Dispersion of APD was assessed in each sample by pacing at a cycle length of 1,000 ms, followed by a premature beat with a coupling interval of 400 ms. Vulnerability to re-entry was assessed with an aggressive pacing protocol with pacing cycle lengths decreasing from 450 to 250 ms in 25 ms intervals for normal tissue and 300–150 ms for remodeled tissue. Simulated fibrosis at smaller spatial scales tended to lengthen APD, increase APD dispersion, and increase vulnerability to sustained re-entry relative to fibrosis at larger spatial scales. This study shows that when fibrosis is represented by smoothly varying tissue diffusion, the spatial scale of fibrosis has important effects on both dispersion of recovery and vulnerability to re-entry.Fibrosis in atrial tissue can act as a substrate for persistent atrial fibrillation, and can be focal or diffuse. Regions of fibrosis are associated with slowed or blocked conduction, and several approaches have been used to model these effects. In this study a computational model of 2D atrial tissue was used to investigate how the spatial scale of regions of simulated fibrosis influenced the dispersion of action potential duration (APD) and vulnerability to re-entry in simulated normal human atrial tissue, and human tissue that has undergone remodeling as a result of persistent atrial fibrillation. Electrical activity was simulated in a 10 × 10 cm square 2D domain, with a spatially varying diffusion coefficient as described below. Cellular electrophysiology was represented by the Courtemanche model for human atrial cells, with the model parameters set for normal and remodeled cells. The effect of fibrosis was modeled with a smoothly varying diffusion coefficient, obtained from sampling a Gaussian random field (GRF) with length scales of between 1.25 and 10.0 mm. Twenty samples were drawn from each field, and used to allocate a value of diffusion coefficient between 0.05 and 0.2 mm Although atrial fibrillation (AF) is a common cardiac arrhythmia, the mechanisms that initiate and sustain it are complex and remain incompletely understood. Circulating waves of electrical activation and recovery, often called re-entrant waves or rotors, are believed to sustain AF Nattel, . The anaFibrosis is hard to study in experimental preparations because it is difficult to control the extent of fibrosis and the size of individual lesions, and so computational models of cardiac cell and tissue electrophysiology have been used to examine how regions of simulated fibrosis affect activation and recovery in cardiac tissue, as well as vulnerability to arrhythmias  with exponential covariance, which is specified by a mean, a variance, and a correlation length. In 2-dimensions (2D), a GRF can be sampled to produce a series of random fields. In each sample, the values of the field will be correlated in both principal directions, and will be normally distributed. However the configuration of fluctuations in the field will vary from sample to sample. This approach has been used in models of groundwater flow with varying hydraulic conductivity , vulnerability to re-entry, and dynamics of re-entry.Electrical activation and recovery were simulated in a 2D sheet of atrial tissue, to enable the effect of cell and tissue electrophysiology to be examined without the additional complexity of anatomical structure. To represent the effect of fibrosis, the diffusion coefficient was set to vary smoothly from a low value representing regions of fibrosis, up to higher levels representing normal excitable tissue. The length scale of fluctuations in diffusion coefficient was varied by taking samples from GRFs with different correlation lengths. The effects of heterogeneities in diffusion at different length scales were examined by simulating normal beats during pacing at a steady cycle length, a single premature stimulus, vulnerability to re-entry during rapid decremental pacing, and behavior of an imposed spiral wave. These simulations were run using model parameters set to represent normal human atrial cells, and cells that have undergone remodeling as a consequence of persistent AF.+]i and [K+]i were fixed at their default initial values of 11.17 and 139.00 mM, respectively. The CRN model was implemented in C, based on code automatically generated from the CellML repository (www.cellml.org).Cardiac cellular electrophysiology was modeled using the Courtemanche-Ramirez-Nattel (CRN) model of human atrial cells , IK, ur was decreased by 49%, and the maximum conductance of IK1 was increased by 110%. These changes are based on those described in a previous study  can represent a smoothly varying field in space, and are composed of a mean and a covariance. The covariance function describes correlations between the value of the field at a single point and within its neighborhood. A squared exponential covariance function includes a correlation length δ, which can be used to vary the length scale of fluctuations in the field.2ms−1, respectively. The value of the diffusion coefficient was capped at 0.2 mm2ms−1.Stationary GRFs with mean of 0, variance of 1.0, and an exponential covariance function were generated with length scales of 1.25, 2.50, 5.0, and 10.0 mm using the method of circulant embedding, which is detailed in Kroese and Botev . Samples2ms−1 were designated areas of fibrosis, and were set to be coupled but inexcitable, with a diffusion coefficient of 0.05 mm2ms−1. Inexcitability was imposed by setting the membrane current term in the monodomain equation to zero, while retaining the term describing voltage diffusion.Regions with a diffusion coefficient less than 0.05 mm2ms−1 as expected, and the effect of truncation at 0.05 and 0.2 mm2ms−1 can be seen.Figures CRNnormal and CRNremodelled variants.Since the configuration of fibrotic regions would be expected to affect simulated tissue behavior, 20 samples of the GRF were obtained at each of the four length scales. The procedure described above was used to generate a set of 20 diffusion fields, and these were then used for simulations with the different pacing protocols described in the next section. Multiple samples enabled the effect of length scale to be assessed independent of the specific configuration of a single sample relative to pacing sites. Each sample was then used to simulate the different pacing protocols specified below, with both 2ms−1 was retained, but elsewhere it was set to 0.2 mm2ms−1.To compare the effect of the smooth variations in diffusion coefficient provided by the GRF with abrupt changes, a further set of diffusion fields were generated. For each length scale, a copy was made of each of the 20 diffusion fields obtained by sampling the GRF. These copies were then further processed so that they contained only two values of diffusion coefficient, with an abrupt change between the two. In the fibrotic regions, the diffusion coefficient of 0.05 mm2ms−1, and the cell model was set to be ineexcitable) was 18.7, 18.0, 19.1, and 17.0% at length scales of 1.25, 2.50, 5.0, and 10.0 mm, respectively. The size and configuration of these regions was different in each of the 20 samples. At a length scale of 10 mm, the proportion of simulated fibrotic tissue ranged between 4.9 and 25.1% across the 20 samples. With a length scale of 1.25 mm, which was a smaller length scale relative to the size of the simulated tissue, the range was 17.9–19.6%.Within the 20 diffusion fields generated at each length scale, the average proportion of simulated fibrotic tissue  had a sharp upstroke, and an amplitude and recovery that were comparable in slope to the other time series shown. In contrast, the corresponding voltage time series shown for a length scale of 10 mm in Figure CRNremodelled variant produced a lower amplitude deflection in the inexcitable regions.A possible explanation for these observations is explored in Figure CRNremodelled variant resulted in a lower amplitude excursion. The lower amplitude excursion in the CRNremodelled variant in turn shortened the recovery of neighboring excitable regions by acting as a current sink. At longer length scales, these neighboring regions were smaller in extent, which could explain in the skewed distribution toward shorter APD seen in the right hand panels of Figure CRNnormal variant could act as a current source during recovery, acting to prolong APD, an effect that would have had more impact on neighboring regions. This could explain the skew toward longer APD values seen in the left hand panels of Figure The interaction of local APD, heterogenous diffusion, and the length scale of heterogeneities therefore appears to be complex, and this is emphasized by the range of action potential shapes seen in Figure CRNnormal variant, some configurations of heterogeneity resulted in one or more of the pacing stimuli being blocked, and so the number of beats recorded was less than 9. However, this effect was not observed with the CRNremodelled variant, which could be attributed to the greater prolongation of APD in heterogenous tissue with the CRNnormal variant described above. Overall, the prevalence of additional beats and re-entry was greater with the CRNremodelled variant, and with shorter length scales. Additional simulations were run using diffusion fields where the smoothly varying diffusion obtained from the GRF was replaced by an abrupt transition, and the maximum number of beats elicited was 10. These data are included as Supplementary Figure The response of this complex tissue model to decremental pacing with 9 stimuli is illustrated in Figure CRNremodelled model variant is illustrated in Video The precise configuration of regions with lower diffusion was important for eliciting re-entry, and this is illustrated in Figure CRNremodelled variant resulted in sustained re-entry of more than 2 beats, whereas simulations with the CRNnormal variant tended to result in non-sustained re-entry  is an important tissue property, and excitable tissue must be large enough to accommodate the wavelength in order to sustain re-entry. Heterogeneous diffusion modulates wavelength by slowing conduction velocity (delaying activation) and modifying APD, and so the behavior of re-entry was found to depend on the configuration of heterogeneities. Figure t Figure , and oney Figure , whereasy Figure , and a mIt is possible to represent heterogeneity in cardiac tissue conductivity at different length scales using samples of a GRF.2ms−1, but larger length scales of 5.0 and 10.0 mm tended to produce a wider spread of activation delays.The length scale of heterogeneity did not markedly affect the median delay in activation time relative to simulations with a uniform diffusion coefficient of 0.2 mmCRNnormal variant rather than CRNremodelled.A delay in recovery relative to simulations with a uniform diffusion coefficient was found. This tended to be greater in simulations with heterogeneity on a smaller length scale, and greater with the CRNnormal variant and negatively skewed toward lower APD with the CRNremodelled variant. This difference was attributed to inexcitable regions of simulated fibrosis acting as a current source during repolarization in simulations with the CRNnormal variant, and as a current sink in simulations with the CRNremodelled variant.Distributions of APD tended to be positively skewed toward higher APD with the CRNremodelled variant were most likely to result in sustained re-entry. However, the configuration of regions of simulated fibrosis also played an important role.With decremental pacing, simulations with smaller length scale heterogeneity and the CRNremodelled variant were also most likely to result in sustained re-entryWith a spiral wave as an initial condition, simulations with smaller length scale heterogeneity and the It is well-known that electrical heterogeneity is an important factor for determining vulnerability to arrhythmias in cardiac tissue, and this has been investigated in experimental preparations (Allessie et al., The interaction of heterogenous diffusion, length scale, simulated fibrosis, and APD is complex and merits further investigation. The relationship of this study to previous work is discussed below.The findings of the present study depend on the way that cellular electrophysiology and regions of fibrosis have been represented. In previous studies, regions of fibrosis have been represented in many different ways including inexcitable obstacles (Ten Tusscher and Panfilov, 2ms−1. The slowed upstroke and lower amplitude of voltage excursion observed in simulated fibrosed regions compared to normal tissue was comparable to other studies that have used more detailed models of fibroblast electrophysiology (Ashihara et al., In the present study regions of diffuse fibrosis were represented by smoothly varying but reduced diffusion, and focal fibrosis was represented as inexcitable tissue with a fixed diffusion coefficient of 0.05 mm2ms−1 in fibrosed regions and 0.2 mm2ms−1 elsewhere (see Supplementary Figure CRNnormal variant, simulations with abrupt changes in diffusion had a longer APD and greater APD dispersion, especially at shorter length scales, compared to simulations with smoothly varying diffusion. With both variants, the delay in activation time was much shorter compared to simulations with smoothly varying diffusion. The overall response to decremental pacing was also similar for the CRNnormal variant, but it was not possible to elicit more than one additional beat with the CRNremodelled variant (see Supplementary Figure An additional set of simulations was done to compare the effect of simulations that used smoothly varying diffusion fields with simulations where the diffusion coefficient was set to 0.05 mmThe length scale of heterogeneity was found to play a role in the prolongation and dispersion of APD, as well as in determining vulnerability to re-entry and rotor stability. A shorter length scale favored a longer APD, greater APD dispersion, and sustained re-entry. The configuration of simulated fibrotic regions was also important because a small isthmus of tissue often formed part of the activation pathway during re-entry as illustrated in Figure 2ms−1, there were discontinuities at the boundary of these regions as illustrated in Figures One of the aims of the present study was to investigate the effect of smooth changes in diffusion. Since diffusion was capped at 0.2 and 0.05 mmCRNnormal representing normal atrial myocytes, and CRNremodelled representing remodeled atrial myocytes (Wilhelms et al., CRNremodelled variant tended to modulate the effects of inexcitable fibrotic regions, and to increase vulnerability to re-entry. A further consequence of the shorter APD was the shorter wavelength of the CRNremodelled variant, which favored sustained re-entry in almost all simulations with the CRNremodelled variant and a spiral wave as an initial condition (Figure Persistent or permanent AF leads to changes in the electrophysiology of atrial myocytes, which act to shorten APD and hence wavelength, and these changes are consistent with the observed progression of AF from a short-lived paroxysmal arrhythmia to a permanent state Nattel, . In the n Figure .2+ handling mechanisms, which are affected by remodeling and also feed back into action potential shape (Grandi et al., It is possible that more complex effects come into play in real cardiac tissue. Isolated atrial myocytes from canine right atrium show a wide range of action potential shapes (Feng et al., The anatomy and configuration of fibrosis has recently been shown to be more important in determining rotor location than cellular electrophysiology (Deng et al., This study used a very simple representation of fibrosis, as discussed above. There was no attempt to represent the detailed anatomy of the human atria, which would add a further layer of complexity to the findings. In particular, diffusion was assumed to be isotropic, although fibrosis is known to have a stronger effect on lateral connections between myocytes, acting to increase anisotropy (Kohl and Gourdie, RC designed the study, wrote and implemented the computational model, ran the simulations, prepared the figures, and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Following publication of the original article , the autThe correct versions of the figures are given below:


In the original publication figures The correct figures are provided in this correction.The original article has been corrected.


CS  and peripartum antibiotics were also associated with greater alpha diversity of the meconium microbiome . Meconium from infants born by CS  had lower relative abundance of the genus Escherichia (p < 0.001). Prenatal antibiotic use and peripartum antibiotic use  were associated with differential abundance of several bacterial taxa in the meconium. Bacterial taxa in the meconium microbiome were also differentially associated with infant excess weight at 12 months of age, however, sample size was limited for this comparison. In conclusion, prenatal and peripartum antibiotic use along with CS delivery were associated with differences in the diversity and composition of the meconium microbiome. Whether or not these differences in the meconium microbiome portend risk for long-term health outcomes warrants further exploration.The meconium microbiome may provide insight into intrauterine and peripartum exposures and the very earliest intestinal pioneering microbes. Prenatal antibiotics have been associated with later obesity in children, which is thought to be driven by microbiome dependent mechanisms. However, there is little data regarding associations of prenatal or peripartum antibiotic exposure, with or without cesarean section (CS), with the features of the meconium microbiome. In this study, 16S ribosomal RNA gene sequencing was performed on bacterial DNA of meconium samples from 105 infants in a birth cohort study. After multivariable adjustment, delivery mode ( The human gut microbiome undergoes rapid dynamic development in the early years of life, reaching a stable state with a diversity and complexity resembling adult gut microbiota generally by the age of three . These dAberrant early intestinal microbiome development has been associated with specific childhood diseases, such as obesity. Childhood obesity has become a critical public health problem ,8, with Early life antibiotic use, including during the prenatal and peripartum period, has been associated with higher obesity risk in studies ,15,16,17The study reported in this manuscript was undertaken to investigate the association of prenatal and peripartum antibiotic exposure and delivery mode on the meconium microbiome, and to determine if the meconium microbiome was associated with excess weight of the infant at 1 year of life.Mothers were recruited prenatally in an ongoing longitudinal childhood health cohort study within the Inova Health System “The First 1000 Days of Life and Beyond”. Informed consent was obtained from all subjects. All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Inova Health System Institutional Review Board . Infants had meconium collected . Meconium was stored in Eppendorf Tubes at −80°C within 12 hours of collection. Only full term infants  were included.Demographic information was collected including infant sex, maternal ethnicity, and gestational age at delivery by questionnaire and the electronic medical record. Detailed information was recorded regarding delivery  and emergency CS . Pregnancy details collected from the electronic medical records included the use of prenatal antibiotics (defined as antibiotics given to mother from conception to 2 days prior to delivery) and peripartum antibiotics (defined as antibiotics given to the mother from 2 days prior to delivery to during the delivery). Weight gain during pregnancy was calculated with pre-pregnancy weight and admission weight at delivery, noted in electronic health records . The Heag for 2 min. Supernatant was removed, avoiding the pellet, and placed in a new ET with one Inhibit X tablet (Qiagen). Samples were vortexed until the tablet was dissolved and incubated at room temperature for 3 min. Samples were centrifuged at 20,000× g for 2 min; supernatant was removed and put into a new Eppendorf Tube and centrifuged again at 20,000× g for 3 min. The Qiagen QIAmp DNA Stool Mini kit protocol then followed (Qiagen). Meconium samples stored at −80 °C were thawed on ice and suspended in ASL buffer  at a ratio of 2.5 mL ASL to 0.5 g of meconium . Tube-stStaphylococcus aureus and Escherichia coli were also included.Sequencing libraries were prepared using a Nextera XT kit  according to the 16S Metagenomic Illumina Library Preparation Protocol for sequencing the variable V3 and V4 regions of the 16S rRNA gene. If samples failed QC, library preparation was completed again with the “PCR 1” using Hemo KlenTaq®  for the PCR reaction. Hemo KlenTaq® is known to work well with sample containing PCR inhibitors, especially bilirubin, which is highly present in meconium samples. Each specimen/condition passing QC was then sequenced on the Illumina MiSeq platform (Illumina) with paired-end reads 301 bp (600 cycles). Only 3 meconium samples failed sequencing, leaving a total of 105 for analysis. Sequencing of negative controls of lysis buffer undergoing the above DNA extraction process was performed. Positive control samples of Demuliplexed reads from each sample were treated as single-ended reads and bacterial operational taxonomic units (OTUs) were picked using the open reference method using QIIME 1.9  with GreThe OTU counts table and the phylogenetic tree created by QIIME were imported into R bioconductor package phyloseq (Version 1.25.2) for data analysis. All the figures in the manuscript were generated by R Version 3.4.0. A single rarefaction at 7324 was performed  to normaBeta diversity was measured by the unweighted unifrac distance in order to capture the differences in the low abundance features. PERMANOVA was performed using the adonis function in the R package Vegan 2.5-3 to compare the beta distances between multiple clinical factors. The adonis function in R package Vegan 2.5-3 was also used to perform permutational multivariate analysis of variance using the unweighted unifrac distance matrices. Alpha diversity was measured by the number of OTUs, and the Fisher, Shannon, and Simpson indices. Two sample Wilcoxon test was used to access differences in alpha diversity between groups. Normalized counts for each sample were used to calculate the abundance of bacteria at the phylum, class, order, family, and genus levels. A two sample Wilcoxon test was used to compare the differences in relative abundance between groups at the phylum and genus levels using the normalized counts. Raw read counts for each OTU for each sample were analyzed by R Bioconductor package EdgeR 3.20.9 to discover differentially abundant OTUs according to clinical factors of interest. Benjamini and Hochberg adjustment was performed for multiple testings . The 16S rRNA sequence data are available in NCBI Sequence Read Archive (SRA), PRJNA600283.p < 0.001). Of those receiving peripartum antibiotics for VD, all were for detected Group B Streptococcus (GBS) prophylaxis. Women without a penicillin allergy undergoing a scheduled CS received a first generation cephalosporin within 60 minutes prior to the start of CS . Those who were penicillin allergic (2/27) received clindamycin (900 mg intravenously) and gentamicin (5 mg/kg intravenously). Women undergoing emergency CS also received a first generation cephalosporin; additional azithromycin was given in 12/16 mothers (500 mg intravenously). All 19 women who had VD and received antibiotics for GBS prophylaxis received penicillin . Dosing for peripartum antibiotics was confirmed in the medical administration record, as these were inpatient medications. Seventeen out of 105 (16%) of mothers received prenatal antibiotics; these were given for urinary tract infections (10/17) and respiratory infections (7/17). Penicillins, cephalosporins, or macrolides were used in all 17 cases. One out of 17 received antibiotics in the 1st trimester, 7/17 in 2nd trimester, 8/17 in 3rd trimester, and 1/17 in both the 1st and 2nd trimester. While these prenatal antibiotics were documented in the electronic medical record, dosing and duration could often not be confirmed, as they were not part of the medical administration record due to being outpatient medications.Meconium was collected within 48 hours of life and was sequenced successfully from 105 infants. Forty-five (43%) were male; 62 (59%) were born by VD and 43 (41%) by CS. Of those delivered by CS, 27/43 (63%) were by a scheduled CS and 16/43 (37%) by emergency CS. p < 0.05). No significant differences were found in the sex, maternal pre-pregnancy BMI, maternal weight gain during pregnancy, weight status of the infant at 12 months of age, and scheduled CS versus emergency CS . In multivariable analyses adjusted for covariates that were significant in univariable models, delivery mode (p = 0.044), prenatal antibiotic use (p = 0.005), and peripartum antibiotic use (p < 0.001) remained significant.In the 105 meconium samples, each of the clinical variables was examined in relation to beta diversity using the Adonis test with 9999 permutations. Beta diversity was significantly associated with delivery mode (CS versus VD), prenatal antibiotic use, and peripartum antibiotic use  and Simpson (p = 0.002) indices compared to VD, but no difference in the number of observed OTUs (p = 0.55) or Fisher (p = 0.58) diversity. Use of peripartum antibiotics was associated with higher Shannon and Simpson diversity, but a lower number of observed OTUs and Fisher diversity . Shannon diversity was also higher in samples from non-Hispanic mothers (p = 0.04), but sample size for ethnicity analysis was limited. No significant differences in any of the alpha diversity measures were observed based on sex, maternal weight gain during pregnancy, and weight status of the infant at 12 months of age.When analyzing the alpha diversity of meconium, infants delivered born by CS had higher Shannon (iversity a. While p < 0.05) among VD infants. Observed OTUs, and Shannon and Fisher indices were also higher in samples from non-Hispanic mothers (p < 0.05), however, the peripartum antibiotic use in infants born by VD was highly skewed between the Hispanic and non-Hispanic populations, with 9 out of 20 (45%) non-Hispanic mothers using peripartum antibiotics compared with only 2 out of 13 (15%) Hispanic mothers. Therefore, whether there was a difference in alpha diversity in the samples from mothers that did not use peripartum antibiotics was further examined (11 Hispanic mothers and 11 non-Hispanic mothers). Two sample t-tests demonstrated that, while the average alpha diversity in the four alpha diversity measures were still higher in samples from infants born from non-Hispanic mothers, the differences were no longer significant. It was therefore concluded that the difference observed between ethnicity in the mothers may be driven by the peripartum antibiotic use.Next, whether these alpha diversity associations remained significant when considering only infants born by VD was investigated b. PrenatEscherichia had the highest mean abundance (28.5%), followed by Methylobacterium (15.7%) and Streptococcus (7.2%).Overall, in full term meconium samples, Proteobacteria had the highest mean abundance at the phylum level (65.2%), followed by Firmicutes (26.3%), and Bacteroidetes (4.2%). At the genus level, Escherichia and Bacteroides were higher in VD (FDR adjusted p < 0.05). Escherichia in particular was markedly higher in VD (38%) vs. CS (15%) infants (FDR adjusted p < 0.001), Methylobacterium and Veillonella were higher in CS meconium samples (adjusted p < 0.05), with Methylobacterium being markedly higher in CS compared to VD . At the genus level, the relative abundance of Streptococcus . Among the top 10 most significant OTUs, 8 were from the genus tococcus , which w infants . SignifiClostridium and 2 OTUs from the family Enterobacteriaceae with unspecified genus were enriched in meconium samples from infants born to mothers who used prenatal antibiotics. Next, antibiotic use was examined. A total of 126 bacterial OTUs were differentially abundant between meconium samples from infants delivered to mothers with vs. without prenatal antibiotic usage. The number of OTUs decreased to 75 if only infants born by VD were considered . The topEnterococcus was lowes in the meconium of infants born to mothers that used peripartum antibiotics, whereas the OTUs belonging to Agrobacterium and Streptococcus were higher.A total of 214 OTUs were significantly different in relative abundance between samples from mothers with vs. without peripartum antibiotics use; the number of significant OTUs decreased to 53 for infants born by VD only . The topStreptococcus, Klebsiella, Escherichia, Veillonella, and Acinetobacter. There were more OTUs from the genera Pseudomonas and Enterococcus and fewer OTUs from Escherichia and Propionibacterium in the meconium of infants born to mothers with excess weight gain. Differentially abundant OTUs were also examined between other clinically relevant factors. Escherichia. Prior studies support the notion that the microbiome present in the first passage of newborn meconium may reflect the infant’s intrauterine and perinatal environmental exposures [In our birth cohort of full-term mother-child dyads, it was found that prenatal antibiotic use, peripartum antibiotic use, and delivery mode were associated with features of the meconium microbiome. The beta diversity of meconium, after multivariable adjustment, was associated with delivery mode, with prenatal antibiotic use and with peripartum antibiotic use. CS and peripartum antibiotics were associated with greater alpha diversity (Shannon and Simpson), and CS samples had a lower relative abundance of the genus xposures . This stPeripartum antibiotics are given to ≥40% of pregnant women in the USA and have successfully decreased the rate of neonatal GBS infection and maternal post CS infection ,35,36. IEscherichia were seen in the meconium from infants born by CS. This is a shift from the expected “normal”, very early colonization pattern where there is a predominance of facultative anaerobes, such as species from Escherichia, as seen in infants born by VD [In examining the association of peripartum antibiotics with the meconium microbiome, it is important to account for delivery mode, which can result in a lack of exposure to maternal vaginal and perineal microbes . Indeed,rn by VD . Prenatal antibiotic exposure was also associated with changes in the meconium microbiome, although to a lesser extent, with differences in beta diversity and relative abundance of certain taxa and a trend towards lower alpha diversity. Prenatal antibiotic exposure has been associated with later obesity in children , howeverPrenatal and peripartum antibiotics, in addition to CS delivery, have been associated with obesity ,18,19,20There are several limitations of our study. The sample size was limited to rigorously examine prenatal antibiotics as well as other factors such as excess infant weight at 1 year of age and ethnicity. Furthermore, although in general there was homogeneity in the type of peripartum antibiotics used, there was a lesser degree of prenatal antibiotics used, and a larger sample size would be needed to determine whether specific antibiotics and doses of antibiotics, broad versus narrow spectrum antibiotics or the specific timing in the prenatal period in which they were received, impacted the microbiome to a greater degree. Additionally, the dosing and duration of all of the prenatal antibiotics could not be confirmed. A further limitation is that meconium samples generally had a low abundance of bacteria and as with any low abundance microbiomes, there was a potential for contamination; our inclusion of both positive and negative controls serves to minimize this concern. Additionally, anthropometrics used were parent reported, which are known to have varying reliability. While measured anthropometrics were preferred, the accuracy of parent-reported anthropometrics was improved in this study by the removal of outliers, which has been previously validated in this cohort .In conclusion, through examination of the meconium, which may provide an insight into intrauterine events and the very earliest pioneering gut microbes, it can be seen that prenatal and peripartum antibiotic use, in addition to CS delivery, were associated with differences in the structure and composition of the very early infant gut microbiome. Prospective longitudinal studies are needed to follow how early life antibiotic exposure may continue to affect microbiome development trajectories and association with diseases, such as obesity, thought to be related to aberrant gut microbiome development.


Despite our understanding of the principal factors that shape bird migration strategies, there is conflicting evidence regarding the role of habitat in shaping migration routes and schedules, including day and night activity and differences between autumn and spring. For fly-and-forage migrants, we predict that habitat characteristics might guide migration speed, route selection and migrating schedules.We use solar-powered GPS transmitters, obtaining high accuracy data, to monitor the migratory movements of Eleonora’s falcon breeding in Cyprus, which is the easternmost breeding population of the species. We tested for potential preferences in habitat characteristics along the migration routes, separately for the northern, drier part and the more vegetated southern part of the trips. We also examined the relationship between migration speed and vegetative cover during day and at night, accounting for wind support.We found that tagged individuals repeatedly exhibited an anticlockwise loop migration pattern with spring routes being more easterly than autumn ones. We identified a preference for migration through vegetation-rich areas, where during daytime tagged individuals travel at slower migration speeds compared to vegetation-poor areas, indicating fly-and-forage activity. Birds roosted during most nights, combining refueling stopovers at selected vegetation-rich areas before or after crossing ecological barriers. Conversely, both during day and night, tagged individuals overflew unsuitable habitats more quickly.Our results suggest that habitat is an important factor in Eleonora’s falcon migratory strategies. Active selection of vegetation rich areas in combination with reduced migration speeds there, allows the migrating falcons to combine migration during the day with fly-and-forage refueling, while roosting most nights. Migration is part of the annual cycle of many bird species that has evolved over millennia –3. The mAvian migration strategies are guided by pre-migratory fuel deposition, but refueling en route is equally critical . IntegraFalco eleonorae) is a complete, long-distance, trans-equatorial migrant species [Eleonora’s falcon  was used as a decoy [Birds were caught using mist nets at nesting and foraging locations . Mist ne a decoy . In addi a decoy  and mark a decoy , while b a decoy . Sex and a decoy . Transmi a decoy . Two dif a decoy , 50, 51. a decoy , with in a decoy .The positional error of GPS locations was less than 20 m for 80% of the retrieved points , we thusBecause of the lower accuracy of PTT obtained locations, we decided to rely only on GPS positions for path metric estimates using all available segments. Segment length between two successive telemetry fixes was calculated based on the geodesic distance , i.e. thBird activity and flying mode between two successive telemetry fixes  and related tortuosity of the tracks affect path metrics . TherefoAs stopovers we considered areas with a 25 km radius where an individual stayed for at least 24 h without exhibiting directional migratory movement, allowing for one outlier point per stopover , 65. ThioN. We used the Corrected Akaike’s Information Criterion (AICc) to select the best models for each test, considering all models with ΔAICc < 4 compared to the best supported model.In order to determine whether Eleonora’s falcon selects specific habitat characteristics within its seasonal migration corridors, we compared actual bird positions with random locations generated within a 50 km buffer either side of each trip . This diBecause migration speed is related to the activity of tracked individuals, for example slower migration speeds are expected when birds are foraging, we investigated which factors affect the migration speed of Eleonora’s falcon during migration with the use of GLMMs with a Gaussian distribution with identity link function using lme4 in R 3.5.1 . Specifio degrees for autumn and 350o in spring, based on the shortest direct line potential migratory route from Akrotiri colony in Cyprus to the point of the narrowest crossing over the Mozambique Channel to Madagascar and vice versa.Knowing that wind will have an effect on migration speeds, and to make sure that the wind effect is accounted for when estimating the independent effect of habitat characteristics, we included wind as a fixed effect in our models. The loggers that we used did not provide flying altitude data, so to calculate tailwind at each GPS location we used winds at 850 hPa pressure level, corresponding to a mean altitude of 1500 m a.s.l.. In previous studies on raptors migrating through Africa a 925 hPa pressure level  has been used, but most of those studies were conducted on the western African flyway e.g. , 68, 79), 7968, 7From the eight Eleonora’s falcons tagged that provided migration data Table , we obtaN = 8, mean = 24.75 days, SD = 11.52) than spring migration , though we note that the confirmed durations in spring came from only one individual (Farofylakas) for which the comparative results indicate the same average duration . In autumn, departure from Cyprus occurred between 4th October and 6th November, with the peak around 26th October . Arrival in Madagascar occurred between 8 and 28th November without any obvious peak. In spring, departure from Madagascar was between 9 and 15th April, and arrival in Cyprus between 25th April and 9th May , compared to 7245 km (SD = 401) in spring. The shortest path distance was 5749 km (SD = 209) in autumn and 5845 (SD = 626) in spring, so actual distances traveled were about 24% further than the minimum possible distance. The longest apparently continuous flight, recorded for Ifigeneia, was 3530 km, covered in 85 h with an average migration speed of 40 km/h (maximum single segment migration speed 73 km/h). We found that during the crossing of the drier (northern) part of their migration routes, the falcons used areas with higher NDVI in lower elevations compared to generated random points within the 50 km buffer zone either side of their path  and 6.41 km/h  respectively. The highest migration speeds though were recorded at night, both in autumn and spring, over the Mediterranean, Sinai and the Red Sea with a maximum migration speed of 89 km/h recorded. Overall, migration speeds were significantly slower over areas with higher NDVI and higher tree cover and also significantly slower in spring compared to autumn because of the relative effect of tailwinds  compared with the day  (N = 901): a) full list of candidate models; b) full model; c) candidate model (ΔAICc <4); d) candidate model (ΔAICc <4); e) candidate model (ΔAICc <4); f) candidate model (ΔAICc <4); g) best supported model. In the drier north (e.g. Sahara desert), the falcons used areas with higher NDVI compared to randomly generated points, avoiding higher elevations. Table S2. GLMM results for comparison between actual positions and random points generated within a 50 km buffer zone either side of the flying path, in relation to NDVI, tree cover and elevation for the south part of the migratory routes (latitude 16 N southwards) (N = 2811): a) full list of candidate models; b) full model, c) candidate model (Δ AICc < 4); d) best supported model. In the more vegetated south they occurred , in areas with higher tree cover, and subsequently higher elevations (particularly in autumn) than what was available to them alongside their routes. Table S3. GLMM results for the effect of all GPS telemetry locations (N = 1435) on migration speed, of NDVI, tailwind, tree cover and season, with interactions of season with the other fixed effects, and individual and year as crossed random factors: a) full list of candidate models; b) full model; c) Candidate model (ΔAICc < 4); d) Candidate model (ΔAICc < 4); e) Candidate model (ΔAICc < 4); f) best supported model. Migration speeds were significantly slower over areas with higher NDVI and tree cover and also significantly slower in spring compared to autumn because of wind effect. Table S4. GLMM results for the effect on migration speed, of NDVI, tailwind, tree cover, season and day/night (excluding transitioning segments between day and night) with the interactions of day/night with the other fixed effects and individual and year as crossed random factors (N = 984): a) full model; b) best supported model (Delta AICc < 4). In this dataset, slower migration speeds were recorded at night, and over areas with higher NDVI. Differences in migration speed between day and night were greater in autumn compared with spring, with significantly slower migration speeds at night during spring. Table S5. GLMM results for the effect on active migratory movements’ migration speed (> 5 km/h), of NDVI, tailwind, tree cover, season and day/night , with interactions of day/night with the other fixed effects and individual and year as crossed random factors: a) full list of candidate models; b) full model; c) Candidate model (ΔAICc < 4); d) Candidate model (ΔAICc < 4); e) best supported model. In this dataset, migration speeds were higher with increasing tailwind. Migration speeds were still slower over higher NDVI, but conversely, higher during the night compared with the day. Also, the effect of increasing tree cover percentage on migration speed was higher during the day compared to the night. A seasonal effect was also identified here, with the difference in migration speed between day and night being again greater in autumn compared to spring with significantly slower migration speeds at night during spring. Figure S1. Migratory routes during (a) autumn 2013 and (b) spring 2014 migration seasons overlaid on to the respective average monthly east – west winds (U wind component) at 850 hPa (c1500 m a.s.l.). Line colors represent the different individuals. East-west wind component is weaker compared to north-south component, facilitating north-south primary movement direction to and from wintering grounds. Data downloaded from ERA5 atmospheric reanalysis product of the European Centre for Medium-Range Weather Forecasts (ECMWF).


The mammalian Notch family ligands delta‐like 3 (DLL3) is reported to be a potential therapeutic target for large cell neuroendocrine carcinomas (LCNEC). The effect of DLL3 expression on LCNEC prognosis has not yet been elucidated.We reviewed the medical records of 70 LCNEC patients undergoing surgical resection between 2001 and 2015 using a prospectively maintained database. We performed immunohistochemistry for DLL3 and investigated the correlation between the sensitivity of LCNEC to platinum‐based adjuvant chemotherapy.P = 0.36, five‐year RFS: 41.7% vs. 35.7% P = 0.74). In contrast, among patients with DLL3‐negative tumors, significantly greater five‐year OS and RFS rates were observed for patients with adjuvant chemotherapy than for those without it . A multivariate analysis for the RFS revealed that adjuvant chemotherapy was a significant independent prognostic factor among patients with DLL3‐negative tumors , although it was not a factor among patients with DLL3‐positive tumors .DLL3 expression was positive in 26 (37.1%) LCNEC patients. A total of 23 patients (32.9%) received platinum‐based adjuvant chemotherapy. Among patients with DLL3 expression‐positive tumors, no difference was found in the five‐year overall survival (OS) or recurrence‐free survival (RFS) between patients with and without adjuvant chemotherapy (surgery + chemotherapy vs. surgery alone, five‐year OS: 58.3% vs. 35.7% Our results revealed that DLL3 is a predictive marker of sensitivity to platinum‐based adjuvant chemotherapy for LCNEC.DLL3 was a predictive marker of sensitivity to platinum‐based adjuvant chemotherapy for LCNEC. Among patients with DLL3 expression‐negative LCNEC, platinum‐based adjuvant chemotherapy significantly improved the OS and RFS, although it did not do so among patients with DLL3 expression‐positive LCNEC.Our results suggest that DLL3 expression‐positive LCNEC may be better treated with other types of adjuvant chemotherapy, such as the anti‐DLL3 therapies if these effects are confirmed by ongoing clinical research. DLL3 was a predictive marker of sensitivity to platinum‐based adjuvant chemotherapy for LCNEC. Among patients with DLL3 expression‐negative LCNEC, platinum‐based adjuvant chemotherapy significantly improved the OS and RFS, although it did not do so among patients with DLL3 expression‐positive LCNEC. Large cell neuroendocrine carcinoma (LCNEC) is categorized as high‐grade neuroendocrine carcinoma (HGNEC) in the fourth edition of the World Health Organization Classification of Lung Tumors.et al. reported that pulmonary LCNEC comprise two genomic subgroups with specific transcriptional patterns, defined as “type I LCNEC” and “type II LCNEC” in the first comprehensive molecular analysis of LCNEC.Delta‐like protein 3 (DLL3) is an atypical member of the Notch receptor ligand family and reported to inhibit Notch signaling.These previous findings suggest that DLL3 expression will play a key role in clinical practice of LCNEC in the near future; however, the association of DLL3 expression with the clinicopathological features of LCNEC has not yet been elucidated. In the present study, we analyzed the effect of DLL3 expression on overall survival (OS) and recurrence free survival (RFS) in LCNEC and further analyzed the predictability of this expression for the efficacy of adjuvant chemotherapy.n = 5), synchronous multiple lung cancers (n = 3), performing induction chemotherapy or radiotherapy (n = 3), and insufficient medical records or pathological samples (n = 6). The 70 remaining patients were analyzed  staging system.Contrast‐enhanced chest and abdominal computed tomography (CT), positron emission tomography (PET)‐CT, and brain magnetic resonance imaging (MRI) were performed for preoperative staging. Patients were evaluated postoperatively at three‐month intervals for two years, at six‐month intervals for the subsequent three years, and annually thereafter. Follow‐up examinations included chest radiography, contrast‐enhanced CT, brain MRI, and bone scintigraphy as well as hematologic and biochemical analyses, including the measurement of the tumor markers. The OS and RFS were calculated according to the Kaplan‐Meier method, and the log‐rank test was used to evaluate differences in the distributions. OS was defined as the time interval between the date of surgery and the date of death. RFS was defined as the time interval between the date of surgery and the date of death without recurrence or the date of the first recurrence detected by a radiological examination.LCNEC was diagnosed based on the histopathological criteria described by the World Health Organization in 2015Immunohistochemical staining was performed on 4 μm thick formalin‐fixed, paraffin‐embedded sections. Sections were deparaffinized in xylene and rehydrated in graded alcohol baths. Antigen retrieval was performed by immersing the sections in 10 mM sodium citrate buffer  for 20 minutes at 98°C in a water bath. Endogenous peroxidase activity was blocked in 3% hydrogen peroxide in water. Immunostaining was performed using the avidin–biotin complex technique . Sections were incubated with anti‐DLL3  at a dilution of 1:300 at 4°C overnight and then incubated with biotinylated goat‐anti‐rabbit IgG, followed by peroxidase‐conjugated streptavidin complex. Complexes were visualized with 3,30‐diaminobenzidine, and the slides were counterstained with Mayer's hematoxylin, dehydrated and mounted. For the neuroendocrine markers, the sections were immunostained with the streptavidin‐biotin technique with an automated immunostainer  according to the manufacturer's instructions. Antibodies against chromogranin A , synaptophysin , and CD56 (NCAM)  were used.All samples were evaluated by an expert pathologist (Y.S.) without knowledge of the patient's outcome. In this study, the cutoff value for the percentage of positive tumor cells was set at 1% for DLL3 Fig. . We set t‐test and the chi‐square test were performed to assess the significance of the differences in age, sex, smoking status, lung function, surgical procedure, adjuvant chemotherapy, pathological stage (p‐stage) and the other pathological factors between the DLL3 expression‐positive and DLL3 expression‐negative groups. The OS and RFS were calculated according to the Kaplan‐Meier method, and differences in the distributions were evaluated by the log‐rank test. The Cox proportional hazards model was used to evaluate the association between prognostic factors and the RFS after pulmonary resection, with the hazards ratio (HR) and 95% confidence intervals (CIs). P‐values of <0.05 were considered statistically significant.Statistical analyses were carried out using the JMP 14 software program . Student's P < 0.01), patients with a better lung function (P < 0.01), and more patients with lymphatic permeation than the DLL3 expression‐negative group (P < 0.01). The five patients with combined LCNEC were all included in the DLL3 expression‐negative group. There was no marked difference in the sex, smoking status, surgical procedure, pathological stage, pathological tumor size, or rate of lymph node metastasis, pleural invasion, or vascular invasion between the patients with DLL3 expression‐positive and expression‐negative LCNEC. Patients who received adjuvant chemotherapy were significantly younger in both DLL3 expression positive and negative groups (P < 0.01). In DLL3 expression‐positive group, significantly more patients who underwent surgery alone were performed sublobar resection (P = 0.04). In the DLL3 expression‐negative group, significantly more patients with pleural invasion received adjuvant chemotherapy (P = 0.04).The median follow‐up period of all patients was 37.8 months. DLL3 expression was positive in 26 37.1%) LCNEC patients. Adjuvant chemotherapy was administered to 12 (46.2%) of the 26 patients in the DLL3 expression‐positive group, and 11 (25.0%) of the 44 patients in the DLL3 expression‐negative group. The patient characteristics are summarized in Table % LCNEC pThe regimens of adjuvant chemotherapies are shown in Table We next examined the correlation between DLL3 expression and three neuroendocrine markers. The comparison of immunohistochemical staining for the three neuroendocrine markers between DLL3‐positive and DLL3‐negative LCNEC are summarized in Table P = 0.73, five‐year RFS: 38.5% vs. 36.4% P = 0.91)   Fig. . We then.01) Fig .P = 0.36, five‐year RFS: 41.7% vs. 35.7% P = 0.74)   Fig. . In cont01) Fig. .P < 0.01), although it was not a significant independent prognostic factor among patients with DLL3 expression‐positive LCNEC   that were considered to have an influence on the recurrence of patients with LCNEC.8) Table .In the present study, we demonstrated that DLL3 expression was a strong predictor of the efficacy of platinum‐based adjuvant chemotherapy for pulmonary LCNEC patients. Among patients with DLL3 expression‐negative LCNEC, platinum‐based adjuvant chemotherapy significantly improved the OS and RFS, although it did not do so among patients with DLL3 expression‐positive LCNEC. These results were confirmed by a multivariate analysis.DLL3 is a unique Notch receptor ligand that inhibits the Notch pathway.There is increasing evidence that surgical resection alone is insufficient for treating LCNEC,et al. revealed that LCNEC could be divided into two subtypes (type I and type II LCNEC) based on the genetic background according to a transcriptome analysis conducted with a next‐generation sequencer.We previously reported that the combination of three neuroendocrine markers was predictive of the sensitivity to platinum‐based chemotherapy.et al. compared the performance of four DLL3 antibodies in HGNEC samples and cell cultures and found poor results concerning the overall agreement.The rate of DLL3 expression by immunohistochemistry was reported to be 65% among LCNEC patients,et al. reported that LCNEC responded better to an SCLC‐targeting regimen (platinum‐etoposide).Several limitations associated with the present study warrant mention. First, this study was a retrospective, nonrandomized single‐center study with a small sample size. There may be some selection bias with regard to the performance of adjuvant chemotherapy, and its selection criteria have not been clarified. Second, the chemotherapy regimens were not standardized in this study. An NSCLC‐targeting regimen (platinum‐vinorelbine) was administered to patients with LCNEC before Rossi There are no potential conflicts of interest.


Somatic cell nuclear transfer (SCNT) has broad applications but is limited by low cloning efficiency. In this review, we mainly focus on SCNT-mediated epigenetic reprogramming in livestock and also describe mice data for reference. This review presents the factors contributing to low cloning efficiency, demonstrates that incomplete epigenetic reprogramming leads to the low developmental potential of cloned embryos, and further describes the regulation of epigenetic reprogramming by long non-coding RNAs, which is a new research perspective in the field of SCNT-mediated epigenetic reprogramming. In conclusion, this review provides new insights into the epigenetic regulatory mechanism during SCNT-mediated nuclear reprogramming, which could have great implications for improving cloning efficiency. Somatic cell nuclear transfer (SCNT) is an assisted reproduction technology for the generation of cloned mammals that involves the culture of donor somatic cells and oocytes, transplantation of donor cell nuclei into enucleated oocytes, activation of reconstructed embryos, and transfer of cloned embryos into surrogates . SCNT enThe first SCNT mammal was a sheep known as Dolly that was born in 1997 , and sinAlthough SCNT has been successfully used to clone many species of mammals with significant improvements in cloning efficiency in more than 20 years since the birth of Dolly, the proportion of cloned embryos that develop to full term remains very low, greatly limiting the application of SCNT technology . To imprDuring development, totipotent embryos differentiate into pluripotent stem cells and subsequently into differentiated cells. Cell fate determination is largely achieved by activating some genes while suppressing other genes through epigenetic modification such as DNA methylation, histone modification, genomic imprinting, and X chromosome inactivation (XCI) . These hde novo DNA methylation  and histone deacetylase (Hdac). Hat opens up chromatin, which allows transcription factor binding and leads to activation of gene transcription, whereas Hdac promotes gene inactivation . After fGenomic imprinting, an epigenetically regulated phenomenon that shows monoallelic parent-specific gene expression, is controlled by the differentially methylated region (DMR) or specific histone modifications. The DMR is protected by DNA-binding complexes composed of Dnmt1, zinc finger protein 57, and tripartite motif-containing 28, and the H3K27me3 mark . In geneFemale mammals have two X chromosomes, whereas only one is present in males. In order to balance gene dosage, female mammals silence one X chromosome through the activity of the Xist gene product, a long non-coding (lnc)RNA on the inactive X chromosome that recruits transcriptional repressors such as PcG proteins . During As epigenetic modification regulates gene expression, disrupted epigenetic modification during SCNT leads to the abnormal transcription of genes related to development in cloned embryos. The persistently high expression of donor somatic cell-specific genes and failure to activate genes related to embryo development are against SCNT-mediated nuclear reprogramming . TherefoPresently, the aim of SCNT-mediated nuclear reprogramming-related research is to improve epigenetic reconstruction in cloned embryos, as the degree of epigenetic reprogramming determines the developmental competence of cloned embryos .Improving DNA methylation reprogramming has been applied in cloned embryos . One wayModifying histone marks is another approach for increasing the development competence of cloned embryos. Hdac inhibitor treatment increases histone acetylation and opens up the chromatin structure, which facilitates the binding of transcription factors that activate genes involved in early embryonic development. Hdac inhibitors have been used to improve the developmental ability of cloned embryos. For example, trichostatin A (the class I and II Hdac inhibitor), scriptaid (a synthetic Hdac inhibitor with low toxicity), and valproic acid all increase histone acetylation levels, especially H3K9ac and H3K14ac, improve gene expression levels in cloned embryos, and thus enhance SCNT-mediated nuclear reprogramming . These rImportantly, with the enhanced development of cloned embryos induced by histone modification improvements, genome imprinting in cloned embryos, fetuses, and offspring is also effectively maintained, suggesting that epigenetic modifications form mutually regulatory networks . H19 knoTargeting XCI is another strategy for improving developmental potential in SCNT. When Xist is deleted, the pattern of X-linked gene expression is corrected in cloned embryos, and the birth rate of cloned mammals is improved . The inhWith the understanding of epigenetic modifications during SCNT, researchers have successfully cloned macaque monkeys . TherefoAn increasing number of studies have shown that the degree of epigenetic reprogramming determines the developmental potential of cloned embryos . HoweverLncRNAs are gene transcripts longer than 200 nucleotides that do not encode proteins but nonetheless play a critical role in gene regulation in nearly all physiologic processes, as well as in cell fate determination during development . A comprRecent studies have shown that lncRNAs participate in many epigenetic modification processes, such as DNA methylation, histone modification, genome imprinting and XCI, and regulate the activation or silencing of genes according to cell function requirements . During After SCNT, epigenetic and gene expression profiles undergo substantial changes in cloned embryos. Naturally, lots of differentially expressed lncRNAs exist during the development of cloned embryos. However, research on key lncRNAs during the SCNT process is very limited. Encouragingly, the existing studies have suggested that lncRNAs can regulate the developmental competence of cloned embryos . StudiesIt is known to be difficult to produce cloned embryos, and their developmental competence remains poor. Moreover, studies on the factors that regulate epigenetic reprogramming during SCNT, especially those investigating lncRNAs, are still limited. To enhance the developmental potential of cloned embryos, a systematic analysis of the factors and mechanisms involved in SCNT is required. In recent years, with technological advancements, particularly the application of single-cell transcriptome sequencing, great progress has been made in discovering and identifying the reprogramming factors related to the development of cloned embryos. To date, lots of novel genes and lncRNAs have been revealed . HoweverEfforts to improve cloning efficiency have also promoted the application of SCNT technology. Presently, a series of agriculturally and economically important animals can be cloned, which could not only enable the protection of endangered species but also accelerate the utilization of livestock. Gland bioreactors can be created through SCNT to produce therapeutic proteins. Animal models can also be generated through SCNT to investigate the pathogenesis of human diseases. Moreover, with the CRISPR/Cas9-mediated genome editing technology, SCNT can produce desired animals or models for specific applications. In short, if cloning efficiency is greatly improved, the application of SCNT technology will be more extensive.SCNT has important theoretical and practical research value. In this review, we present our understanding of SCNT-mediated nuclear reprogramming, especially the factors contributing to low cloning efficiency, and that incomplete epigenetic reprogramming leads to the low developmental potential of cloned embryos. We further demonstrate that the application of epigenetic modification methods can improve cloning efficiency. We also describe the regulation of epigenetic reprogramming by lncRNAs and provide a new research perspective in the field of SCNT-mediated epigenetic reprogramming. The elucidation of these mechanisms has enhanced cloning efficiency and expanded the application of SCNT technology in agriculture, regenerative medicine, and other areas. We believe that with further advances in technology, more molecular mechanisms will be revealed to enhance the development of cloned embryos, and improved cloning efficiency will promote the extensive application of SCNT technology.YH designed the manuscript. XW, JQ, and JL wrote the manuscript. YH, HH, and ZL provided the writing guidance and revised the manuscript. All authors read and approved the final manuscript.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.


Both excipients were added in the composition for their mucoadhesive properties. The formulative space was rationalized based on the drug physicochemical stability and the main critical quality attributes of the formulation, e.g., rheological properties, syringeability, mucoadhesiveness and in vitro penetration of budesonide in porcine esophageal tissue. The obtained results demonstrated that gums allowed a prolonged residence time. However, the concentration of the mucoadhesive polymer has to be rationalized appropriately to permit the syringeability of the formulation and, therefore, easy dosing by the patient/caregiver. Eosinophilic esophagitis (EE) is a chronic immune/antigen-mediated esophageal inflammatory disease for which off-label topical corticosteroids  are widely used in clinic. In general, thickening excipients are mixed with industrial products to improve the residence time of the drug on the esophageal mucosa. The compounding procedures are empirical and the composition is not supported by real physicochemical and technological characterization. The current study aimed to propose a standardized budesonide oral formulation intended to improve the resistance time of the drug on the esophageal mucosa for EE treatment. Different placebo and drug-loaded (0.025%  Eosinophilic esophagitis (EE) is a chronic immune/antigen-mediated esophageal inflammatory disease associated with esophageal dysfunction resulting from severe eosinophil-predominant inflammation ,2. EE trWhen an authorized medicinal product is not available on the market, the compounding of extemporaneous preparations by the community and hospital pharmacists is crucial to meet the special needs of patients ,17. The In the case of the BU for EE, a certain number of studies in the literature suggested the clinical efficacy of viscous preparations. Frequently, they have been prepared by mixing a commercial sterile suspension of BU to be nebulized, which is indicated in the treatment of bronchial asthma and in infants and children with croup, with a thickening agent  ,20,21. AThe development of standardized formulations can be a valid strategy to support pharmacists in their activities, reducing heterogenicity and uncertainty in compounding procedures, improving the quality of the final preparation, at the same time. Indeed, the availability of well set up and validated operating procedures is instrumental in assuring the quality of the magistral preparation.The aim of this work is to propose a standardized BU oral formulation to improve the residence time of the drug on the esophageal mucosa. Starting from a formulation already in use in hospital pharmacies, based on xanthan gum, six pharmacists were enrolled in the compounding process to verify its reproducibility. On the bases of these results, the formulation and the compounding procedures were optimized. Considering the evidence, reported in the literature, on the combined use with galactomannans, guar gum was selected to compare its performances, when in ratio 1:1, as opposed to xanthan gum alone. The formulative space was rationalized based on the drug’s physicochemical stability and the main critical quality attributes of the formulation, such as rheological properties, mucoadhesiveness and in vitro penetration of BU in porcine esophageal tissue. Micronized Budesonide (BU) was obtained from Farmabios, Gropello Cairoli, PV, Italy. Guar Gum  was kindly gifted by Lamberti spa, Albizzate, VA, Italy. All other materials were obtained from the named supplier: Xanthan Gum , (ethylenedinitrilo)tetraacetic acid disodium salt dihydrate (EDTA), sodium benzoate, sodium saccharin , glycerin, sodium dihydrogen phosphate, orthophosphoric acid, ethanol chemical grade .Acetonitrile was HPLC-gradient grade. Purified water was obtained from the purification system Milli-Q, according to Ph. Eur. 10.0 ed.w/w was added. All substances were mixed to form a homogeneous mixture, then, the exact weighted amount of BU (1 mg/4 mL) was added and carefully mixed. Purified water was weighed and added, then stirred until a uniform system was obtained. Different percentages of gums were used: F1P was prepared using XG 2% w/w, F2P with XG:GG 2% w/w, F3P with XG 1.5% w/w and F4P with XG:GG 1.5% w/w. Placebo formulations were prepared for rheological and technological evaluation. BU was added only to obtain final loaded formulations F1, F2 and F4. The composition of the formulations is reported in The exact amount of glycerin (23.6 mL) was weighed on an analytical balance and poured into a beaker. Sodium saccharin, EDTA and sodium benzoate were crushed to a fine powder with mortar and pestle, then, the exact amount of each was weighted and the powders were transferred into the same beaker. Then, XG or the mixture of gums (XG:GG) 1/1 The pH was measured at time T = 0, using a pHmeter CyberScan 1100 .v/v/v) and analyzed [About two grams of each formulation were exactly weighed and transferred into a 10 mL amber glass volumetric flask, bringing up to volume with ethanol. Then, the flask was placed in an ultrasound bath for 20 min. The sample was then centrifuged for 5 min at 3500 rpm. A portion of the supernatant was diluted to 2:5 with the mobile phase composed of phosphate buffer pH 3.2:acetonitrile:ethanol  at 40 °C. The drug content was measured at time T = 0, T = 10, 20, 60 days. Evaluation of BU content was also performed after T = 30 days at room temperature exposed to light.The steady and dynamic shear rheological properties of the formulations were carried out using a controlled stress/strain rheometer Anton Paar MCR 302  equipped with a plate-plate geometry (25 mm diameter and 500 μm gap). The temperature was controlled by a Peltier system on the bottom plate. Each sample was transferred to the rheometer plate and was then equilibrated at 25 °C for 5 min before steady and dynamic shear rheological measurements were taken.−1 at 25 °C. The shear stress-shear rate data were fitted to the well-known power law and Casson models [Flow behavior was evaluated at controlled strain mode to obtain flow rheological data (shear stress and shear rate) over a shear rate range of 0.1–100 sn models  to descr−1 at 2% strain using a small-amplitude oscillatory rheological measurement. The applied 2% strain was confirmed within the linear viscoelastic region by strain sweep measurement. Frequency sweep tests were also performed at 25 °C. The RheoCompass software  was used to obtain the experimental data and to calculate the storage (or elastic) modulus (G′), loss (or viscous) modulus (G″) and loss factor (tan δ = G″/G′).Dynamic rheological data were obtained from frequency sweeps over the range of 0.628–62.8 rad s®, VWR International, Milan, Italy) filled with 9 mL formulation was positioned in the dynamometer holder, downward needle. The plunger end of the syringe was placed in contact with a 50 N loading cell. Testing was carried out at the crosshead speed of 0.5 mm/s. The loading force required to displace the plunger was measured as a function of plunger displacement. The following parameters were also determined from the force-displacement plot:Plunger-stopper break loose force : the force required to initiate the movement of the plunger;Maximum force (MF): the highest force measured before the plunger finishes its course at the front end of the syringe;Dynamic glide force (DGF): the force required to sustain the movement of the plunger to expel the content of the syringe.The measurement of the injection force was performed in compression mode by using a software-controlled texture analyzer . A 10 mL syringe  in A dose of the selected formulation was deposited onto a 3 × 2 cm mucosal surface corresponding to a total amount of about 500 mg. Then, the porcine esophageal membrane was placed on the sample support and pH 6.8 phosphate buffer solution (PBS) was dropped at a rate of 1 mL/min to simulate the physiological environment and the saliva swallowing. The test was performed at room temperature.The residence time was qualitatively estimated by checking the time required for each formulation to be thoroughly washed away from the surface of the mucosa exposed to the washing medium. In all cases, after 30 min from the beginning of the experiment, the apparatus was dismantled, and the applied test sample was peeled away using an adhesive tape strip . The muc2 = 0.99930; 22S: R2 = 0.99999). The analysis was carried out by HPLC-DAD  using an RP-C18 column  with pre-column . The mobile phase was composed of phosphate buffer pH 3.2:acetonitrile:ethanol . The elup < 0.05 level.Tests for significant differences between means were performed by the one-way ANOVA followed by Turkey-Kramer post-analyses. Differences were considered significant at the As the number of patients suffering from EE is increasing, among both adults and young population, the need to have a standardized topical dosage form has grown. Moreover, the widespread habit of mixing the industrial inhalation product with sweeteners  should not be the first choice. Instead, the use of a viscous preparation as a vehicle is well established in improving the efficacy of topical corticosteroid administration. The proprietary blend Mucolox™ gave good results , but it XG is a polysaccharide, largely applied in the food industry. It is highly stable in a wide range of pH and ionic strength, and, dispersed in water at moderate temperatures, it increases its viscosity even at low concentration ,31. Tempw/w, according to the Italian Pharmacopoeia  or the handling during the application, F1P was modified and a mixture of XG and GG was used F2P, . The add−1, which is the highest that can be reached with the experimental setup used. In addition, samples loaded with the active principle were investigated and it was concluded that the behavior was not influenced by the presence of BU  and XG:GG (F2P and F4P), mixed at room temperature. They have different behaviors: formulations containing the mixture of the gums have higher shear stress in comparison to those containing only XG, as already reported in the literature , but theA different behavior among the formulations was also evidenced from the performed dynamic shear measurements. In particular, as shown in p < 0.0001).To identify elastic or viscous behavior, loss factor values can be calculated. When this value is smaller than the unit, a sample is more elastic than viscous, and this has been suggested as a rheological criterion for safe-swallow foods meant for dysphagia . All proThe administration of this preparation in very young patients was performed by caregivers, normally the parents, by means of a big syringe containing 60 mL of the formulation. During this treatment, some difficulties due to the hard extrusion of the formulation from the syringe were described by the users. The values measured for the injection force measured for placebo formulations are reported in In the force vs. displacement plot of low-viscosity formulations , two difp = 0.3890), suggesting that the force required to initiate the movement of the plunger was independent from the formulation.PBF values were not statistically different among the tested products (p = 0.1565). Moreover, in the shear stress plots, F4P showed also a pseudoplastic behavior that could not be detected with the syringeability test, probably because the applied force felt below the useful range of stress. In the case of this pseudoplastic formulation, taking into consideration the loss factor value, another rheological parameter, we can observe that it was higher if compared to the other formulations, suggesting that F4P has a more pronounced viscous component, even if its overall behavior was mainly elastic. However, overall results show that in any case low forces are needed to extrude the formulations from a syringe, despite the differences highlighted in the rheological studies. The measured extrusion forces showed that F4P required a slightly higher extrusion force (in terms of DGF) with respect to the other formulations, even if the differences were not statistically significant  can interact more strongly with mucin glycoprotein [The good mucoadhesive properties can be due to the presence of free –COO¯ groups of XG. As a matter of fact, it is well known that polymers that exhibit a high density of available hydrogen bonding groups (–COOoprotein . MoreoveAs it is known, penetration of BU into the mucosa is the final combination of many events; the physicochemical characteristics of the molecule, and the mucoadhesive properties and viscosity of the formulation. The advantage of increased viscosity of the formulation is a reduced outflow from the mucosal surface, with a consequent increase in the contact time of the drug with the mucous membrane, and therefore, in its penetration into the tissue. On the other hand, an excessive viscosity could obviously create problems in the extrusion from a syringe. The results showed that the use of the two gums in combination can help to modulate the viscosity of the formulation, however, remaining within an optimal range such as to be retained onto the mucosal surface for enough time. Moreover, thanks to its pseudoplastic behavior, the extrusion of such formulations through a syringe can take place in a feasible way. Unfortunately, this aspect could not be fully investigated through the experiments carried out in this work, since the viscosity data should be collected in a higher shear range to highlight a correlation between syringeability test and viscosity measurements.The prepared formulations showed suitable technological and rheological characteristics for the treatment of EE. As the addition of GG to XG caused an increase in viscosity, the choice of a mixture of gums allows the use of thickening agents in a reduced amount. In this case, the obtained results were not significantly different from those measured with the formulation already in use. Considering the limited percentage of BU absorbed in the in vitro penetration experiments, further evaluations should be carried out to rationalize the drug content and reduce the risk of systemic side effects.


The resulting continuous β-sheet nanocrystal network exhibits improved compressive strength and stiffness over the initial network lacking β-sheets of up to 30 MPa  and 6 MPa  respectively. The network demonstrates improved resistance to strong acid, base and protein denaturants over 28 days.The high toughness of natural spider-silk is attributed to their unique β-sheet secondary structures. However, the preparation of mechanically strong β-sheet rich materials remains a significant challenge due to challenges involved in processing the polymers/proteins, and managing the assembly of the hydrophobic residues. Inspired by spider-silk, our approach effectively utilizes the superior mechanical toughness and stability afforded by localised β-sheet domains within an amorphous network. Using a grafting-from polymerisation approach within an amorphous hydrophilic network allows for spatially controlled growth of poly and poly(valine- It is known the β-sheet structures in silk-inspired materials generate increased mechanical properties. Here, the authors report on a method of creating silk-inspired materials using in situ formation of β-sheets in an amorphous polymer to replicate the structure of silk and increase the mechanical properties. The ability to form these secondary structures in response to various environmental cues , as well as their diverse functionality, has made them a cornerstone of biochemical research. The importance of how these secondary structures affect material properties is exemplified in naturally occurring dragline spider-silk; a natural polypeptide-based structure which displays a high tensile strength comparable to high tensile steel10. Specifically, its excellent mechanical properties are attributed to the spatial arrangement of the amino acids within the polypeptide, which arrange to form higher-order β-sheet architectures through hydrogen bonding primarily from the hydrophobic amino acid residues 14. Surrounding these β-sheet architectures is an arrangement of semi-amorphous, highly extendable glycine-rich regions. This specific arrangement  leads to spider silk displaying incredible toughness—a tensile strength between 0.88–1.5 GPa coupled with an extension at break of 21–27 %13. This incredible mechanical potential garners significant interest towards the utilisation of polypeptides in synthetic materials, such as hydrogels, films and fibres19 for a wide range of applications, including tissue engineering and drug delivery22.Polypeptides are the fundamental building blocks of many naturally occurring materials due to their ability to fold into specific yet complex molecular architectures, including α-helices and β-sheets29. N-Carboxyanhydride ring-opening polymerization (NCA ROP) is comparatively a cheaper and a more simple method towards the synthesis of large quantities of polypeptides with a greater potential to create long chain polypeptides, although the method lacks the same level of sequence control observed in solid-state synthesis and genetic engineering35. Despite this lack of control, we have recently shown the star-shaped polymers synthesised from NCA ROP ‘arms’ are able to observe equivalent, if not better anti-microbial activity when compared to sequence-controlled polypeptides )36. Specifically, when designing polypeptides for β-sheet formation, the control of hydrophobic association is fundamental, yet difficult to achieve in the laboratory. This generally leads to ill-defined, uncontrolled and poorly soluble aggregates37, which greatly impacts the mechanical potential of the formed structures (e.g. bulk hydrogels)37.When synthesising β-sheet forming peptides, both the synthetic technique and the molecular components need to be carefully considered. Synthesis via genetically modified microbes and solid-state synthesis are both common methods for producing peptides, but can require acute knowledge of the peptide sequences that the genes are derived from in the case of the former, and in the case of the latter, solid-state synthesis is limited to the fabrication short chain peptides due to decreasing reaction yield with increased coupling cycles for each amino acid22. A grafting-to approach may then be used to subsequently incorporate peptides into a hydrogel39. For example, Clarke, Pashuck40 have reported the use of grafted β-sheet forming peptide sequences to the polymeric backbone of hydrogels to induce self-healing properties, however because the peptides remained short with a high hydrophilic residue content, mechanical performance was limited. When employing NCA ROP to synthesise linear polypeptides, hydrophilic blocks such as poly(ethylene glycol)35 or hydrophilic polypeptides33 have often been used to prevent this uncontrolled association from occurring, but their inclusion is highly detrimental to the polypeptides mechanical potential due to the disruption of the hydrophobic association35. Natural spider-silk uses blocks of alanine to form β-sheets during the natural silk fiber production process which involves specific environmental changes to encourage the formation of the β-sheets10. However, it should be noted that although it is well known that alanine forms β-sheets at low molecular weight , high molecular weight (larger blocks) alanine has been shown to form α-helices43. Our group has had previous success using valine NCA ROP to form β-sheets, making valine a viable option for constructing spider-silk inspired materials44.Generally, attempts commonly used to allay the uncontrolled association of hydrophobic residues and subsequent unusable aggregates introduce a high ratio of hydrophilic components to form polypeptidesHerein, we report a simple method to mimic spider-silk by creating localised β-sheet domains, which endow the formed bulk hydrogels with superior mechanical performance. A grafting-from approach is used to incorporate β-sheet forming polypeptides into a pre-fabricated three-dimensional hydrophilic network, which acts as a template to guide β-sheet assembly to mimic the amorphous matrix found in spider-silk. Free amine groups embedded in the networks are subsequently used as a site for NCA ROP of valine and glycine resulting in the spatial and controlled formation of crystalline β-sheet nanocrystals. The physical characteristics of the β-sheet reinforced networks are reported in terms of compressive characteristics, revealing a three-orders of magnitude increase in toughness attributed to the overall β-sheet network rather than the general effect of increased hydrophobicity. Given the natural ease and versatility of polypeptide synthesis via NCA ROP, this synthetic approach can facilitate the incorporation of β-sheets in a far broader range of materials moving into the foreseeable future.L-valine)  chains are known to result in β-sheet formation, resulting in crystalline and rigid β-sheet regions being observed45. Thus, glycine was introduced to act as a β-sheet destabilizing residue12 and being responsible for amorphous β-sheet regions in spider-silk10. Thus, L-valine NCA  and glycine NCA (Gly NCA) were synthesised and then used to form crystalline and amorphous β-sheet regions respectively.To demonstrate a proof of concept, an initial network was synthesised by free radical polymerization with pendant amine groups followed by directed β-sheet incorporation via NCA ROP Fig. . The iniThe polymerization of β-sheet forming polypeptides via NCA ROP is grown from the available amine groups of the polymer backbone as initiating sites. Hence, Val NCA monomer concentration was varied from 30 to 360 mg/mL to yield increasing pVal chain lengths Table . As expe12, it is used to replace small quantities of Val NCA at 5 and 10 mol % within a constant total molar concentration at 2.51 mmol/mL  in a solution  and insufficient swellability to take on enough overall mass in water  was performed for both hydrogels and cryogels. Examination of the initial networks showed smooth surfaces for both hydrogels and cryogels as expected with highly swellable polymeric networks   binding assay under fluorescence microscopyage Fig. , the labage Fig. , resultiage Fig. . Howeverne) Fig. . In the ne) Fig. . At closne) Fig. , the β-s−1 , indicating successful polymerization48. The amide A band is shown to have a small shoulder at 3300 cm−1 once glycine is introduced, which is more evident in samples containing greater amounts of glycine  has been identified as the most sensitive spectral region corresponding to different polypeptide secondary structures and used to determine their conformations49. Hereby, the strong amide peak around 1627 cm−1 with a weak shoulder at 1690 cm−1 suggests the formation of β-sheet secondary structure adopted in the mixture of antiparallel and parallel conformations, respectively49. The shoulder assigned to the parallel conformations slightly increases with increasing glycine content  as the amide V bond is not diminished in the glycine containing gels51. Other distinguishing features of the β-sheet incorporated networks include amide II band (N–H in-plane bending) near 1530 cm−1, amide V band (N–H out-of-plane bending) around 710 cm−1, and the pVal aliphatic side-chain C–H stretch peaks around 2965 cm−1 (which is diminished slightly one glycine is introduced)50. Also, the characteristic peaks of the initial network are still clearly present in the spectra of β–sheets-incorporated networks, including C–H stretch on the polymer backbone, C = O stretch of ester group and C–O–C stretch in the side chain. The increase in β-sheet content with increasing Val NCA concentrations in the ROP process was also highlighted with the intensity of the β-sheet band (amide I) relative to the polymer backbone stretch in the initial network (C–O–C stretch)  spectroscopy and X-ray Diffraction (XRD). The FTIR spectra Fig.  shows a h) Table . The high) Table . XRD pat35. The stress-strain curves obtained indicate hydrogel stiffening and strength consistently increased with increasing pVal formed β-sheet content with the Young’s Modulus increasing by three orders of magnitude (from 2 kPa to 9.4 MPa)  Fig. . These fPa) Fig.  provides3, implying that the overall integrity of the gel remains intact despite the introduction of β-sheet forming residues. However, a loss in strength and subsequent increase in compressibility defines the tunable nature of these materials  (PMMA), a typical hydrophobic polymer that is hard to crystallize, was synthesised using RAFT  Fig.  shows thThe β-sheets-incorporated networks also exhibited remarkable stability at harsh pH conditions (2 M HCl and 2 M NaOH solution) and in the presence of protein denaturants (6 M GdnCl) Fig. . After 462. Using our grafting-from strategy, β-sheet regions can be loaded into 3D-printed networks, endowing them with the enhanced physical properties defined above  grafts of up to 79 repeat units on average which would proceed to form a continuous β-sheet nanocrystal network. Subsequently, compressive strength was found to increase by 3 orders of magnitude up to 9.9 MPa for the hydrogels and 2 orders of magnitude up to 30 MPa for the cryogels. Furthermore, the properties of these networks can be easily tuned using glycine, with poly grafts introducing an increased compressibility from 40 to 60% without a significant loss in toughness. This easily employable approach provides a powerful tool in the preparation of unique hybrid networks furnished with β-sheets and can be further used to tune the mechanical and degradation properties in other hydrogels for biomedical functionality and materials science.L-valine (≥98%) and glycine (≥98%) were purchased from Merck and used as received. Triphosgene (98%), (+)-α-pinene (99%), anhydrous n-pentane (>99%), anhydrous N,N-Dimethylformamide (DMF) (>99.8%), 2-aminoethyl methacrylate hydrochloride (97%), triethylamine , oligo(ethylene glycol) methylether methacrylate , oligo(ethylene glycol) dimethacrylate , ammonium persulfate , N, N, N’, N′-Tetramethyl ethylenediamine , Diphenylphosphine oxide  Thioflavin T (ThT), Guanidine hydrochloride , phosphate buffered saline (PBS) tablets, protease from streptomyces griseus (Type XIV), N-(3-Dimethylaminopropyl)-N’-ethyl carbodiimide (EDCI), 4-(Dimethylamino)pyridine  and N, N, N’, N”, N”-pentamethyl diethylenetriamine  were all purchased from Sigma Aldrich and used as received. Anhydrous and deoxygenated Tetrahydrofuran (THF)  was obtained by distillation from benzophenone and sodium metal under argon. AR grade hydrochloric acid (HCl), sodium hydroxide (NaOH) and other solvents were purchased from Chem-Supply Pty. Ltd. and used without further purification. L-valine (≥98%) was purchased from Merck and used as received. Anhydrous and deoxygenated Tetrahydrofuran (THF)  was obtained by distillation from benzophenone and sodium metal under argon. Deuterated chloroform (CDCl3) was purchased from Cambridge Isotope Laboratories and used as received. 2-(((butylthio)carbonothiolyl)thio)propanoic acid (TTC-1) was received from Dulux Group Australia and used as received. Methyl methacrylate  was de-inhibited by basic alumina prior to use.L-valine NCA was synthesised using a modified procedure from previously reported literature44. Briefly, L-valine  was suspended within 80 mL of anhydrous THF in a two-necked round bottom flask under argon. Triphosgene  was added and the mixture was continuously stirred 60 °C for 2 h or until all valine had dissolved. The reaction mixture was sparged with argon into a saturated NaOH solution for 1 h after which solvent was removed in vacuo  until equilibrium. The reduced mixture was redissolved in anhydrous 50 mL ethyl acetate which was chilled in 5 °C and then washed with saturated brine at 5 °C and 0.5 w/v % sodium bicarbonate aqueous solution at 5 °C with the organic phase being washed after separation each time. The resulting organic phase was dried using magnesium sulphate with the filtrate being reduced in vacuo  until equilibrium. The residue was recrystallised using n-pentane overnight. The resulting crystals were filtered and reprecipitated to afford a white powder (~60% Yield). 1H NMR : δH 0.92 , 2.00–2.18 , 4.08 , 8.84 .63. Glycine  was suspended within a mixture of 250 mL of anhydrous THF and 21.2 mL (+)-α-pinene (133 mmol)in a two-necked round bottom flask under argon. Triphosgene  was dissolved and added dropwise over 1.5 h while mixture was continuously stirred 70 °C and stirred for a further 1 h. The reaction mixture was sparged with argon into a saturated NaOH solution for 1 h after which the suspension was filtered and the filtrate was reduced in vacuo  until equilibrium. The resulting suspension was redissolved in THF and recrystallized using n-pentane overnight. The supernatant was removed and recrystallization was repeated two more times. The resulting white solid was filtered and washed with n-pentane . 1H NMR : δH 4.33 , 8.84 .Glycine NCA was synthesised using a modified procedure from previously reported literature2 for a few minutes. After addition of APS (20 mg) and TEMED (25 µL) , the solution was sonicated and separated into 1 mL batches in 3 mL syringes and allowed to stand at room temperature for 3 days. The resulting gels were swelled in DI water and washed six times to remove unreacted reactant. They were dehydrated in vacuo  for 2 days and stored in vacuo at room temperature for further use.2-aminoethyl methacrylate hydrochloride  was first deprotonated with TEA  in 20 mL of DI water overnight. The mixture was mixed with co-monomer OEGMEMA  and crosslinker OEGDMA  and degassed with NThe gel precursor solution was prepared and transferred to the syringes in the same procedures as described above. Then the solution in the mould was frozen at −18 °C and kept at this temperature for 3 days. After thawing and being washed with water, the cryogel sample was dried and stored in vacuo for further use.The following synthesis of HB360 is used as an example of the formation of β-sheet incorporated networks  was then switched on, and the reaction mixture was left at room temperature with occasional agitation under positive N2 pressure for 3 days. A small aliquot (~100 μL) of the supernatant was taken for 1 H NMR analysis to check monomer conversion rate, according to an established calibration curve as described below. After the reaction, the supernatant was removed, and the network was washed with DMF (3 × 3 mL) and sonicated in DMF (3 mL) for 30 min. To further remove potential non-grafted monomers/polymers, the network was then washed with THF (6 × 3 mL). Finally, the network was dried and stored under vacuum.The initial network hydrogel or cryogel was swollen in 3 mL of TTC-1 (RAFT agent)  dissolved in anhydrous DMF. EDCI  and DMAP  was then added in the mixture with the reaction sealed at room temperature with occasional agitation under vacuum for 2 days to the RAFT agent. Afterwards, the network was washed with anhydrous DMF for several times to remove the free RAFT agent and remaining catalyst until the network colour did not fade. The network attached with RAFT agent was then transferred to the MMA monomer solutions (in 3 mL anhydrous DMF) at different concentrations  in presence of PMDETA . The reaction mixture was sealed and degassed using nitrogen for approximately 1 h. The UV light source  and TEA (600 µL) overnight and then washed with anhydrous DMF (6 × 10 mL).2-Aminoethyl methacrylate hydrochloride , co-monomer OEGMEMA  and crosslinker OEGDMA  were dissolved in 25.8 mL of DMF: deionised water (7:3), with TPO photoinitiator  dissolved into the solution just prior to printing. The 3D printing was performed on an ANYCUBIC Photon 3D Printer with a λThe swollen networks were transferred to vials containing 50 mL of valine NCA in anh. DMF (120 mg/mL). The vials were then sealed and agitated at room temperature under vacuum for 3 days. The resulting networks were then washed with six aliquots of DI water, followed by subsequent washing with DMF (3 × 3 mL) before being sonicated in DMF (3 mL) for 30 min.1H) Nuclear Magnetic Resonance (NMR) spectroscopic analysis was performed on a Varian Unity Plus 400 MHz spectrometer using deuterated chloroform (CDCl3) as the solvent. Attenuated total reflection- Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy was carried out using a Bruker Tensor 27 FTIR, with GladiATR ATR attachment obtained from Pike Technologies. The FTIR was equipped with OPUS 6.5 spectroscopy software from Bruker Optik GmBH. X-ray diffraction (XRD) spectroscopy was carried out on a Bruker D8 Advance Diffractometer, using standard Ni-filtered Cu kα radiation. Fluorescent images of ThT stained gel networks were acquired on a Leica TCS SP2 confocal laser scanning microscope (CLSM), using excitation wavelength of 490 nm. Scanning electron microscope (SEM) images were acquired using a FEI Quanta 200 ESEM FEG. Samples were pre-coated with gold using a Dynavac Mini Sputter Coater prior to imaging. Thermogravimetric analysis (TGA) was performed on a TGA/SDTA851e, Mettler Toledo in air with heating rate of 10 °C·min−1.Proton  at 5 different concentrations  diluted with CDCl3 (900 µL). Integration of the Val NCA doublet peak at 0.92 ppm relative to the methyl DMF signals at 2.88 ppm and 2.96 ppm determined for the 5 Val NCA concentrations, afforded a calibration curve as shown in Supplementary Fig. A calibration curve was generated from 3 (900 µL). These solutions were analysed by 1H NMR by normalising against the same peaks of DMF, to determine Val NCA concentrations of each solution. This was then used to calculate the monomer conversion , the network was dried under heat in vacuo  for 24 h and then swollen in water until it reached equilibrium swelling. The weight of dry and fully swollen samples was determined by analytical balance and denoted as Wd and Ws, respectively. The equilibrium swell ratio (Q) is defined by Eq. . The samples were removed from each vial at certain time point and washed in deionised water. Then the samples were dehydrated by soaking in ethanol for 1 h followed by drying in vacuo  for 24 h. Finally, the samples were weighed, and the mass values obtained were plotted against time to obtain the degradation profiles.The compressive strain-stress curves were obtained using an Instron Microtester 5848 equipped with a 2 kN static load cell and Bluehill material testing software. cylindrical hydrated network samples  were compressed to their maximum strain between two parallel plates at a crosshead speed of 0.1 mm/second. Engineering stresses and strains were recorded.Supplementary InformationPeer Review File