Anaemia is highly prevalent in cancer patients, adversely affects quality of life and impacts survival. The pathogenesis is multifactorial, with iron deficiency being a major and potentially treatable contributor. This study aimed to assess the effectiveness and economic impact of ferric carboxymaltose in chemotherapy-induced anaemia. This prospective cohort study between 2015–2016 of chemotherapy-treated patients for solid tumours, grade ≥2 anaemia and iron deficiency evaluated hematopoietic response four weeks after ferric carboxymaltose treatment. Transfusion rate of all cancer patients treated at our ambulatory unit during the two-year study period (2015–2016) was compared to a retrospective cohort (2013–2014) who received blood transfusion only. Between 2015–2016, 99 patients were included and treated with ferric carboxymaltose, the majority of whom (n = 81) had relative iron deficiency. Mean haemoglobin concentrations improved from 9.2 [6.7–10.8] g/dL to 10.6 [7.8–14.2] g/dL four weeks after treatment. A 26% reduction in the transfusion rate was observed from control retrospective to the prospective study group including ferric carboxymaltose treated patients [relative risk 0.74 (95% CI:0.66–0.83)]. The cost analysis showed a benefit for the use of ferric carboxymaltose in chemotherapy-induced anaemia. This study shows that ferric carboxymaltose is an effective, cost-saving support treatment, reducing the need for allogeneic transfusions saving blood units which are a limited resource. Anaemia has a negative impact on the quality of life of cancer patients. It is associated with poor performance status, fatigue, and may also jeopardize adherence to treatment, affecting therapeutic results, hospital stay and even survival6. According to the World Health Organization (WHO), anaemia is defined as haemoglobin (Hb) levels <12.0 g/dL in women and <13.0 g/dL in men7. The National Cancer Institute subdivides anaemia into different grades: mild – grade 1 , moderate – grade 2 (8–<10 g/dl), severe – grade 3 (6.5–<8 g/dl) and life threatening (<6.5 g/dl) anaemia8.Quality of life should be a priority in the management of cancer patients. As one of the most common problems reported at diagnosis and during cancer treatment, anaemia is a major concern in any Oncologist’s daily practice. The incidence of anaemia in cancer patients reported in literature varies significantly, ranging from 20% to 60% at the time of cancer diagnosis and reaching 60–90% during cancer treatments10. Anaemia may be attributed to absolute ID that can result from chronic blood loss due to gastrointestinal and gynaecological malignancies or surgery. Although less frequent, it can also derive from nutritional deficiencies due to cancer-induced anorexia, as well as reduced iron absorption due to gastrectomy, or the use of proton pump inhibitors, taken approximately by 20% of cancer patients12. The production of cytokines that leads to chronic inflammation and iron sequestration, and the myelosuppressive effects of chemotherapy or metastatic infiltration of the bone marrow limiting erythropoiesis are also contributing factors14. A variety of factors have been reported to be predictive of cancer and chemotherapy-associated anaemia: recent anticancer therapy, old age, poor performance status, advanced stage of the disease and particular tumour location 6.In cancer, anaemia has a multifactorial etiology. Iron deficiency (ID) is one of the underlying causes as its prevalence varies from 32 to 60%15, or functional , when iron reserves are normal or increased but sequestered inside macrophages and enterocytes16. In the latter case reduction of iron availability for erythropoiesis is observed leading to anaemia.ID can be classified as absolute when iron stores are depleted, mainly due to bleeding, which corresponds to between 7 and 42% of all cases17, whereas The National Comprehensive Cancer Network (NCCN) guidelines targets ferritin level between 30 ng/mL and 500 ng/mL and a TSAT level <50%8.Considering absolute ID, while in normal individuals a serum ferritin level of <30 ng/mL is virtually diagnostic, in cancer patients a higher ferritin cut-off (<100 ng/mL) appears more reliable, due to the chronic inflammation status. In functional ID, the guidelines available recommend testing both serum ferritin and transferrin saturation (TSAT). European Society for Medical Oncology (ESMO) guidelines considers functional ID when TSAT is <20%18. RBC transfusions increase the risk of thrombotic events, may decrease survival19 and must be reserved for patients with severe anaemia symptoms in need of rapid Hb improvement. Safety issues regarding ESA’s link to decreased survival or increased disease progression were raised in the past. However recent data from a meta-analysis and prospective trials did not reveal links to tumour progression or reduced survival prospects21. Venous thromboembolic events are a known risk of ESA use in cancer patients22. Therefore, anaemia treatment guidelines do not recommend transfusions and suggest minimization of ESA dosage17. Some guidelines go as far as limiting their use to patients whose cancer treatment is not curative in intent18. Studies showing that intravenous (IV) iron (with or without concomitant ESA therapy) can improve Hb levels and reduce transfusion requirements in cancer patients support these goals25.Once other causes of anaemia are excluded, most patients with cancer and chemotherapy-induced anaemia (CIA) are treated with red blood cell (RBC) transfusion and/or iron supplement as monotherapy or in association to erythropoiesis-stimulating agents (ESA)26. In Portugal, four are available: iron sucrose, low molecular weight iron dextran (LMW-ID), iron isomaltoside and ferric carboxymaltose (FCM), all studied in CIA (reviewed in27). Because of its stable complex which allows for a slow and prolonged iron release, FCM enables administration of 1000 mg of iron in a 15-minute infusion29. Moreover, as reported by several authors in a variety of anaemia backgrounds, FCM is safe and effective providing a rapid correction of Hb and serum ferritin levels in iron-deficient patients30.There are a number of IV iron formulations in the marketTherefore, the main objective of this study was to assess the effectiveness of FCM in the treatment of CIA.8 were included in the study and treated with FCM infusions according to body weight as described in the methods section . Median age was 66 years [31–84 years] and 49.5% (n = 49) were male  and iron deficiency (defined as ferritin <800 ng/mL and TSAT <50%)The majority of patients received cytotoxic chemotherapy  and 5 (5.1%) participants were not receiving anti-cancer treatment at the start of the study. Treatment intent was curative in 45 patients. During the treatment phase, the median [Interquartile Range (IQR)] total dose of iron received was 1500 (±500) mg. The majority of participants (74.7%) received two infusions.versus baseline) on average to 10.6 [7.8–14.2] g/dL  had functional ID  received RBC transfusion within four weeks after the first FCM administration, and 26 (26.3%) after week four. RBC transfusions did not influence mean Hb concentrations achieved, as the mean post treatment Hb in the 88 patients treated with FCM-only was similar: Hb 10.7 [8.2–14.2] g/dL A total of 319 vials of 500 mg of FCM were used in the study. Treatments were well tolerated, and neither hypersensitivity reactions nor other severe drug-related adverse events (immediate or delayed) occurred.With regard to the secondary endpoint transfusion rate, we compared a cohort that comprises 99 patients treated with FCM and all patients subjected to RBC transfusion in the years 2015–2016 (prospective cohort), to a historical control group from two years before (2013–2014). The control group had received RBC transfusion as the only treatment for anaemia.During the study period (2015–2016), a total of 13221 chemotherapy sessions were performed in 1811 patients, and a total of 517 blood units were used, representing a transfusion rate (number of RBC units per chemotherapy session) of 3.9%  was effective in 85% of cases, improving the mean Hb levels of anaemic cancer patients. These observations are in line with the results published by Steinmetz et al. and Toledano et al., where the supplementation with FCM only, in anaemic cancer patients with absolute iron deficiency under chemotherapy, led to a substantial and sustained increase in Hb levels, suggesting a role for IV iron as first-line treatment for CIA24.IV iron supplementation is widely used for the treatment of chronic iron deficiency anaemia. FCM was shown to be an effective treatment for anaemic patients with chronic kidney disease undergoing haemodialysis, chronic heart failure, post-partum anaemia or inflammatory bowel disease17. Although ESAs are useful, ASCO guidelines limits their use to palliative intent18, and NCCN recommends its use only in patients receiving myelosuppressive chemotherapy or undergoing palliative treatment8.Currently, the treatment of anaemia in cancer patients consists of RBC transfusions and use of ESAs with or without iron supplementationAt recommended doses, IV iron is well tolerated, particularly when compared with oral iron. FCM was a safe treatment as no severe drug-related adverse events were reported in this study.32, and a recent prospective trial33, although no serious adverse events due to low phosphate values were seen32. Therefore, serum phosphate levels should be determined in all FCM treated subjects.Compared to other IV iron formulations, the incidence of transient hypophosphatemia with FCM is fairly high as reported in two retrospective studiesRognoni et al., FCM resulted in a higher increase of serum ferritin levels in comparison to ferric gluconate and showed a high safety profile30, consistent with the other formulations able to be administered as a total iron dose infusion 26.FCM has been prospectively compared to other IV iron formulations and showed similar safety and efficacy levels. As showed in a systematic review and meta-analysis of 21 randomized controlled trials by The main advantage is the administration of a single total dose, which decreases the risk of reactions to multiple courses of treatment. In addition to being a convenient option for the patient, it also reduces the number of interventions from medical staff and reduces ambulatory clinic booking hours, which contributes to a reduction in costs.Additionally, our results showed a significant reduction in the percentage of cancer treatments that needed transfusion support in the group that included patients treated with FCM, reducing the need of allogeneic transfusion, and therefore saving RBC units.For all these reasons, supplementation with IV iron, stands as an attractive therapeutic option for treatment of anaemic cancer patients with iron deficiency.The cost reduction analysis was also favourable, as a direct cost saving was achieved in the FCM treatment group, due to the reduced need for RBC transfusions. More importantly, a number of indirect cost savings were achieved, namely in terms of time spent at ambulatory clinic and number of RBC units saved. From the patient’s perspective, those savings translate into fewer hospital visits and work absences, reduced transportation costs to name a few.34. Cost reduction analysis also favours FCM over iron sucrose for the ambulatory treatment of iron deficiency35. Two studies, from Italy and Brazil, showed that FCM might be a cost-saving option for their health care systems when compared to ferric gluconate or iron sucrose in the treatment of iron-deficient patients37. With regards to the treatment of CIA, no data is available.FCM was already shown to have reduced direct and indirect costs of hospitalization compared with iron sucrose or oral iron6. Iron deficiency is associated with impaired physical function, weakness and fatigue even in the absence of anaemia9.This topic is of major importance in clinical practice as anaemia is highly prevalent among cancer patients and its treatment might have a significant impact on survival and quality of lifeOne potential limitation arising from the design of our study is the lack of long term follow-up data on Hb values or patient-reported outcomes, which would have been helpful in assessing the clinical significance of our findings. Cost issues needs to be considered on a country level, as significant price differences for FCM between Europe and the United States are present.Future studies may assess the benefit of this treatment in patients without anaemia, despite the documented iron deficiency, as it may not only prevent the occurrence of anaemia, but also improve symptoms of iron deficiency.st January 2015 to 31st December 2016. The study was reviewed and approved by the ethics committee of this hospital. All data was collected from electronic medical records. Data regarding the group which includes patients treated with FCM was collected prospectively. Data regarding the control group was collected retrospectively at baseline, for the period between the 1st January 2013 and 31st December 2014. The eligible population consisted of adults (≥18 years old) with a malignant solid tumour and diagnosed with at least grade 2 anaemia (defined by Common Terminology for Adverse Events v.5.0 as haemoglobin inferior to 10 g/dL8) and iron deficiency (defined as ferritin level <800 ng/mL and TSAT <50%)8. All were undergoing anti-cancer treatment and signed an informed consent. Subjects who received more than 50 units of blood or patients treated with ESAs were excluded from the study. Cancer staging was set in accordance to the TNM Classification (AJCC 7th edition).A prospective cohort study was performed in a central Portuguese hospital  from 1st of January 2015 and 31st of December 2016. Treatment was prescribed when ferritin was lower than 800 ng/mL and TSAT was inferior to 50%. If patient body weight was 35 Kg to <70 Kg the dosage recommended was 1000 mg+ 500 mg . If patient weight was greater than 70 Kg the dosage recommended was 1000 mg + 1000 mg . Patients were examined during the first half hour post-administration for any possible side effects such as pain at the site of injection, flushing, allergic reactions, headache, dizziness, nausea, vomiting and a feeling of heaviness in the head. Haemoglobin levels were assessed four weeks after the last treatment dosage. The main aim of this study was to assess the effectiveness of FCM in the treatment of CIA in patients diagnosed with solid tumours. In order to do so, we analysed the hematopoietic response, defined as a positive haemoglobin variation 4 weeks after the post-carboxymaltose treatment.At the start of the study, all patients were analysed for ferritin levels, transferrin saturation, vitamin B12 and folic acid status. The group treated with FCM consisted of subjects who initiated treatment with FCM  between the 1This study also aimed to compare the transfusion rate, defined as the number of RBC units per chemotherapy treatment, between the cohort that included patients treated with FCM and those of a historical control group. The control group fulfilled the same inclusion criteria but had received RBC transfusions as the only form of treatment for anaemia.A safety endpoint was to assess the number of adverse events during FCM treatment. The authors also analysed the economic impact of FCM protocol. Informed consent was obtained from all individual participants prior to the start of the study inclusion.Categorical variables are presented as frequencies and percentages, and continuous variables as means and standard deviations, or medians and interquartile range (IQR) for variables with skewed distributions. Normal distribution was checked using skewness and kurtosis. A dependent t-test for paired samples was used for analysis of Hb variation over time (Hb at baseline vs Hb 4 weeks after treatment). Analysis of the results was performed using SPSS statistical software, version 22.With regards to the cost analysis (Table th December 2014. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.The study was reviewed and approved by the ethics committee of Centro Hospitalar Vila Nova de Gaia/Espinho, where the study was conducted and was approved by unanimity on the 29


TC), first melting temperatures (TM1), heat fusion (Hf), crystallinity, and melt flow rate (MFR), which were measured as responses for the design of experiment (DOE). The adjusted and predicted correlation coefficient (R2) shows good agreement between the actual and the predicted values. To confirm the optimal values from the response model, supplementary experiments as a performance evaluation were conducted, posing better operational conditions. It has been confirmed that the RSM model was adequate to reflect the predicted optimization. The results suggest that the addition of LMPP into iPP could effectively enhance the functionality and processability of blend fibres if correctly proportioned.Optimization of the mechanical and thermal properties of isotactic polypropylene (iPP) homopolymer blended with relatively new low molecular low modulus polypropylene (LMPP) at different blend ratios was carried out via surface response methodology (RSM). Regression equations for the prediction of optimal conditions were achieved considering eight individual parameters: naming, elongation at break, tensile strength and elastic modulus, crystallization temperature ( Isotactic polypropylene (iPP) polymer is a type of broadly utilised polymer that is used for its low cost and appealing mechanical properties, but only when blended with other polymeric materials, as iPP alone has some drawbacks. For example, brittleness at low temperatures , low impPolymers are mainly blended with other polymeric materials to combine existing polymers into new compositions obtaining specific properties, which also allows the faster development of new materials. In polymer blending, miscibility is the most important property, which also refers to the solubility when two polymers dissolve in each other at the right proportion . SometimThe attributes of blend iPP, such as morphology, crystallinity and crystallization control are dependent mainly on the blended components. On one hand, with extensive theoretical and experimental investigation on the miscibility of polymer blends, the nature and characteristics of the amorphous/amorphous or amorphous/crystalline polymer blends are well accepted ,10. HoweTo achieve high performance and productive polymeric fibre, the optimization of the fabricating process is a key factor to be considered. Therefore, the aim of this research is to employ a computational and statistical tool, a response surface method (RSM) as a design of experiment (DOE), and to optimize the processability of fibres by predicting blend ratios of iPP and LMPP (a relatively new polymer). The considered variables are thermal and tensile properties, in order to have a better understanding of LMPPs effects on iPP blend fibres.In recent years, RSM has been widely used for establishing the optimum input parameters in different manufacturing applications, especially in the domain of textiles and polymeric materials. For instance, it has been used in maximizing processing parameters of chemical finishing of flame retardant textiles , eco-friThe conditions for the optimal DOEs are mostly related to the mathematical modelling of a process and, usually, these mathematical models are polynomials having unknown structures, and consequently the corresponding experiments are designed for particular problems only. Moreover, choice of DOEs can largely influence the building of a response surface and accuracy of the approximations. At first, RSM was developed to model experimental responses ,21, late5 g/mol. LMPP (LMPP S901) were provided by Idemitsu Kosan Co., Ltd., Tokyo, Japan. The blend mixing ratios of iPP/LMPP (wt/wt) were used according to the lower and upper limit of DOE to get RSM responses. The basic molecular characteristics of the individual iPP and LMPP are presented in For the blending, iPP, S2040 (Ziegler–Natta) was provided by SECCO Petrochemical Limited, Shanghai, China, with an average molecular weight of 2.0 × 10The iPP/LMPP blend fibres at different compositions were prepared after blending the two polymers for 2 min at room temperature with the help of a mechanical blender, made by Giant Co., Ltd., Kunshan, China. Homopolymers were blended with a twin-screw extruder  for melt blended samples with an L/D value of 40, where the processing speed and temperature were 60 rpm and 210 °C respectively. The samples were then cut into pellet form after drawing.The iPP/LMPP melt-spun fibres were prepared with a rheometer accessory (Haul-off drafting device)  at room temperature 25 °C. The spinning temperature was set at 190 °C, and the drawing and collecting roller speed was 30 m/min. The iPP/LMPP monofilaments were prepared with a linear density of about 0.7 dtex. Other preoperational and experimental details can be found in previous studies [The mechanical attributes of the melt-spun fibres produced were characterised using an Instron 2365 universal testing machine. The testing conditions of iPP/LMPP filament were carried in a standard conditioning lab at a temperature of 25 °C ± 2 °C and relative humidity 62–68%. For the filament mechanical test, the gap between two jaws was set at 10 cm and filaments were extended following the movement of the upper jaw set at a speed of 100 mm/min.TM = 156.6 °C). Samples of 5 mg were loaded onto the sample pan, from room temperature to 200 °C under a nitrogen atmosphere. The first heating rate 10 °C/min was used, whereas isothermal holding was set at 200 °C for 3 min, and then the samples were heated again to 200 °C with heating rate of 10 °C/min.The melting behavior and crystallization of iPP/LMPP were measured with a Perkin Elmer DSC 8000 differential scanning calorimeter , with an intercooler lowermost temperature of −90 °C. The temperature of iPP/LMPP samples was calibrated with pure Indium (TM = 156.6 °C). Five milligrams of the sample was cut from the blended specimen and loaded into the sample pan, from room temperature to 200 °C at the heating rate of 100 °C/min in nitrogen atmosphere. The isothermal holding was at 200 °C for 3 min in order to erase the thermal history, followed by cooling at a rate of 50 °C/min to −30 °C to keep the amorphous part, in order to make the glass transition temperature (Tg) more obvious. The area of the exothermic curve and temperature of the peak were taken as the crystallization temperature (TC). As the temperature reached −40 °C, it was reheated again at a rate of 10 °C/min and the melting thermogram was measured. Tg and temperature of the peak and the area of the endothermic curve were taken as the melting temperature (TM), and the heat of fusion, (△Hf), respectively.iPP and LMPP blend fibre samples were measured for their thermal properties, namely crystallization and melting behaviour, with a Perkin Elmer DSC 8000 differential scanning calorimeter , which has an intercooler lowermost temperature of about −90 °C. Temperature of the fibre samples was calibrated by using pure Indium . Since pure LMPP has a low melting point and low melting strength, it could not be drawn into fibres, therefore its fibre SEM was not feasible. However, morphological studies of pure LMPP in molten and film form can be found elsewhere [TC), first melting temperatures (TM1), heat fusion (Hf), crystallinity, and MFR were measured as responses for the DOEs. The RSM will produce the statistical models for these responses, along with optimized values of variables for the maximization of individual responses. For that, the experiment was designed composing a set of 17 experimental runs. These experiments were separately performed for pure iPP and LMPP, and their blends. However, the pure iPP and LMPP were not included in design run. The effects of several independent input variables (iPP and LMPP blends) on the dependent output response variables  will be investigated through the RSM approach. RSM will define the relationship between these several independent variables and response variables, by employing a series of experiments intended for an optimal response [Thermal and tensile properties with eight individual parameters, namely elongation at break, tensile strength and elastic modulus, crystallization temperature  variables.The regression analysis between independent variables and the response was performed to fit the empirical second-order polynomial model and is shown in the following equation:TC (y5), TM1 (y6), MFR (y7), and Hf (y8) are the predicting responses. The adequacy of the built model was checked with ANOVA computations and various statistical parameters. Probability , Fisher , the correlation coefficient (R2), adjusted correlation coefficient (Adj R2) and predicted correlation coefficient (Pred R2) were used for adequate approximation and validity of model [p < 0.05) are significant for all of the predicted designs based on experimental data.The experimental levels of independent variables and dependent responses are given in of model . P and fTC, TM1 and Hf; while for tensile property 2Fl, they were identified as significant by the software.The present work deals with the concept of RSM with 17 experimental runs carried out to determine the optimum iPP/LMPP blending parameters. The statistical combination of independent variables and analysis of variance for the experimental results with their response are presented in 2 for elongation, elastic modulus, TC, TM1, MFR, crystallinity, Hf, and tensile strength are 0.976, 0.918, 0.938, 0.889, 0.867, 0.869, 0.995, and 0.718, respectively. Apart from the precision, the R2 values of each attribute were within the desirable range; the regression analysis was carried out by evaluating the adjusted R2 and predicted R2. The greater correlation coefficients further confirm the appropriateness of the formulated RSM models. Although the difference between the R2 values and adjusted R2 values for every response was evident, the R2 values were in conceivable agreement with the adjusted R2 values. They were 0.966 for elongation, 0.889 for elastic modulus, 0.905 for TC, 0.838 for TM1, 0.809 for MFR, 0.808 for crystallinity, 0.995 for Hf and 0.607 for tensile strength, respectively. Whereas the coefficient of variation percentage (C.V %) was less than 5% in all responses, indicating the established model is reproducible [The predicted response values from the RSM model and the actual values obtained from the experiments were analyzed as predicted versus actual plots that are shown in oducible ,28. ThisThe generated 3D surface plots by RSM assist in visualizing the effect of parameters on a wide array of responses. Likely, the elastic modulus of iPP/LMPP blend fibres diminishes with the increase of LMPP content, as shown in In Tg and TM of iPP fibre and its fibre blend accurately, which is caused by the weak transition in its molecule structure. The higher the temperature, the greater the molecular motion will be. The freedom of molecular motion makes the crystallization difficult at a given temperature, thus increasing the time needed for crystallization [TM1 and Hf measured. Hf values reduce with the content of LMPP increased in iPP/LMPP blend fibres. The apparent change of Hf values reflects the melting behavior of iPP itself within its structure formed with the addition of LMPP. Hence, change in Hf affects the overall crystallinity of the blends [TM1 [Very often it is difficult to measure the lization . The adde blends . It can nds [TM1 . This innds [TM1 ,32.Generally, polymers with high molecular weight have stronger intermolecular interaction, which causes low MFR and higher viscosity . WhereasTM, Hf of melting, TC, and latent heat of crystallization, were found to decrease evidently in all blends with increasing aPP content [A dramatic change in mechanical properties of iPP/LMPP blends is anticipated if the auxiliary polymer is out of the tested proportion. Likewise, it is considered reasonable to blend iPP and atactic polypropylene (aPP) for their similar molecular structure and good affinity between them ,36, but  content . Hence, Generally, batch and semi-batch reactors are utilised for the production of high-value polymers, for which the optimal recipe is essential. To achieve the optimal recipe, a number of experiments are carried out as a trial and error method. This trial and error method not only is used to optimize operating conditions but also to make assumptions by monitoring the influence of one factor at a time on an experimental response, whereas only one parameter is altered and others are kept constant . In addiIn order to justify the optimization of individual mechanical and thermal properties obtained in Tg of pure LMPP was obvious around −10.2 °C, while for the pure iPP was not, as seen from the DSC of heating curves in Tg signals to be detected by DSC. On the other hand, LMPP has a low crystallization rate and low crystallinity, which can also be confirmed by its cold-crystallization peak at about 24.5 °C. This re-crystallization process of LMPP is carried out when the molecular chain gets more dynamic in the relatively high temperatures [The changes in thermal properties of predicted iPP and LMPP blend ratios are shown in Tg may affect MFR, as well as the flow-ability of a polymer [There were changes in the DSC curves among three predicted blend ratios by RSM, but some similarities and differences were seen as well in their melting peak trends. For instance, in  polymer . On one The SEM images of RSM suggested iPP/LMPP blends with variant LMPP contents are shown in Optimization factors to achieve fibre blend with desired properties were determined from appropriate spinnability with better mechanical and thermal property perspectives. A blend ratio of 82.5 wt iPP and 17.5 wt LMPP was suggested by the developed RSM model. The accordance between the predicted and the actual results with a marginal difference were in the permissible limits, indicating the blend ratios obtained from RSM were practical for attaining optimized mechanical and thermal properties in fibres. It has been approved that the functionality and processability of the blend fibres can be improved with the addition of LMPP into iPP, if correctly proportioned.Response surface methodology (RSM) was used to study the effects of blend polymerization factors of isotactic polypropylene (iPP) and low molecular low modulus polypropylene (LMPP) on the mechanical and thermal behavior of iPP/LMPP blend fibres through a Design-Expert software analysis. Empirical models to simulate the variant blend ratios of iPP/LMPP were developed in order to optimie the output responses of the blend fibres. The models were validated by experimental results, and a satisfactory correlation coefficient was achieved for the evaluated models. Variation in the mechanical and thermal properties was seen by changing LMPP contents from 5 to 25 weight parts in iPP/LMPP blend fibres. Fibres with lower LMPP content showed higher values for elastic modulus (MPa), crystallinity (%), heat fusion (J/g), crystallization and overall melting temperature (°C). Whereas, higher values for tensile strength (MPa), elongation at break (%) and melt flow rate (g/10 min) were obtained in higher LMPP content blend fibres. A blend ratio of 82.5 wt iPP and 17.5 wt LMPP was suggested by the RSM model, to achieve desired properties determined from appropriate spinnability with better mechanical and thermal property perspectives. The results were approved by the experimental results, indicating the validity and reliability of the developed RSM model. This implies that the developed RSM model can be used to predict/estimate the properties of fibre blends to inform industrial practice.


IUHT heifers produced less milk compared to IUCL. The mammary glands of IUHT heifers differed morphologically from IUCL, with the IUHT heifers having smaller alveoli and a greater proportion of connective tissue relative to their IUCL herdmates. However, intrauterine heat stress had little impact on the proliferation and apoptosis of mammary cells during lactation. Our results indicate that fetal exposure to heat stress impairs milk production in the first lactation, in part, by inducing aberrant mammary morphology. This may result from alterations in the developmental trajectory of the fetal mammary gland that persist through the first lactation rather than to alterations in the cellular processes controlling mammary cell turnover during lactation.Earth’s rising temperature has substantial repercussions for food-producing animals by increasing morbidity and mortality, diminishing reproductive potential, and reducing productivity. In the dairy industry this equates to massive losses in milk yield, which occur when cows are exposed to heat stress during lactation or during the non-lactating period between lactations (i.e. dry period). Furthermore, milk yield is significantly lower in first-lactation heifers that experienced fetal heat stress. The mechanisms underlying intrauterine effects of heat stress on the offspring’s future lactation have yet to be fully elucidated. We hypothesize that heat stress experienced through the intrauterine environment will alter the mammary gland microstructure and cellular processes involved in cell turnover during the cow’s first lactation. Mammary biopsies were collected from first-lactation heifers that were exposed to heat stress or cooling conditions while developing in utero (IUHT and IUCL; respectively,  Environmental heat stress negatively impacts livestock health, welfare, and productivity . High prIn dairy cattle, the dry period, a six to eight-week non-lactating state between lactations, coincides with late gestation, an important stage of fetal growth and development. During the last two months of gestation, the fetus grows at the fastest rate and accumulates approximately 60% of its birth weight . Calves The capacity of the mammary gland to synthesize and store milk depends on the maintenance of both the number and activity of secretory mammary epithelial cells (MEC) throughout lactation and the size of the alveoli, the functional units of the mammary gland –18. The The objective of the current study was to evaluate the microstructure and cellular processes of the mammary gland of first-lactation heifers born to dams heat stressed during the dry period (i.e. in late gestation). We predicted that in utero exposure to heat stress would alter mammary gland architecture and cellular processes through the first lactation, thus impairing milk synthesis.All procedures performed in this study were approved by the Institutional Animal Care and Use Committee at the University of Florida.n = 10; 2015, n = 18) or cooled  during their dry period (i.e. last 46 d of gestation) as described in detail in Monteiro et al. [In 2014 and 2015, 51 multiparous dams were either heat stressed  and daily milk yield up to 84 DIM were compiled from AfiFarm Dairy Herd Management Software . Gestation length was calculated as the artificial insemination date subtracted from the calving date. Calves born to IUHT and IUCL heifers were weighed by farm staff at birth prior to colostrum feeding.n = 9) and IUCL heifers (n = 10) in 2016 and 2017. Biopsies were performed following the method described by Farr et al. [Mammary biopsies were collected on 21 and 42 DIM for a subset of IUHT  to be quantified. Masson’s trichrome stain was performed to visualize the connective tissue. Both techniques were performed following standard protocols at the Molecular Pathology Core at the University of Florida.2O2. After washing in 1 x TBS with 0.1% Tween-20, tissue sections were blocked with the UltraV block provided in the kit for 5 min. Sections were then incubated with primary antibody  diluted 1:100 in 1 x TBS for 30 min at room temperature. Sections were incubated with UltraVision One HRP Polymer from the kit for 30 min at room temperature, subsequently washed in 1 x TBS with 0.1% Tween-20, and incubated for 5 min with 3,3’-diaminobenzidine substrate from the kit, diluted according to the manufacturer’s recommendation. Sections were counterstained with hematoxylin, subjected to a series of graded ethanol washes and xylene, and mounted with Permount . Mammary tissue of a cow at 14 DIM (from a separate study) was run without primary antibody as a negative control. Jejunum tissue from a 2-day old calf (from a separate study), expected to have a high proliferation rate, was used as a positive control.Slides were incubated at 60°C for 30 minutes, deparaffinized in xylene, and rehydrated in a graded series of ethanol washes  followed by a rinse in distilled-deionized water. The UltraVision One Detection System: HRP Polymer/DAB Plus Chromogen kit  was used for the Ki67 assay. Antigen retrieval was achieved by incubating slides in a coplin jar with 1 x citrate buffer in a water bath at 95°C for 20 min. Endogenous peroxidases were blocked by quenching slides in Hin situ Apoptosis Kit  was used following the manufacturer’s protocol with a few modifications. Tissue sections were first deparaffinized in xylene followed by a graded series of ethanol washes for rehydration and a rinse in 1 x PBS. Antigen retrieval was conducted via the enzymatic method, incubating sections in 20 μg/ml proteinase K  for 8 min at room temperature. After several washes in distilled-deionized water, tissue sections were quenched in 3% hydrogen peroxide in 1 x PBS for 10 min, and incubated for 60 min at 37°C with TdT enzyme. Sections were then incubated with anti-digoxigenin-peroxidase for 30 min at room temperature followed by 3,3’-diaminobenzidine substrate for 8 min. Methyl green was used for the counterstain and tissue sections were dehydrated with n-butanol and xylene, then mounted with Permount. Mammary tissue of a cow 14 DIM was run with TdT enzyme diluted in stop/wash buffer instead of reaction buffer as a negative control. The Apoptag kit contains slides of involuting rat mammary gland collected 3–5 days post-weaning, which was used as a positive control.ApopTag Plus Peroxidase 2 and at 40X is 96,820 μm2. For H&E and trichrome stained slides, alveoli number, alveoli area, and connective tissue area, respectively, were averaged across the 5 fields measured per section and the averages were used for statistical analyses. Percent of connective tissue in the mammary gland was calculated as connective tissue area divided by total tissue area at 20X, multiplied by 100. To describe the relationship between alveoli area and number of secretory cells, alveoli area was measured and secretory cells were counted across 5 photomicrographs per section taken with a 40X objective from 21 DIM samples.Histological slides were imaged with an EVOS XL Core imaging system . For H&E and trichrome stained sections, 5 photomicrographs were captured per section with a 20X objective for quantification of alveoli number and luminal area. Alveoli were defined as any rounded structure with a lumen enclosed by a single layer of epithelial cells. Alveoli area was measured as the luminal space, not including the area comprised by the surrounding mammary epithelial cells. Ductal structures were not included in counts or area measurements. Fields were selected at random but areas close to the edge of the section were avoided. Alveoli area, connective tissue area, and the number of alveoli and stained cells were quantified using ImageJ . For couFor Ki67 and TUNEL labeled tissue sections, 5 photomicrographs were taken per section using a 40X objective. For Ki67 and TUNEL, every cell was counted in each of the 5 fields per section and scored as positive  or negative . This resulted in a total of more than 3,000 cells counted per section. All cells were classified as either a mammary epithelial cell or a stromal cell. Mammary epithelial cells (MEC) included mammary alveolar cells, ductal epithelial cells, and myoepithelial cells. Stromal cells were considered to be the cells in the area surrounding the epithelium, such as fibroblasts, endothelial cells, blood cells, immune cells, and adipocytes as described in Tao et al. [P-value less than or equal to 0.05 was considered significant. P-values less than 0.1 but greater than 0.05 were considered a tendency. Data are presented as least squares means (LSM) ± standard error of the mean (SEM) unless indicated otherwise.All data were statistically analyzed using SAS v. 9.4 . Colostrum yield, gestation length, and calf birth weight were analyzed by one-way ANOVA (PROC GLM). The model for calf weight included calf sex as a covariate and an interaction between sex and treatment. Models for colostrum yield and gestation length included year as a covariate and an interaction between year and treatment. Correlations between alveoli luminal area and number of secretory cells comprising the alveoli were assessed by Pearson correlation (PROC CORR). Proliferation and apoptosis data were analyzed by generalized linear mixed models (PROC GLIMMIX) with a binomial distribution, logit link function, year, treatment, and DIM as fixed effects, and heifer ID as a random effect. All other variables were analyzed by general linear mixed models (PROC MIXED) with treatment, DIM, year, and interactions as fixed effects, DIM within subject ID as the repeated measure, and compound symmetric or autoregressive covariance structure chosen based on AIC. Data were checked for normality and outliers. A The dams of the heifers had significantly lower respiration rates and rectal temperatures if provided with active cooling during their dry period (i.e. during the last 46 d of gestation). THI of the heat and cooled sides of the barn were similar in 2014 and 2015 across the entire dry period . ThP = 0.003), where IUHT had significantly lower rectal temperatures in 2016, but higher temperatures in 2017 compared to IUCL. IUHT and IUCL heifers had similar gestation lengths, similar body weights throughout lactation, and their calves were similar in body weight at birth . Milk yield increased over the course of lactation (P < 0.0001) and there was a significant year effect (P = 0.01), where heifers produced, on average, 2 kg/d more milk in 2016 than 2017.When heifers born to heat or cooled dams were evaluated at maturity, there were no differences in their rectal temperature or respiration rate during the first 42 DIM . Howeverat birth . IUHT coP = 0.39). However, the alveoli from IUHT were significantly smaller in area compared to alveoli from the IUCL heifers . For both treatment groups, there were more alveoli present in the mammary gland at 21 DIM compared with 42 DIM . Interaction terms were not significant for alveoli area or number. Alveoli luminal area was positively correlated with the number of secretory cells comprising them . This tendency was largely attributed to a lower percent of proliferating cells in the stromal compartment (P = 0.06; P > 0.52, There was a tendency for IUHT heifers to have a lower percent of proliferating cells in the mammary gland compared to IUCL (0.8 ± 0.2% vs. 1.5 ± 0.3%, Maternal heat stress exposure during late gestation reduces milk yield during the first lactation of their offspring . HoweverIUHT heifers had higher colostrum yield but produced 1.3 kg/d less milk through the first 84 days in milk relative to IUCL heifers. A compilation of production data from 5 years of studies employing an experimental design identical to the present study revealed that IUCL heifers produced 5.1 kg more milk per day in the first lactation relative to IUHT heifers . This diMilk yield is a function of the number of secretory cells, the metabolic activity of those cells, and the size of the alveoli formed by the secretory cells , 18, 19.Cell proliferation and apoptosis, the main cellular processes involved in mammary remodeling, together determine the number of cells in the mammary gland throughout lactation . The graMammary development begins in utero. At birth, teats and a rudimentary ductal compartment are present, although the secretory tissue will not form extensively until the first pregnancy , 35. MilOur data suggest that adverse conditions experienced in utero alters mammary morphology in a way that may compromise milk production in the first lactation. However, it is important to note that the majority of mammary growth occurs postnatally. Substantial ductal elongation and branching occurs in the peripubertal period and with each subsequent estrous cycle and extensive lobulo-alveolar development occurs during pregnancy . AlthougIn summary, our data show that morphology of the lactating mammary gland of primiparous cows is altered by exposure to heat stress in utero, which may be associated with the observed phenotype of lower milk production. Deviations from the normal intrauterine environment appear to have consequences for the developing mammary gland that persist into adulthood, almost two years after the heat stress insult occurred. Further studies are needed to elucidate how in utero heat stress affects fetal and postnatal mammary development and to determine the specific mechanisms through which alterations in alveoli development affect milk production. Efforts aimed at improving production efficiency should consider how to modulate not only the ambient environment experienced by the dam but also the intrauterine environment experienced by the fetus.


Global livestock production is facing serious new challenges, including climate-driven changes in parasite epidemiology, and anthelmintic resistance, driving a need for non-chemotherapeutic methods of parasite control. Selecting for genetic resistance to gastrointestinal nematode infection could reduce reliance on chemical intervention and mitigate increases in parasite challenge due to climate change. Ewes of the composite Exlana breed with a range of estimated breeding values (EBVs) based on nematode faecal egg counts (FECs) were monitored during the peri-parturient period on two farms in southwestern England. Ewes with low EBVs (“resistant”) had lower FECs during the peri-parturient period than those with high EBVs (“susceptible”): the mean FEC was reduced by 23% and 34% on Farms 1 and 2, respectively, while the peak FEC was reduced by 30% and 37%, respectively. Neither EBV nor FEC were correlated with key performance indicators . Simulations predict that the reduced FECs of resistant ewes would result in a comparable reduction in infection pressure (arising from eggs shed by ewes) for their lambs. Furthermore, although the reduced FECs observed were modest, simulations predicted that selecting for nematode resistance in ewes could largely offset predicted future climate-driven increases in pasture infectivity arising from eggs contributed by these ewes. Selective breeding of the maternal line for nematode resistance therefore has potential epidemiological benefits by reducing pasture infectivity early in the grazing season and alleviating the need for anthelmintic treatment of ewes during the peri-parturient period, thus reducing selection pressure for anthelmintic resistance. These benefits are magnified under predicted future climate change. The maternal line warrants more attention in selective breeding programmes for nematode resistance. FurtherThe observed impact of selecting individuals with low faecal egg counts (FECs) on gastrointestinal nematode epidemiology, for example to reduce infection pressure in subsequent years , the relEwes are known to increase nematode egg shedding in faeces during the peri-parturient period, around the time of lambing. This peri-parturient rise (PPR) in FECs provides an important source of eggs to contaminate pastures and increase subsequent infection pressure for lambs. Many farms treat ewes around the time of lambing to minimise contamination of lambing paddocks. Due to the contribution of the eggs produced during the PPR to the nematode population infecting lambs each grazing season, however, there are concerns that this strategy may select heavily for drug-resistant nematodes . FurtherEstimated Breeding Values (EBVs) are used by commercial breeders to select for desirable traits as they take into account the effects of genotype and environment on the observed phenotype . Hence, A potential limitation to the strategy of breed improvement for parasite resistance is that it takes many years to attain goals, yet is necessarily tied to performance under current environmental and management conditions. Where the epidemiological situation is changing, as for gastrointestinal nematodes under climate warming , currentIn this paper we aim to determine whether selection of ewe lambs for reduced FEC leads, in later life, to lower egg output during the PPR. Further, we explore the benefit of reduced PPR in terms of lower gastrointestinal nematode infection pressure for lambs under current and future climate scenarios.22.1http://www.sig.uk.com/). The Exlana is a composite hair breed sheep based on more than 14 breeds selected for their favourable or low input features, such as the Wiltshire Horn, Barbados Blackbelly and Lleyn. The dominant gastrointestinal nematode species in this region is Teladorsagia circumcincta. Haemonchus contortus infection was not observed or suspected on these farms.Exlana ewes on two separate farms in southwestern England (referred to as Farm 1 and Farm 2) were monitored in the spring of 2014 as part of an ongoing commercial breed improvement programme (Sheep Improved Genetics Ltd – http://www.signetfbc.co.uk/) using the Best Linear Unbiased Prediction (BLUP) method on FECs taken when the ewes were 5–6 months old. BLUP takes into account individual FECs, environmental effects such as management group, and pedigree  and low (negative) scores. EBVs were estimated by Signet . No supplementary feed was given.All ewes lambed over a 10-day period in mid-March, and within each farm were managed together for the duration of the study. On Farm 2, monitored ewes were all of similar age (2 or 3 years), and gave birth to twins (with the exception of one triplet-bearing ewe). This restriction was intended to reduce variation in FEC due to, inter alia, unequal resource allocation trade-offs among ewes feeding different numbers of lambs, which could modify the expression of immunity and hence FEC during the PPR . EquivalNematodirus spp. eggs and coccidia oocysts were counted separately. Raw counts were used for statistical analysis and transformed to epg for data presentation. For each farm, the week of peak FEC was extracted for individual ewes with at least three FECs over the four sampling periods to estimate the timing and magnitude of the peak FEC during the PPR. For all other analyses, the complete dataset was used, except 11 ewes that were excluded from the dataset for Farm 2 as FECs were only available for the first sampling period. A further four ewes with a single FEC in either week 5 or 7 were also identified on Farm 2. However, these ewes were not excluded as these FECs fell within the plausible peak of the PPR.Around the time of lambing, and on three further occasions in the next eight (Farm 1) and 11 (Farm 2) weeks, individual faecal samples were taken from ewes per rectum by the farmer. Not all ewes provided a sample on each occasion. On Farm 2, lambs were weighed at approximately 8 weeks of age as a measure of dam milk production, and ewes were weighed in late pregnancy and at the end of the study. Faecal samples were sent to the laboratory by post where they were stored at 8 °C, and FECs were conducted within 5 days using a modified McMaster method with a detection limit of 10 eggs per gram of faeces (epg). Trichostrongylid nematode eggs, 2.2The relationship between the FEC-EBV estimated at 5–6 months of age and observed FEC in individual ewes around lambing was assessed by a Spearman rank correlation, using the peak FEC per ewe, and average FEC per ewe over all sampling occasions. One-way Kolmogorov-Smirnov tests were applied to these mean and peak FECs for ewes in low and high EBV groups to determine whether these observed FECs were drawn from the same distribution.P < 0.05 cut-off value.Differences between the average FEC at each sampling point, and over the entire duration of the study, were compared between high (positive or zero) and low (negative) EBV groups using bootstrapping, following 2.3To evaluate whether enhanced immunity against nematodes, manifested as reduced FECs, came at the cost of reduced resources for milk production or for maintenance of body weight, a Spearman rank correlation was used to assess associations between EBVs, FECs, and indicators of performance, i.e. mean lamb weight per dam at 8 weeks of age, and ewe weight loss between late pregnancy and the end of the study.2.4T. circumcincta  using the lsoda function in the deSolve package (The impact of the observed reduction in PPR on pasture contamination and lamb exposure to third-stage infective larvae (L3) arising from eggs deposited by ewes was simulated using the GLOWORM-FL model, which tracks the climate-dependent population dynamics of the free-living stages of umcincta . The mod package .Fig. 1Concdf4  format data files. R package version 1.9. http://dwpierce.com/software) and chron  packages in R. Climatic time series were continuous except for one missing value in the precipitation dataset, which was replaced with 0 mm to match rainfall values on adjacent days.Model input required daily temperature and rainfall data. Mean daily air temperature (°C) and total daily precipitation (mm) for the grazing season (24 March 2014 to 30 November 2014) were extracted from the EOBS gridded dataset v11.0  using thapproxfun function in R, to estimate daily group FEC values. Ewe FEC input after weaning was reduced to 0 to simulate removal of ewes from the pasture. Model simulations track the fate of these eggs excreted by ewes during the PPR in FECs  and provide daily estimates of the number of infective larvae on herbage (L3h) per unit area (per hectare in the present study).Model input also required the number of eggs deposited on pasture per hectare, per day, between lambing and weaning, which was estimated as the daily mean group FEC for the high and low EBV ewes, respectively, multiplied by a representative stocking rate of five ewes per hectare (ha) and 2000 g of faeces produced daily ; Fig. 1B−1(dry matter) ha−1 was estimated using the L3h ha−1 (L3h) from the model output and a representative herbage biomass (B) of 2000 kg DM ha−1 . For this, L3h kg DM DM ha−1 . This wa weaning , and theDM day−1 ; Fig. 1AEggs deposited by lambs were not included in the simulations, since the aim was to track the consequence of eggs produced from ewes on lamb infection pressure.2.5Gastrointestinal nematode infection pressure is predicted to increase under climate change . Additio3 factorial design considering the nematode species (T. circumcincta and H. contortus), host resistance status (susceptible and resistant), and climate (historic and future high emissions scenario). Although T. circumcincta is currently more common in the study area than H. contortus, simulations suggest increasing H. contortus in northern Europe in future , and were removed at weaning on day 181 (end of June) when lambs were approximately 12–14 weeks of age.T. circumcincta were defined using the high and low EBV ewe FECs observed in this study on Farm 2. Haemonchus contortus FECs are typically several-fold higher than T. circumcincta, therefore the same FEC profiles used for T. circumcincta were adapted for H. contortus using a multiplication factor of 5. Linear interpolation was performed on the raw FEC data using the approxfun function in R, to estimate daily group FEC values.Representative FEC profiles for Historic climate data for a baseline period (1970–1998) and future projected data for 2071–2099 under the RCP8.5 scenario were obtained for southwestern England from the Coupled Model Intercomparison Project Phase 5 CMIP5;  as descrThe impact of resistance, climate change, and resistance plus climate change was estimated by calculating the percentage change in annual pasture contamination compared with baseline simulations of susceptible (high EBV) ewes using the historic climatic data.33.1An average of 3.5 faecal samples per ewe on Farm 1 (164 FECs on 47 ewes), and 2.6 on Farm 2 (166 FECs on 65 ewes) were analysed during the peri-parturient period. Individual ewe FEC and EBV data for both farms are provided in Supplementary Tables S1 and S2. On both farms, the average FEC increased after lambing, peaking at subsequent sampling occasions, before declining . FECs pers = 0.31, P = 0.058). On Farm 1, peak coccidian oocyst counts were lower than on Farm 2 , and there was no correlation between them and nematode egg counts.The coccidian oocyst count also peaked on Farm 2 in week 5 at 521 oocysts per gram of faeces (range 0 to 2640), and tended to be higher in ewes also shedding high numbers of nematode eggs  and peak FEC and EBV  on Farm 2, where EBVs ranged from 0.46 to 0.49 , where the range of EBVs was smaller  and peak FECs  between the low and high EBV ewes on Farm 2, but not on Farm 1 .There was a weak, positive relationship between mean FEC and EBV ( to 0.49 . There welations . The diselations . There wP = 0.036) and week 7  on Farm 2, although not on Farm 1. Bootstrapped peak FECs were 30% and 37% lower in low EBV ewes compared with high EBV ewes on Farms 1 and 2, respectively , but was not correlated with EBV  nor average FEC over the PPR .On Farm 2, for which ewe and lamb live-weight data were available (Supplementary Table S3), ewes lost an average of 6.27 kg (±S.D. 5.12 kg) between lambing and weaning (reducing from 56.8 2 kg (±S.D. 4.58 kg) to 50.55 kg (±S.D. 6.00 kg)). Ewe weight loss tended to be higher in ewes that produced higher maximum FECs, though there was substantial unexplained variability , mean ewe FEC , nor FEC EBV .Lambs weighed an average of 21.26 kg (±S.D. 3.41 kg) at 8 weeks of age and ewes produced a total 8 week lamb weight of 35.13 kg (±S.D. 9.28 kg). Eight week lamb weight was not significantly correlated with maximum ewe FEC  of between 34% and 38% .Table 2TH. contortus and T. circumcincta total pasture contamination increased under the future RCP8.5 scenario of climate change (Compared with the baseline simulations of susceptible (high EBV) ewes under historic climatic conditions, both e change .Introducing resistant (low EBV) ewes was predicted to mitigate the impact of climate change, bringing pasture contamination back in line with historic baseline predictions for susceptible (high EBV) ewes , Fig. 6.4Ewes on two commercial farms were evaluated for resistance to gastrointestinal nematode infection as ewe lambs as part of a breed improvement programme, using EBVs based on FECs, and monitored after lambing to assess the impact of genetic status on nematode egg output during this critical period, when egg counts typically show a transient rise (the PPR). The impact of an attenuated PPR on the simulated exposure of lambs to infection arising from eggs excreted by ewes during the peri-parturient period, and the potential for resistant ewes to reduce future impacts of climate change on infection pressure, was simulated.Resistance to gastrointestinal nematodes is largely a result of an acquired IgA-mediated regulation of worm fecundity after 3 months of age whereas variation in worm burden is not thought to be under genetic control in older lambs . As lambAlthough the correlation between EBV and FEC was weak on Farm 2 and non-significant on Farm 1, discretizing the data into low  and high  EBV ewes revealed that a low EBV was associated with reduced egg output during the peri-parturient period. Peak egg output was reduced by 30–37% in low compared with high EBV ewes, and overall egg output during the peri-parturient period was reduced by 23–34%. EBVs evaluated at 5–6 months of age may therefore be an appropriate phenotypic parameter to select for resistance to nematodes in maternal lines.www.signetfbc.co.uk). Previous studies have used a range of methods to evaluate resistance, e.g. FECs taken at 12 months of age  Signet Breeding Services: s of age , and muls of age . HoweverTrichostrongylus colubriformis infective larvae on pasture grazed by susceptible (control) Romney ewes during the peri-parturient period were over four times higher than on pasture grazed by resistant Romney ewes. Subsequent infection of lambs amplifies nematode populations on pasture, and survival of infective larvae over winter carries infection over to the next grazing season, magnifying onward epidemiological impacts of selective breeding on egg output during the PPR  ewes compared with high EBV (susceptible) ewes translated into a similar reduction in exposure of lambs to L3. The small variation observed is due to interactions between egg deposition and weather conditions, which determine the development success of eggs on pasture. The predicted reduction in pasture contamination on fields grazed by “resistant” ewes is supported by the observation of  the PPR .Based on a conservative mean establishment rate of 12.7% (to account for the impact of developing acquired immunity on establishment rates; Mounting an immune response is energetically expensive  and the In this study, the high and low EBV ewes and their lambs were grazed on the same paddocks on both farms and shared a common environment. Therefore, the overall exposure to infection was similar for all lambs and the lambs born to high EBV ewes benefitted from the reduced FEC of co-grazing low EBV ewes. Given the negative association between worm burden and daily live weight gain , selectiPrevious modelling studies have demonstrated the potential for carry-over of L3 pasture contamination from year to year, to magnify the benefits of breeding for resistance . HoweverT. circumcincta. Notably, the observed decrease in egg output achieved by vaccination is greater than the average decrease achieved in the present study through selective breeding. Despite the relatively smaller reduction in FECs in the present study, the predicted benefits of this reduction in the context of reduced pasture contamination and reduced lamb exposure to infection lend support to the future role of vaccination in controlling gastrointestinal nematodes. Selective breeding might also enhance vaccine effectiveness in the field, as indicated by breed differences in responsiveness to a T. circumcincta vaccine (Breeding for resistance shows considerable potential to mitigate the impact of climate change and offers a sustainable alternative to chemical interventions. Furthermore, the predicted benefit of breeding for nematode resistance in the maternal line is relevant to other novel approaches to gastrointestinal nematode control that achieve similar reductions in FECs. For example,  vaccine .The simulations presented here considered only the impact of climate on the free-living component of the nematode life cycle as this is now well-characterised  while thPasture infectivity is highly variable from year to year due to the impact of variable weather conditions on the survival and development of the free-living stages . It is tP values estimated by Monte Carlo simulation tended to be higher than the traditional acceptance threshold of 0.05 on Farm 1. This is reflected in the overlapping distributions of FECs in high and low EBV ewes. This is, to an extent, expected given the tendency for negative binomial parasite distributions within host populations. However, this could also be due to the lower sample size, narrower range of EBVs, and especially the looser inclusion criteria for the observed group on Farm 1, with ewes not managed in groups of similar age or number of lambs born, due to limited availability of ewes. Higher variability between ewes in factors affecting FECs during the PPR, such as individual fitness, nutrition or infection rate, or the resource demands of variable numbers of lambs, is likely to decrease the power of statistical comparisons between groups, and confound results. It is recommended that future studies, as far as possible, match ewes on potentially important criteria such as number of lambs, in order to increase study power. Effects of genetic resistance to nematode infection act through enhanced acquired natural immunity, and are likely to be most evident in relation to less resistant animals when infection pressure and nutritional stress are both high (In this study, similar qualitative patterns were observed on both farms, although differences in FECs between high and low EBV ewes at times of peak egg output on Farm 1 were more elusive. There was a statistically significant difference between the distributions of low and high EBV ewe mean FECs and maximum FECs on Farm 2, but not Farm 1. Similarly, the oth high , and theBenefits from selective breeding for nematode resistance may be realised in the maternal line through reduced egg output during the PPR, with positive epidemiological consequences on infection pressure in lambs, and without negative consequences for other aspects of performance. Measuring FECs at approximately 5–6 months of age for EBVs, as recommended by some breeding services, was observed to be an appropriate phenotypic parameter to select for resistance to gastrointestinal nematodes in maternal lines. Measuring FECs approximately 5 weeks after lambing appears to be an effective way of comparing egg output during the PPR in ewes.T. circumcincta and H. contortus are predicted to improve in many regions as a result of climate change, increasing infection pressure. Predicted pasture infectivity from resistant and susceptible ewes under historic and a future climate change scenario in a temperate region in Europe demonstrated the potential of selection for resistance to gastrointestinal nematodes to mitigate the expected increase in challenge, and may offer a sustainable alternative or complement to chemical interventions.The developmental success and survival of the free-living stages of


A silk-based small-caliber tubular scaffold (SFTS), which is fabricated using a regenerated silk fibroin porous scaffold embedding a silk fabric core layer, has been proved to possess good cell compatibility and mechanical properties in vitro. In this study, the endothelialization ability and the steady-state blood flow of SFTSs were evaluated in vivo by implanting and replacing a common carotid artery in a rabbit. The results of the color doppler ultrasound and angiographies showed that the blood flow was circulated in the grafts without aneurysmal dilations or significant stenoses at any time point, and ran stronger and close to the autologous blood vessel from one month after implantation. The SFTSs presented an initial tridimensionality without being distorted or squashed. SEM and immunohistochemistry results showed that a clear and discontinuous endodermis appeared after one month of implantation; when implanted for three months, an endothelial layer fully covered the inner surface of SFTSs. RT-PCR results indicated that the gene expression level of CD31 in SFTSs was 45.8% and 75.3% by that of autologous blood vessels at 3 months and 12 months, respectively. The VEGF gene showed a high expression level that continued to increase after implantation. Vascular bypass grafting is a major mode of treatment for ischemic heart disease and peripheral vascular disease via autotransplantation, allotransplantation, and artificial vascular transplantation. Large caliber artificial vessels have been a successfully realized clinical applicationfor a long time; however, small-caliber artificial vascular transplantation has not been as successfully applied. In the clinic, small-caliber artificial vascular transplantations have thus far been performed by autotransplantation, such as heart bypass surgery using the saphenous vein or mammary artery ,2. HowevChemically synthesized polymers of expanded poly-tetrafluoroethylene or Dacron have performed very well in large caliber artificial vascular transplantation but have had poor patency and poor histocompatibility in small vascular transplantation ,4. The iThe silk fibroin molecule consists of 20 kinds of α-amino acids, in which the numbers of acidic amino acid (such as Asp and Glu) residues are greater than the numbers of alkaline amino acid (Lys) residues, leading the silk fibroin to exhibit a strong electronegativity. The inner surface of natural blood vessels has a negative charge, which protects blood vessel cells and regulates the balance of the coagulation system. Therefore, silk fibroin is more suitable for fabricating artificial vessels. In recent years, researchers have paid more attention to developing fibroin-based small-caliber artificial vessels using a variety of fabrication techniques, such as electrospinning and salt leaching. These products exhibit good biocompatibility in vivo and in vitro ,15. HoweIn comparison, rapid endothelialization plays a decisive role in realizing the patency of small-caliber vascular grafts. The native blood vessel wall is composed of three different layers: the intima, media, and adventitia . The intBombyx mori silk fibroin solution was prepared as described previously [2CO3 solution was used to treat raw silk to remove sericin. Degummed silk fibers were dissolved in CaCl2 solution. After dialyzing against distilled water at 4 °C for 3 days, the silk fibroin aqueous solution was concentrated to 8% (w/w).eviously . Briefly60 gamma irradiation and kept at 4 °C.SFTSs were fabricated as described previously . A silk n = 6 per time point). The autogenous common carotid arteries of rabbits were used as controls.Vascular grafts (15 mm in length and 3 mm in inner diameter) were implanted to replace the rabbit’s common carotid artery. The rabbits  were anesthetized by pentobarbital sodium  at 30 mg/kg via intravenous injection. The animal model was approved by the ethical committee of Soochow University (No. ECSU-2019000135). A midline incision in the neck was made, and the common carotid artery was dissected. After infusion of 100 U/kg heparin  intravenously, the common carotid artery was excised and replaced by SFTSs using two end-to-end anastomoses, and sutured with 8−0 nylon sutures . The surgical wound was closed in layers with absorbable 3−0 silk sutures  . Rabbits® contrast agent .After implantation, color doppler sonography was used to observe the dynamic blood flow in the SFTSs using the MyLabClassC doppler ultrasound equipment . Digital subtraction angiography was also used to evaluate the patency using an LA 523 contrast probe by injecting a SonoVueThe endodermis of SFTSs taken from rabbit necks at different time points was observed under an SEM  with an accelerating voltage of 3 kV. Samples were soaked three times with phosphate-buffered saline (PBS) (pH=7.4) and dehydrated by exposure to a gradient concentration of ethanol . Finally, the samples were fixed on copper plates and sputter-coated with gold, then observed by SEM.v/v), and then embedded in paraffin and sectioned into 5-μm-thick slices. These sections were used for further histological and immunohistochemical analysis as described below. For hematoxylin and eosin (H&E) staining: after deparaffinization by xylene and hydration by a graded concentration of ethanol , the sections were immersed in hematoxylin  solution for 10 min and 1% ammonia to make them appear blue. Next, the sections were treated in a series of graded alcohol baths  and counterstained in eosin  solution for 2 s, then washed and immersed in 95% ethanol for coloration and were made transparent with xylene for 5 min. Finally, observation was performed with an IX51 inverted microscope .For the histological evaluation, the grafts were harvested at the designed time point and fixed in 10% neutral buffered formalin for 2 days. Specimens were dehydrated in a series of graded ethanol baths, made transparent in a mixture of anhydrous ethanol and xylene  H2O2 to inactivate endogenous peroxidase, then heated at 9 °C in 10 mM sodium citrate (pH = 6.0) for antigen retrieval and blocked using 5% (w/v) bovine serum albumin (BSA). After removing BSA, sections were incubated overnight at 4 °C with a 1:800 diluted mouse anti-rabbit VEGF primary antibody  or mouse anti-rabbit CD31 primary antibody (Abcam). Biotinylated goat anti-mouse IgG (1:500 dilution)  was used as a secondary antibody. Color detection was performed by 3,3-N-diaminobenzidine tertrahydrochloride horseradish peroxidase color development kit (Gene technology) according to the manufacturer’s instructions. Finally, all sections were counterstained with hematoxylin and observed on an IX51 inverted microscope (Olympus). Immunofluorescence analysis was performed with 1:500 diluted Alexa Fluor® 488 goat anti-mouse IgG secondary antibody (Abcam). All sections were counterstained with 4,6-diamidino-2-phenylindole  and observed on a FV1000 confocal laser scanning microscope (Olympus) at an excitation wavelength of 488 nm.CD31 and VEGF were used to mark vascular endothelial cells (VECs). After deparaffinization and hydration, the sections were dipped in 3% . According to the supplier’s instructions, total RNA was extracted from the scaffolds using a total RNA purification kit , then reverse-transcribed using a cDNA synthesis kit . RT-PCR were performed as follows: 95 °C/15 min for pre-denaturation, 40 cycles of 95 °C/30 s for denaturation, (54–60 °C)/30 s for annealing and 72 °C/30 s for elongation, and carried out on a Step One Plus Real-Time PCR System . The primers and the annealing temperature of each gene are shown in Keeping an unobstructed blood flow is the most important factor that determines whether an artificial blood vessel can be applied or not. In our work, firstly, we investigated the endurance of the dynamic patency of SFTSs during and up to 12 months following surgery. We visually observed that the appearance of the lumen surface of the removed tubular scaffolds was very clean and smooth with no signs of aneurysmal dilations or significant stenoses at any time point. The SFTSs retained their initial tridimensionality and smooth inner surface.In the early stages of implantation, the blood flow signals became relatively weak when flowing from natural blood vessels through an exogenous material surface A,B. ThreThe frequency spectrum of blood flow showed that the systolic and diastolic blood flow velocities became strong and stable spontaneously via the inner surface of the SFTSs, close to the autologous blood vessels on the opposite side. The results indicated that the SFTSs had a satisfactory ability against thrombus formation. Regenerated silk fibroin possesses better blood compatibility in comparison with poly-tetrafluoroethylene and Dacron . CertainThe inner surface of the SFTSs removed at each time point after implantation were observed by SEM, as shown in Because rapid re-endothelialization can be of vital importance, the ability of the SFTSs to endothelialize was verified by a histologic analysis and an immunohistochemical analysis. After implantation for 2 weeks, H&E staining showed that a large number of VECs had migrated to and engrafted on the inner surface of SFTSs, as shown in VEGF is a stimulating factor of endothelial cell growth and induces endodermal formation and angiogenesis ,34. We eCD31 is a membrane glycoprotein and is distributed in the endodermis to closely connect VECs to each other and maintain the integrity of the endothelial monolayer’s structure ,36. FiguThe immunohistochemistry results for VEGF and CD31 expression showed that VECs preferred to rapidly adhere to the inner surface of the SFTSs, and exhibited high proliferation activity and a secretory capacity in the extracellular matrix. A complete endodermis was formed at 3 months after surgery, and it self-assembled into an autologous intima-like groove structure at 6 months. These results are consistent with the SEM observation results.In this study, the gene expression levels of VEGF and CD31 were investigated by RT-PCR to evaluate the intima’s formation ability. In the experiment, samples were selected from the whole implant rather than only the intima. The relative expression level of CD31 reached 45.8% at 2 weeks and 75.3% at 12 months after implantation in comparison with the autologous blood vessel A. The reDue to the risk and impact of vascular diseases, such as atherosclerosis and angiomas, and the limited availability of autografts, tissue engineering strategies for vascular engineering have mainly focused on the creation of small-caliber artificial vessels. However, the patency of small-caliber artificial vessels still faces great challenges. A healthy endothelial layer is the only fully blood-compatible surface, and excellent mechanical properties provide a steady blood flow that can completely avoid thrombus development. The employed SFTSs exhibited good biocompatibility and excellent mechanical properties in our previous studies. The present results indicate that VECs or EPCs rapidly proliferated onto the inner surface of SFTSs, an endodermis formed at 1 month after implantation, and the blood flow velocity was able to spontaneously recover to normal and remain stable without thrombosis, restenosis, and occlusion. In addition, the SFTSs had the biodegradability to allow for full regeneration and functional recovery, and hence extend thelife expectancy of patients.


While alternating current stimulation (ACS) is gaining relevance as a tool in research and approaching clinical applications, its mechanisms of action remain unclear. A review by Schutter and colleagues argues for a retinal origin of transcranial ACS’ neuromodulatory effects. Interestingly, there is an alternative application form of ACS specifically targeting α-oscillations in the visual cortex via periorbital electrodes . To further compare these two methods and investigate retinal effects of ACS, we first aim to establish the safety and tolerability of rACS.The goal of our research was to evaluate the safety of rACS via finite-element modeling, theoretical safety limits and subjective report.20 healthy subjects were stimulated with rACS as well as photic stimulation and reported adverse events following stimulation. We analyzed stimulation parameters at electrode level as well as distributed metric estimates from an ultra-high spatial resolution magnetic resonance imaging (MRI)-derived finite element human head model and compared them to existing safety limits.Topographical modeling revealed the highest current densities in the anterior visual pathway, particularly retina and optic nerve. Stimulation parameters and finite element modeling estimates of rACS were found to be well below existing safety limits. No serious adverse events occurred.Our findings are in line with existing safety guidelines for retinal and neural damage and establish the tolerability and feasibility of rACS. In comparison to tACS, retinofugal stimulation of the visual cortex provides an anatomically circumscribed model to systematically study the mechanisms of action of ACS. Non-invasive brain stimulation (NiBS) is an effective method for research, as well as a promising tool for therapy in cognitive and clinical neuroscience . Its effRetinofugal alternating current stimulation (rACS) is a comparably novel form of alternating current stimulation (ACS). In contrast to tACS, rACS is characterized by transmission along retinofugal tracts terminating predominantly in cortical visual areas and neuromodulation of central rhythms . While dBefore any novel method can be employed to its full potential or compared with other methodologies, establishing its safety and tolerability is critically important . The lacRefraining from potentially dangerous invasive measures, the safety of a novel NiBS montage should be assessed in several different ways.2) and charge density (C/m2), although other parameters such as charge per phase (C/ph) have been proposed to account for the shifting polarity of AC stimulation  Stimulation parameters (current and charge densities at the electrode) are within theoretical safety limits, (2) finite element modeling data shows the same for EF estimates and current densities at eye, retina and cortex, and (3) adverse events do not exceed that of other established stimulation methods in rate as well as severity.To address the primary hypothesis, the stimulation parameters of rACS were recorded during stimulation and employed for the calculation of safety limits. Ultra-high resolution topographical finite element modeling was performed to identify regions of critical interest and to calculate theoretical safety parameters. Adverse events were identified with an extended adverse events questionnaire developed for tDCS . For dirTo address the safety profile of rACS, we observed and questioned 20 test subjects during rACS and PS sessions. We assessed cutaneous, retinal and central adverse events and drew a comparison between PS and rACS.We stimulated 20 healthy volunteers (10 men), mean age 25.9 ± 4.95, as part of a study investigating a common framework of action for NiBS. The subjects were interviewed prior to experimentation regarding their state of health. We applied established exclusion criteria for NiBS  and addeTM gold electrodes, Astro-Med, Inc., RI, United States) were contained in foam-padded stimulation goggles and bilaterally made skin contact via small felt buffers (0.35 cm2) superior and inferior to the eye. The return electrode  was fastened on the back of the neck at the midline.Retinofugal alternating current stimulation was applied via a multi-channel low-voltage stimulation device certified for clinical use, which delivered weak oscillatory current sinus-pulses over four individual periorbital electrodes, respectively . The four superficial active stimulating electrodes ” values to describe the resulting electrical power of an electric current stimulation would be misleading.Regarding charge, we decided to refrain from more complex line integral calculations, and instead used the following simple formula:This was done to ensure straightforward comparability of resulting values. It also adds to the rigidity of our safety considerations by rather over-than underestimating the injected charge.Photic Stimulation was applied via two 3 × 5 cm multi-color white LEDs contained in the stimulation goggles, which work via red, green and blue LEDs mixing their emissions to form white light. To be able to compare stimulation intensities with rACS, sinusoidal pulses of white light were applied at an intensity of 120% light threshold and with a frequency of 10 Hz. This threshold was also determined by an ascending method of limits and resulted in an average luminous intensity of 1.24 cd (±0.44) for stimulation.The stimulation was also delivered in 30 s blocks followed by 30 s pauses over 10 min, and the subjects received the same instructions as for rACS.The ultra-high resolution head and neck model  available through the IT’IS Foundation was used in this study . The nifThe stimulation electrodes were created as CAD files mimicking the exact physical geometry and dimensions of the electrodes used in the experiments. The electrodes were positioned interactively within the image data simulating the electrode montage used for rACS see . The adaThe representative isotropic average electrical conductivities assigned to the different tissue compartments and the electrode materials (in S/m) are listed in The Laplace equation was solved and current densities corresponding to 350 μA total current were applied at the anode or active electrode (s). Ground was applied at the return electrode and all other external surfaces were treated as insulated. The linear iterative solver of conjugate gradients with a relative tolerance of 1E-6 was used.Surface as well as cross-sectional EF magnitude maps on the gray matter, retina, and the optic nerve were obtained. For the scalp, the surface current density magnitude plot was obtained.The questionnaire we employed is based on the one proposed by We modified the questionnaire by adding a description of phosphenes. Furthermore, to assess the overall tolerability, we defined the broad category of “Pain” as a summary of all discomforting sensations mentioned above and added a Numeric Rating Scale   as a morThree months after stimulation, the subjects received a second questionnaire to identify late and longer lasting after-effects.P-values of <0.05 were considered significant. All analyses were performed using IBM SPSS Statistics, Version 19.0.0.1 .As the data is not normally distributed and equal variance of residuals cannot be assumed, the severity of pain was analyzed in Wilcoxon Signed Ranks Tests for paired samples. The nominally scaled side effects were analyzed in Fishers Exact Tests, as expected values in several of the cells of a contingency table are below the recommended threshold for a classical Chi-Squared Test . P-value2 (SD 0.28), and the charge density to 0.60 C/cm2 (SD 0.11). As sine waves pulses were employed, we additionally calculated the effective amplitude, resulting in a mean 248.68 μA (SD 47.0). Using effective amplitude, current density amounted to a mean 0.71 mA/cm2 (SD 0.13), and the charge density to 0.42 C/cm2 (SD 0.08). RACS was found to be well within safety limits and the findings comparable to other similar stimulation methods (see 2) is smaller than most tES montages (16–35 cm2), comparable only to Electrosleep and TCES (0.35–1.25 cm2). Regarding stimulation frequencies, rACS was compared to studies using similar frequencies (10–20 Hz), with the exceptions of Electrosleep, which is set at higher frequencies (100 Hz) as well as the non-oscillating tDCS and Gekeler’s TCES. The calculations following these observations place the charge density of rACS just above the TCES of 2) ranks below Ma (1.2 mA/cm2), well below the maximum intensity employed by Gekeler (8.57 mA/cm2) and far below the safety limit proposed by McCreery (25 mA/cm2). These findings are even more pronounced when using effective amplitude.An average 10 Hz phosphene threshold at 290.50 μV (SD 45.36), impedances at 12.05 kΩ (SD 2.89), and an average amplitude of 351.69 μA (SD 63.95) were noted. Calculated from peak-to-peak amplitude, the current density at electrode level amounted to a mean 1.00 mA/cmhods see . RegardiThe EF distributed by rACS is strongest at the eye level, with the highest current density estimates at the retina. Further areas of elevated current densities are optic nerve and cortex .2, while optic nerve (0.33 A/m2) and cortex (0.13 A/m2) were both subjected to less current flow. Regarding the EF, we estimated a maximum of 2.6 V/m in the optic nerve, followed by 1.99 V/m for the retina and 0.47 V/m for the cortex. Finally, current density at skin level underneath the active electrode amounted to a maximum induced value of 14.79 A/m2 , which all occurred exclusively in rACS. For a more detailed view, see In the statistical analyses, rACS and PS showed no significant effect of stimulation type (rACS versus PS) on pain intensity as assessed by Wilcoxon Signed Ranks Tests , fatigueTo address the safety profile of rACS, we assessed theoretical safety limits as well as finite-element modeling data and compared the reported adverse events for rACS and simple PS.The primary findings are that rACS is safe based on the following observations: (1) stimulation parameters (current and charge densities at the electrode) are within theoretical safety limits, (2) finite element modeling data shows the same for EF estimates and current densities at eye, retina and cortex, and (3) adverse events are comparable to PS in direct experimental comparison see , 4 and rTo be efficacious and safe, a stimulation system must stimulate neural tissue without damaging tissue or electrode. Tissue damage is caused when excitable tissue is overstimulated and electrode damage ensues as metal oxidation occurs . CurrentCurrent density is the proposed optimal safety parameter for a constant current stimulation  and can We find that rACS current densities are within reported safety limits for tissue damage .While current density is a well-established safety parameter, it is best suited for assessing the safety of constant current stimulation. ACS injects less charge than constant current stimulation of the same amplitude , dependeWe find that rACS charge densities are also within reported safety limits for tissue damage .While stimulating at higher current and charge densities than most forms of tES, rACS stimulation parameters proved comparable to dose parameters reported for TCES using up to 10 mA per pulse to establish safety guidelines , well beDespite arguable differences between different stimulation techniques, there are remarkable similarities, e.g., comparably distant periorbital montage of electrodes, as well as modeling results for the serial resistance of the skin and eyelid  to motivThis leads to the conclusion that the employed charge injection was safe with regards to possible tissue as well as electrode damage. In the future, studies addressing the calculation of rheobase and chronaxie and stimulation with variable pulse parameters might help to further reduce charge injection to the minimum necessary to efficaciously achieve a neuronal response .–inferior, we see stronger EFs in the temporal regions and at the return electrode. While there is increased flow through the subcortical structures, brain stem and cerebellum, there appears to be no strong current flow to occipital areas, with a maximum current density of 0.033 A/m2 and a maximum EF of 0.1208 V/m   . ConsideAdditionally, all of the EF estimates are at least one OOM below the safety threshold of 42 V/m . It shouNo fatal or serious adverse events  were obsAs the modeling results showed high maximum current densities and EF estimates at skin level, the presence of cutaneous adverse events during and after rACS comes as no surprise. Comparing rACS and PS regarding the summary category of pain, we have to note the complete lack of cutaneous sensations in PS and that multiple aversive sensations may be clustered and perceived in sum total as painful .None of the subjects reported skin rashes or damage. Whereas the applied charge density is clearly strong enough to stimulate C-nociceptors, it is too low and the duration is too short to induce skin damage . For dirElectrical stimulation of skin nociceptors is known to produce itching, burning and tingling sensations in the animal model, as well as in human subjects . While eOne third of the subjects reported pain with a median strength of 2.5 NRS. The sensation of pain during and after electric stimulation is understood to be a combination of several factors, with the terminal branches of C-nociceptors of the stimulated skin acting as the primary central conductor . This maWhile the feeling of pain and discomfort should be monitored closely in future studies, it should be noted that we found no significant difference between rACS and well-established and tolerable PS regarding overall discomfort/pain .This pain during and after PS is most likely a form of “discomfort glare” associated with visual discomfort, annoyance, irritability or distraction without affecting the ability to see, but leading to symptoms of visual fatigue .As we stimulated our subjects at 120% phosphene threshold, all subjects experienced phosphenes. These phosphenes induced by rACS were typically described as flickering at the edges of the field of view and not experienced as painful.Historically, phosphenes induced by alternating current have been seen as a purely retinal phenomenon  resultinYet, due to the respective montages there should be a magnitude of difference between methods  with TCEA previous tACS modeling effort indicated why transcranial stimulation may induce retinal phosphenes  by virtuWhere exactly rACS phosphenes are generated remains subject to further investigation. While we find the highest EF estimates in the optic nerve, other authors  suggesteFatigue, reported by one third of the subjects after rACS, has been suggested in previous research to be an unspecific effect of tES. Similar to rACS, the early approaches to tES involved two “active” electrodes placed directly over the eyes, presumably to facilitate active current delivery through the optic foramina. These montages were first used in Electrosleep research initiated in The findings in this study, that rACS produces more fatigue than PS, support the notion of an indirect and unspecific central (adverse) effect specific to electrical stimulation. This notion is in line with previous findings showing that action potentials induced by electrical stimulation of the retina can propagate directly to the visual cortex , produceBeyond fatigue, the possibility of direct structural damage to central nervous structures by rACS seems low considering the distance between charge injection and brain tissue as well as stimulation strength. Yet, for rhythmic PS the danger of inducing an epileptic seizure is well established. Although not found in this study, for electrical stimulation the danger must also be assumed to be high due to neurophysiological similarities with intermittent photic stimulation (IPS)  and provHaving theoretically and experimentally characterized the relative safety profile of rACS, we believe future studies can further investigate retinal mechanisms of action for ACS effects, especially in comparison with tACS. Additionally, rACS allows for studies addressing the interaction of different signal types entering the visual system through two separate input channels (left and right eye) and converging at the level of the primary visual cortex. This provides an promising tool for studies aiming to address a common framework of action for NiBS with more than one input-signal, e.g., noise and oscillation .The datasets generated for this study are available on request to the corresponding author.All protocols conformed to the Declaration of Helsinki, and were approved by the Ethics Committee of the Charité – Universitätsmedizin Berlin (“Ethikkommission der Charité – Universitätsmedizin Berlin”). Informed consent was obtained from all individual participants included in the study. This study adheres to the principles of good scientific practice of the Charité – Universitätsmedizin Berlin (“Grundsätze der Charité zur Sicherung guter wissenschaftlicher Praxis”).LH, AD, SS, MS, and SB conceived and designed the study. LH, AD, CT, AJ, and SS carried out data acquisition and analysis. LH, AD, and SS drafted the manuscript. CT, AJ, AK, MR, MS, and SB critically revised the manuscript. All authors participated in the interpretation of the data.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.