Seasonal reproduction limits productivity, flexibility, and profitability in commercial sheep production. Hormonal and (or) photoperiodic manipulation can be used to control estrous cycles in sheep and reduce limitations that are imposed by the seasonal anestrous but are often impractical or incompatible with the extensive management systems preferred for ruminant livestock. Thus, the current study investigated the use of selection to improve realized fertility  following an out-of-season spring joining period (May and June) in a crossbred sheep population.Over 17 years, estimated breeding values (EBV) for fertility in selected (S) ewes increased by 0.175 (0.01 per year). The mean EBV for fertility of S ewes was greater than that of control ewes by year 10 (P = 0.02), and the fertility of adult (≥ 3 years old) ewes reached 0.88 ± 0.05 by year 17. Lambing began approximately 140 days after the introduction of rams, and 64% of the S ewes that lambed did so in the first 17 days of the potential lambing season, which indicated that most of the S ewes were cycling at the time of ram introduction and were not induced to cycle by the introduction of breeding males . Animals in the S line had modest increases in body weight and scrotal circumference. A modest negative trend in the additive maternal effect on birth weight was observed but was reversed by additional selection on EBV for maternal birth weight. The heritability of litter size in autumn lambing was low (0.04) and could potentially limit the response to selection for this trait.Selection improved realized ewe fertility in out-of-season mating, with absolute increases of approximately 1% per year in the percentage of joined ewes that lambed in the autumn. Genetic antagonisms with other performance traits were generally small. A modest antagonism with maternal breeding values for birth weight was observed but it could be accommodated by selection on EBV for maternal birth weight. Our results support results of previous studies that indicate that these selected ewes had one of the shortest seasonal anestrous periods reported for temperate sheep breeds and that spring-lambing lactating ewes from the selection line were capable of relatively rapid rebreeding in the spring. Seasonal changes in reproductive activity are commonly observed in sheep and goats exposed to the circannual fluctuations in day length that occur at temperate latitudes . A distiAttempts to develop lines of sheep with improved fertility in autumn lambing –8 generaThis study was conducted in Blacksburg, VA at 37° N and 80° W. The base population was a composite line with 50% Dorset, 25% Rambouillet, and 25% Finnsheep breeding . In autuThe S and EC ewes were joined with rams during 6 weeks from May 1, and GC ewes were joined with rams during 4 weeks from October 1. The selection objective was to improve fertility during the annual spring joining period. However, to avoid keeping potentially large numbers of barren ewes, S and EC ewes were also joined with groups of Suffolk rams during 30 d from August 1. Lambs from this joining period were born in January and weaned in early March. We assumed that S and EC ewes that had lambed in January would not be less fertile than ewes that had lambed in October at the subsequent May joining period. Figure The experiment was divided into three phases: 1, 2 and 3. In Phase 1 (1989 through 1993) , 11, S aPhase 2 (1994 through 1998) included modifications based on the results from Phase 1 . Based oIn Phase 3 (1999 through 2005), selection continued in the S line, but the GC line was terminated, and no new replacements were added to the EC line. Selection procedures in Phase 3 were the same as those in Phase 2. However, subsamples of S and remaining EC ewes were used in several semi-intensive studies –16, in wEwes in all lines were maintained throughout the year on grass-legume pastures that comprise primarily tall fescue, orchardgrass, and white clover. Grass-legume hay was fed as needed in January through March. Ewes received alfalfa hay and a supplement of grain in late gestation and during lactation. Lambs were weighed and docked within 24 h of birth. Male lambs remained intact. Lactating ewes and their lambs were maintained in an elevated feeding facility with an expanded-metal floor. Lambs had access to creep feed after approximately 3 weeks of age and were weighed and weaned at approximately 60 d of age. Lambs remained in the same facility, were fed a high-concentrate diet ad libitum for 60 d after weaning, and were weighed at approximately 90 and 120 d of age. Scrotal circumference was measured on male lambs at approximately 60, 90, and 120 d of age but was not recorded in 1991. Replacement rams and ewes were identified by 150 d of age, removed from the feeding barn, and managed on grass-legume pastures with a supplement of grain as needed to achieve target minimum breeding weights of 30 kg for females and 50 kg for males at approximately 210 d of age. All lambs of S ewes that attained the target breeding weight and were free of obvious structural defects were retained and joined with rams. The number of replacement S ewes was reduced to approximately 42  before the second joining period by removing yearling ewes that did not lamb and had the lowest average dam fertility (Phase 1) or EBV for fertility (Phases 2 and 3). Housing, management, and data collection for lactating GC ewes and their lambs were the same as for autumn-born lambs. Most GC ewe lambs were retained to meet replacement needs for the GC and EC flocks.The intensity of lamb performance recording was relaxed in some years during Phase 3. Lambs were weighed only at 60 and 120 d in 2001, and 60 and 90 d in 2003. In 2002, 15% of the available ram lambs were identified after weaning as possible replacements based on EBV for fertility; 120-d weights were recorded for these ram lambs and all ewe lambs, but the remaining ram lambs were not retained. In 2004, 82% of the male lambs and 30% of the female lambs were removed from the study after recording weaning and 90-d BW. In 2005, 23% of the lambs of both sexes were removed after recording weaning and 90-d BW. Measurement of SC was discontinued after 2000.Autumn-born ram lambs selected for use in the spring at 7 to 8 months of age were subjected to an examination for breeding soundness a few days before joining. In small pens, the ram lambs were exposed to adult ewes that had been brought into estrus. Ram lambs that did not serve any ewes on the first day of testing were held overnight in pens adjacent to those containing estrus ewes and tested again on the following day. Twenty five to 35% of the candidate ram lambs did not serve estrus ewes and were discarded.For this study, data on fertility and litter size came only from spring joining periods of S and EC ewes. Records of reproductive performance for spring-lambing GC ewes were used only to characterize their fertility during the autumn joining period. Likewise, birth weight records came only from autumn-born S and EC lambs; this decision was based on differences in direct and maternal heritabilities for birth weight between lambs born in autumn and spring . PostnatGenetic parameters and EBV were estimated using Bayesian methods, multivariate animal models, and the BLUPF90 software . The addth animal; th animal; th service sire in the th lambing year; and The model for ewe fertility and litter size was:th animal and the th dam; th dam; Lamb birth weight and BW and SC at 60, 90, and 120 days were analyzed as separate traits. Before analysis, birth weights were adjusted for dam age and type of birth and postnatal BW and SC were adjusted for effects of dam age and type of birth and rearing using multiplicative adjustment factors derived from the data and following procedures from . The modEstimated breeding values for fertility were derived for univariate linear and threshold models. Mean EBV for fertility were calculated by year and line and were based on the ewes that were joined in each line and year or, for line S, the average EBV of each joined pair . This apFertility differed with ewe age Fig. . Adult eThe heritability of realized fertility during the spring joining periods was 0.07 ± 0.01 for the linear model and 0.15 ± 0.04 for the threshold model Table . PermaneThe heritability estimate for lamb BW was low (0.05 ± 0.03) at birth but increased with lamb age to 0.19 ± 0.04 at 120 d  for 2-year-old ewes and 0.08 ± 0.05 (P = 0.11) for adult ewes but increased in Phase 2 to 0.11 ± 0.06 (P = 0.08) for 2-year-old ewes and 0.14 ± 0.04 (P = 0.001) for adult ewes. Fertility of 2-year-old and adult S ewes in Phase 2 averaged 0.73 ± 0.09 and 0.86 ± 0.05, respectively. By comparison, fertility of spring-lambing yearling, 2-year-old, and adult GC ewes averaged 0.74 ± 0.04, 0.94 ± 0.02, and 0.93 ± 0.01, respectively. In yearlings, EC ewes had higher fertility than S ewes in Phase 1 (0.35 ± 0.06 versus 0.11 ± 0.03). However, the advantage in fertility for yearling EC ewes declined from 0.24 ± 0.07 (P = 0.001) in Phase 1 to 0.09 ± 0.05 (P = 0.08) in Phase 2, and fertility of yearling ewes in the S line further increased to 0.28 ± 0.04 in Phase 3.Genetic trends in fertility in line S were based on the mean EBV for fertility of each pair of prospective parents Fig. . As expeEBV for fertility of ewes in the S, EC, and GC lines were compared using EBV of ewes joined in each line and year Fig. . The EC Additive genetic trends in EBV for birth weight were not observed in any of the lines Fig. . HoweverThis study documents the additive genetic effects on spring mating behavior in sheep. The heritability estimate for fertility during the spring joining period from the linear model was low (0.07 ± 0.01), but consistent with previous estimates of heritabilities of fertility in sheep . HoweverOver the 17 years of the study, fertility of line S increased by 0.01 per year  and the average EBV for fertility at the end of Phase 3 was 0.15 ± 0.03. Means of EBV for fertility from the linear model at the end of Phase 2 were 0.10 ± 0.01 for the S and − 0.032 ± 0.019 for the EC line. However, the SE for genetic trends and line differences in EBV for fertility did not explicitly account for genetic drift. Al-Shorepy and Notter  used proA limitation of the experimental design was that EC ewes, born in March and joined for the first time in May at 14 months of age, were on average 7 months older than the S ewes at each joining. This problem could have been avoided by transferring frozen embryos from GC ewes into EC donors in the spring, but we rejected this option because of concerns regarding estrous induction and embryo transfer with frozen embryos in unselected ewes in spring. In retrospect, we see no better structure for the control lines.The May joining corresponded to the anticipated maximum depth of anestrous at this location , 15. HowSeveral other studies attempted to reduce seasonality of reproduction in sheep –8, but fChanges in sensitivity to ram effect did not have a major impact on our results. Ewes were exposed to vasectomized rams in Phase 1 of the study, but not in Phases 2 or 3. For S ewes in Phases 2 and 3, most ewes lambed in the first 17 days of the lambing season Fig. . By contmelatonin receptor 1a gene in S and EC ewes [Mechanisms that control seasonal breeding in sheep are relatively well-understood  and invo EC ewes . Similar EC ewes  suggesteThe objective of our study was to genetically improve ewe fertility during the annual spring joining period. However, there has also been interest in identifying methods to improve ewe reproductive efficiency in a wider range of out-of-season and accelerated lambing systems . The potCorrelated responses to selection to improve fertility were modest. EBV for litter size increased by 0.1% per year through Phase 2 but plateaued in Phase 3. The heritability estimate of 0.04 ± 0.02 for litter size in the current study was lower than both the average heritability of 0.11 for litter size in sheep  and the EBV for body weight at 60, 90, and 120 d increased by approximately 0.5%/year, perhaps in association with secondary selection for BW in replacement lambs. Screening ram lambs based on SC and mating behavior did not always favor the largest lambs, but it is likely that some positive selection on BW occurred. There were no significant correlated responses in maternal effects on postnatal BW.Correlated responses in birth weight were more complex than those observed for postnatal BW. EBV for birth weight increased until 1996 but then slowly declined. However, maternal effects on birth weight declined in Phase 1 and, particularly, in Phase 2. Occasional problems with underweight lambs and reduced survival were observed in Phase 2. As a result, some positive emphasis was placed on EBV for maternal birth weight in the selection of ram lambs in Phase 3 and appeared to have a positive effect on lamb birth weight. These results were consistent with seasonal differences in birth weight in these populations and larger estimates of additive maternal variation for birth weight in autumn-born lambs . Our finCorrelated responses in SC were positive and largest (0.5%/year) at 90 d of age. Maximum testicular growth occurs at approximately 90 d of age , suggestOur findings indicate that selection can be used to improve realized fertility  in ewes joined in May and June and that changes in sensitivity to ram effect did not meaningfully contribute to the selection response. Genetic antagonisms with other performance traits were small, although a modest antagonism with maternal breeding values for birth weight may require attention in selection programs to maintain adequate lamb birth weights. The heritability of litter size in autumn lambing was lower than expected and could potentially limit the response to selection for prolificacy. This study is consistent with previous ones, i.e. that selection in these ewes shortened the seasonal anestrous by delaying the onset of anestrous until mid- to late summer with no corresponding delay in the onset of the next breeding season.


Altered walking patterns are often described in individuals with chronic ankle instability (CAI). Contemporary treatment paradigms recommend backward walking (BW) to improve locomotion in people with musculoskeletal disorders. The purpose of this study was to determine whether muscle activity and activation variability during BW differs between subjects with and without CAI.Sixteen participants with CAI and 16 healthy controls walked on a treadmill at their self-selected speed under BW and forward walking (FW) conditions. Surface electromyography (EMG) data for the peroneus longus, tibialis anterior, medial gastrocnemius and gluteus medius muscles were collected. EMG amplitude normalized to maximum voluntary isometric contraction (%MVIC) and the standard deviation (SD) of the %MVIC EMG amplitude was calculated throughout the gait cycle. In addition, the area under the curve (AUC) of the %MVIC EMG amplitude was calculated before and after initial contact .p < 0.001) and gluteus medius (p = 0.01), and increased medial gastrocnemius (p < 0.001) activation were observed during pre- and post-IC under BW condition.No differences between groups were noted in the %MVIC amplitude or activation variability (SD of %MVIC EMG) under BW or FW. In both groups, decreased tibialis anterior (Participants with CAI and healthy controls have similar muscle activity patterns during BW. Yet, the results should be interpreted with caution due to the heterogeneity of the CAI population. Ankle sprains are among the most common musculoskeletal injuries , 2. Up tThe precise analysis of electromyography (EMG) muscle activity during movement is an essential step in understanding sensorimotor control. Studies that assessed amplitude of muscle activity during walking reported differences between individuals with and without CAI. While walking, participants with CAI were reported to have increased peroneus longus activity before and after initial contact (IC) –11, loweAnother factor that seems to be affected by CAI is the variability of muscle function during walking. Up to date, two main techniques were used for assessing variability of muscle function during walking in subjects with CAI. Kautzky et al.  utilize Contemporary treatment paradigms for altered gait in people with CAI recommend gait training . BackwarBW has indeed been shown to improve control of movement in patients with neurological lesions and with musculoskeletal disorders. A recent study reported the effectiveness of BW as a rehabilitation technique for improving knee proprioception in patients after anterior cruciate ligament reconstruction . The benStudies that assessed muscle firing patterns in BW compared to FW indicated that it is not a simple reversal of the EMG pattern of FW , 24. TheThe current study investigated the effects of forward and backward walking conditions on EMG amplitude and variability of the peroneus longus, tibialis anterior, medial gastrocnemius and gluteus medius muscles among subjects with and without CAI. We hypothesized that during BW, compared to healthy controls, individuals with CAI would have increased amplitude and activation variability of peroneus longus and tibialis anterior muscles, as measured by EMG.The study was approved by the University Institutional Review Board approved the study, approval number: AU-HEA-20190213. All participants provided written informed consent.The study sample included 16 participants with CAI and 16 aged-matched healthy controls. The inclusion criteria for the CAI group were in accordance with The International Ankle Consortium recommendations , 28: (i)The control group included healthy participants with no history of ankle sprain. Exclusion criteria from both groups were history of ankle fracture, other pathological conditions or surgical procedures in the lower extremity within 1 year of study participation or had vestibular or neurological disorders.The study was conducted during one visit at the Neuromuscular and Human Performance Laboratory, at XXX University. EMG activity of the peroneus longus, tibialis anterior, medial gastrocnemius and gluteus medius muscles was evaluated under both FW and BW walking conditions, while participants walked barefoot on a treadmill. Participants were instructed to walk at a comfortable, self-selected pace. Before data collection, they were provided with an opportunity to habituate to forward and backward walking on the treadmill. At this time, walking speed was adjusted according to the participant’s comfort. This self-selected, comfortable speed during forward and backward walking was used during the gait assessments. EMG data were collected using a wireless EMG system  at 2000 Hz. Before data collection, participants’ skin was shaved and cleaned with isopropyl alcohol to minimize impedance. Electrode placement was performed according to Surface Electromyography for the Non-Invasive Assessment of Muscles (SENIAM) guidelines . LightweThe raw EMG signal was filtered through band pass filter between 50 and 500 Hz with the use of a fourth-order Butterworth filter, and processed with EMG works software . MVIC EMG signals were subjected to a root mean square (RMS) algorithm to convert all amplitude measures to positive values. Gait events were identified using inertial measurement sensors that were placed near the participant’s heel during FW and near the second metatarsal head during BW. Gait cycles were isolated by identifying consecutive heel or toes strikes. The vertical accelerometer signal from the sensor placed near the heel (for FW) or second metatarsal head (for BW) was used to identify initial contact. A foot contact was characterized as a rapid change in vertical acceleration signal followed by a rather lengthy steady-state period . SubjectThe EMG amplitudes during the gait cycle were transformed to 100 data points for each of the 15 consecutive strides, and their output was displayed as a percentage of the MVIC EMG value (%MVIC), which can be used to establish a common ground when comparing data between participants. In addition, to assess stride-to-stride variability of muscle activity, the standard deviation (SD) of the EMG amplitude (as %MVIC) was calculated for each participant at each of the 100 data points. The %MVIC served as the dependent variable for comparing EMG amplitude across the entire gait cycle between groups, whereas the SD of %MVIC served as the dependent variables for comparing activation variability across the entire gait cycle between groups. Finally, the area under the curve (AUC) of the muscles’ activation (%MVIC) was calculated before and after IC  during both walking conditions. Pre-IC was defined as 90–100% of the gait cycle, and post-IC was defined as 0–10% of the gait cycle.These periods during the gait cycle have previously been demonstrated to be relevant to the study of gait dynamics in people with CAI , 15. TheDescriptive statistics included mean (M) and standard deviations (SD). Simple chi-square and independent t-tests were used to compare baseline characteristics between the CAI and control groups.For between-groups analysis, under each walking condition, group mean muscle amplitude activity (%MVIC) and EMG variability (presented as SD) with their respective 95% confidence interval (CI) were plotted for each muscle throughout the gait cycle , 32. A sTo compare the adaptation of each group to BW, a linear mixed model with group and condition as fixed effects and participants as random effect was conducted to examine the effect of group and condition on the AUC during Pre -and Post-IC of each muscle separately. In addition, EMG amplitude difference between FW and BW was calculated (FW – BW) for each muscle and plotted with the respective 95% CI. As previously described, non-overlapping CIs for more than 3% of the gait cycle was considered as significant difference in adaptation. A significance level of < 0.05 was set. Analysis was conducted using IBM SPSS Statistics for Windows, Version 24.0 .Participant characteristics are presented in Table Figure Figure Figure The differences in EMG amplitudes between FW and BW are presented in Fig. The purpose of this study was to determine whether leg muscle EMG amplitude and activation variability during BW differs between groups with and without CAI. Major differences were found between BW and FW. However, in contrast to our hypothesis, no between group differences were evident. This may indicate that participants with CAI and healthy controls use similar motor adaptations to BW.To the best of our knowledge, no previous study has fully assessed EMG patterns during BW in people with CAI. Therefore, comparison is only possible with earlier reports that evaluated EMG during FW in this population. In a study that assessed EMG amplitude of the peroneus longus, tibialis anterior, lateral gastrocnemius, rectus femoris, biceps femoris, and gluteus medius muscles, no differences were noted between CAI and healthy groups at either 100 msec before or 200 msec after IC, during shod, treadmill walking at fixed 4.8 km/h speed. In this study, the area under the RMS curve was normalized to quiet standing . SimilarConsisted with the reports that evaluated EMG amplitude and found no differences between subjects with and without CAI, some evidence suggests no difference in muscle activation variability between these populations. Kautzky et al.  studied Yet, conflicting results were reported in other studies. Louwerens et al.  evaluateDue to the large discrepancies between studies, it seems that no sound conclusion can be drawn regarding leg muscle EMG amplitude and activation variability during gait and dynamic tasks among individuals with CAI. However, several explanations are proposed for the inconsistencies observed between studies. Some discrepancies might be due to the heterogeneity of the CAI population. The updated paradigm of CAI suggests that there is a list of impairments that people with CAI as a group, are likely to demonstrate; however, each individual may present certain clinical and performance outcomes that are affected by personal and environmental factors . SpecifiAnother aspect that could explain the variation in findings between studies is related to data collection. The reference for EMG normalization differs significantly between studies; thus, it is difficult to conclude whether the differences were due to the group or to the normalization methods. Furthermore, while some studies evaluated discrete EMG signals , 12, othAlthough BW did not distinguish between groups, it influenced muscle activation in both groups compared to FW. This finding is agreement with previous studies that reported changes in lower limb muscle activation patterns during BW, as compared to FW , 23, 27.Interestingly, a systematic review of studies that assessed the effects of CAI on muscle activity during FW concluded that while some studies observed significant differences between the CAI and the control groups, and others did not, it seems that increased activity of the peroneus longus pre-IC is often found in participants with CAI . HoweverThere are large discrepancies in EMG parameters between studies that assessed muscle activity during gait in the CAI population. Based on previous research, in the present investigation we examined both EMG amplitude and AUC, which are two of the most commonly used outcomes . FurtherBackward gait training is becoming a popular treatment method for people with musculoskeletal disorders. Therefore, it is important to understand how the application of BW affects motor control among individuals with CAI. This study indicates that, compared to FW, participants with CAI and healthy controls demonstrate significant changes in mean EMG activation and activation variability in leg muscles during BW. However, there are no differences between groups. Due to the heterogeneity of the CAI population, the results should be interpreted with caution. Future investigations that evaluate motor control during BW with larger and varied cohorts of people with CAI are warranted.


Extracellular signal-regulated kinase (ERK) is a part of the mitogen-activated protein kinase (MAPK) signaling pathway which allows the transduction of various cellular signals to final effectors and regulation of elementary cellular processes. Deregulation of the MAPK signaling occurs under many pathological conditions including neurodegenerative disorders, metabolic syndromes and cancers. Targeted inhibition of individual kinases of the MAPK signaling pathway using synthetic compounds represents a promising way to effective anti-cancer therapy. Cross-talk of the MAPK signaling pathway with other proteins and signaling pathways have a crucial impact on clinical outcomes of targeted therapies and plays important role during development of drug resistance in cancers. We discuss cross-talk of the MAPK/ERK signaling pathway with other signaling pathways, in particular interplay with the Hippo/MST pathway. We demonstrate the mechanism of cell death induction shared between MAPK/ERK and Hippo/MST signaling pathways and discuss the potential of combination targeting of these pathways in the development of more effective anti-cancer therapies. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway represents a highly conserved signal-transduction pathway comprised of three individual kinases, namely RAF, MEK and ERK . MAPK/ERDevelopment of resistance in cancer cells represents major problem during monotherapy with a single MAPK/ERK inhibitor. Resistance development often arises as a result of the MAPK/ERK cross-talk with other signaling pathways such as the PI3K/AKT/MTOR signaling pathway . On the In this review, we tend to discuss the impact of the MAPK/ERK cross-talk with other signaling pathways and MAPK/ERK protein interactors on the regulation of individual cellular processes in cancer cells. We focus mainly on cross-talk between MAPK/ERK, Hippo/MST and PI3K/AKT/MTOR signaling pathways in various cancer cells and discuss interplay between these pathways in the context of current cancer therapies. We highlight similarity between MAPK/ERK and Hippo/MST signaling pathway in regulation of proliferation and induction of cell death in cancer cells. Finally, we discuss combination targeting of MAPK/ERK-Hippo/MST-PI3K/AKT/MTOR signaling pathways for improve outcomes of current cancer therapies.MAPK1/ERK2 and MAPK3/ERK1 represent two well-known serine/threonine kinases involved in the modulation of the MAPK signal transduction pathway. ERK1 and ERK2 kinase acts as the final signal node of the MAPK/ERK signaling pathway and participates in the regulation of key cellular processes such as proliferation, metabolism, differentiation and survival. ERK1/2 kinase phosphorylates broad spectrum of protein substrates which are involved in transcription regulation, cytoskeleton organization, protein phosphorylation, protein translation and cell death induction. The canonical MAPK/ERK signaling pathway is often demonstrated as a positive regulator of cellular proliferation, growth and survival. Selective inhibition of individual kinases of the MAPK/ERK signaling pathway using targeted inhibitors represents a promising method of effective cancer treatment, however, resistance development seems to be serious problem ,5.ERK1/2 kinase shares large number of protein interactors. BioGRID database contains 270 unique interactors of ERK2 kinase . HoweverActivation of ATF2 by ERK kinase promotes growth of intrahepatic cholangiocarcinoma and induces apoptosis in MDA-MB-435 breast cancer cells ,8. A decERK kinase shares several protein interactors with the MST kinase representing the central kinase of the Hippo signaling pathway . ActivitERK kinase regulates the upstream components of the MAP kinase signal transduction pathway as well as its own activity by oneself in feedback loops occurring in internal or external, positive or negative, regulatory feedback loops. In internal negative feedback loops, ERK abrogates SOS (Son of sevenless) complex formation and in internal positive feedback loops it stimulates downstream interactors of growth factor signaling at the growth factor receptor level. In external negative feedback loops ERK inhibits EGFR  signaling and in external positive feedback loops it stimulates the activity of growth factor signaling through external factors. This model can partially explain bistability and oscillations in the ERK signaling as demonstrated using theoretical dynamic modelling performed by Arkun et al. . MTORC1 V12 in HEK293T cells induces NOXA expression and BECLIN1 autophagic cell death in an ERK-dependent manner [Inhibition of the MEK/ERK signaling blocks proliferation and induces apoptosis in cancer cells. Contrary to this, several reports demonstrate activation of MEK/ERK signaling as a key apoptotic mechanism triggered by anti-cancer drugs in cancer cells ,52. Thest manner . Recovert manner . Activatt manner . Galectit manner . ERK andt manner . Activatt manner . Activatt manner . A decret manner . Cross-tt manner . Activatt manner . A critit manner . Cytotoxt manner . Inhibitt manner . Hyperact manner . Expresst manner . Inductit manner . ERK sigt manner . Sustaint manner . ERK alst manner . Activatt manner . Biphasit manner . These rMYC oncogene in T-ALL cells and breast cancer cells as well as in xenograft mice model of breast cancer [The Hippo signaling pathway controls development and organ size in diverse species. Deregulation of this pathway induces tumor formations in model organisms and occurs in a broad spectrum of human carcinomas . MST1/2 t cancer ,77. MST t cancer ,78,79. Mt cancer ,81,82. Mt cancer . Hippo/Mt cancer ,85,86. Mt cancer ,88,89. Mt cancer ,90. Intet cancer ,62,63. Ft cancer .+ breast cancer cells [Hippo/MST signaling and ERK signaling pathways share several targets to regulate proliferation and cell death in cancer cells . Activater cells . Pan-MTOer cells . Rapamycer cells . Severaler cells ,99. Flaver cells . The poler cells ,102,103.er cells ,105. Sever cells ,80. An ier cells . Pyranocer cells . Tetracyer cells . Flavoneer cells .Targeted inhibition of the MAPK/ERK signaling pathway triggers tumor growth suppression and cell death in cancer cell models. The final effect of the MAPK/ERK regulation depends on actual cross-talk with other signaling pathways active in cancer cells. RAS/RAF signaling activates PI3K/AKT as well as MEK/ERK signaling pathways and could be inhibited by ERK and AKT kinase in feedback loop ,111,112.Dual-face activity of the MAPK/ERK signaling has a significant impact on clinical outcomes of the MAPK/ERK targeted therapies. The combination of PI3K/AKT/MTOR and MEK/ERK inhibitors can potentially decrease therapy efficiency due to different impacts on the Hippo/MST signaling pathway. While active PI3K/AKT/MTOR signaling has a negative impact on the Hippo/MST activity, activation of the MEK/ERK signaling potentiates the induction of cancer cell death through the Hippo/MST signaling pathway. Based on this evidence, a combined inhibition of the MAPK/ERK pathway at the levels of RAF and MEK/ERK kinase seems to be effective in cancer cells depending exclusively on the activity of the MAPK/ERK signaling pathway. Reactivation of the MEK/ERK axis in cancer cells addicted to inhibition of the Hippo/MST pathway through the PI3K/AKT/MTOR signaling pathway can be beneficial for therapy efficiency after targeted PI3K blockade. Moreover, activation of the Hippo/MST signaling and chemical regulation of its downstream targets can significantly boost clinical outcomes of targeted therapies based on MAPK as well as PI3K signaling inhibitors. Detailed characterization of the MAPK/ERK regulatory network brings also perspectives for the treatment of several other pathologies such as metabolic syndromes, rare genetic diseases and neurodegenerative diseases .


The effect of rapid maxillary expansion (RME) on the upper airway (UA) has been studied earlier but without a consistent conclusion. This study aims to evaluate the outcome of RME on the UA function in terms of aerodynamic characteristics by applying a computational fluid dynamics (CFD) simulation.ms), and maximum wall shear stress (Pws) were compared by paired t-test and Wilcoxon test according to the normality test at T0 and T1.This retrospective cohort study consists of seventeen cases with two consecutive CBCT scans obtained before (T0) and after (T1) RME. Patients were divided into two groups with respect to patency of the nasopharyngeal airway as expressed in the adenoidal nasopharyngeal ratio (AN): group 1 was comprised of patients with an AN ratio < 0.6 and group 2 encompassing those with an AN ratio ≥ 0.6. CFD simulation at inspiration and expiration were performed based on the three-dimensional (3D) models of the UA segmented from the CBCT images. The aerodynamic characteristics in terms of pressure drop (ΔP), maximum midsagittal velocity (Vms (m/s) decreased from 2.79 to 2.28 at expiration after RME (P = 0.057).The aerodynamic characteristics in UA revealed no statistically significant difference after RME. The maximum VThe aerodynamic characteristics were not significantly changed after RME. Further CFD studies with more cases are warranted. Adenoid hypertrophy (AH) is a common cause of upper airway (UA) obstruction in children and adolescents. Considerable variation in AH prevalence, ranging from 27 to 80%, has been reported between countries and ages . AH may AH may cause abnormal craniofacial development such as a short cranial base, long face, small and narrow maxilla, and mandibular retrusion –12. SomeRME’s possible effect on nasal obstruction has been evaluated by several methods including rhinomanometry, acoustic rhinometry, polysomnography (PSG), cephalometric radiographs, cone beam computed tomography (CBCT) and computed tomography (CT), but with inconsistent conclusions , 16. LabThe CFD method is a well-established technique that has been widely used in mechanical engineering, yet it is quite new to flow analysis in medicine. Based on a three-dimensional (3D) structure segmented from CBCT, CT, or magnetic resonance imaging (MRI), the CFD simulates and calculates the flow of gases or fluids and their interactions with the surrounding surfaces as defined by boundary conditions. At a given inlet pressure, the shape and boundary condition of a pipe-like UA would theoretically determine the aerodynamic characteristics in terms of pressure, velocity, and wall shear stress. The application of CFD in dentistry is nevertheless sparse. Few previous studies have shown that CFD could be applied to evaluate the outcome of mandible advanced devices on respiratory function , 19. RegIn this study, we aim to evaluate the effect of RME on airflow within the UA by investigating the aerodynamic characteristics that result from applying CFD simulation. The null hypothesis is that RME has a positive effect on UA ventilation.This is a retrospective cohort study. All methods were carried out in accordance with the declaration of research involving human subjects and the regional ethical and scientific guidelines in Vestland region, Norway. Data for all patients who had undergone RME were retrospectively collected at the Department of Orthodontics  between January 2013 and December 2016. The inclusion criteria were patients younger than 15 years old who had both pre- and post-CBCT scans due to orthodontic indication. The pre-RME CBCTs were taken within seven days prior to fixing the expander (T0) and the post-RME CBCTs at the removal of expanders (T1). The exclusion criteria were severe abnormalities of maxillofacial tissue, previous surgery on skeletal and soft tissue related to respiration, and previous orthodontic or orthopaedic treatment. Eventually, 17 patients  were eligible for inclusion in the study. An experienced radiologist viewed all CBCT scans and ensured that the images were qualified to construct 3D models of the UA.A fixed Hyrax expander was used for RME, banded to the maxillary first premolars and first molars. The patient, or their guardian, rotated the expansion screw twice a day at home and a clinical check-up was performed by orthodontists once a week. The expansion was terminated when the occlusal aspect of the maxillary lingual cusps of the upper first molars contacted the occlusal aspect of the vestibular cusp of the mandibular first molars. After achieving the desired expansion, the expander remained in place for 5.2 ± 1.7 months to stabilise the expansion.Figure The examination protocol of CBCT scans was as follows: field of view (FOV) 16 × 13 cm; tube potential 120 kVp and tube current 5 mA; scanning time 14.7 s . The voxel size was set at 0.2 mm, and the contrast resolution had a 14-bit depth. All CBCT examinations were performed according to the standardised clinical routine, i.e. with the Frankfort horizontal plane parallel to the floor, teeth in maximum intercuspation, and peaceful nasal breathing without swallowing. We divided the 17 patients into two groups according to the AN ratio at baseline (T0): group 1 was comprised of individuals with an AN ratio < 0.6 and group 2 encompassing those with an AN ratio ≥ 0.6. The measurements of AN ratios were performed aiming to present the geometric obstruction status of the UA following Fujioka’s method , which wMesh generation is the practice of creating a mesh by computer algorithms. The continuous geometric UA space may be subdivided into discrete geometric cells. Mesh cell is the fundamental element of the reconstructed space that contains a local approximation of aerodynamic characteristics, which will be used for a later calculation. We chose tetrahedral and prismatic cells to construct the main body and boundary layer of the UA . Each UA mesh had five boundary layers and an average of 2 million elements. The inlet and outlet of UA were defined at the extended superior and inferior boundary, as earlier described.–6. A radiologist (XF) performed all the simulations under the technical supervision of a fluid engineer (YCC). The CFD simulations were repeated six months later on ten randomly selected cases by the same operator (XF).ANSYS Fluent  was applied to simulate the airflow of UA, and the SST κ-ω model was used to calculate the aerodynamic characteristics of UA. The wall of UA was defined as no-slip, stationary, and rigid, and the temperature and density of air were set as fixed . In the ms), and maximum wall shear stress (Pws) at T0 and T1. Data were processed using the IBM-SPSS version 25.0 . Significance was set at p less than 0.05. Statistical tests for normality were conducted for all variables. Accordingly, paired t-test or Wilcoxon test was used to compare the changes of the aerodynamic characteristics between T0 and T1. Intraclass Correlation Coefficient (ICC) was applied to test the consistency of the CFD simulations.We calculated the aerodynamic characteristics at inspiratory and expiratory phases, including mean pressure at the four planes defined on UA Fig. . The parms and maximum Pws of the UA between before (T0) and after (T1) RME were shown in Table The comparison of aerodynamic characteristics in terms of ΔP, the maximum Vd after T RME werems than group 1 at both inspiration and expiration regardless of T0 or T1; whereas the maximum Pws shows the opposite trend being lower for group 2.Among the 17 patients, ten patients were classified in group 1 (mean age 11.9 ± 1.3 years); seven patients in group 2 (mean age 12.6 ± 1.3 years). Figure ms (m/s) drop (2.79–2.28) at expiration is close to being significant (p = 0.057).Due to the limited number of cases, group 1 and group 2 were merged when performing the statistical analysis on the effect of RME. Except for ΔP at inspiration, all the other aerodynamic parameters decrease after RME treatment  at expiration is nearly significant (p = 0.057). This finding is in line with previous reports where the airflow resistance at expiration was found to be closely related to obstructive severity [In the present study, CFD simulation was applied to elucidate the aerodynamic characteristics of the UA before and after RME. The null hypothesis was rejected, i.e. RME does not have a positive effect on UA ventilation. We failed to observe any statistically significant change in airflow characteristics after RME despite overall declines of ΔP, maximum VE 2.79–2. at expirseverity . Also, Cseverity .The effect of RME on aerodynamics has been investigated sparsely. In contrast to the present study, Iwasaki et al. found significant changes in aerodynamic characteristics in the nasal cavity after RME . This mams seemed to be lower in group 1 as compared to group 2 regardless of T0 or T1 (Fig. ws.Enlarged adenoid is a common cause of nasal obstruction in children. Knowledge of aerodynamics in this group of patients would help understand the disease mechanism, assist diagnostics and evaluate treatment outcomes. Due to the small sample size, we did not perform statistical analysis on the effect of RME for each individual group. Nevertheless, ΔP and maximum V T1 Fig. , indicatws and a pharyngeal jet were observed [ws. Consequently, the maximum Pws was lower in group 2 than group 1 at T0. After RME the adenoid jet may be weakened, resulting in a increased maximum Pws in group 2. However, due to limited cases and the diverse airflow characteristics in group 2, random effect can not be excluded. Therefore, more cases with severely enlarged adenoids are needed to confirm our assumption.It has been reported that one of the most restricted areas in UA was located at the velopharynx where the maximum Pobserved , 32. In CFD is a valuable tool for investigating the aerodynamic characteristics of the UA for better understanding the complex airflow ventilation related to UA morphology. At present, the simulation procedure is not entirely automatic and thus very time consuming. Part of the 3D segmentation and mesh generation needs to be performed manually due to the irregular anatomic structure of the UA. This may be the cause for the limited number of samples in the available CFD studies , 33, 34.The CFD method makes the aerodynamic characteristics within the UA visible. However, due to the intrinsic nature of a retrospective study design, the lack of clinical otolaryngologic examination makes it difficult to conclude whether RME would affect the airflow condition. Nevertheless, the enlarged adenoid may influence the UA’s ventilation. Further perspective study is warranted to identify the specific patients who may benefit from the RME.The aerodynamic characteristics were not significantly changed after RME. Further CFD studies with more cases are warranted.


During the 2010s, fertility rates fell across the Nordic region. The onset of these declines seems linked to the Great Recession of 2008–2009, but their continuation cannot easily be linked to subsequent economic change. The 1990s, too, brought episodes of economic crises to the Nordic region that were followed by different degrees of fertility decline. In this study, we provide an empirical overview of parity-, age- and education-specific fertility developments in the five Nordic countries in the wake of the economic recessions in 2008 and the early 1990s, respectively. We demonstrate a high degree of heterogeneity in fertility developments across countries after 1990, whereas after 2008, the trends are much more similar across the five countries. Likewise, the educational differences in birth hazards that characterized the developments after 1990 were much smaller in the initial years after 2008–2009. This reversal from heterogeneity to homogeneity in the fertility response to recessions calls for an expansion of theories on the cyclicality of fertility in relation to uncertainty and economic and social change. In our discussion, we consider the role of a set of factors that also incorporates the state, crisis management, and perceptions of economic and welfare uncertainty. The Nordic countries—Denmark, Finland, Iceland, Norway, and Sweden—are widely recognized for having generous family policies, protective and employment-oriented welfare systems, high rates of labor-force participation among both men and women, and, until very recently, comparatively high fertility levels. After the large decline in fertility across Western countries in the 1970s, total fertility rates (TFRs) in the Nordic countries remained in the range between 1.7 and 2.3 children per woman for several decades (1980–2010). Only in Denmark and Sweden did it temporarily fall to 1.4 and 1.5 children per woman in the mid-1980s (Denmark) and mid-1990s (Sweden), respectively. During the same period, most other countries in Europe consistently registered fertility rates below 1.7, and many Southern and Eastern European countries even experienced lowest-low fertility rates below 1.3 children per woman , and prolonged unemployment benefits from one to two years . In the years leading up to the crisis, the country saw an increase in private and public indebtedness, with an accumulated foreign debt around eightfold Iceland’s GDP. Housing prices had increased significantly; the trade deficit was large and the currency overvalued. In late 2008, consumption fell by roughly 20 percent, and in 2010, the unemployment rate rose to 7.6 percent Ólafsson . Social The second-most-affected country in the group was Finland. First, when the Great Recession hit, the country had not fully recovered from the previous crisis. Second, idiosyncratic shocks, such as crises in the information- and communication-technology sectors (with Nokia), and the Western embargo on and recession in Russia, one of Finland’s largest trade partners, negatively affected the Finnish economy. Third, a rigid salary structure and rapid decline in competitiveness of Finnish workers relative to other countries made it more vulnerable  as the baseline year from which the deviation in the relative risk is measured. By applying event-history techniques to the population-register data of five countries, we are able to get an accurate picture of similarities and differences in childbearing responses to the two episodes of recession in the countries considered. We thus investigate 5 × 2 country-period cases of childbearing dynamics before, during, and after the crises, running separate but identical models for each country. Our focus is on relative changes in fertility levels of each country in the wake of each recession rather than differences in absolute levels of fertility between countries that belong to the same Nordic fertility regime  and educational level: primary (including lower-secondary level), secondary (upper-secondary level), and tertiary education. Results are mainly presented graphically by parity, age group, and educational level to facilitate the cross-country comparison. Full tables of relative risks are available in Appendix.Figure In all Nordic countries, the relative risk of first birth declined in the years following the recession onsets, and this happened in both recessionary periods. Relative to 1990, the strongest drop in the risk of first birth was witnessed in Sweden, followed by Iceland and Norway. A delayed and milder, but still observable, reduction in the risk of first birth occurred in Denmark and Finland. Ten years after the onset of the crisis, Sweden still had a relative risk of first birth 40 percent lower than in 1990. In the other four countries in 1999, the relative risk of first birth was around 10–20 percent lower. , followed by Iceland. In comparison, after 2008, the same two countries, if anything, witnessed an increase in the relative risk of higher-order births. In Denmark, the opposite held, with higher-order fertility increasing after 1990 and decreasing after 2008. Finally, in Norway and Finland there was basically no change in the risk of third and fourth births in the 1990s, while a drop was observed in third births in both countries, beginning in 2013 in Finland. An analogous drop in fourth births after 2013 was evident in Finland, while in Norway, the decline started much earlier.Overall, we can conclude that, as found in previous studies, lower parities were more uniformly negatively affected by economic conditions across periods and contexts , remained stable (Denmark and Norway), or declined much less (Sweden) than the first-birth risks of low- and medium-educated women. In the first years after 2008, such educational differences in the development of first-birth risks no longer existed; the relative risks of entering parenthood declined at the same pace in each educational group. However, after 2014 primary educated women register a steeper continued drop in their relative risk of becoming a mother. We further notice that while in the 1990s, it was primarily the behavior of tertiary-educated women that differed from the others (in positive terms), after 2014 it was the childbearing of primary educated women that deviated (in negative terms) from those of the mid and high educated. With respect to second births, educational differences after 1990 were only visible in Finland and Denmark. After 2008, the development of second-birth risks across educational groups was more homogenous in all countries, but primary educated women displayed a more negative trend. Only at very high parities did the educational gradient in period changes reverse and tertiary-educated mothers displayed the steepest declines in relative risks of childbearing.income effect) or joblessness (uncertainty effect), they may perceive their futures as less secure if the economy slows down or skids into a recession (perceived economic uncertainty effect). In response, couples may postpone childbearing during a recession, and prefer to wait-and-see  in each Nordic country. Our systematic comparison of parity-, age- and education-specific childbearing risks across two crises and five “similar” welfare states allows us to reflect on existing theories and contemplate neglected theoretical links that may be pursued empirically in future research. Our empirical presentation was mainly descriptive, and we did not provide the rationale for empirical tests of different theoretical hypotheses. We leave for future research to, for example, evaluate the differential role of long-term family change, macroeconomic factors and labor-market restructuring, individual sociodemographic circumstances, perception of uncertainty, and policy changes during recession episodes to disentangle the extent to which the decline in fertility may be related to each component.In our study, we focus on the development of childbearing during a crisis, but even more so on the development The study produced several thought-provoking findings. Finland’s fertility increase among highly educated women during the 1990s mass unemployment is interesting. The countercyclical tendencies in Finland’s fertility during the 1990s recession have been shown elsewhere Vikat . Here, wMore generally, although the five Nordic countries have shared similar macroeconomic outlooks during the last half century, with similar labor-market structures, women’s employment rates, welfare-state orientation, and, as we have shown, homogenous trends in fertility rates in the late 1980s, there was significant country heterogeneity in the fertility developments after the 1990s recession. Moreover, even though in most Nordic countries, the more recent crisis in 2008 was milder and lasted less long than that in the 1990s, the latter seems to have had more persistent and uniform scarring effects on childbearing intensities across countries, parities, ages, and educational groups. Further, our findings show that the decline in first birth rates accelerated in the second half of the 2010s in three out of four countries for which we have data for these years, and, that first and second birth rates to low-educated women dropped at an increasing rate in these more recent years.crisis management—that is, the policies that are introduced to tackle economic turmoil—may be directed at the economic performance of the country—for example, by stabilizing the banking sector or supporting specific industries. However, crisis management may also involve amendments of labor-market, social, and family policies ; Finland reacted less severely and less abruptly; and Denmark expanded its parental-leave program and introduced job-rotation programs, while at the same time imposing shorter durations and stricter eligibility criteria for receiving unemployment benefits. Subsequently, all five Nordic countries mobilized and extended their active labor-market programs  and supported education programs for young people, particularly those with lower levels of training.In 2008, the situation was different. The 2008 crisis also came unexpectedly. However, it had a common external cause and it was handled more quickly and less idiosyncratically with respect to social policies. Furthermore, the countries concerned could rely on the lessons learned during the 1990s , there have been no signs of trend reversals. The same goes for the five Nordic countries examined in this study. The magnitude of the fertility rate contraction in the USA is very similar to that of the Nordic countries, with a TFR declining from 2.12 in 2007 to 1.73 children per woman in 2018 CDC . This siAlthough demographers often view national fertility developments in the context of the nation state, such a confinement may no longer be suitable. Today’s world is much more interconnected than in the early 1990s, economically as well as socially. The availability of mass communication channels that were not yet established in the early 1990s, such as smartphones and social media, may increase the perception that the future bears unknown uncertainties that cannot be controlled locally or nationally. Global changes in production or economic turmoil elsewhere tend to affect national economies and labor markets more than before, while the capacity of states to influence such global events have become more limited. Similarly, other events that happen internationally, e.g., terror acts and armed conflicts, and developments that cannot be contained nationally, such as climate change or pandemics, may increase people’s perception of uncertainty in a global perspective. At the time of the writing (2020), this becomes clearly evident as the ongoing COVID-19 pandemic is not only a global health crisis, but most expert also expect a new long-lasting global economic crisis, despite different national investment programs and social-policy measures.We do not know the extent to which subjective factors of this kind may lie behind the surprisingly uniform 2010s fertility decline in the Nordic countries despite the countries’ underlying steady material circumstances and the stability of the Nordic welfare state. A study on regional fertility trends in Europe by Matysiak et al.  also hig


Both thymic stromal lymphopoietin (TSLP) and dendritic cells (DCs) are involved in many autoimmune diseases, but the potential roles of TSLP and DCs in bullous pemphigoid (BP) have not been clarified. We sought to explore the contributions of TSLP and DCs in patients with BP.  TSLP levels in sera and blister fluids were measured by enzyme-linked immunosorbent assay. The TSLP expression in the BP lesional skin was detected by immunohistochemical staining. Infiltration of DCs, marked by DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), and its relationship with TSLP and TSLP receptors was evaluated by immunofluorescence staining.  We found that TSLP levels in sera and in blister fluids of patients with BP were higher compared to the control groups. In patients with BP, TSLP levels in sera correlated with TSLP levels in blisters. The expression of TSLP in the BP lesional skin was higher compared to the healthy controls' skin. Greater numbers of TSLP-positive cells were observed in the epidermis of patients with BP compared to the healthy controls. Greater numbers of DC-SIGN-positive cells were present in the BP lesional skin compared to the skin of controls. The expression of TSLP was highly upregulated in DC-SIGN-positive cells, and most DC-SIGN-positive cells expressed TSLP receptors.  We conclude that TSLP may activate DC-SIGN-positive DCs directly, which may be involved in the pathogenesis of BP. Bullous pemphigoid (BP) is the most common autoimmune blistering disease, which typically presents as pruritic vesicles and bullae. Distinguishing histological features of BP include subepidermal blisters accompanied by dermal infiltrates of eosinophils, neutrophils, and mononuclear cells, as well as linear deposition of autoantibodies and complement along the basement membrane zone (BMZ) as measured by immunofluorescence . AutoantThere are two main types of APCs in the skin: Langerhans cells (LCs), which are predominantly located in the epidermis, and dendritic cells (DCs), which are located in the dermis . LCs werThymic stromal lymphopoietin (TSLP), an IL-7-like cytokine, participates in several autoimmune diseases, such as eosinophilic esophagitis, inflammatory bowel disease, and rheumatoid arthritis , 17. In In this study, we detected TSLP levels in sera and blister fluids and further determined the TSLP expression, DC-SIGN-positive DCs, and LCs in the skin of patients with BP and healthy controls (HCs). Our result suggested that TSLP may potently activate DC-SIGN-positive DCs in the pathogenesis of BP, which will be helpful for our further in vivo investigation to clarify the mechanism.We retrospectively assessed 35 Chinese patients with BP from 2016 to 2018, in the Dermatological Department of Peking Union Medical Collage Hospital . These pn = 35) and HCs (n = 44); blister fluids were taken from patients with BP (n = 35) and patients with other blister dermatoses (n = 39), such as eczema, toxic epidermal necrolysis, contact dermatitis, herpes, and burns (n = 10) and leftover tissue that remained after excision of nevus or cysts (n = 9). The collected tissues were fixed in 10% neutral buffered formalin for 20 h at room temperature, embedded in paraffin, and then sectioned at 6 μm. Prior to immunohistochemistry and immunofluorescence, the tissues were deparaffinized in xylene, rehydrated in a series of graded ethanol, and then retrieved by heat treatment.Serum samples were obtained from patients with BP , according to the manufacturer's recommendations. Optical densities were measured at 450 nm and 540 nm using the Varioskan Flash Multimode Reader .Paraffin sections were deparaffinized and were then blocked with 3% hydrogen peroxide for 15 min, followed by incubation with the goat serum solution for 1 h at room temperature. Next, rabbit anti-human TSLP  was added and incubated overnight at 4°C, followed by incubation with HRP-conjugated affinipure goat anti-rabbit IgG . The specimens were then stained with DAB , followed by washing and counterstaining with hematoxylin .For quantitative image analysis, the slides were scanned using NanoZoomer 2.0-RS , and five visual fields were selected randomly for each slide. The number of positive cells was counted at ×40 magnification by four dermatologists separately, and each slide's average number of positive cells was recorded. The staining density of the TSLP expression in the epidermis was converted into a numerical value by using the ImageJ analysis system , a public domain Java image processing program .μg/ml, Solarbio, Beijing, China) for 5 min. Slides were viewed in the Vectra 3.0 Automated Quantitative Pathology Imaging System .Paraffin sections were deparaffinized and were then blocked with bovine serum albumin (10%) for 1 h at room temperature. Next, primary antibodies, which consisted of mixtures of either mouse anti-human langerin  or mouse anti-human DC-SIGN  with rabbit anti-human TSLP  or goat anti-human TSLP receptor , respectively, were added, followed by overnight incubation at 4°C. FITC-labeled donkey anti-mouse IgG , AF647-labeled donkey anti-goat IgG , and AF647-labeled donkey anti-rabbit IgG  were used as secondary antibodies, which were incubated for 1 h at room temperature. Finally, the sections were incubated with 4,6-diamino-2-phenyl indole  was used for statistical analysis. Prism 7 software  was used to generate statistical graphs.The number of positive cells in the skin and the density of the TSLP expression in the epidermis were presented as the means ± standard deviations. TSLP levels in sera or blister fluids and the TSLP expression in the skin were expressed as medians and interquartile ranges due to lack of normal distribution, according to the Shapiro-Wilk test. Student's p < 0.001p < 0.001; rs = 0.52, p = 0.0013; p = 0.089; ELISAs were performed in order to examine TSLP levels in sera and blister fluids of patients with BP and controls . The levp = 0.0307, 2, p < 0.001, To evaluate the expression of TSLP in the skin, we measured it in patients with the BP lesional skin and HCs' skin using immunohistochemical technology . CompareTo analyze APCs in BP, we next stained DC-SIGN or langerin in five BP lesional skins and five HCs' skins by immunofluorescence Figures  and 4. TTo investigate whether the TSLP expression in BP is associated with APCs, TSLP was stained with either DC-SIGN or langerin by immunofluorescence experiment on slides of five patients with BP and five HCs . StrongeFinally, to reveal whether TSLP produced by keratinocytes in BP affects DCs' infiltration in the lesional skin directly, TSLPR was stained with either DC-SIGN or langerin by immunofluorescence experiment on slides of five patients with BP . The DC-Our present study showed that the TSLP expression and DC-SIGN-positive DCs' infiltration increased in the samples of patients with BP, and TSLP might affect DC-SIGN-positive DCs in BP. We identified increasing expression of TSLP in sera, blister fluids, and lesional skin of patients with BP. Our results showed that DC-SIGN-positive DCs infiltrated in the BP lesional skin more extensively, compared to the skin of HCs. Furthermore, in this study, we provided evidence that TSLP was relevant to DC-SIGN-positive DCs in BP, and TSLPR was expressed in DC-SIGN-positive DCs. Together, these data strengthen the hypothesis that the interaction of DC-SIGN-positive DCs-TSLP-TSLPR may be involved in the pathogenesis of BP.TSLP, as a Th2-related cytokine, was found to be involved in BP recently –21. By iTSLP is produced by various types of cells, such as epithelial cells, keratinocytes, fibroblasts, and DCs , 25. It εRI+CD207− cells, which were presumed to be inflammatory dendritic epidermal cells, in the BP lesional skin were significantly higher than those in the perilesional skin of patients with BP and the skin of HCs [Both LCs and DCs, as the main professional APCs of the skin, promote production of antibodies by regulating T follicular helper cells , 30. DCsn of HCs . In our n of HCs . In our In previous research, the Th2 immune response was shown to likely play a key role in BP , 34. MorNevertheless, LCs in the epidermis did not present a clear association with TSLP. Unlike our study, Soumelis et al.  found thIn summary, our research demonstrated that the levels of TSLP were higher in the lesional skin, blister fluids, and sera of patients with BP than in HCs or other blister dermatoses. DC-SIGN-positive DCs, which infiltrated near the TSLP-positive epidermis and expressed TSLPR, infiltrated the dermis in BP lesion more extensively than the skin of HCs. Together, our findings add to the accumulating evidence that TSLP expressed by epidermal keratinocyte activates DC-SIGN-positive DCs directly, which may be involved in the pathogenesis of BP. A further study is warranted to reveal specific subtypes of DCs activated by TSLP, clarify the relationship between LCs and TSLP, and uncover the downstream mechanisms of TSLP and DCs.


Among patients with GBS, CSF UA levels were higher in those with demyelination (p = 0.022), although the difference was not significant after multiple testing correction. CSF UA levels in GBS were positively correlated with serum UA levels  and CSF lactate . However, no significant correlations were found between CSF UA levels and GBS disability scores. There were no significant differences in serum UA levels among GBS, NIND, and healthy controls. These results suggest that CSF UA may be related to the pathogenesis of demyelination in patients with GBS and may be partially determined by serum UA and the impaired blood–nerve barrier.Uric acid (UA) is a natural scavenger for peroxynitrite and can reflect antioxidant activity and oxidative stress in several neurological disorders. Changes in serum and cerebrospinal fluid (CSF) levels of UA have been reported in patients with multiple sclerosis and neuromyelitis optica spectrum disorders. The levels of UA in CSF are relatively poorly understood in patients with Guillain–Barré syndrome (GBS). It remains unclear whether UA can play an antioxidant role and reflect oxidative stress in GBS. The purpose of this study is to investigate CSF and serum UA levels in patients with GBS and their relationship with clinical characteristics. The CSF and serum UA levels were detected in 43 patients with GBS, including 14 acute inflammatory demyelinating polyneuropathy (AIDP), 6 acute motor axonal neuropathy (AMAN), 13 with acute motor and sensory axonal neuropathy (AMSAN), 7 Miller Fisher syndrome (MFS), and 3 unclassified, and 25 patients with non-inflammatory neurological disorders (NIND) as controls. Moreover, serum UA levels were also detected in 30 healthy controls. The levels of UA were measured using uricase-based methods with an automatic biochemical analyzer. CSF UA levels were significantly increased in patients with GBS ( Uric acid (UA) is the final metabolite of purine degradation in the human. As a natural scavenger for peroxynitrite and oxygen radicals, UA is thought to be one of the most important antioxidants and responsible for nearly two-thirds of the total plasma antioxidant activity , 2. The Guillain–Barré syndrome (GBS) is an acute onset autoimmune inflammatory peripheral neuropathy. The clinical features of GBS are characterized by rapid progressive symmetrical muscle weakness, decreased tendon reflexes or areflexia, and neuropathic pain . Abnorman = 14), AMAN or AMSAN , MFS (n = 7), and unclassified (n = 3). Twenty-five age- and sex-matched patients with non-inflammatory neurological disorders (NINDs) were enrolled as controls, including seven patients with subacute combined degeneration, four patients with mental disorders, four patients with cerebrovascular disease, two patients with cerebral venous sinus thrombosis, two patients with myasthenia gravis, two patients with nutritional and vitamin deficiency neuropathy, two patients with cervical spondylosis, one patient with migraine, and one patient with spinal cavernous hemangioma. Thirty healthy controls from the Health Center of Tianjin Medical University General Hospital were also enrolled in this study. Cases including patients with GBS, NIND controls, and healthy controls who had been treated with acetylsalicylic acid, antitubercular drugs, thiazide diuretics, or other drugs that could affect UA levels  antibodies status, electrophysiology, and CSF parameters of enrolled GBS patients were obtained from our GBS database. The preceding infections of the GBS patients were determined based on the influenza-like syndrome (such as fever and respiratory signs) and gastrointestinal tract infection  within previous 4 weeks before the onset of weakness. The severity of disease was evaluated using the GBS disability score  at nadirSerum and CSF UA levels were measured using uricase-based methods with a LABOSPECT 008 α Hitachi Automatic Analyzer  according to the manufacturer's instructions. The normal range of serum UA levels in our hospital is 140–414 μmol/L.p < 0.05 was considered statistically significant. Mann–Whitney U-tests were conducted for quantitative data analysis between two groups and Kruskal–Wallis H-tests for three or more groups. Correlation between CSF UA levels and age, serum UA levels, CSF parameters, and GBS disability scores were evaluated by Spearman's rank correlation analysis. The one-way ANOVA was used to compare age or other normally distributed continuous data among three or more groups. The chi-square tests or Fisher exact tests were conducted to compare qualitative variables. The Bonferroni correction was performed to adjust p-values for multiple testing.All analyses were performed using SPSS, version20  and GraphPad Prism, version 7 . The quantitative data were presented as mean ± standard deviation (SD) or median with interquartile range (IQR) depending on whether the data were normally distributed. In summary, age was normally distributed. The UA levels in CSF and serum, GBS disability scores, and CSF parameters were not normally distributed. A p = 0.284 for age and p = 0.906 for gender). In patients with GBS, 55.8% had preceding infections, 44.2% had cranial nerve involvement, 65.1% had antiganglioside antibodies positive, and 90.7% had albuminocytologic dissociation.Demographic and clinical features of enrolled cases in the present study are summarized in p = 0.011, p = 0.004, p = 0.285].In the present study, CSF UA levels were significantly higher in patients with GBS compared with NIND [12.10  μmol/L vs. 7.00  μmol/L, p = 0.175, p = 0.650, The serum UA levels in GBS, NINID, and healthy controls were 302.00  μmol/L, 315.00  μmol/L, and 337.00  μmol/L, respectively. However, there were no significant differences in serum UA levels among these three groups (p = 0.022). The serum UA levels of female GBS patients were significantly lower than male patients (p = 0.011). However, after multiple testing corrections , neither of these differences remained significant. The differences in UA levels were not statistically significant in other clinical characteristics in patients with GBS.To investigate the UA levels in GBS patients with different clinical characteristics, 43 patients with GBS were further divided into two subgroups by gender, preceding infections, antiganglioside antibodies, GBS disability scores, cranial nerve involvement, and nerve conduction study (NCS) results (excluded two patients whose NCS results were not available). As shown in r = 0.455, p = 0.002; r = 0.310, p = 0.043; r = 0.499, p = 0.001; p-value = p-value*11 characteristics), the correlations with serum UA levels (adjusted p = 0.022) and CSF lactate (adjusted p = 0.011) were still significant. However, no significant correlations were found between CSF UA levels and age , GBS disability scores , CSF white blood cells , CSF lactate dehydrogenase , CSF adenosine deaminase , CSF high-sensitive C-reactive protein , CSF glucose , or CSF chloride .In the present study, Spearman's rank correlation analysis was conducted to investigate the relationship between CSF UA levels and age, serum UA levels, GBS disability scores, and CSF parameters in patients with GBS. CSF UA levels were positively correlated with serum UA levels (There has been a growing interest in the role of UA in the nervous system due to the potential for antioxidant therapy and reflection of oxidative stress and purine catabolism in neurological diseases , 32, 33.UA is considered a natural antioxidant and believed to have neuroprotective functions by counteracting the peroxynitrite-mediated oxidative stress , 32. OxiThe CSF UA levels in CNS neuroinflammatory diseases were inconsistent in different studies. Some studies found no significant difference in CSF UA levels between MS patients and controls , 38, 39,Little attention has been paid to UA in peripheral nerves system diseases such as GBS. In this study, the elevated CSF UA levels in patients with GBS may be interpreted as the compensatory reaction of antioxidant activity against the increasing oxidative stress. In the study of Tang et al. , lipophiNotably, unlike other subtypes of GBS, patients with AIDP showed significant increase in CSF UA levels compared with NIND. Within GBS patients, cases whose NCS results indicted demyelination had higher levels of CSF UA than cases without demyelination, although the difference was not significant after multiple testing correction. The pathophysiological differences in subtypes of GBS have been extensively studied . AntibodCSF UA levels are affected by multiple factors , and theSeveral limitations to this study need to be acknowledged. First, the sample size is small in the present study, especially when considering the subtypes of GBS, which may be the limit for firm conclusions. Future research requires a larger sample size and a sufficient number of cases in subtypes of GBS. Second, allantoin, a product of the oxidation of UA, and other products of purine, were not detected in this study. Future work should therefore include allantoin and purine metabolites such as hypoxanthine and xanthine to evaluate oxidative stress and purine catabolism in patients with GBS. Third, although the relationship between CSF UA and demyelination has been proposed with evidence in this study, further studies are needed to verify the lipid peroxidation in GBS probably through estimating malondialdehyde, a biomarker of lipid peroxidation and oxidative stress . Fourth,In summary, this study has shown that the levels of UA in CSF were significantly increased in patients with GBS and AIDP, suggesting that CSF UA may be related to the pathogenesis of demyelination in patients with GBS and may be partially determined by serum UA and the impaired blood–nerve barrier. As a natural antioxidant and the end metabolite of purine catabolism, elevated UA levels in CSF may reflect oxidative stress and enhance purine or ATP metabolism in GBS.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Medical Research Ethics Committee at Tianjin Medical University General Hospital. The patients/participants provided their written informed consent to participate in this study.LY, X-BT, and S-HC contributed to the design and data interpretation. S-HC and X-BT contributed to the drafting of the manuscript. S-HC, JW, and L-JZ contributed to data analysis. M-QL, YQ, C-NH, and C-LG contributed to data collection, case diagnosis, and confirmation. L-SS contributed to sample analysis. LY, X-BT, and D-QZ contributed to critical review. All authors contributed to the article and approved the submitted version.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.


The Wnt signaling pathway plays important roles in organ development and disease processes. Secreted frizzled-related protein 2 (sFRP2), a vital molecule of Wnt signaling, can regulate cardiac development and cardiovascular disease. Recent studies have suggested that sFRP2 is not only an antagonist of the canonical Wnt signaling pathway, but also has a more complex relationship in myocardial fibrosis, angiogenesis, cardiac hypertrophy and cardiac regeneration. Here, we review the role of sFRP2 and Wnt signaling in cardiac development and cardiovascular disease. The heart is one of the most essential organs for maintaining the normal operation of activities of daily living, and the development of the embryo heart and cardiovascular disease are complex processes. Exploring the regulatory mechanisms that underlie these processes will allow a deeper understanding of cardiac development and provide new therapeutic targets for cardiovascular disease.The Wnt signaling pathway is important for maintaining the homeostasis of embryonic development, and participates in the regulation of biological processes that include cell proliferation, differentiation, apoptosis, and cell localization The proteins of secreted frizzled-related protein (sFRP) family are antagonists of the Wnt signaling pathway, and sFRP2 is considered to be the most potent wg), the segment polarity gene of fruit flies, and the mouse proto-oncogene int-1. In 1982, Nusse et al. first discovered a Wnt gene in the study of a viral transcriptional mechanism in the mouse mammary gland that was found to transmit growth and development signals between cells Wnt genes have been found in mammals. Wnt proteins bind receptors on the cell membrane in an autocrine or paracrine manner to regulate target gene expression. The Wnt signaling pathway is widely involved in early embryo development, cell proliferation, migration, tumorigenesis, metastasis, and stem cell growth regulation 2+/ PKC pathway The term “Wnt” is derived from a combination of two words: wingless (Fz) receptor to form a large complex on the cell surface with low-density lipoprotein receptor-related protein (LRP)5/6 to activate scattered protein (Dsh/Dvl), and affect the distribution of intracellular axis protein Axin. Therefore, inhibiting the phosphorylation activity of GSK3-β leads to stable accumulation of β-catenin in the cytoplasm c-Myc and cyclin D1  have been identified in mammals The sFRP family has long been considered a class of antagonists of the Wnt signaling pathway. Five members of sFRP family  pathway, without affecting the canonical Wnt pathway Fzd5) also plays an important role during angiogenesis promoted by sFRP2. Endothelial cell tubes fail to form when Fzd5 is silenced in endothelial cells sFRP2 exerts pro-angiogenic effects through activation of non-canonical Wnt/CaIt is also generally understood that sFRP2 can exert an angiogenic effect through the above pathways in a wide variety of human tumors, including angiosarcoma, prostate cancer, renal cell carcinoma, lung cancer, and pancreatic cancer Cardiac hypertrophy is an adaptive response of heart under many pathological states such as pressure overload and β-adrenergic stimuli The HyperGEN study used linkage analysis in siblings with hypertension to determine that genetic variation in sFRP2 was associated with left ventricular hypertrophy The limited regenerative potential of cardiomyocytes has made cardiac regeneration therapy by stem cell transplantation a hotspot in cardiovascular disease research Sfrp2 can abolish the aforementioned benefits Bcl-2, inhibit cardiomyocyte apoptosis, and improve cardiac function Mesenchymal stem cells (MSCs) are believed to be a promising candidate stem cell for cardiac therapy in vivoApart from MSCs, sFRP2 also has multiple effects on the properties of other types of stem cells. Aubert et al. found that sFRP2 can promote the differentiation of embryonic stem cells into neural progenitors by antagonizing the canonical Wnt signaling pathway 2+/NFAT pathway, while without affecting the canonical Wnt pathway. Furthermore, sFRP2 has been shown to downregulate the canonical Wnt signaling pathway, and protect cardiomyocytes from pathological hypertrophy. In cardiac regeneration therapy, sFRP2 can improve the survival of stem cells in the adverse environment, and enhance the therapeutic effects for myocardial infarction. Therefore, we believe that sFRP2 is a promising therapeutic target for multiple pathophysiological conditions of cardiovascular disease. However, the specific mechanisms of sFRP2 in these pathophysiological processes require further exploration. Interventions in the Wnt signaling pathway using modified sFRP2 expression may be a promising area of research for heart diseases.SFRP2 plays an important role in the process of heart development and a various cardiovascular pathophysiological condition. In myocardial fibrosis, sFRP2 may play a bidirectional effect depending on the different stage of disease and concentration. Subsequent studies had shown that sFRP2 can promote angiogenesis by activating the non-canonical Wnt/Ca