Muscle–tendon power output is commonly assessed in the laboratory through the work loop, a paired analysis of muscle force and length during a cyclic task. Work-loop analysis of muscle–tendon function in out-of-lab conditions has been elusive due to methodological limitations. In this work, we combined kinetic and kinematic measures from shear wave tensiometry and inertial measurement units, respectively, to establish a wearable system for estimating work and power output from the soleus and gastrocnemius muscles during outdoor locomotion. Across 11 healthy young adults, we amassed 4777 strides of walking on slopes from −10° to +10°. Results showed that soleus work scales with incline, while gastrocnemius work is relatively insensitive to incline. These findings agree with previous results from laboratory-based studies while expanding technological capabilities by enabling wearable analysis of muscle–tendon kinetics. Applying this system in additional settings and activities could improve biomechanical knowledge and evaluation of protocols in scenarios such as rehabilitation, device design, athletics, and military training. The study of biomechanics is often conducted with the purpose of understanding human movement as it pertains to everyday life. In pursuit of this knowledge, it is of value to study individuals in an array of activities that reflect their daily tasks or athletic engagements outside of the laboratory environment. While certain tasks such as treadmill walking or jumping may be readily tested and analyzed in a motion capture space, others are more difficult to replicate . Many stWearable technologies have attempted to enable the portable capture of biomechanical measurements. Traditional lab-based assessments of muscle–tendon forces are indirect. Motion capture measures movement kinematics, while force plates record external forces ,9,10. CoThe fusion of wearable kinematic and tensiometry measures could enable muscle power and work analyses during real-world movements such as manual labor, sports, physical training, and rehabilitation ,34,35,36This study aimed to enable field-based estimates of muscle-specific work and power using only wearable sensors. To do this, we fused triceps surae muscle–tendon load assessed via tendon tensiometry with ankle and knee kinematics estimated from inertial pose sensors. Using this system, we investigated stride-by-stride triceps surae work production during outdoor walking on a course with varying slope. Previous laboratory studies have found that with inclining slope, positive ankle power, and work increase, negative power and work decrease in magnitude, and net ankle work increases in response to incline angle ,3,37,39.2).Twelve healthy young adults participated in this study after giving their written informed consent . ParticiThe tendon tensiometer unit consisted of a piezoelectric actuator stack  embedded in a tendon-tapping mechanism and two miniature accelerometers  embedded in silicone, as previously described . The actThe driver excited the piezoelectric tendon tapper at 100 Hz (a 2 ms pulse every 10 ms), and the data logger was comprised of a Raspberry Pi 4B  and an IEPE Measurement HAT , which recorded accelerometer data at the maximum nominal rate of 51.2 kHz  per channel and logged it to a disk through a Python program. For each trial, a trigger signal was sent from the MTw Awinda Station to the data logger to initiate synchronized collection, via a pair of general-purpose programmable wireless modules running a remote pin-mirroring program  .M (units Nm) and wave speed S (units m/s) was:S2, and 17.0 represents the unloaded tendon wave speed. Individual participants’ calibration curves fit their data with moderately strong correlations (R2 = 0.85 ± 0.08)   and thei ± 0.08) .Before heading outside, participants completed a 30-s treadmill walking trial at a self-selected speed (mean 1.2 ± 0.16 m/s). They then traversed an 800-m outdoor course, which consisted of a range of slopes between −10° and +10° ; this waAccelerometer signals were band-pass filtered using a fourth order Butterworth filter with lower and upper cutoff frequencies of 150 and 1500 Hz, respectively. Then tap events were identified across the entire trial. This was done by cross-correlating a representative tap signal with the first accelerometer signal and then finding the times of peak correlation, which corresponded with a tap event. Using the first 4 ms of data following each tap event, cross-correlation of the first and second accelerometer was used to determine the wave travel time between accelerometers ,61. WaveFor each stride, a slope value was assigned by mapping participant position onto a topographical map of course incline . This maInspection of the data revealed a slight delay between the start times of the data collected from the IMU and tendon tensiometry systems. The delay was ultimately traced to non-deterministic timing in the wireless module used to transmit the synchronization trigger  . To retrSpatiotemporal metrics were determined from IMU measures and used in part as criteria for omitting certain strides due to errors in the proprietary IMU motion reconstruction . Stride times were defined by the total duration of each stride according to the IMU timestamps between successive heel strikes. Likewise, stride length was calculated as the total horizontal displacement between heel strikes. Walking speed was then computed as the quotient of stride length and time. If the recorded stride length was greater than 3 m, the stride was omitted. Finally, we computed work against gravity for each step knowing the elevation gain, which was determined by step length and the slope of the terrain. Work against gravity per step was calculated as 50% of the product of body weight with the length of each stride and the tangent of its incline.To estimate the power and work of each unit of the triceps surae, we combined tendon load estimates from tensiometry with muscle–tendon velocity estimates obtained from IMU movement data. Because muscle and tendon interact in series and change length nonuniformly throughout movement, muscle vs. tendon length can only be differentiated via imaging such as ultrasound ,70. Longentified ,73:(2)Rao angles ,73. Fromhe ankle ,73,74: and (3) with respect to measured knee and ankle angles.F (units N), was determined by dividing moment, M (units Nm) (Equation (1)), by instantaneous ankle moment arm, The force in each MTU  was estimated from Achilles wave speed and a simple load distribution model. After converting wave speed values to ankle moment (Equation (1)), Achilles tendon force, m (units kg):This total Achilles force is distributed across the two muscle–tendon units that load the Achilles: the gastrocnemius and soleus. Data-derived apportionment of Achilles force to the individual units would depend on individual-specific and time-varying motor control, muscle mechanics, and posture; accounting for these factors would require adding electromyography, imaging, and modeling. To avoid this complexity and retain a mobile, wearable system for this study, we assumed a constant proportion of load for each muscle–tendon unit based on the physiological cross-sectional area of the muscle . A totalInstantaneous muscle–tendon powers generated by the soleus, Numerical integration of muscle power data yielded net work of the soleus, Similarly, positive and negative work were obtained by numerical integration of positive and negative power, respectively. We also plotted normalized muscle force vs. muscle–tendon excursion for both muscles to visualize the work loop plots of the triceps surae.For each participant, we first normalized the Achilles tendon wave speeds over a gait cycle to participant-specific mean peak wave speed in the level condition. We then averaged the normalized wave speed patterns across participants at different slopes, from −10° to +10° in 2° bins, to produce mean curves for plotting. For each step of each participant, we determined the net, positive, and negative muscle–tendon work and the slope of the terrain. We then subtracted participant-specific mean net work in the level condition to obtain the change in net work from the level condition, accounting for inter-participant differences. Separately for inclines and declines, we fit a line to estimate the sensitivity of the change in net work to the slope angle of each step, for both the soleus and gastrocnemius. Linear regression coefficients were evaluated at the 0.05 statistical significance level.Participants navigated the course in 10.3 ± 0.8 min (mean ± standard deviation) with a speed of 1.31 ± 0.10 m/s and a stride length of 1.60 ± 0.25 m in the level condition (mean ± standard deviation). On average, 63 strides per participant were excluded due to data quality concerns, arising from poor sensor contact and temporary hardware failures. A total of 4777 strides were included for analysis, with a mean of 434 strides per participant.Achilles tendon wave speed measures were sensitive to changes in slope  Figure . Across Both the gastrocnemius and soleus displayed diminished swing-phase power as well as increasing stride time with increasing slope . Soleus Work loop plots exhibited systematic variations with ground incline . All sloTo quantify the effect of slope on individual muscle output, integration of power over time yielded work estimates . Soleus Relative to participant-specific mean net work in the level condition change in net work scaled with slope . Inter-pThis study introduces a wearable system of IMUs and tensiometry to characterize triceps surae kinematics, kinetics, and work production during sloped walking outdoors. Tensiometry tracks tendon loading based on the wave speed propagating in the tissue. The wave speeds and tendon forces measured in this study reflect trends previously demonstrated on mild slopes . At steeTensiometry provides for a continual assessment of muscle–tendon kinetics, including during periods without ground contact. We have previously observed elevated wave speeds during the late swing phase in level walking, corresponding with passive triceps surae stretch due to dorsiflexion motion forced by the antagonist muscles  ,56,61,68The fusion of IMU and tensiometry data enabled an analysis of the variation of gastrocnemius and soleus excursion and work production with slope. Our work loop plots qualitatively agree with previous laboratory work , though Our reported trends in work agree with previous studies of joint-level work. In particular, the steady increase in net soleus work clearly reflects how ankle work scales with ground incline  2,37]. ,37. 2,37Previous laboratory studies have reported shifts in net, positive, and negative work among the lower extremity joints depending on slope. Several studies in similar slope ranges of −10° to +10° depict increasing ankle work with incline, including increased positive work uphill and increased negative work downhill ,37. At sIn this study, magnitudes of soleus and gastrocnemius work were compared to the total work against gravity at each step. Despite experiencing an overall negative work against gravity in negative slopes, both soleus and gastrocnemius net work were positive at all slopes. This observation is consistent with a prior laboratory study which found gastrocnemius work to be positive at slopes ranging from −6° to +6° . At the There were limitations within our methods that should be considered in interpreting the results. The wireless triggering method between the kinematic and kinetic measurement systems resulted in a delay varying between 10 and 100 ms for every trial. At the preferred cadence of the participants in this study, this equates to offsets of approximately 1–10% of the gait cycle. As power and work calculations are highly dependent on the time-sensitive relationship between force and movement, it was thus necessary to correct for these delays. After applying the correction described in the Methods section, a systematic sensitivity study of the effect of incremental shifts in the data on resultant calculations of work output demonstrated that even 1% gait cycle offsets in either direction could affect work estimates by approximately 20%. However, for up to at least 10% gait cycle offsets in either direction, the overall trends in the work output calculations remained unchanged within and across participants. Thus, while a hardwired synchronization will be preferred in future work, we are nevertheless confident in the applied strategy and its ability to yield reasonable absolute outputs with meaningful relative trends.Relative to our previous experiment , data loA final source of potential error lies in the limitations of the data acquisition hardware and processing methods. First, the accelerometers themselves have some inherent noise, in this case approximately 0.0001% to 0.0032% of the signal amplitude (greater for the second than for the first accelerometer due to attenuation as the shear wave propagates) . AdditioInterpretations of the estimated quantities afforded by this wearable system may be applied in numerous contexts. Employing this methodology in healthy athletes and those recovering from an injury such as Achilles tendon rupture could enable the assessment of kinetic limb symmetry in field-based maneuvers; by capturing triceps surae work and moment through our instrumentation, this work could expand and inform the types of tests necessary for a safe return to sport . Toward This work fuses wearable Achilles tendon tensiometry and lower-limb kinematic measures from IMUs to simultaneously assess muscle–tendon kinematics, loads, and work production during outdoor locomotion. By detecting different trends in gastrocnemius and soleus work with changes in ground incline, the combined use of tensiometry and IMUs demonstrates its potential to illuminate muscle-specific behaviors in unconstrained settings. The wearable muscle–tendon power analysis system could enable exciting new applications beyond the traditional laboratory setting, including rehabilitation, sports medicine, and athletic training.


Pearl millet is an important crop for food security in Asia and Africa’s arid and semi-arid regions. It is widely grown as a staple cereal grain for human consumption and livestock fodder. Mechanistic crop growth and water balance models are useful to forecast crop production and water use. However, very few studies have been devoted to the development of the model parameters needed for such simulations for pearl millet. The objectives of the study were to determine crop-specific model parameters for each of three pearl millet varieties , as well as to calibrate and validate the Soil Water Balance (SWB) model for predicting pearl millet production and water use based on weather data. The SWB was chosen because it is widely used in southern Africa; however, the developed parameters should benefit other models as well. The presented crop-specific parameter values were derived from field observations and literature. Varieties with different phenology, maturity dates and tillering habits were grown under well-watered and well-fertilised conditions for calibration purposes. The calibrated model was used to predict biomass production, grain yield and crop water use. The hybrid’s water use efficiency was higher than that of the landrace and improved variety. Pennisetum glaucum (L.) R.Br.) is an important crop in subsistence agriculture in large areas of the semi-arid tropics of Africa and India, where it is widely grown for grain, fodder and fuel [Pennisetum glaucum, a C4 grass, has excellent photosynthetic efficiency and biomass production potential. Millet grain is highly nutritious, with 8–19% protein, low starch, high fibre (1.2%) [Pearl millet  . Despitee (1.2%) ,12,13,14Recently, models have been extensively used to predict future events for different scenarios  and provCrop models have become increasingly useful for different purposes, primarily in education and technology transfer ,23, deciCrop growth simulation models have been used to investigate the effects of crop management options such as irrigation timing, amount and fertiliser applications, under different environmental conditions on long-term mean yield ,40,41,42This paper specifically looked into the Soil Water Balance (SWB) model because it is widely used in southern Africa. The SWB model is a mechanistic crop growth model that is well established and widely used ,52,53,54The components of the soil water balance for December 2017–April 2018 for the three pearl millet varieties are shown in It was observed that volumetric soil water content in the root zone of the landrace variety increased by 21 mm from 328 to 350 mm. Similar trends were observed in both hybrid and improved varieties during the same period. During the seedling stage, there was an increase in soil water content due to irrigation and rainfall.Seasonal ET varied between 598 and 670 mm  and variHigher drainage was observed during the last week of March. In all varieties this was due to a high rainfall event. In March about 208 mm of rain fell during the late growth period, concentrated mainly in three days, when the soil was wet from previous irrigations, and while water consumption by the crop was low. The amount of drainage below the root zone (1 m soil depth) varied from 0 to 88 mm during growing seasons. The total drainage observed in the landrace was higher than improved and hybrid. Total drainage value represents 18.9% (landrace), 14.7% (improved) and 13.4% (hybrid) of the water applied by irrigation and rainfall.2 values (>0.97) were achieved for all three pearl millet varieties  is the slope of the relationship between the total dry matter produced and cumulative intercepted radiation. High Rarieties , highlig−3 kg MJ−1 [−3 kg MJ−1 [−3 kg MJ−1 [−3 kg MJ−1) but well below the 4 × 10−3 kg MJ−1 found by Ram et al. [The values for both the hybrid and improved variety compares well with those found in the literature for pearl millet . Sim kg MJ−1  . The la−3) for landrace biomass was in the range of that reported by Payne [Dry matter water ratio (DWR) values determined for the three varieties are presented in by Payne .−3) was higher than that recorded for both the landrace (1.29 kgm−3) and improved (1.23 kgm−3) variety, but lower than the seasonal average determined by Payne [The grain yield WUE of the hybrid (1.43 kgmby Payne .Growing degree days (GDD) are widely used for describing the developmental response of crops to temperature. GDDs recorded from emergence until senescence for the three varieties ranged between 1480–2124 day degrees . All thrPAR for the hybrid pearl millet variety was 0.43, with an LAI of 6.79 which intercepted 96% of solar radiation. The landrace and improved variety recorded a lower KPAR value than for the hybrid. The extinction coefficient for solar radiation (Ks) was 0.30 for the improved pearl millet variety.Maximum fractional interception of radiation of the three pearl millet varieties was almost identical and reached a maximum of 0.94–0.98, similar to what was reported by Squire et al. . The higPAR (98%) were generally higher for the landrace at the heading stage. Similarly, the improved variety recorded its highest LAI (6.99) and FIPAR (94%) also at heading. However, for the hybrid, the greatest LAI and FIPAR were observed at grain filling. Calculated Ks values for improved, landrace and hybrid varieties for well-watered treatments are reported in The radiation interception and canopy LAI varied from planting to harvesting and showed a similar trend to those observed by other authors . The LAI2 kg−1 for the hybrid, 2.48 m2 kg−1 for the improved variety and 1.27 m2 kg−1 for the landrace  partition parameter was 2.76 mlandrace . The spe2 kg−1 for the hybrid, 19.91 m2 kg−1 for the landrace and 23.34 m2 kg−1 for the improved variety. Specific leaf area (SLA) is vital in leaf and plant functioning and regulates the strategy of resource acquisition and plant growth [2 kg–1) ranged between 19.91 and 23.34 m2 kg−1, which are in accordance with values of 18 and 33 m2 kg−1 reported by Singh [The values of SLA presented in t growth . SLA is by Singh , Van Heeby Singh  and Montby Singh . It is cPAR, biomass produced, grain yield and measured soil water deficits were used to calibrate the model. Root depth was not measured but estimated from depth of soil water extraction. The crop-specific growth parameters of the three varieties are shown in the list of values in The parameters derived from the calibration data set were used to calibrate the SWB model for each of the three pearl millet varieties . Field m2 = 0.70, D = 0.85 and MAE = 47% for Kantana, R2 = 0.62, D = 0.83 and MAE = 44% for Kangara and R2 = 0.70, D = 0.78 and MAE = 93% for Agrigreen  of the varieties grown under different irrigation and weather conditions was obtained from three years of independent experimental data and are presented in The validation of the above-ground dry matter (AGDM) and harvestable dry matter (HDM) yields were predicted with reasonable to good accuracy for pearl millet varieties that were irrigated only once in two weeks c,f,i. Th2 = 0.82, D = 0.72 and MAE = 75% for hybrid; R2 = 0.87, D = 0.81 and MAE = 83% for improved; and R2 = 0.77, D = 0.84 and MAE = 64% for landrace). These findings imply that the SWB model was able to accurately simulate crop growth, water use and yield for any of the varieties studied under various water supply conditions. As a result, the SWB model can be used with confidence to predict growth, water use and yield of these varieties under various scenarios for future planning.The soil water deficits were less accurately estimated, particularly from mid-season onwards, when the crop was rainfed or irrigated less regularly, using the validation data from the 2016–2018 growing season  than the hybrid and improved variety was in part due to late maturity.The top dry matter accumulated was high in the landrace variety as it absorbed more radiation over time. This finding is supported by Monteith , SinclaiDry matter production is very sensitive to the DWR and moderately sensitive to RDMAX and available water, both of which determine the amount of water used by the plant. Therefore, dry matter production can also be estimated by considering water use efficiency, another important model parameter. In SWB, a simple gas exchange model that acknowledges the tight link between transpiration corrected for dry matter production and vapour pressure deficit (VPD) is used . It was −1 when daily maximum saturation vapour pressure deficits did not exceed 3 kPa. However, lower RUEs (0.3 and 0.8 g MJ−1) were reported where daily maximum saturation vapour pressure deficits were between 3 and 5 kPa [The landrace had lower Ks due to its vertical and clumped leaves, which allowed more solar radiation to penetrate through the canopy, leading to more dry matter production . The impnd 5 kPa , less thPAR) and LAI in this study. Photosynthetic active radiation (PAR) is the basic source of energy for dry matter production [Crop genotype influenced fraction of intercepted photosynthetic active radiation  for all varieties differed during the growing cycles and decreased with time due to the senescence of old leaves. However, a calculated seasonal average value of SLA was used in the SWB model, as supported by Rinaldi , that ca2 (5 × 9 m2) each in size, with 1 m paths between plots. Plots were demarcated with raised soil bunds between them to avoid the surface movement of water between adjacent plots. The soil was a Hutton sandy clay loam [1: Agrigreen), open pollinated variety (OPV) landrace (V2: Kantana) and OPV improved (V3: Kangara). The irrigation regimes were well-watered (I1: irrigated every week to field capacity), an intermediate irrigation level (I2: irrigated every second week to field capacity) and a zero-irrigation control (I0: rainfed).The field experiments of a detailed growth analysis and calibration of three pearl millet varieties were conducted in an open field at the Hatfield Experimental Farm  of the University of Pretoria, South Africa. The trial was laid out as a randomised complete block design and treatments were randomly assigned to blocks in all years of the experiment. The field was divided into three blocks with nine plots of 45 mlay loam  , thereafter every week until the plants reached physiological maturity. Plant heights and destructive harvests were executed fortnightly for each replicate. The plant samples were separated into leaves, stems and panicles, whereafter the leaf area (LA) was measured using a belt-driven leaf area meter model LI-3100  calibrated to 0.01 cm2. Leaf area at the plant level was calculated as the sum of the areas of each green leaf on a plant, and leaf area index (LAI) was calculated. An electronic balance was used for weighing samples that were oven-dried at 70 °C to constant mass to determine leaf dry matter (LDM) and stem dry matter (SDM). At maturity, grain yield was harvested from 1 m2 per plot.For calibration of the SWB model, the OPV landrace (Kantana), OPV improved (Kangara) and hybrid (Agrigreen) were planted in a well-managed plot. The well-watered plots were used for growth analysis. The pearl millet crops in the experiment were planted at a density of 18 plants per m−1. The soil water deficit to field capacity was calculated for each layer and the average for the four different layers determined. A profile pit was dug at the experimental site and soil samples were taken at 0.20 m intervals to a depth of 1.0 m to determine bulk density (bρ) soil texture and volumetric soil water content (θ) at field capacity (FC) and permanent wilting point (PWP). The bρ of the soils was determined by gently driving a cylinder of known volume horizontally into the side of the profile pit. The soil was removed and dried at 105 °C for 24 h. Volumetric water content at FC was determined, before planting, by saturating a portion of the field which was then left for 48 h to drain before sampling, while PWP was determined at the end of the season by withholding irrigation on a section of the experimental plot until the plants died. Soil samples of a known volume were taken and θ was calculated from the mass loss before and after the soil samples were dried at 105 °C for 48 h. Bulk density varied from 1152–1506 kg·m−3 for the 0–1 m soil profile [For monitoring the soil water status as part of the soil water balance, a neutron water meter model 503 DR CPN Hydroprobe  that was calibrated for the experimental site was used to measure soil water contents at 0.2 m increments to a depth of 1.0 m. Root-zone soil water deficit calculations for irrigation were made over an assumed rooting depth of 1.0 m. Weekly soil water content measurements were taken, and thereafter the field was watered to field capacity using a high-density drip system with drip lines spaced 0.45 m apart with an in-line dripper spacing of 0.30 m, and a delivery rate of 8.9 mm hS) of the root zone of a crop, and this is equal to the difference between the water content at end of measurement period oQ, and the water content at the beginning of measurement in the root zone, iQ [S shows the change in soil water storage over the 1 m soil profile, P is precipitation, I is irrigation, R is runoff and D is deep percolation, all expressed in millimetres. The term R was assumed to be negligible because the experiment was conducted on flat terrain and no high-intensity precipitation was experienced. In addition, irrigation was applied using a drip irrigation system and the rate of application did not surpass that of the soil infiltrability. A negative value for ΔS shows a decrease in the amount of water stored in the soil.The water applied to the entire root zone by irrigation or rainfall was used to calculate the change in soil water storage at the end of every week in each plot. The soil water balance method ,118,119 zone, Qi , in a giSoil water was measured weekly at 0.20 m intervals from the soil surface down to 1.00 m. Aluminium access tubes that protruded 0.10 m above soil level were covered to prevent rain or soil from entering. Maximum root depth was estimated from weekly measurements of soil water extraction with the neutron water meter model 503 DR CPN Hydroprobe  that was calibrated for the experimental site. Root-zone soil water deficit calculations for irrigation were made over an assumed rooting depth of 1.00 m.Weather data, including daily minimum and maximum air temperature, as well as humidity, wind speed, incoming solar radiation and precipitation, were recorded by a nearby automated weather station. The automatic weather station consisted of an LI-200SA pyranometer  to measure solar radiation, a cup anemometer  to measure average wind speed, a tipping bucket rain gauge , an HMP60 relative humidity and temperature sensor , and a CR200X datalogger . For calibration of the SWB model, weather, soil and crop data collected in the field trial were used to determine crop-specific growth parameters for well-irrigated landrace, improved and hybrid pearl millet varieties for the 2018 growing season.sTmaxe is saturated vapour pressure at maximum air temperature (Pa), sTmine is saturated vapour pressure at minimum air temperature (Pa) and ae is actual vapour pressure (Pa). Saturated vapour pressure (se) at maximum (maxT) and minimum air temperature (minT) was calculated by replacing aT  with maxT and minT (°C) [ae  was calculated from the measured daily maxT, min, RHmaxT and minRH, using the following equation from Allen, Pereira, Raes and Smith [Vapour pressure deficit was calculated according to the following equation by Jovanovic, Annandale and Mhlauli :(3)VPD=emin (°C) , as presnd Smith :(5)ea=esT is actual evapotranspiration (mm), and PET is potential evapotranspiration (mm).As water becomes limiting, actual evapotranspiration will fall below the potential rate. A measure of the ratio of actual to reference evapotranspiration should therefore be an index of crop water status. Water stress occurs when available soil water does not meet potential transpiration demand, and it is calculated as the ratio of actual to potential transpiration. A water stress index can also be used to reduce the accumulation of growing day degrees, depending on phenology calculated with thermal time and modified by water stress. This dry matter is partitioned into roots, stems, leaves, grains, or fruits . Such s−2, which includes root mass. RUE is the slope of the regression between intercepted solar radiation and DM production, forced through the origin (kg MJ−1). RUE is also known as the radiation conversion efficiency (cE), FIRAD is the fraction of daily value incremented each day (i = 1 to n) of solar radiation intercepted by the green, transpiring canopy and sR is the total fraction of daily value incremented each day (i = 1 to n) of incoming solar radiation in MJ m−2 day−1.Many crop growth models are radiation driven and require an estimate of radiation use efficiency (RUE). In SWB, the radiation conversion efficiency is a crop-specific parameter used to calculate dry matter production under conditions of radiation limited growth, from Monteith , as foll2 taken up is converted to organic carbon, and this process which is accompanied by transpirational water loss can be described by a simple gas exchange dry matter production model [T), adjusted to account for vapour pressure deficit (VPD) [−2). In SWB, a simple gas exchange model that acknowledges the tight link between transpiration corrected for dry matter production and vapour pressure deficit (VPD) is used [Transpired water vapour leaves the plant by diffusion through stomata, and carbon dioxide is taken up for photosynthesis following the same pathway but in the opposite direction. The COon model . Transpion model .(8)DWR=uctivity  of the dit (VPD) , and DM  is used .T) is difficult to measure, so ET was used, which also gives a lower DWR than the actual value. Evapotranspiration and top dry matter were therefore used to estimate a lower limit value of DWR. The DWR lower limit value was increased during model parameterisation until simulation data were observed in line with measured data. The estimation of lower limit DWR values was performed using measured top dry matter accumulation data at physiological maturity to avoid errors caused by plants losing leaves, as leaf senescence usually occurs after physiological maturity.VPD is the seasonal average vapour pressure deficit. ET is the seasonal (from sowing to maturity) total evapotranspiration obtained using Equation (1). Both VPD and DWR are in Pa, while ET is in mm. Due to the difficulty in determining root dry matter, only top dry matter was measured, resulting in a smaller DWR than required in Equation (8). Similarly, transpiration  (d °C) were calculated using the daily average air temperature, from Monteith :(9)GDD=∑l millet . If the l millet . GDD wasPAR  was determined using the following formula:A Decagon Sunfleck ceptometer  was used to measured fractional interception of photosynthetically active radiation on a weekly basis. On clear-sky days, measurements were performed between 10:00 and 12:00. Five reference values above and five readings below the canopy were averaged in a series of measurements. FIThe Beer–Bouguer law was used to determine the canopy extinction coefficient for PAR :(11)FIPAPAR. Field measurements of LAI and FIPAR were used to calculate KPAR values. Dry matter production can be estimated using KPAR as a function of intercepted PAR. SWB requires the canopy extinction coefficient for solar radiation (Ks), as fractional interception of solar radiation (FIRAD) is used to predict radiation limited dry matter production [PAR into Ks:bd is the canopy radiation extinction coefficient for black leaves and diffuse radiation, ap is the absorptance of PAR, and as is leaf absorptance of near-infrared radiation . The value of ap was set to 0.8, while the value of an was set to 0.2 [s.The canopy extinction coefficient for PAR is represented by Koduction , and foroduction . The prooduction  was usedt to 0.2 . The geoIn SWB, the daily increment in DM is computed to be either limited by transpiration or limited by radiation , with th2 kg−1. LAI is the leaf area index (m2 m−2), and SLA is the specific leaf area (m2 kg−1). SDM is stem dry matter.The SLA, LAI, and canopy dry matter (CDM) were used to calculate the leaf-stem partitioning parameter (p) . The lea−2.Grain yield, LDM, CDM and SDM are all measured in kg m−1·Pa), radiation use efficiency , leaf-stem dry matter partitioning  and specific leaf area .For SWB model calibration the following crop growth parameters for each of the crops were recorded: canopy extinction coefficient (Ks) for total solar radiation, dry matter: transpiration ratio, adjusted for vapour pressure deficit  until the end of the growing season.These pearl millet varieties were subjected to three different irrigation regimes . The soil was similar to that used to parameterise the model. The experiment was set up in a three-replication randomised complete block. Pearl millet was planted at a 0.45 m inter-row spacing with a 0.14 m intra-row spacing, in plots measuring 4.95 m by 9.5 m. A calibrated neutron probe was used to measure the amount of water in the soil on a weekly basis. Drip irrigation was used to irrigate the plots. The rainfed treatments and every second week irrigated treatments used to validate the model were as follows:All the varieties had almost similar water use efficiency (for grain yield), radiation use efficiency and degree days to emergence. The specific leaf area, canopy extinction coefficient for total solar radiation and flowering day degrees parameters were different among the varieties. The hybrid variety portrayed a determinate growth habit; therefore, if similar varieties grow in this way, hybrid parameters must be used. However, if the variety is late maturing and indeterminate, modellers are advised to use landrace parameters. If the modellers have an early maturity variety that is indeterminate, then the improved variety parameters should be used. The average values for canopy extinction coefficient for photosynthetic active radiation differ largely among the three varieties; therefore, using average values across the varieties is not advisable. The parameters need to be used with the model to evaluate results. Despite the lack of actual long-term yield data to compare to simulations, the authors are confident that the calibrated SWB model will be useful in estimating pearl millet yields under various production conditions. It is, however, recommended that crop parameters should also be determined for other pearl millet varieties to make the SWB model more useful for the management of irrigation, long-term yield projection and planning purposes.


Despite significant advances in treatment of acute coronary syndromes (ACS) many subjects still develop heart failure due to significantly reduced ejection fraction. Currently, there are no commonly available treatment strategies that replace the infarcted/dysfunctional myocardium. Therefore, understanding the mechanisms that control the regeneration of the heart muscle is important. The development of new coronary vessels plays a pivotal role in cardiac regeneration. Employing microarray expression assays and RT-qPCR validation expression pattern of genes in long-term primary cultured cells isolated form the right atrial appendage (RAA) and right atrium (RA) was evaluated. After using DAVID software, it indicated the analysis expression profiles of genes involved in ontological groups such as: “angiogenesis”, “blood vessel morphogenesis”, “circulatory system development”, “regulation of vasculature development”, and “vasculature development” associated with the process of creation new blood vessels. The performed transcriptomic comparative analysis between two different compartments of the heart muscle allowed us to indicate the presence of differences in the expression of key transcripts depending on the cell source. Increases in culture intervals significantly increased expression of SFRP2, PRRX1 genes and some other genes involved in inflammatory process, such as: CCL2, IL6, and ROBO1. Moreover, the right atrial appendage gene encoding lysyl oxidase (LOX) showed much higher expression compared to the pre-cultivation state. Cardiovascular diseases are the most common cause of death worldwide. Coronary artery disease (CAD), manifested frequently by myocardial infarction and, subsequently, end-stage heart failure (HF), are still an unsolved clinical problems that focus the attention of the scientific world . UnfortuNeovascularization is the process of blood vessel development and growth through three distinct biological processes: vasculogenesis (limited to the embryonic development), angiogenesis, and arteriogenesis . It is nHowever, previous studies demonstrate that resident fibroblast lineages mainly mediate cardiac fibrosis ,12, suggThe aim of the present study was to analyze the transcriptomic profile of genes encoding proteins that could be recognized as new molecular markers regulating neovascularization process in the cardiac muscle during in vitro long-term primary cell culture. The pivotal role of the vascular network for development and regeneration of cardiac muscle stimulated research aimed at creating a specific transcriptomic map for cultured porcine myocardial cells. The present study focuses on factors related to the process of creating new vessels, and the expression patterns depending on the duration of the cultivation and the heart compartment from which the cells were isolated  are considered.Daily observation of cell cultures, during 30 days of in vitro culture, was performed, and documented with pictures from inverted microscope employing relief contrast  for RA and 4662 DEGs for RAA. During the initial analysis of the microarray results, to better understand the intergroup differences in the global expression of overall DEGs, principal component analysis (PCA) of the samples was performed to examine variance between the analyzed sample groups. The results presented in p-values are shown in Changes in the transcriptome profile of cultured cells were observed at individual time intervals. Whole transcriptome profiling by Affymetrix microarray allowed us to analyze gene expression changes between the starting point and 7, 15, and 30 days of pig cardiac cells in vitro primary culture. Using Porcine Gene 1.1 ST Array Strip, microarray transcriptome screening was performed. For both cells obtained from RAA and RA, we observed upregulated and downregulated genes. Genes considered as differentially expressed and selected for downstream analysis showed ratio higher than abs |2| and corrected The DAVID software analysis showed that the differently expressed genes belonged also to terms associated with the formation of new vessels, like: “angiogenesis”, “blood vessel morphogenesis”, “circulatory system development”, “regulation of vasculature development”, and “vasculature development” GO BP terms. Hierarchical clusterization procedure was carried out for sets of these genes, with the results presented as heatmaps .Due to the structure of the GO database, single genes can often be assigned to many ontological terms. For this reason, we explore the gene intersections between the selected GO BP terms, and the relationship between genes and GO terms were mapped with circle plots, with visualization of logFC values and gene symbols . All of In order to further investigate the changes within chosen GO BP terms, we measured the enrichment levels of each selected GO BP term. The enrichment levels were expressed as z-score and presented as circular visualization .Furthermore, to better understand the interaction between chosen GO BP terms, hierarchical clusterization of the gene expression profiles was performed. The dendrogram was combined with fold change (FC) of studied gene expressions and gene assignment to studied terms . All genCreated Venn diagram, to compare expression profile of two different compartments of the heart, displays the information about the gene expression patterns, namely we can distinguish commonly upregulated and commonly downregulated genes in RA and RAA . As can The most significantly upregulated and downregulated genes belonging to GO BP terms of interest, presented on the Venn diagram and in p < 0.05). Differentially expressed genes belonging to these pathways were assigned to a predetermined color scale, which was subsequently imposed on the gene/protein symbol field  database. This analysis al-lowed us to determine the biological pathways, and for the present study “signaling path-ways regulating pluripotency of stem cells” and “hypertrophic cardiomyopathy” was chosen. Presented involve a significant enrichment of differentially expressed genes in the examined group . Given these factors, the whole complex process of neovascularization  taking pPorcine hearts were chosen for this study because easy access to them and more importantly due to many similarities to the human beings. Although significant insight into the molecular and cellular basis has come from small animal models, significant differences exist with regard to cardiovascular characteristics between these models and humans. Therefore, large animal models are essential to develop the discoveries from murine models into clinical therapies and interventions . MoreoveSFRP2 gene in all analyzed time periods. Secreted frizzled-related proteins (sFRP) are a family of glycoproteins that can bind to Wnt ligands or frizzled (FzD) receptors, thus the SFRP family may be involved in the regulation of Wnt signaling via both the canonical and noncanonical pathways [In cells isolated from both the right atrial appendage (RAA) and right atrial (RA) wall during long-term culture both exhibited increased expression of pathways . The Wntpathways . Moreovepathways ,21. Givepathways . Secretepathways ,24,25. Hpathways . Additiopathways . Other ipathways . Courtwrpathways . Moreovepathways ,31,32. Opathways .It is difficult to clearly determine the effect of sFRP2 on the Wnt pathway and how it will translate into the formation of new vessels in the heart. Nevertheless, numerous research results suggest a pro-angiogenic effect of sFRP2 overexpression, which may suggest that in the conditions of in vitro culture, the increasing mRNA level of the SFRP2 gene will be a factor promoting the angiogenesis process.A significant increase in the mRNA levels of paired related homeobox 1 (PRRX1), a member of the paired homeobox family, and functions as a transcription co-activator . PRRX1 iIt is well established, that inflammation and angiogenesis are interdependent and highly linked processes in ischemia and tumor formation. A significant upregulation of transcript level of few factors involved in inflammatory process appears to be important. Secretion of C–C chemokine ligand 2 (CCL2) results in the attraction of blood monocytes into sites of inflammatory responses and tumors . This maMYH7, MYL3), actin (ACTC1), actinin (ACTN2) or troponin (TNNC1) were observed. The expression profile at the molecular level appears to promote blood vessel formation when we look at factors positively regulating neovascularization . However, in the case of genes encoding structural proteins, there is a noticeable need to further optimize the culture conditions, because without specific “building blocks” that build vessels, the expression of transcription factors alone that stimulate vascularization will be insufficient for the formation of new vessels.High transcript levels were observed in some genes, but significantly lower mRNA levels for many genes coding mainly structural proteins, such as like myosin (Sus scrofa f. domestica) hearts, delivered on ice in the shortest possible time after slaughter, from a local slaughterhouse were the source of cells for in vitro culture. For our study, a pubertal crossbred Polish Landrace (PBZ × WBP) gilts, bred on commercial local farm were used. They had a mean age of 155 days (range 140–170 days) and the mean weight were 100 kg (95–120 kg). All the animals were housed under identical conditions and fed the same forage.Porcine . The base of the heart is directed dorsally and cranially while the apex of the heart is directed ventrally, caudally and slightly to the left. In this position, the heart is held in place by the cranial vena cava, the caudal vena cava, the aorta and the sternopericardial ligaments. After cutting the sternum and the diaphragm, the heart was removed along with the lungs, trachea, esophagus and tongue. The hearts in pericardium were then severed and transported to the laboratory within 30 min. Each time the delivered hearts will be assessed for their suitability for downstream analyzes. Hearts were disqualified from further study by observations of macroscopic injury at the slaughterhouse; contaminated during transport and preliminary preparation; with evident signs of inflammation (connective tissue adhesions) or ischemia . Then, the hearts were removed from the pericardial sacs. After identification of left and right side of porcine heart, right atrial appendage and piece of its free wall (ca. 1 cm × 1 cm) of whole thickness were extracted. The extracted fragments were manually prepared with surgical instruments to remove the visceral lamina of the serous pericardium (epicardium). As the research material is usually disposed of after slaughter, being a remnant by-product, no ethical committee approval is needed for the project.g, RT), in order to remove the remaining collagenase from the cell environment. Cell pellet obtained was washed with the PBS buffer and then initially placed on 25 mL cultures bottles in culture medium , 20% FBS , 10% HS , EGF , bFGF  1% P/S, and preincubated for 4 h in 37 °C, 5% CO2. This stage aims to deplete the fibroblasts, which show much higher adhesion affinity. After this time, the supernatant (including nonadherent cardiac muscle cells) was pelleted by centrifugation  and transferred to the new 25 mL culture bottle, previously coated with 0.1% gelatin solution. The cells were cultured in DMEM/F12 complemented with 20% FBS, 10% HS, EGF (20 ng/mL), LIF (10 ng/mL) and 1% P/S at 37 °C in a humidified atmosphere of 5% CO2. The culture medium was changed every three days.The right atrial appendage (right auricle) and right atrium were extracted from the delivered material, washed in ice-cold PBS solution, to remove the blood and, in the next stage, after the two-step mincing by sterilized scissors in Petri dishes, the tissue undergoes enzymatic digestion in DMEM + collagenase type II (2 mg/mL) solution conducted in 37 °C for 40 min with gentle mixing. After the end of digestion, the remaining tissue will be separated with nylon strainers of 70 μm pore size. The filtrate (containing cells of interest) was centrifuged  daily observation of cultured cells was performed. In present study, the images obtained during the cultivation period corresponding to the time intervals used in the molecular analysis are shown .Total RNA from all of the samples (both before and after in vitro cultivation) was isolated according to the method published by Chomczyński and Sacchi  employin® WT Expression Kit). The synthesis of cRNA was performed by in vitro transcription . Then, cRNA was purified and re-transcribed into cDNA. Subsequently, cDNA samples were used for biotin labelling and fragmentation using Affymetrix GeneChip® WT Terminal Labeling and Hybridization kit . Next, the biotin-labelled samples were loaded onto and hybridized to the Affymetrix® Porcine Gene 1.1 ST Array Strip. Hybridization was conducted at 48 °C for 20 h, in an AccuBlock™ Digital Dry Bath  hybridization oven. Microarrays were then washed and stained, according to technical protocol, using an Affymetrix GeneAtlas™ Fluidics Station . The strips were scanned using an Affymetrix GeneAtlas™ Imaging Station . The scans of the microarrays were saved on hard drives as *.CEL files for downstream data analysis.In the first step, total RNA (50 ng) from each pooled sample  was subjected to two rounds of sense cDNA amplification  was uploaded to the DAVID software . To retrieve gene ontology biological process (GO BP) terms containing differently expressed transcripts, the DAVID software was employed. The selection of significantly altered GO Term was based on a p-value (Benajamini) <0.05 and volume of at least 5 genes.Quality control (QC) studies were performed using the Affymetrix GeneAtlas™  software, according to the manufacturer’s standards. The generated *.CEL files were subjected to further analysis performed using the R statistical language and Bioconductor package with the relevant Bioconductor libraries. To correct background, normalize and summarize results, we used the Robust Multiarray Averaging (RMA) algorithm. Assigned biological annotations were obtained from the “pd.porgene.1.1.st” library, employed for the mapping of normalized gene expression values with their symbols, gene names, and Entrez IDs, allowing us to generate a complex gene data table. To determine the statistical significance of the analyzed genes, moderated t-statistics from the empirical Bayes method were performed. The obtained It is important to compare the expression profile in RA and RAA to understand the molecular basis for new vessel formation. A Venn diagram that was used to detect relations between lists of differentially expressed genes in both heart’s compartments and to explore the intersection of genes of analyzed terms from the functional analysis. In order to create the diagram, the 50 most altered genes for both heart compartments were analyzed.® 96 Real-Time PCR System, Roche Diagnostics GmbH  with SYBR Green as a detection dye. Levels of analyzed transcripts were standardized in each sample, in reference to hypoxanthine 1 phosphoribosyltransferase (HPRT1) and β-actin (ACTB) as an internal control. For each of the amplification reactions, 1 μL of cDNA solution was mixed with 5 μL of mastermix , 3 μL of PCR-grade water and 1 μL of specific starter pair (10 μM). We have used the Primer3 software for primer design (−ΔΔCq method. In order to confirm the specificity of the results and size of amplified products, 2% agarose gel electrophoresis of the products was performed.Determination of the transcript levels for analyzed genes was conducted using a Light Cyclerr design . The exoThe study evaluated cardiac muscle cells derived from the right atrial appendage and right atrium. The salient of the study was that in vitro condition significantly altered mRNA expression levels in evaluated cells, suggesting that the employed culture conditions may create a favorable environment for neovascularization. The expression profile of factors positively regulating neovascularization such as, SFRP2, PRRX1 or cytokines were upregulated, and ACTN2, ACTC1 or MYH7 were down regulated. Nevertheless, further optimalization of the culture conditions is still needed, because transcript levels of genes coding pivotal structural proteins were significantly lower.


Mercenaria mercenaria. Total and differential hemocyte counts were similar between EPF and hemolymph. Overexpressed genes in the EPF were found to have domains previously identified as being part of the “biomineralization toolkit” and involved in bivalve shell formation. Biomineralization related genes included chitin-metabolism genes, carbonic anhydrase, perlucin, and insoluble shell matrix protein genes. Overexpressed genes in the EPF encoded proteins present at higher abundances in the EPF proteome, specifically those related to shell formation such as carbonic anhydrase and insoluble shell matrix proteins. Genes coding for bicarbonate and ion transporters were also overexpressed, suggesting that EPF hemocytes are involved in regulating the availability of ions critical for biomineralization. Functional assays also showed that Ca2+ content of hemocytes in the EPF were significantly higher than those in hemolymph, supporting the idea that hemocytes serve as a source of Ca2+ during biomineralization. Overexpressed genes and proteins also contained domains such as C1q that have dual functions in biomineralization and immune response. The percent of phagocytic granulocytes was not significantly different between EPF and hemolymph. Together, these findings suggest that hemocytes in EPF play a central role in both biomineralization and immunity.Circulating hemocytes in the hemolymph represent the backbone of innate immunity in bivalves. Hemocytes are also found in the extrapallial fluid (EPF), the space delimited between the shell and the mantle, which is the site of shell biomineralization. This study investigated the transcriptome, proteome, and function of EPF and hemolymph in the hard clam  Bivalves have an open circulatory system populated by hemocytes, the quintessential component of bivalve immunity. Hemocytes function in several additional physiological processes including wound healing, biomineralization, nutrition, and transport –4. HemocMercenaria mercenaria, the hard clam, has recently been assembled at a chromosome level and genes were annotated  and carbonate (CO3)2-, providing a supply of Ca2+ which is a major constraint during calcification  crystals and can function in biomineralization; however, the role of hemocytes in biomineralization is still not well understood. Regulation of pH in the EPF is critical to maintain calcification, and evidence suggests marine invertebrates can elevate pH in this calcifying fluid relative to the seawater and increase CaCO3 saturation through ion transport  and Ruditapes philippinarum (Manila clam), and are often associated with hemocyte infiltration to mantle surfaces and the EPF, suggesting localized-immune response at the site of infection . Clams were scrubbed to remove debris and epibionts upon arrival. They were maintained in four aerated aquaria , Temperature 25°C, pH 7.8) and fed daily with commercial diet . Body fluids were collected five to seven days later.Adult (50-70 mm) EPF and hemolymph samples were individually collected Figure 1FAST™ Protease Inhibitor Tablets; 50 µl/m1) was added. The pelleted cells and tubes containing supernatant were flash frozen and stored at -80°C.Each fluid type was aliquoted, and 100 µl was saved for functional assays and diluted 1:4 with ice cold filtered artificial seawater (FASW 30 PSU). The remaining fluid was centrifuged . The supernatant was transferred to a new collection tube and protease inhibitor cocktail  was used to assess hemocyte activities as described below. Most of these assays rely on the assessment of fluorescent signals emitted by hemocytes following the addition of dyes that target specific pathways or molecules. For all these assays, a minimum of 1,000 hemocytes were assessed, and data was compared between EPF and hemolymph using nested ANOVAs. Fluid type was the main effect and the aquarium was the random or nested factor. Data was generated from a total of 20 clams, with five individual clams sampled from four replicate tanks (n=4). Before analyses, cells were separated from debris by size and intracellular complexity, which is a standard procedure for bivalves that does not require the addition of specific dyes that could otherwise alter cellular activities , 23.Propidium iodide  was added at the final concentration of 20 μg/ml, incubated for 10 min in the dark at room temperature (RT) before flow cytometry readings. PI cannot enter viable cells, but binds to DNA in dead cells, consequently making dead cells more fluorescent in the orange (FL2) channel. Agranulocytes and granulocytes were separated based on light forward (FSC) and side (SSC) scatter parameters and treated separately for the calculation of the percent of dead cells in each hemocyte subpopulation .Fluid samples were transferred to sealed 0.5 ml microcentrifuge tubes to minimize gas exchange and then immediately centrifuged . The supernatant was removed and the pellet was resuspended in FASW containing 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester  at a final concentration of 1μM. Samples were incubated at RT in the dark for 10 min before reading on the flow cytometer.+2 contents in hemocytes were assessed using Fluo-3 (ThermoFisher), a dye that shows increase in green fluorescence (FL1 channel) intensity with increasing levels of Ca+2. Fluo-3 was added at a final concentration of 5 μM and incubated at RT in the dark for 20 min before sample reading.Relative CaYellow-green latex beads  were added to samples (1:10 hemocyte:bead ratio) and incubated at RT for 1 hr before sample reading. Hemocytes associated with beads were considered phagocytic.® Ultra™ RNA Library Prep Kit for Illumina® , following manufacturer’s instructions and with indices added for demultiplexing of samples. Libraries were sequenced on Illumina platform (Novaseq 6000) and 150 paired end (PE) reads were generated. Novogene performed quality control tests, and cleaned reads were used in downstream analyses. RNASequencing of the EPF generated 351,049,084 PE reads  and 355,059,084 PE reads for hemolymph . Cleaned sequence reads were trimmed based on quality scores (limit 0.05), ambiguous nucleotides (max 2 nucleotides per sequence), and adapters ]). Reads were mapped on the predicted genes annotated on the M. mercenaria genome . DNA was removed using DNA-free™ Kit (Ambion), following manufacturer’s instructions. After checking the quality and quantity of RNA (Nanodrop), samples derived from EPF and hemolymph from the same eight clams were selected for sequencing. Extracted RNA was sent for sequencing to Novogene Corporation . One µg RNA per sample was used as input material. Sequencing libraries were generated using NEBNexta genome  using CLa genome  on Stonyonductor  to perfoonductor  were aliere from . Gene On package .Cell-free hemolymph and EPF were solubilized in 5% SDS, 100mM TEAB, 10mM DTT at 55°C for 30 min. Reduced cysteines were alkylated with 20mM iodoacetamide for 30 min at RT in the dark and proteins were acidified with phosphoric acid. Then proteins were micro precipitated with 90% methanol, 50mM TEAB, and bound to S-Trap solid phase cartridges as described elsewhere . ProteintopGO R package  and silica tubing (100µm ID x 20 cm) and were self-packed with 3u Reprosil resin. Peptides were separated using a flow rate of 300 nl/minute, and a gradient elution step changing from 0.1% formic acid to 40% ACN over 90 minutes, followed 90% ACN wash and re-equilibration steps. Parent peptide mass and collision-induced fragment mass information were collected using an orbital trap  instrument followed by protein database searching using Proteome Discoverer 2.4 (Thermo). Electrospray ionization was achieved using spray voltage of ~2.2 kV. Information-dependent MS and MS-MS acquisitions were made using a 100 ms survey scan (m/z 375 – 1400) at 60,000 resolution, followed typically by ‘top 20’ consecutive second product ion scans at 15,000 resolution. Database searching used Proteome Discoverer 2.4 (Thermo). Peptide and spectra false discovery rates were set to 0.05. Sample normalization was based on total peptide amount. Label free quantitation (LFQ) between samples was performed using intensity based pairwise like-peptide comparisons to generate fold change ratios. Protein abundance was based on summed pairwise peptide abundances and t-tests performed. Shared and modified peptides  were excluded from quantitation. Protein and RNA datasets were integrated and analyzed using JMP10 (SAS)  and the percentage of agranulocytes and granulocytes  did not differ significantly between EPF and hemolymph Figure 22+ contents, was significantly higher in granulocytes from the EPF as compared to hemolymph  while there were no differences for agranulocytes   or granulocytes (p=0.51) from both fluids Figure 3Gene transcription profiles were analyzed for hemocytes collected from the EPF and hemolymph. Principal component analysis (PCA) showed separation in gene expression between both fluids Figure 42+ binding domains, three collagen, type VI, alpha genes, two alkaline phosphatase genes, two laminin alpha genes, and a xylosyltransferase. Genes with EF-hand calcium-binding domain, EGF-like domain, and VWA domain made up the genes contributing most to the first and second principal component  hemocytes transport Ca2+ ions after induced shell damage , C. gigas, and Mytulis edulis (blue mussel) during shell repair and proposed the upregulation of bicarbonate transporters was to regulate the availability of bicarbonate ions to the site of calcification. Genes overexpressed in the EPF transcriptome that could potentially be involved in calcification by transporting ions included ion transporters, solute carriers, and bicarbonate exchange genes. Similarly, ion transporters, solute carriers, and Ca2+ binding domains were overexpressed in the EPF proteome. Ca2+ content was higher in granulocytes from the EPF than hemolymph, which supports the idea that hemocytes serve as a source of Ca2+ during biomineralization. In addition to transporting Ca2+ to the site of calcification, some of the aforementioned genes/proteins might be important for acid-base regulation. CA was included in the “tool kit” for biomineralization, because of its role in concentrating inorganic carbon in shell fluid, but it is also very important in acid-base regulation  protein and other complement components are overexpressed in the plasma proteome of clams infected with the fungus-like pathogen Mucochytrium quahogii [formerly QPX,  share many proteins including those related to cytoskeleton, membrane organization, cell motility, cell adhesion, and metabolic processes  in each body fluid. This is not surprising as many previous studies hypothesized or effectively demonstrated that hemocytes actively secrete a broad range of molecules into the surrounding media , 70, 71.rocesses . In factrocesses  and myosrocesses . Interes2+ into the extrapallial cavity. This compartmentalization may allow the use of hemolymph as an important conveyer of elements (e.g. Ca2+) towards calcification sites, while the specific hemocyte activities in EPF may create the microenvironment conducive to biomineralization. Future work is required to further clarify the role of hemocytes in biomineralization, such as their relationship with mantle-mediated calcification. Similarly, studies are needed to determine whether hemocytes in EPF represent a specialized fraction of hemocytes commonly present in the circulatory system or if their unique transcriptomic signature results from a “maturation” process that they undergo when they migrate to the extrapallial space. This study underscores the well-established role of circulatory hemocytes in bivalve defense, while also demonstrating that peripheral compartments including the extrapallial cavity contain hemocytes that function in immune protection of their compartment. Functional analysis of genes that might be important for both biomineralization and immunity, such as genes with C1q and kazal domains, should be further investigated to better elucidate whether a dual function in immunity and biomineralization is truly the case for the same genes or whether different members of these diversified gene families play different roles.Together, these findings demonstrate functional specialization of hemocytes between EPF and hemolymph. Variation in the expression of biomineralization-related genes and proteins between these fluids, along with differences in calcium content, underline the role of EPF in shell formation. Hemocytes in the hemolymph played an auxiliary role, by transporting Cahttps://www.ncbi.nlm.nih.gov/sra under accession number SAMN24368625. Proteomic data can be found at: https://massive.ucsd.edu/ProteoSAFe/static/massive.jsp under accession number MSV000088664.The original contributions presented in the study are publicly available. RNASeq data can be found at: EP and BA led the conceptualization and secured the funding. CS contributed to investigation, methodology, formal analysis, and writing the original draft. SF and JH assisted with the generation and processing of transcriptomic and proteomic data, respectively. All authors participated in manuscript revision. All authors contributed to the article and approved the submitted version.This research was supported by New York Sea Grant program (projects R/XG-24 and R/XG-32).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Common cold symptoms may be mitigated by products in caplet, nasal spray, and oral solution formulations, although variations exist in the bioavailability of the active ingredients contained within these products. Rapid gastric emptying (GE) of these active ingredients is important for reducing the delay between drug absorption and onset of cold symptom relief. Hot drink cold remedies are associated with greater comfort and may enhance the bioavailability of active ingredients. The objective of this study was to characterize the gastrointestinal transit of powder (reconstituted in hot water) and caplet formulations of commercially available multisymptom cold medications.This was an open-label, single-dose, parallel-group study. Healthy male adults under fasted conditions were randomized 1:1 to receive a single dose of radiolabeled Theraflu Daytime Severe Cold and Cough powder for oral solution or radiolabeled Theraflu ExpressMax Daytime Severe Cold and Cough caplet. External gamma scintigraphy was utilized to monitor GE and intestinal transit of two radiolabeled drug formulations.A total of 28 participants completed the study. The mean ± SE GE onset times were 1.1 ± 0.3 min and 8.5 ± 1.8 min for powder and caplet formulations, respectively. The mean ± SE GE completion times were 121 ± 13 min and 65 ± 13 min, respectively. Despite the similar mean times to GE25%, the powder had later mean GE50%  and GE90%  than caplets. Caplets had a shorter overall GE half-life, lower total gastric exposure, and faster transit time through the small intestine versus the powder formulation. No serious safety events were observed.The results of this study in healthy male adults suggest that the Theraflu powder formulation had a more rapid GE onset but longer time to GE completion than the caplet formulation.ClinicalTrials.govNCT03415243The online version contains supplementary material available at 10.1186/s13063-022-06037-x. The common cold is the most frequent acute illness found throughout the industrialized world and is associated with a high economic burden through lost productivity and expenditures for treatment –3. On avTheraflu Daytime Severe Cold and Cough powder for oral solution and Theraflu ExpressMax Daytime Severe Cold and Cough caplets  are among the numerous commercially available multisymptom formulations , 9. BothThis clinical study was designed to characterize the gastrointestinal (GI) transit of powder (liquid) and caplet (solid) formulations of these commercially available multisymptom formulations using gamma scintigraphy. We assessed and quantified GI transit of radiolabeled drug formulations in healthy male adults and evaluated the safety of these products.2 and total body weight > 110 lb (> 50 kg). Advanced aging induces morphological and functional changes to the GI tract that can affect transit time, metabolism, and gut permeability, potentially compromising effective drug absorption in an elderly population ) were collected at visit 1 (screening) and at visit 2 (day 1) after the final scintigraphic image was taken prior to dinner. Laboratory parameters, for which reference ranges were available, were categorized with respect to the reference ranges as high, low, normal, and missing.Adverse events included any abnormal laboratory test results  or other safety assessments , including those that worsened from baseline, and were considered clinically significant in the medical and scientific judgment of the investigator ; an exacerbation of a chronic or intermittent pre-existing condition including either an increase in frequency and/or intensity of the condition; new conditions detected or diagnosed after study treatment administration even though they might have been present before the start of the study; signs, symptoms, or the clinical sequelae of a suspected drug-drug interaction; and signs, symptoms, or the clinical sequelae of a suspected overdose of either study treatment or a concomitant medication. Recording of AEs was conducted at both visits 1 and 2 and until 5 days following the last administration of the powder or caplet formulation.For the analyses, the safety population consisted of all participants who received a radiolabeled treatment irrespective of whether they were included in the scintigraphy analysis. The scintigraphy analysis population included all participants who received radiolabeled treatment, did not vomit within 60 min after study drug administration, had sufficient data to determine the time to onset and completion of GE, and had no major protocol deviations. Scintigraphy images were corrected for radioactive decay and background radiation. The scintigraphy variables were summarized by treatment group using descriptive statistics. There were no formal clinical hypotheses tested in this study; therefore, a formal sample size calculation was not performed although it was planned to screen approximately 42 subjects to allow for at least 12 evaluable subjects per treatment group, a number sufficient to provide descriptive statistics. The statistical analysis software used was the SAS software version 9.4 or higher .A total of 32 participants were screened, 28 of whom were randomized 1:1 to receive the powder or caplet formulation and successfully completed the study  and caplet 12 ± 2.4 min) formulations had similar mean ± SE times to GE25%. The caplet formulation had earlier mean GE50% and GE90% times compared with the powder formulation. Specifically, the mean ± SE GE50% values were 23 ± 3.0 min and 16 ± 3.2 min for the powder and caplet formulations, respectively, and the mean ± SE GE90% values were 85 ± 12 min and 36 ± 9.0 min, respectively , reduced gastric exposure (area under the GE curve), and faster transit time through the small intestine versus the powder formulation , and starting at approximately 8 min post-dose, the 100% GE rate was faster for caplets, resulting in an earlier GE completion time for the caplet versus the powder formulation (65 min vs 121 min). As expected, no serious safety events were observed with either treatment.The powder formulation reconstituted in hot water had a later GE completion than the caplet. The reason for this observation is unclear, but the prolonged GE of the powder in solution may have occurred due to the higher volume of dispersed radioactivity in the solution. It is also possible that, because of the high sugar content present in the powder formulation, the liquid formulation may have had a higher caloric density or greater osmolality, both of which are identified as regulators of GE rates . MoreoveTreatments that most effectively reduce cold symptoms have the potential to ease the economic burden associated with the common cold –3. Hot dDespite the value of this study in examining well-matched treatment groups and a robust assessment technique to evaluate the transport of the formulations through the GI tract, there were several constraints imposed by the study design. First, all participants in this study were male, as the study was designed to exclude females based on potential differences in GE during menstruation. Although the exclusion of females was important to control experimental variables to potentially elucidate GE differences, it limits translation of the findings to a broader population. Second, this study was small in size and descriptive, as no formal hypothesis testing or statistical significance was performed; moreover, at the time of our study, no prior data were available to aid in defining a statistical hypothesis. Third, this study lacked pharmacokinetic and pharmacodynamics data to correlate the timing of GE with the rate and extent of absorption of the active ingredients. Fourth, none of the participants, investigators, or analysts were blinded to the treatment. However, as the scintigraphy outcomes are not subjective, treatment knowledge would be very unlikely to result in bias. Finally, the assessments conducted were of marketed products; therefore, this was not an excipient or formulation study that could allow for formula manipulations to ascertain the precise mechanism(s) responsible for the disparity between GE times of the caplet and powder formulations.This is the first clinical study to characterize the GI transit of two powder and caplet formulations of commercially available cold symptom relief products. Time of onset, completion of GE, and small intestinal transit time were successfully assessed and quantified using gamma scintigraphy. The results suggested that the powder formulation in solution had a more rapid mean time to GE onset, but slower time to GE completion, than the caplet formulation in healthy male adults. Although the powder formulation in solution had a slower GE rate than the caplet, no correlation with the pharmacokinetics or pharmacodynamics profiles is available, or their potential correlation with cold symptom relief. There were no serious safety events observed with either treatment in this small, descriptive, exploratory study. Future hypothesis-driven trials of the GI transit of these formulations in a broader population of subjects affected by the common cold are warranted to establish the clinical relevance of our findings.Additional file 1 Fig. S1. CONSORT 2010 checklist of information to include when reporting a randomised trial*.


System boundaries extended from cropping input manufacture to cattle arriving at the slaughterhouse. Beef systems were modeled using herd population dynamics, feed and performance data sourced from producers in four Brazilian states, with additional data from global databases. Implants were used in calves, growing and finishing cattle at low (LI), medium (MI), and high (HI) levels of performance enhancement, compared to nonimplanted (NI) controls. Feed use results were used in combination with producer-derived input costs to assess the economic impacts of implant use, including production costs and returns on investment. Improved FCE, ADG, and carcass weights conferred by implant use reduced the number of cattle and the time taken to produce 1.0 × 106 kg HCW beef. Compared to NI controls, the quantities of feed, land, water and fossil fuels required to produce 1.0 × 106 kg HCW beef was reduced in implanted cattle, with reductions proportional to the performance-enhancing effect of the implant (HI > MI > LI). Implant use reduced GHG emissions per 1.0 × 106 kg HCW beef by 9.4% (LI), 12.6% (MI), or 15.8% (HI). Scaling up the MI effects to represent all eligible Brazilian cattle being implanted, revealed avoided GHG emissions equivalent to the annual exhaust emissions of 62.0 × 106 cars. Economic impacts of implant use reflected the environmental results, resulting in a greater margin for the producers within each system . The 6.13% increase in kg of HCW beef produced generates a cost reduction of 3.76% and an increase in the return on invested capital of 4.14% on average. Implants offer the opportunity for Brazilian beef producers to demonstrate their dedication to improving environmental and economic sustainability through improved productivity, although care must be taken to avoid negative trade-offs.Brazilian beef systems contribute 14.9% of global beef production, therefore given climate change concerns, there is a clear need to reduce environmental impacts while maintaining economic viability. This study evaluated the hypothesis that steroid implant use in Brazilian beef cattle would reduce resource use, greenhouse gas (GHG) emissions and economic costs of production, thereby improving environmental and economic sustainability. A deterministic model based on beef cattle population demographics, nutrition and performance was used to quantify resource inputs and GHG emissions per 1.0 × 10 The sustainability of animal source foods  is an issue of considerable debate for all food stakeholders, from primary producers, processors, and retailers through to consumers, media, and government. The combination of an ever-expanding worldwide population  and a ri2e, yet significant variation exists within this figure, with regional values ranging from 14 kg CO2e for Eastern European beef to 76 kg CO2e for beef produced in Southeast Asia  emissions on climate change. According to the ast Asia . Given tast Asia , it is tProgress made in cattle genetics, nutrition, management and health over time has been key to reducing the environmental impacts of regional beef production systems  and thisSteroid hormone implants have been used in U.S. cattle production systems for decades, yet, to date, have not been registered in every country worldwide. The active hormones in implants  increase muscle protein synthesis, reduce protein degradation, and improve cattle ADG ; althougBos indicus cattle  of steroid implant use within Brazilian beef production were assessed using a deterministic Microsoft Excel-based model of cattle nutrition, metabolism and herd population parameters founded on life cycle assessment (LCA) principles. Economic costs of production were derived from feed use results produced by the environmental impact model, in combination with producer-derived input costs. This study used production data from commercial Brazilian beef operations collected via questionnaire, therefore approval from an animal care and use committee was not required.The environmental model employed was based on the original models described by 6 kg (one million kg) of hot carcass weight beef (HCW).System boundaries extended from the production of feed and forage crops  through to and including transport of finished cattle to the slaughterhouse door. The impacts of postslaughter transportation, processing, packaging and consumption were not included; neither were specific on-farm technologies and practices  not directly related to cattle feeding, management, and husbandry. A number of co- and byproducts originate from beef cattle production, including (but not limited to) leather, pharmaceuticals and bone meal. Ideally, system environmental impacts would be allocated between the principal product (beef) and all co- and byproducts, however, this was beyond the scope of the current investigation. To ensure that the results of the analysis were as conservative as possible, the decision was therefore made not to apply allocation within this analysis. The functional unit by which environmental impact was assessed was the production of 1.0 × 10Brachiaria brizantha, and signal grass—Brachiaria decumbens) and supplemented with minerals. Nelore cows had a mature weight of 420 kg (MG) or 450 kg (MGS), milk yield, and composition according to The producer questionnaire collated data relating to cattle performance, feed, transportation, crop production and infrastructure, plus economic data relating to costs of production and revenues accrued. The baseline characteristics of these production systems are shown in The majority of bull calves were grown and finished for beef, whereas, due to the relatively late age at first calving, a higher proportion of heifers were retained as herd replacements. Weaned calves destined for beef were either grown and finished on pasture on a finishing farm  characteristic of a significant proportion of Brazilian beef cattle , or grow4) emissions. The fraction of nitrogen emitted as enteric nitrous oxide was calculated from data reported by Within each regional (MG or MGS) simulation, there existed 30 groups of cattle: one group of lactating cows; one group of dry cows; two groups of barren cows; one group of mature bulls; four groups of replacement heifers ; three groups of replacement bulls ; three groups of preweaned calves ; six groups of growing cattle destined for the feedlot ; three groups of feedlot cattle ; and six groups of finishing farm cattle . Agricultural Modeling and Training Systems  ration fImplants were assumed to be used in all eligible cattle destined for beef production, excluding those reared as heifer or bull replacements for the cow-calf operation, i.e., preweaned calves, growing calves and finishing cattle. The aim of this paper was to investigate the environmental and economic impacts of generic implant use on the Brazilian beef system, rather than examining the effects of specific types  or commercial brands of implant; or the impacts on specific breeds or groups of cattle. Changes in ADG, FCE, CW, and dressing % conferred by implant use were derived from those published by 2O) emissions from manure stored as slurry, and the nitrogen losses from manure deposited on soil according to manure nitrogen content and the emissions factors from Manure in grazing operations  deposited onto pasture, whereas feedlot operations employed a combination of spreading on fields and lagoon storage  and 2. MCropping yields were supplied from the commercial beef operations participating in the study (corn silage) or derived from FAOSTAT  based onWith the exception of pasture and corn silage, all feeds were grown off-farm. Feed, fertilizer and pesticide transport was accounted for based on distance data .Carbon dioxide emissions from fertilizer and pesticide manufacture were derived from The savings in water use, energy use and GHG emissions conferred by implant use at the average level of performance enhancement were converted into consumer-friendly metrics in terms of mean annual human use per capita (water and energy), car-equivalents (GHG emissions) and tree-equivalents (GHG emissions). Water savings were transformed into the mean annual usage per capita in Brazil, using data from 2e per car) were based on the average distance travelled for passenger cars per year in Brazil , a fuel efficiency of 18.2 km/L, and GHG emissions for Brazilian fuel of 1.22 kg CO2/L based on 57% ethanol and 43% diesel (2 sequestered by a mature tree per year) were based on the geospatial data published by 2 sequestration rate of 5.06 kg CO2 per mature tree. These equivalents were scaled to up to represent the impacts if all Brazilian growing and finishing cattle were implanted, based on a total Brazilian beef production of 10.2 × 106 metric tonnes in 2019  is equal to sales of cattle and by-products from the operationEffective operating cost (COE) comprises all items considered to be variable costs or direct expenses represented by the cash disbursement recorded throughout the production cycle, being a function of the quantity used and the economic value per unitTotal operating cost (COT) refers to the portion of indirect costs represented by linear depreciation, provision of labor and fees associated with the production process and family labor.After determining revenues and costs, the gross margin (revenue - effective operating cost) and net margin  were calculated, in addition to the return on investment (ROI) of implant use. The ROI acted to quantify the efficiency of an investment or to evaluate the efficiency of a series of different investments, i.e., the capital gained or lost through implant use compared to the amount of capital invested.Livestock industries across the globe are under increasing pressure to improve all three facets of sustainability: environmental responsibility, economic viability and social acceptability. As such, the The role of improved cattle performance in reducing environmental impacts from beef production via the “dilution of maintenance” effect was discussed by 6 kg of HCW beef from 2.55 × 104 total cattle (NI) to a minimum of 2.40 × 104 head (HI), a 5.88% decrease . As shown in decrease .6 kg beef, with up to 0.26 × 108 kg less feed (a 13.3% reduction) and 0.30 × 104 ha less land (a 13.7% reduction) required to maintain production in the HI scenario compared to the control (NI). These results reflect the extensive nature of Brazilian beef production in that pastureland represented 98.5% of the total land savings, with very little supplemental feed across the entire nonfeedlot population, and feedlot cattle only being fed for an average of 3.8 mo in the NI scenario. Similarly, The improvements in beef cattle productivity conferred by implant use are well documented and are summarized in the reviews by The concept of improved productivity reducing deforestation through land sparing was expanded upon by 8 l per 1.0 × 106 kg HCW beef (HI) to 1.27 × 108 L per 1.0 × 106 kg HCW beef (NI), are within the cited limits, although these quantities are considerably lower than those cited by 8 l conferred by implant use with a medium performance impact (scenario MI) is equal to the annual usage of only 377 Brazilian people, yet if this is scaled-up to represent all eligible Brazilian beef cattle being implanted, would be sufficient to supply 3.66 × 106 people with their annual water needs , focusing upon the promotion of holistic approaches to land management, sustainable practices and implementation of the Brazilian Forest Code . Nationa6 kg HCW to total Brazilian beef production, would be equivalent to eliminating the exhaust emissions of 62.0 million Brazilian cars, or planting 11.3 billion trees—a considerable environmental bonus.The GHG emissions per kg HCW beef within the current study are comparable to those published by Capper, 2012; 2e per kg of LW gain. 2e per kg of LW, 45% lower than the control) within the groups studied. Furthermore, in a survey of 40 cattle ranchers within the Brazilian Amazon biome, 2e per kg of CW less than those that did not participate. This 19% decrease increased to 35.8% on farms that had participated in the programs for two years or more.In contrast to the results published by 4 accounted for the greatest share of emissions at 88.8%  and 12.6 kg to –28.1 kg CO2e/kg LW in the integrated crop-livestock-forest system. It is possible, therefore, that given appropriate pasture management, enhanced sequestration would have an additive effect on reducing GHG emissions.Carbon sequestration was not included within the current model, primarily due to lack of accurate data on the potential for sequestration in Brazilian soils under varying management strategies. Given the need to report and reduce national GHG emissions data, measuring and benchmarking soil organic carbon is likely to be mandatory for beef production systems across the globe in future, therefore this knowledge gap will need to be addressed and incorporated into modeling scenarios and carbon tools . SeveralAny change in management practices or technology use designed to improve system sustainability must have a neutral or positive effect upon economic viability over time. Using data from an earlier review of 37 implant trials , DuckettEconomic viability ultimately depends on a continuing demand and willingness to pay for the product and therefore meat quality plays a key role. Meat presentation, color and price were cited by The improvements in environmental and economic sustainability conferred by implant use within the current study reflect results of previous analyses , and strThe potential impacts of PET use on global trade should also be considered, given Brazil’s role as a significant beef exporter. After β-adrenergic agonists (βAA) were approved for use in Brazil, and implants approved in Argentina, some countries responded by demanding beef from cattle that were not given these technologies, thereby requiring supply chain segregation . Given tThe use of implants in Brazilian beef cattle provides clear and significant opportunities to improve resource use, GHG emissions and the economic viability of beef production systems, which all contribute to improving overall system sustainability. Given Brazil’s significant contribution to global beef production, with consequent implications for its role in anthropogenic climate change, it is crucial for beef producers to demonstrate dedication to improving environmental impacts. This may be achieved, in part, by simply improving production efficiency, although this must be done in a conscious manner to make the best use of resources, including previously deforested land. Changes in management practice and PET adoption must be executed in a manner that do not lead to negative economic consequences, e.g., limited access to export markets, or impaired consumer confidence. Brazilian beef producers must therefore focus on improving all three facets of sustainability  in a holistic manner.txab144_suppl_Supplementary_MaterialsClick here for additional data file.