Organophosphorus pesticides (OPPs) cause great risk to human health as they are used globally. Therefore, the purpose of this research was to determine the total phenolics, flavonoids, and antioxidant activity of agricultural waste, as well as to control the pesticide residues  at a laboratory scale level using dried-milled fruit wastes. The pesticide residues parathion and diazinon were used at concentrations of 0.094, and 1.90 mg/mL respectively. The fruit wastes used in this study were orange and banana peels, as well as date stones, and they were used in two concentrations (3 and 9 g/30 mL deionized water). The total phenolic and flavonoid contents and the antioxidant activity were measured in fruit wastes. Also, the Fourier transmitted infrared (FTIR) spectra of fruit wastes were established to figure out the nature of the functional groups found before and after pesticide residues removal. The ability of fruit wastes to remove pesticides residues was determined using Gas Chromatography/Mass spectrometry (GC/MS). Data showed that date stones contained a higher amount of total phenolic content than orange and banana peels. However, orange peels contained a higher amount of total flavonoid contents than those of date stones and banana peels. As for antioxidant activity, banana peels recorded the higher antioxidant activity, followed by orange peels and date stones respectively. Results revealed that there was no relation between total phenolic content, total flavonoid content, and antioxidant activity. Results also indicated that date stones at a concentration of 9 g successfully reduced diazinon (81.18%), followed by banana (63.86%) and orange peels (43.42%) respectively, whereas parathion was reduced by banana peels at a concentration of 9 g (50.34%), followed by orange peels (45.28%), and date stones (39.52%) respectively. This study demonstrated that agricultural wastes were effective in the adsorption of diazinon from water, and their use is considered safe for the environment.  •Date stones successfully reduced diazinon by 81.18%, whereas banana peels effectively reduced parathion by 50.34%.•FTIR spectra of fruit wastes displaced severeal peaks.•Date stones contained a higher amount of total phenolic content.•Orange peels contained a higher amount of total flavonoid contents.•Banana peels recorded the higher antioxidant activity. Occupational exposure to pesticides is ubiquitous among farmers worldwide In recent years, organochlorine pesticides have been gradually replaced in favor of more efficient, safer chemicals with faster biodegradation rates, such as organophosphorus insecticides Orange peels are largely made up of cellulose, pectin, hemicellulose, lignin, chlorophyll pigments, and other low molecular weight hydrocarbons with multiple hydroxyl functional groups, making it a potential sorbent for a variety of contaminants Date stones have been shown to have high nutritional value, particularly in terms of fiber and antioxidant content It could be observed that the removal of diazinon and parathion by orange and banana peels and date stone was not previously studied. Therefore, this study aimed to determine the total phenolics, flavonoids, and antioxidant activity of agricultural waste, as well as to control the pesticide residues  at a laboratory scale level using dried-milled fruit wastes. This experiment was performed as a simulated model to predict the behavior of these agricultural wastes for the removal of pesticide residues from irrigation waste water. The success of this system would provide a novel strategy to solve the pesticide residues issue, as well as, save the environment against contamination.22.1Diazinon and parathion pesticides were purchased from Santa Cruz Biotechnology Inc., .2.2Citrus sinensis L.), and banana (Musa sp) were obtained from the farms of the Egyptian Ministry of Agriculture on December, 30th 2020. Dates (Phoenix dactylifera L.) were obtained from a date farm in El-Ayat, Giza on January 4th, 2021.About 10 kg of each of the following fruits were obtained for the experiments. Ripened fresh fruits of sweet orange of Washington Navel orange  to remove any dust or debris that had adhered to the peels and then wiped dry. Fruit peels were separated manually and cut into small parts for about 2 × 2 cm, then sun-dried for 96 h. The fruit peels were then ground thoroughly by a house mill and passed through a 0.25 mm mesh.2.4Date stones were cleansed with water to remove any adhered date flesh before being sun-dried. A heavy-duty mill  was used to grind the collected date stones. The powder was then passed through a 0.25 mm mesh.2.5g to remove the agricultural waste. The samples were filtered, and the filtrated solutions were analyzed for the pesticide residues using GC/MS/MS Diazinon and parathion were added separately at a concentration of 1.90 and 0.094 mg/mL respectively to water containing each of the following separately; 1) ground/milled orange peels 2) ground/milled banana peels, and 3) ground/milled date stones at concentrations of 3.0 and 9.0 g/30 mL deionized water. Larger agricultural waste quantities were not evaluated to avoid an overabundance of biosorbent, which would limit practical uses. Positive controls were prepared to contain each of the pesticides separately without the ground/milled agricultural wastes. Negative controls were prepared to contain each of the ground/milled agricultural wastes separately, without pesticides. The samples were shaken in a shaking incubator  . The suspensions were centrifuged  at 1750 × 2.6Gas Chromatography system 7890B with tandem mass spectrometer 7010A Quadrupole  was used. Chromatographic separations were achieved using the HP5MS ultra inert capillary column . Mass Hunter software version 7.01  was used for instrument control and data acquisition/processing. Under scanning settings, a mass spectral library (NIST 14) was utilized to confirm pesticides and identify co-extractives. The GC oven temperature was programmed to initially be held at 70 °C for 1 min then ramped to 150 °C at 50 °C/min for 0 min, and raised to 260 °C at the rate of 6 °C/min for 0 min, then ramped from 260 to 310 °C at 20 °C/min for 1.567 min with a total run time of 25 min. In splitless mode, one microliter of each sample was injected, and detection was achieved using electron impact ionization in positive ion mode (70 eV). At a flow rate of 1.654 mL/min ultra-high purity helium (> 99.999%) functioned as the carrier gas. The flow rates of collision cell gases  were 2.25 mL/min and 1.5 mL/min, respectively. The temperatures of the injector, transfer line, ion source, and quadrupole were 250, 280, 300, and 180 °C, respectively. During solvent delay time of 2 min, the filament current (100 μA) was switched off. The acquisition method was utilized in MRM mode, with one MRM transition used for quantification (quantifier peak) and the others for confirmation . Diazinon and parathion standards were used.2.7-1) at 4 cm-1 resolution was used to analyze the functional groups contained in dried orange and banana peel, as well as date stones Absorption spectroscopy in the infrared region (400–4000 cm2.8-2) . The temperature was kept constant (25 °C). A rotary evaporator  was then used to evaporate the extracts at room temperature. The recovered residues were re-evaporated to eliminate contaminants and kept at 4 °C until analysis.The milled peels of oranges and banana, as well the milled date stones were extracted using Ultra-sonic  assist in line with the methodology explained by Vinatoru et al. 2.9Folin-Ciocalteu’s reagent was used for the determination of total phenolic content, with gallic acid as a standard. At 765 nm, the absorbance was measured, and results were articulated as  per g) 2.10The aluminum chloride colorimetric method was used for the determination of total flavonoid contents with quercetin as standard. At 533 nm, the absorbance was measured, and the results were presented as  per g) 2.112.11.150 was also calculated, which indicated the quantity of fruit wastes (mg) in 1 mL of solution needed to reduce the initial concentration of DPPH radicals by 50%, with ascorbic acid as a standard. The results were articulated as  per 100 g).The DPPH was measured according to the method designated by Abdel-Razek et al. 2.11.2ABTS radical scavenging experiment was carried out according to Fitriana et al. 2.11.3A modified spectrophotometric approach according to the method of Ferreira et al. 2.12P < 0.05 was regarded statistically significant. All tests were performed in triplicate (n = 3).The data were presented as mean (95% CI). Statistical analysis was performed using the SPSS software version 16. A one-way analysis of variance (ANOVA) was carried out, in which 33.1Results in 3.2−1 were assigned to O-H stretching, carboxylic acids (–OH), C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="pc-E00C" format="PBM" resolution="300" x-size="12" y-size="9">000000000000000000000000000000000000111111111111000000000000111111111111000000000000000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="20.666667pt" height="16.000000pt" viewBox="0 0 20.666667 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z M0 280 l0 -40 480 0 480 0 0 40 0 40 -480 0 -480 0 0 -40z"/></g></svg>O stretching of amide, alkane (–CH3), C-O stretch, and C-H bending of alkynes, respectively. Similar findings have been stated by El-Nemr et al. The FTIR spectra of orange and banana peels, as well as date stones, were determined to understand the nature of the functional groups existing on the exterior of the date stones and peels , Table 2−1 and were assigned to O-H stretching, carboxylic acids (–OH), CO stretching of amide, alkane (–CH3), C-O stretch, and C-H bending of alkynes, respectively. In agreement, Memon et al. −1. Similar results were reported by Afolabi et al. Results in -1-1 suggests the existence of the carbonyls of phytosterols and fatty acids −1The presence of long-chain fatty acids, waxes, carotenoids, and phytosterols is thought to be consistent with the methylene v(C-H) asymmetric stretch between 2800 and 3000 cm−1 was decreased in the presence of diazinon, which means consumption of (O-H) groups by reaction or binding. The changes in the FTIR spectra of banana peels due to parathion existence were verified in the wavelength of 1630.5 cm−1. The change in the spectra could point out the possible mechanism of fruit waste impact to reduce the pesticide residues. Regarding the dates stones spectra, the changes appeared clearer regarding the presence of diazinon and parathion in date stones. The change of fruit waste spectra means a change in their active functional groups. These slight but significant changes in the FTIR spectrums following pesticide adsorption demonstrated that chemical adsorption of pesticides on fruit waste is achievable It is of importance to mention that the presence of pesticide residues led to a change in the appearance of the FTIR spectra, particularly in the part related to the (O-H) groups. The depth of the FTIR spectra of orange peels wavelength of 3424.96 cm3.3The total phenolic contents of the orange and banana peels and date stones were expressed as mg of gallic acid equivalent per g dry weight . Date st3.4Data in 3.550 value, which was also estimated against ascorbic acid as a standard reference (50 values. The presence of several antioxidant components may be responsible for the remarkable antioxidant capabilities of banana peels Antioxidant activity of the fruit peels and date stones was determined using three different assays , as welleference . ResultsSeveral studies have found that pesticide exposure can cause oxidative stress by increasing the formation of free radicals, which can accumulate in the cell and damage biological macromolecules There has been little research on the use of agricultural wastes for the removal of pesticides residues from water. Water quality has been a major source of concern in recent years, as it is one of the most important needs of humans, and animals The research's strength was studying the removal of pesticide residues by agricultural wastes, which received little attention, in contrast to the use of activated carbon prepared from agricultural wastes which received more attention Future research should include the detection of suitable and lower quantities of agricultural waste, as well as other agricultural wastes than those studied here. Future research should include the application of these agricultural wastes in the soil to study the removal of pesticides residues in soil.4Date stones extract recorded higher total phenolic contents; whereas orange peels extract recorded higher total flavonoid contents. On the other hand, banana peels extract recorded higher antioxidant activity. These results indicated that the enrichment of these agricultural wastes with phytochemicals might have provided a modern approach for reducing pesticide residues. The FTIR of agricultural wastes reflected significant changes after their exposure to the pesticide residues; this might be due to the removal of pesticide residues. More studies are required for the detection of suitable and lower quantities for the application of each fruit waste for the reduction of several pesticide residues. In this regard, the results of this study recommend date stones to be used to solve the commonness of pesticide residues in water.Omaima Hussain: Conceptualization, Methodology. Emam Abdel Rahim: Supervision. Ahmed Badr: Data curation, Visualization. Amal Hathout: Writing – review & editing. Magdy Rashed: Supervision. Ahmed Fouzy: Project administration, Funding acquisition.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Cardiac ventricular aneurysms affect 1 in 200,000 live births. To the best of our knowledge, no reported cases of a left ventricular pseudoaneurym and in utero rupture exist to guide optimal management.We present a case of fetal left ventricular rupture with a large pericardial effusion, cardiac tamponade and subsequent pseudoaneurysm formation with concerns for a poor prognosis. Interventional drainage of the pericardial effusion led to resolution of tamponade and significant improvement in fetal condition. A multidisciplinary team was utilised to plan birth to minimise risk of pseudoaneurysmal rupture and a catastrophic bleed at birth.For similar cases we recommend consideration of birth by caesarean section, delayed cord clamping and a prostaglandin E1 infusion, to reduce the systemic pressures on the left ventricle during transition from fetal to neonatal circulations, until definitive surgical repair. In this case, this resulted in a successful outcome. Cardiac ventricular aneurysms affect 1 in 200,000 live births [A 25-year-old primiparous woman was referred for tertiary specialist fetal assessment at 23 weeks’ gestation following a mid-trimester ultrasound indicating right outflow tract obstruction with pericardial effusion. Her pregnancy was previously uncomplicated. She had no significant past history. On review, a complex epicardial mass associated predominately with the left ventricle and a massive pericardial effusion was seen Fig. , suggestAt 24 + 1 weeks’ gestation, the fetal pericardial effusion appeared stable but with an increasing epicardial thrombus. There was bidirectional flow between a pseudoaneurysmal defect and the left ventricle. An abnormal ductus venosus Doppler waveform, indicating poor ventricular function secondary to cardiac tamponade, and new onset ascites was evident.Drainage of the massive pericardial effusion was deferred at 25 weeks’ gestation, despite worsening hydropic features, due to bleeding into the pericardial space raising concerns for pseudoaneurysmal rupture (Fig. A multidisciplinary team meeting with input from maternal fetal medicine, neonatology, paediatric cardiology, paediatric cardiac intensive care and perinatal physiology was conducted at 31 weeks’ gestation to plan birth and postpartum care. Concerns existed of pseudoaneurysmal rupture and a potential catastrophic bleed during transition, as afterload pressures on the pseudoaneurysm increase due to increased systemic vascular resistance upon cord clamping and the loss of the low resistance placental circulation.A caesarean birth was advised due to concerns of fetal stress and transient increases in placental resistance during labour increasing left ventricular afterload and the risk of pseudoaneurysmal rupture. Birth at term to minimise risks of prematurity and optimise birthweight prior to definitive surgical repair on cardiac bypass soon after birth was planned .A fetal MRI showed normal neuroanatomy at 33 weeks’ gestation. At 35 + 1 weeks’, a large left ventricular pseudoaneurysm persisted, but the pericardial effusion had completely resolved (Fig. An elective caesarean birth occurred at 37 + 5 weeks’ gestation following steroids to minimise the need for respiratory support. Cord clamping was deferred until after respiration was established to reduce pulmonary vascular resistance and allow left-to-right shunting through the ductus arteriosus to minimise the increase in left ventricular afterload associated with cord clamping. A female infant was born weighing 3209 g with Apgars of 9 at 1 min and 9 at 5 min. In the immediate post-partum period, a prostaglandin E1 infusion was commenced to maintain ductal patency and reduce systemic and pulmonary vascular resistance. She remained stable and was transferred on day 1 of age to a quaternary paediatric cardiac centre for surgical management, undergoing aneurysmal resection with patch closure and ligation of a patent ductus arteriosus at 10 days of age. She made a good post-operative recovery and was discharged home at 19 days of age. On follow-up at 4 months of age she is thriving with a repeat echocardiogram showing good biventricular function.Here we describe a case of fetal cardiac left ventricular pseudoaneurysm with in utero rupture that resulted in a successful outcome. Due to the small isolated defect, it is thought to have occurred secondary to a single coronary artery occlusion causing a localised myocardial infarction. Counselling the parents on the likelihood of having a live born infant and the subsequent prognosis following birth was challenging due to the rarity of the condition.In view of the maternal primigravity and age, vaginal birth was considered to minimise maternal surgical morbidity in light of uncertain neonatal outcome. As labour and vaginal birth are known to cause transient episodes of fetal and placental hypoxia, leading to increased fetal acidosis and systemic arterial pressures, this mode of birth was thought to increase the possibility of pseudoaneurysmal rupture . UtilisiMany key cardiovascular changes occur during transition from a fetal to neonatal circulation, including a significant reduction in the pulmonary vascular resistance, an increase in systemic vascular resistance, and closure of the ductus arteriosus . Prior tWe would recommend that in similar cases caesarean birth, deferred cord clamping and immediate commencement of a prostaglandin E1 infusion be considered until definitive surgical repair. Importantly, the possibility of an excellent outcome is also valuable in counselling and guiding families that may be impacted by this rare complication in the future.


The magnitude, distribution, and characteristics of the electric field induced in the brain by application of transcranial electric stimulation depend on the electrode configuration used and the specific parameters of stimulation. An approach to calculate the generated electric field is the computational finite element method. This method enables simulation of the current spread and electric field strength according to the electrode configuration and stimulation parameters used. However, current approaches have several limitations, which constrain the application of tDCS in empirical research and clinical practice. In this study, we provide examples of standard model-based electric field simulations corresponding to motor, dorsolateral prefrontal, and posterior parietal cortex stimulation using twenty typical electrode configurations. Two different current flow-modeling tools were used to compare the results and determine possible differences between both procedures regarding the specificity, as well as the reliability of the estimates. The results were rather consistent between both simulations. Some modest differences of the simulated distribution and intensity of the electric fields between the results of the respective modeling approaches were identified, which might have functional significance and reveal the need to empirically validate these models. The non-availability of quantitative data about the precise electric field distribution beyond the cortical target is a common limitation of both methods, which limits the determination of the spatial specificity of the intervention. These findings help to define future directions of research that allow to exploit the full potential of standard simulation approaches in the field of non-invasive brain stimulation.Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation procedure to modulate cortical excitability and related brain functions. tDCS can effectively alter multiple brain functions in healthy humans and is suggested as a therapeutic tool in several neurological and psychiatric diseases. However, variability of results is an important limitation of this method. This variability may be due to multiple factors, including age, head and brain anatomy , cognitive reserve and baseline performance level, specific task demands, as well as comorbidities in clinical settings. Different electrode montages are a further source of variability between tDCS studies. A procedure to estimate the electric field generated by specific tDCS electrode configurations, which can be helpful to adapt stimulation protocols, is the computational finite element method. This approach is useful to provide a priori modeling of the current spread and electric field intensity that will be generated according to the implemented electrode montage. Here, we present standard, non-personalized model-based electric field simulations for motor, dorsolateral prefrontal, and posterior parietal cortex stimulation according to twenty typical tDCS electrode configurations using two different current flow modeling software packages. The resulting simulated maximum intensity of the electric field, focality, and current spread were similar, but not identical, between models. The advantages and limitations of both mathematical simulations of the electric field are presented and discussed systematically, including aspects that, at present, prevent more widespread application of respective simulation approaches in the field of non-invasive brain stimulation.  In the last two decades, a growing number of publications have provided evidence for the potential of transcranial electric stimulation (tES) techniques to modulate cortical excitability and underlying brain functions ,2. TransA drawback of tDCS effects is its outcome variability ,21, a feA method to calculate and simulate the intensity and directionality of the electric field, current density and spread induced in the brain by tDCS is the finite element model, which is based on magnetic resonance imaging (MRI) data to build anatomically realistic head models ,33. SeveConsidering this background, an area to be explored is the comparison of available standard modeling approaches to analyze the resulting electric field intensities and distributions when default values for respective simulations are taken, and thus to evaluate the predictive potential of these methods. Therefore, the objective of this work is to provide simulations of the electric field induced by twenty typical tDCS electrode configurations using two different current flow modeling tools based on standard head models. We explored simulations of tDCS targeted to three brain regions and compared the results gained by these tools. Respective model-based adjustments of stimulation protocols might be helpful to determine electrode configurations in future tDCS studies. This includes determination of the maximum intensity of the electric field generated in the region of interest (ROI) for each electrode configuration , as well as the distribution of the electric field dependent on electrode position and size, and current intensity. These data provide a priori information about predicted electric field strengths and characteristics, and are a relevant example how to define tDCS electrode configurations for empirical studies. The consistency or inconsistency of the results obtained by both modeling approaches will furthermore help to make assumptions about the level of reliability of these approaches in general, and the need for further empirical validation to exploit their full potential in basic research and clinical domains in future. In short, the simulations provided in this study are practical examples delivering a foundation for discussing the state-of-the-art, to analyze important shortcomings, and develop future research directions.6 tetrahedra and 7 × 105 nodes and was automatically created using the headreco tool of SimNIBS 2.1 [The SimNIBS 3.1.2 free-software , which iNIBS 2.1 . DefaultNIBS 2.1 . Tissue NIBS 2.1 , and theNIBS 2.1 ,52. The results from this simulation approach were compared with those of another electric field modeling tool based on the finite element method and a different head model: Computation of Electric field due to Transcranial current Stimulation (COMETS) v.2.0 . Here, aThe twenty electrode configurations whose electric fields were modeled are shown in 2 vs. 7 cm × 5 cm = 35 cm2), return electrode positions , and current intensities . The combination of these parameters and cortical regions resulted in the following twenty electrode montages: (1–2) anodal left primary motor cortex -cathodal right supraorbital region , at 1 and 2 mA intensity, respectively, and 20 cm2 electrode size; (3–4) anodal lM1-cathodal right M1 (C4 electrode position), at 1 and 2 mA, respectively, and 20 cm2 electrode size; (5–6) anodal lM1-cathodal rSOR, at 1 and 2 mA of intensity, respectively, and 35 cm2 electrode size; (7–8) anodal lM1- cathodal right M1, at 1 and 2 mA intensity, respectively, and 35 cm2 electrode size; (9–10) anodal left dorsolateral prefrontal cortex -cathodal rSOR, at 1 and 2 mA of intensity, respectively, and 20 cm2 electrode size; (11–12) anodal lDLPFC-cathodal right DLPFC , at 1 and 2 mA of intensity, respectively, and 20 cm2 cm electrode size; (13–14) anodal lDLPFC-cathodal rSOR, at 1 and 2 mA of intensity, respectively, and 35 cm2 electrode size; (15–16) anodal lDLPFC-cathodal rDLPFC, at 1 and 2 mA of intensity, respectively, and 35 cm2 electrode size; (17–18) anodal left posterior parietal cortex -cathodal rSOR, at 1 and 2 mA of intensity, respectively, and 20 cm2 electrode size; (19–20) anodal lPPC-cathodal rSOR, at 1 and 2 mA of intensity, respectively, and 35 cm2 electrode size. We explored the results for different electrode sizes  the maximum intensity of the electric field within the ROI = the peak of intensity as measured by V/m; and (ii) the electric field distribution associated with each electrode configuration as a measure of specificity. Contrasting the results of different stimulation protocols allows to evaluate the characteristics of their resulting electric fields (peak of intensity and distribution), which provides information about the specificity of each protocol. Additionally, comparisons between different modeling approaches allow to analyze the consistency of the results, and therefore their reliability. The consistency or inconsistency of the simulation results between both models may serve as a guide to motivate further empirical validation of estimates based on standard models.The maximum intensity values of the electric field generated in the brain for each tDCS electrode configuration based on both software packages are shown in 2 electrode sizes, as compared to lower stimulation intensity, and larger electrode sizes. Considering that the electric field strength induced by a direct current that has been linked to moderate physiological effects on slices of animal brain tissue, i.e., effects on neuronal ion channels and excitability, is about 5 V m−1 (=0.2 V/m) [2-2 mA), ventromedial prefrontal cortex (vmPFC) (configuration 2: lM1/rSOR-20 cm2-2 mA), dorsomedial prefrontal cortex (dmPFC) (configuration 2: lM1/rSOR-20 cm2-2 mA), and DLPFC (configuration 2: lM1/rSOR-20 cm2-2 mA). With the same cortical target, electric field intensity also reached this threshold under the electrode configuration 6 (lM1/rSOR-35 cm2-2 mA), but only in the dmPFC, and under the configuration 4 (lM1/rM1-20 cm2-2 mA), in the anterior parietal cortex (APC). When stimulation was targeting the DLPFC (2-2 mA) and 16 (lDLPFC/rDLPF-35 cm2-2 mA), and only in the vmPFC under the configurations 10 (lDLPFC/rSOR-20 cm2-2 mA), and 14 (lDLPFC/rSOR-35 cm2-2 mA). When targeting the DLPFC, electric field intensity within the orbitofrontal cortex (OFC) reached this threshold under the electrode configurations 14 (lDLPFC/rSOR-35 cm2-2 mA) and 16 (lDLPFC/rDLPF-35 cm2-2 mA). No electrode configuration targeting the PPC was associated to electric field intensities above this threshold outside the ROI  is shown qualitatively by the outputs of both modeling approaches, via whole brain cortical surface images. Variations of the color of the electric field indicate the range of electrical field strengths. Different from the peak electric field intensity, which is quantified by the models, electric field distributions can only be reported qualitatively, based on the outputs of the respective modeling software packages. However, based on the images, we provide estimated ranges of electric field intensity (by reporting lowest and highest estimated intensities for each area) in cortical regions impacted by the current beyond the ROI for each electrode configuration and modeling approach as a measure of electric field distribution . In gene0.2 V/m) ,65,66,67he DLPFC , electri the ROI .2). The electric field was extended beyond the ROI (M1) in both cases, although the extension was slightly larger in the SimNIBS simulation (2 vs. 20 cm2)   (2)  B, compar; 2) (2) D, as rev; 2) (2) A,C. 2), according to the COMETS outputs which revealed a minimum current spread (2 electrode sizes) was somewhat larger compared to the homologous configurations using smaller electrodes, with SimNIBS showing again a wider current spread , minimal differences between the two simulation tools were found regarding maximum electric field intensities . Bilateral stimulation, involving both hemispheres, resulted in more focally restricted effects over M1, particularly for COMETS modeling. The typical left M1 (anode electrode)-right SOR (cathode electrode) configuration resulted in increased current spread from M1 towards prefrontal cortex regions in all cases. However, according to both tools, electric field intensity reached the level of 2 V/m outside the ROI only when applying 2 mA of current intensity. The frontal areas reaching this threshold under this electrode configuration and current intensity were the PMC, vmPFC, dmPFC, and DLPFC. With the bilateral configuration targeting the M1 (lM1-rM1), the threshold of 2 V/m outside the ROI was only reached with 2 mA of current intensity as well, but only in the APC. Similar electric field strengths over the ROI were obtained using both tools for bilateral (lM1-rM1) or unilateral (lM1-rSOR) M1 stimulation, with slightly higher maximum intensities in the unilateral configurations. The results of these electric field calculations for the target M1 can be contrasted with physiological effects of respective stimulation protocols on motor cortex excitability obtained via transcranial magnetic stimulation (TMS)-elicited motor-evoked potentials (MEP) ,74,75,76Similar to the M1 modeling results, for all configurations involving the DLPFC, minimal differences between the two simulation tools were found with respect to the maximum electric field intensity  as well, but an opposite pattern of electrode position-dependent focality (as compared to M1) was observed for configurations targeting this prefrontal region (9–16). Bilateral DLPFC stimulation (anode over the left DLPFC and cathode over the right DLPFC) was associated with larger current spread over the prefrontal cortex, particularly over ventromedial and dorsomedial regions, for all current intensities and electrode sizes. The extension of the current spread was larger for the SimNIBS modeling results. In the bilateral configuration targeting the DLPFC, the threshold of 2 V/m was exceeded outside the ROI in the vmPFC and dmPFC only when applying a current strength of 2 mA, according to both simulation tools, and only in the vmPFC in the case of the unilateral electrode configuration and a current intensity of 2 mA. For both electrode configurations, unilateral and bilateral, this threshold was reached in the OFC only with 2 mA current intensity. The left DLPFC (anode)–right SOR (cathode) configuration showed higher focality in all electrode size/intensity combinations. As compared to available evidence regarding the M1, the physiological changes induced in the DLPFC by tDCS are less well explored , which m2), in which the maximum electric field intensity was 0.25 V/m higher in the COMETS modeling. On the other hand, the focality of the SimNIBS-simulated electric field was similar for all electrode configurations, but evident between-configuration differences in electric field focality were found in COMETS simulations. With SimNIBS, current spread involved parietal, temporal, and frontal regions of the left hemisphere, while it was much more limited when the electric field was calculated by COMETS, with moreover larger focality associated with smaller electrodes (20 cm2). Despite these differences between models, with respect to current spread characteristics, the electric field intensity calculated by both methods did not reach the threshold of 2 V/m outside the PPC with any of the electrode configurations targeting this area. The overall strength and distribution of these electric fields align with physiological results showing increases of PPC excitability and electrical activity under similar tDCS protocols, as measured by TMS-elicited MEP [The most notable differences were found for electrode configurations aimed to stimulate the left PPC (17–20). On the one hand, the largest difference between both simulation tools was found for the left PPC-right SOR configuration , while the SimNIBS results show less focality and larger current spread, particularly when the left PPC was the cortical target. The latter was specifically evident for the electric field computed for the left PPC-right SOR electrode montage, in which anode–cathode electrode distance was the largest of all modeled configurations. These differences might be caused by the different default conductivity values of both modeling approaches, whose effects on the outcomes will increase with larger distances between electrodes, and by the fact that SimNIBS integrates more tissues in respective models, as compared to COMETS. Assumptions about the relative validity of both modeling approaches would require face-to-face experimental approaches involving measures of electrical fields, but respective data are not available at present. The peak of electrical filed intensity was doubled in all electrode configurations when applying 2 mA of stimulation, as compared to 1 mA, which is trivial according to the quasi-static assumption for the electric fields induced by stimulation. Overall, while both simulation approaches revealed generally comparable results, higher values of the maximum electric field intensity resulted from the COMETS simulation (with only two exceptions, corresponding to the left DLPFC-right DLPFC configuration, 35 cmStandardized non-individualized modeling approaches, as shown here, provide quantitative estimations of the peak electric field intensity, and a qualitative overview about electric field distributions achieved with conventional electrode montages. These results are valuable for gaining a general overview about the electrical fields induced by specific tES protocols, but face also some limitations. For validation of the predictions made by the models, a few studies are available ,57,58,59Another limitation of these models is that these are purely based on macroscopic anatomy. They cannot take into account the alignment of electric fields with neuronal orientation in a detailed way, which might however be decisive for physiological and also functional effects ,82,83,84At the level of the individual, gradual misalignment between predicted and empirical electrical fields in case of non-personalized modeling, as conducted here, can be caused by anatomical differences between the head model and individual brains ,71,86,95It should furthermore be noted that these models generate data about electrical fields, but not directly about physiological, psychological, or behavioral effects. Pure physical models do not necessarily predict cognitive and behavioral effects of an intervention. For improving the correspondence between simulated electric fields and cognitive and behavioral effects of tDCS, brain physiology, including task-related physiological alterations during stimulation, has to be considered. This is particularly relevant since task- ,97,98,99As proposals for future studies, the use of modeling for multielectrode stimulation approaches, which may induce more spatially restricted electrical fields , may be In summary, standard modeling, as shown here, can be a useful approach for initial screening of tDCS protocols. However, since the resulting electric field estimations are not based on all potentially relevant anatomical factors, quantitative information about the spatial dispersion of the electrical fields is not available in these models, and simulations do not include physiological models, this strategy faces relevant limitations for making detailed decisions about stimulation protocols. Some fundamental aspects should be stressed in this regard. First, the simulation of the electric fields in these models is not based on individual brain data , and botWe provide examples about how current flow can be calculated using two different modeling tools for tDCS configurations varying with respect to electrode positions, current intensity, and electrode sizes. Based on this approach, we discussed advantages and limitations of those simulations in which individual MRI is not required, with respect to basic experimental research and clinical applications. We furthermore discuss future perspectives of electric field simulations of non-invasive brain stimulation.The general outcomes of the simulation procedures were relatively similar between models, although higher values of the maximum intensity of the electric field were generally obtained by COMETS modeling. Less focality and extended current spread were observed when the electric field was simulated by SimNIBS, particularly with those configurations in which the anode electrode was positioned over the PPC region. Between-model differences were thus apparent for quantitative measures of electric field intensity and qualitative overviews of the electric field distribution. However, it can be concluded that, in general, both simulation approaches deliver roughly comparable results for a standard head model. Unresolved issues of these modeling approaches are the limited physical and physiological validation of simulations, and the transfer of results to behavioral and clinical levels. These shortcomings do currently prevent the application of modeling for routine clinical applications, which would be however a principally important field for personalization of therapeutic neuromodulation in neuropsychiatric disorders to reduce interindividual variability of results ,112. A o


Arabidopsis thaliana, like suppression of primary root growth, increase of lateral roots or root hairs formation. However, the most proficient PGPR displayed specific ability in changing a single root morphological trait. This ability was related not only to bacterial genotype, but also with the environment where bacteria thrived and isolation temperature. Bacteria from semi-arid environments (mainly Bacillus megaterium isolates) could hold a promising tool to enhance plant development. Other isolates (Serratia quinivorens or B. cereus) could be further explored for biocontrol purposes.Plant growth promoting rhizobacteria (PGPR) are in increasing demand due to their role in promoting sustainable practices, not only in agriculture but also in forestry. Keeping in mind the future application of PGPR for increasing cork oak sustainability, the aim of this study was to find cork oak PGPR isolates with increased nutrient solubilisation traits, able to promote root morphological changes and/or antagonize cork oak bark phytopathogens. Soils from three cork oak forests with distinct bioclimates  were used for isolating bacteria. From the 7634 colony-forming units, 323 bacterial isolates were biochemically assayed for PGPR traits , and 51 were found to display all these traits. These PGPR were able to induce root morphological changes on  Quercus suber L.) is a slow-growing and long-lived evergreen tree, native to western and central Mediterranean region, being mainly distributed along coastal regions of southwest Europe  and northwest Africa  Kuntze; Xylariales) and bot canker . Both B. mediterranea and D. corticola have been found in cork oak trees in a latent endophytic phase  × 100, where AC is the area of pathogenic fungal growth in the control plate, and AB is the area of pathogenic fungus growing in the presence of PGPR.The same previously selected PGPR isolates, characterized by displaying multiple PGPR traits, were grown in liquid LB medium, at 30 °C, for 24 h. PGPR growth was evaluated by densitometry (OD600) and adjusted to 1.0. Isolates were pre-screened in vitro for antifungal activity against the cork oak phytopathogens, a region . Fresh Pe method . For thiDifferences between isolated bacteria obtained from distinct isolation temperatures and bioclimates were determined by two-way ANOVA and Tukey’s multiple comparison tests, using the Windows GraphPad Prism 6.01 program . Evolutionary distances between identified species and phylogenetic tree construction were based on the Maximum Composite Likelihood method  and evolp ≤ 0.001) when compared to sub-humid (LI) and humid forests (ER).A total of 7634 colony-forming units (CFU) were isolated from the cork oak soil samples from GR , LI  and ER . As expected, the number of CFUs decreased with increasing temperatures used for growing bacteria, being the highest number of CFUs registered at 30 °C and the lowest at 45 °C . RegardiSerratia quinivorans) from LI forest soil was capable of producing HCN. From the 51 bacterial isolates, only 43 were able to be sequenced, the remaining being hereafter referred to as Unidentified. From the sequenced samples, 42 were successfully identified up to at least genera level, sample AJ46 being hereafter named as Unknown. Identified bacterial isolates (Firmicutes (24 isolates), which was represented exclusively by Bacillus genus . The remaining isolates belonged to Proteobacteria (18 isolates), comprising three bacterial families . Enterobacteriaceae was represented by three genera , Yersiniaceae by two genera [Rouxiella (4) and Serratia (2)], while Pseudomonadacae was individually represented by Pseudomonas mohnii (1). ER presented a total of six genera identified , followed by LI and GR, with three  and two (Klebsiella and Bacillus) genera, respectively. While Bacillus was present in all sampled forests, Cedecea (LI), Serratia (ER), Ewingella (ER) and Pseudomonas (ER) were exclusively present in most humid forests. On the other hand, the semi-arid forest was highly enriched in Bacillus megaterium .From each forest and isolation growth temperature group, a subsample of bacterial colonies was randomly isolated for further studies. At the end, a total of 324 single colonies were isolated  and used for studying PGPR features . From alisolates  mainly bBacillus genus (clade 1) and the other with similarities to species from other genera , while other Bacillus species clustered in a single subclade (subclade 1.3). AJ46 isolate, previously named as Unknown, clustered into subclade 1.2, making it a potential member of Bacillus genus, closely related to B. megaterium species. Phylogenetic evaluation results have also revealed that clustering patterns did not form according to forest geographic location and bioclimates.Phylogenetic analysis of 16S gene from identified bacterial species was performed to analyse the pattern of bacterial distribution according to cork oak forests. Phylogenetic tree presented two clades, one with similarities to species from clade 2; . Twenty-A. thaliana primary root architecture was evaluated by a dual culture method. To better understand the dynamics over time, primary root length, number of lateral roots and presence/absence of root hairs were evaluated at 3-, 6- and 9-days after PGPR inoculation. From all tested PGPR, 33.33% displayed a consistent suppressive effect on primary root length , Serratia sp.  and Cedecea neteri (3 isolates reducing at least 60%). Some Unidentified isolates (4) were also capable of promoting at least 60% root length reduction.Specific effects of each PGPR (51 isolates) on A. thaliana , C. neteri (AJ15), Rouxiella sp. (AJ22) and an Unidentified isolate (AJ13) induced the lateral root number during the experiment. For not underestimating PGPR with non-significant differences in the early stages of inoculation, but that still promoted lateral root formation by the end of the assay, lateral roots induction (LRI %) was calculated in relation to control and over time. It was possible to understand that besides the previous selected PGPR, other B. megaterium (AJ40 and AJ61) isolates were able to highly increase the lateral roots formation. Besides these, Klebsiella aerogenes (AJ45) and P. mohnii. (AJ25), as well as an Unknown  and Unidentified (AJ51 and AJ62) isolates, also highly induced the lateral roots formation.PGPR also modulated lateral root formation in B. megaterium (AJ50 and AJ53) and an Unidentified isolate (AJ56). The ability of inducing root hairs increased with incubation time. At the end of the experiment (9 dpi), from the PGPR isolates that promoted root hairs formation in more than 75% of A. thaliana seedlings, most were B. megaterium (7 out of 8).Compared to the control, where no root hairs ever developed during the entire assay, the promoting effects of PGPR on root hairs incidence became evident over time . By 3 dpp ≤ 0.05). On the other hand, lateral root development was not dependent on PGPR isolation temperature (p > 0.05). Lastly, analysing the PGPR effects on root hairs formation, a high increase of root hairs presence was more evident in higher isolation temperatures (37 °C or 45 °C), as weaker root hair inducers were isolated at 30 °C. Therefore, out of the three evaluated root-parameters, the development of primary root and root hairs seemed to be the most affected by PGPR isolation methods.Taken together, most PGPR (31 out of 51) induced great outcomes on a single root parameter, but only 3 could simultaneously modulate two root parameters . None ofB. mediterranea and/or D. corticola fungal growth inhibition  than semi-arid forest (1 isolate from GR). In contrast, PGPR presenting antifungal activity against D. corticola were similarly isolated from semi-arid  and humid forests . PGPR isolates that presented antifungal activity against both phytopathogens were isolated from moister forests .From all 51 PGPR isolates, only 15 presented antifungal activity against hibition . ResultsB. mediterranea with time (p ≤ 0.05) at 5 dpi, but from 7 dpi on, only S. quinivorans maintained a significant (p ≤ 0.01) antagonistic activity against B. mediterranea. Also, from all assayed PGPR, Serratia genus (Serratia sp. and S. quinivorans) presented the highest growth inhibition (PGI) at 5 dpi . The antifungal activity of S. quinivorans was further studied by following the cultural features over time (p ≤ 0.001), where the highest antagonistic activity was displayed 5 days after incubation  significantly inhibited D. corticola mycelial growth (p ≤ 0.0001) at 5 dpi. However, at 7 dpi, most assayed PGPR (7 isolates) presented significant antagonistic activity (p ≤ 0.05). Among these, Rouxiella badensis AJ21, B. cereus AJ24 and Unknown AJ46 isolates revealed the highest inhibitions (p ≤ 0.01). In any case, due to the high variation detected, further investigation is necessary for supporting and complementing these findings. Unknown AJ46 and B. cereus were further studied regarding their cultural features over time  was the only identified genus in all sampled forests. Among these, B. megaterium was the most frequent species identified in semi-arid forest (GR) and also the single one to be identified when using heat-forcing selection. Due to the ability of producing endospores, Bacillus species have been reported to exhibit an increased resilience to extreme conditions, such as water deficiency, high temperatures and high levels of UV radiation, having been isolated from arid, semi-arid and desertic climates [Taking into consideration the creation of a PGPR collection that could provide multifunctional services to cork oak stands, soil bacterial isolates were obtained from cork oak forests in distinct bioclimates and using different temperatures for selecting bacteria. The idea was to get the most diversified isolates, already adapted to different environmental conditions for foreseeing future biocontrol and biofertilizer strategies for plant protection. As expected, the highest number of CFU was detected in 30 °C, as the most soil microorganisms are mesophilic, presenting the maximal growth temperatures between 25 °C and 35 °C . Neverthclimates ,35.Bacteria displaying all assayed PGPR traits  were also able to change plant root-architecture, namely by reducing primary root growth, or by increasing lateral root or root hairs formation. However, only three isolates were proficient in inducing more than a single root morphologic trait. Furthermore, the most proficient PGPR in promoting a specific root alteration rarely significantly affected other morphological aspect, suggesting specificity and even antagonism among suppressing/promoting abilities of PGPR on root architecture. This behaviour could be related with the inducing factors that are behind root morphological changes. Alterations in root morphology have been related with modulation of auxin levels, namely in indole-3-acetic acid (IAA) concentrations . Indeed,Bacillus and Pseudomonas genera [Bacillus spp. with inhibitory effects on primary root growth. Unlike primary root, lateral root development has been described to be stimulated through high IAA levels [A. thaliana primary root length, possibly due to high IAA concentrations, would be expected to significantly promote the formation of lateral roots and/or root hairs. In the present work, this was not the case and even a significant positive correlation was found between primary root length and formation of lateral roots . This result suggests that the production of IAA by itself does not fully explain the growth-suppressing/stimulating abilities by PGPR, and other phytohormone levels  and/or factors could also modulate root development and elongation [B. megaterium) revealed different suppressing/stimulating root morphogenic features, which were dependent from bacteria background (forest and/or isolation temperature). For example, B. megaterium isolated at lower temperatures (30 °C and 37 °C) displayed lower primary root suppression rates, when compared to B. megatarium isolated at 45 °C (B. megaterium in inducing root hair formation were found in semi-arid forest (6 out of 7 isolates).Several bacteria from plant rhizosphere possess the ability to produce IAA, including bacteria from s genera ,41. ThisA levels ,43. Therongation . Most inat 45 °C . Also, fB. mediterranea and D. corticola). Several PGPR presented an in-vitro inhibitory effect against B. mediterranea that decreased over time, probably due to the lapse production of antifungal compounds or to the defensive mechanisms created by the fungus. Nevertheless, S. quinivorans (AJ11) revealed an inhibitory activity that lasted throughout the full antagonist assay, possibly due to the production of the volatile HCN. Indeed, in this study, only S. quinivorans was identified as a HCN producer, a volatile antifungal compound capable of inhibiting growth of several plant pathogens [Serratia genus members have been also reported as active producers of different lytic enzymes and several antibiotics, as well as HCN producers [B. mediterranea growth has been described to be affected by microbial volatiles produced by the cork oak beneficial endophytes, Coniothyrium carteri and Fusarium oxysporum [B. mediterranea, the antifungal activity of PGRP against D. corticola suggested increasing antagonistic effect with time that could be explained by a dose-dependent effect of antimicrobial compound(s). The isolates Unknown  and B. cereus (AJ24) resulted in D. corticola hyphal modifications, similar to those reported with known synthetic antimicrobial compounds [Besides biochemical traits and root phenotypic alterations, PGPR were also evaluated for their antifungal activity against bark cork oak disease agents  and by inducing root beneficial morphological changes. Being already adapted to cork oak forests, these isolates could hold a promising tool for enhancing cork oak development and sustainability. Members of Bacillaceae were recently described to play an essential role on cork oak forests mycorrhiza establishment.The use of PGPR as biofertilizers has been increasing because they facilitate the overall plant growth and yield of multiple crops in an eco-friendly manner . AlthougBacillus genus, could hold a promising tool to enhance plant development, even under stressful environmental circumstances. Such natural occurring plant beneficial microorganisms are in increasing demand due to their role in promoting more sustainable practices, not only in agriculture and crop production, but also in forestry.This work resulted in the creation of a PGPR collection from cork oak forests that display increased nutrient solubilisation traits , induce a root phenotype as described for other plant-beneficial microorganisms, and are able to antagonize cork oak bark phytopathogens. Suppressing/promoting abilities on root architecture were found to be specific from each PGPR and depended on the environment where bacteria reside, and method used for their isolation. PGPR communities, mainly from semi-arid environments and from


The aim of the study is to compare structure tensor imaging (STI) with diffusion tensor imaging (DTI) of the sheep heart (approximately the same size as the human heart).MRI acquisition on three sheep ex vivo hearts was performed at 9.4 T/30 cm with a seven-element RF coil. 3D FLASH with an isotropic resolution of 150 µm and 3D spin-echo DTI at 600 µm were performed. Tensor analysis, angles extraction and segments divisions were performed on both volumes.A 3D FLASH allows for visualization of the detailed structure of the left and right ventricles. The helix angle determined using DTI and STI exhibited a smooth transmural change from the endocardium to the epicardium. Both the helix and transverse angles were similar between techniques. Sheetlet organization exhibited the same pattern in both acquisitions, but local angle differences were seen and identified in 17 segments representation.This study demonstrated the feasibility of high-resolution MRI for studying the myocyte and myolaminar architecture of sheep hearts. We presented the results of STI on three whole sheep ex vivo hearts and demonstrated a good correspondence between DTI and STI.The online version contains supplementary material available at 10.1007/s10334-021-00913-4. Myocardial structure plays an important role in the normal function of the heart, by facilitating efficient contraction and significantly influencing electrical propagation . VariousThe myocardium consists of branching myolaminae, or sheetlets, which are 4–6 cells thick. The long axis of the myocytes within these laminae, also referred to as fiber orientation, shows a regular low-order organization in the ventricular wall often described as a helical transmural arrangement, which was described previously in detail –4. The lVarious techniques have been used to visualize the myocardial architecture, including histology , extende3 to visualize and quantify cardiac microstructure in ex vivo rat hearts [3 provided accurate information on sheetlet orientation and cardiac orthotropy and was superior to DTI [Different MRI techniques can provide information on myocardial–myocyte orientation and myolaminar/sheetlet structure in the intact heart. Over the last 15 years, diffusion tensor imaging (DTI) has been applied to investigate cardiac fiber orientation in vivo –10 and et hearts . They der to DTI . Our gror to DTI . HoweverThe aim of this study was to present a pipeline acquisition of DTI and STI, and compare the two modalities to assessing cardiac microstructure by analysis fiber orientation and sheetlet structure on three ex vivo sheep hearts at 9.4 T with a large bore access of 30 cm.3, heart weight = 150 ± 10 g) were perfusion-fixed in 10% formaldehyde  containing 2 mL of gadoterate meglumine (Gd Dotarem), a gadolinium-based contrast agent  for 12 h. This method provides enhanced contrast between the myolaminae and cleavage planes and a greater signal-to-noise ratio [Hearts (hearts 1–3) were explanted from three female sheep (weight: ~ 50 kg) via sternal thoracotomy under general anesthesia. This protocol was approved by the Animal Research Ethics Committee in accordance with the European rules for animal experimentation. The three hearts  with an open bore access of 30 cm , a 200-mm inner diameter gradient (300 mT/m), and a shim system. Hearts were placed in the center of the magnet in the base–apex axis along the gradient coil.A dedicated RF volume coil for imaging large samples designed by Bruker was used for scanning. The RF coil was a cylindrical volume array  including seven equally spaced overlapping loop elements . In this design, no exactly opposing elements occur, but each element is opposed to a gap between two other elements, thus strongly reducing the degree of coupling between the critical elements without the need of reducing the loop size. Phase setting of each element was shifted of 45°–50° to avoid destructive interference. Then, scout images were obtained, 3D B1 maps were acquired within 20 min using the Bloch Siegert shift  method fb value of 1000 s/mm2, as described previously [Diffusion tensor MRI was carried out using a 3D diffusion-weighted spin-echo sequence with TE = 22 ms, TR = 500 ms, FOV = 100 × 80 × 110 mm, matrix = 166 × 133 × 183 and an isotropic resolution of 600 μm. Each diffusion gradient had a duration of 4.5 ms, and gradients were separated by an 11-ms delay: three b0 maps were generated and six gradient directions were applied with a eviously , and an b = 0 s/mm2 to avoid the segmentation of regions showing some B1 inhomogeneity and signal drop-off associated with RF coil sensitivity profile in the region of the cardiac apex and base  along each direction were computed: first a 5 × 5 × 5 point derivative template was defined . The derThe structure tensor (the outer product of the intensity gradient vectors) was computed for each voxel and smoothed at a progressive resolution from 150 to 600 µm isotropic (731 × 665 × 532 tensors to 183 × 166 × 133 tensors). Each structure tensor component at resolution doubling was determined using level 4 binomial low-pass filters to smooth from one level of resolution to the next.The principal directions of the structure tensor at each discrete point were extracted using eigenanalysis at isotropic resolution 600 µm. The 600 µm smoothed structure tensor data set was used to best-match the expected diffusion tensor resolution. , which is the angle between the short axis plane and the projection of the fiber vector onto the wall tangent plane. The fiber transverse angle (TA) is the angle between the wall tangent plane  and the projection of the fiber vector onto the transverse   angle is the angle between the short axis plane and the projection of the vector onto the radial orientation. The sheetlet azimuth (SA) angle is the angle between the local radial orientation and the projection of the vector onto the short axis plane .Each segment for each sample contains a mean of 16,032 ± 2270 voxels.x = 0) and epicardium . We performed a local unwrap for pixels up to 90°.Mean transmural evolution was plotted  for each angle for 17 segments between the endocardium  [Short axis registration of the parametric volume maps was performed. Rigid registration using 3D Slicer (cer.org)  between cer.org) . We usedcer.org) , in whicp < 0.05 are considered as statistically significant. One asterisk (*) identifies p values between 0.01 and 0.05, two asterisks (**) identify adjusted p values between 0.01 and 0.001.Scatter and Bland–Altman plots in whole three hearts using GraphPad Prism  were performed. Mean of HA angles for DTI and STI in endocardium, mid-ventricular and epicardium were calculated to analyze the reproducibility of measured between samples and similarities between methods. Statistical analysis are performed for comparison between groups using nonparametric test  with R2 linearity and slope) and curve fitting were calculated for HA and TA transmural profile.Descriptive statistics from linear regression  [Image reconstruction was performed using ParaVision 6.0 (Bruker) on a workstation with 512 GB of accessible memory to process the large data matrices. Visualization of 3D volume renderings was performed using Volview and Paraview software . Short axis registration of the parametric volume maps was performed using 3D Slicer software and (cer.org)  and paracer.org) .3. In these images, the interstices between sheetlets, as well as vessels, appear bright due to the presence of residual Gd Dotarem. The fine details of the laminar architecture  can be seen in these images. Raw images extracted from 3D volumes after MRI acquisitions are displayed in Fig. Figure The tissue FA for the first heart is extracted from the 3D DTI acquisitions. Yellow and red areas are more anisotropic than blue areas Fig. a. FigureHA, TA, SE and SA maps are represented in Fig. A qualitative interpretation of angles maps is presented in the following part: maps are consistent across the three hearts. In general, a larger noise level is observed in the STI maps. We identify the well-known transmural profile in the LV, where the HA varied smoothly from a negative value (blue–green) at the epicardium to a positive value (red) at the endocardium. There is a smooth transition between the RV and LV in the posterior position, and an abrupt change of orientation in the same area in the anterior view.TA maps are mainly close to zero in short axis view. The angle distribution appeared to be more heterogeneous in the septum. TA maps derived from DTI and STI appear to be similar; short axis images of the left free wall for all hearts for both acquisitions showed negative values in the same areas.There are similarities between STI and DTI for images of the LV, with SE angles < 0° in the LV and septum. In long axis views, the SE angle is positive at the apex and negative at the base for the three hearts. On SA angle maps, STI and DTI data are similar with abrupt angle changes in the LV in short axis view but heterogeneous angle distribution in the long axis view in the septum. In the RV, some differences can be seen between DTI and STI, with negative values derived from DTI and positive values derived from STI in short axis view, especially for SE angles in all samples. Abrupt changes in short axis orientation were seen in the ventricles and also at the LV–septum–RV insertions.Difference angles maps between DT and ST images are represented in the last column of Fig. A more quantitative approach is described in the next Fig. In the endocardium, the mean of HA is in the same range for DT with difference in mean of 3.9 ± 16.1° between hearts #1 and #2, 8.3 ± 16.2° between hearts #2 and #3 and 4.4 ± 14.9° between hearts #1 and #3. STI displays differences in mean of 12 ± 13.7°, 14.6 ± 13.4° and 2.6 ± 14.6° between hearts #1 and #2, between hearts #2 and #3 and between hearts #1 and #3, respectively.p > 0.12 between hearts #1 and #2, hearts #1 and #3 and hearts #2 and #3. For the STI method, the differences between samples are more pronounced: with a p = 0.02 and p = 0.003, respectively, for hearts #1 and #2 and hearts #2 and #3; no significant difference is observed in STI between hearts #1 and #3.In the mid-ventricular area, heart #2 displays higher values in DT (25.5 ± 25.6°) and ST (30.1 ± 17.3°) for HA angle in comparison with heart #1 and heart #3. In this area, higher standard deviation is calculated due to the transition of positive to negative HA angles. Then, in the epicardium, we can see that HA from DTI displays lower values (between − 27.5 ± 14.9° and − 33.8 ± 11.1°) than for STI . Statistical results are presented in in Fig. R2 calculated ranges from 0.63 to 0.97 using DTI and from 0.66 to 0.95 for STI. Across the 17 different regions, slopes means for HA are − 129 ± 34° and − 121 ± 43° for DTI and STI, respectively. Linearity for TA is more variable across segments with R2 = 0.53 ± 0.38 for DTI and for STI R2 = 0.61 ± 0.29. A difference in slope is noticeable between STI and DTI in segment 2 and 8 in RV/LV junction. A mean difference between DT and STI slopes for TA is 10.4 ± 5.7°, and we notice a higher difference for segment 2 of 50.1° (slope(DTI) = 21.3°, slope(STI) = − 28.8°) and for segment 8 of 41.5° (slope(DTI) = 4.8°, slope(STI) = − 36.7°) between DTI and STI.Transmural evolution for mean and standard deviation of HA and TA angles for three hearts are plotted between − 90° on the endocardium to 90° on the epicardium in Fig. Figure For TA distribution, difference maps between DTI and STI distribution are always below 10° except for segments 2 and 8 closed to RV with a difference higher than 15°. For SE, DTI displays globally negative values ranging for − 35.6° (segment 15) to 14.7° (segment 6). Absolute difference maps angles are close or above 20° in segments 6, 13, 16 and 17 . For the SA angles, DTI and STI bullseye representation displays maps close to 0°. Absolute difference maps between DTI and STI displayed difference below 15° in absolute value except for in segments 2, 8, and 13 close to RV with difference of 27.9°, 24.4° and 18.4°, respectively, between |DTI-STI|.x axis with a HADTI-STI of − 2.2 ± 17.5° (mean ± 1.96 standard deviation in y axis). TA mean varies between − 45° and 45° in x axis with TADTI-STI centered to zero of − 2.2 ± 20.0°. For sheetlet comparison, SE points tend to be negative in mean with a SEDTI-STI of − 3.6 ± 51.5°. SA mean varies between − 45° and 45° in x axis with SADTI-STI centered to zero of − 9.1 ± 30.5°.Figure We presented a detailed investigation and comparison between DTI and STI on several hearts from a large mammalian species (sheep) with same age, size, and cardiac fixation and without any cardiac pathologies, to study robustness of STI acquisition and post processing on whole hearts. This allowed us to successfully analyze myocardial structure using a combination of 3D STI (150–600 µm) and 3D DTI (600 µm) within a total acquisition time of ~ 2 days.In the current study, we have used the same acquisition and a similar post processing pipeline as those presented in the 2019 FIHM conference [The fibers followed the widely described organization with transmural fiber rotation; whereas, sheetlets in the three ventricles appeared macroscopically to be mainly aligned along two distinct perpendicular orientations across the LV wall. Previous studies have demonstrated the utility of DTI , 29, 30 We demonstrated that the DTI and STI myofiber orientation exhibited similar rotation along the short and long axes, and both imaging modalities produced analogous maps of fiber orientation consistent with a previous study on rat hearts . HA mapsFLASH images at 150 µm on sheep hearts at 9.4 T includes a lot of information like vessels, fiber and sheet orientation, etc. We noticed that on FLASH images, we can both identify bright and black interstices in specific area. The black interstices’ meaning remains speculative, but it could indicate either less well-perfused regions during the fixation protocol or larger extracellular clefts where less Gd is accumulated. However, it probably did not affect quantitative analysis, given the good correspondence between DTI and STI (at least in fiber orientation).Bland–Altman Fig.  plots asOverall, the differences between STI and DTI were larger for SE and SA (~ 6.3°) than for HA and TA (~ 2.2°). Similar results were found by Bernus et al. , showingMazumder et al.  demonstrIn this study, the three hearts were perfusion fixed in 10% formaldehyde  containing 2 mL of gadoterate meglumine (Gd Dotarem), during 12 h. Then, hearts were immersed inside container with perfusion solution and were stored in a cold room. Scanner room temperature was monitored by constant air temperature around 19 °C. For three hearts the same protocol of fixation and storage have been applied . During acquisition, an optical temperature probe is placed close to the sample and connected to a computer, the fiber monitors change of temperature. We measure an increase of temperature of 2° ± 0.5 °C using SE DTI for 6 h of recording. No temperature variation was measured during FLASH acquisition.The acquisition of data from large-sized samples using ultra-high field HR MRI is still challenging due to B0 and B1 homogeneities. B0 could influence MR signal and create susceptibility artifacts and affect image quality. In this study, we used fomblin to avoid susceptibility effects in ventricular cavities. A 3D B0 mapping was performed, using a dual-echo steady-state sequence and performed a global shim to reach a full-width at half-maximum of 50 Hz. B1 could influence RF pulses efficiency and create signal drop-off associated with RF coil sensitivity. To achieve homogenous B1 field and excitation, B1 maps were acquired within 20 min and a shim box in 3D was placed in the septum. Local B1+ shimming was performed by determining a set of transmit phases and amplitudes that will maximize the homogeneity within a region of interest and avoid RF destructive effects. Moreover, in post-processing pipeline, a N4 filter was also used to correct low frequency intensity non-uniformity present in 3D volumes (due to coil sensitivity or B1 effect). B0 and B1 have been optimized for each acquisition. However, some studies present results a study based on SNR and the impact of DTI results on large mammalian hearts at 3 T and 7 T  and on eA large amount of data produced by HR 3D imaging require adequate processing hardware and dedicated software. The acquisition time to obtain 3D images of the whole hearts is several hours, and if data for both DTI and STI are acquired, the total acquisition time could exceed several days (acquisition time in this study was 2 days). Moreover, Teh et al.  used tenMRI allowed unprecedented visualization of cardiac structure of the ex vivo sheep heart with a high level of structural accuracy. In particular, new areas of interest, such as the RV and RV/LV junction, can be observed directly (segments 2 and 8). We also noticed that angles in the RV present abrupt orientation changes. Indeed, the importance of using an appropriate coordinate system to investigate the RV fiber and sheetlet orientations, as using an LV centroid may give rise to artifactual measurements in fiber helix/transverse angles and sheetlet orientations , 39.In conclusion, results of STI on three whole ex vivo sheep hearts demonstrated a good correspondence with DTI and opens new perspectives for HR 3D structural characterization of normal/pathological cardiac structure. These results applied on healthy and pathological hearts will provide new insight for cardiac modeling research groups and it will improve developments of mathematical cardiac model. The objective is to gain a better understanding of the links between structural remodeling and electrical disorders of the heart. These results hold promise for the development of new noninvasive imaging methods to better characterize the cardiac microstructure in healthy and pathological human hearts.y axis in function of LV wall thickness normalized between 0 and 1 corresponding to the LV epicardium and endocardium, respectively, in x axis. (TIF 78667 KB)Supplementary file1. S1: Transmural variation in sheetlet orientations comparison between mean of DTI and STI for all hearts. Regions were defined by the 17-segment AHA model. Segments 1–17 are resented for DTI (violet dots) and STI (blue dots). Helix angle (HA) and Transverse angle (SA) are displayed. Mean of SE and SA for three hearts is shown between − 100° and 100° in Below is the link to the electronic supplementary material.


Vigilancia de Embarazadas con Zika (VEZ), an intensified surveillance of pregnant women with symptoms of the Zika virus disease (ZVD) in Colombia, aimed to evaluate the relationship between symptoms of ZVD during pregnancy and adverse pregnancy, birth, and infant outcomes and early childhood neurodevelopmental outcomes. During May–November 2016, pregnant women in three Colombian cities who were reported with symptoms of ZVD to the national surveillance system, or with symptoms of ZVD visiting participating clinics, were enrolled in Project VEZ. Data from maternal and pediatric (up to two years of age) medical records were abstracted. Available maternal specimens were tested for the presence of the Zika virus ribonucleic acid and/or anti-Zika virus immunoglobulin antibodies. Of 1213 enrolled pregnant women with symptoms of ZVD, 1180 had a known pregnancy outcome. Results of the Zika virus laboratory testing were available for 569 (48.2%) pregnancies with a known pregnancy outcome though testing timing varied and was often distal to the timing of symptoms; 254  were confirmed or presumptive positive for the Zika virus infection. Of pregnancies with a known outcome, 50 (4.2%) fetuses/infants had Zika-associated brain or eye defects, which included microcephaly at birth. Early childhood adverse neurodevelopmental outcomes were more common among those with Zika-associated birth defects than among those without and more common among those with laboratory evidence of a Zika virus infection compared with the full cohort. The proportion of fetuses/infants with any Zika-associated brain or eye defect was consistent with the proportion seen in other studies. Enhancements to Colombia’s existing national surveillance enabled the assessment of adverse outcomes associated with ZVD in pregnancy.Project  Aedes Stegomyia species [The Zika virus is a flavivirus transmitted to humans primarily by the bite of infected mosquitoes of the  species  and can  species ,3. A con species ,5,6,7 an species ,19,20,21 species ,22,23.Sistema de Vigilancia en Salud Pública (Sivigila). The Zika virus outbreak peaked in Colombia in February 2016, coinciding with the request by the government of Colombia for technical assistance from the United States Centers for Disease Control and Prevention (CDC) to strengthen Colombia’s ongoing surveillance of symptomatic ZVD in pregnant women. Colombia declared an end to the outbreak in July 2016, with over 100,000 cases of symptomatic ZVD reported to Sivigila. Among them were 18,117 pregnant women; one-third of symptomatic ZVD cases in pregnant women in Colombia were laboratory-confirmed with the Zika virus infection [Colombia began national surveillance of the symptomatic Zika virus disease (ZVD) in August 2015 through its public health surveillance platform, nfection .Instituto Nacional de Salud, INS)-led Project Vigilancia de Embarazadas con Zika (VEZ), which was an intensified surveillance of pregnant women with symptomatic ZVD and their children at clinical sites in three cities in Colombia—Barranquilla, Cúcuta, and Cali. These sites were selected because of their high burden of symptomatic ZVD at the beginning of the outbreak. The aim of this first analysis of data from Project VEZ is to evaluate the relationship between ZVD during pregnancy and a spectrum of adverse pregnancy, birth, and infant outcomes and early childhood neurodevelopmental outcomes, considering the timing of symptom onset during pregnancy and the presence of laboratory evidence of confirmed or presumptive Zika virus infection during pregnancy.CDC supported the national public health institution (Women were enrolled in Project VEZ if they were (a) currently pregnant, (b) receiving prenatal care or delivering at the selected clinical sites within the three cities, and (c) either experiencing two or more ZVD-compatible symptoms at enrollment or reported two or more ZVD-compatible symptoms since their last menstrual period (LMP). Alternatively, women could be identified through Sivigila with laboratory-confirmed or clinically compatible ZVD since LMP and were enrolled if they received prenatal care or delivered at the selected clinical sites. Enrollment took place between May–November 2016. After enrollment, a trained field staff abstracted data from prenatal care (including ultrasound reports), delivery hospitalization, and infant records with standardized data collection forms. Pediatric follow-up data from medical records were abstracted on the children if available until they reached up to two years of age.Field staff captured additional pediatric follow-up data through six health brigades (two in each of the three cities) conducted by INS between April–August 2018 to conduct screenings and evaluations based on the nationally recommended guidelines for the assessment of children born to mothers with symptoms of ZVD during pregnancy. Assessments included a general pediatric and neuropediatric evaluation, a hearing screen using auditory brain stem responses, an ophthalmological exam assessing external structures of the eye as well as eye movement and alignment, and a fundus examination through indirect ophthalmoscopy, and developmental milestone screenings. Mothers were asked to bring the child’s medical record to the brigade; any information relevant to Project VEZ that was either in the child’s record or captured during the clinical encounters that were part of the brigade was abstracted by field staff onto the standardized data collection forms.Specimen collection and Zika virus testing were based on Colombian national guidelines . For allLaboratory procedures for detecting possible Zika virus infections evolved over time as assays and training became available in Colombia. For maternal serum samples that were collected as part of Sivigila, INS protocol was to collect and test serum samples within five days of symptom onset using one of two nucleic acid amplification testing (NAAT) procedures to detect viral ribonucleic acid (RNA)—a single target real-time reverse transcription polymerase chain reaction (rRT-PCR) assay (singleplex assay), based on a published method with reflex testing for dengue and chikungunya for those with negative Zika rRT-PCR results  or the TAll specimens collected as part of Project VEZ were sent to INS for processing in its virology or pathology laboratories; aliquots were immediately prepared and stored at −80 °C. It was not feasible to limit collection and testing to within five days of symptom onset for these specimens. INS laboratory scientists performed NAAT on a subset of 50 maternal serum, urine, and tissue samples using the singleplex assay. One aliquot of all available Project VEZ samples was subsequently sent to CDC. INS also created a biorepository of samples related to the Zika virus outbreak in Colombia, in which all remaining samples from Project VEZ are stored.http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 18 October 2018). Serologic testing of all maternal serum samples to detect the Zika virus IgM antibodies was performed using the Zika virus MAC-ELISA  [CDC laboratory scientists tested all available specimens of maternal serum and maternal urine for the presence of Zika viral RNA using the Trioplex assay. Frozen placental, umbilical cord, and fetal tissues were thawed, fixed in 10% neutral buffered formalin, and then embedded in paraffin. CDC laboratory scientists also tested fixed tissues for the presence of Zika viral RNA using a conventional Zika virus RT-PCR, followed by Sanger sequencing of the PCR products . The seaGA, USA) ,30. DengAdditionally, any results of Zika virus laboratory testing conducted during pregnancy found in the medical records of participants in Project VEZ were abstracted.Using all available laboratory results , an algorithm to define laboratory evidence of Zika virus infection during pregnancy was developed by the Colombian INS in consultation with CDC .Project VEZ applied the updated CDC surveillance case definition of Zika-associated birth defects: brain abnormalities with or without microcephaly and structural eye abnormalities. Infants with early brain malformations such as neural tube defects and holoprosencephaly were excluded in accordance with the case definition . MicroceOther pregnancy outcomes assessed were pregnancy loss <20 weeks’ gestation and pregnancy loss ≥20 weeks’ gestation. Among singleton, liveborn infants without Zika-associated birth defects, we also assessed preterm delivery (<37 weeks’ gestation), low birth weight (<2500 g), and small for gestational age . Gestational age at delivery was assigned based on a hierarchy; the estimated date of delivery based on first trimester ultrasound data was prioritized. If first trimester ultrasound data were unavailable, dating based on second trimester ultrasound, clinician estimate at delivery, or reported date of last menstrual period, was used. We also assessed deaths among all live-born infants in the first year of life.Escala Abreviada de Desarrollo (EAD-1) [The definition of follow-up was the presence of clinical information in the medical record after two weeks post-birth. Among this group of children, we explored early childhood neurodevelopmental outcomes. Neurodevelopmental outcomes were categorized as the following neurodevelopmental sequelae documented in the medical record or clinical notes at a health brigade: seizures, swallowing abnormalities, tone abnormalities, movement abnormalities, arthrogryposis, visual impairment, or hearing abnormalities, or potential delay in achieving developmental milestones. Potential delay in achieving developmental milestones was based on documentation of developmental “alert scores” or areas of concern requiring further assessment, based on the Colombian national, standardized, validated developmental screening assessment normed on a diverse sample of Colombian children, the  (EAD-1) . The EADp < 0.05 was used to assess differences in proportions. For statistical testing of the differences in Zika-associated birth defects by trimester of ZVD symptom onset, we compared the first and second trimesters only due to limited sample sizes during the periconceptional period and third trimesters; for other adverse pregnancy outcomes, we compared across the three trimesters. When the expected cell size was less than five, Fischer’s exact testing with significance level at p < 0.05 was used. All data analyses were conducted in SAS 9.4 .All abstracted data were entered into INS/CDC-developed forms in REDCap Version 6 . We calculated the proportion of adverse pregnancy outcomes and Zika-associated birth defects among completed pregnancies with a known outcome, as well as in the subgroup of completed pregnancies with a confirmed or presumptive Zika virus infection. We also stratified the analyses of adverse pregnancy outcomes and Zika-associated birth defects by trimester of ZVD symptom onset, including periconceptional ZVD symptom onset (defined as up to 56 days before the estimated date of conception). In the cohort of children with data abstracted after two weeks post-birth, we calculated the proportion with neurodevelopmental sequelae and the proportion of children with EAD-1 alert scores by domain separately among the children with Zika-associated birth defects and those without. Chi-square testing with the significance level at The enrollment of pregnancies and follow-up of children up to two years of age is shown in More than half of the Project VEZ population of pregnant women were aged 20–29 years and had at least a secondary, technical, or professional education. Just over a quarter of the women were experiencing their first pregnancy. The most common ZVD symptoms reported were rashes (83.3%), fever (66.7%), and joint pain (58.6%). Just over half of the women (52.2%) reported ZVD symptom onset during the first trimester. In general, characteristics of the subcohort with laboratory evidence of a Zika virus infection were similar to the entire cohort .There were 50 completed pregnancies (4.2%) resulting in an infant or fetus with evidence of Zika-associated birth defects; when restricted to the 254 completed pregnancies with laboratory evidence of Zika virus infection, the proportion of fetuses/infants with Zika-associated birth defects was 8.7%. Seventeen additional infants had postnatal-onset microcephaly with no report of brain abnormalities and no evidence of microcephaly at birth. Additional clinical details for these 67 fetuses and infants with evidence of a Zika-associated birth defect or postnatal-onset microcephaly are in There were no statistically significant differences by trimester of ZVD symptom onset in the proportion of pregnancies with fetuses or infants with selected eye anomalies, microcephaly only at birth, postnatal-onset microcephaly, preterm delivery, low birth weight, or small for gestational age. However, there is a pattern suggesting increased risk with earlier trimester of ZVD symptom onset for preterm delivery and low birth weight. Statistically significant differences in the proportion of selected brain anomalies with or without microcephaly were observed by trimester of ZVD symptom onset .Among the 1180 completed pregnancies, a total of 569 (48.2%) had Zika virus testing results. Pregnant women without any laboratory testing results were more likely to have secondary and technical education than those with laboratory testing results. They were also more likely to be multigravida compared to those with laboratory testing results and more likely to have delivered by Caesarean section. Finally, those without any laboratory testing results were more likely to have had periconceptional (up to 8 weeks before the estimated date of conception) ZVD symptoms compared to those with laboratory testing results .n = 220) or presumptive (n = 34)). As noted in the Methods, VEZ participants originally identified through national surveillance (Sivigila) had a blood sample collected as soon as possible after symptom onset and per INS protocol, testing was to be conducted within five days of symptom onset. However, for VEZ participants not identified through Sivigila, the time between symptom onset and sample collection could be much longer. The time between the date of symptom onset and date of sample collection was up to 155 days among the sample of Zika-positive pregnancies, with one or both dates missing for nearly half of the samples. There were 315 pregnant women (55.4% of those with testing results) that tested negative for Zika virus infection. Of the 611 (51.8%) participants in Project VEZ with no Zika virus testing results available, 597 (97.7%) had documentation of a sample collected during pregnancy. Of these, 184 (30.8%) had only a tissue sample collected as part of Project VEZ  for which testing was not performed due to constraints in resources. The remaining 413 participants did not have a sample collected as part of Project VEZ but did have documentation in either the medical record or in Sivigila that a specimen had been taken during the pregnancy; however, there were no test results available for abstraction (There were 254 pregnant women (44.6% of those with testing results) with laboratory evidence of Zika virus infection  had documentation of any brain imaging including a cranial ultrasound, computed tomography, or magnetic resonance imaging, 605 (51.4%) had documentation of at least one ophthalmologic exam, 655 (55.6%) had documentation of at least one hearing screening, 886 (75.2%) had documentation of at least one developmental screening, and 1125 (95.8%) had at least one physical examination (data not shown).n = 58), the proportion with any neurodevelopmental sequela documented was 44.8%; the proportion among those with neither Zika-associated birth defects nor postnatal-onset microcephaly (n = 932) was 10.7%. In the subgroup of infants with laboratory evidence of confirmed or presumptive Zika virus infection during the pregnancy and neither Zika-associated birth defects nor postnatal-onset microcephaly (n = 203), the proportion with any neurodevelopmental sequela was higher  than among those without Zika-associated birth defects overall (10.7%). Overall, 22.4% (222/990) of children had a potential delay in achieving at least one developmental milestone. Among the 58 children with Zika-associated birth defects or postnatal-onset microcephaly, 34.5% (n = 20) had a potential delay in achieving at least one developmental milestone; 31.0% had a potential delay in achieving a developmental milestone in the gross motor domain, 27.6% had a potential delay in the fine motor domain, 24.1% had a potential delay in the personal and social domain, and 27.6% had a potential delay in the hearing and language domain based on the Escala Abreviada de Desarrollo (EAD-1) [There were 990 children in the Project VEZ cohort who had information beyond their birth hospitalization or after 14 days after birth  in their medical records for whom early childhood neurodevelopmental outcomes could be assessed. Of them, 126 children had documentation of any neurodevelopmental sequelae in the medical record or clinical notes from a health brigade (12.7%). Among those with Zika-associated birth defects or postnatal-onset microcephaly  investigation in Brazil showed that among 19 children aged 19–24 months with Zika-associated microcephaly, 100% had a least one adverse health or developmental outcome including feeding challenges, sleeping difficulties, severe motor impairment, vision and hearing abnormalities, and seizures . In a coAmong children without Zika-associated birth defects, there was limited evidence of adverse early childhood neurodevelopmental outcomes; nearly 22% of children screened with EAD-1 had a potential developmental delay in one or more domains (202/932) and this was somewhat higher than expected in the Colombian population . HoweverThe findings in this report should be evaluated in light of several limitations. First, more than half of the pregnancies in Project VEZ did not have any laboratory testing results. For 427 of these pregnancies, there were no blood or tissue samples taken during the course of the project, despite specimen collection being a component of the surveillance protocol. For the remaining 184 pregnancies, the only specimen collected was placental or umbilical cord tissue taken at delivery, for which capacity and resources were not available to complete testing. However, for nearly 50% of the Project VEZ cohort, there were laboratory testing results, either from a specimen collected as part of the enhanced surveillance protocol, or from a specimen collected as part of national surveillance. The subpopulation with laboratory results is qualitatively similar and largely representative of the full Project VEZ cohort, although some differences have been noted. The limitations of laboratory testing in general are important to note. Optimal timing of NAAT to detect Zika viral RNA in maternal serum and urine samples is seven days after symptom onset . HoweverThis analysis had several strengths. First, Project VEZ was a large cohort of pregnant women with symptomatic ZVD during the height of the Zika virus outbreak in Colombia. Of the over 1200 pregnancies enrolled in Project VEZ, a pregnancy outcome was ascertained for 97%, and over 75% of the infants had some clinical follow-up documented beyond the birth hospitalization. Second, because Project VEZ was built on the foundations of a strong public health surveillance infrastructure in Colombia, there was interest and engagement in Project VEZ from public health authorities at the jurisdictional and national levels. Third, because INS wanted to ensure that as many infants as possible received the recommended screenings and assessments through health brigades, additional data were captured for 139 children that would not have otherwise been available. Finally, although EAD-1 is a developmental screener rather than a diagnostic tool, it is standardized, validated, and normalized on a diverse sample of Colombian children. Our results can be compared to results from other Colombian cohorts of children in which the EAD-1 has been implemented.Critical enhancements to Colombia’s existing national surveillance early in the outbreak allowed for the implementation of Project VEZ. In this large surveillance cohort of pregnant women with symptoms of ZVD during the height of the outbreak in Colombia, the proportion of fetuses/infants with any Zika-associated brain or eye defect, including microcephaly at birth, was 4.2%. This is consistent with the proportion seen in other studies to date. Among completed pregnancies of mothers with confirmed or presumptive Zika virus infection, the proportion was doubled. Despite the limitations in the completeness of Zika virus testing in this cohort, data from Project VEZ contribute to our understanding of the impact of ZVD during pregnancy. The relatively high proportion of children with a potential delay in achieving a developmental milestone in the cohort overall suggests that even among infants without Zika-associated birth defects or postnatal-onset microcephaly, exposure to ZVD in pregnancy warrants careful and comprehensive follow-up of the infant into early childhood.