Transient reflectancespectroscopy enables the direct probe of the separated charge carriersresponsible for water reduction to follow their kinetics. Comparisonwith transient photocurrent measurement allows the direct probe ofthe initial charge separation quantum efficiency (ϕCS) and provides support for a transient photocurrent model that dividesIPCE into the product of quantum efficiencies of light absorption(ϕabs), charge separation (ϕCS),and photoreduction (ϕred), i.e.,IPCE = ϕabsϕCSϕred. Our study shows that there are two general key loss pathways: recombinationwithin the bulk GaP that reduces ϕCS and interfacialrecombination at the junction that decreases ϕred. Although both loss pathways can be reduced at a more negative appliedbias, for GaP/TiO2, the initial charge separation lossis the key efficiency limiting factor. Our combined transient reflectanceand photocurrent study provides a time-resolved view of microscopicsteps involved in the overall light-to-current conversion processand provides detailed insights into the main loss pathways of thephotoelectrochemical system.Photoelectrochemical solar fuel generation at the semiconductor/liquidinterface consists of multiple elementary steps, including chargeseparation, recombination, and catalytic reactions. While the overallincident light-to-current conversion efficiency (IPCE) can be readilymeasured, identifying the microscopic efficiency loss processes remainsdifficult. Here, we report simultaneous  Thus, the developmentof operando time-resolved spectroscopic techniquesthat can directly probe these steps is essential to identify key efficiencylimiting factors and to discover strategies for designing more efficientPEC systems. Recently, transient reflectance spectroscopy (TRS) hasbeen applied to the study of carrier dynamics of photoelectrodes,including GaInP2, GaAs, Si, and GaP, with surface modificationranging from metal oxide protective layers to molecular species.28 In our previous work, we have successfully characterized GaP electrodesprotected with TiO2 layers of varying thickness (GaP/TiO2) in air without external bias by TRS and showed that TRScan directly probe the kinetics of photogenerated carriers at theGaP/TiO2 junction.28 However,so far, these studies were carried out mainly ex situ, in the absence of applied bias and electrolyte, which cannot bedirectly connected to the performance of these materials for light-drivensolar fuel generation reactions in photoelectrochemical cells.Although thetransient photocurrent model has been used to rationalizethe in situ transientreflectance spectroscopy and photocurrent measurementof a photoelectrochemical cell with a 5 nm TiO2-protectedGaP photocathode . We investigate how thecharge separation efficiency (ϕCS) and photoreductionefficiency (ϕred) depend on the applied bias andexcitation fluence, and we identify key loss pathways of the overalllight-driven HER on GaP/TiO2 photocathodes.In this work, we report a simultaneous 2 electrodesas a function of applied potential under modulated CW illumination.The details of photoelectrode preparation, TEM characterization , photocurrent measurement,and Mott–Schottky characterization  can be found in SI S1–S3. For GaP/TiO2 electrodes under these conditions, the photocurrent can be attributedto proton reduction.30 Shown in j(0) and thendecays to a steady-state value j(∞) on a ∼10 s time scale. IPCE values can be calculated from j(∞) according to 2 illumination power density and as a function of applied biasis shown in 2 are shown in Figure S4. Within the range of CW illumination power densities,the IPCE values show negligible dependence on power density.We firstcarried out transient photocurrent study of GaP/TiOrization S3 can berization S3 can be2 electrodes ϕabs is measuredto be 75% under our experimental conditions for both 400 and 405 nmillumination light . Following CS and ϕred asa function of applied bias measured at an illumination power densityof 1 mW/cm2 are shown in 2 electrode increases from ∼0 at a more positive appliedpotential of −0.4 V (Ag/AgCl) to a plateau value of ∼20%at a more negative potential of −1.2 V (Ag/AgCl). ϕred decreases from ∼100% at −1.5 V to ∼0%at −0.3 V, while ϕCS shows less pronouncedbias dependence, decreasing from ∼30% at −1.5 V to ∼15%at 0 V. Comparison of red.For GaP/TiOight see S5. Folloight see S5. Folloight see S5. Folloight see S5. Folloight see S5. Folloin situ transient reflectance spectroscopy and transient photocurrent measurementsof GaP/TiO2 photocathodes, as shown in CS by transient reflectance spectroscopyand compare with those determined from the transient photocurrentmethod. We first discuss the photocurrent results and will discussthe TRS results in the next section. Under the TRS conditions, thephotocurrent is generated mainly by the pump beam at 400 nm becauseits pulse energy is much larger than the white light probe. A typicalphotocurrent transient is shown in Figure S6a. The measured IPCE values as a function of applied bias at indicatedaverage illumination power densities (400 nm) are displayed in 2, significantly smallerthan that of CW illumination  were carried out at an averagepulse excitation power density of 1 mW/cm2 and higher.These results show that compared to CW illumination, pulsed laserexcitation reduces the absolute value of IPCE but does not affectits dependence on the applied bias. As will be discussed below, thekey difference between the CW and pulsed illumination can be attributedto increased charge recombination loss caused by large transient photocarrierdensity within the ∼100 fs pulse width of the intense femtosecondillumination.To provide experimental support for the three-stage transient currentmodel described above, we carried out simultaneous 2 electrodeunder −0.5 V applied bias as a function of probe wavelengthand pump–probe delay time after 400 nm excitation. For a delaytime up to 100 ps, the TR spectra show two main features: (1) thedecay of a broad negative ΔR/R signal from 1.4 to 3 eV and (2) the growth of an oscillatory signalcentered at around 2.78 eV, the direct band gap of GaP.31 After 100 ps, only the oscillatory signal remains.The broad feature can be attributed to the reflectance decrease causedby the increase in free carrier density (ΔN) in the GaP, and the normalized reflectance change is given by 33 The oscillatory signal is assigned to the suppressed Franz–KeldyshOscillation (FKO) signal, which is caused by the photoinduced changeof the built-in electric field at the surface.37 Similar FKO signals have beenreported recently on related TiO2-protected photoelectrodes.28SI S6.1), making TR spectroscopyan interface sensitive approach. The FKO signal amplitude is directlyproportional to the surface density of transferred electrons acrossthe GaP/TiO2 junction , andthe free carrier signal amplitude is proportional to free carrierdensity within the GaP. These signals provide a direct probe of thekinetics of the initial transfer of free electrons in the GaP acrossthe GaP/TiO2 interface. The transient reflectance kineticsof the free carrier decay (monitored at 1.5 eV) and FKO formation(monitored at 2.78 eV) are compared in Table S2. The time constants (and amplitudes)are 0.40 ± 0.02 ps (37.2%) and 32.6 ± 2.3 ps (62.8%) forthe free carrier decay and 0.40 ± 0.02 ps (65.0%) and 32.6 ±2.3 ps (35.0%) for the FKO growth. Similar agreement between the kineticsof free carrier decay and FKO signal formation is observed at allmeasured biases (see below and Table S2). These results confirm that the FKO signal growth can be attributedto the electron transfer across the GaP/TiO2p-n junction. It should be noted that we have used bi-exponential fitsto obtain apparent time constants and confirm the agreement betweenthe FKO and free carrier kinetics, while the actual kinetics of carriertransport and interfacial transfer is a much more complex processthat requires numerical solution of transport equations to fit.28The probe penetration depth on the GaP ranges from8 nm at 3.1eV and 10 nm at 2.78 V to 21 nm at 1.5 eV , and the amplitude of the FKO signalis given by ΔR/R (ℏω) ∝ EDCEAC.39 Because of the short penetrationdepth of the probe beam at 2.78 eV (∼10 nm), we have used thesurface electric field amplitude as an approximation of the averagedfield strength in the probe region for the FKO signal. With this approximation, EDC is the built-in electric field in the depletionregion in the dark at the GaP surface and EAC is the light-induced change of the built-in field at the GaP surface. EDC can be calculated by eq S5 from the measuredflat band potential and dopant density (SI S3). EAC at the GaP surface is proportional to the amount ofseparated photogenerated charge carriers across the GaP/TiO2p-n junction per unit electrode area (EAC ∝ σ), following the Gauss law.28 This suggests that the FKO signal amplitudedepends linearly on both the DC electrical field strength (or bandbending) and the amount of separated carriers. This model providesreasonable fits to the FKO spectra at all applied biases, as shownin Figure 3c. Similar linear dependence of the ΔR/R (ℏω) signal oncarrier density was also observed in a previous study.21 It is worth noting that this signal differsfrom strong field conditions, in which the FKO amplitude was reportedto depend on the carrier density logarithmically.22 Furthermore, the presence of free carriers at early delay timescan lead to band state filling and bandgap renormalization effects,which can also lead to transient spectral change.40 However, as shown in 2 sample, the TR spectral feature at the direct bandgap isdominated by the electric field-induced FKO signal caused by chargeseparation across the GaP/TiO2 interface.Transient reflectance spectra and kineticswere measured as a functionof applied potentials with an average excitation fluence of 15 mW/cm0–200 ps 3c shows EAC as a function of appliedbias, assuming EAC = −1 at −1.5V. The determination of the absolute value of EAC is not possible in this experiment because of the unknownproportionality constant that relates the FKO signal amplitude withthe product of EDCEAC. EDC can be calculated fromthe applied potential according to eq S5. The calculated EDC and fitted relative EAC is shown in EDC (by 1.4 times)and EAC (by 2.5 times), with the latterindicating a 2.5-fold increase in the charge separation yield. Thisobservation is consistent with the expected increase in charge extractionefficiency at a more negative applied bias, qualitatively similarto the charge separation efficiency increase observed under CW illuminationin EDC valuesestimated using eq S5 is not accurate andshould be corrected to obtain more reliable charge separation efficiencies.The fitof the FKO spectra in Table S2 and Figure S7). Thus, the bias-dependent TR kineticsreveal that at more negative applied potentials, the built-in electricfield strength increases, which drives faster and more efficient separationof photogenerated electrons across the GaP/TiO2p-n junction.The bias dependence of the TR kinetics of the freecarrier andFKO signals is compared in Figure S8, respectively. A comparisonof the FKO spectral lineshape  shows that they are independent of the excitation fluence, consistentwith the weak field limit described above, and the FKO signal amplitudeat long delay times saturates at large excitation fluence. This fluencedependence can be seen more clearly in p-n junction. This effect can be attributedto band flattening caused by a large transient carrier concentration.42 The band flattening effect originates from the high peak power ofpulse laser excitation (SI S5), which introducestransient carrier density several orders larger than the dopant level.This undesirable effect can be mitigated by a larger initial bandbending, as shown in 2, while the 0 V curve saturates at 5 mW/cm2.To investigate the effect of the photogeneratedcarrier densityon charge separation, transient reflectance spectra and kinetics weremeasured as a function of excitation fluence at fixed applied biases.The averaged TR FKO spectra at 100–200 ps and the transientFKO kinetics at 2.78 eV measured at −1.5 V for selected fluencesare compared in CS), i.e., the probability of transferring an electron acrossthe GaP/TiO2 junction per absorbed photon, can be approximatelydetermined from the FKO signal amplitude at 50 ps when the initialcharge separation has reached its peak amplitude and slow interfacialcharge recombination losses are small (EAC (related tothe amount of separated photogenerated electrons) and EDC, the charge separation efficiency (ϕCS′) is proportionalto the FKO amplitude at 50 ps after normalizing by the number of absorbedphotons and EDC, according to The initial charge separation efficiency , which scales with the numberof absorbed photons, EDC is the DC field strength calculated from eq S5 (in kV/cm), α (≤1.0) is acoefficient that accounts for the decrease in DC field due to chargeaccumulation under transient reflectance measurement conditions (seebelow), and A (=1031 kV/cm·mW/cm2) is a scaling factor. The value of the scaling factor A is determined such that the calculated IPCE using ϕCS′ accordingto calc. = ϕabsϕCS′ϕred), where αis set to 1. The α value is allowed to change at higher excitationdensity to achieve the best match of calculated and measured IPCEvalues. In calculating IPCE, we have used the reduction efficiencyϕred obtained from CW excitation .This difference can be attributed to the much larger peak power andtransient carrier density under the pulse excitation conditions. Withthe same average power density of 1 mW/cm2 (average carriergeneration rate of 1.5 × 1020 cm–3 s–1 at 400 nm), the total number of generatedcarriers are the same under CW and pulsed illumination averaged overthe repetition period of pulsed illumination (2 ms). Under CW illumination,the carriers are generated continuously, and the average photogeneratedcarrier density within the semiconductor can be estimated to be 1.5× 1010 cm–3, assuming a transporttime of 100 ps to sweep the carriers from the depletion region intoTiO2 (see 16 cm–3) and has a negligibleeffect on the built-in electric field. Under pulsed illumination,one excitation pulse arrives at the sample every 2 ms and the carriersare generated within the 100 fs pulse width, corresponding to a transientcarrier density of 2.9 × 1017 cm–3 in GaP, which is higher than its dopant density. As discussed inour previous study, the high transient carrier density under pulsedillumination leads to significant excitation power-dependent bandflattening and recombination loss in GaP on the sub-picosecond timescale.28 Similar ultrafast recombinationunder pulsed illumination has also been reported in a previous transientoptical study of Si nanowires and Si wafer.44 Thus, the reduced IPCE and ϕCS′ under pulsed illumination can be attributedmainly to the enhanced charge recombination loss within the GaP duringthe initial separation stage caused by the large transient carrierdensity and band flattening effect.49 Because such loss is likely caused by an Auger recombination process,which depends on the concentrations of electrons (n) and holes (p) to the third power, n2p and p2n, the recombination loss under pulsed illuminationincreases at higher average power.44The measured IPCE under pulsed illuminationcontinues to increaseuntil an average pulsed excitation power density of 1 μW/cm2 2d,while2 S4.This 2 average power density, the photocurrent densities are 0.015 and0.062 mA/cm2 under pulse and CW illumination, respectively. Because the steady-statephotocurrent densities are dependent on the surface electron density,these results suggest that with the same average illumination power,the steady-state surface electron density is smaller under pulsedillumination conditions than CW, which is consistent with a lowerinitial charge separation efficiency under pulsed illumination. Furthermore,because the photoreduction quantum efficiency ϕred is determined by the competition of surface reaction and interfacialrecombination at the steady state, it should be the same under CWor pulse illumination conditions, independent of illumination power.On the time scale of catalysis (>2 ms),the average carrier densityat the electrode surface is similar under similar average excitationpower densities. For example, at −1.5 V and 1 mW/cmCS′decreases at higher excitation power densities, indicating a largerdeviation of the DC field from that calculated from the applied biasaccording to eq S5. This can be attributedto the accumulation of photogenerated electrons in TiO2 under femtosecond laser excitation. In the TRS measurement, we comparedthe reflectance between the pump (light) and unpumped (dark) electrodes,and the time window between light and dark probe pulses (1 ms) islikely too short compared to the water reduction time. As a result,there is a possibility of accumulated electron in TiO2,and the GaP/TiO2 has not decayed to the true dark statefor the “dark” probe signal. This is supported by thelong photocurrent decay time (in the order of seconds) of the transientphotocurrent measurement, as shown in EDC values that are smaller than the true dark ones. Suchaccumulation effect increases with the average laser fluence, leadingto the observed fluence-dependent α values shown in 2 not only increases the charge recombinationloss but also decreases the initial charge separation efficiency.Future experiments at lower repetition rates and lower excitationpowers may help to decrease this effect. Furthermore, simultaneousTRS and TPC measurements with the devices already under 1 sun CW illuminationmay provide a way to study these charge separation and water reductionsteps under conditions more similar to device working conditions.50As shown in 2 electrodes: (1) the competitionof interfacial electron transfer across the GaP/TiO2 junctionwith the electron–hole recombination within the GaP that limitsthe initial charge separation efficiency, ϕCS; (2)the competition of water (proton) reduction by the transferred electronsin TiO2 and their interfacial charge recombination withthe holes in the GaP limits the reduction quantum efficiency, ϕred, as shown in CS increasesat a more negative applied bias because the increased built-in fieldmore effectively drives charge separation and suppresses charge recombinationof photogenerated carriers within GaP. It is important to note that,at an applied bias of −1.5 V (vs Ag/AgCl) and CW illumination,although the ϕred reaches ∼100%, IPCE is stilllow (∼20%) because the main loss occurs at the initial chargeseparation process (ϕCS ∼ 30%). The chargeseparation efficiency further decreases to ∼4% at higher peakpower under pulsed excitation conditions. For GaP/TiO2,the initial charge separation efficiency may be limited by the lowextinction coefficient of GaP compared to other semiconductors, whichresults in a long optical absorption path. This leads to a large transportdistance of the minority carriers to the interface, increasing thecharge recombination losses within GaP.The results described above suggestthat there are two main loss pathways in the overall photon-to-currentconversion process in GaP/TiOred) increases rapidly at more negative potentials.ϕred is determined by the relative rates of interfacialreduction of proton and electron recombination with GaP valence bandholes across the GaP/TiO2 junction. Because proton reductioninvolves electrons from the TiO2 conduction band or surfacetrap states, their energetics are independent of the applied potentialon the GaP, which is dropped mainly within the GaP depletion region.As a result, we can assume that the proton reduction rate is independentof the potential and the observed potential dependence of ϕred reflects the change of the interfacial charge recombinationrate. At less negative potentials, the extent of band bending is smalland the concentration of the majority carrier (holes) at the surfaceis large, which increases the recombination loss. At potentials >−0.4 V, while IPCE approaches 0, ϕCS doesnot. This indicates that at this potential range, the GaP/TiO2 junction can still support initial charge separation, butthe interfacial recombination of the separated electrons in TiO2 with the holes in GaP is much faster than the photoreductionreaction. At higher negative potentials (∼ −1.5 V),ϕred increases because the large band bending inGaP can slow down the recombination such that it is outcompeted bythe slow proton reduction process on TiO2, even in theabsence of catalyst layers.The second recombinationloss can be effectively suppressed by decreasing the interfacial recombinationrate and/or increasing the catalytic reaction rate. As shown in 2 interface. This feature overcomes the technicalchallenge of transient absorption measurements in which all carriersare probed and it is difficult to isolate the small population ofbias-dependent separated carriers from the total carrier population.Furthermore, because the transient reflectance technique does notrequire optically transmissive electrodes, it can be applied to awider range of photoelectrodes under device operation conditions.Because of these advantages, we believe that transient reflectancespectroscopy is a useful technique for operando studyof charge carrier dynamics of photoelectrodes. Although our initialstudy is focused on GaP/TiO2, it can be extended to fullphotoelectrode systems with surface-attached catalysts, such as asemiconductor/TiO2/Pt photoelectrode, with better HER performance,as long as the surface catalyst does not block light penetration intothe light-absorbing semiconductor. We will explore this capabilityin future work.It is interesting to note that eventhough the charge separationefficiency under femtosecond pulsed excitation conditions is small,∼1–7% as shown in in situ transientreflectance spectroscopy and transient photocurrent measurements ofphotoelectrochemical cells with TiO2-protected GaP photocathodesfor water reduction. TR spectroscopy enables the direct probe of thekinetics of the free carriers inside the GaP and separated carriersacross the GaP/TiO2 junction. Comparison of the bias- andexcitation fluence-dependent carrier kinetics and IPCE enables thedirect measurement of the initial charge separation efficiencies ϕCS and provides support of a transient photocurrent model thatdecomposes the overall IPCE into the product of the quantum efficienciesof three stages (IPCE = ϕabsϕCSϕred). According to this model, there are two key loss pathwaysin the overall light-to-photocurrent conversion process: first, chargerecombination within the GaP that competes with the initial built-infield-driven charge separation across the GaP/TiO2 junctionto reduce the initial charge separation efficiency (ϕCS), and second, the interfacial recombination of the separated electronsin TiO2 with the holes in the GaP that competes with protonreduction, limiting the photoreduction quantum efficiency (ϕred). Our results show that both loss pathways can be reducedat a more negative bias to increase the built-in electric field thatfacilitates charge separation and suppresses recombination. However,at an applied bias of −1.5 V (vs Ag/AgCl) and CW illumination,although the ϕred reaches ∼100%, the initialcharge separation process ϕCS is ∼30%, whichlimits the overall IPCE to ∼20%. Our study gives a detailed,sequential time-resolved view of charge carriers dynamics from generation,separation, and recombination to reaction in a model photoelectrochemicalsystem and provides helpful insights into key efficiency limitingfactors.In summary, we report simultaneous


Multiples of the normal median (MoM) of free βHCG is a valuable parameter in evaluation of risk of adverse pregnancy outcomes. In the current retrospective study, we assessed the maternal and fetal outcomes in pregnant women having free βHCG MoM levels < 0.2 or > 5 in their first trimester screening (FTS). Relative risk of trisomy 21 was significantly higher in patients having free βHCG MoM > 5. On the other hand, relative risk of trisomies 13 and 18 and Turner syndrome were higher in those having free βHCG MoM < 0.2. Other chromosomal abnormalities were nearly equally detected between those having free βHCG MoM < 0.2 or > 5. Relative risk of hydrocephaly and hydrops fetalis was higher when free βHCG MoM was below 0.2. On the other hand, relative risk of low birth weight was higher when free βHCG MoM was above 5. Moreover, frequency of gestational diabetes mellitus, preeclampsia, preterm delivery and vaginal bleeding increased with levels of free βHCG MoM. However, polyhydramnios had the opposite trend. Frequencies of premature rupture of membranes and pregnancy induced hypertension were highest among pregnant women having levels of free βHCG MoM < 0.2. The current study indicates importance of free βHCG MoM in identification of at-risk pregnancies in terms of both fetal and maternal outcomes. In fact, βHCG MoM < 0.2 or > 5 can be regarded as risk factors for adverse maternal or fetal outcomes irrespective of the presence of other abnormalities in the FTS results. Maternal serum levels of free β-hCG has been found to be increased in the first trimester of pregnancy in trisomy 21, while being decreased in trisomies 18 and 134. Moreover, this serum marker has been shown to be increased in pregnancies with established preeclampsia and in the third trimester prior to clinical manifestations, yet being decreased or unaltered at 11–13 weeks of gestation8. Thus, along with assessment of PAPP-A, evaluation of serum levels of free βHCG can enhance the prediction of preterm preeclampsia9.Maternal serum levels of free beta human chorionic gonadotrophin (βHCG) have been regarded as markers for evaluation of the presence of chromosomal abnormalities in the fetus for a long time10. Another large-scale study has approved association between first-trimester free βHCG levels higher than 95th percentile and fetal loss after 24 weeks of gestation11. More importantly, low maternal serum βHCG levels have been recently found to be associated with increased risk of having pathogenic findings in chromosomal microarray analysis12.Besides, extreme levels of free βHCG in the first trimester have been correlated with increased frequencies of adverse perinatal outcomes, including intrauterine growth restriction (IUGR) and low birth weight (LBW)13. Thus, we designed the current retrospective study to assess the maternal and fetal outcomes in pregnant women having these free βHCG MoM levels in their first trimester screening (FTS). The current study aimed at investigation of the association between extreme values of βHCG MoM level in FTS and adverse obstetric outcomes. Since fetal sex can affect maternal and obstetric outcomes14 as well as βHCG levels15, we assessed association between fetal sex and these factors in our study.Our recent study in a large cohort of Iranian pregnant women came for amniocentesis has led to identification of the importance of free βHCG multiples of the normal median (MoM) levels < 0.2 or > 5 as indicators of adverse pregnancy outcomesThe findings of our study can be used for prediction of risk of trisomies as well as pregnancy complications such as preeclampsia, small for gestational age (SGA), gestational diabetes mellitus (GDM) and premature rupture of membranes (PROM).In this retrospective study, we assessed data of 327,291 pregnant women referred for FTS to Nilou Laboratory, Tehran, Iran during 2018–2020. Multiple pregnancies were excluded from the study. FTS was based measurement of levels of pregnancy-associated plasma protein-A (PAPP-A) and free βHCG in the maternal blood as well as an ultrasound exam to measure the size of nuchal translucency (NT). The time at which patients referred was between 11 weeks of gestation and 13 weeks and 6 days.This study included 832 patients who had free βHCG MoM levels < 0.2 and 1447 patients with free βHCG MoM levels > 5 in their FTS assessment. A total of 7494 pregnant women with 0.2 < free βHCG MoM levels < 5 were also included in this study as controls. All cases and controls were Persians. We called all women via phone call in a period of 8–12 months after delivery and collected all relevant data regarding maternal and foetal outcomes. Data was collected in a standardized proforma which was filled by trained staff. Hospital records were also assessed in cases that had abnormal findings. The study protocol was confirmed by the host ethical committee. Maternal demographic features, the results of first trimester sonography and biochemical parameters of all pregnant women at 11 to 13 + 6 weeks of gestation were collected. NT thickness and the presence/absence of nasal bone were also recorded. Serum levels of β-hCG and PAPP-A were assessed by immunofluorescence technique  and used for assessment of risk of trisomies. Levels of serum markers were converted to multiples of the median (MoM) for adjustment of errors associated with race, gestational age, maternal demographic data and mode of conception. The study protocol was approved by Ethical Committee of Shahid Beheshti University of Medical Sciences and all methods were performed in accordance with the relevant guidelines and regulations. Informed written consent forms were signed by all study participants.Adverse fetal outcomes were defined as follow: stillbirth is a fetal death that occurs at or greater than 24 weeks gestation; intrauterine fetal demise is a fetal death before 23 weeks gestation; fetal morbidity included all problems that have been seen during the newborn period—the first 28 days of life (excluding respiratory problems and jaundice that were reported separately). These problems include seizure, hypoglycemia and infections, any cardiac disorders, grades 3 and 4 intraventricular hemorrhage , and vision and hearing impairment .Preterm delivery was defined as spontaneous delivery before 37 weeks of pregnancy.Respiratory problems included all types of breathing problems that affect newborns, such as transient tachypnea of the newborn, neonatal respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), meconium aspiration syndrome, persistent pulmonary hypertension of the newborn, pneumonia, and apnea.Death after delivery include death during the newborn period (the first 28 days of life) for any reason. Large and small for gestational age were defined as birth weight above the 90th percentile and below the 10th percentile, respectively.SPSS version 21  was used for assessment of data. Chi-2 test was used for assessment of association between categorical variables. The relationship between quantitative variables was evaluated using ANOVA test. P values less than 0.05 were considered as significant. Tukey test was used as a nonparametric equivalent for ANOVA for comparison of two or more subgroups within each group. This test was used for comparison of median values of circulatory markers.The results of comparison of means showed no significant difference between groups in terms of gestational age . Yet, there was significant difference in weigh and age of patients within these groups  Table .Table 1MTotally, 19.6% of pregnant women had a consanguineous marriage. This rate was not different between different groups of women categorized based on the levels of free βHCG MoM . Frequency of GDM, preeclampsia, preterm delivery and vaginal bleeding increased with levels of free βHCG MoM. However, polyhydramnios had the opposite trend. Frequencies of PROM and pregnancy induced hypertension (PIH) were highest among pregnant women having levels of free βHCG MoM < 0.2 Table .High risk for each trisomy was considered as having risk > 1/250 in FTS. Intermediate risk was defined as having 1/1500 < risk < 1/251 for trisomy 21 and 1/1000 < risk < 1/251 for trisomies 13 and 18. Finally, low risk was defined as risk < 1/1500 for trisomy 21 and risk < 1/1000 for trisomies 13 and 18.Kruskal–Wallis test and Tukey test for intra-group comparisons showed significant differences in odds of being affected given a positive result (OAPR) in high risk groups (P < 0.001) Table . In factWhen free βHCG levels are below 0.2, risk of trisomies 13 and 18 and other chromosomal abnormalities increase in all three categories of high, intermediate and low risks based on the results of FTS (P < 0.001). If we include all chromosomal anomalies in the analyses in this group, an OAPR value of 1/7.7 is calculated .Besides, risk of structural abnormalities is higher in patients having free βHCG levels are below 0.2 (P < 0.001). Moreover, both adverse fetal outcomes and adverse maternal outcomes  are higher in patients having free βHCG levels are below 0.2.In pregnancies with free βHCG levels between 0.2 and 5, OAPR was different between intermediate and low risk groups (P < 0.001). OAPR was calculated to be 1/39.5 in this group.Finally, free βHCG MoM > 5 has importance only in high risk group. In the intermediate risk group, an OAPR value of 1/65.5 was obtained which is not appropriate.We also assessed association between adverse fetal outcomes and calculated risk of trisomy 18 Table . Fisher Moreover, risks of PROM, abortion, preeclampsia and HELLP syndromes were increased with increased risks of trisomies 18 and 21.Frequencies of trisomies 21 and 18 were significantly higher in male fetuses Table .PROM, fetal morbidity, stillbirth, hospitalization and LBW were higher in female fetuses. On the other hand, gestational diabetes mellitus, PIH, spontaneous abortion, IUFD and structural had higher frequencies in male fetuses Table .Table 5A13. In the current study, we have collected data about perinatal complications and adverse fetal/maternal outcomes from patients with free βHCG MoM > 5 or < 0.2 in a period of 8–12 month after delivery.In our recent study, we have suggested high risk non-invasive prenatal testing (NIPT), PAPP-A MoM < 0.2 and free βHCG MoM > 5 or < 0.2 as important indications for necessity of invasive testing in pregnancy with appropriate OAPR values16. Similarly, significant differences have been observed in European and Asian MoM values17. Thus, adjustment for ethnicity is important in FTS programs.It is worth mentioning that evaluation of serum levels of βHCG and other biochemical factors in each ethnic group has practical significance. This supposition is supported by previous studies. For instance, Chinese women have been shown to have higher maternal serum levels of free βHCG and PAPP-A in the first trimester, after correction for the maternal weightWe found that relative risk of trisomies 13 and 18 and Turner syndrome were higher in those having free βHCG MoM < 0.2. Relative risk of hydrocephaly and hydrops fetalis was higher when free βHCG MoM was below 0.2. On the other hand, relative risk of low birth weight was higher when free βHCG MoM was above 5. Moreover, frequency of GDM, preeclampsia, preterm delivery and vaginal bleeding increased with levels of free βHCG MoM. However, polyhydramnios had the opposite trend. Frequencies of premature rupture of membranes and pregnancy induced hypertension were highest among pregnant women having levels of free βHCG MoM < 0.2. Thus, levels of this serum marker can predict occurrence a wide array of maternal/fetal adverse outcomes.18. Thus, it is not surprising that abnormal levels of free βHCG is associated with a wide range of maternal and fetal adverse outcomes. In fact, hCG contributes to the several functional and structural aspects of healthy pregnancy.From a mechanistic point of view, numerous functions of hCG can be related with these adverse outcomes. For instance, hCG has a role in enhancement of production of progesterone by corpus luteal cells and induction of angiogenesis in uterine vasculature. Moreover, it promotes the fusion of cytotrophoblast cell and differentiation to make syncytiotrophoblast cells. Most importantly, hCG contributes to the blockage of maternal immune or macrophage actions on foreign invading placental cells and causes uterine growth parallel to fetal growth20, this rate was not different between different groups of women categorized based on the levels of free βHCG MoM.In our cohort of patients, the rate of consanguinity was 19.6. Although there is evidence to suggest association between consanguinity and genetic and congenital abnormality as well as adverse pregnancy outcomes21.Sharony et al., have detected low maternal serum concentrations of hCG in 0.57% of the assessed women. Approximately, 16% of these women have been found to have missed abortions. They have not detected any case of trisomy 18 or other chromosomal abnormalities in their patient population. Yet, they have reported perinatal complications in these pregnancies22, maternal serum free βhCG below 10th centile of the reference range was detected in about 15% of the pregnancies that afterwards led to miscarriage or resulted in pregnancy induced hypertension or growth restriction, and in 20% of pregnancies that developed GDM, indicating the importance of this marker in the prediction of pregnancy complications. In fact, low maternal serum free βhCG below might indicate insufficiency of placenta.In the study conducted by Ong et al.In patients having βHCG levels below 0.2, if we include all chromosomal anomalies in the analyses, an OAPR value of 1/7.7 is calculated. Considering the acceptable value of OAPR for FTS , this OAPR indicates that free βHCG levels < 0.2 can be used as a novel indication for amniocentesis. Since this result is found in 0.25% of total patients, this indication can result in improvement of final OAPR in screening of chromosomal abnormalities. Besides, the observed higher risk of structural abnormalities in patients having free βHCG levels below 0.2 indicates necessity for precise follow-up of these patients using higher standards anomaly scans. Moreover, both fetal and maternal adverse outcomes were higher in patients having free βHCG levels are below 0.2. Thus, these pregnancies should be considered as high risk pregnancies receiving higher levels of care, including follow-up of pregnant women for monitoring blood glucose and blood pressure, exact monitoring of fetal weight and delivery at more specialized centers.23. The latter finding is in accordance with our finding regarding the higher frequencies of PROM among pregnant women having levels of free βHCG MoM < 0.2. However, we found association between GDM and high levels of free βHCG MoM. There are studies showing lower risk of GDM in pregnancies with high level of βhCG (≥ 2.0 MoM)25. Two other studies have reported no association between serum free βhCG levels and GDM27.Recently, a large-scale study in Turkish population has reported association between serum levels of PAPP-A and SGA, while GDM, PROM and preterm PPROM have been more common in pregnant women with low serum free β-hCG28. βhCG has functional interactions with a number of these pathways. For instance, it attenuates NF-κB activation and cytokine expression29. Moreover, PI3K/mTOR signaling pathway is involved in the hCG-mediated induction of vascular endothelial growth factor30. Although the exact mechanism of contribution of abnormal levels of βhCG in the pathogenesis of GDM is not clear, abnormalities in the regulation of these pathways might contribute to this pathology.Mechanistic studies have indicated that a number of signaling pathways and biomolecules including NF-κB, PPARs, SIRTs, AMPK, GSK3, PI3K/mTOR, inflammasome and endoplasmic reticulum stress are implicated in the pathogenesis of GDM31. This finding is in accordance with our results.High level of βhCG has been shown to be a predictor of preeclampsia in another study in Iranian pregnant women. In fact, βhCG > 3 MOM has been associated with more than five-fold increase in the risk of developing preeclampsia in the mentioned studyth or 95th percentile and free βHCG MoM/PAPP-A MoM > 3 in assessment of patients. Based on these parameters, second trimester screening, sequential combined tests or NIPT can be recommended for pregnant women.In pregnancies with free βHCG levels between 0.2 and 5, OAPR was different between intermediate and low risk groups. Since OAPR was calculated to be 1/39.5 in this group, we recommend inclusion of other parameters such as maternal age > 35, fetal NT ≥ 99Finally, since free βHCG MoM > 5 has importance only in high risk group based on the FTS results, we recommend using the calculated risk of FTS for further assessments. Risk of adverse maternal/fetal outcomes in this category was significantly different from control group in all three high, intermediate and low risk groups indicating the importance of higher care systems for this group of pregnant women.13.We also found associations between some parameters of adverse maternal/fetal outcomes and fetal sex as well as calculated risk of trisomies 18 and 21 in FTS which is in line with our previous study32 have reported no correlation between paternal age and risk of trisomy 21, other studies have reported positive and inverse associations between this factor and risk of trisomy 21, respectively17.Future studies are needed to assess the role of other factors such as paternal age, residential area, nutritional deficiency and chemical or biological contamination on the results of FTS and interpretation of βHCG MoM levels. In the current study, we did not assess the possible impact of these factors on the obtained results of FTS. The role of these factors in risk of trisomies is controversial. For instance, while Quattrocchi et al.Taken together, the current study indicates importance of free βHCG MoM in identification of at-risk pregnancies in terms of both fetal and maternal outcomes and proposes extreme βHCG MoM values as indicators of necessity of invasive testing during pregnancy. In fact, βHCG MoM < 0.2 or > 5 can be regarded as risk factors for adverse maternal or fetal outcomes irrespective of the presence of other abnormalities in the FTS results. A possible explanation for association between adverse outcomes and both low and high levels of βHCG is that these extreme values reflect inappropriate function of placenta.Our study has some limitations. We did not analyze the effects of socioeconomic status in this study. Although our study was conducted in a large group of pregnant women, the results should be confirmed in multicenter prospective studies. Finally, in our study, patients reported outcomes following a telephone call at 8 to 12 months post-delivery. This may not be reliable and potentially subject to recall bias.Supplementary Tables.


Photosynthetic capacity is usually affected by light intensity in the field. In this study, photosynthetic characteristics of four different Triticeae crops  were investigated based on chlorophyll fluorescence and the level of photosynthetic proteins under high light. Compared with wheat, three cereals  presented higher photochemical efficiency and heat dissipation under normal light and high light for 3 h, especially highland barley. In contrast, lower photoinhibition was observed in barley and highland barley relative to wheat and triticale. In addition, barley and highland barley showed a lower decline in D1 and higher increase in Lhcb6 than wheat and triticale under high light. Furthermore, compared with the control, the results obtained from PSII protein phosphorylation showed that the phosphorylation level of PSII reaction center proteins (D1 and D2) was higher in barley and highland barley than that of wheat and triticale. Therefore, we speculated that highland barley can effectively alleviate photodamages to photosynthetic apparatus by high photoprotective dissipation, strong phosphorylation of PSII reaction center proteins, and rapid PSII repair cycle under high light. In this process, carbon dioxide and water are converted into organic carbohydrates and oxygen is released . In high complex . Among trs (RCs) . HoweverThe growth and development of vascular plants are closely involved in photosynthesis; however, excessive light absorption by pigment molecules usually causes serious oxidative damage to photosynthetic apparatus . High liTriticum aestivum L.) is the most widely planted food crop and mainly grows in temperate regions in the world [Triticosecale sp.) is a cereal that was intentionally developed by crossing diploid rye and hexaploid wheat in order to combine the positive quality attributes of wheat with durability and hardiness of rye [Hordeum vulgare L.), as one of the earliest domesticated crops of Old World agriculture and the fourth most abundant cereal in the world, is widely used as animal feed, malt, or as a component of various health foods [Hordeum vulgare Linn. var. nudum Hook. f) has been known as hulless barley and the major staple food of Tibetans for generations [Triticeae, as a sub−category in Poaceae, is an important tribe in the grass family and contains many important agricultural crops such as wheat, triticale, rye, barley, and highland barley as well as a large number of wild species . Among the world . Octoplos of rye . Barley th foods . It has th foods . Highlanerations . Highlanerations . It is gerations . A previerations . Arenas−erations . In addierations . AlthougIt has been known that different plant species probably adopt different mechanisms in response to high light exposure. In this study, we investigated the adaptive responses of four different Triticeae crops  to high light by comparing the difference in chlorophyll fluorescence, gas exchange parameters, and the levels of photosynthetic proteins, PSII protein phosphorylation, and thylakoid membrane complexes. Our results suggested that highland barley possesses better photosynthetic capacity and photoprotective mechanisms relative to other three Triticeae crops under high light.Triticuma estivum L., Chinese Spring, CS), octoploid Triticale , barley , and highland barley  were presented in The differences in photosynthetic pigment contents among wheat (Pn), transpiration rate (Tr), and stomatal conductance (Gs) compared to other three Triticeae crops under normal and stress conditions. When compared with the control, intercellular CO2 concentration (Ci) significantly decreased in four Triticeae crops exposed to high light for 3 h, especially XZ31  was significantly higher in H30 and X19 as compared with CS and XZ31 under normal and stressful conditions  and state transition were determined under high light. Compared with CS, other three Triticeae crops  showed higher transitory increase in fluorescence when the far−red light was turned off under high light for 1 h and 3 h, especially X19 . In addiVO−P under high light , octoploid Triticale , barley , and highland barley  were disinfected with 2% sodium hypochlorite (NaClO), washed with deionized water, and germinated in a Petri−dish with wet filter paper for 48 h at 25 °C in the dark. The germinated seedlings were cultured in sterilized sand with 1/2 Hoagland nutrient solution in a climate chamber (25 ± 1 °C) under illumination with 250 μmol photons m−2 s−1 at the 16/8 h light/dark photoperiod for two weeks. Then, nine seedlings were subjected to high light (1200 μmol photons m−2 s−1) for 0 h (HL 0 h), 1 h (HL 1 h), and 3 h (HL 3 h) in the greenhouse. After the treatments, the second leaves from the top were used for the following measurements.Seeds of wheat (v/v) acetone and then measured with a UV−visible spectrophotometer  according to the previous method [Carotenoid and chlorophyll of fresh leaves were extracted with 80% (s method . UV−Vis s method .Pn), intercellular CO2 concentration (Ci), transpiration rate (Tr), and stomatal conductance (Gs) of the intact leaves (2nd leaf) were estimated with the GSF−3000 photosynthesis system . The measuring conditions were illumination of 1000 µmol photon m−2 s−1, 360 µmol mol−1 CO2 concentration, and 60−80% relative humidity at 25 °C. Net photosynthetic rate  was determined with a dual PAM−100 fluorometer  according to the method of Lin et al. [−2 s−1) for 0.5 s. The O, J, I, and P steps were the minimal fluorescence intensity when all the reaction centers (RCs) of PSII are open, the intensity at 2 ms, the intensity at 30 ms, and the maximal intensity when all the RCs of PSII are closed, respectively. To obtain VO−P curves, OJIP curves were standardized by O−P as described previously [The fast phase of Chl n et al. . To measeviously .p−benzoquinone , 0.2 M sucrose, 5 mM CaCl2, and 10 mM NaCl at 20 °C.A Clark−type electrode  was used to measure the oxygen−evolving activity of thylakoid membranes obtained from the leaves as the previous method . The ass−2 s−1 and 8000 μmol m−2 s−1, respectively. Then, the maximum efficiency of PSII photochemistry (Fv/Fm), effective PSII quantum yield (Y(II)), quantum yield of non−regulated energy dissipation (Y(NO)), and coefficient of photochemical quenching (qP) were imaged and calculated .Chlorophyll fluorescence was analyzed using an imaging PAM M−series chlorophyll fluorometer  based on our previous method . Prior tDual PAM−100 fluorometer  was used to analyze state transition and NPQ kinetics of the plants following established protocols . The samThe redox state of P700 was determined with a Dual PAM−100 fluorometer following the manufacturer’s instructions. After 30 min of adaptation in the dark, the P700 parameters including the photochemical quantum yield of PSI (ΦPSI), the reduction status of PSI receptor side (ΦNA), and the oxidation status of PSI donor side (ΦNA) were calculated as described previously .−2 s−1 for 4 h followed by low light (10 µmol photons m−2 s−1) for 24 h. The Dual PAM−100 fluorometer was used to determine Fv/Fm at a regular time. To analyze D1 damage during photoinhibition, detection of D1 protein was performed by immunoblotting with specific D1 antibody.The sensitivity of PSII to high light was determined based on the changes in Fv/Fm in the intact leaves of the control and 3 h of HL plants with or without lincomycin . To measThylakoid proteins were extracted from the leaves in the presence of 10 mM NaF under dim light , and thew/v) n−dodecyl−β−D−maltoside was used to solubilize thylakoid membranes (20 μg Chl) for 10 min in the dark on ice. After centrifugation  at 4 °C, BN−PAGE was done using a gradient of 5–12.5% acrylamide in the separation gel and a gradually increasing voltage (75–200 V) for 4 h at 4 °C.Blue native−polyacrylamide gel electrophoresis (BN−PAGE) was carried out as previously described by Chen et al. . 1% . All results were expressed as the mean ± standard deviation (SD) of at least three independent replicates. Different lowercase letters in the figures indicate to be significant differences among treatments when In the present study, we found that four Triticeae crops  showed obviously different photosynthetic acclimation to high light. Our results indicated that highland barley and wheat exhibited the highest and lowest photosynthetic capacity and light energy dissipation under high light, respectively. In highland barley, the effective photosynthetic acclimation to high light is mainly involved in the rapid repair cycle of PSII and strong phosphorylation of PSII RC proteins. Taken together, we propose that highland barley possesses better photoprotective mechanisms under environmental stresses and should be more suitable for planting in some regions with unfavorable environments.


There is an error in in vitro culture.”No drug” and DMSO were the negative controls. Pooled data obtained from 3 experiments per drug are displayed.Methylene blue, pyronaridine, and praziquantel (positive control) were exposed to the respective concentrations for 7 days (* for 24 h) followed by a 7 days drug wash-out


Studies from the past two decades have demonstrated convincingly that cells are able to sense the mechanical properties of their surroundings. Cells make major decisions in response to this mechanosensation, including decisions regarding cell migration, proliferation, survival, and differentiation. The vast majority of these studies have focused on the cellular mechanoresponse to changing substrate stiffness (or elastic modulus) and have been conducted on purely elastic substrates. In contrast, most soft tissues in the human body exhibit viscoelastic behavior; that is, they generate responsive force proportional to both the magnitude and rate of strain. While several recent studies have demonstrated that viscous effects of an underlying substrate affect cellular mechanoresponse, there is not a straightforward experimental method to probe this, particularly for investigators with little background in biomaterial fabrication. In the current work, we demonstrate that polymers comprised of differing polydimethylsiloxane (PDMS) formulations can be generated that allow for control over both the strain-dependent storage modulus and the strain rate-dependent loss modulus. These substrates requires no background in biomaterial fabrication to fabricate, are shelf-stable, and exhibit repeatable mechanical properties. Here we demonstrate that these substrates are biocompatible and exhibit similar protein adsorption characteristics regardless of mechanical properties. Finally, we develop a set of empirical equations that predicts the storage and loss modulus for a given blend of PDMS formulations, allowing users to tailor substrate mechanical properties to their specific needs. We have generated novel formulations of polydimethyl siloxane with varying viscoelastic properties that can be used to study cellular response. We present equations that can be used to predict the storage and loss moduli of these polymers. Indeed, studies have shown that this is the case in many pathologies: tumor stiffness correlates with aggressiveness in cancer,7–10 liver stiffness correlates with liver dysfunction in patients with chronic liver disease,11–13 and increased stiffness in kidneys predict chronic kidney disease earlier than other markers.14–16 These findings suggest a significant clinical implication of cellular response to stiffness.The field of mechanobiology has become a burgeoning field of research. Discoveries that showed that the stiffness (elastic modulus) of substrates can drive cell migration, cell proliferation, and cell differentiation17–19 and the majority of mechanobiology research has focused only on the elastic component. Viscoelastic tissues have viscous properties, meaning that the tissue will continue to deform over time for a given load , and elastic properties, where the tissue strain is only proportional to the force applied . Viscoelastic materials can be quantified by the loss modulus, which represents the viscous component, and the storage modulus, which represents the elastic component. Most tissues in the human body exhibit viscoelastic behavior, with a loss modulus roughly 10 percent of the tissue's storage modulus.20 Most soft tissues in the body demonstrate viscoelastic properties that are subcategorized as “strain-stiffening materials”, where as the material stretches, it becomes effectively stiffer.17–19,21 Loss moduli have been quantified for various tissues, including lung (∼600 Pa),22 brain (∼1 kPa),23 cornea (∼12 kPa),24 and liver (∼17 kPa),25 suggesting that the loss modulus of soft tissues varies greatly throughout the body.However, there is a significant concern with drawing correlative conclusions from these studies: soft tissue in the human body is viscoelastic,26 At early stages of fibrotic diseases, where there is significant assembly of de novo extracellular matrix, the tissue does not exhibit an increased stiffness, or storage modulus, but does exhibit a significant change in loss modulus.26 There has been a growing interest in the role of the loss modulus in modulating cellular response: several studies have probed the effects of altered creep or viscoelasticity in hydrogels and demonstrated that an altered viscous component can affect cell size, focal adhesion formation, and proliferation.27–30While the majority of work in the field of mechanobiology has only probed the effects of the elastic modulus on cellular response, studies have shown that increases in tissue elastic modulus occurs late in disease progression.31 Polyacrylamide gels have a loss modulus near zero,32 and as such, these studies have examined the cellular response to an almost purely elastic substrate. These gels can results in substrates with elastic moduli ranging from the order of 0.5 Pa to 300 kPa,33,34 which does not span the full range of biologically relevant stiffnesses.35 Cellular mechanoresponse studies have also utilized PDMS, which is capable of generating surfaces with a much larger range of elastic moduli, ranging from 1 kPa to 1.5 MPa.36The majority of work investigating cellular responses to substrate mechanics have utilized polyacrylamide gels, where the elastic modulus can be regulated by changing the concentration of the bis-acrylamide crosslinker.27,37 The loss modulus of polyacrylamide gels can also be altered by fine-tuning of the ratio of acrylamide to bis-acrylamide;29 however, this approach results in small changes in the loss modulus (1 to 130 Pa) and can only be utilized for low storage modulus (4 kPa) gels. As such, there is not a currently available approach that can span the ranges of both physiologically relevant storage moduli and loss moduli.While most cellular mechanoresponse work has focused on purely elastic surfaces, a few experimental methodologies have been developed for altering the loss moduli of cell substrates; these have primarily focused on modifying polyacrylamide gels to increase the loss modulus. One approach is to add long linear polyacrylamide chains, which are trapped within the polyacrylamide gel. Substrates generated using this approach exhibit storage moduli in the range of 1 kPa to 6 kPa and loss moduli in the range of 1 to 500 Pa.While experimental methodologies exist to vary either the storage modulus or the loss modulus of cellular substrates, there is not an established system to vary both. Here we present a system that allows for control of both, with little to no experience with generating polymer substrates. We have generated surfaces comprised of two formulations of PDMS, namely Sylgard 184 and Sylgard 527, and demonstrate that by changing the base : crosslinker ratio of Sylgard 184 and the ratio of Sylgard 184 to Sylgard 527, we can generate surfaces with a wide range of both storage and loss moduli. These surfaces adsorb protein at similar rates, have comparable biocompatibility, and are easily fabricated in research labs without specific expertise in biomaterials. We have developed empirical equations for the storage and loss moduli as a function of Sylgard 184 base : crosslinker ratio and Sylgard 184 : Sylgard 527 ratio, which will allow researchers to formulate substrates to meet specific experimental needs. PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>CH2 bond randomly along the polymer and form a network of varying chain lengths of PDMS. Previous studies have shown that the elastic modulus of PDMS can be altered by changing either the base : crosslinker ratio of Sylgard 184 PDMS38 or by changing the ratio of Sylgard 184 : Sylgard 527.36 To the best of our knowledge, no studies have reported the effects of altering the base : crosslinker ratio nor the Sylgard 184 : Sylgard 527 ratio on the loss modulus of substrates, nor have studies investigated the combinatorial effect of altering both the base : crosslinker and 184 : 527 ratios. To investigate the combinatorial effects of these two on surface mechanical properties, we fabricated an array of PDMS blends. The base : crosslinker ratio of Sylgard 184 was varied from 5 : 1 to 30 : 1 (w : w), and the ratio of 184 : 527 was varied from 1 : 5 to 1 : 30 (w : w). Each formulation uses the notation (X : 1) : Y in which X : 1 is the base : crosslinker ratio of Sylgard 184, and Y represents the ratio of Sylgard 527 to the Sylgard 184. Sylgard 184 base and crosslinker, and Sylgard 527 base and crosslinker were mixed separately and then degassed under vacuum. The two mixtures were then combined at the appropriate ratio, degassed again, and then cured at 110 °C for 18 h. Note that for all formulations, Sylgard 527 was mixed at a 1 : 1 (w : w) ratio of base : crosslinker as recommended by the manufacturer.PDMS polymers are generated by mixing a base and crosslinker at various ratios and curing at 110 °C. This can be done using either Sylgard 184 or Sylgard 527; both share the same basic chemical structure, with the major difference between the two polymers being the chain length of each polymer: Sylgard 184 has 184 repeated mer units while Sylgard 527 has 527 repeated mer units. While the exact chemical structures of the curing agents in both Sylgard 184 and Sylgard 527 are not known due to the proprietary nature of the polymer, we can hypothesize that polymer chains of PDMS having either 184 or 527 mer units as the base interact with dimethylvinylsiloxy-terminated chains of the same length. Multiple catalyst and filler chemicals interact with the Sivia parallel plate rheometry. Samples with a 20 mm diameter and a height of 5 mm were fabricated from each formulation, with a minimum of 3 samples per formulation. Samples were compressed with a 3 N axial force, and were subjected to 5% oscillating rotational strain over a range of oscillating frequencies from 1 to 100 rad s−1. The resulting amplitude and phase shift of the force was measured and converted to values for G′ and G′′ using the TA Instruments Trios software package. G′ and G′′ for formulations with: a 5 : 1 base : crosslinker 184 ratio . Taken together, these data indicate that we are able to fabricate PDMS formulations with storage moduli ranging from 3 to 130 kPa and loss moduli ranging from 1500 to 3000 Pa, which spans a much broader range of physiological and pathological values than other existing methods. Mean values for each blend are shown in After fabricating the substrates, we quantified the storage and loss moduli 84 ratio ; a 10 : 84 ratio ; a 20 : 84 ratio ; and a 384 ratio . All valin vitro cellular assays, cells cannot typically attach directly to a surface. Instead, they must bind to extracellular matrix proteins that have been adsorbed onto the surface. In order to ensure that protein adsorption was consistent across all formulations, we microcontact-printed substrates with a stamp coated in 200 ng mL−1 of the extracellular matrix protein fibronectin that had been labeled with rhodamine isothiocyanate (RITC). Microcontact printing is widely used for facilitating cell attachment onto PDMS substrates, and as such, we utilized this technique as we have previously done.39 Coated stamps were brought into conformal contact with UV-treated samples for 30 seconds before peeling. Four substrate formulations that span the range of material properties were selected. Following microcontact printing, substrates were rinsed in PBS, and adsorbed protein was recovered by treating samples with 0.05% of the proteolytic enzyme trypsin, which cleaved attached protein, for 1 hour. Recovered solutions were analyzed for red fluorescence via 555 nm absorbance on a spectrophotometer  and fluorescently labeled with a solution of 2 μM calcein AM and 4 μM ethidium homodimer III (EthD-III). Calcein AM is plasma membrane permeable and will enter both living and dead cells passively, but only gets activated when cleaved by intracellular esterases. EthD-III labels DNA but is plasma membrane impermeable; as such, only dead cells with compromised plasma membranes can uptake the dye. We imaged labeled cells on an Axiovert inverted fluorescence microscope (Zeiss). Representative images from each of 6 formulations selected from the spectrum of mechanical properties are shown in G′ and the ratio of Sylgard 184 to Sylgard 527  and loss modulus (viscous component) of an underlying substrate, and that cellular mechanoresponse to these mechanical inputs can dictate cellular proliferation, migration, differentiation, and apoptosis. Studies have also demonstrated that disrupted mechanoresponses can drive disease progression in a host of pathologies. While studies have highlighted the importance of these roles, there are limited experimental tools to generate substrates with varied storage and loss moduli that span physiologically and pathologically relevant values. Here we demonstrate the generation of substrates with varying loss and storage moduli that will allow researchers to assess the independent effects of these two mechanical properties on cellular response. The substrates are shelf stable, biocompatible, and can be fabricated easily by researchers with limited biomaterial fabrication experience. Furthermore, we have developed a set of empirical equations that allows a user to tailor the mechanical properties to their specific needs. As such, this provides a vital new resource in future studies of the effects of storage modulus and loss modulus on cellular responses.As discussed above, both Sylgard 184 and Sylgard 527 have the same basic chemical structure, but with varying numbers of the repeated units. We hypothesize that it is the mixture of the two bases with differing lengths that generate the unique loss modulus properties of the blends. As the primary resistance to deformation in a polymer is due to chain entanglement, it stands to reason that different concentrations of bases with two distinct lengths will yield different energy loss, and that changing the density of crosslinks will further alter the mechanical properties.vice versa. This will allow for future studies in which the effects of storage modulus and loss modulus can be independently isolated. For example, it has been established that human mesenchymal stem cells and hepatic stellate cells both differentiate into osteocytes40 or myofibroblasts41 when exposed to substrates of increased substrate stiffness. However, a recent study has shown that osteocyte differentiation increased with increased loss modulus,29 while another study showed myofibroblast differentiation decreased with increased loss modulus.27 It is possible that these cell lines respond differently; however, these studies also used substrates of differing storage modulus, and as such, the independent effects of each variable cannot be isolated. Systems to independently tune the storage and loss modulus, such as the system presented here, will become critical to more deeply explore the effects of viscoelastic properties on cellular responses .A major benefit of the presented work is that the generated PDMS substrates can be tuned such that a pair of substrates is generated with comparable storage moduli but distinct loss moduli, or 2 under standard culture conditions.ASC52-telo, hTERT immortalized adipose derived mesenchymal stem cells were purchased from American Type Culture Collection and cultured at 37 °C and 5% COTo make the different formulations of PDMS, Sylgard 184  was thoroughly mixed at a ratio of 5 : 1, 10 : 1, 20 : 1, or 30 : 1 (w/w) base to crosslinker. This mixture was then degassed under vacuum. Separately, Sylgard 527  was blended at a ratio of 1 : 1 (w : w) base to crosslinker and degassed. After the Sylgard 184 and Sylgard 527 polymers were both mixed thoroughly, Sylgard 184 was mixed with Sylgard 527 at a ratio of 1 : 5, 1 : 10, 1 : 15, 1 : 20, or 1 : 30. Final formulations were cured for 18 hours at 110 °C.For cell experiments, prior to curing, 1 mL of the final PDMS blend was pipetted onto 25 mm diameter coverslips that had been cleaned with 70% ethanol. The coverslip was then placed on a Laurell spin processor (Laurell Instruments) using the following parameters: (1) 900 rpm for 10 seconds, (2) 500 rpm for 10 seconds, (3) 300 rpm for 10 seconds, and finally (4) 100 rpm for 15 seconds to generate a coating layer of approximately 100 μm. Coverslips were then cured at 110 °C for 18 hours.−1 with a 5% strain applied to the samples.Parallel plate rheometry was performed using a Discovery HR-3 Hybrid rheometer (TA Instruments) on the PDMS formulations. Samples were prepared as described above and cured in a 65 mL aluminum weighing dish then cut into cylinders 20 mm in diameter with a height of 5 mm. Samples were mounted into the parallel plate rheometer and compressed with an axial force of 3 N. Data was collected at room temperature over a frequency range of strain oscillations from 1 to 100 rad s−1 rhodamine-labeled fibronectin (Cytoskeleton Inc) 1 : 20 with 200 ng μL−1 fibronectin (R&D Systems). Spin-coated substrates were cleaned in 70% ethanol and then exposed to UV ozone for 10 minutes with a UV ozone cleaner (Novascan) in order to generate reactive oxygen groups to bind protein. Each substrate was then microcontact printed with 100 μL of the labeled fibronectin using a stamp cut to be 1 cm2. After microcontact printing, the substrates were placed in 1× PBS for 30 minutes at 37 °C. After 30 minutes, protein was released from the surface with 2 mL of 0.05% trypsin and incubated at 37 °C for another 30 minutes. Absorbance of the collected samples was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher).Protein adsorption onto the surface of the different PDMS formulations was tested by first preparing rhodamine-labeled fibronectin by diluting 50 μg mL−1 fibronectin for 1 h. 40 000 ASC52-telo human adipose-derived mesenchymal stems cells were seeded onto the substrates. Cells were incubated for 72 hours on the substrates and were then labeled using a cell viability assay (Biotium) per manufacturer's instructions. Briefly, a staining solution of 2 μM calcein AM and 4 μM EthD-III were prepared immediately before imaging. After rinsing twice with 1× PBS, samples were incubated at room temperature in the dark for 30 minutes. After labeling, cells were rinsed again with 1× PBS before imaging.Substrates for cell viability experiments were prepared as describe above, and rinsed in isopropanol. Substrates were then exposed to UV ozone for 10 minutes as described above, before being incubated with 200 ng μLvia Zen Blue software (Zeiss). Red cells per image were counted manually and averaged over 25 images.Fluorescence images of cells from the live/dead assay were imaged on an Axiovert inverted fluorescence microscope (Zeiss). Images were acquired using a 20× objective There are no conflicts to declare.RA-011-D1RA03548G-s001


Hydrophobic hollow silica particles are desirable for several applications such as hydrophobic coatings, thermal insulation, and thermally resistant insulative paints. However, converting hydrophilic particles into hydrophobic particles without compromising their structural integrity is challenging. In this work, we present a low cost strategy to modify the surface of hollow silica particles from hydrophilic to hydrophobic without compromising their structural integrity. A low cost strategy is developed for hydrophobization of hollow silica particles without compromising with their structural integrity. Although these solution-phase strategies are suitable for solid silica particles, they are difficult to apply to hollow silica particles because solution-phase coating of hydrophobic silanes requires mixing and stirring for several hours. Solid particles can withstand this process, however, we noticed that a fraction of hollow silica particles breaks . Therefore, there is a need for an approach for making a hydrophobic silane coating on hollow silica particles without compromising their shell integrity while keeping the costs lower.Additionally, solution-phase grafting of hydrophobic silanes is an expensive process because it wastes large amounts of solvents  and very low vacuum, and is applicable to both flat surfaces and particle powders. This approach is the first vapor-based approach to modify the surface of hollow silica particles from hydrophilic to hydrophobic.Though, a similar technique, chemical vapor deposition (CVD) of silanes has been investigated before for making thin films on flat surfaces,3 in 100 mL of water), and styrene were purchased from Sigma Aldrich. 2,2′-Azobis(2-methylpropionamidine)dihydrochloride was purchased from ACROS-Organics – Fisher Scientific. Isopropanol was purchased from Fisher Scientific.Tetraethyl orthosilicate (TEOS), trimethoxymethylsilane (TMMS), triethoxymethylsilane (TEMS), trimethoxyoctylsilane (TMOS), ammonium hydroxide (28–30% of NHStyrene (4.54 g) was added to the water (200 mL). The reaction mixture was allowed to heat for 30 minutes at 68 °C. Afterward, 2,2′-azobis(2-methylpropionamidine)dihydrochloride (320 mg) was added to this stirring (500 rpm) reaction mixture, and the reaction continued for approximately 12 hours. The final concentration of styrene and 2,2′-azobis(2-methylpropionamidine)dihydrochloride was 0.21 M and 5.8 mM, respectively. Monodisperse particles approximately of 315 nm in diameter were obtained. Fig. S1 ESI shows th3 isopropanol, 40 cm3 water) solution. 2 mmol of tetraethyl orthosilicate was added to make shells around the polystyrene cores. Ammonium hydroxide solution  was used as a catalyst. The core–shell particles were heated at 550 °C for 4 hours to remove the polystyrene core. To scale up the production of hollow particles, the synthesis was proportionately increased. Fig. S2 (ESI0.5 g of polystyrene particles were mixed in an isopropanol/water (160 cmg. S2 ESI shows thScanning Electron Microscopy (SEM) imaging was performed by using a Merlin 200 microscope. Both polystyrene and hollow particles samples were deposited on a silicon wafer, which was in turn attached to the SEM stubs by using carbon tape, before performing the SEM imaging. Carbon tape was used in order to minimize the charging on particle surface.3 of hydrophobic silane and the other 15 cm3 of hollow silica particles. The flask with the hydrophobic silane was heated to 100 °C to vaporize the silane. The flask with the hollow particles was heated to ≈50 °C to enhance the conjugation process. We shook the samples every 10 minutes to make sure that most of the particles were coated. Other similar setups can be used for vapor formation, deposition, and shaking.Thermal conductivity measurements were performed using a Transient Plane Source (TPS 2500 S) instrument with manufacturer provided sample holder and sensor C5465. All measurements were done on loose particle powders without additional compacting.Moisture absorption/desorption experiments were performed using a dual vapor gravimetric sorption analyzer. Samples were dried overnight at 50 °C and placed in the measurement chamber of the instrument. The moisture adsorption and desorption isotherms were generated at room temperature conditions (25 °C and 1.0 atm). The initial mass of the samples was about 2.0 mg. Each data point  represenWe tested three silanes: triethoxymethylsilane (TEMS), trimethoxymethylsilane (TMMS), and trimethoxyoctylsilane (TMOS). TEMS and TMOS didn't provide any coating. 3CH2O− in TEMS has higher −ve charge on ‘O’ due to electron donating inductive effect of –CH2CH3. The higher −ve charge on ‘O’ makes the anion unstable and thus slows the conjugation with ‘–OH’ groups on particle surface. Additionally, the higher −ve charge on the ‘O’ atom in CH3CH2O–Si, makes the ‘Si’ atom less electronegative, i.e., less positively charged.27 This lowered electronegativity makes it more difficult for the nucleophilic attack of ‘–OH’ groups attached to hollow particles surface on the ‘Si’ atom. Thus, lowered electronegativity of ‘Si’ and instability of leaving anion CH3CH2O− makes the TEMS unsuitable for our vapor-phase coating approach. Similarly, in case of TMOS, the increased electron donating inductive effect of the octyl chain makes the ‘Si’ atom less electronegative and thus difficult to attach to the –OH groups of the hollow silica particles, making TMOS also unsuitable for our purposes. Because of the lesser electron donating inductive effect of the –CH3 group, the negative charge on the ‘O’ atom in the CH3O− anion is lesser than its counterpart (CH3CH2O−) in TEMS and TMOS, which makes it a stable leaving group compared to CH3CH2O−. Similarly, the lesser electron donating inductive effect of the –CH3 group directly attached to the ‘Si’ atom and lesser −ve charge on ‘O’ atom of CH3O− anion, makes the ‘Si’ atom more electronegative and more susceptible to nucleophilic attack of –OH groups attached to particle surface, thus making the conjugation process possible under normal conditions.28The inability of TEMS to produce a hydrophobic coating results from its higher stability, which makes it difficult for –OH groups on hollow particles to conjugate with the TEMS molecules. Because leaving anion CHWe observed that conjugation of hydrophobic silane was incomplete when the as synthesized hollow silica particles were used. This occurred because heat treatment at 550 °C resulted in removal of most of the –OH groups from the surface of the particles, which indirectly decreases the number of silane molecules that can attach to the particles' surfaces. In contrast, leaving the particles overnight at ambient conditions results in regeneration of –OH groups on the surface of the particles, which leads to efficient conjugation of TMMS molecules on the surfaces of the particles.To investigate if the particles retained their structural integrity after coating with hydrophobic silanes, we gathered SEM images that showed that both uncoated  and coatTherefore, we conclude that our vapor-based approach is superior than the solution-based approach in preserving the structural integrity of the hollow silica particles.To investigate the effectiveness of our vapor-based hydrophobization, we added coated and uncoated particles to flasks with water. Coated particles stayed on the surface of water for at least a month , because−1) with missing shoulder, and other Si–O–Si peaks at 778 cm−1 and 475 cm−1 was observed. Similar to the previous studies,29 we also couldn't see a distinct peak of –CH3 group because of very low concentration of –CH3 groups compared to ‘Si–O–Si’ network.To further investigate the formation of hydrophobic silane deposition, we performed infrared spectroscopy studies on uncoated and coated samples. Infrared spectra of coated particles showed a subtle difference from uncoated particles  conditions and evaluated the amount of adsorbed water vapors. 30,31 of glass slides coated with hydrophobic silane modified hollow particles and unmodified hollow particles. Glass slide with hydrophobic particles showed a water contact angle of 147.6° ± 2.7°, while glass slide coated with unmodified particles had a contact angle of 25.6–3.1° . We assume, almost no effect on thermal conductivity arises from very thin nature of coating (monolayer of TMMS molecules). Therefore, our coating process does not interfere with the required qualities such as thermal conductivity and structural stability while it lowers the moisture adsorption by particles.We also measured the thermal conductivity of coated and uncoated particles. The coated particles and uncoated particles showed almost similar thermal conductivity  that are prone to structural disintegration when coated using solution-phase processes. Additionally, this approach is cost effective since it does not include wastage of solvents. The process can be used for large scale and continuous processes in which silane vapors are introduced into a chamber having material to be coated continually. We anticipate that this study will entice the research community for vapor phase deposition of different coatings on materials that are not suitable for solution-phase coatings.There are no conflicts to declare.RA-010-D0RA06114J-s001


The dorsal DG had significantly more reelin within the subgranular zone compared to the ventral DG (p < 0.01). There was no difference in reelin between nicotine (n = 5) and controls (n = 5). IHH exposed piglets (n = 10) had significantly lowered reelin in the CA1 (p = 0.05), specifically the stratum pyramidale (p = 0.04) and the hippocampal fissure (p = 0.02), compared to their controls (n = 7); the duration of IHH had no effect. No exposure was associated with an alteration in DG morphology. This study shows that postnatal IHH exposure decreased reelin expression in the developing piglet hippocampal CA1, suggesting that IHH may result in altered neuronal migration.This study evaluated the expression of reelin, an extracellular protein involved in lamination and migration of neurons, in the hippocampus of young piglets, and quantified to examine the following: (i) baseline levels within layers of the hippocampus and dentate gyrus (DG); (ii) differences between ventral and dorsal hippocampi; and (iii) changes attributable to postnatal exposure to continuous nicotine for 12 days, or intermittent hypercapnic hypoxia (IHH), with further analysis according to duration of IHH (1 vs 4 days). Additionally, we analysed whether any exposure altered DG morphology and whether it is related to altered reelin expression. Reelin was visualised via immunohistochemistry, and the number of positive reelin cells/mmThe online version contains supplementary material available at 10.1007/s12640-022-00598-0. The migration and formation of granule cells in the brain is a process that is vulnerable to insults during early development, whether these are experienced prenatally  and Cornu ammonis (CA) 1–4 . Reelin, an extracellular matrix protein secreted by Cajal-Retzius (CR) cells, is critically involved in the cellular organisation and development of the hippocampus and DG  . Piglets were alternately subjected to 6 min of either exposure for a total of 48 min per day. Controls were placed in the same experimental conditions but only exposed to air for a 1- or 4-day period  and another of repeated exposure over 4 consecutive days (4D IHH). The methodology of the live piglet work was previously detailed in Waters and Tinworth . BrieflyRegarding nicotine exposure, this was previously detailed in Machaalani et al. . PigletsAll piglets were euthanized at the age of 13–14 days, through an overdose of pentobarbital (200 mg/kg piglet body weight), the whole brain removed, and immersion fixed in 10% neutral buffered formalin for 14 days. Regions of interest from the left hemisphere were separated and sectioned into 4-mm slices and returned to the 10% neutral buffered formalin for another 5 days, before being processed to paraffin , with sections mounted onto salinized microscopic slides, dried overnight at 37 °C, and stored at room temperature for a minimum of 1 week before staining.Immunohistochemistry (IHC) was applied using a standard protocol from our laboratory  for 14 min. Endogenous peroxidase activity was blocked by incubating sections in 50% methanol, 50% phosphate buffered saline (PBS), and 3% hydrogen peroxide (H202) for 20 min. To avoid non-specific staining, sections were blocked using 10% normal horse serum (NHS) for 30 min and then incubated with the primary antibody reelin , Millipore, 1:1000 dilution in 1% NHS) overnight. Sections were washed with PBS and incubated with biotinylated secondary anti-rabbit/anti-mouse IgG made in horse  for 45 min and then with avidin–biotin complex  for 45 min. Reaction was visualised via incubation with 3,3’ diaminobenzidine tetrachloride  for a maximum of 8 min, and counterstained with Harris Haematoxylin. Sections were then dehydrated through graded ethanol to xylene, mounted and cover slipped with dibutylphthalate polystyrene xylene (DPX).All steps were conducted at room temperature unless otherwise indicated. Briefly, after de-paraffinisation in xylene and rehydration through a graded series of ethanol to distilled HAll hippocampal sections were captured at × 20 using a slide scanner  and a single section per case was imaged and quantified using the supporting software (Olympus OlyVIA 3.2).After sub-group analysis to examine for differences in reelin expression in ventral and dorsal hippocampal structures Fig. A, the fo2 per region analysed. This analysis was undertaken using a set box and quantifying 1–2 fields per layer, rather than the whole layer, based on a pilot study on two tissue sections which showed within 20% variability amongst the fields of the whole hippocampal formation which equated to 10–15 boxes pending on the size of the formation from one piglet to another.The quantitative method we applied was the one we applied recently in our human study determining the effects of aging on reelin in the hippocampus , subgranular zone (SGZ), granule cell layer (GCL), inner molecular layer (IML), and the outer molecular layer (OML). Positive reelin cells were counted within their respective layers. The ventral DG was counted at two separate locations along the straight limb and averaged. Adjacent to the SGZ of the DG, the CA4/Hilus was quantified separately. A box was drawn directly underneath the SGZ to determine if there is any significance with reelin expression in neighbouring layers and overall neurogenesis of the DG.The straight limb of the ventral DG Fig. A was ide2 was drawn including the layers of stratum oriens (SO), stratum pyramidale (SP), stratum radiatum (SR), stratum lacunosum-moleculare (SLM), and hippocampal fissure (HF). Positive reelin cells were counted within their respective layers.Next, the CA1 was differentiated by its cell type and a box with an area of 0.5mmn = 3 saline controls, n = 3 nicotine exposed) had both the dorsal and ventral hippocampus within the same section. In these cases, the DG and CA4/Hilus were quantified except that due to the differing DG structures and size, only one boxed area of 0.3mm2 of the dorsal DG was quantified.A total of 6 cases  for statistical analyses. All values are expressed as mean ± standard error of the mean (SEM). Student’s t-test was used when comparing reelin expression between the paired groups of dorsal to ventral hippocampi, saline and nicotine, and IHH and air . Subgroup analysis to compare the individual effects of 1D IHH and 4D IHH to their corresponding control groups was performed via ANOVA using post hoc Tukey. A p-value ≤ 0.05 was considered statistically significant.Raw counts and areas were collated in Microsoft Excel, converted to positive reelin expression/mmp = 0.04, Table A total of 27 piglets were included in this dataset and belonged to one of the 6 exposure groups. The piglets were derived from a total of 18 litters all mixed; thus, each exposure group was made up of a minimum of 2 litters and maximum of 5. All piglet characteristics are reported in Table Reelin-positive cells were identified as having brown cytoplasmic staining and were seen throughout all hippocampal layers and DG Fig. A–E. CellWithin the dorsal DG, distinct layers of the SGZ, GCL, IML, and OML were visible Fig. B. Reelin2) and internal (3.4 ± 3.4 positive reelin cells/mm2) limb in the control groups (n = 7), reelin expression was greater in the external limb (p = 0.004). None of the morphological features reported in human infant DG structures was observed in the ventral DG of any piglet sections in any treatment groups, whereas GCD was seen in the dorsal DG of 2 piglets: one each of a nicotine and saline group   and their respective controls (n = 3), so the groups were combined for regional analyses. Reelin expression in the ventral DG was highest in the GCL followed by the SGZ  expression of reelin in the SGZ is higher in the dorsal than the ventral DG, (ii) nicotine exposure had no significant effect on reelin expression in the hippocampus and DG, (iii) IHH exposure decreased reelin expression in the SP and HF layers of CA1, and with (iv) no additional impact on reelin expression due to duration of IHH exposure.Consistent with published literature, reelin was expressed in all layers of the CA1 and DG, and the distribution and cellular expression are similar to those seen by Ábrahám et al.  in the aThere do appear to be species differences in expression amongst the layers. We reported that reelin expression in the SP of the human was minimal  projects fibers through the perforant path and these are crossed through the HF Ramón . StudiesThis is the first study to report on regional reelin expression in the young piglet hippocampal CA1 and DG. We found reelin expressing cells in all layers of CA1 and DG, with greater expression in the dorsal than ventral regions. While nicotine exposure did not affect reelin expression, IHH was associated with decreased reelin expression in the SP and HF layers of CA1, independent of the duration of IHH exposure. We postulate that reduced reelin expression after hypercapnic hypoxic exposures could translate to reduced cell migration.Supplementary file1 (DOCX 288 kb)Below is the link to the electronic supplementary material.