We aimed to assess the effects of renal denervation (RDN) on systemic and pulmonary hemodynamics in a swine model of thromboxane A2- (TXA2-) induced pulmonary arterial hypertension (PAH).  The study protocol comprised two PAH inductions with a target mean pulmonary artery pressure (PAP) of 40 mmHg at baseline and following either the RDN or sham procedure. Ten Landrace pigs underwent the first PAH induction; then, nine animals were randomly allocated in 1 : 1 ratio to RDN or sham procedure; the second PAH induction was performed in eight animals . In the RDN group, ablation was performed in all available renal arteries, and balloon inflation within artery branches was performed in controls. An autopsy study of the renal arteries was performed. P > 0.05). In the RDN group, the second PAH induction resulted in a numerical but insignificant trend toward a decrease in the mean systemic blood pressure and systemic vascular resistance, when compared with the baseline induction . No difference in hemodynamic parameters was noted in the sham group between the first and second PAH induction. Autopsy demonstrated artery damage in both groups, but RDN resulted in more severe lesions.  At baseline, the target mean PAP was achieved in all animals with 25.0  mcg of TXA2. The second PAH induction required the same mean TXA2 dose and infusion time. There was no statistically significant difference in the mean PAP at second PAH induction between the groups (39.0 ± 5.3 vs. 39.75 ± 0.5 mmHg,  According to our results, RDN does not result in significant acute pulmonary or systemic hemodynamic changes in the TXA2-induced PAH model. The potential chronic effects of RDN on PAH require further research. Pulmonary arterial hypertension (PAH) is a fatal disease characterized by a progressive increase in pulmonary vascular resistance, leading to right ventricular failure and earlier death. This condition is caused by vascular cell proliferation, intimal and medial hypertrophy, inflammation, and fibrosis. The sympathetic nervous system and renin-angiotensin-aldosterone system are involved in PAH pathogenesis , 2.Several approaches to decrease sympathetic nervous activity in subjects with PAH have been reported, including transcatheter or surgical pulmonary artery denervation (PADN) , direct Although the effects of RDN in earlier studies on systemic hypertension have been questioned, the results of recent studies seem more optimistic , 7. ThusTheoretically, the course of pulmonary hypertension may be aggravated by the activation of the efferent sympathetic bundles, leading to vasoconstriction, sodium retention, and release of renin from juxtaglomerular cells, and promoting the production of angiotensin II and aldosterone. Thus, deactivation of the afferent sympathetic bundles of the renal nerves may modulate the overall sympathetic nervous system activity and have positive hemodynamic effects in PAH.RDN in induced PAH using dehydromonocrotaline in dogs has been previously described . A few pIn a previous pilot experimental study, a decrease in pulmonary artery pressure (PAP) and pulmonary artery resistance has been detected in normotensive swine following extensive renal artery (RA) ablation . WhetherAmong several PAH models, intravenous thromboxane A2 (TXA2) infusion has been tested for the evaluation of acute hemodynamic changes after PADN . PreviouThis study is aimed at assessing the effects of RDN on systemic and pulmonary hemodynamics in a swine model of TXA2-induced PAH.This was a randomized experimental study with a sham procedure to evaluate acute hemodynamic changes after extended RDN. Ten normotensive breed Landrace swine  were included in the experiment. The Institutional Animal Care and Use Committee reviewed and approved all procedures and protocols for this study.2 0.3, tidal volume 10 mL/kg, and peak end-expiratory pressure 6 cmH2O. Vascular access was obtained through the right femoral artery and vein using the Seldinger technique. An 8F multipurpose sheath  was placed into the right femoral artery, and 7F and 6F sheaths  were inserted into the right femoral vein. Immediately after obtaining vascular access, heparin was delivered to reach an activated clotting time (ACT) over 300 s; ACT was assessed every 30 min, and additional intravenous heparin was administered, if necessary.All procedures were performed under general anesthesia. Sedation of the animals was performed using an intramuscular injection of 1.5 mL Zoletil 100 , and the outer ear vein was cannulated for drug infusion. Then, intubation was performed using mechanical ventilation. Anesthesia was maintained by ventilation with 1% isoflurane  using an anesthesia machine  with the following parameters: FiOContinuous invasive blood pressure monitoring was performed during the procedure. Measurement of hemodynamic parameters was described in detail earlier .The baseline hemodynamic parameters were considered after a 20 min waiting period following vascular sheath placement. A Swan-Ganz catheter  was used for right heart catheterization, and the following parameters were assessed: heart rate (HR), invasive blood pressure (BP), PAP, pulmonary artery wedge pressure (PWP), RV pressure, and right atrial pressure (RAP). Arterial blood samples were obtained from the abdominal aorta through a multipurpose sheath, and venous blood samples were obtained from the PA. Blood tests were performed using a portable analyzer . Cardiac output (CO) was calculated using Fick's equation. Pulmonary vascular resistance (PVR) and systemic vascular resistance (SVR) were calculated using established formulas as previously reported .During the induction of PAH, a constant recording of systemic BP and PAP was performed. After the first PAH induction, a time interval of 20–30 min was allowed until the baseline PA pressure was completely restored. Then, the RDN or sham procedure was performed according to the allocation group. A second PAH induction protocol was then performed after at least 30 min. The study flowchart is shown in PAH induction was performed twice, at baseline and following RDN or the sham procedure, depending on the allocation group, using a synthetic analogue of TXA2 . During induction, the mean PA pressure was targeted at 40 mmHg. Continuous administration of TXA2 was gradually increased every 5 min according to a previously established protocol . Electron = 6), bilateral RDN with repeatedly induced PAH, or group #2 (n = 4), a sham procedure. Blocked randomization was performed using an online randomization service .After the first PAH induction and spontaneous normalization of hemodynamic parameters, experimental animals were randomly allocated to group #1  ablation balloon catheter  was placed into each RA via a 0.014 in ×185 cm guidewire .Selective angiography of the right and left RAs was performed using 20 mL of contrast medium . After the first PAH induction, PA pressure restoration to a baseline level, and group allocation, isosorbide dinitrate 0.1%  was delivered selectively into both RAs through a vascular sheath at a dose of 50 In the RDN group, ablation was performed bilaterally in all available RA branches and segments. Each RF application was set at 30 s in duration, temperature 65–68°C, and automatically adjusted power of 0.5–1.0 W.In the sham group, the procedure was performed using the same protocol, including angiography, nitrate injection, and the number and duration of balloon inflation. However, no RF energy was delivered. After RDN or balloon inflation, a waiting period of 30 min was considered, and the PAH induction procedure was repeated.Hemodynamic parameters were compared between phases 1 and 2 of the study in each group and between the groups.μm thick and stained with hematoxylin and eosin.At the end of the experiment, all animals were euthanized using a lethal dose of intravenous potassium chloride solution . After the onset of biological death, the kidneys and abdominal aorta with the RAs were excised as a single unit and fixed in a solution of 10% buffered formalin for further gross examination. The RAs were cut longitudinally from the ostia to the renal parenchyma and evaluated macroscopically. Visually detected lesions were calculated and excised within normal tissue (3–5 mm borders around a lesion) through all artery wall layers  and prepared in paraffin blocks. Then, the items were sampled with a 1 mm step into slices 3 Unaltered and damaged nerves were calculated for each sample, the median number of total nerve fibers was calculated per lesion, and the percentage of damaged nerves was calculated per lesion. Nerve damage was considered when a nerve fiber was affected by a lesion, compressed by a hematoma, or totally destroyed.The primary endpoint was a change in TXA2 dose required to achieve a mean PAP of 40 mmHg after RDN in comparison with the sham group. The secondary endpoint was a change in the time required for TXA2 infusion until the target mean PAP was reached following RDN.U-test. Correlations were analyzed using Pearson's test. A two-sided P value < 0.05 was considered statistically significant. The analysis was performed using Statistica 12.0 .Continuous variables are presented as mean ± standard deviation or median with interquartile ranges, and categorical variables are presented as percentages. Group comparisons were performed using the Mann-Whitney Two out of ten animals died during the procedure and were excluded from the analysis. One animal died of acute right ventricular failure with a decrease in systemic BP and ST-segment depression on the ECG during the first PAH induction protocol. The other death occurred 5 min after renal denervation: complete atrioventricular block developed with further asystole, despite temporary RV pacing, and the animal died of electromechanical dissociation. In both cases, resuscitation measures were unsuccessful. In the first case, autopsy revealed a massive pulmonary embolism. Therefore, data analysis was performed on eight animals (four in each group).The total number of RF ablation points in the RDN group was 87 ; the range of ablation temperature was 67.1–68.5°C. The median number of balloon inflations inside the RAs was 4  per animal in either group.The target PAP was achieved in all eight animals before and after the RDN and sham procedures. The hemodynamic parameters in the RDN and sham groups are shown in P = 0.28). Similarly, the level of systemic vascular resistance (SVR) showed a trend towards a lower mean value (2433.7 ± 1176.7 vs. 1995.3 ± 494.3 dyn∗sec∗cm−5) after RDN at PAH induction, but the difference was not statistically significant (P = 0.46). There was also no statistically significant change in hemodynamic parameters in the sham group before and after the procedure. There was no statistically significant difference in the doses of TXA2 between the groups, either at the initial administration or during PAH induction following the procedure. Within each group, TXA2 doses for the first and second PAH induction were statistically equivalent (At PAH induction before and after RDN (phase 1 and phase 2), a statistically insignificant decline in the mean level of BP was noted   vs. 3 in total  ( < 0.05) . The shaNerve damage was detected in both animal groups, with a significant prevalence in the RDN group: 34% vs. 3% of nerve fibers visualized by microscopy .No blood clot was detected in any RA.In our experimental study, the primary outcome was not achieved; no change in the dose of TXA2 required for the increase of mean PAP up to 40 mmHg was observed after RDN. The secondary endpoint, the mean time of TXA2 infusion until the target PAP, showed no difference between the RDN and sham groups. Therefore, the main finding of this study is that RDN does not significantly influence hemodynamic parameters in TXA2-induced acute PAH. Although there was a trend toward a lower systemic BP after RDN at repeated induction of PAH, the difference was not statistically significant.The long-term clinical effects of RDN on BP have been widely demonstrated, and the major results of RDN are prominent several weeks following the procedure , 15. TheOur theoretical suggestion that RDN resulting in an overall sympatholytic effect might influence the inducibility of PAH in an animal model has not been proven in this experimental study. This may be explained by the following two assumptions. First, the TXA2 model of PAH induction is not suitable for our analysis because TXA2 is a direct vasoconstrictor and its effect cannot be attenuated by autonomic nervous tone. Second, the pathogenesis of acute PAH may not be related to sympathetic activity in animal models. The design of our study did not include remote assessment of RA denervation on pulmonary hemodynamics, and the chronic effects of the procedure on PAH inducibility should be evaluated in further studies.In a recent report by our group, RDN in normotensive pigs led to rapid and significant changes in systemic BP and PVR . These rIn one animal, a lethal massive pulmonary embolism possibly associated with TXA2 infusion was detected. The thrombogenic effect of TXA2 despite adequate anticoagulation might be responsible for this complication, as shown in a previous report .In our study, all procedures were performed under the same conditions, namely, all animals were of approximately the same age and similar morphometric indicators, and general anesthesia with intubation was performed. The only difference between the groups was the number of RF applications.Damage to the vascular wall of the RA was detected in all animals in the RDN group and in two animals in the sham group, suggesting that balloon inflation was performed adequately in both groups. These results confirm the previous results obtained in normotensive animals, where RA wall damage has been detected in RDN and sham-operated animals . Here, wPrevious studies in rodents have shown positive effects of renal denervation on monocrotaline-induced PAH. Thus, Liu et al. have found that earlier RDN treatment significantly decreases sympathetic nervous activity renin-angiotensin-aldosterone system (RAAS) activation and significantly improved the survival rate of experimental animals and attenuated cardiopulmonary fibrosis . SimilarIn our study, 34% of nerves were found affected in the acute period following ablation, and one might find this a low damage incidence. Thus, in a study by Cohen-Mazor et al. where a very similar ablation protocol has been used, necrotic changes at 7 days following the procedure were detected in 29 and 44% of nerves following single-ablation and full-artery length ablation, correspondingly. The additional damage of 45 and 59% of nerves was characterized as “degenerative” and “chronic/reactive” reflecting delayed changes. Considering the difference in time of necropsy with our study, we speculate that the rate of nerve damage should be comparable. However, the analysis of nerve damage was different and certain diversity in local results of ablation may present. Therefore, there is a potential possibility that the lack of a BP drop or hemodynamic effects might be related to insufficient denervation rather than a mechanistic finding .Since our neutral results of RDN on the inducibility of reversal of PAH using TXA2 and positive previous studies on chronic PAH models, we suggest that TXA2 might not be fully suitable for neuromodulation interventions for PAH and other models should be preferred. Indeed, acute hemodynamic changes following RDN detected in normotensive pigs were not reproduced in the TXA2-induced PAH model . AnotherWe evaluated the acute cardiovascular effects of RDN and did not assess its long-term effects.Another limitation of this study is the small sample size of animals on which experimental studies were conducted. Thus, this study is exploratory and shows the effects of RDN on the inducibility of acute PAH in animals in an experimental operating room. However, the use of a sham group in the course of research makes the conclusions of the experimental work more significant.Our protocol did not include high-frequency electrical stimulation of the RAs before and after RF ablation in the RDN group. Given the inconsistent results of this approach in the identification of successful denervation from previous studies, including our recent report , we deciAdditionally, our study protocol did not include the analysis of norepinephrine spillover before and after RDN. However, previous studies reported a significant drop in its level after ablation in the distal and proximal RA branches, as we implemented in the current study .In a previous report of Tzafriri et al., where a novel spiral multielectrode ablation system was implemented, a systematic correlation of 7d histological nerve effects with norepinephrine reduction determined >46% affected nerves as the threshold for statistically significant norepinephrine reduction . While tAccording to the results of our experimental study, RDN does not lead to significant hemodynamic changes in an animal model of acute TXA2-induced PAH. The potential chronic effects of RDN on pulmonary hemodynamics require further research.


Pseudomonas fluorescens biofilmsgrown on glass substrates by an external liquid is employed as a meansto investigate the complex morphology of these biofilms along withtheir capability to interact with external fluids. The porous structureleft behind after dehydration induces interesting droplet spreadingon the external surface and imbibition into pores upon wetting. Staticcontact angles and volume loss by imbibition measured right upon dropletdeposition indicate that biofilms of higher incubation times showa higher porosity and effective hydrophilicity. Furthermore, duringsubsequent rotation tests, using Kerberos device, these propertiesdictate a peculiar forced wetting/spreading behavior. As rotationspeed increases a long liquid tail forms progressively at the rearpart of the droplet, which stays pinned at all times, while only thefront part of the droplet depins and spreads. Interestingly, the experimentallydetermined retention force for the onset of droplet sliding on biofilmexternal surface is lower than that on pure glass. An effort is madeto describe such complex forced wetting phenomena by presenting apparentcontact angles, droplet length, droplet shape contours, and edgesposition as obtained from detailed image analysis.Wetting of dehydrated  Fundamental understanding of biofilmsrepresents strategic knowledge for public health, because of theirstrong resistance to antimicrobial agents. In industrial settings,biofilms are known for their ability to promote microbiofouling resultingin the clogging of hydraulic systems with consequent energy loss andpossible cutbacks and shutdowns and in the reduction of conductiveheat transfer across surfaces.5 In thecase of the food industry, biofilms can contaminate processing equipmentwhich are directly in contact with food, by also causing cross-contamination.6 However, biofilm formation is encouraged in someapplications like in wastewater treatment7 and bioremediation processes.9Biofilms are communitiesof microorganisms enclosed by a matrixmade of extra-cellular polymeric substances .10 It is known that chemical and mechanical stimuli  playa key role in biofilm development at different stages of growth, byregulating microbial transport, attachment on substrata and theirsubsequent adhesion, nutrients and oxygen delivery, and metabolitesdisposal through biofilm matrix and cell detachment.17 In addition, environmental conditions are also responsiblefor altering bacteria phenotype and genotype,19 bacterial quorum sensing,20 and morphologyof biofilms.22Biofilm formationis considered as a multistage process startingwith microbial surface attachment and adhesion, followed by the productionand accumulation of EPS that leads to the growth of sessile populationsinto mature biofilms and, eventually, concluded by cell detachmentand dispersion.23 It must be stressed thatbacterial adhesion, proliferation, and spreading over a surface tocreate a biofilm, which for brevity in literature is sometimes calledwetting of biofilm, is different from the wetting of a biofilm byan external liquid that we deal with herein. In our case, the biofilmitself is the substrate on which wetting occurs. The latter sharesseveral common features with the wetting of nonbiological porous layersby external liquids.28 It is recognized that biological surfaces are far from being idealsolid surfaces and as such experimentally measured quantities describingtheir wetting behavior represent apparent quantities, e.g., apparent contact angles.29 Thereare only a few systematic quantitative studies focusing on the physicsof the wetting of biofilms by liquids and these studies refer mainlyto Bacillus subtilis colonies grown on agar substrates.32 Attention to such biofilms stems from their extremely nonwettablecharacter and the technological crucial interest in understandinghow hydrophobicity can be reduced to remove deleterious biofilms bycleaning solutions.32 It was found that such biofilms are resistant towardwater and nonpolar liquids,31 showing hydrophobicor even omniphobic behavior.32 Werb et al.31 and Falcón García et al.32 demonstrate that biofilm colonies show variouswetting behavior depending on the selected system and conditions , i.e., from hydrophilicto superhydrophobic behavior. In this latter case, either lotus-likeor rose petals-like wetting regimes are obtained; generally, the wettingbehavior is strongly affected by the biofilm surface topography.31 The literature is rich on phenomena relatedto wetting of heterogeneous  substrates by droplets,where the effect of surface topography and chemical heterogeneitiescharacteristic of the micropatterned surfaces have been experimentallyand numerically investigated regarding the pinning/depinning behavior.40 The overall conclusions are that the presence of surface heterogeneityaffects the contact angle hysteresis and when external forces areemployed, overcoming pinning is strongly influenced by the natureof microscale defects,33 which act likeenergy barriers, hampering droplet motion.36 Interestingly, it is found that, in the case of the wetting of liquidson tilted patterned surfaces, the critical Bond number, seen as thedimensionless force needed to initiate droplet sliding, is stronglyaffected by the shape and spatial arrangements of surface patterningas shown in refs ,simultaneously with (re)hydration of dried solid matter. Even in theabsence of solid mass rehydration, wetting together with imbibitionis a quite complex process where several wetting states can be distinguisheddepending on the applied conditions. For instance, the Cassie–Baxterimpregnating wetting state corresponds to the classical imbibition into the porous layer.42 Moreover, wetting and imbibition by a finite volume sessile dropletover a thin dry porous layer is different from the cases where eitherthe liquid comes from an infinite liquid reservoir or the dropletlies on a thick dry porous layer.41 Whenstudying the wetting of dehydrated hydrophilic biofilms by liquids,the concomitant imbibition and solid mass rehydration processes implythat the standard definition of thermodynamic wetting arising betweenliquid and solid interfaces, is difficult to realize and the phenomenologyof biofilm wetting is quite complex to interpret. To our best knowledge,Recupido et al.,43 were the first to examine static (equilibrium) wetting properties on dehydrated hydrophilicbiofilms of Pseudomonas fluorescens AR 11 cultivatedon real (non-nutrient) substrates, i.e., glass coupons, under differentflow conditions. Biofilms grown at different conditions exhibit differentinternal structure and morphology which need be known to allow strategieseither for their effective removal from surfaces if they representa contamination (“bad” biofilms) or for intensificationof their development if they represent a means for nutrients production(“good” biofilms). To do so, hydrophilic biofilms aredehydrated to leave behind a solid porous structure which then mustbe analyzed. These authors found that wetting properties do not varysignificatively as a function of the different biofilm morphology,induced by growing them at different flow conditions. Their dehydratedbiofilms showed both hydrophilic and oleophilic behavior. To expand43 in investigating biofilm morphology effects,it is fundamental to also consider the application of external bodyforces on sessile droplets (forced wetting), which in principle maybe hydrodynamic, electrical, or mechanical ones and can drive thesystem beyond thermodynamic equilibrium. As far as we know, thereis no evidence in the literature about forced wetting on dehydratedhydrophilic biofilms.Wetting of dehydratedhydrophilic biofilms by external liquidsconstitutes an interesting but very complex case. Contact angles ofsessile droplets on hydrophilic microbial lawns, after an initialdehydration step, have frequently been used in physicochemical modelsas a measure of microbial cell surface structure.Pseudomonas fluorescens AR 11 bacteriacultivated on glass substrates is used as a model microorganism toform biofilms. Wetting properties on dehydrated biofilms are investigatedusing a custom-made device, named Kerberos, dedicated to the studyof physics of forced wetting of droplets deposited on different substrates.47 Kerberos employs independent control of centrifugal and gravitationalforces acting on droplets by combining rotation and tilting whileat the same time monitoring the droplet shape deformation and motionby three cameras viewing the droplet at the three orthogonal directions.The present experiments refer exclusively to rotation of horizontalsubstrates (no tilting). For porous/micropatterned surfaces like biofilms,it is well-known that wettability is strongly affected by surfaceproperties such as morphology, which, however, is driven by the differentbiofilm growth conditions i.e., nutrient composition, shear flow,or incubation time. The goal of this study is to identify basic featuresof the forced wetting phenomena of dehydrated porous biofilms up tothe onset of droplet sliding as a tool to examine biofilm morphologyand describe an appropriate image analysis scheme for data extractionand evaluation. The investigation of the droplet contact angles aswell as of depinning thresholds during rotation tests, is devotedto estimate the retention forces needed for droplet onset for sliding,which actually represent an indirect measure of biofilm surface adhesion.In this work, Pseudomonas fluorescens AR 11 bacteria are provided by DSMZ-GermanCollection of Microorganisms and Cell Cultures and reactivated according to the manufacturer protocol.48 The rehydration is performed in 0.5 mL of liquidcomplex medium  and subsequent mechanical agitationfor 30 min. The bacterial suspension is grown at 30 °C overnightat 80 rpm and then diluted to achieve an optical density (OD600 nm) ≈ 0.5 cm–1, which corresponds to around107 cells/mL. Biofilms are developed using M9 minimal mediumcontaining the following constituents, while pH is adjusted at 7:6.8g/L of Na2HPO4, 3 g/L of KH2PO4, 0.5 g/L of NaCl, and 1 g/L NH4Cl, 0.24 g/L MgSO4·7H2O, 0.04 g/L CaCl2·2H2O, 0.05 g/L EDTA, 8.3 mg/L FeCl3, 0.84 mg/L ZnCl2, 0.1 mg/L CuCl2·2 H2O, 0.1 mg/LCoCl2·2H2O, 0.1 mg/L H3BO3, and 0.016 mg/L MnCl2·4 H2O and0.1% glucose.Lyophilized 3) placedinside polystyrene Petri-dishes (Φ = 90 mm) .Glass coupons are cleaned with commercial anionic surfactant P1, rinsedwith tap water, then immersed in 95% ethanol solution, rinsed againwith distilled water, and finally dried at 200 °C for 2 h. Ineach Petri dish, a microscope slide is placed horizontally and coveredwith 1.5 mL of bacterial suspension and 13.5 mL of sterile minimalmedium (1:10 dilution). The resulting overall thickness of the fluidlayer is 2.3 mm. Biofilms are formed on both sides of the glass substrate.The total area of the glass is 38.7 cm2, and the totalarea of the Petri dish exposed to the medium is 70.22 cm2. The selected operative conditions are meant to ensure aerobic conditionsfor bacterial growth. Biofilm formation and development is carriedout at 30 °C for 7 days. Nutrient replacement is performed after24 h from the inoculation and then, every 48 h, in order to promotebacterial viability. The evolution of glucose concentration withinbacterial suspensions is estimated during the first 24 h after inoculationand displayed in S1 (SI).Biofilm formationand growth isconducted under stagnant fluid conditions. Biofilms are formed onmicroscope slides . Before OD600 nm measurement,bacterial suspensions are gently homogenized and then 2 mL are sampledand measured. Biofilm growth is determined by scratching biofilm fromglass surfaces using a sterile toothbrush and resuspending it in 15mL of sterile M9 phosphate solution. First, the OD600 nm turbidity of the mechanically removed and resuspended biofilm ismeasured. Then, the Total Organic Carbon (TOC) and Dissolved OrganicCarbon (DOC) of the resuspended scratched sample are also determinedaiming to quantify the biofilm organic matter. TOC is measured byusing a TOC analyzer . TOC measurementsare obtained indirectly by subtracting the Inorganic Carbon (IC) fromthe Total Carbon (TC) as reported by ref . Images are acquired using a 40×/0.70PL Fluotar objective. In order to increase the contrast between thebiofilm covered surfaces and the surrounding blank glass area, glasssubstrates are stained with Crystal Violet solution (0.1% in bidistilledwater). Briefly, 3 mL of Crystal Violet solution is added to eachsample. Samples are incubated for 10 min. Rinsing with bidistilledwater is performed to remove the excess of Crystal Violet solution.At each incubation time, the morphology of three independent glasssamples is examined, by acquiring, for each sample, images at threeindependent positions. Biofilm coverage area over the total area ofthe image (%) is evaluated using the commercial software Image ProPlus 6.1 . Covered area normalized withrespect to the entire image size is considered as an estimate of biofilmcoverage of the glass sample. Measurements of averaged covered area%are reported for different incubation times, Standard Deviation (SD)provides an estimate of samples variability.47 The device combines simultaneous rotation and tilting to provideindependent control of tangential and normal forces acting on a dropletplaced on a solid substrate. Kerberos allows monitoring of the 3Ddroplet shape as a function of time, by recording images with threeindependent wireless cameras that observe the droplet simultaneouslyalong the 3 orthogonal directions X, Y, Z. Cameras rotate and tilt together with the sample.In particular top, side, and back views visualize the droplet on the, , and  plane, respectively.During an experiment, the chamber containing the sample and the camerasallows to control the temperature and air humidity surrounding thesample. Detailed description of the main features and capabilitiesof the device along with analysis of spreading/sliding of dropletson different solid substrates at varying tilting angles and rotationspeeds can be found in.47 In this work no tiltingis done, while centrifugal forces are employed to induce droplet deformation,spreading, and sliding depending on the applied conditions  is the distance betweenthe droplet and the axis of rotation. It is noted that for the employedexperimental configuration αc takesa value equal to the gravitational acceleration at about 60 rpm. Wettingexperiments of water solution on the uncoated glass (no biofilm) underthe application of centrifugal forces are used as negative controls.During tests, biofilm coated and blank glass substrates are placedinside a test cell  described in detail inref . The cod51 Although Image ProPlus software requires more effort by the user compared to the automaticMatlab code, it allows versatility in selecting the background forthe determination of droplet contour even at the level of single frameswhich cannot be provided by the automatic Matlab code.Apart from the aforementionedMatlab code (which runs without possibility for interventions), detailedanalysis of droplet images is conducted to identify the complex wettingbehavior of biofilm-coated surfaces. Every frame is converted fromRGB to gray scale and analyzed using a commercial image analysis software(Image Pro Plus 6.1). Image analysis measurements include image-histograms,(reporting the percentage of pixels of the image as a function ofthe gray intensity); and linear intensity profiles . These tools are used to identifydroplet edges and, therefore, the distribution of water along sample.Alternative visualization of droplet edges is also obtained by applyingedge detection filters (Sobel), based on the calculation of intensitygradient profiles.600 nm, due to the medium replacement and thus to therestoration of the initial conditions, which suggests the achievementof a good physiological state of the planktonic cells, typical ofthe exponential phase of bacterial growth, since OD600 nm is lower than 1 cm–1.52 Biofilm OD600 nm represents an indirect measureof the whole biofilm mass, which increases during the testing period.53 Biofilm TOC and DOC also increase over time.This implies that, at higher incubation times biofilm formation isenhanced, primarily due to the food replacement. More specifically,it is found that the TOC measured at 7 days, is about 5 times higherwith respect to the value measured at 3 days, while the DOC valueis about 4 times higher. It is important to point out that biofilmTOC refers to the overall available organic matter within the biofilmstructure, which includes both the part contained in bacterial cellsand that contained in the dissolved matter surrounding the cells.DOC corresponds exclusively to dissolved organic carbon that is predominantlyfound in EPS; therefore, this parameter represents a quantitativeestimation of biofilm EPS. The observed increase in the differencebetween TOC and DOC with the incubation time, indicates a progressiveincrease of the number of bacterial cells attached to biofilm.Inthe present work,biofilms are grown on glass substrates under stagnant fluid conditionsat two incubation times: 3 and 7 days, respectively. In Under stagnant fluid conditions,biofilms are in the form of patchy like clusters consisting of rod-shapedbacteria cells and EPS, surrounded by empty spaces 3. Cells/25 This preliminary observation might indicatethat the biofilm-coated surfaces show a higher effective hydrophilicitycompared to pure glass. However, the possibility exists that partof the droplet edges get pinned soon after deposition because of localheterogeneities and defects of the biofilm surface. In such case,the observed progressively lower contact angles might be also theresult of volume reduction due to imbibition, at least down to anangle where depinning occurs. This is in line with other studies relatedto contact line pinning on heterogeneous substrates.57 These observations are qualitatively comparable with the resultsobtained by Recupido et al.43 but are oppositeto the case of B. subtilis colonies grown on Agarsubstrates,32 which show different wetting behavior i.e., fromhydrophilic to superhydrophobic depending on growth conditions. Dropletsgently deposited on uncoated glass show always sharp circular edges,typical of a spherical cap  was 54 ±2 rpm. On the present biofilm-coated substrates, already at 40 rpmthe droplet moves. This suggests that the force needed for the inceptionof droplet motion is lower compared to the case of pure glass. Itis probable that due to imbibition a thin moist biofilm layer surroundsthe droplet which facilitates motion. This is in line with the notionof capillary forces continuing to elongate the droplet, although ata much lower speed, after its initial fast spreading upon deposition.Wettingexperiments arecarried out under the application of centrifugal forces (Kerberos)on glass substrates coated with biofilms cultivated in stagnant fluidconditions at two different incubation times: 3 days and 7 days, respectively.Experiments are performed with the rotation speed increasing from0 to a 100 rpm following a ramp with a slope of 1 rpm/s. Before presentingdetailed information on how apparent contact angles, contact lengths,droplet contours, and droplet edge positions vary with rotation speed,a few representative droplet images are displayed to allow describingsome qualitative features of wetting of dehydrated biofilms. Thisis done in ical cap 4 A,i–ii.ical cap 4B,i-ii. During motion of a droplet ontothe biofilm, its rear edge neverdepins from its original site but instead a liquid tail develops atthe rear side of the droplet, where a translucent residual moist layerseems to be left on the biofilm surface 4B,iii–iv57 that calculates local light intensity gradients along theimage plane. Objects edges, corresponding to maxima in the gray gradients,are visible as bright lines. Sobel filtered images of top and sideviews are reported in Amax. Image histogram of top viewin In Amax is reported as the gray area in Gray intensity profile of the image in Amax, x axis (αA) shows theprojection of droplet length along the moving direction (x), which is the quantity typically measured as contact length fromthe side view. The observed angular deviation αA with respect to the x direction, isthe result of a minor imperfection in leveling the substrate withrespect to the horizontal because of which the gravitational accelerationcreates a lateral force component affecting the direction of dropletmotion or due to heterogeneities and micropatterns of biofilms surface,which may create preferential paths in droplet motion. This minordiscrepancy is not relevant for the scopes of our work. The two measurementsof the droplet length obtained from the top view, identified by thered and green edges, are defined as LT1 and LT2 and referto the whole droplet and the dark bulky region, respectively. It isworth mentioning that the difference between jagged (continuous) andellipsoidal (dashed) edges lead to a less than 2% discrepancy in theestimated droplet area and a less than 10% discrepancy in the estimateddroplet length. In our analysis we refer to top lengths LT1 and LT2, as measured by the ellipsoidaledges. By observing the droplet from the side and for the dark bulky region (green edge), respectively. Similarly,two different rear contact angles are defined as θr1 and θr2. Additionally, two droplet lengths are measured fromside images defined as Ls1 and Ls2. The definition of two dropletlengths implies the existence of two different baselines for the darkbulky region and for the whole droplet, respectively increases along with the rotation speed and the dark bulky droplet region (green lines) mentioned before.For the dark bulky region (45x) of the droplet on 3-days old and 7-daysold biofilms, respectively. As with Figure S2. The motion ofthe droplet edges gives additional information with respect to thedroplet length evolution by revealing the pinning behavior of thedroplet and allowing better identification of spreading and sliding.The edge positions are calculated from the top and side views, forboth the entire droplet and the dark bulky droplet region  never move during theentire rotation test, corroborating the above-mentioned results reportedin The position of the front and the rear dropletedges are also derivedfrom the above measurements. Inthis work, wetting properties of dehydrated hydrophilic biofilmsby an external liquid under the application of centrifugal forcesare examined for the first time. A specialized device, Kerberos, capableof studying the wetting properties of sessile droplets under the actionof body forces, i.e., gravitational and centrifugal ones, is employed.Biofilms are cultivated in stagnant fluid conditions at two incubationtimes: 3 and 7 days, respectively. An accurate analysis of wettingproperties is conducted by means of an automatic and a manual computercode that combines fast treatment of long videos with the versatilityin resolving contrast problems of single frames. It is found thatthe porous structure left behind after dehydration leads to simultaneouswetting and imbibition of the biofilm by the liquid of the droplet.This peculiar behavior affects the droplet shape right upon its depositionon the biofilm surface leading to small apparent contact angles asa result of liquid being sucked into the pores. As a result, dehydratedporous biofilms when compared to bare glass appear effectively morehydrophilic in the sense that contact angles on the biofilm surfaceare lower because of the volume loss by imbibition. This effectivehydrophilicity increases at higher incubation times because the biofilmis thicker with more pores. Furthermore, during rotation tests therear edge of the droplet never depins leading to the formation ofa severely elongated droplet with a long thin tail at its back. Thisis attributed to imbibition which keeps the liquid sucked in the poresat all times. All in all, forced wetting tests reveal features ofthe dehydrated biofilm morphology that dictate their interaction withexternal fluids and so can be valuable for a wide range of applications,including biofilm removal or prevention i.e., optimization of detergentsolution formulations or design of antimicrobial/antibiofouling surfacesimpairing microbial attachment.


In case of extreme premature delivery at 24 weeks of gestation, both early intensive care and palliative comfort care for the neonate are considered treatment options. Prenatal counseling, preferably using shared decision making, is needed to agree on the treatment option in case labor progresses. This article described the development of a digital decision aid (DA) to support pregnant women, partners and clinicians in prenatal counseling for imminent extreme premature labor.This DA is developed following the International Patient Decision Aid Standards. The Dutch treatment guideline and the Dutch recommendations for prenatal counseling in extreme prematurity were used as basis. Development of the first prototype was done by expert clinicians and patients, further improvements were done after alpha testing with involved clinicians, patients and other experts (n = 12), and beta testing with non-involved clinicians and patients (n = 15).The final version includes information, probabilities and figures depending on users’ preferences. Furthermore, it elicits patient values and provides guidance to aid parents and professionals in making a decision for either early intensive care or palliative comfort care in threatening extreme premature delivery.A decision aid was developed to support prenatal counseling regarding the decision on early intensive care versus palliative comfort care in case of extreme premature delivery at 24 weeks gestation. It was well accepted by parents and healthcare professionals. Our multimedia, digital DA is openly available online to support prenatal counseling and personalized, shared decision-making in imminent extreme premature labor.The online version contains supplementary material available at 10.1186/s12911-021-01735-z. A so-called “gray zone of parental discretion” exists, in which parents and physicians make treatment decisions together –5. The etologist –12. Givetologist , 9–12. TDecision aids (DAs) are used to assist in shared decision-making, as they provide objective information and visual material in a comprehensive way. DAs have been proven to be effective to enhance communication between patient and physician by eliciting situation-specific values and preferences. Moreover, they improve patient knowledge and satisfaction in the shared decision-making process for both patients and care providers , 14.Internationally, some tools have been developed to help parents in the situation of imminent extremely premature labor. An overview of these tools is provided in the supplementary material  fulfill the need for visual supportive material, (b) take into account the Dutch situation, guideline, and language, and (c) to better involve parents in decision-making, using value elicitation. Aim of this research was to describe the development  of this DA to support prenatal counseling in imminent extreme premature labor.0/7 and 246/7, but counseling can be offered from 230/7 onwards [0/7 and 246/7 weeks GA [In the Netherlands, nine perinatal care centers with NICU  facility exist. In Dutch practice since 2010, the defined gray zone of parental discretion lies between gestational age (GA) of 24 onwards , 16. EacThe digital DA was developed using a model development process that includes all original elements of the systematic process established by the International Patient Decision Aid Standards (IPDAS).This pro0/7 weeks GA. Considering potential birth at 240/7- 246/7 weeks, counseling and thus use of the DA may take place from 230/7 weeks onwards [The purpose of this digital DA was to support both expectant parents and physicians in prenatal counseling regarding treatment options for imminent extremely premature birth. Next to obstetricians and neonatologist, the target audience comprises expectant parents of the general Dutch population, who will receive prenatal counseling before 25 onwards . The inf onwards . So, thi+0/7 – 24+6/7 weeks GA between 2010 and 2013 and received counseling in 5 different hospitals. Regarding the 6 experienced (pairs of) parents, palliative comfort care was chosen once and early intensive care was chosen 5 times. From their 9 infants, 3 survived. All 9 tertiary perinatal care centers in the Netherlands were represented by at least an obstetrician or neonatologist or both in the steering group. Both the Delphi consensus study, and earlier work among Dutch parents and physicians [For the development of the Dutch counseling recommendations, an expert panel was formed for a Delphi consensus study – which can be regarded as steering group 1. It consisted of 6 (pairs of) parents, 7 obstetricians and 8 neonatologists . Those pysicians , 22, 23,Patients’ views on prenatal counseling were assessed before in a survey (n = 61) and interviews (n = 13) , 23, jusNext to their general views and preferences regarding prenatal counseling, both clinicians and parents were specifically assessed on their preferences regarding a DA in the interviews as referred to in design 1 and 2, 18. As In a Delphi consensus method, evidence from international literature was combined with Dutch results (patients’ and clinicians’ views from Design 1–2) to develop cultural sensitive, nationwide recommendations for prenatal counseling in extreme prematurity , 22, 23.For the development of the DA, a new steering group was created. This group consisted of 2 obstetric professionals  and 2 neonatal professionals , representing two tertiary perinatal centers, and one professional in development of DAs. Steering group 2 drafted the first version of the DA see . This fi0/7 weeks and 246/7 weeks, previously involved in stage Design 1). Furthermore, six other experts were involved: two obstetricians , one neonatologist, an expert in quality of care improvement, a professional in DA development and one Dutch language expert of the Easy Reading Platform (in Dutch: Stichting Makkelijk Lezen). The latter was involved to assure readability of the (Dutch) text following their “Quality Seal for comprehendible texts”. All participants in alpha testing  reviewed the content of the DA several times, leading to multiple rounds of revision. Steering group 2 reviewed all comments and finalized the process of alpha testing. The first prototype was subsequently build in the online module, including graphics, for beta testing.To check comprehensibility and usability of the first draft, several rounds of alpha testing were performed, in an iterative process during development with members of our Steering groups. Involved in this stage were six parents of children born extremely premature  signs of prematurely delivery (n = 4). The second group involved pregnant women, who were admitted for imminent preterm labor < 246/7 weeks GA and received antenatal counseling (n = 3). Clinician participants were obstetricians and neonatologists recruited amongst two Dutch university hospitals with NICU facility . All reviewed the online module of the digital DA and subsequently filled out a questionnaire. This questionnaire included 7 propositions to be rated on a 5 point Likert scale (very much disagree – very much agree) and two open questions, asking for three positive points of the concept DA and three improvement points. Answers to the multiple choice and open questions were reviewed by Steering group 2 and discussed in order for improvement of the final version of the DA.Feasibility of the DA was checked during one round of beta testing with clinicians and pregnant women, in a “real-world” setting, outside of the developmental process. All participants  were not previously involved in the processes of the DA development. Patient participants comprised two groups. One group involved pregnant women between 24Recommendations regarding important content for decision-making  were inc6/7 weeks GA were based on the national perinatal registry (Perined) between 2011–2017 [Mortality and survival rates of children born before 24011–2017 , 23. Lon011–2017 , 25, 26 011–2017 , 27, andThree additional steps were developed to aid in decision making Table . In stepAlpha testing comprised multiple rounds with 12 participants for revision of the text of the main body and the illustrations. The revisions concerned mostly clarity of wording, phrasing and shortening without compromising the actual content. The level of readability was comparable to eight grade level in English language.Two visual aspects of the DA raised discussion in the rounds of alpha testing. Regarding the report of numbers of mortality and morbidity, two options were explored. The first option was to show all numbers, including the pictographs, directly in the main body of the DA. The second option was to explain the possibility to review numbers and pictographs, followed by a mouse-click in an expanding drop-down box. There was consensus on the second option after exploration of both parents’ and clinicians’ views. This is consistent with previous literature describing that many, but not all, parents would like to review statistics , 18, 28.After alpha testing, the first prototype of the DA as website was tested (n = 15). All 15 participants reviewed the digital DA and answered the usability questionnaire. Results are shown in Table Results from field testing including all open comments were reviewed by the research group and several textual changes were made, which led to the final version of the DA. All IPDAS criteria were fulfilled . The DA was also approved by the Dutch patient organization (CARE4NEO). Furthermore the DA was given the “Quality seal of comprehendible texts” by the Dutch Easy Reading Platform.www.keuzehulpvroeggeboorte.nl in the autumn of 2019.The DA (in Dutch) was made available on This study describes the development of a DA to support prenatal counseling in extreme prematurity with involvement of healthcare professionals, parents who experienced extreme prematurity, and  future users. Its content is in line with the Dutch guideline for perinatal management of extremely preterm delivery and the nationwide recommendations for counseling on this matter , 16. We The following strengths of this study need to be discussed. This DA is developed with a systematic approach as recommended by IPDAS . Parts oThere are limitations to this study. Due to the national setting, it is unknown to which extent our content is exemplary for other, international settings. The use of Dutch outcome data can be regarded as cultural-sensitive, since the target population is Dutch, and Dutch health care providers wanted national data. However, others may be worried that these outcome data introduce a bias since the restrictive Dutch approach could influence outcome data , 30. I.epreference sensitive decision. Parents need to understand the options, the risks and benefits of each option, and the related information about potential outcomes. This information must be presented in a balanced way and physicians should elicit and clarify individual values of the parents facing this decision [In the counseling of parents facing imminent extreme premature labor, practice variation exists as a result of different health care systems and various cultural, social and individual preferences . The decdecision –12. Decidecision , 18. ParIn contrast, restraints have been described for the use of DAs in this setting. Although parents are usually involved in development and design of DAs, as in ours, it is unsure whether its information will match the information need of all parents , 32. FurFinally, research on how to elicit patient values in this setting is scarce but growing, and should be incorporated in future updates of our DA , 10, 37.The overview of existing DAs, as shown in Additional file The effectiveness of DAs in decision-making has already been proven on many different levels , 14. FutA digital DA to support prenatal counseling regarding the decision on early intensive care versus palliative comfort care at imminent extreme premature delivery was developed and was well accepted by parents and health care professionals during beta testing. By following a systematic developmental process with critical involvement of both clinicians and (future) parents, the digital DA is anticipated to support the decision-making process. Implementation of this digital DA, as well as its effects on shared decision making, is the focus for future studies.Additional file 1. Overview of existing DA tools for imminent extremely premature delivery.Additional file 2. Supplemental DA figures.Additional file 3. International Patient Decision Aid Standards instrument.


An experiment was conducted to investigate the continuous and intermittent lighting program effects on terms of the productive performance, carcass traits, blood biochemical parameters, innate immune and oxidative status in broiler chicks.A total of 600 Cobb-500 one day old chicks were randomly allocated into six equal groups (100 chicks per treated group with five replicates of 20 chicks each) based on lighting program; 22 continuous lighting (22 C), 11 h lighting+1 darkness twice daily (11 L/1 D), 20 h continuous lighting (20 C), 5 h lighting+1 darkness four times daily (5 L/1 D), 18 h continuous lighting (18 C) and the final group subjected for 3 h lighting+1 h darkness six times daily (3 L/1 D). The experimental period lasted 42 days.Compared with those under the intermittent light program, broiler chicks exposed to continuous lighting for 22 h had significant improvement in live body weight and carcass (dressing and breast percentage) measured traits. Though reducing lighting hours significantly reduced feed intake and feed conversion ratio values. Different lighting programs revealed no significant effect on all blood biochemical parameters. Oxidative stress and innate immunity parameters significantly enhance by reducing lighting hours (3L/1D).The findings suggest that reducing lighting hours up to 3L/1D would be more useful in enhancing feed efficiency, innate immunity, and oxidative status compared with continuous lighting programs on broilers. Globally, demand for producing high-quality broilers with lower costs has increased the emphasis on enhancing bird health and immunity to optimum performance. Rapidly growing broiler chickens, especially in early stages, are associated with several health problems such as sudden death syndrome (SDS), ascites and skeletal abnormalities . DuratioBroiler producers extensively use an intermittent lighting (IL) programs on broiler farms . AccordiWorldwide, the Cobb 500 broiler is a recent commercially available broiler strain. This strain has made an impression on the commercial poultry market and has a unique growth rate and yielding ability characteristics compared to other available strains . TherefoTherefore, the propose of the current study was not only to compare the continuous and IL programs but also to evaluate their effect on growth performance, carcass traits, blood parameters, oxidative stress and innate immunity in cobb broiler.All experimental procedures were followed and approved by the Ethics Committee of the Local Experimental Animal Care Committee, Department of Animal Husbandry and Animal Wealth Development, Faculty of Veterinary Medicine, Damanhour University.The present study was carried out at the Research Poultry Farm, Faculty of Veterinary Medicine, Damanhour University, Egypt. A total number of 600 one-day-old of both sexes Cobb-500 broiler chicks were purchased from El-Watania Hatcheries, km 59, Alexandria - Cairo Desert Road, Alexandria, Egypt. Chicks were individually weighed to make uniform replicate groups. The chicks were distributed in a completely randomized design into six equal experimental groups (n = 100) with five replicates (20×5). The six groups were as follows: T1, birds subjected to 22 continuous lightings (CL22); T2, birds subjected to 11 h lighting and 1 h darkness (11 L/1 D) twice daily (IL22); T3, birds subjected to 20 h continuous lighting (CL20); T4, birds subjected to 5 h lighting and one h. darkness (5 L/1 D) 4 times daily (IL20); T5, birds subjected to 18 h continuous lighting (CL18); and T6, birds subjected to 3 h lighting and 1 h darkness (3 L/1 D) six times daily (IL18).ad libitum consumption. For the first three weeks, chicks were fed on starter ration  and followed by grower ration  for the remaining period of the experiment as presented in The facility was equipped with supplementary pan feeders and drinkers for the brooding period and then birds were moved to the cages. The birds had free access to feed and water for Chicks were brooded in floor pens (covered with wood shaving litter) during the first two weeks with using supplemental feeders and drinkers and then moved to cages, each replicate housed in 2 separate pens (each pen 100 cm length×90 cm width×45 cm height). The cages provided with separate lines of nipple drinkers for each pen and separate feeders. Chicks were raised under identical hygienic, managerial, and environmental conditions throughout the study conforming to manual guide recommendation for the strain. Briefly, the temperature was set constant 33°C at the birds’ level during first three days of age then gradually reduced with 1°C per two days until 25°C at 21 days and then remained at 25°C up to the end of the rearing period. The lighting schedule was applied, as mentioned before, from hatch to the end of the rearing period (42 days). The intensity of measured light in the middle of the room was ranged between 5 and 10 lux. Routine vaccination schedule was administered and necessary medication when needed based on diagnoses and symptoms shown by the birds.During the experimental period, all birds were subjected to the same method of data collection. Chicks in each replicate were individually weighed at weekly intervals with electric balance until 6 weeks of age. Weighing of the birds was done every week in the early morning before receiving any feed or water. Also, weekly FC, total feed consumption (TFC), and dead birds (if any) were recorded.Growth traits include the determination of BW, body weight gain (BWG), and FCR, mortality (%) and livability (%). After calculation of livability % and FCR performance index of European Production Efficiency Factors (EPEF) were used to evaluate the growing performance index of broilers as suggested by Aviagen . The EPEAt the end of the trial (42 days), 20 birds  from each group were randomly chosen around the average weight of the group to determine the carcass characteristics and internal organs. Birds were slaughtered with a knife , allowed to bleed for 150 s, scalded at 60°C×90 s, de-feathered and manually eviscerated. Following evisceration, all carcasses were chilled in cold water for 15 min. Hot carcass, breast, thigh, shoulder, left filet, liver, heart, gizzard, intestine, and abdominal fat were weighed. The blood, viscera, lungs, limbs, head, and neck were termed as the offal’s and they were discarded. The abdominal fats in the pelvic and abdominal cavity were collected separately from the carcass and weighted. The technological division of the carcass was performed and calculated according to Xie et al .The blood samples (5 samples from each replicate) were collected from the wing vein at 42nd day of the experiment. Blood tubes were placed at a slant position at room temperature for 30 min and then separated through centrifugation at 3,000 rpm×15 min. The separated serum samples were collected, frozen and stored at −20°C until subsequent analysis of blood biochemical parameters.Total protein and Albumin were determined using the Bio-diagnostic colorimetric kit according to the method of Apanius et al . The valThe malondialdehyde (MDA) concentration concentrations in homogenates were evaluated by the method of Jo and Ahn . Serum bCandida albicans culture was added to 1 mL of citrated blood from each group and incubated in the water bath at 25°C for 5 h, and then blood smears from each tube were stained with Giemsa stain. Phagocytosis was estimated by determining the proportion of macrophages which contained intracellular yeast cells in a random count of 300 macrophages and expressed as percentage of phagocytic activity (PA). The number of phagocytized organisms was counted in the phagocytic cells and called PI.Blood samples were collected at 42 days of age (5 samples per replicate) as mentioned above. Phagocytic activity and phagocytic index (PI) were determined as described by Kawahara et al . Brieflyijkl = μ+Aj+ei; where: Xij = observational data, μ = overall mean, Aj = effect of lighting program, ei = random error.Data obtained were analyzed by a one-way analysis of variance using Statistical Analysis System  . The maiResults of the lighting program’s effects on growth performance parameters  are presented in Furthermore, regarding feed utilization, different lighting programs significantly (p<0.01) affect the TFC of all groups. It was found that broilers reared in CL programs  consumed significantly more feed than their comparable exposed to IL programs , respectively. At the same time, broiler groups exposed to long photoperiod for 22 h (T1 and T2) whether CL or IL consumed significantly more feed than those in medium photoperiod for 20 h (T3 and T4) or short photoperiod for 18 h (T5 or T6). Equally essential results showed that lighting programs significantly (p<0.05) affect the total FCR of all broiler groups. It was found that chickens raised in most prolonged light intervals T1 and T2 (for 22 h whether CL or IL) recorded significantly higher and lowest total FCR (1.58) values than those in medium (1.65 and 1.54 for T3 and T4) or short photoperiod for 18 h (1.55 and 1.54 for T5 or T6), respectively. Besides, broilers reared in IL programs  recorded better FCR than others exposed to CL programs , respectively.Regarding the mortality rate, the results presented in Results of dressing %, carcass traits and internal organs as affected by lighting programs are presented in Results of the lighting program’s effects on blood biochemical, blood kidney and liver function parameters are presented in Results presented in Additionally, regarding the PI and PA data presented in When the birds’ environment is taken into consideration, lighting program is one of the most critical of all environmental factors affecting performance. The lighting program affects the birds’ metabolism, which in turn is responsible for maximizing growth performance and maintaining normal physiological processes and functions. The results of the present study indicated that broiler performance in terms of BW and BWG was significantly affected by differences in lighting intervals (continuous or intermittent). Reducing lighting interval (from 22 h to 20 h or 18 h) and using intermittent light programs (IL) caused a significant reduction in BW and BWG, especially at older ages of the broiler. This reduction in BW and BWG could be explained by the reduction in time of feeding which causes a decrease in feed consumption by birds in the shortest light intervals where there was a highly positive correlation between BW and FC.The present results are in agreement with Schwean-Lardner et al  who compIn fact, broiler chickens do not feed or drink during a dark period . Thus, fDuration of lighting is a major factor affecting broiler per formance. Several investigations showed that a CL regime is not recommended as an optimal program ,38. The Regarding the mortality rate, the application of graded levels of lighting programs on broilers indicated that extended lighting programs increased mortality, reduced livability and changed the behavior as found by Schwean-Lardner et al . High moSimilar findings to our results were supported by Farghly and Makled . MoreoveIn regard to carcass traits, lighting programs had an effect on dressing, breast, abdominal fat, liver and intestine % in the present study, especially for the group which was exposed to 22 h (CL22). Increased dressing, breast, abdominal fat, liver and intestine % in CL22 group may be due to the increase in pre-slaughter weight which highly correlated with dressing yield. Significant correlations between pre-slaughter, carcass and breast weights were observed in this study. Similar to the current study, pre-slaughter weight was highly correlated with dressing yield in Cobb 500 and Hubbard broiler strains . ReducinThese results were supported by the data from Rahimi et al  who obseIn general, blood biochemical parameters are used as an indicator to detect disorders due to incorrect lighting programs effecting metabolic, nutritional and welfare conditions of broilers. In the present study, there were no changes in biochemical parameters. It might be due to chronically stressed after exposure to different light programs in broiler birds. It is well known that intermittent light decreases physiological stress, improved immunity and bone metabolism .The results of the present study were in full agreement with those obtained by El-Slamoney et al  who founParameters of oxidative stress such as SOD, GPx, and T-AOC are indicators for assessing oxidative stress status . Also, M4+ cells and T lymphocytes cells which enhanced by melatonin levels.Phagocytic cells (non-lymphoid cells), including macro phages, neutrophils and heterophils play a crucial role in immunity which digests foreign materials, such as bacteria, viruses, denatured proteins and lipids, and apoptotic cells . In thisAnother possible reason is that long photoperiod and CL programs decrease the opportunity for rest and sleep, thereby increasing fear reaction and physiological stress. So, birds exposed to long CL regimes have a decrease in PA and PI due to higher plasma concentrations of corticosterone, indicating the high level of physiological stress and foreign bodies which consequently cause inflammation, ultimately resulting incidence of metabolic disorders and increase MR % in the broiler. Therefore, groups reared under short intermittent light programs have lower physiological stress, improved specific and non-specific immune responses which emphasizes the importance of dark periods during the production cycle of broiler chickens . The resIn conclusion, the results from our study revealed that, although conventional CL programs for 22 h provide an enhancement in final BW, BWG, and dressing % of broilers it still had adverse effects on FCR values, innate immunity, oxidative status, and livability ratio. In conclusion, these results suggest that reducing lighting hours up to 3L/1D seems to be more beneficial in enhancing feed efficiency, livability, antioxidant activity and immune responses as an alternative to the conventional CL programs. Finally, it is believed that a further study is needed to evaluate the effects of different photoperiods and IL programs on economic consideration in a commercial production setting.


A well-developed canopy structure can increase the biomass accumulation and yield of crops. Peanut seeds were sown in a soil inoculated with an arbuscular mycorrhizal fungus (AMF) and uninoculated controls were also sown. Canopy structure was monitored using a 3-D laser scanner and photosynthetic characteristics with an LI-6400 XT photosynthesis system after 30, 45 and 70 days of growth to explore the effects of the AMF on growth, canopy structure and photosynthetic characteristics and yield. The AMF colonized the roots and AMF inoculation significantly increased the height, canopy width and total leaf area of the host plants and improved canopy structure. AMF reduced the tiller angle of the upper and middle canopy layers, increased that of the lower layer, reduced the leaf inclination of the upper, middle and lower layers, and increased the average leaf area and leaf area index after 45 days of growth, producing a well-developed and hierarchical canopy. Moreover, AMF inoculation increased the net photosynthetic rate in the upper, middle and lower layers. Plant height, canopy width, and total leaf area were positively correlated with net photosynthetic rate, and the inclination angle and tiller angle of the upper leaves were negatively correlated with net photosynthetic rate. Overall, the results demonstrate the effects of AMF inoculation on plant canopy structure and net photosynthetic rate. Canopy structure affects the effective photosynthetic area of the leaves and aspects of the microenvironment such as temperature, humidity and CO2 concentration inside the canopy, thereby affecting crop photosynthetic efficiency and yield4. Canopy structure can be measured directly or indirectly based on leaf area index (LAI) and leaf angle distribution (LAD). Direct measurement is time-consuming, cumbersome and destructive to plants5. Using three-dimensional modeling methods to study the characteristics of crop canopy structure can avoid the problems mentioned above, So it gets many scholars's favor. Riczu et al.6 established several plant branch models using an on-board laser scanner and compared the columnar branch model established in the Leica system, the 3D mesh tree model established using Geomagic software, and the trunk model using 3D shaping software. They considered the latter two models to be more realistic and more sophisticated. Chang et al.1.established a three-dimensional canopy photosynthesis model of rice plants and used it to study the effects of three canopy structural parameters, namely tiller number, tiller angle and leaf angle, on the efficiency of canopy radiation. Xiang et al.7 established a non-destructive 3D scanning system using a commercial depth camera and sorghum (Sorghum bicolor) as an experimental model to continuously monitor plants of different heights and automatically extract morphological characteristics of sorghum.Crop canopy structure is a critical factor affecting photosynthesis and commonly refers to the geometry, quantity and spatial distribution of various aboveground parts of the crop. More than 90% of the dry matter accumulated in plants is derived directly or indirectly from photosynthesis and the remainder from nutrients taken up by the roots10. AMF can regulate chloroplast enzyme activity, reduce chlorophyll decomposition rate, accelerate the synthesis of important enzymes required for the chlorophyll peptide chain, promote chlorophyll synthesis, increase chlorophyll content plants11, increasing the intensity of photosynthesis12, promoting plant growth, increasing leaf area13, and then increase the absorption and utilization efficiency of light energy15, thereby significantly promoting plant biomass16.Arbuscular mycorrhizal fungi (AMF) are a common group of soil microorganisms. They can form potentially symbiotic relationships with most terrestrial plant species and have the potential to promote plant growth and increase net photosynthetic rateWe used peanut as the experimental material and a three-dimensional laser scanner and an LI-6400 XT photosynthesis system to monitor the dynamic effects of AMF on canopy structure and photosynthetic characteristics to investigate the role of AMF on plant growth, canopy structure, photosynthesis and yield and to estimate plant photosynthetic capacity at later growth stages. The results provide information for improving crop canopy structure and increasing material accumulation and yield through AMF inoculation in nutrient-poor environments.After 70 days, we calculated the root colonization rate of each plant, including hyphae, arbuscules, vesicles of AMF, calculated the average colonization rate of the same treatment, and calculated the variation with standard error, and found that the percentage of peanut root length colonized by the AMF was 95.4 ± 1.1(%). No AMF colonization was found in the control plants. The results confirm that peanut plants formed mycorrhizal relationship. This relationship may have influenced plant development and growth.17.Canopy structure refers to the relative position and number of stems and leaves above the ground. There are two types of indicators, namely non-complex traits and complex traits. The former are the features extracted from the whole plant such as height, width, volume, and rough leaf area estimates, and the latter describe traits at the organ level such as accurate leaf area, leaf inclination, tiller angle, and fruit countAt 30 days the growth rates of mycorrhizal and uninoculated control plants were similar. At 45 days the growth rate of the mycorrhizal plants was significantly higher than that of the controls. The mycorrhizal plants continued to grow after 70 days. In contrast, the plant height of the control treatment increased but the leaves withered due to severe nutrient deficiency. The height, canopy width and total leaf area of mycorrhizal plants were 68.7, 49.7 and 71.1% greater than those of the controls after 45 days of growth and 179, 187 and 1020% greater at 70 days. AMF promoted plant growth and increased the total leaf area, and provided a prerequisite for increasing photosynthetic accumulation and further promoting plant growth.. The results are similar to those of Xiao et al.19 and in line with the changes in canopy structure. The canopy structure of mycorrhizal plants become more developed than that of the controls. The leaf angles of the upper and lower layers adjusted to receive more light and increased the photosynthetic rate to increase plant yield  is the ratio of net photosynthetic rate to transpiration rate and is an important index of the drought resistance of plants. When water resources are scarce, WUE is an important way to coordinate the contradiction between plant productivity and water consumptionThe AMF colonized the peanut roots and may have formed a potentially symbiotic association. AMF inoculation increased nutrient uptake, plant height, crown width, total leaf area, dry matter accumulation and yield. The tiller angle of inoculated plants showed the trend: upper layer < middle layer < lower layer, modifying the canopy structure to a more hierarchical configuration, and there was no significant effect among different layers in the uninoculated controls. AMF inoculateion changed the structure of the canopy and further affected photosynthesis. AMF inoculation increased the net photosynthetic rate of leaves in different layers, and the net photosynthetic rate was significantly positively correlated with leaf height, crown width, total leaf area and leaf area index, and significantly negatively correlated with leaf upper dip angle and tiller angle. The results indicate that the use of AMF may have some potential in practical agriculture. This study has concentrated on canopy structure but has not explored the effects of AMF on root traits, a topic that merits future investigation.The authors of the above articles promise to: all plant studies were carried out in accordance with relevant institutional, national or international guideline in the methods section.Funneliformis mosseae provided by the Laboratory of Microbial Reclamation, China University of Mining and Technology (Beijing), was used here. The mycelial length was 3.12 m g-1 and the spore density 26 g−1 substrate. The peanut variety used was Arachis hypogaea cv. The Variety was Fuyu Siguanhong and the Registration number was GDP Peanut (2018) 220,156, Silihong purchased from China Seed Trading Network. A high temperature-sterilized river sand was selected as the experimental soil to explore the effects of AM fungal inoculation on peanut growth in nutrient-poor conditions. The river sand hads content of available phosphorus and available potassium of 1.25 and 41.3 mg kg−1, respectively, and the organic matter content was 2.71 g kg−1.A strain of the AMF The experiment was conducted outdoors at the Microbial Reclamation Laboratory, China University of Mining and Technology (Beijing). In detail, a total of 16 pots were randomly assigned into AMF-inoculated and uninoculated control treatments with eight replicate pots each. Each inoculated pot contained a mixture of 5 kg river sand and 50 g of AMF inoculum composed of AMF root segments and rhizosphere soil. Each control pot contained a mixture of 5 kg river sand and 50 g of sterilized AMF inoculum. Five peanut seeds were sown in each pot on 25 May 2019. At the true leaf stage only one seedling was retained in each pot and the pots were watered to maintain soil water content at 70–80% of the water holding capacity (WHC) based on weight.4NO3, KH2PO4, and KNO3 to maintain the contents of N, P, and K per kilogram of soil at 100, 25, and 150 mg, respectively. The plants were observed after growth for 30, 45 and 70 days and harvested on day 70. The above- and below-ground dry weights of peanut were determined using the oven dry method. In addition, 0.1 g of the ground, dried roots, stems and leaves were digested in a mixture of sulfuric acid and hydrogen peroxide. A portion of each sample was used to determine the total nitrogen content using a Kjeldahl instrument, and the remainder was used to determine total nitrogen content using an ICP-OES spectrophotometer. Some fresh fine root samples were collected and used for determination of total phosphorus and total potassium37. A small amount of fresh fine root sample was randomly collected. After immersion in 10% KOH (w/w) for 24 h and rinsing with water they were stained using acid fuchsin:lactic glycerol solution as reported previously. Fifteen root sections were randomly selected, prepared on slices, and observed under an optical microscope to determine the percentage of root length colonized38.In addition, each pot was supplied with nutrient solution containing NHhttps://www.creaform3d.com.cn/zh/ji-liang-jie-jue-fang/bian-xi-shi-3d-sao-miao-yi-goscan-3d) with a scanning accuracy of 0.5 mm after 30, 45, and 70 days of growth in an indoor windless environment. After the scanning was completed the grid data were created and exported in the STL format. The data were then processed using VXelements with the main steps of coordinate transformation, noise removal, model repair and data extraction to obtain an independent and complete three-dimensional peanut model39.The point cloud data of the three-dimensional canopy structure of peanut were obtained using a portable HandySCAN 700 three-dimensional laser scanner 41. In the intermediate and later growth stages the canopy was divided into upper, middle and lower layers according to tiller height and leaf age  and crown width (cylinder diameter) were extracted using the cylinder in the "Feature" tool as the smallest cylinder surrounding the plant2 concentration, transpiration rate and net photosynthetic rate of peanut leaves at different layers were therefore measured on a clear and cloudless day using an LI-6400 XT portable CO2/H2O analysis system  . The effective radiation of the light source was PAR 1000 m2 mol/(m2 s), the leaf chamber used was 2 × 3 cm, and the gas flow rate was 500 mmol s-1. Three groups of leaves were determined randomly at each layer of each plant.Peanut reached peak photosynthesis at 10:00–13:00 on a day with sunny and stable weather. The stomatal conductance, intercellular COThe yield of each peanut plant was determined and then the average yield of mycorrhizal and control peanut was obtained.Microsoft Excel 2010 was used for data collation and PowerPoint 2019 was used for drawing the Figures. The SPSSwas used for one-way analysis of variance (ANOVA) and correlation analysis and the significance level was P < 0.05.The AMF and peanut formed a potentially mutualistic relationship. The AMF increased plant growth and made the plant canopy structure more hierarchical than the control. The changes in canopy structure of mycorrhizal plants are attributable to increased leaf area, chlorophyll content and photosynthetic rate and to plant nutrient uptake, leading to higher peanut biomass.There was a reciprocal relationship between the AMF and the plants that promoted plant nutrient uptake and made the plant canopy well developed and layered. AMF increased net photosynthetic rate and photosynthetic leaf area, thereby increasing plant biomass and yield.The authors of the above articles promise to: all plant studies were carried out in accordance with relevant institutional, national or international guideline in the methods section.


Traditional body measurement techniques are commonly used to assess physical health; however, these approaches do not fully represent the complex shape of the human body. Three-dimensional (3D) imaging systems capture rich point cloud data that provides a representation of the surface of 3D objects and have been shown to be a potential anthropometric tool for use within health applications. Previous studies utilising 3D imaging have only assessed body shape based on combinations and relative proportions of traditional body measures, such as lengths, widths and girths. Geometric morphometrics (GM) is an established framework used for the statistical analysis of biological shape variation. These methods quantify biological shape variation after the effects of non-shape variation–location, rotation and scale–have been mathematically held constant, otherwise known as the Procrustes paradigm. The aim of this study was to determine whether shape measures, identified using geometric morphometrics, can provide additional information about the complexity of human morphology and underlying mass distribution compared to traditional body measures. Scale-invariant features of torso shape were extracted from 3D imaging data of 9,209 participants form the LIFE-Adult study. Partial least squares regression (PLSR) models were created to determine the extent to which variations in human torso shape are explained by existing techniques. The results of this investigation suggest that linear combinations of body measures can explain 49.92% and 47.46% of the total variation in male and female body shape features, respectively. However, there are also significant amounts of variation in human morphology which cannot be identified by current methods. These results indicate that Geometric morphometric methods can identify measures of human body shape which provide complementary information about the human body. The aim of future studies will be to investigate the utility of these measures in clinical epidemiology and the assessment of health risk. BMI provides a measure of relative obesity and stratifies individuals into categories  according to World Health Organization (WHO) guidelines [Anthropometry refers to the scientific procedures and processes of acquiring surface anatomical measures of the human body–height, weight, lengths, and girths–by means of specialist equipment . Medicalidelines , 10. Howidelines  and distidelines , 12, whiidelines , 13.0.5 ratios (WHT.5R) [Waist girth is also commonly used in health assessments, due to its ease of capture, as well as stronger relationships with visceral adiposity volume , lean ma(WHT.5R) , 21–23. (WHT.5R) . WHO sug(WHT.5R) , 10. How(WHT.5R) –28. More(WHT.5R) , 29, 30 (WHT.5R) .Three-dimensional (3D) imaging systems capture detailed and accurate point cloud data that provides a representation of the surface of a 3D object. This point cloud data contains the differential geometric properties of the objects surface, from which both size and shape characteristics can be extracted. 3D imaging is an established anthropometric tool within several applications, including apparel sizing –33, and Geometric morphometrics (GM)—methods for analysing variations in the shape of biological organisms —quantifyThe aim of this study was to determine whether shape measures, identified using geometric morphometrics, can provide information that cannot be obtained using current techniques. The objectives were to determine the inter-dependence between traditional body measures and features of torso shape within a large sample and identify variations in torso shape which are unexplained by current anthropometric techniques.In this study 3D imaging data collected in the Leipzig Research Center for Civilization Diseases LIFE-Adult study was analysed. LIFE-Adult is a population-based cohort study, which collected phenotype data of over 10,000 randomly sampled individuals from the city of Leipzig, Germany, covering a main age range from 40–79 years of age, with a subset of 400 participants aged 18–39 years. Comprehensive details of the study design can be found in Loeffler et al. . As a prBasic characteristics of participants considered in the present analysis are shown in 3D imaging data of participants was acquired using a commercial Vitus Smart XXL 3D laser scanner , in accordance with ISO 20685–1:2018, international standards for 3D scanning methodologies for internationally compatible anthropometric databases . DetailsAll extracted body measures related to body segments outside of the torso region of interest  were removed from the analysis. The resulting data matrix consisted of 34 body measures for each participant see . To faciA modified version of the lower trunk segment proposed by Wicke  was usedThe following procedure was used to determine the circumferential location of the xiphoid process landmark on the anterior torso aspect and a landmark corresponding to the spinous process on the posterior aspect. To identify the circumferential location of the xiphoid process and spinous process a band of data points was extracted from each 3D image at the height of xiphoid process . The locY = X×Z.To remove non-shape variation  from the data, an anatomical coordinate axis system was created at the centre of each torso segment, according to the convention defined in ISO 20685–1:2018 . The x-aX) is denoted mathematically as:Following alignment, each 3D image was constrained to only include the coordinate points relating to the torso segment region of interest . Twenty-jC is the location of the jth component of the centroid. The centroid size of all extracted shape profiles along the length of each torso was scaled by a single scale factor, making the sum of distances from each point to the centroid for all slices along the torso segment equal for all participants. This removed any differences in scale between participants, enabling the analysis of torso shape according to statistical shape theory.Where K is the number of landmarks, M is the number of coordinates and Following alignment, cubic smoothing splines  were fitPrincipal component analysis (PCA) was used to further reduce the dimensionality of the torso segment’s 3D data. Previous studies have demonstrated that body shape variations are subtle, requiring a greater number of principal components (PCs) to describe them fully , 35, 41.r, were used to assess associations between individual body measures, as well as between body measures and shape PCs. P-values < 0.001 were considered statistically significant, to account for the effect of large sample sizes . Independent t-tests were conducted to determine if there were statistically significant differences between male and female participants in all 34 body measures and shape PCs see . In addi of 0.05 . The coeple size . CorrelaPLS regression models were used to determine how the torso shape PCs related to the 34 body measures. Since torso shape PCs were unrelated, separate PLS regression models  were created for each one shape PC, following recommendations by Wold , 52. TenSex-specific models were created so that trends between traditional body measures and shape PCs could be compared for each sex. Separate PLS regression models were created to estimate changes in each torso shape PC from unit changes in waist girth. Changes in torso shape were estimated across the entire range of waist girth measures within the sample, with height measures held constant at 180 cm and 160 cm for the male and female models, respectively.www.life.uni-leipzig.de/en/).Pre-processed torso shape PCs of all participants analysed in this study, together with their age, sex, BMI and WHR are provided in PCA produced 9 PCs that captured 90.6% of the total torso shape variation within the sample . Fig 3 sr) matrices of relationships between body measures, as well as relationships between body measures and shape PCs are provided as r: median = 0.88; IQR = 0.13) and females  (p < 0.001). While torso length and distance measures, such as side upper torso length and waist-buttock distance, showed the weakest correlations with other body measures, both for males  and females  (p < 0.001). Shape PC3 demonstrated the strongest correlations with body measures; in particular, with girth measures around the abdominal region, such as waist girth and middle hip girth, for males  and females  (p < 0.001). Shape PC’s 1, 2 and 5 demonstrated weak to moderate correlations with torso girth and width measures, males  and females  (p < 0.001). The remaining torso shape PCs demonstrated only weak linear correlations with body measures. Though the original PCs detected within the entire sample of participants were orthogonal and therefore uncorrelated due to the nature of PCA, some weak-moderate correlations were observed between the shape PCs for males and females separately. The PC scores of the male and female participants were recentred as separate z-scores, due to observed sex differences, the subsequent change in the correlational structure between individuals’ shape PCs caused some correlation to emerge between them. However, this change was not deemed to have affected the subsequent analysis, because the ordinal association between body measures and shape PCs were not affected.Pearson correlation coefficient  and (R2 = 0.73) for males and females, respectively. Conversely, variations in shape PC4, which represent tilting of the torso from side to side and are unlikely to be captured by body measures, were almost completely unexplained by body measures, for both males (R2 = 0.03) and females (R2 = 0.04).It was determined by estimating the mean squared prediction error by 10-fold cross-validation that six PLS components were required to maximise the prediction accuracy, Ricipants . Shape PWithin the male PLS regression models, between 44–65% of body measures demonstrated VIP values ≥ 0.8, while in the female PLS regression models between 41–82% of body measures had VIP values ≥ 0.8 see . Within Figs As shown in Figs Variations in shape of the human torso cannot be wholly explained by variations in traditional body measures. This investigation demonstrated that 49.92% and 47.46% of the observed torso shape variation within the LIFE dataset were explained by changes in body measures for males and females, respectively. Linear combinations of body measures in the PLS regression models explained over 50% of the variation in each of shape PCs 1, 2, 3 and 5 for males (PC1 (65%), PC2 (58%), PC3 (84%), PC5 (67%)) and females (PC1 (66%), PC2 (51%), PC3 (73%), PC5 (62%)). The VIP values of body measures in the PLS regression models suggest that torso girth measures contributed most to the prediction of torso shape. This would suggest that traditional body measures are a good predictor of torso shape for approximately 50% large proportion of the population that follow the expected trend.However, the results of this investigation also show that nearly 40% of the observed torso shape variation within the LIFE dataset could not be explained by changes in body measures. PCs 4 and 8, represented torso features which were almost completely unexplained by changes in body measures. From visualisations of these shape features, PC4 appears to represent lateral asymmetries of the torso. These variations could be the result of participants leaning to the left or right during the scanning procedure, a common issue within digital anthropometry, or could indicate issues such as scoliosis, which size measures would likely not capture. PC8 appears to represent subtle, localised variations in body shape on the inferior anterior aspect of the torso, which could relate to distributions of body fat or lean mass along the length of the torso that are not captured by current techniques. This would suggest that there are individuals within the population that exhibit torso shape characteristics which do not match the predicted trend according to existing body measures alone, with additional measures therefore required to evaluate other aspects of their morphology.2 = 0.001, p = 0.024), suggesting that variations within the unexplained component of PC2 were not due to body shape differences between males and females. PC2 appears to correspond to the observed thoraco-pelvic covariation observed by Torres-Tamayo et al., [2 = 0.002, p < 0.001) and , respectively. This may suggest that the unexplained torso shape variations within these PCs could be driven primarily by differences in torso shape between males and females, rather than other health-related factors. What is not yet known is whether the more subtle, largely unexplained shape PCs  are more significant in describing an individual’s health profile (for example) than the proportion of unexplained variance within the larger PCs .Shape PCs 1, 2, 3 and 5 had the strongest associations with body measures and represented over 76% of the total torso shape variability within the LIFE dataset. In comparison, the remaining PCs 4, 6, 7, 8 and 9 only accounted for 14.5% of the total torso shape variability in the LIFE dataset. Though large proportions of PCs 1, 2, 3 and 5 are explained by changes in body measures, the variance within these PCs that remains unexplained still accounts for over 26% of the total shape variability within the sample—which is greater than the variation within the other PCs combined. In addition, greater levels of agreement were observed between VIP values in the PLS regressions for first few PCs between males and females, while the higher order PCs showed less agreement. A possible reason for this could be that the first few PCs represent large-scale, high amplitude shape variations that vary in a consistent manner between males and females, while the higher order PCs represent more subtle, lower amplitude variations which characterise sexually dimorphic aspects of torso shape that differ in an unexplained manner between males and females. When assessing the relationships between unexplained variations in torso shape and sex, it was demonstrated that the unexplained variance within some of the torso shape PCs were more related to sexual dimorphism than others. For example, the regression of unexplained torso shape variations within PC2 on sex was not statistically significant Click here for additional data file.S1 Table2 values and variable importance in projection (VIP) statistic scores for the predictor size variables used within each PLSR model for males and females.Information about body size measures included in the torso segment analysis; statistical difference testing between males and females for all size and shape anthropometrics; correlations between size and shape anthropometrics; as well as R(XLSX)Click here for additional data file.S2 TableContains participant characteristics, size measures and body shape principal component scores of all 9,209 participants.(XLSX)Click here for additional data file.