dry bulb temperatures in the quarantine facility were 28–31 °C during the day and 22–24 °C at night; relative humidity was 80%. On 6 March, 2016, 24 heifers were randomly selected and blood samples (≈10 mL) were taken by coccygeal venepuncture into blood collection tubes. Serum samples were separated by centrifugation at 1600 × g for 10 min. The plasma clots as well as the serum were separated in replicates of 1.5 mL aliquots and stored at −80 °C until analysis.The sea voyage to Pasir Gudang Port in Johor, Malaysia, covered approximately 3362 km at a speed of 10 knots, over a duration of 14 days. On the route to Malaysia, the vessel transited at Brunei for 48 h. Upon arrival (07:00 h) at Pasir Gudang Port, Johor, the animals were inspected by quarantine officers and remained in the vessel for 12 h before being unloaded. Feed and water were available until disembarkation. At the port, dry bulb temperatures on that day were maximum 35.3 °C and 23.4 °C minimum, with a relative humidity of 81.6% . Prior to disembarkation from the vessel, 24 animals were randomly selected and blood samples taken by coccygeal venepuncture into blood collection tubes for serum and plasma (with heparin as an anticoagulant). Samples collected were stored temporarily in crushed ice for about an hour and later centrifuged at 1000 × 2 per animal. The trucks travelled the highway at an average speed of 80 km/h. Upon arrival at UPM (24.5–25.5 °C with relative humidity of 84.4%), the animals were unloaded and 24 animals were randomly selected for blood sample collection by the same technique as the initial sampling.Following blood sampling, the animals disembarked and were loaded onto trucks at 22:00 h, and then transported for 7.00 h (330 km) by road to the Animal Research Centre of the Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia (UPM), Serdang, Selangor. The animals were unloaded from the ship and loaded onto the trucks using non-slip loading ramps with stair-steps built from wood. The angle of the ramp was about 20°. Similar ramps were used to unload the animals from the truck at UPM. The transportation of animals took place in two different trucks, each with a carrying capacity of 30 cattle, at a space allowance of 0.96 m2 and it had a concrete floor and aluminum roofing. All the 60 animals were subjected to a similar travel chain from Australia to UPM and, thus, it is likely that the animals in each pen were familiar with each other. The animals had access to commercial beef cattle feed , grass pellets, rice straw, and drinking water. The feeding space allowed was 120 cm per animal. Supplementary lighting provided ensured continuous lighting. Over the seven days post-road transport, the mean maximum and minimum daily dry bulb temperatures and relative humidity were 35.3, 23.4 °C and 81.6%, respectively . Blood sampling procedures detailed earlier were repeated at 4 and 7 days post-road transport. Twenty-four randomly selected animals were used for each occasion of blood sampling. The sampling intervals were chosen based on earlier work [On arrival at the Animal Research Centre, the animals were randomly assigned to groups of 15 to four separate pens in a naturally ventilated house. The floor area of each pen was 52.5 mier work , which dSerum concentrations of cortisol (CORT) , alpha-1 acid glycoprotein (AGP) , amyloid-A (AA)  were determined using the appropriate Enzyme-Linked Immuno Sorbent Assay (ELISA) kits. An automatic analyzer  was used to determine serum levels of alkaline phosphatase (ALP), creatine kinase (CK), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and alanine aminotransferase (ALT). All the reagents used were obtained from Roche .® 3700, Abbott, Lake County, IL, USA), the constituent white blood cells (WBC), red blood cells (RBC), packed cell volume (PCV), haematocrit, haemoglobin, platelets, and neutrophils and lymphocytes counts were ascertained using the Veterinary Package software. All reagents used were from Abbott  ratio was calculated.On a microscope glass slide (25 mm × 75 mm and thickness of approximately 1 mm), a thin layer of blood was smeared and stained with methylene blue. The glass slide was then observed and assessed under an electronic microscope, and with the help of an automatic haematology analyzer  were placed on the zygomatic process of the frontal bone (the inverting–electrode) and the right mastoid process (the non-inverting + electrode) after shaving these areas and cleaning them with 70% alcohol, as recommended by Gibson et al. [TM data acquisition system, Sydney, Australia). This software identified potential interferences from concurrent electrocardiograph signals, which were digitally minimized from the raw EEG recordings. Signals were then processed for consecutive non-overlapping 1 s epochs, yielding 60 epochs/min. The root mean square (RMS) of the alpha, beta, delta and theta waves were determined. Two other parameters that were determined were the median frequency  and total power .Electroencephalograms recorded electrical activity at different locations on the scalp, using carefully positioned electrodes . This enn et al. . The elen et al. . Precedip < 0.05.The structure of the experiment was based on a complete randomized design (CRD). Statistical analysis was carried out by using the General Linear Models (GLM) methodology of Statistical Analysis System (SAS) package Version 9.4 software  . IndividAfter sea transport, ALT, AST, and CK were increased and LDH decreased compared to before sea transport . After rCompared with samples taken after sea transport, those taken after the subsequent road transport had increased WBC, neutrophils and N:L ratio, and decreased HB and HCT . After 4CORT was increased after road transport, when compared to other sampling times . Sea andThe effects of road transport on the alpha, beta, delta and theta waves are illustrated in When discussing the CORT results of this experiment, it should be remembered that, because of technical constraints, blood samples were collected at different times of the day and, thus, there is a possibility that the diurnal secretion of the hormone  may haveAs part of the acute phase response (APR), the serum proteins that are mainly synthesized by hepatocytes  are released as part of the early defense mechanism. This is induced by a range of challenges, such as tissue injury, endotoxin exposure, bacterial infection and inflammation ,42, to pPacked cell volumes ,54, HCT In the present study, because of technical constraints, we were unable to determine haematological parameters before sea transport. Thus, the baseline values were based on Jackson and Cockroft . AccordiElevated amounts of AST, ALT and CK serum activities suggest cell muscle damage and muscle fatigue . It has The electroencephalogram (EEG) records the electrical activity of the cerebral cortical neurons, believed to play a role in pain perception . ActivitSea transport of cattle from Darwin, Australia, to Johore, Malaysia, sufficiently affected the concentration of various physiological and biochemical variables. To conclude, stress was induced, particularly noted in the elevation of acute phase proteins, even though blood samples were taken 12 h after arrival. The lack of circulating cortisol in reaction to sea transport could be attributed to the transient responses of the hormone . The phy