In May of 2010, two communities (Truenococha and Santa Marta) reported to be at risk of vampire bat depredation were surveyed in the Province Datem del Marañón in the Loreto Department of Perú. Risk factors for bat exposure included age less than or equal to 25 years and owning animals that had been bitten by bats. Rabies virus neutralizing antibodies (rVNAs) were detected in 11% (7 of 63) of human sera tested. Rabies virus ribonucleoprotein (RNP) immunoglobulin G (IgG) antibodies were detected in the sera of three individuals, two of whom were also seropositive for rVNA. Rabies virus RNP IgM antibodies were detected in one respondent with no evidence of rVNA or RNP IgG antibodies. Because one respondent with positive rVNA results reported prior vaccination and 86% (six of seven) of rVNA-positive respondents reported being bitten by bats, these data suggest nonfatal exposure of persons to rabies virus, which is likely associated with vampire bat depredation. Lyssavirus. Rabies virus  is the most prolific of the 12 viral species classified within the genus, and it is responsible for greater than 55,000 human deaths annually.Desmodus rotundus), although several other Neotropical bat species play a role in RABV circulation.12Rabies is caused by single-stranded negative-sense RNA viruses in the genus D. rotundus.D. rotundus.In Perú, outbreaks of rabies linked to vampire bat bites have been documented among populations living in the Amazon region over the past several decades.Rabies has the highest case fatality rate of any conventional infectious disease, approaching 100%. The likelihood of a productive rabies infection after exposure to a lyssavirus is known to depend on a variety of factors, including but not limited to dose, route of exposure, site of exposure, variant, host genetic makeup, pre- and/or post-exposure prophylaxis , etc.38The objective of this study was to investigate risk factors for bat and RABV exposure in Amazonian communities that were suspected to be at high risk of vampire bat depredation based on their proximity to recent outbreaks in Perú and rural living conditions. A survey questionnaire was used to capture demographic information of the study populations, salient details of any previous bat exposure, and self-reported vaccination history among respondents sampled.Two communities were surveyed in May of 2010 in the Province Datem del Marañón in the Loreto Department of Perú . Samples50]). Sera that exhibited complete neutralization of RABV at a 1:5 dilution were considered rVNA positive using the recommendations of the Advisory Committee on Immunization Practices (ACIP).−1 by comparison with the US Standard Rabies Immune Globulin  diluted to 2 IU mL−1.Sera were screened for rVNA by the rapid fluorescent focus inhibition test (RFFIT) as described.Sera were screened for RABV ribonucleoprotein (RNP) immunoglobulin M (IgM) and IgG antibodies by the indirect fluorescent antibody (IFA) test as describedSeparate analyses were conducted with survey data. The first analysis stratified all respondent data by exposure history to bats  to evaluate risk factors for bat exposure. The second analysis focused only on individuals from whom a serum sample was obtained and tested, with stratification of respondent data regarding bat exposure by serological status to evaluate risk factors for rabies virus exposure. All statistical analyses were performed using SAS v.9.3 . Fisher exact test was used to evaluate associations (α = 0.05) between the response (stratification) variable  and factors such as community of residence, age, sex, education level, and self-reported bat exposure history, which includes subcategories of bite, scratch, and/or touching a bat with unprotected skin.A total of 92 persons were interviewed from 51 households and represented a total community population of 316 persons . The meaP < 0.001). Other significant factors included age, with persons aged 25 years or younger having a greater risk of bat exposure , and households reporting pets or livestock bitten by bats . Furthermore, households with more than five family members  were at higher risk of bat exposure. Contrastingly, persons who reported living at their house for less than 1 year were at a lower risk for bat exposure compared with other respondents .Among the surveyed populations, several risk factors for exposure to bats were identified . The mos−1). From the IFA test, RABV RNP IgM and IgG antibodies were detected from 4 of 63 samples , 11% (7 of 63) showed an rVNA titer (range = 0.1–2.8 IU mL samples . The rVN samples . Only onP = 0.01)  . Seropos = 0.01) .−1).−1). A later study among fox trappers in Alaska reported rVNA among 12% (3 of 26) of individuals.−1), perhaps associated with a 47-year history of trapping and skinning foxes  and a cumulative harvest of over 3,000 foxes. During a human rabies outbreak investigation in the Department of Amazonas in Perú in 1990, 17% (8 of 48) of persons in two affected communities were seropositive for rVNA, one of whom later died.−1 (range = 0.14–0.66 IU mL−1), whereas the person who died had a titer of 7.6 IU mL−1 at the time of sampling. Because the study by Lopez and others−1, and given that six of seven rVNA titers were greater than 0.2 IU mL−1, these data do not suggest a high potential for nonspecific inhibition. The single respondent with an rVNA titer below 0.2 IU mL−1 (and RNP IgG titer of 1:8) in this study also reported a history of vaccination  of sera from raccoon hunters in Florida, although at low titers . Both RABV antibodies to the glycoprotein and RNP have a proven role in the immune response after vaccination.The presence of rVNA in unvaccinated subjects implies prior viral exposure but not necessarily viral replication, which can be shown by the induction of rVNA responses to even a single dose of inactivated rabies vaccine.in vivo, thus facilitating entry into the CNS.Innate immunity is typically another important component in combating viral infections. Prior studies have suggested that street RABVs tend to evade induction of the host innate immune response and particularly, interferon and inflammatory pathways.Rather than invoking peripheral viral replication as a requisite to the induction of rabies-specific serum antibody, one could also consider a dirty bite hypothesis. Little is known about (1) the population of RABV particles transmitted in the saliva during an animal bite and (2) what other substances or organisms may also be present. It is unrealistic to assume that homogenous populations of completely intact RABV virions are passed in the saliva, particularly given reports of defective interference (DI) particles.Prior vaccination history could confound the interpretation of the serological data in this study. Human rabies cell culture and nerve tissue vaccines are inactivated and do not replicate in recipients,70Prior reports of human rabies outbreaks in the Amazon, including 11 cases in the Department of Loreto in Perú in 1995, the results of this study, and nearby recent vampire bat-associated outbreaks suggest a high rabies risk in the Peruvian Amazon .12,15 VaThrough evidence presented in this study and one earlier report,Lyssavirus genotypes , whereas there are now 12 recognized species within the genus, 11 of which are presumed to have bats as the primary reservoir host.In closing, it is relevant to recognize that the number of newly discovered lyssaviruses has increased significantly in recent decades. Pre-1980, traditional nomenclature recognized just four


Ceriporiopsis subvermispora. Concentrations of glucose, ammonium tartrate and Polysorbate 80 were varied to establish the optimal composition of liquid media (OLM), where the highest experimentally obtained activities and productivities were 41 U L−1 and 16 U L−1 day−1 for laccase (Lac), and 193 U L−1 and 80 U L−1 day−1 for manganese peroxidase (MnP). Considering culture growth in OLM on various types of immobilization support, the best results were obtained with 1 cm beech wood cubes (BWCM). Enzyme activities in culture filtrate were 152 U L−1 for Lac and 58 U L−1 for MnP, since the chemical composition of this immobilization material induced higher Lac activity. Lower enzyme activities were obtained with polyurethane foam. Culture filtrates of OLM and BWCM were applied for dye decolorization. Remazol Brilliant Blue R (RBBR) was decolorized faster and more efficiently than Copper(II)phthalocyanine (CuP) with BWCM (80% and 60%), since Lac played a crucial role. Decolorization of CuP was initially faster than that of RBBR, due to higher MnP activities in OLM. The extent of decolorization after 14 h was 60% for both dyes.Response surface methodology  was employed to simultaneously optimize enzyme production and productivities of two ligninolytic enzymes produced by  C. subvermispora is a well-known fungus in the bio-pulping and paper industry because it is a selective lignin degrader )max and ([EMnP])max are the highest values of the experimentally obtained maximal Lac and MnP activities ([ELac] and [EMnP] in U L−1) for all settings presented in w is the weighting factor , (d[ELac]/dt)max and (d[EMnP]/dt)max are the highest values of experimentally obtained maximal Lac and MnP production rate (d[ELac]/dt and d[EMnP]/dt in U L−1 day−1) for all settings presented in OFMnP could not be optimized due to the absence of local maxima within reasonable substrate concentration values (the combined measurement and estimation error was too large). Nonetheless, a new Objective Function (OFLac/MnP) for batch experiments was defined as:where ()/productivity (d[ELac]/dt) at the specific glucose (x1) and ammonium tartrate (x2) concentrations at the fixed Polysorbate 80 level , meaning that at lower concentrations both medium components promote enzyme production, whereas an additional increase in concentrations results in an inhibitory effect. The research of C. subvermispora in biotechnological applications revealed the obtained Lac activities around 75 U L−1 and MnP activities around 40 U L−1 after 5 and 14 days of incubation, respectively, in a MPG  medium at 25 °C  on x3, but x3 could have been approximated by linearization. Another feature of interest lies in the fact that the influences of carbon source, nitrogen source, and surfactant seem to be independent, as the interdependence terms  in x1x2 (+)) being the largest of them. The dependences on glucose (x1) and ammonium tartrate (x2) in OFLac) as a function of glucose (x1) and ammonium tartrate (x2) concentrations in y2, d[ELac]/dt). Lac productivity is thus noticeably affected by the inhibitory effect of glucose (x1x1 (−)) and the positive combined glucose/ammonium tartrate effect (x1x2 (+)) (x3) principally affects productivity, as well as activity; the productivity both in the linear (−) and quadratic (+) terms. The overall effect of the surfactant on productivity is the same as that which it asserts on activity, namely negative. Other effects had negligible influence.The maxima in Lac activity/productivity (x2 (+)) . The latw. Parameters  were estimated by nonlinear regression, namely the Levenberg–Marquardt algorithm (10−5 tolerance).The optimization of the quadratic model utilized ensued, where the model parameters were estimated by linear regression. The objective function, constituted from enzyme (Lac and MnP) activity and production rate, was maximized for variable portions of either, OFLac) Lac activity and productivity, and (OFLac/MnP) Lac and MnP activities, which were maximized simultaneously, with variable contributions of Lac activity (w) to the objective function. In the first case one may observe that although the overall objective function OFLac assumes its greatest value (1.17) at w = 0 due to high Lac productivity, Lac activity is greatly decreased . As the contribution to Lac activity in OFLac, w, increases, Lac activity  first abruptly and then gradually increases towards 1.14, while concurrently Lac productivity  exhibits a rather small decrease, from 1.17 to 1.00. This would imply that, with the exception of very rare cases when the maximal productivity would be attained regardless of the relatively low activity , continuous systems should preferably be operated in conditions of the highest activity and only slightly decreased productivity , or at least in this vicinity. Of course, another aspect to consider is the concentration of medium components at which distinct levels of activity or productivity may be achieved.OFLac/MnP) reveals that it is difficult to obtain high levels of both Lac and MnP, respectively, since for high MnP concentrations (w→0), Lac activity decreases basically to zero. On the other hand, upon obtaining high Lac concentrations , MnP activity may be maintained at an intermediate level .Simultaneously optimizing Lac and MnP activities (OFLac) and Lac and MnP activities (OFLac/MnP) are presented in Examples of initial glucose, ammonium tartrate, and Polysorbate 80 concentrations for the optimal Lac activity and productivity max at CP = 0 g L−1 arise because of the broadly examined range of factors  and Q2 (the latter indicates predicted variability) coefficients, which were 0.875 and 0.872 (R2), and 0.608 and 0.623 (Q2) for Lac activity and productivity, correspondingly. A poorer predictability of the model for MnP activity and productivity may be observed through lower values of R2 and Q2 parameters, which were 0.686 and 0.795 (R2), and 0.339 and 0.468 (Q2) for MnP activity and productivity, correspondingly. The objective function (used in the model), using which we aimed to optimize the production, is indeed extremely useful, but in point of fact implies good predictability of the model . The reported coefficients demonstrate this for Lac activity and productivity, but not for the case of MnP.According to the model there should preferably not be any surfactant in the medium. The implication of the fact that the highest Lac activity and productivity are predicted by the model in the absence of Polysorbate 80 is on the one hand favourable, thus avoiding an additional component in the medium and directly decreases cost; furthermore, this could decrease downstream processing and potential contamination of crude filtrates as well. Nonetheless, verification experiments showed a discrepancy between model and experimental data, implying that the range of Polysorbate 80 concentrations examined may have been too broad. Consequently, a series of experiments was undertaken varying only the surfactant concentration in the region below the lowest concentration of 0.3 g L w = 0.5 , clearlyC. subvermispora cultivation experiments on various immobilization supports using the optimized liquid medium (OLM). Beech wood cubes (BWCM) and pine wood cubes (PWCM) containing natural lignin as inducer and inert polyurethane foam cubes (PUFCM) were used to immobilize the culture growth and to control the immobilization effect in comparison to the combined effect of the other supports that act as carriers as well as inducers. The composition of the liquid media was prepared according to the optimum settings for OFLac/MnP at w = 0.5 obtained from the response surface methodology experiment. In addition, the OLM was used as a reference medium with free mycelium. The results are presented in −1 for MnP and 19 U L−1 for Lac. It is evident that the use of wood supports affected the enzyme activities. Beech wood and pine wood induced higher Lac activities. Maximal values using BWCM and PWCM, achieved after eight days of cultivation, were 150 U L−1 and 135 U L−1, respectively. In contrast, MnP activities using wood supports were lower than the results with only liquid medium OLM, namely 60 U L−1 for BWCM and 20 U L−1 for PWCM. Production of enzymes was the lowest under PUFCM cultivation conditions: MnP activity was only 40 U L−1, while Lac activity was close to zero (2 U L−1). In the literature it was reported that inert materials such as PUF affect fungal growth as an immobilization surface, but alone cannot increase enzyme synthesis [The second phase of the study continued with ynthesis ,20. In cynthesis . Hence, ynthesis . Presentynthesis .C. subvermispora could be used in environmental applications, in the paper industry and also for the decolourization of synthetic dyes [Dichomitus squalens [et al. [D. squalens [C. subvermispora ligninolytic enzyme system show the potential use of this fungus for environmental protection and bioremediation of pollutants. However, in the future during the enzymatic degradation studies, more attention should be paid to the analysis of the products appearing during enzyme degradation.Further, the mixture of ligninolytic enzymes produced by the white rot fungus tic dyes ,4,15,17.squalens . A simil [et al. . The decsqualens . The extCeriporiopsis subvermispora CBS 347.63 was obtained from the CBS culture collection . The strain was maintained on 5% malt extract agar (MEA) at 4 °C as described previously [The fungus strain eviously .v/v) of the mycelial suspension. Glucose, ammonium tartrate and Polysorbate 80 concentrations in the basal culture medium were varied as shown in The mycelial suspension was prepared as previously described by Babič and Pavko . The shaLaccase (Lac) activity was measured by monitoring the oxidation of a 5 mM solution of 2,2′-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS) as the increase in absorbance at 420 nm , and man−1 and 10.0 g L−1 for glucose, 0.1 g L−1 and 2.0 g L−1 for ammonium tartrate, and 0.1 g L−1 and 1.0 g L−1 for Polysorbate 80, the real values being presented in To optimize the medium composition with regard to Lac and MnP activity and their productivity, surface response methodology for enzyme activity and productivity was utilized. Central composite design (CCD)  was used applying full factorial, the inscribed type of design, and no limitations of the maximal block size. Although CCD is a really useful approach for the optimization of enzyme production processes it cannot always be successfully applied using fungi; to overcome this possible problem we repeated in triplicate the experiment of the CCD. Modelling, sensitivity analysis and optimization was performed with Matlab R2011a software. The coded values in the central composite design matrix were transformed into real values using minimal and maximal concentration values of 1.0 g LA complete description of the process behavior requires a quadratic model :yi is either the maximal Lac activity (i = 1) or its maximal production rate (i = 2) and either the maximal MnP activity (i = 3) or its maximal production rate (i = 4), ai0, aij, ai12, ai13, ai23 and aijj are respectively, the constant term (ai0), linear (aij), mixed  and quadratic (aijj) coefficients for a complete description of the behavior of either the maximal Lac activity (i = 1) or its maximal production rate (i = 2) and either the maximal MnP activity (i = 3) or its maximal production rate (i = 4). The coefficients aij, ai12, ai13, ai23 and aijj correspond to independent variables; specifically, the initial glucose , ammonium tartrate  and Polysorbate 80  concentrations. Both the independent (yi) and dependent (xj) variables were normalized, that is divided by their maximal values, so that they would be within the range of 0–1.where 3 pine (PWCM), beech (BWCM) and polyurethane (PUFCM) cubes for 14 days. The OLM contained 10 g L−1 glucose, 1 g L−1 ammonium tartrate and 0.05 g L−1 Polysorbate 80 in the basal culture medium. In order to prepare the solid support for fungal colonization, PUF cubes were washed three times with hot distilled water to remove all foreign matter and air dried. The cubes were autoclaved in 150 mL of OLM in 250 mL Erlenmeyer flasks. After autoclaving, 50 mL of the medium was removed and the rest of it was inoculated with 5% (v/v) of the mycelium suspension. Aliquots of the culture liquid were collected for determination of extracellular Lac and MnP activities.Immobilized cultures were grown in optimized liquid medium (OLM) on 1 cmmax = 592 nm, and Copper(II)phthalo-cyanine , λmax = 694 nm. Both dyes were purchased from Sigma (USA).Dye decolourization was calculated by comparing the absorbance, measured at the maximum absorbance wavelength for each compound, during the decolorization treatment. The dyes used in this study were Remazol Brilliant Blue R , λin vitro dye decolorization by the crude culture liquid, culture filtrates obtained from cultivation cultures of C. subvermispora in OLM and in BWCM were used. The reaction mixtures consisted of 100 mM Na-tartrate buffer, pH 4.5, 50 mg L−1 dye and 100 μL of crude culture liquid in a final volume of 1 mL. Dye decolorization was measured continuously using a Perkin Elmer spectrophotometer, type Lambda 25 (USA) [For 25 (USA) .C. subvermispora in submerged cultures and their dependence on medium composition can be well described by response surface methodology (RSM) over a broad range of concentrations. For better accuracy, some repetition of the RSM procedure should be performed.In fungal cultures medium optimization should play an important role in the upstream process due to the consequent lowering of process costs. The activity and productivity of ligninolytic enzymes from A two-step strategy, that is, liquid media optimization first and then selection of an appropriate immobilization support to induce the enzyme activities, was successful in increasing Lac activities. It was shown that the methodology of liquid medium optimization can be readily utilized for a wide range of compositions for predicting both ligninolytic enzyme activity and production rate simultaneously and relatively accurately. Therefore, a major advantage of the proposed approach is that it may be usefully applied for systems in which as high activity as possible is desired to avoid downstream separation and purification costs, or for those processes in which maximizing the enzyme production rate is imperative, namely in continuous processes. Balancing these two contributions allows for intermediate regimes in which both the activity and production rate play an important role. Despite the fact that the methodology was applied for a specific medium  its applicability may be extended to other components as well.The natural tendency of filamentous fungi to adhere to surfaces was successfully applied to increase Lac activity in the second step of the study. Naturally an immobilization material with appropriate chemical composition which affects fungal activities and induces enzyme activities should be used for this purpose. This principle, which was used here for enzyme production in batch stationary cultures, is already well recognized for enzyme synthesis in various types of bioreactors with immobilized biomass, for example in packed bed bioreactors, rotating disc bioreactors and biofilm reactors.C. subvermispora can be successfully used for decolorization of synthetic dyes. In addition, they also have good potential for use in other bioremediation and environmental protection applications, as well as in lignin breakdown.It was shown that ligninolytic enzymes, namely manganese peroxidase (MnP) and laccase (Lac), produced by the white rot fungus


Considering the high rate of neuropsychiatric symptoms found in ALS patients, this paper examines whether caregiver burden is associated with behavioral changes 140 caregivers of patients with ALS participated in a postal survey investigating patients’ neuropsychiatric symptoms (Cambridge Behaviour Inventory Revised CBI-R), motor function , caregiver burden (Zarit Burden Interview), and caregiver mood . Seventy four percent of them were caregivers of patients with limb onset and 25.7% were caregivers of patients with bulbar onset.Moderate to severe behavioral changes were reported in 10-40% of patients with ALS. The levels of depression, anxiety and stress in the caregivers reached 20%. Burden was high in 48% of the caregivers. The strongest predictor of high caregiver burden was ALS patients’ abnormal behavior rather than physical disability, with an odds ratio of 1.4, followed by caregivers’ stress.Our study has identified that behavioral changes  and caregiver stress have greater impact on caregiver burden than level and pattern of physical disability. Over the past decade there has been increasing awareness of behavioral changes in patients with amyotrophic lateral sclerosis (ALS), which mirror those found in frontotemporal dementia (FTD),2. Caregover and above the physical disability of patients with ALS.Caregiver burden is a complex construct involving the caregiver’s feelings about the emotional/physical health, social life and financial status that they suffer as a result of caring for their relatives. CaregivThe aims of this study were: 1) to assess the impact of ALS patients’ behavior and motor disability on caregiver burden, 2) to assess the frequency of depression, anxiety and stress of the caregivers and possible impact on caregiver burden.Caregivers of patients with ALS were contacted through the Motor Neurone Disease Association Australia, who kept their identities anonymous. Of 354 ALS patients registered as members of the association in NSW, 114 of their caregivers (32%) accepted an invitation to participate in a postal survey evaluating neuropsychiatric symptoms and motor function in ALS patients and burden of the caregiver. Additionally, 56 caregivers from other Australian states accepted the invitation through the local MND associations. Of the final one hundred and seventy caregivers who agreed to participate, 30 (17.6%) of them did not continue the study due to patients’ health issues or personal problems. Finally, data from the caregivers of 140 ALS patients was collected ; 104 (74.3%) of them were caregivers of patients with limb onset, whereas 36 (25.7%) were caregivers of patients with bulbar onset. Ethics approval was obtained from the Human Research Ethics Committee of South Eastern Sydney/Illawarra Area Health Service. Signed consent was obtained from each participant.A self- administered version of the Amyotrophic Lateral Sclerosis Functional Rating Scale Revised (ALSFRS-R) was completed by ALS patients and/or their caregivers. It contains 4 sub scores: bulbar, fine, gross and respiratory function,20. A toChanges in ALS patients’ behaviour were measured with the Cambridge Behavioural Inventory Revised (CBI-R), a caregiver-based measure of neuropsychiatric symptoms and daily activities, which correlates highly with the Neuropsychiatric Inventory (NPI).It contains 10 domains: memory, everyday skills, self care, mood, beliefs, sleep, eating habits , abnormal behavior (e.g. disinhibition and impulsivity), stereotypic and/or motor behaviours and lack of motivation (apathy). The behCaregivers completed the Depression, Anxiety, Stress scale (DASS 21). The folThe Zarit Burden Interview (ZBI) was used to measure caregiver burden by evaluating disease impact on caregivers’ quality of life, psychological suffering and impact on social and family relationships. Caregivers completed a self-administered short version of 12 items, which has shown to have comparable psychometric properties to the full version. The ZBI has a 0–4 point score with a maximum score of 48. A score ≥17 indicates a high burden,24,25.t-tests or Mann Whitney U tests were used where appropriate.SPSS Statistics 17.0 was used for analysis of results. To examine clinical features reported by caregivers, independent X2 tests with Yates’ correction for continuity and Fisher’s exact test. A logistic regression was used to analyse the impact of caregiver’s mood, patients’ motor function, and behavioral changes on caregiver burden.Comparison of behavioral changes across motor onset groups was analysed using Pearson’s The caregiver group (n = 140) was predominantly composed by females (69.3%). The mean age was 60.8 ± 12 years. The majority of caregivers were the patient’s spouses (90%), followed by patient’s children (7%); other relatives or friends were occasionally caregivers (3%).ALS patients’ mean age was 63.5 ± 9 years, with a median of disease duration of 3 years (interquartile range 2–5). The mean ALSFRS-R total score was 30.4 (S.D. 9.7). Distribution of patients by motor onset showed a predominance of limb (73.6%) over bulbar 26.4%) onset. The age range and pattern of disease onset (limb vs. bulbar) mirrors closely that found in large cohorts.4% onset.p > 0.1). As expected, patients with bulbar onset had lower scores on bulbar function than patients with limb onset . Likewise, patients with limb onset had lower scores in fine motor  and gross motor function . No significant difference was found for respiratory function across groups .Comparison of ALSFRS-R total scores between patients with limb and bulbar onset showed no significant difference .Comparison of CBI-R scores across patients with limb and bulbar onset showed no significant association between motor onset and behavioral changes .The percentage of caregivers with anxiety and stress was lower. Overall, 16% had moderate to extremely severe anxiety and 21% equivalent levels of stress. Comparison of the results between caregivers of patients with limb and bulbar onset showed no significant association between motor onset and presence of depression, anxiety and stress (p > 0.1).The Zarit Burden Interview showed that high levels of burden (score ≥ 17) were reported by 48% of the caregivers. There was no significant difference when comparing caregivers’ burden of patients with limb and bulbar onset  and behavioral changes . Caregivers’ variables considered were depression, anxiety and stress .Logistic regression was performed to assess the impact of different factors on the likelihood of caregivers in presenting with high burden. The patient related variables considered were motor function , and explained between 45% (Cox and Snell R Square) and 60% (Nagelkerke R squared) of the variance in caregiver burden scores. Of the caregivers predicted to have high levels of burden, the model correctly classified 83.1%. As shown in TableThe full model containing all predictors was statistically significant,  and caregivers’ level of stress have greater impact on caregiver burden than any other patient variables, including level and pattern of physical disability.Behavioral symptoms, as measured by the Frontal Systems Behavioral Scale (FrSBe), have previously been reported to be associated to higher caregiver burden,,18 althoA number of studies have shown that caregivers of patients with ALS report high levels of burden, anxiety and depression in association with the patient’s loss of physical functions,15. In oThe levels of anxiety and stress (around 20%) in our caregivers were lower than reported previously,34. AgaiA longitudinal study identified that, initially, caregiver distress was best predicted by the psychosocial impact of ALS symptoms and emotional lability. Later, caregiver distress related to satisfaction with social relationships and social support rather tThe fact that ALS patients presented similar behavioural symptoms to patients with FTD has been reinforced by a recent genetic discovery. The presence of a hexanucleotide repeats in the intron of C90RF72 in almost a quarter of cases with familial ALS and 12% of the cases with familial FTD,36 suppoIn summary, behavioral changes appear to lead to considerable caregiver burden, highlighting the importance to screen for neuropsychiatric symptoms in MND Clinics. Importantly, these symptoms could be modified by psychological or pharmaceutical therapies in an attempt to reduce caregiver burden. Caregivers’ emotional state also affects burden, with stress (but not depression or anxiety) playing major roles. Professionals involved with the care of ALS patients should be aware of these specific caregivers’ needs, which once addressed, are also likely to benefit the wellbeing of the patients.Dr Lillo is supported by a CONICYT scholarship (Government of Chile) and Faculty of Medicine, University of Chile.Dementia and Geriatric Cognitive Disorders and is a recipient of a National Health and Medical Research Council of Australia Early Career Fellowship and an Motor Neurone Disease Research Institute of Australia Grant.Dr. Mioshi serves on the editorial board of Prof Hodges is funded by the Australian Research Council Federation Fellowship, FF0776229.Aphasiology, Cognitive Neuropsychiatry, and Cognitive Neuropsychology; receives royalties from publication of Cognitive Assessment for Clinicians  and Frontotemporal Dementia Syndromes ; and receives fellowship support from the Australian Research Council Federation.Prof. Hodges serves on editorial boards of In order to give appropriate credit to each author of a paper, the individual contributions of authors to the manuscript should be specified in this section. PL: 1) have made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data; 2) have been involved in drafting the manuscript and revising it critically for important intellectual content; and 3) have given final approval of the version to be published. EM: 1) have made substantial contributions to conception and design, analysis and interpretation of data; 2) have been involved in revising it critically for important intellectual content; and 3) have given final approval of the version to be published. JRH: 1) have made substantial contributions to conception and design, analysis and interpretation of data; 2) have been involved in revising it critically for important intellectual content; and 3) have given final approval of the version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/12/156/prepub


Eurytemora affinis on predation risk, foraging, growth, metabolic activity and antioxidant capacity. Using pigmented and unpigmented specimens, we compared (1) predation and selectivity by the invertebrate predator Cercopagis pengoi, (2) feeding activity of the copepods measured as grazing rate in experiments and gut fluorescence in situ, (3) metabolic activity assayed as RNA:DNA ratio in both experimental and field-collected copepods, (4) reproductive output estimated as egg ratio in the population, and (5) total antioxidant capacity. Moreover, mitochondrial DNA (mtDNA) COI gene variation was analysed. The pigmented individuals were at higher predation risk as evidenced by significantly higher predation rate by C. pengoi on pigmented individuals and positive selection by the predator fed pigmented and unpigmented copepods in a mixture. However, the antioxidant capacity, RNA:DNA and egg ratio values were significantly higher in the pigmented copepods, whereas neither feeding rate nor gut fluorescence differed between the pigmented and unpigmented copepods. The phenotypic variation in pigmentation was not associated with any specific mtDNA genotype. Together, these results support the metabolic stimulation hypothesis to explain variation in E. affinis pigmentation, which translates into beneficial increase in growth via enhanced metabolism and antioxidant protective capacity, together with disadvantageous increase in predation risk. We also suggest an alternative mechanism for the metabolic stimulation via elevated antioxidant levels as a primary means of increasing metabolism without the increase in heat absorbance. The observed trade-offs are relevant to evolutionary mechanisms underlying plasticity and adaptation and have the capacity to modify strength of complex trophic interactions.Intraspecific variation in body pigmentation is an ecologically and evolutionary important trait; however, the pigmentation related trade-offs in marine zooplankton are poorly understood. We tested the effects of intrapopulation phenotypic variation in the pigmentation of the copepod  Variation in pigmentation among individuals, populations and species has been associated with various adaptations to predation, ultraviolet radiation (UVR) and diet, but the ultimate mechanisms and trade-offs regulating the observed patterns in the field are still unclear. Studies on pigment variation and its adaptive function in zooplankton have been focused on carotenoids in calanoid copepods Cercopagis pengoi and Bythotrephes longimanus have been implicated in the reduction of zooplankton diversity and abundance in the Laurentian Great Lakes In addition to the photoprotective function of pigments, a metabolic stimulation hypothesis has been advocated to explain the adaptive value of pigmentation by providing warmth through absorption of solar radiation Many zooplankton pigments are important quenching agents and antioxidants that neutralize reactive oxygen species; they also have other biological functions, such as regulatory effects on cell signaling Daphnia, both genetic and environmental effects were found to influence pigmentation As far as we know, no studies have addressed pigmentation-mediated trade-offs between fitness parameters and vulnerability to predation by invertebrate predators in marine zooplankton. Moreover, whereas genetic basis and developmental mechanisms generating the diversity of pigmentation and color patterns are well appreciated in vertebrates Eurytemora affinis Poppe, raising questions about the factors that influence pigmentation in this species. This copepod is one of the few dominant mesozooplankton species in the northern Baltic and a preferred prey for the dominant zooplanktivores C. pengoiE. affinisEurytemora, differences in pigmentation between the cryptic species and association between pigmentation and certain mtDNA haplotypes in E. affinis are possible.In our fieldwork in different areas of the Baltic Sea, we were puzzled by variability in pigmentation in the calanoid copepod Eurytemora affinis and a morphologically similar species E. carolleeae recently reported from the Gulf of Finland E. affinis in our collections was confirmed, we examined the intrapopulation variation of the mitochondrial cytochrome oxidase c subunit 1 (COI) gene in relation to pigmentation, and tested effects of this pigmentation on the predation and selectivity by C. pengoi and on the variations in antioxidant capacity, feeding, metabolic activity and reproduction of the copepods.In this study, we investigated whether the observed variation in pigmentation is related to interspecific variation between The sampling was conducted within national Finnish monitoring in the Gulf of Finland and no specific permissions were required for the sampling locations of this study. Also, we did not require ethical approval to conduct this study as we did not handle or collect animals considered in any animal welfare regulations and no endangered or protected species were involved in the samplings or the experiments.E. affinis. There was a clear distinction between pigmented and unpigmented individuals, with the latter appearing opaque or transparent in transmitted light, without visible dark coloration of any specific area, apart from the eye, ovaries and egg sacks . By contrast, the pigmented individuals had dark brown coloration on either one side of the body or on the both sides; the pigmentation was particularly strong on antennae, ventro-lateral part of cephalothorax and furcal rami  in August 2011 in the eastern and central Gulf of Finland, the northern Baltic Sea  equipped with a cod end. The samples were used to obtain animals for the experiments and to preserve them for different measurements. For predation and grazing experiments, E. affinis individuals with contrasting pigmentation were sorted with a pipette under a dissecting microscope and kept in groups of 10 individuals in 0.2 µm-filtered seawater (FSW) in 0.1 L vials. For the predation experiments, C. pengoi were carefully picked under a dissecting microscope with forceps and wide-mouth pipettes and transferred individually to FSW in 0.1 L vials; only actively swimming individuals were used in the experiments. All specimens were kept at 16°C approximating the temperature in the mixing layer until the experiments commenced. The remaining zooplankton collections were treated as follows. The water was removed as much as possible with a filter paper and the samples were preserved by: (1) freezing at −80°C for measurements of gut fluorescence and antioxidant capacity; and (2) adding RNAlater 10∶1 (v/v) for genetic analysis and measurements of nucleic acid concentrations; these samples were also used for microscopic analysis to estimate sex- and age-related difference in pigmentation and to calculate egg ratio.The experimental animals were collected and the experiments were conducted on board R/V ‘ltic Sea . Eurytemlater  and mature (CVI) individuals using the same counts.To test whether the proportion of pigmented individuals differed between males and females (CIV-VI), 25 individuals of each sex per sample were counted in the 6 field samples preserved with RNACercopagis pengoi (instars II and III) as predators and older copepodites  of Eurytemora affinis as a prey. In Experiment 1, predation rates (PR) of C. pengoi offered either pigmented or unpigmented copepods were determined, whereas in Experiment 2, prey selection using a mixed (1∶1) assemblage of pigmented and unpigmented copepods was determined. The experiments consisted of 6 replicate incubations for each treatment and were conducted in either 0.1 (Experiment 1) or 0.2 (Experiment 2) L vials under constant illumination with green light at 15°C and lasted ca. 20–22 h. Incubations were started by adding the known number of prey (10 in Experiment 1or 20 in Experiment 2 to keep the same prey concentration in both experiments) to a single C. pengoi. In each experiment, there were two predator-free control incubations with the same number of prey to correct for prey recovery . Recovered prey were counted directly upon the experiment termination and the pigmentation status of each copepod was noted. Body length  of every C. pengoi and prosome length (PL) in a subsample of 20–25 individuals for each E. affinis group were measured under a dissecting microscope. PR was calculated as the difference between the initial and final numbers of prey and scaled to 24 h.We conducted two predation experiments using parthenogenic females of Rhodomonas salina  grown on f/2 medium at 17°C and 8‰ salinity in artificial seawater . To minimize algal growth during the experiment and increase accuracy of the grazing rate (GR) estimates, the algae were pre-treated with γ-radiation to prevent cell division −1; 6 replicates per treatment) were incubated during 22–24 h in 0.2 L of FSW with 600 µgC L−1 of food; light regime was the same as in the predation experiments. This food concentration is above the saturation level for E. affinis–1), 15 mL of the media were preserved with acid Lugol’s solution and analyzed using a laser particle counter Spectrex PC-2000 (Spectrex Corp.). The individual GR was determined as a difference between the initial and final algal concentrations in each vial and scaled to 24 h and individual copepod.Experiment 3 was conducted to test for differences in grazing, metabolic activity and antioxidant capacity between the copepods with contrasting pigmentation. As food, we used later and stored at 4°C Upon termination of the experiment, the copepods were examined with a dissecting microscope, and dead individuals were noted. In all incubations, survivorship was ≥95%, with no significant difference between the pigmented and unpigmented copepods . Live copepods recovered from each vial were split in two groups: (1) 5 individuals designated for RNA:DNA ratio assay were transferred to Eppendorf tubes containing 100 µL of RNAEurytemora affinis  was analyzed fluorometrically. After thawing on ice, the copepods were quickly sorted in groups of 5 individuals per pigmentation type; 2 replicate samples per station, 5 stations in total (–1 of chlorophyll-a (Chl-a) and 0.08 µg L–1 of phaeophytin-a. Fluorescence was measured before and after acidification with HCl and GPC, and Chl-a was calculated as ng Chl-a eq ind –1Using the frozen zooplankton samples collected in the field, gut pigment concentration (GPC) in in total . Gut piglater  using microplate fluorometric high-range RiboGreen  assay Eurytemora affinis of similar size −1, 10 measurements well−1). The DNA:RNA standard curve slope ratio was 1.76.This ratio was used as a proxy for overall metabolic activity in the copepods −1) were homogenized in Tris buffer with ∼50 µg of glass beads (<100 µm) for 2 min at 4°C using FastPrep, centrifuged at 15000×g for 15 min at 4°C and then 10 and 30 µL of the supernatant were taken for measurements of ORAC and protein content, respectively. Protein concentration (mg mL−1) was measured using the bicinchoninic acid assay  according to the manufacturer’s instructions. The ORAC values were expressed in Trolox-equivalents, µg mg protein−1. All measurements were conducted in duplicates that were averaged for statistical analysis.These measurements were done using copepod samples obtained in the Experiment 3 (6 replicates) and frozen samples of field-collected copepods that were also used for the GPC  and GR (Experiment 3) between the pigmentation types were evaluated by an unpaired t-test. To test for differences in prey preference, Manly’s α index Pearson’s product-moment and correlation coefficients were calculated for all pairs of measured physiological and biochemical variables. To further evaluate effect of pigmentation on RNA:DNA ratio and ORAC, 2-way ANOVA was used with pigmentation status (pigmented or unpigmented) and animal origin (field or experiment) as categorical predictors. To compare egg ratio and GPC between the pigmentation types, paired t-test was used with pairing by station. Finally, to establish a relationship between the metabolic status and antioxidant capacity and to evaluate effect of pigmentation on this relationship, we used a general linear model (GLM) with the pigmentation status and the animal origin as the categorical variables and ORAC as continuous variables and RNA:DNA ratio as the dependent variable. The data for the correlation and regression analyses were Box-Cox transformed to improve distribution of the residuals. If not specified otherwise, data are presented as means and standard deviations (SD). Statistica version 8.0  was used for statistical analyses; the differences were considered significant at p<0.05.Eurytemora affinis from coastal areas of the Gulf of Finland , but also from other parts of the Baltic Sea ; see E. carolleeae of North American origin . Therefore, all species in our collections were assumed to belong to the Baltic lineage of E. affinis.All 50 sequences had high identity (98–100%) to COI sequences reported for the Baltic lineage of The aligned COI sequence dataset contained 16 unique haplotypes with 20 polymorphic sites sampled across 571–574 bp. In both pigmentation groups, many closely related haplotypes co-existed, with high haplotype diversity and low nucleotide diversity . Four haNeither sex- (Wilcoxon matched-pairs signed-ranks test: p>0.8) nor age-related (p>0.5) differences in pigmentation were significant. The proportion of the pigmented individuals varied from 25% to 42% among the stations.10 = 3.503, p<0.006; 5 = 5.000, p<0.003; 10 = 2.72, p<0.03; C. pengoi BL: t48 = 0.97, p>0.43) or the prey .In the single prey type experiment, the effect of pigmentation on PR was significant , whereas no significant differences in ORAC values between the animals used in the experiment and those in the field samples were observed . In no case the interaction term was significant .Pigmentation had significantly positive effects on egg ratio in the field-collected 1,19 = 4.98, p<0.04), with a slope being significantly higher in animals used in Experiment 3 compared to the field samples , whereas pigmentation did not affect the relationship .There were significant positive correlations between ORAC and growth indices, but not with food intake . FinallyEurytemora affinis, pigmentation carries both penalty in terms of the greater vulnerability to predation and fitness advantage in terms of enhanced growth, reproduction and metabolism. Three crucial results support this conclusion. First, higher predation rates and positive selectivity for the pigmented copepods were observed in the predation experiments with the visual predator Cercopagis pengoi. Second, growth-related variables (egg ratio and RNA:DNA ratio) were consistently higher in the pigmented copepods, whereas the food intake was similar between the pigmentation types. Consequently, the higher growth output in the pigmented individuals at a given feeding rate implies higher growth efficiency, the capacity to convert ingested energy into biomass. Third, the pigmented copepods had higher antioxidant capacity, suggesting that this phenotype is superior in handling excessive formation of free-radicals, which is often a primary cause or a downstream consequence of tissue damage due to various environmental stressors, including predation pressure In Eurytemora affinis, juvenile development is highly temperature dependent in the range of 7 to 18°C Our results that pigmented individuals have higher egg production, metabolic activity and antioxidant capacity lend support to the metabolic stimulation hypothesis We suggest an alternative mechanism for the metabolic stimulation that could involve elevated antioxidant levels as a primary means of increasing metabolism without temperature increase. As physiological functions of pigments include antioxidant protection, the observed higher total antioxidant capacity  in the pin situ growth conditions for copepods were suboptimal as indicated by the higher RNA:DNA ratio of the copepods incubated at ad libitum food levels in the grazing experiment compared to the field-collected copepods  and unpigmented (B) Eurytemora affinis.(PDF)Click here for additional data file.Figure S2Maximum Likelihood (ML) tree for Eurytemora affinis sequences reported from the Baltic Sea.(PDF)Click here for additional data file.


Caries, enamel hypoplasia, molar incisor hipomineralization, amylogenesis imperfecta, dentine dysplasia, hypophosphatasia and other dental disorders lead to tooth mineralization disturbances and structural abnormalities, decreasing masticatory organ functions. Dental disorders in sheep can lead to premature slaughter before they have attained final stage of their reproductive life and induce economic loss due to high flock replacement costs. Growth rate, health status and meat quality of sheep depends on tooth properties and quality determining in large extent efficiency of the masticatory apparatus and initial food break up. Considering lack of basic anatomical and physiological data on teeth properties in sheep, the aim of the study was to evaluate morphometric, densitometric and mechanical traits of deciduous mandibular incisor, canine and the second premolar obtained at the slaughter age of 5 months of life.The obtained results have shown the highest values of weight, total tooth volume, enamel volume and dentine volume in second premolar. Morphometric and mechanical parameters of incisors reached the highest values in first incisor and decreased gradually in second and third incisor, and in canine. Densitometric measurements have not revealed significant differences of the volumetric tooth mineral density in hard dental tissues between the investigated teeth.In conclusion, proposed methodological approach is noninvasive since the deciduous teeth undergo physiological replacement with permanent teeth. Deciduous teeth can be easy collected for analyses from large animal population and may reflect mineral status and metabolism resulting from postnatal growth and development of the whole flock. In individual cases, evaluation of properties of deciduous teeth may serve for breeding selection and further reproduction of sheep possessing favorable traits of teeth and better masticatory system functions. Bone, dentine, cementum and enamel are considered as four types of hard tissue. Teeth are the most durable structures of mammalian body . Except Dental disorders in sheep can lead to premature slaughter before they have attained final stage of their reproductive life and induce economic loss due to high flock replacement costs. It must be underlined that growth rate, health status and meat quality of sheep depends on tooth properties and quality determining in large extent efficiency of the masticatory apparatus and initial food break up. While incisor disorders reduce dramatically intake of forage, cheek tooth dysfunction has negative impact on food grinding ,4. Thus,Etiology of tooth disorders is multifactorial. Defects of the tooth structure may result from soil ingestion and following abrasion combined with chemical attack from acids present in the ingested soil ,8. BacteConsidering lack of basic physiological data on tooth properties in sheep, the aim of the study was to evaluate morphometric, densitometric and mechanical traits of deciduous mandibular incisors, canine and the second premolar - the analyzed teeth had completely developed all their anatomical structures by the slaughter age of 5 months of life.th February 2008).This experiment was conducted in accordance with national guidelines for animal experimentation and the “Guide for the Care and Use of Laboratory Animals”. The experimental procedures used in this study were approved by The Local Ethics Committee on Animal Experimentation of Medical University in Lublin, Poland , canine (fourth incisor – i4) and second premolar (p2) were isolated from the left part of the mandible. All isolated teeth were cleaned from soft tissues and cementum, weighed and their length was measured. Teeth samples were kept at room temperature in saline solution that was changed regularly until further analyses.This study was performed on 7 healthy male Polish Merino lambs born physiologically to ewes. Lambs and ewes were kept indoors in pens under standard rearing conditions and provided with drinking water Using quantitative computed tomography (QCT) technique and SOMATOM EMOTION SIEMENS apparatus  equipped with Somaris/5 VB10B software , the determination of mean volumetric tooth mineral density (MvTMD) and total tooth volume (Tvol) was performed. Mean vTMD was measured for whole sample, including all the anatomical structures and reflects mean density of all hard tissues in a single tooth. For Tvol and MvTMD determinations, the volume-of-interest was defined at the values between minimum and maximum density of the investigated samples at 0 and 3071 Hounsfield units (HU), respectively .Microtomography (μCT) technique and SkyScan 1174 apparatus  was used to determine total enamel volume (Evol), volumetric enamel mineral density (vEMD), total dentine volume (Dvol) and volumetric dentine mineral density (vDMD). Images for analysis were obtained at 50 kV and 800 μA with a 0.25 mm aluminum filter. For each tooth, 300 projection images were obtained with a rotation of 0.70°. An image pixel size corresponded to 21.72 μm. The samples were rotated over 180°. Reconstruction of the teeth was performed with the use of NRecon 1.6.1.5 software . The software allows three-dimensional reconstructions of the investigated sample from the stack of two-dimensional sections  were subjected to a three-point bending test and maximum elastic strength (Wy) and ultimate force (Wf) of the teeth were determined. The distance between tooth supports was set at 40% of tooth length, and the measuring head loaded the investigated samples on the concave (posterior) surface with a constant speed of 10 mm/min. The second premolar tooth (p2) was subjected to a compression test and ultimate force (Wf) was determined. Tooth sample was placed on the buccal  surface and the loading head compressed tooth on the lingual  surface with the speed of 10 mm/min [Mechanical properties of teeth were determined in Zwick/Roell Z010 apparatus . Incisors and canines . All data are presented as means ± SEM. The data were found to be normally distributed in accordance with Kolomogorov-Smirnov test. The differences of means between the investigated teeth were tested for statistical significance with the use of one-way ANOVA. Post hoc comparisons of the differences were performed using Duncan’s test. Differences showing 1 – i4) in the three-point bending test (P < 0.05). The highest value of ultimate force was obtained executing compression in second premolar when compared to the other teeth (P < 0.001). The three-point bending test of i1 – i4 revealed higher value of Wf in first incisor than in second and third incisors while the lowest value of this parameter was stated in canine. The differences of Wf in all these teeth were found to be significant (P < 0.05).Results of morphological, densitometric and mechanical properties of the investigated teeth in 5 month old sheep are shown in Table Standard veterinary examination of teeth in sheep would be an essential part of the clinical investigation procedures which enables age estimation or poor productive potential indication . Even th2 and i3 to the lowest value in i4 (canine). The differentiated weight of the teeth resulted from analogical differences of the length and total tooth volume of the incisors. Considering significant difference of weight, length and tooth volume, it is surprising that total enamel volume was comparable in i1 and i2 as well as in i3 and i4, even though i1 and i2 possess approximately 40 – 68% more enamel than i3 and i4. Gradual reduction of total dentine volume was observed between i1 – i2, i2 – i3 and i3 – i4, that reached 23%, 32% and 25%, respectively. However, densitometric analysis has shown similar values for all the evaluated deciduous teeth in this study when analyzing both MvTMD and vEMD. The only one difference obtained from densitometric evaluation of teeth in sheep has shown significantly higher vDMD in i1 when compared to i3 and i4. Thus, the performed densitometric measurements in both compartments have not shown considerable differences of volumetric tooth mineral density between several deciduous teeth, confirming similar course of mineralization processes of hard dental tissues in all the investigated teeth. Thus, it may be postulated that proper mineral metabolism and mineralization processes during first months of life in sheep lead to comparable mineral density threshold attainment in hard dental tissues of all teeth. The results of densitometric analysis in our study are in accordance to the other reports confirming significantly higher mineral density in enamel than in dentine. It was shown that enamel consists of 95% inorganic substance, 4% water and 1% organic substance, while in dentine all these constituents represent 70%, 20% and 10%, respectively [1 – i2, i2 – i3 and i3 – i4 and it reached 23%, 20% and 34%, respectively. Similar decrease of Wf was stated evaluating deciduous incisors and the differences between i1 – i2, i2 – i3 and i3 – i4 reached 24%, 25% and 21%, respectively. In case of the second premolar, its large crown and multi-root anatomical structure contributed to significantly higher values of tooth weight, volume (both in enamel and dentine) and ultimate force when compared to these parameters in incisors and canine. However, tooth length, MvTMD, vEMD and vDMD of p2 were similar when compared with the other investigated teeth. It should also be explained here that mechanical analysis of i1 – i4 and p2 was performed with two different methods such as three point bending and compressive tests and thus the assessment of Wy for p2 was not possible. Even though the current study was not performed on large animal population, the obtained values of morphological, densitometric and mechanical parameters may be deemed as reference data for deciduous teeth in sheep since the investigated teeth were completely developed and their growth was finished. In case of the other deciduous mandibular teeth that were not evaluated in this study, longer breeding procedure is required to finish their growth and development and receive meaningful data [This study presents basic morphological, densitometric and mechanical properties of completely developed deciduous teeth such as mandibular incisors, canine and the second premolar in sheep at the age of 5 months of life for the first time. The analyses performed have shown significant differences of weight of teeth between all incisors reaching the highest value for the first incisors and diminishing gradually in iectively -21. Analful data .β-hydroxy-β-methylbutyrate (HMB) in lambs significantly increased serum concentrations of bone-specific alkaline phosphatase (BAP) and osteocalcin (OC) – the bone tissue formation markers reflecting osteoblastic activity. Moreover, growth hormone (GH) and insulin-like growth factor-1 (IGF-1), the hormonal factors inducing improved bone tissue metabolism and mineralization, were significantly increased as the consequence of 21-day neonatal administration with HMB. All these effects were associated with permanent changes of bone morphology, mineral density and mechanical endurance. β-hydroxy-β-methylbutyrate administered in young lambs improved bone weight, length, volume, vBMD of the cortical bone as well as geometrical and mechanical properties of femur and lumbar spine [Precise evaluation of teeth properties in terms of morphology, microstructure and mechanical properties would be much more effective than simple clinical examination to provide data on whole body and calcified tissues metabolism during crucial periods of life. Considering very rapid growth rate during neonatal and postnatal development in sheep, morphometric , densitometric and mechanical evaluation of deciduous teeth would be informative in terms of physiological, nutritional, pharmacological, toxicological and environmental conditions taking place during first months of life. Experimental studies on growing sheep have shown that advantageous nutritional manipulations during early stages of neonatal development positively influence mineral metabolism and bone tissue properties. Oral administration with alpha-ketoglutarate (AKG) during the first two weeks of life induced improved morphological traits (weight and length), volumetric bone mineral density (vBMD) and mechanical properties (maximum elastic strength and ultimate strength) of ribs in 5 month old ram lambs . Similarar spine . Even thar spine .in vivo determination of skeletal system properties. Furthermore, in vivo evaluation of bones in sheep with microtomography and mechanical tests is not possible due to limitations resulting from ability to scan small samples in microtomographic devices, as well as consideration of ethical issues [An advantage resulting from morphometric, densitometric and mechanical dental evaluation in animals is the fact that the methodological approach is noninvasive since the deciduous teeth undergo physiological replacement with permanent teeth, taking place at several stages and does not require substantial intervention, sedation or general anesthesia, when compared to l issues ,28. Thesβ-hydroxy-β-methylbutyrate; BAP: Bone-specific alkaline phosphatase; OC: Osteocalcin; GH: Growth hormone; IGF-1: Insulin-like growth factor-1; BMP: Bone morphogenetic proteinsMIH: Molar incisor hypomineralization; EDJ: Enamel-dentin-junction; i1: First incisor; i2: Second incisor; i3: Third incisor; i4: Fourth incisor (canine); p2: Second premolar; QCT: Quantitative computed tomography; MvTMD: Volumetric tooth mineral density; Tvol: Total tooth volume; HU: Hounsfield units; μCT: Microtomography; Evol: Total enamel volume; vEMD: Volumetric enamel mineral density; vDMD: Volumetric dentine mineral density; Dvol: Total dentine volume; ROI: Region of interest; Wy: Maximum elastic strength; Wf: Ultimate force; AKG: Alpha-ketoglutarate; HMB: The authors declare that they have no competing interests.MRT was responsible for the concept of the study, supervised all stages of the study, participated in animal management, collection and interpretation of data, as well as writing the manuscript. AS was responsible for the concept of the study, collection and interpretation of data from mechanical analyses, statistical analysis and writing the manuscript. WK performed QCT analysis of teeth and interpretation of data and supervised all stages of the study. BT was responsible for references collection, interpretation of data and writing the manuscript. IŁ-S was responsible for teeth preparation, interpretation of data and writing the manuscript. JB was responsible for micro CT analysis of teeth, participated in interpretation of data and critical revision of the manuscript. MO was responsible for micro CT analysis of teeth and participated in interpretation of data. All authors have read and approved the manuscript.


P < 0.0001). Damage in follicular quality, that is, increased oocyte vacuolization and detachment of the oocyte from granulosa cells, was found after chemotherapy. Ovarian tissue cryopreservation, preferably performed before initiation of chemotherapy, should be offered to pediatric patients, including prepubertal girls, at risk of sterility.Cancer treatments improve the survival rate of children and adolescents; however chemo- and radiotherapy result in gonadal damage leading to acute ovarian failure and sterility. Ovarian tissue cryopreservation allows long-term storage of primordial follicles and represents the only possibility of preserving the potential fertility in prepubertal girls. The aim of the present study is to describe our experience in ovarian tissue cryopreservation in 45 pediatric patients. The number of follicles per square millimeter of the overall section area and follicle quality were evaluated histologically. A strong negative correlation was found between age and follicular density in patients both prior to and after chemotherapy ( The survival rate of children and adolescents with cancer has increased markedly over the past 30 years as a result of advances in supportive care and changes in cancer therapies. The use of cancer multimodal therapy  is now routine in the treatment of children with neoplastic disease. However many children treated for cancer suffer from side-effects of curative chemo- and radiotherapy, such as the risk of Acute Ovarian Failure (AOF) or early menopause, then sterility . GonadalThe level of ovarian damage varies according to the cancer treatment protocol: ovarian integrity is affected by the type of chemotherapeutic agent, the cumulative dose of chemotherapeutic agents, treatment duration, and total dosage effect. In general alkylating agents such as cyclophosphamide are associated with the highest risk of infertility –5. OvariPrevious studies indicate that radiation affects the ovaries and the degree of impairment depends on the radiation dose, fractionation schedule, and radiation field. Doses as low as 400–600 cGy in adults and 1000–2000 cGy in children have been noted to decrease ovarian function . HoweverThe most well-established options for female adult fertility preservation are embryo and oocyte cryopreservation. Unfortunately, in the pediatric setting embryo cryopreservation is inappropriate and ovarian tissue cryopreservation maybe an alternative option for fertility preservation in these patients: with ovarian tissue freezing, no ovarian stimulation is needed and ovarian tissue can be harvested laparoscopically without any preparation. Furthermore ovarian tissue cryopreservation has the greatest fertility potential in children since they have a high number of primordial follicles , 7–15. After thawing, cryopreserved ovarian tissue can be grafted into its normal anatomical position or into a heterotopic site. Alternatively the thawed tissue can be placed in culture with the aim to achieve antral follicle development and to avoid the risk of reimplantation of malignant cells . HoweverUntil today all pregnancies obtained after transplantation of frozen-thawed ovarian cortex were observed in adult patients at the time of harvesting; however there is no reason to doubt the capacity of prepubertal ovarian cortex to develop and function correctly after reimplantation . Animal The purpose of this paper is to describe our experience in ovarian tissue cryopreservation in pediatric patients focusing on the clinical characteristics of the patients, the technical procedures, and strategies used to harvest and preserve the tissue. In adolescent patients, the opportunity to preserve their fertility enabling a future normal life is a psychological support allowing the patients to overcome the difficulties that the disease entails.From January 1999 to August 2011, 51 pediatric patients with malignant and benign diseases approached the Gynecology and Reproductive Medicine Unit, Department of Obstetrics and Gynecology, S. Orsola-Malpighi Hospital, Bologna, for consultation about the possibility of ovarian tissue cryopreservation. The ovarian tissue of 45 pediatric patients, including pre-and postmenarcheal girls, was collected and cryopreserved. Parents of the patients gave their consent for both laparoscopy and cryopreservation of ovarian tissue. Ovarian tissue cryopreservation was not carried out for six patients: in two cases patients did not fit the inclusion criteria; in two cases parents refused because the procedure was too new or they did not want their daughter to be subject to further surgery; in the last two cases the patients suffered from Turner Syndrome with very small ovaries and a menopausal value of FSH, so, given the possibility of cryopreservation of few follicles, their parents refused consent.At our centre, the exclusion criteria for ovarian tissue cryopreservation included patients positive for Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Human Immunodeficiency Virus (HIV), or Treponema Pallidum (TP); patients with ovarian cancers; and patients who had contraindications for surgery. Regarding patients with leukaemia could be removed and cryopreserved in the hope that in vitro maturation of the oocytes will be possible in the future . As listed in Ovarian tissue cryopreservation was carried out within a maximum of 20–30 days from the last chemotherapy cycle, while within 10–15 days in patients not undergoing chemotherapy before ovarian tissue recovery. The ovarian preservation procedure was not associated with any delay in the commencement of anticancer treatment. Ovarian tissue was collected at our department by laparoscopy. In all cases laparoscopy was carried out as a day surgery procedure under general anesthesia. A carbon dioxide pneumoperitoneum at a pressure of 10 mmHg was created via a Verres needle inserted into the peritoneal cavity through an umbilical incision. A three-port laparoscopy was performed. The primary 5 mm port was inserted through the umbilicus for the telescope; two secondary 3 mm or 5 mm ports were inserted to the left and the right, respectively, lower quadrants at the same level lateral to the rectus sheath. Ovarian tissue was removed with scissors proceeding along the connecting line between two atraumatic graspers positioned in the ovary just above the infundibulopelvic and utero-ovarian ligaments first on the left and then on the right. Each ovarian fragment was placed in an Endobag  and retrieved through the umbilical 5 mm port to avoid tissue damage. The harvested tissue was then transferred to the laboratory for immediate cryopreservation. In the majority of cases, there was negligible bleeding of the ovaries; haemostasis was carried out by bipolar current. The port sites were closed using 4–0 resorbable (Biosyn) sutures. The median duration of this technique was 30 minutes (range: 15–45 min). All patients were discharged on the day of surgery or at most the day after, with the discomfort experienced being no greater than after diagnostic laparoscopy. No complications occurred due to anesthesia or surgery in any patient.Forty-three patients underwent laparoscopic harvesting of ovarian tissue from both ovaries, while in two patients the ovarian tissue was harvested from only one ovary because the other ovary was too small (patient with Wilms Tumor) and in the other case unilateral ovariectomy was performed (patient with Hodgkin Lymphoma). The amount of ovarian tissue removed was subordinated to the anticancer treatment protocol and to the size of the ovaries: in premenarcheal patients, due to the small size of the ovary and a high density of primordial follicles, the biopsy was smaller compared to the postmenarcheal ones.n = 19, n = 9, 10 and 17, When pelvic radiotherapy is indicated, ovarian transposition can be performed in order to displace the ovaries away from the radiation field –5, 20. IThe cryopreservation protocol used by our group was approved by the Ethics Committee of S. Orsola-Malpighi Hospital .After biopsy, the ovarian tissue was placed in Dulbecco's phosphate-buffered solution (PBS)  with 10% human serum added  and cut into pieces (mean size 1 × 0.3 × 0.1 cm) using a scalpel. The mean number of pieces obtained was 23.1 ± 6.8 (mean ± SE) in premenarcheal patients and 27.6 ± 2.9 (mean ± SE) in postmenarcheal patients. One or two pieces (1–3 mm) from each ovarian sample were immediately fixed for histological analysis. The remaining tissue was maintained in PBS + 10% HS solution until cryopreservation. Samples were cryopreserved using a slow-freezing/rapid-thawing protocol, according to Fabbri et al. . At the For each patient, a cryopreserved sample from each ovary was thawed after one month and fixed for histological analysis in order to evaluate the preservation of cortical tissue.μm thick section out of every 50 was collected and stained with toluidine blue for light microscopy (LM) examination (Leitz Diaplan light microscope equipped with a CCD JVC video camera). Semithin sections were examined, double blind by two trained pathologists, to (i) identify and count follicles, (ii) establish the number of damaged follicles, and (iii) assess follicular density, expressed as the number of follicles per square millimeter of the overall section area. Measurements were performed on digitalized images using ImageProPlus software. Fresh and frozen/thawed tissue samples were fixed in a freshly prepared solution of 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.4, overnight at 4°C. After osmium tetroxide postfixation and alcohol dehydration, the samples were embedded in Araldite epoxy resin  and then sectioned with an ultramicrotome . For each sample one 0.5 Semithin stained sections were initially observed at 10x to determine whether tissue was appropriate for histological examination: artefacts, erroneous sampling, presence or absence of follicles, and pathological involvement of ovarian tissue were recorded. Sections were then observed at medium (25x) and high magnification (40x). Follicles were counted and classified as resting type , growing type , and atretic type, according to Gougeon classification . At 40x Blood samples were analyzed for FSH, LH, Estradiol, Estrone, Prolactin, Testosterone, and 17-Hydroxyprogesterone one or two days before laparoscopy. Follow-up was performed six months after the end of therapy, and then every year, using ultrasound and hormonal tests.t-test. A value of P < 0.05 was accepted as significantly different. We studied the follicular density of the pre- and post-chemotherapy groups and, within each group, the follicular density of pre and postmenarcheal patients. The relationship between age and follicular density was evaluated using a linear regression model. Follicular density value used for statistical analysis was obtained by averaging the follicular densities of the left and right ovary. Results were statistically analysed by unpaired n = 22) aged 53–214 months  (n = 23) aged 19–215 months  (Ovarian tissue was collected and cryopreserved from 45 pediatric patients aged 160.9 ± 6.9 months (mean ± SE). Patients were grouped into two categories: pre-chemotherapy patients (n  ± SE)  and postan ± SE) . The speIn both groups, menarcheal age was 150 ± 3.8 months (mean ± SE). In the pre-chemotherapy group 80% of patients had regular menstrual cycles and normal flow, whereas 20% had slightly irregular menstrual cycles of up to 35 days. In post-chemotherapy patients, it was not possible to evaluate menstrual cycles because they had been treated with pills or GnRH analogues. n = 2, A total of 5749 follicles from pre-chemotherapy patients and 2684 follicles from post-chemotherapy patients were analyzed; in both series LM showed that 99% of follicles were of the resting type and 1% distributed among primary, secondary, and preantral follicles. In one patient n = 2, , the ovay = −0.378x + 87.124, R2 = 0.354, P < 0.0001; y = −0.637x + 126.63, R2 = 0.776; P < 0.0001)  as well as in the post-chemotherapy group   . Follicuan ± SE) .n = 1, Follicular density results were slightly higher in the post-chemotherapy group than in the pre-chemotherapy group  . This fiHormone levels of pre-chemotherapy patients were within the reference range of our laboratory for age and phase of menstrual cycle. Hormone levels of post-chemotherapy patients were not considerable because these patients were receiving pills or GnRH analogue treatment to protect the ovarian function.Median follow-up time was 30 months (range 10–50 months). Thirty-six of the girls are still alive. Nine patients died of the primary pathology during the study period: three of these patients suffered from Acute Leukaemia, two from Hodgkin's Lymphoma, one from Wilms Tumor, one from PNET, one from Thalassemia Major, and one from Synovial Sarcoma. To date we have not received any request for ovarian tissue reimplantation because most of the patients are very young, many are still undergoing treatments, and others do not currently need or desire motherhood.Ovarian tissue cryopreservation offers great promise for fertility preservation among young patients affected by cancer and represents the only option for prepubertal girls. The human ovary has a fixed number of primordial follicles which progressively decrease with increasing age in a biexponential fashion, culminating in the menopause at around 50 years of age. Both chemotherapy and radiotherapy quicken follicle depletion leading to premature menopause . OvarianTo date many authors have written about availability, feasibility, safety, and efficiency of ovarian tissue cryopreservation in pediatric patients. Jadoul et al.  performeThis study reports on a large number of pediatric girls undergoing ovarian tissue cryopreservation to preserve fertility. Our data show a high correlation between follicular density and age, and a decrease in follicular quality after chemotherapy. In light of these results and those reported in literature, the orientation of our group is in favour of ovarian tissue cryopreservation in pediatric patients because the procedure has a high potential of maintaining endocrine function and preserving fertility. It is well known that the optimal timing of ovarian cryopreservation is before sterilizing treatment. However, the live births obtained in adults after transplantation of ovarian tissue cryopreserved after chemotherapy and the high number of healthy follicles found in histological sections of tissue after chemotherapy in pediatric patients indicate that ovarian tissue cryopreservation could be offered after chemotherapy to young girls. In addition, the indication for fertility preservation in children can be extended to patients genetically predisposed to premature ovarian failure , with acceptable results. Although no births have yet resulted from freeze-thawing of prepubertal ovarian cortex, expectations of this approach are encouraging, considering the high follicular pools of these patients.